untitled document prof. dr. mirnawati sudarmanto, faculty of veterinary medicine, bogor agricultural university, bogor (ipb), indonesia dr. ratih dewanti hariyadi, faculty of agricultural technology, bogor agricultural university (ipb), bogor, indonesia prof. dr. m.a. chozin, faculty of agriculture, bogor agricultural university (ipb), bogor, indonesia dr. soekisman s. tjitrosemito, faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. dahlia sukma, faculty of mathematics and natural sciences, the state university of jakarta (unj), jakarta 13220, indonesia dr. lisdar m. sudirman,  faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. nurita l. toruan-mathius, tissue culture laboratory, smart research institute (smartri), bogor, indonesia ir. soemaryono m.sc., biotechnology research unit for estate crops, bogor, indonesia dr. okky s. dharmaputra, seameo biotrop/faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. gayuh rahayu, faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. yadi haryadi, faculty of agricultural technology, bogor agricultural university (ipb), bogor, indonesia dr. zainal mahmud, indonesian agency for agricultural research and development (puslitbangbun), bogor, indonesia  biotropia no. 6, 1992/1993: 66-70 the adsorption of imazapyr by three soil types in indonesia s. tjitrosemito seameo biotrop, p.o. box 116, bogor, indonesia s. matsunaka and m. nakata department of biotechnology, faculty of engineering, kansai university, yamate-cho, suita, osaka 564, japan and lab. pesticide science, faculty of agriculture, kobe university, rokkodaicho, nadaka, kobe 657, japan, respectively abstract the adsorption of imazapyr in three indonesian soil types was investigated with labelled 14 c-imazapyr using freundlich adsorption isotherm. the availability of adsorbed imazapyr to plants as affected by washing and liming was assayed using root elongation of rice seedlings. red-yellow podsolic soil adsorbed imazapyr more than andosol and sandy soil of laladon. the adsorption was greater at lower ph. washing seemed to reduce the concentration of imazapyr as shown by the increasing length of rice roots. on the other hand liming facilitated higher concentrations of imazapyr in the solution as shown by the reduction of rice root length. the practical implication is discussed. introduction upland rice established in pot experiment by zero tillage technique on alang-alang sprayed with imazapyr at 1.5 kg a.i./ha died immediately after germination (tjitrosemito and purwanto 1991). however, imazapyr applied at 2.0 kg a.i./ha to soybean, planted using zero tillage technique in the field during the wet season, did not show any phytotoxic effect (tjitrosemito and suwinarno 1988). to understand its fate in soil, imazapyr adsorption in various soils and the effect of liming and washing were investigated. materials and methods the experiment was carried out at the laboratory of pesticide science, faculty of agriculture, kobe university, japan. three soil types, i.e. red-yellow podsolic soil (ryp), andosol and sandy soil of laladon were air dried, and sieved through a 1 mm sieve. the ph was measured in a 1:1 (w/v) soil: deionized water/slurry 66 the adsorption of imazapyr by three soil types s. tjitrosemito, s. matsunaka and m. nakata using ph meter (model com-10, dkk denki kagaku keiki co. ltd.). the ph values were 3.75; 4.83 and 5.40 for ryp, andosol and sandy soil of laladon, respectively. adsorption commercially formulated imazapyr (assault) was combined with 14 c-ring labelled imazapyr (specific activity: 43.916µci/mg) to obtain initial concentration of 5, 25, 45 and 65 µm. five grams of air dried soils (sieved through 1 mm) and 10 ml of herbicide solution were placed in 30 ml vial. the samples were equilibrated by shaking on a wrist action shaker for 24 hours at room temperature. the mixtures were transferred to centrifuge tubes and spun at 15 000 x rpm for 30 minutes. a 2-ml aliquot was removed from each tube and placed in 20 ml of scintillation cocktail and the 14 c-activity was determined by scintillation spectrophotometry. adsorption isotherms were constructed using best-fit regression equations. freundlich constant kf and 1/n were calculated from the equation: log (x/m) = log kf + (1/n) log c where, x/m = the amount of herbicide adsorbed (µmole/kg) c = herbicide concentration (µmole/i) in solution after equilibrium kf = freundlich constant, an indicator of relative adsorption of herbicide at unit concentration (i.e. c = 1 µmole/1) (1 /n) = indication relative linearity between adsorption and concentration. the phytotoxicity of imazapyr a rice bioassay was conducted by placing 5 g of air dried soil into 30-ml tubes containing commercially formulated imazapyr. the mixtures were allowed to equilibrate for 24 hr by shaking in a shaker. after equilibration, washing treatment was done by replacing the solution with deionized water and later on, 10 pre-soaked rice seeds were put in each tube. after 3 days, the rice root length was measured and analysed statistically. the treatments consisting of soil (red-yellow podsolic soil, andosol and laladon sandy soil), imazapyr (0, 0.1 and 0.2 ppm), liming (addition of caco3 at 0.1 % ( / + )) and washing (/ +) were combined factorially and randomized completely. 67 biotropia no. 6, 1992/1993 results and discussion the relationship between the amount of adsorbed imazapyr and its equilibrium concentration in 3 types of soil is presented in figure 1. red-yellow podsolic soil, the highly weathered soil with low ph adsorbed more imazapyr than andosol or sandy soil of laladon. the addition of caco3 somehow reduced the adsorption considerably. the calculation of freundlich equation showed the characteristic of adsorption (table 1). figure 1 . the relationship between the amount of imazapyr adsorbed and its equilibrium concentration from table 1 the value of (1/n) ranged from 0.67 to 1.10 indicating a nonlinear relationship between the amount adsorbed and the equilibrium concentration. freundlich constant of ryp showed the highest value (246.0µm/kg) indicating that it has the highest affinity for imazapyr. in ryp soil alang-alang is usually found abundantly. 68 the adsorption of imazapyr by three soil types s. tjitrosemito, s. matsunaka and m. nakata table 1. relative adsorption of imazapyr (kf) and coefficient of regression (r 2 ) on soils. soil lime kf (µ mole/kg) 1/n r 2 red-yellow podsolic soil 246.0 0.84 0.99 (ph 3.75) + 98.0 0.67 0.99 andosol 120.5 0.98 0.99 (ph 4.83) + 34.7 1.05 0.98 sandy soil of laladon 17.4 1.10 0.92 (ph 5.40) + 15.9 0.95 0.77 when the values of kf and ph were examined, there was a linear relationship, i.e. y = 756.22 134.4x (r 2 = 0.98), where y = kf and x = ph value. the adsorption of imazapyr increased with the decrease in soil ph, similar results were reported by stougaard et al. (1990), wehtje et al. (1987) and arnold (1981). when the soil was limed, soil ph increased and presumably more imazapyr molecules stayed in the solution. phytotoxicity of imazapyr rice (norin no. 8) as a test plant in this bioassay showed a severe root length reduction when exposed to the soil treated with 0.1 or 0.2 ppm of imazapyr solution (table 2 a). table 2. root length (mm) of rice as affected by imazapyr and washing, liming, and soil types. treatments imazapyr (ppm) 0 0.1 0.2 a . washing no washing 32.7 18.3 14.7 lsd (5%): 5.3 with washing 35.1 28.2 20.5 b . liming no liming 35.1 27.2 18.7 lsd (5%): 6.1 with liming 31.2 19.4 15.6 c . soil redyellow podsolic 31.8 28.7 20.7 andosol 31.0 20.3 16.8 lsd (5%): 5.3 sandy soil of laladon 23.8 20.8 15.3 69 biotropia no. 6, 1992/1993 the reduction of root length was modified by washing. root length was increased from 18.3 mm to 28.2 mm and from 14.7 mm to 20.5 mm for 0.1 ppm and 0.2 ppm imazapyr treatment, respectively. it seems that washing reduced the concentration of imazapyr in the solution, so less phytotoxicity was experienced by the rice root. it indicates that imazapyr can be leached away by the flowing water. on the other hand liming (0.1% caco3) increased the phytotoxicity to the rice plant as shown in table 2 b. the soil also contributed differential response to the rice root elongation. the availability of imazapyr seems greater under the sandy soil of laladon (table 2 c). the results of this experiment may explain the different results of imazapyr on crop growth, when imazapyr is applied during or at the end of the dry season. the crop introduced in the following wet season may suffer from imazapyr phytotoxicity. however, when imazapyr is applied in the wet season, crops established one month later will escape imazapyr injury because imazapyr molecules would have been leached by the rainfall. liming will further speed up leaching if carried out long enough before crops are planted. references arnold, p.w. 1981. surface electrolyte interaction. in d.j. greenland and m.b. hayes, eds. the chemistry of soil constituents, john wiley & sons, new york: 355-404. stougaard, r.n., p.j. shea and a.r. martin. 1990. effect of soil type and ph on adsorption, mobility and efficacy of imozaquin and imazethapyr, weed sci, 38: 67-73. tjitrosemito, s. and d. suwinarno. 1988. the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides. biotropia (2): 1217. ______, purwanto. 1991. the performance of upland rice established by zero tillage technique on imperata field. biotropia (5): 1014. wehtje, g., r. dickens, j.w. wilcut and b.f. hajek. 1987. sorption and mobility of sulfometuron and imazapyr in five alabama soils. weed sci. 35: 858-864. 70 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf biotropia no biotropia no. 15, 2000 : 1 – 7 control of bagras (eucalyptus deglupta) damping-off by fungicides emilio o. anino northern mindanao state institute of science and technology 8600 ampayon, butuan city, philippines abstract selected fungicides were tested to control damping-off affecting bagras seedlings in the central nursery of the paper industries corporation of the philippines (picop), surigao del sur, philippines. the fungicides, at three concentrations each, were applied once before seed sowing to control pre-emergence damping-off and applied again after germination to control post-emergence damping-off. ajax detergent (2g/l h2o), benlate (0.5 g/1 h2o), brassicol (1.5 gv'l h2o), and fungitox (1.0 g/1 h2o) provided the best level of control against the disease. ajax detergent is the most practical among the best chemicals because it is cheap, locally available, not a health hazard, and less polluting. key words : eucalyptus deglupta/ seeds/nursery/flwzoctoma so/am'/fungicides/application introduction anino (1983) reported that the fungus causing damping-off in bagras seedlings in the central nursery of the paper industries corporation of the philippines (picop), surigao del sur, philippines is rhizoctonia solani. r. solani is a soil-inhabiting fungus belonging to class deuteromycetes (fungi imperfecti) under order mycelia sterilia and genus rhizoctonia (bernett and hunter 1972). the pathogen also caused web blight in falcata (paraserianthes falcataria), mangium (acacia mangium) and bagras (eucalyptus deglupta). the fungus started inciting post-emergence damping-off when bagras germinants were about 3 days old. for about 20 days, thereafter, the pathogen fatally infected 60% of all seedlings in a lightly irrigated sowing bed (anino 1983). the objective of this study is to screen for chemicals that can control the damping-off disease. 1 biotropia no. 15, 2000 materials and methods treatments the chemicals and concentrations tested were as follows : the soil was sterilized by fire-heating for 24 hours. experimental design the treatments, in three replicates, were arranged in crd at the shedhouse in the picop experimental nursery. evaluation parameters were as follows: a. number of emerging seedlings for determining the effects of the treatments on pre-emergence damping-off. b. total number of healthy seedlings for determining the effects of the treatments on pre-post and post-emergence damping-off. data in percent were transformed first into arc-sine values before analysis in crd. all treatment means were compared using duncan's multiple range test. 2 control of bagras (eucalyptus deglupta) emilio o. anino seed box preparation seventy-eight seed boxes for 26 treatments in 3 replicates were prepared. seventy-five boxes were filled with unsterilized soil, while three boxes for control b were filled with fire-sterilized soil. the boxes were arranged in crd, 30 cm apart atop the sowing table in the shedhouse. the table was rid of ants. the soils in all boxes (except control b) were sprayed with mycelial suspension of r. solani and watered daily for 15 days to successfully contaminate the soil with live pathogen. chemical application fifteen days after inoculation, the soils in test boxes (except controls) were drenched with one liter of their prescribed treatments. the treated boxes were not watered for 5 days. thereafter, about 1100 bagras seeds taken from seedlots stored at 0°c with germination capacity of 90% were sown into each box. the approximate viable number of seeds was 1056. the boxes were then watered once daily. the amount of water was just enough to wet the seeds and the soil medium at about 2-cm deep to avoid leaching of the chemicals. determining treatment effects on pre-emergence damping-off five days after sowing, the number of emerging seedlings on each box was determined by: a) establishing three 5-cm wide sampling strips; b) making 100% tally of seedlings in each strip; c) taking the area (cm2) of the strip and converting the average number of seedlings to per cm2, and d) taking the inner area of the box and multiplying it with seedlings/cm2 to get the total number of seedlings on the box. the percentage of healthy seedlings on each box was used as indicator of the effects of the chemicals on the pathogen and was based on the 1056 viable seeds, which were equivalent to 90% germination capacity. determining treatment effects on post-emergence damping-off the same seedlings on the boxes were used as samples for testing the effects of the treatments on post-emergence damping-off. when most of the treated boxes showed one or more infected seedlings, the boxes were rid of the diseased seedlings and re-sprayed with 130 ml of their assigned chemicals. re-spraying was conducted 13 days after the first spraying. for 20 days, the infected seedlings per box per treatment were counted and subtracted from the number of emerging seedlings to get the total number of plants protected by the treatments after two spraying schedules. 3 biotropia no. 15, 2000 result and discussion protection on pre-emerging seedlings table 1 shows the treatments and their effects on damping-off table 1. effects of test chemicals on damping-off cv = 30% cv = 32 % note : 1. control a-soil not sterilized and not chemically treated but inoculated with r. solani. 2. control b-soil was sterilized but not chemically treated and inoculated with r. solani. 3. means followed had by the same latter(s) are not significantly different at the 5 % confidence level. the seedlings, which had successfully amerged from below or had grown at the soil surface, were considered to have been protected from the pre-emergence damping-off pathogen (r. solani) by the test chemicals. the numbr of healthy 4 control of bagras (eucalyptus deglupta) emilio o. anino seedlings per box per treatment concentration was, therefore, indicative of the fungicidal effectiveness of the test chemicals. table 1 shows that among 8 chemicals, 4 were effective as shown below: the number of seedlings protected by the above treatments was about equal because they were not significantly different at 5% confidence level. the above results suggest that the four chemical treatments were highly effective in sterilizing the r. solani infested soils, thus allowing a high percentage of seeds to germinate without infection. protection on 5-to 20-day-old seedlings table 1 also shows the effects of the chemicals on post-emergence damping-off after re-spraying 130 ml of each of the said chemicals onto the 5-day-old seedlings. as expected, the treatments effective in controlling pre-emergence damping-off had consistently protected significant number of growing seedlings. for 20 days, the infection incidence in treated seedlings ranged from 0 to 8.20%, while the incidence in the untreated ones (control-a) reached as high as 9.24%. based on the number of seedlings protected after two spraying schedules, the best fungicides were as follows: note : values followed by the same letters are not significantly different at 5% confidence level. 5 biotropia no. 15, 2000 in the fire-sterilized but not chemically-treated soil (control b), the number of emerging and healthy seedlings was 74.16% only or 11.36% lower than the 85.52% in the non-sterilized but pathogen-inoculated soil and subsequently treated with 2 g/l/h2o of ajax detergent. the sterilized soil was apparently contaminated later with r. solani because of the relatively lower number of emerging seedlings and subsequent infection of 0.56% of the plants (table 1). moreover, the current practice of sterilizing soil with fire will not always protect the seedlings from severe infection if greater amount of pathogen propagules were inadvertently introduced into the soil during routine watering. in fact, the incidence of damping-off, which occurred at a seed box in the greenhouse, reached as high as 95%. conclusion beniate (0.5 g/i h2o), ajax detergent (2 g/1 h2o),'brassicol (1.5 g/1 h2o), and fungitox (1.0 g/1 h2o), in that order, are effective fungicides against damping-off pathogen (r. solani) infecting bagras seedlings. spraying 130 ml of ajax detergent and beniate solutions onto a sowing box with slight infection incidence can protect 85.52% and 88.51% of the seedlings, respectively, for 20 or more days. being very cheap, less polluting, and locally available, ajax detergent is the practical fungicide for nursery use. it is recommended that ajax detergent (2 g/1 h2o) can be used for sterilizing soil medium for seed sowing. methods of application are as follows: spray evenly 1 liter of water mixed with completely melted 2 g ajax detergent onto the soil inside a sowing box up to dripping point. let the treated soil stand for 3 days before seed sowing. after sowing, water the seed boxes once a day and just enough to wet about 2 cm deep of the soil. irrigate only when absolutely necessary and allow soils to partially dry before re-irrigating. inspect daily all sowing boxes for any symptom of damping-off. if 1 or 2 seedlings were infected, spray 130 ml of ajax solution (about 4 passes of a plastic mist sprayer) onto all seedlings in the box. do not wait for the disease to spread because the seedlings could not be saved any more once infected. seedlings affected by damping-off fall into the soil. their stems and leaves are rotten, watery and "melting". do not water the seedlings on the day treatment is applied. water on the next day when absolutely necessary. if infection recurred, spray again the detergent solution. the same techniques, as enumerated, shall be followed if beniate, bassicol, and fungitox at their prescribed concentrations were applied instead of ajax detergent. acknowledgements the author is grateful to the paper industries corporation of the philippines for giving him the chance to conduct this experiment. 6 control of bagras (eucalyptus deglupta) emilio o. anino references anino, e. o. 1983. control of bagras damping-off by fungicides. progress report of frd-picop study number 303-04-04 (unpublished). bernett, h. l. and b. b. hunter, 1972. illustrated genera of imperfect fungi. 3rd ed.burgess publishing company, minneapolis, minnesota. 241 p. 7 biotropia vol. 28 no. 3,2021: 274 277 d o i : 10.1 1598/btb.2021.28.3.1276 beardless barb cyclocheilichthys apogon (vazenciennes, 1842) (cypriniformes: cyprinidae) i n madura island, indonesia veryl hasanl*, soemarn02, maheno sri widod03 and dewa gede r a i a wiadnya4 'department offisb health management and aquaculture, uniuersitas airlanga, surabaya 601 15, indonesia 2agmecotecbnology department, agriczilture faculty, uniuersitas bramjaya, malang 65145, indonesia 'aquatic resources management department, fisheries and marine science facult), uniuersitas brawqaya, malang 65145, indonesia 4fisheries and marine resources utilixation department, fisheries and marine science facalty, universitas brawjqa, malang 6 5 145, indonesia received 25 june 2019/accepted 11 december 2019 abstract the freshwater fish beardless barb cyclocbeilichtbys apogon (valenciennes, 1842) is a native species of southeast asia, including western indonesia area (borneo, sumatra and java). previously, the species was found only in the mainland of java island. this paper provides the first record of c. apogon in the lembung river, one of the major rivers in madura island, indonesia, thereby extending the species distribution up to 150 krn northeast from the place of earlier record. the specimens of c. apogon were characterized as follows: dorsal fin rays 12; anal fin rays 8 9; pectoral fin rays 17 18; lateral line scales 34 35. a detailed description of the morphological characters of the specimen is provided in this manuscript. keywords: cyprinid, distribution, freshwater fish introduction in the last glacial era, southeast asia and western indonesia (sumatera, borneo and java) were still connected as a single area called sundaland, where many blocth rivers were connected to each other, extendmg from indochina to the java sea (voris 2000). major rises in sea level in the south china sea and java sea that occurred in that era had chided the sundaland into several archipelagos (pubellier & morley 201 4). this geographical change has resulted in the isolation of several freshwater fishes (hubert e t al. 2015), one of which was the beardless barb cyclocheilichtt5y.r apogon (weber & de beaufort 19 1 6). c. apogon (class: actinopterygii; order: cypriniformes; family: cyprinidae) is a freshwater fish native to southeast asia and western indonesia (rainboth 1996; icottelat 'corresponding author, email: veryl.hasan@fpk.unair.ac.id 2001; i<enthao & jearranaiprepam 2018). the species is distributed widely in rivers across mainland java, including east java, central java and west java (weber & de beaufort 1913; roberts 1993). as reported in this paper, c. apogon was recently sighted in lembung river, madura island, thereby widening the previously known distribution range of this species. materials a n d methods nineteen (19) live specimens of c. apogon were obtained from local fishermen during a fieldwork carried out on 22 23 march 2019 in lembung river (7o02'21" s, 113°46'35" e) (fig. 1). administratively, the site is located on madura island, sumenep regency, east java province, indonesia. the fishng gear used by the fishermen was a small hook fastened with molluscs as the baits (stein eta/. 2012). cyclocheilichtbys apogon on madura island hasan et al. figure 1 lembung river, madura island where the cyclocheilichthys qbogon were collected on 23 march 2019 to ensure the identity of c. apogon, its of the skin above the upper lip. other morphological characters were analysed using morphological characters of the 5 preserved the weber and de beaufort's (1916) model. specimens are as follows: 12 dorsal fin rays; 8 9 anal fin rays; 17 18 pectoral fin rays; 34 35 results and discussion the nineteen (19) live specimens of c. apogon had total lengths between 7.8 and 16.1 cm and total weights between 55.5 and 204 g. five (5) of these were preserved in 96% alcohol solution (hasan e t al. 2019) and deposited as laboratory specimens at the hydrobiology laboratory, universitas brawijaya, malang, indonesia (ub.0002). the remaining fourteen (1 4) were transported in polyethylene bags with oxygen and were kept as livestocks at the fish reproduction laboratory, brawijaya university, malang, indonesia. identification qclocheikichth_ys apogon was distinguished from the other species of qclocheidichth_ys by having no barbels in the snout but with conspicuous folds lateral line scales; head pointed, lips swollen, with both lips evenly curved; dorsal deeply concave, origin of dorsal is opposite to 13th scale of lateral line and nearly in the middle of a line, connecting the end of the snout and the end of the shortest caudal rays, the location of which is nearer to snout in young specimens, far behind the origin of ventrals; anal concave, with a rather weak third spine that is longer than half head; pectorals reach the ventrals; ventrals are about equal to the pectorals, reaching or surpassing the anal; caudal deeply incised, while the lobes are rounded. coloration in fresh specimen: yellowish brown, upper parts are dark brown, and each scale has a dark spot at the base; vertical fins have darkish colors, the others more or less hyahe. a blotch was also found at the end of the lateral line. all of these characters were found in every specimen collected from lembung river, madura island (fig. 2). biotropla vol. 28 no. 3,2021 figure 2 specimen of cyclocheilichth_ys ajbogon captured on 23 march 2019 from lembung river, madura island distribution the discovery of c. apogon in the lembung river, madura island, is the first record of this species beyond its type locality (mainland java: batavia, buitenzorg, ngawi, surabaya and pasuruan) (weber & de beaufort, 1916), and represents the easterly extension of its previously known distribution that is about 150 km away (fig. 3). this new record of native species cyclocbeilichth_ys apogon in the lembung river, madura island, whch is its first outside mainland java is an important contribution in understanding species diversity and biogeography (souto-santos 2019). this new record of c. apogon has improved one's knowledge and understanding of the species particularly on its extended distribution range toward further northeast. several studies on indonesian freshwater fishes were focused on single rivers. crossocheilzls obsczlrms was recorded in sumatra (tan & i<ottelat 2009), and on batang hari basin (tan & kottelat, 2008). c. obscnm also observed on musi basin where the &stance between the location of the first and the second recording was more than 250 km (iqbal e t a l (2017). the presence of c. apogon on madura island is probably due to the previous connection of lembung river to east sunda river during the last glacial era (hanebuth et al. 2000; sathamurthy & voris 2006). the connection was then being cut off and lembung river became isolated due to rising sea levels. besides geological factors, the spread of freshwater fishes outside the mainland could occur due to human induced factors. figure 3 known distribution of cyclocheidchthys apogon. black square are the previous records of the species based on weber and de beaufort's (1916) observation, mainland java. the black triangle is the recent record on lembung river, madura island cyclocheilichth_ys qbogon on madura island hasan et al conclusion cyclocbeilicht&r upogon is a southeast asian native freshwater fish that is not only found in mainland java, but also in the island of madura which is at the eastern end of java. the existence of c. upogon in such a remote area is an added information on the distribution of freshwater fishes in indonesia. references hanebuth t, stattegger i<, grootes pm. 2000. rapid flooding of the sunda shelf: a late-glacial sea level record. science 288(5468):1033-5. hasan v, tamam mb. 2019. first record of the invasive nile tilapia, oreocbromis niloticus (linnaeus, 175 8) (perciformes, cichlidae), o n bawean island, indonesia. check list 15(1):225-7. https://doi.org/ 10.15560/15.1.225 hubert n, icadarusman, wibowo a. 2015. d n a barcoding indonesian freshwater fishes: challenges and prospects. d n a barcodes 3:144-69. https://doi.org/l0.l515/dna-2015-0018 iqbal m, yustian i, indriati w, setiawan d, setiawan a. 2017. crassocheihs obscums tan & kottelat, 2009 (teleostei, cyprinidae): distribution extension and first record for musi basin, south sumatra, indonesia. check list 13(6):1121-4. https://doi.org/l0.15560/13.6.1121 kenthao a, jearranaiprepam p. 2018. morphometric variations and fishery unit assessment of cyclacheilichth_ys qbogon (actinopterigii: cyprinidae) from three-different rivers in north-eastern thailand. pakistan journal zoology 50(1):111-22. icottelat m. 2001. fishes of laos. sri lanka &i<): wildlife heritage trust. rainboth wj. 1996. fishes of the cambodian mekong. f a 0 species identification field guide for fishery purposes. italy (it): fao. pubellier m., morley ck. 2014. the basins of sundaland (seasia): evolutionand boundary conditions: marine and petroleum geology 58:555-78. roberts tr. 1993. the freshwater fishes of java, as observed by kuhl and van hasselt in 1820-1823. zoologische verhandelingen 285:l-94. sathiamurthy e, voris hk. 2006. maps of holocene sea level transgression and submerged lakes o n the sunda shelf. the natural history j chulalongkorn university 2:l-44. souto-santos ica, ferraro ga, jennings wb, vergara gls, buckup pa. 2019. geographic distribution of phallocems eigenmann, 1907 (cyprinodontiformes, poeciliidae) in the ilha grande bay hydrographic region, rio de janeiro, brazil. check list 15(1):181-92. https://doi.org/10.15560/15.1.181 stein ja, shultz ad, cooke sj, danylchuk aj, hayward i<, suski cd. 2012. the influence of hook size, type, and location o n hook retention and survival of angled bonefish (albula uzllpes). fisheries research 113:147-52. voris hk. 2000. maps of pleistocene sea levels in southeast asia: shorelines, river systems and time durations. journal of biogeography 27(5):1153-67. weber mcw, de beaufort lf. 1916. the fishes of the indo-australian archipelago 111, ostariophysi: i1 cyprinoideaapodes, synbranchi. netherlands (nl): brill. biotropia no biotropia no. 14, 1999: 10-16 morphological comparison of three asian native honey bees (apis cerana, a. dorsata, a. florea) in northern vietnam and thailand n.v. niem and l. q. trung vietnam bee research and development center long ha, dongda, hanoi, vietnam abstract three species of asian native honey bees (apis cerana, a. florea and a. dorsata) from northern vietnam and thailand were morphologically analyzed for investigations on their geographic variations and relations. in vietnam, samples were collected from feral and managed colonies. in thailand, the collections were from feral colonies or from field bees on flowers. morphological analysis was carried out, using measurements common to honeybee taxonomy. measured characters were done under stereomicroscope with an ocular micrometer. anova program and multivariate statistical analyses were applied for treating the data. overall, a. cerana populations in northern vietnam are significantly morphologically different than from those in thailand. it may be due to their different geographic locations between the thai and vietnamese populations of a. cerana. a. florea bees from vietnam are generally bigger in size than those from thailand, but the differences are uncertain. in contrast, the body size of a. dorsata populations from thailand are bigger than those from vietnam. however, these differences are also not significant. it is necessary to take further comparative investigations of these bee species from both countries. key words: honey bee/apis cerana/apis dorsata/apisjlorea/moif>ho\ogy introduction in the genus apis, eastern honey bees (apis cerana), giant honey bees (apis dorsata), and dwarf honey bees (apis florea and apis andreniformis) are native to many countries in southeast asia. these bee species play an important role in producing large amount of valuable products for people and in pollinating agricultural crops. however, their value is not well documented. the studies of their biological and economic characteristics being useful for strains improvement are less than those of european honey bees (a. mellifera). a few publications relate to geographical variations and distribution of these honey bee species in different parts of southeast asia. ruttner (1988) reviewed the geographical distribution of a. dorsata, a. cerana, a. florea in asia. verma (1990) made an extensive investigation on the distributions of a. cerana in india. peng et al. (1989) reviewed studies on the biology and distribution of races of a. cerana in china. a morphological analysis of a. cerana and a. nigrocincta populations from southeast asia has been taken by damus and otis (1997). rinderer et al. (1995) published data on comparative morphology of the dwarf honey bees in southeast thailand and palawan, philippines. wongsiri et al. (1993) carried out a comparative investigation of some biological characteristics of a. cerana bees in china, thailand and their hybrids for the purpose of using biological measures to control varroa parasitic mites. 10 biotropia no. 14, 1999 therefore, at present, there is a big gap in information on the genetic biodiversity of asian native honey bees. it is not clear how many subspecies and ecotypes of these native bee species exist in southeast asia and how they are related to each other. the purpose of this paper is to present some preliminary results on comparative morphology of the three honey bee species: a. cerana, a. florea and a. dorsata in vietnam and thailand to contribute basic information on certain morphological parameters. materials and methods in vietnam, samples of the three honey bee species were collected from locations of four provinces in the north (haiphong, laichau, hoabinh, nghean). at each location, four to six colonies were sampled. from each single colony, a sample of 40 '-60 worker bees was collected. in thailand, a. cerana samples were collected in bangkok, partly from a feral colony, partly from field bees on flowers in the gardens. a. florea bees were also collected on flowers. a. dorsata bees were sampled in nan province by using high voltage light trap to attract the bees from 14 colonies in one tree to the light. this sample should contain bee individuals from different colonies. the sampled bees were killed with hot water (around 90°c) to obtain their stretched proboscis, then they were preserved in 70% ethanol. thirty worker bees of each location (five to ten bees per sample) were selected and analyzed morphologically on temporary slide preparations. the following morphological measurements were recorded: 1. length of proboscis 2. width of prementum 3. length of forewing 4. width of forewing 5. cubital index 6. length of femur 7. length of tibia 8. length of basitarsus 9. width of basitarsus 10. tergite 3, longitudinal 11. tergite 3, transverse 12. stemite 3, longitudinal 13. stemite 3, transverse 14. wax plate of sternite 3, longitudinal 15. wax plate of sternite 3, transverse the measurement was done according to the ruttner's methods (1988), using a stereomicroscope with an ocular micrometer. for most parts, numbers presenting lengths or widths of various structures are in millimeters. the data were analyzed using anova program, and the student's t test was used to test the null hypothesis from the two equal sources of data. a clustering analysis was also applied to analyze the relationship between two honey bee populations, and establish their tree diagrams. 11 morphological comparison of three asian native honey bees n.v. niem & l.q. tiling results the results of morphometric measurements from these honey bee species are presented in table 1. the results show that the body size of a. cerana in vietnam was bigger than that in thailand. of the 15 measured characters, the values of 11 characters were significantly bigger (1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14) particularly the cubital index, while the width of prementum and forewing were smaller. the body size of a. florea in vietnam was also bigger than those from thailand. however, these differences were not clear. six characters (3, 4, 5, 6, 12, 14) of the bees from vietnam were significantly larger than those from thailand. the remaining characters were slightly larger (10, 13), equal (2,7,9, 15) or smaller (1, 8, 11). in contrary to a. cerana and a. florea, the body size of a. dorsata in thailand was bigger than those from vietnam. qf 15 measured characters, the value of 11 characters of a. dorsata in thailand were higher (2, 3, 4, 5, 6, 7, 8, 11, 12, 14, 15), one character was equal (10) and three characters were smaller (1, 9, 13). interestingly, while the cubital index of a. cerana and a. florea in thailand were significantly bigger than those in vietnam, this character in a. dorsata of both countries was not significantly different. discussions the differences in body parts between a. cerana populations from vietnam and thailand are reasonably significant. twelve larger measured characters of a. cerana in vietnam indicate that these two honey bee populations may be considered as separating geographical groups. according to ruttner( 1988), the characters that can be considered to distinguish bee populations into subspecies levels are lengths of basitarsus, tergite 4, sternite 3, wax plate transverse of sternite 3, and cubital index. in this study, all these characters are significantly different between the two populations. the tree linkage diagram analysis (figure 1-a) shows the bee population in vietnam is also clearly distinguished from that in thailand. all the vietnamese bee populations are ranged in a distinct group (from 1 to 15) separated from the thai group ( from 16 to 30) and there is no overlap between them. our data support the ruttner's (1988) statement that a. cerana can be distinguished into two subspecies: a. cerana cerana and a. cerana indica. the former includes the bees of vietnam, afghanistan, pakistan, north india and china. the later includes the bees of thailand, malaysia, myanmar, bangladesh, sri lanka and south india, indonesia, philippines. comparing these results to the data of peng et a/.(1989) and verma (1990), it is recognized that the a. cerana bees from vietnam are closer to the bees in southern yunnan (china) and nepal, while a. cerana bees from thailand are closer to the bees in southern india. 12 13 figure la tree linkage diagram of apis cerana bee populations from northern vietnam (1-15) and thailand (16-30). overall, the body size of a. florea from vietnam is slightly bigger than those from thailand. however, the differences between these bee populations are too small to consider them as different ecotypes. the tree diagram (figure 1-b) shows an overlap between these two populations. therefore, it is necessary to take further studies on the relationship of these bee populations between vietnam and thailand. figure ib. tree linkage diagram of apis florea bee populations from northern vietnam (1-15) and thailand (16-30). 14 biotropia no. 14,1999 contrary to a. cerana and a. florea, a. dorsata bees in thailand are bigger in body size than those in vietnam, but the differences of the measured characters and the tree diagram analysis (figure 1-c) could not support any distinction at the species levels between these two bee populations. regarding the giant honey bees (a. dorsata), maa (1953) created a genus megapis with four species: megapis breviligula, m. binghami, m. dorsata and m. laboriosa. on the other hand, ruttner (1988) stated that there is very little evidence to support the idea of distinguishing a. dorsata into different subspecies, although its distribution is in a large range of the southeast asian region. however, according to his opinion, a. dorsata in this area can be separated only into two subspecies: a. d. breviligula and a. d. binghami. in this study, the differences in morphological characteristics of a. dorsata populations from vietnam and thailand are rather low to make any conclusion at the species level of classification. figure ic. tree linkage diagram of apis dorsata bee populations from northern vietnam (1-15) and thailand (16-30). acknowledgments we would like to thank prof. dr. siriwat wongsiri, mrs. wandee wattanachaiyngcharoen (bee biology research unit, chulalongkorn university, thailand) and dr. det wattanachaiyangcharoen, mr. sawang piyapichart (faculty of agriculture, natural resources and environmental science, naresuan university, thailand) for organizing a field trip to collect samples of a. dorsata. we are grateful to prof. nguyen dinh hien (hanoi agricultural university) for helping in statistical analysis. we thank prof. t. q. hoc (vietnam national university, hanoi) for helpful suggestions on the manuscript and the correction. 15 morphological comparison of three asian native honey bees n.v. niem & l.q. trung references damus, m.s. and g.w. otis. 1997. a morphological analysis of a. cerana f. and a. nigrocincta smith populations from southeast asia. apidologie 28:309 319. maa, tsing-chao. 1953. an inquiry into the systematics of the tribe apidini or honey bees (hym.) treubia21(3)525-640. peng, y.s., m.e. nasr and s.j. loske. 1989. geographical races of a. cerana fabricius in china and their distribution. review of recent chinese publications and a preliminary statistical analysis. apidologie 20:9 -12. rinderer, i.e., b. p. oldroyd, s. wongsiri, h.a. sylvester, l.l.de guzman, j.a. stelzer and r.m. riggio. 1995. a morphological comparison of the dwarf honey bees of southern thailand and palawan, philippines. apidologie 26:387-394. ruttner, f. 1988. biogeography and taxonomy of honeybees. springer-verlag, berlin. verma, l.r. 1990. honey bee resources: biology and management: 56 69. in beekeeping in integrated mountain development. oxford & ibh publishing, new delhi. wongsiri, s,, l. chariya, p. sureerat. 1993. biological control of varroa mite: mass rearing biological control agent by crossing the chinese strain apis cerana with the thai strain a. cerana indica by instrumental insemination. asian apiculture, p. 148 -155. 16 biotropia no. 8, 1995: 23-29 sources of mycorrhizal infection of shorea acuminata seedlings under laboratory conditions*) lee su see forest research institute of malaysia kepong 52109 kuala lumpur malaysia abstract uninoculated dipterocarp seedlings raised in normal field soil in nurseries were always found to have mycorrhizas after a few months. this study set out to determine whether dipterocarp seedlings could continue to grow and develop in the absence of mycorrhizas and also to determine possible sources of mycorrhizal infection of dipterocarp seedlings raised under laboratory conditions using shorea acuminata as a typical example. seedlings were planted in capped or uncapped perspex boxes containing sterile or non-sterile field soil and watered daily with sterile water or tap water. seedling growth and development of mycorrhizas were monitored at monthly intervals for up to seven months. seedlings grown in sterile soil remained uninfected after seven months while infection was found in some of the seedlings grown in normal soil regardless of whether they had been watered with tap water or sterile water. this showed that field soil (i.e. under grass) far from the forest contained suitable inoculum for forest tree seedlings. tap water and the air were not important sources of infection. however, mycorrhizal infection was very uneven indicating that the inoculum was probably very unevenly distributed in the soil or that the inoculum density was rather low. seedlings grown in sterile soil showed better growth than those grown in normal soil and infection of roots by parasitic fungi in the latter was also observed. key words: mycorrhizas/plant pathology/lnfections/shorea acuminata/seedlings. introduction enrichment planting is often conducted in logged forests in malaysia to ensure adequate stocking of dipterocarps for future rotations. for this purpose, dipterocarp seedlings mostly of shorea species are raised in nurseries whenever seed is available. shorea species, especially of the red meranti group are important for their valuable hardwood timber. under normal practice, freshly collected seeds with their wings removed are sown into boxes or beds containing washed sand or a soil/sand mixture. the seeds germinate very quickly, usually after five to seven days, and at the age of one month are transplanted into polybags containing an unsterilised mixture of *)paper presented at the second asian conference on mycorrhizae, acom '91, march 12-17, 1991. chiang mai, thailand. 23 biotropia no. 8, 1995 sand and top soil. the seedlings can be maintained in the nurseries without any fertilizer application for quite some time before outplanting without any visible symptoms of poor health. plants of over one year-old have been observed to do well in the nursery. no mycorrhizal inoculum is added but the roots of the seedlings are usually found to be ectomycorrhizal by about three months (unpubl. data; lee 1988). an account of the mycorrhizal association of the dipterocarpaceae is given by lee (1990). smits et al. (1988) reported that dipterocarp seedlings could not be successfully raised in the nursery in east kalimantan and that seedlings transplanted into secondary forest usually died. they stated that dipterocarps are obligately ectomycorrhizal and are thus unable to survive unless associated with their specific fungal partner(s) (smits 1983, 1985; noor and smits et al. 1987). moreover, smits et al. (1988) advocated various methods of inoculation of dipterocarp seedlings to ensure survival and successful establishment in the nursery and forest. this study was conducted to determine whether dipterocarp seedlings could continue to survive and develop in the absence of mycorrhizas and also to determine possible sources of mycorrhizal infection of seedlings raised under laboratory conditions similar to those found in the nursery. materials and methods experimental design the experimental design was a randomised 2.2.2 factorial: ± steamed soil, ± cap, ± watering with sterile tap water. each treatment consisted of five replicates. plant material shorea acuminata dyer seeds were obtained from trees growing in field 3 in the compound of the forest research institute of malaysia (frim) and germinated in boxes containing washed sand. s. acuminata is common in mixed dipterocarp forest and yields timber classed in the light red meranti group. soil mixture and planting top soil was collected from under grass at the farm in universiti pertanian malaysia and sieved to remove stones, roots and other organic debris before mixing with vermiculite in the ratio of 7:3. one portion of the soil mixture was stored for later use while the other portion was steamed in a soil steamer for one hour 24 sources of mycorrhizal infection of shorea acuminata seedlings lee su see on two consecutive days. the steamed and cooled soil mixture was then incubated in a tightly closed black plastic bag in the greenhouse for two weeks before further use. perspex boxes consisting of two sheets of clear perspex measuring 30 cm x 20 cm separated by a 1.5 cm polystyrene spacer and bound together with "cling film" were used as pots for the seedlings. a one month-old seedling was transplanted into each box with its roots in contact with one sheet of perspex. the boxes were then wrapped in black plastic and placed in plastic basins in the laboratory under a bank of fluorescent tubes emitting cool white light at an intensity of about 2000 lux. the daily temperature ranged from a maximum of 32°c to a minimum of 23°c. caps for the boxes were made from aluminium foil with a perforation to allow the seedling to emerge. caps were also fitted with a glass tube to facilitate watering. the plants were watered daily with either sterile or normal tap water. measurement and analysis at fortnightly intervals seedling height was measured and the roots inspected for formation of ectomycorrhizas. when present mycorrhizas were clearly visible through the clear perspex under the stereomicroscope. the experiment was terminated after seven months. relative growth rates were used to compare the performance of the seedlings under the various treatments. results and discussion all s. acuminata seedlings grown in steamed soil remained non-mycorrhizal but healthy after seven months. leaving the boxes uncapped did not produce any infection in the seedlings; neither did watering with normal tap water. it appeared that tap water and the air were not important sources of infection as had earlier been postulated and that steaming the soil mixture was effective in preventing infection. the results indicated that dipterocarp seedlings could easily survive and develop normally for up to seven months in the absence of ectomycorrhizas and fertilizer application. ectomycorrhizal infection of seedlings grown in normal soil appeared to be a random process. at least one seedling in each treatment (± cap, ± sterile tap water) was infected after seven months (table 1). since the soil mixture was the only common factor in all the treatments, and steaming the soil eliminated ecto-mycorrhizal infection, it appeared that the soil was the main source of infection. 25 biotropia no. 8, 1995 the random pattern of infection and the low infection rate was probably an indication of the random natural distribution and low density of the mycorrhizal inoculum in the field soil. it also appeared that soil under grass located several kilometers from the forest contained suitable dipterocarp mycorrhizal inoculum. it had previously been assumed, and in fact smits and co-workers (1988) reported, that such areas would not contain any suitable dipterocarp inoculum in the absence of dipterocarp host trees. earliest infection occurred at week 10 while infection was last observed to occur at week 17 (table 1). two main types of ectomycorrhizas were observed. these mycorrhizas have also been commonly observed on roots of nursery seedlings (un-publ. data). uninfected seedlings continued to grow normally but were generally smaller than the infected seedlings as reflected in the relative growth rates shown in figure 1. it appeared that ectomycorrhizas enhanced the growth of infected seedlings, the effect being more dramatic the earlier the infection. table 1. ectomycorrhizal infection of s. acuminata seedlings grown in normal soil under various treatments treatment number of seedlings infected infected at week tap water, uncapped 1 10 1 (2/5) 17 tap water, capped 1 d/5) 11 sterile tap water, capped 1 (1/5) 17 sterile tap water, uncapped 1 13 1 (2/5) 17 * figures in parentheses indicate the total number of infected seedlings in each treatment at the end of 7 months. roots of a number of seedlings grown in normal soil were attacked by root pathogenic fungi such as cylindrocladium sp. resulting in death of roots, the cessation of growth and yellowing of three seedlings after 10 weeks and the death of one seedling after 17 weeks. cylindrocladium spp. are relatively common in local soils and have been observed to be associated with death of dipterocarp seedlings in the natural forest (unpubl. data). attack by such root pathogenic fungi could have caused the death of the dipterocarp seedlings reported by smits and co-workers (1988) in indonesia. in comparison, all seedlings grown in steamed soil were healthy with normal green foliage until the end of the experiment and had much more extensive but 26 sources of mycorrhizal infection of shorea acuminata seedlings lee su see figure 1. relative growth rates of shorea acuminata under different treatments in normal unsterilised soil: -i tap water, capped; * tap water, uncapped; x sterile water, capped; . sterile water, uncapped; mean (± s.d.) for uninfected seedlings. normal uninfected root systems than seedlings grown in normal soil. steaming the soil before planting probably killed all potentially pathogenic organisms resulting in better seedling survival. the mean relative growth rates of seedlings grown in unsterilised or steamed soil were not significantly different (p ≤ 0.05) after 29 weeks (fig. 2). although the use of steamed soil for planting may results in higher survival rates and generally produce healthier plants, the economics of the practice must be given serious consideration before it can be adopted on a routine basis. based on the results of this study it would appear that the potting medium used for planting dipterocarp seedlings in the nursery is the main source of ecto-mycorrhizal inoculum and that earliest formation of ectomycorrhizas in the field normally takes a period of about 10 to 12 weeks. the inoculum found in field soil was also compatible with the seedlings and appeared to promote better growth. therefore, there appears to be no absolute necessity to inoculate dipterocarp seedlings in the nursery unless local conditions are such that absolutely no suitable inoculum 27 biotropia no. 8, 1995 figure 2. mean relative growth rates of shorea acuminata in steamed soil (•) and in normal unsterilised soil ( + ). is present. this would be highly unlikely since the results of this study show that soil located several kilometers from forest areas without dipterocarp host trees could still initiate ectomycorrhizal infection producing enhanced growth in infected plants. it should further be pointed out that nursery-raised dipterocarp seedlings offfopea and shorea spp. have been successfully planted, growing into normal trees in parks and urban areas. this further suggests that survival and normal development of dipterocarp seedlings can be achieved without inoculation with specific fungi. however, this does not mean that mycorrhizal inoculation of dipterocarps should be ignored as it has been shown that mycorrhizal infection definitely enhances the growth of seedlings. efforts should be made to find the most suitable mycorrhizal fungi and methods of inoculation for the economic production of dipterocarp seedlings suitable for planting in logged-over forests and other cleared areas to ensure the best chances for their survival and growth. 28 sources of mycorrhizal infection of shorea acuminata seedlings lee su see acknowledgements i would like to thank seameo biotrop for sponsoring my attendance at this conference, the international foundation for science for a grant that made this study possible, the dean of the faculty of forestry, universiti pertanian malaysia for use of facilities during my tenure there, and the director-general of the forest research institute of malaysia (frim) for permission to present this paper. references lee, s.s. 1988. do mycorrhizas play a role in the growth and development of parashorea densiflora sloot & sym.? in: m. mohinder singh (ed.). agricultural and biological research priorities in asia. proceedings of the ips symposium of science asia '87. 14-17 october, kuala lumpur, p. 167-171. lee, s.s. 1990. the mycorrhizal association of the dipterocarpaceae in the tropical rain forest of malaysia. ambio 19(8): 383-385. noor, m. and w.t.m. smits. 1987. pengaruh intensitas cahaya dan suhu tanah terhadap ektomikoriza dan pertumbuhan anakan shorea polyandra. in: komar soemarna, harun alrasyid, ishemat surianegara dan achdiat (eds.). presiding simposium hasil penelitian silvikultur dipterocarpaceae, 24 november 1987. jakarta, indonesia, p. 11-30. smits, w.t.m. 1983. dipterocarps and mycorrhiza: an ecological adaptation and a factor in forest re generation. flora malesiana bull. 36: 3926-3937. smits, w.t.m. 1985. specificity of dipterocarp mycorrhiza. in: r. molina (ed.). proc. 6th nacom. 25-29 june, 1984. bend, usa. smits, w.t.m., d. leppe and m. noor. 1988. metode inokulasi untuk persemaian dipterocarpaceae. edisi khusus no. 5, balai penelitian kehutanan samarinda. 29 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf biotropia no biotropia no. 12, 1999 : 25 30 the effects of a-chlorohydrin on the gestation of the wistar rat (rattus norvegicus) rahmaniah department of biology, university ofhaluoleo, kendari, indonesia and lien a.. sutasurya department of biology, bandung institute of technology, bandung, indonesia abstract the investigation of the effects of a-chlorohydrin on the gestation of the wistar rat has been carried out to determine whether a-chlorohydrin could interfere with the gestation of the rat and gestation period for optimal sensitivity. alpha-chlorohydrin was administered by gavage to three different groups of pregnant rats, namely on the first (pre-implantation period), sixth (early postimplantation period) or fourteenth (late post-implantation period) gestation day, at a single dose of 62.5; 75 and 90 mg/kg body weight. rats were sacrificed on the is* gestation day. number of implantations, gestation loss and post-implantation mortality were recorded. the results revealed that 75 and 90 mg/kg body weight of a-chlorohydrin significantly interfered with all the gestation periods used. for the preimplantation group, a-chlorohydrin decreased significantly the implantation number, but increased the gestation loss as well as the post-implantation mortality compared to the control groups. for the early post-implantation and late post-implantation groups, a-chlorohydrin caused a significant increase in post-implantation mortality compared to the control groups. from these results it is concluded that achlorohydrin at 75 and 90 mg/kg body weight influences the gestation of the wistar rat administered during the pre-implantation period as the most sensitive gestation period. key words : a-chlorohydrin//tattu? norvegi'ctu/rat/pre-implantation/early post-implantation/late postimplantation/implantation/gestation loss/post-implantation mortality. introduction rats comprise a major pest that often causes substantial financial loss to agriculture. there are several ways to control rat populations. for example, exposure of rats to rodenticides to kill the rats. the disadvantage of this approach is that rats will stay away from sites populated with dead rat remains. as a result, rats will move to other areas. another disadvantage is that rodenticides are likely to kill other animals. to control rat populations effectively and without detriment to other animals, it is essential to explore alternative approaches. one of the best alternatives is to use a chemical agent that would reduce fertility in rats without the need to kill them. investigations conducted during the past 35 years were carried out with steroid and non-steroid agents. among the non-steroid compounds, only a-chlorohydrin produced the ideal contraceptive effects in male rats (jones 1983). the results 25 blotropia no. 12, 1999 indicated that low dosages of a-chlorohydrin reduced fertility in males but this effect was reversible. at relatively high dosages, a-chlorohydrin effectively caused sterility in male rats. successful infertility has also been reported for a-chlorohydrin in boar (stevenson and jones 1982; 1985), rats (tsunoda and chang 1976; brown-woodman et al. 1979; paz and homonnai 1982), and macaca mullata (kirton el al. 1970). to achieve optimization of control of rat populations, successful contraception of females is essential. paz and homonnai (1982) reported that oocytes in female rats inseminated with sperm carrying low levels of a-chlorohydrin did not develop. although gao and short (1993) could not demonstrate that a-chlorohydrin produced an antifertility effect in female rats, we decided to investigate the effect of achlorohydrin on pregnancy. our results show that a-chlorohydrin significantly interfered with rat gestation. methods and materials this investigation was carried out on female rats, rattus norvegicus wistar, 90100 days old and a body weight of 180-200 gram. the antifertility drug, achlorohydrin, used in this study was supplied by sigma chemical at a concentration ofl.32g/ml. the general procedure used was as follows. female rats were mated in the afternoon, and the following day, a vaginal smear was obtained to determine the presence of sperm. when sperm was detected, the rats were designated to be in their first day of gestation. pregnant rats were divided into four groups of six rats per group. a single dose of a-chlorohydrin was administered by gavage on the first day of gestation (pre-implantation period), repeated on the sixth day of gestation (early post-implantation period) and again on the fourteenth day of gestation (late postimplantation period). one group of rats received treatments of 62.5 g/kg body weight of a-chlorohydrin, the second group received 75 mg/kg body weight and third group received 90 mg/kg body weight of the drug. the control group received distilled water. the rats were sacrified on the eighteenth day of gestation. the ovaries were removed and placed in 9% nacl. the uterus was cut opposite the implantation sites. the number of implantations, live fetuses, dead fetuses and resorption of embryos were recorded. the ovaries were microscopically examined and the corpus lutea were recorded. from these data, the following reproduction parameters were calculated for every rat: number of implantations percentage of implantation = —————————————— x 100% number of corpus lutea 26 the effects of a-chlorohydrin on the gestation of the wistar rat rahmaniah and l.a. sutasurya number of corpus lutea number of implantations percentage of gestation loss = ——————————————————————— x 100% number of implantations number of live fetuses percentage of post-implantation mortality = ——————————————— x 100% number of implantations the data were statistically analyzed for significance in difference by the lsd test. results and discussion pre-implantation period the pre-implantation period showed that at dosages of 75 and 90 mg/kg body weight gestation loss and post-implantation mortality increased significantly compared to the controls. whereas implantation showed a significant decrease (see table 1 and figure 1), the dosage of 62.5 mg/kg body weight ct-chlorohydrin produced no difference in implantation, gestation loss, and post-implantation mortality from the controls. table 1. percentage of implantation, gestation loss (gl), and post-implantation mortality (pim) for achlorohydrin treatments compared to control for all of the gestation periods gestation period dose (mg/kg b.w.) number of rats examined % implantation (x±sd) %gl (x±sd) % pim (x±sd) pre implantation (1" gd) 0 62.5 75 90 6 6 6 6 93.8 ± 0.3' 93.3 ± 0.31 79.6 ± 0.4" 77.7 ± 0.6" 6.2 ±1. 2" 6.7 ±1.3 20.4 ± 0.7" 22.3 ±1.0* 1.9 ±1.0" 6.1 ± 1.3* 16.7±1.5bc 25.3±l.lc early post implantation (e^gd) 0 62.5 75 90 6 6 6 6 94.7 ± 0.3" 94.8 ±0.3' 92.6 ± 0.8' 89.8 ±0.9* 5.3±1.5a 5.1 ± 1.4 8.0 ± 2.2' 1 0.2 ±2.3' 4.7 ±1.4* 5.4 ± 1.4' 25.0 ±1.0" 39.8±1.6c late post implantation (141" gd) 0 62.5 75 90 6 6 6 6 93.8 ± 0.3" 90.2 ± 0.71 87.6 ± 08* 89.6 ± 0.3' 6.1 ±1.4' 9.8 ± 2.1' 12.4 ±2.4* 10.4 ±1.3' 4.3 ±1.4" 5.4 ±1.4' 20.7 ±uc 24.3 ±1.0cd b.w = body weight gd = gestation day number followed by the same letter indicate that they are not significantly different (lsd test at p<0.05). 27 blotroplano. 12, 1999 figure 1. fetuses and embryo resorption for a-chlorohydrin treatments compared to control for preimplantation period (la gestation days). early post-implantation period at dosages of 75 and 90 mg/kg body weight, post-implantation mortality increased significantly, but implantation and gestation loss were not statistically different, compared to the controls (see table 1). at a dosage of 62.5 mg/kg body weight, a-chlorohydrin produced no difference in implantation, gestation loss, and post-implantation mortality from the controls. late post-implantation period at dosages of 75 and 90 mg/kg body weight, post-implantation mortality increased significantly compared to the controls. however, at these dosages implantation and gestation loss were not affected by a-chlorohydrin. at 62.5 mg/kg body weight, a-chlorohydrin had no effect on implantation, gestation loss, and postimplantation mortality. in all three implantation periods, a-chlorohydrin produced no difference in number of corpus lutea, live fetuses, and dead fetuses compared to the controls. 28 the effects of a-chlorohydrin on the gestation of the wistar rat rahmaniah and l.a. sutasurya the results obtained in this investigation clearly showed that a-chlorohydrin at 75 and 90 mg/kg body weight interferes with pregnancy when administered at all three gestation periods used, and administration during the pre-implantation period generally as the most effective stage. the agent's antifertility effect was most pronounced at 90 mg/kg body weight. at 62.5 mg/kg body weight the result was not significantly different from that of the control groups. alpha-chlorohydrin significantly decreased implantation and increased both gestation loss and postimplantation mortality. alpha-chlorohydrin decreased fertility by more than 50% and, therefore, qualifies as an antifertility drug. the mechanism of action of a-chlorohydrin in female rats is not known. in male rats, it is converted to 3-chlorolactaldehyde, which inhibits glycolysis and reduces atp synthesis in the sperm mitochondrion because less pyruvic acid is available for import into the organelle (stevenson and jones 1985). administration of a-chlorohydrin during the pre-implantation period decreased implantation and increased gestation loss. it is conceivable that the effects were caused by the drug as a result of impairment of transport of the zygote via the oviduct to the uterus, facilitated by muscle contraction and ciliar movement. this requires expenditure of a considerable amount of energy by way of atp supply (johnson and everitt 1988). this might reduce the transport rate of fertilized eggs and cause arrival of the zygote at implantation sites beyond the requisite period of 5th to 6th day of gestation. since administration of a-chlorohydrin during the pre-implantation period resulted in a decrease in implantation and increased post-implantation mortality in the course of eighteen days, this long-term effect may be attributed to storage of achlorohydrin in body fat to prolong its effect (paz and homonnai 1982). to account for the higher values of post-implantation mortality when the drug was presented in the early post-implantation period as compared to the results obtained when treatment occurred in the late post-implantation period, it is conceivable that the placental barrier, once completely formed during post-implantation treatment, may have reduced transfer of the drug and lower embryo resorption (juchau 1981). the present study supports the role of a-chlorohydrin as an effective antifertility drug in female rats. references brown-woodman, p.d.c., i.g. white and d.d. ridley, 1979. the antifertility activity and toxicity of achlorohydrin derivatives in male rats. contraception. 19: 517-527. gao, y. and r.v. short. 1993. use of estrogen, androgen or gestagen, a potential chemosterilant for control of rat and mouse population. j. reprod. pert. 97: 30-49. johnson, m. and b. everitt. 1988. essential reproduction. blackwell scientific publications. oxford. jones, a.r. 1983. antifertility actions of a-chlorohydrin in the male. aust. j. biol. sci. 36: 333-350. juchau, m.r. 1981. the biochemical basis of chemical teratogenesis. elsevier/north-holland. amsterdam. p.3 & 85. 29 biotropla no. 12,1999 kirton, k.t., r.j. ericson, j.a. ray, and a.d. forbes. 1970. male antifertility compounds, efficacy of u-5897 in primates (macaca mullata). j. reprod. pert. 21. 275-278. paz, g.f. and t.z. homonnai. 1982. a direct effect of a-chlorohydrin on rat epididymal spermatozoa. int. j. androl. 5:308-316. stevenson, d. and a.r. jones. 1982. inhibition of fnictolisis in boar spermatozoa by male antifertility agents (s>o.-chlorohydrm. aust. j. biol. sci. 35: 595-605. stevenson, d. and a.r. jones. 1985. production of 3-chlorolactaldehyde from a-chlorohydrin by boar spermatozoa and inhibition of glyceraldehyde-3-phosphate dehydrogenase in vitro. j. reprod. pert. 74: 157-165. tsunoda, y. and m.n. chang, 1976. fertilizing ability in vivo spermatozoa of rat and mice treated with achlorohydrin. j. reprod. fert. 46: 401-406. 30 biotropia no biotropia no. 12, 1999 : 53 58 in vitro inoculation of asparagus officinalis tissue culture shoots with fusarium prolifera tum a.k.mohd omar', n.a.r. nik norulaini2, b. salleh3 and r.a.r. iskandar' 1school of industrial technology, 2school of distance education, 3school of biological sciences, universiti sains malaysia, 11800, penang, malaysia abstract artificially inoculated asparagus tissue culture plantlets with a virulent fungus, fusarium proliferatum showed signs of infection as early as 4 days after inoculation. macroscopic observations revealed presence of early symptoms such as necrotic lesions at the affected area and light microscopic examinations clearly revealed the post-penetration events that took place including the destruction of surrounding cells. however, little is known of the hyphal activity or advancement on the host's surface at the initial stage after inoculation. scanning electron microscopic examination clearly revealed the hyphal advancement on the surface and the mode of entrance into the host tissues beneath. four days after inoculation, the fungi proceeded to spread out from the inoculation point onto the host surface which eventually developed into a sparse network of both aerial and non-aerial hyphae. non-aerial hyphae form a network of mycelium that adheres to the surface and it's movement appeared to be oriented towards the stomata. hyphal penetration occurs more often through the stomata, natural openings or wounds. in some cases, the hyphae crossed over the stomatal opening without entering the host tissues. at places where the cuticle layer is absent or not well developed the hyphae successfully grew in between the epidermal cells into the tissues beneath. key words: tissue culture/asparagus officinalis/shoots/artificial inoculstion/fusarium proliferatum. introduction in pathogen infected tissues, the preand post-penetration as well as results of infection can provide valuable knowledge regarding the virulence and epidemiology of the fungus involved. as a result of infection the physiological changes in diseased plants are usually manifested in visible symptoms which may be mild or severe, depending on the nature and aggressiveness of the pathogen and the susceptibility of the host plant. the physiological changes occurring in the diseased plant can be witnessed in the anatomical and morphological changes ('morbid anatomy') which constitute the visible symptoms. the visible symptoms such as necrosis or rot are in part related to the pathogenicity mechanisms of the pathogen, at least at the early stages of infection. histological examination very often can reveal the post-penetration development of the fungus and histological changes of the infected cells (jewell et al. 1979; nik norulaini 1992 a,b). scanning electron microscopy observations are useful in studying the development of the fungus on the epidermal surface, it's subsequent colonization on the surface and mode of penetration into the tissues beneath (ab. rahmane/a/. 1987; jacobiefa/. 1982). 53 biotropia no. 12, 1999 fusarium has been known to infect a wide range of crops in malaysia (sabariah 1987). one of the most serious diseases, namely crown rot, was described and the pathogen was identified as f. proliferatum (salleh 1990). the other casual pathogens include f. solani, f. nygamai and f. oxysporum. in the field, primary infection of f. prolifeatum is through wounding particularly those caused by insects (salleh et al. 1998). field symptoms, however, were reproduced by an artificial inoculation of asparagus in the greenhouse. the symptoms-include irregular reddish brown lesions on infected stems followed by stem girdling, crown and root rot and finally the entire plant die (salleh et al. 1998). the objective of this paper is to study the pre-penetration development of f. proliferatum, the causal agent of crown and root rot, into asparagus tissue culture shoots. materials and methods production of tissue culture shoots as hosts one year-old asparagus (asparagus officinalis l.) plants cv. mary washington 500 (mw 500) raised from seeds in the greenhouse (salleh et al. 1996) were used as source of explants. spears about 20 cm long and 0.25 cm in diameter were cut into 2.5 cm in length and surface-sterilized with 10% bleach (v/v) (5.25% sodium hypochlorite). a few drops of tween 20 emulsifier was added to enhance spread of the disinfectant. the segments were rinsed three times with sterile distilled water and blotted on sterile filter papers to remove the bleach. the sterilized segments were cut into one node segments of 1 cm each and cultured on murashige and skoog (1962) medium with the following concentrations in mgl"1 : naa 0.3, kinetin 0.1, thiamine-hcl 1.0, pyridoxine-hcl 5.0, nicotinic acid 5.0, myo-inositol 100.0, adenine sulphate dehydrate 40, sucrose 25 000 and supplemented with difco bacto malt extract 500, nah2po4.h2o 170. each culture bottle (500 ml) contained three segments and incubated at 27°c with continuous lighting at 1000 lux. multiple shoots that formed from 1 to 6 weeks were used as hosts. preparation of inoculum the fungus, f. proliferatum culture no. 851, was collected from infected asparagus plants cv mw 500 from the field. the fungus was kept in liquid nitrogen (-196°c) for several months (deposited at the fusarium culture collection unit, usm, penang) before culturing on potato dextrose agar (pda) (salleh & sulaiman 1984). four-day old cultures on pda were used as inoculum. 54 in vitro inoculation of asparagus officinalis tissue culture shoots a.k.. mohd. omar el al. inoculation of tissue culture shoots one to six weeks old shoots produced were used as hosts. three to four mm of the epidermis were carefully removed from the shoots as inoculation sites. the sites of inoculation were at the middle, basal and apex of the plantlets. mycelial plugs, about 1 mm3 taken from the periphery of the fungal colony were placed on the inoculation sites. scanning electron microscopic examination of infected shoot segments showing pathological changes were fixed in 5% glutaraldehyde for 1 hour, washed with phosphate buffer, fixed in 4% osmium tetroxide for 3 4 hours and then washed in phosphate buffer. the tissues were dehydrated through an alcohol series and critical point-dried using a critical point drier. they were then coated with gold in an ems-76m gold coater and examined with a jeol jsm35c scanning electron microscope. results and discussion twenty four hours after inoculation the hyphae began to spread progressively from the inoculum. four days later, the hyphae ramified into sparse network of mycelia that grew close to the surface of the tissue (fig. 1a). little aerial hyphae were observed. colonized stems will be overwhelmed by mycelia after six days of inoculation. removal of the mycelia from infected stems revealed early stages of necrotic lesions even at areas away from the inoculated sites (fig. ib). likewise, inoculation with conidia on mature asparagus stems with f. proliferatum showed occasional lesions (tan 1991). these reddish lesions are normally small, about 0.5 mm in length and can only be detected with a dissecting scope. the emergence of hyphae from beneath the tissues through necrotic spots were also observed, indicating existence of mycelial network within the cells. mature asparagus stems have developed a cuticle layer that inhibits the penetration of the fungus. penetration can, however, take place in cracks and crevices (nik norulaini & salleh 1990). this was observed in the basal part of the stem where the tissues were well covered with cuticle layer, or waxy deposits. the hyphae were seen entering the tissues via the stomatal opening (fig. 1c) or the natural cracks (fig. id). movement of hyphae in the groove in between cells was also apparent. no direct penetration in between cells was discovered even after exhaustive examination. mature tissues also offer resistance to any mycotoxins that might be released by the fungus. in a study by tan (1991), an incorporation of culture filtrate that contains toxins into a culture caused lesions on asparagus stems. the toxins were translocated through the base of the stem from the media. direct inoculation of the culture filtrate on the mature stem failed to induce any lesion. a significant difference in the cuticle development was observed at the apical areas of the inoculated shoots, where ramification of hyphal networks on the surface show the mycelia moving close to the surface (fig. ie). the lack of waxy deposits may influence penetration of the fungus as observed, since penetration between 55 biotropia no. 12, 1999 epidermal cells took place (fig. if). stomatal penetration of a single hypha was observed near the apical region, where the tissues were younger with little mechanical barrier, since there were only scant deposits of wax in the region. figure 1: growth and penetration of f. proliferatum on asparagus tissue culture shoots. a: ramifications of hypha into sparse network 4 days after inoculation (dai) b: necrosis (n) on inoculated stem 8 dai c: a single hypha entering the stomata d: hypha entering the tissue via natural cracks e: hypha network moving close to the surface f: penetration in between epidermal cells 56 in vitro inoculation of asparagus officinalis tissue culture shoots a.k. mohd. omar et al. although field infection is known to be via wounding, the in vitro sem examinations indicated that penetration can occur quite commonly through stomatal openings in the basal region. in younger tissues penetration can take place both between cells and through the stomata. in more mature parts of the tissue, the cuticle layer forms an effective mechanical barrier to the penetration of the fungus. conclusion although fusarium spp. have been associated with and caused many plant diseases, little has been studied on the host defence mechanisms or the fungus activities on the host. extensive review revealed the importance and potential of fusarium as disease-causing organisms, especially in the tropics (salleh & sulaiman 1984). with this preliminary finding it is hoped that this will offer a better understanding of the infection mode in nature and the pathogen's infectious capabilities on a large number of plants. further work is needed to understand the biochemical and physiological aspects of fusarium-related diseases. this approach could also lead to a screen for asparagus varieties resistant to fusarium-related diseases, such as have been used to screen non-fusarium resistant plant varieties (utkhede 1986), and to study resistance mechanisms (dolan & coffey 1985; diner et al. 1984), evaluation of susceptibility (sylvestre-guinot & delatour 1983) and surface interaction (nik norulaini & salleh 1990). the in vitro approach may offer a simplified and rapid screening method. references ab. rahman, n.n., a.m. diner, skillino, d.d. and d.f. karnosky. 1987. in vitro responses of conifer adventitious shoots and call! inoculated with gremmemella abietina. forest science 33:4: 1041-1053. diner, a.m., r.l. mott, and h.v. amerson. 1984. cultured cells of white pine show genetic resistance to axenic blister rust hyphae. science 24:407-408. dolan, t.e., and m.d. coffey. 1986. laboratory screening technique for assessing resistance of four avocade to phytophtora cinnamoni. plant disease. 70: 115-118. jacobi, w.r., h.v. amerson and r.l. mott. 1982. microscopy of cultured loblolly pine seedlings and callus inoculated with cronartiumfusiforme. phytopathology 71:138-143. jewell, f.f., p.c. jewell and c.h. walkinshaw. 1979. histopathology of the initiation of resistance-zones in juvenile slash pine to cronartium quercuum. f. sp. jusiforme. phytopathologisch meditterranica 19: 8-12. murashige, t. and t. skoog. 1962. a revised medium for rapid growth and bioassays with tobacco tissue culture. physiologia plantarum . 15:473-479. nik norulaini n.a.r. 1992a. assessment of in vitro inoculation of asparagus with conidia and culture filtrate of fusarium proliferation. proceedings of the 4* jsps-vcc seminar on integrated engineering, 13th 14th october 1992, kyoto, japan. nik norulaini n.a.r. 1992b. in vitro inoculation of asparagus with conidial suspension and culture filtrate of fusarium oxysporum schlect. f. asparag. proceedings of asia-pacific conference on agricultural biotechnology, august 20-24 1992, beijing, china. 57 blotropla no. 12, 1999 nik norulaini, n.a.r and b. salleh. 1990. in vitro inoculations of asparagus tissue culture of plantlets with vegetative hyphaof fusarium proliferatum. proceedings of the 3"* international conference on plant protection in the tropics. 20-23 march 1990, genting highlands, malaysia. sabariah, h. 1987. diseases of asparagus. b.sc. thesis, school of biological sciences, universiti sains malaysia, penang, malaysia. salleh, b. 1990. crown rot caused by fusarium proliferatum (matsushima) nirenberg; a new disease of asparagus in malaysia. proceedings of the 3rt international conference on plant protection in the topics. genting highlands, malaysia, p. 343 (abstract). salleh, b. and b. sulaiman. 1984. fusaria associated with naturally diseased plants in penang. journal of plants protection in the tropics 1: 47-53. salleh, b., a. safinat, l. julia and c.h. teo. 1996. brown spot caused by curvalaria spp., a new disease of asparagus. biotrop1a9: 26-37. sylvester-guinot, g. and c. delatour. 1983. possibility's di'appreciation de la sensibiliti'e du genre larix au lachnellula willkommii (hartig) dennis par inoculations artificielles. full annales scientia forsswiststand., 40(4) 337-354. tan, l.h. 1991. penginolculatan batang dan pucuk kultur tisu muda asparagus secara in vitro dengan ampaian konidia dan turasan kultur fusarium proliferatum (matsushima) nirenberg. b.sc. thesis, school of biological sciences, universiti sains malaysia, penang, malaysia. salleh, b., s.m. hakam, d. fachri, b.a.y. insanul, a. k.asmal, l. lahmuddin and s. nurduati. 1998. current status of asparagus diseases in southeast asia (sea). asean food journal (submitted). utkhede, r.s. 1986. in vitro screening of the world apple germplasm collection for resistance to phytophthora cactorum crown rot. scientia horticulturae, 29:205-210. 58 biotropia no. 8, 1995: 53-59 response of two sunflower (helianthus annuus l.) genotypes to va-mycorrhizal inoculation and phosphorus levels c.p. chandrashekara, v.c. patil and m.n. sreenivasa university of agricultural sciences, dharwad-580 005, karnataka, india abstract the performance of two sunflower genotypes (morden and msfh-8) with and without va-mycorrhizal fungi at three p levels (38, 56 and 75 kg p2o5 ha -1 ) in vertisol of dharwad was studied to determine the effect of mycorrhizal inoculation on plant growth, yield and p uptake. the results showed that the vam inoculation increased sunflower yield (14%), total biomass (16%), oil content (3.1%) and p uptake (30.5%) over uninoculated control. the percent root colonization and chlamydo-spore count decreased with increasing p levels. the total biomass production, seed yield and p uptake of mycorrhizal plants at 38 kg p2o5 ha -1 more than the non-mycorrhizal plants at 75 kg p2o5 ha -1 . the biomass and seed yield of mycorrhizal plants at same p level were more than the non-mycorrhizal plants. mycorrhizal plants of morden at 38 kg p2o5 ha -1 and msfh-8 at 56 kg p2o5 ha -1 produced higher seed yield, oil content and total biomass than non-mycorrhizal plants supplied with 75 kg p2o5 ha -1 . the results indicated that, va-mycorrhizal inoculation helps in saving 25 and 50 percent of recommended dose of phosphatic fertilizer (75 kg p2o5 ha -1 ) in msfh-8 (single cross hybrid) and morden (open pollinated variety), respectively. key words: mycorrhizas/plant nutrition/inoculum/glomus fasciculatum/helianthus annuus/phosphorus fertilizers/metabolism. introduction the current trend is to explore the possibility of supplementing chemical fertilizers with organic ones, more particularly biofertilizers of microbial origin. in this context, vam fungi are receiving greater attention in their beneficial effects on plant growth. vesicular-arbuscular mycorrhizae (vam) are widespread in soils, and often the growth of mycorrhizal plants will be higher in comparison to non-mycorrhizal plants. this beneficial effect on plant growth has largely been attributed to higher phosphorus (p) uptake and consequently better p nutrition of mycorrhizal plants (sanders and tinker 1973). sunflower, one of the potential oilseed crops of india has a higher p requirement. morden (open pollinated variety) and msfh-8 (single cross hybrid) are the two predominant genotypes of sunflower in karnataka. vam association have been 53 biotropia no. 8, 1995 reported in sunflower (cabello 1987), and it becomes mycorrhizal as early as at two leaf stage (iqbal and qureshi 1977). roots are heavily infected with glomus macro-carpum var. geospora under greenhouse conditions (ross and harper 1973). the sunflower roots were more colonized in bioassay tests than love grass (anderson and liberia 1987). phosphorus is usually considered to be the major problem when va-mycorrhizal infections are poor. high p levels in soil or p additions are known to reduce vam colonization of roots and sporulation (sreenivasa and bagyaraj 1989). studies have indicated that addition of an excess of readily available p eliminates beneficial mycorrhizal effects (sreenivasa et al. 1993). generally, research work on the use of mycorrhizae has been carried out under controlled conditions. pot culture trials have proved substantially that sunflower responds to inoculation with efficient strains of vam fungi at lower levels of phosphorus (jones and sreenivasa 1992). however, very few field experiments have been carried out so far. hence, the present study was undertaken to study the response of two sunflower (helianthus annuus l.) genotypes to the inoculation of vam fungus (glomus fasciculatum) and p levels, and to explore the possibility of substituting phosphatic fertilizer through vam inoculation. materials and methods a field trial was conducted at main research station, university of agricultural sciences, dharwad, on medium black soil (ph 7.6; organic carbon 0.63%; available n-0.063%; p2o5-32 kg ha -1 and k2o-295 kg ha -1) under irrigated conditions during summer season of 1993. the experiment consisted of twelve treatments comprising three p levels, two levels of vam inoculation (inoculated and un-inoculated) and two sunflower genotypes (morden and msfh-8) forming a 3 x 2 x 2 factorial experiment in a randomised complete block design with four replications each. plots of size 5.0 m x 3.6 m were prepared and spacing of 45 cm x 20 cm for morden and 60 cm x 20 cm for msfh-8 were followed with 200 and 150 plants per plot (18 m 2 ), respectively. the recommended dose of n and k were given (62.5 kg and 51.25 kg ha -1, respectively) in the form of urea and muriate of potash. the p levels consisted of 38, 56 and 75 kg p2o5 ha -1 (equivalent to 50,75 and 100% of recommended dose, respectively) as single super phosphate. glomus fasciculatum inoculum was multiplied in sterilized sand: soil mixture (1:1 by volume) using rhodes grass (sreenivasa and bagyaraj 1988). the inoculum (@ 30 g/spot) was placed 2 cm below the sunflower seeds. the inoculum consisted of colonized root fragments, hyphae and chlamydospores (126 per 50 g inoculum). 54 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa irrigation was given at is days interval to maintain soil moisture near field capacity. five plants were uprooted 90 (morden) and 105 (msfh-8) days after planting. shoots were severed at ground level, dried at 70°c, weighed, ground to pass a 0.5 mm sieve and analysed for p by vanadomolybdate phosphoric yellow colour method (jackson 1967). roots were washed with water and representative fresh samples were stained with trypan blue and percent root colonization was estimated (phillips and hayman 1970). chlamydospore count per 50 g rhizophere soil was taken by wet-sieving and decanting technique (gerdemann and nicolson 1963) and oil content was determined by using nuclear magnetic resonance (nmr) spectrophotometer. the seed yield (kg ha-1) was computed by using net plot yield (7.2 m 2 ). the data were analysed by mstat statistical program and least significant difference was used for mean separation. results and discussion sunflower genotypes responded well to the inoculation of va-mycorrhizal fungi. vam inoculation resulted in increased total biomass (16%), seed yield (14%) and oil content (3.1 %) (table 1). vamycorrhizal fungi are associated with increased growth of many plant species, mainly through increased uptake and translocation of not only p but also other nutrients (abbott and robson 1982). in addition to p, vam also help in uptake of zn, cu, mn and fe etc., (sreenivasa et al. 1993). iqbal and qureshi (1977) reported that mycorrhizal sunflower plants were taller, stouter, had more numerous larger leaves and bore bigger capitulum producing viable seeds than non-mycorrhizal plants. va-mycorrhizal plants recorded 30 percent higher p uptake (19.91 kg ha-1) than uninoculated plants (15.25 kg ha-1), which ultimately enhanced the oil content of the seed. the percent root colonization and spore count was higher in mycorrhizal treatments than in uninoculated controls. sunflower roots were heavily infected with glomus macrocarpum var. geospora under greenhouse conditions (ross and harper 1973). this probably resulted in greater fungal host contact and greater uptake of nutrients and hence better plant growth and yield. gerdemann (1964) and daft and nicolson (1966) correlated the increased growth and yield of crops to the extent of mycorrhizal infection. the percent root colonization and spore count decreased with increasing p levels in both inoculated and uninoculated plants. bagyaraj and powell (1985) have also observed depressed vam development at recommended p level due to higher availability of phosphorus. in a pot culture study, jones and sreenivasa (1992) observed increased percent root colonization of sunflower with increase in p level up to 50% of recommended dose of p, which decreased thereafter. the total bio 55 biotropia no. 8, 1995 56 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa mass, seed yield and p uptake of mycorrhizal plants at lower phosphorus levels (38 and 56 kg p2o5 ha 1 ) was more than non-mycorrhizal plants at 75 kg p2o5 ha -1 , which may be ascribed to higher vam-fungus root association and sporulation at lower p levels than at higher p level (table 1). the total biomass, seed yield and p uptake of mycorrhizal plants at same p level was more than the non-mycorrhizal plants. the higher efficiency of vam at lower levels of p has been ascribed to exploration of greater soil volume by mycorrhizal plants and faster movement of p ions into mycorrhizal hyphae (bolan 1991). mycorrhizal plants of morden at 38 kg p2o5 ha -1 and msfh-8 at 56 kg p2o5 ha -1 produced higher yield, oil content and total biomass than non-mycorrhizal plants supplied with 75 kg p2o5 ha -1 (table 2). this can be ascribed to the higher percent root colonization and spore count recorded with consequent increase in uptake of phosphorus. siqueria and paula (1986) found low efficiency of indigenous vam fungi. they also found maximum dry matter at 30 ppm of p2o5 in mycorrhizae inoculated soybean plants as compared to 120 to 240 ppm of p2o5 with native fungi. the maximum effect of vam fungi was found at 50 and 75% of recommended dose of p in morden and msfh-8, respectively. smith and gianinazzi-pearson (1988) found direct effect of soluble p on fungal metabolism, 57 biotropia no. 8, 1995 mainly by regulating enzymatic activities related to phosphate transfer to the host. therefore, it could be noted that further increase in p level did not enhance growth or yield of sunflower. the relative benefits of mycorrhizal fungi decreased with increasing p supply. conclusion the present study demonstrated that, the percent root colonization and spore count decreased with increasing p levels. the growth and yield of mycorrhizal plants at same p level are higher than in uninoculated plants. the differential response was obtained among the genotypes to vam inoculation. glomus fasciculatum was efficient at 38 and 56 kg p2o5 ha -1 in morden and msfh-8, respectively as compared to at 75 kg p2o5 ha -1 (100% of recommended dose); which suggests that, va-mycorrhizal inoculation helps in saving of 25 and 50 percent of recommended dose of phosphatic fertilizer (75 kg p2o5 ha -1) in msfh-8 (single cross hybrid) and morden (open pollinated variety), respectively. literature cited abbott, l.k. and a.d. robson. 1982. the role of vesicular-arbuscular mycorrhizal fungi in agriculture and the selection of fungi for inoculation. aust. j. agric. res. 33: 389-408. anderson, r.c. and a.e. liberta. 1987. variations in vam relationships of two sand praharie species. am, midland natural, 118 (1): 56-63. bolan, n.s. 1991. a critical review of the role of mycorrhizal fungi in the uptake of phosphorus by plants. plant soil. 134: 183-207. bagyaraj, d.j. and c.li. powell. 1985. effect of vesicular-arbuscular mycorrhizal inoculation and fertilizer application on the growth of marigold. new zealand. j. agric. res. 28: 169 -173. cabello, m.n. 1978. vesicular-arbuscular mycorrhizae in sunflower (helianthus annuus l.) crop. resista. de la facutad de agronomia, universidad national de la plata, 63: 46-52. daft, m.j. and j.h. nicolson. 1966. the effect of endogone mycorrhizae on plant growth. new phytol. 65: 345-350. gerdemann, j.w. 1964. the effect of mycorrhizae on the growth of maize. mycologia. 56: 342-349. gerdemann, j.w. and j.h. nicolson. 1963. spores of endogone species extracted from soil by wet sieving and decanting. trans. br. mycol. soc. 46: 235-244. iqbal, j.h. and k.s. qureshi. 1977. the effect of vam association on growth of sunflower (helianthus annuus) under field conditions. biologia (pakistan). 28: 189-196. jackson, m.l. 1967. soil chemical analysis. prentice hall of india (ltd.), new delhi. jones, n.p. and m.n. sreenivasa. 1992. response of sunflower to the inoculation of va mycorrhiza and phosphate solubilizing bacteria in black clayey soil. j. oilseeds res. 10: 86-92. 58 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. br. mycol. soc. 55: 158-161. ross, j.p. and j.a. harper. 1973. hosts of vesicular arbuscular endogone sp. j. elisha mitchell sci. 89 (1/2): 1-3. sanders, f,e. and p.b. tinker. 1973. phosphate flow into mycorrhizal roots. pestic. sci. 4: 385-395. siqueria, j.o. and m.a. paula. 1986. effect of vam nutrition and phosphate use by soybean in cerado soil. revista. brasileria. de gencia do solo. 10 (2): 97-102. smith, s.e. and v. gianinazzi-pearson. 1988. physiological interactions between symbionts in vam plants. ann. rev. plant physiol. 39: 221-244. sreenivasa, m.n. and d.j. bacyaraj. 1988. chloris gayana kunth (rhodes grass) a better host for mass production of glomus fasciculatum inoculum. plant soil. 106: 289-290. sreenivasa, m.n., p.u. krishnaraj, g.a. gangadhara and h.m. manjunathaiah. 1993. response of chilli to the inoculation of an efficient vesicular-arbuscular mycorrhizal fungus. scientia hortic. 53: 45-52. sreenivasa, m.n. and d.j. bagyaraj. 1989. suitable form and level of phosphorus for mass production of va-mycorrhizal fungus, glomus fasciculatum. zentralbl. mikrobiol. 144: 33-36. 59 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf blotropia no blotropia no. 11, 1998: 1 8 studies on natural products of albizia sp. hilman affandi, arifnuryadin seameo biotrop, p.o. box 116, bogor 16001, indonesia and roger w. read department of organic chemistry, the new south wales university, p.o. box 1 kensington, n.s.w. 2033, australia abstract the bark of albizia lebeckioides and albizia falcataria have been examined for their chemical constituents. a. lebeckioides has yielded the steroidal ketone, stigmasta-4,22-dien-3-one, and a triterpene alcohol as the major neutral components. a. falcataria has yielded a similar triterpene and fatty ester as its major constituents. the toxicity of the compounds was evaluated in bioassay against termites. the results showed that lupenone and stigmastadienone were toxic ioneotermes dalbergiae species. in contrast these compounds were less toxic to cryptotermes cynocephalus species. key words: insecticidal plants / albizia lebeckioides i albizia falcataria i bark / extracts / chemical constituents / toxicity / termites introduction some substances are produced by plants reasons for such as defence, growth regulation, hormonal action or as waste products. in teak (tectona grandis l.f), for example, tectoquinone is produced to protect these trees against termite attack (romanis 1887; oshima 1919; wolcott 1946). resin ofpinus possesses a number of biologically active natural products. a major constituent, a-pinene is well known for its antimicrobial and insecticidal properties (bridges 1987). among fast growing trees, albizia falcataria was reported to resist termite attack (kalshoven 1950; griffioen 1954). laboratory tests designed to study the resistance of albizia falcataria against dry wood termites (cryptotermes cynocephalus) revealed that the resistance of albizia falcataria lies somewhere between that of teak and hevea brasiliensis or even closer to teak (tarumingkeng and martawidjaja 1969). another species of albizia e.g. albizia lebeckioides was reported earlier to contain toxic alkaloids (greshoff 1914). these reports suggested that both species of albizia may contain useful chemicals that resist termite attack, therefore a search for biologically active 1 biotropia no. 11, 1998 compounds from the bark of albizia falcataria and albizia lebeckioides was undertaken. the objective of this study was to isolate and characterize the main chemical components and to determine their toxicity to termites. materials and methods general 'h nmr spectra were recorded at 500 mhz and 13c nmr spectra were measured at 125.6 mhz on a bruker am 500 spectrometer. mass spectra were recorded at 70 ev with an a.e.i. ms 12 spectrometer. all measurements were performed at the school of chemistry, the university of new south wales, australia. initial column chromatographic separations were made on merck 60 silica gel. merck kieselgel 60 f254 was employed for preparative tlc with a 1 mm layer of adsorbent on 20 x 20 cm glass plates and 200 mg of material applied to each plate. analytical tlc was performed on commercial aluminium-backed merck kieselgel 60 f254 art. 5554 plates, visualizing under uv 254 nm light or charring after applying a 4% h2so4/meoh spray. plant material samples of bark of albizia falcataria were collected from kesatuan pemangkuan hutan (kph), kediri while the bark of a. lebeckioides was taken from batutulis, bogor. extraction and isolation dried powdered bark of albizia lebeckioides (1.50 kg) was extracted with methanol to give crude extract (78.15 g). the crude extract was then re-extracted with ether to generate an ether soluble fraction (27.77 g) and an insoluble residue (50.37 g). the ether soluble fractions were carefully fractionated by column chromatography (silica gel 60) eluting with hexane containing increasing amounts of ethyl acetate to give 6 fractions. fraction 4 (1.12 g) and fraction 5 (0.98 g), as two major fractions, were subjected to purification by preparative thin-layer chromatography (ethyl acetate hexane, 1 : 9). fraction 4 gave a solid material 2 studies on natural products ofalbizia sp.hilman affandi et al. (0.85g) which crystallized from chloroform to give stigmasta-4,22-dien-3-one (2) (0.67 g) m.p. 181-184°. fraction 5 generated a crystalline solid (0.76 g). recrystallization of the latter gave lupeol (1) (0.51 g) m.p. 198-199°. the bark of a. falcataria (1.50 kg) was treated in a similar manner to the method above. re-extraction of the crude extract (65.83 g) with ether gave an ether soluble fraction (16.20 g) and a residue (49.62 g). fractionation of the ether soluble portion by column chromatography eluting with hexane generated 6 fractions. purification of the fastest moving fraction (1.28 g) by preparative thin-layer chromatography (ethyl acetate-hexane, 1:9) gave a solid material (0.93 g) which crystallized from chlorform to give fatty ester (4) (0.82 g). fraction 2 (1.04 g), upon thin layer chromatography (ethyl acetate-hexane, 1:9) gave a crystalline solid (0.78 g). recrystallization of the solid from chloroform gave lupenone (3) (0.68 g) m.p. 160-161°. termite bioassays the toxicity of the pure constituents was evaluated by bioassay using the termite neotermes dalbergiae, obtained from commercial sources in bogor, and cryptotermes cynocephalus, collected from the forest product research institute, bogor. pure compounds (25 mg) were diluted with dichloromethane (0.5 ml). the solutions were applied to 4.5 cm whatman no. 1 filter paper dish. after the solvent had been evaporated at ambient temperature, the filter papers were placed in 5 cm glass petri dishes, and 25 termites added to each container. control chambers were treated in a similar manner using pure chloroform in place of solutions of the test subtances. each pure compound was tested in four replicate experiments. at daily intervals the number of termites surviving was recorded, dead termites were removed, and the filter papers were moistened with distilled water. dishes were kept covered in a darkened room at ambient temperature. results and discussion structural analysis methanolic extracts of the bark of a. lebeckioides and a. falcataria were reextracted with ether. the ether soluble extracts were carefully fractionated by column chromatography (silica gel) and purificed by preparative thin-layer chromatography 3 biotropia no. 11, 1998 to yield lupeol (1) and stigmata-4, 22-dien-3-one (2) from die bark of a. lebeckioides, and lupenone (3) and fatty ester (4) from the bark ofa.falcataria. the structure of compound (1) was established from its microanalytical and spectroscopic data. elemental analysis and mass spectrometry indicated the molecular formula c30h50o. the 'h n.m.r. data showed six singlet resonances at 5 0.82, 0.89, 0.93, 0.98, 1.02 and 1.07, arising from six methyl groups attached to quaternary carbons. there also appeared one isolated singlet at 5 1.68 due to a vinylogous methyl group, and since the olefmic proton signals at 8 4.56 (dd, j 2.2, 1 4 hz) and 4.68 (d j 2.2 hz) were consistent with a 1,1-disubstituted alkene, a propenyl group was likely. low field signals resonated at 5 3.18 (dd, j 10,.9, 5.3 hz), 2.38 (dt, j 5.7, 11.1 hz) and 1.91 (m), but there were no other olefmic resonances. the n.m.r. signal at 8 3.18 was suggestive of an axial proton adjacent to a quarternary centre and attached to a carbon bearing an hydroxyl group. the i3c n.m.r. spectrum (table 1) -supported the structural assignment of (1). in particular there appeared seven methyl signals, only two olefmic carbon signals (8 109.3 (ch2) and 151.0 (c), and a low field methine resonance (8 78.9) for c3. the identity of compound (1) as lupeol was confirmed by melting point and mixed melting point with an authentic sample. lupeol has been found in many plant extracts, including the resin of pistacia leuticus (marner et al. 1991). compound (3) was obviously closely related to lupeol (1). its mass spectrum gave a molecular ion at m/z 424, corresponding to two fewer hydrogens than lupeol. the 'h n.m.r. spectrum (table 2) of compound (3) resembled that of (1), including the appearance of coupled olefmic signals at 8 4.57 and 4.70, and a deshielded methyl signal due to h3 in lupeol (1) was absent. instead, there appeared additional signals at 8 2.4, corresponding to protons adjacent to a multiple bond. the compound was thereby identified as lupenone (3) and the structure was confirmed by mixed melting point with an authentic sample. compound (2) differed from compounds (1) and (3). elemental analysis and mass spectrometry indicated through its molecular formula, c29h46o, that it was a nortriterpene. there appeared in its 'h n.m.r. spectrum two methyl singlets, 8 0.57, 1.01, three methyl doublets, 8 0.79, 0.84, 1.03, and a methyl triplet, 8 0.80. also there appeared a three proton multiplet corresponding to two internally coupled, trans olefmic protons (8 5 03, dd, j 15.1, 8.4 hz, and 5 15 (dd, j 15.1, 8.4 hz) and an isolated olefmic proton (8 5.18, m) with long range coupling, but no other signals at higher chemical shift than 2.5 ppm. the presence of a 1,2-disubstituted and a 1,1,2-trisubstituted olefin was supported by the presence of signals at 5 117.0 (ch), 129.6 4 studies on natural products ofalbizia sp.hitman affandi el al. (ch), 138.1 (ch) and 139.5 (c) in the 13c n.m.r. spectrum. the presence of a quaternary signal at 8 212.0 also confirmed the occurrence of a carbonyl group in the compound. structure (2) was assigned from these data and was confirmed by mass spectrometric fragments at m/z 367 (m-c3h7), 298 (m-c8hi6), 271 (m-cioh,9), 269 (mci0h2i), and comparative melting point. figure 1. structures of chemical constituents isolated fro falcataria (3) table 1. 13c n.m.r. chemical shift data of compounds (l)-( c 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 39.3 34.3 55.3 47.0 59.3 19.4 33.0 39.9 50.5 37.1 21.8 26.4 39.7 41.2 26.5 30.1 44.7 37.6 52.5 150.6 30.2 23.5 2. stigmasta 4,22-dien-3 3. lupenone m the bark of a lebeckioides (1 and 2) and a. 3) (ppm in cdclj) 2 3 37.3 31.6 212.2 71.8 42.3 121.1 33.9 31.7 50.2 36.5 20.9 38.6 42.2 56.6 24.1 28.2 56.1 11.6 19.4 40.8 21.5 138.8 39.7 34.1 218.4 47.3 54.9 19.6 33.0 40.3 50.6 36.9 21.9 26.7 39.5 41.0 26.4 30.1 44.9 37.7 52.5 150.8 30.3 23.2 5 biotropia no. 11. 1998 table 1. continued c 1 2 3 23 24 25 26 27 28 29 30 26.8 20.9 16.2 15.4 14.3 151.0 101.0 20.6 129.5 51.4 32.2 19.2 21.3 25.7 12.5 26.8 20.9 16.3 15.4 14.2 151.1 101.8 20.7 table 2. 1h n. m.r spectral data of compound 1-3 (ppm in cdc13) h 1 2 3 h-2 h-3 h-6 h-18 h-19 h-22 h-23 h-24 h-25 h-26 h-27 h-28 h-29 2.37 – 2.55 m 3.85 m 1.2 – 2.0 m 1.07 s 1.02 s 0.93 s 0.98 s 0.89 s 4.56 dd, 4.68 d 5.45 d 0.57s 1.01 1 5.15 dd 5.03 dd 0.79 d 0.84 d -1.50m 0.80 1 2.36 2.60 m 1.5 -2.0m 1.07s 1.19s 0.94s 0.97s 0.89s 4.57 dd, 4.70 d compound (4) could not be obtained pure and its structure could not be fully elucidated. from its 'h n.m.r. spectrum the substance was not terpene derived but was probably a fatty ester. there appeared in the low field region of the spectrum two triplets of equal intensity corresponding to methylene protons adjacent to oxygen (8 4.05) and a carbonyl group (8 2.28), respectively. in addition, there was a triplet resonance at 8 0.88 that could be assigned to coincident signals from the methyl groups at the ends of aliphatic chains. the remaining signals comprised two narrow multiplets at 8 1.25 and 8 1.57 corresponding to groups of similar methylene protons. the mass spectrum gave significant ions at m/z 425 and 205 that could not be assigned to any sensible structure or fragments. toxicity of compounds (l)-(3) a laboratory bioassay was conducted to assess the toxicity of pure compounds (l)(3), the tentatively assigned fatty ester (4), and the remaining extracts, to termites. the results showed that 53%, 48%, 42%, 54%, and 64%, respectively, of neotermes 6 studies on natural products ofalbizia sp.hitman aff'andi et al. dalbergiae termites that were fed these substances remained alive after the fourth day of the experiment (diagram 1). while termites in control groups moved actively when the dish was uncovered for counting, temites in treatment groups did not. this observation may suggest that termites receiving chemical treatment responded in a general rather than specific manner to pure compounds. specific responses such as death caused by direct application onto the termites were not observed. in addition the total termite mortality was not as high as expected from related studies using cryptotermes cynocephalus termites (diagram 2). this may have been due to insufficient doses of the pure compounds rather than low activity. comparison of the relative toxicity of the compounds to neotermes dalbergiae showed that lupenone and stigmastadienone were the most toxic compounds (diagram i). in contrast lupenone and stigmastadienone were less toxic against the cryptotermes cynocephalus species. this might be due to the fact that neotermes dalbergiae, a drywood termite, does not attack living trees as does the cryptotermes cynocephalus. therefore, the former are more susceptible to offensive chemicals, because they have not developed resistance mechanisms (messer 1990). % t er m in re m ai ni ng diagram 1. toxicity of the compounds against neotermes dalbergiae 7 biotropia no. 11, 1998 % t er m in re m ai ni ng diagram 2 toxicity of the compounds againts cryptotermes cynocephalus conclusion this study has shown that lupenone and stigmastadicnone, contained in the bark of albizia falcataria and albizia lebeckioides respectively, have biological activity against the drywood termite neotermes dalbergiae and that in function they may play a defensive role. toxic alkaloids, previously reported to be present, (heyne 1984) have not been found in the bark of albizia falcataria. references apsimon, j. 1973. the total synthesis of natural products, vol 2, john wiley & sons, london. bridges, j.r. 1987. effect of terpenoid compounds on growth of symbiotic fungi associated with southern pine beetle. phytopathology, p. 77. fffiser, l.f. and m. fffiser. 1959. steroids, van nostrand reinhold company, london. griffioen, k. 1964. albizia falcataria sen goede industrie houtsoort. tectona 63: 97-100. greshoff, m. 1914. indische vergiftrapporten, no. 3 in heyne, k. 1987. tumbuhan berguna indonesia, ruygrok & co., jakarta, p. 1021-1040. kalshoven, l.c.b. 1950. de plagen van de cultuurgewassen in indonesia. deel i: 145-150. marker, f.j., freyer, a., lex, j. 1991. interpenoid from gum mastic, the resin of pistacia leuticiis. phytochemistry, 30 (11). messer, a.c. 1990. chemical ecology in an indonesian context, ph.d. thesis, cornell university. oshima, m. 1919. formesan termites and method of preventing their damage. philip. sul. 15: 319-386. romanis, r. 1887. certain products from teak. chem. soc. 54: 868. tarumingkeng, r.c. and a. martawidjaja. 1969. pengujian kayu djeundjing (albizia falcataria backer) terhadap rayap kayu kering (cryptotermes cynocephalus light) secara laboratories. rimba indonesia, xiv (1-2) wolcott, g.n. 1946. what to do about pollila? univ. puerto rico, agric. exp. sta., bull. no. 68. 8 3 intraspecific (rika).cdr biotropia vol. 18 no. 1, 2011: 24 30 intraspecific variations of 16s mitochondrial gene sequences of yellow rice stem borer, (lepidoptera: crambidae) from west java scirpophaga incertulas rika raffiudin , ruth martha winnie , i made samudra yellow rice stem borer ( ) is one of the most important rice pest insects in asia, including indonesia. however, there is a lack of genetic data for this important agricultural insect. therefore, this study was conducted to explore intraspecific differentiation of partial 16s mitochondrial gene from bogor, karawang, indramayu and cirebon (west java, indonesia). here, we reported a total of 325 bp of 16s mitochondrial gene of from the obtained samples. among all dna sequences, three haplotypes of 16s mitochondrial gene were observed and submitted to genbank under accession number of gu191881, gu191882, gu191883, respectively for haplotype 1, 2, and 3. the haplotype 1 was found in all surveyed locations, except bogor. haplotype 2 and 3 were found only in from cirebon and bogor samples. these haplotype variations can be applied as dna markers for early larva detection method among other rice stem borers. hence, further explorations of the mitochondrial variations of in java and other parts of indonesia are needed moth, haplotypes, genetic differentiations, molecular identification 1* 1 2 (1 (2 department of biology, bogor agricultural university (ipb), darmaga, bogor 16680, indonesia indonesian centre for agricultural biotechnology and genetic resources research and development (icabgrd) scirpophaga incertulas s. incertulas s. incertulas s. incertulas s. incertulas s. incertulas abstract introduction . key words: yellow rice stem borer ( ) is one of the most important rice pest insects in asia, including indonesia, that highly reduces rice yield. they are dominantly spread in the north coast of java (or region, known as rice production regions), i.e. karawang, subang, indramayu and cirebon (hattori & siwi 1986). larvae of yellow rice stem borer damage the rice stem, hence disturb nutrient translocation from scirpophaga incertulas pantura * corresponding author : rika_r@cbn.net.id 24 root to leaf (pathak 1975). as a result, tillers at vegetative stage die, called dead hearth. larvae of also infest the generative stage of rice; therefore producing empty panicles, called white head. yield loss at the rice generative stage was 1-3% higher than that of vegetative stage. several methods were used to control the yellow rice stem borers such as by using pesticide, cleaning the infected area and controling their eggs. almost 57 kinds of insecticides were prohibited to be applied for rice plant because of polluting the environment and decreasing natural enemies. white rice stem borer ( ) was reported resistant to carbofuran insecticide (soejitno 1994). mitochondrial dna is a suitable tool to examine interand intraspecies genetic differences due to higher evolution rate of genes compared to those in nuclear dna (li 1997). moreover, the abundance of mitochondria in the cell provides sufficient materials for the analysis (crozier 1977). current study explores 16s mitochondrial gene of from regions. however, partial gene of 16s available in genbank are only from , the sugarcane stem borer from papua new guinea (genbank acc num ay32046) and from india acc num ay320460). those two records were published by lange (2004) to clarify the phylogenetic relationship of moth members from pyralidae and crambidae in sugarcane stem borers based on 16s and cytochrome oxidase ii (coii) mitochondrial genes (lange 2004). they also suggested that many taxonomy data based on morphology need to be re-organized. yellow rice stem borer was previously classified in the family pyralidae (pathak 1975). currently, is classified in the suborder pyraloidea; family crambidae; subfamily schoenobiinae (lange 2004). four species of rice stem borers commonly found in java are yellow rice stem borer ( ), white rice stem borer ( ), stripe rice stem borer ( ), and pink rice stem borer ( ). alteration of species domination was detected in a certain period, i.e. before 1995, in subang, west java, the dominant species was white stem borer; but later, yellow stem borer dominates the population (suharto & usyati 2005). in an attempt to build database of mitochondrial dna of yellow rice stem borers in indonesia, here we commenced the exploration of intraspecific differences of based on 16s mitochondrial gene in west java (bogor, karawang, indramayu and cirebon). the results of 16s variations of can be applied as dna markers for early larva detection method among other rice stem borer. yellow rice stem borer were collected from four localities in west java, indonesia. those were from several subdistricts in bogor, karawang, indramayu and cirebon (table 1). s. incertulas s. innotata et al. s. incertulas pantura s. excerptalis et al. et al. scirpophaga et al. s. incertulas s. innotata chilo suppressalis sesamia inferens s. incertulas s. incertulas materials and method s. incertulas collection intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al 25 table 1. sample locations of moth in west java, indonesia.s. incertulas biotropia vol. 18 no. 1, 2011 locations (subdistrict, district) latitude longitude sindang barang, bogor 6o34’24.61” 106 o45’50.9” pedes, karawang 6o04’13.26” 107 o22’32.61” arjawinangun, cirebon 6o39’08.63” 108 o24’40. 47” lelea, indramayu 6o24’14.75” 108 o11’12.59” dna extraction and 16s mitochondrial dna amplification dna sequencing and analysis yellow rice stem borer dna was extracted by using ctab extraction and ethanol precipitation, using thoraces as tissue source (raffiudin & crozier 2007). the 16s of mitochondrial gene of yellow rice stem borers were amplified by using forward primer 16scbf: 5'-aagattttaatgatcgaacag-3' and reverse primer 16scbr 5'tgactgtacaaaggtagcata-3' (simon . 1994). amplifications were carried out in conditions of: initial denaturation at 94 c for 2 min. then 40 cycles at : 92 c for 45s; 50 c for 60s, 72 c for 90s, and final extension at 72 c for 2 min (lange 2004). amplicon of 16s gene was sequenced by using the same primer as in the amplifications. homology of the dna sequences was analysed by using blast (http:/blast.ncbi.nlm.nih.gov/blast.cgi). dna alignments were carried out by using clustal x (thompson . 1997) and sugar stem borer 16s gene gen bank acc. num ay320460 from india and ay320461 from papua nugini (png) were used as the outgroups. all haplotypes obtained from this research were submitted to genbank. tamura-nei distance parameter implemented in mega 5.05 (tamura . 2011) was used to reveal interand intraspecific genetic distance among and 16s mitochondrial gene. this study revealed the first mitochondrial data of yellow rice stem borer 16s mitochondrial gene from west java, which was approximately 325 bp. the moth 16s rna gene sequences were at rich with 65% at. however, the value was lower than that of sugarcane stem borer 16s gene (79.01% at) (lange 2004). blast analysis of 16s gene showed 89% homology with sugarcane stem borer of 16s mitochondrial gene (e-value: 9e ). among all 16s mitochondrial gene sequences, we observed three haplotypes from bogor, karawang, cirebon and indramayu samples (table 2, fig. 1). the first 16s mitochondrial gene haplotype was the common haplotypes found from in three locations (karawang, indramayu and cirebon), i.e samples number sc14kr, sc3id, sc2cb, respectively (table 2). the second 16s mitochondrial et al et al. s. incertulas et al s. excerptalis s. excerptalis et al s. incertulas s. excerptalis s. incertulas s. excerptalis et al. s. incertulas s. excerptalis s. incertulas s. incertulas o o o o o -104 results and discussion 26 haplotypes locations s. incer tulas sample code nucleotide position (see figure 1 ) nucleotide variations genbank acc num 1 karawang sc14kr indramayu sc3id gu191881 cirebon sc2cb 2 cirebon sc15cb 154 a gcirebon sc17cb gu191882 3 bogor sc20bg 129 t ” –“ (dele tion) gu191883 table 2. haplotype variations of 16s mitochondrial gene of from bogor, karawang, indramayu, and cirebon (west java) s. incertulas sc: ; notation followed “sc” = sample number and sample location, bg = bogor, kr = karawang, id= indramayu, cb= cirebon. s. incertulas 27 table 3. genetic distance based on 16s mitochondrial gene among and corrected with tamura-nei distance s. incertulas s. excerptalis gene haplotype was found in both samples from cirebon, having transition substitution at the nucleotide number 154 (fig. 1, table 2). compared to 16s mitochondrial gene haplotype 1 and 2, haplotype 3 in sc20bg sample showed deletion of one nucleotide (number 129) (fig.1). these three haplotypes were submitted to genbank under accesion number gu191881, gu191882, g 191883. intraspecific genetic distance among in west java based on 16s mitochondrial gene revealed 0.003. further analysis of intraspecific genetic distance of 16s gene between and from india and png showed 0.145 and 0.133, respectively (table 3). these values were higher than genetic distance between and , i.e. 0.063 (unpublished data). result of this study can be used for identification of yellow rice stem borer in the early instar larvae. this is due to the morphology of rice stem borers larvae which show slightly differences between yellow and white rice stem borers, hence careful examination is needed (amir 2004). phylogenetic analysis frequently establishing interand intra specific relationship between taxa and within populations as well. this was shown in several mitochondrial genes i.e. coi, nd1, and 16s that were used s. incertulas s. incertulas s. incertulas s. excerptalis s. incertulas s. innotata et al. u intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al [1] sc15cb.h2 [5] sc3id.h1 [2] sc17cb.h2 [6] sc14kr.h1 [3] sc20bg.h3 [7] se.india.ay320460 [4] sc2cb.h1 [8] se.png.ay320461 figure 1. dna alignments of 16s mitochondrial gene of from bogor (bg), karawang (kr), indramayu (id), cirebon (cb), and from india and png; “ = homology of nucleotides in the same column. sc: (yellow rice stem borer), se: (sugar cane stem borer). notation followed “sc” = sample number, sample location and haplotype number; ay320460 and ay320461 mentioned after “se” = genbank (www.ncbi.nlm.nih.gov) acc. num. for from india and png s. incertulas s. excerptalis scirpophaga incertulas scirpophaga excerptalis s. excerptalis ·” 28 biotropia vol. 18 no. 1, 2011 to construct a molecular phylogeny of butterflies belonging to the genus s.l.. s.l. has been divided into four genera in an earlier revision. the current results showed three well-supported groups within the genus, corresponding to three of the four proposed genera (zimmermann 2000). another example was on the basis of 16s ribosomal dna and coi-coii regions that revealed restriction-site variations in geographically divergent collections of tobacco budworm, (f.) tennesse, mississippi, oklahoma, and texas (roehrdanz 1994). euphydryas euphydryas et al. heliothis virescens et al. 29 s. incertulas et al. s. incertulas et al s. incertulas s. incertulas s. incertulas s. incertulas this study explored three haplotypes of in several regions in west java, however, a further exploration is needed to reveal the complete genetic intraspecific variations of this agricultural importance insect. database of mitochondrial variations from rice stem borer can be applied for further monitoring strategy to examine the dynamic population of the stem borer, to seek both the origin of this pest and the relationships among other species of rice stem borer. moreover, this result can be used as a basic data to establish enviromental safely control method such as by disrupting the mating time of the stem borer (samudra 2002) and characterise the sex pheromone of to disrupt the mating behaviour (tatsuki . 1985). this study was the first report for intraspecific differentiation of basedon 16s mitochondrial gene obtained from bogor, karawang, indramayu and cirebon (west java, indonesia) samples. three haplotypes of 16s mitochondrial gene were observed and submitted to genbank under accession number: gu191881, gu191882, gu191883, respectively for 16s mitochondrial gene haplotype 1, 2, and 3. haplotype 1 of 16s mitochondrial gene was found in almost all locations, except bogor. haplotype 2 and 3 were found only in from cirebon and bogor samples. further explorations of 16s mitochondrial gene in java and other parts of indonesia are needed to reveal complete 16s gene intraspecific differentiations of this agricultural importance insect. . funding for this research was provided by seameo-biotrop and is gratefully acknowledged. conclusions acknowledgements references amir m, kartohardjono a, siwi s, ubaidillah r. 2004. morphological species variability in the stemborer genus (lepidoptera:pyralidae) on graminous crops. treubia 33:147-163 crozier rh. 1977. evolutionary genetics of the hymenoptera. annual review of entomology 22:263-288 hattori i, siwi s 1986 rice stemborers in indonesia. 20:25-30 lange cl, scott kd, graham gc, sallam mn, allsopp pg. 2004. sugarcane moth borers (lepidoptera: noctuidae and pyraloidea): phylogenetics constructed using coii and 16s mitochondrial partial gene sequences. bulletin of entomological research 94:457-464 li wh. 1997. molecular evolution. sinauer associates, inc. publishers, massachusetts pathak mb. 1975. insects pest of rice. international rice research institute, los banos raffiudin r, crozier rh. 2007. phylogenetic analysis of honeybee behavioural evolution. molecular phylogenetics and evolution 43:543-552 scirphopaga . jarq intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al 30 biotropia vol. 18 no. 1, 2011 roehrdanz rl, lopez jd, loera j, hendricks de. 1994. limited mitochondrial dna polymorphism in north american populations of heliothis virescens (lepidoptera: noctuidae). annals of the entomological society of america 87:856-864 samudra i, emura k, hoshizaki s, ishikawa y, tatsuki s. 2002. temporal differences in mating behavior between riceand water-oats-populations of the striped stem borer, chilo suppressalis (walker) (lepidoptera: crambidae). applied entomology and zoology 37:257-262 simon c, frati f, beckenbach a, crespi b, liu h, flook p. 1994. evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers [review]. annals of the entomological society of america 87:651-701 soejitno j, samudra im, kilin d 1994. kajian ketahanan penggerek padi putih, walker terhadap insektisida karbofuran. penelitian pertanian 14:78-83 suharto h, usyati n. 2005. the stem borer infestation on rice cultivars at three planting times. indonesian journal of agricultural science 6:39-45 tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution 28:2731-2739 tatsuki s, sugie h, usui k, fukami j, sumartapura mh, kuswadi an. 1985. identification of possible sex pheromone of the yellow stem borer moth, scirpophaga incertulas (walker) (lepidoptera: pyralidae). . applied entomology and zoology 20:357-359 thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acid research 25:4876-4882 zimmermann m, wahlberg n, descimon h. 2000. phylogeny of euphydryas checkerspot butterflies (lepidoptera: nymphalidae) based on mitochondrial dna sequence data. annals of the entomological society of america 93:347-355. . scirpophaga innotata biotropia no biotropia no. 15, 2000 : 36 47 genetic diversity of ampicillin-resistant vibrio isolated from various stages of tiger shrimp larvae development widanarni a and antonius suwanto b c "department of aquaculture, faculty of fisheries and marine science bogor agricultural university, bogor 16680, indonesia b department of biology, faculty of science and mathematics, and iuc biotechnology, bogor agricultural university, bogor 16680, indonesia 'seameo-biotrop, ji. raya tajur, km 6 bogor, indonesia abstract this research was carried out to study genetic diversity of ampicillin-resistant vibrio from various stages of tiger shrimp larvae (penaeus monodon) development from,tambak inti rakyat hatchery, near labuan, west java, indonesia. a total of 25 ampicillin-resistant vibrio isolates were isolated using thiosulphate citrate bile-salt sucrose agar (tcbs-agar) and seawater complete agar (swc-agar). physiological and biochemical characterization showed that the isolates could be grouped into only two species, i.e. v. harveyi from the egg stage; and v. metschnikovii from larvae and post-larval stage (i.e nauplius, zoea, mysis, pli, pl5, pl,0, and pl,5). these isolates were also present in their respective rearing water of each stage and some natural feed. schizotyping analysis employing restriction endonuclease noll (5'-gc4ggccgc) indicated that the isolates could be grouped into at least 13 different genotypes. therefore, schizotyping was more discriminative than physiological characterization. this study showed that particular groups of vibrio colonized all stages of shrimp larvae and demonstrated closed phylogenetic relationship. these groups of vibrio might be the dominant microbiota which could suppress the development of other vibrio including the pathogenic vibrio. key words : shrimp/ampicillin-resistant k/fcno/schizotyping introduction tiger shrimp (penaeus monodori) culture has developed towards intensive levels due to high demand and significant price of this commodity. however, recently shrimp production has decreased significantly because of diseases and poor environment quality (anonymous 1994). one of the most serious problems is the disease caused by vibrio (lavilla-pitogo et al. 1990). the disease greatly influenced the sustainable supply of healthy fry (lightner et al. 1992). hameed (1993) observed that the decrease of survival rate of larvae and postlarvae was due to the concomitant increase of the bacteria population. generally, shrimps at the stadia of zoea, mysis, and the beginning of post-larvae were vulnerable to vibrio infection (rukyani et al. 1992). it was believed that these bacteria contaminated the hatchery through broodstock feces because of their presence in significant number in the midgut (lavilla-pitogo et al. 1990). therefore, * corresponding author : email address : asuwanto@indo.net.id;fax : 62-251-315107 36 biotropia no. 15, 2000 contamination of the eggs and nauplius by these bacteria was highly probable. besides the infected shrimp, these bacteria could also be isolated from seawater used for rearing water in the hatcheries (tjahjadi et al. 1994). recently, suwanto et al. (1998) demonstrated that vibrio harveyi isolated from different broodstocks were genetically very diverse and different to each other. therefore, it can be expected that vertical infection from broodstock to eggs is more likely to occur rather than horizontal transfer from larvae to larvae. in order to understand genetic diversity of vibrio at various stages of shrimp larvae development, a study on genetic diversity of vibrio isolated from egg, larvae, and post-larvae of shrimp originated from the same broodstock was carried out. a number of techniques have been developed to identify bacteria at the subspecies level. these include phage typing (stringer 1980), analyses of plasmid dna (davies et al. 1981), ribotyping (olsen et al. 1986), random amplified polymorphic dna (rapd) (welsh and mcclelland 1990), and schizotyping by pulsed-field gel electrophoresis (pfge) (suwanto and kaplan 1992). pfge analysis of large sizes of dna molecules requires a technique to isolate intact genomic dna as well as the availability of rare-cutting restriction endonucleases and the appropriate mega-base molecular size markers. under optimized condition, pfge can efficiently separate dna fragments of 100 to 10,000 kbp to distinguish strains of microorganisms which otherwise exhibit similar or identical physiological and morphological characteristics (suwanto 1994). genetic diversity analysis based upon genomic dna profile utilizing pfge has been performed for various bacteria for various aims, such as vibrio anguil-larum, a causative agent of vibriosis in fish (skov et al. 1995), genomic dna analysis of xanthomonas campestris, a causative agent of root-pustule disease of soybean (rukayadi 1995) and vibrio harveyi, a causative agent of "luminous-bacterial disease" (suwanto et al. 1998). pfge genomic dna analysis used in the experiments above have proved to be more discriminative to visualize phylogenetic diversity rather than analysis based on phenotype characterization. the purpose of this research was to study the genetic diversity of ampicillin-resistant vibrio from various stages of shrimp larvae development by pfge analyses. materials and methods isolation and identification of vibrio sp. vibrio were isolated from eggs, larvae (nauplius, zoea, and mysis), and post-larvae (pl,, pl5, pl10, and pl15) of tiger shrimp (p. monodori), natural shrimp feed (i.e. anemia and skeletonema), and rearing water obtained from tambak inti rakyat hatchery, near labuan, west java, indonesia. shrimp larvae were collected in the month of march-april, 1998. five eggs and shrimp larvae were washed gently in sterile seawater and collected into a test tube containing 1 ml sterile seawater, and homogenized by vortex agitator before it was spread on tcbs agar. appropriate 37 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto dilution was performed to obtain single isolated colony on tcbs agar by adding sterile seawater. the culture was incubated at room temperature, (28-31)°c, for 24 hours. isolated colonies were randomly selected for further study. biochemical and physiological characterization were conducted with microbactr analysis kit (medved science pty. ltd. australia). identification of vibrio isolates were done as described by baumann et al. (1984). seawater complete agar (5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water) supplemented with ampicillin (50 ug/ml) (swc-ap) was employed as a selective media to screen for ampicilin-resistant vibrio isolates. the isolates were subsequently suspended in sterile seawater containing 15% glycerol (v/v) before they were stored in the freezer at -50°c. preparation of intact genomic dna and digestion purified isolates of ampicillin-resistant vibrio were grown in swc agar. one separated colony was regrown in 10 ml lb medium (10 g tryptone, 5 g yeast extract, 25 gnacl and 1 l distilled water) at 28°c overnight. one ml of bacterial suspension was centrifuged at 5,000 rpm for 30 seconds. the bacterial cell pellet was suspended in sterile piv solution (10 mm tris-cl ph 7.5, 1 m nacl). preparation of intact genomic dna was performed by embedding the bacterial cells in low melting aga-rose blocks as described previously (schwartz and cantor 1984). digestion of the intact genomic dna was done using restriction enzyme as described by suwanto et al. (1998) as follows: digestion with 10 units of not\ were performed in 150 ml of appropriate restriction buffer (1335 ul distilled water, 150 \i\ lox restriction buffer, 15 ul of 10 mg/ml bovine serum albumine). the mixture was incubated at 4°c for 15 minutes followed by incubation in 37°c overnight. dialysis of the gel plugs were performed by immersing the gel plugs in excess of ix te buffer solution (10 mm tris-cl ph 8.0, 1 mm edta ph 8.0) before placing the gel plugs into the wells of the running gel. separation of dna fragment using pfge the running gels of 1% (w/v) agarose (pharmacia) were prepared in 100 ml of 0.5x tbe (50 mm tris-borate buffer, 0.1 mm edta ph 8.0). pulsed-field gel electrophoresis was performed using chef-drii (bio rad, richmond, ca). the gels were run in 0.5 x tbe buffer, 5 volt, 14°c with ramping pulse time from 10-80 seconds for 20 hours. as molecular marker, rhodobacter sphaeroides 2.4.1 genomic digested with asel was routinely used for pfge (suwanto and kaplan 1989). dna visualization gels were stained by submerging in ethidium bromide solution (1 ug/ml) for 10 minutes. destaining were done in distilled water for 20-30 minutes. transillu-minator with uv length of 280 nm (hoefer scientific instrument, san francisco) 38 biotropia no. 15, 2000 was used to visualize the gel. photographs were taken with fast film polaroid (type 667, japan polaroid company). statistical analysis a matrix was constructed as the basis for determining the presence or absence of schizotyping bands at a given position over the size range from 30-500 kb. a cluster analysis was carried out using the unweighed pair group method with arithmetic means (upgma clustering with simple matching coefficient) of similarity coefficient for all pairs of strain and a dendrogram was generated using a computer-based taxonomy program (numerical taxonomy system, ntsys-pc version 1.60) (rohlf 1990). results and discussion physiological and biochemical characterization of vibrio isolates twenty five ampicillin-resistant isolates have been isolated and selected randomly from either tcbs or swc-ap for further study (table 1). of these isolates, 20 isolates were found to be associated with eggs, larvae, post-larvae, and the rearing water of each stage; two isolates were isolated from seawater reservoir; and the other isolates were obtained from natural feed (i.e. artemia and skeletonema). table 1. codes and sources of vibrio isolates no. code source no. code source 1. e, egg 14 st broodstock tank water 2. e2 egg 15. et spawning tank water 3. n nauplius 16. nt nauplius tank water 4. z zoea 17. zt zoea tank water 5. m, my sis 18. mt mysis tank water 6. m2 mysis 19. pl,t post-larvae 1 tank water 7. m3 mysis 20. p15t post-larvae 5 tank water 8. pl, post-larvae 1 21. pl,0t post-larvae 10 tank water 9. pl5 post-larvae 5 22. pl15t post-larvae 1 5 tank water 10. pl,o post-larvae 10 23. skt skeletonema 11. pl,, post-larvae 15 24. at, artemia 12. sw, sea water 25. at2 artemia 13. sw2 sea water 39 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto physiological and biochemical characterization showed that the isolates shared several similar characters. the cell was rod shape, produced lysine decarboxylase and indole, and all were able to ferment glucose (table 2). the isolates could be classified into three groups. first group, composed of e,, e2, m,, sw,, sw2, and mt shared the same characters such as: forming green colonies on tcbs, oxidase-positive, produce protease and chitinase, and able to reduce nitrate. a number of isolates were also able to utilize arabinose as a carbon source. the second group of vibrio composed of only one isolate, at2 isolated from anemia had the same characters as first group except that it was luminous. the third group (n, z, m2, pl,, pl,5, et, zt, pl5t, pl10t, skt, at,), was the dominant group of the isolates found in every larval stage except the eggs possessed completely different characters from the two groups in having nonluminous yellow colonies on tcbs, showed negative oxidase reaction, did not produce protease and chitinase, and were able to use sucrose as a carbon source. based upon baumann et al. (1984) identification, the first group was non-luminous v. harveyi, the second was luminous v. harveyi, and the third was v. metschnikovii. therefore, physiological characterization of vibrio isolated from each stage from tambak inti rakyat hatchery, west java, showed that two species of vibrio had been found. v. harveyi were associated with eggs, while v. metschnikovii were found in larvae and post-larvae (nauplius, zoea, mysis, pl,, pl5, pl,o, and pl15), these isolates were also present in their respective rearing water of each stage and some natural feed. schizotyping analysis pfge electrophoresis of the 25 vibrio isolates (obtained in this study) and of v. harveyi s14b (suwanto et al. 1998) using notl restriction enzyme (figure 1) showed discrete profiles of genomic dn a of the isolates based upon the number of fragments and migration distance (table 3). restriction with notl produced 10-16 discrete dna fragments with sizes ranging from 31-910 kb. genomic dna analysis of 25 isolates showed 13 different dna profiles. these dna profiles could be used as a unique fingerprint of each of those isolates. dendrogram of genomic dna digested with notl (figure 2) showed genetic relatedness of the isolates, which could be divided into 13 different subgroups. the sum of horizontal lines connecting the two isolates indicated genetic distance of those isolates. for example, the distance between isolate pl| and pl5 is zero indicated that these isolates are identical. of these, 13 subgroups were divided into 2 major groups. the first major group consisted of 20 isolates associated with all stages of shrimp larvae and their rearing water. the second group consisted of 5 isolates, i.e. 2 isolates from seawater reservoir, 1 isolate from broodstock rearing water, and 2 isolates from natural feed (skeletonema and anemia). some vibrio isolates originated from seawater and natural feed also could not be isolated from all larvae stages and eggs of shrimp. this result suggested that eggs were exposed to bacterial contamination not only from broodstock feces as reported by lavilla 40 biotropia no. 15, 2000 41 genetic diversity of ampiciloin-resistand vibro – widanarni & antonius suwanto 42 biotropia no. 15, 2000 43 genetic diversity of ampicillin-resistant vibrio – widanarni & antonius suwanto 44 biotropia no. 15, 2000 table 3. grouping of genomic dna profiles of 25 vibrio isolates generated by noll schizo typing no schizotype profile isolates exhibited similar schizotype 1. e, e, 2. m, m, 3. pl, pl, pl,5, pl,t, pl,,t 4. pl5t pl,t 5. z zt 6. at2 at2 7. pl,o pli(1t 8. et nt 9. m2 mt, m, 10. e2 ea 11. n n 12. sw, sw2 st 13. sk.t at, pitogo et al. (1990), but also when they were still in the ovary. our study also demonstrated that vibrio isolates from sea water or natural feed possessed distance genetic relationship to that of vibrio from eggs and larvae, indicating that vibrio from eggs and larvae may be the dominant microbiota and might play a role in suppressing the growth of vibrio from seawater or natural feed. compared to the results of suwanto et al. (1998), all of vibrio isolates in this experiment showed relatively similar genetic background. this might be due to the fact that the broodstocks in this study were obtained from the same area (aceh province), while suwanto et al. (1998) used broodstocks from different areas and provinces. this study showed that genetic analysis by pfge was more discriminative than that of physiological and biochemical analysis. from 25 isolates characterized by physiological analysis, they could be grouped into two species, while using schizotyping analysis, there were 13 different groups of genotypes. furthermore, with this analysis, v. metschnikovii, that did not produce protease and chitinase was apparently closely related to v. harveyi, a shrimp pathogen. therefore, the existence of v.metchnikovii might influence shrimp larval fitness and survival. in conclusion, physiological and biochemical characterization were able to classify 25 isolates vibrio (both luminous and non-luminous) into two species, i.e. v. harveyi and v. metchnikovii. notl schizotyping analysis showed that the isolates consisted of at least 13 different genotypes, which indicated that schizotyping was 45 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto more discriminative than physiological characterization. the result also demonstrated that vibrio isolates present in all stages of shrimp larvae showed close phylogenetic relationship. these groups of vibrio might be dominant and might play a role in suppressing the development of other vibrio originated from sea water, broodstock feces or natural shrimp feed. acknowledgements we express our thanks to tambak inti rakyat hatchery, labuan, west java, for supplying shrimp larvae and to allow us to use the field laboratory facility. we would like also to thank prof. dr. maggy t. suhartono and dr. dwi andreas santosa for their invaluable advice during the experiment. this research was supported by riset unggulan kemitraan (ruk) research grant to antonius suwanto and biaya pendidikan pasca sarjana (bpps) research grant to widanarni. references anonymous. 1994. problem solution alternatives of shrimp culture in java. directorate general of fisheries. jakarta. 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(eds). diseases in asian aquaculture 1. fish health section. asean fisheries society. manila, philippines p.57-80. olsen gj, lane dj, gionvannoni sj, pace nr. 1986. microbial ecology and evolution: a ribosomal rna approach. ann rev microbiol 40: 337-365. rohlf fj. 1990. nt-sys-pc, numerical taxonomy and multivariate analysis system, version 1.60. exeter software, new york. rukayadi y. 1995. dna profile analysis of the genomes of a number of xanthomonas campestris pv. glycines isolates using pulsed-field gel electrophoresis. thesis pascasarjana program, ipb. bogor. 69p. rukyani a, taufik p, taukhid 1992. luminous vibrios in tiger shrimp hatchery and the control of the disease in shrimp larvae. j. litbang pertanian 2:1-17. 46 biotropia no. 15, 2000 schwartz dc, cantor cr. 1984. separation of yeast chromosome-sized dna by pulsed-field gradient gel electrophoresis. cell 37:67-75. skov mm, pedersen k, larsen ja. 1995. comparison of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling for typing of vibrio anguillantm serovar ol. app and env microbiol 61:1540-1545. stringer j. 1980. the development of a phage typing system for group b streptococci. j med microbiol 13:133-143. suwanto a, kaplan s. 1989. physical and genetic mapping of rhodobacler sphaeroides 2.4.1 genome: genome size, fragment identification and gene localization. j bacteriol 171:1135-1145. suwanto a, kaplan s. 1992. chromosome transfer in rhodobacter sphaeroides: hfr formation and genetic evidence for two unique circular chromosomes. j bacteriol 174:1135-1145. suwanto a. 1994. pulsed-field gel electrophoresis: a revolution in microbial genetics. as pac j mol biotechnol 2:78-85. suwanto a, yuhana m, herawaty e, angka sl. 1998. genetic diversity of luminous vibrio isolated from shrimp larvae in: flegel tw (ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok, p 2-9. tjahjadi mr, angka sl, suwanto a. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial disease in tiger shrimp larvae (penaeus monodon, fab.). as pac j mol biol biotechnol 2:347-352. welsh j, me clelland m. 1990. fingerprinting genomes using pcr with arbitrary primers. nucleic acids res 18:7213-7218. 47 biotropia no. 9, 1996: 53 61 growth inhibition of pathogenic root fungi by extracts of ectomycorrhizal fungi or picea glehnii inoculated with ectomycorrhizal fungi* maria catarina megumi kasuya dep. de microbiologia, universidade federal de vicosa, vicosa, mg, 36570-000, brazil satoshi tahara dept. of applied bioscience, faculty of agriculture, hokkaido university, kita-ku, sapporo 060, japan and tsuneo igarashi dept. of forest science, faculty of agriculture, hokkaido university, kita-ku, sapporo 060, japan abstract this work sought to verify the presence of compounds with antimicrobial properties in extracts of ectomycorrhizal fungi or in picea glehnii inoculated with ectomycorrhizal fungi. extracts from pisolithus tinctorius, scleroderma flavidum, amanita pantherina and paxillus sp., grown in liquid culture media, and from p. glehnii seedlings inoculated or not with the above ectomycorrhizal fungi and cultivated in in vitro condition, were processed to obtain two fractions, water and ethyl acetate solubles. these fractions were tested for the presence of inhibitory constituents against fusarium roseum, pythium sp. and rhizoctonia solani. direct bioautography technique on tlc or paper disc technique was used, depending on the extract and pathogenic fungi tested. the results showed the production on inhibitory components, not only by ectomycorrhizal fungi, but also by p. glehnii inoculated or not with ectomycorrhizal fungi. the sensitivity varied considerably according to the type of fungus or extract. key words: japan/mycorrhizas/inh|bition/extracts/antimicrobial compounds/amanita pantherina/pisolithus tinctorius/paxillvs spjscleroderma flavidum/fusarium roseum/pythium spjrhizoctonia solani. introduction damping-off has been responsible for considerable losses of tree species in nurseries. research has shown that these losses can be reduced by inoculation of effective * paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19 21 april 1994, yogyakarta, indonesia 53 biotropia no. 9, 1996 ectomycorrhizal fungi (sinclair et al 1982, chakravarty and unestan 1987; sampangi et al. 1985; duchesne et al. 1989). it has been postulated that root protection by ectomycorrhizal fungi results from antibiotic synthesis which could either be by the myco-symbiont or by the host plant; a barrier effect caused by the presence of a fungal mantle around roots; or a nutrient competition in the rhizosphere (zak 1964; and marx 1973). none of these can be viewed as universal mechanism (harley and smith 1983), but several mechanisms may act simultaneously and/or synergistically to suppress disease (schisler and linderman 1987). the synthesis of antimicrobial substances by plants in response to ectomycorrhizal fungi has been reviewed and the use of ectomycorrhizal fungi in biological control has been reported (duchesne et al. 1987: 1989b). since the protection provided by these fungi is also influenced by environmental conditions including tree species and pathogens involved (marx 1969; stack and sinclair 1975; sylvia and sinclair 1983a,b; perrin and garbaye 1983; sampangi and perrin 1986; chakravarty and unestan 1987a,b; duchesne et al. 1989), a screening of ectomycorrhizal fungi that is able to produce antimicrobial substances is useful for a better understanding of the mechanism of disease suppression by ectomycorrhizal association. a better understanding of involved mechanisms may lead not only to field manipulations of environmental conditions to enhance disease protection, but also help the design of optimal screening programs. it may also favour the discovery of new pesticides which lead to novel methods of disease control (duchesne et al. 1989b). this study sought to verify the presence of compounds possessing antimicrobial properties in the extracts of ectomycorrhizal fungi or in p. glehnii inoculated with ectomycorrhizal fungi. materials and methods fungal cultures the ectomycorrhizal fungi, pisolithus tinctorius (pers.) coker & couch, scleroderma flavidum e.&e., amanita pantherina (dc.:fr.) krombh. and paxillus sp., were isolated from basidiocarps collected under different forest species in hokkaido. all of them were proven to be mycorrhizal fungi by in vitro condition with p. glehnii (unpublished data). the phytopathogenic fungi, fusarium roseum, rhizoctonia solani and pythium sp. were isolated from p. glehnii seedlings, collected from nursery beds, and kindly provided by y.y. cha, of lab. of sylviculture, fac. of agriculture, hokkaido university. the ectomycorrhizal fungi were maintained on modified melin norkran 54 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. (mmn) solution a (kottke et al. 1987) and the pathogenic fungi on pda (potato dextrose agar), both on petri dishes at 26°c in the dark. extracts from ectomycorrhizal fungi the ectomycorrhizal fungi were grown in mmn at 16°c, in the dark. after 34 days, a volume of 90% methanol (meoh) equal to that of growing cultures was added to each culture. this mixture was filtered after 24 hours, and the meoh in the filtrate material was evaporated under vacuum condition at 40°c. the constituents in the concentrate were partitioned between water and ethyl acetate (etoac). these extracts were stored in the dark at-10°c. in vitro seedlings cultivation the growth system for the in vitro seedlings cultivation consisted of a modified method of sylvia and sinclair (1983a) and duchesne et al (1988a). the test tubes (200 x 30 mm) were lined with filter paper (100 x 100 mm), immersed in mmn solution a (kottke et al. 1987). seeds of p. glehnii were superficially disinfested for 30 min. in h2o230%, rinsed with autoclaved distilled water, and germinated for 7 days in petri dishes before transferring to the test tubes containing the nutrient solution. three seedlings per tube were transferred to each tube and the roots were placed between the filter paper and test tube wall. a week after the seedlings had been transferred, they were inoculated by using one disc (7 mm diameter) containing ectomycorrhizal fungi's myce-lia. the discs were placed between filter paper and test tube wall close to the roots. twenty-five replications per treatment were taken. extracts from seedling the extraction was done on six-month-old seedling. the in vitro synthesis material was divided into two parts: (a) the liquid part, containing nutrient solution and exudates from root and fungi, and (b) the solid part, containing root systems, mycelia and filter paper. an equal volume of etoac was added to the liquid part, and the mixtures were shaken to yield water and etoac soluble fractions. to the solid part, initially about 20 ml of 90% meoh was added per synthesis in vitro. twenty-four hours later, the mixture was filtered. the meoh of the filtrate was evaporated under vacuum condition, at 40°c, and the constituents in the concentrate were partitioned between water and etoac soluble fractions. 55 biotropia no. 9, 1996 direct bioautography technique on tlc this technique was applied to etoac soluble fractions and pathogenic fungi, fusarium roseum and pythium sp. that produce large quantities of spores. the spores of cladosporium herbarum were used as the control to verify the presence of fungitoxic substance due to its high sensibility. samples of etoac extracts equivalent to 10 and 20 mg of mycelia or seedling (dry weight basis) were subjected to tlc using silica gel 60 f254 plates with a thickness of 0.25 mm and a mixture of chloroform-meoh (20:1). the constituents were viewed under uv 245nm (quenching) and 356nm (fluorescent) light, and then the chromatograms were sprayed with a spore suspension of c. herbarum, f. roseum and pythium sp. (romans and fuchs 1970). the plates were incubated in a moisture chamber at 28°c for 5 days to detect growth inhibition spots. the relative distance (rrf) of each inhibition spot was calculated as the relation between the distance of inhibition spot from the starting line and the distance of remarkable fluorescent spot presented by the extract from p. glehnii (b) from the starting line, which was detected close to the front line. paper disc technique the effects of etoac soluble fractions on r. solani, and on all water soluble fractions on phythium, f. roseum and r. solani were examined using paper disc technique. extracts equivalent to 10 and 20 mg (dry weight basis) of seedling material and a. pantherina, and 10 and 13.8; 10 and 16.6; and 10 and 11.8 mg for p. tinctorius, s. flavidum and paxillus sp., respectively, were aseptically imbibed in 8 mm paper discs. the smaller quantities (<20 mg) were used since the densities of extracted fraction for those fungi exceeded the absorption capacity of the paper discs. to verify the inhibition zone produced by the extracts, two doses of each extract were placed on the same petri dish but opposite each other, and the pathogenic fungus mycelia disc (7mm) was located in the center of the dish (fig. 1). the incubation time defined according to the growth rate of pathogenic fungi in previous assays was 3, 8 and 20 days for pythium, f. roseum and r. solani, respectively. the relative inhibition radius (rlr) was calculated as the relation between the radius of inhibition and the radius of paper disc (fig. 1). there were 2 replications per treatment. results and discussion some constituents of etoac extract inhibited the growth of pathogenic fungi. however, these substances acted differently on the individual pathogens, as verified 56 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. figure 1. scheme of the paper disc technique. a and b are 8 mm disc imbibed, respectively, with equivalent to 10 and 20 mg of ectomycorrhizal fungi or p. glehnii extract. c is 7 mm containing pathogenic fungi inoculum; a and b are the radius of inhibition zone. shaded area represents colony growth of pathogenic fungi. from the different rrf on inhibition spots (table 1), except for the extract of a. pantherina x p. glehnii. for this host-fungus combination the same spot inhibited both c. herbarum and f. roseum. some spots visible under uv 254nm and 365nm were fungitoxic, but not all fungitoxic spots were detected under uv light. the fungitoxic substances were more frequently detected in the liquid part of extracts obtained from the seedlings, suggesting that many antibiotic components are present in the exudates. duchesne et al. (1988a) observed that fungitoxic materials were present in the rhizosphere but not in plant tissue of pinus resinosa inoculated with p. involutus. in the water fractions (table 2), fungitoxic compounds were detected in the solid materials (root system + filter paper + mycelia). all ectomycorrhizal fungi tested were capable of producing some kind of fungitoxic substance (table 1). inhibition of phytopathogenic fungi by cell free culture media of ectomycorrhizal fungi was tested by kope and fortin (1989). the authors verified that 7 ectomycorrhizal fungi inhibited 20 out of 24 pathogenic fungi tested. it is possible to produce fungitoxin againts pythium when p. glehnii inoculated with p. tinctorius was synthesized by the latter, since the same spot was present in the extract of this fungus (table 1). the presence of the host plant could have stimulated the synthesis of the fungitoxin, considering the smaller quantity of mycelia present in the 57 biotropia no. 9, 1996 table 1. inhibition spots (rrf*) against cladosporium herbarum, fusarium roseum and pythium sp. when extracts equivalent to 10 and 20 mg of mycelia or plant material (dry weight basis) was applied to direct bioautography on the tlc plates. extract of the inoculated seedling. duchesne et al. (1988b) observed that sporulation of f. oxysporwn was depressed by p. involutus and this suppression was more pronounced when the exudate of pinus resinosa was added, suggesting that the presence of root exudate can stimulate synthesis of antimicrobial substances produced by ectomy-corrhizal fungi. in in vitro studies, perrin and garbaye (1983) verified a strong suppression of pythium ultimum, possibly as an effect of diffusible or volatile inhibitory substances produced by ectomycorrhizal fungus hebeloma crustuliniforme. although we are unable to determine whether ectomycorrhizal fungi or host plant or both were responsible for the synthesis of antimicrobial compounds, the results from this study suggest that mycorrhizal association stimulated production of new fungitoxic compounds (table 1). laccaria laccata was able to inhibit fusarium spp. in vitro, but production of phenolics, induced by inoculation of these ectomycorrhizal fungi might be the basis of root protection of pseudotsuga menziesii (sylvia and sinclair 1993b; and chakravarty and hwang 1991). according to buscot et al. (1992) not only production of 58 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. fungistatic phenolics compounds, but also the greater vigor of mycorrhizal plants is perhaps the cause of resistance to phytopathogenic fungi. earlier work by stack and sinclair (1975) showed that inoculation of laccaria laccata spores protected p. menziesii against f. oxysporum, even without mycorrhizae. complementary results obtained by the paper disc technique (table 2) suggest that some fungitoxins were also present in the aqueous extracts, and the degree of inhibition varied according to the origin of extract. table 2. inhibition radius (rrf) * of fusarium roseum, pythium sp., and rhizoctonia solani, when extracts equivalent to 10 and 20 mg ** of mycelia or plant material (p.m.) (dry weight basis) were applied using paper disc technique. p. glehnii could produce antimicrobial compounds in the absence of ectomycorrhizal fungi (table 2). since these compounds were not detected when the plant was associated with ectomycorrhizal fungi, it is speculated that the mycosymbionts degraded these compounds or transformed them into other metabolite(s). 59 biotropia no. 9, 1996 the growth of r. solani was not affected by any etoac fraction tested. however, the water fraction presented some compounds that delayed pathogenic mycelial growth. no antagonistic effect on the growth of r. solani was observed when a. pantherina or p. tinctorius were confronted on the pda media (zhao et al. 1989). kope and fortin (1989) verified that p. tinctorius cell free media showed high inhibition against r. pra-ticola, but such inhibition was very low against r. solani (kope and fortin 1989). although the methodologies used to test inhibition by zhao et al. (1989) and kope and fortin (1989) differed from those used in this study, sensitivity of r. solani to compounds produced by ectomycorrhizal fungi seems very low especially in comparison with pythium (table 2). the results of this study although not field tested suggest that inoculation of ectomycorrhizal fungi even when the association with the host plant is not established, may protect plants from pathogenic fungi by production of antibiotic substances. it was also verified that new antimicrobial compounds are produced directly by the ectomycorrhizal fungi or by the host plant in response to mycosymbiont association as reported by zak (1964) and marx (1973) for other mycorrhizal systems. further research is needed : to detect whether these fungistatic compounds are produced in the field; and to monitor whether these ectomycorrhizal fungi are still able to protect plants against pathogenic fungi tested when they are interacting with other microorganisms (krupa et al 1973; schisler and linderman 1989; and chen et al. 1988) or with different environmental conditions (harley and smith 1983). acknowledgements the authors sincerely thank r.m.c. muchovej for her invaluable suggestions as well as for improving the english, and to cnpq (conselho nacional de desenvolvimen-to cientifico e tecnologico brazil) for the scholarship granted to the first author. references buscor, f, g. weber and f. oberwinkler. 1992. interactions between cylindrocarpon destructans and ectomycorrhizas ofpicea abies with laccaria laccata and paxillus involutes. trees, 6: 8390. chakravarty, p. and s.f. hwano. 1991. effect of an ectpmycorrhizal fungus, laccaria laccata, on fusarium damping-off in pinus banksiana seedlings. eur. j. for. path., 21: 97-106. chakravarty, p. and t. unestan. 1978a. mycorrhizal fungi prevent disease in stressed pine seedlings. j. phytopathology, 188: 355-340. chakravarty, p. and t. unestan. 1987b. differential influence of ectomycorrhizae on plant growth and disease resistance in pinus sylvestris seedlings. j. phytophatology, 120: 104-120. 60 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. duchesne, l.c., r.l. peterson and b.e. ellis. 1987. the accumulation of plant-produced antimicrobial compounds in response to ectomycorrhizal fungi: a review. phytoprotection, 68: 17-27. duchesne, l.c., r.l. peterson and b.e. ellis. 1988a. interaction between the ectomycorrhizal fungus paxillus involutes and pinus resinosa induces resistance to fusarium oxysporum. can. j. bot., 66: 558-562. duchesne, l.c., r.l. peterson and b.e. ellis. 1988b. pine root exudate stimulate the synthesis of antifungal compounds by the ectomycorrhizal fungus paxillus involutus. new phytol., 108: 471476. duchesne, l.c., r.l. peterson and b.e. ellis. 1989a. the time course of disease suppression and antibiosis by the ectomycorrhizal fungus paxillus involutus. new phytol., i l l : 693698. duchesne, l.c., r.l. peterson and b.e. ellis. 1989b. the future of ectomycorrhizal fungi as biological control agents. phytoprotection, 70: 51-57. harley, j.l. and s.e. smith. 1983. mycorrhizal symbiosis. new york, academic press inc., 483 p. homans, a.l. and a. fuchs. 1970. direct bioautography on thin layer chromatograms as a method for detecting fungitoxic substances. j. chromatog., 51: 327-329. kope, h.h. and j.a., fortin. 1989. inhibition of phytopathogenic fungi in vitro by cell free culture media of ectomycorrhizal fungi. new phytol., 113: 57-63. kottke, i., m. guttenberger, r. hamp and f. oberwinkler. 1987. an in vitro method for establishing mycorrhizae on coniferous tree seedlings. trees, 1: 191-194. marx, d.h. 1969. the influence of ectrotophic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. i. antagonism of fungi to root pathogenic fungi and soil bacteria. phytopathology, 59: 153-163. marx, d.h. 1973. in: marks, g.c. and kozlowski (eds.). ectomycorrhizae: their ecology and physiology. academic press, new york. pages 351-382. perrin, r. and j. garbaye. 1983. influence of ectomycorrhizae on infectivity of fyf/z/wm-infested soils and substrates. plant and soil, 71: 345-351. sampangi, r. and r. perrin. 1986. attempts to elucidate the mechanisms involved in the protective effect of laccaria laccata against fusarium oxysporum. in : gianinazzi-pearson and s. gianinazzi (eds), proceedings of the 1st european symposium on mycorrhizae. dijon, france, july 1-5, 1985. p. 799-806. schisler, d.a. and r.g. linderman. 1989, response of nursery soil microbial populations to volatilize purge from soil around douglas-fir ectomycorrhizae. soil biol. biochem., 21: 397-401. sinclair, w.a., d.m. sylvia and a.o. larsen. 1982. disease suppression and growth promotion in douglas-fir seedlings by the ectomycorrhizal fungus laccaria laccata. for, sci., 28: 191-201. stack, r.w. and w.a. sinclair. 1975. protection of douglas-fir seedlings against fusarium root rot by a mycorrhizal fungus in the absence of mycorrhizal formation. phytopathology, 65: 468472. sylvia, d.m. and w.a. sinclair. 1983a. suppressive influence of laccaria laccata on fusarium oxysporum and douglas-fir seedlings. phytopathology, 73: 384-389. sylvia, d.m. and w.a. sinclair. 1983b. phenolics compounds and resistance to fungal pathogens induced in primary roots of douglas-fir seedlings by the ectomycorrhizal fungus laccaria laccata. phytopathology, 73: 390-397. zhao, z.p., kuo s.c. and bi. k.c. 1989. selection of ectomycorrhizal fungi with resistance to rhizoctonia solani. agriculture, ecosystems and environments, 28: 575-579. zak, b. 1964. role of mycorrhizae in root disease. ann. rev. phytopath., 2: 377-392. 61 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf biotropia no biotropia no. 13, 1999: 37-48 morphometric study for identification of the bactrocera dorsalis complex (diptera : tephritidae) using wing image analysis a. adsavakulchai', v. baimai', w. prachyabrued2, paul j. grote' and s, lertlum" ' department of biology and2 department of physics, faculty of science, mahidol university, rama iv road, bangkok, 10400, thailand e-mail address: toung@ait.ac.th 3 school of biology, institute of science, suranaree university of technology, thailand 4 faculty of computer science program, chulachomklao military academy, thailand abstract the bactrocera dorsalis complex (diptera: tephritidae) used in this study included b. dorsalis, b. arecae, b. propinqua, b. pyrifoliae, b. verbascifoliae, and three new species complexes are species e, species k and species p. bactrocera tau was used as an out-group. a total of 424 adults, which emerged from pupae collected from natural populations in thailand, were prepared for wing measurements. morphometric analysis was performed on measurements of wing vein characters. wing images were captured in digital format and taken through digital image processing to calculate the euclidean distance between wing vein junctions. discriminant and cluster analyses were used for dichotomy of classification processes. all 424 wing specimens were classified to species in terms of the percentage of "grouped" cases which yielded about 89.6% accurate identification compared with the formal description of these species. after clustering, the percentage of "grouped"cases yielded 100.0%, 98.9%, 98.1%, 95.2% and 84.6% accurate identification between the b. dorsalis complex and b. tau; b. arecae and species e; b. dorsalis and b. verbascifoliae; b. propinqua and b. pyrifoliae; and species k and species p, respectively. this method of numerical taxonomy may be useful for practical identification of other groups of agricultural pests. key words: bactrocera dorsalis complex/wing image processing/morphometric/discriminant and cluster analyses. introduction the oriental fruit fly, bactrocera dorsalis (diptera: tephritidae) group of the subgenus bactrocera, has been considered one of the most important groups of agricultural pest in southeast asia because some of these species attack seed bearing organs of plants, including soft fruits and flowers (mcpheron and steck 1996). the b. dorsalis group comprises about 52 closely related species in asia, mostly southeast and south asia, with additional species in the south pacific region (drew and hancock 1994). some of these species are mophologically similar. drew (1989) postulated that the dacinae fruit flies originated in the papua new guinea area and speciated prolifically throughout the region. drew and hancock (1994) listed fourteen closely related species of the b. dorsalis complex from thailand on the basis of morphological characters. these species are b. arecae, b. carambolae, b. dorsalis, b. irvingiae, b. kanchanaburi, b. melastomatos, b. osbeckiae, b. papayae, b. propinqua, b. pyrifoliae, b. raiensis, b. thailandica, b. unimaculata and b. verbascifoliae. recently, we provided population genetic data of some members of the b. dorsalis complex (baimai et al. 1995, 1998 unpublished data; satayalai 37 biotropia no. 13, 1999 1996). nonetheless, most species of the b. dorsalis complex have limited distribution within the tropical and subtropical regions (drew 1989). the limitation of the distribution range of these species is due in part to physical, climatic and gross vegetation factors. however, it is more likely that the distribution range is correlated with the specificity on fruits of particular host plants, yet little information is available on the range of host plants of these species. the b. dorsalis complex is systematically one of the most interesting groups of insect pest (ibrahim and ibrahim 1990). because of similarity in external morphology among the members of the b. dorsalis complex and the geographic variation in morphology within each species, it has been very difficult to separate these species. consequently, such morphological variation has caused taxonomic problems (hardy 1977). thus, the most common errors are synonyms, homonyms, misidentifications and establishment of supra-specific groups based on questionable morphological characters (white and elson-harris 1992). there is still a major need for more taxonomic study in correlation with population genetic investigations of the b. dorsalis complex to address some sibling species problems. in most countries, a complete list of reference collections for identification purposes can be found, resulting from the work of trained taxonomists. it has long been obvious that fruit flies of the subfamily dacinae have major economic effects on society. therefore, economic entomologists were needed to identify the various species involved. in spite of this tireless work, however, many taxonomic problems and misidentifications accrued (mcpheron and steck 1996). some of these systematic problems can be elucidated with the aid of the recent development of taxonomic techniques such as cytotaxonomy and molecular biology as well as improved numerical taxonomy (sneath and sokal 1973). yu et al. (1992) studied morphometric analysis of linear wing measurements for identification of ichneumonid wasps using image analysis of wings. the authors outlined the procedure to digitize and to measure various wing elements with an image analyzer and the wing specimens were assigned to species by discriminant analysis and independent univariate comparisons of wing measurements. recently, weeks (1996) developed daisy (digital automated identification system) based on the idea that the pattern of veins and pigments on insect wings are distinct-like fingerprints. much of the acquisition by computer of morphological characters of insects (white and scott 1994) has involved measurement of projected images on digitizing tablets (howell et al. 1982). use of computers efficiently provides accurate measurements and saves development time. in addition, these methods can be easily repeated and made available to any user and reworked with a minimum of effort. however, these methods are costly hi terms of software development and maintenance. image analysis of morphological characters of wings may be the first step towards a completely automated insect identification technique cfuetal. 1992). in our ongoing research on the biology of fruit flies hi thailand, we attempt to employ ecological observations in the field and genetic investigations in the laboratory coupled with morphological examination of larvae, pupae and adults to help solve the problems of identifying some cryptic or isomorphic species. thus some new sibling species of the b. dorsalis complex have been found through 38 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. allozyme electrophoresis (satayalai 1996) and cytogenetic studies (baimai et al. 1995, 1998 unpublished data). in this paper we describe the methodology of image analysis to acquire and quantify morphological characters of the wing veins of eight species of the b. dorsalis complex in a computer compatible form for suitable identification of these species. materials and methods specimen collection eight species of the b. dorsalis complex used in this study include b. dorsalis (hendel), b. arecae (hardy and adachi), b. propinqua (hardy and adachi), b. pyrifoliae drew and hancock, b. verbascifoliae drew and hancock, and three new species complexes are species e, species k and species p with morphological characters different from the record of drew and hancock (1944). in addition, bactrocera tau (walker), of the subgenus zeugodacus, was used as the out-group. larval specimens of these members of the b. dorsalis complex were obtained from a wide variety of infested fruits from various parts of thailand (table 1). some larvae were processed for mitotic karyotype study which provided useful information for table 1. specimens of the eight species of the bactrocera dorsalis complex and bactrocera tau collected from different localities in thailand 39 biotropia no. 13, 1999 species identification. most of the larvae were reared either in the field or in the laboratory allowing them to pupate and finally emerge as adults. some adults were processed for electrophoretic study to confirm the genetic species as determined by mitotic chromosome markers. adults from each collection were examined morphologically for species identification in correlation with chromosomal evidence and electromorphic allozyme patterns. some adults were kept for wing specimens preparation. the framework of the research is shown in figure 1. wing preparation wings of individual adults were detached from the thorax and they were placed on a microscope slide. the wings were secured under a coverslip with canada balsam. image processing wing image processing procedure is shown in figure 2. the microscope slide with wing samples was positioned on a nikon smz-2t stereomicroscope with a low objective lens (ix). the vertical tube has a control light path switchover which allows the diversion of the right eye image to the camera. a nikon e2s digital still camera was attached with a cf projection lens (4x) that captured the whig image on the memory card. digital imaging with high-resolution of 1.3 million pixels was then transferred to application handling jpeg (joint photographic experts group) files (fig. 3). 40 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. the original color image (jpeg : raster format) was transformed into gray scale in the form of bmp (bitmap) raster file format that allows efficient localized image processing. from bmp, the image was pre-processed by low-pass filtering with average of 3x3 to create a smooth image (gonzalez and woods 1992); then the image was transformed by applying a linear function to enhance the image by calculating the ratios from pixel values of the original image divided by pixel values of the smooth image. then, the image was vectorized to create vector file format in dxf (drawing exchange file) file format and manually adjusted until suitable for measuring the 30 wing vein distances (fig. 4). the vector file was used to create 41 biotropia no. 13,1999 figure 3. showing digital image of a wing in the form of jpeg file format; 1. bactrocera arecae 2. b. dorsalis 3. b. propinqua 4. b. pyrifoliae 5. b. verbascifoliae 6. species e 7. species k 8. species p 9. bactrocera tau figure 4. diagrammatic representation of a wing of the b. dorsalis complex showing the 30 wing vein measurements. 42 b un a # 15 0 9/ 23 /9 7 4: 52 p m p ag e 10 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. automatically the coverage that contained 30 data sets of each sample in terms of euclidean distances. euclidean distance is the measurement between the 2 coordinates and is computed as the square root of the sum of the squared differences as shown in the following formula: euclidean distance (d) = [ (x-s)2 + (y-t)2 ] vl where euclidean distance is the distance between two points : (x,y) is co-ordinate of p; (s,t) is co-ordinate of q. statistical analysis spss for windows version 6.0 (george and paul 1995) was used for data analysis. discriminant and cluster analyses were used for dichotomy of the classification processes. all 424 wing specimens were classified to species in terms of the percentage of "grouped" cases. results and discussion our data show that the length of a vein is correlated with the size of the wing. ratios of the vein lengths provide a very effective means for recognizing trends in variation and for a quick diagnostic character. discriminant function analysis was used to derive a function as a criterion for separation of the eight species used in this study. the percentage of "grouped" cases correctly classified with the accuracy of 89.6% is shown in table 2. the linear discriminant function can completely discriminate members of the b. dorsalis complex from b. tau as follows: y = 2.76xj +9.85x2-16.74 where x! = vein2/vein22, and x2 = vein3/vein8. specimens are identified by the following rule : if y < 0 then species is bactrocera dorsalis complex if y > 0 then species is bactrocera tau the eight species of the b. dorsalis complex can also be separated by using cluster analysis (fig. 5). the results from cluster analysis show that the eight members of the b. dorsalis complex can be classified into 4 classes : (1) b. arecae and species e; (2) b. verbascifoliae and b. dorsalis', (3) b. propinqua and b. pyrifoliae; and (4) species k and species p. bactrocera tau was used as the out-group. discriminant function analysis was used to derive a function to provide maximum values for separation of these 4 classes. the "grouped" cases were correctly classified with an accuracy of 98.9%, 98.1%, 95.2% and 84.6%, respectively (table 3). the stepwise 43 biotropia no. 13,1999 table 2. classification results of the 8 species of the bactrocera dorsalis complex and bactrocera tau and percent of "grouped" cases correctly classified: 89.6%. figure 5. dendrogram using average linkage (between groups); 1. bactrocera arecae 2. b. dorsalis 3. b. propinqua 4. b. pyrifoliae 5. b. verbascifoliae 6. species e 7. species k. 8. species p 9. bactrocera tau 44 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. table 3. classification results in 4 groups of the b. dorsalis complex revealing percentage of "grouped" cases correctly classified as 98.9%, 98.1%, 95.2% and 84.6% for classes 1, 2, 3 and 4, respectively discriminant analysis procedure has certain advantages in reducing the use of a large number of variates to a small number of canonical variables. these selected variables were calculated as a ratio between variables, which were used in discriminant analysis for classification in each group afterwards. the stepwise discriminant analysis procedure was performed to take data correlation and to select variables for transformation in the next step. finally, the best ratio was selected as an index for each species as shown in table 4. the results appear to be generally satisfactory in separation of these species of the b. dorsalis complex compared with the genetic data (baimai et al. 1995, 1998 unpublished; satayalai 1996) and classical taxonomy (drew and hancock 1994). some adults were processed for electrophoretic study to confirm the genetic species as determined by mitotic chromosome markers. adults from each collection were examined morphologically for species identification in correlation "with chromosomal evidence and electromorphic allozyme patterns. however, correct 45 biotropia no. 13,1999 table 4. index for classification of the 8 species of the bactrocera dorsalis complex and bactrocera tau. (sd) = standard deviation; + = 2 or more indices must be used for classification species index value %accuraccy bactrocera tau (out-group) b. dorsalis complex vein2/vein22+ vein3/vein8 vein2/vein22+ vein3/vein8 2.82 (0.33) 0.83 (0.06) 4.59 (0.48) 0.97 (0.05) 100.0% 100.0% b. arecae species e vein!8+ vein7/vein!4+ veinl8/vein20 vein!8+ vein7/vein!4+ vein!8/vein20 25.8 (2.5) 0.39 (0.03) 1.55(0.09) 31.0(1.7) 0.4 (0.01) 1.63(0.08) 98.9% 98.9% b. dorsalis b. verbascifoliae veins /vein 17+ vein8/vein!6+ vein8/vein29+ vein!3/vein24 vein5/vein!7+ vein8/vein!6+ vein8/vein29+ veinl3/vein24 0.83 (0.05) 1.75(0.07) 1.93 (0.26) 1.15(0.04) 0.88 (0.05) 1.57(0.08) 1.85(0.26) 1.22(0.04) 98.1% 98.1% b. propinqua b. pyrifoliae vein8+ vein26+ vein7/vein!8+ vein20/vein24+ vein!3/vein20 vein8+ vein26 vein7/veinl8+ vein20/vein24+ vein!3/vein20 91.0(7.2) 10.28 (1.57) 3.09 (0.23) 0.6 (0.05) 2.02 (0.14) 85.0 (7.9) 12.92(1.27) 3.03(0.12) 0.6 (0.04) 2.03 (0.22) 95.2% 95.2% species k species p vein 23/perimeter+ veinl8/vein21+ veinl3/vein24 vein23/perimeter+ vein!8/vein21+ vein 13^6^4 0.036 (0.002) 0.77 (0.06) 1.16(0.04) 0.034 (0.002) 0.63 (0.03) 1.21 (0.04) 84.6% 84.6% identification of some species is rather low, especially species k with a correct classification of only 75%, although this species is quite distinct in external morphology. in addition, "cluster membership of cases using average linkage (between groups)" among the eight species of the b. dorsalis complex and b. tau showed some overlapping characters since there are mixed characteristics which lead to difficulty in classification (fig. 3). moreover, the dendrogram shows how the species could overlap as a result of genetic differentiation during the speciation processes (ashlock 1979). the methodology used for discrimination of members of the b. dorsalis complex proposed and employed in this study has some advantages over other tedious taxonomic techniques (e.g. cytotaxonomy and electrophoresis) for separation 46 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai etal. of closely related species of insects. first, this method does not require fresh specimens. second, it can be operated by a person who has a minimal knowledge of taxonomy or a non-taxonomist. finally, the methodology described in this study seems to be promising for further development of on-line identification systems. it is clear that the data in the form of numerical tables can be easily stored and the computations can be rapidly made (frampton et al. 1991). the methodology of morphometric analysis described here also illustrates the rapid advance in automated methods of on-line biological classification schemes which may have implications in the field of agricultural entomology, particularly in the tropical regions. acknowledgments we wish to thank s. tigvatananont for providing most of the dried specimens of the fruit flies used in this study. we are grateful to hollywood international ltd. for providing access to use nikon e2 series (nikon digital still cameras). this work was supported by the thailand research fund (rta 3880008). references ashlock, p.o. 1979. an evolutionary systematist's view of classification. systematic zoology, 28: 441 50. baimai, v., w. trinachartvanit, s. tigvattananony, pj. grote, r. poramarcom. and i). kijchalao. 1995. metaphase karyotypes of fruit flies of thailand i. five sibling species of the bactrocera dorsalis complex. genome, 38: 1015-1022. drew, r.a.i. 1989. the taxonomy and distribution of tropical and subtropical dacinae (diptera: tephritidae). world crops pests (ed. by a.s. robinson and g. hooper). amsterdam, elsevier. p. 13-66. drew, r.a.i. and d.i. hancock, 1994. the bactrocera dorsalis complex of fruit flies (diptera: tephridae: dacinae) in asia. bulletin of entomological research supplement series: supplement, 2, 1-68. frampton, e.r., j. fry, b.p. stephenson, and j.m. cowley, 1991. a computerised system for data management during a fruit fly outbreak. the international symposium on the biology and control of fruit flies. george, d. and m. paul, 1995. spss/pc + step by step: a simple guide and reference. ca: duxbury press. belmont. gonzalez, c.r. and e.r. woods, 1992. digital image processing. addison-wesley publishing company. inc. hardy, d.e. 1977. family tephritidae. catalog of the diptera of the oriental region (ed. by delflnado m.d. and hardy d.e.). honolulu, the university press of hawaii. 854 p. howell, v.d., k. hoelmer, p. norman and t. allen, 1982. computer-assisted measurement and identification of honey bees (hymenoptera: apidae). annals of the entomological society of america, 75, 591-5944. ibrahim, r. and g.a. ibrahim, 1990. handbook on identification of fruit flies in the tropics. universiti pertanian malaysia press. mcpheron, b.a. and g.j. steck, 1996. the fourth international symposium on fruit flies of economic importance. a world assessment of their biology and management. st. lucie press. 47 biotropia no. 13, 1999 satayalai, o. 1996. electrophoretic study of natural populations of the bactrocera dorsalls complex (diptera: tephritidaej) in thailand. ph.d. thesis, bangkok. faculty of graduate studies, mahidol university. sneath, p.h.a. and r.r. sokal. 1973. numerical taxonomy: the principles and practice of numerical classification. w.h. freeman and company. weeks, p. 1996. daisy takes wing to sort out the bugs of the world. the times. july 17. white, i.m. and m.m. elson-harris, 1992. fruit flies of economic significance: their identification and bionomics. wallingford, uk, cab international. white, i.m. and p.r. scott, 1994. computerized information resources for pest identification: a review, p.129-137 in: hawksworth, d.l. (ed.). the identification and characterisation of pest organisms. wallingford, uk, cab international. yu, d.s., e.g. kokko, j.r. barron, g.b. schaalje, and b.e. gowen, 1992. identification of ichneumonid wasps using image analysis of wings. systematic entomology, 17, 389-395. 48 biotropia no biotropia no. 15, 2000 : 26 35 presence of hema-like and hemt-llke genes in a jsumber of anoxygenic photosynthetic bacterial isolates from indonesia and soil samples from bogor area nurul aini'and antonius suwanto' 2 -1 'laboratory of microbiology and biochemistry, inter university centre for biotechnology, bogor agricultural university, bogor-16680 indonesia. department of biology, faculty of science and mathematics, bogor agricultural university, bogor 16144, indonesia. 'south-east asian regional center for tropical biology, bogor-16001, indonesia. abstract the rhodobacter sphaeroides hema and hemt are known to encode a distinct 5-aminolevulinic acid (ala)-synthase isozyme. this enzyme catalyzes the first and rate limiting step in ala biosynthesis through the c4 pathway. this study was carried out to detect hema-\\ke and hemt-\\ke genes in twenty anoxygenic photosynthetic bacterial (apb) isolates from several wetland areas in indonesia, and four dna samples that were isolated from four soil samples obtained from bogor area. hybridization techniques of southern and dot blot were used, using hema and hemt fragment as probes. southern hybridization analyses indicated the presence of hema-\\ke gene in five of apb isolates, i.e., mb15, mb16, mb21.2, mb55 and mb6, whereas hemt-\\ke gene was detected only in mb15. dot blot hybridization analyses suggested that the soil samples from waterlogged paddy-field, dry paddy-field as well as a mud pond were predominantly occupied by prokaryotic organisms which harboured hema-]\ke gene. however, /iem7"-like sequences were also found in soil sample from dry paddy-field. key words: hema-\\ks gene / hemt-\\ke gene / southern hybridization analysis / dot blot hybridization analysis. introduction the rhodobacter sphaeroides hema and hemt genes encode a distinct 5aminolevulinic acid (ala) synthase isozyme (neidle & kaplan 1993). alasynthase catalyzes the first and rate-limiting step in ala biosynthesis through the c4 pathway. ala is the first committed precursor in the common tetrapyrrole pathway (goodwin & mercer 1986; beale & weinstein 1991; beale 1995). recently, ala has received attention as a new biodegradable herbicide (sasaki et al. 1987) and insecticide (sasaki et al. 1990). the dna sequences of hema and hemt genes and their location on r. sphaeroides physical map have been determined. the hema gene is located on the large chromosome whereas hemt gene is found on the small chromosome (neidle & kaplan 1993a). the hema and hemt genes encode peptides that are 53% similar to each other, and these peptides are also significantly similar to ala-synthase from several bacteria and eucaryotic species (neidle & kaplan 1993). the hema fragment has been cloned in r. sphaeroides (tai et al. 1988) as well as in escherichia 26 biotropia no. 15, 2000 coli (werf & zeikus 1996). the cloned hema fragment is expressed well in e. coli and able to enhance ala production (werf & zeikus 1996). this evidence shows that hema can be used as a genetic material for enhancing ala production. since hema and hemt genes have high homology to gene encoding alasynthase from other organisms, hema-v\ke and hemt-v\ke genes might be found in other bacteria that form ala through the c4 pathway. in this study, we detected the presence of hema-\\ke and hemt-\ike genes in anoxygenic photosynthetic bacterial (apb) isolates, because most member of apb use the c4 pathway to produce ala. soil, especially paddy-field soil, is known as a common habitat of apb (habte & alexander 1980: gest et al. 1985). therefore, we also carried out an experiment to detect these genes in four soil samples. the results of this study would be expected to generate some insights on the distribution and population density of apb as well as r, sphaeroides strains. in addition, the specificity of hemt would be assessed to be used as a specific molecular marker for r. sphaeroides isolates. materials and methods bacterial strains, plasmids, growth conditions and soil samples the bacterial strains and plasmids used are listed in table 1. apb isolates were grown photoheterotrophically in sistrom's minimal medium (lueking et al. 1978) in full filled screw-cap tubes, ph 7.2. e. coli were grown at 37°c in luria bertani (lb) table 1. bacterial strains and plasmids 27 presence of hema-like and hemt-like genes – nurul aini & antonius suwanto table 1. continued (sambrook et al. 1989) supplemented as needed with antibiotics. antibiotics were added at the following concentrations: 100|ag ampicillin/ml (for maintaining puc19, pui1004, pui1014, and pui612), 25|ig chloramphenicol/ml (for maintaining phfl.l). soil samples were taken from waterlogged paddy-field, dry paddy-field, lsi pond and grawida yard, bogor agricultural university, darmaga campus. all sampling areas were located at darmaga, bogor. the description of the soil samples are listed in table 2. table 2. soil samples dna isolation plasmids dna were isolated by using wizard miniprep dna purification system (promega, wise.) according to the manufacturer's instruction. genomic dna was extracted from each apb isolate using the phenol extraction method with slight modification as follows. the cell pellet was suspended in edta solution containing 15 mg lysozyme/ml, and incubated at 37°c for 1 hour. the lysis was accomplished by adding 300 ul sds buffer (0.1 m nacl; 4% sds; 0.5m tris-hcl, ph 8). the extract was freeze-thawed. the dna was phenol-extracted and ethanol 28 biotropia no. 15, 2000 precipitated as in standard protocol (sambrook et al. 1989). the e. coli genomic dna was isolated as described previously (leach et al. 1994). the dna was extracted from soil by using modified tiedje method (keller 1997, unpublished). the soil sample (10 g) was finely grinded. the 5 g grinded soil was mixed with 13.5 ml tiedje buffer (100 mm tris-hcl, ph 8; 100 mm naedta, ph 8; 100 mm na2po4, ph 8; 1.5 m nacl; 1% ctab) in 100 ml centrifuge tube, and freeze-thawed 3x. after freeze-thawing, 100 ul proteinase-k (20 mg/ml) (sigma, singapore) was added and incubated at 37°c for 30 minutes, then 10 ml 10 % sds was added and incubated at 65°c for 2 hours. the mixture was centrifuged at 6000 g for 10 minutes. the supernatant was extracted with 1 volume of chloroform (merck, jakarta) and centrifuged at 6000 g for 1 minute. the aqueous phase was transferred to a new 100 ml centrifuge tube and 0.6 volume of isopropanol (merck, jakarta) was added at room temperature. the dna was recovered by centrifugation at 16,000 g for 20 minutes at 4°c. the supernatant was discarded and the pellet was washed with 70 % ethanol. the dna was dried at room temperature and dissolved in 100 ul te buffer (omm tris-hcl, ph 8; i m m edta). southern hybridization analysis the 1.2 kb bamttl (neb, singapore) fragment from p u i 1 0 1 4 and 1.8 kb bam\\\ (neb, singapore) fragment from pui1004 were isolated for preparing hema and hemt probes. the fragments were purified from agarose gel using the gene clean kit (bio 101 inc, la jolla, calif.), and labeled with biotin-14-atp using nick translation system (gibco/brl, grand island, ny) according to the manufacturer's instruction. the unincorporated nucleotides were removed from the probes with nuctrap push columns (stratagene, la jolla, calif.). the genomic dnas extracted from apb isolates were digested with ecorl (neb, singapore), except the dna from mb 15 isolate, which was digested with bam\\\ (neb, singapore). digestions were performed in appropriate buffer at 37°c for 12 hours. the digested dna was electrophoretically separated. the dna fragments were transferred to a nylon membrane (photogene, gibco/brl, grand island, ny) by capillary action with standard method (sambrook et al. 1989). hybridization was carried out as described previously at 42°c for 12 hours (sambrook et al. 1989), followed by washing at 37°c for 2x 15 minutes each, and detection using a chemiluminescent method (photogene detection system, gibco/ brl, grand island, ny). dot blot hybridization analysis four biotinilated dna probes, i.e. hema, hemt, pucba, and i6s rrna genes were used. the probes were prepared as described above. to avoid bias in calculation, the population density of prokaryotes was based on the same amount of soil samples, and not on the same amount or standardization of dna concentration. dna isolated from soil samples were denatured as described previously (keller and 29 presence of aema-like and hemt-\ike genes nurul aini & antonius suwanto manak 1992), and applied to nylon membrane (photogene, gibco/brl, grand island, ny) by spotting directly onto the membrane. high stringency hybridization and washing conditions were used. hybridization was carried out at 42°c for 12 hours, with washing temperature of 55°c (sambrook et al. 1989). detection was done using the photogene detection system (gibco/brl, grand island, ny). results and discussion southern hybridization analyses southern hybridization analyses were performed to determine the presence of hema-like and hemt-\\ke genes in twenty apb isolates from indonesia (table 1). r. sphaeroides 2.4.1 (rsp 2.4.1) hema and hemt genes were used as probe. the analyses identified some homologous regions the homology to hema in the genomic dna of mb6, mb 15, mb 16, mb21.2 and mbs 5. the region of homology to hema probe in the genomic dna of each apb isolate is shown in figure la, lanes 3-7. the hemtprobe hybridized only to 4.8 kb bamhl fragment in mb15 genomic dna (fig. ib, lane 3). a b figure 1. a. southern hybridization analyses using hema probe. lanes (1) rhodobacter sphaeroides 2.4.1, (2) kbslell, (3) mb15, (4)mb16, (5) mb21.2, (6) mb55, and (7) mb6 b. southern hybridization analyses using hemtprobe lanes (1) rhodobacter sphaeroides 2.4.1, (2) kbslell, and (3) mb 15 30 biotropia no. 15, 2000 the results implied the presence of hetna-like gene in mb6, mb15, mb16, mb21.2 and mb55, whereas hemt-like gene was only implied in mb 15. the presence of hema-like and/or hemt-like genes in the five apb isolates indicates the ala biosynthesis in these apb isolates is employed through the c4 pathway, because the c4 pathway is mediated by ala synthase which encoded by hema and/or hemtgenes. moreover, the presence of hema-like and/or hemt-\ike genes in the five apb isolates indicates that these apb isolates could be classified under sub group cc-proteobacteria. in photosynthetic bacteria, c4 pathway is utilized by purple non-sulfur bacteria, especially sub group a-proteobacteria (avissar et al. 1989; beale 1995). the sub group a-proteobacteria contains species of genera rhodospirillum, rhodopila, rhodopseudomonas, rhodomicrobium and rhodobacter (imhoff 1995). neither hema-like gene nor hemt-like gene was identified in the other 15 apb isolates. the data suggested that the c4 pathway is not utilized by these apb isolates to produce ala. the 15 apb isolates might produce ala through c5 pathway, which does not require ala synthase. these apb isolates used are not the member of sub group a-proteobacteria, although all apb isolates studied here belong to purple non-sulfur bacteria. the hemt-like gene was only identified in mb 15. this apb isolate also carries hema-like gene. interestingly, the hema-like gene in mb 15 was detected on the same locations with rsp. 2.4.1 hema, i.e., at 1.2 kb and 2.4 kb bamhl fragments (fig. la, lane 1 and 3). the strong intensity of the hybridization signals revealed that the similarity between mb 15 hema-like gene and rsp.2.4.1 hema was very high. moreover, the color comparison of mb 15 culture with rsp. 2.4.1 culture also showed a high similarity. based on these findings, it is very likely that mb 15 was rhodobacter sphaeroides. we tentatively conclude that hemt-like gene harbors specifically in r. sphaeroides. neidle and kaplan (1993) reported that the r. sphaeroides is the only bacterial species that produces two ala synthase isozymes. however, previously ala synthase isozymes are found in some vertebrates (dierks 1990), while no previous information on bacterial ala synthase isozymes have been reported. dot blot hybridization analysis dot blot hybridization analysis was employed to detect the presence of hemalike and hemt-like genes in dna extracted from four soil samples (table 2). four dna probes, i.e., hema, hemt, pucba, and 16s rrna gene were used. this analysis also revealed the relationship of the activity of prokaryote and apb in the soil samples with the presence of hema-\ike and hemt-like genes. figure 2 shows the results of dot blot hybridization using the four probes. interpretation of these results is described in table 3. based on the hybridization using hema probe (fig. 2a), the presence of hema-like gene was detected in soils taken from waterlogged paddy field, dry paddy field and lsi pond. the presence of hema-like gene in these soil samples indicates the activity of organisms producing 31 presence of aema-like and hemt-\\ke genes nurul aini & antonius suwanto ala through the c4 pathway. the c4 pathway is utilized by animals, fungi, protozoa and sub group cc-proteobacteria (avissar et al. 1989; beale & weinstein 1991; beale 1995). figure 2. dot blot hybridization analyses using hema (a), hemt(b), 16s rrna (c), and pucba (d) as probes. dots (1) rhodobacter sphaeroides 2.4.1., (2) soil sample from, waterlogged paddy field, (2) soil sample from dry paddy field, (3) soil sample from lsi pond, and (5) soil sample from grawida yard table 3. interpretation of the result of dot blot hybridization + : detected : nodetected the number of (+) represent the degree of intensity of hybridization signal 32 biotropia no. 15, 2000 using 16s rrna probe, we identified the presence of prokaryotic microorganisms in all four soil samples (fig. 2c). the results of hybridization to pucba probe indicated that the apb could be detected in all soil samples (fig. 2d). thus, it is possible to find the ct-proteobacteria in the soil samples, because some members of oc-proteobacteria are classified as apb group. the possibility for the ct-proteobacteria to exist in the four soil samples correlates with the presence of hema-l\ke gene in these soil samples. however, in spite of the detection of the apb activities in soil from grawida yard, hema-\ike gene was not found in this soil. the intensity of hybridization signal to both 16s rrna (fig. 2c) and pucba (fig. 2d) probes shows that the population densities of both prokaryote and apb in soil from grawida yard are much lower than those found in the other three soil samples. the a-proteobacteria might not exist in the soil from grawida yard or the ccproteobacteria were actually present in this soil, but the population density is very low. therefore, presence of hema-yike gene could not be detected. soils obtained from waterlogged paddy field, dry paddy field and lsi pond were found to have high population density of prokaryotes and apb. the high population density of apb enables the a-proteobacteria to proliferate. the presence of hema-v\ke gene in these soil samples is related to the population density of apb. the population density of apb as well as other microorganisms in the soil is dependent on the availability of growth nutrient, o2 and water (brock & madigan, 1991). the fertility and the soil texture are responsible for the availability of these factors. therefore, the presence of hema-\ike gene in soil is indirectly affected by the fertility and the soil texture based on the results of hybridization to hema probe, the hema-\ike gene might be present in the soil taken from apb habitat. in nature, apb occur in moist soil, paddy field, sewage water, fresh water, brackish water, waste water, marine habitat and in extreme condition of the antartic (sasikala et al. 1985). the presence of hema-\ike gene in the soil is related to the fertility of the soil, because the availability of growth nutrient affects apb growth. the dot blot hybridization using hemt probe identified the presence of hemtlike gene only in soil taken from dry paddy field (fig. 2b, dot 3). by comparing the intensity of hybridization signal to hemt probe with the hybridization signal to hema probe, it is clear that the homologous sequence to hemt probe is present in relatively fewer amount than the homologous sequence to hema probe. the results indicate that not all organisms carrying hema-\\ke gene also carry the hemt-\ike gene. these findings confirm the data obtained from southern hybridization analyses that the presence of hemt-\ike gene is more specific than that of hema-\ike gene. the hemt or hemt-\\ke gene might be specific to r. sphaeroides. however, further studies to determine the nature of specificity of hemt or hemt-\\ke gene to r. sphaeroides should be carried out to develop in situ hybridization method to examine directly the distribution and density of r. sphaeroides in the soil or water ecosystem. 33 presence of aema-like and hemt-\\ke genes nurul aini & antonius suwanto ackowledgments this research was supported by a competitive grant (hibah bersaing v, grant 08/p21 pt/dppm/96/phbv/i/v/1996) to antonius suwanto. references avissar, y.j., j. ormerod, and s.i. beale. 1989. distribution of 6-aminolevulinic acid biosynthetic pathway among photosynthetic bacteria and related organism. arc. microbiol. 151:513-519. beale, s.i. 1995. biosynthesis and structure of porphyrine and hemes. in: r.e. blakenship, m.t. madigan, and c.e. bauer (eds). anoxygenic photosynthetic bacteria. kluwer academic publisher. netherlands, p. 847-870. beale, s.i., and j.d. weinstein. 1991. biosynthesis of of 8-aminolevulinic acid in phototropic organism. in. h. scheer (ed). chlorophylls. crc press. boca raton. p. 368-392. brock, t.d., and m.t. madigan. 1991. biology of microorganism. prentice-hall international inc. usa. p. 566-569, 703-726. dierks, p. 1990. molecular biology of eukaryot 5-aminolevulinate synthase. in h.a. dailey (ed). biosynthesis of hemes and chlorophylls. mcgraw-hill. new york. p. 210-234. gest, h., j.l. favinger, and m.t. madigan. 1985. exploitation of n2 fixation capacity for enrichment of anoxigenic photosyntetic bacteria in ecological studies. fems microbiol. ecol. 31: 317-322. goodwin, t.w., and e.i. mercer. 1986. introduction to plant biochemistry. pergamon press. oxford, p. 465-467. habte, m., and m. alexander. 1980. nitrogen fixation by photosynthetic in lowland rice culture. appl. environ. microbiol. 39: 342-347. imhoff, j.f. 1995. taxonomy and physiology of photosynthetic purple bacteria and green sulfur bacteria. in: r.e. blakenship, m.t. madigan, and c.e. bauer (eds). anoxygenic photosynthetic bacteria. kluwer academic publisher. netherland. p. 1-15. irawan, a. suwanto, p.d. tjondronegoro. 1998. isolation and screening of anoxygenic photosynthetic bacteria producing extracellular l-aminolevulinat acid. hayati. 5: 98-102. keller, g.h., and m.m. manak. 1992. dna probes. stockton press. uk. p. 1-16. kiley, j. p, and s. kaplan. 1987. cloning dna sequence and expression of the rhodobacter sphaeroides light-harvesting 8800-850-a and b800-850-p genes. j. bacteriol. 169: 3268-3275. leach, j. e., f.f. white, m.l. rhoads, and h. leung. 1994. a repetitive dna sequence differentiates x. campestris p.v. oryzae from other patovar of x. campestris. mol. plant-microb. interact. 98: 238246. leuking, d.r., r.t. faley, and s. kaplan. 1978. intracytoplasmic membrane synthesis in synchronous cell population of rhodopseudomonas sphaeroides. j. biol. chem. 253: 451-457. neidle, e.l., and s. kaplan. 1993. expression of the rhodobacter sphaeroides hema and hemt genes, encoding two 5-aminolevulinic acid synthase isozymes. j. bacteriol. 175: 2292-2303. sambrook, j.e., f. fritsch, and t. maniatis. (1989). molecular cloning. cold spring harbor laboratory press. usa. p. 9.31-9.58. 34 biotropia no. 15, 2000 sasaki, k., s. ikeda, y. nishizawa, and m. hayashi. 1987. production of 5-aminolevulinic acid by photosynthetic bacteria. j. ferment. technol. 65: 511-515. sasaki, k., n. noparatnaraporn, s. nagai. 1990. production of 5-aminolevulinic acid as a herbicide from swine waste by rhodobacter sphaeroides. ann. rep. 1c biotech. 13: 277-281. sasikala, k., ch. v. ramana, p.r. raghureer. and k.l. kovacs. 1985. anoxygenic photosynthetic bacteria: physiology and advances in hydrogen production technology. adv. app. microbiol. 28: 211-295. schleifer, k.h., w. ludwig, j. krauss, and h. festl. 1985. cloned ribosomal rna genes from pseudomonas aeruginosa as probe for conserved deoxyribonucleic acid sequences. int. j. syst. bact. 38:231-237. suwanto, a., and s. kaplan. 1989. physical and genetic mapping of the rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. j. bacteriol. 171: 5850-5859. tai, t.n., m.d. moore, and s. kaplan. 1988. cloning and characterization of the 5-aminolevulinic synthase gene(s) from rhodobacter sphaeroides. gene. 70: 139-151. werf, m. j., and j. g. zeikus. 1996. 5-aminolevulinat production by escherichia coli containing the rhodobacter sphaeroides hema gene. appl. environ. microbiol. 62: 3560-3566. 35 biotropia no biotropia no. 14, 1999: 1-9 genetic diversity analysis of thermophilic bacteria from candradimuka crater in central java employing pcr-rflp of 16s-rrna gene temmy desiliyarni1+, antonius suwantoaaao2, 3 4*, maggy t. suhartono3, and tresnawati purwadaria5 'graduate school, bogor agricultural university, bogor, indonesia 2 department of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, indonesia 3 inter university center for biotechnology, jl. puspa tromol pos 1 darmaga, bo gor agricultural university, bogor, indonesia 4seameo-biotrop, jl. raya tajur, km. 6 bogor, indonesia ''research institute for animal production, po box 221 bogor 16002, indonesia abstract the specific primers for bacteria (63f and 1387r) were used to amplify the 16s-rrna genes from total community genomic dna of thermophilic bacteria. the total community genomic dna was obtained from muds and water samples of candradimuka crater, dieng plateau, central java. pcr products were cloned into vector pcr*2.1-topo (3.9 kb) and transformed into escherichia coli topic. two tetrameric restriction endonucleases rsal and hhal were employed to generate restriction fragment length polymorphisms (rflp) paterns. these enzymes yielded 10 and 9 groups of 16s-rrna profiles or otu (operational taxonomic units) from 27 16s-rrna gene clones. rsal was found to be more discriminative in differentiating the clones than hhal. rsal-rflp indicated that otu 7 and otu 3 represented the most abundant clones, i.e. 6 and 5 clones respectively. the distribution of 16s-rrna gene clones could indicate relative distribution of specific groups of thermophilic bacteria in their natural habitat. analysis of diversity at the dna level could represent both culturable and unculturable bacteria in the environment. similarity analysis showed that at level 0.600 there were 8 different groups from 10 rflp profiles generated by rsal digestion. this study indicated that there were at least 8 groups of different thermophilic bacteria occupying candradimuka crater. key words: thermophiles, 16s-rrna, candradimuka crater. introduction sustainability of the biosphere on our planet depends on microbial activities. however, we know very little about the microbial world. the reason for this poor understanding lies in the fact that microbes are tiny and individually invisible to the eye. until recently, microbial identification required isolation of pure cultures followed by a series of physiological tests and biochemical characterization. the pure culture approach has limited our view of microbial diversity in the world, since ' corresponding author; e-mail address : asuwanto@indo.net.id * present address : mercu buana university, jl. meruya selatan, jakarta. 1 biotropia no. 14, 1999 it has been known that approximately only 1% of microorganisms in the environment could be cultured (amann et al. 1995; pace 1997; borneman et al. 1996). microorganisms are involved in many important activities such as soil formation, toxin removal, biogeochemical cycles of carbon, nitrogen, phosphor and others, conservation of more complex species, and to serve as valuable materials to many industries. environmental stresses can alter microbial population and therefore endanger biosphere health. in the absence of systematic approaches to studying and ensuring survival of diverse microorganisms, perhaps the best approach is to preserve as many habitats as possible, especially those from extreme environment which may have unique microorganisms (fox 1994; borneman et al. 1996). one of the extreme environments is thermal environment which could be found in natural and artificial systems such as volcanic region, coal refuse piles, hydrothermal vents and geothermal power plants (stetter 1995). species diversity of the thermophilic organisms is found in both the domain of bacteria and archaea. thermophilic bacteria exhibit a wide range of nutritional capability such as phototrophy and chemotrophy; autotrophy and heterotrophy; chemolitotrophy and chemo-organotrophy; aerobiosis and anaerobiosis (brock 1986). the application of molecular approaches to assess bacterial diversity is now entering the exponential growth. these approaches have overcome the requirement for prior cultivation. the most widely used technique to amplify the gene coding for 16srrna from environmental samples relies on the application of pcr (service 1997; marchesi et al. 1998). for identification purposes, hypervariable regions of rkna molecule are particularly useful due to relatively high differences between species but relatively low variability within species. currently, there are more than 4000 16srrna entries in the database, covering about 1800 species which continue to grow (bottger 1996; amann et al. 1994). hypervariable regions are also the regions with the most concentrated polymorphism for restriction fragment length polymorphisms (rflp) purposes (green 1998). the highly conserved regions of the rrna molecule can serve as primer binding sites for in vitro amplification by pcr (amann 1994). pcr primers designed and evaluated by marchesi et al. (1998) i.e. . 63f (5'-cag gcc taa cac atg caa gtc) and 1387r (5'-ggg cgg wot gta caa ggc) could amplify 1300 bp of a consensus 16s-rrna genes from bacteria. in our study, we attempt to explore genetic diversity of thermophilic bacteria with pcr-rflp of 16s-rrna genes from environmental dna. rflp analysis was conducted using tetrameric endonucleases rsal (5'-gtlac) and hhal (5'-gcgic). these enzymes along with bstul were described by moyer et al. (1996) as the most efficacious at detecting and differentiating bacterial small sub unit rrna genes on the basis of their ability to correctly classify operational taxonomic units (otu). in this study, we have also analyzed partial 16s-rrna sequences of two dominant otu in candradimuka crater. 2 genetic diversity analysis of thermophilic bacteria temmy desiliyarni et al. materials and methods sample collection candradimuka crater is located in dieng plateau (above 2000 m from sea level), central java, indonesia. sample was collected from a small pool (cl site) characterized by almost boiling hot spring with temperature of 88°c and ph7 (fig. 1). water and sandy-mud were collected in a 250 ml sterile bottle, tightly sealed, and transported within 24 hours for immediate processing. figure 1. profile of cl site in candradimuka crater, dieng plateau, central java. extraction and purification of genomic dna approximately 5 g of sample was extracted as described by zhou et al. (1996) and modified by tiedje (1997, unpublished). the crude dna obtained was purified with the prep-a-gene kit (biorad, richmond, ca). amplification and cloning of 16s-rrna genes the 16s-rrna genes were pcr-amplified by specific primers for bacteria (63f and 1387r) from purified genomic dna (200 ng) using ready-to-go pcr 3 biotropia no. 14, 1999 beads (pharmacia-biotech). total volume of pcr reaction contained 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in gene amp pcr system 2,400 thermocycler (perkin-elmer cetus, norwalk, conn.)the pcr protocol was: pre-pcr at 94°c for 2 min, denaturation at 92°c for 30 s, annealing at 55°c for 30 s, elongation at 75°c for 1 min and post-pcr at 75°c for 5 min with a total of 30 cycles (marchesi et al. 1998). the pcr product was cloned into pcr®2.1-topo (3.9 kb), and transformed into competent e. coli top 10 using topo ta cloning kit (invitrogen corp.). the positive clones were screened for a-complementation and selected as white colonies. rflp analysis of 16s-rrna genes recombinant plasmids were isolated using the wizard plus sv miniprep system (promega, madison, wi). £cori and rsal were employed to digest the plasmids in order to verify the cloned dna and to classify according to their restriction patterns. the reason for selecting these two enzymes was based on the fact that the insert dna was cloned between two sites of ecori and there are 3 rsal sites in the vector plasmid. clones that contained approximately 1,300 bp of 16s-rrna genes were chosen for rflp analysis. these clones were subsequently pcr-amplified, restricted by tetrameric endonuclease rsal and hhal to detect the different restriction profiles. restriction enzyme digestion was performed at 37°c for 6 hours before they were separated by gel electrophoresis in 2% agarose gel, stained with 0.5 ug of ethidium bromide per ml and visualized by uv transilluminator (sambrook et al. 1989). rflp profiles obtained from rsal digestion were used to generate dendrogram reflecting their genetic relationship employing upgma clustering method from ntsys program (rohlf 1990). dna sequencing of 16s-rrna genes genes for 16s-rrna from two dominant otus were partially sequenced to infer the closest related organism from rdp database. the sequencing reactions were done using the big dye ready reaction dye deoxy terminator kit and purification using ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn.). results and discussion screening for a-complementation yielded 36 white colonies (transformants). recombinant plasmids from all of these transformants were extracted. verification of the clones containing an insert dna (16s-rrna gene) using rsal generated 19 groups of restriction profiles. thus, we used these 19 groups for subsequent rflp 4 genetic diversity analysis of thermophilic bacteriatemmy desiliyarni et al. analysis. a total of 16 groups (representing 27 clones) contained the entire 1.3 kb to 1.35 kb 16s-rrna insert after pcr-amplification using amplimers 63f and 1387r. two tetrameric restriction endonucleases were employed to differentiate rflp profile from 16s-rrna genes of 16 groups mentioned above in order to identify every object to be classified as otu or a strain in most bacteriological works (logan 1994). the rflp profiles resulted from rsal digestion generate 10 otus (fig. 2) which were: otu 1 (clone number 1, 2, 5, 31), otu 2 (number 3), otu 3 (number 4, 10, 14, 21, 32), otu 4 (number 6), otu 5 (number 9), otu 6 (number 7, 8), otu 7 (16, 18, 19 25, 26, 34), otu 8 (number 17, 20), otu 9 (number 12, 15, 27, 36) and otu 10 (number 30). two dominant otus are otu 7 (6 clones) and otu 3 (5 clones). in evaluating these profiles, we identified discrete patterns that are specific for a clone and could be distinguished one from another as an otu. results from these data infer that bacteria belonging to otu 7 and otu 3 were distributed relatively wide and dominant at the habitat of candradimuka crater. figure2. rflp profile of 16s-rrna gene digested with rsal. molecular standard is 100 bp dna ladder (m). the second digestion of 16s-rrna gene using hhal produced 9 otu (fig. 3). the grouping of restriction profiles was the same as rsal except that otu 7 and otu 8 (from foal-rflp) were in the same group in ///zal-rflp. the result of rflp using these two enzymes indicated that rsal was more discriminative than hhal to differentiate the clones, in this case for bacterial strains from candradimuka crater belonging to otu 7 and otu 8. this result was in agreement with the one conducted by moyer et al. (1996) that hhal, rsal and bstul were the most 5 m 17 18 20 21 27 30 34 36 biotropia no. 14, 1999 figure 3. rflp profile of 16s-rrna gene digested with hhal. molecular standard is 100 bp dna ladder (m). efficacious enzymes at detecting and differentiating 16s-rrna genes originated from environmental dna. our result indicated that at least 10 otus were present in candradimuka site cl which reflected the relative abundance of bacteria in this extreme habitat, in contrast to only two culturable clones obtained on thermophilic maintenance medium incubated at ph 1 and temperature of 70°c under aerobic condition. research conducted by huber et al. (1991) with samples taken from three sites of candradimuka crater obtained two hyperthermophilic archaea, i.e. thermoproteus and desulfurococcus and novel anaerobic obligate bacteria having chemolitotrophy h2/no3". the use of amplimers 63f and 1387r in this study would limit the amplification of 16s-rrna genes only for bacteria. consequently, one should keep in mind that assessment of prokaryote diversity in this study did not include members from archaea. the reduction of 16 groups of restriction profiles to 10 otus from /faal-rflp profiles could be explained with the possibility that the gene insert may have two different orientations when ligated to the vector. the difference could be determined from recombinant plasmid restriction profiles, but not for intact 16s-rrna genes obtained from pcr. the 10 otus of thermophilic bacteria were analysed further using cluster analysis (fig. 4). this dendrogram showed that at level 0.600 there were 8 groups from 10 otus, in that otu 4 and otu 5 were in one cluster as well as otu 7 and otu 8. from this dendrogram, it could also be shown that at level 0.205, there were two groups of genetic relationship. one group consisting of otu 7, 8, 9 and 10, were in the same group with it-08 isolate obtained from gunung pancar, west java 6 m 2 3 < 6 9 1 0 16 m 17 ib 20 2t 27 30 31 34 36 8 bp (tan 1999) and cr1.2 (isolate from 16s-rrna gene library of candradimuka crater at site 4). isolate of it-08 has the dna sequence similar to bacillus thermoleovorans (98%) whereas cr1.2 has the dna sequence similar to brevibacterium thermoruber (99%). the other group shows that otu 1 was closely related to e. coli (16s-rrna genes in pkk3535 constructed by brosius et al., 1981 in herawati, 1996). one possible explanation is the construction for otu 1 and e. coli involved 3 restriction fragments which have two similar band sizes. another explanation 'is that otu 1 is a new bacterial isolate which yielded dna profile similar to e. coli when restriction enzyme rsal was employed in this study. in a habitat like candradimuka crater, there may be some dominant bacteria occupying that extreme habitat. they are physiologically active among the slow grower and dormant bacteria. therefore, estimating genetic diversity through isolation of dna from environmental sample is able to represent all of the organisms including unculturable ones. although there are some biases such as different cell lysis methods and amplimer sets resulting in limited comparability to other studies, the data of 16s-rrna genes from environmental libraries demonstrated the presence of hitherto unidentified bacteria. only a minority of sequences retrieved from directly isolated soil dna were reported to be closely related to cultured organisms so that bacterial communities in the environment were composed mainly of uncultured species (felske et al. 1998). partial sequencing of otu 3 (520 bp) and comparison to ribosomal database project (rdp) database from the university of illinois indicate that otu 3 demonstrated 96% similarity to ocrobactrum anthropi (iam 14119) whereas otu 7 (450 bp) had 86% similarity to pseudomonas flavescens (b62(t)). dna sequencing provides more accurate data because nucleotides are the basic unit of information, straightforward approaches for inferring phylogenetic history and very reliable to compare specific dna fragment. besides the advantages, this technique carries inherent drawbacks, such as relatively high cost operation and impractical for 7 biotropia no. 14, 1999 routine application long dna molecule. on the contrary, pcr-rflp technique is simple, rapid, low cost, for a 50 3000 bp dna fragment range could be assayed, and especially more appropriate when many individuals need to be sampled (hillis et al. 1996). to assess the diversity of an environmental sample using pcr-rflp study, we do not have to use the most accurate technique like dna sequencing. therefore, the simpler and faster method could be valuable in taxonomic study for bacterial in situ detection and routine investigation. acknowledgments this research was financially supported by hibah tim project (urge) no. 005/htpp/iv/urge 1999/2000, and dip seameo-biotrop bogor 1998/1999 to antonius suwanto. references amann, r.i., w. ludwig, and k.h. schleifer. 1994. identification of uncultured bacteria: a challenging task for molecular taxonomists. asm news. 60:360-365. amann, r.i., w. ludwig, and k.h. schleifer. 1995, phylogenetic identification and in situ detection of individual microbial cells without cultivation. microbiol. rev. 59:143-169. anonim. 1997. topo ta-cloning® manual. invitrogen corp. san diego, california. bomeman, j., p.w. skroch, k.m.o'sullivan, j.a. palus, n.g. rumjanek, j.l. jansen, j. nienhuis and e.w. triplett. 1996. molecular microbial diversity of an agricultural soil in wisconsin. appl. environ. microbiol. 62:1935-1943. bottger, e.c. 1996. approaches for identification of microorganisms. asm news. 62:247-250. brock, t.d. 1986. an overview of the thermophiles. in brock, t.d. thermophiles: general, molecular and applied microbiology. john wiley & sons. new york. herawati, e. 1996. physiology and genetic characterization of luminous vibrio causing disease on tiger prawn (penaeus monodon fab.). thesis, aquaculture program, faculty of fisheries, bogor agricultural university, bogor. 'felske, a., a. wolterink, r. van lis and a.d.l. akkermans. 1998. phylogeny of the main bacterial 16srrna sequences in drentse a grassland soils (the netherlands).appl. environ. microbiol. 64:871-879. fox, j.l. 1994. microbial diversity: low profile, immense breadth. asm news. 60:533-536. green, e. k. 1998. restriction fragment length polymorphisms. in rapley, r. and j.m. walker. molecular biology methods handbook. humana press. totowa, new jersey. hillis, d. m., b. k. mable, a. larson, s. k. davis and e. a. zimmer. 1996. nucleic acids iv: sequencing and cloning. in d. m. hillis, c. moritz and b. k. mable (ed.). molecular systematics. sinauer associates, inc. massachusetts. huber, g., r. huber, b.e. jones, g. lauerer, a. neuner, a. segerer, k.o. stetter and e.t. degens. 1991. hyperthermophilic archaea and bacteria occurring within indonesian hydrothermal areas. system. appl. microbiol. 14:397-404. 8 genetic diversity analysis of thermophilic bacteria temmy desiliyami et al. logan, n. a. 1994. bacterial systematics. blackwell scientific publ. london. marchesi, j.r., t. sato, a.j. weightman. t.a. martin, j.c. fry, s.j. hiom and w.g. wade. 1998. design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacterial 16srrna. appl. environ. microbiol. 64:795-799. moyer, c.l.. j.m tiedje, f.c. dobbs, d.m. karl. 1996. a computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rrna genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. appl. environ. microbiol. 62:2501-2507. pace, n. r. 1997. a molecular view of microbial diversity and the biosphere. science. 276:734-739. rohlf, c. l., c.c. yeo and l. tay. 1990. ntsys-pc, numerical taxonomy and multivariate analysis system, version 1.60. exeter software. new york. sambrook, j., e.f. fritsch and t. maniatis. 1989. molecular cloning. a laboratory manual. second edition. cold spring harbor laboratory press. service, r. f. 1997. microbiologists explore life's rich, hidden kingdoms. science. 275:1740-1742. stetter, k. o. 1995. microbial life in hyperthermal environments. asm news. 61:285-290. tan, i. 1999. characterization of a thermophilic xylanolytic bacterium isolated from gunung pancar hotspring. bogor. master thesis, graduate study, bogor agricultural university, bogor zhou, j., m.a. brunns, and j. m. tiedje. 1996. dna recovery from soils of diverse composition. appl. environ. microbiol. 62:316-322. 9 biotropia no. 9, 1996: 1-14 natural products in organic synthesis robert h. burnell departement de chimie, universite laval, quebec quebec, canada g1k 7p4 abstract a resume of four lectures presented by the author during a one month visit to biotrop in 1995. the seminars were held at the university of indonesia at depok, the technical institute in bandung, the bogor agricultural institute and at biotrop, bogor. the talks were grouped under the general title "the use of natural products in organic synthesis". key words: organic compounds/natural products. the venerable art of the chemical synthesis of naturally occurring organic compounds which has inspired and fascinated chemists for roughly one hundred and fifty years continues to be necessary today for several reasons. first, and traditionally the most important reason, is that synthesis provides the ultimate proof of structure of a substance by showing that it can be prepared in an unambiguous fashion from simple building blocks. in principle the structures of all the hundreds of new compounds isolated from nature every year should be synthesized. however, if new substances are very similar in structure they can be interrelated by conversion to one another or perhaps to products that have already been synthesized. a second reason for practicing this demanding art we call synthesis is the challenging necessity of preparing increasingly complex substances in more efficient ways and this is the most important source of innovation and invention that assures progress in the organic field. of course, synthesis can also provide an alternate to nature as the source of useful compounds. for instance, despite the effectiveness of the anti-cancer natural product taxol, it is a sobering thought that all the taxus trees in the world could not supply at present enough of the drug to treat the women actually suffering from breast and cervical cancer. and to make matters worse, the trees take almost a century to mature. furthermore it has been estimated that by the year 2000 there will be a world wide short fall of several tons of the natural anti-malarial obtained from artemesia species. the difficulty of shortages can sometimes be alleviated by synthesizing simpler and cheaper analogs. 1 biotropia no. 9, 1996 (burnell et al. 1985), maytenoquinone (burnell et al. 1988) and coleon b (burnell et al. 1983), while occasionally we were able to correct the published structures, as in the case of the nellionols (burnell et al. 1984). for structure confirmation, the simpler synthesis of the left and right handed mixture (the racemic substance) is quite adequate because as mentioned, with the exception of the optical property of rotating the plane of light, the left hand form, the right hand form or the 50:50 mixture of the two, all show the same spectroscopic behavior and these are the characteristics we use to identify them (ultra-violet, infra-red, nuclear magnetic resonance and mass spectra). strictly for proofs of structure, we have prepared molecules by total synthesis from smaller symmetrical, easy to buy or make, starting materials (burnell et al. 1993). the general strategy is shown in the upper part of scheme 6. to the rather commonplace aromatic compound 30 we attached a long chain of carbons derived from natural geraniol 29. this flexible substituent has two "double" bonds in critical positions enabling it to form two rings when attacked by a suitable "positively charged" reagent (ph-s + in our case). both the ph-s and the cn groups in the product 32 can be exchanged by oxygen and this completed a rapid synthesis of methyl nimbinone 33, a diterpenoid from the "neem" tree, azadirachta indica (ira et al. 1988 (1)). the final product from the rapid synthesis was "racemic", a 50:50 mixture of the natural substance and its mirror image and all the physical properties of our sample (except the rotation of light) were identical with those in the literature. obviously the proposed structure is correct! to compare the two approaches we also prepared methyl nimbinone from natural podocarpic acid (burnell et al. 1993). this far more arduous route is summarized in the lower part of scheme 6. the key step in this sequence was the formation and subsequent opening of the three-membered ring as a method of installing the two methyl groups on the first ring of the skeleton. in this case when the synthesized methyl nimbinone 36 was examined in the polarimeter, it did rotate the plane of polarized light in the same direction and to exactly the same extent as the product isolated from nature. this synthesis not only confirms the general structure forwarded for the diterpene, it also tells us that the substance elaborated by the neem tree is precisely and exclusively as represented in formula 36. that is, if the three rings of the skeleton are in the plane of the paper, the methyl group identified by the dark wedge between the rings, protrudes towards the reader. choosing between the rather simple straight-forward synthesis that leads to a mixture of the two image forms of methyl nimbinone or the more difficult and less rewarding preparation of the real natural diterpene from natural podocarpic acid depends on the real goal of the synthesis. but it would obviously be an enormous advantage to be able to combine the simplicity of the former with the specificity of the latter. to investigate this possiblity we selected another relatively simple compound, methyl nim 10 natural products in organic synthesis robert h. bumell bionone 44 to experiment with this "enantiomeric" style of synthesis. this diterpene is similar in name and in structure to methyl nimbinone 36 and it was also extracted from the "neem" tree (ira et al. 1988(2)). crucial to this approach is the incorporation of an asymmetric unit (technically a "chiral auxilliary") into the starting material at the outset. in theory, the "left or right-handedness" of this appendage will just be borrowed temporarily during the synthesis and it should impose its asymmetry on the reactions to induce the preferred formation of just one of the mirror image forms of the final product. at a propitious moment this helpful but perhaps costly auxilliary can be removed and recovered to be used in another synthesis. 11 biotropia no. 9,19 96 we chose to attach to the necessary aromatic starting product 43, the asymmetric unit 42 (our auxilliary) invented by evans (evans el al. 1985) which we had previously prepared from the abundant natural amino acid, l-valine 41. as in the earlier synthesis, we added the ten carbon bromide 29 and immediately we knew the auxilliary was performing its function. while there are two three-dimensional structures possible for the product 45, only one of the two was formed in quantity in the reaction ! 12 natural products in organic synthesis robert h. burnell employing roughly the same steps as in the methyl nimbinone sequence (above), the intermediate 45 was cyclized to compound 46 and the two oxygen functions were introduced to afford a product which showed all the spectroscopic attributes of methyl nimbionone 47 (dumont 1993). it was encouraging that examination of the optical properties of the product in the polarimeter showed that the substance did indeed rotate the plane of light. however, the magnitude of the rotation was much lower than that recorded for the natural product which indicated that the high "left hand, right hand" selectivity we had achieved early in the synthesis (only one form of 45 was isolated), had been scrambled or lost in the later steps. we suspect that the conditions for the cyclization (45 to 46) are too severe and our efforts are now aimed at finding milder alternate methods of performing this step. the improved conditions we are searching for must provide the product in high yield, sufficient to enable the completion of the synthesis which still involves several steps. unfortunately, as yet, we have not found this more gentle procedure. acknowledgements the work described was financed partly by nsecc (the natural sciences and engineering council of canada), partly by the government of quebec (f.c.a.r.) and more recently by generous personal funding (emeritus professor charles r. engel). my sincere appreciation is extended to the following graduate students whose collaboration and talent were crucial to the study : sonia desfosses, nathalie dumont, nathalie the-berge, michel jean and david miller. the author wishes to thank the staff at biotrop for their hospitality (in particular dr. hilman affandi and dr. gloria enriquez) and the canadian international development agency (cida) for making the visit to indonesia possible. literature cited barton, d.h.r., a.g. brewster, s.v. ley, c.m. read and m.n. rosenfeld. 1981. oxidation of phenols, pyrocatechols and hydroquinones to orthoquinones using benzeneseleninic anhydride. j. chem. soc., perkin. trans. i., 1473-1476. brewster, j.h. 1959. a useful model of optical activity. j. amer. chem. soc., 81: 5475-5483. burnell, r.h., a. andersen, m. neron-desbens and s. savard. 1981. approaches to the synthesis oflyco-xanthol and similar natural products. synthesis of coleon u. can. j. chem., 59: 28202825. burnell. r.h., m. jean and s. savard. 1983. synthesis of coleon b tetramethyl ether. can. j. chem., 61: 2461-2465. 13 biotropia no. 9,1996 burnell. r.h., m. jean, d. poirier and s. savard. 1984. the structures of the nellionols. synthesis of model abieta-8,11,13 trien-7-ones. synthesis of 5-dehydronellionol trimethyl ether. can. j. chera., 62: 2822-2829. burnell, r.h., a. andersen, m. neron and s. savard. 1985. synthesis of coleon c tri-o-methyl ether. can. j. chem., 63: 2769-2776. burnell, r.h., m. jean and d. poirier. 1987. synthesis of taxodione. can. j. chem., 65: 775-781. burnell, r.h., m. jean and s. marceau. 1988. synthesis of maytenoquinone. can, j. chem., 66: 227-230. burnell, r.h., n. dumont and n. theberge. 1993. synthesis of omethyl-nimbinone. j. nat. prod., 56: 1930-1936. carriere, y., j. millar, j.n. mcneil, d.j. miller and e.w. underbill. 1988. identification of the female sex pheromone in alfalfa blotch leafminer, agromyzafrontella (rondani). j. chem. ecol., 14: 947-956. dumont, n. 1993. synthese de diterpenes naturels isoles de 1' azadirachta indica (neem). m.sc. thesis, university laval, canada. evans. d.a., d.j. mathre and w.l. scott. 1985. asymmetric synthesis of the enkephalinase inhibitor thiorphan. j. org. chem., 50: 1830-1835. furia, t.e. and n. bellanca. 1971. fenaroli's handbook of flavor ingredients. the chemical rubber co., cleveland, ohio. ira, b.s., s. siddiqui, s. faizi and b.s. siddiqui. 1988 (1). tricyclic diterpenes from the stem bark of azadirachta indica. j. nat. prod., 51: 1054-1061. ira. b.s., s. siddiqui, s. faizi and b.s. siddiqui. 1988 (2). teipenoids from the stem bark of azadirachta indica. phytochemistry, 27 : 1801-1804. king, f.e., t.j. king and j.g. topliss. 1956. synthesis of podocarpic acid. chem. and ind., 113. kupchan, s.m., a. karim and c. marcks. 1969. tumor inhibitors. xviii. taxodione and taxodone, two novel diterpenoid quinone methide tumor inhibitors from taxodium distichium. ]. org. chem., 34: 3912. miller, d., f. bilodeau and r.h. burnell. 1991. stereoselective synthesis of isomers of 3,7-dimenthyl-nonadecane, a sex pheromone of the alfalfa blotch leafminer (agromyza frontella (rondani)). can. j. chem., 69: 1100-1106. 14 biotropia no. 9, 1996 yet another motivation for synthesis stems from the fact that nature very often provides only very small quantities of the substances of the greatest physiological interest. modern analytical instruments enable the chemist to elucidate the structures of these unknown, potentially useful biological, medicinal or phytochemical compounds even if the elaborate extraction procedures afford only a few milligrams of material. however, for proper assessment of their physiological activities and their mechanisms of action, much greater amounts are probably needed and only synthesis could provide the necessary quantities. most of the interesting molecules of nature are asymmetric rather like our hands. each hand is made up of the same elements such as fingers, thumbs, palms, nails and so on and while they are very similar, they are not identical. a right-hand glove does not fit on the left hand ! in fact our hands are mirror images of one another. natural molecules made of the same atoms could be assembled in two fashions, one being the image of the other. unlike our hands, however, nature usually synthesizes only one of the two forms (chemically we call the two images "enantiomers"). since all "our human" metabolic intermediates and enzymes are also asymmetric, the physiological activity of a natural substance may be quite different from that of its mirror image. the "enantiomeric" form could just be inactive or less active than the natural product but it could have harmful effects or be highly toxic. the much quoted case of thalidomide springs to mind where one enantiomer was a benign sedative recommended for women suffering nausea during pregnancy whereas the mirror image of exactly the same molecule was teratogenic producing babies with serious physical defects. so synthesis must not only aim at preparing the target molecule with the right general structure but the methodology should also lead to the right three-dimensional structure (or stereochemistry). in simpler terms, the synthesis must give exclusively the "left" or exclusively the "right" hand molecule ! chemically producing this "left or right" hand configuration is extremely difficult. the problem is further compounded by the fact that virtually all the physical properties of the two image forms are identical which means we cannot easily tell them apart and, furthermore, we cannot separate one from the other !! however, two characteristics of the enantiomers (images) are different. first, as we have seen, they can vary greatly in their physiological activities. secondly, like all asymmetric substances, they rotate the plane of polarized light but the images turn the light in opposite directions. fortunately we can measure this in a polarimeter and under the conditions defined by the test, a fresh solution of the more prevalent form of the common sugar glucose rotates light through 112 degrees in a clockwise or right-handed fashion while its image would also turn light 112 degrees anti-clockwise or to the left. in fact we often say that a substance such as glucose is dextro-rotatory (from the latin dextrus : right) or we just abbreviate this to d-glucose where the small "d" shows that light passing through a solution of the product 2 natural products in organic synthesis robert h. burnell would be turned to the right. the image form would be levo-rotatory and called 1-glucose. (another convention describes rotation to the right as positive (+) and rotation to the left as negative or (-)). one way of assuring that the molecule targeted in a synthesis will have the right stereochemistry (left or right-handedness) is to start the synthesis from an asymmetric substance available in pure form in nature. many potentially useful, commercially available compounds exist from simple amino acids or sugars to complex steroids, alkaloids and terpenes. our work on an insect pheromone is a simple example of such an "enantio-selective" synthesis (as reaction sequences aimed exclusively at the right or left handed structures are called). the insect, the small fly agromyza frontella was accidentally introduced into canada from europe in the 1960's and its presence was rapidly detected because in its larval stage it destroyed the leaves of the popular cattle fodder, alfalfa. in their breeding ritual, the female insects attract the males by emanating a minute quantity of a chemical messenger or pheromone and although the quantity of this substance available was infinitesimal, structure 1 (or 3,7-dimethylnonadecane) was advanced for the pheromone. the structure elucidation was made possible by a skillful study of the mass spectral fragmentation after rigorous purification by preparative gas chromatography (carriere et al. 1988). potentially this natural chemical could be used to control the population of the insect by using it to divert the male flies away from the females and thus preventing reproduction ! 1 3 7 ch3-ch2-ch-ch2-ch2-ch2-ch -ch2 11 ch3 ch3 ch3 at first glance the substance is chemically uninteresting. it is just a member of the stubbornly unreactive class of organic substances known as paraffins or saturated acyclic hydrocarbons. on reflection, however, the pheromone is a synthetic challenge because the positions of the two methyl groups (chs or me groups on the third and seventh carbons of the long chain) make the molecule asymmetric so in fact there are four spatial arrangements possible, as shown in structures 2 to 5. only one will be the active pheromone but which one ? our synthetic strategy should enable us to prepare one (or better all) of them specifically. 3 biotropia no. 9, 1996 in designing a synthesis we normally work backwards from the target, hypothetically disconnecting parts we feel we can graft back onto the remaining core using chemical methods of proven specificity and geometry. our first idea for the synthesis of the agromyza pheromone 1 arose from the "retro-synthesis" shown in scheme 1. disconnections leave a central portion which contains both of the methyl branches on the long chain. many naturally occurring oils (commercially available at low price) contain compounds with this skeleton and our final choice of a "starting material" would be motivated by the need for elements of structure permitting the easy grafting of the other units. and, of most importance, the molecule should be asymmetric with at least one "left" or "right" hand centre. after considering several possibilities, pulegone 6, the main constituent of the "oil of european pennyroyal" from mentha pulegioides l. (furia and bellanca 1971) seemed ideal for the purpose. 4 natural products in organic synthesis — robert h. burnell without commenting at length on the chemical reactions involved, we constructed the pheromone as shown in scheme 2. for those interested in how the transformations were achieved, the reagents (indicated by the small letters on the arrows) are, of course, published in detail (miller et al. 1991). the steps from 6 to 11 not only add the "one carbon unit" but also induce the stereochemistry (left or right-handedness) at c.3. none of these chemical transformations could have altered the configuration of the methyl substituent of the original pulegone so the geometry at the other methyl branch, now further in the chain at c.7, survives unchanged from beginning to end. mechanistic and theoretical arguments told us that the final product from our synthesis was actually structure 3, the drawing of which implies that if the long chain zig-zags in the plane of the paper then both methyl groups represented by the dotted wedges, stick out to the rear. other considerations told us that of the four possible structures this was actually the active pheromone. when analyzed by polarimetry and using rules formulated by brewster for calculating the optical properties of hydrocarbons 5 (brewster 1959), we felt the synthetic oil also contained lesser quantities of structure 4 in which the c.3 methyl has the other configuration. 6 biotropia no. 9, 1996 natural products in organic synthesis robert h. burnell we had chosen pulegone as the starting material for the synthesis for another reason. if the disconnetion suggesting possible synthetic approaches is envisaged in another fashion as shown in scheme 3, the pheromone could be prepared by just grafting an eleven carbon fragment onto the pulegone 6. so, to check both the structure and the purity of the end product from our first synthesis, we prepared the pheromone a second time by this alternate route which as shown in scheme 4, starts from compound 8, an early intermediate in the previous synthesis (miller et al. 1991). this second sequence gave exactly the same pheromone 3 but in higher yield. remarkably in this case and as intended, the original asymmetry of pulegone (the methyl branch) is now at c.3 rather than at c.i as in the first synthesis. this "left hand : right hand" selection was only possible because the natural pulegone is asymmetric and the pennyroyal plant synthesizes only one of the two possible mirror image forms. interestingly, the image form of pulegone can be isolated in pure form from other plant species so our synthesis could also lead to the image of pheromone 3 represented by structure 2. a second example of synthesis in our laboratory starting from commercially available, naturally occurring asymmetric substances involves podocarpic acid 23 which was itself synthesized as early as 1956 (king et al. 1956). the world source of this acid is the resin obtained from podocarpus trees found here in java and new zealand. one of the molecules we hoped to synthesize was taxodione, an anti-tumoral substance shown to possess structure 28 (kupchan et al. 1969) and at that time was considered a promising drug for human therapy. when we started, other syntheses had already been reported by other groups but we felt that we could improve on existing syntheses in elegance and efficiency. podocarpic acid was a natural choice as starting material because even uninformed inspection shows that it already possesses many of the structural 7 biotropia no. 9,1996 elements of taxodione including the three angularly attached six-membered rings. but even more important, the absolute stereochemistry (the handedness) of the centres identified as c.10 and c.5 is exactly the three dimensional geometry required for "natural" taxodione ! scheme 5 is a brief outline of the crucial intermediates in our synthesis (burnell et al. 1987). the white arrows merely draw attention to the part of the molecule that will be modified in the next transformation. the key step was the use of a selenium reagent (barton et al. 1981) to oxidise the phenol 25 to the ortho-quinone 26. some of the reactions were most gratifying. for instance, while conventional wisdom would say such a change was unlikely, compound 26 rearranged spontaneously to 27 and the latter was oxidized to the final taxodione 28 by merely percolating its solution through a column of silica gel. the product was, of course, pure natural taxodione containing none of the 8 natural products in organic synthesis robert h. burnell mirror image form and this was confirmed by measuring the direction and magnitude of its ability to rotate the plane of polarized light. if we had started the synthesis from simple symmetrical chemicals, the final substance would only be half taxodione and half its mirror image and would show no rotation of the plane of light. the taxodione preparation was part of a much broader study to corroborate the structures of several naturally occurring aromatic diterpenes by synthesis. since in all cases we started from podocarpic acid (or the very similar equally affordable dehydro-abietic acid) the final products also possessed the exact stereochemistry (left or right handedness) of the natural substances. most of the structures proposed in the literature were shown to be correct as in the case of coleon u (burnell et al. 1981), coleon c 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no. 8, 1995: 11-22 effects of soil sterilization on the formation and function of two strains of pisolithus tinctorius on eucalyptus urophylla*) nelly s. aggangan, bernie dell biological and environmental sciences, murdoch university, perth, western australia 6150 nick malajczuk csiro division of forestry, private bag p.o. wembley, western australia 6014 reynaldo e. de la cruz biotech, university of the philippines at los ratios, college, laguna, philippines 4031 abstract to examine the effects of soil microbial population on mycorrhizal development and function, eucalyptus urophylla seedlings were inoculated with two pisolithus tinctorius isolates and grown in sterile, partly sterile and non-sterile soil. the two isolates of pisolithus were an effective isolate (h445) collected from under eucalypts in australia and an isolate (h615) collected from under eucalypts in the philippines. soils used were infertile acid soils collected from field sites in pangasinan, luzon and surigao, mindanao. in both soils, the australian pisolithus h445 improved the growth of e. urophylla seedlings more than philippine isolate h615. the uninoculated seedlings exhibited stunted growth typical of p deficiency. height at 8 weeks was significantly taller in sterile than in non-sterile soil. a significant interaction effect of inoculation and soil sterilization on height at harvest was observed only in surigao soil. soil sterilization had a varied effect on mycorrhizal formation. in pangasinan soil, root colonization by h445 was significantly greater in non-sterile soil than in sterile soil. whereas in surigao soil, root colonization was significantly reduced by 54% from partly sterile to non-sterile soil. on the other hand, h615 showed significant mycorrhizal colonization in non-sterile soil compared from those in partly sterile and sterile soils. the degree of infection did not necessarily correspond to growth promotion in e. urophylla seedlings. these results indicate that the performance of the h445 was markedly affected by the microbial flora of the two soils. thus, its potential use in the philippines needs to be thoroughly tested in the field before its widespread use in any inoculation program. key words: mycorrhizas/soil sterilization/pisoto/ws tinctorius/eucalyptus urophylla. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference in mycorrhizae (acom hi), 19-21 april 1994, yogyakarta, indonesia. 11 biotropia no. 8, 1995 introduction eucalypts are important reforestation species grown in the philippines to provide raw materials for the pulp and lumber industries. their fast growth and adaptability to low nutrient soils might be dependent on the presence of symbiotic mycorrhizal associations. for example, previous inoculation work carried out in the philippines on pisolithus and eucalypts indicate growth enhancement with a pisolithus isolate collected from under pinus in the philippines (de la cruz et al. 1990, 1991). pisolithus species have been shown to increase survival and growth of a wide range of tree species such as oaks, pines and eucalypts. in the tropics, lee su see et al. (1994) found that a pisolithus isolate colonized the roots and subsequently improved the growth of two dipterocarp seedlings grown in the nursery. recent work conducted by burgess et al. (1994a) found that growth stimulation of e. grandis seedlings varied greatly among 20 isolates of pisolithus. an isolate (h445) collected from under eucalypts was one of the best growth promoters. this same isolate has also been reported to dramatically increase the growth of e. globulus and e. diversi-color in other studies (burgess et al. 1993). however, not all isolates are beneficial to plant growth. for example, tonkin et al. (1988) observed severe root reduction and retarded shoot growth of e. marginata when inoculated with certain pisolithus isolates in aseptic condition. one factor influencing the response of eucalypts to inoculation with pisolithus is the host from which the isolate was isolated. pine isolates of pisolithus, for example, have been shown to form poor associations with eucalypts (malajczuk et al. 1982, 1990; burgess et al. 1994a). in the development of an inoculation program for nurseries in the philippines, it is essential to introduce compatible ectomycorrhizal isolates that can improve the growth of eucalypts. isolate h445 presents a possible candidate for such introduction. introduction of pisolithus isolates into the philippines from australia requires a thorough testing of the behaviour of these fungi on roots of eucalypt seedlings growing in soil collected from field sites in the philippines. in addition, it is necessary to compare the performance of introduced australian isolates with local philippine pisolithus isolates. the glasshouse screening experiments conducted, so far, for pisolithus and eucalypts have used pasteurized soil (burgess et al. 1993, 1994a). it is important to understand factors that affect inoculum survival and persistence of fungi on eucalypt roots in the field. predicting the effectiveness and competitiveness of introduced ectomycorrhizal fungi in the field, and comparison with previous glasshouse experiments, requires preliminary testing of the fungi in the presence or in the absence of native soil microflora. in a sterile condition, all the native microorganisms 12 effects of soil sterilization nelly s. aggangan et al. are eliminated by sterilization, thus leaving the introduced mycorrhizal fungi to function freely without interference from other microbes. in non-sterile soil, on the other hand, the introduced fungi must compete with other microorganisms in order to survive and function. this would represent conditions similar to those in the field. it is also possible that partial sterilization such as incorporation of a small amount of non-sterile soil to a fumigated one, can stimulate an isolate to develop and absorb more nutrients. in this paper, the effectiveness of an australian (h445) and a philippine pisolithus isolate (h615) on the formation of ectomycorrhizas and growth stimulation of e. urophylla seedlings was compared. plants were grown in soils collected in the philippines and subjected to three sterilization treatments (non-sterile, partly sterile and sterile soil). materials and methods seed germination and mycorrhizal synthesis seeds of e. urophylla (seedlot # 18094 from mt. egon, flores island, indonesia), obtained from csiro australian tree seed centre (canberra, australia), were aseptically germinated on agar plates. seeds were washed with 70% ethanol containing tween 20 for one minute, surface sterilized with 10% sodium hypochlorite for 5 minutes and washed three times with sterile water. two isolates of pisolithus were selected: a) h445 an isolate provided by csiro division of forestry, collected under e. marginata in western australia and which has been shown to stimulate the growth of eucalypts in field trials, and b) h615 collected under e. camaldulensis in the philippines but probably associated with pinus (burgess et al. 1994b). ectomycorrhizas were synthesized aseptically in petri dishes by transferring 7 day-old germinants onto the surface of 10-day old hyphal mats growing on modified melin norkrans medium (marx 1969) with low glucose concentration (1.75 g/l) for two weeks (malajczuk et al. 1990). plates were incubated in a growth room set at 12 hrs photoperiod and temperature of 25°c. at 10 days they were transplanted into undrained pots containing 2 kg soil. the surface of each pot was covered with aluminium foil with four holes where the seedlings were inserted. the foil reduced water loss from the soil and minimized spore contamination. after four weeks, the seedlings were thinned to two plants per pot. 13 biotropia no. 8, 1995 soil treatments soils (015cm depth) were collected at sites in: a) labrador, pangasinan, luzon and b) in bislig, surigao sur, mindanao, referred to in figures as pangasinan and surigao soil, respectively. both sites are grasslands which previously were monsoon forests dominated with dipterocarps. the pangasinan soil has a ph of 4.1 (1:1 soil, 0.005m cacl,), 2.02% organic matter (walkey-black method), 0.11% total n (modified kjeldahl method), and 0.84 ppm available p (bray no. 2) while the surigao soil has a ph of 5.9, 2.48% organic matter, 0.06% total n and 0.21 ppm available p. textural class of the two soils is silt loam. the soils were air dried, pulverized and passed through a 2 mm screen. two kg dry soil were dispensed into plastic pots with polyethylene bag liners. soil treatments were: sterile, partly sterile and non-sterile. pots designated as sterile and partly sterile were fumigated with methyl bromide (one canister per m 3 soil) in a fumigation chamber for 3 days. the non-sterile designated pots were left unfumigated. the partly sterile soil treatment was prepared by mixing 1% unfumigated soil with 99% methyl bromide, fumigated soil. addition of fertilizer one week after fumigation, all pots received the following basal nutrients (per kg soil): 16 mg ca(h2po4)2.h2o, 270 mg nh4no3, 233 mg k2so4, 71.3 mg cacl2, 21.4 mg mg so4.7h2o, 10 mg znso4.7h2o, 5 mg cuso4.5h2o, 0.36 mg coso4.7h2o, 0.7 mg h3bo3 and 1.62 mg na2moo4.2h2o. manganese and iron were not included because the soils had high concentration of these elements (dell, unpublished). the nutrient solutions were added evenly on the soil surface after sterilization. the soil was mixed thoroughly and watered to 80% field capacity and incubated in benches inside a screenhouse for five days prior to planting. seedling maintenance and growth monitoring two weeks after transplanting, 2.5 ml nitrogen solution (54g nh4no3/l h2o) pot -1 week -1 was added. the same rate was added for the next two weeks after which the rate of nitrogen was increased to 5 ml pot -1 week -1 for a period of 7 weeks. all pots were watered to field capacity by weight when necessary. height was monitored twice a month. 14 effects of soil sterilization nelly s. aggangan et al. harvest and mycorrhizal assessment after 12 weeks, the seedlings were harvested. shoots were cut 1 cm above the soil surface and the root systems were gently washed under running water. fine roots (diameter less than 0.5 mm) were separated from the coarse roots, blotted dry in between paper towels and cut into 1 cm lengths. fine root samples of 0.2 g fresh weight were further chopped into 1-2 mm lengths and were fixed in 70% ethanol for counting mycorrhizal root infection. fine roots were cleared and stained as described by phillips and hayman (1970). stained roots were spread evenly over a petri dish and were examined under a stereomicroscope. ten randomly selected fields of view were chosen and all roots crossing a hair-line were examined for the presence or absence of infection. fully colonized root tips were scored as mycorrhizal. experimental design and statistical analysis the experiment was conducted in a screenhouse at biotech, up los banos, philippines following a two factor (fungi and soil treatment) factorial in randomized complete block design (rcbd) with three replicates. pots were rearranged in benches once a week. within each soil type, all data collected were analyzed statistically using two way anova. treatment means were compared using duncan's multiple range test at p< 0.05. results plant growth in pangasinan soil, inoculation (as factor a) significantly (p< 0.01) affected heights at 6, 8,10 and 12 weeks (at harvest) after planting, and dry matter yields (shoot, root, fine root, coarse root and total biomass). generally, the australian pisolithus h445 was more effective in increasing height growth of e. urophylla seedlings from 6 weeks to 12 weeks after planting, dry matter yields and phosphorus uptake (data not shown) than the philippine isolate h615. inoculation with the australian isolate increased (p< 0.05) height at 6 weeks until 12 weeks (harvest) after planting. h615 significantly increased seedling heights from 6 weeks to 10 weeks after planting compared with the uninoculated plants but significantly shorter than those inoculated with h445. at harvest (12 weeks) however, height of seedlings inoculated with the philippine isolate was not significant compared with the uninoculated counterpart. likewise, soil sterilization (as factor b) significantly affected 15 biotropia no. 8, 1995 height at 6 weeks (p< 0.01) and at 8 weeks (p< 0.05) and fine root dry matter yield (p < 0.05). height at 6 and 8 weeks after planting and fine root dry weight obtained in sterile soil were significantly higher than in non-sterile soil. heights and fine root dry weight in partly sterile soil were intermediate and these were not significant compared with those in sterile soil as well as those in non-sterile soil. there was no significant interaction effects of inoculation and soil sterilization on any of the periodic height measurements (fig. 1a). height at harvest due to inoculation with h445 was increased by 4x, 5x and 8x in sterile, partly sterile and in non-sterile soil, respectively, relative to the uninoculated seedlings. on the other hand, h615 increased seedling heights by 1.2x, 2x and 3x in the same soil treatment order. although the mean height of h445 plants were two times taller than those of h615, there was no significant differences among treatments. the uninoculated seedlings had the shortest height in all the three soil treatments. height of uninoculated plants was reduced by 2 folds while total dry weight (data not presented) by 10 folds from sterile to non-sterile soils. whereas, the inoculated seedlings did not differ much irrespective of soil treatment. shoot dry weight (data not presented) showed similar trends to plant height. a significant interaction effect was observed only on fine root dry weight. fine root dry weight was dramatically increased (p< 0.05) by h445 in sterile (fig. 2a) compared with h615 and uninoculated treatments. fine root dry weight was greater (p< 0.05) in sterile than in non-sterile soil. in surigao soil, inoculation (as factor a) significantly affected height at 6 to 12 weeks after planting, coarse root dry weight, root p concentration and uptake and shoot p uptake. h445 and h615 inoculated seedlings were of similar height which was significant compared with height of the uninoculated ones from 6 to 10 weeks after planting. at harvest, h445 inoculated plants were significantly taller than those inoculated with h615 and the uninoculated ones. although, height at harvest, coarse root dry weight and root p concentration obtained by h615 inoculated plants were twice those of uninoculated counterpart, the differences were not statistically significant. soil sterilization (factor b) significantly affected height at 8 weeks and shoot p concentration. height in sterile soil was significantly taller than in partly sterile and non-sterile. the latter soil treatments (partly sterile and non-sterile soil) yielded similar height growth response. shoot p concentration in partly sterile soil was significantly higher than in non-sterile soil. unlike in pangasinan soil, there was a significant interaction effect of inoculation and soil treatment. in sterile, h445 was consistent in promoting a significant increase in height (p<0.01) from 6 weeks until harvest (fig. 1b) compared with the uninoculated seedlings. two way analysis revealed that height growth obtained by seedlings inoculated with h445 in sterile soil was significant compared with those inoculated with h615 and the uninoculated ones. in partly sterile soil, height obtained by h445 16 effects of soil sterilization nelly s. aggangan et al. figure 1. effects of mycorrhizal inoculation on the accumulated height growth of e. urophylla seedlings grown in pangasinan (a) and surigao (b) soils subjected to three sterilization treatments. letters represent results of duncan's multiple range test after two way anova on height at 12 weeks on each soil treatment. lsd bar = 56. 17 biotropia no. 8, 1995 inoculated seedlings was significantly taller than the uninoculated seedlings but not significant as compared with those inoculated with h615. in non-sterile soil, height differences between the inoculated and uninoculated seedlings was not significant. h615 inoculation gave intermediate height and shoot dry weight in sterile and in partly sterile soil which were not statistically different from the uninoculated seedlings. unlike seedlings grown in pangasinan soil, there was no significant interaction effect of inoculation and soil sterilization on fine root dry weight (fig. 2b). however, plants had much larger shoot and root systems in the surigao than in the pangasinan soil. mycorrhizal development in pangasinan soil, there was a significant difference between the three inoculation treatments and soil sterilization significantly affected mycorrhizal development. mycorrhizal infection by h445 was significantly higher (41%) than h615 (21%). the uninoculated seedlings had 7% infection. mycorrhizal infection in non-sterile soil was significantly higher (30%) than in sterile soil (18%). root colonization in partly sterile soil was intermediate which was not significant compared with those obtained in sterile or in non-sterile soils. two way analysis indicates that in sterile and non-sterile soils, h445 and h615 had similar infection levels (fig. 2c). however, in partly sterile soil, h445 had greater (p<0.01) root colonization than h615. root colonization by h445 was significantly higher in non-sterile than in sterile soil but not significant compared with those in sterile and in non-sterile soils. uninoculated seedlings had low levels (not more than 11 %) of infection in the two soils (fig. 2c). in surigao soil, mycorrhizal development formed by h445 and h615 (as factor a) was not significantly different from each other but significant compared with the uninoculated treatment. root colonization was significantly higher in non-sterile and in partly sterile soils than in sterile soil. there was a significant interaction effect of inoculation and soil sterilization treatments. in non-sterile soil, h615 formed higher (p<0.05) infection compared with h445 (fig. 2d). greatest mycorrhizal formation by h445 was observed in partly sterile soil which was reduced (p<0.05) in sterile and in non-sterile soils. percent mycorrhizal infection by h445 in non-sterile soil was similar to that of uninoculated plants grown in either of the three soil treatments. root colonization by h615 was higher (p<0.01) in non-sterile (65%) than in partly sterile soil (34%). infection in sterile soil was intermediate and not significantly different to percent mycorrhizal infections observed in non-sterile and in partly sterile soils. correlation analyses revealed a low relationship (r 2 = 0.6) between percent mycorrhizal infection and height or shoot and fine root dry weight. 18 effects of soil sterilization nelly s. aggangan et al. figure 2. fine root dry matter weight (a and b) and mycorrhizal infection (c and d) of e. urophylla seedlings inoculated with two pisolithus isolates grown on sterile, partly sterile and non-sterile acid soils. letters represent results of duncan's multiple range test after two way anova on each soil. lsd bar = 0.116. 19 biotropia no. 8, 1995 discussion growth of e. urophylla seedlings in the two philippine soils was stimulated by inoculation with two isolates of pisolithus. growth of plants inoculated with the australian eucalypt isolate, h445, was enhanced more than those inoculated with the philippine isolate, h615. previously, h615 was classified as a pine isolate (burgess et al. 1994b) on the basis of polypeptide patterns identified by id sds-page. the effectiveness of a pine pisolithus isolate in promoting growth of eucalypts has been demonstrated in field trials (de la cruz et al. 1990, 1991). potentially, the introduction of australian isolates in any nursery inoculation programs in the philippines would provide an advantage for growth promotion of eucalypts. it remains to be determined whether these specific eucalypt isolates would persist and outperform the indigenous pine pisolithus isolates in the field. it is interesting to note that garbaye et al. (1988) observed the replacement of an introduced pine isolate (marx strain 270), by an indigenous scleroderma species within 2 years after outplanting. in this experiment, mycorrhizal root development was markedly affected by sterilization treatment in one philippine soil (surigao). mycorrhizal infection by h445 was reduced by 54% in non-sterile soil. in contrast, h615, was unaffected by soil sterilization treatments. this would suggest a possible role of soil microflora in affecting the infection process. several studies have suggested that soil microflora play an important role in influencing the inoculation process of seedling roots by mycorrhizal fungi (garbaye and bowen 1987; marx et al. 1982, 1984). such effect may be inhibitory or stimulatory to mycorrhizal formation (bowen and theodorou 1979; garbaye and bowen 1989; fitter and garbaye 1994). it would appear that the introduction of new strains of ectomycorrhizal fungi from australia and screening of endemic strains requires a thorough testing of the performance in a range of soils. it is anticipated that no single species would be appropriate for all soil types in the philippines and the introduction should include a wide range of physiologically distinct species. introduced ectomycorrhizal fungi should be able to survive and tolerate ecological variation within a site and progressively colonize the new roots after outplanting (garbaye 1982). most importantly, the introduced fungi must enhance host performance to a greater degree than the native fungi (danielson 1988). competitiveness or persistence of the association would depend on three factors: the host, mycorrhizal fungus and soil microbes wherein each factor is subject to environmental influences. further work is required to examine the influence of the composition of the microbial population on ectomycorrhizal formation on a range of philippines soil. 20 effects of soil sterilization nelly s. aggangan et at. acknowledgements this work was supported by the australian international development bureau and the australian center for agricultural research while dr. nelly s. aggangan was doing her postgraduate degree. references burgess, t., n. malajczuk and b. dell. 1994a. variation in mycorrhizal development and growth stimulation of 20 pisolithus isolates inoculated onto eucalyptus grandis w. hill ex maiden. new phytol (in press). burgess, t., n. malajczuk and b. dell. 1994b. variation in pisolithus based on basidiocarp and basidiospore morphology, culture characteristic and polypeptide analysis using id-sds page (in press). burgess, t., n. malajczuk and t.s. grove. 1993. the ability of 16 ectomycorrhizal fungi to increase the growth and phosphorus uptake of eucalyptus globutus labill and e. diversicolor f. muell. plant and soil 153: 155-164. bowen, g.d. and c. theodorou. 1979. interactions between bacteria and ectomycorrhizal fungi. soil. biol. and biochem. 11: 119-126. danielson, r.m. 1988. mycorrhizae in forestry: the state-of-the-art in land reclamation. in canadian workshop on mycorrhizae in forestry, 1 -4 may 1988, universite laval, ste-foy, que. edited by m. lalonde and y. piche. universite laval, ste-foy, que. p. 39-41. de la cruz, r.e., n.s. aggangan, j.t. zarate and r.p. yecyec. 1991. improved reforestation technologies in the philippines. pcarrd book series 121: 65-78. de la cruz, r.e., e.b. lorilla and n.s. aggangan. 1990. ectomycorrhizal tablets for eucalypthus species. in fast growing tress and nitrogen fixing tress. eds. d werner and p muller, gustav fisher verlag, stuttgart, new york. p. 371. fitter, a.h. and garbaye, j. 1994. interactions between mycorrhizal fungi and other soil arganisms. plant and soil 159: 123-132. garbaye, j. 1982. quelques aspects de la competitive des souches ectomycorhiziennes. in les mycorhizes, panic integrante de la plante: biologic et perspectives d'utilisation. eds. s gianinazzi, v gianinazzi-pearson and a trouvelot. pp. 303-312. proceedings of a seminar, 5-6may 1982 atm dijon, france, les colloques de finra, no.13. garbaye, j. and g.d. bowen. 1987. effect of different microflora on the success of ectomycorrhizal inoculation of pinus radiata. can.j.for. res. 17: 941-943. garbaye, j. and g.d. bowen. 1989. stimulation of ectomycorrhizal infection of pinus radiata by some microorganisms associated with the mantle of ectomycorrhizas. new phytol. 112: 383-386. garbaye, j., j.c. delwaulle and d. diangana. 1988. growth response of eucalypts in the congo to ectomycorrhizal inoculation. forest ecology and management 24: 151-157. lee su see, f. lapeyrie and mohd. yazid zanip. 1994. techniques for controlled ectomycorrhizal inoculation of dipterocarp seedlings and cuttings. in programs and abstract third asian conference on mycorrhizae, 19-21 april 1994, yogyakarta, indonesia, p. 4-6. 21 biotropia no. 8, 1995 malajczuk, n., f. lapeyrie and j. garbaye. 1990. infectivity of pine and eucalypt isolates of pisolithus tinctorius on roots of eucalyptus urophylla in vitro. new phytologist 114: 627-631. malajczuk, n, r. molina and j. trappe. 1982. ectomycorrhizal formation in eucalyptus. i. pure culture synthesis, host specificty and mycorrhizal compatility with pinus radiata, new phytol. 91: 467-482. marx, d.x. 1969. the influence of ectotropic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. i. antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. phytopathology 59: 153-163. marx, d.h., j.l ruehle, d.s. kenny, c.e. cordell, j.w. riffle, r.j. molina, w.h. pawuk, s. navratil, r.w. tinus and o.c. goodwin. 1982. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on container-grown tree seedlings for. sci. 28(2): 373-403. marx, d.h., c.e. cordell, d.s. kenny, j.g. mexal, j.d. arthman, j.w. riffle and j.w. molina. 1984. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. for. sci. monogr. no. 25. phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. brit. mycol. soc. 55: 158-160. tonkin, n.c., n. malajczuk and j.a. mccomb. 1988. ectomycorrhizal formation by micro-propagated clones of eucalyptus marginata inoculated with isolates of pisolithus tinctorius. new phytologist 111: 209-214. 22 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf biotropia vol. 28 no. 1,2021: 84 91 doi: 10.11598/btb.2021.28.1.1159 harvesting time a n d viability of ixora coccinea 'dwarf red coccinea' pollen saowaros phanomchai', i(itt1 bodhipadmai, s o w o c h noichindai, luepol punnai(anta2 and david w.m. leung3* 'division ofagro-industrial technology, facalg *plied science, king mongkat's university o f technology no& bangkoh bangsue, bangkok 10800, thailand 2faculty o f environment and resource stua'ies, mahidol universig, salaya, nakhon pathom 7 3 170, thailand 'school ofbiological sciences, universig o f canterbuy, christchurch 8 140, new zealand received 1 december 2018/accepted 14 february 2020 abstract the cultivated variety of the non-native i. coccinea, dwarf red coccinea @rc), is most popular and widely spread all over thailand. however, knowledge about its pollen morphology and fertility for plant breeding purposes, is limited. this study aimed to investigate the quantity, viability and germinability of pollen grains collected from the flowers of drc at different times o n a summer day, particularly from 8 am to 4 pm. pollen quantity was determined using a haemacytometer while its viability and germinability were examined after staining with 1% acetocarmine and allowing the pollen to germinate o n a modified agar-gelled germination medium. the pollen collected at 10 am had the highest pollen density (53.3x104 pollen/ml) and viability percentage (72.05'/0). when these pollen were allowed to germinate o n an artificial medium supplemented with various sucrose concentrations, the highest in vitro pollen germinability was found at the medium containing 10% sucrose. hence, the best time to collect the i. coccinea, cv. 'dwarf red coccinea' pollen was at 10 am. however, further investigations are recommended on the effects of daily or hourly environmental changes particularly, ambient temperature and humidity, o n the quantity and quality of harvestable pollen as well as o n the pistil phenology, to develop a more complete breeding strategy for the ixora species. keywords: plant breeding, pollen fertility, rubiaceae introduction ixora is one of the pantropical genera in the rubiaceae farmly comprising at least 500 species (mouly e t al 2009). among the 28 cultivated varieties, the non-native i. coccinea or dwarf red coccinea (drc) is the most popular and widely grown all over thailand. it is used as flower bed border, living fence, pot plant or individual shrub (puff e t al 2005; mouly e t al: 2009; chamchumroon 2014). drc has a superior character as a year-round and non-stop bloomer. its weak characters include a requirement of full s u d g h t to partial shade and excellent drainage in pots. both the quantity and quality of pollen collected from flowers are important in plant breedmg (ashman e t al 2004; colling e t al *corresponding author, e-mail: david.leung@canterbury.ac.nz 2004). both the abiotic (humidity and temperature) and biotic (pollinator) components of the environment also influence the pollen amount and viability of the flowering plant (aronne 1999; d e luca e t al. 2013). hence, it is of fundamental interest to study pollen. few studies have focused on pollen morphology of ixora spp. (de block & robbrecht 1998; sreekala e t al 2003). however, the pollen quantity and quality of i. coccinea, cv. dwarf red coccinea, have never been described. thus, the objective of this study was to determine the morphology, and changes in viabhty, density, and germinability of drc pollen collected at different times of the day when the flowers were in full bloom. under a light microscope, the pollen of this species has a generally prolate shape which is different from that of i. congesta and i. arborea. ixora pollen morphology and fertility phanomchai e t al, materials and methods plant material inflorescences were collected from the blooming ixora coccinea, cv. dwarf red coccinea (drc) plants at the garden of king mongkut's university of technology, north bangkok at 2 hour intervals from 8 am to 4 pm on a sunny day in three consecutive weeks of march, the summer season in thailand. pollen size, shape and viability the drc flowers were randomly selected at different times of the day (8 am, 10 am, 12 midday, 2 pm and 4 pm). the pollen grains were released by holding each flower upside down over a glass slide and by tapping (fig. 1). overall, pollen from 20 anthers from 5 different plants of different populations were placed on a glass slide for observation of pollen size and shape under a light microscope. the pollen viability was then investigated by staining with 1% (w/v) acetocarmine. the unstained pollen grains were non-viable whde the red stained grains were considered viable. before and after pollen staining, its shape was clarified by using the p / e ratio (punt e t al. 2007; hesse e t al. 2009). all pollen grains were randomly selected with 50 and 30 replications to determine pollen size and viability, respectively. pollen density the density (or quantity) of drc pollen grains was investigated at different times (8 am, 10 am, 12 am, 2 pm and 4 pm) using a modified method based on bunderson e t al. (2012). pollen grains from ten flowers were placed in a microcentrifuge tube. some 60 pl glycerol and 40 pl distilled water were then added into the tube and were mixed for 30 s using a vortex mixer. then, 8 pl of the pollen suspension was placed in a haemacytometer (improved neubauer rulings, boeco, germany) using a micropipette. the pollen grains were then covered with a glass and spread over the grid which was divided into nine large squares. only the pollen grains in the grid number 1, 2, 3 and 4 were counted (fig. 2). those pollen grains that touched the line on the bottom and right of the grid were omitted from counting. figure 1 ixora coccinea, cv. 'dwarf red coccinea' flower notes: a = top view (an arrow pointed at an anther); b = side view (bar = 1 cm). figure 2 grid layout of haemacytometer illustrating the position of number 1, 2, 3 and 4 pollen grains that were counted (modified from legresley and mcdermott, 2012) notes: the formula for pollen density calculation (legresley and mcdermott, 2012) is as follows: the average number of pollen per ml = [@ollen number in chamber 1+2+3+4)/4] x lo4 = average count per large square x lo4; data from this experiment were collected from 6 replications. biotropia vol. 28 no. 1,2021 pollen germination results and discussion in vitro pollen germination test was carried out using the modified mercado e t al. (1994) medium which consisted of 0.1 mm boric acid, 1 mm calcium chloride and various sucrose concentrations [o, 5, 10 and 20% (w/v)]. the medium was adjusted to ph 5.7, gelled with 0.9% (w/v) agar and sterilized at 121 oc, 15 psi for 20 min. later, pollen grains from ten flowers were brushed over the surface of the germination medium. the grains were incubated at 25k5 "c for 24 h in a dark room. pollen grains were considered to have germinated when pollen tube length was twice longer than the diameter of pollen grain. percentages of germination data were averaged from 20 replications. scanning electron microscope analysis pollen samples were collected at 10 am and sent to the scientific and technological research equipment centre, chulalongkorn university for morphological analysis using a scanning electron microscope (sem-eds, model jsm-6610lv, jeol ltd., tokyo, japan). data analysis anova of all data were carried out and the mean values in pollen size, viability, density and germination were compared using the duncan test at p < 0.05. one of the characters used to describe and identify a pollen grain of a flowering plant is its size and shape. for this purpose, measurements of the polar axis q and equatorial axis q have been broadly used (groppo e t a/. 2010; chwil 2015). in the case of i. coccinea, cv. dwarf red coccinea (drc), most pollen grains were probably shed from the anther before 4 pm as there was insufficient number (less than 10 grains per flower) by that time. therefore, pollen at this time was excluded in the experiments. under the light microscope, the drc pollen had polar axis longer than equatorial diameter and the p / e ratio was around 1.80 to 1.94 before staining (table 1). pollen with a polar axis longer than equatorial diameter or p / e ratio of 1.33-2.00 was described as prolate p u n t e t al. 2007; hesse e t al. 2009). moreover, the largest diameter was generally used for specifying the size of pollen. pollen diameter between 26 and 50 pm was categorized as a medum size (hesse e t al. 2009). thus, the drc pollen from 8 am to 2 pm was generally prolate in shape and had a me&um size before staining (fig. 3; table 1). when compared with other ixora species observed under the light microscope, the shape of drc pollen was dissimdar to those of ixora conge& (suboblate) and i. arborea (oblate spheroidal) (ibrahim e t al. 2012; prabhakar & ramakrishna 2014). table 1 pollen size and shape of ixora coccinea, cv. 'dwarf red coccinea' (under a light microscope) at different times before staining length of the axis (pm) time p / e ratio shape p e 8 am 39.4 k 0.210b 21.8 k 0.238a 1.8 k 0.012b prolate 10 am 39.6 k 0.261b 22.0 k 0.178a 1.8 k 0.008b prolate 12 am 39.1 k 0.169b 20.2 k 0 . 1 3 0 ~ 1.9 k 0.011a prolate 2 pm 40.2 k 0.135a 21.0 k 0.176b 1.9 k 0.014a prolate notes: values are means of 50 replications k se. data marked by the same letter in a column are not significantly different (p < 0.05). ixora pollen morphology and fertility phanomchai e t al, figure 3 pollen morphology of ixora coccinea, cv. 'dwarf red coccinea' before staining note: bar = 20 ym. t o examine more closely, the drc pollen were also investigated under a scanning electron microscope. the polar shape was tri-lobulate and the equatorial shape of this monad pollen was prolate (fig. 4). the grain had tricolpate aperture and psilate-perforate sculpturing. this was different from i. congesta whose pollen had suboblate shape, microreticulate sexine ornamentation, pericolpate aperture and quadrangular o u h e (ibrahim e t al. 201 2). these results suggested that pollen from the genus ixora may have divergent forms in different species. pollen staining with acetocarmine is one of the most widely used technique in estimating pollen viability (malayeri e t al. 2012). after the drc pollen grains were hydrated with 1% (w/v) acetocarmine, the shape of the dry pollen changed from prolate to spheroidal (fig. 5) and the diameter was around 32.6 36.7 pm (table 2). moreover, the viable pollens, as manifested by those exhibiting acetocarmine staining, were most numerous at 10 am (72.05%) (table 2). in another study, (sreekala e t al. 2003) the anther dehscence in ixora agastlyamaiqana occurred at 7 am to 1 pm and the peak anther dehiscence was also at 10 am. in the current research, the experiment was done until 4 pm when there was a noticeable decline in pollen quantity and quality. figure 4 polar (a) and equatorial (b) views of ixora coccinea, cv. 'dwarf red coccinea' pollen under a scanning electron microscope biotropia vol. 28 no. 1,2021 figure 5 pollens of ixora coccinea, cv. 'dwarf red loccmea arter stamng w t n 170 (w/ v) acetocarmine notes: pollens were collected at different times: a) 8 am, b) 10 am, c) 12 noon; and d) 2 pm. bar = 20 pm. table 2 pollen density, diameter and viability after staining of ixora coccinea, cv. 'dwarf red coccinea' at different times time diameter' ('pm) viability2 (oh) density3 (pollen per rnl) 8am 36.4 f 0.5a 61.8 f1.7b 8.9x104f 4.6x103c 10 am 36.7 + 0.3a 72.0 + 1.0a 5 3 . 3 ~ 1 0 ~ + 0.2x103a 12 am 36.6 + 0.3a 60.7 + o.6b 18.7x1o4 + 3.8x103b 2 pm 32.6 + o.lb 36.0 + 1 . 0 ~ 7.7x104+ 1.9x1o3c notes: 'values are means of 50 replications f se; 2values are means of 30 replications fse; 3values are means of 6 replications fse. data marked by the same ietter in a column are not significantly different (p < 0.05). although the thai rubiaceae plants are mainly dependent on animal-assisted pollination (puff etal. 2005), the best time to collect highly viable pollen of i. coccinea, cv. 'dwarf red coccinea7 for artificial breeding would be at 10 am. this was also the time of the day that the highest number of pollen per ml (53.292~10~) was collected (fig. 6; table 2). possibly, the pollen density was reduced after 10 am because of the continuous shedding of pollen from the anther to the external environment. pollen numbers are effectively counted using a haemacytometer (godini 1981; kelly e t al. 2002; bunderson e t al. 2012), and in the current study, a neubauer improved haemacytometer was used to successfully determine the pollen density. density of pollen collected at different times throughout the day was rarely investigated. considering the findings of the present study, it is possible that not only different amounts of pollen are produced at different flowering seasons (piotrowska 2012; peel e t al. 2014), but also different quantities of pollen may be obtained at different times of the day. hence, further studies are recommended to investigate the effects of environmental conditions particularly, ambient temperature and humidity of pollen shedding/collection at different times of the day. figure 6 pollen density of ixora coccinea, cv. 'dwarf red coccinea' on a neubauer improved haemacytometer notes: pollens were collected at different times: a) 8 am, b) 10 am; c) 12 noon; and d) 2 pm; all figures from grid a. ixora pollen morphology and fertility phanomchai et al. since little information is available on the germination potential of i. coccinea, cv. 'dwarf red coccinea' pollen, the pollen collected at 10 am was used in this study to determine the effect of different sucrose concentrations on pollen germination on an agar medium (modified based on mercado e t al. 1994). the highest percentage of pollen germination (about 34%) occurred at the medium containing 10% sucrose (table 3). at higher sucrose concentrations (20% sucrose) fewer than 10% of pollen germinated. this is consistent with other studies showing that sucrose concentration is an important factor for in vitro pollen germination (fig. 7; table 3). the ixora pollen may lose their viability quickly after collection. the fresh pollen exhibited 72% viability as revealed by acetocarmine staining. however, during in vitro germination, more pollen could have lost viability. another possibility is that sucrose may not be the only factor to enhance ixora pollen germination. to increase the germinability, other factors (for example, boric acid concentrations, fragdah e t al 2019) could also be studied in the future. the optimal sucrose concentration for in vitro pollen germination appears to depend on the plant species. for example, cannonball tree pollen needed a high level of sucrose (20% w/v), while two forms of day-blooming native thai waterlily responded very well on a low sucrose concentration (5% w/v) (bodhipadma e t al 2013; 2016). in contrast, the germination percentage of drc pollen was sharply reduced at sucrose concentrations that were lower and higher than 10%. table 3 percentage of ixora coccinea, cv. 'dwarf red coccinea' pollen germination at 10 am on modified mercado et all (1994) medium supplemented with different sucrose concentration sucrose concentrations germination (yo) 0.0 + o.od 3.2 f 0 . 5 ~ 33.8 f 1.7a 6.6 f 0.56b notes: values are means of 20 replications + se. data marked by the same letter in a row do not significantly differ (i? < 0.05). figure 7 ixora coccinea, cv. 'dwarf red coccinea' pollen germination on modified mercado e t al. (1994) medium notes: a = 0%; b = 5%; c = 10%; d) 20% (w/v) sucrose. bar = 50 pm. biotropia vol. 28 no. 1,2021 conclusion the quantity of i. coccinea, cv. 'dwarf red coccinea' (drc) pollen produced was related to the different production times during the day. the number of drc pollen grain peaked at 10 am which also exhbited the hghest estimated viability based on 1% (w/v) acetocarmine staining and density measurement with a neubauer improved haemacytometer. this is probably related to the varying environmental conditions like ambient temperature and hurni&ty at different times of the day of pollen collection. moreover, the optimal sucrose concentration of 10% (w/v) was essential for the drc pollen germination. since other studies on ixora pollen were m a d y focused on pollen morphology, it would be of interest to study the effect of sucrose level on the germination of pollen from other ixora spp. another implication of the present study is that for the breeding of ixora coccinea 'dwarf red coccinea', the critical time to collect pollen would be 10 am. to develop a more complete breeding strategy, further study is recommended on the 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predicting and quantifying pollen production in janipems ashei forests. phytologia 94:417-38. chamchumroon v. 2014. two new species of ixora (rubiaceae) from thailand. tfb (botany) 42535-90. chwil m. 2015. micromorphology of pollen grains of fruit trees of the genus pmnus. acta sci pol-hortoru 14:115-29. colling g, reckinger c, matthies d. 2004. effects of pollen quantity and quality on reproduction and offspring vigor in the rare plant scoqonera hz~milis (asteraceae). am j bot 91 :1774-82. de block p, robbrecht e. 1998. pollen morphology of the pavetteae (rubiaceae, ixoroideae) and its taxonomic significance. grana 37:260-75. de luca pa, bussie're lf, souto-vilaros d , goulson d , mason ac, vallejo-marl'n m. 2013. variability in bumblebee pollination buzzes affects the quantity of pollen released from flowers. oecologia 172:805-16. fragallah sada, lin s, li n, ligate ej, chen y. 2019. effects of sucrose, boric acid, ph, and incubation time on in vitro germination of pollen and tube 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palmer-maloney cell and sedgewick-rafter cell. in: icarlson b, cusack c, bresnan e, editors. microscopic and molecular methods for quantitative phytoplankton analysis (ioc manuals and guides, no. 55). paris (fr): unesco. p.25-30. malayeri be, noori m, jafari m. 2012. using the pollen viability and morphology for fluoride pollution biomonitoring. biol trace elem res 147:315-9. mercado ja, fernandez-muzor r, quesada ma. 1994. in vitro germination of pepper pollen in liquid medium. scientia horticulture 57:273-81. ixora pollen morphology and fertility phanomchai e t al. mouly a, razafimandimbison gs, ichodabandeh a, bremer b. 2009. phylogeny and classification of the species-rich pantropical showy genus ixora (rubiacrae-ixoreae) with indications of geographical monophyletic units and hybrids. am j bot 96:686-706. peel rg, 0 r b y pv, skj0th ca, kennedy r, schliinssen v, smith m, sommer j, hertel 0. 2014. seasonal variation in diurnal atmospheric grass pollen concentration profiles. biogeosciences 11:821-32. piotrowska k. 2012. meteorological factors and airborne h e x l. pollen concentration in lublin. acta agrobot 65:45-52. prabhakar r, ramakrishna h. 2014. palynodiversity in boath mandal forest division of adilabad district, telangana state, india. int j pharm life sci 5:3685-93. puff c, chayamarit i<, chamchumroon v. 2005. rubiaceae of thailand: a pictorial guide to indigenous and cultivated genera. bangkok ph): the forest herbarium, department of national parks, wildlife and conservation. punt w, hoen pp, blackmore s, nilsson s, le thomas a. 2007. glossary of pollen and spore terminology. rev palaeobot palyno 143:l-81. sreekala ai<, rajkumar g, pandurangan ag. 2003. studies on floral biology of ixora agastbamaiayana sivadasan & mohanan a rare southern western ghats endemic. zoos print j 18:1041-2. biotropia no. 10, 1997 : 14-28 influence of insect and seed sample size and heat treatment on the infestation of callosobruchvs chinens1s (l.) on mungbean, vigna radiata (l) wilczek *) elisa m. bucruanon 1 and belen morallo-rejesus 2 1 crops research division, philippine council for agriculture, forestry and natural resources research and development, 4030 los banos, laguna, philippines 2 department of entomology, college of agriculture, university of the philippines at los banos, 4031 college, laguna, philippines abstract the influence of different insect and seed sample size and heat treatment on the infestation of bean weevil, callosobruchus chinensis on mungbean,vjg/m radiata (l.) wilczek, was studied. insect and seed sample size as well as varieties/genotype had significant influence in obtaining large responses in the number of eggs and progenies of the bean weevil. use of at least 10 adult weevils to infest test samples containing at least 40 seeds for a 5-day oviposition period should produce reliable results when infesting mungbean seeds with unsexed weevils. dry heat treatment was very effective in disinfesting mungbean seeds from the bean weevil in different developmental stages. it improved germination depending upon the condition of the seed before tr eatment and certain temperature limits. a suggested treatment for mungbean dismfestation using dry heat would be 60 °c and 70°c for two-and one-hour treatments, respectively at 12% moisture content. for seeds in bulk, 60°c is much preferred. key words: callosobruchus chinensis/ insect and seed sample size/mungbean infestation/heat treatment introduction bruchids are world-wide pests of mungbean (vigna radiata (l.) wilczek) in storage. the bean weevil, c. chinensis is one of the three bruchid species that infests mungbean in the field and during storage. of the two given periods of attack, it is during storage that infestation results in greatest loss. measures to control the pest include among others the use of bruchid resistant cultivars and selective insecticides. much effort has been directed towards the screening or evaluation of chemical insecticides, vegetable oils and plant products effective hi controlling bruchids, as well as in the development of resistant varieties (duguet and wu 1986; davis et al 1984; varma and pandey 1978; pandey et al. 1981; epino and morallo-rejesus 1982). * paper presented at the symposium on pest management for stored food and feed, 5 -7 september 1995, bogor, indonesia. 14 biotropia no. 10, 1997 during the process of screening, however, certain ecological and biological factors may have their individual and interactive effects on the test insect. current practice in routine screening studies particularly against bean weevils differs largely in the number of insects and seeds used and the length of time the weevils are allowed to oviposit on the seeds. the optimum number of insects and seeds to use and the minimum oviposition period that should produce reliable results when infesting mung-bean seeds with bean weevils need to be determined. this has been established for maize and rice weevils (widstrom el al. 1972) but not for bean weevils in mungbean. inasmuch as seeds should be clean and free from insects before use in all screening and other related studies, disinfestation is of prime importance and it must therefore be very effective to ensure that the seeds used for screening are clean. it becomes even more important with the bean weevil which has a particular way of development that prevents easy detection. heat treatment at 70°c for 1 hour has been reported to be very effective in disinfesting maize seeds in china (dr. gonzalo de leon, personal communication, 1988, cimmyt entomologist). nevertheless, it needs to be further tested for its possible effect on the general condition of seed especially with legumes. this study therefore, was undertaken to determine the influence of insect and seed sample size and heat treatment on the infestation of bean weevil hi stored mungbean. materials and methods influence of insect and seed sample sizes on infestation of bean weevil on stored mungbean two mungbean genotypes, v2802 and vc 1973a, which had shown resistance and susceptibility to bean weevil attack, respectively were used in the experiment (talekar 1988; avrdc 1986). four sample sizes of mungbean seeds (10, 20, 40 and 60 seeds per sample) were tested. likewise, four weevil sample sizes (10, 20, 30 and 40 per sample) were tested. weevils were introduced into cups containing seeds of different sample sizes and held at 25°c, 75% rh. a designated male/female ratio of adult weevils was not used hi this experiment because earlier studies reported a sex ratio (males:females) of 1.06:1.00 (bato and sanchez 1972) and 6:5 (raina 1970) and therefore, it was assumed that there was a high probability of equal number of males and females in the weevil samples. moreover, emergence of adult weevils took place at night and mating occurred within an hour after emergence from the seed. one mating is enough to ensure egg fertilization and laying starts a day after emergence. the weevils were allowed to oviposit for three periods: 3, 5 and 10 days. after each oviposition period, 15 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus the weevils were removed and the eggs were counted. the cups were checked daily for progeny emergence. the two mungbean genotypes, four bean weevil sample sizes, tiiree oviposition periods, and four mungbean seed sample sizes were combined in a factorial arrangement requiring % treatment combinations per replication. three replications provided three blocks for analysis under a completely randomized design. seed damage and number of eggs and progeny per adult weevil in all treatments were determined. results from the analysis of variance were used to determine which combination(s) of the main effects influenced the measurement of bean weevil biology and mungbean quality. comparisons between the responses within each factor were performed using the least significant difference (lsd). effect of dry heat treatments on different stages of bean weevil mungbean varieties, v2709 and mg-9, a resistant and a susceptible line, respectively were used. each sample consisted of 2000 seeds which were exposed to 1000 adult bean weevils for oviposition. for the egg stage disinfestation experiment, mungbean seeds were exposed to oviposition by the weevil for five days. after oviposition, seeds were mixed thoroughly and divided equally, and packed in small cotton bags before heat treatment. for the larval stage disinfestation test, the eggs laid on the seeds were allowed to hatch. the seeds were then mixed thoroughly, and divided equally into a sample size consisting of 100 seeds before dry heat treatment. for the adult stage disinfestation study, twenty-five bean weevil adults were placed in small bottles containing 100 seeds per replication. the bottles were then placed in the oven for heat treatment. heat treatments for disinfestation of all the developmental stages of the bean weevil were at 70°c and 80°c for one hour, and 60°c and 50°c for two hours. the treatments including the untreated control were replicated 4 times. mungbean seeds had 12% moisture content before heating. each of the samples was transferred into small bottles with a screen cover and arranged hi a completely randomized manner under room conditions at 25°c and 75% rh. effect of dry heat treatments on mungbean seed vigor germination tests according to the aosa (1981) "rules for testing seeds" was conducted to evaluate the damage on the seeds because of the exposure to heat. seedling growth rate (sgr) vigor test was carried out. 16 biotropia no. 10, 1997 results and discussion influence of insect and seed sample sizes on the infestation of bean weevil on stored mungbean main effects and first-order interactions that significantly influence the number of eggs laid by the bean weevil, progeny produced per adult parent weevil, and percent damaged seeds are shown in table 1. of the four principal factors tested in this study, mungbean variety or genotype (v), bean weevil sample size (b), and seed sample size (s) were the most significant factors influencing weevil infestation. table 1. summary of f values for die main effects and interaction in the analysis of variance of factors mat influenced the number of bean weevil eggs and progeny per adult and percent damaged seeds 1 . source of variation df no. of eggs peradulr no. of progeny peradulr 2 percent damaged seeds 3 variety (v) bean weevil samples (b) seed samples (s) oviposition period (o) v x b 1 3 3 2 3 462.05** 36.94** 41.04** 2.63ns 15.3** 2720.19** 225.18** 181.54** <1 ns 83.87** 4217.7** 4.1** 2.69* <1 ns 4.1** v x s 3 2.43ns 61.64** 2.69* v x o 2 <1 ns <1 ns <1 ns bxs 9 <1 ns 3.57** 1.87ns b x o 6 <1 ns <1 ns 1.23ns s x o 6 2.61* 1.05ns 1.87ns v x b x s 9 <1 ns 1.53ns <1 ns v xb x o 6 <1 ns <1 ns 1.23ns v x s x o 6 <1 ns <1 ns <1 ns b x s x o 18 <1 ns <1 ns <1 ns v x b x sx o 18 <1 ns <1 ns <1 ns error 192 * = significant at 5% level ** = significant at 1 % level ns = not significant 1 = average of the three replicates 2 = based on data analyzed using the log (x + 1) transformation 3 = based on data analyzed using the arc sine transformation four significant interactions involved seed sample size. the number of eggs per adult parent weevil was significantly influenced by the interaction of seed sample size and duration of oviposition (fig. 1). eggs per adult markedly increased with larger 17 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus seed sample size. this is probably due to the greater number of seeds available for oviposition with more seeds thus lessening competition among the weevils. comparison among the three oviposition periods (3, 5 and 10 days) revealed that there was no significant differences between 3-day and 10-day oviposition period. the highest number of eggs laid by the weevils occurred at the 40-seed and 60-seed samples and at the 5-day oviposition period. although the highest egg count was obtained at the 60-seed sample this was comparable with that of the 40-seed sample at 5-day oviposition period and the latter was preferred. it was the minimum seed sample size and shortest duration of oviposition that yielded a high number of eggs per adult parent weevil. fig. 1. number of bean weevil eggs laid per adult with increasing mungbean seed sample size for three oviposition periods the interaction of the varying level of mungbean seed and parent bean weevils strongly influenced the number of progeny obtained per adult (fig. 2). the highest number of progeny (14/parent weevil) was obtained from 10 weevils/60 seeds. nevertheless, at 10 weevils/40 seeds, the same trend was observed, i.e. number of 18 biotropia no. 10. 1997 progenies was still high (8/parent weevil). the highest number of progenies per adult weevil obtained at 10-weevil sample size was suspected to be due to competition between larvae that existed at higher weevil sample size (20, 30 and 40). this resulted in only a few surviving adults. fig. 2. number of bean weevil progenies per adult with increasing seed sample size for four bean weevil sample sizes a highly significant effect was obtained in the number of progenies per adult as a result of the interaction between seed sample size and genotype (fig. 3). many bean weevil progen ies emerged from the susceptible seeds. however, it was quite interesting to note that both genotypes received, more or less the same number of eggs as shown by their nonsignificant f -value (table 1). the marked difference in the number of progenies suggests failure of eggs to hatch or failure of larvae to develop into adult weevils in the resistant mungbean genotype. the effect of seed genotypes and seed sample size was also significant in terms of percent damaged seeds (fig. 4). complete damage in susceptible seeds indicated that all seeds had eggs deposited on them. in the resistant genotype, however, percent 19 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus fig. 3. number of bean weevil progenies per adult with increasing seed sample size for two mungbean varieties damaged seeds significantly decreased at higher seed sample size. this shows that some seeds of the resistant genotype were missed by the females or eggs were laid but failed to develop into adult weevils. the proportion of these seeds without eggs increased as the number of seeds increased. the absence of eggs or progeny on some seeds of the resistant genotype, however, indicates that they are unsuitable for oviposition or larval growth and development. the reason why some seeds of the resistant genotype are unsuitable for oviposition is not yet clear. four highly significant interactions involved bean weevil sample size (table 1). the seed genotype x bean weevil sample size interaction (fig. 5) strongly influenced the number of eggs per adult. as the number of adult parent weevils increased, the number of eggs laid decreased in both susceptible and resistant genotypes. interference among weevils or eggs laid early by the weevils could make the bean testa less attractive (rougher) for oviposition. probably, it could be due to the effect of crowding of weevils or competition for oviposition sites. ten weevils/40 or 60 seeds seem to be the ideal insect and seed sample sizes to use in the interaction/screening studies for 20 biotropia no. 10, 1997 fig. 4. mean percent of damaged seeds as influenced by increasing seed sample size for two mungbean varieties bean weevils in mungbean since the highest number of eggs and progenies per adult occurred in this level. the degree of egg aggregation at the 10, 20 and 30-weevil samples on the resistant genotype was less than the susceptible counter part but rose significantly between 30and 40-weevil samples. the reason for this significant in crease is not yet clear. the number of progeny per adult weevil was strongly affected by bean weevil sample size and genotype interaction (table 1). the numbe r of adult progeny emerging decreased as bean weevil number increased in both susceptible and resistant geno types (fig. 5b). the result is consequent with the trend of result on the number of eggs (fig. 5a). adult weevil production depends on the hatchability of eggs which in turn is influenced by collisions between adults and laid eggs (nwanze el al. 1975). egg hatchability and larval growth and development may have been affected also by some resistance factors present in the seed. the susceptible and re sistant genotypes va ried in their reaction to bean weevil numbers as indicated in the percentage of dam aged seeds (fig. 5c). the seeds of the susceptible genotype were completely 21 influence of insect and seed sample size and heat treatment – e. m. buctuanon and b. morallo-rejesus 22 biotropia no. 10, 1997 damaged. in the resistant genotype, the percent damaged seeds in the 10-, 20and 30-weevil samples were invariably less than the 40-weevil samples. it was noted, though, that there were significantly more eggs laid at 40-weevil sample in the resistant genotype (fig 5a), yet the resulting number of progenies did not give the same trend of result (fig. 5b). not many weevil progenies were produced at 40-weevil sample. this result is consistent with the previous discussion that progeny production on resistant seeds was suppressed due to some resistance factors present in the seeds. effect of dry heat treatments on the different stages of bean weevil in the egg stage disinfestation experiment, results show that heat treatment significantly influenced the number of adult bean weevils that emerged from the disinfested mungbean seeds. no adult weevils emerged from seeds treated at 60°c, 70°c and 80°c, and exposed for 2-hour and 1-hour, respectively (fig. 6a). weevils survived from dry heat treatment at 50°c for two hours. likewise, temperatures at 70°c and 80°c applied for one hour and 60° c for wo hours killed all the larvae (fig. 6b). higher number of larvae survived from 50°c for 2-hour exposure which was comparable to the control. moreover, no adult weevils survived after exposure to 70°c and 80°c for one hour and at 60°c for 2 hours (fig. 6c). only 64% and 79% of adult weevils were killed at 50°c for 2 -hour exposure in susceptible and resistant lines, respectively. these results indicated that effective control was achieved using dry heat treatments at 60°c, 70°c and 80°c for twoand onehour exposure, respectively. insect eggs, larvae and adults were completely killed at these temperature levels and duration of exposure, thus indicating that this temperature range could be used in disinfesting mungbean seeds depending upon its effect on the viability and vigor of seeds which is discussed in the succeeding topic. effect of dry heat treatments on mungbean seed vigor the effect of dry heat temperatures on the vigor of seeds of two mungbean genotypes based on the percentage of normal seedlings, plumule length and dry matter weight is shown in table 2. results showed that dry heat treatments increased the percentage of normal seedlings in both susceptible and resistant genotypes (fig. 7a). highest and lowest percentages of normal seedlings from the resistant genotype were obtained at 70°c and 80°c, respectively for one hour exposure. lower temperatures (50°c and 60°c) but of longer duration of exposure (two hours) gave results comparable to that of the control and those exposed at 70°c. the most pronounced in crease in the percentage of normal seedling (50.5%) with the susceptible genotype was 23 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus 24 biotropia no. 10, 1997 table 2. summary of f values of the analysis of variance on the effect of the different treatment levels and varieties on the percentage of normal seedlings, mean plumule length and weight 1 obtained from seeds exposed to 80°c. no germination occurred from among those seeds in the control. there were hard seeds recovered from the susceptible genotype indicating the seeds to be probably dormant were broken by heat treatment specifically at higher temperature. results clearly indicated that dry heat treatment tremendously improved germination of seeds. this may probably be due to the increased water permeability at the strophiole of the mungbean seeds. mott and mckeon (1979) observed this phenomenon with dry heat treatments of stylo seeds, s. humilis. the effect of different dry heat temperatures and genotype interactions on plumule length was highly significant (table 2). differences in plumule length were markedly evident between genotypes (fig. 7b). in the resistant genotype, the plumule length tended to be longer, comparable with the control treatment, from seeds exposed to higher temperatures (70°c and 80°c) for one hour. seeds from the control treatment failed to germinate. there was no significant difference in the dry matter weight obtained between genotypes, and in all levels of dry heat temperatures in the resistant genotypes (fig. 7c). highest dry matter weight was obtained in 60°c treatment for two hours in the susceptible genotype as compared to the 80°c-1 hr treatment. heating mungbean seeds with dry air generally improved germination. however, certain important factors and/or seed conditions should be considered before treatment. in this particular study, resistant variety seemed to respond well to heat treatments especially at higher temperatures, but only to a maximum of 70°c. dry heat temperatures of 80°c significantly damaged the seeds as indicated by the lowest percentage of germination and normal seedlings obtained from this treatment level. variety can be a factor in heat damage resulting perhaps from differences in hardness, protein content or the variety as such (ghaly and taylor 1982). 25 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus 26 biotropia no. 10, 1997 mungbean seed vigor as evaluated, based on the four parameters, seemingly is not affected detrimentally by dry heat treatments up to certain limits. dry heat treatment of seed at 60°c is safer to use. the 60°c temperature level was found to be equally effective when compared with 70°c and 80°c in disinfesting mungbean seeds from the bean weevil, c. chinensis. summary and conclusion the influence of insect and different seed sample sizes and heat treatment on the infestation of bean weevil, callosobruchus chinensis (l.) on mungbean was determined. insect and seed sample sizes as well as variety/genotype had significant influence in obtaining high production of eggs and progenies of the bean weevils. these different parameters are tp be considered in screening studies. bean weevil eggs laid per adult increased with high seed numbers and the number of progenies decreased with increasing numbers of parent weevil. the weevils per 60 or 40 seeds in 5 -day oviposition periods provided the greatest frequency of significant responses in the number of eggs and progeny per adult weevil and percentage of damaged seeds. these are the minimum number of weevils necessary in a parent population and the practical seed sample size that would support the minimum number of days of oviposition to obtain maximum oviposition and/or infestation for screening studies. the number of weevil progenies differed in the resistant and susceptible genotypes, but no variation was observed in the number of eggs laid. the number of eggs laid and emerging progenies decreased as the number of parent weevils introduced was increased in both susceptible and resistant genotypes. seeds of susceptible genotypes were completely damaged in all seed density levels. however, percent damaged seeds decreased at higher seed densities in resistant genotypes. dry heat treatment was found to be very effective in disinfesting mungbean seeds from the different development stages of bean weevil. dry heat treatment improved germination and is not detrimental to the seeds regardless of genotype, depending however, on the condition of the seed before treatment which affects the dry heat temperature requirement and/or limits of the seeds. dry heat treatment of seeds at 60°c for 2 hours and 70°c for 1 hour exposure at 12% moisture content give complete insect control and is safer to use. heat treatment at 60°c however, is much preferred in disinfesting big bulk of seeds. 27 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus literature cited aosa. 1981. rules for testing seeds. 6(2): 30, 113. avr dc, 1986. avrdc progress report. 1984. asian vegetable research and development center (avrdc), shanhua. taiwan, 480 p. bato, s.m. and f.f. sanchez. 1972. the biology and chemical control of callosobruchus chinensis (linn.) (coleoptera: bruchidae). philipp. ent. 2(3): 167-182. davis, r., j. boczek, d. pankiewicz-nowicka and m. kruk. 1984. efficacy of tricalcium phosphate as a legume grain protectant. proc. third int. working conf. on stored-product entomology. kansas state university, manhattan, kansas, p. 256-261. duguet, j.s. and g.x. wu. 1986. assessment of activity of deltamethrin against callosobruchus chinensis l. and callosobruchus maculatus fab. (bruchidae). int. pest control. 28: 36-41. epino. p.b. and b. morallo-rejesus. 1982. mechanisms resistance of mungbean (vigna radiata (l.) wilczek) to callosobruchus chinensis (l .). philipp. ent. 5: 447-462. ghaly. t.f. and p. a. taylor. 1982. quality effects of heat treatment of two wheat varieties. jour. agric, and eng'g. res. (27): 227-234. mott. j.j. and g.m. mckeon. 1979. effect of heat treatments in breaking hardseededness in four species of stylosanthes. seed sci. & tech. 7: 15-25. nwanze. f.f., e. horber and c.w. pms. 1975. evidence for oviposition preference of c. maculatus for cowpea varieties. environ. ent. 4: 409-412. pandey. g.p.. r.b. dohar ey and b.k. var ma. 1981. efficacy of some edible oils for protecting greengram against the attack of callosobruchus maculatus i.fabrj. indian jour. agric. sci. 51: 910-912. raina, a.k. 1970. callosobruchus spp. infesting stored pulses (grain legumes) in india and a comparative study of the biology. indian jour. entomol. 32: 303-310. talekar. n.s. 1988. biology, damage and control by bruchid pests of mungbean. proc. of the second int. symp. on mungbean. p. 329-342. varma, b.k. and g.p. pandey. 1978. treatment of stored greengram seed with edible oils for protection from callosobruchus maculatus (fab.). indian jour. agric. sci. 48: 72-75. widstrom. n.w., l.m. redlinger. and w.j. wiser. 1972. appraisal of methods for measuring com kernel resistance to sitophilus zeamais. jour. econ. ent. 65: 790-2. 28 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 6 effect of plant (deden) pak... biotropia vol. 18 no. 1, 2011: 50 60 effects of plant growth regulators on shoot multiplication and root induction of cassava varieties culturein vitro deden sukmadjaja and herni widhiastuti a study on propagation of three superior cassava ( crant) varieties i.e. darul hidayah, malang-6 and adira-4 through tissue culture technique was conducted at the tissue culture lab of seameo biotrop, bogor. the objective of the experiment was to study effect of plant growth regulators on propagation, which can be used in cassava micropropagation protocol. plant materials used were auxiliary shoots of a stem node. the experiment consisted of (i) shoot multiplication, (ii) roots induction, and (iii) acclimatization. the multiple shoot regeneration was observed by using murashige & skoog (ms) basal media supplemented with 0, 0.1, 1.0 and 5.0 mg/l of benzylaminopurine (bap) combined with 0.0, 0.1 and 1.0 mg/l of thidiazuron. the root induction was observed by using 0.1 and 1.0 mg/l of iba and naa. the resulting plantlets were transplanted into plastic polybags containing soil mixed with organic fertilizer (1:1) covered with plastic sheets and transferred to a greenhouse. the result of the study showed that the highest number of shoots for darul hidayah, malang-6 and adira-4 varieties were 4.93 shoots treated with bap 1 mg/l + thidiazuron 0.1 mg/l, 4.20 shoots at bap media of 1 mg/l, and 7.20 shoots at the media of bap 1 mg/l + thidiazuron 0.1 mg/l respectively. the highest number of nodes produced was 2.9 nodes for darul hidayah at bap 5 mg/l, 5.13 nodes for malang-6 at bap 0.1 mg/l, and 6.18 nodes for adira-4 at bap 5 mg/l + thidiazuron 1 mg/l. the utilization of auxin iaa or naa could induce and accelerate the growth of roots which finally could increase the success of acclimatization process. with an average of four multiplication factors of each culture period, the potency of each cassava shoot propagated through tissue culture could produce around 37 000 plants/year. , shoot multiplication, root induction, ba, thidiazuron, iba, naa 1* 2 1 2 indonesian center for agricultural biotechnology and genetic resources research and development, jl. tentara pelajar no.3a, bogor, indonesia 16111 seameo biotrop, jl. raya tajur km.6, po box 116, bogor, indonesia manihot esculenta in vitro manihot esculenta abstract key words: * corresponding author : 50 introduction materials and method cassava ( crantz) is one of the food crops as a source of carbohydrates. in addition , cassava has a purpose used not only as a source of carbohydrates but also as raw materials for industry, cosmetics, feed and energy. as a raw material for industry, cassava could be processed into tapioca, glucose and fructose syrup, citric acid, monosodium glutamate, plywood, maltose, sorbitol and ethanol (sani 2006). meanwhile, with the increasing world prices of gasoline, cassava has now become the second source of bioethanol for premium mixture. in developing cassava production for agro-industry, the government of indonesia has issued regulations among others to optimize and to use genetic sources from the available varieties and clones of cassava. a few superior cassava varieties such as darul hidayah, adira and malang could produce 22-49 ton/ha fresh root crop. it has been reported that the productivity of darul hidayah could reach 102 ton/ha fresh root crop. the need of plant shoots exploited on a large scale will be difficult to fulfill using conventional propagation. cassava plant materials are commonly derived from vegetative propagation using stem cuttings. presently tissue culture technology is used for mass propagation of plant materials. propagation of cassava has been conducted either with shoots multiplication method or somatic embryogenesis. somatic embryogenesis was proposed as an alternative for mass cassava propagation (raemakers 1993; mathews 1993; konan 1994; ma and xu, 2002). however, it has not been really applied for large scale propagation because the frequency of plant regeneration from cassava somatic embryos is usually low. shoot multiplication method is the simplest and the safest method if observed from the point of genetic stability. this method becomes the most commonly used in the initiation step of plant propagation experiment. many authors such as kartha (1974) and konan (1997) explored the possibility to multiply cassava using microcuttings. nodal explants onto solid media provide an average of 3 to 4 microcuttings in 2 to 3 months. the objective of this study was to investigate the effects of plant growth regulators on plant regeneration of three cassava varieties in media culture. a study was conducted at the tissue culture laboratory of bioresources management center (brmc), seameo biotrop bogor. the experiment was conducted to observe the effects of several concentrations of bap combined with several concentrations of thidiazuron on multiple shoot formation and of auxin (iaa and naa) on rooting formation. plant materials applied this research were the auxiliary shoots derived from stem nodes of superior cassava clones, i.e. darul hidayah, malang and adira. isolated auxiliary shoots as explant sources were sterilized by soaking in the detergent solution for 15 min. then the explants were rinsed and soaked in 2 g/l of manihot esculenta in vitro et al et al. et al. et al. et al. in vitro explant source: 51 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . both fungicide and herbicide solution for 2 hrs, respectively. the soaked explants were rinsed with distilled water three times, then soaked in 2% na-hypochlorite solution for 30 min. explants were rinsed again by distilled water which contained 50 mg/l ascorbate acid for 10 min. explants ranging in size from 2 to 3 mm were cut aseptically without damaging the apical dome (shoots growth spot). the auxiliary shoot explants were inoculated onto sterilized solid basal ms medium (murashige & skoog's 1962) supplemented with different concentrations and combinations of different plant growth regulators. aseptically explants were inoculated onto initiation shoot ms media i.e. supplemented with 30 g/l sucrose and 100 mg/l polyvinyl pirolidone (pvp). explants which have been planted in initiation media for 1-2 weeks were transferred into regeneration and shoot multiplication media. the regeneration and multiplication medium was used with ms basal media supplemented with 30 g/l sucrose, 80 mg/l adenine sulfate and different range of bap (0, 0.1, 1, 5 mg/l) combined with thidiazuron (0.0, 0.1 and 1.0 mg/l). as long as the explants were in these media, they were sub cultured into the same media every 2 weeks within 4-6 weeks. elongated micro shoots on shoot regeration media were excised and transferred to ms basal media supplemented with 30 g/l sucrose, 2 g/l activated carbon (charcoal) and two different concentrations of iaa and naa (0.1 and 1.0 mg/l) either individually. the ph of the medium was adjusted to 5.8 before gelling with agar (8 g/l) and prior to autoclaving for 15 min at120 c and at 15 lbs psi pressure. sugar was added at the concentration of 30 gr/l. medium of 25 ml was dispensed into the culture jar and plugged with autoclavable plastic cap. all the cultures were incubated in a growth room with a 16 hour photoperiod (cool, white fluorescent light -1000-1500 lux) and the temperature was maintained at 25 ± 3 c with 70-80% relative humidity in the culture room. each treatment consisted of 10 replicates and repeated three times. plantlets with well-developed roots were removed from the culture medium. the roots were washed gently under running tap water and transferred to plastic pots (polybag) for hardening which contain mixed soil and manure (1:1). the harden plantlets in the plastic polybag were covered with transparant polyethylene sheets (for about 15 days) to maintain high humidity and were kept under shade in a net house for further growth and development. the calculation of the potential number of cassava plants produced through tissue culture followed the formulation of pennell (1987): where : y = number of plantlet/plants that could be produced a = number of shootss produced at each subculture period (multiplication factor) 2 o o culture medium and condition: shoot initiation medium: shoot regeneration and multiplication medium: rooting medium: environmental condition: acclimatization and transfer of plantlets to soil: potential of cassava plant production: y = a x b x f1 x f2 x f3 n biotropia vol. 18 no. 1, 2011 52 b = number of initial explant which grow n = number of subculture at a certain period (per year) f1 = the percentage of successful culture at the stage of shoot induction f2 = the percentage of successful culture at the stage of shoot elongation f3 = the percentage of successful acclimatization experimental design: experiments were set up in a completely randomized design (crd) and each experiment usually had 10 replicates and was repeated three times. 10 explants were used per treatment in each replication. observations were recorded on the percentage of shoots, number of shoots or nodes per explant, shoot length, numberof roots per shoot and root length respectively. statistical analysis was limited to the calculation of standard errors of means. the first step of shoot initiation, after one week explants were cultured on initiation media where var. darul hidayah produced the highest number of aseptically explants compared to the other four varieties. this is assumed to be related to the source of explants. the source of darul hidayah explants was taken from the green house which has low contamination due to controlled sanitation and environmental condition compared to the two other sources of explants which were taken from the field. results and discussion shoot initiation from source of explants 53 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . (a) (b) (c) (d) figure 1. (a) auxiliary shoots on ms shoot induction media, (b-d) shoots of three cassava varieties on multiplication media: var. darul hidayah, var. malang-6, and var. adira-4, respectively. 54 biotropia vol. 18 no. 1, 2011 auxillary shoot will appear after one-week old explants were cultured on ms shoot induction media supplemented with 30 g/l sucrose and 100 mg/l polyvinyl pirolidone (pvp) (fig. 1a). pvp was used to inhibit necrosis of the plant tissues which were always found in woody plants according to zdravkovic 2004. the growth rate of shoot elongation with the best visual appearance was shown by adira-4 and darul hidayah, while malang-6 showed a lower growth rate of its shoot elongation. in general, shoot induction and elongation occurred after the explants were one-week old and cultured on ms media. at the age of 4 weeks the shoots had between 3-4 nodes and sufficient enough to be sub-cultured on propagation media. among three cassava varieties, adira-1 was not used because most of the explants were contaminated after sub-cultured on regeneration and multiplication media. two steps of experiments were conducted i.e. shoot multiplication and root induction. another activity is acclimatization of plantlet in the green house. the experimental results of the role of bap and thidiazuron on the number shoots produced on regeneration and multiplication media are shown on table 1. all of bap and thidiazuron concentrations examined on darul hidayah and adira-4 showed to produce an average of higher number of shoots compared to the non plant regulators media (control). on the other hand, malang-6 explant produced higher number of shoots only on 1 mg/l bap and 0.1 thidiazuron compared to the control media. bap could encourage cell division which stimulates formation of multiply shoots. according to lu (1993) the addition of thidiazuron in media containing bap could increase the explant ability to produce shoots. in perennial woody plants among others and , thidiazuron could stimulate the formation of shoots. kerns and meyer (1986) reported that a good result was obtained at the stage of acer shooting by using combination of bap and thidiazuron. the highest shoot number of darul hidayah produced was about 4.93 shoots on media supplemented of 1 mg/l bap combined with 1 mg/l thidiazuron. the use of high concentration of 5 mg/l bap without thidiazuron resulted in high number of shoots (4.80 shoots). adira-4 has a better response and produce the average number of shoots compared to the other two varieties in all treatments examined. the average highest number of shoots of adira-4 was about 7.20 shoots on media containing 1 mg/l bap combined with 0.1 mg/l thidiazuron. the number of shoots decreased without addition of thidiazuron. the addition of thidiazuron at low concentration (0.1 mg/l) tended to increase the number of shoots produced. the average number of malang-6 shoots produced was lower than of the two varieties at all similar treatments. the highest number of shoots (4.20) was produced when treated with bap 1 mg/l. the effects of growth regulator bap and thidiazuron on the lenght of shoots are shown on table 2. the highest lenght of shoots of malang-6 were 4.33 cm obtained on media without plant growth regulator. no significant measurable effect of bap and thidiazuron were obtained on lenght of malang-6 shoots. meanwhile, on darul hidayah, the use of bap at low concentration (0.1 mg/l) with or without the addition of thidiazuron at low concentration (0.1 mg/l) resulted in higher growth of et al, in vitro prunus rhododendron the effect of media composition on shoot and root regeneration 55 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . shoots compared to the other treatments. on the other hand the highest growth of shoots on adira-4 was obtained if treated with bap at 5 mg/l concentration with or without thidiazuron, i.e. between 3.13-4.19 cm (table 2). a high growth of shoots was also observed in media containing thidiazuron 1 mg/l combined with or without bap 1 mg/l, i.e. 3.44 and 3.66, respectively. nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on ms medium containing 10 mg/1 bap. this latter structure, when cultured on ms medium supplemented with 0.1 mg/1 naa, 1 mg/1 bap and 0.1 mg/1 ga , produced multiple shoots. rooting of regenerated shoots exceeded 95 % in phytohormone-free ms medium. (konan . 1997). cytokinin could interact with other growth regulators to stimulate the vegetative growth of plants (maxwell & keiber 2004). in the plant physiology process, cytokinin influences cell division in order to broaden the area of the tissues and plantlet height (davies 2004). 3 et al table 1. the effect of bap combined with thidiazuron in ms media on the shoot number of darul hidayah, malang-6 and adira-4, 30 days after planting plant growth regulator (mg/l) number of shoots darul hidayah malang-6 adira-4 without plant growth regulator 2.07 ± 0.21 2.79 ± 0.30 3.00 ± 0.70 bap 0 + thi 0.1 3.93 ± 1.02 3.44 ± 1.61 6.20 ± 1.30 bap 0 + thi 1 2.87 ± 1.30 1.59 ± 0.45 5.50 ± 1.29 bap 0.1 2.20 ± 0.45 2.25 ± 0.58 4.20 ± 0.84 bap 0.1 + thi 0.1 3.06 ± 0.90 2.86 ± 1.07 3.40 ± 0.89 bap 0.1 + thi 1 3.60 ± 1.14 2.71 ± 0.90 3.60 ± 1.14 bap 1 4.75 ± 1.69 4.20 ± 0.86 6.41 ± 1.52 bap 1 + thi 0.1 4.20 ± 1.00 2.35 ± 1.44 7.20 ± 3.27 bap 1 + thi 1 4.93 ± 1.18 2.35 ± 0.91 6.00 ± 0.81 bap 5 4.80 ± 1.12 2.15 ± 0.48 5.40 ± 2.08 bap 5 + thi 0.1 3.34 ± 0.63 2.45 ± 0.58 5.20 ± 1.93 bap 5 + thi 1 3.48 ± 0.87 2.35 ± 0.59 3.67 ± 1.16 note: means are calculated by standard errors in general, the number of nodes produced in malang-6 and adira-4 varieties was higher compared to darul hidayah. in malang-6 the use of media with or without low concentration of bap combined or not with thidiazuron 0.1 mg/l produced higher number of nodes compared to other treatments (3.98-4.61 nodes). in adira-4, the use of thidiazuron l mg/l with or without addition of bap produced high number of nodes, except for bap 0.1 mg/l + thidiazuron 1 mg/l (4.83-6.18 nodes). in darul hidayah, the highest number of nodes was obtained if treated with high concentration of bap combined with or without thidiazuron, i.e. between 2.49-2.90 nodes (table 3). according to mingxia ., 2011, the cytokinin n-benzyladenine (ba) (02.0 mg l )et al −1 biotropia vol. 18 no. 1, 2011 was effective on cassava shoot regeneration. konan (1997) has conducted mass propagation system of some cassava cultivars through auxiliary shoots multiplication and stem nodes which showed that the addition of bap on basal ms media was more efficient if compared to the addition of other kinds of cytokinin. the effectiveness on organogenesis was different based on different based on different cytokinins and in combination with auxins. benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and n-isopentenyladenine (ma 1998). et. al. table 2. the effect of growth regulator in ms media on the lenght of cassava shoot var. hidayah, malang-6 and adira-4 at 30 days after planting. plant growth regulator (mg/l) lenght of shoots (cm) darul hidayah malang-6 adira-4 without plant growth regulator 1.50 ± 0.68 4.33 ± 1.29 1.05 ± 0.37 bap 0 + thi 0.1 2.54 ± 0.71 2.18 ± 0.98 2.77 ± 0.40 bap 0 + thi 1 2.16 ± 1.52 2.31 ± 1.19 3.66 ± 0.40 bap 0.1 3.85 ± 1.49 2.17 ± 1.54 1.80 ± 0.82 bap 0.1 + thi 0.1 3.90 ± 1.50 2.36 ± 0.92 1.11 ± 0.39 bap 0.1 + thi 1 1.44 ± 0.69 2.65 ± 0.83 1.02 ± 0.33 bap 1 1.61 ± 0.76 2.69 ± 0.87 2.17 ± 0.47 bap 1 + thi 0.1 1.61 ± 0.45 1.60 ± 1.52 2.61 ± 1.16 bap 1 + thi 1 2.26 ± 0.75 2.06 ± 1.16 3.44 ± 0.58 bap 5 2.04 ± 0.28 1.24 ± 0.44 3.13 ± 1.09 bap 5 + thi 0.1 1.14 ± 0.24 1.73 ± 0.73 4.19 ± 1.85 bap 5 + thi 1 1.75 ± 0.20 1.61 ± 0.45 3.78 ± 1.25 note: means are calculated by standard errors marked differences were observed among the three varieties in the auxin requirements for root induction (table 4). darul hidayah and adira-4 showed increase of both root number and length resulted from addition of iaa to the nutrient media. no significantly effect of iaa and naa were obtained on number and lenght of root in malang-6. the length of roots of darul hidayah culture showed the highest growth compared to malang-6 and adira-4. in darul hidayah, the use of iaa and naa at the same concentrations did show a significant difference in the number of roots (table 4). the use of naa relatively produced higher number of roots compared with iaa. on the contrary, the use of iaa on malang-6 produced slightly higher number of roots compared to the use of naa. while in adira-4, the use of iaa (01 and 1 mg/l) gave a significantly different result compared to the use of naa 0.1 mg/l. ogburia (2003) has conducted a faster propagation on a number of cassava cultivars with 90 % efficiency by using ms media of 0.1 mg/l naa and 0.05 mg/l bap. the use of 0.1-1.0 mg/l iaa in rooting media of darul hidayah and adira-4 produced longer roots compared to the treatment with naa. the longest root was produced in darul hidayah treated with iaa l mg/l (5.43 cm), and for adira-4 treated 56 57 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . with iaa 0.1 mg/l (3.23 cm). meanwhile, the longest root of malang-6 was produced if treated with naa 0.1 mg/l, i.e. 2.37 cm (table 5, fig 2 (a-c). mingxia ,(2011) reported the auxi ) proved to be effective on cassava root development. aladele and kuta (2008) reported that the growth rate of cassava plantlet is significantly different on various genotypes and growth environment. in acclimatization stage, rooted shoots were transplanted in the green house for hardening and their survival rate was 90% under the field condition (fig. 3). . et al n α-naphthalene acetic acid (naa) (02.0 mg l −1 table 3. the effect of growth regulator in ms media on the number of cassava nodes in darul hidayah, malang-6 and adira-4 at 30 days after planting plant growth regulator (mg/l) number of nodes darul hidayah malang-6 adira-4 without plant growth regulator 2.10 ± 0.22 4.61 ± 0.06 2.53 ± 1.52 bap 0 + thi 0.1 2.20 ± 0.65 4.78 ± 1.42 4.65 ± 0.30 bap 0 + thi 1 1.23 ± 0.28 3.44 ± 1.24 5.76 ± 0.76 bap 0.1 2.48 ± 0.84 5.13 ± 1.04 2.56 ± 1.11 bap 0.1 + thi 0.1 1.77 ± 0.84 3.98 ± 1.09 2.70 ± 1.36 bap 0.1 + thi 1 1.22 ± 0.48 3.33 ± 0.30 2.64 ± 0.43 bap 1 2.04 ± 0.58 4.08 ± 0.86 3.92 ± 0.90 bap 1 + thi 0.1 1.83 ± 0.63 3.23 ± 1.82 4.09 ± 1.91 bap 1 + thi 1 2.21 ± 0.58 3.47 ± 1.51 4.83 ± 0.96 bap 5 2.90 ± 0.50 2.86 ± 0.42 3.75 ± 0.72 bap 5 + thi 0.1 2.49 ± 0.37 3.43 ± 0.93 4.52 ± 0.62 bap 5 + thi 1 2.85 ± 0.43 3.21 ± 0.83 6.18 ± 3.48 note: means are calculated by standard errors table 4. the effect of auxin growth regulator in ms media on the number of cassava roots of darul hidayah, malang-6 and adira-4 at 15 days after planting. plant growth regulator (mg/l) number of roots darul hidayah malang-6 adira-4 iaa 0.1 4.40 ± 1.42 2.30 ± 1.77 3.60 ± 1.51 iaa 1 3.70 ± 0.89 3.30 ± 2.06 4.30 ± 1.57 naa 0.1 4.86 ± 2.19 2.00 ± 1.63 1.90 ± 1.37 naa 1 3.80 ± 1.92 2.50 ± 1.84 3.20 ± 2.10 note: means are calculated by standard errors (a) (b) (c) fig. 2. t . he occurrence of the root growth of three cassava varieties in rooting media, (a) darul hidayah; (b) malang-6; and (c) adira-4 (a) (b) fig. 3. seedlings in the green house after one week old acclimatization (a) and the performace of seedling roots (b). 58 biotropia vol. 18 no. 1, 2011 table 5. the effect of auxin growth regulator in ms media on the length of roots (cm) of darul hidayah, malang-6 and adira-4 at 15 days after planting. plant growth regulator (mg/l) length of roots (cm) darul hidayah malang-6 adira-4 iaa 0.1 4.88 ± 1.43 0.77 ± 0.62 3.23 ± 1.04 iaa 1 5.43 ± 2.49 1.88 ± 1.55 2.65 ± 0.67 naa 0.1 3.60 ± 0.76 2.37 ± 1.55 1.24 ± 1.14 naa 1 3.25 ± 2.02 1.61 ± 0.84 1.50 ± 1.54 note: means are calculated by standard errors 59 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . potential of cassava plant production y = a x b x f1 x f2 x f3 one shoot explant (b) could produce an average of 4 (a) nodes or shoots for each culture period, with a frequency of 8 subcultures per year (n), and assumed that the success a the stage of shoot induction (f1) = 90%, the success at the stage of rooting (f2) = 80%, then the number of plants that could be produced per year (y) from each shoot is as follows, using the formulation of pennell (1987) : 4 x 1 x 0.9 x 0.8 x 0.8 = 37 748 plants if there are 10 initial shoot explants (b) available then the number of plants that could be produced is about 377.480. it should be noted that the number of plants produced is based on theoretical calculation; however, the implementation depends on some other related factors such as number of manpower and available facilities. each variety of clone cassava needed a different protocol to get optimum shoot initiation, shoot multiplication, root induction and elongation. the maximum number of axillary shoots and stem nodes of cassava var darul hidayah, malang-6 and adira-4 were obtained on shoot multiplication media containing bap and thidiazuron at different concentrations. the use of iaa and naa media induced and accelerated the growth of roots and increased the percentage of survival plantlet in acclimatization process. the potency of each cassava shoot could be propagated to produce about 37000 plantlets per year. the authors are grateful to seameo-biotrop for providing financial support and facilities. thanks are due to the technicians of the brmc, seameo-biotrop plant tissue culture laboratory for their assistance. n 8 conclusions acknowledgements references davies pj. 2004. the plant hormones: their nature, occurrence and function in: davies pj (editor) plant hormones biosynthesis, signal transduction, action. kluwer acad press. p 1-15 kartha kk, gamborg ol, constabel f, shyluk jp. 1974. regeneration of cassava plants from apical meristems. plant sci lett 2: 107-13 konan nk, sangwan rs, sangwan bs. 1994. somatic embryogenesis from cultured mature cotyledons of cassava crantz). plant cell tiss org cult 37: 91-102 konan nk, schapke c, carcamo r, beachy rn, fauquet c. 1997. an efficient mass propagation system for cassava ( crantz) based on nodal explants and auxiliary shoot-derived meristems. plant cell rep 16(7):444-49 (manihot esculanta manihot esculenta 60 biotropia vol. 18 no. 1, 2011 kerns hr, meyer mm. 1986. tissue culture propagation of acer x freexmani using thidiazuron to stimulate shoot tip proliferation. hort sci 21(5):1209-10 lu cy. 1993. the use of thidiazuron in tissue culture. cell dev biol 29:92-96 ma gh. 1998. effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants. plant cell tissue org cult 54: 1-2 ma gh, xu qs. 2002. induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava. plant cell tissue org cult 70: 281-282 mathews h, schopke c, carcamo r, chavarriaga p, fauquet lb. 1993. improvement of somatic embryogenesis and plant recovery in cassava. plant cell rep12: 328-33 maxwell bb, keiber jj. 2004. cytokinin signal transduction. in davies, pj (editor) plant hormones biosynthesis, signal transduction, action. kluwer acad press. p 321-349 pennell d. 1987. micropropagation in horticulture. growerguide no.29. grower books, london raemakers cjjm, schavemaker cm, cobsen e, visser rgf. 1993. improvement of cyclic embryogenesis of cassava crant). plant cell rep12: 226-229 sani s. 2006. kebijakan dalam strategi pengembangan ubi kayu untuk agroindustri. prospek, strategi, dan teknologi pengembangan ubikayu untuk agroindustri dan ketahanan pangan. puslitbangtan, bogor. hlm: 20-28 zdravkovic-korac s, muhovski y, druort p, calic d, rabjevic l. 2004. agrobacterium rhizogenes mediated dna transfer to l. and the regeneration of transformed plant hormones. plant cell rep 22:698-704. in vitro (manihot esculenta dalam aesculus hippocastonum biotropia no. 6, 1992/1993: 45-54 review of aquaculture genetic researches in thailand uthairat na-nakorn department of aquaculture, faculty of fisheries, kasetsart university, bangkok 10903, thailand aquaculture business has been well established in thailand for more than 40 years. the most recent data indicated a total production of 260 380 tons. sixty-five percent of the total production came from coastal aquaculture, mainly tiger prawn (penaeus monodon) culture. other important species for coastal aquaculture are banana prawn (p. merguensis), cockle (anadara granosa), green mussel (perna viridis), oyster (crassostrea belcheri, saccostrea commercialis), sea bass (lates calcarifer) and grouper (epinephelus tauvina). freshwater aquaculture, although produced only 35% of the annual production, provides major protein source for people in rural areas. important freshwater species are nile tilapia (oreochromis niloticus), tawes (puntius gonionotus), sepat siam (trichogasterpectoralis), walking catfish (glorias spp.), stripped catfish (pangasius sutchi) and giant freshwater prawn (macrobrachium rosenbergii). optimum aquacultural practises, namely stocking density, nutrition requirement and water quality have been obtained in most cultured species. but genetic approach has not been considered, thus resulting in deterioration in economic traits which might be due to excessive inbreeding (reviewed by uraiwan 1989) and/or negative selection (wongsangchan 1985). the history of researches on genetics in aquaculture in thailand started in 1982 when the aquaculture genetic programme in form of a network has been established at the national inland fisheries institute, department of fisheries. this programme was supported by the international development research centre (idrc, canada) in cooperation with dalhousie university, canada (uraiwan 1989). in the same year a genetic improvement programme aiming at improving economic characters of some economic fish species has been conducted at the department of aquaculture, kasetsart university. paralelly a course in fish genetics has been offered. since then different approaches of genetics have been applied with final objectives on improving aquaculture production of the country. researches being conducted are reviewed according to the following fish species. 45 biotropia no. 6, 1992/1993 nile tilapia (oreochromis niloticus) nile tilapia was first introduced to thailand in march, 1965 as a present from the crown prince of japan to his majesty king bhumipol (duangsawasde et al. 1982 cited by uraiwan 1989). the original stock consisted of 50 pairs of fish. nowadays, it has been cultured all over the country and ranks the first in annual production in the recent years (department of fisheries 1991). in fact, most of the cultured stocks are contaminated by hybridizing with o. mossambicus introduced earlier. the only one pure strain is maintained at chitralada royal palace. main genetic problems in tilapia culture in thailand are: 1. slow growth which might be a result of inbreeding depression caused by the small number of foundation stock. unintentional negative selection might also be a cause of the slow growth (wongsaengchan 1985). 2. unacceptable appearances, namely dark colour and long shape, resemble o. mossambicus. therefore, genetic improvement programmes have been conducted to solve these problems. selection programmes using within family selection have been conducted since 1986 aiming at improving the growth of nile tilapia. sixteen families were produced from each of the chitralada strain and chitralada x nakornpathrom cross. the rotation crossing between these families was applied in each generation. after 3 generations of selection the selected lines grew an average of 17 and 13% faster than those of the control and the station seed production lines, respectively (uraiwan et al. 1989 and uraiwan et al. 1982). moreover, indirect selection for increasing growth rate has been performed by uraiwan (1989). in this selection programme offsprings of each of 5 different families were grouped into 3 categories : early maturing (21-23 weeks), medium maturing (25 27 weeks) and late maturing (> 27 weeks). selected fish from the five families were mixed together according to maturing classification. results revealed that the direct response to selection was significant. the fish selected for early maturing matured 11 to 14 days earlier than those selected for late maturing. the selection indirectly resulted in a genetic growth gain. the early maturing selected line was significantly larger and grew faster than the late maturing selected line (size 19 26% larger and growth 5 9% faster). phenotypic correlation coefficient between age and size at maturity ranged between 0.3-0.9. there were no differences in prematurity growth rates between the selected and unselected lines (uraiwan and virasith 1989). 46 review of aquaculture genetic researches in thailand u. na-nakorn thai red tilapia thai red tilapia is a hybrid of oreochromis niloticus and o. mossambicus. it has been originally observed at ubonratchathani fisheries station, in north-eastern thailand (jarimopas 1988). selection programme for better growth rate of thai red tilapia has been conducted since 1982 (jarimopas 1989). size-specific selection was used to prevent effects of asynchronous spawning and variation of mouth-brooding duration on size differences among offspring obtained from mass spawning. in each selection generation twenty-five pairs of parents were mass spawned. fry were collected for a week to obtain approximately 2000 fry. after a rearing period of about 6 weeks fry were graded. only the medium size group was kept and reared for about 8 more weeks. selection was performed when the fish were 14 weeks old. after 6 generations of selection, the selected strain of thai red tilapia was in weight and length 6.76% and 4.09% greater (p<0.05), respectively, compared to control line at 12 weeks of age (jarimopas and nukwan 1989). realized h 2 were 0.44 for weight and 0.53 for length. this selection programme is continuing. thai walking catfish (clarias spp) there are 3 species of clarias being cultured in thailand. c. macrocephalus is the most acceptable due to good meat quality, thus commands high price. culture of this species faces problems of slow growth and high disease susceptibility. c. batrachus is an alternative species for farmers who could not cope with the risk. although its growth and disease resistance is better than c. macrocephalus, it is not so acceptable due to poor meat quality, thus its price is very low. recently, african catfish, c. gariepinus has been introduced to thailand because of its high growth rate. but, again its meat is not acceptable among thai consumers. therefore, an attempt was made to combine the advantages of c. gariepinus and c. macrocephalus by hybridizing these 2 species. successful cross has been obtained between female c. macrocephalus x male c. gariepinus but not the reciprocal one (nukwan et al. 1990). hybrid has improved growth and disease resistance compared to female parent. although its meat quality and appearances could not totally replace that of c. macrocephalus, nowadays it comprises more than 90% of clarias production of the country. this hybrid has been found to be partly fertile. further studies are going on at the department of fisheries in order to study genetic properties of the hybrid for its genetic improvement in the future (lawanyavudth personal communication). 47 biotropia no. 6, 1992/1993 in fact, hybridization experiments attempting to solve the problems of c. macrocephalus were conducted before. tarnchalanukit (1986) made crosses between c. macrocephalus and pangasius sutchi which has faster growth rate and more resistance to disease than c. macrocephalus. the author reported 4 hybrid morphotypes which were described later to be actually 2 morphotypes found to be diploid and triploid hybrids (na-nakorn et al. 1992a). moreover, the authors also reported gynogenetic offsprings arisen from this cross. although these hybrids were not useful for aquaculture, they might contribute knowledge on the phylogenic relationship of these 2 species which belong to different classes (na-nakorn et al. 1992a). at present, although problems have been partly solved by culturing of hybrid clarias, more serious problems emerge which are shortage of female c. macrocephalus brooders. therefore, genetic improvement programmes of c. macrocephalus have been continued. jarimopas et al. (1989) conducted mass selection for fast growing c. macrocephalus starting in january 1986. after 3 successive selection generations the selected line was 11.8% heavier and 2.35% longer than the control line. selection response in weight and length were 26.21 g and 0.66 cm, respectively. realized heritability of weight and length was 0.47 and 0.30 respectively. selection programmes for resistance to aeromonas hydrophila which is the most serious disease were conducted at the department of aquaculture, faculty of fisheries, kasetsart university during 1987-1989 (na-nakorn et al., 1992b). c. macrocephalus was collected from 5 different localities in thailand. their performances including resistance to a. hydrophila were compared but only slight differences were observed. mass selection was performed in the f2 generation arisen from all possible crosses of the 5 strains. the 104 day-old fish were intraperitoneally injected with a. hydrophila suspension and survivors were kept as selected brooders. after one generation of selection, little improvement was observed in resistance to a. hydrophila. heritability of liability for this trait was 0.17. unfortunately the selected fish died of flexibacter columnaris which always occurred in winter and made a second generation of selection impossible. during 19871988 a cross breeding experiment was conducted hoping to benefit from heterosis. semi-diallel crosses were performed using fish collected from the southern and central parts of thailand. results showed that progenies of the cross between females from the south and males from the central grew better than the pure-breds and reciprocal cross. heterosis was shown for body weight and length at the age of 2, 3 and 3.5 months. there were no differences in disease resistance among progenies of different crosses (na-nakorn et al. undated). since this experiment was done in tanks, therefore it is being repeated in earthen ponds which is a normal practise for clarias culture. 48 review of aquaculture genetic researches in thailand u. na-nakorn chromosome manipulation has been applied to improve growth of c. macrocephalus. triploid fish were induced using cold shock (7°c, 25 minutes) applied immediately after fertilization. the shock resulted in 80% triploid fry. growth comparison showed that triploid fish grew slower than diploid ones. the survival rate of triploids was lower than that of diploids (na-nakorn and lakhaanantakun 1992). attempting to combine techniques in biotechnology and quantitative genetics na-nakorn et al. (1992d) and na-nakorn et al. (1992c) induced gynogenesis in c. macrocephalus to produce highly inbred lines for further cross breeding. meiotic gynogenesis was induced by activation of c. macrocephalus eggs using irradiated sperm of pangasius sutchi. sperm dilution (1:100 in ringer's solution) was uv-irradiated (30 w, 30 cm, 2 mins). after the activated eggs were subjected to cold shock, best results were achieved at shock temperature between 6 11 °c with shock duration of 14 minutes and starting 3.5 4.5 minutes after activation. gynogenetic fish were all females. thus, sex reversal to maleness is needed to produce male parents. recently, in my laboratory 17 alpha methyltestosterone has been orally administered to feeding fry of c. macrocephalus. results showed that administration of 30 or 60 mg mt/kg feed for either 30 or 60 days reduced growth at 60 days of mt treated fish as well as survival rate at 30 days. no changes in sex were observed, except for intersex fish found in both mt treated groups being treated for 60 days. paradoxical feminization was presumably accounted for the intersex fish. at present five different stocks of gynogenetic c. macrocephalus have been produced. sex reversal experiments are continuing dealing with lower doses of mt being administered to fry of different ages. a different kind of hormone, 11 b androstenedione is being used. thai silver barb or tawes (puntius gonionotus) being accepted among thai consumers, tawes produces an average annual production of more than 11 000 tons between 1987 1989 (department of fisheries 1991). inferior growth rate of males has been widely accepted which may be a result of their early maturing (4 months compared to 8 months in females). in normal rearing conditions females reach marketable size (500 700 g) within 8-12 months while males grow to only 300 g in the same period. thus, the production of sterile triploids, which could potentially have better growth than diploids at time of sexual maturation, may provide a practical solution to this problem. na-nakorn and legrand (1992) induced triploidy in tawes by subjecting fertilized eggs to cold shock (15°c). they found that cold shock applied immediately after fertilization 49 biotropia no. 6, 1992/1993 lasting 15 and 30 minutes resulted in 95.45 and 90.45% triploid fry, respectively, hatching percentage relative to control was 46.53 and 16.01% for shock durations if 15 and 30 minutes. triploid fish were sterile. however, growth of triploids and liploids has not yet been compared due to the small sample size. all female stock of tawes have been attempted by crossing sex-reversed male genetic female) with normal female as well as by inducing gynogenesis. sangsri 1991) reported sex-reversed tawes to maleness by oral administration of 17 alpha nethyltestosterone to 1 and 2 weeks old fry. dosages of mt used were quite high ^30 120 mg/kg feed) and numbers of sterile gonads were observed in the groups ireated for 30 days. some sex-reversed testes were histologically observed. treatment period of 60 days caused high proportion of sterile gonads. progeny testing of the presumed sex-reversed male is being performed at the department of aquaculture. stock of tawes with high percentage of females (98.7%) was produced by roongratri et al. (1992) using gynogenesis induction techniques. eggs were activated with uv-irradiated sperm of tawes 11.8 mjcm -2 ). at 1.5 minute after activation eggs were subjected to cold shock (2±0.1°c) for 10 minutes. a maximum survival rate of 61.3% of viable diploid gynogenetic offsprings (relative to control) was obtained. the gynogenetic offsprings will be sex-reversed to produce sex-reversed males which will be crossed with normal females to produce inbreeding-free all-female stock. crustaceans application of genetics to aquaculture of crustaceans is limited due to lack of techniques in broodstock rearing of most species such as penaeus monodon. however, population genetics of p. monodon in the gulf of thailand and the andaman sea was studied (sodsuk et al. 1992). this may provide useful information for genetic improvement programmes in the near future. in giant freshwater prawn (macrobrachium rosenbergii) which has been successfully cultured in central thailand, the genetic aspect of this species has recently been a matter of concern. meewan et al. (1992) estimated the heritability of carapace length of this species and reported h 2 -value of 0.04 ±0.22, 0.13 ±0.07 and 0.26 ±0.11 based on paternal, maternal and fullsib analyses, respectively. this study provided useful information for further selection programmes for fast growing stock, provided that carapace length and growth are positively correlated. 50 review of aquaculture genetic researches in thailand u. na-nakorn mollusks culture of marine mollusks in thailand is solely based on naturally produced seeds. hatchery seed production is limited at laboratory scale, thus limited genetic study of these species. however, genetic improvement of oyster has been carried out at the department of marine science, chulalongkorn university (jarayabhand and jindalikit 1992) attempting to produce a triploid small oyster, saccostrea cucullata. thermal shocks (4°c and 35°c) were applied to fertilized eggs 20 minutes after fertilization. results showed that shock duration of 6 minutes for cold shock and 6 or 15 minutes for heat shock gave an average 40% of triploids. growth and other economic important characters were not investigated. basic researches relevant to aquaculture cytogenetic researches especially karyotype studies of various fish species were conducted (tabel 1). unfortunately the application of the results was not discussed in most of the reports. biochemical genetic studies have been conducted in clarias spp. serum protein of c. batrachus and c. macrocephalus was analysed using sds-polyacrylamide gel electrophoresis. the electrophoresis patterns were found to be species-and sex-specific (jondeung and na-nakorn 1986). lawanyawut et al. (1992) conducted allozyme table 1. chromosome number of some fishes of thailand species chromosome no. (2n) references osteochilus hasselti 46 magtoon et al. (1988) puntius daruphani 50 magtoon et al. (1988) pangasius larnaudii 60 magtoon and donsakul (1988) p. sutchi 60 magtoon and donsakul (1988) p. gonionotus 50 na-nakorn and legrand (1992) clarias macrocephalus 54 donsakul and magtoon (1989) c. batrachus 100 donsakul and magtoon (1989) notopterus chitala 42 donsakul and magtoon (1990) n. blanci 42 donsakul and magtoon (1990) n. notopterus 42 donsakul and magtoon (1990) channa striata 44 donsakul and magtoon (1991) c. micropeltes 44 donsakul and magtoon (1991) c. lucius 48 donsakul and magtoon (1991) c. gachua 112 donsakul and magtoon (1991) 51 biotropia no. 6, 1992/1993 studies in c. gariepinus, c. macrocephalus and their hybrids. sixteen protein loci were resolved. three loci, pgi-2, ldh-1 and mdh-2, showed fixed differences between the parent species while the hybrids patterns were always heterozygotes. for gene transferring the growth hormone gene of the giant freshwater catfish (pangasianodon gigas), the world biggest catfish, has been sequenced, and cloned at the department of biochemistry, mahidol university (panyim personal communication). the gene has been transferred to bacteria and expression was observed. successful cloning of this gene encourages scientists to transfer the gene to economic fish species. future studies during the past 10 years aquaculture genetics was applied to only a few of completely domesticated species. genetic research and genetic improvement programmes are urgently needed to solve different problems occurring in different species, for example slow growth in sand goby (oxyeleotris marmoratus), inferior growth and less acceptability of male sepat siam, deterioration of growth of freshwater prawn, etc. intensive studies must be conducted in order to solve problems which cause a bottle neck in breeding and propagation of some economic species such as the problem of broodstock rearing in p. monodon or artificial breeding of snakehead (channa striata) etc. without these techniques genetic improvement of those species is impossible. genetic conservation of natural stocks must be emphasized. genetic stocks of wild fish are being destroyed due to intentional stocking of hatchery stock to natural water as well as unintentional escaping of hatchery stocks. genetic diversity is affected and hence, particular gene pool useful for genetic improvement programmes may be destroyed. in order to prevent undesirable genetic change, effects of introduction of new species in natural water have to be studied in the field before introduction. references department of fisheries. 1991. fisheries statistics of thailand 1989. fisheries statistics sub-division, tech. pap. no. 5/1991. dept. of fisheries, bangkok. 92 p. donsakul, t. and w. magtoon. 1989. a chromosome study of walking catfish, claries batrachus and c. macrocephalus in thailand. in: proceedings of the 27th kasetsart university conference: 426-428. kasetsart university, bangkok. (in thai). 52 review of aquaculture genetic researches in thailand u. na-nakorn donsakul, t. and w. magtoon. 1990. a chromosome study on three species of featherbacks, notop-terus chilata (hamilton), n. bland d'aubenton and n. notopterus (pallas), from thailand. in: proceedings of the 28th kasetsart university conference, 29 31 january 1990:459 466. kasetsart university, bangkok. (in thai). donsakul, t. and w. magtoon. 1991. a. chromosome study on five species channid fishes (channa, family channidae), from thailand. in: proceedings of the 29th kasetsart university conference, 4-7 february, 1991: 561-574. kasetsart university, bangkok. (in thai). jarayabhand, p. and j. jindalikit. 1992. chromosome manipulation in small oyster, saccostrea cucullata, by thermal shock : evidences from trochophore larvae (abstract). abstract of the international workshop on genetics in aquaculture and fisheries management, 31 st august 4th september 1992. asean-eec aquaculture development and coordination programme. university of stirling, scotland. jarimopas, p. 1988. thai red tilapia. thai fisheries gazette. 41 (1): 41 -43. jarimopas, p. 1989. realized response of thai red tilapia to 6 generations of size-specific selection for growth. in: final report, fish genetics project (phase ii) : 147 -164. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jarimopas, p., a. kumnan and j. wongchan. 1989. mass selection of clarias macrocephalus gunther for growth (3 generations). in: final report, fish genetics project (phase ii): 177 191. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jarimopas, p. and s. nukwan. 1989. evaluation on growth rate of selected strain thai red tilapia. in: final report, fish genetics project (phase ii) : 165176. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jondeung, a. and u. na-nakorn. 1986. analysis of serum proteins of clarias batrachus (linnaeus) and c. macrocephalus (gunther) by electrophoresis. the kasetsart j. (nat. sci.) 20 (1): 108 111. magtoon, w. and t. donsakul. 1988. karyotypes of pangasiid catfishes, pangasius larnaudii and p. sutchi. in: proceedings of the 26th kasetsart university conference, 3-5 february, 1988 : 239 244. kasetsart university, bangkok. (in thai). magtoon, m., u. tipsaena and y. taki. 1988. karyotypes of cyprinid fishes, osteochilus hasseltii and puntius daruphani. in: proceedings of the 26th kasetsart university conference, 3 5 february, 1988 : 235-238. kasetsart university, bangkok. (in thai). meewan, m., c.k. lin, s. tumwasorn and r.w. doyle. 1992. growth heritability of giant freshwater prawn (abstract). in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore: 81. na-nakorn, u. and a. lekhaanantakun. 1992. the effects of triploidy on survival, growth and feed conversion ratio of clarias macrocephalus. manuscript submitted to symposium on fish genetics and its application to aquaculture and fishery management, 8-10 december 1992, bogor, indonesia. na-nakorn, u. and e. legrand. 1992. induction of triploidy in puntius gonionotus (sleeker). fishery research bulletin no. 18. kasetsart university, bangkok. 10 p. na-nakorn, u., p. sidthikraiwong, w. tarnchalanukit and t.r. robert. 1992a. chromosome study of hybrid and gynogenetic offspring of artificial crosses between members of the catfish families clariidae and pangasiidae. manuscript submitted to environmental biology of fishes. 53 biotropia no. 6, 1992/1993 na-nakorn, u., s. chantsawang and w. tarnchalanukit. 1992b. response to mass selection for disease resistance in clarias macrocephalus. manuscript submitted to journal of applied aquaculture. na-nakorn, u., w. luangpromporn and r.a. dunham. 1992d. induction of gynogenesis in clarias macrocephalus eggs using irradiated sperm of pangasius sutchi and cold shock. paper presented in the third asian fisheries forum, october 26 30, 1992, singapore. na-nakorn, u., w. rangsin and s. witchasunkul. 1992c. suitable conditions for induction of gynogenesis in clarias macrocephalus using sperm of pangasius sutchi. paper presented in international workshop on genetics in aquaculture and fisheries management, 31st august 4 th september 1992, university of stirling, scotland. na-nakorn, u., w. tarnchalanukit and c. limsuwan: undated. genetic improvement of walking catfish (clarias macrocephalus gunther) for bacterial disease resistance. report submitted to the national research council of thailand. 163 p. (in thai). nukwan, s., m. tangtrongpairoj, k. lawanyawut and p. veerasidth. 1990. cross breeding between clarias macrocephalus and clarias gariepinus. in: proceedings of the 28th kasetsart university conference, 29-31 january, 1990: 553-567. kasetsart university, bangkok. roongratri, n., d.j. penman and t. viputhanumart. 1992. induction of diploid gynogenesis in java carp, puntius gonionotus (bleeker). (abstract) in: abstracts of the third asian fisheries forum. october 26-30, 1992. singapore : 83. sanosri, j. 1991. effects of methyltestosterone on sex reversal of puntius gonionotus. master thesis, kasetsart university. bangkok. 79 p. (in thai). sodsuk, s., b.j. mcandrew and d.j. penman. 1992. genetic population structure of the giant tiger prawn (penaeus monodon fabricus, 1978) in the gulf of thailand and the andaman sea. (abstract). abstracts of the international workshop on genetics in aquaculture and fisheries management, 31 august-4 september, 1992. asean-eec aquaculture development and coordination programme, university of stirling, scotland. tarnchalanukit, w. 1986. experimental hybridization between catfishes of the families clariidae and pangasiidae in thailand. env. biol. of fishes 16(4) : 317-320. uraiwan, s. 1989. aquaculture genetics improvement programs appropriate for thailand and other developing countries in asia. in: final report, fish genetics project (phase ii): 279-384. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. uraiwan, s., m. meewan and a. kumnane. 1989. within-family selection for increasing growth rate of oreochromis niloticus (linn.). in: final report, fish genetics project (phase ii): 26-36. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. uraiwan, s. and p. virasith. 1989. selection in thai oreochromis niloticus (linn.) by line performance growth test, (abstract) in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore : 81. uraiwan, s., r.w. doyle and m. meewan. 1992. evaluation of response to within-family selection of oreochromes niloticus (linn.) by line performance growth test, (abstract) in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore: 81. wongsangchan, a. 1985. broodstock selection and management study. technical paper in fish genetic (thailand) project. national inland fisheries institute, department of fisheries, bangkok. 36 p. 54 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf biotropia vol. 28 no. 3,2021: 231 238 doi: 10.11598/btb.2021.28.3.1344 growth inhibition of fungal plant pathogens by antagonist bacteria using dual culture assays yan ramona* ztchool $biology, facalg of mathematics and sciences, uniuersitas udqana " integrated laborat09 for biosciences and biotechnology, uniuersitas udqana received 6 april 2020 /accepted 26 july 2020 abstract there has been excessive use of chemical-based pesticides globally resulting in significant environmental consequences with adverse effects o n human health. therefore, more sustainable environment friendly alternative solutions are needed to counter such environmental and health challenges. development of biocontrol method is a sustainable solution to these challenges. the main objective of this research was to investigate the efficacy of potential antagonist bacteria to inhibit important fungal plant pathogens in uitro by applying dual culture assays o n potato dextrose agar f d a ) or trypticase soya agar (tsa) media, with a view to isolate and screen potential antagonists for development of future biocontrol agents. the target pathogens were fasariam o y p o r m , ceratoystis sp., apergilasj7au.w and apergillz~s niger, commonly found to infect horticultural plants in bedugul village in north bali island. the antagonist bacteria were isolated from various sources such as soil of rhizosphere zone and roots of lettuce plants and mature compost. the potential of antagonist candidates was screened o n the basis of inhibitory activity against targeted fungal pathogens. bacterial antagonists with highest zone of inhibition were identified up to genus level using biochemical tests, and the results were matched with those specified in the bergey's manual of determinative bacteriology. fifteen bacterial isolates were successfully isolated from various sources, and 60% of these isolates showed antagonistic activity in vitm against fungal pathogens with various degree of inhibition. this indicated the initial potential to develop as biocontrol agents. based on preliminary identification, genera of baciiias and psezldomonas were found to be the predominant isolates and in addition genus acinetobacter was also identified in this study. keywords: antagonistic, a.pergiilas $au.us, aspergiiias niger, ceratoystis sp., dual culture assay, fmrizlm 0 ~ ~ p 0 m / m introduction chemical-based pesticides have been used to control plant pathogens in farming practices for decades, and its application in agricultural sector into the future wdl continue in the short to the medium term (bueno e t al. 2017), despite the pollution challenges. these chemical-based solutions such as pesticides are used to protect agricultural crops from attack by fungi, insects, weeds, and rodents as well as lengthen the lifespan of farming products or prevent spoilage (i<umar e t al. 2012). application of chemical pesticides is also common in non-agricultural *corresponding author, email: yan-ramona@unud.ac.id sectors, such as sport facilities, shampoos for animals, and boats (nicolopoulou-stamati e t al. 2016). long term application of chemical based pesticides in agricultural and non-agricultural sectors has resulted environment and health impact in everyday life. the residue of such harmful compounds is now found in soil, waterways, agricultural crops, milk and meat causing human health challenges (kim e t al. 2016). the presence of these chemical contaminations in soil also frequently kills beneficial microbiota of soil, such as mycorrhiza and plant growth promoting rhzobacteria beyond the target pathogens. furthermore, increase in resistance of plant pathogens may occur as a result of excessive application of such biotropia vol. 28 no. 3,2021 chemical compounds in farming practices (lucas e t al. 2015). this may also result in increased dose or innovations in new pesticides that are needed to control the same pathogens (fernandes et al. 2010). other studies have also reported negative impacts of pesticide exposure to the consumer and community (andersson 2014). in terms of health impacts pesticides studies indicate neurological effects, diabetes, respiratory diseases, fetal diseases, genetic ksorders and cancer in humans (andersson 2014; iom et al. 2017). methyl bromide is an example of a widely used broad spectrum pesticide which was phased out in developed countries without an alternative (unep 2014). in addition it is claimed that these chemicals contribute to greenhouse gas emission (heimpel et ai. 2013). although above serious challenges exist, pesticides use in farming practices will continue and totally phasing out of such compounds will take time until better sustainable non-toxic alternatives are found (bueno et al. 2017). biological based sustainable technologies are being intensively researched towards the development of alternative methods to control plant pathogens worldwide. integrated pest management (ipm), including the use of biological control agents to control pests and pathogens is one of the most favorable and sustainable solution paths globally. in bali, particularly in bedugul village, crop rotation in -farming practices has been-applied for years. horticultural crops planted in this village include tomato, lettuce, cabbage, potato, and broccoli. at the time of sample collection, t h s farm was planted with lettuce plants, and therefore, the bacterial antagonists were isolated from soil in the rhizosphere zone and roots of lettuce plants from this area. in addition to these sources, we also isolated antagonists from vermi compost collected from a composting site in denpasar city where earth worms were used as starter in the process of composting. previous literatures for example in gehan e t a/. (2018) have reported that mature composts (including vermi compost) are rich in antagonists of fungal pathogens. based on the above rationale, the goal of t h s study focused on isolation, screening, and identification of potential of bacterial biocontrol agents to inhibit plant pathogens, with a view toward the development of biocontrol agents, so that chemical-based fungicide use in the future can be reduced. the primary objective of t h s study was to investigate the effectiveness of bacterial antagonist isolates to inhibit the in vitro growth of several fungal plant pathogens (fasam'am oqporam, cerato ystis sp., aspepiiias flavas, and aspegjills niger), commonly found to infect horticultural plants (including lettuce plants) in bali, particularly in bedugul area (suryanti et al. 2013) and neighboring villages of bedugul, including i<embang merta village (wulansari et al. 201 5). materials and methods fungal pathogens fungal pathogens (fasam'am oypomm, cerato ysth sp., a.pe@i/as yavzls, and ape@iias niger) were obtained from stock culture collections of the mycology laboratory, school of biology, universitas udayana, bali. these fungal isolates were identified in our previous study from samples collected in bedugul village (suryanti et ai. 2013). for regular use, these pathogens were subcultured on fresh pda. for long-term storage, they were grown for 48 hours at ambient temperature on potato dextrose agar, cut into 1 x 1 cm2, placed in sterile distilled water, and stored at 4 "c. sample collection and isolation of potential bacterial antagonists antagonists of plant pathogens were isolated from several sources, namely mature vermi compost (a composting site in denpasar city), soils sampled from rhizhosphere in lettuce farm in bedugul, bali, and rhizoplane (roots) of lettuce plants from bedugul, bali. isolation of bacterial antagonists from these sources was conducted by applying serial dilution and plating method on potato dextrose agar (pda) or trypticase soya agar (tsa). samples weighing 10 g were dissolved in saline solution (0.85% w/v nac1) and volume was adjusted to 100 ml to obtain dilution rate of lo-', and stomached for 20 minutes to release attached microbes on particles of soil or other samples. these samples were then further diluted in saline solution up to and 0.1 ml of sample from each dilution growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona was then spread onto either potato dextrose agar (pda) or trypticase soya agar (tsa), followed by incubation at ambient temperature for 24 -72 hours. colonies that could be distinguished appeared on media (at dilution rates of 10" following 48 hours incubation at ambient temperature and were streaked for single colony isolation to obtain pure culture isolates. these pure cultures were then suspended in trypticase soya broth (tsb) or potato dextrose broth (pdb) with 30% glycerol in the medium, and stored at -80°c (as stock cultures) until needed for further experiments. in vitro dual culture assay for screening potential antagonists isolates of bacteria obtained previously were spot inoculated in triplicates onto the periphery of pda or tsa plates (corresponding to the medlum on which they were isolated). plugs (1 cm2) of fungal pathogens were then placed in the center of the plates followed by incubation at ambient temperature or approximately at 25 "c for 1 to 7 days, after which time the inhibition zone between the antagonists and pathogens were measured from three different angles using a vernier calipers, and then averaged. three replications were made in the experiment to obtain representative data. isolates showing inhibition zone were subcultured on fresh pda or tsa and stored at 4 oc for further study. for long term storage, these isolates were stored in tsb with 30% glycerol and stored at -80 "c. preliminary identification of biocontrol agent candidates bacterial isolates showing in vitro antagonistic activity against tested plant fungal pathogens were preliminarily identified up to genus level on the basis of their morphological characteristics and specific selected tests (for soil bacterial identification) and the results were matched with those indicated in bergey's manual of systematic bacteriology (krieg & holt 1984; holt e t al. 1994). the tested characteristics of bacterial antagonist candidates were: gram stain and cell morphology, oxidative/fermentative properties, spore stain, motility, flagella stain, oxidase, urease, and catalase production, ability to hydrolyze casein, uv fluorescence, levan production, gelatin hydrolysis, casein hydrolysis, and starch hydrolysis. the procedures (preliminary identification of soil bacteria up to genus level) of all tests followed the steps specified in ramona (2003), suryanti e t al. (2013) and wulansari e t al. (201 5). results and discussion a total of fifteen (15) distinguishable bacterial isolates were successfully isolated from this study. of these, 60% of the isolates showed antagonistic activity in vitro against one or more tested fungal pathogens with various degree of inhibition (fig. 1 and table 1). rhizosphere zone of lettuce plants appeared to be the best source of potential antagonists. most bacterial antagonists isolated in this study were obtained from such zone and they showed various degree of in vitro inhibition of plant pathogens (table 1). surprisingly, only 2 isolates from lettuce roots inhibited the plant pathogen in vho. bacterial isolates obtained from vermi-compost inhibited the in vitro growth of all fungal pathogens used in the study with various degree of inhibition (table 1). it can be seen in table 1 that isolates a4, a5, t i , and t2 which were identified as baciiizts or p~eztdomonas (table 2 and 3) appeared to have the widest spectrum of in vitro inhibition. these isolates inhibited the growth of all tested fungal pathogens with various degree of inhibition, depending on the species of pathogens. these results provided us with initial indication of antagonists with potential to be developed as biocontrol agents. t h s can now be targeted for further greenhouse or field trial scale studies for validation and effective application. the inhibition zone produced by these bacterial antagonists against the targeted fungal pathogens varied between 0.86 f 0.11 cm and 3.20 f 0.00 cm (table 1). isolate a1 whch was identified to belong to genus of pseudomonas only inhbited the growth of fzt~aiztm o.yqbormm, with inhibition zone of 1.83 f 0.15 cm (table 1 and 2). the o r i p of this isolate was the rhzosphere zone of lettuce plants. the limited number of isolates obtained in t h s study (table 1, 2, and 3) was partly due to the primary focus on budding overall knowledge and basic tools related to in uitro cultivation of biotropia vol. 28 no. 3,2021 beneficial antagonistic microbes against fungal pathogens of horticultural crops and from this foundation then subsequently build a wider screening system for extensive isolation of biocontrol agents. previous reports indicated that an optimal optimized screening system is essential for in vitro cultivation from wider substrates (weller 1988) and this study advanced this objective. our results from this initial study were in line with malleswari (2014) and suthar e t al. (2016) who reported similar numbers of antagonist isolates from soil samples. in synthetic media, such as tsa or pda (considered as general media for bacteria and fungi, respectively) genera of bacillus, pmdomonas, and some genera belonging to enterobacteriaceae commonly grow well on such media and dominate during culturing. bacteria or fungi with specific or unidentified need will not show growth response on such media and has been reviewed previously by pham and kim (2012) and as suggested only 1% of soil bacteria may be culturable on synthetic media. therefore, there is a need for further improvement of methods as well as content and quality of synthetic media so that wider range of soil bacteria become culturable and therefore increase the probability to obtain potential isolates for biocontrol development. pham and igm (2016) proposed a novel method for bacterial cultivation by applying polycarbonate permeable membrane of trans well plates in liquid cultures with common synthetic media to mimic the actual environmental conditions. by applying such method they increased numbers of culturable isolates by more than 20%. such methods will be built on the foundations of this study in future strategies. the dual culture assays applied in our study (following isolation of antagonist candidates) appeared to be very convenient to initially indicate antagonism of bacterial antagonists against plant fungal pathogens. these results will have to be evaluated and correlated for their effectiveness in further greenhouse or field scales experiments. overall, this is a classical method and has been used effectively to isolate and initially screen bacterial antagonists of fungal plant pathogens in vitro. other studies recently applied this method to obtain bacterial antagonist candidates and subsequently identified the same to target fungal pathogens (suthar e t al. 2016; wang e t a l 2019); and manandhar e t al. 2019). this indicates that dual culture method is still a reliable approach to initially screen and identify antagonistic activity of a bacterial isolate against fungal or bacterial pathogens. digwe 1 some examples of ia .uitw antaganism between bacterial isolates obtained in this study and the tested pathogens. (a) p s 8 ~ h m o w sp. vs c ~ ~ k o p i f sp., @) ps81domona sp. vs aspergdihs niggt", (c)~ b~ilzfs sp. vs rfpqdii~@aas and (d). b(dn'&s sp. vs ffian'itrn o x ~ p m r n note: arrowheads show the inhibition zones between the bacterial antagonists and tested fungal pathogens. growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona table 1 average diameter of in vitro inhibition zones (on pda or tsa media) of bacterial antagonist candidates on several plant pathogens ( f m v i u m o q ~ o r z l m , ceratoystis sp., a.pergilusj7auas, and a.pegiiias niger) diameter of inhibition zones o n fungal pathogens (cm)* isolate codes source of antagonists f. oq.porzlm cerato ystis sp. a. j7auu.s a. niger a1 (pda) lettuce rhizosphere 1.83 f 0.15 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 a4 (pda) lettuce rhizosphere 1.06 f 0,06 2.20 f 0.10 1.50 f 0.10 1.60 f 0.11 a5 (pda) lettuce rhizosphere 3.20 f 0.00 2.46 f 0.1 1 1.33 f 0.11 1.30 f 0.10 a6 (pda) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 t i (pda) vermi-compost 0.96 f 0.06 1.80 f 0.06 1.13 f 0.11 1.13 f 0.11 t2 (pda) vermi-compost 0.86 f 0.11 1.83 f 0.06 1.26 f 0.06 0.90 + 0.10 s1 (tsa) lettuce rhizosphere 1.63 f 0.06 0.00 f 0.00 3.36 f 0.30 0.00 f 0.00 s2 (tsa) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 s3 (tsa) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 1.13 f 0.15 f1 (tsa) roots of lettuce 1.50 f 010 0.00 f 0.00 2.70 f 0.10 0.00 f 0.00 f2 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 f3 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r1 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r2 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r3 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 1.60 f 0.17 0.00 f 0.00 notes: * values in table 1 f standard deviation are averages of triplicates; pda (potato dextrose agar) and tsa (trypticase soy agar) were media used in the dual culture assays. the relative average inhibition zones shown in table 1 varied from 0.0 to 3.36 cm, and these apparently were affected by types and the origin of the isolates. the zone of inhibition was also determined by species of fungal pathogens tested. generally, bacterial isolates obtained from rhizosphere of lettuce plants was found to be antagonistic against one or more fungal pathogens tested in this study (table 1). similar results were also reported by ramona (2003) and wang et al. (2019) who found abundant antagonist isolates in the rhizosphere zones of various plants. this indicates that rhizosphere area can still be considered as a reliable source of bacterial antagonists of plant pathogens for development of biocontrol agents. in addition to rhizosphere zone, antagonists can also be isolated from many sources, such as mature compost or surface of plant roots (rhizoplane) (table 1). some researchers have also often isolated these antagonists from suppressive soil (schlatter e t al. 2017), suppressive compost (gehan eta/. 2018), or from contaminated media in the laboratory (ramona 2003). the origin of the antagonist isolates is not the most important aspect in the development of biocontrol agents. the effectiveness of these antagonists in protecting horticultural plants from attack by certain pathogens either in greenhouse or field applications is apparently more important consideration than their origins. to increase the effectiveness of antagonist isolations, ideally these antagonists should be isolated from places where these agents are to be applied, as they are already well-adapted in such areas of plant systems (tvtupps 2001; ganeshan & i<umar 2005; dukare e t al. 201 8). spectrum of fungal pathogen inhibition in vitro of the bacterial isolates obtained from this study varied among species (table 1). some isolates were able to control only one tested fungal pathogen, while the other controlled two or more pathogens (table 1). generally, bacterial antagonists isolated from verrni compost and rhizosphere zone of lettuce plants appeared to have wider spectrum of fungal pathogen inhibition in vitro when compared to those isolated from other sources. surprisingly, isolates obtained from rhizoplane (hair-root surfaces) of lettuce plants were found to be less effective in inhibiting the four fungal pathogens in vitro than isolates obtained from other sources (table 1). this phenomenon was contradictory to that reported by wei et a/. (1996) and walia et al. (2013), but in line with that reported by oka (2004). although some isolates in table 1 showed a wide spectrum of fungal pathogen inhibition, these isolates will have to be further b i o t r o p i a vol. 28 no. 3 , 2 0 2 1 tested to study their effectiveness when applied in glasshouse or field trials. ramona (2003) reported that a positive correlation between the results of in vitro bioassay and those of glasshouse or field trials may not be necessary. the reasons for these were extensively reviewed by whipps (2001) and o'brien (2016) and wider testing is essential following the screening for practical application. although observable inhibition zones were clearly indicated in the in vitro bioassay fable 1 and fig. i), the mechanisms by which bacterial antagonists may inhibit the fungal pathogens (for examples: antibiosis, siderophore production, or other mechanisms) was still inconclusive. besides that, the effect of this antagonism (whether it was lethal or fungi static) has not yet been answered and w d be considered in future studies. bacterial isolates with abllity to inhibit the growth of at least one tested fungal pathogen were initially identified up to genus level, based on the method of soil bacterial identification specified in ramona (2003) and suryanti e t al. (2013). the results are shown in table 2 and table 3. based on these results, the genus name of the isolates was determined. one of the pseadomonas isolates showed fluorescence properties under uv light, in addition to the results of other tests generally used in the initial preliminary identification of soil bacteria, and therefore, it was identified as p. flzlorescence (table 2). among the 9 bacterial antagonists, 5 isolates belong to baciiias sp. as all of them produce endospore and showed characteristics of genus baciiias as shown in table 3. p r e h n a r y identification of the isolates obtained from this study showed that genera of baciiias and pseadomonas were predominant bacterial isolates contained in all samples (table 2). this is in line with that reported by icumari and iulanna (2016) and soare e t al. (2019). the presence of these two bacterial genera in the soil or other samples is relatively more abundant than others and is partly due to simpler requirements for growth of these groups when compared to others (raaijmakers e t al. 2010). the ability of baciiius sp. to produce endospore also contributes to the survival of this genus in adverse environmental conditions (icumari & iulanna 201 6). besides ban'iias and pseadomonas, genus of acinetobacter was also isolated in this study (table 2). table 2 preliminary identification of gram-negative bacterial antagonists isolate gram flagella uv starch levan gelatin preliminary o / f motility oxidase pigment codes stain position fluorescence hydrolysis production hydrolysis identity a1 negative 0 + polar + + pseudomonas sp. a4 negative 0 + polar + + + p.~uorescence ti negative 0 + polar + + pseudomonas sp. s1 negative 0 nd + creamy acinetobacter sp. note: o/f: oxidative/fermentative. table 3 preliminary identification o f gram-positive bacterial antagonists isolate gram flagella starch casein preliminary o / f motility catalase pigment spore urease colony codes stain position hydrolysis hydrolysis identity a5 positive 0 + polar + + + + + dry baciiius sp. t2 positive 0 + polar + + + + + dry baciiius sp. s3 positive 0 + polar + + + + dry ban'iius sp. f1 positive 0 + + + + dry baciiius sp. r3 positive 0 + polar + + + + + dry baciiius sp. note: o/f: oxidative/fermentative. growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona conclusion nine out of 15 (60 yo) bacterial isolates showed antagonistic activities against several tested fungal pathogens in the in vitro dual culture assay, although the mechanisms by which these biocontrol candidates inhibit the fungal pathogen is still inconclusive, and therefore further study is needed to specifically elucidate the mechanisms (such as antibiosis, production of siderophore, etc.) of biocontrol. in the initial and preliminary identification of these antagonists, it was found that genera of baciiius and p~eudomonas were predominant isolates found in all samples. besides these, genus of acinetobacter was also isolated and identified in this study. acknowledgements the author wishes to acknowledge laboratory of mycology, school of biology, universitas udayana for the provision of cultures of fungal pathogens. the laboratory of illicrobiology, school of biology, universitas udayana, and the integrated laboratory for biosciences and biotechnology, universitas udayana should also be acknowledged for the provision of facilities and equipment during this project. special gratitudes also go to prof i<alidas shetty (ndsu-usa) for his proofreading and criticism on this manuscript. references andersson h, tago d, treich n. 2014. pesticides and health: a review of evidence o n health effects, valuation of risks, and benefit cost analysis. advances in health economics and health services research. p.1-61. bueno adf, carvalho ga, dos santos ac, 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(root special issue):487-511. wulansari n t, ramona y, proborini mw. 2015. identification of xantbomonas campestns isolated from rhizosphere zone of broccoli farm at icembang merta village, tabanan-bali. jurnal metamorfosa vol ii(1):29-33. biotropia vol. 28 no. 3,2021: 214 220 doi: 10.1 1598/btb.2021.28.3.1333 effectiveness of indigenous endomycorrhizal biofertilizer prototype o n organic salak leaves a n d fruits i n bali i nyoman rai i*, i i(etut suada', i wayan wiraatmajal and ni icomang alit astiari2 'stuaj program ofagroecotechnology, faculty afagriculture, uniuersitas udgana, kampzls unud bukit jimbaran, kuta selatan, badung 80361, bali, indonesia * s t u h program ofagrotechnology, faculty ofagriculture, uniuersitas warmadewa, denpasar 80235, bali, indonesia received 14 february 2020 /accepted 1 july 2020 abstract organically cultivated salak (salacca xalacca) o n dry land has limited production in bali. typically, fertilization is carried out using leaf litter or other organic fertilizers because soil fertility is low for salak plantations. the present study analyzed the effectiveness of an indigenous endomycorrhizal biofertilizer o n the nutrient and total carbohydrate content of salak leaves and fruits. the study used a randomized block design with nine replicates. the treatment consisted of three levels, i.e., 1. fertilization with leaf litter only, as is practiced by the farmers and as control (c); 2. fertilization with indigenous endomycorrhizae biofertilizer prototype (p); and 3. combined fertilization with leaf litter and indigenous endomycorrhizae biofertilizer prototype (pm). spores of indigenous endomycorrhizae from salak plantations were used for making a biofertilizer prototype. the results showed that p and cp treatments provided beneficial results such as: 1. significantly increased the chlorophyll, relative water content of leaves, as well as the number and weight of fruits per tree; 2. improved fruit quality by increasing sweetness and weight per fruit; and 3. had a positive effect o n water uptake and nutrient absorption as indicated by high n and p of leaf tissue and high carbohydrate content of leaves. keywords: biofertilizer, endomycorrhizae, organic, prototype, salacca xalacca introduction bali province is a major tourist destination in indonesia. increasing the number of domestic and international tourists leads to an increased demand for fruits, including organic salak fruits. salak cultivation in bali has been carried out organically since the 1500s and is hugely beneficial to the farmers; however, the quantity, quality, and continuity of organic salak fruit produced is low (rai e t al. 2014). this low productivity can be attributed to the nature-dependency of the farmers, who have not yet implemented adequate agricultural practices. typically, fertilization only uses leaf litter, while irrigation relies only on rainfall. thus, the ferulization of organic salak renders low soil fertility thereby decreasing the plant productivity *corresponding author, email: rainyoman@unud.ac.id over time (rai e t al. 201 4). rai e t al (201 0) found that the nutrient content of n, p, and i< in the leaf tissue of organic salak in bali was low; also, the soil fertility was low as indicated by the levels of c-organic and the content of n, p, and k in the soils. in order to maintain the soil fertility and environmental sustainability for high organic salak productivity, the cultivation should be carried out with indgenous endomycorrhizae biofertilizer based on the fact that the diversity of indigenous endomycorrhizae fungal species in nature is very large paslam e t al. 201 1; proborini 2013; suamba e t al 2014; sarah & ibrar 2016; invam 20 1 7). igm e t al. (2017) established a close correlation between soil, plants, and endomycorrhizae. indigenous endomycorrhizae isolated from plants in certain locations, will be more effective if applied directly to the target plant concerned where it is taken as compared effectiveness of indigenous endomycorrhizal biofertilizer prototype o n organic salak leaves rai e t all to the endomycorrhizae brought from outside (non-indigenous endomycorrhizae) . the arbuscular mycorrhzal symbiosis between plants and fungi exerts a positive impact on improving the soil structure, expanding the water absorption areas, increasing the plant tolerance to biotic and abiotic stresses, increasing the nutrient uptake, and increasing the plant growth and yield (nikhat 2014; olagunju et al. 2014; soka & ritchie 2016). in addition, endomychorrizae increases plant tolerance to various biotic and abiotic stresses, including alkalinity and metal toxicity (abbasi et al. 2015; bitterlich et al. 2018). the endomycorrhizae fungus whch is also called arbuscular mycorrhizae is beneficial to the host plants as it plays a role in increasing nutrient absorption through the endomycorrhizal structures that form large root surface areas so that the plant roots can absorb the nutrients (baslam et al. 2011; sasvari 2012; brundrett & tedersoo 2018). endomycorrhizae plays a role in increasing plant resistance to root pathogen attack through antibiotics produced during symbiosis with plant roots that weaken and also kill the pathogenic bacteria, viruses, and fungi (brundrett 2017; sasvari, 2012). furthermore, the diversity and activity of indigenous endomycorrhizae in plant roots are mainly determined by the type of the host plant (ningsih et al. 2013; sadhana 2014), environmental growth factors, such as climate and soil moisture/drought (hernadi 2012; quiroga et al. 2017; mathimaran et al. 2017; i<ehri et al. 2018), and the level of soil fertility (tahat & sijam 2012; kavitha & nelson 2013; mo et al. 2016). rai et al. (2019a) explored and identified morphologically and genetically the indigenous endomycorrhizae from salak root areas in three regencies in bali: icarangasem, gianyar, and tabanan. in our study, two genera of indigenous endomycorrhizae in salak were identified i.e., glomzls and entrophospora. the genus glomzls consisted of three species (glomzls cabens, glomzls czlstos, and glomzls indicum) , while genus entrophospora consisted of only one species (entropbospora-sp-sh 197095.06fu). after propagation, the spores of these species were formulated into prototypes of biological ferulizers/bioferulizer with carrier medla zeolite, quartz sand, sea sand, and volcanic sand. the most effective method to be applied in salak seedling is a prototype composed of a mixture of 100 spores from glomzls species in 500 g volcanic sand carrier media. this phenomenon was shown by the optimal growth of salak seedling and the maximum (100%) colonization of endomycorrhizae at the roots (rai et al, 2019b). positive test results on salak seedling growth need to be followed up by evaluating the efficiency of the prototype of indigenous endomycorrhizae biofertilizer on organic salak plantations in the field. thus, the present study aimed to determine the effect of prototype indigenous endomycorrhizae biofertilizers on organic salak production and its effect on the nutrient and carbohydrate content of the leaves. materiaj3 and methods the study was conducted from february to december 2019 in organic salak farms owned by farmers in sibetan village, bebandem district, icarangasem regency. the study used 15-year old salak trees and a randomized block design with nine replicates. three levels of fertilization were applied as follows: 1. fertilized with leaf litter only, as is practiced by the farmers and as control (c); 2. fertilized with prototype indigenous endomycorrhizae biofertilizer 0; and 3. combination of leaf litter and prototype indigenous endomycorrhizae bioferthzer (cp) . therefore, 27 salak trees with a total plot size of 216 m square were needed. the spores of indigenous endomycorrhizae were taken from salak roots in sibetan village. the isolation of the spores was carried out using the wet filtering technique, followed by centrifugation as described by brundrett (2017). these spores were propagated according to the method described by rai et al. (201 8), using corn as a host plant with water stress treatments. topping or shoot cutting was carried out at the age of 4 weeks. watering to field capacity was carried out from the beginning of seed planting to 1 week of age, followed by watering to 50% of field capacity until 4 weeks of age. after topping at the end of the 4th week, watering was stopped so that the plant experienced stress. then, the plant was dismantled to harvest the spores from the multiplication at the end of the 5th week. subsequently, the resulting spores were placed on volcanic sand: 100 spores/500 g sand. biotropia vol. 28 no. 3,2021 fertilization was conducted by making a hole, 30 cm wide and 20 cm deep, around the tree at a distance of 40 cm from the base of the tree. the salak trees in treatment c were fertilized with 4 kg leaf litter of salak/tree. the leaves were cut into 20-cm-long pieces, placed in a hole, and covered with soil. o n the other hand, the trees in the p treatment were fertilized with prototype indigenous endomycorrhizae made from a mixture of 100 spores of three glomas species (glomas cabense, glomtls czlstos, and glomas indicum) in 500 g volcanic sand carrier media per plant. the prototype of the biofertilizer was evenly spread around the tree and covered with soil. interestingly, the cp treatment was carried out by spreading a prototype of the biofertilizer made from a mixture of 100 spores of three glomzts species in 500 g of volcanic sand carrier media per plant, followed by spreading the salak leaf litter on it evenly around the tree. the chlorophyll content and the relative water content (rwc) of the leaves, the percentage of fruit set, the number and weight of fruits per tree, weight per fruit, fruit diameter, fruit sweetness level, n and p content of the leaves, and total sugar, reducing sugar (r-sugar) and sucrose content of the leaves, and root colonization by indigenous endomycorrhizae were recorded. the colonization of roots by indigenous endomycorrhizae was observed by the slide method of giovannetti and mosse (1980) using the following formula: percentage of colonized roots = the number of colonized roots/the total number of roots observed x 100%. the percentage of fruit set was calculated as follows: the number of flowers developed into fruits/the number of flowers x 100%. the leaf chlorophyll content measured three times by chlorophyll meter spad-502 in april, june, and august, and the average was calculated. the total sugar was analyzed by anthrone method, r sugar by nelson-somogyi method, and the sucrose content was calculated by subtracting the value of total sugar content from that of r sugar, and multiplied by 0.95. the rwc was measured from matured leaves. after being cut from the tree, the mature leaves were then immediately wrapped in aluminium foil, stored in an icebox, and transported to the laboratory for further analysis. thirty pieces of leaf samples (10 pieces from the tip of the leaf, 10 pieces from the middle, and 10 pieces from the bottom), with a diameter of 1 cm, were taken from leaf sheaths using a round punch and weighed ( w i ) . these leaf samples were immersed in water and irradiated with 40 w fluorescent light at room temperature for 5 h. then, each piece of leaf sample was carefully dried with paper towel, and weighed (wz), followed by oven-drying at 70 oc for 24 h before being estimated ( w 3 ) . the value of rwc was calculated by the formula: p ~ w z ) / (w3-wz) x 100%. the nutrient content of n and p leaves was analyzed by kjeldahl method and the p-availability was analyzed by olsen method. the number and weight of fruits per tree were calculated cumulatively at the end of the study. moreover, the weight of each fruit was calculated as follows: total weight of fruit per tree/the number of fruits per tree. these data were analyzed using analysis of variance with spss (statistical product and service solution) software version 26. if the f test showed a significant difference, the least significant difference (lsd) test was performed to differentiate the average value among tretaments. results a n d discussion the results of the analysis of variance (anova) showed that the fertihzation treatment significantly affected all the observed variables. the weight and number of fruits per tree (790.80 g and 16.81 fruits, respectively) in the treatment of indigenous endomycorrhizae biofertilizer prototype (100 spores/500 g volcanic sand carrier me&a) were sipficantly hgher than those fertilized with leaf litter/control (653.81 g and 14.94 fruits, respectively), but were not significantly different from cp (790.14 g and 16.56 fruits, respectively) (table 1). the quality of salak fruit in the p and cp treatments was significantly improved as compared to that in treatment c, as shown by the increased sweetness of the fruit (table 2) and the weight per fruit (table 1). the high number and weight of fruit per tree, as well as the increased fruit quality due to the administration of indigenous endomycorrhizae biofertilizer prototype, elevated the n and p content of the leaves as compared to the control (table 2); also, the chlorophyll content and rwc of the leaves were increased (table 1). visually, the trees treated with c and cp treatments had dark green and fresh leaves as compared to the control. effectiveness of indigenous endomycorrhizal biofertilizer prototype on organic salak leaves rai e t al: table 1 effect of prototype indigenous endomycorrhizae biofertilizers on the leaf chlorophyll content, rwc of leaves, number and weight of fmits per tree, weight per fruit, and fruit sweetness in salak plants leaves chlorophyll leaves relative number of weight of weight per fruit treatments content water content fruit per tree fruit per tree fruit sweetness (spad) ("0) (unit) 0 0 (% brix) lsd 5% 0.67 0.98 1.59 96.34 7.27 0.51 note: numbers followed by the same letter in the same column did not differ significantly in the lsd 5% level. higher production and yield quality in p and cp treatments as compared to control (c) were related to h g h leaf chlorophyll content (table 1) and the ability of salak tree to absorb nutrients and water (table 2). the high p and n content in the leaves resulted from the p and cp treatments was significantly associated with root colonization as compared to the c treatment. table 2 displayed that root colonization by indigenous endomycorrhizae in the p and cp treatments was 100%, but only 23.14% in treatment c. the high root colonization in p and cp revealed a mutualism symbiosis between indigenous endomycorrhizae and salak trees, which in turn, increased the p and n content and the rwc in the leaves. the endomycorrhizae infects the roots of plants but does not cause injury, whch in turn causing mutual reciprocal processes. host plants obtain nutrients from endomycorrhizae, while endomycorrhizae obtain carbohydrates or food from host plants (hernadi e t al. 2012; zasvari e t al. 2012). h ~ g h nutrient content and rwc in the leaves resulted from the p and cp treatments increased the chlorophyll content, which improved the photosynthesis as indicated by elevated total sugar, r-sugar, and sucrose content in the leaves resulted from the p and cp treatments as compared to that in c (table 2). the present study showed that indigenous endomycorrhizae fungus isolated from root of salak trees is suitable to be used as a biofertihzer. the administration of indigenous endomycorrhizae resulted in the absorption of nutrients and water through the endomycorrhizae structure that enlarges the surface area of salak root, so that the plant roots can absorb the nutrients and water (baslam e t al. 201 1; zasvari 2012; brundrett & tedersoo 2018, bitterlich e t al. 201 8). spore density and root colonization of host plants are largely determined by the compatibility of endomycorrhizae with host plants, environmental factors, and interactions between endomycorrhizae and chemical compounds produced by host plants (beltrano e t al. 2013; sarah & ibrar 2016). thus, a correlation between indigenous endomycorrhizae biofertilizers prototype and salak trees can be suspected, which increases the yield, the quality of the yield, and the physiological processes of salak trees. the results of this efficiency test are in accordance with those of previous studies wherein endomycorrhizal fungi biofertilizer can increase the growth, production and quality of pineapple yield (nurhandayani e t al. 2013), chill (tanwar e t al. 2013), teak (proborini, 2013), tea (nepaleon e t al. 2012) and apple (fediala e t al. 2018). endomycorrhizae plays a role in increasing plant resistance to drought or lack of water in the dry season because the root of the plants possess mycelium that can reach water in the wider rhizosphere area of the soil and adsorb water despite h t e d availabihty (sasvari 2012). furthermore, it is involved in producing various growth regulators such as auxins, cytokmins, and gibberellins and vitamins that can increase the growth of plant organs and roots that do not rapidly age and hence can function effectively in the absorption of nutrients and other solutes (baslam e t al. 201 1; tanwar e t al 2013). the endomycorrhizae also improve the soil structure because the mycelium on the outside of the roots of plants (covering soil grains) produces polysaccharide gels that increase the stability of soil aggregates (io-uger 2011; sasvari 2012; sadhana 201 4; jansa e t al. 201 6; i<lm e t al. 201 7). biotropia vol. 28 no. 3,2021 table 2 effect of prototype indigenous endomycorrhizae biofertilizer application on the n and p nutrient, total sugar, r-sugar, and sucrose content of the leaves and root colonization on salak plants total sugar r-sugar n content p content of of sucrose content root content of treatments of leaves leaves of leaves colonization leaves ("4 ("/o) leaves ("10) ("10) ("10) lsd 5% 0.03 0.01 0.51 0.22 0.79 3.16 note: numbers followed by the same letter in the same column did not differ significantly in the lsd 5% level. although c was not given indigenous endomycorrhizae bioferulizer, root colonization occurred albeit with significantly lower intention as compared to that in p and cp treatments. the data showed that indigenous endomycorrhizae were naturally present in salak root area; however, for a positive influence on the production and quality of salak, the population needed to be increased by giving indgenous endomycorrhizae biofertihzer for the optimal production of the plants. the indigenous endomycorrhizal fungi, also known as arbuscular mycorrhizal fungi, in nature is diversified (baslam e t al. 2011; proborini 2013; suamba e t al. 2014), with a positive effect on the salak trees (juliadewi e t a/. 2014). indigenous endomycorrhizae is naturally associated with plant roots without human intervention and has a high potential for extensive colonization because it recognizes the host plant and has a higher tolerance to environmental conditions, such as high stress (sadhana 2014; quiroga e t al. 2017). based on these characteristics, it can be speculated that indigenous endomycorrhizae isolated from salak roots has great potential to be developed as biofertilizer because it adapts to host plants to be fertilized (sasvari 2012; sadhana 2014; hohmann & messmer 2017). conclusions acknowledgements our gratitude goes to universitas udayana for financial support through udayana invention grants in 2019. the authors also thank the directorate general of higher education, ministry of research, technology and higher education for financial support through the higher education applied grants 2018. references abbasi, hisamuddin, akhtar h, sharf r. 2015. vesicular arbuscular mycorrhizal (vw fungi: a tool for sustainable agriculture. amer j plant nutr fert techno 5(2):40-9. baslam m, garmendia i, goicoechea n. 2011. arbuscular mycorrhizal fungi (amf) improved growth and nutritional quality of greenhouse grown 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kadian n, gupta a. 2013. arbuscular mycorrhizal inoculation and super phosphate application influence plant growth and yield of capsicum annuum. j soil sci plant nutr 13(1):55-66. biotropia vol. 28 no. 2, 2021: 173 180 doi: 10.11598/btb.2021.28.2.1296 173 environmental stress on the reproduction of non-human primates mashitah shikhmaidin*,1,2, nur hafizah mohammed1 and ahmad ismail1 1department of biology, faculty of science, universiti putra malaysia, 43400 upm serdang, selangor, malaysia 2institute of tropical agriculture and food security (itafos), universiti putra malaysia, 43400 upm serdang, selangor, malaysia received 9 january 2020/accepted 29 may 2020 abstract this paper aims to review some highlights on the effects of environmental stresses on the non-human primate population, particularly, climate change and food limitation that may have resulted in their poor reproductive performance. the international union for conservation of nature (iucn) lists more than a third of the world’s primates as critically endangered or vulnerable. non-human primates, which are the closest biological relatives of humans, are threatened with extinction from human activities and environmental stress. deforestation is the main problem that intercalates with climate change. either, indirectly or directly, those extinction factors could interrupt the physiological basis of reproduction among non-human primates. researches on other species showed that high ambient temperature causing heat stress had harmed the reproductive performance by interfering with the hypothalamic-pituitary-gonadal axis. therefore, the survival, conservation and sustainability of nonhuman primates growing in captivity and in the wild, require more works and researches to be done. keywords: climate change, conservation, heat stress, primate, reproductive introduction the non-human primate populations are increasingly endangered as they experienced warnings and threats from abrupt climate changes that slowly result in their population extinction. the intergovernmental panel on climate change (ipcc) has warned about the impacts of the escalating global temperatures. moreover, global warming will continue even though it is not regionally uniform and the nations’ potential for adapting to this situation is limited in terms of protecting the natural systems. this situation is more complex than just rising temperature because climate change is an environmental stress causing changes in the body physiological metabolism in a short-term to long-term fight-or-flight response (schulte 2014). the intense increases in humidity and ambient temperature had resulted in animals and plants being exposed to adverse circumstances that threaten their survival, and ultimately, their biodiversity. climate change would directly or indirectly cause reduction and shifting of wildlife populations and habitats, including the nonhuman primates, thereby eventually inhibiting the survivability and sustainability of the species. most primates are widely distributed throughout the tropical and subtropical regions of africa, america and asia. the majority of primates live in tropical areas that are rainfed and rain forested (reed & fleagle 1995), but they also exist in tropical dry forests, mangrove vegetation above high-tide levels, savannas, grasslands, inland wetlands and rocky areas (mittermeier et al. 2013). habitat for this group of animals is widespread but some of the areas are no longer able to accommodate these primates’ populations, as the areas have been mostly disturbed by human activities. about 80% of the malaysian rainforests are degraded *corresponding author, email: mashitah@upm.edu.my biotropia vol. 28 no. 2, 2021 174 by logging and largely covered with oil palm plantations (sophie 2013). logging had threatened the biodiversity of those areas, including the nonhuman primate populations. deforestation is also a result of human overpopulation happening in some places in the african and asian countries. the populations of nonhuman primates are disturbed by human population growth, economic expansion and other human activities, such as those in the agricultural industry. the significant thermal impact that obviously elevated the effects of climate change has led to phenological changes in the wildlife habitats. extreme irregularities in phenological events have also resulted in shifting food sources that has changed many natural cycles of pollinators and plants, predators and prey, and of primate and their food sources (visser 2008; both et al. 2009; butt et al. 2015). all these disturbance factors threatening the nonhuman primate species have forced them to move out of their habitat range to get clean water, food, and to raise their offspring. these external factors, such as climate change, demanded an adaptive response from the animals to a new environment (altmann et al. 2002; beehner et al. 2006a; estrada et al. 2017; fairbanks et al. 2011). the critical challenge facing the global population of nonhuman primates is therefore, survival or extinction, because as animals lose the ability to control their environment (for examples, habitats lost and climate change), their physiological structure ultimately change. moreover, it could cause adverse effects on their reproduction and development. this paper briefly reviews and discusses the impact of physical disturbances such as feed restriction and climate change on the reproductive physiology as these factors slowly create stress affecting reproduction and eventually, the survival of the nonhuman primates. review does climate change affect the reproductive performance of nonhuman primates? in the last few decades, the effects of environmental stress to animals, mainly climate change, has become a major concern. however, still sparse works are done on the sustainability of wildlife, particularly the nonhuman primates, in this complex stress phenomenon. many researches showed that continuous increase of the ambient temperature resulting in heat stress had significantly affected the reproductive physiology of animals, including nonhuman primates (beehner et al. 2006ab; sharpe, 2010; rylander et al. 2013; wang et al. 2014). an acute increase of ambient temperature generally affect the breeding patterns of animals through physiological changes in the brain, the center of the nervous system. studies among livestock animals showed that extremely high temperatures strongly affected the hypothalamic-pituitary-gonadal axis (shelton & huston 1968; wolfenson et al. 2000; santolaria et al. 2014). these deleterious effects of heat stress could lead to disruptions in the reproductive performance of nonhuman primates. about 419 primate species, such as lemurs, lorises, tarsiers, monkeys and apes, would experience 10% more warming than the global average (taylor 2016). meanwhile, some primates would experience increases of more than 1.5 oc in annual average temperature for every degree in global warming. therefore, primates that show high sensitivity to little changes on ambient temperature could experience more intense effects in their reproduction as compared to other primates. the consequence of climate change on the reproductive performance of nonhuman primates not only occurs in females but also in males. a 43 ˚c heat stress of about 30 minutes could induce apoptosis of germ cells in the testis of macaca fascicularis, a non-human primate, (zhang et al. 2005). this situation was described in studies where suppression of testicular function under heat stress had led to a decrease in fertility in ruminants (hamilton et al. 2016), and humans (garolla et al. 2013; rao et al. 2015). those testis exposed to high ambient temperature could constantly effect the dna fragmentation of sperms; thus, reduces the quality of sperms. an elevated surrounding temperature causing a rise in testicular temperature result in a reduction in sperm output, low sperm motility and an increase of morphologically abnormal spermatozoa (hansen 2009). germ cell depletion and increase in dna damage are induced by heat stress (lue et al. environmental stress on reproduction of nonhuman primates – shikhmaidin et al. 175 1999; paul et al. 2008). oxidative stress clearly disturbs the functionality of sperm and leads to sperm damage, deformity, and eventually, male infertility (halliwell & gutteridge 2007; mahfouz et al. 2010). animals vulnerable to environmental changes, particularly heat stress, also exhibited changes in their mating behavior (deutsch et al. 2008; mcfarland et al. 2014). in female, heat stress had significant adverse effects on the reproductive performance particularly, delayed estrus cycle, lower embryo survivability, slow fetus development and early pregnancy abortion (beehner et al. 2006b; hansen 2009; wiederholt et al. 2011). documented information on the effects of these environmental stressors towards reproduction patterns of the female and male nonhuman primates is still sparse. hence more studies are needed on understanding the impact of heat stress or climate change particularly on reproductive-physiology and reproductive behavior. identifying anthropogenic stressors on nonhuman primates is also essential for conservation planning and management of these primates (graham et al. 2016). importance of nutrition and reproduction the overall functions of the reproductive system are largely controlled by the hormonal interactions of the hypothalamic-pituitaryovarian axis, but the final output also depends on extrinsic factors such as nutrition. the diet that is easily digested and rich in fibre, carbohydrates and lipid from fruits, leaves, buds and insects, constitute an immense food source for most nonhuman primates (strier 2007; lambert 2010; moges et al. 2014; kassim et al. 2017). however, habitat fragmentation and climate change could harm food availability. captive orangutans fed with high-quality feeds have shorter inter-birth intervals as compared to wild orangutans (kuze et al. 2012). therefore, with a good quality food sources, the orangutan population will result in an increased reproductive performance and eventually increased population density. food availability and nutritional composition are also important to the population density of nonhuman primates (hanya & chapman 2013). two factors that contribute to pregnancy failure in females are the inadequate food intake and increased energy expenditure. it is important for pregnant females to meet the basic metabolic requirements mainly during early pregnancy to avoid negative energy balance. studies done on the nutritional status and reproduction of laboratory and livestock animals recorded that underfed animals experienced delayed puberty period, decreased embryo survival rate, suppressed spermatogenesis and reduced number of sperm motility. moreover, females that were deprived of food or underfed were less fertile and this reduces the chances of fertilisation; thus, affecting the population birth rate and eventually causing a decrease in population. this probably occurs severely among subordinate females of a nonhuman primate. dominant females of marmoset monkey produced more offsprings as compared to subordinate females in a healthy environment (abbot 1987). in the physiological level, dominant females can defeat the reproductive performance of subordinate females. the impact of a threatening activity, such as forest abuse, can damage the individual survival of nonhuman primate species, decrease fertility, and will shrink the population through time. climate disaster also leads to the extinction of plants and animals. a study on wild baboons documented that pregnancy failure was high if the females conceived after the drought (beehner et al. 2006b). during the 1990s, the decline in the orangutan population by at least 30% of its total population was caused by fires and drought in the bornean forests (gould et al. 1999). in addition, wildfires in mexico also affected the nonhuman primates, whereby the habitats of new world monkeys were destroyed by drought and el nino. reduction of food availability as a result of el nino event caused the population size reduction of atelines one year after the event (wiederholt & post 2010). extreme temperatures can decrease the food production and drinking water availability of the existing nonhuman primates. high ambient temperature could decrease the concentration of fibre and protein contents of leaves due to high atmospheric carbon dioxide. in addition, a rising level of heat and carbon dioxide could make the leaf or green resources less nutritious (gray & brady 2016) and could also affect the plant physiology resulting in changes in the number and size of leaves (taylor et al. 2003; dermody et biotropia vol. 28 no. 2, 2021 176 al. 2006). higher temperatures also increased the plant matter toxicity resulting in reduced leaf sizes (moore et al. 2015). decreasing food source could also increase the competition within the population. in the long run, this indirect effect might impact the evolutionary and ecological processes of a certain population. obviously, several factors contribute to food limitation in nonhuman primate habitats, such as forest exploitation, forest clearing for agriculture and climate change. as mentioned earlier, nutritional restriction can interrupt the breeding pattern, fertility, puberty development, embryo growth and development, and can increase susceptibility to disease and predation, intensify mortality of infants and mothers, and can delay reproductive maturity to a much later age (korstjens & hillyer 2016; chaves et al. 2019; laver et al. 2020). the synchronization of reproductive events of primate to fit the availability of the resources was also affected (van schaik et al. 1993; post 2013). climate change occuring in the african regions has cause significant changes in fruit production, as it was lower between 1988 and 1993 than between 1994 and 2003 (erhart and overdorff 2008). this event negatively affected the red-fronted lemurs (eulemur fulvus rufus), specifically their reproductive patterns, sex ratio, group sizes and population density. early embryo wastage, one of the key factors towards species extinction, is a particularly critical issue for nonhuman primates,. the nonhuman primates in captive management may need to refine the feed intake that would satisfy the dietary needs and take into account to have an optimal reproductive functions. reproductive and stress hormones on reproduction it is well-known that progesterone is the main reproductive hormone that is essential to maintain pregnancy in animals, including in nonhuman primates and humans. meanwhile, testosterone is important in mating behaviour and confounded with dominance rank in male primates (dixson 1998; wallen & zehr 2004). expressions of reproductive hormones depend on maternal responsiveness with the environments and differ between young and experienced female primates (rangel‐negrín et al. 2009; saltzman & maestripieri 2011). the understanding of neuroendocrine and primate maternal behaviour has increased, but of the physiology mechanism underlying the effects is still limited. during early pregnancy in nonhuman primates, progesterone secretion is solely from corpus luteum and it is replaced by the placenta during mid-pregnancy (beehner et al. 2006a). an in vitro study on macaque recorded the direct actions of progesterone and oestradiol on primate pre-antral follicle development (ting et al. 2015). androgens appeared to be a survival factor but hindered antral follicle differentiation; oestradiol appeared to be a survival and growth factor at the pre-antral and early antral stages, whereas progesterone may not be essential during early folliculogenesis in primates. macaque and baboon are nonhuman primate models used for studies related to human reproductive health, such as contraception, reproductive aging, infertility, ovarian function, and reproductive tract disorder (wandji et al. 1997; d'hooghe et al. 2004; ting et al. 2015). maternal stress during pregnancy that is controlled by the hypothalamus–pituitary– adrenal (hpa) axes could produce disruptive effects on embryo and fetal growth and development (del giudice 2012; laver et al. 2020). among wild baboons, food limitation due to drought contributed to fetal loss in the amboseli population (beehner et al. 2006b). a study on orangutans in southeast asian tropical forests showed that solitary lifestyle is relative to late weaning which is a consequence of their ecological environment (vogel et al. 2015). faecal glucocorticoid (fgc) is a survival probability indicator of many nonhuman primates, such as ring-tailed lemurs and redbellied lemurs. it shows high mortality rate due to habitat degradation that would cause reduction in fruit availability (pride 2005; tecot 2013; balestri et al. 2014). the non-invasive technique was used to measure reproductive hormones and cortisol through urine and faeces during a normal reproductive cycle (rangel-negrín et al. 2009). chaves et al. (2019) used non-invasive biomarkers, fecal glucocorticoid metabolites to assess the physiological stress of adult wild brown howlers and the food availability in brazilian atlantic forest fragments, and found environmental stress on reproduction of nonhuman primates – shikhmaidin et al. 177 that nursing females are highly nutrition demanding for their pregnancy and lactation it shows that hormones obsess and affect the appetite throughout pregnancy, explaining the consistent association between undernutrition and reproductive failure. studies on brain activity via hypothalamic-pituitary-ovarian axis and stress hormones on embryo and fetal losses are still scarce. we suggest that researches need to focus on this because as observed, maternal hormonal patterns and external factors are important in sustaining embryo survivability. conclusion and future perspectives this article has focused on how environmental stress, mainly climate change and poor dietary intake, can lead to reproductive stress and failure among nonhuman primates. these factors, known to be either directly or indirectly link to the hypothalamic-ovarian axis, harm the reproductive sites. furthermore, the anthropogenic stressors on primate populations also contribute towards species extinctions, particularly in animals with slow reproductive rates. all these factors are closely related to the extinction of this vulnerable species. primates are a flagship species for entire ecosystems, so its conservation also present important consequences for many other species. the success of its conservation and preservation programs are dependent on the ability of the species to reproduce successfully and to minimize offspring loss. thus, important information on the fitness and success of its reproduction provides an understanding of the physiological-environment interactions of the nonhuman primates reproduction. moreover, the successful reproduction is fundamental to the survival and evolution of all species. 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expression of hsp105 and hsp60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (macaca fascicularis). front biosci 10(1):3110-21. biotropia vol. 28 no. 2, 2021 180 biotropia no biotropia no. 12, 1999 : 59 77 the effectiveness of some ectomycorrhizal fungi in alginate beads in promoting the growth of several dipterocarp seedlings supriyanto seameo b1otrop, p.o box 116, bogor 16001, indonesia abstract the effectiveness of six species of ectomycorrhizal fungi (scleroderma columnare, s.dictyosporum, laccaria toccata, rhizopogon luteolus, amanita umbronata and descomyces sp.) in alginate beads in promoting the growth of four species of dipterocarp seedlings (shorea pinanga, s.leprosula, s.ovalis and hopea odorata) were studied. different sodium alginate concentrations of 5 g, 10 g, 15 g and 20 g/l were tested to find out the best bead's elasticity and spore germination. seedling height, relative field mycorrhizal dependency, percentage of mycorrhizal colonization, nutrient absorption, and the formation of hartig's net and mantle structure of dipterocarp seedlings were observed 4 months after inoculation. the best elasticity of alginate beads was found in the concentration of sodium alginate of 15 g/l. the best growth increment was found in hopea odorata inoculated with amanita umbronata (126.60 %) followed by shorea pinanga inoculated with descomyces sp. (27.10%), shorea ovalis inoculated with amanita umbronata (26.90 %) and shorea leprosula inoculated with descomyces sp. (24.20 %) over the control. the highest relative field mycorrhizal dependency was found in hopea odorata followed by shorea ovalis, s. pinanga and s. leprosula. the highest mycorrhizal colonization was obtained in shorea pinanga inoculated with descomyces sp. (75%), while inoculation with amanita umbronata on s.leprosula, s.ovalis and hopea odorata increased mycorrhizal colonization i.e. 64.5 %, 52.5%, and 46.2 %, respectively. hartig's net and mantle structures were well formed in shorea leprosula as well as s.ovalis seedlings with all mycorrhizal fungi tested, while in s.pinanga seedlings these structures were only well formed with descomyces sp. there is no clear difference in p levels in the leaves following inoculation as compared to the controls. key words: mycorrhizas/alginate beads/scleroderma columnare/scleroderma dictyosporumllaccaria laccatalrhizopogon luteoluslamanita umbronataldescomyces sp./growth/dipterocarpaceae/seedlings. introduction background industrial forest plantation (iff) is one of the government programs to improve the reduced forest productivity due to logging activities (logged-over areas) and deforestation (critical lands). during the period of the fourth to sixth five-year development plan of the government of indonesia, the area of ifp was projected to be 4.6 million hectares (departemen kehutanan 1994). high quality seedlings to replant these areas are needed. dipterocarp species make up one of the recommended species for sawn-timber production. dipterocarp is an important timber species in southeast asian countries (prosea 1994). seventy five percent of indonesian 59 biotropia no. 12, 1999 wood trading comes from dipterocarp species. currently, the rate of logging is higher than the rate of replanting. there are some problems in planting dipterocarp species. among these are the irregular flowering season, recalcitrant seeds, and the need of mycorrhizae for promoting their growth during the seedling stage. mycorrhizal research in indonesia has been promoted by the government, because of its beneficial contribution to increase the quality of seedlings via growth acceleration (marx 1973; supriyanto et al. 1992), to control root pathogenic microorganisms (marx 1973), to improve soil structure (de la cruz 1982), to improve rooting system due to hormone production (gay and debaud 1987), to increase nutrient and water uptake (boyle et al. 1987), to increase drought resistance (boyle et al. 1987; supriyanto et al. 1992), and to increase the survival rate and accelerate the xylem formation of seedlings obtained by tissue culture techniques (supriyanto 1989; and chang 1993). inoculum improvements of the potential ectomycorrhizal fungi have been achieved in some species by single spore culture, cell fusion (mating type) and protoplast culture. these techniques can be used for mass production of vegetative inoculum (fortin et al. 1988). the inoculum can be packed in several ways such as tablet, capsule, spore powder or mycelium suspension (supriyanto et al. 1992) and in alginate beads (supriyanto et al. 1994; supriyanto 1996). there are some advantages and disadvantages for each type of packaging system. alginate beads are simple, easily stored, free from climatic influences, inexpensive and are available at any time. recent technological improvements of the vegetative inoculum production promote the use of the inoculum packaging system. it is important to evaluate the effectiveness of selected tropical mycorrhizal fungi packed in alginate beads to promote the growth of dipterocarp species recommended for the industrial forest plantation program. objective the objective of the research was to compare the effectiveness of alginate inoculum of six species of ectomycorrhizal fungi on the growth of four species of dipterocarp seedlings. materials and methods seed germination dipterocarp seeds (s.pinanga, s.leprosula, s.ovalis, h.odoratd) were collected from dipterocarp experimental areas, jasinga, forest research institute and were dipped in dithane m-45 at 1% concentration for 10 minutes to eliminate the pathogenic fungi. seeds were germinated in sterilized sand medium. germination and transplanting media were autoclaved at 1.5 bar, 120°c for 30 minutes. dipterocarp seedlings, two weeks old, were transplanted into poly-bags containing sterilized sand: soil: compost (1:1:1). 60 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto aiginate bead elasticity ectomycorrhizal fungi of scleroderma columnare, laccaria laccata, amanita umbronata, descomyces sp. were collected from dipterocarp plantation. scleroderma dictyosporum was collected from pinus merkusii plantation, while rhizopogon luteolus was collected from eucalyptus urophylla plantation. the spores of ectomycorrhizal fungi were packed in alginate beads based on le tacon et al. method (1985) modified by supriyanto et al. (1994). the elasticity tests of alginate beads were done in different concentrations of sodium alginate (5, 10, 15, 20 g/l). an amount of 2 g of ectomycorrhizal fungi spores were extracted from mature fruit bodies and diluted in each different concentration of sodium alginate (an algal extract). spore suspension was trapped in sodium alginate to form beads in 0.7m calcium chlorite solution. the alginate bead elasticity was classified into very soft, soft, elastic and hard by pressing the beads in between two fingers. to evaluate the spore viability in different concentrations of sodium alginate, 10 beads were placed in a petri dish containing modified melin-nokran medium. five replicates were used for the evaluation of each sodium alginate concentration. germination percentage of the spores in alginate beads was recorded. inoculation technique three-week old dipterocarp seedlings (one week after transplanting) were then inoculated with 5 alginate beads at a concentration of 15g/l sodium alginate. a pipette was used to aspire the alginate beads. the alginate beads were then injected into transplanting media close to the rooting system (at a depth of 2 cm). eight replicates were used for each treatment. parameters mycorrhizal seedlings were evaluated 4 months after inoculation on the basis of several parameters to determine the effectiveness of selected mycorrhizal fungi as follows: hthe high growth increment (hgi, %) = ————— x 100 %, where: he ht = height of mycorrhizal seedlings, he = height of non-mycorrhizal seedlings (control) relative field mycorrhizal dependency / rfmd (plenchette et al. 1983 in bagyaraj 1994). dwt-dwc rfmd (%) = ———————— x 100 %, where : dwc 61 blotropla no. 12,1999 dwt = dry weight of inoculated seedlings (treatment), dwc : dry weight of noninoculated seedlings (control) seedlings were dried in an oven at 70°c for 48 hours and their dry weights were determined. the leaf dry weights were separated for nutrient content analysis. number of colonized roots percentage of colonization (fortinef a/. 1982)= ——————————————xloo% total number of root samples the colonized roots were determined by the presence of hyphae enveloping the roots (external hyphae). structural analysis of mycorrhizal and non-mycorrhizal roots was done to observe the presence of mantle and hartig's net formation based on the sass microtechniques method (1958) in berlyn and miksche (1976). toluedine blue 0 of 0.1% was used for root staining. toluedine blue 0 stains specifically the mycorrhizal hyphae either mantle or hartig's net in blue pinkish color. the presence of mantle (m), hartig's net (hn) or none in the rooting system were used to determine the compatibility of inoculated mycorrhizal fungi and host plant. it was classified into semi-compatible (mantle only), compatible (hartig's net and mantle) or incompatible (no mantle and no hartig's net). good formation of mantle and hartig's net will influence the effectiveness of mycorrhizal fungi in nutrient and water absorption. the criteria of mantle formation are as follows: (negative) for no mantle formation, + (positive) for one layer or poor formation, ++ for two layers or good formation and +hfor three layers or best formation. the criteria of hartig's net formation are as follows: (negative) for no hartig's-net formation, + (positive) for one series or poor formation, ++ for two series or good formation, +++ for three series or best formation. leaf content of n, p, k, ca, fe, mg were used as a nutrient parameter. all of the seedlings were dried at 70 ° c for 48 hours for nutrient analysis. dried leaves were powdered and 250 mg leaf powder was digested with concentrated h2so4 and 30% h2o2. the leaf nutrient content was analyzed as follows: nitrogen nitrogen content was analyzed using the titration method. to 20 ml of filtered solution in the nitrogen distillate apparatus was added 15 ml of 30% naoh. nitrogen distillation was done for 10 minutes. an amount of 20 ml of 1% boric acid added to the distillate solution were combined with 3 drops of bcrm (brom cresol green and red methyl) indicator. titration of the ammonium solution was done using 0.05n h2so4. 62 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanlo phosphate phosphate content was determined spectrophotometrically at x 693 nm. phosphate solutions of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ppm were used as standards from which the phosphate content was determined. iron the wet destruction method of powdered leaf was done using hno3 + hc1o4 + hjsc^. the iron was analyzed spectrophotometrically at a. 508 nm. an amount of 0.25 ml of metal reduction solution was added to the destructed solution thoroughly to homogenize the solution. an amount of 0.5 ml of orthophenontroline and 7 ml of sodium citrate were combined. standardized iron solutions of 0, 1.0, 2.0, 4.0, 6.0 and 8.0 ppm using (nh4)2fe(so4)2 . 6 h2o were used. potassium, calcium and magnesium atomic absorption spectrophotometry was used to determine the potassium, calcium and magnesium content. standardized solutions of potassium and calcium each of 0, 2.5, 5.0, 10.0, 15.0, 20.0 and 25 ppm were prepared and used to determine potassium and calcium content. to determine magnesium content, standardized solutions of 5 ml magnesium were made from mgso4 .7 h2o, i.e. 0, 2.5, 5.0 ppm to which 5 ml lacl3 containing 4000 ppm la were added. r e s u l t s alginate bead elasticity the spore suspension was trapped using sodium alginate and formed to beads in calcium chloride solution. bead elasticity depended on the concentration of sodium alginate used. the best concentration is determined by the criteria for bead elasticity and germination percentage of trapped spores. this was 15g/l sodium alginate with the average spore germination percentage of 77% (table 1). the concentration of sodium alginate of 20 g/l produced harder beads but the germination percentage of spores trapped in alginate beads decreased. concentrations of sodium alginate lower than 15 g/l produced soft or very soft beads and can easily be dried by airflow even when its spore germination was higher. the concentration of sodium alginate at 15 g/l will be used for inoculum packaging system at seameo biotrop. this packaging system is easily handled, with high productivity and high inoculum viability. although le tacon et al. (1985) used the sodium alginate at the concentration of 10 g/l, vegetative mycelia were used instead of spores. 63 biotropia no. 12, 1999 table 1. degree of bead elasticity and germination percentage of scleroderma columnare ectomycorrhizal fungi at different concentrations of sodium alginate. no. 5g/ls.a. vs %g l o g / l s.a s %g 15 g/l s.a. els % g 20 g/l s.a. h %g 1 2 3 4 5 vs vs vs vs vs 70 85 80 75 8 0 s s s s s 75 75 80 90 80 els els els els els 80 75 80 75 75 h h h h h 60 65 60 50 60 average vs 78 s 80 els 77 h 59 note: vs: very soft, s: soft, els: elastic, h: harder, % c: germination percentage, s.a.: sodium alginate. high growth increment (hgi) the percentage of high growth increment (hgi) of mycorrhizal seedlings over the control (not inoculated) is presented in table 2. the best hgi was obtained at hopea odorata seedlings inoculated with amanita umbronata (126.60%). all of the mycorrhizal fungi stimulated the growth of hopea odorata seedlings, ranging from 40.6 % to 126.60 % and the growth of shorea pinanga ranging from 5.70 % to 27.10 %. the hgi of shorea pinanga and s. leprosula was promoted by inoculation with descomyces sp., whereas the hgi of shorea ovalis and hopea odorata was promoted best by amanita umbronata. scleroderma columnare, rhizopogon luteolus and laccaria laccata were not able to stimulate the hgi of shorea leprosula, and laccaria laccata was not able to stimulate the hgi of shorea ovalis. scleroderma dictyosporum consistently stimulated the hgi of four dipterocarp seedlings tested, ranging from 10.30 % to 75.00 %. table 2. percentage of high growth increment over the control of 4-month old dipterocarp seedlings inoculated with different species of ectomycorrhizal fungi. species of ectomycorrhizal fungidipterocarp species sd sc r ll am de shorea pinanga 10.30 5.70 12.90 13.60 15.50 27.10 shorea leprosula 11.10 -3.00 -13.70 -18.00 23.80 24.20 shorea ovalis 18.30 11.60 14.10 -10.80 26.90 24.10 hopea odorata 75.00 54.70 49.60 40.60 126.60 84.40 note : sd: scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. the growth response of each dipterocarp seedling inoculated with different mycorrhizal fungi can be seen in figure 1 (s. pinanga), figures 4 and 6 (s. leprosula). 64 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 1: shorea pinanga seeds (a, x 0.1), alginate beads (b, x 0.18), mycorrhizal seedlings (sc, sd, r, ll, am, de) and nonmycorrhizal seedlings (c) (c, x 0.06) and the colonization of scleroderma columnare mycorrhizal fungi at the rooting system of shorea pinanga seedling (d, x 0.2). relative field mycorrhizal dependency (rfmd) the influence of mycorrhizal inoculation can also be determined from their relative field mycorrhizal dependency (rfmd). rfmd is the dependency of a plant species on the mycorrhizal fungi at a given soil fertility. rfmd is determined on the dry weight of mycorrhizal seedlings. it is important because dry weights may reflect the efficiency of physiological processes due to the mycorrhizal inoculation and photosynthetic activity. regarding the rfmd value (table 3), hopea odorata was very dependent on the inoculated mycorrhizal fungi, ranging from 82.45% to 416.10%. shorea pinanga was dependent on scleroderma dictyosporum (17.87%), rhizopogon luteolus (8.6%), laccaria laccata (27.82%), amanita umbronata (16.82%) and descomyces sp. (34.89%). shorea leprosula was dependent on descomyces sp. (34.32%) and on amanita umbronata (27.86%), while shorea ovalis was dependent on all of 65 biotropia no. 12, 1999 inoculated mycorrhizal fungi ranging from 32.10 % to 88.20 %. since some values are negative, it is possible that in this stage of colonization, mycorrhizal fungi consumed a high amount of carbohydrate for their energy and development. generally, those carbohydrates are translocated for plant organ development. table 3. percent relative field mycorrhizal dependency over the controls (not inoculated) of 4-month old dipterocarp seedlings inoculated with different species of ectomycorrhizal fungi. ectomycorrhizal fungidipterocarp species sd sc r ll am de shorea pinanga shorea leprosula shorea ovalis hopea odorata 17.87 -25.10 43.50 394.20 -12.28 -15.30 32.10 221.10 8.60 -33.90 32.50 128.10 27.82 -225.30 7 3.20 82.45 16.82 27.86 88.20 416.10 34.89 34.32 63.20 321.40 note : sd : scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. percentage of mycorrhizal colonization percentage of mycorrhizal colonization is defined as the ability of inoculated mycorrhizal fungi to form a mantle sheet and hartig's net in the rooting system of certain host-plants. the presence of the mantle sheet can be observed by macroscopic or microscopic observation (external hyphae), while the presence of hartig's net can be observed by a microscope (internal hyphae). the presence of mantle and hartig's net in the rooting system explains the compatibility status between host plant and inoculated mycorrhizal fungi. technically, the percentage of mycorrhizal colonization is obtained from the hyphal extension that can be observed visually, while the hartig's net structure must be observed using microscopic magnification. the extent of mycorrhizal colonization of inoculated mycorrhizal fungi on four species of dipterocarp seedlings is shown in table 4. the extent of mycorrhizal colonization is classified in five categories: excellent (75 100%), good (50 74%), moderate (24 40%), poor. (1 24%) (marx and bryan 1969). based on this classification, the colonization by scleroderma dictyosporum, s. columnare, rhizopogon luteolus, laccaria laccata, amanita umbronata and descomyces sp. are table 4. percentage of mycorrhizal colonization in the root system of 4-month old seedlings of shorea pinanga, shorea leprosula, shorea ovalis and hopea odorata. mycorrhizal colonization for each mycorrhizal fungus dipterocarp seedlings sd. sc. r ll am de shorea pinanga shorea leprosula shorea ovalis hopea odorata 51.87 63.00 43.10 37.50 ,54.37 41.50 49.40 45.00 64.37 39.00 50.00 46.20 66.26 42.50 52.70 30.70 67.50 64.50 52.50 46.20 75.00 . 55.50 44.00 37.50 note : c: control, sd : scleroderma dictyopsorum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. 66 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto moderate and good (30.70% to 75.00 %). excellent mycorrhizal colonization was found in shorea pinanga inoculated with descomyces sp. the ability of scleroderma dictyopsorum, s. columnare, rhizopogon luteolus, laccaha laccata, amanita umbronata and descomyces sp. to form the hartig's net and mantle structure is shown in table 5. mycorrhizal colonization in shorea pinanga and s. leprosula seedlings were generally classified in term of good and best formation, while s. ovalis and hopea odorata were classified as poor. table 5. the presence of mantle and hartig's net in the root system of mycorrhizal and non-mycorrhizal seedlings of 4-month old seedlings of shorea pinanga, s. leprosula, s. ovalis and hopea odorata. note: : no formation, + : poor formation, -h-: good formation, +++ : best formation. c: control, sd : scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. the extent of mycorrhizal colonization of the inoculated mycorrhizal fungi in dipterocarp seedlings is shown in figure 2 (s. pinanga), figures 5 and 6 (s. leprosula), figure 7a, 7b (s. ovalis). the transversal section showing the mantle and hartig's net formation in each host plant is shown in figure 3 (s. pinanga), figure 8 (s. ovalis). leaf nutrient content nutrient concentration in the leaves of s.pinanga, s. leprosula, s. ovalis and h. odorata is presented in table 6. effect of mycorrhizal inoculation on the leaf nutrient content was obtained by comparing the nutrient content of mycorrhizal and non-mycorrhizal seedlings. table 6 shows that the increment of leaf nutrient content varied depending on the host plant species and the inoculated mycorrhizal fungi. 67 biotropia no. 12, 1999 figure 2: non-mycorrhizal and mycorrhizal root system of shorea pinanga. a: non-mycorrhizal root system (x 10), b: root system inoculated with s.columnare (x4), c: root system inoculated with s.dictyosporum (x 5), d: root system inoculated with l laccata (x 5), e: root system inoculated with a. .umbronata (x 5), f: root system inoculated with descomyces sp. (x 5). 68 the effectiveness of some ectomycorrhizal fungi in alginate beadssupriyanto figure 3 : transversal section of shorea pinanga mycorrhizal roots inoculated with s.columnare (b, x 400), a. umbronata (c, x 400), rhizopogon luteolus (d, x 400), descomyces sp. (e, x 400) and non-mycorrhizal roots (a, x 400). cc = cortical cells, reec= radially elongated epidermis cells, m= mantle, hn= hartig's net. 69 biotropia no. 12, 1999 figure 4 : the growth of non-mycorrhizal seedling (a,c) and mycorrhizal seedlings (sd: s.dictyosporum, sc: s.columnare, r: r. luteolus, ll: l.laccata, am: a.umbronata, de: descomyces sp.) of 4 month old shorea leprosula (a, x 0.07) and the colonization of rhizopogon luteolus (b x 0 3 c,xl5). ' ' ' 70 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 5 : the development and colonization of mycorrhizal fungi hyphae at the root system of 4-month old shorea leprosula seedlings. a (x 0.1) and h (x 10): non-myconrhizal roots (control), b: inoculated with s. dictyosporum (x 0.1) and 5". columnare (c, x 0.1), l .laccata (d, x 0.1), descomyces sp. (e, x 0.1), rhizopogon luteolus(f,\q.l),a. umbronata(g,xq.l;l,x 15). 71 biotropia no. 12, 1999 figure 6: root system of 4-month old shorea leprosula seedlings inoculated with amanita umbronata (a, x 0.2 , b, x 0.5). figure 7a: degree of mycorrhizal colonization of s.dictyosporum (c, \ 0.4; d, x 8), s. columnare (e, x 0.4; f, x 10), rhizopogon luteolus (g, x 0.4; h, x 10), at root system of 4-month old shorea ovalis. a, b: non-mycorrhizal roots. 72 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 7b : degree of mycorrhizal colonization of i .laccata (i, x 0.4; j, x 10), a.umbronata (k, x 0.4; l, x 10), and descomyces sp. (m, x 0.4; n, x 10). figure 8 : transversal section of non-mycorrhizal and mycorrhizal roots of 4-month old shorea ovalis seedlings. a. structure of non-mycorrhizal roots (x 400), and inoculated with s.columnare (b, x 200), 5. dictyosporum (c, x 400), a.umbronata (d, x 400), descomyces sp. (e, x 400) and rhizopogon luleolus(f,x 1000). 73 biotropia no. 12, 1999 table 6. leaf nutrient content of shorea pinanga, shorea leprosula, shorea ovalis and hopea odorata mycorrhizal and non-mycorrhizal seedlings. note : values in bold means nutrient content increment and the values not in bold means no nutrient increment. c: control, sc: scleroderma columnare, sd.: scleroderma dictyosporum, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. in s. pinanga seedlings, calcium concentration in the leaves was increased by the inoculation of laccaria laccata (1.085%) and descomyces sp. (1.111%), while the concentration of iron (fe) was increased by the inoculation of amanita umbronata ( 511.2 ppm) and rhizopogon luteolus (410.6 ppm). the concentration of n, p, k and mg was not significantly influenced by mycorrhizal inoculation. in shorea leprosula, the concentration of nitrogen in the leaves was increased by the inoculation of scleroderma columnare (1.737%), laccaria laccata (1.659%) and amanita umbronata (1.708%) and the concentration of calcium and magnesium was increased by the inoculation of scleroderma columnare (0.904%) and laccaria 74 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto laccata (0.204%). the concentration of p, k and fe in the leaves was not influenced by the mycorrhizal inoculation. in shorea avails, the nitrogen concentration in the leaves was increased by the inoculation of scleroderma dictyasporum (1.317%), amanita umbronata (1.356%), rhizopogon luteolus (1.756%) and descomyces sp. (1.542%). the concentration of p, k, ca, fe, mg in the leaves of shorea ovalis was not influenced by the inoculation of mycorrhizal fungi. in hopea odorata, the nitrogen levels increased with inoculation of rhizopogon luteolus (2.049%) and the level of calcium in the leaves was increased by scleroderma columnare (0.903%), laccaria laccata (0.909%) and amanita umbronata (0.934%). magnesium level increased with inoculation of laccaria laccata (0.207%), amanita umbronata (0.201%) and descomyces sp. (0.200 %). iron levels in the leaves of hopea odorata was increased by the inoculation of scleroderma dictyosporum (1154.7 ppm), laccaria laccata (825.1 ppm) and rhizopogon luteolus (661.2 %). there is no clear pattern of correlation between inoculation with mycorrhizal fungi and leaf nutrient content. there is also no clear difference in p concentration in the leaves following inoculation as compared to the controls. discussion method of inoculum production of ectomycorrhizal fungi should be easily handled, cheap and effective to increase the seedling quality. alginate beads, as a carrier of inoculum of ectomycorrhizal fungi, produces an inoculum of high quality and large scale (9,000 100,000 beads/hour/machine). feldman and idczak (1994) specified two characteristics of high quality inoculum. high quality means firstly that the inoculum is able to colonize rapidly the plant root system. this is guaranteed by an inoculum of high infection. it does not necessarily mean large number of spores. secondly, the characteristic of a high quality inoculum is that it should be aseptic or contaminated with only small quantities of plant pathogenic microorganisms. low level of contamination can be ignored, for example for planting stock production in the nursery. regarding the percentage of germination of ectomycorrhizal fungi packed in alginate beads, the concentration of sodium alginate of 5 20 g/l does not prevent the spore germination, but its elasticity was different. based on our results, a concentration of sodium alginate of 15 g/l was selected because of the high spore germination percentage and good bead elasticity associated with this. s. columnare, s. dictyosporum, rhizopogon luteolus, laccaria laccata, a. umbronata, descomyces sp. that were packed in alginate beads, were able to colonize the root system of shorea pinanga, s. leprosula, s. ovalis and hopea odorata. their colonization is shown to be moderate to good. the mycorrhizal fungi formed mantle and hartig's net structures in the root system of the plant. it means that the alginate bead as a carrier of inoculum does not prevent the spore germination. their degree of 75 biotropia no. 12, 1999 compatibility can be evaluated on their capability to form the mantle and hartig's net structure. a mycorrhizal species able to form mantle and hartig's net structures in several plant species means that this mycorrhizal species has high compatibility. likely, it is reflected on its natural distribution. descomyces sp. and a. umbronata were the most possible to form those structures in the root system of dipterocarp species tested. their degree of compatibility should be followed further on their growth effect and their mycorrhizal dependency value. plants differ greatly in their need for, and response to, mycorrhizal infection. relative mycorrhizal dependency (rmd) as degree to which a plant is dependent on the mycorrhizal condition at a given level of soil fertility had been defined (gerdeman et al. 1975 in bagyaraj 1994). the rmd concept was also improved to define the mycorrhizal dependency of crops on the mycorrhizal fungi in field conditions, called relative field mycorrhizal dependency (plenchette et al. 1983 in bagyaraj 1994). based on the first and second rank of rfmd value, the dipterocarp mycorrhizal dependencies are as follows: 5. pinanga to descomyces sp. and l. lacata, s. leprosula to descomyces sp. and a. umbronata, s. ovalis to a. umbronata and l. laccata, h. odorata to a. umbronata and s. dictyosporum, respectively. those mycorrhizal fungi are important for improving the growth of dipterocarp seedlings. mycorrhizae have long been known to affect the nutrient cycling especially of p nutrition of the host plant. phosphate is the major form of p available in the soil solution and, therefore, it is not readily transported by mass flow. thus, as mycorrhizal hyphae explore the bulk soil beyond the root hairs, additionally p is taken up by hyphae and transported to the host (alien 1996). mycorrhizal fungi can also increase the uptake of mineralized p by occupying the microsites of active decomposition 'and, possibly, by being involved in the degradation of litter. mycorrhizal hyphae on a decomposing leaf and rapid transport of32 p from that leaf to a host plant via mycorrhizal hyphae had been observed (herrera et al. 1978 in alien 1996). by their role in the acquisition of nutrient resources, mycorrhizae are critical components of nutrient cycling. nutrient levels in the leaves of dipterocarp tested showed that mycorrhizal fungi could increase the nutrient concentration of some of the elements tested in the host plant. based on our results, there is no clear difference in p levels in leaves following inoculation as compared to the controls. references allen, m.f. 1996. the ecology of mycorrhizae, cambridge studies in ecology. cambridge university press. bagyaraj, d.j. 1994. vesicular-arbuscular mycorrhiza: application in agriculture. in j.r. morris, d. read and a.k.. varma (eds) : techniques for mycorrhizal research, methods in microbiology. academic press, harcourt brace & company, publishers, london-san diego-new york-bostonsydney-tokyo-toronto, p. 819-834. berlyn, g.p. and j.p. mlksche, 1976. botanical microtechniques and cytochemistry. iowa state university press, ames, iowa. 76 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto boyle, c.d., w.j. robertson and p.o. salonius, 1987. use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree seedling nurseries. can. j. for. res. 17: 1480-1486. chang, d. 1993. the study of va mycorrhizal effects on horticultural crops. in proc. of second acom, 11-15 march 1991, chiang mai, thailand. eds. i. soerianegara and supriyanto. biotrop special publication no. 42. de la cruz, r.e., 1982. tree nutrition and fertilization . lecture note in the training course on biological aspects of silviculture, seamed biotrop, bogor, indonesia. departemen kehutanan, 1994. rencana pembangunan kehutanan pelita vi. jakarta, indonesia. feldmann, f. and e. idzcek, 1994. inoculum production of vesicular-arbuscular mycorhizal fungi use in tropical nurseries. in j.r. norris, d. read and a.k. varma (eds) : techniques for mycorrhizal research, methods in microbiology. academic press, harcourt brace & company, publishers, ondon-san diego-new york-boston-sydney-tokyo-toronto. fortln, c., j.a. fortin, g. gaulin, n. jomphe dan s. lemay, 1988. large scale ectomycorrhizal inoculation of containerized grown seedlings. in proc. of canadian workshop on mycorrhizae in forestry, 1-4 may 1988. gay j.c. and j.c. debaud 1987. genetic study on indole-3-acetic acid production by ectomycorrhizal hebeloma species : inter-and intra specific variability in homo and dikaryotic mycelia. appl. microbiol. biotechnol. 26:141-146. le tacon, f. , g. jung, g. mungier, p. michelot and c.maperin, 1985. efficiency in a forest nursery of an ectomycorrhizal fungus inoculum, produced in fermentor and entrapped in polymeric gels. can. j. bot. 63: 1644-1668. marx, d.h. and w.c. bryan, 1969. studies on ectomycorrhizae of pine in an electronical air filtered, air conditioned plant growth room. can. j. bot. 47 : 1903-1909. marx, d.h. 1973, mycorrhizae and feeder root diseases. in ectomycorrhizae : their ecology and physiology. eds. g.c. marks and t.t. kozlowski. academic press, new york. prosea, 1994. plant resources in south-east asia 5 : (1) timber trees : major commercial timbers. eds. i.soerianegara and r.h.m. lemmens, prosea foundation, bogor, indonesia pudoc-dlo, wageningen, the netherlands. supriyanto, 1989. micropropagation de pinus nigra et pinus sylvestris', application a leurs hybrides interspecifiques. phd dissertation, university of nancy i, nancy, france. supriyanto, 1996. conservation and utilization of mycorrhizal fungi genetic resources. seameo forum, vol. 3 no. 2, seameo bangkok. supriyanto, i. setiawan dan m. harahap, 1992. usaha peningkatan kualitas bibit tanaman kehutanan melalui inokulasi mikoriza (pengalaman dan program penelitian seameo biotrop). pros. seminar nasional status silvikultur di indonesia saat ini. dept. kehutanan aphi fak. kehutanan, ugm. wanagama, 27-29 april. supriyanto, i. setiawan, r.m. omon, dan e. santosa, 1994. effects of scleroderma dictyosporum obtained by protoplast culture on the growth of shorea selanica and shorea leprosula cuttings. bio-refor expert meeting. jica-frim, kangar, malaysia, 28 nov-1 77 biotropia vol. 27 no. 1, 2020: 69 79 doi: 10.11598/btb.2020.27.1.1122 69 association of tree communities with soil properties in a semi deciduous forest of perlis, peninsular malaysia radhiah zakaria1*, mohd nizam mohd said2,3 and faridah hanum ibrahim4 1faculty of public health, universitas muhammadiyah aceh, banda aceh 23123, indonesia 2school of environmental and natural resource sciences, faculty of science and technology, universiti kebangsaan malaysia, 43600 bangi, selangor, malaysia 3institute of climate change, universiti kebangsaan malaysia, 43600 bangi, selangor, malaysia 4faculty of forestry, universiti putra malaysia, 43400 serdang, selangor, malaysia received 20 august 2018 / accepted 16 november 2018 abstract plant community distribution is associated with environmental factors, particularly, the soil properties of habitats. this study was conducted to determine the effect of soil properties on the association of tree communities within three distinct habitats in a semi-deciduous forest in perlis state park (psp), perlis. eighteen plots of 40 × 60 m (0.24 ha each) with sampling areas of 1.92 ha (8 plots) in setul formation, 0.96 ha (4 plots) in granite and 1.44 ha (6 plots) in kubang pasu formation (totalling 4.32 ha) were established at the psp. all trees with 5.0 cm and above diameter at breast height (dbh) were enumerated, while the top soil samples were collected from each plot for soil analyses. a total of 412 tree species, 207 genera, and 68 families were recorded; 270 tree species from 152 genera and 57 families in the setul forest; 204 tree species of 130 genera and 50 families in the granite forest; and 109 tree species from 76 genera and 31 families in the kubang pasu forest. euphorbiaceae was the most represented family at setul, granite and kubang pasu with 36, 19 and 12 species, respectively. soil properties significantly varied among the study sites. setul had loam, kubang pasu had clayloam, and granite had the sandy-loam texture. the soils were acidic and had low to high concentrations of available nutrients. ordinations using canonical correspondence analysis indicated that the soil factors play an important role in the distribution and diversity of plants in these forest habitats. keywords: canonical correspondence analysis, perlis state park, semi-deciduous forest, vegetation– environment relationship introduction in tropical rain forest, the tree species composition varies with the type of habitat (richards 1952) and their distributions are often associated with environmental factors (newbery & proctor 1984; fujii et al. 2018). studies conducted in different tropical regions had emphasized the influence of environmental factors, especially soil properties, on plant species distributions (wentworth 1981; oliveirafilho et al. 2001; nizam et al. 2013). relationships between soil variables and plant species distribution had been discovered in various habitats, such as in granite and limestone areas (nizam et al. 2013), grasslands (cachovanová et al. 2012), savannas (barruch 2005) and the tropical rain forests (silk et al. 2010; sukri et al. 2012). peninsular malaysia is predominantly of the rainforests type and comprise only a small restricted part of a semi-deciduous forest. the semi-deciduous forest in peninsular malaysia is located near kra isthmus, a transitional zone where malesian and indochinese floristic regions intersect (van steenis 1979; whitmore 1984; middleton 2003). the state of perlis, which is situated in the northernmost of peninsular malaysia, is the only part in this country with a semi-deciduous forest (faridah-hanum 2006). *corresponding author, e-mail: radhiah@unmuha.ac.id https://link.springer.com/article/10.1007/s12224-012-9131-3#auth-1 biotropia vol. 27 no. 1, 2020 70 the perlis state park (psp) exhibits a semideciduous forest with most areas consisting of limestone hill forests and a small portion of granite-based parent material. this study aimed to determine the association between the tree communities and their soil properties under different forest habitats with different rock formations. this study is timely because the increasing human pressures on limestone and granite habitats compromise their integrity, diversity and functions. limestone forests are being replaced by plantations, agricultural crops and forages to increase their productivity. materials and methods study site and tree sampling a semi-deciduous forest in psp, perlis, peninsular malaysia, was selected as the study site (latitude 634'n to 643'n, longitude 10010'e to 10013'e) (fig. 1). tree samplings were conducted in 18 plots of 40 × 60 m each, covering a survey area of 4.32 ha. due to different topography of the selected forest habitats, several plots were selectively established to avoid rocks and huge boulders. as such, the number of plots that were established also varied between forest habitats of different geological formations; eight plots (1.92 ha) in the setul, six plots (1.44 ha) in the kubang pasu, and four plots (0.96 ha) in the granite. all trees with 5 cm and above diameter at breast height (dbh) were enumerated and identified using the published keys (whitmore 1972; whitmore 1973; ng 1978; ng 1989). since the number of plots in each forest habitat was unequal as mentioned earlier, the rarefaction analysis was conducted using ecosim software program (gotelli & entsminger 2003) to produce the rarefaction curves. rarefaction allows comparison of the observed richness and diversity between sites even though sampling efforts are not equal, or samples differ in the total number of individuals (lee & chao 1994). the rarefaction analysis of the study site confirmed that although the sampling efforts between habitats were unequal, nevertheless the species richness was of the same trend with the actual observation in the survey plots (zakaria et al. 2015). perlis state park figure 1 location of the perlis state park in the state of perlis, peninsular malaysia association of tree communities with soil properties – zakaria et al. 71 soil sampling and analysis using the soil corer, the top soil samples at 0-15 cm depth were collected from 18 established plots of various habitats within the psp. five replicate samples were obtained from each plot and air dried at room temperature. the replicates were then homogenized together to represent one composite sample from each plot. root fragments and unwanted materials were removed and the soils were lightly ground and passed through a 2 mm mesh sieve before analysis. the soil samples were analyzed for physical properties, i.e., particle size distribution and organic matter (om) content as well as chemical properties, i.e., exchangeable acid cations (al3+ and h+), exchangeable base cations (k+, mg2+, na+ and ca2+) and available nutrients (phosphorus (p), potassium (k) and magnesium (mg). om content was measured using loss on ignition techniques, while the soil ph was recorded based on soil–water ratio of 1:2.5 (mclean 1967). by titration, the exchangeable acid cations were analyzed in 1 m kcl while the exchangeable base cations used the 1 m ammonium acetate extract (black 1967; shamshuddin 1981). to determine the cation concentrations, the extracts were run using the perkin-elmer atomic absorption spectrophotometer (aas) model 3300. available nutrients (p, k and mg) in the soil samples were extracted using 1 m ammonium acetate–acetic acid. the concentration of p in the extract was recorded using ultraviolet (uv) spectrophotometer, whilst k and mg concentrations were measured using the aas. data analysis the enumerated trees in the plots were investigated for overall floristic composition. the soil parameters among the study plots were tested for significant difference using t-test. association between tree communities and soil variables was analyzed using the multivariate techniques of canonical correspondence analysis (cca) (ter braak 1987; ter braak & prentice 1988; ter braak 1992), which is available in canoco for windows 4.56 (ter braak 2009). tree species with four occurrences, or less, were not included in the analysis because they weaken the uniformity when assigned to the groups (legendre & gallagher 2001; barruch 2005). the species with four occurrences or less were disregarded to limit the number of species to less than 200 and consequently increase the accuracy of the results of cca ordination diagram. thus, 161 tree species were selected for the cca. detrended correspondence analysis (dca) was first conducted on the species data to confirm their unimodality and subsequently justify the appropriateness of using cca (lepš & šmilauer 2003). soil variables selected for the cca were soil ph, available p, available magnesium (mg2+), calcium (ca2+), natrium (na+), potassium (k+) content, ammonium-n (nh4-n) and nitrate-n (no3-n). the abundance values were log-transformed prior to matrix processing because their distributions were skewed toward extremely large values (ter braak 1995). the correlation significance between matrices was tested using monte-carlo permutation test based on 499 random trials at a 0.05 significant level (lepš & šmilauer 2003). the ordination diagrams were subsequently plotted by canodraw 4.14 to illustrate the association patterns of tree communities in relation to soil properties. results and discussion tree species composition a total of 4,300 trees were recorded within the sampling area of 4.32 ha at perlis state park. the floristic composition included 412 tree species, 207 genera and 68 families. the setul habitat showed high species richness of 270 species from 1,722 trees; belonging to 152 genera and 57 tree families and the plots in granite habitat showed 204 tree species, from 1,245 trees; belonging to 130 genera and 50 families and the plots in the kubang pasu habitat contained 109 tree species from 1,333 trees, belonging to 76 genera and 31 families. in all three habitats, euphorbiaceae was the most represented family with 36 species in setul, 19 species in granite and 12 in kubang pasu. on the contrary, 11 families were represented by only one species with a single individual in setul, granite and kubang pasu habitats. these families include actinidiaceae (saurauia pentapetala), ancistrocladaceae (ancistroclados biotropia vol. 27 no. 1, 2020 72 tectorius), anisophylleaceae (anisophyllea apetala), convolvulaceae (erycibe albida), ctenolophonaceae (ctenolophon parvifolius), hypericaceae (cratoxylum formosum), magnoliaceae (magnolia elegans), oleaceae (chionanthus macrocarpus), opiliaceae (milientha suavis), oxalidaceae (sarcotheca griffithii) and rhizophoraceae (gynotroches axillaris). hence, these species were not considered in the analysis of species association with soil variables. soil properties the three habitats have different soil properties as follows: the soil in setul was loam whereas those in granite and kubang pasu were sandy and silty-loam, respectively. granite habitat had the lowest silt and clay contents but had the highest sand content at more than 69% among all study sites (table 1). the high sand content in granite is related to sandy parent material; the quartz sand derived from granitic hill was washed out and deposited a long time ago as reflected by the high sandy materials and extremely low clay and silt contents (osumi 1979; zaidey et al. 2010). in terms of om content, the granite soil had the significantly lowest om content (3.12 ± 0.08) followed by the setul soil (6.97 ± 1.96) and kubang pasu soil (10.72 ± 2.01) (table 1). the lowest om content in granite is related to the lower percentage of silt and clay in the soil; because the absence of silt and clay generally increases decomposition rates and decreases om for a particular set of environment interactions (paul & clark 1996). overall, it is apparent that the amount of om in the study sites is generally low. this low om content is due to the high decomposition rate of om in tropical rainforest soils (longman & jenik 1987). the low soil ph in granite (4.39), setul (5.64) and kubang pasu (5.98) indicated that the soils were strongly to moderately acidic. the soil ph in granite was significantly lower than those of setul and kubang pasu (table 1). granite soil has the highest acidity probably because it originated from the acidic parent rocks (juo 1981) and the decomposition of om also adds to the acidification of surface soil (zaidey et al. 2010). a similar study recorded that most soils in peninsular malaysia tropical rainforests are acidic with ph values between 4.5 and 5.5 (othman & shamshuddin 1982). table 1 summary of soil variables in setul, granite and kubang pasu habitats at perlis state park, perlis soil parameters granite (mean ± s.e.) setul (mean ± s.e.) kubang pasu (mean ± s.e.) p value organic matter (om) (%) 3.12 ± 0.08a 6.97 ± 1.96b 10.72 ± 2.01b p < 0.05 sand (%) 69.75 ± 2.12a 41.45 ± 4.68b 36.60 ± 8.52b p < 0.01 silt (%) 14.15 ± 0.77a 35.61 ± 3.89b 44.20 ± 2.17b p < 0.01 clay (%) 16.05 ± 1.42 21.42 ± 2.80 19.20 ± 2.23 ns soil ph 4.39 ± 0.12a 5.64 ± 0.35b 5.98 ± 0.30b p < 0.01 exchangeable cations (meq/100g) ca2+ 0.94 ± 0.27a 5.5 ± 2.05b 7.87 ± 2.18b p < 0.05 mg2+ 0.41 ± 0.02a 0.75 ± 0.16b 0.95 ± 0.44a p < 0.05 na+ 0.20 ± 0.04 0.13 ± 0.02 0.13 ± 0.02 ns k+ 0.32 ± 0.03a 0.50 ± 0.06ab 0.57 ± 0.12b p < 0.01 al3+ 0.60 ± 0.16 0.19 ± 0.14 0 ns h+ 0.50 ± 0.03 0.35 ± 0.04 0.34 ± 0.02 ns cec 2.92 ± 0.2a 7.58 ± 2.09b 9.87 ± 2.54b p < 0.05 available nutrients (ug/g) phosphorus (p) 9.04 ± 0.31a 7.18 ± 0.69b 7.27 ± 0.37b p < 0.05 nitrate-n (no3-n) 27.00 ± 1.5 34.39 ± 6.45 30.96 ± 4.94 ns ammonium-n (nh4-n) 3.59 ± 0.46a 18.64 ± 3.57b 30.81 ± 3.57b p < 0.01 magnesium (mg) 28.56 ± 0.51 105.35 ± 48.56 139.49 ± 66.78 ns potassium (k) 175.02 ± 8.43a 227.27 ± 32.4b 253.80 ± 26.35ab p < 0.05 note: mean values in a row with the same letter were not significantly different. association of tree communities with soil properties – zakaria et al. 73 the cation exchange capacity (cec in granite was the lowest (2.92 ± 0.20 meq/100 g,) was significantly different from those in setul (7.58 ± 2.09 meq/100 g) and kubang pasu (9.87 ± 2.54 meq/100 g). cec varies with the clay–humus content of the soil. clay soils usually have large cec, whereas sandy soils have low cec (cruickshank 1972). the sites showed various concentrations of available macronutrients, namely magnesium (mg), potassium (k) and phosphorus (p), and soluble nutrients, i.e., ammonium-n and nitrate-n (table 1). the phosphorus concentration in granite plots (9.04 ± 0.31 μg/g) was significantly higher than those in setul (7.18 ± 0.69 μg/g) and kubang pasu (7.27 ± 0.37 μg/g). the available magnesium (mg) in granite (28.56 ± 0.51 μg/g), setul (105.35 ± 48.56 μg/g) and kubang pasu (139.49 ± 66.78 μg/g) did not differ significantly. the available inorganic soil nitrogen is in the form of nitrate-n (no3-n) and ammoniumn (nh4-n). the concentrations of these two soluble nutrients showed a consistent trend; the granite habitat had lower concentration than the setul and kubang pasu habitats. ammonium-n was significantly low at granite (3.59 ± 0.46 μg/g), higher at setul (18.64 ± 3.57 μg/g) and the highest at kubang pasu (30.81 ± 3.57 μg/g). nevertheless, the concentration of nitrate-n was not significantly different among the habitats. the lowest availability of inorganic soil nitrogen in granite habitat was in line with the om in the habitat. tan (2009) mentioned that soil inorganic n content increases linearly as organic matter increases in the soil. the inorganic n was characterized by a large no3-n, which exceeds the nh4-n at all the habitats; this might be attributed to the drier conditions in the study sites (yamashita et al. 2003). relationships between tree communities and soil variables dca confirmed that the data on tree communities were unimodal with the length of gradient of the first axis at 5.758 (table 2), which was greater than 4 standard deviation (sd) before being analyzed by cca (lepš & šmilauer 2003). the eigenvalue produced by the dca axis 1 (0.758) was high, indicating an environmental gradient where most species vary essentially in their abundance (ter braak 1995). plots in granite (symbol x) were clustered together, while plots in setul (symbol ○) and kubang pasu (symbol +) exhibited the gradient that occurs from granite to limestone (fig. 2). these dca ordination plots indicated the approximate locations of the sample plots. the plots that clumped together represented the plots with relatively similar floristic attributes, while the separated plots indicate dissimilar floristic composition. the unclearly separated plots in kubang pasu and setul proved that kubang pasu formation is overlain by setul formation (basir & zaiton 2002). therefore, these habitats shared similar needs for mineral nutrient elements and other edaphic variables. table 2 summary of the detrended correspondence analysis (dca) of the vegetation data in 18 plots at the perlis state park, perlis axes 1 2 3 4 total inertia eigenvalues 0.758 0.464 0.393 0.174 6.527 length of gradient 5.758 3.879 3.012 2.509 cumulative percentage variance of species data 11.6 18.7 24.7 27.4 sum of all eigenvalues 6.527 biotropia vol. 27 no. 1, 2020 74 -1 6 -1 4 figure 2 dca ordination diagram of plots based on the abundance of tree species at perlis state park, perlis notes: x = plots in granite; ○ = plots in setul; + = plots in kubang pasu. based on the cca analysis, the species– environment correlations were high on the first and second axes with values of 0.988 and 0.974, respectively (table 3). the eigenvalue was 0.561 for the first axis and 0.444 for the second axis. additionally, the cumulative variation explained by the first three axes of the speciesenvironment relationship was 55.2%. the monte-carlo permutation test also indicated a significant difference on the eigenvalues among the ordina-tion axes (p = 0.002). the cca ordination plot showed the approximate locations of sample plots and the locations, lengths and directions of soil chemical variables (fig. 3). plots in granite (1-4) were clustered together and associated with available phosphorus. meanwhile, setul and kubang pasu plots did not cluster into the well-defined group. their plots were mostly assembled on the bottom right part of the diagram and were strongly correlated with several soil variables, i.e., magnesium (mg), potassium (k), calcium (ca), ph, available nitrate-n (no3) and available ammonium-n (nh4). the cca biplot of species and soil variables showed the species occurrence in relation to soil variables (fig. 4). most species were clumped on the centre part of the diagram and correlated with several soil variables such as available phosphorus (p) and available ammonium-n (nh4). some species such as antidesma cuspidatum (47) were clearly associated with calcium, whilst ficus annulata (102) and duabanga grandiflora (88) were strongly associated with ammonium-n (fig. 4; table 4). furthermore, hopea ferrea (34) had strong association with sodium. aglaia affinis (93) had strong association with magnesium, whilst bombax valetonii (23) was strongly associated with potassium and magnesium. however, alstonia macrophylla (19), terminalia subspathulata (30), senna timorensis (87), ficus aurata (103) and colona merguensis (155) showed a weak association with soil factors when they are farther away from all the soil gradients (fig. 4; table 4). table 3 summary of the canonical correspondence analysis (cca) of the vegetation and soil chemical properties in the 18 plots at perlis state park, perlis axes 1 2 3 4 total inertia eigenvalues 0.561 0.444 0.356 0.301 4.238 species-environment correlations 0.988 0.974 0.961 0.987 cumulative percentage variance of species data 13.2 23.7 32.1 39.2 cumulative percentage variance of species-environment relation 22.7 40.8 55.2 67.5 sum of all eigenvalues 4.238 sum of all canonical eigenvalues 2.464 association of tree communities with soil properties – zakaria et al. 75 the variations in species distribution within the study plots were strongly correlated to edaphic factors and vegetation distribution pattern. calcium and ph were some factors that strongly influenced the vegetation pattern in the limestone study area (setul and kubang pasu), whereas sodium and phosphorus strongly affected the vegetation pattern in granite area. soils which developed on granite are low in calcium and magnesium (burnham 1974), whereas soils that developed on limestone parent material such as in setul and kubang pasu are mainly high in calcium and ph (gauld & robertson 1985). -1.5 1.5 -2 .0 3 .0 php ca mg na k no3 nh4 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 figure 3 cca ordination plot showing the approximate locations of sample plots and location, length and directions of soil variables notes: x = plots in granite (1-4); ○ = plots in setul (5-12); + = plots in kubang pasu (13-18). -0.8 1.0 -1 .0 1 .0 1 8 9 12 16 19 23 30 31 34 37 40 47 56 58 62 66 75 83 87 88 90 91 92 93 101 102 103 105 109 110 115 132 134 138 143 144 147 155 157 161 php ca mg na k no3 nh4 figure 4 canonical correspondence analyses of the biplot for tree species and soil variables at the perlis state park note: numbers denote the species in the plots as listed in table 4. biotropia vol. 27 no. 1, 2020 76 table 4 list of species with code numbers referring to diagram in fig. 4 species code species species code species 1 alangium kurzii 61 sapium baccatum 2 boea oppositifolia 62 sauroupus suberosus 3 buchanania arborescens 63 trigonostemon villosa 4 dracontomelon dao 64 lithocarpus cantleyanus 5 gluta elegans 65 casearia capitellata 6 gluta velutina 66 homalium longifolium 7 parishia insignis 67 hydnocarpus castanea 8 pentaspodon curtisii 68 hydnocarpus curtisii 9 semecarpus cochinchinensis 69 hydnocarpus filipes 10 semecarpus curtisii 70 osmelia maingayi 11 swintonia floribunda 71 garcinia eugeniifolia 12 alphonsea curtisii 72 garcinia hambroniana 13 goniothalamus tenuifolius 73 garcinia nigroleniata 14 mitrephora maingayi 74 garcinia parvifolia 15 orophea cuneiformis 75 kayea kunstleri 16 polyalthia glauca 76 mesua ferrea 17 xylopia ferruginea var. oxyantha 77 stemonurus malaccensis 18 xylopia magna 78 cryptocarya ferrea 19 alstonia macrophylla 79 cryptocarya rugulosa 20 kibatalia maingayi 80 litsea machilifolia 21 ilex cymosa 81 barringtonia scortechinii 22 radermachera glandulosa 82 abdulmajidia rimata 23 bombax valetonii 83 leea aequata 24 durio lowianus 84 callerya artropurpurea 25 canarium littorale f. rufum 85 cynometra malaccensis 26 dacryodes rubiginosa 86 saraca cauliflora 27 dacryodes rugosa 87 senna timoriensis 28 lophopetalum javanicum 88 duabanga grandiflora 29 parastemon urophyllus 89 lagerstroemia floribunda 30 terminalia subspathulata 90 lagerstroemia ovalifolia 31 tetrameles nudiflora 91 memecylon caeraleum 32 dipterocarpus costatus 92 memecylon minutiflorum 33 dipterocarpus fagineus 93 aglaia affinis 34 hopea ferrea 94 aglaia argentea 35 hopea latifolia 95 aglaia simplicifolia 36 parashorea stellata 96 aglaia spectabilis 37 shorea macroptera 97 aphanamixis polystachya 38 shorea siamense 98 chisocheton ceramicus 39 vatica cinerea 99 chisocheton patens 40 diospyros andamanica 100 chukrasia tabularis 41 diospyros buxifolia 101 artocarpus dadah 42 diospyros scortechinii 102 ficus annulata 43 diospyros sumatrana 103 ficus aurata 44 diospyros venosa 104 ficus fistulosa 45 elaeocarpus rugosus 105 ficus microcarpa 46 erythroxylum cuneatum 106 ficus oligodon 47 antidesma cuspidatum 107 ficus sundaica 48 aporosa aurea 108 ficus variegata 49 baccaurea griffithii 109 streblus elongatus 50 chondrostylis kunstleri 110 streblus ilicifolius 51 cleistanthus hirsutulus 111 streblus macrophyllus 52 croton argyratus 112 streblus toxoides 53 croton cascarilloides 113 horsfieldia polyspherula 54 croton laevifolius 114 honsfieldia sucosa 55 dimorphocalyx muricatus var. minor 115 horsfieldia tomentosa 56 drypetes longifolia 116 knema laurina 57 koilodepas longifolium 117 knema patentinervia 58 macaranga andamanica 118 ardisia crassa 59 macaranga lowii 119 ardisia pachysandra 60 mallotus peltatus 120 maesa ramentacea association of tree communities with soil properties – zakaria et al. 77 tabel 4 continued species code species species code species 121 syzygium cerasiforme 141 pometia pinnata 122 syzygium glaucum 142 xerospermum noronhianum 123 ochna integerrima 143 chrysophyllum roxburghii 124 galearia fulva 144 palaquium hexandrum 125 galearia maingayi 145 palaquium microphyllum 126 xanthophyllum affine 146 eurycoma apiculata 127 xanthophyllum griffithii 147 pterocymbium javanicum 128 prunus grisea 148 pterospermum javanicum 129 aidia densiflora 149 pterospermum pectiniforme 130 diplospora malaccensis 150 pterygota alata 131 ixora pendula 151 sterculia cordata 132 morinda elliptica 152 sterculia gilva 133 psydrax sp. 7 153 sterculia parviflora 134 psydrax sp. 8 154 tetramerista glabra 135 dimocarpus longan ssp. longan 155 colona merguensis 136 guioa bijuga 156 grewia viminea 137 harpullia cupanioides 157 pentace strychnoidea 138 nephelium costatum 158 schoutenia accrescens 139 nephelium lappaceum 159 teijsmanniodendron coriaceum 140 paranephelium macrophyllum 160 vitex pinnata 161 vitex siamica conclusion the differences in floristic patterns between limestone (setul and kubang pasu) and granite habitats at psp suggested that environmental gradients influenced floristic composition. the soil properties such as organic matter, moisture, clay, silt, calcium and ph were among the factors that strongly associated with the limestone habitats, while sand and available phosphorus were strongly correlated with granite. differences in the availability of elemental mineral nutrient between the soils of granite and limestone may have also contributed to the contrasting vegetation. in relation to soil properties, the different habitats displayed varied spatial distributions among the tree species. identifying the key underlying gradients, abiotic conditions and major soil influences on the vegetation pattern is essential to gain information about the ecology of particular species and to formulate plans to protect and conserve this fragile habitat. acknowledgements the authors would like to thank the perlis forestry department, malaysia for the cooperation and support throughout the study. 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hanum i. 2015. floristic, diversity and structure of tree communities in three distinct forest habitats of perlis state park, perlis, peninsular malaysia. j sci 5(11):1016-23. biotropia vol. 29 no. 1, 2022: 47 55 doi: 10.11598/btb.2022.29.1.1623 47 genetic variability of arrowroot (maranta arundinacea l.) in yogyakarta province, indonesia based on issr analysis bernardinus danang krisna aji p.1 purnomo2,* and budi setiadi daryono3 1master’s program in biology, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2laboratory of plant systematic, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 3laboratory of genetics and breeding, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia received 29 july 2021 / accepted 3 february 2022 abstract the cultivation of arrowroot (maranta arundinacea l.) in indonesia, particularly in yogyakarta province has a great potential to be developed. this study aimed to determine genetic variability and analyze the intraspecific relations of arrowroot using inter simple sequence repeats (issr) markers to be used as basic information in considering characters selection of arrowroot cultivation in yogyakarta. exploratory survey method was conducted in gunungkidul, kulon progo, sleman, and bantul districts to collect cultivar accessions. accessions were replanted in sawitsari research station. dna isolation from the leaves of 7-month-old accessions was carried out using ctab buffer solution. the dna fingerprinting analysis was carried out using the result of dna amplification with 4 issr markers. the polymorphism data were then used for phenetic analysis using upgma algorithm and baroni-urbani busser similarity coefficient to form a dendrogram. a total of 5 local cultivars were found, identified as ‘sili’, ‘sembowo’, ’sugo’, ‘kebo’, and ‘teropong’. each cultivar showed distinct rhizome morphological characteristics. the issr-pcr analysis resulted in high polymorphism with a 68.17% polymorphism mean. the mean of polymorphic band was 6.75. the dendrogram was developed based on the analyses and consisted of 4 clusters with 80% similarity index. cluster a, b.i. and b.ii.b consisted of ‘sili’, ‘teropong’, and ‘kebo’ cultivars, respectively, while cluster b.ii.a gathered ‘sugo’ and ‘sembowo’ cultivars. keywords: cluster analysis, intraspecific classification, issr markers, polymorphism introduction arrowroot cultivation in indonesia has a great potential to be developed. flour made from arrowroot rhizome has high economic and nutritional values (madineni et al. 2012; guilherme et al. 2017). arrowroot flour contains high protein, even greater than that of cassava flour (aprianita et al. 2014), low glycemic index value and high fiber content. therefore, food products made from arrowroot flour are easier to digest compared with those made from other types of flour (lestari et al. 2017). food products made by arrowroot fluor is also recommended for people with digestive disorder and the elderdy (silva et al. 2000; heredia-zárate & vieira 2005; mason 2009; silveira et al. 2013) furthermore, arrowroot flour has long been used traditionally by the household and by the food industries as a basic ingredient for baby food. further research revealed that arrowroot flour is potential to be used as a thickening agent in various industries, such as cosmetics, pharmaceutical and food industries (kitahara et al. 2007). in addition to these benefits, the widespread arrowroot population in indonesia, especially in yogyakarta province, is the main reason for developing arrowroot cultivation (djaafar et al. 2010; masitoh 2014). information obtained from molecular analysis of a plant population can identify genotype differences among individuals as a selection step in plant breeding programs. an efficient character selection process by relying on the information about the genetic variation of a wild plant population with a suitable *corresponding author, email: purnomods@ugm.ac.id biotropia vol. 29 no. 1, 2022 48 breeding method has succeeded in developing modern cultivars, which has increased the yield of various food crops since the mid-20th century. molecular markers applied to reveal genetic variabilities can thus, provide promising information in agriculture as well as in protecting crop variabilities (govindaraj et al. 2015; perez-de-castro et al. 2012; faraldo et al. 2003). inter simple sequence repeat (issr) is one of molecular markers commonly used in the study of genetic diversity, phylogeny, gene detection, genome mapping and evolutionary biology in various types of plants (reddy et al. 2002). the combination of using issr in pcr has been done in genetic fingerprinting (blair et al. 1999), cultivar identification (wang et al. 2009), phylogenetic analysis (gupta et al. 2008) and assessment of hybridization (wolfe et al. 1998). the issr marker uses a 16-25 bp single primer that is specific to target identical regions among microsatellites. primers composed of eight repeating dinucleotide units, six repeating trinucleotide units or several repeating tetraor pentanucleotide units with or without nucleotide anchorage system that targets the microsatellite region (zietjiewicz et al. 1994). various types of molecular markers were used to reveal the genotype diversity of arrowroot, especially between the local varieties and cultivars. two of the molecular markers are rapd and issr markers (pinto 2015; asha et al. 2015). this study aimed to determine genetic variability and analyze the intraspecific relationship of arrowroot population in yogyakarta province, indonesia by using the issr markers. the expected results were the diversity of arrowroot germplasm in indonesia which can be used to strengthen the phenotypic characters variations that have been used in arrowroot cultivation and breeding. materials and methods plant materials sample collection was carried out during the dry season, from august to september 2019 in 18 subdistricts in sleman, gunungkidul, kulon progo, and bantul districts in yogyakarta province using the exploratory survey method. the whole arrowroot rhizome was taken from each accession and then replanted in the sawitsari research center plantation, faculty of biology, universitas gadjah mada, yogyakarta. species identification was carried out using the key of determination from van steenis (1975), woodson and schery (1980), wu and kennedy (2000), hammel et al. (2014), lim (2016). meanwhile, the cultivar identification was carried out through interviews with arrowroot farmers. fresh leaf from each 7-month-old accession was used for dna extraction. dna isolation genomic dna from the finely ground leaves (0.5 g) was extracted using the modified ctab method (deswina et al. 2019). extraction buffer consisted of 50 mm tris-hcl (ph 8), 0.7 m nacl, 0.1% β-mercaptoethanol, 10 mm edta, 0.1% cetyltriethylammonium bromide (ctab). the dna quality was checked in an agarose gel through the electrophoresis and then quantified by measuring the od at 260 nm and 280 nm through the spectrophotometry. purity of dna sample was calculated from od at a 260/280 ratio. issr analysis in this recent study, a total of 4 issr primers were used for analysis. the primers are ubc 811, ubc 827, ubc 818 and ubc 825 (table 1). table 1 issr markers details markers sequence (5’ 3’) annealing temperature (oc) size range (bp) ubc 811 (ga)8c 53 300-2,000 (asha et al. 2015; kambale et al. 2018) ubc 827 (ac)8g 52 300-1,600 (asha et al. 2015; kambale et al. 2018) ubc 818 (ca)8g 45 350-1,600 (asha et al. 2015) ubc 825 (ac)8t 52 400-2,000 (asha et al. 2015; kumar et al. 2010) genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 49 the extracted dna was amplified in a thermal cycler (bio-rad). the reaction mixture consisted of 8.5 µl ddh2o, 2.0 µl of each primer, 12.5 µl mytaq polymerase and 2 µl dna sample. the total reaction volume was made up to 25 µl using sterile distilled water. pcr was carried out in a master cycler gradient with a certain temperature setting (table 2). table 2 pcr amplification setting (asha et al. 2015) steps temperature (oc) time (minutes) cycles pre-denaturation 94 4 1 denaturation 94 1 annealing 45-55 0.5 40 extention 72 1 post extention 72 4 1 hold 4 ∞ 1 the annealing temperature was standardized for each primer. the results of amplification were separated by gel electrophoresis in 2% agarose gel with 1x tbe buffer and were imaged using gel documentation with observation through uv vis transilluminator. data analysis the analysis was carried out only with the unambiguously and reproducibly amplified issr bands. those dna bands were scored as present (1) or absent (0). smeared and weak bands were excluded. the resulting binary data matrix was analyzed using mvsp (multivariate statistical program) version 2.0. the analysis aimed to examine genetic relationship among accessions by estimating the similarity index using the baroni-urbani busser similarity coefficient. cluster analyses were carried out on the same similarity coefficient with the unweighted pair-group method of average (upgma) (rohlf 2000). results and discussion plant collection and identification identification of local cultivars succeeded in revealing the existence of 5 cultivars, namely 'sembowo', 'sili', 'sugo', 'kebo', and 'teropong'. the 'sili' and 'sembowo' cultivars were found to be widely distributed in 4 districts (table 3). dna extraction dna extraction using the ctab method was carried out on the accessions 'sembowo' (represented by seng1), 'sili' (represented by sipe3), 'sugo' (represented by subr1), 'teropong' (represented by teng1) and 'kebo' (represented by keke1). the quantity of isolated dna was tested by using spectrophotometry and showed a fairly high level of purity in four of the five accessions. keke1 accessions were known to be contaminated, but based on the results of the electrophoresis tests, this dna sample could still be used. results of dna concentrations varied. each accession had a good dna concentration and met the standards. the results of the spectrophotometric test are shown in table 4. table 3 sampling collection no sampling location accessions type accessions code district subdistrict village, subvillage 1 bantul sedayu argodadi, brongkol ‘sili’ (besar) sibb1 2 bantul sedayu argodadi, brongkol ‘sili’ (kecil) sikb1 3 bantul sedayu argodadi, brongkol ‘sili’ (bengkok) sieb1 4 bantul sedayu argodadi, brongkol ‘sugo’ subr1 5 sleman pajangan triwidadi, ngincep ‘sembowo’ seng1 6 sleman pajangan triwidadi, ngincep ‘sili’ sing1 7 bantul pajangan triwidadi, ngincep ‘teropong’ teng1 8 bantul pleret wonolelo, kedungrejo ‘kebo’ keke1 9 gunungkidul wonosari gari, gondangrejo ‘sembowo’ sego2 10 kulon progo pengasih pengasih, pengasih ‘sembowo’ sepe3 11 kulon progo pengasih pengasih, pengasih ‘sili’ sipe3 12 kulon progo pengasih sendangsari, gegunung ‘sembowo’ sege3 13 kulon progo pengasih sendangsari, gegunung ‘sili’ sige3 14 sleman moyudan sumberagung, kaliduren iii ‘sembowo’ sekd4 15 sleman prambanan sumberharjo, sengir ‘sembowo’ sese4 biotropia vol. 29 no. 1, 2022 50 table 4 spectrophotometry results accession code od 260 od 280 od at 260/280 ratio concentration (ng/µl) seng1 7.03 3.67 1.929 365.52 sipe3 2.54 1.35 1.898 127.32 subr1 4.62 2.43 1.917 231.11 keke1 5.57 9.95 1.371 678.87 teng1 3.82 1.95 1.970 191.00 each sample of isolated dna concentrations showed various values ranging from 127.32 ng/µl to 678.87 ng/µl. the results indicated that isolated dna from 'sili' leaves produced the lowest concentration. however, the results also showed a fairly high absorbance ratio value of 1.898 at od ratio 260/280. the od value shows the purity of the dna sample. there was only dna sample of the local cultivar 'kebo' which had a very low absorbance ratio value of 1.371 at od ratio 260/280. the quantity of dna concentration and quite low purity could be caused by polyphenol contamination. the quality of the dna sample from ‘kebo’ cultivar was still good and clearly showed the dna bands. the absorbance purity ratio of 260/280 is a very important measurement for estimating polyphenol contamination in the extracted dna samples. ratio of a260/a280 below 1.8 shows a poor dna extraction result and is not recommend for molecular analysis (sambrook & russell 2001). a high purity ratio of a260/a280 also indicates the purity of dna from rna contamination (koetsier & cantor 2019). the use of high enough mercaptoethanol can replace the role of rnase in degrading and removing rna from dna samples, even though this reducing agent is toxic and pungent (mommaerts et al. 2015). isolation of plant dna using the ctab protocol has many advantages and is proven to produce more sample volumes with high dna concentrations. ctab buffer solutions generally play a role in damaging cell structures, from cell walls to cell membranes and also the nuclear membrane. this is solely done to release the genetic material from the nucleus (amani et al. 2011). ctab buffer solution contains 2-βmercaptoethanol which has been shown to completely remove polyphenol components in cells (horne et al. 2004; li et al. 2007). in this study, a buffer solution with a concentration of 0.1% mercaptoethanol was used and proved to be optimal in removing polyphenol contamination. meanwhile, another study revealed that the use of 0.3% mercaptoethanol could improve the quality of dna pellets from precipitation (suman et al. 1999). analysis of issr-pcr molecular analysis used four issr markers, namely ubc 811, ubc 818, ubc 825 and ubc 827. pcr temperature optimization for these four markers resulted in different optimum annealing temperatures between ubc 827 & ubc 825 and ubc 818 & ubc 811. ubc 827 and ubc 825 markers require an annealing temperature of 50 °c, while the other two markers require an annealing temperature of 55 °c. electrophoresis of pcr-issr results showed variations in the number of dna bands, polymorphic bands and different monomorphic bands among issr markers (table 5). table 5 number of dna bands and polymorphism (%) in each molecular marker issr markers sequences (5’3’) total dna bands total polymorphic dna bands polymorphisms (%) ubc 811 (ga)8c 11 8 72.7 ubc 818 (ac)8g 8 4 50 ubc 825 (ca)8g 10 8 80 ubc 827 (ac)8t 10 7 70 average 9.75 6.75 68.17 genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 51 visualization of the electrophoresis results using the gel-doc transilluminator showed variations among these molecular markers (fig. 1). the average polymorphism appeared was 68.17% with the mean number of polymorphic bands of 6.75 and the average number of dna bands of 9.75. ubc 811 primer produced the highest number of dna bands, while ubc 811 and ubc 825 primers produced the highest number of polymorphic dna bands with eight bands. amplification using ubc 825 primer resulted in the highest polymorphism at 80%. electrophoresis using a dna ladder with a size of 100 bp and a buffer solution of 2% tbe 1x showed that the size of the dna bands in each primer was different, but there were several monomorphic bands with the same size between one marker and another. the dna bands were 750 bp, 600 bp, and 450 bp. overall, the size of dna bands appeared to be ranging from 180 bp to 1,000 bp. meanwhile, the polymorphic bands showed by each marker were diverse. in ubc 811, polymorphic dna bands were found to have sizes of 200-300 bp, 300-400 bp and 800 bp. ubc 818 primer amplification resulted in the lowest polymorphism value, with only 50% polymorphism showed by the dna bands 250 bp, 650 bp and 500-600 bp. issr marker is an ideal genetic marker for various studies, most notably on genetic variation (shafiei-astani et al. 2015) and dna fingerprinting (shen et al. 2006). as a good genetic marker, issr produces high genetic variability and shows multilocus data due to the use of the highly variable microsatellite sequences that are ubiquitously distributed across the genome (anne 2006; tautz & renz 1984; wolff et al. 1995). the addition of one or more nucleotide anchors in issr marker to target the end of the microsatellite region can prevent primer dimerization (bani et al. 2017). based on comparison with other molecular markers, the issr marker has higher reproducibility compared to the random amplified polymorphic dna (rapd). the issr marker is more time and money efficient compared to the amplified fragments length polymorphism (aflp) (phong et al. 2011; ng & tan 2015). electrophoresis of issr-pcr products generally uses 1.5-2.0% agarose gel by weight/volume (w/v) in order to achieve good dna band separation. when the agarose gel concentration is higher, reaching 3% w/v or more, the gel will break more easily as it hardens (ng & tan 2015). according to bornet & branchard (2001), 2.0% w.v agarose gel showed the best performance in resolving the issr band compared to other concentrations of agarose gel. figure 1 amplification results notes: amplification with 5 accessions (seng1 (lane 2); sipe3 (lane 3); teng1 (lane 4); keke1 (lane 5); and subr1 (lane 6)) using issr markers (ubc 811 (group a); ubc 818 (group b); ubc 825 (group c); and ubc 827 (group d)) with 100 bp dna ladder (lane 1). biotropia vol. 29 no. 1, 2022 52 plant systematic studies of several plant taxa based on the molecular characteristics using issr-pcr revealed a greater number of polymorphic loci than the use of rapd-pcr, because the abundant formation of issr primer target sequences throughout the eukaryotic genome, which evolves rapidly (ansari et al. 2012; phong et al. 2011; moulin et al. 2012). previous study on genetic variability of arrowroot in yogyakarta using rapd markers showed a low polymorphism and required further study (setyowati 2013). in this recent study, the use of issr was proven to show the genetic variability of arrowroot in yogyakarta. therefore, issr marker proved to be better and more suitable in the analysis of arrowroot genetic variability than rapd. issr is a recent genetic marker that overcomes many technical limitations in other methods such as rflp and rapd analyses. issr markers have higher reproducibility than that of rapd and have been successfully used to estimate the extent of genetic diversity at intra and inter-specific levels in a wide range of crop species (asha et al. 2015). hence, the issr markers were used in our study to assess the genetic diversity of arrowroot. the amplification results of the 4 primers with 7 arrowroot accessions in india showed high polymorphism values (asha et al. 2015), which were in agreements with the results of this study. however, the number of total dna bands and the average percentage of polymorphisms obtained were lower than the previous research conducted by asha et al. (2015). the differences were due to genetic diversity among plant population influenced by geographical condition and also appeared as molecular characteristics of each sample. based on both studies, the polymorphic dna bands obtained from issr analysis ranged from 10 to 60 fragments from various loci. the use of issr ubc 825 primer in this study resulted in the highest polymorphism values and the greatest number of polymorphic dna bands. this showed the effectiveness and the high reproducibility of these primers in the analysis of genetic diversity of arrowroot plants. the primary use of ubc 825 in the previous study conducted by asha et al. (2015) was also known to produce the greatest number of polymorphic dna bands and the highest polymorphism. based on the electrophoresis, ubc 825 indicated the presence of microsatellite dna sequences with sizes ranging from 800 bp to 180 bp. phenetic analysis based on molecular characteristics phenetic analysis based on the molecular characteristics can separate five local cultivars of arrowroot in yogyakarta into four clusters. dendrogram branching resulted in four clusters which were determined with the help of phenon lines at 80% similarity index. three of the four clusters were outliers, each consisted of accessions of sipe3, teng1 and keke1. one cluster consisted of accessions seng1 and subr1 (fig. 2). figure 2 dendrogram of arrowroot phenetic relationship based on molecular characteristics note: molecular characteristics were the result of dna fingerprinting analysis with issr markers, using the baroni-urbani buser similarity coefficient. genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 53 table 6 similarity matrix for cluster analysis of arrowroot based on molecular characteristics keke1 seng1 subr1 teng1 sing1 keke1 1 seng1 0.741 1 subr1 0.745 0.86 1 teng1 0.698 0.672 0.629 1 sing1 0.654 0.676 0.726 0.534 1 dendrogram branching was determined by the similarity matrix between accessions. the similarity values between one accession and the other accessions were different (table 6). analysis using the baroni-urbani buser similarity coefficient showed that the accessions of subr1 and seng1 had the highest similarity coefficient, which was 0.86. the lowest similarity coefficient was shown by sing1 and teng1, which is 0.534. the dendrogram showed 4 branches (fig. 2). the first branch separated the sing1 accession, representative of the local cultivar 'sili', from the other four local cultivar accessions at 0.64 point. it indicated the high dissimilarity between ‘sili’ local cultivar with the other five local cultivars based on its molecular genetics. the genotypic characters obviously made distinct phenotypic characters of ‘sili’ local cultivars. based on interviews with arrowroot farmers, it was found that the 'sili' cultivar was not very attractive for cultivation because it produced rhizomes containing higher fiber and smaller size than the other four local cultivars rhizomes. the second branch of the dendrogram separated the cultivar of 'teropong' (teng1) from 'sugo', 'sembowo', and 'kebo' cultivars at 0.68 point. then, the keke1 accession separated from the cluster consisting of subr1 and seng1 accessions at 0.86 point. the highest similarity that showed by ‘sugo’ cultivar and ‘sembowo’ cultivar was supported by the similarity of rhizome morphologies from both cultivars, which information was provided by the farmers during the interviews. however, around 10% of dissimilarity index obtained based on molecular characteristics still showed great differences on the morphological and or anatomical characters. further studies of the use of issr markers in dna fingerprinting techniques, cultivation or revealing genetic variation can provide several advantages. genotype-specific issr markers can be sequenced to be used as a basis for the synthesis of sequence characterized amplified region (scar) primers, which can then be used to determine genotype taxonomy. in addition, markers associated with high agronomic value characters can be sequenced to be used as sequence tagged sites (sts) markers that are useful in cultivation and breeding programs. meanwhile, although the microsatellites that play a role in issr-pcr are probably nonfunctional, they are known to be associated with region coding. therefore, issr can be used to construct gene-rich regions (vijayan 2005). conclusion the study used 5 local cultivars of arrowroot (m. arundinacea l.) from four districts in the yogyakarta province consisting of five local cultivars namely 'sembowo', 'sili', 'kebo', 'teropong' and 'sugo'. molecular analysis using issr markers was successful in showing high polymorphism and explaining genetic variabilities in the five local arrowroot cultivars. the analysis of the phenetic relationship using the mvsp program, the upgma algorithm method and the baroni-urbani buser similarity coefficient showed the formation of four clusters on the dendrogram based on molecular characteristics. clustering based on molecular characteristics was able to separate local cultivars from one another, but showed a close relationship between the local cultivars 'sembowo' and 'sugo'. acknowledgments the author would like to thank the farmers who provided great assistance in collecting arrowroot accessions. references amani j, kazemi r, abbasi ar, salmanian ah. 2011. a simple and rapid leaf genomic dna extraction method for polymerase chain reaction analysis. iran j biotech 9:69. biotropia vol. 29 no. 1, 2022 54 anne c. 2006. choosing the right molecular genetic markers for studying 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marantaceae. in: wu zy, raven ph, editors. flora of china, vol 24, flagellariaceae through marantaceae. beijing (cn)/st. louis (us): science press/missouri botanical garden press. p. 27-30. zietjiewicz e, rafalski a, labuda d. 1994. genome fingerprinting by simple sequence repeat (ssr): anchored polymerase chain reaction amplification. genomics 20(1):176-83. biotropia vol. 28 no. 2, 2021: 128 140 doi: 10.11598/btb.2021.28.2.1180 128 sea ranching of holothuria atra: stocking density and time retno hartati1*, ambariyanto1, muhammad zainuri2, and widianingsih1 1department of marine science, faculty of fisheries and marine science, universitas diponegoro, kampus tembalang, semarang 50275, indonesia 2department of oceanography, faculty of fisheries and marine science, universitas diponegoro, kampus tembalang, semarang 50275, indonesia received 28 december 2018/accepted 28 january 2020 abstract strong market demand, uncontrolled exploitation and/or the inadequate fisheries resource management have caused the overexploitation of sea cucumbers. hence, sea ranching is suggested as an intervention to overcome, if not minimize, this problem. since stocking density is the most important consideration in sea cucumber rearing, this study is aimed at discovering the best stocking density for the ranching of holothuria atra. h. atra individuals were taken from the panjang island, jepara waters and reared in cages at the bottom of teluk awur waters, jepara with a density of 30, 20, or 10 individuals per cage measuring 2 × 2 × 1.8 m (with bottom area of 4 m2). the stocking of h. atra was carried out three times, starting from the time of cage installation, then at the second, and finally at the third months after installation. characteristics of the bottom sediment (i.e., chlorophyll a, b, phaeophytin, and total carotene) of the sea cucumber habitat and water quality in the cages were also measured monthly. this study showed that the growth of h. atra fluctuated. the low stocking density yielded a higher weight gain per individual than the high stocking density. the highest weight gain was observed at the stocking density of 10 individuals/cage in the second stocking month. the highest survival rate was recorded at stocking density of 30 individuals/cage (93%) in the third stocking month, considering that these sea cucumbers were only reared for three months. the highest mortality occurred at stocking density of 20 individuals/cage. low survival rate of 45% occurred at the first stocking time or after the fifth month of rearing. fission was observed among h. atra reared in the cages, resulting in smaller organisms. among the water quality parameters, the concentration of chlorophyll a, b, phaeophytin, and carotene in the sediment fluctuated with the rearing duration due to the feeding and bioturbation of sea cucumbers. the results of this study suggested that low stocking density of h. atra during the second stocking month yielded a higher growth rate. keywords: fission, growth, sea cucumber, sediment, survival introduction utilization of invertebrate fisheries resources have recently expanded in catch and value, worldwide (anderson et al. 2011). one increasingly harvested marine invertebrate species is the sea cucumber, locally known in indonesia as “teripang”, “trepang", “timun laut”, or “gamat” (hartati et al. 2015). strong market demand resulting in uncontrolled exploitation, and/or inadequate fisheries management have led to the overfishing of many marine species, including the sea cucumber (ambariyanto 2017). hence, to overcome this problem, sea ranching was suggested. sea ranching is essentially an effort to release/grow cultured or wild juveniles into the natural habitat and harvest them when they reach optimum commercial size (purcell et al. 2012a, b). some advantages of sea ranching include nominally lower inputs, as the processes between release and harvest are largely left to food availability in the habitat, and the minimum level of care in sea cucumber rearing. these practices were able to yield a marketable size of the sea cucumber. however, published studies corresponding author, email: retnohartati.undip@yahoo.com holothuria atra stocking density and stocking time – hartati et al. 129 on sea ranching of sea cucumber in indonesia is very limited. attempts at culturing sea cucumber is mainly grow-out practices, especially for h. scabra (pangkey et al. 2012). similar studies on the ranching of sea cucumber had been done in several countries mainly for h. scabra, such as; in philippines (juinio-meñez et al. 2012), australia (bowman 2012), and papua new guinea (hair et al. 2016). studies for other species were also done in ecuador and mexico for isostichopus fuscus (mercier et al. 2012) and portugal for h. arguinensis (domínguez-godino et al. 2015). consequently, to avoid the overexploitation of the natural populations of h. atra, further research on its sea ranching prospect is necessary. such study would also provide knowledge to ensure a better understanding and application of programs in marine conservation, population genetics, and connectivity patterns. a new trend emerging among indonesian fisherfolks is the cultivation of sea cucumbers in sea pens. sea ranching practices provide a way to restore damaged fisheries without having to legalize no-take zones or establish fishing rights for sea cucumbers. the sea ranching of sand fish h. scabra sea cucumber has given the fisherfolks additional activity i.e., rearing small sea cucumbers harvested from the wild in sea pens until they reach a marketable size (juiniomeñez et al. 2012; hair et al. 2016). among the recent published and unpublished data on sea cucumber from mariculture programs in the indo-pacific providing hatchery production, the use of juveniles (for experimental sea or pond farming, sea ranching, and/or stock enhancement reasons), and proponents information, very little came from indonesia (purcell et al. 2012a,b). this might be due to a lack of international publications on sea cucumber research in indonesia. moreover, for conservation purposes, no published work is available on the sea ranching of sea cucumbers in indonesia. with the declining natural stock of sea cucumbers (hartati et al. 2015), sea ranching technology has become urgent for the continuity of its production and conservation considering that h. atra, locally known as “teripang hitam”/loly fish, provides a source of protein for human consumption as well as bioactive molecules for marine pharmaceuticals. the species is also ecologically important for their sediment bioturbation and remineralization, and also demonstrate a specific reproduction scheme (i.e., asexual reproduction through natural fission). since stocking density plays a very critical role in aquaculture growth, the objectives of this study, therefore, was to determine the best stocking density and stocking time on the rearing of sea cucumber h. atra, and their specific effects on the species performance and characteristics of the bottom sediment in the cages used. materials and methods some 180 individuals of h. atra, weighing 100 150 g, were taken from panjang island, jepara waters. bottom cages measuring 2 × 2 × 1.8 m were installed in teluk awur waters, jepara with a geographic position of 06o37'43.8" s and 110o38'31.7" e. the habitat has a muddy sand substrate with sea grasses and seaweeds and is known to be a suitable location for the rearing of h. atra (hartati et al. 2017a). the stocking densities applied were 30, 20, and 10 individuals/cage (equivalent to 7.5, 5, and 2.5 individuals/m2), with three different stocking times i.e., when the cages were first installed, then at the second and the third months after installation. the stocking density applied in the study is based on the works of lavitra et al. (2010a) on h. scabra and xing et al. (2012) on apostichopus japonicas. stocking density is affected by behavioral performance due to competition for area and food. the sea cucumber weights were measured monthly to determine their growth performance and their numbers were counted at a determined time interval to calculate the survival rate. samples of bottom sediment were taken monthly to measure the biomass of the micro phytobenthic organisms through their photosynthesis pigment (chlorophyll a, b, phaeophytin; and carotene). biomass of microphytobenthos were measured as concentration of chlorophyll a, b based on jonge et al. (2012) and kuczynska et al. (2015), phaeophytin (method of montani et al. 2012) and carotene (method of androuin et al. 2018). water quality parameters (e.g., temperature, salinity, ph, and dissolved oxygen) biotropia vol. 28 no. 2, 2021 130 were also measured using a water quality checker in situ. the growth rate (weight gain) was calculated following xing et al. (2012): weight gain = w2 − w1 (1) w1 = weight of h. atra at tn-1 (grams) w2 = weight of h. atra at tn (grams) the survival rate was calculated as follows: sr (%) = (nt/n0) × 100% (2) nt = number of h. atra alive at the end of the experiment n0 = number of h. atra stocked at the beginning of the experiment results and discussion the growth and survival rates of h. atra weights of h. atra were measured at the beginning of the study and every month thereafter, until the study was completed. weight gains were influenced by both the stocking density and time. notably, high stocking density affected the weight of the reared sea cucumber. likewise, stocking on the second month after installation of the bottom cage also resulted in higher weight gains than at other time of stocking (fig. 1). the growth of h. atra was calculated based on weight gain at the end of the study. the low stocking density yielded a greater weight gain than did the higher stocking densities. the highest increase was observed at the density of 10 individuals/cage at the second stocking. the lowest weight gain was recorded at the stocking of 30 individuals/cage at the first stocking (fig. 2). stocking density remarkably affected the aquaculture growth. at all times of stocking, the lower stocking density (i.e., 10 and 20 individuals/cage) produced higher weight gains than the higher stocking density (e.g., 30 individuals/cage). the highest increase in weight (19.69 g) occurred at the stocking of 10 individuals/cage and the lowest (2.1 g) occurred at the stocking of 30 individuals/cage. stress due to high stocking density greatly affected the growth performance of the sea cucumbers h. arguinensis and h. mammata (domínguezgodino & gonzález-wangüemert 2018). stocking density had also adversely affected the growth of sea cucumber apostichopus japonicas, not by decreasing the water quality or by food competition, but rather through the role of density as an environmental stress factor (pei et al. 2012). this impact of high density led to greater variations in body weights, which also occurred in this study. stress caused by crowding or overcrowding can stimulate the endocrine system of smallsized individuals, increasing cortisol levels in coelomic fluid and, in turn, accelerating energy consumption, modifying the energy budget model, and ultimately playing a negative role in the growth and composition biochemistry of these individuals (pei et al. 2012). in this study, the growth was also lowered by the effects of fission, which made the body size smaller. at high enough densities, sea cucumbers stop growing. the density value at which sea cucumbers can no longer grow is called the critical biomass value (cbv) (lavitra et al. 2010a). the cbv for h. scabra was 650 g/m2, whereas that for juvenile h. atra is 250 g/m2 to 350 g/m2. cbv is strongly influenced by the availability of food. h. atra is a deposit feeder (asha et al. 2015; hartati et al. 2019b), so these organisms digest organic materials largely from sediments, which continue to decrease during maintenance. sea cucumbers mainly eat microphytobenthos (algal epiphyte) (paga´njime´nez et al. 2019) and bacteria (lavitra 2010b) and the presence of sunlight reaching the bottom of the waters will support rapid growth of microphytobenthos which in turn will support sea cucumber growth (mactavish et al. 2012). in this study, overcrowding caused fission which in turn slowed the growth. hence, this study suggests the use of optimum stocking density to prevent problems of overcrowding, which will result in low growth and/or variations in body size, deformation of body shape, and the phenomenon called "rotten stomach" (thickened stomach wall) (seeruttun et al. 2008). the stocking time generally influenced the growth of h. atra which for the first, second and third stocking periods, were reared for five, four, and three months, respectively. longer rearing of five months led to less weight gain of 2.1 12.52 g versus the second stocking time of holothuria atra stocking density and stocking time – hartati et al. 131 four months which recorded 13.1 19.69 g weight gain. the second stocking provided an opportunity for the cage sediments to be overgrown with the microphytobenthos thereby preparing the sediments for sea cucumber habitation. at the third stocking, the maintenance duration of three months presented an average weight increase ranging from 5.43 g to 8.86 g. b a biotropia vol. 28 no. 2, 2021 132 figure 1 average weight of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. figure 2 weight growth of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. the number of live h. atra differed according to stocking density. remarkable fluctuations occurred in the number of sea cucumbers calculated as survival rates that registered 100% at the 10 individuals/cage stocking density at the third stocking (fig. 3). this likely occurred because of the fission phenomenon (asexual reproduction) among the sea cucumbers reared in cages. the highest survival rate of h. atra occurred in the 30 individuals/cage stocking density, including 93% in the third stocking, which meant that sea cucumbers can be maintained for only three months (fig. 4). the lowest survival rate of 45% occurred at the density of 20 individuals/cage in the first stocking or in the fifth rearing month. during their rearing period in cages, the survival rates of sea cucumbers fluctuated. survival rate at the 30 individuals/cage stocking density was higher than that at the other stocking densities. the third stocking time also provided a high survival rate because rearing took place for only three months. the high survival rate might be caused by the existence of a fission process that increased the number of living individuals (fig. 3), but resulted in smaller physical sizes (fig. 2). c holothuria atra stocking density and stocking time – hartati et al. 133 figure 3 survival rates of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. a b c biotropia vol. 28 no. 2, 2021 134 figure 4 survival rate of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. table 1 number of h. atra fissions during cage rearing stoking time stocking density 30 20 10 month 0 8 11 2 month 1 8 9 8 month 2 8 10 5 total fissions 24 30 15 during its rearing period in cages with different stocking densities and stocking times, the sea cucumbers did participate in asexual reproduction or fission across all conditions (table 1). although most of these phenomena were not directly observed, the organisms that are results of fission can be distinguished from the original organisms (fig. 5). sea cucumbers naturally have a great potential to regenerate after the process of evisceration (expulsion of their internal organs). evisceration occurs due to physiological changes or in response to various external factors. during evisceration, sea cucumbers secrete a large portion of their internal organs and, within a certain period of time, such will grow back and continue to live normally (hartati et al. 2016). regeneration also occurs after the process of natural asexual reproduction by fission as well as during fission stimulation (hartati et al. 2016). this process produces new individuals or regeneration in the posterior or anterior part. fission is a naturally occuring process (fig. 5); first is the narrowing of the part that will divide into two portions (fig. 5a). after splitting (body fission), the new individual closes its wound in the division section (fission plane) (fig. 5b) and the initial regeneration appears as a bulge in the fission plane. then, regeneration of the body part happens (fig. 5c). ultimately, this fission process contributed to high survival rates but produced smaller individuals of sea cucumber. generally, in this study, the more densely populated cages experienced more fission than did the non-dense ones, but stocking time did not significantly affect the number of fission organisms (table 1). fission is a phenomenon that is highly dependent on population density (asha & diwakar 2015; asha et al. 2015; hartati et al. 2019a). in nature, fission is vital in maintaining populations of some types of holothurians to compensate for their mortality and migration (dolmatov 2014). holothuria atra stocking density and stocking time – hartati et al. 135 figure 5 sea cucumbers that are undergoing fission (a); those that have just undergone fission, where the cleavage wound has been closed (b); and the regenerated missing part of the body (c) note: → arrow mark k is the position of fission/fission plane and regeneration the biomass of the microphytobentic food sources of h. atra were measured as concentration of chlorophyll a, b, phaeophytin, and total carotene levels in the sediment (figs 6 & 7). the concentration trends of chlorophyll a, b, phaeophytin, and total carotene were almost the same in all cages; high in the first month of rearing, decreasing in the second and third months of rearing, increasing in the fourth month, and then decreasing again in the last or fifth month (figs 6 & 7). microphytobenthic or benthic microalgae are groups of photoautotrophic microorganisms that inhabit the surface layers of shallow aquatic ecosystems sediments such as diatoms, cyanobacteria, and other chlorophytes (paga´njime´nez et al. 2019). in shallow coastal waters, the microphytobenthos significantly contribute to the metabolic systems of primary producers (hardiso et al. 2013). these microphytobenthos include important food sources for meiofauna such as sea cucumbers (mfilinge & tsuchiya 2016; hartati et al. 2017a,b; paga´n-jime´nez et al. 2019). holothurians can assimilate low content of organic matter as live diatoms, bacteria and detritus by passing sediment through their gut system (tolon et al. 2015), thereby causing fluctuations in the concentrations of microphytobenthos (hartati et al. 2019b). the microphytobenthos biomass is measured as chlorophyll content in the sediments (du et al. 2017) and the ratio of total chlorophyll c and b to chlorophyll a can be a good indicator of the amount of diatomic and green algal biomass (cartaxana et al. 2016). a b c https://www.researchgate.net/profile/paulo_cartaxana?_sg%5b0%5d=6lvu8fypltbuqidrhjgvtjfryd4-ilb81vuxj9myqegscm11utvueceurbrvun2sdkjhbwc.ojkg9uwa7kzscjnmj2vdjbtfvtf2tppfhfytdgi7yspm2jmnfz1d25t1ynaujhb27zl5bmngbhtp-rea9nloag&_sg%5b1%5d=ab-ekfl8kpt3ciboo-qvandlajy_mpx_kep7-wpas5dkhrkavanhfjxz9drcregd55z-eou.wgm2xnnwemrdtdo_xkgoowamilljsiz3ipjjrpitsa0inmevjeusidps4llpjumcmxkmi5yw9vqueqguuicsia biotropia vol. 28 no. 2, 2021 136 figure 6 concentration of chlorophyll-a (a) and chlorophyll-b (b), in the cage sediments of h. atra reared at different densities figure 7 concentration of phaeophytin (a) and total carotene (b) in the cage sediments of sea cucumber h. atra reared at different densities bottom sediment is one of the basic components influencing sea cucumber habitat preferences. hence, studies of its characteristics are primarily important (slater & jeffs 2010). rocky sediments should be avoided because sea cucumbers live in mud or sand and remain in the organic material for its lifetime. h. atra is a deposit feeder that swallows sediment along with organic matter (robinson et al. 2013; asha et al. 2015) and the microbial composition in the intestine of these adult holothurians constituted a greatly diverse microorganisms, such as bacteria, viruses, protozoa, and fungi colonizing their intestines (paga´n-jime´nez et al. 2019). studies have also shown that sea cucumbers swallow marine animal feces, including even their own feces (barrio & tuya 2013). in shallow waters (<10 m depth), h. atra prefers sea grass habitats with sediments of 15% to 25% gravel and coarse sand (0.7 1.2 mm), but not mud (dissanayake & stefansson 2012). h. atra was also found mainly in sand-dominated habitats (hartati et al. 2017a). its preference for specific habitat characteristics seems to be related to the feeding and security needs of h. atra (hartati et al. 2017a,b). water quality parameters (e.g., temperature, salinity, ph, and dissolved oxygen) were also measured during the rearing of h. atra (fig. 8). water temperature is one most important parameter affecting growth and development of sea cucumbers as well as their distribution in the sea. temperatures in seawaters tend to be stable at 28 30 °c throughout the study period (fig. 8). the temperature between cages were not significantly different because the cages were close to one another. since h. atra is a tropical species, they perform optimally in the higher temperature environments between 23 31 oc (buccheri et al. 2019). the aestivation process (adaptation to high temperatures) has not been widely studied in h. atra, but the species was a b a b holothuria atra stocking density and stocking time – hartati et al. 137 observed to usually cover its body with sand during the day, which is thought to be a response to higher temperatures (hartati et al. 2019a). sea cucumbers are stenohaline invertebrates that are unable to tolerate a wide range of salinity. therefore, their settlement in river mouths, estuaries, and other bays where salinity falls below 10‰ during the monsoon season should be avoided. although there is a small river that goes into the teluk awur waters, it did not greatly affect the salinity around the cages which were located 500 m from the river mouth. salinity levels in the teluk awur waters ranging from 27.5‰ to 30.5‰ were still suitable for the sea cucumbers (fig. 8). besides temperature and salinity, the water ph can also affect h. atra. fluctuations in ph occur seasonally, in that the dry season is characterized by high water temperatures and low water movements/currents resulting in low ph values between 7.3 and 7.5, while ph increases to between 8.4 and 8.6 during the rainy season because of high water influx (gullian & preciat 2017). generally, vertebrates have the ability to regulate osmotic pressure and create very good acid-base conditions and therefore, are able to cope with changes in ph better than invertebrates which have a lower regulatory capacity (wittmann & portner 2013). however, information about the effects of ph on sea cucumber is still limited. figure 8 average temperature, salinity, ph, and dissolved oxygen of the sea water surrounding the cages of h. atra reared with different stocking densities sea cucumbers respire with their tentacles, skin, and respiration trees by consuming a significant amount of water and absorbing the dissolved oxygen inside. the hypoxia (minimum amount of oxygen) limit of h. leucospilota was 3 mg o2/l, wherein the sea cucumber released its cuverian tube to lower its energy needs in this aesthetic process (zamani et al. 2018). furthermore, the normal conditions of dissolved oxygen levels at >6 mg/l provide a biotropia vol. 28 no. 2, 2021 138 100% survival rate. for a. japonicas, dissolved oxygen was maintained at >5 mg/l (xie et al. 2013). during the rearing period in this study, the dissolved oxygen content in the cages ranged from 6 mg/l to 7 mg/l (fig. 8) and no signs of h. atra evisceration due to hypoxia was observed. sea cucumber rearing in bottom cages has been carried out previously for both h. scabra and stichopus hermannii with no food addition (purcell & agudo 2013; hartati et al. 2016). the maintenance of sea cucumbers with feeding greatly affected their growth performance (i.e., sea cucumbers grow faster when given additional feed) (yokohama 2013) and mahmoud et al. 2017). in this study, however, the h. atra were not given additional feed considering that the sediment/substrate would provide ample feed for the species. in sea ranching, the current sea cucumber management practices do not apply additional input so the existence of sea cucumbers depends largely on the availability of natural feed (purcell et al. 2012). therefore, choosing the right location for the rearing of h. atra is very crucial. conclusion h. atra reared at low stocking densities of 10 individuals/cage at the bottom cages of the teluk awur waters of jepara, exhibited higher growth and survival rates. stocking time in the second month after the cages were installed also resulted in higher growth, as the food organisms for the sea cucumber were already developed and thus, were made available. moreover, the chlorophyll a, b, phaeophytin, and carotene levels fluctuated during the time of sea cucumber rearing due to their feeding and bioturbation activities. acknowledgments the authors would like to thank seameobiotrop for partly funding this present work through the dipa biotrop 2018 of phd grant and universitas diponegoro for also funding the chemical analyses through the international publication research (rpi) grant of other than apbn lppm rkat universitas diponegoro year 2018-2019. our gratitude also goes to robertus tm, elsa la, fabian pa and mustagpirin for their help during the field work. references ambariyanto. 2017. conserving endangered marine organisms: causes, trends and challenges. in iop conference series: earth and environmental science, 55(1): 012002. iop 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vol. 27 no. 1, 2020: 41 50 doi: 10.11598/btb.2020.27.1.1030 41 the physicochemical properties of several indonesian rice varieties susiyanti1,5*, rusmana1,5, yeyen maryani2,5, sjaifuddin3,5, nanang krisdianto4,5 and mohamad ana syabana1,5 1department of agroecotechnology, faculty of agriculture, universitas sultan ageng tirtayasa, serang 42123, indonesia 2department of chemical engineering, faculty of engineering, universitas sultan ageng tirtayasa, serang 42435, indonesia 3department of science education, universitas sultan ageng tirtayasa, serang 42117, indonesia 4department of agribusiness, faculty of agriculture, universitas sultan ageng tirtayasa, serang 42123, indonesia 5 indonesia center of excellent for food security, universitas sultan ageng tirtayasa, serang 42123, indonesia received 15 march 2018 / accepted 20 december 2018 abstract rice has different varieties, with each variety possessing diverse physical and chemical characteristics. the objective of this study was to analyze the physicochemical properties of several indonesian local rice varieties. the experiment was conducted from march to april 2017 at the agriculture applied technology laboratory of the faculty of agriculture, universitas sultan ageng tirtayasa and at the laboratory of food analysis services in the department of food and technology, ipb university. nine local rice varieties from several areas in indonesia were used as samples, namely jalahawara, ciherang, pandan wangi, rojolele, sokan, bendang pulau, batang piaman, cisantana and sidrap. their physicochemical characteristics were analyzed based on some criteria such as: physical quality (weight, length, width, form and percentage of chalkiness), chemical content, water content, ash content, fat content, protein content, carbohydrate content, crude fiber content, starch, amylose and amylopectin content. data obtained were analyzed by one-way anova using a randomized block design. jalahawara has the highest percentage of chalkiness. based on the ratio of length and width, sidrap and ciherang were categorized as medium type and the others were oval/round. the heaviest and lightest based on the 1000grain weight of rice were ciherang and bendang pulau, respectively. the water content was about 2-4% for all samples. the highest and lowest amount of ash and fat content were found in sidrap and sokan, respectively. the highest and lowest amount of protein content were found in batang piaman and sokan, respectively. the highest and lowest starch content were observed in pandan wangi and ciherang. the content of amylose and amylopectin were the highest in batang piaman. the rice samples were categorized into two groups of low and medium levels of amylose. the low level of amylose were observed in cisantana, ciherang, pandan wangi and sidrap, while the medium level of amylose were observed in jalahawara, sokan, bendang pulau, batang piaman and rojolele. keywords: indonesian local rice, physicochemical properties introduction food, a basic need of human beings, is a source of energy for humans to do their activities and survive in life. the quality of both husked and un-husked rice becomes the priority of rice cultivation which is related to consumer satisfaction. more than half of world population consumes rice as the main daily source of calories and protein (tonini & cabrera 2011; rohit & parmar 2011). industrial development has affected the determination of rice taste and quality. a dominant factor in determining the taste of rice is amylose content in the rice (setyowati & kurniawati 2015). rice quality is a combination of its physical and chemical (physicochemical) characteristics particularly needed by consumers. rice tastiness is closely related to rice quality and its complex physicochemical characteristics, namely amylose content, starch, gel consistency, gelatin temperature and protein level. rice quality is also related to stickiness, sweetness, transparency and palatability. *corresponding author, e-mail: susiyanti@untirta.ac.id biotropia vol. 27 no. 1, 2020 42 palatability (good taste) is a characteristic of rice related to quality, determined by smell, appearance, taste and texture. in addition to genetic factors, such as those in the synthetic processing of starch and protein, rice quality is affected by environmental conditions, such as water availability, temperature, fertilizer utilization, drought and salinity stresses (chen et al. 2012). the physical and chemical properties of rice depend on the variety used (rolando et al. 2013). consumers have the right to information about physical characteristics and chemical content of consumable goods, including rice. sitaresmi et al. (2013) stated that a large amount of germplasm of good quality local rice variety had been identified as vulnerable and tolerant to biotic and abiotic stresses. the indonesian agricultural research and development agency had released tungro-resistant varieties, such as tukad unda, tukad petanu, tukad balian, bondojudo, kalimas, inpari 7 lanrang, inpari 8 and inpari 9 elo (suprihatno et al. 2009; mejaya et al. 2014). these variety had not been fully adopted by farmers because of its limited seeds availability, undesirable taste and poor quality which affected the selling price (muliadi et al. 2015). however, ciherang varieties have advantages in terms of its productivity, tastiness, market segments and relatively early maturity. new superior varieties are difficult to develop and market, if they do not have the previously mentioned potential characteristics. indonesia possesses a very wide rice diversity comprising around 17,000 germplasm accessions including several wild varieties (maulana et al. 2014). indonesia is the centre of rice origin. many places in indonesia still have good potential sources of the local rice seeds. each variety has different and unique characteristics, namely unique taste, color, nutrients and chemical composition (yang et al. 2010). hence, it is necessary to gather basic data related to the physicochemical properties of these local rice varieties, particularly related to plant cultivation processes that affects rice quality. the objective assessment of rice quality includes its physical and chemical characteristics, while the subjective assessment focuses on the individual and community preferences. the objective of this study was to analyze the physicochemical properties of several indonesian local rice varieties. materials and methods plant genetic materials nine varieties of the indonesian local rice were used as samples, namely java varieties (jalahawara, ciherang, pandan wangi, rojolele), sumatra varieties (sokan, bendang pulau, batang piaman) and sulawesi varieties (cisantana, sidrap). the research was conducted from march to april 2017 at the agriculture applied technology laboratory of the faculty of agriculture, universitas sultan ageng tirtayasa and food analysis laboratory, department of food technology, ipb university. methodology milled rice grains from 9 local rice varieties from several places in indonesia were used as research samples. their quality was identified based on the following criteria: physical quality, water content, ash content, fat content, protein content, carbohydrate content, crude fiber content, starch, amylose and amylopectin content. the data acquired were then analyzed using the one-way analysis of variance (anova) and a randomized block design for all the physicochemical observations. the mean values were compared using duncan's new multiple range test (dnmrt) to confirm the differences among the varieties. physical quality of rice physical quality of rice was analyzed according to utami et al. (2019). the weight of un-husked rice was measured by measuring the weight of 1,000 undamaged grains using analytical scales of each sample. moreover, the length and width of 10 rice grains from each sample were measured by using calipers. the rice form was determined by the ratio of length and width of the rice grain. furthermore, the percentage of chalkiness was obtained by observing at the opacity proportion of the endosperm. water and ash content the water and ash content were analyzed using the gravimetric method (longvah & prasad 2020). for water content, it was measured by comparing the weight of rice physicochemical properties of indonesian rice – susiyanti et al. 43 before and after being heated. the difference is assumed as the decreased water content in the local rice after being heated at 105 oc, above the boiling point of water. for ash content, an empty cup was heated in the oven and then cooled in the desiccator for 30 min. the cup was then filled with 5-gram rice of each sample. each sample was weighed and then heated in the furnace at 550 °c for 2-3 h until it turned into ash. the cup was then cooled in the desiccator and weighed subsequently. the percentage of ash content was calculated as follows: ash content (%) = [ash weight (g)/sample weight (g)] × 100%. fat content the fat content was measured using soxhlet method (sultana et al. 2018; choi et al. 2015). the fat was extracted using the non-polar fat solvents (hexane). the solvent was then distilled and evaporated; the fat extract stayed in the tube and was measured as fat weight. protein content the protein content was measured using the kjeldahl method (wang et al. 2016), assuming that all ns in the sample are protein. the observation process was done in three stages: first, destroying the sample in acid atmosphere to free the n from the sample then binding n in the form of ammonium sulfate, while c and h were oxidized into h2o and co2; second, distilling the sample to break the ammonium sulfate into ammonia based on the different boiling points; third, titration was done to measure the n content in the sample (legowo 2005). carbohydrate analysis carbohydrate analysis was done using “by difference” method (shakappa & talari 2016). the following is the equation used in calculating carbohydrate levels: carbohydrate content (%) = 100% (% water content + % ash content + % protein content + % fat content). analysis of crude fiber using the soxhlet method (sultana et al. 2018; choi et al. 2015), a 0.4 g of fat sample was extracted and then filtered with filter paper and wrapped. the filter washed with acid and alkali solvents (0.3 n naoh and 1.5 n h2so4) and placed in boiling water and was finally cleaned with distilled water. afterwards, the filter was washed again with solvent and heated to reach its boiling point, and then put in the oven for 1 hour at 105 °c, and finally, the crude fiber content was calculated. starch, amylose and amylopectin contents the starch content was analyzed according to the luff schoorl method (aoac 1995), using reagent luff schoorl from 0.1 g of ground rice sample which was then measured by the titration process. the starch content was measured based on the difference between titration on the blank paper and sample titration. sugar content was reduced after inversion (after hydrolysis using hcl 25%) inside the compound, which could be found in the table of standards. the starch content of the compound was calculated from the difference between the sugar content before and after inversion, then multiplied with 0.9. furthermore, the measurement of amylose content was conducted through iodometry based on the reaction between amylose and iodine compound which produced the blue color (juliano & villareal 1993). each sample was taken from 100 mg of rice dissolved with 1 ml of 95% ethanol and 9 ml of naoh 1 m and boiled for 10 min with water boiling at 95 c until it turned into a gel. then, 100 ml of water was added into the gel and stirred. five ml of sample solvent was homogenized with 1 ml of acid acetate 1 n, 2 ml of iodine solvent 0.01 n (step by step) and aquadest, then heated at 30 c for 20 min and measured with spectrophotometer uv-vis in 620 nm wavelength. amylopectin content was calculated by reducing the starch content with amylose, as starch basically consists of amylose and amylopectin. results and discussion determining the rice physical quality grain characteristics such as weight, length and width are prominent criteria in determining rice quality (table 1). the width, length, and shape of rice grain affect the ease of the grinding biotropia vol. 27 no. 1, 2020 44 process. jalahawara has the highest percentage of chalkiness compared to other rice and has small grains with a less transparent appearance. on the other hand, although its nutrients disappear after cooking and do influence the food quality, rice with transparent grain are still preferred by consumers. most rice consumers in principle, choose uniform and translucent grains (xi et al. 2014). rice consumers also prefer a product of satisfactory appearance even though most of the nutrients have disappeared (abbas et al. 2011). the genetic and environmental factors influenced the appearance of the rice grain which is assessed as the percentage of grain chalkiness. chalkiness is an unwanted trait that negatively affects the milling, cooking, eating, and grain looks and that reflects the main concern in many producing areas of rice (siebenmorgen et al. 2013). nevame et al. (2018) stated that the happening of the chalkiness in rice is linked to both the genetic and environmental element, notably high temperature. the highest percentage of chalkiness was found in jalahawara rice and the lowest in pandan wangi. based on its length, the local rice was classified into extra-long grain at >7 mm (cisantana, ciherang, sidrap), long grain at 6.06.99 mm (jalahawara, sokan, pandan wangi, rojolele) and medium grain at 5.5-5.99 mm (bendang pulau). based on the ratio of length and width, sidrap and ciherang rice were included in the medium size category, while the other types were categorized as short grain rice with oval or round shaped. most people in india prefer long grain, but people in south east asia prefer medium grain (jewell et al. 2011). furthermore, the heaviest 1000-grain weight was that of ciherang variety, followed by jalahawara rice, while the lightest weight was that of the bendang pulau rice. the weight of the 1000-grains of rice determined the amount of rice production. the weight of rice is affected by the environmental and atmospheric factors at the moment of grain harvesting. patindol et al. (2015) said that the environmental elements (such as air temperature, atmospheric carbon dioxide, light, water, and soil nutrients) are important for plant growth and reproduction, temperature is the most explored environmental aspect in relation to rice production and grain quality. table 1 chalkiness, grain weight, length, width and the ratio of length/width of indonesian rice varieties no rice sample % chalkiness 1,000 grains weight (g) length (cm) width (cm) ratio of length/width 1 jalahawara 89 a 24.715 b 0.655d 0.255 f 2.57 d 2 cisantana 25 f 19.575 f 0.769 a 0.261d 2.95 c 3 ciherang 62 c 25.098 a 0.742 c 0.221 i 3.36 a 4 sokan 35 e 16.890 h 0.617 g 0.254 g 2.43 e 5 bendang pulau 14 h 11.554 g 0.532 h 0.258 e 2.06 h 6 batang piaman 22 g 21.693 d 0.618 g 0.302 a 2.05 i 7 pandan wangi 5 i 19.596 f 0.624 f 0.266 c 2.35 f 8 rojolele 72 b 21.013 e 0.644 e 0.282 b 2.28 g 9 sidrap 56 d 21.995 c 0.744 b 0.222 h 3.35 b note: means with different letters inside a column are significantly different (p < 0.05, dmrt). physicochemical properties of indonesian rice – susiyanti et al. 45 figure 1 the appearance of indonesian local rice grains the opaque areas in the endosperm of the rice grains formed the starch and protein particles that break easily at grinding (fig. 1). compared to rice grains with transparent endosperm, their selling point is low. the different colors of rice are arranged genetically as a result of genetic differences of aleuron color, endosperm color and starch composition in the endosperm. white rice has white and transparent colors because it has only a little aleuron color and amylose content of about 20%. the outermost layer of the endosperm consists of aleurone cells and starch inside (ishimaru et al. 2015). the aleurone layer accumulates lipids, while the starchy endosperms mainly accumulate starch. during the ripening stage, the degree of starch accumulation is known to be out of sync, depending on the location of the starchy endosperm. water content based on the indonesian national standard no. 6128:2015 (bsn 2015), the maximum water content of premium rice is 14%, while that of medium rice is 15%. the water contents of the different rice varieties varied between 12% and 14%, and only jalahawara had the water content of above 14% (table 2); so almost all of the local rice samples are included under the category of premium rice. one of the benefits of water content standardization of the local rice is the prolonged rice storage time. if the rice has low water content, the development of microbes in the rice can be slowed down. moreover, moisture also plays an important role in determining the rice quality (tamrin et al. 2017). water content affects the physical and chemical appearance of rice. if the water content is high then the rice will turn yellow with an unpleasant aroma and cannot be stored for a long period. table 2 level of water, ash, fat, protein and carbohydrate contents of indonesian local rice varieties no rice sample water content (%) ash content (%) fat content (%) protein (%) carbohydrate (%) 1 jalahawara 14.44 a 0.54 g 0.24 h 9.41 e 75.36 h 2 cisantana 12.35 f 0.88 b 1.15 b 8.79 g 76.83 d 3 ciherang 12.68 d 0.80 c 0.66 f 9.98 b 75.88 e 4 sokan 13.01 c 0.45 i 0.21 i 7.32 i 79.01 a 5 bendang pulau 12.25 g 0.57 f 0.35 g 8.21 h 78.63 b 6 batang piaman 12.10 h 0.52 h 0.74 e 10.93 a 75.71 f 7 pandan wangi 13.71 b 0.72 e 1.06 c 9.68 c 74.84 i 8 rojolele 12.17 i 0.74 d 0.96 d 8.82 f 77.32 c 9 sidrap 12.38 e 1.00 a 2.12 a 9.57 d 74.94 g note: means with different letters inside a column are significantly different (p < 0.05, dmrt). biotropia vol. 27 no. 1, 2020 46 ash content the lowest ash content was found in sokan (0.45%), while the highest was in sidrap (1.00%). based on indonesian national standard no. 3549:2009 (bsn 2009), the maximum ash content of rice starch is 1%; hence, all the samples have fulfilled the indonesian national standard. ash content is related to the mineral content of rice because when rice is heated until 1,000 oc, only the mineral is not oxidized. ash content is also influenced by the environment where the rice grows. the composition of rice varieties differs according to the soil in which rice is grown, together with manuring (abbas et al. 2011). the minerals most frequently found in rice husk ash are p, mg, and k. in addition, it may consist of ca, cl, na, si and fe. the minerals are distributed over the outer layer of the rice grain, and the concentration decreases as they move towards the internal part of the rice. oko and ugwu (2011), said that the mineral compositions in rice are: n (0.1-1.08%), p (0.52-0.54%), k (0.15-0.20%), na (0.13-0.17%), ca (0.07-0.11%) and mg (0.19-0.26%). fat content the fat content varied from 0.21% to 2.12%. the large amount of fats (above 0.5%) was found in cisantana, ciherang, batang piaman, pandan wangi, rojolele and sidrap rice. the study results differed from that of tarigan and kusbiantoro (2011), which recorded a fat content between 0.2-0.3%. the amount of fat content in rice is influenced by the environment where the rice plant grows. kang et al. (2011) stated that the main component of fatty acid in rice was oleic acid (18:1), linoleate (18:2), and palmitate (16:0), and the composition of those three components reaches 90% of the total fatty acid in rice. protein content the lowest protein content was observed in sokan (7.32%) and the highest in batang piaman (10.93%). the highest amount of amino acids found in the rice protein were aspartate and glutamate, while the acids with the smallest amount were cysteine and tryptophan (kang et al. 2011). protein content has a close relationship with palatability or taste quality rice (song et al. 2012). protein also supports rice texture by making the barrier which results in less leaching of rice component during the cooking process. variation in protein content causes differences in the viscosity of rice paste due to its effect on the expansion of starch granules and on the character of its stiffness. protein plays an important role in rice quality based on a water-producing agent that occurs during cooking rice and that increases the stickiness of cooked rice. carbohydrate content carbohydrate was the most abundant nutrient in all the samples. the lowest (74.84%) was found in pandan wangi and the highest (79.01%) in sokan. carbohydrate content in rice is mostly in the form of starch, consisting of amylose and amylopectin. fiber is also found in rice, including hemicellulose (food fiber) and lignin (crude fiber). the shape and size of starch granules are affected by the source and the condition around the growing zone (omar et al. 2016). fiber content adequate amounts of dietary fiber are also found in rice grains (aune et al. 2011), thus rendering rice a healthy food (thomas et al. 2015). the crude fiber content varied from 8.91% to 24.51% among the rice varieties. the crude fiber content is influenced by the variety of rice. fiber is a plant component and a type of carbohydrate. fiber is not a nutrient and cannot be digested by the human system. based on its structural characteristics, fiber consists of two kinds, food fiber, and crude fiber. food fiber cannot be digested by amylose enzyme, while crude fiber can be hydrolyzed by acid and strong alkaline such as cellulose, hemicellulose, and lignin. starch, amylose and amylopectin content the starch content in the rice sample largely contributed (72.92% to 88.18%) to its dry weight (table 3). starch is composed of amylose and amylopectin (bertoft 2017). starch is one kind of carbohydrate in plants that functions as an energy reserve. starch in rice and other seeds is the major carbohydrate, so that the amount of starch is more prominent than other kinds of physicochemical properties of indonesian rice – susiyanti et al. 47 carbohydrate. the histochemical investigation of the caryopsis indicated that starch was mainly accumulated in the endosperm (yu et al. 2014). amyloplasts in endosperm contained many starch granules that were spherical during the early stages but turns polyhedric at the later stages. structurally, the starch arrangement of monosaccharide units is like glucose. glucose molecules are bonded with each other, producing two kinds of starch, amylose and amylopectin. amylose has a linear chain structure bound by α-(1,4)-d-glycoside bonds, while amylopectin has a branching structure linked by α-(1,6)-d-glycoside bonds that comprise around 4% to 5% of the total weight. the starch content varied depending on the rice variety and the environment where the rice grew. the highest amount of starch was found in pandan wangi (88.18%), and the lowest was in ciherang (72.92%). the amylose content was around 17% to 24%, and amylopectin was around 54% to 70%. the ratio of amylose/amylopectin is stated only in amylose content because amylopectin can be obtained from the reduction of starch by amylose. the ratio of amylose/amylopectin determines the texture of rice, whether hard or not, getting hard easily or not, sticky or not. the smaller the content of amylose or the higher the content of amylopectin, the stickier the rice will be. for example, when sticky or glutinous rice is cooked, physically it is really sticky because the amylose content is very low, around 1% to 2% (in waxy/glutinous rice). however, the amylopectin is very high. this study showed that the rice samples were not glutinous. non-sticky rice are classified into 4 groups based on its amylose content: 1) rice with high amylose content (25% to 33%); 2) rice with medium amylose content (20% to 25%); 3) rice with low amylose content (9% to 20%); and 4) rice with very low amylose content (2% to 9%) (masniawati et al. 2018). based on this classification, the rice samples in this study belonged to 2 groups; that is rice with low amylose content for cisantana, ciherang, pandan wangi and sidrap rice; and rice with medium amylose content for jalahawara, sokan, bendang pulau, batang piaman and rojolele. comparison between those two groups of starch (amylose and amylopectin) determines the color of rice (transparent or not) and also the texture of rice (sticky, soft, hard or really hard). sticky rice is dominated with amylopectin so that it is really sticky, while hard rice (‘pera’ rice) has an amylose content of more than 25% resulting in the seeds of rice not sticking to each other when they are well cooked. the main component of rice is starch, while the minor components are protein (4.5% to 10%) and fat (0.5%). amylose content determines the physicochemical characteristics of rice starch. starch granule which has high amylose is strong and stiff and will result in hard rice (‘pera’ rice). on the other hand, if the starch granule is soft, it can result in soft and tasty rice when it is well cooked (li et al. 2017). table 3 amounts of crude fiber, starch, amylose and amylopectin no rice sample crude fiber content (%) starch (%) amylose (%) amylose criteria amylopectin (%) 1 jalahawara 11.65 d 78.88 e 20.89 e medium 57.99 e 2 cisantana 24.51 a 77.39 g 18.31 h low 59.07 d 3 ciherang 18.67 b 72.92 i 18.48 g low 54.44 i 4 sokan 12.62 c 85.90 c 23.29 b medium 62.61 c 5 bendang pulau 8.91 h 87.39 b 23.19 c medium 64.20 b 6 batang piaman 9.39 g 79.25 d 24.61 a medium 54.63 h 7 pandan wangi 9.53 f 88.18 a 17.74 i low 70.45 a 8 rojolele 9.72 e 77.67 f 21.78 d medium 55.90 g 9 sidrap 11.73 d 76.74 h 19.30 f low 57.44 f note: means inside a column with different letters are significantly different (p < 0.05, dmrt). biotropia vol. 27 no. 1, 2020 48 conclusion the jalahawara variety was moderately transparent and had a high percentage of chalkiness compared to other varieties. the ratio of the length to width of rice grain for sidrap and ciherang varieties were considered mediumsized, and the other types as short/oval/round size. ciherang is the heaviest rice based on the 1,000-grain weight, and the lightest rice was bendang pulau. the water content in the sample of the rice was between 12-14%. the lowest ash content was found in sokan (0.45%) and the highest was in sidrap (1.00%). the fat content varied from 0.21% (sokan) to 2.12% (sidrap). the lowest protein content was found in sokan (7.32%), and the highest fat content was in batang piaman (10.93%). the highest starch content was observed in pandan wangi (88.18%), and the lowest is in ciherang (72.92%). amylose content among the varieties was around 17% to 24%, and amylopectin was around 54% to 70%. the studied local rice varieties were not sticky rice. the amylose content categorised the rice varieties into two; the first group included the varieties with low amylose content (cisantana, ciherang, pandan wangi, and sidrap); and, the second group included those varieties with medium amylose content (jalahawara, sokan, bendang pulau, batang piaman, and rojolele). acknowledgments the authors would like to express their gratitude to the islamic development bank who gave financial support as part of the research grant of the indonesia center of excellence for food security of universitas 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by scanning electron microscopy. plant prod sci 17(4):285-90. yang ds, lee ks, kays sj. 2010. characterization and discrimination of premium-quality, waxy and black pigmented rice based on odor-active compounds. j sci food agric 90(15):2595-601. doi: 10.1002/ jsfa.4126 yu x, zhou l, xiong f, wang z. 2014. structural and histochemical characterization of developing rice caryopsis. rice sci 21(3):142-9. biotropia no biotropia no. 12, 1999 : 19-24 the establishment of procecidochares connexa in west java, indonesia : a biological control agent of chromolaena odorata soekisman thtrosemito seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract kirinyu (chromolaena odorata (l.) r.m. king and h. robinson) was reported for the first time in 1934 from lubuk pakam north sumatera. it grows vegetatively during the wet season, flowers and sets a high number of fruits at the end of the wet season, and senesces during the dry season. it may be controlled manually by uprooting the weed or by slashing, and chemically by spraying with herbicides. however, this has not been successful. recently a biological control agent, procecidochares connexa was introduced at parung panjang, west java, as a biological control agent of c. odorata. this paper reports the successful establishment of procecidochares connexa. two releases of p. connexa colonies were made in parung panjang at the end of 1995. the colony was able to survive through a harsh dry season of 1997. when c. odorata was swept by fire, the emerging shoots soon were attacked by p. connexa. the population of c. odorata went down to 37.2% with 31.8% of its emerging shoots attacked by p. connexa in a 2-year period. when twigs were attacked by p. connexa, the production of cypsellas was reduced by about 50% in one season. however, the survival of emerging flies was also affected by the death of twigs upon maturation of the cypsellas. key words: biological controuchromolaena odoratalprocecidochares connexa introduction the first herbarium record in indonesia of chromolaena odorata was from lubuk pakam, north sumatera, reported in 1934 (tjitrosoedirdjo 1990). the specimen was identified as eupatorium repandum, but re-identified by van steenis as eupatorium odoratum, and is now called chromolaena odorata. it is a shrub, capable of growing up to 7 m tall when a tree is available to climb on. normally it forms a thicket of branches of 1.5-3.0 m height. when cut the stem will regrow. being a member of the compositae, flowers are arranged into clusters consisting of 30-35 uniform flower units, with 3-5 composit flowers at one terminal point. in an established community, flowers may number 3200/m2, to yield a total of 960 000/ha flowers at one time, so in one season 1 ha of c. odorata may produce more than 10 billion seeds. as a result c. odorata will spread very fast. gauitier (1993) estimated that the weight of each dried fruit is approximately 0.2 mg. with its long papus, this fruit will be able to float and reach a very high altitude as reported by white (1970) and at 500 m it will be able to spread as far as 5 km from its point of origin. c. odorata was shown to have a high relative growth rate (rgr), although it has low net assimilation rate (nar) (tjitrosemito 1996). nar is a physiologica 19 biotropia no. 12, 1999 index indicating the plant's efficiency in converting solar energy for the reduction of co2 to carbohydrate. this low nar was more than compensated for by a high value of leaf area ratio (lar). it shows that, although the nar is low, the carbohydrates produced are utilized to produce leaves. therefore, it has a very high rgr. ischaemum timorense, being a c4 photosynthetic plant has a high efficiency for its physiological activities, but a low rgr, because most of the photosynthate was used for the generation of stolon, a plant part which is not very productive. in java the common method of controlling c. odorata in plantation is by uprooting the weeds and placing the stem upside down above the soil, because if it contacts the soil, it will regrow. other methods are either chemical control or slashing conducted at prohibitively high cost (tjitrosemito 1996). considering the characteristics of c. odorata, it seems that biological control agents that attack leaves, branches, and flowers may be a good approach to control this weed. accordingly, the biocontrol agent pareuchaetes pseudoinsulata was introduced. however, despite its successful establishment in sumatera, it does not do well in java. on the other hand another biocontrol agent, procecidochares connexa established more readily, and since its release in 1995 survives in west java. rearing of p. connexa has been reported elsewhere (tjitrosemito 1996). release was also readily achieved and spread in terms of areas covered by recorded galls, occurred logarithmically. it showed an intrinsic rate of increase in the area covered as r = 1.3. this paper reports the establishment of p. connexa and its impact on the population growth of c. odorata. methodology population of procecidochares connexa the population of p. connexa was estimated by taking samples using line transects starting from the center of the field, drawn in 4 cardinal directions. along each of the line transects at intervals of 20 m, quadrants measuring 2 x 2 m2 were established. in each quadrant, the number of shoots and galls were counted. a sample of 20 shoots taken at random was obtained from each quadrant, brought to the laboratory and the presence of eggs was recorded under a binocular microscope. monitoring was carried out at 2 monthly intervals. the extent of population spread was estimated by the presence of galls. from the release sites, a line was followed to the farthest distance covered by the gall and this distance was used as the measure of spread in october 1997. 20 the establishment of procecidochares connexa in west java, indonesia soekistnan tjitrosemito impact of p. connexa on the chromolaena odorata population to evaluate the impact of p. connexa on c. odorata, the population of c. odorata in the permanent plots was counted and compared with that at the time of release, including the number of galls found on the plot. the effect of galls on seed production was measured by randomly selecting 65 c. odorata plants free of p. connexa and compared to another batch of 65 plants selected at random from those attacked by p. connexa. the number of twigs, flowerheads and cypsellas were recorded. the effect of flowerheads on the development of galls for fly emergence batches of galls with and without flowers were observed on 8 populations. the numbers of successful emergence of flies from galls were recorded and statistically evaluated. results and discussions population of p. connexa the recorded number of shoots carrying eggs of p. connexa, and the number of galls are presented in table 1. table 1. the percentage of shoots carrying eggs and noticeable galls time jan. feb. apr. jul. aug. oct. dec. eggs 30.1 27.9 27.4 31.1 52.2 39.5 34.0 galls 27.1 25.9 17.1 25.7 40.7 34.7 29.2 the precentage of shoot bearing eggs went down slightly from january (30.1%) to april (27.4%), but went up again to reach a peak in august (52.2%). it seems that during the wet season, when growth of c. odorata was rapid and shoots were abundantly available, the number of emerging flies was still too low to visit the available shoots, thus accounting for the low precentage of shoot carrying eggs. when the dry season approached c. odorata did not produce new shoots, because it began to flower. the percentage of shoot bearing eggs increased to reach a maximum value of 52.2% in august 1997, but went down again to 34.0% in december. it is interesting to note that percentage of galls was always lower than that of eggs. 21 biotropia no. 12, 1999 population spread the spread of the population was recorded in october 1997 and is presented in table 2. table 2. the population of galls in a 2 x 2 m2 quadrant at various distances measured from point or release assuming that the spread of the bioagent was exponential, when calculated from the time of release in december 1995 and an initial area of 25 m2, the rate of spread was approximately 0.69/m2/m2/month. at this rate, the spread will double the size of the area each month. it was slower than previously reported when it was observed that doubling in size of the infested area occurred each 0.5 month. since spread is also affected by the availability of c. odorata plants, this was not unreasonable. impact on c. odorata the permanent plot established in 1995 contained a very dense vegetation of c odorata. in 5 x 5 m2, 145 plants with 2333 shoots were present. the initial releases in december 1995 were made with 75 pairs of flies and repeated again with 100 pairs. in 1997 the site was swept by fire and the population of c. odorata became 54 plants/5 x 5 m2 containing 891 shoots. although the plot was burnt down, the surviving plants quickly took over, and only 4 new recruits were recorded in the permanent plot, eventhough 90 plants disappeared during a 2-year period. the new recruits were still small in december 1997, and an old surviving stump was able to regrow bearing 74 new shoots (buds) in the plot. the infestation of p. connexa remained high, as indicated by the 277 galls recorded on the permanent plot. this meant that 31.08% of all shoots were infected by flies. this was much higher than 5% reported in december 1995. the population of c. odorata was reduced leaving 22 the establishment of procecidochares connexa in west java, indonesia soekisman tjitrosemito only 37.2% of the original population, and the shoot was reduced to the same extent i.e. only 38.2% remained, with 31.08% infested by p. connexa. here, biological control was effective. the effect of galls on seed production while the number of twigs did not differ significantly among plants attacked or not by p. connexa the flower heads differed significantly in numbers. formation of galls seemed to draw nutrition from the plants, reducing flower head development from 40.95/plant to 27.7/plant (see table 3). the reduction of seed production was even more conspicuous, i.e. from 1391.5 cypsellas/plant down to 647.6 cypsellas/ plant, a reduction of about 50%. table 3. cypsellas/plant production as affected by p. connexa recorded on 65 plants characters plants with galls plants without galls statistical test number of plants 65 65 ns number of twigs 264 316 ns flowerheads/plan 27.7 40.95 p <. 0.05 cypsellas/plant 647.6 1391.5 p ^ o . o l the effect of flowerheads on the development of galls leading to fly emergence comparison of population of galls having flowerheads and those that did not show a very significant difference in gall development at the 1% level. galls developed on shoots without flowerheads produced 0.8 successful emergences, while galls developing on growing flowerheads produced only 0.3 successful emergences (see table 4). this is caused by the fact that twigs bearing flowerheads will soon dry up following maturation of fruits. table 4. the emergence of flies from galls (8 populations of 20 colonies of galls, with and without flowers) no. colony with flowers colony without flowers statistical test 1 0.40 0.90 2 0.30 0.70 3 0.20 0.80 4 0.35 0.90 5 0.30 0.75 6 0.35 0.90 7 0.40 0.65 8 0.40 0.95 mean 0.33 0.82 ps 0.01 23 blotropla no. 12, 1999 this finding has a practical implication as the survival of emerging adults is reduced significantly by drying out of the bearing flowerheads. when all twigs are dried up it destroys the colony of p. connexa. the condition in parung panjang seems to support the perpetuation of the colony of p. connexa, since the population has survived the harsh dry season in 1997. acknowledgement this research has been funded by aciar through aciar project no. 9110. biological control of chromolaena odorata in indonesia and the philippines. references gauitier, l. 1993. reproduction of a pantropical weed: chromolaena odorata (l.). king & robinson. condollea48: 179-193. tjitrosemito, s. 1996. the management of chromolaena odorata (l.) king & robinson in indonesia. in: prasad, muniappan, ferrar, aeschliman, de foresta (eds.). agric. ex. station. univ.guam. pub. 202: 135-142. tjitrosoedirdjo, sri. s. 1990. some notes on chromolaena odorata. proc. 10th indonesian weed sci. conf.: 40-46. white, t.c.r. 1970. dispersal of exotic weeds. austral. j. sci. 32(9): 370-378. 24 biotropia no. 7, 1994: 30-40 notes on some growth characteristics of mikania cord at a (burm. f.) b.l. robinson*) b.t. mercado institute of biological sciences, university of the philippines at los banos, college, laguna, philippines abstract mikania follows a sympodial dichotomy pattern of branching. both stem (branches) and leaves give rise to new plants with relative ease. internodes also root easily but do not give rise to new plants. flower formation and seed germination are strongly influenced by light. numerous seeds are produced but only few are filled; still fewer are the seeds that germinate. the period from early emergence to about the 3-leafed stage is most critical for survival of the new plant. keywords: mikania cordata, growth, weed physiology introduction mikania cordata (burm. f.) b.l. robinson, a creeping woody perennial is popularly known by its local names of climbing hempvine and mile -a-minute plant. it is described systematically in the compendium of the world's worst weeds (holm et al. 1977). the stem (and its branches) and the mature leaves easily form roots when these come in contact with the soil (figs, la & b). the leaves on the creeping stem or branch are strongly negatively geotrophic. if a portion of the stem or branch is twisted to bring the leaves to face the soil, overnight the leaves would be upright, held up by the petioles that had curved upward (fig. 2). pattern of branching branching follows a sympodial dichotomy (fig. 3). the stem produces a terminal shoot which dries up after some time. at its closest node emerges the first pair of branches which elongates. on these branches are borne several nodes. the *) the project was supported by a grant from the national research council of the philippines (nrcp). 30 notes on some growth characteristics of mikania cordata b.t. mercado la ib fig. 1. new plants arising from stem (a) and leaf (b) cuttings fig. 2. the upright bended petioles and nodal roots pointing upward indicate the strong negative geotrophic characteristic of leaves and stem 31 biotropia no. 7, 1994 fig. 3. the sympodial dichotomous branching in mikania. the numbers indicate approximate length of the branches axillary buds on these nodes emerge to form new shoots. in most cases however, only one of the pair develops into a new branch, on whose nodes again emerge pairs of shoots, again with only one of the pairs elongating into new branches. this pattern is repeated thereafter, ultimately resulting in the entangled mass of branches which may be circular to the right or circular to the left (holm et al. 1977). the terminals of the main branches, on the other hand, die off after some time, permitting the side branches to elongate rapidly. thus this pattern of branching makes possible the continuous growth and rejuvenation of the plant. with time the stem progressively becomes woody. considering the numerous branches formed, and the bulk of leaves borne by these branches, this growth pattern allows the plant to produce a dense mat of foliage in quite a short time. 32 144.2 notes on some growth characteristics of mikania cordata b.t. mercado rate of stem elongation of plants from leaf and stem cuttings the data in figure 4a represent the observations from 10 plants grown from leaf cuttings, while those in figure 4b, from 13 plants grown from stem cuttings. in both cases, the first branch that came out from the rooted cutting was tagged for the daily or weekly measurements. the branches of the leaf cuttings elongated much more rapidly than the similar branches of the stem cuttings. these were at its longest on the 26th day after the first measurement. these ceased to elongate after this date. three (3) tagged branches of the stem cuttings died off 39 days after the initial measurement, while the rest, after the 68 th day. fig. 4 rate of elongation of the first branch from leaf (a) and stem cutting (b) 33 biotropia no. 7, 1994 effect of sunlight on rate of stem elongation three plants raised from seeds were grown under direct sunlight and another three plants, under partial shade. (a makeshift enclosure roofed by 2 layers of fishnets served this purpose). the plants were observed daily for 21 days after the 5-leafed stage. as before, the first branch that emerged was tagged and measured daily until the elongation of the branch decreased or stopped in any of the light treatments. under partial shade (fig. 5a) the branches elongated much more rapidly than those under direct sunlight (fig. 5b). its elongation continued even after the 20th day, while the elongation of branches under direct sunlight completely stopped on the 18th day after the initial measurement. the number of nodes borne on the tagged branches of both sets of plants did not differ very much from each other, although node count was slightly higher in the branches of the plants under partial shade. fig 5. elongation of the first branch from a seed-grown plant exposed to (a) partial shade and (b) direct sunlight 34 notes on some growth characteristics of mikania cordata b.t. mercado the biomass productions after a six-month growth period under partial shade or direct sunlight of plants grown from cuttings are given in table la (stem cutting) and in table 1b (leaf cutting). in both light treatments, the shoot dry weight of the plants under partial shade was 2-5 times higher than those of the plants grown under direct sunlight. light apparently did not influence the dry matter production of the roots. surprisingly, however, the root system of the plant was not profuse and long as might be expected of a plant capable of producing such dense vegetative growth (fig. 6a). this may be due in part to the fact that the creeping stem or branch readily produces two kinds of roots: the usual nodal roots, and the internodal roots (fig. 6b). undoubtedly, anchorage as well as absorption of nutrients and water is a function well carried out by these stem-borne roots. there are not very many creeping plant species, succulent or woody, that develop internodal roots. on the whole, the plants grown under partial shade are much more vegetative, luxuriant and green than the plants grown under direct sunlight. the plants under partial shade remain vegetative. on the other hand, those which are exposed to sunlight start to flower in mid november until late january. field observation also showed that even in the same plant, the shaded portion remains vegetative while the upper portion exposed directly to sunlight produces flowers profusely. the seed the seeds are elongated, 1.5-1.8 mm in length and about 0.3-0.4 mm at its widest. these are black, with a longitudinal ridge in the middle, tufted at one end with pappus (about 40 50 hairs). the pappus adds about 3.0 mm to the length of the seed. the seed is filled with white endosperm. red ants are common foragers of ungerminated seeds. the seed dries up easily if kept in the open air. it has a very short period of viability. table la. dry weight (g/p) of mikania grown from stem cuttings under partial shade or direct sunlight (6 feb.-sept. 1991) plant partial shade direct sunlight no. shoot root shoot root 1 135.7 3.2 38.5 2.2 2 136.0 2.3 17.2 1.4 3 176.6 3.1 27.0 2.1 4 95.6 2.2 30.0 3.0 5 176.6 1.4 25.6 1.4 average 144.1 2.44 27.66 2.02 35 biotropia no. 7, 1994 table 1b. dry weight (g/p) of mikania grown from leaf cuttings under partial shade or direct sunlight (11 march-9 sept. 1991) plant partial shade direct sunlight no. shoot root shoot root 1 170.5 2.5 46.0 2.0 2 81.5 2.3 40.0 1.3 3 63.5 1.4 43.0 2.3 4 65.6 2.2 35.0 1.6 5 85.3 3.8 43.5 2.9 average 93.28 2.44 41.5 2.02 36 fig. 6a. the main root system of mikania plant fig. 6b. the nodal and internodal roots of a creeping stem of mikania seed count and seed germination a panicle has as many as 134-428 seeds (table 2), but the number of filled seeds is very low (6.52 27.62%). similar observation was reported by wirjahardja (1976) in the three mikania species common in indonesia. fifteen (15) filled seeds were sown in petri dish lined with moist filter paper. four (4) dishes were completely wrapped with two layers of carbon paper, while another four (4) uncovered dishes were placed on a lighted laboratory table. germination took place within three days in the light-exposed dishes. the carbon paper-wrapped dishes, opened seven days after sowing the seeds, showed complete failure of germination. the germination of the light-exposed seeds (table 3) was very low. repeated trials failed to exceed 50% germination even when the germination period was extended for another week. soil germination trials showed that seeds slightly buried into the soil (sticking the whole 1.5 — 1.8 mm length of the seed into the moist soil) failed to germinate, while seeds, laid flat on its side on the surface of moist soil germinated within a few days. . , • 37 notes on some growth characteristics of mikania cordata b.t. mercado biotropia no. 7, 1994 table 2. number of seeds (cypsela) per panicle panicle no. filled seeds unfilled seeds total % filled 1 21 301 322 6.52 2 41 227 268 15.29 3 25 183 208 12.02 4 25 317 342' 7.31 5 37 97 134 27.61 7 43 385 428 10.04 8 39 239 278 14.03 table 3. germination of mikania seed (15 seeds per petri dish) replication no. of seeds germinated germination % 1 4 2 4 3 6 4 6 26.6 26.6 40.0 40.0 the above observations suggest that although a mikania plant (one exposed to sunlight) has the propensity to produce numerous seeds which could be dispersed at great distance, only a handful of these seeds are filled, and only a few of them are viable. for the viable seeds to germinate light is indispensable. the seedling the seedling emerges from the pole directly opposite the pappus-bearing pole of the seed. the thin, long primary root comes out first. the root tip is distinctly covered with a black root cap. conspicuous is the tuft of short, fine and sticky roots borne at the base of the hypocotyl. these roots would later on constitute the root system of the plant, replacing the long primary root that dies off after some time. the hypocotyl is much bigger in diameter than the spindly primary root but a little shorter. the hypocotyl of the newly emerged seedlings had an average length of 0.97 cm. in another 12 days, however, it had already an average length of 21.83 38 notes on some growth characteristics of mikania cordata b.t. mercado cm, about twice longer than the primary root. at one end of the hypocotyl the pair of round, green, succulent cotyledons is borne. the cotyledons remain intact on the seedling until the young plant has attained the 3-leafed stage. the seedling, from germination to the early early 3-leafed stage is most vulnerable to wilting. however, once it has survived this critical period it develops rapidly into a mature and highly vegetative plant. summary mikania has a well developed and efficient means of vegetative reproduction (fig. 7). a portion of the mature stem or branch (iv-1), as long as it bears a node, easily gives rise to a new plant. with similar ease, the mature leaves can also give rise to new plants (iv-3). an internodal stem or branch (one which does not bear a node), easily forms roots but does not give rise to a new plant (iv-2). fig. 7. the life cycle of mikania cordata 39 biotropia no. 7, 1994 the development of the plant, whether derived from cuttings (i) or from seeds (iii) is strongly influenced by light. the plant grown under partial shade elongates very rapidly, is dark green and luxuriant. it remains however, vegetative (i-b). in contrast, the plant grown under direct sunlight, develops less densely but comes to profuse bloom in mid-november until late january (i-a). numerous seeds are produced but only a few are filled, and fewer still are viable. the pappus-bearing, minute seeds (ii) are dispersed by wind. for the seed to germinate, light is indispensable. for the seedling to survive it must be located in a moist and shaded area. references . holm, l.g., d.l. plucknett, j.v. pancho, and j.p. herberger, 1977. the world's worst weeds. university press. hawaii, honolulu. wirjahardja, j. 1976. autecological study of mikania spp. proceedings of the fifteenth asian-pacific weed science society conference. tokyo, japan. asian-pacific weed science society. 40 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf biotropia no. 8, 1995: 45-52 effects of pisolithus tinctorius and laccaria fraterna on the growth and mycorrfflzal development of pinuspatula seedlings*) m. sudhakara reddy and k. natarajan centre for advanced studies in botany, university of madras, guindy campus, madras 600 025, india abstract vegetative inoculum of pisolithus tinctorius and laccariafraterna were inoculated to pinuspatula seedlings grown in both steam sterilized and unsterilized shola soil. after 4 months of seedling growth, 10 seedlings from each treatment were harvested and various growth parameters were studied. inoculation of these two fungi resulted in the production of ectomycorrhizas and increase in growth of p. patula seedlings when compared to uninoculated seedlings. laccariafraterna inoculated seedlings showed more number of mycorrhizas than p. tinctorius inoculated seedlings at the end of one year. both these fungi poorly colonized the root system in both soil treatments. there was no significant difference between these two fungi in improving the seedling growth in the nursery. key words: pisolithus tinctorius/laccaria fraterna/pinus patula/inoculum/seedlings/growth. introduction extensive work on nursery inoculation has been done with pisolithus tinctorius because of its ecological adaptation to adverse soil conditions, wide geographic distribution and broad host range, tolerance to a variety of environmental conditions and its easy propagation and manipulation in pure culture (marx et al. 1984). eventhough p. tinctorius is worldwide in distribution and has a broad tree host range it has not been found in the nilgiri hills either in pine or eucalypt plantations. the other potential ectomycorrhizal fungus laccariafraterna is wide spread throughout the world where eucalyptus and other ectomycorrhizal hosts have been introduced (tommerup et al. 1991). at certain places in nilgiri hills where eucalypt and pine plantations occur side by side, l. fraterna is mostly found to be associated with the eucalypts but rarely with pines (natarajan 1977). but under in vitro condition these two fungi were able to form mycorrhizas with p. patula seedlings. the present research study was undertaken to study the effects of these two fungi on the growth and mycorrhizal development of p. patula seedlings in the nursery. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19-21 april 1994, yogyakarta, indonesia. 45 biotropia no. 8, 1995 materials and methods pisolithus tlnctorius culture was isolated from basidiomata collected from eucalyptus tereticornis plantations, near madras coast, tamilnadu and the culture of l. fraterna was isolated from the basidiomata collected from e. globulus plantations in nilgiri hills, tamilnadu, south india. the cultures were maintained at 25°c on potato dextrose agar medium. the mycelial inoculum of p. tinctorius and l. fraterna were prepared according to the method of marx and bryan (1975). inoculum of each fungus was grown aseptically in one litre erlenmeyer flasks containing 750 ml of vermiculite moistened with 375 ml of mmn liquid medium. the flasks were incubated at 25°c in dark. after 12 weeks of incubation, inoculum was removed from the flask and leached with cool running tap water to remove the unused nutrients. excess free water was removed by gently squeezing the inoculum wrapped in cheese cloth. fungus free vermiculite wih mmn served as control. the shola soil collected under natural vegetation of the upper region of nilgiri hills was used in the present study. the chemical constituents of the soil are as follows: ph 5.3; organic matter content -4.79%; npk levels 245, 5.2 and 35 kg/acre, respectively. the nursery experiment was conducted at the forest department nursery, ootacamund, nilgiri hills during july 1992 to june 1993. both steam sterilized and unsterilized soils were filled in polybags (about 2 kg/bag) and 10% inoculum by volume was added to the polybags separately and mixed into the upper 8-10 cm of the soil. fungus free vermiculite added to the polybags served as controls. the surface sterilized seeds of p. patula (30% h2o2 for 30 minutes) were sown in all the bags. five (5) seeds were sown in each bag. after germination, seedlings were thinned to one per bag. ten (10) replicates were kept for each treatment. after 12 months of seedling growth, 10 seedlings from each sample were harvested and the growth and mycorrhizal development were studied. the shoot height, root length, and root collar diameter were measured. after counting the mycorrhizal and non-mycorrhizal tips, the shoots and roots were dried in a hot air oven at 85°c for 48 hours. dry weights of shoot and root were determined by obtaining constant weights. the method used by zak (1973) and agerer (1986) were followed for studying the macroscopic and microscopic features of ectomycorrhizas. the colour terminology used is that of kornerup and wanscher (1978). all the data were analysed by analysis of variance and the means were compared by least significant difference (snedecor and cochran 1967) at p = 0.05 level. 46 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan results the root examinations revealed that both the ectomycorrhizal fungi viz., p. tinctorius and l. fraterna were able to form mycorrhizas with p. patula seedlings in the nursery. the p. tinctorius type of mycorrhizas are mostly dichotomous, tetrapodials and coralloid forms, 3-6 mm long and 0.4-0.5 mm in diameter. colour of mycorrhizas are brownish yellow (5c8) to light brown (6d6). loose hyaline hyphae are associated with the surface of the mycorrhizal system. the rhizomorphs are light brown (6d6) in colour and 150-550 µm wide, composed of closely packed parallel hyphae and covered with radiating hyphae (fig. la). the transverse section showed that the mantle is 15 20µrn thick. it consists of a simple prosenchymatous tissue. cystidia, setae or sclerotia are not observed. the hartig net is composed of one or two rows of oval to globose hyphal cells which measure 5 — l0 µm in thickness and penetrate up to 4 cortical cell layers deep (fig. ib). laccaria fraterna type of mycorrhizas are mostly bipodial, rarely monopodial, 3-5 mm long and 0.3 0.4 mm in diameter. colour of the mycorrhizas are orange white (6a2) when young and brown (6d7) when old. the surface is smooth. rhizomorphs are absent (fig. ic). the transverse section showed a mantle of 1015µm thick and consists of a simple prosenchymatous tissue. the hartig net is composed of a single row of oval to globose hyphal cells which measure 3 6 µm in thickness and penetrate up to 3 cortical cell layers deep (fig. id). inoculation of p. tinctorius and l. fraterna increased the number of mycorrhizas both in sterilized and unsterilized soil. the number of mycorrhizas were more in sterilized inoculated soil than in unsterilized soil. laccaria fraterna produced more number of mycorrhizas than p. tinctorius. but there was no significant difference between these two fungi. the percentage of mycorrhizas did not differ significantly between p. tinctorius and l. fraterna inoculated seedlings in both soil treatments. the percent colonization of both inoculated fungi were lower in both soil treatments. when compared to p. tinctorius, l. fraterna inoculated seedlings showed higher percent colonization at the end of one year. no basidiomata production was seen in the case of p. tinctorius whereas l. fraterna inoculated seedlings produced basidiomata in the bags at the end of one year. the growth of p. patula seedlings was improved in both sterilized and unsterilized soil by the inoculation of the fungi. the shoot height was more in sterilized inoculated soil than in unsterilized inoculated soil. but there was no significant difference between the two fungi in improving the shoot height. the root length has increased in both soils by the inoculation of these two fungi. laccaria fraterna inoculated seedlings showed a higher root collar diameter when compared to p. tinc 47 figure 1. ectomycorrhizas of pinus patula produced by pisolithus tinctorius and laccaria fraterna. a. morphology of p. tinctorius ectomycorrhizas x 240. b. transverse section of p. tinctorius ectomycorrhizas x 320. c. morphology of l. fraterna ectomycorrhizas x 200. d. transverse section of l. fraterna ectomycorrhizas x 325. m mantle; h hartig net. 48 biotropia no. 8, 1995 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan torius inoculated seedlings. the shoot dry weight was more in sterilized soil than in unsterilized soil in inoculated seedlings. the seedlings inoculated with l. fraterna showed maximum shoot dry weight in both soils. but there was no significant difference between the two fungi in improving the shoot dry weight. the root dry weight also did not differ significantly between the two fungi. the shoot/root ratio differed significantly in control seedlings than in inoculated seedlings in sterilized soil. in unsterilized soil there was no significant difference between the two fungi and control seedlings with respect to the shoot/root ratio (table 1). seedlings grown in sterilized soil inoculated with the two fungi showed more growth and mycorrhizal development than the seedlings grown in unsterilized soil inoculated with these fungi. laccaria fraterna and p. tinctorius did not differ significantly in improving the growth and mycorrhizal development of p. patula seedlings in the nursery. discussion since p. tinctorius and l. fraterna are known to be early stage ectomycorrhizal fungi (marx 1991; tommerup et al. 1991), these two species have been selected to evaluate their effects on the growth and mycorrhizal development of p. patula in the present study. vegetative inoculum of these two fungi improved the growth and mycorrhizal development of p. patula seedlings when compared to uninoculated seedlings in the nursery in both steam sterilized and unsterilized soil. but the difference in various growth parameters is not significant in the inoculated seedlings. the seedlings inoculated with l. fraterna seem to be marginally better than the seedlings inoculated with p. tinctorius. tommerup et al. (1991) reported that l. fraterna, an early colonizing mycorrhizal fungus of eucalyptus is a potential competitor when the seedlings were inoculated with other selected ectomycorrhizal fungi. it is interesting to note that basidiomata of l. fraterna very rarely occur in p. patula plantations in nilgiri hills and found mostly associated with eucalyptus plantations which are adjacent to the p. patula plantations. it has also been noticed that basidiomata of l. laccata, the most predominant fungus in p. patula plantations, were seldom found in the eucalyptus plantations. the results of the present experiment suggest that l. fraterna and p. tinctorius are capable of improving the growth of nursery seedlings in nilgiri conditions. the colonization of these two fungi was poor in both treatments. marx and cordell (1987) found that ph between 4.5 and 5.5 is adequate for p. tinctorius, but that a ph above 6.0 inhibits ectomycorrhizal formation. the ph of the soil used in the present study is 5.3 and hence it may not be the factor for 49 biotropia no. 8, 1995 50 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan poor colonization of p. tinctorius in the nursery. marx et al. (1970) reported that p. tinctorius grew rapidly at 28 30°c and capable of growing at 40 42°c in pure culture and formed more mycorrhizas with p. taede at 34° c than at lower temperatures. marx and bryan (1971) reported that aseptically grown p. taede seedlings with p. tinctorius ectomycorrhizae had better survival and growth at 40°c in laboratory tests than non-mycorrhizal seedlings or those mycorrhizal seedlings with t. terrestris. temperature may be the main reason why p. tinctorius is not occurring in nilgiri hills where the temperature ranges between 5 and 20° c during different parts of the year. the isolate of p. tinctorius used in the present study was obtained from the basidiomata associated with e. tereticornis in coastal madras where the temperature in summer will raise up to 40°c. further research is needed to evaluate different environmental conditions on the growth and mycorrhizal development of p. patula by p. tinctorius. in spite of the positive growth response shown by the seedlings in the present study only further research will reveal whether these two fungi will survive when seedlings are outplanted since they are conspicuous by their absence in p. patula plantations in the nilgiri hills. references aoerer, r. 1986. studies on ectomycorrhizae. ii. introducing remarks on characterization and identification. mycotaxon, 26: 473-492. kornerup, a. and j.h. wanscher. 1978. methuen handbook of colour 3 rd ed. methuen and co. ltd. london, p. 243. marx, d.h. 1991. the practical significance of ectomycorrhizae in forest establisment. in: ecophysiology of ectomycorrhizae of forest trees. the marcuswallenberg foundation, symposia proceedings, 7: 54-90. marx, d.h. and w.c. bryan. 1971. influence of ectomycorrhizae on survival and growth of aseptic seedlings of loblolly pine at high temperature. for.sci. 17: 37-41. marx, d.h. and w.c. bryan. 1975. growth and ectomycorrhizal development of loblolly pine seedlings in fumigated soil infested with the fungal symbiont pisolithus tinctorius. for. sci. 21: 245254. marx, d.h. and c.e. cordell. 1987. ecology and management of ectomycorrhizal fungi in regenerating forests in the eastern united states. in: mycorrhizae in the next decade: practical applications and research priorities. seventh nacom, may, 3-8, 1987. gainesville, fl. ed. d.m. sylvia, l.l. hung and j.h. graham. int. of food and agric. sciences, univ. of florida, gainesville. marx, d.h., w.c. bryan and c.b. davey. 1970. influence of temperature on aseptic synthesis of ectomycorrhizae by thelephora terrestris and pisolithus tinctorius on loblolly pine. for. sci. 16: 424-431. marx, d.h., c.e. cordell, d.s. kenney, j.g. mexal, j.d. artman, j.w. riffle and r.j. molina. 1984. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. for. sci. monogr. 25: 1-101. 51 biotropia no. 8, 1995 natarajan, k. 1977. south indian agaricales iii. kavaka. 5: 35-39. snedecor, g.w. and w.g. cochran. 1967. statistical methods. the iowa state university press. iowa usa p. 593. tommerup, i.e., n.l. bougher and n. malajczuk. 1991. laccaria fraterna a common ectomycorrhizal fungus with mono-and bisporic basidia and multinucleate spores: comparison with quadristerigmate, binucleate spored l. laccata and the hypogeous relative hydnangium cameum. mycol. res. 95: 689-698. zak, b. 1973. classification of ectomycorrhizae: in: ectomycorrhizae: ecology and physiology. g.c. marks and t.t. kozlowski eds. academic press, new york and london, p. 43-78. 52 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf biotropia vol. 29 no. 3, 2022: 185 192 doi: 10.11598/btb.2022.29.3.1393 185 postharvest quality improvement of nutmeg (myristica fragrans) okky setyawati dharmaputra*, santi ambarwati, ina retnowati and nijma nurfadila phytopathology laboratory, science innovation and technology department, seameo biotrop, bogor 16134, indonesia received 11 august 2020/accepted 19 september 2021 abstract nutmeg (myristica fragrans) or fragrant nutmeg is an important commodity that has been used in the food and pharmaceutical industries, hence its quality should be monitored. the objectives of this study were to: 1) identify critical control points (ccp) in nutmeg’s postharvest handling process and prepare nutmeg haccp (hazard analysis and critical control point) system and 2) provide a recommendation on ghp (good handling practices) of nutmeg in order to maintain its quality in relation to food safety issue which is very important for international trade. ripe fruits of nutmeg were collected after the fruits had reached maturity and fallen from their trees. a paranet was placed under each nutmeg tree to prevent the ripe nutmeg fruits from falling on the ground. the subsequent processes were taking out the nutmeg seeds from the fruits and separating the nutmeg seeds from the pulps and maces. after that, the nutmeg seeds underwent the drying process by using the smoke and oven-dried methods until the moisture content of the nutmeg seeds was reduced by 10%. subsequently, the nutmeg seeds were divided into two parts, prior to the storing process. the first part was fumigated by using phosphine (2 g/m3) for eight days and the second part was not fumigated. the sampling of nutmeg seeds was conducted at the beginning of storage and after four months of storage. the parameters observed were moisture content, percentage of damaged kernels, the population of each fungal species, and aflatoxin content. the results showed that moisture content, fungal population, aflatoxin b1, and total aflatoxin contents of nutmeg kernels having been dried by using the smokeand oven-dried methods with and without fumigation still complied with the requirements related to food safety, although the nutmegs were stored for four months. the results of this research could also determine the critical control point (ccp) in the postharvest handling process of nutmegs, i.e., 1) choosing only ripe nutmeg fruits to be harvested; 2) harvesting method by preventing the ripe nutmeg fruits from falling on the ground; 3) drying process of nutmeg seeds should be conducted immediately after separating the nutmegs from the maces by using the smokeor oven-dried methods; and 4) nutmeg seeds were stored with the shells. keywords: nutmeg, postharvest, quality, shells, storage introduction nutmeg (myristica fragrans) or fragrant nutmeg is an important commodity widely used in the food and pharmaceutical industries, hence its quality should be monitored (punnathara 2011). nutmeg is native to the moluccas islands of indonesia, but nowadays nutmeg is also grown on penang island in malaysia, in the caribbean (particularly grenada), in the southern state of kerala in india, and on the island of zanzibar. djaelani (2018) reported that north moluccas is the largest nutmeg producer in indonesia. according to cbi (2015), indonesia and grenada dominate nutmeg production and export to european countries with world market shares of 75% and 20%, respectively. india, malaysia, papua new guinea, sri lanka, and caribbean islands, such as st. vincent are also producers and exporters of nutmeg. during the postharvest period (including storage), nutmeg could be infested by insects and microorganisms. among microorganisms, fungi are the most important cause of stored *corresponding author, email: okky@biotrop.org biotropia vol. 29 no. 3, 2022 186 foodstuffs deterioration. fungal infection in foodstuffs can cause discoloration, a decrease in physical quality and nutritional contents, and mycotoxin contamination. aflatoxins are toxins produced by aspergillus flavus and a. parasiticus. aflatoxins are considered dangerous due to their association with various diseases in humans and animals, such as aflatoxicosis and liver cancer. there are four naturally occurring aflatoxins in many commodities, i.e., aflatoxins b1, b2, g1, and g2. the most toxic aflatoxin is aflatoxin b1 (basappa 2009). according to fao (2004), european union has determined the maximum tolerable limits (mtl) of aflatoxin b1 and total aflatoxins in nutmeg as 5 and 10 ppb, respectively. dharmaputra et al. (2015) reported that the postharvest handling method of nutmeg conducted by farmers and collectors in north sulawesi province was not appropriate. as the postharvest handling method of nutmeg can affect the quality of nutmeg, it is important to conduct research on the effect of some methods of postharvest handling on the quality of nutmeg, especially on fungal infection and aflatoxin contamination. the objectives of the research were to: 1) identify critical control point (ccp) in the nutmeg postharvest handling process and prepare the nutmeg haccp (hazard analysis and critical control points) system and 2) provide a recommendation on ghp (good handling practices) of nutmeg in order to maintain its quality in relation to food safety issue which is very important for international trade. materials and methods time and location of research collection of nutmeg fruits and drying of nutmegs with their shells were conducted in the location where nutmeg trees were cultivated, i.e., in kauditan subdistrict, north minahasa regency, north sulawesi province. storage of nutmeg took place in a warehouse located in bitung municipality, north sulawesi province. the determination of moisture content, percentage of damaged kernels, the population of each fungal species infecting kernels, and aflatoxin content were conducted at the food and feed as well as the phytopathology laboratories, seameo biotrop, bogor. collecting nutmeg fruits, nutmegs drying and fumigation ripe fruits of nutmeg were collected one week after they had fallen from nutmeg trees. a paranet was placed under each nutmeg tree to prevent the ripe nutmeg fruits from falling on the ground. the paranet was placed at 1 m above ground (fig. 1). figure 1 a paranet installed under each nutmeg tree to catch the ripe nutmeg fruits that naturally fell from the nutmeg trees the pulps and maces of nutmeg fruits were then separated from the whole nutmeg seeds. nutmeg seeds were dried until the moisture content was reduced by 10%. the drying process was conducted by using: 1) smoke-dried and 2) oven-dried methods (fig 2). subsequently, the nutmegs were divided into two parts prior to storage, i.e., 1) fumigated by using phosphine (2 g/m3) for 8 days to prevent the occurrence of insect infestation during storage and 2) not fumigated. the drying methods and fumigation were replicated three times. paranet postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 187 (a) (b) figure 2 drying of nutmeg using: (a) smoke-dried and (b) oven-dried methods packaging and storing of nutmeg the fumigated and non-fumigated nutmegs in the shell were packed in gunny bags. each bag contained 5 kg of nutmegs-in-shells and was stored for four months under warehouse conditions (fig. 3). in three replicates, each bag containing nutmegs was treated as follows: (a) drying methods; (b) fumigated and not fumigated; and (c) storage durations, i.e., at the beginning of storage and four months of storage. the sampling of nutmegs was conducted at the beginning of storage and after four months of storage. the number of experimental units was 24, i.e., 2 drying methods x 2 fumigated and not fumigated x 2 storage durations x 3 replications. the temperature and relative humidity of the storage were recorded using a thermohygrograph. sampling and obtaining working samples the sampling of nutmegs was conducted at the beginning of storage and after four months of storage. insects found in nutmeg were separated from nutmeg using a sieve. the insects were then preserved in vials containing 70% ethanol. each sample of nutmeg seeds was mixed homogeneously. nutmegs-in-shell were then shelled using a hammer to get nutmeg kernels. after that the nutmeg samples were separated into eight parts i.e., two parts were used for determining the percentage of damaged kernels, while the other six parts were used for determining the moisture content, fungal population, and aflatoxin content. subsequently, the six parts were ground using mill powder tech model rt 04 and mixed homogenously on a plastic tray (40 x 30 x 5 cm). the ground nutmeg was then divided into eight parts to be used as working samples, i.e., one part for determining moisture content, three parts for determining fungal population, and four parts for determining total aflatoxin content. (a) (b) figure 3 condition at the outside (a) and the inside (b) of the warehouse used for storing nutmeg seeds for four months biotropia vol. 29 no. 3, 2022 188 determination of moisture content, percentage of damaged kernels, fungal population, and aflatoxin content the moisture content of nutmeg kernels (based on a wet basis) was determined based on iso 939, i.e., the distillation method (sni 2015). two replicates were used for each sample. damaged kernels included shriveled, cracked, broken kernels, moldy and insect-damaged kernels. the percentage of damaged kernels was determined using the following formula: weight of damaged kernels (g) x 100% weight of working sample used for damaged kernel analysis (g) fungi were isolated using the serial dilution method, followed by the pour plate method on dichloran 18% glycerol agar (dg18) (pitt & hocking 2009). each fungal species was identified following pitt and hocking (2009) and samson et al. (2010). aflatoxin contents were determined using high-performance liquid chromatography (hplc) method (vicam 2007). two replicates were used for each sample. statistical analysis the data were analyzed using a completely randomized block factorial design with three factors, i.e., the drying methods, fumigated and not fumigated nutmeg seeds, and storage durations, respectively. results and discussion moisture content one of the important factors causing the deterioration of foodstuff during storage is moisture content. sni (2015) determined 10% as the maximum moisture content of nutmeg seeds during storage. based on the analysis of variance, there were no significant differences in moisture content among the applied treatments and their interaction (drying methods, fumigation, and storage duration). ranges of moisture content of nutmegs with various treatments at the beginning of storage and after four months of storage were 7.4 7.6% and 7.1 7.7%, respectively. those percentages were lower than the maximum limit of moisture content determined by sni (2015) (table 1). table 1 moisture content of nutmeg caused by various treatments during storage treatment moisture content (%) storage duration (months) 0 4 smoke-dried and fumigated 7.4 ± 0.3a 7.4 ± 0.3a oven-dried and fumigated 7.5 ± 0.3a 7.1 ± 0.3a smoke-dried and unfumigated 7.6 ± 0.1a 7.7 ± 0.3a oven-dried and unfumigated 7.5 ± 0.3a 7.3 ± 0.4a moisture content is always in equilibrium with the relative humidity of a storage room. the humidity of the storage environment will be absorbed by foodstuff stored in the storage room having high relative humidity. on the other hand, foodstuff will lose its humidity if it is stored in a storage room having low relative humidity. moisture content is also affected by the temperature of a storage room. in this study, the mean and range of temperature and relative humidity of the storage room decreased after four months of storage. the mean and range of temperature and relative humidity of the storage room at 0 4 months of storage were 27.9 ± 1.6 oc (23.2 32.8 oc) and 73.7 ± 4.2% (59.0 84.6%). percentage of damaged kernels sni (2015) determined damaged kernels including damages caused by insects and fungal attacks, cracked, broken, and shriveled kernels. based on the analysis of variance, storage duration contributed to the significant differences in the percentage of damaged kernels, while drying methods, fumigation and their interactions did not contribute any significant differences. the percentage of damaged kernels of nutmeg increased after four months of storage (table 2). table 2 percentage of damaged kernels of nutmeg during storage storage duration (months) damaged kernels (%) 0 40.5 ± 5.3a 4 45.7 ± 3.8b postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 189 in this study, the damaged kernels were arguably caused by the occurrence of insects in nutmeg during storage. there were three insect larvae in the fumigated nutmegs. as many as 20 adult insects and 7 insect larvae were found in nutmeg that were not fumigated. these findings indicated that some insects were resistant to phosphine. according to gautam et al. (2016), phosphine resistance in stored product insects occurs worldwide and is a major challenge to the continued effective use of this fumigant. phosphine resistance is present in tribolium castaneum and plodia interpunctella populations in california almond storage and processing facilities. dharmaputra et al. (2018) reported that the dominant insect in nutmeg after being stored for four months was araecerus fasciculatus. haines (1991) and rees (2004) also reported that a. fasciculatus is the most important insect infesting spices, including nutmeg. according to childers and woodruff (1980), a. fasciculatus is a primary insect pest in stored products, such as nutmegs in north and south america, africa, asia, australia, and europe. total fungal population as many as eight fungal species were isolated in nutmeg in this research. yeast was the dominant fungal species and was often isolated in nutmeg samples at the beginning of storage (table 3). in all treatments, the population of each fungal species (except yeast) was relatively low (< 10 cfu/g wet basis). aspergillus flavus was not found. these findings indicated that the postharvest handling method conducted in this research, from harvesting up to storing was appropriate to ensure good quality of nutmegs. based on analysis of variance (data transformed in log (x+1)), the interaction between drying methods and fumigation contributed to significant differences in total fungal population in nutmeg, while storage duration contributed to very significant differences. fumigated nutmeg, dried using smokeand oven-dried methods did not show any significant differences in total fungal population. on the other hand, the total fungal population in unfumigated nutmeg, dried using the smokedried method was lower than that of using the oven-dried method (table 4). the total fungal population in nutmeg after four months of storage was lower than that of the population at the beginning of storage (table 5). it was assumed that the yeast did not grow in nutmeg having moisture content suitable for storing (< 10%) during four months of storage. table 3 population of each fungal species treatment fungi fungal population (cfu/g) storage duration (month) 0 4 smoke-dried and fumigated aspergillus flavus 1 0 a. chevalieri 0 3 a. ochraceus 1 0 cladosporium cladosporioides 4 1 penicillium citrinum 0 1 p. islandicum 0 1 yeast 12 0 smoke-dried and not fumigated c. cladosporioides 2 1 p. citrinum 1 0 yeast 2 0 oven-dried and fumigated a. niger 1 0 c. cladosporioides 1 1 p. citrinum 0 1 yeast 23 0 oven-dried and not fumigated c. cladosporioides 0 2 p. citrinum 0 2 yeast 4,660 0 biotropia vol. 29 no. 3, 2022 190 table 4 total fungal population in nutmeg drying method total fungal population (cfu/g wet basis) fumigated unfumigated smoke-dried 13 ± 9b 3 ± 3a oven-dried 14 ± 16b 2,332 ± 5,395c table 5 total fungal population in nutmeg during storage storage duration (months) total fungal population (cfu/g wet basis) 0 1,178 ± 3,832a 4 4 ± 4b according to dharmaputra et al. (2015), the dominant fungal species in nutmeg collected from farmers and collectors in north minahasa regency were penicillium citrinum, a. niger, eurotium repens, a. flavus, and endomyces fibuliger. ichinose et al. (2006) reported that eurotium spp. was the predominant fungi found in 12 powdered nutmeg samples collected from retailers in indonesia. aspergillus flavus (1.0 x 102 cfu/g) was detected in one sample. according to mandeel (2005), peeled seeds of nutmeg imported from india, sri lanka, indonesia, and brazil were found to be infected by aspergillus niger, a. flavus, and rhizopus stolonifer. the predominant species was a. flavus. toma and abdulla (2013) reported 20 fungal and one yeast species isolated from 16 samples of spices and herbal medicines in shekalla market, erbil city, iraq. five of the 20 fungal species that contaminated nutmeg were a. flavus and a. niger (1 x 103 cfu/g, respectively), a. ochraceus (2 x 103 cfu/g), a. versicolor (6 x 103cfu/g), and a. wentii (2 x 203 cfu/g ). aflatoxin content aflatoxin b1 and total aflatoxin of nutmeg in various treatments were lower than the limit detection determined by hplc (< 0.92 µg/kg). these findings indicated that the postharvest handling implemented in this research was appropriate to ensure the good quality of nutmeg seeds on the occurrence of fungi and aflatoxin contamination during four months of storage. studies by dharmaputra et al. (2018) found that: a) the total aflatoxin content of nutmegs originating from hand-picked ripe fruits was lower than that from ripe fruits fell on the ground; b) total aflatoxin content in nutmegs with shells was lower than that in nutmegs without shells; and c) total aflatoxin content in nutmegs was lower for those dried using the smokeand oven-dried methods compared to those dried using the sun-dried method. the smoke-dried method could prevent toxin production produced by toxigenic a. flavus (uraih & ogbadu 1982). tabata et al. (1993) reported that aflatoxin was found in 3,054 of foodstuffs and their processed products, among others in nutmeg. the highest aflatoxin contamination was found in nutmeg (80%), while aflatoxin b1 was found in pistachio (1,382 ppb). takahashi (1993) also reported that in 1986 1991, as much as 29 (43%) of 67 samples of nutmeg collected in japan were contaminated with aflatoxin. according to martin et al. (2001), three nutmeg samples contained aflatoxin b1 from 1 to 5 ppb, three other samples 6 20 ppb, and 2 samples with 54 and 58 ppb, respectively. aflatoxin is also detected in spices, aromatic herbs, and medicinal herbs collected from common markets, supermarkets, shops, and warehouses in italy in the period of 2000 2005. nutmeg was one of the six spices which were analyzed for aflatoxin content. one of the three nutmeg samples was contaminated with aflatoxin. the contents of aflatoxin b1 and b2 in nutmeg were 2.27 and 0.47 ppb, respectively, while aflatoxin g1 and g2 were not detected (romagnoli et al. 2007). as many as 52 samples of nutmeg were imported from india and indonesia. twenty-two samples were heat treated, while the other 30 samples were not heat-treated. the heat-treated samples were less contaminated by aflatoxin than those in the untreated samples. nutmeg in powder form had more contamination than that of the whole nutmeg. of the powdered nutmeg subjected to steam treatment, 72.5% of samples were positive for total aflatoxin contamination, with a range of 0 17.2 ppb (pesavento et al. 2016). conclusion the moisture content, fungal population, aflatoxin b1, and total aflatoxin contents in nutmeg dried using smokeand oven-dried methods, fumigated and not fumigated, still complied with requirements related to food postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 191 safety, although they were stored until four months. the results of this research could also determine the critical control point (ccp) in the postharvest handling process of nutmegs, i.e., 1) choosing only ripe nutmeg fruits to be harvested; 2) harvesting method by preventing the ripe nutmeg fruits from falling on the ground; 3) drying process of nutmeg seeds should be conducted immediately after separating the nutmegs from the pulps and maces by using the smokeor oven-dried methods; and 4) nutmegs were stored with the shells. the ccp can be used as a recommendation for farmers, collectors, and exporters concerning appropriate postharvest handling methods (good handling practice) to ensure the good quality of nutmeg during storage. acknowledgments the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the cv multi rempah sulawesi for the cooperation in conducting the preparation of the research, storing, and sampling of nutmeg; to mr. sunjaya, mr. edi suryadi, mrs. ratnaningsih and mrs. syifa fauzia for their assistance. references basappa sc. 2009. aflatoxins; formation, analysis, and control. new delhi (in): narosa publishing house. cbi. ministry of foreign affairs. 2015. cbi product factsheet: nutmeg in europe. the hague (nl): cbi market. childers cc, woodruff re. 1980. a bibliography of the coffee bean weevil araecerus fasciculatus (coleoptera: anthribidae). bull entomol soc america 26 (3): 384-94. dharmaputra os, ambarwati s, retnowati i, nurfadila n. 2015. fungal infection and aflatoxin contamination in stored nutmeg (myristica fragrans) kernels at various stages of delivery chain in north sulawesi province. biotropia 22(2): 129-39. dharmaputra os, ambarwati s, retnowati i, nurfadila n. 2018a. determining appropriate postharvest handling method to minimize fungal infection and aflatoxin contamination in nutmeg (myristica fragrans). int food res j 25(2): 545-52. dharmaputra, os, sunjaya, retnowati i, nurfadila n. 2018b. keanekaragaman serangga hama pala (myristica fragrans) dan tingkat kerusakannya di penyimpanan. [diversity of insect pest and percentage of damaged kernels in stored nutmeg (myristica fragrans)]. jurnal entomologi indonesia 15(2): 57-64. djaelani ki. 2018. enam provinsi penghasil pala terbesar di indonesia bertemu di ternate. http:// kieraha.com/6-provinsi-penghasil-pala-terbesar-diindonesia-bertemu-di-ternate [26 october 2018]. fao. 2004. worldwide regulations for mycotoxins in food and feed in 2003. fao food and nutrition paper 81. rome (it): food and agriculture organization of the united nations. gautam sg, opit gp, hosoda e. 2016. phosphine resistance in adult and immature life stages of tribolium castaneum (coleoptera: tenebrionidae) and plodia interpunctella (lepidoptera: pyralidae) populations in california. j economic entomol: 1-19. doi: 10.1093/jee/tow/221 haines cp. 1991. insects and arachnids of tropical stored products: their biology and identification (a training manual). kent (gb): natural resources institute. ichinoe m, takahashi h, ikeda n, kanai y, kiraku y. 2006. occurrence of aspergillus flavus and storage fungi in nutmeg. proceedings 2nd international symposium on mycotoxicology. bangkok, thailand, 13-14 december 2006. mandeel qa. 2005. fungal contamination of some imported spices. mycopathologia 59: 291-8. pesavento g, ostuni m, calonico c, rossi s, capei r, lo nostro a. 2016. mycotic and aflatoxin contamination in myristica fragrans seeds (nutmeg) and capsicum annum (chili), packed in italy and commercialized worldwide. j prev med hye 57: e102-e109. pitt ji, hocking ad. 2009. fungi and food spoilage. new york (us): springer. punnathara cj. 2011. campaign launched to check aflatoxin in spices. http://www. peppertrade.com. br/vernoticia08b1g09.php?idn=2062. [6 jun 2011]. rees d. 2004. insects of stored products. collingwood (au): csiro publishing. romagnoli b, menna v, gruppioni n, bergamini c. 2007. aflatoxins in spices, aromatic herbs, herb-teas and medicinal plants marketed in italy. food control 18: 697-701. samson ra, houbraken j, thrane u, frisvad jc, andersen b. 2010. food and indoor fungi. cbs laboratory manual series. utrecht (nl): cbsknaw fungal biodiversity centre. biotropia vol. 29 no. 3, 2022 192 sni. 2015. pala. sni 0006-2015. jakarta (id): badan standardisasi nasional. tabata s, kamimura h, ibe a, hashimoto h, iida m, tamura y, nishima t.1993. aflatoxin contamination in foods and foodstuffs in tokyo: 1986-1990. j aoac int 76(1): 32-5. takahashi t. 1993. aflatoxin contamination in nutmeg: analysis of interfering tlc spots. j food sci 58: 197-8. toma fm, abdulla f. 2013. isolation and identification of fungi from spices and medicinal plants. res j environ earth sci 5(3): 131-8. uraih n, ogbadu l. 1982. influence of woodsmoke on aflatoxin production by aspergillus flavus. european j appl microbiol biotechnol 14: 51-3. vicam. 2007. aflatest instruction manual for hplc. watertown (us): vicam. p 14. biotropia vol. 29 no. 1, 2022: 69 80 doi: 10.11598/btb.2022.29.1.1627 69 litterfall, litter decomposition and nutrient return of rehabilitated mining areas and natural forest in phangnga forestry research station, southern thailand jetsada wongprom1*, roongreang poolsiri2, sapit diloksumpun2, chatchai ngernsaengsaruay3, samita tansakul2 and wasan chandaeng1 1forestry research center, faculty of forestry, kasetsart university, bangkok 10900, thailand 2department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand 3department of botany, faculty of science, kasetsart university, bangkok 10900, thailand received 3 august 2021 / accepted 20 december 2021 abstract litterfall and litter decomposition play important roles in the maintenance of nutrient cycling and rehabilitation of degraded lands. litterfall, litter decomposition and nutrient return were investigated in a 27-yearold acacia mangium plantation on sandy and clay sites, and in a mixed plantation at the phangnga forestry research station, phangnga province, thailand. additionally, secondary and primary forests were investigated and compared with the values obtained from the acacia mangium and the mixed plantations. the results indicated that litter production in a. mangium plantation on sandy and clay sites, and in mixed plantations (15.47, 11.68 and 7.89 t/ha/yr, respectively) was higher than that in the secondary and primary forests (6.34 and 6.92 t/ha/yr, respectively). the rate of litter decomposition was the greatest in the secondary forest (3.01/yr) and the lowest occurred in the primary forest (1.15/yr). the decomposition rate of the mixed leaf litter between native trees and a. mangium in plantations was higher than that of only a mangium leaf, except in the mixed plantations. a high initial nitrogen concentration in a. mangium could accelerate litter decomposition and improve litter quality in the mixed litter. in addition, the nutrient return in plantations was higher than that in the secondary and primary forests, especially for n. increased litter production, high decomposition rate and nutrient return from a. mangium plantation had important roles in nutrient cycling, suggesting that a mixed plantation consisting of a. mangium and native trees should be considered for the reclamation of mining land. keywords: litter decomposition, litterfall, mining rehabilitation, nutrient return, tropical forest introduction mining operations have been undertaken on a global scale as the raw minerals obtained play an important role in economic and infrastructure development. a high level of mineral production has been reported in asian countries (reichl et al. 2017). the ill-effects of mining cause severe impacts on the ecosystem, including soil degradation, water and air pollution, and loss of wildlife habitat. the vegetation richness and soil properties are destroyed by mining activities, resulting in the degradation of the ecosystem (bell & donnelly 2006; tripathi et al. 2016). improving environmental conditions in mining areas using natural methods takes a long time (oktavia et al. 2015). barriers to natural regeneration include environmental factors such as water, soil ph and soil properties (thaiutsa & rungruangsilp 1990; tripathi et al. 2016). forest plantations have been established on degraded lands to improve the soil properties and ecosystem function (singh et al. 2004; zhang et al. 2014; oktavia et al. 2015). rehabilitation in such areas by planting nitrogen-fixing trees has been recommended and they have been planted on many sites as *corresponding author, email: fforjdw@ku.ac.th mailto:fforjdw@ku.ac.th biotropia vol. 29 no. 1, 2022 70 they grow well and have a high aboveground biomass and litter production (dutta & agrawal 2003; singh et al. 2004). acacia mangium is the most preferred species for reclamation efforts in mining land (martpalakorn 1990; oktavia et al. 2015) and other degraded lands (kamo et al. 2008). soil properties and microclimate under such plantations are improved, resulting in the accelerated natural regeneration of native forest species (parrotta 1999; inagaki et al. 2010). litter production and decomposition play important roles in nutrient cycling and the improvement of soil fertility in terrestrial ecosystems (goma-tchimbakala & bernhardreversat 2006; paudel et al. 2015). on degraded lands, litterfall and litter decomposition are keys to restoring the ecology and soil properties because the litter is a nutrient source (lugo 1992; parsons & congdon 2008). nutrient contents of plants are closely related to tree species, especially nitrogen which is abundant in nitrogen-fixing trees (parrotta 1999; singh et al. 2004). tree composition and forest type are the main factors influencing the quality of the litter produced (parrotta 1999; zhou et al. 2006; tang et al. 2010; paudel et al. 2015). in addition, climatic factors can significantly affect litterfall (zhou et al. 2006; scherer-lorenzen et al. 2007; triadiati et al. 2011). litter fractions that fall onto the forest floor are decomposed by organisms living in the soil, and nutrients are subsequently released to the forest floor, resulting in the improved soil properties and a better forest community, which maintain forest function (parrotta 1999). litter quality and microclimate are related to the litter decomposed by soil microbes (parsons & congdon 2008; cizungu et al. 2014; zhong et al. 2017). furthermore, litter decomposition varies depending on the tree species, successional stage, and forest type (lugo 1992; parsons & congdon 2008; tang et al. 2010). the efficiency of plantations in degraded land rehabilitation not only builds productivity and improves forest structure but also increases forest function such as nutrient cycling (lugo 1992; parrotta 1999). a high litter decomposition rate and nutrient return encouraged natural succession and increased soil nutrients (lugo 1992; celentano et al. 2011), which reduced the time required for the restoration process. therefore, the objective of the current study was to evaluate the potential of a. mangium plantation for mining rehabilitation, focusing on litter production, decomposition and nutrient return in comparison to secondary and primary forests. an effort has been made to compare the rehabilitated plantation area to secondary and primary forests. materials and methods study sites this study was carried out in an abandoned tin mining area, in the phangnga forestry research station, phangnga province, southern thailand (8˚46′5″ n, 98˚16′7″ e) (fig. 1). exotic trees, such as acacia mangium and eucalyptus camaldulensis were commonly planted for tin mining reclamation in thailand. the mining was operated by the gravel pumping method. landform after mining was divided into clay, sand, and gravel areas. soil nutrients were observed to be very low (thaiutsa & rungruansilp 1990). the experimental plots were established in a 27-year-old a. mangium plantation, planted in a sandy soil area (ams), in a clay soil area (amc), and mixed plantation (mp). the planted trees in mp consisted of a. mangium, eucalyptus camaldulensis and dipterocarpus alatus. litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 71 figure 1 location of study sites in phangnga province, southern thailand in addition, the experimental plots were established in a secondary forest (sf, located at 8˚39' 28″ n, 98˚24′ 35″ e) and a primary forest (pf, located at 8˚51' 11″ n, 98˚20′ 5″ e) as reference sites. the sf plot was in an approximately 30-year-old site abandoned after shifting cultivation. the pf plot was classified as a low tropical rainforest. dominant native trees in the 27-year-old plantations and the secondary forest were pioneer tree species including carallia brachiata, aporosa planchoniana, bridelia tomentosa, vitex pinnata, microcos paniculata and eurya acuminata. meanwhile, dominant trees in pf were swintonia schwenckii, dipterocarpus kerrii, mesua ferrea, hopea griffithii and gluta elegans. tree composition and dominant trees in ams, amc, mp, sf and pf were reported by wongprom et al. (2020). climatic conditions during the study were recorded at the climate station in the takuapa district. the rainfall was 3,260.10 mm with the rainy season occurring from april to november and the dry season happening from december to march. the mean relative humidity was 83% and the mean temperature was 27.58 °c. data on rainfall, mean temperature and relative moisture recorded during the study are shown in figure 2. figure 2 climatic conditions at takuapa district, phangnga province during the study phangnga province bangkok takuapa district pf pfrs sf primary forest (pf) secondary forest (sf) phangnga forestry research station (pfrs) n biotropia vol. 29 no. 1, 2022 72 litter production three experimental plots (each 40 x 40 m) were established in each ams, amc, mp, sf, and pf for estimating the amount of litterfall production. five litter traps (each 1 x 1 m) with a 2 mm mesh size were placed 1 m above the ground in each plot. litterfall in the traps was collected monthly for 1 year. litterfall samples were sorted into leaf, branches, reproductive parts and miscellaneous. a. mangium litter was separated from that of the other tree species. all samples were oven-dried at 80 °c for 48 h until reaching constant weight. the samples (leaf, branches, reproductive parts and miscellaneous) were analyzed during both the rainy and dry seasons. the organic debris obtained from each site was analyzed as a single sample, as it was difficult to identify the components as they were very small and had a similar texture. litter decomposition and nutrient return nylon bags (50 x 50 cm) with a 2 mm mesh size were used for investigating litter decomposition. leaf having the top five importance value index (ivi) trees in ams, amc, mp and sf, and the top seven ivi trees of pf were selected to investigate litter decomposition. litter decomposition was divided into two classes: 1) mixed leaf litter in ams, amc, mp, sf and pf; and 2) pure a. mangium leaf litter in ams, amc, and mp. in each case, 30 g samples of air-dried leaf litter were filled into the nylon bags according to the ivi ratio of the dominant trees for each site. the ivi of trees in ams, amc, mp, sf and pf was reported by wongprom et al. (2020). the litter water content was determined by first, ovendrying subsamples of air-dried litter at 80 °c for 48 h to obtain the initial dry mass. twelve litter bags from each treatment with three replications were randomly placed in each plot. three samples of both mixed leaf litter and pure a. mangium leaf litter were retrieved monthly. the leaf litter remaining in each bag was brushed to remove any soil particles and roots. the remaining litter was oven-dried at 80 °c for 48 h until a constant weight was recorded. the annual litter decomposition rate (k) was calculated according to the negative exponential decay model (olson 1963) given as k = ln (x/x0)/t, where x0 is the initial dry weight, x is the dry weight remaining at the end of the study, and t is the time period in years. nutrient return (n, p, k, ca, and mg) to the forest floor (kg/ha) was estimated by multiplying the amount of litter production by the nutrient concentration of litter for each site. chemical analysis of litter nutrient concentrations of nitrogen (n), phosphorus (p), potassium (k), calcium (ca) and magnesium (mg) of leaf, branches, reproductive parts and miscellaneous obtained from the study sites were determined during the rainy (april to november) and dry (december to march) seasons. samples of each litter type in the rainy and dry seasons were mixed and analyzed. the concentration of n was measured based on dry combustion using a cnhs analyzer, while the concentrations of p, k, ca, and mg were determined by wet washing with nho3-hclo4 acid (hno3: hclo4; 5: 2). the concentration of p was analyzed using the vanadomolybdate yellow color method with a spectrometer at a wavelength of 440 nm, while the concentrations k, ca, and mg were analyzed using atomic absorption spectrometry. data analysis nutrient concentrations of each litter sample during the rainy and dry seasons were averaged to calculate the amount of nutrients return. the annual litter production, nutrient return and litter decomposition rate among sites were analyzed using a one-way anova and the means were compared with the tukey hsd test at a 5% probability level. results and discussion litter production the annual litter production was significantly different among sites (p < 0. 05). ams had the highest litterfall followed by amc, mp, pf, and sf (table 1). litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 73 table 1 litter production (leaf, branches, reproductive parts and miscellaneous) (t/ha/yr) of a. mangium (am) and native trees and planted trees (npt) in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site leaf branches reproductive parts miscellaneous total am npt am npt am npt ams 5.90a 2.85b 0.74 1.67 2.86a 0.57 0.88a 15.47a amc 3.56ab 3.83ab 0.56 1.20 1.60b 0.50 0.43b 11.68b mp 1.52b 2.86b 0.59 1.06 0.84b 0.51 0.51ab 7.89c sf 4.12ab 0.76 0.74 0.72ab 6.34c pf 4.72a 0.89 0.72 0.59ab 6.92c f value 11.59** 8.03** 1.83ns 0.87ns 16.55** 2.02ns 4.58* 27.21** notes: * = significant difference; ** = very significant difference; ns = non-significant difference; a c = different superscripts in the same column indicate significant differences at p < 0.05. litter production peaked in the dry season (december to march) (fig. 3), as a response to the water stress, which was similar to that of other tropical forests (triadiati et al. 2011; cizungu et al. 2014). leaf component was the main litter falling onto the forest floor in ams, amc, mp, sf and pf, and made up around 54 to 62% of the total litter. however, the amount of reproductive parts in ams and amc was relatively high compared to branches, especially the reproductive parts of a. mangium as it fell almost all year. a large amount of litter from a. mangium in ams was resulted from the high tree density and crown cover, while a. mangium in amc and mp had a low crown cover due to the high mortality rate. litters from other trees in ams, amc, and mp came from native tree species. planted trees in mp such as d. alatus and e. camaldulensis were also a main source of litter. leaf litter is a significant contributor of annual primary production and nutrient capital in a terrestrial ecosystem (cizungu et al. 2014; paudel et al. 2015). figure 3 total monthly litter production (t/ha/yr) of the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) biotropia vol. 29 no. 1, 2022 74 this study showed that the amount of litter production in ams and amc was higher than that in both sf and pf. a forest plantation with fast-growing trees is highly effective at building litter production compared to a natural forest (goma-tchimbakala & bernhard-reversat 2006) and secondary forest (lugo 1992; kamo et al. 2008). post mining reclamation should focus on increasing the aboveground biomass and litter production to improve the ecosystem. a. mangium is one species recommended for degraded land restoration. acacia grows well and has high aboveground biomass compared to native trees (martpalakorn 1990; kamo et al. 2008). however, the total litter production in mp was similar to that in pf and sf, although a. mangium had less litter production. the canopy cover of a. mangium can promote a high litterfall. fast nitrogen-fixing tree plays important role in the early stage by rapid growth to create a canopy in order to suppress the weeds and shrubs that could obstruct the establishment of native tree understory vegetation and to increase nitrogen availability (ingaki et al. 2010; lanuza et al. 2018). however, the litterfall in ams, amc and mp was not only sourced from planted trees but also native trees through natural succession. old plantations have high tree diversity and richness (lugo 1992; koonkhunthod et al. 2007), as litterfall is contributed to by native trees. native tree species are usually pioneer trees having high growth rates (chazdon 2014; chen et al. 2017) and their litter production increases with succession age (zhou et al. 2006; feng et al. 2019). litterfall production plays an important role in forest functions such as carbon and nutrient cycling (moura et al. 2016; paudel et al. 2015; lanuza et al. 2018) because litter is a source of nutrient pools, which releases nutrient to the forest floor via litter decomposer activities (lugo 1992; fisher & binkley 2000), suggesting that a. mangium should be recommended for reclamation in a mining area due to the capability of a. mangium in bringing out high litter production. litter production in the sf and pf study sites was not different, resulting from pioneer trees producing litter in natural succession. forest structure and tree composition of succession forests affected litter biomass (lanuza et al. 2018). long-lived pioneer trees and high tree diversity were observed in sf. the litter production in the sf and pf study sites was similar to that in many evergreen forests in thailand (6.42 to 7.85 t/ha/yr) (bunyavejchewin 2001; glumphabutr et al. 2007). however, litter production in our study was lower than that in other tropical forests (8.30 13.67 t/ha/yr) (anderson et al. 1983; tang et al. 2010; triadiati et al. 2011; paudel et al. 2015). litter decomposition litter decomposition of pure litter and mixed litter was significantly different among treatments (p < 0.05). the litter decomposition rate (k) ranged between 1.15 and 3.01/yr, which was classified as medium to high levels. the decomposition rate in sf was the highest, indicating litters were rapid litter decay, but not different in amc (2.91/yr) (table 2). the dominant trees in sf and amc were pioneer tree species and were of similar tree composition (wongprom et al. 2020), having thin and flexible leaves without a prominent skeleton, and so could decay easily. on the other hand, a low rate of decomposition was found in climax forest trees due to their thick, tough leaf and prominent midribs and veins. the leaf morphology of trees (leaf thickness, roughness, toughness) significantly affects the rate of litter decomposition (liao et al. 2006; cizungu et al. 2014). in addition, the chemical properties of leaf litter, including high n, n/ p, low c/ n and low lignin, are generally positive contributors to decomposition (xuluc-tolosa et al. 2003; liao et al. 2006; cizungu et al. 2014; rai et al. 2016). similarly, leaf litter in sf, ams, amc, and mp had a high nitrogen content, which could explain their faster rate of decomposition. leaf litter in ams, amc, and mp was rich in nitrogen due to the litter produced by a. mangium. in addition, leaves of dominant pioneer trees in sf, ams amc and mp have thin and less rigid leaves leading to a high decomposition rate compared to pf. however, the litter decomposition of pf was of moderate level (1.15/yr), which was similar to other climax tropical forests (hättenschwiler et al. 2011). litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 75 table 2 decomposition rate of pure a. mangium and the mixed leaf in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site type of leaf k (constant) ams pure a. mangium leaf 1.56bc mixed leaf 1.64bc amc pure a. mangium leaf 1.88bc mixed leaf 2.91a mp pure a. mangium leaf 2.05b mixed leaf 1.87bc sf mixed leaf 3.01a pf mixed leaf 1.15cd f value 7.905* notes: * = significant difference; a d = different superscripts in the same column indicate significant differences at p < 0.05. climate conditions and soil microbials significantly affect litter decomposition (berg & mcclaugherty 2008). this study indicated that rainfall and relative humidity in the study sites were of similar conditions. however, soil microbials could be different because soil properties in ams, amc, and mp were of poor soil compared to that in sf and pf, especially in terms of total n and om (wongprom et al. 2020), leading to low diversity and abundance of soil microbials (zhang et al. 2016). soil nutrients, n and p and organic matter (om) are significantly correlated with soil microbials (zhang et al. 2016; ngugi et al. 2020). low decomposition rate of litter was found in the pf study site. microclimate conditions may slightly affect litter decomposition. litter quality of the restored forest may significantly determine litter decomposition. meanwhile, litter decomposition rate changes depending on successional stage and tree composition (moura et al. 2016; lanaza et al. 2018). a study conducted by xuluc-tolosa et al. (2003) indicated that pioneer trees in the early succession phase has a higher decay rate than tree species in the late succession phase because pioneer trees have a high initial n concentration and low c/n. pioneer tree species have high nutrient resorption efficiency and high leaf nutritional quality, especially n concentration (gomes & luizão 2012). our study showed that a litter mixture of a. mangium and native trees generally resulted in a faster decay than the decay of pf litter. this result could be affected by synergistic effects of mixed litter with high litter quality and a high initial n concentration. mass loss is positively correlated with initial n concentration (liu et al. 2016). thus, poor litter quality is improved. mixing litters from various trees accelerate the mass loss, enhance the nutrient release and nutrient cycling (liu et al. 2016; trogisch et al. 2016; cizungu et al. 2016) and different litter components may change the litter chemical components and the decomposer community (gartner & cardon 2004). nutrient concentration in litter and nutrient return nutrient concentration in litter produced by a. mangium in this study varied among components (leaf, branches and reproductive parts). however, the n concentration was greater than that of p, k, ca, and mg (fig. 4). nutrient concentration in the leaf and branches litters of a. mangium in all sites followed the order of n>ca>k>mg>p, except for the reproductive parts, where the k concentration was higher than that of ca. biotropia vol. 29 no. 1, 2022 76 figure 4 nutrient concentration of pure a. mangium leaf, branches and reproductive parts of the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc) and mixed plantation (mp) n concentration in the litter of other trees (native tree species in ams, amc, mp, sf and pf, and planted trees in mp) was the highest for all litter components, and was especially high in the litter miscellaneous component. the p concentration was relatively high in the miscellaneous and reproductive parts components, while the concentrations were similar for k, ca and mg in the leaf, branches, reproductive parts and miscellaneous components. the nutrient concentration of leaf, branches, reproductive parts and miscellaneous for the other tree species in ams, amc, mp, sf, and pf are shown in figure 5. figure 5 nutrient concentration of mixed leaf, branches, reproductive parts and miscellaneous litter of native tree species in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), secondary forest (sf), and primary forest (pf) and planted trees and native tree species in mixed plantation (mp) litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 77 the nutrient return of n, p, k, ca and mg from litters to forest floor was significantly different among sites. n contributed the highest nutrient return to the forest floor in all sites. the large amounts of n, p, k, ca and mg returned in ams, amc, and mp were mostly from the litter of the planted trees, especially a. mangium (table 3). a. mangium is the dominant tree with a large size and crown cover, although the tree density is low, particularly in the amc and mp sites (wongprom et al. 2020). litters from planted trees in a plantation are still the main nutrient source to the forest floor, although the vegetation composition are shifted after restoration. leaf litter was the main source of nutrient return in all sites. however, nutrient return from miscellaneous and reproductive parts of a. mangium was relatively high. the annual nutrient return of ams, amc and pf followed a pattern of n>ca>k>mg>p, while that of mp and sf followed a pattern of n>k>ca>mg>p (table 4). the annual nutrient return of n, p, ca and mg to forest floor in ams and amc was significantly higher than that in sf and pf, especially for the nitrogen. meanwhile, the annual nutrient return of n, p, ca, and mg in sf was similar to that in pf. forest community on a restored site and in a secondary forest can accelerate litter decomposition and nutrient cycling because the litter quality is improved. nutrients returned to the forest floor can promote natural regeneration and tree growth, resulting in a complex forest structure in the long term, especially as n is a significant nutrient in developing a forest community and establishing seedlings and saplings during mining restoration and natural succession in a degraded land (zhao et al. 2013; lei et al. 2015). fast litter decomposition of restored sites resulted in high nutrient depositions, especially n. in the current study, n was the major nutrient return in ams, amc and mp, and was significantly higher than that in sf and pf. the high n return may be resulted from a. mangium due to the acacia being a nitrogen-fixing tree. moreover, the return of p, k, ca and mg to the soil of the rehabilitated sites was greater than that in pf. soil nutrients of a. mangium plantation in an abandoned mining area are higher compared to that in an abandoned mining area without the a. mangium plantation, especially in terms of n and soil organic matter (wongprom et al. 2020). therefore, the study result indicated that a. mangium improves soil chemical properties through nutrient cycling processes. n flux of nitrogen-fixing tree plays an important role in the early succession (moura et al. 2016). table 3 nutrient return (kg/ha/yr) of a. mangium (am) litter and native trees and planted trees (npt) litter in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), and mixed plantation (mp) site n p k ca mg am npt am npt am npt am npt am npt ams 18.96a 7.12 0.94a 0.49 2.07a 1.48b 2.92a 2.14ab 1.91a 1.20b amc 10.97a 6.52 0.68a 0.52 1.37a 2.93a 2.08ab 2.64a 1.41a 1.94a mp 5.47b 6.42 0.30b 0.49 0.85b 2.73a 1.11b 1.91b 0.62b 1.56ab f value 26.37** 1.09ns 18.39** 0.37ns 14.95** 29.01** 14.48** 6.19* 15.66** 12.36** notes: * = significant difference; ** = very significant difference; ns = non-significant difference; a b = different superscripts in the same column indicate significant differences at p < 0.05. table 4 nutrient return (kg/ha/yr) of litter to the forest floor in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site n p k ca mg ams 26.09a 1.43a 3.56b 5.06a 3.12a amc 17.49b 1.19a 4.30a 4.72a 3.36a mp 11.89c 0.79b 3.58b 3.03b 2.18bc sf 8.26c 0.63b 4.21ab 2.72b 1.74bc pf 7.47c 0.61b 2.77c 3.20b 1.28c f value 53.60** 33.78** 15.87** 22.75** 34.63** notes: ** = significant difference; a c = different superscripts in the same column indicate significant differences at p < 0.05. biotropia vol. 29 no. 1, 2022 78 fast-growing tree plantations are commonly planted for wood production. however, they are also planted for forest restoration in degraded lands. high litter production and litterfall are significant parts in restoration processes (parrotta 1999). the n nutrient returned to the forest floor from nitrogen-fixing tree plantations was significantly higher than that from a nonnitrogen-fixing tree plantations and for other nutrients (bernhard-reversat 1996). the litter decomposition of nitrogen-fixing trees positively affect the acceleration of nutrient release and nutrient deposition to promote vegetation development and improve soil properties (lugo 1992; ruiz-jaén & aide 2005; lanuza et al. 2018), suggesting that a. mangium plantation should be used for improving nutrient cycling in mining reclamation. nutrient cycling is a key process for mining rehabilitation, relating to vegetation succession and soil development in the mining area. conclusion a. mangium plantation is an effective way for mining rehabilitation compared to the reference sites. a. mangium plantation showed relatively high litter production and litter decomposition rate which could modify the litter quality in the litter mixture. mixed litter sourced from nitrogen-fixing trees and native trees in plantations can enhance litter decomposition, which can result in higher levels of soil improvement and revegetation. ams, amc and mp had high levels of nutrient return compared to that in sf and pf, leading to contributing high levels of soil nutrients to forest floor, particularly n. the litter mixture in mp decomposed more rapidly compared to that in pf with improved the litter quality. a mixed plantation consisting of a. mangium and native tree species could be considered for reclamation efforts in mining area and other similar degraded lands. acknowledgments financial support for this study was provided by the kasetsart university research and development institute (kurdi) in bangkok, thailand. we are grateful to the staff at the phangnga forestry research station for their assistance during the field work. references anderson j, proctor mj, vallack hw. 1983. 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doi: 10.11598/btb.2021.28.2.1277 149 agronomic traits of local wetland rice varieties in jambi province julistia bobihoe*, jumakir, araz meilin, and endrizal balai pengkajian teknologi pertanian jambi (jambi assessment institute for agricultural technology/aiat), jambi 36128, indonesia received 26 june 2019/accepted 23 july 2019 abstract indonesia’s swamplands are among areas earmarked for future agricultural development. a s a t y p e o f w e t l an d s , swamplands are inundated and have soil properties that are uniquely different from other agroecosystems. i n indonesia, some of these areas a r e currently u s e d f o r r i c e cultivation of the country’s very diverse genetic resources of local rice varieties. most o f t h e farmers continue to plant and cultivate the local rice swampland varieties because of the abilities to adapt to extreme environments. this study on the agronomic traits of the local swampland rice varieties was carried out to evaluate their agronomic characters and identify varieties having superior quality traits. the research was carried out from april to october 2016 using a single plot method at the rantau kapas mudo village, batanghari regency, jambi province. eleven (11) rice varieties were planted in 10 x 5 m single plots, with a spacing of 25 x 25 cm and 1 m distance between plots. these 11 genetic resources of the local swampland rice varieties, include the serendah halus, rimbun daun, karya, serendah bawang, sereh aek, botol, pontianak, semut, dawi, ketan itam and di. the observed characters consisted of the plant height at harvest, number of productive tillers, age of harvest, number of grains per panicle, number of filled grains per panicle, number of empty grains per panicle, weight of 1,000 grains, seed shape and the production volume. there were differences among the local swampland rice varieties with the highest production volume of 3.32 tonnes/ha obtained from the rimbun daun variety, followed by 2.86 tonnes/ha from the dawi variety. these two varieties had shown potential to become the leading regional swampland rice varieties. keywords: agronomic traits, local rice, swampland introduction swampland is a very unique agro-ecosystem with characteristically inundated soils. in indonesia, it is one of those areas designated for future agricultural development (noor & fadjry 2008). swampland is also distinguished by the presence or absence of the influence of a surrounding river. those inundated by the surrounding river is called the river swamp, while the free or unaffected swampy area is called a caged or half-caged swamp (kosman & jumberi 1996). increasing rice production in swampland had increased farmers' income, and had supported food self-sufficiency (djafar 1992). however, the main obstacle in swamp rice cultivation is the uncontrolled water flow that during the rainy season the entire area is inundated quite deep for quite a long time. this makes it difficult for farmers to estimate the rice planting period. puddles that are too high during the vegetative phase due to flooding and heavy rains, occur after the seedlings are moved into the field. these are growth constraints that result in low production of even the low-yield rice variety. almost synchronously, the risk of crop failure due to drought can also occur if there is no rain when the rice plants bloom (swamp research center 2008). hence, farmers generally plant rice varieties that are genetically adapted to the local growing environment in swamplands. germplasm is the fourth natural resource in addition to water, land and air resources which need to be conserved. hence, preservation of *corresponding author, email: julistia117@gmail.com biotropia vol. 28 no. 2, 2021 150 germplasm as a genetic source will affect the success of agricultural development programs. the desired food sufficiency will depend on the regions’ own germplasm diversity, because in reality superior varieties, which have been, are being, or will be produced are a collection of specific genetic diversity that is expressed in the desired superior qualities. the regional genetic resource is the starting point in the formation of plant varieties that produce high yield, are pests and diseases resistant, and are tolerant to environmental stress (situmeang 2015). agricultural genetic resources (agrobiodiversity) are one of the most important germplasm to be protected from extinction and potential genetic erosion (diwyanto & setiadi 2008). in reality, these resources have been and will continue to be utilized for the survival and welfare of the community, at the local, regional, national, and global levels. as a mega-biodiverse country, indonesia is currently thought to be rich in germplasm collection. however, indonesia actually has inadequate collection of germplasm for producing superior varieties. genetic diversity is an economic, tourism, health and cultural resource. however, it is not evenly distributed in each region as it depends on the regional ecosystem (wardana 2002). natural resources utilization, accompanied by preservation of region-owned diversity and uniqueness, can be directed toward human welfare and carried out continuously from one generation to the next. the archipelagic nature of indonesia with its various tribes and cultures, is closely related to genetic resource utilization which may vary between regions and agroecology. cultural diversity together with agricultural genetic resources diversity will produce diversified community knowledge on resource utilization for food, shelter, clothing, medicines and industrial raw materials (diwyanto & setiadi 2008). among the various wetland agroecosystems (irrigation, rainfed and swamp) in jambi province, the swampland agroecosystems are the widest, comprising some 137,132 ha with the swampland area occupying 25,157 ha currently planted with local rice varieties (cpm 2016). these varieties are potential alternative food resources that need to be inventoried and conserved for the production of local superior varieties. local rice varieties are those varieties that have long been adapted in certain areas and are generally consumed as food in its original form (hajoeningtijas & purnawanto 2013). these local varieties need to be preserved as regional genetic resources and as a source of breeding material for future varieties improvement programs (arda 2013; rais 2004). the characteristics of these local rice varieties are largely not well identified, so knowledge on their potential and development opportunities as superior local rice varieties is still lacking. in the field, their local population still looks diverse, particularly the plant height, ripening or harvest age, shape, and grain color. the local varieties have adapted to certain environmental conditions and were characterized based on their low production rate, high and strong trunk, chronic unresponsiveness to input or fertilization, diverse physical characteristics, delicious taste, popularity among many consumers, and high market price. this will affect farmers’ production of rice. seeds of local rice varieties used by farmers are also of low quality because they are continuously obtained from farmers’ rice crops and inherited from generation to generation (bobihoe 2014). the purpose of this study is, therefore, to determine the characteristics of local rice in swampland and to discover the varieties that have superior qualities based on their agronomic characteristics. these results are expected to help expand opportunities to produce novel rice varieties with better qualities thereby improving rice productivity in the swamplands. materials and methods the research activities were carried out from april to october 2016 at rantau kapas mudo village, batanghari regency, jambi province. eleven (11) local varieties of swampland rice were studied, namely serendah halus, rimbun daun, karya, serendah bawang, sereh aek, botol, pontianak, semut, dawi, ketan itam, and di. using the single plot method, each variety was planted in a 10 x 5 m single plots with 25 x 25 cm spacing and 1 m distance between plots. the study location was classified as low-lying, lower topography with a pool of water between agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 151 50 and 100 cm deep during a period between three and six months. the swampyland is generally muddy and has a medium to high soil fertility (waluyo & suparwoto 2016). the observed parameters included those at the vegetative and reproductive phases. the vegetative characters consisted of the plant height at harvest (measured from the base of the stem to the highest tip of panicle), the number of productive tillers (calculated at the time of harvest). the generative characters were measured after harvest time and consisted of the harvest age (calculated from the number of days after planting), number of grains (counted number of grains) per panicle, number of filled grain (calculated number of filled grains) per panicle, number of empty grains (calculated number of empty grains) per panicle, weight of 1,000 grains (weight of 1,000 grains of grain content), and yield (tonnes/ha). plant data were observed agronomically and tabulated for 10 clumps of rice plants. results and discussion eleven (11) local swamp rice varieties characterized in terms of agronomical traits, consisted of serendah halus, leaf rimbun, karya, serendah bawang, serehaek, bottle, pontianak, semut, dawi, ketanitam, and di (table 1). these were chosen from the farmers’ fields located at rantau kapas mudo village, muaro tembesi district, jambi province, indonesia. vegetative characteristics of the rice plants plant height the height of the tested local rice varieties ranged between 86.2 and 147.6 cm with serendah bawang being the tallest at 147.6 cm, and botol the shortest at 86.2 cm (table 1). the crops were classified into three categories based on their height, i.e., short (<110 cm), medium (110 to 130 cm), and tall (>130). the local rice varieties were generally tall (>130 cm) (scale 5) except botol, dawi, and di (<110 cm) (scale 1). the plant heights were measured at the start of the harvest. medium-height plants (110 130 cm) more likely developed more productive tillers than taller crops, as tall crops use up photosynthates for vegetative growth, making photosynthesis less efficient. the results showed that the rice cultivation system in the swampland produced taller plants than that in the upland cultivation system. rice plant heights can be used as a parameter of rice growth, but taller plants do not guarantee greater yields (kristia & iskandar 2018). plant height is one component that influences a plant’s weight. the desired characteristics in the development of superior rice varieties are stems that are short and stiff, considering that plants with this characteristic will be resistant to fall, be responsive to fertilization, and have a more balanced proportion between grain and straw. another characteristic is the plants’ absorption of n; the higher the performance of a plant, the higher the likelihood of its vulnerability (kristia & iskandar 2018). table 1 vegetative characteristics of the local swampland rice varieties no. variety name plant height* (cm) number of productive tillers** (tiller) 1. botol 86.2 10 2. pontianak 139 10 3. semut 120.8 10 4. dawi 91.3 13 5. ketan hitam 111.4 9 6. di 104.7 10 7. serendah halus 141.4 7 8. rimbun daun 132.9 10 9. karya 140.6 9 10. serendah bawang 147.6 6 11. sereh aek 143.6 9 notes: * = average plant height at harvest (cm); ** = number of productive tillers at harvest (tillers). biotropia vol. 28 no. 2, 2021 152 among the selection criteria for rice plants, height is related to the length and shortness of the panicles and to the shedding resistance of plant stems. plants tend to be shorter at locations higher than sea level (simanulang 2001). although plant height is one selection criteria for rice plants, nevertheless, taller stems do not influence the level of production (suprapto & dradjat 2005). high plant growth does not guarantee high plant productivity. optimal plant growth has a large influence on the relationship between panicle length and yield. plants that grow well absorbs large quantities of nutrients. the availability of these nutrients in the soil influences photosynthetic activity in a way that plants can increase crop growth and yield (aribawa 2012). number of productive tillers the number of productive tillers varied from 6 to 13 with the highest number produced in the karya variety (13 tillers) and the lowest number in serendah bawang (6 tillers). this low number of tillers is probably due to the submersion of the initial seedling for about a week, thus inhibiting the formation of saplings (icr 2016). high and low plant growth and yield are also influenced by internal factors which include genetic traits and plant derivatives, and the external or environmental factors, such as soil regime and biotic factors (cepy & wayan 2011). the different number of tillers produced by each variety is thought to be due to the influence of these factors. the number of tillers and plant height differs because the genetic properties of each variety also differ (manurung & ismunadji 1988). productive tillers directly affect the high and low yields of grain as it greatly determines the number of panicles (simanulang 2001). more productive tiller means more number of panicles. in this study, a correlation exists between the number of panicles and yield; the higher number of panicles resulted in higher yield of the plants. generative characteristics of the rice plants in the generative growth phase, the characteristics observed included the harvest age, number of grains, number of filled grains, empty grains, weight of 1,000 grains, age of harvest and yield (table 2). harvest period (number of days after seedling) generally, the cultivation ages for rice crop are categorized into early age (about 110 days) and old age (more than 120 days). the local rice varieties are generally old at >151 days after seedling stage, while high yielding varieties mature at 105 124 days after seedling stage (icrr 2016). the local swamp rice cultivars are generally cultivated within 150 days, while the local variety is cultivated at an early age (120 days for karya). obviously, shorter harvest age is preferred by farmers because it means shorter harvest time gaps and more frequent harvest periods. agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 153 table 2 yield components of the local landraces of swampland no. variety name harvest age1 (dap) number of grain per panicle2 (grain) number of filled grains per panicle3 (grain) number of empty grain per panicle4 (grain) weight of 1,000 grains5 (g) volume production6 (tonnes/ha) 1. botol 150 132 74 58 22 2.32 2. pontianak 150 122 88 34 23 2.24 3. semut 150 113 49 64 14 1.26 4. dawi 150 172 80 92 16 2.86 5. ketan hitam 150 136 82 54 21 2.05 6. di 150 132 78 54 23 2.42 7. serendah halus 150 151 61 90 21 1.77 8. rimbun daun 150 173 86 87 24 3.32 9. karya 120 137 47 90 19 1.97 10. serendah bawang 150 170 103 67 25 2.04 11. sereh aek 150 165 73 83 20 2.37 notes: 1 = average age of harvest (day after planting); 2 = number of grain per panicle (grain); 3 = number of filled grains per panicle (grain); 4 = number of empty grain per panicle (grain) (empty grains were generally found at the base of panicles); 5 = weight of 1,000 grains (g); and 6 = volume production (ton/ha) of local rice swamp varieties. number of grains per panicle (grains), number of filled grains per panicle (grains), number of empty grains per panicle (grains). the dawi and rimbun daun varieties produced the highest number of grains per panicle, 172 and 173 grains per panicle, respectively, while the semut variety produced the least numbers of grains per panicle at 113 grains/panicle. moreover, serendah bawang variety had the highest grain content at 103 filled grains, while semut variety had the least grain content at 42 filled grains. the lowest number of empty or unhulled grains was produced in the pontianak variety at 34 grains while the highest number was found in dawi variety at 92 grains. this high number of empty grains showed the inability of those plants to fill their own grains. such a low yield can be due to genetic or environmental factors. empty grains affect the rice yield; the higher the percentage of empty grains, the lower the rice yield would be. consequently, higher proportion of empty seeds would result in lower rice production. the number of filled grains per panicle correlates with rice yield, hence, it is one of the criteria for a high yielding rice variety. although the number of filled grains is markedly correlated with rice crop yield, the latter variable is still strongly influenced by the number of empty grains. likewise, the weight of grain contents also determines the weight of the yield. empty seeds also affect the rice yield; the higher the percentage of empty grains, the greater the effect on rice yields. new types of rice varieties with high yield potential, generally possess the following characteristics, namely (1) the number of grains per panicle is between 150 and 250 grains; (2) the percentage of filled grains is between 85% and 95%; and (3) the percentage of empty grains is between 5 and 15% (abdullah 2008). generally, the number of grains per panicle is positively correlated with panicle length. the longer the panicle is formed, the more amount of grain is accommodated by the concerned panicle. meanwhile, the number of filled grains and the weight of 1,000 grains formed in one panicle is highly dependent on the photosynthesis (seed filling) of the plants during their growth and on the genetic properties of the cultivated rice plants (ariwibawa 2012). weight per 1,000 grains (g) and weight of yield (tonnes/ha) the weight per 1,000 grains of each variety varied between 14 g and 25 g. the highest weight per 1,000 grains was produced in the serendah bawang variety at 25 g, while the lowest was found in the semut variety at 14 g. weight per 1,000 grains indirectly describes the biotropia vol. 28 no. 2, 2021 154 size or enormity of a rice grain. the higher the weight per 1,000 grains of a rice variety, the larger is its grain size, and vice versa. the grain size is affected by its genetic properties and adaptability to its growing environment. during the dry season, the highlands with their low temperatures, affect the weight of the 1,000 grains. the difference in weight per 1,000 grains of plants determines the ability of a variety to produce a lot of grains which is often inversely proportional to its ability to produce large and heavy grains, nevertheless, a high production rate can still be achieved with a large number of grains, even though the grain size is smaller (simanulang 2001). weight of the 1,000 grains indirectly illustrates the grain size of a rice variety. strains/varieties with large grains produce higher weights of the 1,000 grains and vice versa. grain size is influenced by genetic traits and adaptability to the growing environment. hence, rice yield is also affected by the the weight of the 1,000 grains. finally, of all the varieties tested, rimbun daun produced the highest yield at 3.32 tonnes/ha. this result coincides with that of the number of grains per panicle where the rimbun daun variety produced the highest yield of 173 grains, while the lowest yield was at 1.26 tonnes/ha in the semut variety. this result also confirmed the effects of both the lowest number of filled grains per panicle and the lowest weight per 1,000 grains on the production volume of the semut variety. plant yield is determined by its components’ yield. hence, an imbalance in the components will greatly influence the expected potential result. similarly, rice yield is determined by its yield components, such as the number of filled grains per panicle and the weight per 1,000 grains. correlation of the plant’s tangible yield with its weight per 1,000 grains and its number of filled grains per panicle is one of the criteria for selecting the rice variety which can produce a high yield (manurung & ismunadji 1988). the yield per hectare of the eleven tested local swamp rice varieties planted on lebak swamp showed that two local varieties produced the two highest yields of more than 2.5 tonnes per ha, namely; rimbun daun at 3.32 tonnes/ha and dawi at 2.86 tonnes/ha. these two varieties have shown some potential for becoming the superior local rice varieties. finally, with the use of appropriate cultivation technology, these two local rice swamp varieties will produce their expected potential yield. conclusion in summary, two local rice varieties have exhibited high yield potential of above 2.5 tonnes per hectare, namely; rimbun daun at 3.32 tonnes/ha, and dawi at 2.86 tonnes/ha. these two varieties have shown some potential for large scale production and prospects of becoming the leading rice varieties in the swampy areas. acknowledgments the author would like to thank the technicians ms. hasniarti, ms. kusningsih, and mr. joko supriyanto; batanghari district extension instructor sp, muhammad; jambi bptp worker yatmi; and driver dendy; who helped in the implementation of the local rice characterization activities in batanghari regency, jambi province. references abdullah b, tjokrowidodo s, sularjo. 2008. the development and prospect of assembling new types of rice in indonesia. agricultural research and development journal 27:1-9. agricultural research and development agency (arda). 2013. inventory and/or collection of plant genetic resources in indonesia. center for biotechnology research and development and agricultural genetic resources. agricultural research and development agency. ministry of agriculture. ariwibawa ib. 2012. effect of planting systems on increasing rice productivity in wetland wetlands paper presented at the national seminar on food sovereignty and energy. faculty of agriculture, university of trunojoyo madura, june 2012. bobihoe j, hernita d, salvia e, muliyanti k, hayanti sy, supriyanto j, hasniarti, … , endrizal. 2014. management report on genetic resources. agricultural technology assessment center (bptp) jambi. center for agricultural technology agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 155 research and development (bbp2tp). agricultural research and development agency. ministry of agriculture. cepy, wayan w. 2011. growth and yield of rice plants (oryza sativa l.) in vertisol media and entisol in various water management techniques and types of fertilizers. crop agro journal 4(2): 49-56. cpm. 2016. paddy production in jambi province. agricultural survey. jambi (id): central statistics agency (cpm). diwyanto k, setiadi b. 2008. germplasm in the management of utilization and conservation of sources agricultural genetic power. jakarta (id): national commission on germplasm. djafar zr. 1992. the potential for swampland to achieve and preserve food self-sufficiency. proceedings of the national seminar: utilizing the potential of swampland for achieving and preserving food self-sufficiency. palembang (id): faculty of agriculture, universitas sriwijaya. hajoeningtijas od, purnawanto am. 2013. diversity of local rice in banyumas regency, central java. agritech. xv (2):69-77. icrr. 2016. research institute for rice planting in sukamandi. agricultural research and development agency. kodir ka, yuana j, triyandara. 2016. inventory and characteristics of swamp local rice morphology in south sumatra. germplasm bulletin 22 (2):101108. bb biogen. agricultural research and development agency. kosman e, jumberi a. 1996. display of farming potential in swampy land. in b. prayudi et al. (eds.). proceedings of the sut technology seminar on swamp and dry land. book i. balittra banjarbaru. kristia a, iskandar l. 2018. growth and production of several kalimantan local rice cultivars. agroforti bulletin 6 (2):260-70. manurung so, ismunadji m. 1988. rice: morphology and physiology of rice. agricultural research and development agency. bogor (id): food crop research and development center. noor m, fadjry. 2008. opportunities and constraints of agricultural development in the swampy agroecosystem: the case of primatani village in south kalimantan. proceedings of the national workshop on the acceleration of the application of science and technology and technological innovations in supporting food security and revitalization of agricultural development. jambi, 11–12 december 2007. jambi aiat, food security guidance board jambi province. bbp2tp. research and development agency. silitonga ts, somantri ih, daradjat aa, kurniawan h. 2003. guide to rice crop characterization and evaluation systems. national germplasm commission. agricultural research and development agency. agriculture department. simanulang za. 2001. selection criteria for agronomic and quality properties. training and coordination of participatory breeding programs (shuttle breeding) and multilocation tests. sukamandi, 9– 14 april 2001. research institute for rice, sukamandi. suprapto, dradjat a, 2005. plasma nutrition bulletin 11(1). swamp research center. 2008. increased productivity of lowland land through rice planting tolerant to drought and drought. http://balittra.litbang. deptan.go.id. waluyo, suparwoto. 2016. the role of new superior rice variety in increasing productivity and income of wetland farmers in oganilir regency, south sumatra. proceedings of the national seminar on the 53rdanniversary of the faculty of agriculture, sriwijaya university, palembang, 14 september 2016. isbn 978-979-8389-24-5. wardana hd. 2002. utilization of germplasm in natural herbal and cosmetics industries. germplasm bulletin 8(2): 84-5. blotropla no blotropla no. 11, 1998: 9-21 integrated management of chromolaena odorata emphasizing the classical biological control soekisman tjitrosemito seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract chromolaena odorata, siam weed, a very important weed of java island (indonesia) is native to central and south america. in the laboratory it showed rapid growth (1.15 g/g/week) in the first 8 weeks of its growth. the biomass was mainly as leaves (lar : 317.50 cm'/g total weight). it slowed down in the following month as the biomass was utilized for stem and branch formation. this behavior supported the growth of c. odorata into a very dense stand. it flowered, fruited during the dry season, and senesced following maturation of seeds from inflorescence branches. these branches dried out, but soon the stem resumed aggressive growth following the wet season. leaf biomass was affected by the size of the stem in its early phase of regrowth, but later on it was more affected by the number of branches. the introduction of pareuchaetes pseudoinsulata to indonesia, was successful only in north sumatera. in java it has not been reported to establish succesfully. the introduction of another biological control agent, procecidochares conneca to indonesia was shown to be specific and upon release in west java it established immediately. it spread exponentially in the first 6 months of its release. field monitoring continues to evaluate the impact of the agents. other biocontrol agents (actmole anteas and conotrachelus) wilt be introduced to indonesia in 1997 through aciar project on the biological control of chromolaena odorata in indonesia and papua new guinea. keywords: indonesia / north sumatra / west java / biological control / chromolaena odorata i pareuchaetes pseudoinsulata i procecidochares conneca. introduction the nature of c. odorata (siam weed) as a weed in agricultural production systems is well recognized (tjitrosemito 1996; tjitrosoedirdjo et al. 1991) not only for perennial plantation crops (syamsudin et al. 1993), for forest teak plantations in java island (setiadi 1989), but recently also for forest plantations outside of java island. sagala (1994), for example, pointed out the role of c. odorata in providing inflammable fuel, leading to 2-4 m high flames in forest fires. moses (1996) also emphasized the important infestation role of c. odorata in reducing pasture productivity in east nusa tenggara in a recent regional symposium in kupang, east nusa tenggara. in baluran national park, east java, the presence of the weed caused a severe reduction in herbage yield in the park, leading to the reduction of population of protected wild animals such as banteng (bosjavanicus) and deers (cervus timorensis, 9 biotropia no. i i , 1998 muntiacus muncak). the situation here is quite severe, since the infestation of acacia nilotica, reaching a population of 1337 trees/ha (alikodra 1987) covered a major part of the grassing land. the park manager, following extensive mechanical control of a. nilotica using bulldozers, anticipated the return of native grasses, but c. odorata instead dominated the open space. another line of approach has recently been explored by the agroforestry research institute. following the report by slaats (1995) in africa, c. odorata may be useful as a fallow crop . the report by kasniari 1996, indicated a high yield of shoot biomass when the soil was fertile (12.44 mg ha"1), but not as high when the soil was less fertile (9.04 mg ha"'). when burnt, most of these biomass was reduced (0.77 mg ha'1). it further stressed the role of c. odorata as a weed in agricultural production systems with a very strong competitive power. the role of c. odorata as a weed is particularly severe in the newly planted or established plantations (oil palm, rubber, coconut) particularly in sumatera and kalimantan (sipayung and de chenon 1995) with quite substantial loss of investment. various control techniques have been pursued such as physical (manual, mechanical) as well as chemical approaches, and both are costly. in an attempt to find a better method for c. odorata management, biological control of c. odorata in indonesia has been done since 1989 (sipayung and de chenon 1985), supported further through the aciar project no. 9110: biological control of chromolaena odorata in indonesia and the philippines from january 1, 1993 to december 31, 1995. pareuchaetes pseudoinsulata rego barros (lepidoptera: arctiidae) was imported from guam, and after sufficient testing, this biocontrol agent was allowed to be released in the field in 1992. p. pseudoinsulata did establish in north sumatera, but not in java. although it was established, its effect was not as expected and another agent, i.e. procecidochares connexa (diptera: tephritidae) was introduced in indonesia in 1993. following extensive testing it was allowed to be released in 1995 by the minister of agriculture. this paper reports the results of procecidochares connexa (diptera: tephritidae) introduction to java island and its spread in the field. materials and methods a. the growth of c. odora ta the experiments were carried out in: parungpanjang, west java, in a forest area developed for bee farming. the total area was about 1000 ha planted mainly with 10 integrated management ofchromolaena odorata soekisman tjitrosemito acacia mangium and about 10 ha was planted with kapok trees (ceiba petandra) infested with shrubs of various kinds including c. odorata, mimosa invisa, hyptis sp., melastoma affine, ficus sp., with coppice schima walichii, and lianas such as meremia sp., mikania micrantha, passiflora sp., etc. experiment i: plant structure since october 1995 plots measuring 4 m2 were slashed at monthly intervals, and the stumps were mapped. the diameter was measured in mm, and recorded. the biomass was separated into leaves and stems, dried at 80°c for 48 hours or until the weight was constant and the weight recorded. the data were analyzed to characterize the plant structure of the established mature c. odorata population. experiment ii: the growth of c. odorata coppice the growth of coppice was observed at monthly intervals on plots following slashing treatment described in experiment i without destroying the plants. particular attention was given to emergence of coppice from small diameter stumps. the data were recorded in terms of coppice number and height of each stem, and number and position of branches. the data were analyzed statistically. b. rearing and release of procecidochares connexa b.l. rearing a. gall collection the stem cuttings bearing gall were collected from marihat research station, north sumatera, indonesia, on my 18, 1995. at biotrop, bogor, the galls were dissected. there were 17 galls altogether producing 48 pupae. the pupae were yellowish white when young and turned dark brown when mature. the pupae were subsequently reared on petridishes lined with moist filter paper and put in a rearing cage made of a wooden frame with fine plastic mash measuring 0.4 x 0.4 x 0.4 m3 kept in an air conditioned room. from 48 pupae, 38 imago emerged consisting of 19 females and 19 males. female iinagos were easily differentiated from the males by its conspicuous ovipositor. b. oviposition the emerged imago was soon paired up, in an 8 cm test tube to mate, and released into caged potted chromolaena odorata plants the following day in the 11 biotropia no. 11, 1998 green house. the cages of medium size, 0.5 x 0.5 x 1 m, were made of wooden frames with fine plastic mash, housing plastic pots of 51 capacity containing soil mixed with worm casting at 1:1 ratio as medium to support the growth of c. odorata. the potted c. odorata plant should have at least 20 shooting buds, preferably more, upon which the female fly will oviposit. the cages potted c. odorata with the pair of flies were kept approximately one week or until the flies died to ensure that the female oviposits its eggs on the c. odorata plant. the potted plants were then immediately removed and put under the sun with plenty of water and fertilizer. c. life cycle it takes 50 days for the gall to develop and mature. the maturity of the gall was indicated by the appearance of a window opening on the gall, which was still closed by a very thin cover of transparent lining gall epiderm used to allow emerging fly to escape. when most of the galls showed the window opening, the galls were harvested and dissected. from 19 pairs of flies, 53 galls were produced and after dissection yielded 142 pupae. from 142 pupae, 118 emerged (83%) in a period of 11 days (sept. 26 oct. 6, 1995), with a female to male ratio of 64 : 54. d sex ratio from 64 females of the first generation in the laboratory, 407 galls were produced yielding more than 1200 flies comprising 640 males and 629 females. in the second generation, the mature galls were not dissected, but the harvested gallbearing stem cuttings were collected and reared in the rearing cages by dipping the stem cutting in jars of water to keep the cuttings fresh. e. mass production the emergence took place from december 12, 1995 up to january 12, 1996. some emerged later. the 3rd generation of p. connexa in biotrop produced 3232 flies consisting of a female to male ratio of 1646 to 1586. the rearing procedure in the laboratory at biotrop was slightly modified and standardized as follows: the emerging flies were collected by sucking them with a small modified vacuum cleaner and reared for at most 2 days in a rearing cage. the population of images in 2 days can reach 100 pairs of flies. these paired imagos must be released immediately to new potted c. odorata plants having at least 20 shooting buds each. the potted c. odorata plants were housed in a big cage made of iron frame with a 12 integrated management of chromolaena odorata ~ soekisman tjitrosemito fine plastic screen measuring 2 x 3 x 3 m3. the plastic screen was lifted one week later, or until all flies died. the potted c. odorata plants were provided with adequate water and fertilizer. approximately 50 days later the emerging flies were sucked with the modified vacuum cleaner as mentioned above. about 250 pairs of flies obtained that way were once more released in parungpanjang and 32 galls were sent to yogyakarta. since emergence took more than 1 month, between february 26, 1996 and april 7, 1996 at biotrop, everyday there were new emerging imagos of p. connexa. the 4th generation emerged from may 10, 19% to june 28, 1996. there were more than 3835 galls, producing more than 4000 flies. b.2. release experiment hi. release a. permanent plot permanent 5 x 5 m plots were made in the area densely populated by c. odorata, marked with wooden poles of 4 m at the corner of the plot, delineated with steel wire. inside the plot individual plants of c. odorata were tagged with numbered alluminium tags attached to the base of the plants. the precise location of c. odorata plants was plotted on grid paper. the number of plants and their growing shoots were recorded. the permanent plots were made in parungpanjang and sukabumi. b. release release of p. connexa was done in the permanent plots at parungpanjang and sukabumi. b.l parungpanjang the release was conducted on december 19, 1995 with 75 pairs of mated imagos, and repeated again on december 30, 1995, with 100 pairs of additional mated imagos. b.2. sukabumi the release was carried out directly from the cage containing 100 pairs of mated imagos in the permanent plots on may 28, 1996, and when no gall was 13 b10tropia no. 11,1998 observed in the field on june 17, 1996, the following day, an additional 65 pairs of mated imagos were released. c. evaluation the evaluation was carried out by observing the presence of galls in the permanent plots and in the surroundings. c. 1. permanent plots in parung panjang every single plant was inspected on december 30, 1995, while in sukabumi this was done on september 14, 1996. the galls were counted and recorded. c. 2. surrounding permanent plots outside the permanent plots the observation were out using the line transect method. four lines at four cardinal directions were followed using measuring tape. along the way any c. odorata plants directly under the line was inspected for the occurrence of any galls. the number of galls and the distances were recorded and used to estimate the area infected by p. connexa these data indicated the population growth and dispersal of the fly. c. 3. data a nalyses data on weed growth were analyzed using multiple regression, while the area infected by the fly was analyzed using exponential growth to estimate the rate of spread. results and discussion a. the growth of c. odora a.l. the plant structure the data of the mature, well established c. odorata community slashed in october were analyzed to see how the leave biomass was affected by other variables (i.e. plant height, shootbase diameter, number of branches, and stem dry weight). using linear multiple regression analysis with data from 52 plants, the following regression equation was obtained : 14 integrated management of chromolaena odorata — soekisman tjitrosemito y= i.2258 2.8075 x 10'3 x, + 4.561 x 10'2 x2 3.2034 x 10'3 x3 + 0.18373 x< (t2 = 0.7349) where: y = leaf biomass(g) (= 3.252 g) xi = plant height (cm) (=159.100 cm) x2 = shootbase diameter (mm) (= 4.981 nun) x3 = number of branches (=10.520) x< = stem dry weight (g) (= 14.000 g) further analysis of the regression coefficient indicated that only the x4 variable was significant in affecting the total leaf biomass, because this population of c. odorata just sprouted to produce coppice along the denuded stem after undergrowing leaf shading during the dry season following flowering and fruiting. it is interesting to compare the growth behavior of c. odorata in the laboratory, where in the first two months of growth most of the photosynthate is utilized to produce leaves consequently it has a relatively high relative growth rate (rgr) =1.15 g/g/weeks (tjitrosemito 1996). it seems that this character is consistent whether it grows from seeds or when recovering from drought. at this time of the year the growth of c. odorata was still recovering from drought. the tops of stem and some branches that were carrying inflorescences were dried off following the dispersal of seeds as indicated by the negative sign of the coefficients. the greater the number of branches the lesser the biomass of leaf because those branches did not carry leaves just yet since the top part of those branches were dead. the situation was similar for plant height, as the higher the plant the lesser the leaves, as the top part was still dried off. the plant size was relatively small having an average height of 159.10 cm with leaf biomass of only 3.253 g/plant and total stem dry weight of 14.00 g/plant. the number of branches was relatively high i.e. 10.520 g/plant, but still small and some were only carrying 2 leaves. when slashing was done in may, the following regression line was obtained: y = -12.567 + 3.6413 x 10'2 x, + 6.8146 x 10'1 x2 + 0.9645 x3 + 2.9480 x 10'2 x4 where: y = leaf biomass (g) (= 5.583 g) x, = plant height (cm) (=199.1cm) x2 = number of branches (= 7.243) x3 = stem diameter (mm) (= 5.608 mm) x, = stem dry weight (g) (= 28.53 g) further analysis of regression coefficients indicated that the x2 variable, i.e. number of branches, was highly significant in affecting the biomass of the leaf. x3 15 biotropia no. 11, 1998 and x4 were also significant in affecting the biomass of the leaf. the number of branches represent new growth, where leaves are located. therefore, the greater the number of branches the higher the biomass of the leaves they carry and the sign is positive. stem dry weight and stem diameter were indicating the size of the plant. the taller the plant the higher the biomass of the leaves, and the signs are positive. however, the constant carried a negative sign, indicating that the growth of this population was very thick with many leaves at the top of the population so that some leaves at the lower level were shaded and some of them already senesced. this plant structure was different from those in october when the population was still recovering from the drought. the size of plant already reached a mature stage. stem dry weight reached 28.53 g/plant with the leaf biomass of 5.839 g/plant, and the average plant height was 199.1 cm. the number of branches stabilized at an average of 7.243 g/plant and these branches were effective viable branches which will bear inflorescences when mature. a.2. the growth of c. odorata coppice the growth of coppice was recorded on all stumps from as small as 2 mm to 10 mm when slashing was done during the wet season, i.e. from october june, while from july to september regrowth was recorded only on plants with a diameter of 5 mm or greater. at this period, most of seedlings (if seeds happen to germinate) will also die due to the drought. it seems that the regrowth of coppice of c. odorata is mostly affected by soil moisture. it was unfortunate that we did not collect data on soil moisture. this information will be collected in future observations. the above data revealed the plant structure in terms of biomass distribution. to understand the plant structure in physical terms the data on coppice growth was quite revealing. when observations were carried out at 2 and 3 months after slashing (during the wet season) the following data were obtained (average of 78 plants) (table 1). at two months after slashing as the space was stil available, the plant utilized its energy for producing branches, reaching a total of 26.07 branches/plant. the plant height varied reaching a maximum of 240 cm but the average was 162.5 cm, while the branches were distributed slightly skewing to the left, i.e. indicating that the plants were still investing their energy for growth. the peak of branch number was observed at a height of 80-100 cm above ground. at three months after slashing, however, there was a stabilization in number of branches. leaves of lower branches were shaded out and the branches were sacrificed 16 integrated management of chromolaena odorata — soekisman tjitrosemito by the plant, in favor of the new branches at the top of the plants. the plants grew taller reaching a maximum of 300 cm but the average was 220 cm. the branches were distributed slightly skewing to the right since at this time the lower branches and leaves were dying off. the plants formed a population architecture in such a way that most of the leaves were grown on the top branches to harvest sunlight as much as possible at die expense of the lower leaves and branches. this is the final structure when the population will produce flowers. when the time permits and moisture is available the plant will grow to form a very dense thicket of c. odorata. table 1. the distribution of branches in the c. odorata community at different stages of growth harvested 2 months after slashing harvested 3 months after slashing plant height (cm) 162.50 220.600 total living branches 26.07 18.95 branches from : 0-20 cm height 0.5714 0.1818 20-40 cm height 1.9290 0.3939 40-60 cm height 8.6790 1.0150 60-80 cm height 17.0000 2.3030 80100 cm height 22.4000 3.3940 100-120 cm height 20.7100 6.4550 120-140 cm height 18.2500 9.1360 140-160 cm height 11.9300 12.0000 160-180 cm height 6.8570 12.5200 180-200 cm height 3.0360 13.7000 200-220 cm height 0.9286 10.9500 220-240 cm height 0.4286 7.0450 240-260 cm height 2.9090 260-280 cm height 0.6364 280-300 cm height 0.1212 b. laboratory rearing of p. connexa from table 2, it appears that the time of emergence varies considerably from 9-25 days (from pupation/dissection to emergence). the emergence was about 79% with a sex ratio of 1 : 1. so this initial culture of p. connexa at biotrop was 19 pairs of imago. 17 biotropia no. 11, 1998 table 2. the emergence of imago of p. connexa from pupae no. date of emergence total female male 1. 07-27-1995 3 2 1 2. -282 1 1 3. -294 2 2 4. -303 1 2 5. -31 6. 08-01-1995 7 4 3 7. -02 8. -033 1 2 9. -041 1 . 10. -05. . 11. -06. . 12. -073 2 13. -082 1 14. -093 : 1 15. -103 2 16. -112 1 17. -122 1 total : 38 19 19 assuming that pupae represented the egg laid by a female, each female in generation i, on average, laid 7.5 eggs, this was lower than reported by sipayung and de chenon (1995), being 16, and much lower than the total number of eggs that may be laid by a female of p. connexa (69 eggs). however, for the following generation the estimated eggs laid was higher i.e. 19 per female. for the initial colony each gall contained about 3 pupae per gall. this was maintained up to generation n at biotrop. it dropped at generation iii and iv to about 1 pupae per gall. this condition may be attributed to the stage of the plant occurring mostly in the flowering stage at this time of the year (table 3). table 3. the outcome of the rearing in the laboratory emergence imago remarks gall pupa period total female male initial colony 17 48 17 38 19 19 dissected generation i 53 142 11 -118 64 54 stem cutting in jars modified vacuum cleaner generation ii 407 (nr) 30 1269 640 629 generation hi 3177 (nr) (nr) 3232 1586 1646 generation iv 3835 (nr) (nr) 4027 2023 2004 nr: not recorded 18 integrated management of chromolaena odorata soekisman tjitrosemito c. field experiment c.i. parungpanjang the population of c. odorata in parungpanjang, west java, a permanent plot of 5 x 5 m2, was 145 c. odorata plants with 2333 + 8.7 shoots. the c. odorata vegetation had a very dense thicket of shoots. in the permanent plot of 5 x 5 m2, on december 30, 1995, 128 galls were found. it is about 5% from the available shoots. this infestation is still low, in terms of the efficiency of controlling c. odorata. the spread of p. connexa into the surrounding area may be judged from data in table 4. table 4. the distribution of galls in parungpanjang the northern side of the permanent plot was bordered by a forest of young eucalyptus, mixed with calliandra callothyrsus and ceiba petandra. this forest seems to form a barrier for p. connexa to spread. the rate of spread may be estimated from the distance where galls were recorded. assuming that the forest on the northern side of the plot constitutes a barrier, the available area of spread was half circular as in figure 1. so the area is approximately '/i m2 (where r = distance of galls found). in the first month, the area covered by the infestation of p. connexa estimated from the distance of recorded galls, was about 150 m2. the following 2 months the spread covered an area of approximately 2203 m2 and at 6 months after release the area covered was 120 580 m2. the mode of spread seems to follow an exponential pattern, with relative spread rate of 1.1 in a month. 19 figure 1. diagram of the position of the permanent plot in parungpanjang c.2. sukabumi the population of c. odorata in the permanent plot of 5 x 5 m2, consisted of 82 c. odorata plants with 2140 + 18.5 shoots. this c. odorata vegetation was under a coconut plantation. it seems that this weed has been neglected for some reason. although the population was less than that of parungpanjang, the number of shoots was almost the same. in fact, the average for sukabumi was 26.1 shoots/plant, higher than that of parungpanjang (16.1 shoots/plant). two weeks after the release in sukabumi no gall was observed, since c. odorata plants were in the process of fruit maturation, where shoots were dried out. so c. odorata plants were slashed and p. connexa was released again on june 18, 1996 with 65 pairs of flies. on august 8, 19%, 120 galls were recorded. the rate of infestation is about 5%, similar to the infestation rate in parungpanjang, while the observation outside the permanent plot is still being evaluated. in contrast to pareuchaetes pseudoinsulata, p. connexa survived and reproduced well at java island. this result is very encouraging. although the rate of infestation is still low (5%), it is hoped that with the coming wet season the infestation will be higher. the impact of p. connexa on the performance of c. odorata such as its growth, seed production and seed viability are still under investigation and it is expected that one or two more biological control agents could be introduced in indonesia. references aldcodra, h.s. 1987. the exotic plantation of acacia nilolica and its problem on the ecosystem of savana baluran national park. duta rimba 79/80/xiii/1987: 30-34. kasniari, d.n. 1996. the role of chromolaena odorata in increasing soil fertility in alang-alang field. msc. thesis. university of brawijaya, malang. 20 integrated management of chromolaena odorata soekisman tjitrosemito moses, b.c. 1996. a. concept of integrated agriculture, with reference to the subsector of animal husbandry. a, paper presented to the regional symposium. university of nusa cendana, march 26, 1996. saoala, ap.s. 1994. land fire control, experience from riam kiwa plantation area established on alang-alang grassland. in: tampubolon el al. (eds.) from grassland to forest: profitable and sustainable reforestation of alang-alang grassland in indonesia. 97-108. setiadi, d. 1989. notes on weeds under teak forest. proc. 9*. indonesian weed sci. conf. ill : 74-78. sffayung, a. and r. desmier de chenon. 1995. procecidocrtares connexa to control chromolaena odorata. a paper presented to the meeting on release of procecidochares connexa, july 17, 1995. research centre of oil palm, medan. slaats, jj.p. 1995. chromolaena odorata fallow in food cropping system: an agronomic assessment in south-west ivory coast. phd. thesis. agricultural university, wageningen. the netherlands. 117 p. syamsuddin, e., t.l. toeing and r.a. lubis. 1993. integrated pest management in oilpalm plantation in indonesia. biotrop spec. publ. 50. 137-145. tjitrosemrro, s. 1996. the management of chromolaena odorata (l.) r.m. king & h. robinson in indonesia in: prasad et al. (eds.). 1996. proc. 3rd. international workshop on biological control and management of chromolaena odorata: distribution, ecology and management of chromolaena odorata. agricultural experiment station, univ. guam. mangilao, guam, usa.: 135-142. tjmosoedirdjo, s., s.s. tjitrosoedirdjo and r.c. umaly. 1991. the status of chromolaena odorata (l.) r.m. king & h. robinson in indonesia. biotrop spec. publ. 44: 57-66. 21 biotropiano. 7, 1994: 12-17 bioecology of dioryctria abietella denis and schiff. a pest of conifers in the north-western himalaya t.d. verma and r.k. gaur department of entomology dr. y.s. parmar university of horticulture and.forestry nauni-solan-173 230, h.p., india abstract cones and seeds of conifers, such as pinus roxburghii, p. wallichiana, p. gerardiana, cedrus deodara, abies pindrow and picea smithiana are seriously damaged by dioryctria abietella denis and schiff. (lepidoptera : pyralidae) in the north-western himalayan region of india. bioecological studies carried out during 1991 '92 revealed that the females laid whitish, elliptical eggs singly on the depressed surface of the young cones. the average egg size was 1.00 ± 0.11 x 0.60 ±0.08 mm and this stage lasted for 3 5 days. the larval stage passed through five instars. all the instars differed in appearance, size and duration and larvae became full-fed in an average of 24.8 ± 1.9 days. the full-fed larva spun a cocoon around itself, sealed it with white papery membrane and pupated inside the cone or any other outside protected place. the prepupal and pupal periods lasted for 7-8 and 10-14 days, respectively. the pupa was dark brown, 13.8 ± 0.07 mm in length. total period from egg to adult varied from 46 to 59 days (52.7 ±4.8 days). adults were dirty brown in appearance and were 13.59±0.115 mm long with an average wing expanse of 29.0 ± 1.00 mm. they lived for 4 to 5 days. under laboratory conditions, the pest completed two generations per year and full-fed larvae of 3rd generation overwintered during september october. two larval parasites belonging to order hymenoptera and diptera, and a fungal pathogen (fusarium sp.) were found associated with this pest. the detailed biology, nature and extent of damage, along with its distribution have been discussed and methods of control suggested. key words: insect biology, insect ecology, dioryctria abietella, coniferae, north-western himalaya, forest pests introduction production of healthy seed is the backbone of afforestation programmes. unfortunately this important aspect has so far been ignored in india. a high percentage of cone and seed crop is destroyed by insects every year resulting in great losses and adverse effect on the natural regeneration. at times the insect attack is so severe that the whole seed crop is destroyed. cone and seed insects of indian conifers have not been studied in detail. some of the important insects are the cone worms, dioryctria abietella denis and shiff. (lepidoptera : pyralidae) and cateremna cedrella hampson (lepidoptera : pyralidae). beeson (1941) and browne (1968) gave brief accounts of the insects 12 bioecology of dioryctria abietella t.d. verma and r.k. gaur damaging cone and seed of conifers. recently, bhandari (1988) discussed the insect pests of cone and seeds of conifers and their control. in this paper an account of the life processes of d. abietella is given. materials and methods insect culture was raised from field collected overwintering larvae. the adults emerging from these larvae were held on 2nd year green cones in wooden cages for mating and oviposition. observations on the pattern, time and number of eggs laid and their incubation period were recorded. freshly emerged larvae were carefully transferred with camel hair brush to small plastic jars, provided with fresh green cones. to avoid excess moisture inside the jars, a circular hole was cut in the centre of the lid and a piece of muslin cloth was fixed. the morphological characters and behaviour of the larval instars and the duration of each instar was recorded. the size of head capsule was also measured with the help of calibrated ocular micrometer which was taken as a criterion for differentiation of different instars. full-fed larvae were transferred to glass jars (15 x 20 cm) containing soil and cone scales and needles, for pupation. parasites and pathogens associated with the insect were recovered from the culture in the laboratory and the percent parasitization was recorded. in addition, field surveys were also carried out at monthly intervals in coniferous forests to record the incidence, distribution and level of infestation of the pest. to determine the level of infestation fifteen cones were randomly observed from twenty trees selected at random in each locality. results and discussions d. abietella is a borer of cones and seeds of pinus roxburghii, p. wallichiana, p. gerardiana, picea smithiana, cedrus deodara and abies pindrow in himachal pradesh. the biology of this pest on p. gerardiana, p. roxburghii, p. wallichiana, p. smithiana and c. deodara studied under laboratory condition revealed that the eggs were singly glued to the surface of cones generally in the depressions on the scales. mathur et al. (1958) also reported that d. abietella eggs were whitish, elliptical and flattened, turning reddish brown after two days. the length and breadth of the eggs ranged from 0.86 1.10 mm (av. 1.00 ± 0.11 mm) and 0.54 0.73 mm (av. 0.60+0.08 mm), respectively. the egg stage lasted for 3 5 days (av. 4.4 ± 0.22 days). the larvae came out of the eggs by making a hole in the chorion in 30 40 minutes and entered the cone in about four hours. the larva is reddish brown with black head capsule. the width of the head capsule ranged from 0.31 -0.34 mm 13 biotropia no. 7, 1994 (av. 0.33 ±0.00 mm) (table 1). the first instar larva measured 1.2-1.5 mm (av. 1.34±0.11 mm) in length and the average duration was 3.6 days (table 2). the second instar larva was light brown, 3.5-4.0 mm (av. 3.72 + 0.18 mm) in length. the head capsule measured 0.51-0.56 mm (av. 0.53±0.01 mm) in width. the second instar lasted for 4-5 days (av. 4.5±0.16 days). the third and fourth instar larvae were dark brown with reddish brown head capsule. the width of head capsule of 3rd and 4th instar ranged from 0.80-0.95 mm (av. 0.87 ±0.06 mm), respectively. the body length varied from 9.5012.00 mm (av. 10.59±0.85 mm) and 14.00-16.00 mm (av. 15.30±0.67 mm) in two instars, respectively. the third instar lasted for 45 days (av. 4.70±0.15 days) and fourth instar for 5 -6 days (av. 5.40±0.16 days). the fifth instar larva was dirty brown along with dark brown head capsule. the width of head capsule ranged from 2.37-2.45 mm (av. 2.42±0.01 mm) and measured 22.00-25.00 mm (av. 23.55± 1.21 mm) in length. this stage lasted for 6-7 days (av. 6.7±0.52 days). table 1. the width of head capsule of larval instar of d. abietella (data based on 10 observations) width of head capsule (mm) ratio of head instar range mean capsule width i 0.31-0.34 0.33 ±0.00 11 0.51-0.56 0.53 ±0.01 1.60 iii 0.80-0.95 0.87 ±0,01 1.63 iv 1.39-1.47 1.45 ±0.06 1.65 v 2.37-2.45 2.42 ±0.07 1.67 table 2. duration of different developmental stages of d. abietella (mean of 10 observations) mean duration (days) stage range mean ± s.e. egg 3-5 4.4 ±0.22 larva (instar) i 3-4 3.6±0.16 ii 4-5 4.5±0.16 iii 4-5 4.7±0.15 iv 5-6 5.4±0.16 v 6-7 6.7±0.15 prepupa 7-8 7.6±0.16 pupa 10-14 12.5±0.40 adult 4-5 4.3±0.15 14 bioecology of dioryctria abietella t.d. verma and r.k. oaur the larvae became full-fed in 22 27 days (av. 24.8 + 1.93 days). mathur et al. (1958) reported that mature larvae were about 20 mm long, light reddish with blackish yellow head. the full-fed larva constructed a cocoon before entering the pre-pupal stage. the cocoon was later sealed from all sides by means of white papery membrane. pupation took place inside or on the surface of infested cones or on soil surface. the cocoon measured 16.00-20.00 mm (av. 18.80±2.20 mm) in length and 8.0010.00 mm (av. 9.00± 0.81 mm) in width. the pre-pupal period lasted for 7 8 days (av. 7.60 ± 0.16 days) and pupal period for 10-14 days (av. 12.50 ± 0.40 days). the pupa was dark brown, 13.50+14.10 mm (av. 13.80±0.07 mm) in length. however, mathur et al. (1958) reported it to be light brown and about 10 mm long. adults were dirty brown in appearance and were 13.00 14.00 mm (av. 13.59 ± 0.11 mm) long with wing expanse of 28.00-30.00 mm (av. 29.00 ± 1.00 mm). beeson (1941) reported the wing expanse of the moth as 22 34 mm. adults lived for 4 5 days (av. 4.30 ±0.15 days). the egg laying started 1 -2 days of their emergence. a female laid 30-50 eggs (av. 42.40 ± 6.78 eggs) during her life time. total period from egg to adult varied from 46-59 days (av. 52.7 ±4.80 days). the insect completed two generations annually and full-fed larvae of third generation overwintered during the last week of september. the sex ratio in the laboratory reared population was 1 male to 1.5 female. the natural enemies of the pest included two larval parasites (one each belonging to hymenoptera and diptera). out of 160 larvae of the host, 39 hymenopteran and 15 dipteran parasites were collected which accounted to 24.37 and 9.38 percent infestation. a fungal pathogen fusarium sp. was also isolated from the full-fed larvae. the pest was found to be distributed throughout the inner and outer himalayas of himachal pradesh. it infested c. deodara, p. wallichiana, p. smithiana, p. gerardiana, p. roxburghii in the region (table 3). the infestation was maximum (89.06%) in cedrus deodara cones around shimla and by 70% at chail in solan table 3. incidence of d. abietella on cone and seed of different conifers in himachal pradesh district locality/ percent infestation forest division c. deodara p. wallichiana p. smithiana p. gerardiana p. roxburghii shimla kasumpati 89.06 thanedhar 52.00 11.00 mashobra 30.00 25.00 35.00 solan chail 70.00 nauni 14.72 kinnaur sharbo 30.43 15 biotropia no. 7, 1994 district. in p. wallichiana, the maximum infestation (52%) was recorded at thanedhar. however, in p. smithiana the infestation level was only 11% in this locality. whereas, at mashobra in the shimla district the infestation was 35% in p. smithiana, 30% in c. deodara and 25% in p. wallichiana. the infestation in p. gerardiana was 30.43% in the kinnaur district of inner himalayas. the incidence of this insect was comparatively low in p. roxburghii cones throughout the outer himalayas, being maximum (14. 72%) at nauni in the solan district (fig. 1) figure 1. incidence of dioryctria abietella in coniferous forests 16 bioecology of dioryctria abietella t.d. verma and r.k. gaur it is practically difficult to control cone and seed insects in the natural forests. however, the pest population can be managed by collecting the infested cones and disposing them off together with the use of bio-control agents under the northwestern himalayan conditions. conclusion a high percentage of cone and seed crop is destroyed by insects every year resulting in great losses and adversely affecting the natural regeneration of conifers. numerous insects have been observed feeding casually on cone and seeds. but exceptionally d. abietella was found to be dominant. under laboratory conditions, the insect had a total life span of 46 59 days and completed two generations in a year. during the course of investigation the larval parasites of hymenoptera and diptera suppressed the population to a great extent. survey studies of this pest species revealed that d. abietella was widely distributed throughout the inner and outer himalayan ranges of himachal pradesh especially on cones of cedrus deodara and pinus gerardiana. continuous surveillance of this pest and its natural enemies is important in order to manage this pest. acknowledgements the authors are thankful to the indian council of forestry research and education, dehradun for funding the present studies and dr. o.p. bhalla, head, department of entomology for providing the necessary facilities. references beeson, c.f.c. 1941. ecology and control of forest insects of india and neighbouring countries. vasant press, dehradun: 1007 p. bhandari, r.s. 1988. insect pests of cone and seed of conifers and their control. tree protection: 342-347. browne, f.g. 1968. pests and diseases of forest plantation trees. clarendon press oxford: 1330 p. mathur, r.n., b. sinoh, and k. lal. 1958. insects pests of flowers, seeds and fruits of forest trees. indian for. bull. no. 223 (new series) entomology: 103 p. 17 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf biotropia no. 9, 1996: 15 25 fungi isolated from groundnuts in some locations of west java o.s. dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia i. retnowati seameo biotrop, p.o. box 116, bogor, indonesia abstract one hundred and ninety eight groundnut samples were collected from freshly harvested groundnuts (fhg), farmer storage systems (fss), middlemen warehouses (mw), whole salers (ws) and retailer sample (rs) during the dry and wet seasons from cidolog, cianjur, sukabumi and bogor, west java, indonesia, in 1990/1991. the moisture content (m.c.), intactness of kernels, and the percentages of groundnut kernels infected by each species of fungi were analyzed. in genera), the m.c. of the samples collected during the dry season was lower than of those collected during the wet season. also, the m.c. of samples collected from fhg, fss and mw was higher than of those collected from ws and rs. the m.c. of samples collected from fhg was the highest (12.5-45.75%), but the percentages of damaged kernels were the lowest (2.5-13.8%), because the samples were shelled manually. a total of 25 species of fungi were isolated from samples collected from the 4 localities. they were acremonium strictum, aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, c. sphaerospermum, eumtium chevalieri, e. repens, e. rubrum, fusarium equiseti, f. longipes, f. oxysporum, f. semitectum, mucor sp., papulaspora sp., pestalotia sp., penicillium aethiopicum, p. citrinum, rhizapus sp., r. stolonifer and syncephalastrum sp. the predominant fungi in samples collected from cidolog and sukabumi during the dry season were aspergillus wentii, while those collected from cianjur and bogor were a. niger. the percentages of kernels infected by a. wentii in samples collected from cidolog and sukabumi were between 30-100% and 36-100%, respectively, while those of kernels infected by a. nigerin samples collected from cianjur and bogor were between 34-93% and 14-98%, respectively. the predominant fungi in samples collected from each location during the wet season were a. flavus. the percentage of kernels infected by the fungus in samples collected from bogor was the highest (83-100%). key words: indonesia/west java/stored product pests/groundnut/fungi/acremonium strictum/aspergillus sp./botryodiplodia theobromae/cladosporium sp./eurotium sp./fusarium sp./mucor sp./papulaspora sp./pestalotia sp./spenicillum sp/rhizopus sp./syncephalastrum sp./moisture content. 15 biotropiano. 9, 1996 introduction groundnut is a major food legume and an important secondary crop after maize and soybean in indonesia. since indonesia has a humid tropical climate, this commodity could be easily infected by fungi during storage when freshly harvested or even before harvest. there are various practices of post-harvest processing and storage that could affect the moisture content and the intactness of the kernels and thus their susceptibility to fungal infection. aspergillus and penicillium are the two common genera of fungi found on stored products. they can cause weight loss, seed discolouration, heating and mustiness, and production of mycotoxins, specially aflatoxins. the latter are toxic metabolic substances produced by a. flavus and a. parasiticus and are known to be carcinogenic agents (butler 1974). in indonesia, very few studies have been conducted on storage fungi of groundnuts and their ability to produce aflatoxin. this study shows the presence of fungi infecting goundnuts. information on the percentages of groundnut kernels infected by each species of fungi, moisture content and damaged kernels of freshly harvested, post-harvest, stored and marketed materials during dry and wet seasons are also reported. materials and methods sample collection a total of 198 groundnut samples were collected in cidolog, cianjur, sukabumi and bogor, west java, indonesia, in 1990/1991. of the total, 100 were collected during the dry season (august, september and october, 1990) from farmer storage systems (fss), middlemen warehouses (mw), wholesalers (ws) and retailer samples (rs), while 98 samples were collected during the wet season (february and march 1991) from freshly harvested groundnuts (fhg), fss, mw, ws and rs. the samples consisted of shelled and unshelled groundnuts with the latter shelled manually. about 1 kg of each sample was then divided into 4 subsamples for (1) moisture content analysis, (2) damage kernels analysis, 3) fungal analysis and 4) reserve sample. 16 fungi isolated from groundnuts os. dharmaputra & i. retnowati moisture content analysis percent moisture content of kernels (on the basis of water loss) was determined by the oven method (iso 1968). three replicates were used for each sample. the kernels were ground and dried in the oven at 130 c for 2 hours. the moisture content was determined using the formula: 100 moisture content = (mo ml) x ———— mo where : mo is the initial mass, in gram, of the test portion ml is the mass, in gram, of the dry test portion damaged kernel analysis the kernels were classified as either intact or damaged. damaged kernels are those cracked, broken, porous, shrivelled or insectdamaged. three replicates were used for each sample. the percentage of damaged kernels was determined using the formula: weight of damaged kernels damaged kernels = ———————————————————— x 100 % weight of the kernels analyzed for intact and damaged kernels fungal analysis fungi were isolated using direct plating methods on dichloran 18% glycerol agar (dg 18). according to hocking and pitt (1980), dg 18 was especially used for the isolation of xerophilic fungi. before plating, samples were individually disinfected using 1% sodium hypo-chlorite for 2 minutes. one hundred kernels were then plated (10 kernels/plate) on the medium and incubated at 25 c for 7 days. the fungi were identified using the publications of samson et al. (1984), and pitt and hocking (1985) as the main references. the percentage of kernels infected by fungal species on each medium was determined. 17 biotropia no. 9, 1996 results and discussion moisture content moisture content (m.c.) is the most important factor determining the development of storage fungi in seeds (christensen & kaufmann 1975; neergaard 1979). according to who (1979) the minimum m.c. of groundnuts for the development of aspergillus flavus was 9.0 10.0%. the m.c.'s of samples collected from different localities during the dry season are presented in table 1. the m.c.'s of samples from cidolog, cianjur, sukabumi and bogor were between 9.9 14.1%, 7.9 10.5%, 7.5 11.2% and 6.1 10.7%, respectively. it seems that the m.c.'s of samples collected from the same location were not affected by their source (fss and mw from cidolog, ws and rs from cianjur, sukabumi and bogor). the m.c.'s of samples collected from different localities during the wet season are presented in table 2. the m.c.'s of the samples from cidolog were between 12.5 -45.7% (fhg) and 10.8 18.1% (samples from fss and mw), while those from cianjur, sukabumi and bogor were between 8.7 13.9%, 7.5 10.3% and 7.7 11.1%, respectively. table 1. moisture contents and percentages of damaged kernels of groundnuts collected during the dry season 18 fungi isolated from groundnuts os. dharmaputra & i. retnowati table 2. moisture content and percentages of damaged kernels of groundnuts collected during the wet season in general, the m.c.'s of the samples collected during the dry season were lower than of those of the wet season. the moisture content of samples collected on 30 march 1991 was the highest (12.5 45.7%), because they were not yet dried. according to bulog (1981) the maximum moisture content of stored groundnuts should be 7%. in this study, the moisture contents of the samples collected from farmers storage systems and middlemen warehouses were higher than 7%. damaged kernels damaged kernels gave a chance for fungi to infect the seeds. according to christensen (1980) physical damage of seeds is one of the factors that affects fungal infestation. the percentages of damaged kernels of samples collected from different localities during the dry season are presented in table 1. the percentages of damaged kernels from cidolog, cianjur, sukabumi and bogor were between 12.0 51.8%, 10.2 38.0%, 8.7 27,9%, and 1.5 26.0%, respectively. 19 biotropia no. 9, 1996 the percentages of damaged kernels of samples collected from different localities during the wet season are presented in table 2. the damaged kernels from cidolog were between 2.5 13.8% (fhg) and 9.9 31.7% (samples from fss and mw), while those from cianjur, sukabumi and bogor were between 13.0 61.0%, 18.0 55.0%, and 6.5 -56.0%, respectively. the percentage of damaged kernels of samples collected on 30 march 1991 was the lowest because they were shelled manually, while the other samples were shelled either manually or using wooden/zinc sheller. it was assumed that handshelling prevented the occurrence of damaged kernels, but this method is time consuming. proper tools should be used for shelling groundnuts to minimize the occurrence of damaged kernels. fungal analysis twenty three species of fungi were isolated from samples collected from cianjur, sukabumi and bogor during the dry and wet seasons. the isolated fungal species belonged to the field fungi (i.e. botryodiplodia theobromae, cladosporium cladospo-rioides, c. sphaerospermum, fusarium equiseti, f. longipes, f. oxysporum, f. semitectum and papulaspora sp.), the storage fungi (i.e. aspergillus candidas, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, eurotium chevalieri, e. repens, e. rubrum, penicillium aethiopicum and p. citrinum), and fungi belonging to the mucorales (i.e. mucor sp., rhizopus sp., r. stolonifer and syncephalastrum sp.). aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a, wentii, botryodiplodia theobromae, eurotium chevalieri, e. repens, papulaspora sp. and penicillium citrinum were always isolated from samples collected in each location during the dry season, while those isolated from samples collected during the wet season were always a. flavus, a. niger, a. tamarii, a. wentii, b. theobromae, e. chevalieri, papulaspora sp., penicillium citrium and r. stolonifer. according to dharmaputra and rahayu (1988) the predominant fungal species isolated from groundnut samples collected from a market in bogor, west java, indonesia belonged to the genera aspergillus, eurotium and penicillium, i.e. a. candidus, a. flavus, a. niger, a. restrictus, eurotium spp. and penicillium spp. the fungal species and the percentage of kernels infected by each species of fungi from samples collected from the three localities are presented in table 3. the percentages of kernels infected by the field fungi i.e. f. longipes (collected from cianjur during the dry season), c. cladosporioides and f. semitectum (collected from bogor during the dry season), f. equiseti and f. oxysporum (collected from, cianjur during the wet season) and c. cladosporioides, c. sphaerospermum and f. semitectum (collected from sukabumi during the wet season) were very low. they were between 0 4%, 0 8%, 0 5%, 0 1%, 0 9%, 0 3%, 0 5% and 0 2%, respectively. 20 fungi isolated from groundnuts os. dharmaputra & i. retnowati 21 biotropia no. 9,1996 among the storage fungi, a. niger was the predominant species of fungi infecting kernels collected from cianjur and bogor during the dry season. the percentage of kernels infected by the fungus in samples collected from both localities was between 3493% and 14 98%, respectively. the percentages of kernels infected by a. flavus in samples collected from both localities were between 9 98% and 8-95%, respectively. it was assumed that the moisture contents of the samples collected from the two localities were still favourable for their growth. they were between 7.9 10.5% and 6.110.7%, respectively. the predominant species of fungi infecting kernels collected from sukabumi during the dry season was a. wentii. the percentages of kernels infected by the fungus in samples collected from that location were between 36 100%, while those of a. flavus were between 10 85%. the m.c. of the samples was between 7.5 11.2%. aspergillus flavus was the predominant species of fungi infecting kernels collected from cianjur, sukabumi and bogor during the wet season. the percentages of infected kernels collected from the three localities were between 41 100%, 36 96% and 83 -100%, respectively. pitt et al. (1993) reported that the predominant fungus infecting groundnut collected from farmer storage, middlemen and retailer in thailand was a. flavus, followed by a. niger. the percentages of kernels infected by r. stolonifer and syncephalastrum sp. could not be determined, because their growth was very vigorous. twenty species of fungi were isolated from samples collected from cidolog during the dry and wet season. they were acremonium strictum, aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, b. theobromae, c. cladospo-rioides, e. chevalieri, e. repens, f. equiseti, f. longipes, f. semitectum, papulaspora sp., penicillium aethiopicum, p. citrinum, pestalotia sp., rhizopus stolonifer and syncephalastrum sp. (table 4). the predominant species of fungi infecting kernels collected from cidolog during the dry season was a. wentii, with an infected percentage bertween 30 100%, while the percentage of kernels infected by a. flavus was between 0 91%. the m.c. of the samples collected from that location was between 9.9 14.1%. during the wet season, the predominant species of fungi was a. flavus. the percentages of kernels infected by the fungus in samples collected from fss and mw were between 17 96%, while those in fhg were between 51 99%, and their m.c's were between 10.8 18.1% and 12.5 45.7%, respectively. according to christensen and kaufmann (1969) a. flavus can grow in seeds which have a moisture content of at least 18%. on the other hand martin and oilman (1976) reported that optimum moisture contents of natural substrate, which permitted the growth of a. flavus, were between 15 -25%. 22 fungi isolated from groundnuts os. dharmaputra & i. retnowati table 4. the fungal species and range of precentage of infected kernels of groundnut samples collected from cidolog in different storage systems the percentages of kernels infected by a. flavus in fhg were higher than in samples collected from fss and mw, because it was assumed that a. flavus infected the pods of groundnut before harves. according to kozakiewicz (1989) a. flavus is cosmopolitan in distribution. it is found soil, and decomposes vegetation in stored cereals and other seeds as well as variety of food products. dickens (1977) and pitt (1989) reported that the invasion of groundnut by a. flavus take place before groundnuts are harvested. damage of peanut kernels caused by insects and other small animals forces soil into close contact with kernels, and so facilitates the invasion of the fungus. 23 biotropia no. 9, 1996 conclusion fungi isolated from groundnuts collected during the dry and wet season from the farmer storage system (fss), middlemen warehouses (mw), wholesalers (ws), retailer samples (rs), and freshly harvested groundnut (fhg) in cidolog, cianjur, sukabumi and bogor showed the presence of a large number of species. a total of 25 fungal species were isolated from the 4 locations. the total fungal species isolated from samples collected from cianjur, sukabumi and bogor during the dry and wet season were 23, while those from cidolog were 20. there were variations in the percentage of kernels infected by each fungal species from different sources and localities. the predominant fungi in samples collected from cidolog and sukabumi during the dry season were aspergillus wentii, while those collected from cianjur and bogor were a. niger. the predominant fungus in samples collected from the 4 locations during the wet season was a. flavus. the moisture contents of groundnuts collected from fhg, fss and mw were higher than of those collected from ws and rs. there were also variations in the percentages of damaged kernels of samples collected from different sources and localities. acknowledgement the authors gratefully acknowledge the australian centre for international agricultural research for their financial support. the authors are also thankful to dr. j.i. pitt, leader of aciar project 8806; dr. ruben c. umaly and dr. gloria l. enriquez, the former deputy director and deputy director of seameo biotrop, respectively, who gave advice and suggestions; mr. sunjaya, scientist of the tropical pest biology (tpb) programme, seameo biotrop; the technicians of the laboratory of plant pathology, tpb programme, seameo biotrop; and to dr. a.d. hocking, for the confirmation of fungal identification. references bulog, 1981. bulog procurement on quality and purchasing cost of secondary crop for fy 1981/1982. letter of appoinment, head of national logistics agency, no. kep-311/ka/10/-1981. butler. w.h. 1974. aflatoxin. in purchase, i.f.h. (ed.). mycotoxins. elsevier scientific publishing company, amsterdam: 128. 24 fungi isolated from groundnuts os. dharmaputra & i. retnowati christensen, c.m. 1980. needed: research on storage molds in grains, seeds and their products. plant disease 64(12): 10671070. christensen, c.m. and h.h. kaufmann. 1969. grain storage; the role of fungi in quality loss. university of minnesota press, minneapolis. christensen, c.m. and h.h. kaufmann. 1975. control of postharvest losses caused by fungi in food, feed and grains. feedstuffs 47 (10). dharmaputra, o.s. and g. rahayu. 1988. inventory of fungi on various stored grains. biotrop special publication no. 32:55-61. hocking, a.d. and j.i. pitt. 1980. dichoran-glycerol medium for enumeration of xerophilic fungi from low moisture foods. appl. environ. microbial. 39: 488-492. iso, 1968. cereals and cereal products; determination of moisture content. iso recommendation, r712. iso/r712 1968(f). kozakiewicz, z. 1989. aspergillus species on stored products. mycol. pap. no. 161. cab international mycological institute, p. 148-158. martin, p.m.d. and g.a. oilman. 1976. a consideration of the mycotoxin hypothesis with special reference to the mycoflora of maize, sorghum, wheat and groundnuts. the ecology of mycotoxin formation. tropical product institute, london: 23-30. neergaard. p. 1979. seed pathology. vol.1. the macmillan press ltd. pitt, j.i. and a.d. hocking. 1985. fungi and food spoilage. academic press, sydney. pitt, j.i. 1989. field studies on aspergillus flavus and aflatoxins in australian groundnuts. aflatoxin contamination of groundnut: proceedings of the international workshop, 6-9 october 1987, icrisat center, india, p. 223-235. pitt, j.i., a.d. hocking, k. bhudhasamai, b.f. miscamble, k.a. wheeler and p.t. ek. 1993. the normal mycoflora of commodities from thailand. 1. nuts and oilseeds. int. j. food. microbiol. 20: 211-226. samson, r.a., e.s. hoekstra and c.a.n. van oorschot. 1984. introduction to food-borne fungi. centraal bureau voor schimmelcultures, baarn, the netherlands. who. 1979. environmental health criteria ii: mycotoxin. geneva. 25 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf biotropia no. 9, 1996: 26 37 brown spot caused by curvularia spp., a new disease of asparagus b. salleh 1 , a. safinat 1 , l. julia 2 and c.h. teo 3 1 school of biological sciences, universiti sains malaysia, 11800 penang, malaysia 2 laboratory of plant pathology, agricultural research centre, p.o. box 3, 89207 tuaran, sabah, malaysia 3 laboratory of plant pathology, agricultural research centre, semogok, p.o. box 977, 93720 kuching, sarawak, malaysia abstract the distribution, aetiology and symptomatology of a new disease on asparagus ferns, which we have termed brown spot, is described. descriptions of and a key to identification of the causal organisms, curvularia brachyspora, c. eragrostidis, c. lunata and c. pallescens, are also presented. pathogenicity tests showed that c. lunata was the dominant and most virulent of the four species. inoculation with conidial suspensions or mycelial transfers through wounded ferns were more effective in inducing the disease than inoculations on unwounded ferns. this is the first record of c. brachyspora in malaysia and the first report of this disease on asparagus. key words: malaysia/plant diseases/brown spotjcurvularia brachyspora/curvularia eragrostidts/curvularia lunata/curvularia pallescens/asparagus. introduction asparagus (asparagus officinalis l.) was introducted into malaysia from taiwan in the 1950s. although it is a fairly new crop, asparagus has become a preferred vegetable especially by malaysians in the higher income groups. its caloric and nutrient contents are comparable to those of the four most popular local vegetables i.e. kailan (brassica alboglabra bailey), chinese cabbage (b. chinensis l.), cauliflower (b. oler-cea l. var. botrytis) and sawi (b.juncea bailey) (anon. 1985). during a series of investigations on causes of degeneration in asparagus farms established for experimental or commercial purposes throughout malaysia in 1989-90 (salleh 1990), a new disease was observed on all varieties. in this paper we report the distribution, aetiology and symptomatology of the disease. 26 brown spot caused by curvularia spp., b. salleh et at. materials and methods disease survey and aetiology eighteen asparagus farms established for experimental or commercial purposes throughout malaysia were visited and sampled at least twice in 1989-90 (fig. 1). the last three of the farms are situated in the east malaysia (malaysian territory of the borneo island). figure 1. asparagus disease sampling sites throughout malaysia (1989-1990) plant parts showing brownish lesions were collected and the causal organisms were isolated as previously described (salleh & sulaiman 1984). single conidia of the dominant isolates (curvularia spp.) which developed from the diseased materials were placed on potato-sucrose-agar (psa) plates and incubated under standard incubation conditions described earlier (salleh & sulaiman 1984). growth of the colonies was 27 biotropia no. 9,1996 measured after four days of incubation. curvularia species were identified using the diagnostic characters proposed by ellis (1971; 1976) and sivanesan (1987), and later confirmed by the commonwealth mycological institute, uk. conidial suspensions from monoconidial stock cultures were preserved in 15% glycerol in liquid nitrogen (salleh & strange 1988), and fresh monoconidial cultures on potato-dextrose-agar (pda) slants were covered with sterile mineral oils and kept at 4°c. pathogenicity test pathogenicity tests of selected isolates of all four species of curvularia isolated from asparagus were carried out on healthy 9 month-old asparagus seedlings, varieties mary washington (mw) and uc 157. the seeds were tested for fungal contamination by plating on psa plates. apparently healthy and non-cracked seeds were sown in sterile soil in polyethylene bags (diameter 20 cm) in a greenhouse with natural lighting for nine months. the seedlings were kept healthy by spraying a mixture of a fungicide (benomyl or propineb) and an insecticide (diazinon or aldrin) monthly until the seventh month. the plants were fertilized bimonthly with a granular npk (15:15:15) fertilizer. the first method of inoculation was conducted as follows : conidial suspensions of curvularia spp. were prepared by flooding 7 day-old psa plates with 10 ml sterile water containing 0.05% tween 80. after gentle agitations, the liquid was decanted and the concentration adjusted to 1 x 10 conidia/ml. inocula were sprayed in the evening onto aseptically wounded or unwounded ferns at the base, middle, branch and tip of plants at growth stage 5 or 6 (fully developed ferns) (bansal et al. 1986). wounds were made by pricking the plants with sterile needles. parts of the ferns were surface sterilized with 70% alcohol prior to wounding. control wounded or unwounded ferns were sprayed with sterile water. a second method of inoculation was carried out by placing mycelial plugs from the plates onto similar inoculation points of the aseptically wounded ferns. the inoculation points were wrapped with sterile moistened cotton wool and tied with an adhesive plastic for 48 h. similar points on unwounded stems and branches wrapped in the same manner served as controls. for both conidial spray and mycelial plug inoculations, mixed inocula which comprised of all four species of curvularia were also used on different set of plants throughout the experiments. plants in each polyethylene bag were covered with transparent plastic bags overnight. the plastic chambers were kept moist by spraying 10 ml sterile water into each chamber after inoculation. all treatments were replicated ten times (10 plants; one in each polyethylene bag). symptom development was observed every other day. the 28 brown spot caused by curvularia spp., — b. salleh et al. length of the lesion was measured four weeks after inoculation. reisolations were made from all infected and control ferns and the resulting fungi subcultured on psa and reidentified. results and discussion disease survey and aetiology a new disease with typical greyish brown spots was observed on the ferns of all varieties of asparagus planted in all 18 sampling sites throughout malaysia (table 1). the disease was apparently more severe on asparagus planted at lower altitudes (16 sites) where mean daily temperatures were significantly higher than those of the same variety at higher altitudes (2 sites: cameron highlands and kundasang). young (<2 years) asparagus fields were generally found with only a few infected ferns while in older fields, particularly those at the lower altitudes, 10 25% of the older ferns had been attacked. it was not clear whether the increase in disease at higher temperature resulted from increased pathogen virulence or increased plant susceptibility. as is common for many necrotic diseases, both relative humidity and temperature probably play important roles in disease development. sixty percent of the fungal isolates recovered from asparagus ferns with small oval brown spots were curvularia spp., while alternaria spp. and fusarium spp. comprised the remaining 40%. the role of alternaria spp. and fusarium spp. in the asparagus brown spot complex is presently being studied and will be described elsewhere. based on cultural characteristics and conidial structures, the curvularia isolates were identified as curvularia lunata (wakker) boedijn, curvularia pallescens boedijn, curvularia eragrostidis (henn.) mayer and curvularia brachyspora boedijn. perfect stages of the fungi were not observed either in culture or on infected asparagus plants in nature. amongst the species of curvularia isolated from asparagus in malaysia, c. lunata was the most frequent (85 %), followed by c. pallescens (32.0%), c. eragrostidis (18.2%) and c. brachyspora (11.5%). the description and a key for identification of the curvularia species isolated from asparagus in malaysia are as follows : curvularia brachyspora boedijn (fig. 2) colonies blackish grey, black, cottony or hairy; growth rate 5.5 6.1 cm after four days. conidia 16.5 26.4 x 9.9 16.5 um, dark or light brown, solitary, simple, some slightly curved, clavate, ellipsoidal, broadly fusiform, 3-septate, dark bands at the septa, mostly truly median and asymmetrical. the third cell from the base is conspicuously 29 biotrop1a no. 9, 1996 larger, broader and darker than the others. conidiophores macronematous, monone-matous, straight, flexous, often geniculate, sometimes nodose, brownish and smooth. although the type species of curvularia brachyspora was originated from java, the presence of this species has never been reported in malaysia. elsewhere, it was isolated from agave, digitaria, dracaena, olea, oryza, saccharum, triticum and air (ellis 1971; sivanesan 1987). table 1. distribution of asparagus brown spot caused by curvularia spp. at 18 sampling sites in malaysia 1989-90) curvularia eragrostidis (henn.) meyer (fig. 3) colonies blackish grey, black, hairy or cottony; growth rate 5.7 6.4 cm after four days. conidia 16.5 24.75 x 9.9 16.5 um, dark brown, solitary, simple, broadly ellipsoidal, 3-septate, symmetrical, dark bands at the septa are quite thick and truly 30 brown spot caused by curvularia spp., b. salleh et al. figure 2. curvularia brachyspora boedijn a: conidia; b: conidiophores; c: cmamydospores figure 3. curvularia eragrostidis (henn.) meyer a: conidia; b: conidiophores; c: chlamydosporcs 31 31 biotropia no. 9, 1996 median. usually the second and third cells are the same size, and broader than the others. conidiophores macronematous, mononematous, mostly straight, often branched, geniculate, often nodose, brown and usually smooth. c. eragrostidis has been isolated from a wide variety of plant hosts and other substrates worldwide, including malaysia. it causes minor leaf spots on several grami-nicolous hosts (hawksworth 1990). figure 4. curvularia lunata (wakker) boedijn a: conidia; b: conidiophores; c: chlamydospores curvularia lunata (wakker) boedijn (fig. 4) colonies are dark grey or black, effuse, cottony or velvety; mycelium immersed; growth rate 6.0 7.6 cm after four days. stroma dark brown. conidia 14 28 x 7 13 µm, dark brown, solitary, often curved on the middle septum (not median); predominantly 3-septate and rounded at the apex. very few (<3%) triradiate stauroconidia. conidiophores dark gray or brownish black, macronematous and mononematous, po-lytretic, straight or flexous and smooth. c. lunata is often associated with grain moulds of sorghum and millet, and also causes a leaf spot on sorghum (singh 1982). c. lunata is common and widespread in the tropics, including malaysia, and may be recovered from many different substrates (ellis 1971). 32 figure 5. curvularia pallescens boedijn a: conidia; b: conidiophores; c: chlamydospores curvularia pallescens boedijn (fig. 5) colonies usually grey or pale grey, effuse, cottony or velvety; mycelium immersed; growth rate 7.0 7.7 cm after four days. stroma gray or pale gray. conidia 7 9 x 3 4 µm, pale or very pale brown, solitary, usually straight or only slightly curved; predominantly 3-septate. conidiophores gray or pale gray, macronematous or monone-matous, polytretic, straight or flexous, smooth and brown. c. pallescens is common on many different substrata especially in the tropics (ellis 1971; sivanesan 1987). key for the identification of curvularia spp. isolated from asparagus ferns showing brown spot in malaysia conidia remaining smooth-walled and predominantly 3-septate ............................................................................................................................ 1 1. conidia middle septum truly median ....................................................................... 2 conidia middle septum not median ........................................................................ 3 33 brown spot caused by curvularia spp., b. salleh el al. biotropia no. 9, 1996 2 conidia symmetrical, dark bands at the septa are quite thick, usually the third and the second cells are of the same size ............................................................ eragrostidis conidia asymmetrical and mostly truly median .................................... brachyspora 3. conidia straight or slightly curved, 7-9 x 3-4 u,m ....................................................................................................... pallescens conidia straight or curved, sometimes have triradiate stauroconidia, 14-28 x 7-13 (im .............................................................................................. lunata pathogenicity test pathogenicity tests showed that all isolates of the four curvularia species obtained from asparagus showing brown spot symptoms in malaysia were pathogenic to both varieties of asparagus i.e. mw and uc 157 tested. once the symptoms occurred, there was no difference in the visible symptoms caused by the four species of curvularia. the first symptom of asparagus brown spot appeared as a water-soaked lesion which later developed into a brown spot with a brownish red margin and brownish grey center. the slighty sunken spot, particularly that caused by c. lunata, gradually became dark brown and developed from 0.7 to 5.0 cm within 28 days. the spot developed upwards and sideways along the circumference and coalesced, hence girdling the stem. at this stage, the entire stem appeared brown with irregular dark-brown patches (fig. 6), quite similar to the symptoms observed on rice stem infected by blast (pyricularia oryzae). under favourable conditions, heavily infected ferns were easily broken and eventually killed. no symptoms were observed on the spears and crowns. inoculations of wounded or unwounded plants with either conidial suspensions or mycelial plugs on fully developed asparagus ferns resulted in symptoms identical to those seen in the field. c. eragrostidis, c. pallescens and c. brachyspora only caused milder symptoms on ferns inoculated through wounds and the first symptoms appeared later ( ± 2 3 weeks) than those induced by c. lunata. isolates of these three species did not produce visible symptoms on unwounded plants even up to 4 weeks after inoculation. reisolation from artificially infected ferns showing brown spot symptoms yielded only the pathogens that were used for inoculation. reisolation from plants infected with a mixture of all four species always yielded c. lunata. therefore we concluded that c. lunata was the most dominant and virulent species of curvularia in this context. these greenhouse results are similar to the disease incidence and severity seen under field conditions. wound inoculation using fungal mass was the best (p<0.01) of the four methods tested since the first typical symptom of brown spot i.e. watersoaked spot, caused by c. lunata was observed within seven days of inoculation. the ino 34 figure 6. an advanced stage of brown spot on asparagus stem caused by curvularia lunata. note the irregular dark-brown patches. culation was more effective on the upper parts and branches than on the base and middle parts of the ferns. a wound was not an absolute requirement for disease development since the pathogens also may enter through stomata, and/or natural and accidental wounds. in other diseases of asparagus, e.g. dead stem caused by fusarium culmorum, inoculation succeeded only when the stems were wounded (van bakel & krom-kerstens 1974). infection by the purple,spot pathogen, stemphylium vesicarium on asparagus cv. mw was more numerous arfd occurred at shorter wetting durations on wounded than on 35 brown spot caused by curvularia spp., b. salleh et al. biotropia no. 9,1996 unwounded plants (johnson & lunden 1986). they also showed that wounds produced by blowing sand could serve as entry points for the infection of the pathogen. in scanning electron micrographs of fusarium proliferation, the causal agent of reddish-brown rot of asparagus, nik norulaini & salleh (1990) clearly showed that this pathogen successfully penetrated its host only through wounds and stomata. in all asparagus fields visited during the present survey, we observed numerous bark-eating caterpillars and mollusks that could easily have created wounds that curvularia spp. and other pathogens could use to enter the host. therefore, effective pest control probably will lead to significant reduction of brown spot and other diseases of asparagus in the field. yield losses occurring as a result of infections by curvularia spp. on asparagus in malaysia have not been ascertained, but based on our present survey we would estimate between 15 35%. based on these symptoms and aetiology, we proposed that this disease complex on asparagus ferns denoted as brown spot. this report is the first on brown spot of asparagus in malaysia, and to our knowledge, anywhere in the world. acknowledgements this research was supported by the government of malaysia (irpa 123/31037 2401). we thank the owners and persons in charge of the eighteen asparagus sampling sites. references anon. 1985. tanaman asparagus tanah rendah. risalah pertanian bil. 55. jabatan pertanian semenanjung malaysia, kuala lumpur. 27 p. bansal, r.k., s.a. menzies and p.o. broadhurst. 1986. screening of asparagus species for resistance to stemphylium leaf spot. new zealand journal of agricultural research 29: 539-545. ellis, m.b. 1971. dematiaceous hyphomycetes. commonwealth mycological institute, london. 452-459 pp. ellis, m.b. 1976. more dematiaceous hyphomycetes. commonwealth mycological institute, london. 404-411pp. hawksworth, d.l. 1990. cmi description of fungi and bacteria. mycopathologia 111: 109-130. johnson. d.a. and j.d. lunden. 1986. effects of wounding and wetting duration on infection of asparagus by stemphylium vesicarium. plant disease 70: 419-420. 36 brown spot caused by curvularia spp., b. salleh et al. nik norulaini, n.a.r. and b. salleh. 1990. in vitro inoculation of asparagus tissue culture plantlets with vegetative hyphae of fusarium proliferatum. in: proceedings of the 3 rd international conference on plant protection in the tropics, genting highlands, malaysia, pp. 127-129. salleh, b. 1990. crown rot caused by fusarium proliferatum, a new disease of asparagus in malaysia. in: proceedings of the 3 rd international conference on plant protection in the tropics, genting highlands, malaysia, p. 343 (abstract). salleh. b. and b. sulaiman 1984. fusaria associated with naturally diseased plants in penang. journal of plant protection in the tropics 1: 47-53. salleh, b. and r.n. strange. 1988. toxigenicity of some fusaria associated with plant and human diseases in the malaysian peninsula. journal of general microbiology 134: 841-847. singh, r.s. 1982. plant pathogens-the fungi. oxford and ibh publishing, london. 342pp. sivanesan, a. 1987. graminicolous species of bipolaris, curvularia, drechslera, exserohilum and their teleomorphs. mycological papers 158. pp. 261. van bakel, j.m.m. and j.j. krom-kerstens. 1974. dead stem disease of asparagus caused by fusarium culmorum. netherlands journal of plant pathology 80:104-109. 37 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf biotropia no. 6, 1992/1993: 1-32 notes on the family ampullariidae (gastropoda: prosobranchia) in the philippines: i. digestive, circulatory, and excretory systems roberto c. paoulayan and elpidio a. remigio institute of biology, college of science, university of the philippines, diliman, quezon city, philippines abstract a total of 232 ampullariid snails collected from 23 sites covering 7 islands in the philippines were compared conchologically and 200 alcohol-preserved specimens were dissected for anatomical characteristics. conchological comparison of the shells of the collected snails with that of identified lots from the senckenberg natur-museum, frankfurt, germany, the british museum for natural history, london, england, the koninklijk belgisch instituut voor natuurwetenschappen, brussels, belgium, and the rijksmuseum voor natuurwetenschappen, leiden, netherlands, revealed the presence of 5 species in the collected samples. these are: p. conica, p. ampullacea, p. mainitensis, p. quadrasi, and p. vittala. the latter 3 species were previously reported as being indigenous to the philippines. aside from characteristics of the shell, the morphology of the stomach may be useful for species discrimination. p. quadrasi and p. vittata, however, do not seem to differ anatomically from p. conica. introduction information on the taxonomy of the philippine snails belonging to the family ampullariidae (pilidae) is very deficient. there is a dearth of critical scientific data available aside from the initial reports on pilids from the philippines written more than a century ago by philippi (1851) and reeve (1856). although a number of species has been named and described, an accurate account of the number of existing species and a more reliable system of identification have yet to be clearly established. species differentiation has been based primarily on a very limited set of shell samples with rather polymorphic conchological features. examination of type and collected specimens in the following museums: the senckenberg natur-museum (smn), frankfurt a.m., germany; the british museum of natural history (bmnh), london, england; the koninklijk belgisch instituut voor natuurwetenschappen (kbin), brussels, belgium; and the rijksmuseum voor natuurwetenschappen (rvn), leiden, netherlands, as well as extensive literature search revealed that 9 nominal species of pila have been recorded from the 1 biotropia no. 6, 1992/1993 figure 6. a. the alimentary tract of p. conica. bar = 3 mm. b. jaws of p. conica. bar = 1 mm. abbreviations to figures see appendix 2. also roughly the boundary of the buccal mass with the esophagus. at this region, the roof of the buccal cavity is slightly thickened and is furrowed longitudinally. from this region the cavity leads to the esophagus dorsally and into the radular sac ventrally. the junction between the two entrances being an extended, furrowed flap (ff), corresponding to the elliptical pad of p. globosa (prashad 1925). the chitinous jaws (fig. 6b) are connected to each other on their dorsal edges by a very thin membrane (jm). each jaw is roughly oval in outline, with a thickened slightly serrated cutting edge (jce) which is also the most anterior edge of the jaw. a ridge (jr) is present that serves as the attachment of the jaws to the sphincter muscle. the cutting edge itself is slightly curved inwards. the more posterior portion of each jaw is thinner and bears a slight projection at its ventral edge. except for differences in size, which is dependent on the size of the snail, the jaws of the other species examined are similar to that of p. conica. there are two pairs of cartilages, the superior and lateral cartilages. the superior cartilages (fig. 8d: sc) are small somewhat triangular structures on the lateral sides of the anterior portions of the radula. medial and posterior to them 10 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 7. representative radula of the tentatively identified species. a. p. conica: lake mainit, surigao del norte. b. p. ampullacea: lake manguiao, palawan. c. p. ampullacea: magallanes, sorsogon. d. p. mainitensis: oacao, palo, leyte. e. p. vittata( = p. conica): sta. cruz, marinduque. f. p. quadrasi ( = p. conica): lamluad valley, lake sebu, so. cotabato. bar = 100µm. 11 biotropia no. 6, 1992/1993 figure 8. buccal mass musculature of p. conica. a. side view b. dorsal view c. ventral view d. longitudinal section abbreviations in figures see appendix 2. see text for numbered muscles. bar = 1 mm. are the bigger lateral cartilages (lc). these are somewhat s-shaped and obliquely situated structures, having a thickened ventral edge. the relatively thinner upper portion curves inward and partly overlaps that of the opposite side. externally the lateral cartilages can be seen as ridges on the ventro-lateral sides of the buccal mass between the muscles, the bulk of the cartilages being embedded in the buccal mass. in all the specimens examined, the cartilages are of similar shape and number. the radula (fig. 8d: r) is a ribbon shaped structure, located on the floor of the buccal cavity, bearing the teeth that are arranged in a definite pattern in 33 41 similar transverse rows. the radula is taenioglossate (fig. 7a) consisting of seven teeth in the transverse row, one central (rhachidian) flanked on each side by one lateral and two marginals. the general radular formula is 2-1-1-1-2. the marginals, laterals, and to a certain extent the rhachidians overlap and obscure from view their structures. during preparation, individual teeth were deflected in order to reveal and clearly observe their fine features. the rhachidian teeth have horizontally elongated body from which 7 cusps project. the anterior margin of the body is grooved. the central cusp is the largest, triangular, spade-like, or dagger-like in form. there are two lateral teeth per row. each has a base that is elongated obliquely, with prominent ridges on the dorsal surface. the body is basically shouldered and typically bears five cusps: a large mesocone, a small endocone and ectocone, a basal 12 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio cusp at the antero-medial part of the base and hidden from view by the body, and another cusp lateral to the ectocone. the mesocone is the largest cusp, dagger-like in form, and has smoothly curved or straight lateral edges terminating in a pointed structure. there are four marginal teeth to a row. they are smaller than the laterals. immediately outer to the laterals are inner marginal teeth with narrow bases and longitudinal ridges. an inner marginal tooth has two cusps, a small inner and a large outer cusp. a basal lobe is present at the base projecting posteriorly and firmly attached to the redular membrane. the outer marginal is essentially identical to the inner one but lacks a basal lobe and is smaller. the radular sac (fig. 8a: rs) is a short, rather tubular, and somewhat rigid structure projecting ventrally out of the posterior end of the buccal mass from the midventral line. internally, the more posterior portion of the radula lies in this sac as a broad ribbon whose posterior end reaches the bottom of the sac. the inner wall of the radular sac facing the teeth appears somewhat fleshy and is in close contact with it. this part of the wall also extends to the buccal cavity terminating as the elliptical pad that forms the junction between the entrances to the esophagus and radular sac. the muscular system of the buccal mass (fig. 8) is highly organized in accordance with its complex movements. the action of some of the muscles is however sometimes difficult to interpret. as with the buccal cartilages, the musculature of the buccal mass in all specimens examined is similar. they are listed in table 1 according to the terminology of prashad (1925) and demian (1964). there are two salivary glands (fig. 8: sgl), one on each side, which lie on the postero-dorsal limit of the buccal mass. a short duct (sold) starts near the ventral, anterior edge of each gland and enters the buccal mass muscles. the ducts open into the buccal cavity near the flap forming the junction of the esophagus and the radular sac. the cream colored esophageal pouches (esp), one on each side, lie ventrolateral to the salivary glands. by careful dissection, they can be separated from the salivary glands. the pouches on each side has an opening into the buccal cavity near the junction of the buccal cavity and the esophagus. the internal walls of the pouches are simple with few, if any, longitudinal ridges. the esophagus (fig. 6a: eso) is a thin-walled tube arising from the postero-dorsal edge of the buccal mass. it continues posteriorly below the floor of the pallial cavity, arching to the left in a wide bend as it enters the visceral mass, slightly narrowing as it continues to the stomach at the level just below the pericardium. no difference in the form of the salivary glands, esophageal pouches, and esophagus was noted for the other species examined. the stomach (figs. 6a: st & 9a) is a somewhat complicated pouch located behind the posterior kidney chamber. it is seen externally as a maroon patch partly 13 biotropia no. 6, 1992/1993 table 1. terminology of the muscular system of the buccal mass according to prashad (1925) and demian (1964). 14 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio table 1. continued 15 biotropia no. 6, 1992/1993 figure 9. stomach of representative species (opened along the exposed margin). a. p. conica: u.p. lagoon, quezon city b. p. ampullacea: lake manguiao, palawan c. p. mainitensis: gacao, palo, leyte d. p. vittata( = p. conica): marinduque e. p. quadrasi ( = /". conica): lamluad valley, lake sebu, so. cotabato abbreviations in figures see appendix 2. 16 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio embedded in the digestive gland. upon exposure by slitting through the patch, the stomach shows a broad j-shaped cavity with a very muscular wall. the innerside is lined by longitudinal ridges. the cavity is divided into two areas, the gizzard (gi) at the anterior portion where the esophagus enters and the narrower, tubular, and relatively straight posterior style sac (ss). the esophagus enters the stomach near the midsection of the smaller (inner) curvature. posterior to the entrance of the esophagus, at the bend of the curvature, is the semilunar pit (ve), whose wall is elaborated by ridges.. in this pit are openings for the two ducts of the digestive gland (dgo). the margin of the semilunar pit arises as a prominent gastric shield (gs). the gastric shield, together with the wall of the stomach on the other side, prevents food coming from the esophagus, from entering the semilunar pit. the gastric shield at its posterior end continues to the style sac as the major typhlosole (mat). the wall of the style sac is marked by transverse ridges. running parallel to the major typhlosole is another ridge, the minor typhlosole (mit). the major and minor typhlosole border the intestinal groove (ig) between them. dorsal to the minor typhlosole, at the level of the beginning of the pyloric portion of the stomach, lie two stomach pouches (sp), one smaller than the other. the stomach pouches are somewhat flattened towards the external wall of the stomach and their lumina are separated by a relatively thin vertical septum. no crystalline style in the style sac was found in any of the specimens examined. the digestive gland (fig. 4: dg) is a conspicuously greenish mass occupying the upper portions of the spire and partly covering the stomach. externally, the digestive gland seems to be made up of two distinct lobes, an anterior and posterior lobe, the demarkation made by the position of the stomach as well as that of the two main ducts. upon dissection, however, the apparently "distinct" lobes are actually fused, having no internal demarkation between them. the border between the stomach and the intestine is situated just below the pericardium. this junction is a sharp u-shaped loop with a pleated caecum (fig. 6a & 9a: 1c). the intestine then runs between the stomach and the posterior chamber of the kidney, following the latter's margin to its posterior end. at this point, the intestine turns back and upwards ventral to the posterior kidney chamber. the intestine then makes several loops and seems to lie inside the cavity of the posterior kidney chamber (fig. 11b: in). the intestine then unloops, turning anteriad, running along the opposite margin of the posterior kidney chamber and along the gonoduct on the dorsal side as the rectum. before the rectum terminates into the rectal papilla, the duct of the rectal gland (fig. 6a: rcg) enters it on its dorsal side. the gland is branched and is situated at the dorsal side of the rectum on the level of the anterior portion of the gill. the anal papilla (anp) is a cylindrical 17 biotropia no. 6, 1992/1993 tube with a fringe opening projecting into the mantle cavity dorsal to the genital opening, as previously described above. interspecific variation in the alimentary tract: in p. ampullacea, each spadeshaped rhachidian tooth has a central cusp flanked by two pairs of smaller lateral cusps (figs. 7b & 7c). aberrant rhachidian teeth without lateral cusps were also observed in some snails. the lateral teeth have relatively smaller mesocones with smooth lateral edges. there are 35 37 rows of teeth in the radula. the rhachidian teeth of p. mainitensis (fig. 7d) is similar to that of p. ampullacea but the central cusps are more triangular, resembling an arrow head. the lateral teeth have larger dagger-like mesocone with straight lateral edges. there are 35-36 rows of teeth in the radula. the radular teeth of p. vittata and p. quadrasi (fig. 7e & f) are similar to those of p. conica. with respect to the morphology of the stomach, in p. ampullacea (fig. 9b), the more anterior of the stomach pouches (sp) is not markedly demarkated, giving the stomach an appearance of having only one pouch. it also has a relatively shorter style sac. the stomach of p. mainitensis (fig. 9c) is similar to that of p. ampullacea, although, the two stomach pouches are distinct. the stomach of p. vittata and p. quadrasi (fig. 9d & 9e) is similar to that of p. conica. no difference in the morphology of the intestine, rectum, and anal papilla was noted between the species examined. 3. the circulatory system of p. conica the pericardial cavity (fig. 10a: p) is situated on the left side of the body whorl lying between the posterior limit of the pulmonary sac and the kidney on the anterior and the stomach and the digestive gland on the posterior. its cavity communicates with that of the posterior kidney chamber through the renopericardial duct (fig. 1 ie: rpo) which runs in the septum that separates the pericardial cavity and posterior kidney chamber. the opening of this duct into the pericardial cavity can best be seen by deflecting the heart to the more anterior side of the pericardium. the opening of the duct is at the anteroventro-lateral wall of the pericardium. as the other monotocardians, pila conica has a single auricle and a single ventricle (figs. 10a & 10b). the auricle (a) is a thin walled sac capable of great distensions. internally, fine muscle fibers (am) in the form of thin bundles runs in various directions. however, most of the muscle fibers run longitudinally from the wall of the entrance to the auricle wall. the ventricle (v) is ovoid in form with a thick muscular wall. the auricle opens into the ventricle through the auriculoventribular opening. this opening is guarded by two valves (aw) attached by thin muscular fibers to the wall of the ventricle. as seen from the inner side, the wall of the ventricle is crisscrossed by large muscle bundles forming a coarse meshwork. 18 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 10. a. general scheme of the circulatory system of p. conica. vessels drawn in solid lines are the more superficial vessels. vessels drawn in broken lines are the deeper vessels passing through the head foot region and the visceral mass. bar = 5 mm. b. heart (opened to show the internal structures). bar = 1 mm. abbreviations in figures see appendix 2. the cavity of the ventricle appears to be reduced, because of these muscular bundles. the ventricle in turn leads to the very short aortic trunk. the entrance of the ventricle to the aortic trunk is also guarded by two smaller valves (vav). the morphology of the pericardial cavity and the heart in the other species do not differ from that of p. conica. the ventricle leads to a short aortic trunk at its posterior end. this trunk then divides into the anterior (aa) and posterior aorta (ap). the former turns anteriad and expands into a pouch-like ampulla (am) occupying the most posterior end of the pericardium. the anterior aorta, shortly after leaving the ampulla, continues in its course anteriad, embedded in the muscular tissue and forming the leftventrolateral wall of the pallium. as the aorta reaches the level of the anterior portion of the esophagus, it splits into two big branches, one branch (aa2) turning right, making an exit from the wall, and emerging into the head sinus (the space surrounding the buccal mass and the anterior portions of the esophagus), the other big branch (aa1) continues its course anteriad eventually breaking into branches that supply blood to the left mantle wall, the osphradium, the left siphon, as well 19 biotropia no. 6, 1992/1993 philippines by several workers (philippi 1851; reeve 1856; kobelt 1911; bequaert and clench 1939). these species are: 1. pila conica (gray 1828) 2. pila luzonica (reeve 1856) 3. pila lubrica (reeve 1856) 4. pila vittata (reeve 1856) 5. pila ampullacea (linnaeus 1758) 6. pila moellendorffi (kobelt 1911) 1. pila mainitensis (kobelt 1911) 8. pila quadrasi (kobelt 1911) 9. forbesopomus atalanta (bequaert and clench 1939) up to the present, there are few local studies which have dealt with the other aspects of the snail's biology. palomino and jueco (1983) published data on the radula of some freshwater snails including pila luzonica. they showed the conservative structure of the ampullariid radula but the description lacks important structural detail. garibay et al. (1987) studied the life history as well as early embryogeny of laboratory-reared pilid snails which were identified as p. luzonica. this study hopes to give additional anatomical data which may be of use towards a better understanding of the ampullariid snails in the philippines. materials and methods the study was based on specimens collected in 1986, 1988, 1989 and 1990 from different geographical areas in the philippines as shown in figure 1. brief descriptions of these sites are listed in appendix 1. the shells of the snails collected in this study were compared with those of the identified museum lots in order to come up with a tentative identification of the former. such identification was based on ocular comparison of the shells. snail relaxation prior to dissection snails were first menthol-relaxed before killed and dissected or preserved in 70% ethyl alcohol. this was done by placing the snails in a small basin with two inches of water. a plastic cover was then placed on top. this plastic cover had a gauze sachet filled with menthol crystals taped on the underside. caution had been applied to prevent the menthol crystals from falling on the water as the snails are very sensitive and upon contact with menthol will immediately withdraw inside their shells. relaxation of the snails through "breathing" menthol vapor may be slow but produces the desired result. normally overnight relaxation was enough. bigger snails required more time. relaxed snails were then killed by immersion in 80°c water for 30 seconds prior to dissection or preservation. 2 biotropia no. 6, 1992/1993 as to the surrounding tissue of the left side of the mantle. the branch of the anterior aorta that emerges into the head sinus turns obliquely anteriad and after a short distance crosses the esophagus dorsally as it also gives branches to it. shortly after crossing to the right side of the esophagus, it subdivides into two branches. the right branch penetrates the right side of the sinus and within this wall, it turns anteriad to supply branches to the right siphon (egestion siphon), the tissue of the right side of the mantle, as well as the copulatory organs. the other branch turns ventrad and supplies blood to the buccal mass, the snout, and the foot region. the posterior aorta subdivides immediately after leaving the aortic trunk. one branch (fig. 10a: api) leads to the stomach and, through smaller branches, supplies blood to the various parts of the stomach. the other branch (ap2) can be seen just underneath the mantle epithelium, crossing the junction between the stomach and the intestine, running a course posteriorly between the intestine and the posterior kidney chamber. this artery gives off branches to the intestine, digestive gland, and the gonoduct in this region. the arterial system of the other species examined is similar to that of p. conica. the venous system made up of sinuses consists of the head-foot sinus, (fig. 10a: hf). the main trunk of the foot sinus can best be seen by removing the pedal-pleural ganglia complex. ventrally lies the opening of the foot sinus to the large head sinus. the main trunk of the foot sinus is rather large and situated medially collecting tributaries of smaller sinuses from all parts of the foot. the head sinus is relatively the largest of all sinuses in the snail. it lies above the foot and below the floor of the mantle cavity surrounding the anterior part of the alimentary tract. some of the blood from this large sinus goes to the tributaries of the pallial vein, through small openings along the left and right walls. these tributaries then unite, on each side, to form the main pallial veins (pv). the right pallial vein joins the afferent gill vein (agv) and the left pallial vein opens into the afferent lung vein (alv). the rest of the blood exits from openings on the more posterior portion of the right wall of the head sinus. these openings lead to the sinus through which the sub-intestinal-visceral connective nerve also passes. this sinus (fig. 1 ib: akv) continues as the main branch of the afferent kidney vein as it enters the viceral mass. afferent kidney vein: (fig. 11b: akv). the main branch of this vein turns obliquely inward, becoming larger in diameter. it follows a course along the posterior wall of the columellar muscle, just anterior to the digestive gland where it receives branches from the digestive gland, stomach, and the anterior portions of the intestine, to a level between the junction of the posterior and anterior renal chambers. here it then divides, one branch to become the afferent vessel of the posterior kidney chamber (akpv) and the other to become the afferent vessel of the anterior kidney chamber (akav). 20 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 11. the excretory system of p. conica. a. dorsal view of the anterior and posterior chambers. bar = 5 mm. b. posterior chamber (opened along its margin). bar = 1 mm. c. anterior chamber (opened to show the internal structure). arrow points to the extension of its base. bar = 1 mm. d. lamella of the anterior chamber showing position of blood vessels. bar = 1 mm. e. opening between the anterior and posterior chambers cut to show the opening of the reno-pericardial canal. bar = 1 mm. abbreviations in figures see appendix 2. 21 biotropia no. 6, 1992/1993 branchio-renal complex: this is a system of vessels leading to the gill or in part to the pulmonary sac (fig. 10a: agv & alv). there are two efferent branches from the anterior kidney chamber (fig. 10a, 11c & 1 id: ekav). one branch can be seen prominently along its median axis and exiting to the afferent gill vessel. the other branch is located on the posterior edge of the chamber and receives blood from this chamber as well as some blood from the posterior kidney chamber and the intestines. at the anterior tip of the anterior kidney chamber, this branch receives blood coming from the rectum through a short vessel (rcv), then exits to the afferent gill vessel. the afferent gill vessel (agv) is a prominent vessel with a relatively large diameter that runs alongside the gill, giving branches to the gill leaflets. at its main trunk it receives two to three short vessels from the rectum on the right side (rcv). at the most anterior end of the afferent gill vessel, it receives blood from the genital and rectal papilla as well as the right pallial and visceral vein. the visceral vein (vv) collects blood from the intestines, and the digestive and genital glands then runs alongside the gonoduct ventrally, collecting blood from it along the way. at its anterior end, it seems to join with the pallial vein shortly before entering the afferent gill vein. the afferent gill vein has also an anterior connection with the afferent pulmonary vessel, running along the margin of the mantle. at the left and right side, the afferent pulmonary vessel (apv) receives branches coming from the osphradium (osv), the copulatory organ (cov), and branches from the mantle edge (mv). the vessels leading to the auricle of the heart are as follows: a), the efferent vessel of the posterior kidney chamber (ekpv), whose main trunk and tributaries can be best seen at the inner side of the roof of the chamber. it runs a course through the pericardium entering the extreme right edge of the auricle, its entrance (fig. 10a & b: ekpv) being partly obscured by the fine longitudinal muscles of the auricle; b). the efferent lung-gill vessel (fig. 10a: elgv) this is a relatively big vessel located beneath the skin on the dorsal side running between the edges of the gill and the pulmonary sac. it collects aerated blood from the pulmonary sac and the gill leaflets through a series of small vessels opening on its left and right walls, respectively. it enters the auricle prominently more or less at its tip. some differences in the venous system were noted in the other species examined. in p. mainitensis and p. ampullacea, one of the branches of the efferent branch from the anterior kidney chamber (ekav1) runs along the margin of the anterior kidney, somewhat alongside the afferent gill vessel. as to the vessels entering the auricle, in p. mainitensis, there seems to be a short efferent lung vessel that collects blood from the more posterior portion of the sac, aside from the efferent 22 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio lung-gill vessel. this vessel may, however, be simply a part of the efferent lung-gill vessel as it joins the latter before the latter enters the auricle. the venous system of p. vittata and p. quadrasi do not differ from that of p. conica. 4. the excretory system of p. conica the renal organ or kidney (fig. 11) consists of two chambers an anterior and a posterior chamber. the anterior chamber (fig. 11 a: ak) could be seen on the upper side of the animal as a reddish brown elongated structure lying to the right of the pericardium after removal of the shell. its floor projects somewhat into the mantle cavity, and opens into it to the right of the epitaenia. it can be differentiated from the posterior chamber by the lamellated appearance of its inner wall which is seen through the thin mantle epithelium at its dorsal surface. on opening the chamber by making a slit along the roof margin and then deflecting the resulting flap, two longitudinal, whitish, and medially placed structures with triangular lamella arising from their sides could be observed (fig. 1 ib). these were the main branches of the afferent vessel of the anterior kidney chamber (akav1 & akav2) coming from the main trunk of the afferent vessel of the kidneys described above. one branch (akav1) is placed on the floor of the chamber while the other (akav2) is located at the inner dorsal roof. these vessels divide into smaller branches as they enter the lamellae (fig. 1 id). at the periphery of the lamellae, the tiny branches enter the efferent kidney veins of the chamber (ekav1 & ekav2). one of these (ekav1) is found along the posterior margin of the chamber forming a part of the branchio-renal complex. the other (ekav2) courses along the dorsal surface of the anterior kidney chamber. the anterior kidney chamber also forms a branched appendage, projecting from its posterior edge near the junction with the posterior kidney chamber (fig. 11 a: arrow). it is also lamellated and forms the dorsal roof of the opening of the anterior kidney chamber into the mantle cavity (fig. 11b: ok). at this level, but more medially placed on the floor of the chamber, is a longitudinal slit (o). this is the opening through which the posterior kidney chamber communicates with the anterior chamber. the posterior kidney chamber: (fig. 11 a: pk). the roof of the posterior kidney chamber prominently lies to left of the rectum as a broad brownish structure with a hooked posterior portion. on making a cut along the margin of the roof and deflecting the flap anteriorly, the spacious cavity of the chamber can be seen (fig. 1 ib). the soils of the intestine, parts of the genital duct, and portions of the digestive gland can be seen projecting into the cavity. the floor of the chamber consists of a very thin membrane separating the cavity from the organs projecting into it. in 23 biotropia no. 6, 1992/1993 contrast, the roof of the cavity appears to be thicker and spongy due to the thick plexus formed by the relatively large and branched posterior kidney vessels. there are two main vessels the afferent (akav) and efferent (ekav) vessels of the posterior chamber, and a minor vessel system (fig. 10a: arrow) that is found at the right margin of the organ and exits into the branch of the efferent vein of the anterior kidney chamber on that side. the main trunk of the afferent and efferent vein of the posterior chamber is seen prominently placed at the median portion of the chamber's roof. the posterior kidney chamber is separated from the pericardial cavity by a thin vertical septum. the renopericardial canal connecting the pericardial cavity to the posterior kidney chamber is found in this septum. the canal opens at the tip of a very short papilla very near the longitudinal slit communicating the posterior with the anterior kidney chamber described above (fig. 1 ie: rpo). except for differences in size, which is dependent upon the size of the snail, the morphology of the kidney of the other species examined does not differ from that of p. conica. discussion distinct difference in mantle or body pigmentation was not observed. although some snails have shells that have lighter color, yellow or orange without bands, have been observed, such seem to be independent of mantle pigmentation. snails with lighter colored shells still possess normal black pigmentation of the mantle and the other parts. all members of the family ampullariidae are amphibious. such an adaptation is due to their possession of a ctenidium and a lung sac which are both functioning. this has enabled some members, especially pila, lanistes, and pomacea, to exist in a wide range of habitats (mozley 1939; mandahl-barth 1954; andrews 1965a). they can, in addition, withstand seasonal drought by aestivating (meenakshi 1956). their distribution, however, seems to have a latitudinal limit, being widely distributed only along the tropical and subtropical belt. in turbinicola, asolene, lanistes, pomacea, and marisa (scott 1943; kretschmann 1955; demian 1958; andrews 1965a), the downgrowth of the mantle which forms the lung sac is relatively extensive. the same observation has been noted in the present study on pila. in afropomus balanoides, the lung-sac is formed by a simple circular fold of the roof of the mantle cavity (berthold 1988). as previously observed in other pila species , the lung sac of the snails in this study has a wide slitlike aperture, unlike the small aperture reported for pomacea and lanistes. the 24 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio development of a small lung sac aperture thus seems to represent convergence towards a small and more efficiently manageable aperture (andrews 1965a). due to its peculiar position, the gill seems to correspond to the osphradium on the left with which, however, it is not homologous (prashad 1925). as with other monotocardian prosobranchs, the gill of ampullariid snails is actually the left gill which is shoved to the right side by the development of the extensive pulmonary sac (fretter & graham 1962). one structure that probably plays an important role in the direction of water currents in the mantle cavity, as well as in separating the pulmonary sac from the gill and in providing protection by effectively closing of the ctenidium when the water condition turns foul, is the epitaenia (andrews 1965a). the general morphology of the alimentary tract as described is similar in all the species of pila examined. the characteristics of the radular teeth, especially the differences seen in the number and form of the cusps, have a limited use for species differentiation. this is due to (1) the high probability of the mechanical destruction or modification in the form of the cusps and (2) the observed fusion of cusps in some individuals of the same species. the basal lobe at the inner marginal tooth was described by moretto and nahabedian (1983) for pomacea canaliculata and by berthold (1988) for afropomus balanoides. it was observed in all the snails here examined. such observation supports berthold's statement that a basal lobe is present in the inner marginal teeth of all the other pilid genera. the general morphology of the rest of the buccal mass as well as the salivary glands, esopha geal pouches, and the anterior portion of the esophagus is also similar to that described for p. globosa, m. cornuarietis, p. canaliculata, and a balanoides (prashad 1925; scott 1957; demian 1964; berthold 1988). there are two stomach pouches in all the snails examined. the presence of two stomach pouches has also been observed in marisa cornuarietis (demian 1964) and afropomus balanoides (berthold 1988) and in the thai ampullariidae (keawjam 1987). the stomach pouches in pila, in this study, were not as big as keawjam's illustration for the thai pilidae. andrews (1965b) reported only one pouch ("glandular pouch") in pomacea canaliculata. it is possible that the anterior pouch, as from my observations in p. ampullacea, is not so extensive. rod-like fecal material in the dissected style sac of some of the snails examined were noted. compaction of fecal and excretory material seems to occur at the style sac (andrews 1965b). as previously observed in other genera, there is no crystalline style in the style sac of pila. a caecum which seems to be divided into transversely positioned chambers was found in all the snails dissected in this study. it was also described in the other ampullariid genera such as in afropomus (berthold 1988) and in pila, turbanicola, 25 biotropia no. 6, 1992/1993 and pomacea (andrews 1965b). it may be that the presence of this anterior caecum is a synapomorphy shared by the members of the family ampullariidae. short, semi-compact, pellet like material were noted in the portion of the intestine just after the caecum, giving credence to the suggestion that pellet formation is brought about in the caecum in pila (andrews 1965b). the coiling of the intestine, seen only when the edges of the posterior kidney chamber is cut open, probably needs more attention. in pila, here examined, and in afropomus (see berthold 1988, p. 154,f.bc), the pattern of coiling is similar, while that of pomacea (see andrews 1965b, p.21,f.l) is simpler. the rest of the posterior portion of the alimentary tract shows no difference from that described by andrews (1965b) for pomacea. an anal gland has not been described for afropomus by berthold (1988). it may have been overlooked, as was the case for p. globosa by prashad (1925) and for the thai ampullariidae by keawjam (1987). a similar gland appears in some members of the prosobranchia, including archeogastropods and neogastropods (hyman 1967). the description of the anal glands of some stenoglossans (fretter 1946) resembled that of pila. the anal glands probably play a role in the elimination of excess calcium and iron salts and purines, of which its development in ampullariids may be correlated with their freshwater habits (andrews 1965b). as a final note on the digestive gland of pila, ranjah (1942) proposed that originally there were two lobes of the digestive gland but the anterior (post-torsional left) lobe is supressed at an early stage in development. two distinct ducts were observed in all the specimens dissected. their separated location from each other precludes the presumption that they represent two main branches from a single lobe of the digestive gland. andrews (1965b) observed that in young pomacea, there were two distinct lobes, although a single lobe was observed in mature snails. apparently, the single lobe seen is the result of fusion of the two lobes, such that any border which formerly existed became indistinguishable due to the very close contact between the two lobes. such a close contact is expected when one considers a very limited space available in the visceral hump for the development of a large structure such as a multifunctional digestive gland. in all the species examined, the outline of the venous system appeared to be similar, except for that minor difference in the efferent lung-gill vein of p. mainitensis where a short "pulmonary sinus" is located very near the entrance to the auricle. such a peculiar position gives it an appearance of an independent pulmonary vein. this peculiar sinus, however, opens into the efferent lung-gill vein and not directly into the auricle; therefore it may still be a part of the latter. prashad (1925) described a distinct pulmonary vein for p. globosa, but this was later refuted by andrews (1965a). observations on the venous system of the species of pila examined here are similar to that described by andrews (1965a). 26 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio blood from the posterior kidney chamber exits from two points: (1) the larger efferent vessel of the chamber that opens directly into the auricle and (2) two to four small sinuses from the more anterior right margin of the chamber and joining the efferent vein of the anterior chamber at that area. andrews (1965a) postulates that the larger efferent vein of the posterior chamber is actually the vein from the degenerate nephridial gland. this vein has been enlarged to drain a larger area of the chamber and the actual efferent vein of the chamber is that vessel that joins the second branch of the efferent vessel that drains the anterior chamber. the basic plan of the arterial system of pila here examined is similar to that described for other pilids like p. globosa (prashad 1925), p. canaliculata, t. saxea, l. ovum bangweolicus (andrews 1965a) and a. balanoides (berthold 1988). berthold, however, described a slightly thickened ampulla for afropomus and added that such a form seems to be plesiomorphic compared to the bigger globular ampulla of the other species mentioned above. the members of the family ampullariidae seem to be peculiar in having a kidney made up of two chambers which are morphologically distinct from each other. the general shape as well as position with each other of the anterior and posterior kidney chambers in all the species of pila here examined is similar to that described by prashad (1925) and illustrated by michelson (1961) for p. globosa. summary 1. species identification of the ampullariid snails in the philippines was previously based only on characteristics of the shell, hence, an approach was initiated in this study towards basing identification on as many characters as possible. 2. tentative identification of the collected snails based on conchological comparison with museum specimens were done and five species from the collection were identified. these are: p. conica, p. vittata, p. quadrasi, p. mainitensis, and p. ampullacea. 3. dissection of the soft anatomical parts revealed few character states in the organ systems here studied which may be useful for species identification. these are: (a) the number of osphradial and gill leaflets, (b) the morphology of the cusps of the rachidian teeth (although caution must be exercised in using this character as the radula is prone to mechanical modification), (c) the morphology of the stomach, and (d) the position of the efferent branch of the anterior kidney chamber (ekav1). no difference was observed in the other organs here studied. 4. p. vittata and p. quadrasi have been observed to be anatomically indistinct from p. conica based on comparison of the digestive, circulatory, respiratory, and excretory systems. 27 biotropia no. 6, 1992/1993 acknowledgements during these studies the authors had the support of several private persons and institutions. technical assistance or advise was received from mr. manny sapuay of nsri (photography) and the staff of u.p. n.e.c. (scanning microscope). prof. dr. edmund gittenberger, dr. jackie van goethem, dr. ronald janssen, and dr. peter mordan for allowing me to examine their collection of philippine ampullariid snails. dr. rita triebskorn for sending me much needed literature and dr. tomas berthold for sharing his insights on the group of snails studied. tus and macy mamaril and our assistant, richard kho for the company and lively discussions during the collection trips. dr. gloria l. enriquez, dr. claus meier-brook, and dr. ruben umaly for their critical advices and encouragements. the german academic exchange service (daad) for supporting the senior author's research scholarship in germany. the office of research coordination for the research grant that allowed us to collect snails. we also wish to express our gratitude to our countless colleagues in the institute of biology and outside the university who have in one way or another contributed to this work. literature cited andrews, e.b. 1965a. the functional anatomy of the mantle cavity, kidney, & blood system of some pilid gastropods (prosobranchia). j. zool. 146: 70-94. ______. 1965b. the functional anatomy of the gut of the prosobranch gastropod pomacea canalicuta and of some other pilids. proc. zool. soc. london 145: 19-36. bequaert, j.c. & w.j. clench. 1937. forbesopomus, a new genus in the family pilidae (ampullariidae), from the philippine islands. proc. new engl. zool. club 16: 53-56. berthold, t. 1988. anatomy of afropomus balanoides (mollusca: gastropoda: ampullariidae) and its implication on phylogeny and ecology. zoomorphology 108: 149159. demian, e.s. 1958. on the respiratory system and the mechanisms of respiration in lanistes bolteni chemnitz. ain shams sci. bull. cairo, 3: 301-316. ______. 1964. the anatomy of the alimentary system ofmarisa comuarietis (l.) goetoborgs vetensk. vitterhets-samh. handh. 6 ser. b 9(2): 1-75. fernando, w. 1931. the development of the kidney in ampullaria (pila) gigas. proc. zool. soc. london 62: 745-750. fretter, v. 1946. the pedal sucker and anal gland of some british stenoglossa. proc. malac. soc. london 27: 126-130. fretter, v. and a. graham, 1962. british prosobranch mollusk, their functional anatomy and ecology. ray society, london. garibay, j.l., r.c. pagulayan and b. cruz. 1987. studies on pila luzonica reeve: hatchability and growth under laboratory conditions. nat. appl. sci. bull. 39(1): 81-90. 28 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio hyman, l.h. 1967. the invertebrates. volume vi. mollusca i. mcgraw hill, new york. keawjam, r. 1987. the apple snails of thailand: aspects of comparative anatomy. malacol. rev. 20: 69-90. kobelt, w. 1911. die gattung ampullaria. in: vol. i, pt. 29 ii (neue folge) of systematisches conchylien-cabinet von martini und chemnitz. ed. w. kobelt, von bauer, und raspe. nurnberg: 76-99. kretschmann, i. 1955. untersuchungen ueber bau und function der atmungsorgane bei ampullarien. wiss. z. humboldt-univ. berlin 4: 109-119. mandahl-barth, g. 1954. the freshwater mollusks of uganda and djacent territories. ann. mus. roy. congo bel. tervuren, sciences zool. 32: 1-206. meenakshi, v.r. 1956. physiology of hibernation of the apple snail pila virens (lam.). curr. science 25: 21-322. michelson, e.h. 1961. on the generic limits in the family pilidae (prosobranchia: molluska). breviora 133: 1-10. moretto, h.j.a. and d.e. nahabedian, 1983. the radula of ampullaria canaliculata (prosobranchia: mollusca). comun. mus. arg. cienc. nat. bernardino rivadaria inst. nac. invest. cienc. nat (arg.) (hydrobiol.) 2: 107-117. mozley, h. 1939. the freshwater molluska of the tanganyika territory and zanzibar protectorate and their relation to human schistosomiasis. trans. roy. soc. edinb. 59: 687-744. palomino, m.l.p. and n.l. jueco. 1983. radular pattern in three edible freshwater snails. kalikasan, phil. j. biol. 12: 174-176. philippi, r.a. 1851. die gattung ampullaria. in: vol. 1, pt. 20 of systematisches conchylien-cabinet von martini und chemnitz. h. kuster, von bauer, und raspe (eds.) nurnberg. prashad, b. 1925. anatomy of the common indian apple snail, pila globosa. mem. indian mus. 8: 91-152. ranjah, a.r. 1942. the embryology of the indian apple snail pila globosa (swainson), mollusca, gastropoda. rec. indian mus. calcutta 44: 217-322. reeve, l. 1856. monograph of ampullaria. conchologica iconica 10. london. scott, m.i.h. 1943. sobre la organizacion de ampullaria (asolene) megastoma sowerby. not. m\is. la plata 8, zool. no. 70: 269-280. _______. 1957. estudio morfologica y taxonomica de los ampullaridos de la republica argentina. rev. mus. argent. cienc. nat. (zool.) 3: 233-333. 29 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio both menthol-relaxed, alcohol preserved snails and menthol-relaxed, freshly killed snails were used for anatomical studies. the effect of alcohol fixation, particularly on the size of the organs and tensus were noted accordingly. dissection of the soft parts approximately 200 snails were dissected and anatomically examined. the following steps were done in the removal of the soft parts from the shell without crushing the shell. to remove the operculum, a blunt edged, spatula-like probe was inserted in the space between the operculum and its attachment on the dorsal side of the foot. while holding the snail's body steady, the operculum was loosened by pressing at the attachment area with the probe. for the rest of the soft body, the probe had been inserted in the space between the columellar muscle and the shell. by exerting a little pressure, without puncturing the tissue, the muscle was pried loose from its attachment. a pair of forceps was used to grasp the snail's foot and to pull the body out while rotating it, following the coiling of the shell. dissection was done under a wild zoom stereomicroscope m7. exposure of the mantle cavity was accomplished by a cut made beginning at the right mantle edge, dorsal to the exhalant siphon, between the gill and the rectum up to the level of the anterior chamber of the kidney. in exposing the anterior portions of the digestive tract, a longitudinal slit was made starting from the dorsal tip of the snout and continued posteriorly to the area of exit of the esophagus to the visceral mass. after these two major incisions, most of the organs could be seen through the thin mantle and could be reached without much difficulty. they could, therefore, be exposed in situ. camera lucida drawings were done with a wild drawing tube. radulae were obtained by extracting buccal masses from the snails and immersing them in 10% sodium hydroxide for 24 hours. the radulae were further cleaned by sonication while immersed in 70% ethanol and subsequently processed for microscopic examination. some of the radulae were immersed in 10°7o sodium hypochlorite to dissolve the radular membrane and facilitate the observation of the lateral lobes of the inner marginal teeth. the rest were eventually air-dried, mounted in specimen blocks, sputter coated with gold using a jeol fine coat ion sputter jfc-1100, and examined under a jeol jsm-35c s.e.m at the national engineering center, in u.p. diliman. voucher specimens are presently lodged at the invertebrate museum, institute of biology, u.p. diliman, q.c. and at the mollusc division, national museum, manila. 3 biotropia no. 6, 1992/1993 appendix 1 30 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio appendix 1. (continued) 31 biotropia no. 6, 1992/1993 appendix 2. list of abbreviations a = auricle of the heart j = jaws aa = anterior aorta jce = cutting edge of jaw aabm = branch of the anterior aorta to the buccal jm = membrane connecting the two mass jaws agv = afferent gill/ctenidial vessel jr = ridge of jaw ak = anterior kidney chamber lp = labial pulps akav = afferent kidney vein to the anterior chamber ls = lung/pulmonary sac akl = lamella of the anterior kidney chamber lso = opening of the lung sac into akpv = afferent kidney vein to the posterior the mantle cavity chamber mat = major typhlosole akv = main vessel of the afferent kidney vein mit = minor typhlosole alv = afferent lung vein me = mantle edge am = auricular muscles mo = mouth amp = ampulla of the anterior aorta mv = mantle veins anp = anal/rectal papilla o = slitlike opening between the ap = posterior aorta anterior and posterior kidney aw = auriculo-ventricular valves chambers bm = buccal mass ok = opening of the kidney bmc = cavity of the buccal mass chamber into the mantle c = columellar muscle cavity co = copulatory organ opl = operculiferous lobe on dorsal cov = vessels from the copulatory organ side of the foot do = digestive gland os = osphradium dgd = duct of the digestive gland osv = osphradial vein e = eyes ov = ovary ekav = efferent kidney vein from the anterior p = pericardium chamber pk = posterior kidney chamber ekpv = efferent kidney vein from the posterior pv = pallial vein chamber r = radulla elgv = efferent lung-gill vein rcg = rectal/anal gland ep = epitaenia/mantle fold rcv = rectal vein es = egestion/exhalant siphon rpo = renopericardial opening eso = esophagus rs = radular sac esp = esophageal pouches sc = superior cartilage of the buccal mass sgl = salivary gland f = foot sgld = salivary gland duct g = gill/ctenidium so = subradular organ gi = gizzard sp = stomach pouches gp = genital papilla ss = style sac of the stomach gs = gastric shield st = stomach hf = head-foot sinus t = testis hm = muscles of the internal wall of the heart te = tentacle 1c = intestinal caecum v = ventricle ig = intestinal groove va = ventriculo-aortic valve in = intestine ve = vestibule is = ingestion/inhalant siphon vm = ventricular muscles vv = visceral vein 32 biotropia no. 6, 1992/1993 results species found in the collection sites are map-pinpointed in figure 1. representative photographs of the shells of the snails examined are presented in figures 2 & 3. figure 1. species noted in the collection sites. a-paoay, ilocos norte; b-tuguegarao, cagayan; c-san isidro, isabela; d-caraen, pangasinan; e-u.p. lagoon, quezon city; f-tanay & montalban, rizal; g-magallanes, sorsogon; h-pinamalayan, oriental mindoro; i-boac & sta. cruz, marinduque; j-alimodian, iloilo; k-palo, leyte; l-mainit, surigao del norte; m-lake sebu, marbel, & koronadal, south cotabato; n-lake manguiao, palawan. 4 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 2. representative shells of the snails identified as pita conica from the following sites: a-tuguegarao, cagayan; b-paoay lake, ilocos norte; c-san isidro, isabela; d-carmen, pangasinan; e-u.p. lagoon, quezon city; f-tanay, rizal; g-pinamalayan, oriental mindoro; h-gacao, palo, leyte, 1-hubang, palo, leyte; j-alimodian, iloilo; k-lake mainit, surigao del norte; l-lake sebu, so.cotabato; m-lamluad valley, so. cotabato; n-marbel, so. cotabato; o-koronadal, so. cotabato. bar = 5 mm. 5 figure 3. representative shells of the other species collected. a.-p. ampullacea: lake manguiao, palawan; 3-p. ampullacea: magallanes, sorsogon; c-p. mainitensis: gacao, palo, leyte; d-p. vittata ( = p. conica): sta. cruz, marinduque; e-p. quadrasi ( = p. conica): lamluad valley, so. cotabato. bar = 5 mm. anatomical studies 1. external morphology and description of the mantle cavity of p. conica the body of the animal is divisible into three regions (fig.4) head, foot, and visceral mass. the head is prolonged into a contractile snout. the elongated snout terminates anteriorly on its two sides into a pair of short labial pulps (lp) or tentacles. posteriorly the head bears a pair of long contractile tentacles (te). when fully extended the tentacles measure about 50 mm in length, thicker at its base but gradually tapering to a filamentous structure at the tip. when contracted, as when the animal is disturbed or as in the case of preserved specimens, the tentacles are reduced to about one-fourth of their length. the eyes (e) are situated on small stalks situated on the outer sides of the bases of the tentacles. projecting anteriorly on the two sides of the head are two lobes. the left lobe forms the inhalant siphon (is) and the right lobe forms the exhalant siphon (es). 6 biotropia no. 6, 1992/1993 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 4. external features of the body of p. conica. a. left view; b. right view female; c. right view male; d. view of the mantle cavity. bar = 3 mm. abbreviations in figures see appendix 2. the foot (f) is triangular in outline with the apex directed backwards. the sides are more or less arched in broadly rounded angles. the operculum is attached to the dorsal surface of the posterior half of the foot (fig. 5c:opl). the columellar muscle (fig. 5c) is seen as a broad band arising from its attachment to the columella and running longitudinally through the upper part of the foot to the operculiferous lobe (opl) on the postero-dorsal surface. 7 r . eigv agv ak co iso os dg biotropia no. 6, 1992/1993 figure 5. a. osphradium of p. conica. bar = 1 mm. b. gill leaflet of p. conica. bar = 1 mm. c. collumellar muscle of p. conica. arrows point to area of attachment to the shell. bar = 6 mm. abbreviations in figures see appendix 2. dorsally, the mantle encloses the mantle cavity in which the structures of the pallial complex are situated (fig. 4d). the mantle is thick along its free edges (me). as seen dorsally, pigmented streaks run longitudinally from this free edge eventually coalescing to form a more or less united pigmented patch covering the mantle roof. the pedunculate osphradium (fig. 4d: os & 5a) hangs from the roof of the left side of the mantle close to the edge. it is elongate-oval in shape, somewhat pointed along its left margin and rounded on the right inner end. it is bipectinate and consists of an average of 35 fleshy leaflets arranged along its broad axis. the osphradium hangs transversely to the course of the respiratory water current as it flows into the mantle cavity. posterior to the osphradium and occupying the greater part of the mantle cavity is the pulmonary or lung sac (fig. 4d: ls), arising from the ventral mantle wall as a more or less circular unpigmented folding with a ventral opening into the mantle cavity (lso). the tissue of the pulmonary sac is moderately thickened and is richly supplied with blood sinuses. 8 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio the gill or ctenidium (fig. 4d: g & 5b) is composed of approximately 170 triangular leaflets running alongside and to the right of the pulmonary sac. each triangular gill leaflet is attached along the ctenidial axis and has two free sides of unequal sizes, consequently, the slightly rounded apex is skewed to the right. the epitaenia (fig. 4d: ep) arises from the floor of the mantle cavity. it runs from the left side to the right, ending at the base of the left margin of the exhalant siphon, anteriorly. inside the right wall of the mantle, the rectum runs parallel to the gill on the right side opening to the anal papilla (fig. 4d: anp), and extended projection of the mantle roof with a fringed opening. to the right of the rectum running parallel to it, lies the pallial portion of the reproductive duct terminating into the genital papilla (gp). the genital papilla in males is a somewhat conical structure crossing the anal papilla ventrally. its tip, in which the genital opening is located, is in close contact, but not attached, to the base of the copulatory organ. in females, the pallial oviduct opens into a broad and short genital papilla located just beside the anal papilla. in egg-laying females, the genital opening is enlarged and the papilla is almost obliterated. in all snails examined, a copulatory structure (co) consisting of a penis enclosed in a penis sheath hangs from the right mantle roof. although present, the copulatory organ is degenerated in females. interspecific variation in the structures of the pallial complex: in p. ampullacea, an average of 260 gill leaflets were counted, and in p. mainitensis, an average of 245 leaflets. as to the number of osphradium leaflets, p. ampullacea has about 58 68 leaflets, and p. mainitensis has about 52 54 leaflets. no apparent difference in pigmentation of the head-foot region was observed between the species, although, in p. ampullacea and p. mainitensis, pigmentation of the penis sheath was observed. such pigmentation was not observed in the other species. the pallial complex of p. vittata and p. quadrase do not differ from that of p. conica. 2. the alimentary tract of p. conica the buccal mass which forms the first part of the alimentary tract (fig. 6a) is found inside the head sinus. the buccal cavity (fig. 8d: bmc) is subdivided into different regions. the anterior region is rather small, bounded anteriorly by the mouth opening (mo) and posteriorly by the jaws (j). the skin forming its walls are thickened, partly pigmented, and with longitudinal folds. from the opening of the jaws, the cavity continues as a very short tube, then flattens with its margins arched ventrad and posteriad following the countour of the subradular organ (so) and the more anterior portions of the radula. the cavity then widens a little on the level of the junction of the opening of the esophageal pouches. this region is 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 2.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 3.pdf 30.pdf 31.pdf 32.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no. 7, 1994: 41-46 chromosomal characters of the indonesian sand goby, oxyeleotrismarmorata blkr. 1874 (eleotridae)*) jimmy t. masagca catanduanes state colleges college of agriculture and fisheries, virac, catanduanes 4800, philippines and komar sumantadinata faculty of fisheries, bogor agricultural university, darmaga campus, bogor, indonesia abstract karyomorphological investigation of sand goby or marble sleeper (oxyeleotris marmorata blkr.) from cirata reservoir, west java, indonesia was undertaken to determine the modal chromosome number and fundamental number, and to construct the karyotype from somatic metaphase cells of head kidney. a total of 30 fish samples from cirata reservoir was sacrificed for direct chromosome preparation by colchicine-citrate-aceto-methanol-giemsa staining-air drying technique. chromosome set analysis showed that the modal chromosome number of the test fish is 2n = 46, confirming previous studies. fundamental number is 50. two karyotypic formulas were found, i.e. 1) 2n = 46 (4sm + 42a); and 2) 2n = 46 (2m + 2sm + 42a). key words: freshwater fishes, chromosome analysis, oxyeleotris marmorata, indonesia introduction sand goby or marble sleeper (o. marmorata blkr.) is an important freshwater food fish in most southeast asian countries notably in thailand, indonesia and singapore (tan & lam 1973; tavarutmaneegul & lin 1988). this eleotridine gobioid is the largest in the world (smith 1965) and found naturally in streams, rivers, lakes or reservoirs and old mining pools in southeast asia (weber & de beaufort 1953; noerdin & sidik 1979; mohsin & ambak 1983). in the philippines, this fish was collected in laguna de bay lake by meyer in 1885 but no other account has been made about its range in this country (herre 1927). o. marmorata (locally known in indonesia as "ikan betutu") has been the focus of significant research because of the increasing market demand as an export *) part of a long-term research fellowship work undertaken at seameo biotrop in 1991. 41 biotropia no. 7, 1994 commodity to singapore, hongkong and taiwan. it has been cultured in thailand (supamataya 1988), vietnam (pantulu 1976) and other countries in southeast asia since the 1970's because of its suitability for aquaculture. it has wide tolerance to ph (lie 1968) and can survive at low oxygen tensions. it has a high reproductive rate and is a multiple spawner (tavarutmaneegul & lin 1988). it was introduced in saguling reservoir of west java, indonesia in 1986 (munro et al. 1990) and has spread to the adjacent reservoir of cirata. the commercial value of sand goby is increasingly recognized in many parts of southeast asia, but very little is known about its basic genetics. extensive literature search only identified the studies of arai and fujiki (1979) and manna (1989) on the chromosomal complement of sand goby and both needed further investigation. these studies utilized the gill epithelial cells as the source of chromosomes, while the present study used the kidney. this study reports on the results of karyological investigation of sand goby obtained from a population in cirata reservoir, bogor, indonesia. specifically, this study provides information on the somatic chromosome number, fundamental number or number of chromosome arms and the karyotypic formula. materials and methods live samples of sand goby were obtained from cirata reservoir in cianjur, bogor, west java and were temporarily stored at the indoor tanks of seameo biotrop laboratories. the fish specimens were identified based on the taxonomic descriptions of mohsin & ambak (1983). the methodology of chromosome preparation followed that of rivlin et al. (1985) and reddy & john (1987), with very minor modifications. six hours prior to sacrifice, the test fish was intramuscularly injected with 0.05% colchicine in 0.9% nacl at a dose of 1 ml/100 g body weight. the chromosomes were prepared directly using head kidney tissue. hypotonization was done using 0.5% sodium citrate at a duration of 30 to 40 min. and at room temperature of 25.5 to 26.5°c. prepared slides were conventionally stained with 4% giemsa at a ph of 6.5. the different chromosomes were designated as proposed by levan et al. (1964). results from 303 metaphase kidney cells of 30 pre-adult sand goby samples collected from cirata reservoir it revealed that the modal chromosome number (mcn) of 42 chromosomal characters of the indonesian jimmy t. masagca and komar sumantadinata this eleotridine goby is 46. chromosome counts varied from 38 to 50. out of the 303 metaphase cells analyzed, 117 cells (38.61%) have the characteristic count of 2n = 46; 44 cells (14.52%) have 2n = 44; 36 cells (11.88%) have 2n = 43; 24 cells (7.92%) have 2n = 42; 14 cells (4.62%) have 2n = 41; 10 cells (3.30%) have 2n = 40 and 2 cells (0.66%) have each 2n = 39 and 2n = 38 (fig. 1). frequency distribution of diploid chromosome counts of marble sleeper collected from cirata reservoir eighteen (60%) out of the 30 fish samples analyzed showed a modal chromosome number (mcn) 2n = 46; 4 samples (13.3%) have mcn 2n = 44; 3 samples (10%) have 2n-42; and only 2 samples (6.66%) have each 2n = 41 and 2n = 40. karyotypes were made based on mitotic figures in which the chromosomes were clearly recognizable. the homologous chromosome pairs were identified based on the procedures of levan et al. (1964). the chromosome set of this fish mostly consists of 2 pairs of bi-armed and 21 pairs of mono-armed chromosomes. the karyotype showed that 2 pairs of chromosomes were sub-metacentric (sm), where the first pair was larger than the second one. the test of chromosome pairs showed all acrocentrics (a). in some cells the second pair seems to have a metacentric (m) chromosome, but this was not constant. another karyotypic formula was also found in this study having 2n = 46 (2m + 2sm + 42a). 43 figure 1. biotropia no. 7, 1994 discussion previous data on the diploid chromosome number of o. marmorata analyzed from fish samples obtained in thailand (arai & fujiki 1979) and from india (manna 1989) are the same with the present investigation. a diploid number of 2n = 46 and fundamental number (nf) of 50 confirm their previous findings. the diploid number of 46 recorded in sand goby has also been recorded in' another species of the same genus, o. acanthopomus (2n = 46) from japan (arai & sawada 1974). the same diploid number was also recorded in other gobioid species, dormitator maculatus from mexico (del carmen-maldonado et al. 1985), boleophtalmuspectinorostris and periophthalmus cantonensis (nogusa 1960, cited by denton 1973). however, d. maculatus has a fundamental number of 90, which is higher than o. marmorata having an nf = 50. in the gobiidae, most of the genera have the diploid chromosome number of 2n = 44 to 2n = 48 as shown in bathygobius fuscus (2n = 48), glossogobius giurus (2n = 46) and chaetogobius annularis (2n = 44). based on cytological examination variable counts of 42, 43, 44 and 46 were also known in this study. this could be due to the technical limitations. the suggestion that possible chromosonal re-arrangement by robertsonian translocations may have occurred was discounted since the missing chromosomes in the metaphase spreads are different from each other and cannot be considered as inherent property of the test fish. some metaphase spreads have a small metacentric chromosome, but it was not constant in the cells analyzed. the earlier work of arai & fujiki (1979) reported two formulas: 1) 2n = 46 (2m + 2sm + 42st,a) and 2) 2n = 46 (1m + 3sm + 42st,a). although a metacentric chromosome was seen in few spreads, majority of the 303 cells analyzed have 2 pairs of sub-metacentric chromosomes. this study reports a new karyotypic formula of sand goby as 2n = 46 (4sm + 42a). after careful examination of the chromosome set, it seems that no heteromorphic pairs could be referred to as sex chromosomes. acknowledgement the author is grateful for the long-term fellowship granted by seameo biotrop and the permission of the catanduanes state colleges in virac, catanduanes, philippines for allowing the author to travel to indonesia while on a pasuc scholarship at de la salle university. this study is a part of the masteral thesis submitted to dlsu graduate biology department in manila. profound thanks are due to dr. ruben c. umaly (the initial supervisor of this research), dr. soekotjo 44 chromosomal characters of the indonesian jimmy t. masagca and komar sumantadinata (seameo biotrop director) and dr. sutrisno sukimin (tab programme manager). special thanks are due to mr. a.b. djunaedi, mr. widodo, ms. wati and other staff of biotrop for the assistance provided. the assistance of mr. harton of ipb is greatly appreciated. references arai, r. and a. fujiki. 1979. chromosomes of japanese gobioid fishes (iv). bull. natl. sci. mus. ser. a, 5(2): 153-159. arai, r. and y. sawada. 1974. chromosomes of japanese gobioid fishes (i). bull. natl. sci. mus. tokyo, 17: 97-102. del carmen-maldonado, m., m. uribe-abrocer, j. arreouin-espinosa and a. castro-perez. 1985. karyotypical studies on dormitator maculatus bloch and gobiomorus dormitatur lacepede (gobioidei: perciformes). cytologia, 50(4): 663-667. denton, t.e. 1973. fish chromosomes methodology. charles c. thomas publisher, springfield, illinois. herre, a. 1927. gobies of the philippines. monog. 23 bur. sci. manila. levan, a., k. fredga and a. sandberg. 1964. nomenclature for centromeric position on chromosomes. hereditas, 52: 201. lie, s.f. 1968. a study of some biological aspects of o. marmorata found in singapore. part 2. dept. of zoology, univ. of singapore. 26 p. manna, o.k. 1989. fish cytogenetics related to taxonomy, evolution and monitoring genotoxic agent. p. 21-46. in: das and jhingran (eds). fish genetics in india. today and tomorrows printers and publishers, new delhi. mohsin, m.a.k. and m.a. ambak. 1983. freshwater fishes of peninsular malaysia. penerbit universiti pertanian, malaysia, kuala lumpur. munro, j., iskandar and b.a. costa pierce. 1990. fisheries of the saguling reservoir and a preliminary appraisal of management options, p: 285-328. in: b.a. costa pierce and o. soemarwoto (eds.). reservoir fisheries and aquaculture development for resettlement in indonesia. iclarm tech. rep. 23. noerdin, n. and a. sidik. 1979. survei ikan bakut (o. marmorata blkr.) di danau jempang dan semanikarya. dinas perikanan propinsi dati i kalimantan timur. (in indonesian). pantulu, v.r. 1976. floating cage culture of fish in the lower mekong basin, p. 416-423. in: t.v.r. pillay and w.a. dill (eds.). advances in aquaculture. fao tech. conf. on aquaculture. fishing news int. redpy, p.v.g.k. and g. john. 1987. a method to increase mitotic metaphase spreads in permanent chiomosome preparations for karyotype studies of fishes, p. 199-205. in: proceedings of world symposium on selection, hybridization and genetic engineering in aquaculture, berlin, 27 30 may 1986. rivlin, k., j.w. rachlin and g. dale. 1985. a simple method for the preparation of fish chromosomes applicable to fieldwork, teaching and banding. j. fish biol., 26: 267 -272. 45 biotropia no. 7, 1994 smith, h.m. 1965. the freshwater fishes of siam or thailand. tropical fish hobbyists publication, inc. usa. supamataya, m. 1988. the study of disease in sand goby (oxyeleotris marmoratus bleeker) in cage culture and some environmental factors related to infection. m.s. thesis. kasetsart university, thailand. tan, o. and t.j. lam. 1973. induced breeding and early development of the marble goby (oxyeleotris marmorata). aquaculture, 2: 411-423. tavarutmaneeoul, p. and c.k. lin. 1988. breeding and rearing of sand goby (oxyeleotris marmorata) fry. aquaculture, 69(3/4): 299 306. weber, m. and l.f. beaufort. 1953. the fishes of the indo-australian archipelago. vol. x. e. j. brill ltd., leiden. 46 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf biotropia no. 6, 1992/1993: 33-44 effects of the heavy metal, zinc, on the freshwater fish tilapia nilotica l. virginia s. caring institute of biology, university of the philippines, diliman, quezon city, philippines abstract gills, gonads, and blood of tilapia nilotica exposed to different concentrations of zinc sulfate (znso4. 7h2o) exhibited histological effects. gills of posthatch larvae exposed chronically for 21 days to 2 ppm zinc sulfate and fingerlings to 10 ppm sublethal zinc concentrations exhibited hyperplasia that resulted in fusion of adjacent secondary gill lamellae. the same effects were observed in 4-hour short-term exposure to 30 ppm lethal dose. posthatch larvae subjected to 2 and 5 ppm sublethal levels of zinc for 30 days retained undifferentiated gonads with differentiation with oogonial proliferation. ovaries of control fish demonstrated healthy oocyte growth and other normal histological features after 57 days. in contrast, ovaries in treated groups exhibited excessive amounts of connective tissue, hyperemia and markedly reduced oocyte number. oocytes had wavy irregular surface outlines. deviation from normal was observed to be dose dependent. in juvenile tilapia, spermatogenesis was observed in control testes. testes of zinc-exposed fish, on the other hand, remained immature. hyperemia was markedly pronounced in both testes and ovary after 90 days exposure to zinc. blood of tilapia nilotica fingerlings exposed to sublethal concentrations of 2, 5, and 10 ppm zinc for 30, 60, and 90 days exhibited anisocytosis and poikilocytosis. there was an increase in hematocrit values in zinc-reared fish which, however, reverted to control/near control levels at day 90. hemoglobin values were inversely proportional to the level of zinc in the rearing water. the marked reduction in hemoglobin values in fish reared at the higher zinc concentrations of 5 and 10 ppm suggests the development of some degree of anemia which is also supported by the observations of anisocytosis and poikilocytosis. introduction heavy metal contamination of aquatic environments has become a current serious problem because of increased industrialization. in the philippines, data gathered by the national environmental pollution commission (nepc 1980) on eleven river systems and of researches monitoring levels of heavy metals in fishes indicate high concentrations of these substances in aquatic bodies (nepc 1980; pcarrd 1982). compared to seas and oceans, fresh water environments are more vulnerable to pollution stress inasmuch as they are smaller systems and have more limited numbers and kinds of organisms. pollution of lakes and rivers, thus, pose alarming dangers to aquatic life, as for example to fish. one fresh water fish which is presently being studied intensively insofar as its response to heavy metals contamination, is tilapia nilotica l. economically 33 biotropia no. 6, 1992/1993 important as a poor man's protein source, it is easily available and is a hardy fish species. thus, it is expected that less sturdy fishes would exhibit any effect manifested by this particular species. most of the studies that will be reported in this paper deal with the effects of zinc on posthatch larvae or fry of tilapia nilotica. this stage of development, is a good investigative material since a number of organs, e.g., the gonads have not completed full development and differentiation. the earlier developmental stages are interesting subjects, but in t. nilotica they are not too exposed to the environment inasmuch as they brood in the tilapia mother's mouth and are hence semi-protected. materials and methods preliminary 96 hr ld 50 determination was performed to obtain the approximate sublethal levels of zinc on the t. nilotica stage that would be tested. during the four days period the fish were kept in aerated water with different concentrations of znso4 and were starved to minimize the elimination of the metal together with waste products. dechlorinated tap water was used in all tests. hardness, alkalinity, and ph readings were taken twice a week on the test water in the aquarium. hardness and alkalinity were determined by titration method (apha 1971). temperature was monitored daily. zinc sulphate (znso4. 7h2o, ar merck ® ) was dissolved in deionized water and acidified to ph 2 with nitric acid (hno3). the plastic jars with 3.0 1 test water were spiked with zinc solution to bring the concentration to the desired level. posthatch fry of t. nilotica were obtained from a single source and acclimated in well aerated dechlorinated tap water for 7 days prior to experimentation. chronic as well as acute exposures to zinc were done at different time exposures. the organs to be reported on are the gills, gonads, and blood. for light microscopy, tissues were dehydrated in a series of graded ethyl alcohol, cleared in cedarwood oil and xylene, infiltrated and embedded in paraffin, and cut in ao ® rotary microtome. paraffin sections of 7 urn thickness were stained with hematoxylin-eosin and mounted in entellan ® mounting medium. resin sections were also done. tissues dissected were fixed in 2.5% glutaraldehyde in phosphate buffer, ph 7.2, followed by post-fixation in 1% osmium tetroxide. the samples were dehydrated in ethanol and propylene oxide, embedded in araldite ® , sectioned 1 2 urn thick with carl zeiss ® ultratome, stained with 1 % toluidine blue and examined under the light microscope. ultrathin sections were stained with uranyl acetate and lead citrate for electron microscopy. 34 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. cariffo for blood parameters, fingerlings were reared in zinc concentrations of 2, 5, and 10 ppm for 30, 60, and 90 days (cariño and casauay 1991). blood samples were collected by gill puncture using heparinized capillary tubes and analyzed for haematocrit and haemoglobin content using capillary method (hesser 1960) and als biochemical haemoglobin procedure ® , usa, respectively. blood smears were made and stained with giemsa for erythrocyte morphology. results and discussion effects on gills in tilapia fingerlings exposed to 10 ppm and 20 ppm zinc sulfate for 21 days, the main effect was hyperplasia of the primary gill epithelia resulting in fusion of adjacent secondary lamellae (cariño & puzon 1986). hyperplasia was noted in all treatment groups, both short-term exposure (4 hours) to 30 ppm lethal concentration of zinc and long-term treatment (21 days) to 20 ppm chronic and 10 ppm sublethal levels of the metal (figs. 1 & 2). extensive cell proliferation was noted along the entire length of the gill filaments of fish exposed to the highest zinc concentration figure 1. longitudinal section of control gill filament. cc, chloride cell; sec, secondary epithelial cell; sl, secondary lamellae; pc, pillar cell; bc, blood cell; gl, gill ray. x960. 35 figure 2. longitudinal section of gill filament from 10-ppm exposed fish showing fusion of adjacent secondary gill lamellae. cc, chloride cell; or, gill ray. x480. used, 30 ppm. the overall effect of the zinc salt on the gill structure is the decrease in surface area which would otherwise make for an efficient transport of oxygen in the blood. other investigations showed that chemically induced hyperplasia of the primary gill epithelia is generally due to the increase in the number of chloride cells (ahuja 1970; crespo 1981; tuurala & soivio 1982; crespo 1984). zinc was particularly shown to induce proliferation of the chloride cells in the dogfish synliorhinus canicula (crespo 1981). increase in chloride cells have likewise been noted in the study of cariño & puzon (1986) with t. nilotica fingerlings. this indicates to invigorate osmoregulatory capacities. chloride cells are large salt secretory cells (karnaky et al. 1977) found at the base of the secondary gill filament and believed to be responsible for osmoregulation (karnaky 1980; laurent & dunel 1980). they have been shown to excrete univalent ions, though they might also be able to excrete bivalent ions (ahuja 1970). na-k-atpase is the major enzyme found in chloride cells. thickening or hypertrophy of the secondary gill epithelia was a less observable effect of zinc exposure in t. nilotica fingerlings. this was observed with 20 ppm treatment (figs. 3 & 4). 36 biotropia no. 6, 1992/1993 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino figure 3. longitudinal section of gill filament from 20-ppm exposed fish showing fusion (f) of secondary gill lamellae and hypertropy (arrow) of secondary epithelial cell. cc, chloride cell; sec, secondary epithelial cell; gr, gill ray. x480. figure 4. longitudinal section of gill filament from 20-ppm exposed fish showing hyperplasia of chloride cell and fusion of secondary gill lamellae. cc, chloride cell; pc, pillar cell; bc, blood cell; gr, gill ray. x960. 37 biotropia no. 6, 1992/1993 hypertrophy of secondary gill epithelia has been reported as a response of fish to industrial pollutants. hughes et al. (1979) noted that there was increase in the volume of secondary epithelia in nickel exposed salmo gairdneri. chromium has similar effect on the secondary epithelia of this species (putte et al. 1981). tuurala and soivio (1982) observed that excessive hypertrophy of the secondary epithelial cells was induced by sublethal exposure of s. gairdneri to dihydroabietic acid, a major kraft pulping effluent (leach & thakore 1973). similar results have been obtained with exposure of chanos chanos fingerlings to ammonia (cruz & enriquez 1982). secondary lamellar hypertrophy increases the diffusing distance from the surrounding water to capillaries, but decreases the respiratory dead volume between the secondary lamellae and, thus, would compensate for the increase in the diffusion distance (tuurala & soivio 1982). histological observations indicate that zinc impairs gaseous exchange in fishes by increasing diffusion distance and decreasing absorption surface area. electron microscopic structure of the secondary lamella of control fish is shown in fig. 5. the lamellar blood sinus is large and circular in outline. the pillar cell and its nucleus is very distinct. fish exposed to 2 ppm zinc for 21 days were observed to have lamellar blood sinus dilation and epithelial lifting of the secondary gill lamella (fig. 6). detachment of pillar cell from the epithelium occurred at 20 ppm zinc exposure. epithelial lifting results in the enlargement of the non-tissue spaces. 38 figure 5. electron micrograph of control secondary gill lamellae. x5000. figure 6. electron micrograph of gill lamellae from 2-ppm exposed fish showing dilated blood sinus epithelial lifting. x5000. effect on the gonads gonads of 30-day posthatch control fry exhibited gonial multiplication (cariño & cruz 1990). no distinction can yet be made as to whether the gonad is testis or ovary. in contrast, all experimental fish reared for 30 days in three zinc concentrations had gonads that contained only primordial germ cells. gonial multiplication was delayed. in fish harvested after 57 days in culture, oocyte growth is observed in both control and zinc-treated fish (figs. 7a, b). a notable difference is the presence of promiscuous and dilated blood vessels densely filled with blood cells in the ovaries of zinc-treated fish. oocytes of zinc-treated fish have more nucleoli than control fish. a number of these nucleoli-studded nuclei bear on one side a quarter moon-shaped region of intensely darkly stained material which is most probably extruded rna. accumulation of maternal rna is characteristic of most growing oocytes. graded distinctions between ovaries treated with 2, 5, and 10 ppm appear inconspicuous. tests of 57-day reared control and zinc-treated fish showed spermatogonial multiplication. as noted by casauay & carifio (1988), spermatogenesis in tilapia nilotica commences later than oogenesis. testes of juvenile t. nilotica showed spermatogenic stages. in contrast, zinc-treated fish showed delay in spermatogenesis (figs. 8a, b). 39 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino biotropia no. 6, 1992/1993 figure 7. oocyte growth from 57-day cultured fish (a) control ovary, (b) zinc treated fish ovary showing abundant blood cells. x5000. 40 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. cariffo figure 8. spermatogenic stages in control juvenile t. nilotica; b. delay in spermatogenesis in zinc treated fish. x400. 41 figure 9. blood smear from a contol tilapia nilotica; b. blood smear showing "anisocyclosis", a term applied when the rbcs vary in size (arrow) after exposure to 2, 5, and 10 ppm zinc concentration, c. "poikilocytosis" exhibited by abnormal variation in shape of rbc after 2, 5, and 10 ppm exposure to zinc. xi000. 42 biotropia no. 6, 1992/1993 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino effects on the blood anisocytosis and poikilocytosis were noted in the blood smears of zinc-treated fish (9a, b, c). in all zinc treated fish, an increase in hematocrit (%) was observed at 30 days. with the exception of fish reared at 10 ppm zinc, hematocrit values dropped to control/near control levels after 90 days. haemoglobin (g/1) content in fish reared in zinc-treated water for 90 days was inversely related to zinc concentration. exposure of tilapia nilotica fingerlings for 90 days to zinc concentrations of 5 and 10 ppm induced anemia reducing haemoglobin content of erythrocytes and not by reduced number of erythrocytes. references ahuja s.k. 1970. chloride cell and mucus-cell responses to chloride and sulphate enriched media in the gills of gambusia affinis (baird and girard) and cat/a catla (hamilton). j. exp. zool. 173: 231-250. american public health association, american water works association, and water pollution control federation 1971. standard methods for the examination of water and wastewater. 13th ed., american public health association, ny, usa. cariño, virginia s. and armando g. puzon, jr. 1986. toxic effects of zinc sulphate to tilapia nilotica l. fingerlings. natural and applied science bulletin 38(2):127134. cariño, virginia s. and arsenia a. casauay. 1991. the haemocytology of tilapia nilotica and blood studies on tilapia nilotica fingerlings after sublethal exposure to zinc. science diliman 25(1):6 22. cariño, v.s. and n.c. cruz. 1990. effects of low levels of zinc on the ovarian development of tilapia nilotica l. sci. diliman 3:34-45 casauay, a.a. and carino, v.s. 1988. gonadal sex differentiation in oerochromis niloticus. in: r.s.v. pullin, t. bhukasawan, k. tonguthai, and j.l. maclean (eds.). the second international symposium on tilapia in aquaculture. iclarm conference proceedings, department of fisheries, bangkok, thailand and international center for living aquatic resources management, manila, philippines: 121 124. crespo, s. 1982. surface morphology of dogfish (scyliorhinus canicula) gill epithelium, and surface morphological following treatment with zinc sulphate: as canning electron microscope study. mar. biol. 67:159-166. crespo, s. 1984. an in vitro study of the effects of zinc on the osmoregulatory processes. mar. pollut. bull. 15:341-342. cruz, e.r. and g.l. enriquez. 1982. gill lesions associated with acute exposure to ammonia. nat. appl. sci. bull. 34:1-13. hesser, e.f. 1960. methods for routine fish hematology. prog. fish cult. 22(4):164171. hughes, g.m., s.g. perry, and v.m. brown. 1979. a morphometric study of effects of nickel, chromium, and cadmium on the secondary lamellae of rainbow trout gills. water res. 13:665 679. 43 biotropia no. 6, 1992/1993 karnaky jr. k.j., j. karl, jr., and w.b. kinter. 1977. killifish opercular skin. a flat epithelium with a high density of chloride cells. j. exp. zool. 199:355-364. leach, j.m. and a.n. thakore. 1973. identification of the constituents of kraft pulping effluent that are toxic to juvenile coho salmon (onchorynchus kisutch). j. fish res. board can. 30:479-484. national environmental protection council. 1980. the philippine environment. manila: 52-56. philippine council for agriculture and resources research and development. 1982. state of the art environmental protection research: 1 — 11. putte, i.v.d., m.a. brinkhorst, and j.h. koeman. 1981. effecf-of ph on the acute toxicity of hexavalent chromium to rainbow trout (salmo gairneri). aquat. toxicol. 1:129— 142. tuurala, h. and a. soivio. 1982. structural and circulatory changes in the secondary lamellae of salmo gairdneri after sublethal exposures to dehydroabietie acid and zinc. aquat. toxicol. 2:21 29. 44 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf biotropia vol. 29 no. 1, 2022: 39 46 doi: 10.11598/btb.2022.29.1.1615 39 mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran margareta rahayuningsih*, yanuar revandi and siti harnina bintari department of biology, faculty of mathematics and natural sciences, universitas negeri semarang, semarang 50229, indonesia received 10 july 2021 / accepted 17 october 2021 abstract wreathed hornbill (rhyticeros undulates) is known to build nests in three cavities where they managed to live and breed. this edifice is predicted to contain various molds needed to maintain micro-environmental steadiness. this study was aimed to identify molds diversity in the wreathed hornbill’s nest, using samples collected from empty structure with no bird activity. the samples were obtained from the kalisidi and nglimut observation stations on two occasions, i.e., in 2016 and 2017. furthermore, the samples comprised cover soil, wood and inner material, which were collected aseptically and placed in sterile ziplock plastic bags. these samples were then diluted in sterilized distilled water to attain 10-3 mg/ml, and subsequently inoculated on potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). the inoculants were incubated at 37 °c, followed by the observation of mold colony after the 11th day. the results identified seven and nine species of molds in the kalisidi and nglimut observation stations, respectively. the most abundant species was penicillium sp. which was found in composted nest materials for the whole observation periods. keywords: mold diversity, mount ungaran, nest, rhyticeros undulates, wreathed hornbill introduction wreathed hornbill or rhyticeros undulauts shaw 1811 is a protected bird, which is categorized into apendix ii, according to the convention on international trade of endangered spesies of wild fauna and flora (cites). appendix ii lists "all species which although not necessarily now threatened with extinction may become so, unless trade in specimens of such species is subject to strict regulation". this is an indication that the species is tradable under certain conditions, e.g., for scientific research purposes (rahayuningsih & kartijono 2013). in addition, breeding and nesting occur during fruiting season. the wreathed hornbill selects nest location based on the availability of fruiting trees and a conducive environment (rahman et al. 2019). the nest selection process is followed by the nest building process, which involves the use of existing cavities from other birds and cracked branches (rahayuningsih et al. 2017). generally, the nest selection and building process are initiated in dry season, characterized by low humidity, which is suitable for breeding and protecting the eggs from parasites. (supaamornkul et al. 2011). however, the r. undulatus in khao yai, thailand is capable of completing the breeding process before the heavy rains. the created nests provide a suitable physical environment, alongside microorganisms needed for the development of eggs and chicks (rahayuningsih et al. 2017; utoyo et al. 2017). in addition, the strength of the physical structure is ensured by building the inner and outer parts of the nest using different soil sources, while the outer cover comprises high mircroorganism diversity, including mold. there have been minimal studies on mold diversity in r. undulatus nests present on mount ungaran, although supa-amornkul et al. (2011) reported the importance of micro-fungus in breaking down wood and organic materials. this phenomenon is exploited in nest cavity expansion; hence the study aimed to determine the diversity of molds in r. undulatus nests on mount ungaran. *corresponding author, email: etak_sigid@mail.unnes.ac.id biotropia vol. 29 no. 1, 2022 40 materials and methods this research was an observational exploratory study on the diversity of mold present in the nest of r. undulatus. the identification process was conducted in the microbiology laboratory, department of biology, universitas negeri semarang (unnes). samples were obtained from the kalisidi observation station located in the kalisidi village, west ungaran district, semarang, and nglimut observation station located in nglimut village, gonoharjo district, kendal district, central java. the first observation period was conducted in 2016, and the last observation period was conducted in 2017. samples were taken at each observation period. nest sampling the nest sampling process was performed at the end of the nesting season, marked by the absence of female and juvenile birds. samples were collected aseptically, using sterile pinset and spatula, and then placed in ziplock plastic bags. the collected samples included nest cover, internal wood and nest materials of each nest in the two observation periods. all of the prepared samples were then stored in a freezer at -20 °c, prior to further analysis. three samples were collected at each observation period. thus, our study collected six samples altogether for the two observation period. each of the six samples were mashed up aseptically, followed by taking 10 g of each sample and dissolving the 10 g of each sample by using 100 ml of distilled water. this mixture was then homogenized and kept as a sample solution, from which 10% was diluted to attain 10-3. subsequently, the inoculation step was performed by spreading 1 ml of the sample solution onto culture media, i.e., potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). the isolates were then incubated at room temperature for 3-7 days in an incubator. a microscope was used for identification purposes, followed by the observation of wide mold colony growth. sterilization and medium preparation the culture media used were potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). each of the culture media was measured for 250 ml and dissolved with distilled water. as much as 0.05 g of chloramphenicol was added into the culture media as antibacterial agent, followed by sterilization using an autoclave at 121 °c and 2 atm, for 15 min. subsequently, the culture media were poured into different petri dishes under aseptic conditions, stored inside the biological safety cabinet (bsc), and allowed to solidify, before being placed in a media cooler. calculating and identification of mold colony the grown mold colonies were stained using lactophenol, prior to observation with a microscope at magnifications of 100x 1,000x. subsequently, identification was carried out based on the guidelines of mold morphological structure developed by samson et al. (2019) and robinson (2011) to determine the genus and species of the samples. results and discussion environmental conditions of the kalisidi and nglimut observation stations are presented in table 1. table 1 enviromental condition of the observation stations enviromental conditions kalisidi station nglimut station light intensity (c) 613-1,740 656-1,480 soil ph 5.2-6.2 3.7-4.5 soil humidity (%) 25-55 70-90 air humidity (%) 71-75 75 air temperature °c 29.4-29.9 28.9 tree type dead tree (unidentified) weinmannia fraxenia (nuts) mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 41 despite the similarities, there was a variation in light intensity, with nglimut station amounting 656 c to 1,480 c, due to the presence of tighter and thicker canopies. high and low light intensities impacted the air temperature around the nest, which was relatively higher in kalisidi station. kalisidi station featured higher soil ph and lesser soil humidity (table 1). after reaching a diameter of 1.5 cm, the mold colonies were transferred into the subsequent culture medium, followed by staining and observation by a microscope for identification purpose. mold colonies having a diameter less than 1 cm were re-incubated to ensure further growth for easier identification. table 2 shows the relatively higher density of samples obtained in 2017, compared to those obtained in 2016. within the incubated conditions, the mea culture medium was colonized by various molds, where four and seven species were observed in samples obtained in 2016 and 2017, respectively. on the other hand, two and seven mold species grew in the pda culture medium, while two and four mold species were recognized in cda culture medium. aspergillus was successfully identified as the only genera present in all medium, across both sampling years. furthermore, aspergillus niger occurred less frequently than a. terreus, despite the fact that a. niger is commonly found in general environment. meanwhile, acremonium sp. and p. variabile were both identified in the mea medium of 2016, while rhizopus sp. was only seen in the cda medium of 2017. based on the collection period, there was a possibility that the mold species abundance was affected by the growth medium applied (mahadevan & shanmugasundaram 2018). these mold species generally grow better in the mea culture medium, made from wheat extract, maltosa, long chain carbohydrate and glycerol. the next culture medium preferred by mold to grow is pda and followed by cda. in addition, the composition of mea provides sugar and nutrients as a source of energy for molds and yeast (aziz et al. 2018; cvetkovic & markov 2002). the pepton content in mea functions as a nitrogen source, presents in higher amounts compared to other media. pepton is important for amino acid synthesis, which is required in the production of various functional protein, cell structure and hyphae conformation (wang et al. 2010) and is responsible for the relatively higher amount of essential amino acid in mea, including triptophan and tyrosin. these are important components in the conformation of metabolic protein, and also during cell communication. pda media includes the semi synthetic variety, resulting from the natural and synthetic material (dextrose and agar) components. in addition, complex carbohydrate molecules have been identified as the main source of carbon in potato. carbon is the base material for pda production, needed for the growth of molds and yeast. pda is used because of the vitamin and mineral contents required to support fungi growth. also, the dextrose component in pda provides additional sugar as an energy source. table 2 types of mold grown in the three culture media species 2016 2017 pda mea cda pda mea cda aspergillus sp. √ √ √ √ √ √ aspergillus niger √ aspergillus terreus √ √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ geotrichum sp. √ √ neosartorya fischeri √ √ penicillium sp. √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. √ √ total spesies 2 4 2 7 7 4 notes: pda = potato dextrose agar; mea = malt extract agar; cda = czapek dox agar; 2016 and 2017 = sampling years. biotropia vol. 29 no. 1, 2022 42 on the other hand, cda medium consists of various nutrient molecules, of which sucrose has been identified as the main energy source, while nitrogen is obtained from the natrium nitrate component. furthermore, other constituents, including dipotassium phosphate is known to serve as a buffer solution, while magnesium sulfate and iron sulfate are essential ions. however, both mea and pda used in this study are manufactured medium, ready to use, while cda was created using a formula according to the manufacture’s protocol (himedia laboratories, mumbai), hence the tendency for unclear and unstandardized composition accuracies and capabilities. the molds obtained in 2016 at the kalisidi station were successfully identified, and three species were observed in the first collection (p1), with two in the second collection (p2). two species were accumulated in p1, and four species in p2 for samples collected in 2017. however, the nglimut station portrayed a relatively higher diversity (table 3). aspergillus sp. was the only species present five times in the r. undulatus nest, i.e., two times in kalisidi station and three times in nglimut station, while penicillium sp. were observed two times in each station. in addition, a. terreus was identified on three instances, i.e., one and two times for the respective stations. on the other hand, curvularia sp., fusarium sp., geotrichum sp., n. fischeri and trichoderma sp. were only identified twice in both stations, while a. niger, acremonium sp., p. variabile, and rhizopus sp. were rarely present (table 4). table 3 types of molds in the cover wreathed hornbill (rhyticeros undulatus) obtained in the 2016 and 2017 sampling period species kalisidi station nglimut station 2016 2017 2016 2017 p1 p2 p1 p2 p1 p2 p1 p2 aspergillus sp. √ √ √ √ √ aspergillus niger √ aspergillus terreus √ √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ geotrichum sp. √ √ neosartorya fischeri √ √ penicillium sp. √ √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. total spesies 3 2 √ 2 √ 4 3 5 7 notes: p1 = first collection; p2 = second collection. table 4 types of molds in r. undulatus nest during sampling in 2017 types of molds kalisidi station nglimut station p1 p2 p1 p2 cn wm cm cn wm cm cn wm cm cn wm cm aspergillus sp. √ √ √ √ √ √ aspergillus niger √ √ aspergillus terreus √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ √ √ √ geotrichum sp. √ √ neosartorya fischeri √ √ √ penicillium sp. √ √ √ √ √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. √ √ √ total spesies 1 1 1 3 2 4 3 5 5 3 6 notes: p1 = first collection; p2 = second collection; cn = cover nest; wm = wood material; cm = compost material. mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 43 samples obtained from the nests were divided into three parts, i.e., cover, internal compost and wood material. the most abundant molds identified at the kalisidi station included geotrichum sp., n. fischeri and trichoderma sp. the trichoderma sp. was observed in the first and second collection periods, while the wood material containing n. fischeri and p. variabile was observed in the second collection period. this result indicated that n. fischeri is the most widespread mold species. the high diversity shown in nglimut station included four mold species in the nest cover, with three in the wood material and four species from the compost. in addition, aspergilus sp. and penicillium sp. were identified as the most common molds in all parts, during both collection periods, while a. niger was only found in the nest cover, with a. terreus in the nest inner material for both collection periods. figure 1 molds types identified in r. undulatus nest notes: a = aspergillus sp.; b = acremonium sp.; c = curvularia sp.; d = fusarium sp.; e = geotrichum sp.; f = neosartorya fischeri; g = penicillium sp.; h = rhizopus sp.; i = trichoderma sp. (b = branch; f = phialid; k = conidiophore; kon = conidia; m = metula; mc = microconidia; s = sporangiophore; spo = spora; v = vesicle). microscope magnification: 1,000x. biotropia vol. 29 no. 1, 2022 44 the morphologycal identification process was based on two main characteristics, including: 1) the colony formation and color and 2) the morphological structure. based on these two main characteristics, the molds were identified into genus and species (table 5). table 5 description of molds types identified in the nest of r. undulatus mold description aspergillus sp. the fruit body consisted of aspergillus formed conidiophores (non-septate), vesicles, metula, phialid, stolone (vegetative hyphae) and conidia. the identified aspergillus sp. possessed radiateand biseriate-conidial heads (sideways/ deviate), with phialid organs that grow in the metula, as seen in figure 1.a. hence, molds with similar criteria were designated as aspergillus (diba et al. 2007) acremonium sp. the typical organs present included a cluster of aerial hyphae, conidiophores, phialids and ellipse extended conidia. furthermore, the phialid grew directly on aerial hyphae, and was tapered in the form of a needle, while the conidiophores were single-celled, erected and unbranched condia (fig. 1.b) (samson et al. 1984). the microscopic size range of these components were 17.5-37.5 (-50.4) × 3.2-4 µm, and 6-8.5 (-9.3) × 2.1-2.8 (-4) µm, respectively (gräfenhan et al. 2011; hill et al. 1990). curvularia sp. the conidiophores were branched, brown, and tightly arranged in groups. in addition, the conidia were elliptical with 3-4 bulkheads in each, with brownish white coloration, and comprising of 4-5 cells. the colonies were dark black in color and round, with cotton texture (fig. 1.c). this was in accordance with the description by kusai (2015) and hosokawa (2003), except with the addition of velvet pigmentation. the conidiophores appeared singly or in groups, simple or branched, straight or crouched, with pale brown or young cones, while the conidia had 3-4 septa. the specimen was thin-walled, measuring 20-30 x 9-15 µm (hosokawa et al. 2003; kusai et al. 2016). fusarium sp. the fusarium genera was identified using the method by (bashyal et al. 2016; gräfenhan et al. 2011). the microconidia appeared as fusiform and ovoid form, with 0-1 septate, while the conidiophorous structure present was insulated. in addition, phialid and macroconidia were not seen under microscopic observations, although microconidia were recognized (fig. 1.d). geotrichum sp. conidia were cylindrical, oval, and tubular (barrel) in shape, with green-blue coloration, and also a chain-like and clustered arrangement. in addition, the upper part of this mold was formed from broken fertile hyphae (fig. 1.e), with conidia diameter of 3.7-4.8-12.5 (-13.8) x (1.7-) 2.4-5 µm, and no conidiophores. also, the fertile hyphae present was branched off dichotomous and insulate. neosartorya fischeri the morphological structure of neosartorya fischeri was similar with aspergillus, characterized by vesicles, phialid and conidia, with seemingly insulated hyphen, alongside blue densely arranged conidiophores and hyphae. in addition, the vesicles were slightly elongated in shape, with conidial columnar head, which was also uniseriate (direct phialid growth in vesicles) (fig. 1.f). the colony was white in color, with cotton-like texture, and ± 0.2-2 cm in diameter. the conidia of neosartorya fischeri was round in shape, half round and elliptical, with slightly coarse wall, at ± 2-3 µm diameter, while the conidiophores ranged from 300-500 µm, with characteristic smooth walls (udagawa et al. 1996). penicillium sp. the morphological structures possessed insulated vegetative hyphae, alongside conidiophores, branches, metula, phialid and conidia. the conidiophores were of the two-stage branched (biverticillate-asymmetrical) type, while the conidia appeared round (fig. 1.g). in addition, the colonies were grayish and light green-old, with ± 0.2–2 cm diameter. also, penicillium is included as a deuteromycota, characterized by fast growing colonies, which is green in appearance and sometimes white. the conidiophores had several branching pattern forms, including one to three-stages and more-stage branched (visagie et al. 2014). rhizopus sp. the morphological structure had sporangiofor, sporangium, featuring the release of spores (sporangiospor). in addition, the mold contains the non-septate stolone (vegetative hyphae), alongside the rhizoid, although only the columella covered by sporangium. the sporangiofor stands tall, with a round shape (fig. 1.h) (hartanti et al. 2015). trichoderma sp. morphological structures are similar with trichoderma, featuring vegetative hyphae (aerial hypha), conidiophores with side branches, slim and elongated phialids, and also round conidia, with white and dark green colonies (fig. 1.i). according to gusnawaty et al. (2014), trichoderma sp. has branched conidiophores resembling pyramids, with more to the end, and the branching becomes shorter. also, the conidia are smooth walled and semi-round to oval in shape. this species have green colonies that were initially white (supa-amornkul et al. 2011). mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 45 the nest cover collected in this study consisted of soil and wood. molds identified from the nest cover were of various species, although the more abundant molds were observed in the organic material of the nest’s inner part. this was possibly caused by composted organic materials, including feces, fermented fruit, e.g., ficus, insects and decayed wood. particularly, the ficus fruit or fig (ficus carica) contains 8.98% protein, 6.57% fat, 10.26% moisture content, 18.23% ash content, 20.31% crude fiber, 0.0395% calcium, 0.002% phosphor, 25.48 mg/100 g and 1.64 mg/100 g of vitamin c and e, respectively. in addition, fig also contains various minerals needed by r. undulates, including n, p, k, ca, mg and others (mendoza-castillo 2019). aside from fruits, the fecal matter was high in n for mold protein synthesis, while the soil and wood were characterized by water, fat, carbohydrate and protein. this results were confirmed with the results of previous studies on the nest of r. undulatus containing 53.30 mg/ml of water, 39.02 mg/ml of fat, 35.03 mg/ml of carbohydrates, 5.82 mg/ml of ash and 20.12 mg/ml of protein. in addition, the humidity and the warm and dark conditions of the inner part of the nest form an appropriate and suitable environment for mold growth. conclusion mold species obtained from r. undulatus nests consisting of cover, composted material and wood material in kalisidi and nglimut stations during the sampling in 2016 comprised 6 mold species including aspergillus niger, aspergillus terreus, aspergillus sp., penicillium sp., penicillium variabile and acremonium sp. on the other hand, 9 mold species were reported during the sampling in 2017, including aspergillus terreus, aspergillus sp., curvularia sp., fusarium sp., penicillium sp., rhizopus sp., geotrichum sp., trichoderma sp., and neosartorya fischeri. acknowledgment special thanks are dedicated to the directorate of research and community service, deputy for strengthening research and development, ministry of research and technology of the republic of indonesia/national research and innovation agency for providing research funding scheme penelitian dasar unggulan perguruan tinggi 2018. special gratitudes are also presented to my field research team who provided assistance in data collection and sample analysis. references aziz nha, yousef ns, el-hadded me, el-tayeb ts. 2018. influence of nutritional and climatic conditions on mycelial growth of three oyster mushroom strains. arab univ j agric sci 26: 1165-73. bashyal bm, aggarwal r, sharma s, gupta s, rawat k, singh d, singh 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nesting cycle and nest tree characteristics of the helmeted hornbill rhinoplax vigil, compared to the wreathed hornbill rhyticeros undulatus, in sumatran lowland rainforest. kukila 20:12-22. visagie cm, hirooka y, tanney jb, whitfield e, mwange k, meijer m, …, samson ra. 2014. aspergillus, penicillium and talaromyces isolated from house dust samples collected around the world. stud mycol 78:63-139. wang h, ma f, cheng l. 2010. metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of “honeycrisp” apple (malus domestica borkh) with excessive accumulation of carbohydrates. planta 232:511-22. biotropia vol. 28 no. 2, 2021: 92 101 doi: 10.11598/btb.2021.28.2.1078 92 growth of black soldier fly larvae (hermetia illucens) fed with pak choi (brassica chinensis) and carp (cyprinus carpio) residues agus dana permana1, ramadhani eka putra1,2*, auliya nurulfah2, mia rosmiati1, ida kinasih3, and dian anggria sari2 1school of life sciences and technology, institut teknologi bandung, bandung 40132, indonesia 2department of biology, institut teknologi sumatera, lampung, indonesia 3departement of biology, universitas islam negeri sunan gunung djati bandung, bandung 40614, indonesia received 8 june 2018/accepted 12 february 2020 abstract one main drawback of the local animal industry is the inavailability of affordable and sustainable protein supply for the livestock. insect larvae, such as the black soldier fly (hermetia illucens) larvae (bsfl), have been considered as a protein source which can be produced at a large scale using low cost organic wastes as feeding material. this study was designed to determine the response of bsfl to various waste combinations of vegetable and animal remains, pak choi (brassica chinensis) residues (s) and carp (cyprinus caprio) fish offal (i)). a total of 540 bsfl were fed with 100 mg/larvae/day combination of vegetable wastes: animal waste 70%: 30% (s > i), 50%: 50% (s = i), and 30%: 70% (s < i). among the feed combinations, the s < i group showed the best results as it produced the significantly highest weight of bsfl at 122.8 mg/larvae and approximate digestibility of 62.01%, with the least pupae mortality rate at 4.29%. keywords: bioconversion, biomass, brassica chinensis, cyprinus carpio, hermetia illucens introduction as the communities develop and increase in size, the amount of waste generated by the human population also increases. in the year 2000, about 49% of the total world population lived in cities and generated more than three million metric tons of daily waste (e.g., household items, food waste, packaging, ash) and this number is predicted to double in 2025 (hoornweg et al. 2013). in 2007, the amount of food waste generated worldwide as the result of economic activities, from production to consumption, was estimated at 1.6 gtonnes (fao 2013). these wastes are taking up space in landfills, as the most common waste management practice, thereby contributing to the spread of pathogens, production of noxious odors, and a significant amount of co2 (zhang et al. 2019). for several decades, researchers worldwide have developed a method to process organic matter away from landfills using biotic decomposer such as black soldier fly larvae (bsfl), earthworm, house fly, and mealworm (beard & sands 1973; el boushy 1991; ndegwa & thompson 2001; ramos-elorduy et al. 2002; elissen et al. 2006; diener et al. 2009), in which bsfl is considered as the best candidate. the black soldier fly is of neotropic origin and now spread in all warmer regions through natural and human-mediated dispersal (callan 1974; marshall et al. 2015). this species can colonize a wide range of organic wastes, including agricultural wastes (manurung et al. 2016; supriyatna et al. 2016), animal and human remains (tomberin et al. 2005; pujol-liz et al. 2008), fish offal (st-hilaire et al. 2007a), food waste (diener et al. 2011; nguyen et al. 2013; *corresponding author: ramadhani@sith.itb.ac.id growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 93 oonincx et al. 2015a), as well as human and livestock feces (myers et al. 2008; banks et al. 2014; oonincx et al. 2015b). due to its biological characteristic and being easily mass-produced (sheppard et al. 2002), this species has been studied to recycle the nutrients found in organic wastes to be converted into protein-rich and fatrich biomass (sheppard et al. 1994; diener et al. 2009; li et al. 2011; surendra et al. 2016). through the bioconversion process, the species is applied as a feed ingredient for aquaculture, livestock, and poultry industries (newton et al. 1977; st-hilaire et al. 2007b; li et al. 2016; magalhaes et al. 2017; renna et al. 2017; schiavone et al. 2017). however, the heterogeneity of available organic material created a challenge to the optimization and implementation of this system, especially in the municipal areas. restaurant waste, for example, containing animal and plant matters which are rich in carbohydrate and a similar amount of protein and fat, while mixed fruits and vegetables are rich in carbohydrate with significantly low-fat content. in indonesia, most organic wastes are produced through economic activities in the traditional and modern markets which are dominated by vegetables and animal remains. applying these heterogeneous resources as diet for black soldier fly would affect the development, productivity, some life-history traits, and chemical composition of the biomass (tomberlin et al. 2002; oonincx et al. 2015a; tschirner & simon 2015; cammack & tomberlin 2017). this study was designed to imitate the real condition in indonesia as a model for other similarly developed tropical countries in which different organic wastes are produced. the objectives of this experiment were 1) to compare the consumption efficiency of bsfl to diet combination of vegetable waste and animal residues, and 2) to determine the effects of diet composition on its growth, development time, pupae survival, and on the adult sex ratio. the results of this study could be used as the basis for diet manipulation in optimizing waste reduction, converting organic materials to insect biomass, and sustainability of the bioconversion system using municipal organic wastes. materials and methods animal specimen this study used the seven-day old larvae of the black soldier fly that were obtained from eggs purchased from a bsf farm in sumedang, west java. all the eggs were kept on the substance made of commercial chicken feed (60% moisture) and kept at constant temperature (28 oc, 70% rh) in a container (50 x 25 x 10 cm) at the laboratory of environmental toxicology, school of life sciences and technology, bandung, indonesia. treatment each treatment (with nine replicates) contained 60 larvae fed with 100 mg/day/larvae (wet weight, 60% moisture content) of a diet combination of vegetable wastes (brassica chinensis) and carp (cyprinus caprio) fish offal. the treatments were composed of diet combination ratios of fish offal: vegetable wastes, namely; 30 : 70 ( s > i), 50 : 50 (s = i), and 70 : 30 (s < i), and replicated 3 times. the seven-day old larvae were initially placed into a plastic cup (with a height of 12 cm, upper diameter 7 cm, lower diameter 5 cm) filled with feeding material, and covered with a black sheet. the lid of the cup contained holes to allow air circulation. to prevent oviposition of other flies and parasitoids, a round dark cloth with diameter 0.01 mm was clamped between box and lid. the diet for larvae was prepared, weighed, and kept frozen 24 hours before the treatment to prevent the decomposition process. all cups were kept in a shady area. sampling and feeding were conducted every three days (diener et al. 2009; lalander et al. 2019) during which period the remaining larvae were transferred into another glass already filled with the next feed. the residual material of the previous glass was dried at 60 oc for dry mass determination. feeding was conducted until more than 40% of all larvae metamorphosed into prepupae (tomberlin et al. 2002; lalander et al. 2019) while weighing was conducted until all larva metamorphosed into prepupae. all prepupae were removed daily from each container and weighed, then placed in a plastic container for biotropia vol. 28 no. 2, 2021 94 further rearing process into an adult. prepupae and pupae were held in the same incubator in which the larvae were reared and monitored for adult emergence daily (cammack & tomberlin 2017). data analysis larvae growth rate and productivity future production of insect larvae through the bioconversion method highly depends on the larvae growth rate. in this study, the growth rate of each larva was determined by daily biomass change (waldbauer 1968), with the following formula: growth rate = 𝐵/t (1) where: b = weight gain (mg) t = development time (days) on the other hand, the productivity of determined by formula: productivity = [dry weight of larvae/(t x v)] (2) where: t = larvae rearing period (day) v = volume of rearing container (dm3) in this study, the volume of reactor applied was 0.414 dm3. consumption ability the ability of larvae to consume diet was determined by ad (approximate digestibility), ecd (efficiency of conversion of digestedfeed), wri (waste reduction index), and proportion of diet used for metabolism, converted into biomass, and undigested. ad parameter was used to determine the effectiveness and larvae ability to digest the diet which could be measured by the formula: ad = (i-f)/i x 100% (3) where: ad = approximate digestibility i = initial weight of diet (mg) f = weight of residue (undigested food + excretions) (mg) the ability of larvae to digest each diet composition was measured by ecd based on the formulae of scriber and slansky (1982) and modified by dienar et al. (2009): b = (i – f) – m0 (4) ecd = b/(i – f) (5) where: b = the total amount of food use for growth i = total amount of food offered during the experiment f = total amount of residue (undigested food + excretory food) m = amount of food metabolized by larvae (calculated by mass balance) dry weight was used in all calculations. to measure overall material reduction, the time of larvae development was required to reduce the amount of food included in calculation along with overall degradation (d) of waste. all of those variables were defined as waste reduction index (wri) which was determined by the formula: wri = [(i-f)/i x 100]/t (6) where: i = initial weight of diet (mg) f = total amount of residue (undigested food + excretory), and larvae rearing period (day) mass balance mass balance is one approach to design the biomass production system and to predict the digestibility of the diet. in this approach, the total amount of feed consumed by larvae was divided into three outputs: the mass of diet material that is used to maintain homeostasis of larvae, the mass of undigested diet material, and the harvested biomass (fig. 1). statistical analysis one way anova (p ≤ 0.05) with subsequent tukey hsd tests were applied to detect the difference of the means among all treatments. growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 95 figure 1 mass balance model of organic waste bioconversion into body biomass of the bsfl results and discussion black soldier fly larvae growth the pattern of larval growth among treatments was relatively similar as all prepupae reached pupal stage on day 21 for all treatments. moreover, the larval weight of group s < i was the highest, followed by s = i and s < i. on average, the final weight of harvested prepupae was 122.80 mg for group s < i which was significantly higher than group s = i (113.88 mg) and s > i (106.89 mg) (fig. 2). diet quality affects the growth and development time of bsfl (furmant et al. 1959; myers et al. 2008; diener et al. 2009; oonincx et al. 2015a,b). the development time to reach the prepupae stage could range from two weeks, under optimal condition, to more than 3 months if the food is limited (table 1). in this study, the development time of bsfl to reach the prepupae stage was shorter than most of the other studies (tabel 1). higher protein and fatty acid content in the fish offal might have provided the necessary nutrients for the larval growth and metabolism (cammack & tomberlin 2017). on the other hand, those nutrients might have also encouraged the diversity of bacterial species, some of which might be associated with bsfl and promote larval growth and development (dong et al. 2009; yu et al. 2010, 2011; jeon et al. 2011; zheng et al. 2013). furthermore, other bacteria unassociated with bsfl, such as escherichia coli, salmonella enterica, and pseudomonas marginalis, could have been killed by the antimicrobial substance produced by bsfl and then used as a source of nutrition (erickson et al. 2004; liu et al. 2008; park et al. 2015). figure 2 growth pattern of bsfl biotropia vol. 28 no. 2, 2021 96 table 1 comparative data on development time and efficiency of conversion of digested-feed (ecd) for black soldier fly larvae on various substrates reference substrate development time (days) ecd (%) may (1961) housefly medium 18 myers et al. (2008) dairy manure 28-30 diener et al. (2009) chicken feed 16-42 24.4 38.0 sealey et al. (2009) dairy manure 120 li et al. (2011) dairy manure < 31 gobbi et al. (2013) meat meal 33 gobbi et al. (2013) hen feed 15 manurung et al. (2016) rice straw 38-52 5.69 10.85 supriyatna et al. (2016) cassava peel 20-54 12 21 abduh et al. (2017a) rubber seed 12.5 25.9 abduh et al. (2017b) pandanus tectorius 6.3 27.4 this study combination of vegetables and fish offal 21 17.33 22.53 productivity among all treatments, larvae of group s < i had the significantly highest productivity (11.15 mg/larvae/day/dm3) compared to group s > i (9.69 mg/larvae/day/dm3) and group s = i (9.42 mg/larvae/day/dm3) (fig. 3). the larva of group s < i which contained more protein has produced significantly larger prepupae (anova, p < 0.05). this conformed with earlier studies of diener et al. (2009), cammack & tomberlin (2017). furthermore, the higher moisture content might have also contributed to the higher prepupae biomass of group s < i. consumption ability the consumption-ability of the larva was determined by waste reduction index (wri), efficiency of conversion of digested-feed (ecd), and approximate digestibility (ad). among treatments, the group s < i produced the significantly highest wri (3.13) which indicated a higher preference of larvae to richer proteinand-lipidcontaining diet (fig. 4). the level of waste reduction was similar to chicken feed (diener et al. 2009) however, it was higher than rice straw feed (manurung et al. 2016) and rubber seed feed (abduh et al. 2017a) which indicated the effect of diet composition to the level of consumption by bsfl. the effectiveness of larvae to convert digested diet into biomass was measured by ecd. in this study, the ecd ranged between 17.33 to 22.53% with group s < i showing the lowest ecd (17.33%) while also recording the significantly highest ad at 62.01% (table 2). figure 3 productivity of bsfl note: * = significant at p < 0.05). growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 97 figure 4 waste reduction index of diet note: * = significant at p < 0.05). table 2 ecd and ad among different diet regimes diet (100 mg/larva/day) ecd (efficiency of conversion of digested-feed) ad (approximate digestibility) s = i (50 : 50) 22.53% 49.46% s > i (70 : 30) 21.56% 45.45% s < i (30 : 70) 17.33% 62.01% the ecd level recorded in this study was relatively higher than most of the previous studies (table 1). ecd decreased when the quality of diet decreased (higher proportion of undigested material, such as cellulose and hemicellulose). however, in this study, the larval group that consumed the protein has showed lower ecd than the group that consumed a cellulose-rich diet. higher ecd manifested by bsfl receiving an inferior diet could be a strategy to obtain more nutrients by increasing the consumption rate (couture et al. 2016). on the other hand, as shown by ad, a diet with higher protein content was more readily digested by the larvae which compensated for its lower ecd. this compensation allowed the s < i group to produce heavier harvested prepupae. mass balance of bioconversion process around 4.45 to 6.66% of the substrate was transformed into biomass, 40.99 to 55.35% was used for metabolism, and 37.99 to 54.55% was undigested. the highest transformation rate of the substrate into biomass was recorded on the group s < i (fig. 5). high transformation rate of the substrate to biomass in group s < i indicated the importance of food digestibility in the production of more biomass. pupae mortality and adult sex ratio the level of pupae mortality was low at all groups, around 4.29 to 10.71%. the highest mortality was recorded at group s > i (10.71%) while the lowest in group s < i (4.29%) (fig. 6). the level of pupae mortality in this study was lower than those of tomberlin et al. (2002) and gobbi et al. (2013) and of cammack and tomberlin (2017). generally, the larvae of black soldier fly are fed on a wide variety of substrates on the larval stage and accumulate a large store of fat to reduce and/or eliminate the need for the adult to feed (sheppard et al. 2002). for this reason, the quality of diet plays a key role in the development of adults during the pupae stage. to produce high quality food, the larvae of insects tend to consume a balanced diet that is optimum for its growth and development (gobbi et al. 2013). this study showed that larvae reared with the high protein diet combined with complex carbohydrates (from vegetable wastes) could significantly reduce the level of pupae mortality. furthermore, the juice produced by the degradation of fish offal maintained the diet moisture which highly influenced pupae mortality, particularly for fly species (cickova et al. 2012; cammack & tomberlin 2017). biotropia vol. 28 no. 2, 2021 98 figure 5 proportion of digested feed used for metabolism, converted into biomass, and left undigested (residue) note: * = significant at p < 0.05). figure 6 percentage of pupae mortality note: * = significant at p < 0.05). figure 7 proportion of adult female and male more female adults were produced than the males for all groups although the proportion was more balanced in s < i group with strong differences manifested by the group receiving the balanced feed (fig. 7). the female-biased sex ratio showed in this study confirmed some earlier studies (tomberlin et al. 2002; zarkani & miswati 2012; gobbi et al. 2013). however, other studies reported a more male-biased adult proportion when larva were fed with artificial feeds (ma et al. 2018; meneguz et al. 2018). variables like ph of the substrate, nutritional variability, and feeding regime were probably the factors that govern the sex ratio of adult flies growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 99 (quezada-garcia et al. 2014; ma et al. 2018; meneguz et al. 2018). strong differences on the sex ratio related with substrate differences in this study are against some previous studies reporting the insignificant effect of diet quality and content to the sex ratio of black soldier fly (tomberlin et al. 2002; gobbi et al. 2013; diener et al. 2015). however, this result agrees with the study of zarkani & miswati (2012) in the tropical region which provides some evidences of the effect of environmental factor to the final stage of the developmental period of bsfl. conclusions diet combination consisting of a higher proportion of protein and lipid has resulted in a higher weight of harvested prepupae and the lowest mortality of the black soldier fly which was probably due to a higher consumption rate and approximately higher digestibility. to improve the sustainable production of the feed, further studies are suggested on the rearing environment of larvae particularly, on reducing the mortality rate of pupae brought about by the bioconverted waste that is high in protein and lipid; on the effect of various waste materials to biomass production and sustainability; and on the composition of biomass. acknowledgments this study was partly and equally supported by riset itb 2013 and hibah kompetensi 2018 granted to the corresponding author, p3mi funding granted to the first author, and dipa boptan uin sunan gunung djati bandung granted to the last author. references abduh my, jamilah m, istiandari p, syaripudin, manurung r. 2017a. bioconversion of rubber seeds to produce protein and oil-rich biomass using black soldier fly larva assisted by microbes. j entomol zool stud 5(4):591-7. abduh my, manurung r, faustina a, affanda e, siregar irh. 2017b. bioconversion of pandanus tectorius using black soldier fly larvae for the production of edible oil and protein-rich biomass. j entomol zool stud 5(1):803-9. banks ij, gibson wt, cameron mm. 2014. growth rates of black soldier fly larvae fed on fresh human faeces and their implication for improving sanitation. trop med int health 19(1):14-22. barnard dr, harms rh, sloan dr. 1998. biodegradation of poultry manure by house fly (diptera: muscidae). environ entomol 27(3):600-5. barry t. 2004. evaluation of the economic, social, and biological feasibility of bioconverting food wastes with the black soldier fly (hermetia illucens) [dissertation]. texas (us): university of north texas. beard rl, sands dc. 1973. factors affecting degradation of poultry manure by flies. environ entomol 2(5):801-6. callan em. 1974. hermetia illucens (diptera: stratiomyidae), a cosmopolitan american species long established in australia and new-zealand. entomol mon mag 109:232-4. cammack ja, tomberlin jk. 2017. the impact of diet protein and carbohydrate on select life-history traits of the black soldier fly hermetia illucens (l.) 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browning of coconut endosperm culturein vitro lazarus agus sukamto* the possibility of physiological age and growth regulators affecting callus browning of coconut endosperm was investigated. solid endosperm explants of four coconut fruits from same brunches of two coconut cultivars “samoan dwarf ” were grown on modified murashige and skoog (ms) formula with addition of 10 mg l putresine, 2.50 g l activated charcoal (ac), 1.70 g l phytagel, 0, 10 , 10 , 10 , 10 m 2,4-dichlorophenoxyacetic acid (2,4-d) or 4-amino3,5,6-trichloropicolinic acid (picloram) combined with 10 m 6-benzylaminopurine (ba). callogenesis occurred on 98.83% of explants. callus browning between different physiological ages (antipodal and micropylar tissues) of coconut endosperm at 9, 26 and 31 weeks of culture (woc) was significantly different, but not at 16 and 21 woc. auxins of 2,4-d and picloram did not affect significantly callus browning of endosperm cultures. auxin doses at 10 , 10 , and 10 m decreased significantly callus browning at 9 and 16 woc, respectively, but at 10 m browning was less significant compared to other doses at 21 woc. auxin dose at 10 m caused less significant browning compared to other doses at 31 woc. the addition of ba decreased significantly callus browning at 9 woc, but did not affect callus browning thereafter. coconut, , picloram, 2,4-d, ba research center for biology indonesian institute of sciences in vitro abstract introduction -1 -1 -1 -6 -5 -4 -3 -5 -6 -5 -4 -6 -3 key words: coconut ( l.) is a long-lived tree with a very long juvenile phase (3 5 years), generally cross-pollinated and very heterozygous. vegetative propagation through the use of various explants has been attempted but the success is mostly limited to immature unselected plants and produced very few plants. coconut endosperm is considered a mature tissue which provides large and uniform explants without damaging mother plant for culture. cocos nucifera in vitro in vitro cibinong, indonesia * corresponding author : lazarus_agus@yahoo.com 31 in vitro et al in vitro et al et al citrus grandis in vitro et al et al in vitro et al et al gasteria haworthia et al et al et al et al et al et al et al physiological age is a critical factor for the success of culture (ozyigit . 2007). the age of the endosperm at the time of culture is critical for growth (chen . 1990). explants from younger fruits responded better to culture (karunaratne . 1991). however, coenocytic (free-nuclear) endosperm from very young fruits did not survive in culture because of lack of starch (srivastava 1982). young endosperm at celliferous stage was responsive to culture in and apple (mu and liu 1978; wang and chang 1978). endosperm of 6 7 months postanthesis in coconut which solid endosperm started to form in the antipodal end of coconut fruit (tammes & whitehead 1969) was responsive to culture (kumar . 1985). whole plants are autonomous with regard to growth regulators but isolated tissues or cells require auxins or cytokinins to initiate and maintain growth until they become habituated or organized (everett . 1978). morphogenesis can be regulated by regulators (christianson & warnick 1983), skoog and miller (1957) found that the balance of auxin and cytokinin in culture medium governed morphogenesis. auxins induced callogenesis, adventitious plantlets and roots in palms (tisserat 1979; paranjothy & rohani 1982; reynolds 1982; paranjothy 1986). among auxins, 2,4-d was the most effective compared to the others in coconut culture (blake & euwens 1982; pannetier & buffard-morel 1986; karunaratne & periyapperuma 1989). auxin at high doses (10 m 10 m) was necessary for callus induction in palm, especially on medium supplemented with 1 3 g l ac (jesty & francis 1992). another auxin with properties similar to 2,4-d is picloram. picloram has been successfully applied for callogenesis in date palm (omar & novak 1990), embryogenesis in pejibaje palm (valverde . 1987), and maintained regenerative callus over long time in sugarcane (fitch . 1983). picloram was faster than 2,4-d for callogenesis, embryo induction, and final yield of embryos in and (beyl & sharma 1983). on the other hand, picloram produced more phenolics from cut surfaces and was slower for callogenesis than 2,4-d in sugarcane (fitch . 1983). the presence of cytokinins, auxins, and high doses of sucrose (0.2 m) stimulated growth of coconut and date callus (euwens 1978). combination of cytokinin at low dose with auxin at high dose was necessary for callogenesis of coconut embryos (bhaskaran 1985). srinivasan . (1985) successfully induced somatic embryos of christmas palm with 5 50 x 10 m 2,4-d and 50 x 10 m ba. ba was more effective than kinetin in stimulating callus growth in longan culture (litz 1988), increased greatly fresh weight of coconut callus and date palm callus (euwens 1978; kuruvinashetti & iyer 1980; sharma . 1984). explant browning is often associated with physiological age and failure of explants survival (krishna . 2008; guo . 2010). browning in some explants may be very severe and causes inhibition or cessation of growth (abdelwahd . 2008; misra . 2010). phenolics, tannins or oxidized polyphenols are synthesized through shikimic acid, phenylpropanoid, flavonoid, and terpenoid pathways. these substances are abundantly present in some plants and act as inhibitory agents (preece & compton 1991). phenolics, especially the most common polyphenol cause oxidative browning in explants, which lead to discoloration of the culture medium (forrest 1969; davies 1972; babbar & gupta 1986; tang & newton 2004). oxidized phenolic -5 -3 -1 -5 -5 32 biotropia vol. 18 no. 1, 2011 compounds are frequently exuded into the medium by injured woody tissues causing lethal browning or blackening of explants (alderson 1987; bhat & chandel 1991; trautmann & visser 1991). immature endosperms were more responsive in culture than mature ones (cheema & mehra 1982). this was partially due to oxidation products which were more abundant in the older explants (sugimura . 1988; preece & compton 1991; wu . 2010). activated charcoal can promote cell growth and development, it may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substantially decreasing toxic metabolites, phenolic exudation and browning (zhu . 1997; thomas 2008). browning can be reduced or eliminated through the use of liquid media, ac, silver nitrate, ascorbic acid, citric acid, sodium hydrosulfite, cystein, diethyldithio carbamate (dtt), potassium ethylxanthate, thiourea, benzimidazole, sodium bisulfit, polyvinylpyrrolidone (pvp), polyclar, glutathione, and bovine serum albumin, more frequent transfers, incubation with reduced illumination or in complete darkness, presoaking explants in sterile water, sealing the cut ends with paraffin wax, changing medium, avoiding high temperatures, discarding explants which show browning, reducing explants thickness, and choosing the most suitable stage (chang . 2001; wu & du toit 2004; mitsukuri . 2009). addition of auxins, particularly 2,4-d at high doses, led to browning of coconut leaf (pannetier & buffard-morel 1986). sugimura and salvana (1989) observed that 2,4-d at 2.26 x 10 m 4.52 x 10 m caused severe browning of tissues regardless of the stage and size of coconut inflorescence culture. 2,4-d at levels higher than 30 x 10 m inhibited callusing and enhanced browning of coconut embryos (karunaratne & periyapperuma 1989). callus precociously isolated from explants also caused browning and necrosis in coconut inflorescence (verdeil . 1993). addition of cytokinin, such as kinetin also caused more browning than the use of auxin in palm cultures (reynolds 1982). the objective of the study is to know the effect of physiological age (antipodal and micropylar) and growth regulators (2,4-d, picloram, and ba) on callus browning of coconut endosperm culture plant materials of four seven-month old coconut fruits were taken from two bunches of two coconut trees cultivars “samoan dwarf ” after opening inflorescences. these fruits immediately were disinfested by using alcohol 95%. the fruits were opened and their water decanted, cut horizontally with a sterile big knife. solid endosperms were aseptically cored with cork borer and scooped with a sterile spoon in laminar air flow. these endosperms were taken from either the micropylar region (upper half of fruit where embryo located) or the antipodal region (bottom half of fruit). cylindrical endosperm shapes with 8 mm diameter and 4 mm thick, used as explants, were grown on various media treatments. in vitro et al et al et al et al et al et al in vitro . -4 -4 6 materials and method explant material callus browning of coconut endosperm culture l.a. sukamtin vitro o 33 media culture experimental design callogenesis of coconut endosperm physiological age and calli browning media cultures were a modification of branton and blake formula (1986) added with 10 mg l putrescine, 2.50 g l ac, 1.70 g l phytagel, hormones 2,4-d or picloram at 0, 10 , 10 , 10 and 10 m concentrations, respectively, were combined with or without 10 m ba at 16 woc. the ph of the media were adjusted to 5.70 before they were autoclaved. the media were poured into 2.5 x 15 cm test tubes (14 ml) and autoclaved at 121 c temperature and 1 kg cm pressure for 15 minutes. media were stored for one week before use. single explant was placed into test tube with the uncut surface upright. cultures were incubated at approximately 31 c in the dark room. the experimental design was a randomized complete block design (rcbd) with each fruit as a block. treatments were factorial combinations of endosperm region (micropylar and antipodal), auxins (2,4-d and picloram) and their doses (0, 10 m, 10 m, 10 m, 10 m) and cytokinin (with or without 10 m ba) with 12 replications. callogenesis of coconut endosperm was counted as percentage on 31 woc. the tissues browning were evaluated visually at every transfer using numerical scores ranging from 0 to 3 (0 : no browning, 1 : little browning, 2 : medium browning, and 3 : high browning) at 9, 16, 21, 26 and 31 woc. data were analyzed with the general linear models (glm) and non parametric one way (kruskal-wallis test) procedure of statistical analysis system (sas). coconut endosperm explants formed callus after approximately 3 woc. callus grew predominantly on the uncut surface and on the side of endosperms. eventually, the callus grew and covered the entire explants. callogenesis occurred on almost all explants with an average of 98.83% (table 1). these results were better than those obtained by kumar . (1985) i.e. 30% of coconut endosperms, gmitter . (1990) i.e. 8 25% of endosperms, chen . (1990) i.e. 1.60 3.74% of endosperms; pannetier and buffard-morel (1982) i.e. 20% of mature coconut leaves and 50% of juvenile coconut leaves, and verdeil . (1989) i.e. 45% of coconut inflorescence. the good result could be due to genetic and sterilized factors. in addition, the plant materials used in this study were different in explants type, cultivars or plants than those used by other researchers. sterilized method was different than of kumar . (1985), this experiment only sterilized the outer skin of the fruits not the explants. therefore, the explants were more responsive in promoting callogenesis. oxidative browning (brown to black) of endosperm calli occurred during culture. browning varied within or among treatments, ranging from 0: no browning, 1: slight -1 -1 -1 -6 -5 -4 -3 -5 0 -2 0 -6 5 -4 -3 -5 results and discussion et al et al citrus maxima et al c. sinensis et al et al 34 biotropia vol. 18 no. 1, 2011 source fruit 1 fruit 2 fruit 3 fruit 4 average z y means standard error of 12 measurements means in the same group followed by the same letter in a column except for the average are not significantly different at the 5% level (based on a comparison of possible combinations between the averages of treatments) table 2. browning levels of coconut endosperm callus (0 : no browning, 1 : light brown, 2 : brown 3 : dark brown ) z y means standard error of 12 measurements means in the same group followed by the same letter in a column except for the average are not significantly different at the 5% level (based on a comparison of possible combinations between the averages of treatments) table 1. percentage of callogenesis of coconut endosperm after 31 weeks of culture 35 callus browning of coconut endosperm culture l.a. sukamtin vitro o browning 2:medium browning , and 3: severe browning (fig. 1). tissue browning by all treatments was slight (score = 1.18) at 9 woc. tissue browning increased substantially (browning level = 1.75) after 16 woc and reached maximal browning (browning level = 2.25) on 21 woc. thereafter browning decreased slightly on 26 woc (browning level = 2.23) and 31 woc (browning level = 2.18). compton and preece (1988) found that phenolic compounds exuded from excised explants were oxidized by peroxidases or polyphenolo-xidases, causing browning of both plant tissues and media. this might reduce growth or kill the tissues (preece and compton 1991). severe browning of coconut endosperm still occurred even though in young explants which were not treated by disinfectants and incubated in the dark room. whereas this condition was expected to prevent browning of ova (dias . 1994). the antipodal and micropylar tissues browning showed a steady increase at 21 woc (table 2). statistical analysis showed that browning of antipodal tissues was more significant than in micropylar tissues at 9, 26, and 31 woc. it could be due to antipodal tissues which were earlier formed (older) and thicker than micropylar tissues. these results support the findings of murashige (1974) that the influence of geonoma gami et al figure 1. browning of coconut endosperm callus at various levels i.e. 0, 1, 2, and 3 (from left to right) figure 2. a). varied colors of coconut endosperm callus, b). new yellowish white callus grown from the black callus of coconut endosperm a b 36 biotropia vol. 18 no. 1, 2011 physiological age on explants responses was not significantly different at 16 and 21 woc (table 2). explants thickness influenced browning, antipodal explants were thicker and showed more browning compared to micropylar explants. similar results were obtained by sugimura and salvana (1989) in coconut inflorescence explants of 1 mm which had 32% browning compared to 11% browning in 0.5 mm thick.coconut inflorence explants. the two types of auxins (2,4-d and picloram) did not cause any significant difference in browning of endosperm tissues. different results were reported by fitch . (1983) that picloram caused more browning than 2,4-d in cultures. the browning levels of tissues initially treated with 10 m auxin were significantly higher at 9 woc, the highest at 16 woc, and the lowest at 31 woc than those of the other auxin concentrations (table 2). the browning levels of control were higher at 9 16 woc than of those tissues treated with 10 m 10 m auxins (table 2). the tissues initially treated with 10 m auxin, showed significantly less browning than control or other auxin concentrations at 21 woc. the tissues initially treated with 10 m auxin caued significantly more browning than of the other treatments at 26 woc. this result disagreed with the findings of pannetier and buffard-morel (1986), karunaratne and periyapperuma (1989), and sugimura and salvana (1989) that 2,4-d levels higher than 3 x 10 m caused more browning than lower concentrations in coconut explants. this result agreed with the findings of phillips and henshaw (1977) in cell cultures. addition of ba significantly decreased browning at 16 woc. similar result was reported by herve . (2001) in culture. however, those of addition did not affect significantly callus browning thereafter (table 2). calli color changed progressively, from white to brown, to dark brown and to black (fig. 2a). then new yellowish white callus grew from the black callus and this sequence was repeated through many cycles (fig. 2b). severe browning did not inhibit the growth of endosperm cultures. similar result was reported by jones (1974) in oil palm and ettinger and preece (1985) in cultures. coconut endosperm probably tolerated high level of 2,4-d or picloram, even their browning were less than other treatments including control at 10 m on 31 woc (table 2), due to the presence of ac at 2.5 g l in the medium and dark incubation. it agreed with the findings of wang and huang (1976), fridborg . (1978), tisserat (1979), blake and eeuwens (1982), rao . (1987), sugimura and salvana (1989), and krikorian (1994). callogenesis occurred on 98.83% of coconut endosperm explants. calli browning increased significantly from 9 to 21 woc but not thereafter. browning of antipodal tissue-derived calli was more significant at 9, 26 and 31 woc but not at 16 and 21 woc compared to micropylar tissues. treatments of 2,4-d and picloram did not growth regulators and calli browning et al saccharum spontaneum acer pseudoplatanus et al eucalyptus gunnii hododendron et al et al -3 -6 -4 -6 -5 -5 -3 -1 r conclusions 37 callus browning of coconut endosperm culture l.a. sukamtin vitro o affect calli browning. auxins at 0 and 10 m produced significantly more callus browning than other doses at 9 and 16 woc. auxins of 10 m produced significantly less callus browning than other doses at 21 woc. calli browning was more intense at 10 m auxins at 26 woc. auxins of 10 m produced significantly less callus browning than other doses at 31 woc. addition of cytokinin ba produced significantly more callus browning at 16 woc but not thereafter. the author would like to thank dr. y. sagawa and dr. d.t. webb for their advice, while mr. r. wutzke for providing the experiment the overseas training office/ badan perencanaan pembangunan nasional (bappenas) indonesia is acknowledged for the funding support. -3 -6 -5 -3 acknowledgements references abdelwahd r, hakam n, labhilili m, udupa sm. 2008. use of an adsorbent and antioxidants to reduce the effects of leached phenolics in plantlet regeneration of faba bean. african j biotech 7(8):9971002 alderson pg. 1987. micropropagation of woody plants. in: alderson pg and dullforce wm (editors). micropropagation in horticulture, practice and commercial problems. the univ. nottingham trent print unit. p 37-52 babbar sb, gupta sc 1986. induction of androgenesis and callus formation in in vitro cultured anthers of a myrtaceous fruit tree ( l.). bot mag tokyo 99:75-83 bhaskaran s. 1985. tissue culture technology for higher vegetable oil production. in: srivastava, hc, bhaskaran s, vatsya b, menon kkg (editors). oilseed production: constraints and opportunities. oxford & ibh publishing co., new delhi. p 537-544 beyl ca, sharma gc. 1983. picloram induced somatic embryogenesis in and . plant cell tissue organ cult 2:123-32. bhat s.r, chandel kps. 1991 a novel technique to overcome browning in tissue culture. plant cell rep 10:358361. blake j, eeuwens cj. 1982. culture of coconut palm tissues with a view to vegetative propagation. in: rao a.n. 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buffard-morel j, pannetier c. 1989. embryogenese somatique du cocotier ( l.) a partir de tissues foliaires et inflorescenciels. bilan des recherches et perspectives. oleagineux 44(8-9):403-11 verdeil jl, huet c, grosdemange f, buffard-morel j. 1993. plant regeneration from cultured immature inflorescences of coconut ( l.): evidence for somatic embryogenesis. plant cell rep 13:218-21 wang ty, huang lc. 1976. beneficial effects of activated charcoal on plant tissue and organ cultures. 12(3):260-62 wang ty, chang cj. 1978. triploid plantlet from endosperm culture. in: proc symp on plant tissue culture. science press, peking, china. p 463-67 wu,hc, du toit es. 2004. reducing oxidative browning during establishment of . scientia horticulturae 100(1-4):355-58 wu qy, xu ly, gong lg. 2010. qing qian liu different explants on tissue culture and the prevention of browning. . accessed on december 21, 2010 zhu,ym, hoshino y, nakano m, takahashi e, mii m. 1997. highly efficient system of plant regeneration from protoplasts of grapevine ( l.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. plant sci 123(1-2):151-57 bactris gasipaes cocos nucifera cocos nucifera in vitro citrus in vitro protea cynaroides vitis vinifera http://eng.hi138.com/?i131358 41 callus browning of coconut endosperm culture l.a. sukamtin vitro o biotropia no biotropia no. 15, 2000 : 58 75 stored cocoa beans quality affected by fermentation and ephestia cautella walker (lepidoptera: phycitidae) infestation ok.ky s. dharmaputra' \ sunjaya', ina retnowati' and santi ambarwati' 'seamed biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the effects of fermentation on ephestia cautella population and cocoa beans quality in terms of moisture content, fungal population, the percentage of insect-damaged and mouldy beans, lipid and free fatty acid contents during storage were investigated together with the effects of £. cautella infestation on the quality of stored cocoa beans and weight loss. fermented and unfermented cocoa beans with initial moisture contents of 7 or 9% were placed in ventilated plastic jars (ikg/jar) and stored for 6 months under room conditions. seven larvae of £. cautella instar iv (2 males and 5 females) were introduced in each jar at the beginning of storage. untreated jars contained only cocoa beans. population of £. cautella on fermented cocoa beans with either initial moisture content of 7 or 9% was lower than that on unfermented beans during storage. the population either on fermented or unfermented cocoa beans with initial moisture content of 7% was lower than that of 9%, and the population of all treatments increased during storage. moisture content of all treatments either on cocoa beans with initial moisture contents of 7 or 9% had the same pattern. the percentage of insect-damaged beans on fermented cocoa beans was lower than that on unfermented cocoa beans after 5 to 6 months of storage. the damaged beans on fermented cocoa after 6 months of storage was not different than on unfermented beans after 4 months of storage. the weight loss either on fermented or unfermented cocoa beans with initial moisture content of 9% was higher than that with initial moisture content of 7%. the weight loss on fermented cocoa beans either with moisture content of 7 or 9% was lower than that on unfermented beans during storage. the weight loss either on fermented or unfermented cocoa beans increased during storage. the percentage of mouldy beans on cocoa infested with £. cautella tended to increase during storage, while on beans not infested with the insect it fluctuated during storage. the highest percentage of mouldy beans was on unfermented and infested cocoa beans. twenty-one fungal species were isolated from all treatments of cocoa beans during storage. the total fungal population on fermented and unfermented beans had the same pattern. the population on fermented cocoa beans was lower than that on unfermented beans. total lipid content on fermented cocoa beans either infested or not with £. cautella having initial moisture content of 7 or 9%, was lower than that of unfermented beans. the content either on fermented or unfermented cocoa beans and either infested or not decreased during storage. free fatty acid content on cocoa beans infested with £. cautella was higher and significantly different than that on not infested. the content for both types increased during storage. key words : cocoa beans / fermentation / ephestia cautella i moisture content / fungal population / insect-damaged beans / mouldy beans / lipid / free fatty acid. introduction according to the international cocoa organization (1996), indonesia ranks third among the cocoa producing countries of the world after ivory coast and ghana. it has been predicted that the production of indonesian cocoa beans will 58 biotropia no. 15, 2000 increase by 4.76% per year for the period of 1995-2005. the increase is especially due to the policy which has been determined by the government of indonesia through the development and quality improvement of plant material programs (susila 1996). siswoputranto (1997) reported that in indonesia cocoa is now the fourth most important export commodity after palm oil, rubber and coffee. compared to west african cocoa beans, indonesian cocoa beans have an excessive acidic flavor, low chocolate flavor and often certain other objectionable off-flavors. consequently, they are less desired by cocoa importing countries (duncan 1990). the imperfections in the fermentation process in particular by smallholders should be improved correctly, if better markets and higher prices for well fermented beans are expected through maintaining the quality of indonesian (smallholders') cocoa (siswoputranto 1997). according to zaenudin and wahyudi (1996), insect and mould attacks were the problems of exported cocoa beans derived from smallholders, so that they were subjected to automatic detention. consequently they should be fumigated, which will need additional expenses. the problem of automatic detention could be minimized by improving the method of postharvest handling from farmer to exporter levels, and thus the quality of indonesian cocoa beans will also be improved. with regard to the low quality of cocoa beans, importing country (usa) slapped a fine of 165-180 usd per ton. south sulawesi exported 120,000-140,000 tons of cocoa beans per year with an export value of 130 million usd (kompas 1996). kalshoven (1981) and wood (1985) reported that ephestia cautella walker (lepidoptera: phycitidae) (tropical warehouse moth) is associated with stored cocoa beans. according to dharmaputra et al. (1999) e. cautella was found in some samples of cocoa beans at exporter level in south sulawesi. there is an urgent need for indonesia to improve the quality of smallholders' cocoa beans for having good markets with better prices, and for anticipating whatever changes of markets in this coming era of global economy and international free trade, where severe competitions will be faced by cocoa producing countries, despite deficits of supply which may occur. the objective of this study was to investigate the effects of fermentation on e. cautella population and cocoa beans quality in terms of moisture content, fungal population, the percentage of insect-damaged and mouldy cocoa beans, lipid and free fatty acid content during storage. the effects of e. cautella infestation on the quality of stored cocoa beans and weight loss were also analyzed. materials and methods storage of cocoa beans and insect infestation fermented and unfermented cocoa beans (bulk type) obtained from rajamandala estate crop (ptpn viii), cipatat, bandung, were used in this study. 59 stored cocoa beans quality okky s. dharmaputra et al. soon after harvest, the pods of cocoa beans were opened and removed. the first category of cocoa beans was fermented using the standard method of the state estate crops soon after removing the beans from the pods. after fermentation process, they were washed and sundried until about 7% of moisture content was reached. the second category of cocoa beans was not fermented, but they were washed directly after removing from the pods. prior to storage, cocoa beans were fumigated with phosphine at dosage rates of 2g/ton with an exposure period of five days in order to kill any stage of insect that may exist. after fumigation, the moisture content of each cocoa bean was adjusted (7 and 9%). they were then placed in ventilated plastic jars (1 kg/jar) and stored for 6 months under room conditions. seven larvae of e. cautella instar iv (2 males and 5 females) were introduced in each jar at the beginning of storage. untreated jars contained only cocoa beans. three replications were used for each treatment. methods of sampling a sample consisting of the whole content of each jar was taken before storage, and subsequently after 1, 2, 3, 4, 5 and 6 months of storage. twenty-four samples were taken at each month of storage. consequently, the number of all experiment units was 168. insects were separated from cocoa beans using graded sieves. each cocoa bean sample was then divided several times using a sample divider to obtain working samples for analyzing insect-damaged and mouldy beans, fungal, moisture, lipid and free fatty acid contents. insect and fungal population determinations insect population (larva, pupa and adult) per kg of cocoa beans derived from each jar was determined by counting the number of dead and live insects in the outer and inner parts of the beans after separation from the cocoa beans. fungal population was determined based on dilution method followed by pour plate method using dichloran 18% glycerol agar (dg18) (pitt and hocking 1997). fungal species was identified using the publications of samson et al. (1996), pitt and hocking (1997) as the main references. moisture, lipid and free fatty acid contents analyses moisture content (wet weight) of cocoa beans was determined based on sni 01-2323 (isc 1998). two replicates were used for each sample. the beans were ground and dried in the oven at 103° ± 2°c for 16 hours. the moisture content was determined using the following formula: me = ( m , m 2 ) x 10° m| mo me = moisture content (%) 60 biotropia no. 15, 2000 m0 = the mass, in grams, of the empty dish and its lid mi = the mass, in grams, of the dish and its lid, and the test portion before drying m2 = the mass, in grams, of the dish and its lid, and the test portion after drying total lipid content was determined using soxhlet extraction method (isc 1998). the principle of this method is the extraction of free oil from the cocoa bean sample using non polar organic solvent («-hexane) which has been hydrolyzed. total lipid content was expressed as the percentage of mass and calculated on a dry weight basis, using the following formula: % total lipid content = (m1 – m2) 100 x 100 m0 100 mc me = moisture cpntent of the test sample mi = the mass, in grams, of the flask and lipid after drying m2 = the mass, in grams, of the dry flask mo = the mass, in grams, of the test sample free fatty acid content was determined using titration method (isc 1998). fat obtained from extraction is dissolved in warm ethanol and then titrated using alkali solution (naoh 0.1n). free fatty acid was calculated and expressed as the percentage of mass per mass using the following formula: % free fatty acid content = v x n x 100 m 100 mc v = the volume, in ml, of naoh n = the normality of naoh solution m = the mass, in grams, of cocoa bean lipid me = moisture content insect-damaged and mouldy beans, and weight loss determinations insect-damaged beans are those the internal parts of which contain dead insects at any stage of development, or which show damage by insects, visible to the naked eye. mouldy beans are those with fungi (mould) in the inner part. the percentages of insect-damaged and mouldy beans were determined according to sni 01-2323 (isc 1998). the percentage of dry weight loss was determined based on harris and linblad (1977) using the following formula : % weight loss = (u.nd) – (d.nu) x 100 u (nd = nu) 61 stored cocoa beans quality okky s. dharmaputra et a/. u = weight of undamaged beans nu = number of undamaged beans d = weight of damaged beans nd = number of damaged beans experimental data experimental data were analyzed using completely randomized factorial design with 4 factors. the 1st, 2nd, 3rd and 4th factors were fermentation, initial moisture content, e. cautella infestation and duration of storage, respectively. results and discussion e. cautella population the population of e. cautella on fermented cocoa beans with either initial moisture content of 7% or 9% was lower than that on unfermented beans during storage (figure 1). preliminary study on the preference test of e. cautella on fermented and unfermented cocoa beans showed that the number of insects found on fermented cocoa beans was lower than that on unfermented cocoa beans (table 1). it seems that the insects prefered unfermented cocoa beans. according to wood (1985) the unpleasant smell is caused by high content of acetic acid in fermented cocoa beans. 62 biotropia no. 15, 2000 table 1. preference test ofephestia cautella on cocoa beans treatment number of larvae found (insect/replication) fermented beans i ± 0.89 unfermented beans 10 ± 2.37 the population of e. cautella either on fermented or unfermented cocoa beans with " initial moisture content of 7% was lower than that of 9% during storage (figure 1). the change of moisture content during storage affected the growth and the development of insects. in foodstuff storage, moisture content is one of the important factors related with the growth development of insects (sinha and muir 1973;haines 1991). populations of e. cautella of all treatments increased during storage. after 6 months of storage, the lowest population was found on fermented cocoa beans with initial moisture content of 7% (24 insects/kg), while the highest was on unfermented cocoa beans with initial moisture content of 9% (122 insects/kg). moisture content moisture content of all treatments on cocoa beans with initial moisture contents of 7 or 9% had the same pattern. it decreased until 4 months of storage and then increased after 5 to 6 months of storage (figure 2). the moisture contents of fermented and unfermented beans with insects tended to decrease until 5 months of storage and increase after 6 months of storage. it was assumed that the change of the moisture content was affected by the insect activity, the presence of fungi and biochemical reactions of the commodities (sinha and muir 1973). insect-damaged beans insects are the most important cause of deterioration of stored grain ecosystems. e. cautella belongs to one of internal feeder insects, because it can move inside of the beans and cause damage to the inner parts of the beans. deterioration caused by e. cautella was determined by the presence of hole on beans, feces, silk webbing, dead bodies and frass. after 1 month of storage, the percentage of insect-damaged beans on fermented cocoa beans was not different than on unfermented ones. however, the percentage on fermented cocoa beans was lower (p < 0.05) than that on unfermented cocoa beans after 5 to 6 months of storage (figure 3). the population of e. cautella on fermented cocoa beans was lower than that on unfermented cocoa beans, consequently the insect-damaged beans on fermented cocoa beans was lower than that on unfermented cocoa beans. kresnowati (1999) reported that the percentage of damaged beans infested by araecerus fasciculatus on fermented cocoa beans was lower than that on unfermented ones. 63 stored cocoa beans quality – okky s. dharmaputra et al. storage duration (month) figure 2. moisture content of cocoa beans during storage figure 3. insect-damaged beans of cocoa during storage (note : se mean = 1.2554370) 64 biotropia no. 15, 2000 the damaged beans on fermented cocoa after 6 months of storage was not different than on unfermented beans after 4 months of storage. this showed that fermented cocoa beans could be stored for a longer period. weight loss caused by e. cautella infestation the loss agents generally fall into three classes: unavoidable (e.g. weather), human-induced (e.g., contamination, spillage, theft), and pest-induced (e.g. insects, mites, rodents) (haines 1995). the weight loss either on fermented or unfermented cocoa beans with initial moisture content of 9% was higher than that with initial moisture content of 7%. nevertheless, the weight loss of fermented beans with the two initial moisture contents was not significantly different. the highest weight loss was found on unfermented cocoa beans with initial moisture content of 9%. the weight loss on fermented cocoa beans either with moisture content of 7 or 9% was lower than that on unfermented beans during storage (figure 4). the weight loss either on fermented or unfermented cocoa beans increased during storage (figure 5). the increase of weight loss was related to the percentage of damaged beans caused by e. cautella infestation. figure 4. percentage of weight loss on fermented and unfermented cocoa beans with defferent initial moisture contents 65 stored cocoa beans quality – okky s. dharmaputra et al. figure 5. percentage of weight loss on fermented and unfermented cocoa beans during storage mouldy beans the highest percentage of mouldy beans was on unfermented and infested cocoa beans (figure 6). the percentage on fermented cocoa beans increased during storage. the percentage of mouldy unfermented cocoa beans increased after 1 month of storage and decreased until after 3 months of storage, and then increased until after 6 months of storage (figure 7). the percentage of mouldy cocoa beans infested with e. cautella tended to increase during storage, while the percentage of mouldy cocoa beans that were not infested with the insect fluctuated during storage (figure 8). species and total fungal population twenty-one fungal species were isolated from all treatments of cocoa beans during storage. fungi isolated from all treatments during storage are shown in tables 2, 3, 4 and 5. cladosporium cladosporioides. eurotium chevalieri and penicillium citrinum were always isolated from all treatments during storage. the total fungal population from all treatments during storage was relatively low (tables 2,3,4 and 5). it was due to the good quality of cocoa beans used in this study. retnowati et al. (2000) reported that the low total fungal population was also found on fermented and unfermented cocoa beans obtained from rajamandala estate crop (ptpn viii), introduced or not with a. fasciculutus. 66 biotropia no. 15, 2000 table 2. species and total fungal population of unfermented cocoa beans, infested with ephestia cautella with initial moisture content of 7 % i = infested ni = not infested table 3. species and total fungal population or fermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 7 % i = infested ni = not infested 67 stored cocoa beans quality – okky s. dharmaputra et al. table 4. species and total fungal population of unfermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 9 % i = inferted ni = not infested table 4. species and total fungal population of fermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 9 % i = inferted ni = not infested 68 biotropia no. 15, 2000 figure 6. percentage of mouldy beans on fermented and unfermented cocoa infested and not infested with ephestia cautella during storage (note : se mean = 0.19107623) storage duration (month) figure 7. percentage of mouldy beans on fermented and unfermented cocoa during storage (note : se mean = 0.35747093 69 stored cocoa beans quality – okky s. dharmaputra et al. storage duration (month) figure 8. percentage of mouldy beans on cocoa infested and not infested vvith ephestia caulelta during storage (note: se mean = 0.35739599) the total fungal population on fermented and unfermented beans had a similar pattern. it decreased after 1 month of storage and increased after 2 to 5 months of storage, and then decreased again after 6 months of storage. it was assumed that the decrease of total fungal population on unfermented beans after 1 month of storage was due to the interactions among fungi infecting the beans. nevertheless, the population of fungi on fermented and unfermented beans showed no significant difference (p>0.05) during storage, except on unfermented cocoa beans after 5 months of storage (figure 9). the fungal population on fermented cocoa beans was lower than that on unfermented beans. retnowati et al. (2000) also reported that total fungal population on fermented cocoa beans was lower than unfermented ones. figure 9. total fungal population of fermented and unfermented cocoa beans during storage (note : se mean = 6.59485150) 70 biotropia no. 15, 2000 total lipid content lipids are triglycerides composed of glycerol and 3-oh group which bind 3 fatty acids (sherman and sherman 1989). according to belitz and grosch (1987) lipids are important food flavour substances and the most important component in cocoa. wood (1985) reported that the total lipid content of good cocoa beans quality is between 56 58%. total lipid content of fermented cocoa beans either infested or not with e. cautella having initial moisture contents of 7 or 9% was lower than that on unfermented beans (figure 10). it was due to the presence of fatty acids produced from fermentation process that stimulate lipid hydrolysis into fatty acids and glycerol. according to wood (1985) during fermentation process, lactic acid bacteria homofermentor transformed glucose into lactic acid. lactic acid bacteria hetero-fermentor transformed glucose into lactic acid, alcohol, acetic acid and cc>2. winarno (1991) revealed under certain conditions, such as the presence of acid, lipids are hydrolyzed into fatty acids and glycerol. this study supports the findings of retnowati et al. (2000) that the total lipid content of fermented cocoa beans was lower than that of unfermented beans. figure 10. total lipid content of fermented and unfermented cocoa beans infested and not infested with ephestia cautella having different initial moisture contents (note: se mean = 0.0648736) the lipid content of either fermented or unfermented cocoa beans with or without e. cautella decreased during storage (figure 11), due to the transformation of lipids into free fatty acids (pomeranz 1992). retnowati et al. (2000) also reported that total lipid content decreased during storage. 71 stored cocoa beans quality okky s. dhurmaputra el al free fatty acid content the presence of free fatty acids (ffa) gives an indication of the quality of cocoa beans in producing cocoa butter. cocoa butter with high levels of ffa tend to be soft, have poor crystallization properties, contain off-flavour, and have a poor shelf life (nickless 1994). figure 11. total lipid content of fermented and unfermented cocoa beans infested and not infested with ephestia cautella during storage (note: se mean = 0.1213675) ffa content of cocoa beans infested with e. cautella was significantly (p< 0.05) higher than that without insects (figure 12). the content for both types increased during storage (figure 13). the content on fermented cocoa beans with initial moisture content of 7% was significantly (p< 0.05) lower than that on the beans with initial moisture content of 9%, while the level on unfermented cocoa beans with initial moisture content of 7% was significantly (p< 0.05) higher than that on the beans with initial moisture content of 9% (figure 14). on fermented cocoa beans lipids are easier to be hydrolyzed into ffa, because there are many fatty acids produced from fermentation process. consequently, ffa on fermented cocoa beans was higher than that on unfermented beans, and it increased during storage. 72 biotropia no. 15, 2000 figure 12. free fatty aced content of cocoa beans infested and not infested with ephestia caoutella ( note : se mean = 0.00267261 storage duration (month) figure 13. free fatty acid content of cocoa beans during storage figure 14. free fatty content of fermented and unfermented cocoa beans with different initial moisture contents (note : se mean =0.00377976) 73 stored cocoa beans q u a l i t y okky s. dharmaputra et al. conclusions in relation to e. cautella population and total fungal population during 6 months of storage, the quality of fermented cocoa beans and initial moisture content of ± 7% was better than that of unfermented cocoa beans and initial moisture content of ±9%. infestation of e. cautella decreased cocoa beans qu ality in terms of the percentage of mouldy beans, free fatty acid content, damaged beans and weight loss caused by e. cautella during 6 months of storage. fermented cocoa beans not infested with e. cautella having initial moisture content of 7% could be stored for a longer period than unfermented cocoa beans infested with the insect having initial moisture content of 9%. fermentation, insect control and initial moisture content of 7% are the important factors for maintaining the quality of cocoa beans during storage. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the indonesian research institute for coffee and cocoa, jember, indonesia in providing e. cautella for mass production. the authors are also grateful to the rajamandala estate crop (ptpn viii), cipatat, bandung, for cocoa beans preparation, and to ms diana puspitasari for her assistance in conducting the experiment. references belitz, h.d. and w. grosch. 1987. food chemistry. springer verlag berlin. heidelberg. 128 p. dharmaputra, o.s., sunjaya, m. amad, i. retnowati and t. wahyudi. 1999. the occurrence of insects and moulds in stored cocoa beans at south sulawesi. biotropia (12): 1 1 8 . duncan, r.j.e. 1990. the sime-cadbury process, background and development. paper presented at the seminar on improvements of cocoa beans processing. jakarta. 30 october 1989. haines, c.p. 1991. insects and arachnids of tropical stored product: their biology and identification (a training manual). natural resources institute. uk. haines, c.p. 1995. grain storage in the tropics. in d.s. jayas, n.d.g. white and w.e. muir(eds.). stored grain ecosystems. marcel dekker inc., new york.p. 55-100. harris, k.l. andc.j. lindblad. 1977. postharvest grain loss assesment methods. a manual of methods for the evaluation of postharvest losses. office of nutrition, agent for international development, us. international cocoa organization. 1996. production of cocoa beans by country, 1986/87-1995/96. quart. bull, of cocoa statistics 22(3): 6. international cocoa organization, london. indonesian standardization council. 1998. indonesian national standard, sn1 01-2323, rev.1995: cocoa beans. 74 biotropia no. 15 ,2000 kalshoven, l.g.e. 1981. the pest of crops in indonesia. revised by p.a. van der laan. p.t. ichtiar baru van hoeve, jakarta. kompas. 14 november 1996. kakao indonesia belum ditangani secara profesional. kompas 1996. kresnowati, k. f. 1999. kualitas biji kakao akibat fermentasi dan serangan araecerus fasciculatus de geer (coleoptera: anthribidae) selama penyimpanan. (cocoa beans quality affected by fermentation and araecerus fasciculatus de geer (coleoptera: anthribidae) infestation during storage). department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor. (in indonesian). nickless, h. 1994. cocoa butter quality. in j. selamat, b.c. lian, t.k. lai, w.r.w. ishak and m. mansor. malaysian cocoa board. proceedings of the malaysian international cocoa conference, p. 322-336. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. black academic and professional, cambridge. pomeranz, y. 1992. biochemical, functional, and nutritive changes during storage. in d.b. sauer (ed.). storage of cereal grains and their products. fourth edition. american association of cereal chemists, inc., minnesota, p. 55 142. retnowati, i., o.s. dharmaputra, sunjaya, k.f. kresnowati. 2000. keberadaan jamur pascapanen pada biji kakao yang difermentasi dan terserang araecerus fasciculatus de geer (coleoptera: anthribidae) selama penyimpanan (the occurrence of storage fungi on fermented cocoa beans and infested by araecerus fasciculatus de geer (coleoptera: anthribidae) during storage). proc. 15* national congress and scientific seminar of the indonesian society for phytophatology. purwokerto, 16-18 september 1999. p. 519-526 samson, r.a., e.s. hoekstra, c.j. frisvad, and o. filtenborg. 1996. introduction to foodborne fungi. centraalbureau voor schimmelcultures, baarn, the netherlands. sherman, a. and sherman j.s. 1989. chemistry and our changing world. second edition. prentice hall, inc., englewood cliffs. new jersey. sinha, r.n. and w.e. muir. 1973. grain storage: part of a system. the avi publishing company, inc. connecticut. siswoputranto, p.s. 1997. indonesian cocoa. in report of the 2"d meeting of national focal point for asean cocoa club on asean cooperation and joint approaches in agriculture and forest products promotion scheme. jakarta, indonesia, 4-5 march 1997. ministry of agriculture, republic of indonesia. susila, w.r. 1996. prospek pasar kakao dunia. warta puslit kopi dan kakao 12(1):1 11. winarno, f.g. 1991. kimia pangan dan gizi. pt. gramedia pustaka utama, jakarta. 253 h wood, g.a.r. 1985. from harvest to store. in g.a.r wood and r.a. lass (eds.). cocoa. fourth edition. longman, london, p. 444 504. zaenudin and t. wahyudi. 1996. laporan kunjungan tim askindo ke amerika serikat dalam upaya meniadakan automatic detention terhadap kakao indonesia. warta puslit. kopi dan kakao 12(1): 44-47. 75 biotropia no biotropia no. 14, 1999 : 36 51 litter fall in a primary and two logged-over lowland tropical rainforests in pasirmayang, jambi. upik rosalina wasrin* and agus eka putera ** 'senior scientist and "research assistant, remote sensing and ecology laboratory, seamed biotrop, p.o. box 116, bogor 16001, indonesia abstract litter accumulation in a primary and a logged-over lowland dipterocarp forest at pasirmayang, jambi was measured using the litter trap method. in the primary forest, traps were placed in four distinct areas, reflecting the succession stages of the forest from building to maturation. in the logged-over forest, litter production was measured at two different sites, one cut in 1979/1980 and the second in 1983/1984. in the primary forest, average litter production during the observation period was 925 g m"2yr'. in the logged-over forest, average litter production was 721 g m'2 yr1 for the site cut in 1979/1980 and 706 g m'2 yr1 for the site cut in 1983/1984. leaves comprised the major contributor of litter with 67% of total litter produced in the primary forest, 67% of total litter in the 1979/1980 cut logged-over forest, and 65% of total litter in the 1983/1984 cut logged-over forest. the purpose of the study was to use litter fall as a measure of forest productivity to assess the recovery of logged-over forests and, to provide a basis for comparison of forest-derived land practices for appropriate forest management strategies. key words: litter production/primary forest/logged-over forest/forest productivity/pasirmayang/jambi. introduction knowledge of forest productivity is a basic requirement for determining appropriate management strategies for forests, to maintain or even increase productivity. forest productivity is based on the capture of solar energy and soil-derived nutrients to form chemical compounds used to synthesize various compounds for plant growth and reproduction (binkley 1986). therefore, litter fall can be used as an indicator of productivity (medwecka-kornas 1970). forest productivity is influenced by a range of factors such as nutrient status of the ecosystem, organic matter decomposition, nutrient cycle in the plant (biochemical), nutrient cycle in the ecosystem (biogeochemical), and nutrient cycle between ecosystems (geochemical), which are very complex, since it is also influenced by climate and the availability of nutrients from parent rocks. present address : * lecturer, faculty of forestry, bogor agricultural university, campus darmaga, bogor, indonesia email : wasrinsy @ indo.net.id " ** staff, bali barat national park, bali, indonesia 36 biotropia no. 14, 1999 twigs, bark, flowers, and fruit or seed (small to fine litter), and branches and fallen stems (big litter) to a lesser extent (medwecka-kornas 1970). litter fall can be measured by using litter traps in the form of a basket, box or funnel (medwecka-kornas 1967; newbould 1967; korcagin 1960). for tropical forests, kira & schidei (1967) suggested to use baskets with a circular diameter of 50 cm (medwecka-kornas 1970). the frequency of litter collection from the trap depends on the phenology and the method used. nevertheless, measurement of litter production should represent a minimum period of one year of the total growth season, and should be monitored continuously over several years for at least 3 years (medwecka-kornas 1970). although there is hardly any primary lowland forest left in sumatera, we aimed at collecting litter fall data in one of the last remnants, to provide a basis of comparison for forest-derived land use practices. selective cutting to harvest forest timber practiced up to recent days has resulted in a mostly degraded vegetation structure such as pioneer trees with low canopy (dynamic /), a group of young trees with low canopy (dynamic 2), trees with low branches and small crowns (homeostatic 1 ), and if left undisturbed for sometime (10 years ), this yiejds tall trees with high branches and large crowns similar to the primary forest (homeostatic 2). based on the different vegetation structures following various logging practices and its impacts, we hypothesize that litter fall will differ in quantity and quality (plant components) among various gaps of successional stages of lowland forest, while the litter fall of old logged-over forests (10 years old) equals that of the primary forest. materials and methods to measure litter production, 4 traps were made of nylon with a 1 mm x 1 mm mesh. the circular trap mouth has a diameter of 1 meter and is fixed by 3 iron pipes about 100 cm from the ground, and the lowest part of the trap located about 20 cm from the ground (fig. 1). the circular form avoids the margin effect of the plot. trap mouth nylon wire iron pipe figure 1. illustration of the li 37 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra in the primary forest, measurement was conducted inside a 3 ha area (100 m x 300 m) which is divided into 20 m x 20 m plots, arranged systematically at 25 traps per hectare (fig. 2) fig. 3. vegetation dynamics phases inside the litter production observation plot at a primary forest site after laumonier (1997) fig. 2. location of litter traps inside the 3 ha plot of a primary forest litter trrap arrangement of traps was based on the four vegetation dynamics phases used b laumonier (1997) and defined as follows : dynamic 1 : dominated by pioneer trees with very low canopy. dynamic 2 : dominated by trees of the future with low canopy. homeostatic 1 : dominated by trees of the present with low branches and small crowns. homeostatic 2 : dominated by tall trees of the present with high branches and large crowns. 38 biotropia no. 14, 1999 in the logged-over forest, litter production was assessed in two different forest areas, one cut in 1979/1980 and the second in 1983/1984. the traps were systematically placed inside a square plot with distance between traps of about 50 m. the total number of traps was 25 representing about a 200 m x 200 m area (fig. 4). it was assumed that the logged-over forest only had one vegetation dynamic phase. litter collection from traps was .carried out monthly. collection from the primary forest began from august 1991 until november 1993 and was resumed during the period of june 1994 until july 1996. collections from the 1979/1980 cut logged-over forest were made from july 1994 july 1996, and from the 1983/1984 cut logged-over forest during march 1994 july 1996. figure 4. location of litter traps inside a 200 m x 200 m plot of a logged-over forest site. litter was classified as leaves, flowers, fruits, stipules, twigs, bark and others. litter was oven-dried at a temperature of 105°c for 48 hours and dry weight was measured according to wasrin et al. 1995. results and discussion a. litter production at the primary forest measurement of litter production at the primary forest was conducted from august 1991 november 1993 interrupted from october 1993 to may 1994, due to a technical problem. a total of 54 measurements were obtained that fluctuated during the observation period, as shown in fig. 5. litter production in gm'2 was obtained upon conversion of litter production in g trap"1, by dividing the amount by 39 litter trap litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra trap size (0.785 m2). litter production shown is the monthly average value collected from 75 traps. the highest amount of litter production recorded was 169 gm~2 in october 1991. the smallest amount was 39.22 gm"2 in february 1996. average litter production during the observation period was 77 gm~2month~' or 925 gm~2year~'. this value is lower compared to the number given by wanner in jordan (1983) in the rain forest of north borneo (1070 gnv2year') and those of kira in jordan (1983) in a tropical rainforest of malaysia (1055 gnv2year~'), but higher compared to the rainforest in java (810 gm"2year~'). in general, leaves contributed the largest portion of monthly litter production with an average of 48 gm^month"1 or 62 % of total litter production, followed by twigs (22 %), others (litter components that cannot be identified or classified further due to their small size) (6 %), bark (4 %), fruit (3 %), stipules (2 %), and flowers (1%). month figure 5. total litter production per month (average of 75 traps) in the primary forest during 54 months of observation (august 1991 j u l y 1996) fig. 6, 7, and 8 show fluctuations in litter production attributed to specific plant parts over the observation period. leaves significantly influence the results for total litter production as leaves comprise the major constituent. thus, the high amounts attributed to leaves (fig. 6) correspond to those recorded for total litter (fig. 5) with highest values in august 1991, october 1991, september 1993, august through october 1994, and september 1995. compared to rainfall levels at the study site (fig. 9) there is a good correlation between high amounts of leaf litter and relatively low levels of rainfall. on the other hand, leaf litter decreased during months of high levels of rainfall. 40 biotropia no. 14, 1999 figure 6 . leaf and twig litter production during 54 months of obserbation in the primary forest site. figure. fruit and flower litter production during of months of observation in the primary forest site. 41 litterfal in a primary and two logged-over forest – upik r. wasrim & agus eka putra figure 8. stipule, bark and other litter component production during 54 months of obserbation in the primary forest site figure 9. average variability rainfall per month derived from monthly rainfall data between 1985 -1994. total litter production is also high, albeit less than half of the highest total value for january 1992, march 1993 and december 1994. this could be attributed to the increase in fruit litter in january 1992 and twigs litter in march 1993 and december 1994. fig. 7 compares litter attributed to flowers to that of fruit. the pattern of accumulation for each over time is relatively similar where periods of increased 42 biotropia no. 14, 1999 flower litter are followed by periods of increased fruit litter three months later as would be expected. for example, flower litter was high in october 1991 and october 1994 and fruit litter was optimal in january 1992 and february 1995. similarly, a relatively high level of fruit litter occurred from january 1995 till april 1996, preceded by a moderate accumulation of flower litter in november and december 1995. litter from other plant parts like twigs, stipules, and bark did not follow clear patterns of accumulation. fig. 10 compares litter production to the vegetation dynamics phases of a primary forest. the average monthly data of litter produced was obtained from 11 traps for the dynamic 1 phase, 26 traps for the dynamic 2 phase, 11 traps for the homeostatic 1 phase, and 27 traps for the homeostatic 2 phase (see fig. £ and fig. 3). in general, litter produced in the homeostatic 2 phase was higher than that produced in the other phases, with a total amount of 68 gm'2 month"1 compared to 62 gm"2month'' for homeostatic 1, 58 gm"2 month"1 for dynamic 2, and 53 gm"2 month"' for dynam ic 1. figure 10. litter fall in a primary forest based on vegetation dynamics phase. to assess the relationship between litter production, the rate of litter decomposition, and the vegetation dynamics phases, the data were statistically analyzed using anova (setyawan 1999). the results indicated that the data for the four vegetation dynamics phases, the logged-over forest cut in 1979/1980, and the logged-over forest cut in 1983/1984 were significantly influenced by litter production. when the data for the vegetation dynamics phases of the primary forest are 43 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra compared to data for the logged-over forests, the highest amount of litter was produced in the homeostatic 2 phase, followed by the homeostatic 1, dynamic 2, and dynamic 1 phases, the logged-over forest cut in 1979/1980, and the loggedover forest cut in 1983/1984 with the lowest amount (setyawan 1999). the results suggest that litter production increases with stability or maturity of the forest ecosystem, considering that the vegetation dynamics phases of the primary forest from dynamic 1 to homeostatic 2 reflect a succession process toward stability or maturity characterized by the increase in tree structure. increased stability or maturity of a forest ecosystem results from the increase in forest structure, both horizontally (species distribution) and vertically (stratification). according to ewel (1983) forest structure increases with maturity. this increase in forest structures characterized by an increase in tree crown density, species diversity, tree age, and biomass that influences increased litter production (setyawan 1999). odum in jordan (1983) suggested that net production of litter increases as vegetation becomes larger and leaf area index goes up. litter production is optimal during the climax vegetation stage, and changes in the course of the succession process. litter production varies as it can be influenced by season, tree species, crown density, light, temperature variation during day and night, nutrient availability, disease, age of trees, and forest habitat size (alrasjid 1986; heald 1971). the lower value obtained for litter production in a logged-over forest compared to the primary forest, suggests that the logged-over forest structure is still in the building phase. this would mean that the logged-over forest structure is still unstable, and that its vegetation dynamics phase is more juvenile compared to the primary forest. the lower tree biomass resulting from the logging activity may also account for the lower value of litter production. soerianegara in setyawan (1999) suggested that logging activity might change the forest environment from climax to the unstable condition. in turn, this could affect tree growth. ecologically, logging activity could reduce biodiversity and soil fertility, increase soil erosion and surface run-off, thus decreasing the environmental quality in general. nutrient supplies decrease and forest stability is disturbed. b. litter fall in logged-over forest. the observation period for litter fall in a logged-over forest was shorter than in the primary forest, i.e. 29 months for the logged-over forest cut in 1983/1984 and 24 months for the logged-over forest cut in 1979/1980. litter fall in the logged-over forest cut in 1979/1980 and 1983/1984 are 60 gnrtnonuy1 or 721 gm^year1 and 59 gm"2month"' or 706 gm~2year~', respectively. if we compare litter production of the primary forest to that of the logged-over forest over the same observation period (fig. 11), the logged-over forests show relatively small differences in litter fall compared to the primary forest. litter fall amounts for the logged-over forest cut in 1979/1980 approach those for the primary forest, whereas litter fall levels for the logged-over forest cut in 1983/1984 are lower. this would suggest that the longer 44 biotropia 14, 1999 figure 11. litter fall of a primary forest and logged-over forest between july 1994-july 1996. time period following logging activity allowed the logged-over forest to recover its ondition toward climax, and begins to resemble the primary forest. the proportional distribution of litter components from the total in the loggediver forest cut in 1979/1980 and cut in 1983/1984 are presented in figures 12 and figure 12. percentage of litter component (average monthly dry weight) in a logged-over forest out in 1979/1980 45 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra 13. like in the primary forest, leaves and twigs still comprise the major form of litter in the logged-over forest. however, the proportion of leaf litter to the other components in the logged-over forest is slightly higher than in the primary forest. the dynamics of leaf litter production in the logged-over forest cut in 1979/1980 and cut in 1983/1984 are shown in figures 14 and 15. in general, the highest production of leaf litter occurred in september october, while the lowest amount was in february april. this pattern was reflected in the total litter production,dynamics of both forest types (figures 16 and 17) as leaf ijtter yields the largest contribution to total litter produced. if we compare this with the average monthly rainfall data recorded between 1985 1994 (fig. 9), highest leaf litter production coincides with months of relatively low rainfall or toward the end of the dry season, while the lowest levels occurred in months with relatively high rainfall. this pattern is similar with that for the primary forest. this relates to the increase in availability of sun light in the dry season that increases photosynthetic activity. photosynthesis produces carbohydrate as a source of biomass increase and maturation of leaves and other parts of the plant, thus contributing to the increase in litter weight. the relation between flower and fruit litter production in the logged-over forest cut in 1979/1980 is not obvious from the data obtained. fig. 18 shows that figure 13. percentage of litter component (average monthly dry weight) in a logged-over forest out in 1983/1984 46 biotropia no. 14, 1999 figure 14. leaf and twig litter production between july 1994 – july 1996 in the logged-over forest cut in 1979/1980 figure 14. leaf and twig litter production between march 1994 – july 1996 in the loggedover forest cut in 1983/1984 47 litterfall in a primary and two logged-over – upik r. wasrin % agus eka putra figure 16. total litter production between july 1994 – july 1996 in the logged-over forest out in 1979/1980 figure 17. total litter production between march 1994 – july 1996 in the logged-over forest 1983/1984 48 biotropia no. 14, 1999 figure 18. fruit and flower litter production between july 1994 july 1996 in the logged-over forest cut in 1979/1980. relatively high production of fruit litter occurred in march 1995 and march 1996, whereas peak production of flower litter was recorded in august 1995 that might be responsible for the increase of fruit litter production in march 1996. referring to the rainfall data in fig. 9, highest flower litter production occurred in the months with the lowest monthly rainfall between october december, while highest fruit litter production occurred in the months with the highest rainfall between january march or during the wettest months. there is a similarity with the primary forest. however, to observe the dynamics of fruit and flower litter production in more detail, longer period of observation of the logged-over forest is needed for at least the same period of observation as that for the primary forest. in the logged-over forest cut in 19&3/1984 the highest amounts of leaf litter were produced between august october 1994 and in september 1995, while the lowest amounts were produced in june 1994, april 1995 and march 1996. the highest amounts of fruit litter were produced in march 1994, march 1995, and march 1996,.whereas the highest amounts of flower litter were produced in october 1994, july 1995 and november 1995. fig. 19 also shows that the increased production of flower litter influences fruit litter production proportionally. 49 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra figure 19. fruit and flower litter production between july 1994 july 1996 in the logged-over forest cut in 1983/1984. acknowledgments this research is part of a joint research program between biotrop and barito pacific timber group (bptg) at the pasirmayang permanent plot, jambi. the authors gratefully acknowledge mr. husin and mr. harmen and their staff at pt. ifa-bptg for their assistance and support during the field work. we also wish to thank mr. musa for his assistance in the field and mr. waluyo for his assistance in the laboratory. our sincere thanks are also due to prof. dr. ralph ockerse, academic advisor and team leader hep-2 yogyakarta, for his generous assistance in rewriting the manuscript. references alrasyid, h. 1986. pelepasan unsur c organik dan unsur hara mineral l a i n n y a selama pelapukan serasah di areal tegakan sisa hutan alam mangrove, sungai sepada, kalimantan barat. bu let i n penelitian hutan 503 : 29-44. binkley, d. 1986. forest nutrition management. a wiley-lnterscience publication. new york. 290 p. ewel, j. 1983. succession. in. golley, f.b. (eds.). 1983. ecosystem of the world 14a. tropical rain forest ecosystems : structure and function. elsevier scientific publishing company. amsterdam. 381 p. jordan, c.f. 1983. productivity of tropical rain forest ecosystems and the implications for their use as future wood and energy sources. in : ecosystems of the world 14a.tropical rain forest ecosystems : structure and function. elsevier scientific p u b l i s h i n g company. amsterdam, p. 117-132 kira, t. & t. schidei. 1967. primary production and turn over of organic matter in different forestecosystem. ecology. vol. 17, no. 3 : 70 -87. 50 biotropia no. 14, 1999 korcagin, a.a. 1960. methods of determination of the seed productivity of forest trees and forest communities. field geobotany ii. acad. of science of the ussr press. laumonier, y. 1997. the vegetation and physiography of sumatra. geobotany 22. kluwer academic publisher. medwecka-kornas. 1967. ecosystem studies in a beech forest and meadow in the ojcao national park. in: studia nature. ser. a medwecka-kornas. 1970. plant litter production. unesco. newbould, p.j. 1967. methods for estimating the primary production of forests. ibp handbook no. 2. blackwell scientific publications. oxford. 62 p. setyawan, t.p. 1999. hubungan produktivitas dan laju dekomposisi serasah pada hutan alam primer dan hutan bekas tebangan di hutan alam produksi pasirmayang, jambi. skripsi. jurusan manajemen hutan, fakultas kehutanan, institut pertanian bogor. bogor. 67 p. wasrin, u.rosalina, a. eka putra and i. setiawan. 1995. optimalisasi produktivitas lahan hutan alam produksi di areal pengusahaan hutan pt. ifa-barito pacific timber group, di pasirmayang, jambi. laporan penelitian. kerjasama antara seameo-biotrop dengan barito pacific timber bogor. 52 p. 51 biotropia vol. 28 no. 2, 2021: 117 127 doi: 10.11598/btb.2021.28.2.1174 117 natural habitat of bali starling (leucopsar rothschildi) in bali barat national park, indonesia sutomo1 and eddie van etten2 1research centre for plant conservation and botanic garden, indonesian institute of sciences (lipi), candikuning, baturiti, tabanan, bali 82191, indonesia 2school of science, edith cowan university, joondalup, perth, western australia 6027, australia received 18 december 2018/accepted 26 march 2020 abstract the indonesian tropical savannas and dry forests provide habitats to various endemic wildlife. unfortunately, a few of these endemic species are now seriously threatened and are red listed in the conservation status of international union for conservation of nature (iucn). among these species, the bali starling or bali mynah leucopsar rotschildi, locally known as jalak bali, is now mostly restricted to the bali barat national park. given the high extinction risk faced by such species, conservation programs require multidisciplinary approaches that would address both the biological attributes of the species itself and their habitat requirements. regrettably, for many species, their habitat ecology remains inadequately understood. hence, this study aimed to: 1. characterize the bali starling habitat in terms of structure and floristic composition; and 2. document evidences of vegetation cover changes in the bali barat national park. analysis of remote sensing imagery and field sampling for vegetation attributes were conducted to address these objectives. normalized difference vegetation index (ndvi) was calculated from landsat imageries using red and near infrared bands. tree cover percentage data were downloaded from vegetation continuous fields (vcf) of the university of maryland’s website. results showed that forest and savanna are the dominant land cover types in the bali barat national park. however, their distribution is somewhat dynamic with changes in vegetation cover and greenness found across the years which increase the cover of woody plants is the general trend. the bali starling in the bali barat national park is mostly found at or near distinct vegetation boundaries, such as the borders between savanna and forest, savanna and cropland, savanna and shrubland, settlement and cropland and, between forest and shrubland. although cekik in jembrana, bali and brumbun bay in west bali, as the conservation sites for bali starling, are both planted with tree species providing shelter and food for bali starling, the bird has not been seen in the two areas since the 1990s. these results further confirm the importance of examining the habitat patterns of endemic birds within a landscape that are influenced by multiple factors interacting in space and time. addressing data inadequacy in habitat patterns of endemic species distribution is crucial in developing conservation management strategies. hence, evaluating the habitat remnants of the bali starling is vital for its conservation and needed reintroduction and eventual release to its natural habitat. keywords: bali starling, habitat suitability, savanna introduction tropical savannas and dry forests are important ecosystems which comprise those habitats supporting various endemic wildlife of indonesia. a few of these species are now under serious threat of extinction and, consequently, have high conservation status according to the iucn (iucn 2014), including banteng (bos javanicus) that is now mostly confined to the savanna in the baluran national park of east java province, the komodo dragon (varanus komodoensis) which is endemic to the komodo islands of east nusa tenggara province, and the endemic bali starling bird (leucopsar rotschildi) which is now mainly found in the savanna of bali barat national park (bbnp) on the northwest tip of bali province. unfortunately, the rapid and widespread habitat loss and variable management capacities in natural reserves had posed considerable risk to biodiversity (purwandana et al. 2014). *corresponding author, email: tommo.murdoch@gmail.com biotropia vol. 28 no. 2, 2021 118 the critically endangered bali starling (leucopsar rothschildi) is the only endemic bird found in bali. the first bali starling known to science was collected near bubunan, bali and was described by stresemann (1912). the bali starling is an attractive aviary bird being largely white, with black wings, tail tips and bare skin of a turquoise-blue color on the lores and behind the eye. on account of its restricted range, extremely small numbers growing in the wild and persistent pressures on the last free ranging birds, the bali starling is considered critically endangered according to the latest international union for conservation of nature (iucn) threat categories (iucn 2014). habitat destruction and bird capture for the pet trade brought the species to the verge of extinction (van balen et al. 2000). in 1998, estimates showed that less than 20 birds remained in the wild within a small area on the northwest tip of bali island within the boundaries of bali barat national park (bbnp) (collins et al. 1998). although the wild population is near extinction, bali starlings have been successfully bred in captivity (collins et al. 1998). the species’ original habitat in bali was described as ‘dry savanna and shrub woodlands’ and ‘tall and dense forest’ in the 1920s (van der paardt 1926), and until this time it was believed to be historically restricted to a narrow belt of dry monsoon climate in northern bali and east java (van balen et al. 2000). from 1920-1960, its range had shrunk to the fire-induced open shrub and savanna woodland, found below an elevation of 150 175 masl in the northeast part of the prapat agung peninsular within the bbnp (van balen et al. 2000). bird distribution and abundance patterns within a landscape are influenced by multiple factors interacting spatially and temporally (orians & wittenberger 1991). habitat structure and floristic composition, such as percentage of canopy cover, tree species diversity and the distribution of specific plant taxa, are known to have significant roles in defining the spatial occurrence of bird species (james & wamer 1982; rice et al. 1984; wiens & rotenberry 1981). with the high extinction risk faced by such species, intended conservation programs are likely to require multidisciplinary approaches that would address both the biological attributes and resource requirements of the species, such as their habitat requirements and conditions (estoque et al. 2012). therefore, habitat evaluation is important in the conservation of bali starling and its reintroduction and eventual release back to their natural habitat. moreover, knowledge and better understanding on the potential distribution and habitat suitability of the bali starling is important in selecting potential sites for future ex-situ conservation and breeding programs designed to save this endemic bird from its extinction. several studies on the bali starling have focused on the bird itself; ranging from its behavior, reproduction, breeding, genetics, taxonomy, demography and reintroduction, among others (collins & smith 1994; collins et al. 1998; de iongh et al. 1982; dirgayusa et al. 2000; seibels et al. 1997; williams & feistner 2006). however, studies on the habitat of bali starling are scarce (widodo 2014), considering that these habitats are vital to the ongoing maintenance of its viable populations, and yet are also very prone to conversion, disturbances and degradation. therefore, this study aimed to: 1. describe the current distribution of bali starling and characterize its habitat structure at the bali barat national park in terms of its plant community structure and composition, and 2. assess the cover and greenness index (ndvi) to quantify the dynamics of vegetation cover in these habitat areas; and 3), assess the degree of recent habitat changes broadly affecting the bali starling species at bali barat national park (bbnp). materials and methods the bali barat national park (bbnp), located on the northwestern side of bali, indonesia, covers around 19,000 ha (~ 5% of bali's total land area) comprising 15,588 ha of terrestrial areas and 3,415 ha of marine habitats. a seaport at gilimanuk is situated on the west of the park. bbnp is also bordered with several villages and can be reached by roads from gilimanuk and singaraja, or by using ferries from ketapang, east java. several major habitat types are found in the national park; savanna, mangroves, montane and mixed-monsoon forests, and, coral islands. bali barat was bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 119 designated as a national park in 1984 based on the ministry of forestry decree (no. 096/kptsii/1984). the area is predominanly of the latosol soil type, reddish in color, weakly crumbed and sticky when wet although hardened and cracked when dry. the park is topographically varied, ranging from plains near the coast to steep hills and mountains. it has four mountains, namely prapat agung, banyuwedang, klatakan and sangiang (the highest peak at 1,414 m). off the coast, four islands are also under the bbnp jurisdiction, namely menjangan, burung, gadung and kalong islands. bbnp has a moderate climate seasoned with monthly rains, but higher rainfall occurs during the wet season in december to february. the average annual rainfall ranges from 900 1,500 mm and average temperature is at 33°c (masy'ud et al. 2008; masy'ud et al. 2007; whitten et al. 1996). reports on the bali starling population, distribution and their habitat were obtained through the published literature and also via personal communications with bbnp rangers and managers. data on its local distribution were obtained from de longh et al., (1982), whitten et al., (1996), van balen et al., (2000) and bbnp manager, wiryawan, (2014, pers.comm.). based on these studies, the bali starling occurrence data were divided into three eras of distribution, namely 1984, 1994 and 2010, considering the only 3 years of reliable and accurate surveys done on these years. an overlay analysis of these bali starling location data was done using indonesia’s topographical/ earth surface map (rupa bumi indonesia/rbi) for the year 2001 (scale 1 : 80,000) obtained from the indonesian geospatial agency (big/bakosurtanal). the 2001 land use data was the most recently available. to avoid misalignment, all the data used the same datum and the same map projection within the gis (wgs 1984-utm, zone_50). the vegetation continuous fields (vcf) collection contains proportional estimates for vegetative cover types: woody vegetation, herbaceous vegetation, and bare ground. the product is derived from all seven bands of the moderate resolution imaging spectroradiometer (modis) sensor onboard nasa's terra satellite. this continuous classification scheme of the vcf product may depict areas of heterogeneous land cover better than traditional discrete classification schemes. while traditional classification schemes indicate where land cover types are concentrated, this vcf product is best for showing how much of a land cover such as "forest" or "grassland" exists anywhere on a land surface (dimiceli et al. 2011). ndvi is an index derived from remotely sensed imagery which can differentiate between vegetation types by showing the difference between near infrared (which is strongly reflected by vegetation) and red light (which is absorbed by vegetation). ndvi is correlated to vegetation biomass, vigour and photosynthetic activity. this index exploits the reflectance patterns of ground elements in the red (r) and near-infrared (nir) bands of the electromagnetic spectrum to distinguish green vegetation from its background soil brightness, and is calculated as (nir r)/ (nir + r). ndvi values range from -1 to 1, with positive values representing vegetated areas and negative values representing non-vegetated regions (sankaran 2001). the ndvi ratio approach usually adopted for land cover change estimation is used here in preference to the more commonly employed post-classification pixel-by-pixel comparison method (lillesand et al. 2008) since it also permits identification of areas where changes in vegetative cover have been significant, but insufficient to cause change in class membership (sankaran 2001). in order to generate normalized difference vegetation index (ndvi), a number of landsat images were used. landsat images were downloaded from http://earthexplorer.usgs. gov/path 117, row 066. the chosen downloaded images are those with minimal cloud cover percentage by selected scenes with at least image quality level 9 (no errors detected, perfect scene) (table 1). biotropia vol. 28 no. 2, 2021 120 table 1 details of downloaded images for ndvi analysis images source date acquired spatial resolution image quality cloud cover 1 landsat 4 21/03/1989 30 x 30 m 9 20 2 landsat 7 12/11/1999 30 x 30 m 9 8.63 3 landsat 7 31/05/2003 30 x 30 m 9 7.53 4 landsat 8 11/06/2016 30 x 30 m 9 24.99 ndvi was generated using the ndvi feature in arcmap (arcgis 10.1) image analysis toolbar. band 1, 2, 3, and 4 were chosen for landsat 4 and 7, whereas band 2, 3, 4, and 5 were chosen for landsat 8 as input images in arcmap which represent the blue, green, red and near infrared (nir) bands. by choosing the image analysis tab, all the bands layers were merged into one composite layer and then the rgb (red-green-blue) channels were adjusted to show just the nir, red and green bands to extract the ndvi values. once ndvi images were generated, different levels of a green color scheme was applied for easier interpretation. these ndvi values must be used carefully as they represent only one time in the chosen years (due to the limited availability of good images for the chosen years) and as these will mainly reflect recent rainfall, especially in terms of groundcover. then the data points for the 1984, 1994 and 2010 bali starling locations were overlaid on the ndvi images from different years (1989, 1999, 2003 and 2016). these years were chosen because these years were the closest years to the years of bali starling location (as clear image of the exact years of bali starling location cannot be found). the mean and sd from nine pixel values surrounding the exact coordinate locations of the bali starling was then calculated from the ndvi images. changes in mean ndvi of starling locations between different years were tested for significance using anova in spss and, when significant differences were detected, a post-hoc test was performed. from 1979 to 1994, the bali starling was regularly observed in brumbun bay, west bali. from 1995 to 2009, no survey was done in the area, therefore no bali starling data were obtained from these locations. in 2010, the surveys conducted by bbnp has recorded bali starling population in this location again. at another site, cekik in jembrana, west bali, the bali starling was also observed during the period 1979-1994, but in the 2010 survey, bbnp did not find the starling in the area. fieldwork was again conducted in november 2014 in these two locations, namely cekik and brumbun, and these areas were cross-checked with a fire map based on modis burned areas produced from year 2000 to 2013 to obtain information on fire history. however, no fire occurred at these localities. the landsat imagery during this period also confirmed that no major fires occurred at bbnp. during the dry season in september to november 2014, ten sampling plots of 50 x 50 m were established randomly in each savanna sites (cekik and brumbun). in each of the 50 x 50 m plots, smaller plots of 5 x 5 m were nested randomly. inside the 50 x 50 m plot, all the tree species ≥ 10 cm diameter at 1.3.m height (dbh) were identified, measured and recorded. in the smaller-nested plots, all the groundcovers species were noted (grasses, herbaceous and ferns) and their coverages were estimated. species in each site were identified in the field where possible and a field herbarium was created for easier identification at the species level during subsequent field works. the identification was assisted by personnel from herbarium bogoriense and herbarium baliensis of the indonesian institute of sciences (lipi) who used flora books such as the “flora of java” (backer & van den brink 1963), “mountain flora of java” (van steenis 1972), “weeds of rice in indonesia” (soerjani et al. 1986), “ ecology of java and bali” (whitten et al. 1996) and “ecology of nusa tenggara and maluku” (monk et al. 2000) and names were standardized based on the plant list (www.theplantlist.org). the importance value index or ivi (kent 2011; kent & coker 1992) was calculated for each species in each plot to understand the structure and plant community composition of each savanna. ivi was used to describe the quantitative structure of the community (curtis & mcintosh 1950; kent & coker 1992). this value represents the contribution of a species to the community in terms of the number of plants within the quadrats (density), its contribution to the community through its distribution (frequency), and its influence on the bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 121 other species through its dominance. the importance value index was calculated for each tree species and ground cover in each of the study sites. the differences in plant community composition between brumbun and cekik savanna were tested using abundance data (cover). the data were square-root transformed prior to constructing a resemblance matrix based on the bray-curtis similarity index (valessini 2009). a non-metric multidimensional scaling (nmds) ordination diagram was then generated based on the resemblance matrix. the difference in species composition between savannas was then tested for significance using one-way anosim (analysis of similarity). the ranosim statistic values, generated by anosim, are a relative measure of separation of the a priori defined groups. a zero (0) value indicates that no significant difference existed among the groups, and one (1) value indicates that all samples within groups are more similar to one another than any samples from different groups (clarke 1993). this multivariate analyses made use of the primer v.6 package (clarke & gorley 2005). results and discussion based on the bird observations analysis, the starling is mostly found in a relatively open vegetation (such as savanna and open shrubland) and along their boundaries with other vegetation types in the bali barat national park (bbnp) (fig. 1). this confirms the reports of (de iongh et al. 1982; dirgayusa et al. 2000). based on the bbnp manager’s report, the bird was observed at or near the ecotones between savanna and forest, savanna and cropland, savanna and shrub land, settlement and cropland, and finally, the forest and shrubland. this report is supported by the ndvi analysis for the three-year record of the species locations in 1984, 1994 and 2003 which showed a shift in the preferred habitat of the bali starling from primary forest habitat in the 1980s to more open vegetation areas including, but not limited to, dry forest/monsoon forest, secondary forest and savanna (fig. 2). these results further confirmed those of van balen et al. (2000) that the bali starling range on bali island has shrunk to the fire-induced open shrub and savanna woodland below elevations of 150-175 m in the northeast part of the prapat agung peninsular of the bali barat national park. biotropia vol. 28 no. 2, 2021 122 figure 1 overlay of bali starling occurences with the 2001 land use map of bali barat national park notes: ‘sawah tadah hujan’ refers to a rain fed paddy field. ‘sawah irigasi’ refers to irrigated paddy field. 1984 bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 123 1994 2010 figure 2 bali starling habitat distribution based on their normalized difference vegetation index (ndvi) range class notes: habitat and localities are presented for 1984 (upper, aligned with ndvi image data 21/03/1989), 1994 (middle, aligned with ndvi image date 12/11/1999) and 2010 (lower, aligned with the ndvi image data 11/06/2016); ranges of ndvi: 0.2 0.3 = cropland-grassland-savanna; 0.301 0.4 = savanna shrubland-mangrove; 0.401 0.5 = dry forest-monsoon forest; >0.5 = primary forest-broad leaves evergreen forest. this ndvi classes follow siswoyo (2014). this apparent shift in the habitat preference of bali starling is likely to have been influenced by several interacting factors, primarily, the lack of fire in the bbnp. changes in fire regime has resulted in the presence of more shaded habitat as very little grasslands/savannas have become available. secondly, the increased human population and associated infrastructure and land use (dwellings, roads, croplands) in the areas adjacent to bbnp, that led to a decreasing available habitat for the bali starling. the cekikgilimanuk area has experienced a major increase in human-dominated land uses, mainly through conversion of savanna. accounts of the local inhabitants indicate that the conversion of monsoon forest to agricultural land had a negative impact on bali starlings (van balen et al. 2000). lastly, the shift in habitat preference is perhaps due to changes in plant species composition and vegetation structure (which is also related to the lack of fire) which has affected the bali starling utilization of plants for food and nesting. some 22 plant species belonging to 14 families were recorded in the two savannas. at cekik 10 species belonged to eight families, whereas at brumbun 20 species were from 12 families. significant differences were observed using the bray-curtis species similarity index biotropia vol. 28 no. 2, 2021 124 between the savanna sites (ranosim = 0.228; p<0.003) (fig. 3). eight species were present in both savannas (cekik and brumbun sites) namely chromolaena odorata, lantana camara, desmodium laxiflorum, grewia eriocarpa, bridelia stipularis, cynodon dactylon, calamagrostis australis, and ziziphus mauritiana (synonym z. jujuba). using the shannon-wiener species diversity index, no significant difference (p>0.05) was observed between brumbun and cekik, however species richness was significantly different (p<0.05) between the two savannas (fig. 3). brumbun had more species than cekik. plant species composition was categorized based on their different uses by the bali starling, namely: food, shelter (nesting) or a combination of both (table 2). as plant-food source, cekik has generally higher cover. six species used as a food source for bali starling were ziziphus mauritiana, grewia eriocarpa, schleichera oleosa, streblus asper, azadirachta indica, (tree species) and lantana camara (herbaceous). two species, borassus flabellifer (arecaceae) and acacia leucophloea (synonym vachellia leucophloea) (fabaceae), were both used for shelter. the latter (a. leucophloea) was also utilized as a food source (combined). some of the plant species used, s. oleosa and b. flabellifer, were present only in cekik whereas s. asper, a. indica and a. leucophloea were found only in brumbun. some plant species were present in both of the locations such as z. mauritiana, g. eriocarpa (tree species) and the invasive exotic climber l. camara (table 2). transform: square root resemblance: s17 bray curtis similarity locations brumbun 2010 cekik 1984 3d stress: 0,06 figure 3 non metric multi dimensional scaling (nmds) ordination based on the bray-curtis similarity index on plant species abundance and composition between brumbun and cekik savanna areas at bali barat national park table 2 plant species used by bali starling at the sampling sites (brumbun and cekik) in bali barat national park species family habitus usage found at and ivi ziziphus mauritiana rhamnaceae tree food cekik (32.5) and brumbun (32.5) grewia eriocarpa malvaceae tree food cekik (16.25) and brumbun (16) schleicera oleosa fabaceae tree food cekik (16.25) borassus flabellifer arecaceae tree nest cekik (61.25) streblus asper moraceae tree food brumbun (16) azadirachta indica meliaceae tree food brumbun (16) acacia leucophloea fabaceae tree nest, food brumbun (16) lantana camara asteraceae herb food cekik (26.14) and brumbun (9.8) the bali starling was not observed in cekik since the mid 1990s, although this area has plant species (schleicera oleosa and borassus flabellifer) that are known to provide shelter and food for the bird (widodo 2014). unlike similar areas, i.e., brumbun with its acacia leucophloea, that have bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 125 been successfully re-colonized by bali starling in recent times. this is probably related to several factors like plant species diversity and richness, increases in human habitation that might have led to the decrease in plant species richnessdiversity as well as the increasing risk of poaching. plant species in brumbun is richer compare to cekik. different species of tree dominate in cekik and brumbun however, the composition of the groundcover between the two locations was relatively similar where exotic invasive species such as chromolaena odorata and lantana camara dominate the understory. however, the bali starling habitat in the late 1990s were open woodlands which were dominated by a. leucophloea trees with an undergrowth of l. camara and c. odorata shrubs, and imperata cylindrica grass, and intersected by moister and more densely forested valleys with dominant trees of grewia eriocarpa, vitex pubescens, b. flabellifer and schoutenia ovata (van balen et al. 2000). this vegetation type might, however, be sub-optimal habitat for the bali starling and the bird might have been driven there by poaching pressure (van balen et al. 2000). in west java, a relatively low bird species diversity on the southern bandung (urbanized areas) was attributable to humans (fardila & sjarmidi 2012). land use and other aspects of the environment were interrelated to such an extent with bird communities distribution in north bandung, west java (fardila & sjarmidi 2012). in other studies, bird species richness was significantly higher in natural than in urban habitats in spain (palomino & carrascal 2006). one factor in the decline of the bali starling have been the conversion of savanna and forests to non-native tree plantations, crop land and villages (collins & smith 1994). this was clearly observed in the cekik area. the absence of the bali starling in cekik might also be due to land use changes (fragmented landscape) and the increasing human presence in the area. cekik is located near gilimanuk, a busy port of bali that connects the island with java island. whereas brumbun, is located on the prapat agung peninsula, a more remote area of the national park located near the ranger’s outpost in the northern tip of the national park. in fragmented landscapes, species persistence depends on their ability to use different habitats, so that less suitable habitats may still favour the connectivity of the most suitable habitats in the landscape (calviño-cancela et al. 2012). remaining fragments of native habitats such as forests are often surrounded by a matrix of modified seminatural habitats, such as tree plantations and croplands, which can still provide habitat for species associated with natural forests (lindenmayer & hobbs 2004). in spain, fragmented land, eucalypt plantations has lower species diversity than native forest, but eucalypt plantations provide habitat for species typical of shrublands when young, but do not contribute significantly to the maintenance of the understory biodiversity associated with native forests (calviño-cancela et al. 2012). both locations, cekik and brumbun, provide suitable habitats for bali starlings, although cekik is decreasing into a savanna-forest size, it is more open and more human-populated. most of the bird’s former habitat has been converted into coconut and kapok plantations, and human settlements. cekik ability to provide habitat for the species, the relatively similar species richness with brumbun, and the distribution of specific plant taxa that influence the availability of shelter, feeding and breeding resources (ziziphus mauritiana, grewia eriocarpa, schleicera oleosa, and borassus flabellifer), distinctly contribute to their importance for bali starlings. poaching is the major threat to bali starlings. in the 1960s, the birds were trapped intensively to provide the demands of indonesian, american and european private aviculturists. in 1966, the iucn listed the species as endangered (collins & smith 1994). the indonesian government responded with the 1971 law prohibiting the hunting, capture and export of the bird (van balen et al. 2000). however, poaching has continued in spite of efforts to increase patrols by the park rangers (collins & smith 1994; dirgayusa et al. 2000). in the bali barat national park, specifically in cekik, the local rangers often encountered illegal poachers checking the handcrafted “pigeon holes” made by the bali barat management to facilitate the survival of post-captivity bred starlings that were recently released in the area. this study further confirmed the importance of examining habitat characteristics and dynamics of endemic birds within landscapes that are influenced by multiple factors interacting in space and time (fardila & sjarmidi biotropia vol. 28 no. 2, 2021 126 2012; orians & wittenberger 1991). habitat structure and floristic composition, such as percent canopy cover, tree species diversity and the distribution of specific plant taxa, are known to exhibit a significant role in defining bird species’ occurrences in space (james & wamer 1982; rice et al. 1984; wiens & rotenberry 1981). conclusion in summary, this study suggests that both forest and savanna are important land cover types and habitat for bali starling at bali barat national park considering that the changes in the relative proportions of these types, as well as availability of ecotones between them, are likely to be particularly important for this species. the increase in woody plant cover in the remaining savannas of northern bbnp, which may reflect a lack of burning in the area where the bali starling is known to currently occur, is therefore of primary concern. in cekik, it is suggested to intensify protection efforts and, where feasible, to restore the native species of bali starlings to its best or much improved conservation stautus. acknowledgments the authors would like to thank edith cowan university and rufford foundation for funding this research. special gratitude for wiryawan who had provided permission to conduct this research at bali barat national park and his tremendous assistance during the research. special thanks are also extended to i ketut sandi and i.b.k. arinasa from bali botanical garden for plant sample identification. the authors agreed that each author contributes equally to the paper. references backer ca, van den brink rcb. 1963. flora of java. leiden (nl): the rijksherbarium. calviño-cancela m, rubido-bara m, van etten ejb. 2012. do eucalypt plantations 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(2) there was no difference between distilled water and 1% potato dextrose broth (pdb) as a medium for fungal spores production. (3) the combined use of chevroned water hyacinth weevil and the water hyacinth blight produces a better suppressing on water hyacinth growth. (4) there was no significant effect of heavy infection by the water hyacinth blight on oviposition habit of chevroned water hyacinth weevil. heavy fungal infection only affected feeding habit of the adult chevroned water hyacinth weevil. (5) establishment of the chevroned water hyacinth weevil is in progress at situ bagendit lake, garut regency, west java. key words: indonesia/biological coraro\lneochetina bruchi/'alternaria eichhomiaeleiahhomia crassi-pes/establ ishment. introduction background the water hyacinth (eichhorniae crassipes [mart.] solm.) originated from brazil and has spread out to many subtropical and tropical countries. the plant has been considered as a noxious weed in those countries. the water hyacinth is one of the most serious aquatic weeds in indonesia (soerjani et al. 1976; tjitrosoedirdjo et al. 1993) as well as in other southeast asian countries such as malaysia (ismail 1994). recently, as a control tehnique, biological conntrol has received more attention. however, the objective is not to eradicate but to suppress the target weed population at a tolerable level. biological control can be considered as the most important component of integrated pest management (mangoendihardjo 1995). introduction 1 biotropia no. 13, 1999 of specific and potential natural enemies is the best technique for the control of exotic weeds (andreas et al. 1976; simmons 1970). according to nag raj & ponnappa (1970) and dharmaputra (1997), alternaria eichhorniae is host specific to water hyacinth and monochoria. learning from other countries, water hyacinth weevils have shown a partial control only to water hyacinth. the combined use of the two agents has produced very good results in india (gopal, personal communication 1996). for this reason, the integrated use of weevil and pathogen in indonesia was investigated. objectives the objectives of this study were (1) to investigate the possible better effect of the integrated use ofneochetina bruchi (hustache) and alternaria eichhorniae (nag raj & ponnappa) in suppressing the growth of water hyacinth; and (2) to examine the possible change of feeding and oviposition habit of the chevroned water hyacinth weevil caused by the heavy infestation of the fungus also, observations were made to monitor the establishment of the weevil in situ bagendit, garut, west java. methodology rearing of the water hyacinth weevil the chevroned water hyacinth weevil (neochetina bruchi hustache) was introduced from australia into indonesia in 1995. after a series of tests under quarantine conditions at biotrop, the release permit was issued by the government of indonesia on june, 3 1996. the weevil was then mass reared on water hyacinth grown in concrete ponds at biotrop in bogor. preparation of fungal culture and spore suspension the fungus, a. eichhorniae used in this study was an isolate collected from situ bagendit lake at garut regency, west java. to obtain fungal spores for research purposes, the fungus was grown on rice husk yeast extract agar (rhya) for at least 7 days at room temperature. spore mass was first separated from their mycelia and medium by scrubing it using a fine brush. the spore mass was then filtered using a muslin cloth of about 50 micron pore size. experiment 1. the effect of sticking agent (tween 80) on fungal spore germination the effect of tween 80 (polyoxyethylensorbitanmonooleate) in the aqua spore suspension on fungal spore germination was carried out under laboratory conditions. various concentrations of tween 80 i.e. 0, 1, 2, 3 and 4% were evaluated. the spore concentration used was 1 x 106 spores/ml. spore germination was observed under 2 integrated use otneochetina bruchi and altemaria eichhorniae kasno et al. the microsope (magnification 10 x 10) one hour after addition of tween 80. the optimum concentration with minimum side effect was used in further experiments. experiment 2. optimum spore concentration required to cause the disease (blight) various concentrations of fungal spore i.e. 2.5 x 10s, 5.0 x 105, 7.5 x 105 and 1.0 x 106 spores/ml were evaluated under greenhouse conditions. aside from spore suspension, two concentrations i.e. 2 and 3% of tween 80 as sticking agent, as well as 1% potato dextrose broth (pdb) and distilled water as carrying agents were also evaluated as to their ability to support the fungus to cause the disease in water hyacinth. inoculation of the spore suspension (1 ml/plant) was carried out on the water hyacinth surface using an atomizer at about 04.00 pm. soon after inoculation, water hyacinth was placed in the gauze cage in the greenhouse. the cages were then covered with damp jute bags for 24 hours to create a suitable condition for spore germination and penetration. the parameter used to evaluate the test was the damage severity of the water hyacinth plant due to fungal infectionn. the damage severity score was measured at 7 days after treatment. the scoring system was based on a 0 to 4 damage severity scale. the damage severity score of 0 means no infection, while score of 1, 2, 3 and 4 indicate 1-25%, 26-50%, 51-75% and more than 75% caused leaf damage or death, respectively. experiment 3. combined use of tv. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under greenhouse conditions the target of this experiment was to obtain the optimum number of weevils in combination with spore suspension that would bring about a better effect in suppressing water hyacinth growth. the number of weevils released was 0, 1, 3 and 5 pairs/plant, while concentrations of inoculated spore were 0, 0.5, 1.0 and 1.5 times, respectively from the optimum concentration in experiment 2. water hyacinth with 6 leaves and ± 70 g wet weight was used in this experiment. the selected plants were then grown on plastic pots (1 plant/pot) containing tap water with mud at the bottom. to prevent the weevil from moving away, each pot was enclosed in transparant plastic cages with appropriate ventilation. seven days after the weevils were released, spore suspension was used to inoculate the water hyacinth (3 replications/treatment). the treated plants were then placed in cages enclosed with damp jute bags for 24 hours. parameters used to evaluate the effect of the treatments were damage severity and biomass. the scoring scales as applied in experiment 2 was also applied in this experiment. 3 biotropia no. 13, 1999 experiment 4. combined use of n. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under field conditions a procedure similar to experiment 3 was set up under field conditions during dry and wet seasons. the number of released weevils was 0, 1, 3 and 5 pairs/plant, while the concentration of spore suspensions for inoculation was 0 and 1 x 106 spores/ml/plant. three replications were set up for each treatment (3 plants/ replication) in two seasons. selection of water hyacinth to nearly the same size was made prior to setting up the experiment. observations on the effect of the two control agents were made at 14 and 21 days after weevils were released. experiment 5. possible change in adult weevil habit by selecting feeding and oviposition sites normally, the weevil fed on the leaf surface of the water hyacinth and either the leaf tissue or petiole was used as sites for oviposition. an experiment was set up to investigate the possible effect of heavy fungal infection on the weevil in selecting sites for oviposition and feeding. three pairs of adult weevil per plant were released, while the concentrations of inoculated spore suspension were 0 and 1 x 106 spore/ml/plant. five replications were set up for each treatment. an observation was done 14 days after weevils were released. parameters used to measure the possible effect were the number of feeding scars and eggs on healthy and heavily infected water hyacinth. experiment 6. monitoring of weevil establishment in situ bagendit of garut regency, west java n. bruchii was released in situ bagendit lake on august, 28 1996 at 3 points. the number of weevils released at the first, second and third point was 75, 120 and 192 pairs respectively. release was repeated on october, 16 1977. one hundred and sixty pairs of weevil were released in the center of the lake. monitoring in the first year was done to record the dispersion of weevil from the initial point. further monitoring in the second year was done to record the presence of weevil and feeding scars. therefore, 10 samples for 0.5 m x 0.5 m of water hyacinth showing feeding scars were collected. results and discussion experiment 1. the effect of tween 80 on fungal spore germination under field conditions, rain fall as well as wind may cause fungal spores fail to penetrate into the leaf surface before germination. tween 80 may help spores to fix 4 integrated use ofneochetina bruchi and altemaria eichhomiae kasno et al. on the leaf surface for a certain period of time to avoid possible dispersal of spores due to rain fall and blowing wind. however, tween 80 may cause reduction in spore germination. such agents are often necessary to disperse fungal spores within a formulation and to support the spread and adherence of spores on the plant surface (mitchell 1988). the use of certain concentrations of such agents may influence spore germination (daigle & cotty 1991). a test to evaluate the possible negative effect of tween 80 on spore germination was made using 0, 1, 2, 3 and 4% concentrations. however, there was no significant difference in spore germination. the percentages of spore germination at concentrations of 0, 1, 2, 3 and 4% of tween 80 were 96, 95, 94, 93 and 92, respectively. considering the cost of tween 80, a 2 and 3% concentrations was selected. experiment 2. optimum spore concentration required to cause a disease (blight) based on statistical analysis, the effect of spore and tween 80 concentrations gave very significant differences to the severity score of water hyacinth leaves, while the effect of media was not significantly different (appendix 1). the severity score on leaves when measured 7 days after treatment tended to increase with the increase of spore concentration. at spore concentrations of 7.5 x 105 and 1 x 106/ml, the damage severity score was not different, i.e. 3.5 (table 1). the damage severity score on leaves increased with increase of tween 80 concentration. the scores for 0, 2 and 3% tween 80 concentration were 3.0, 3.1 and 3.7, respectively (table 2). the use of 1% pdb did not show a significant difference in the damage severity score when compared to distilled water as a medium (table 2). aqueous spore suspensions containing about 1.0 x 106 spores/ml and 3% tween 80 was selected for further experiments. table 1. the effect of a. eichhomiae spore concentration, tween 80 concentration and type of media on the severity score of water hyacinth treatment scoring spore concentration (/ml) 2.5x10' 2.8 b 5.0 x l o 5 3.3 ab 7.5x10' 3.5 a 1.0 x l o 6 3.5 a tween 80 concentration (%) 0 3.0 c 2 3.1 c 3 3.7 d type of media distilled water 3.3 e 1%pdb 3.3e numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. 5 biotropia no. 13, 1 999 according to daigle & cotty (1991) addition of 1% pdb to each surfactant concentration increased spore germination capability compared to surfactant or 1% pdb alone. in this experiment spore germination with 1% pdb was not observed. the result showed that there was no difference in the damage severity score between distilled water and 1% pdb as the medium, and the use of 1% pdb was discontinued. experiment 3. combined use of n. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under greenhouse conditions based on statistical analysis the variation in number of adult weevil showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the pathogen, weevils and their combination when measured at 14 days and 21 days after weevil release (appendices 2 and 3). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (tables 2 and 3). the damage severity score of water hyacinth leaves increased with the increase of weevil numbers and spore concentrations. the damage severity score due to pathogen infection was lower than that caused by weevil infestation. nevertheless, the damage severity score caused by the combination of the two agents was higher than that caused by each agent (table 3). the damage severity score of combination between spore concentrations of 1.0 x 106 and 1.5 x 106/ml; and between weevil numbers of 3 and 5 pairs were not significantly different (tables 2v and 3). these facts indicated that the optimum combination in suppressing about 70 gram size of water hyacinth growth under greenhouse conditions was 1.0 x 106 spores/ml and 3 pairs of adult weevil. there was an indication of faster infection of pathogen on undamaged leaf tissue of water hyacinth if compared to the injured one. in other case, there was an indication of feeding preference of the adult weevil on the healthy tissue rather than on infected leaf tissue of water hyacinth. table 2. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 days after weevil release damage severity score spore cone. (/ml) pathogen no. of weevils (pairs) weevil combination 0 0 a 0 0 d 0 h 5.0 x 10s 2.4 b 1 1.8 e 2.5 i 1.0x10' 2.7 be 3 2.9 f 3.7j 1.5x10' 2.9 c 5 3.8 g 3-9j numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. 6 integrated use ofneochetina bruchi and alternaria eichhomiae kasno et al. table 3. the effect of adult n. bruchi and a. eichhomiae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 21 days after weevil release damage severity score spore cone. (/ml) pathogen no. of weevils (pairs) weevil combination 0 0 a 0 0 e 0 i 5.0x10' 2.9 b 1 2.7 f 2.8j 1.0 x 106 3.3 cd 3 3.6 g 4.0k l. s x l o 6 3.4 d 5 3.9 h 4.0k numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. experiment 4. combined use of n. bruchi weevil and spore suspension of a. eichhomiae to suppress water hyacinth growth under field conditions a. dry season based on statistical analysis, the variation in number of adult weevils showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the weevils and combined use of the two agents when measured at 14 days and 21 days after weevil release (appendices 4 and 5). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (table 4). b. wet season based on statistical analysis, the number of adult weevils showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the weevils and combination of the two agents when measured at 14 days and 21 days after weevil release (appendices 6 and 7). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (table 5). the damage severity score either due to weevil, pathogen or their combination during wet season (table 5) was lower than that during the dry season (table 4). during the wet season the higher water nutrient content may provide better growth to water hyacinth plant, therefore the plant may be more resistant to the two suppressing agents. tables 4 and 5 show that the damage severity score due to combined use of the two agents, between 3 and 5 pairs of adult weevils was significantly different. this result differed from the result in experiment 3. the different result in this experiment was assumed due to the difference in the number of plants for each 7 biotropia no. 13, 1999 replication. one plant was used in experiment 3, while 3 plants were used in this experiment. charudattan et al. (1976) reported that infection of plants inoculated with acremonium zonatum was more severe in the presence ofn. eichhorniae. sanders et al. (1982) also reported that in the panama canal a positive correlation exists between a. zonatum and n. eichhorniae. table 4. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 and 21 days after weevil release during dry season damage severity score pathogen weevil combination spore cone, (/ml) 14 days 21 days no. of weevil, (pairs) 14 days 21 days 14 days 21 days 0 l . o x l o 6 0 a 2.7 b 0 a 3.4 c 0 1 3 5 0 d 1.9 e 2.4 f 3.4 g 0 d 2.3 f 2.9 g 3.6 h 0 i 2.9 jk 3.3 kl 3.61 0 i 3.4 kl 3.81m 4.0 n numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. table 5. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 and 21 days after weevil release during wet season damage severity score pathogen weevil combination spore cone, (/ml) 14 days 21 days no. of weevil (pairs) 14 days 21 days 14 days 21 days 0 1.0 x l o 6 0 a 2.3 b 0 a 3.1 c 0 1 3 5 0 d 1.3 e 2.3 g 3.2 i 0 d 2.8 f 2.9 h 3.6j 0 k 2.81 3.3m 3.5m 0 k 3.3m 3.8 n 4.0 o numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. the change of water hyacinth wet weight at 21 days after weevil release in each treatment is presented in figure 1. the results show that the increase of adult weevils and concentration of spore pathogen reduced the increment of wet weight. it means that both water hyacinth control agents could suppress the growth of water hyacinth in the field. 8 integrated use ofneochetina bruchi and altemaria eichhomiae — kasno et al. figure 1 . the change of water hyacinth wet weight at 21 days after weevil release in each treatment. c=spore concentrations (/ml) s=adult weevils (pairs) (1.0 x 106 spore/ml) 0, 1, 3, 5=number of pairs based on statistical analysis, number of adult weevils and concentration of pathogen spore caused very significantly different effect on the change of water hyacinth wet weight (appendix 8). conway et al. (1978) reported that pathogen (cercospora rodmanii) could influence the growth of water hyacinth and reduce the biomass. according to galbraith (1987) acremonium zonatum caused extensive infection of individual leaves of water hyacinth, but the whole plant survived since disease development did not keep up with the production of new leaves. experiment 5. possible change of adult weevil habit in selecting feeding and oviposition sites figure 2 shows that the pathogen infection on water hyacinth did not reduce the production of weevil eggs. weevils preferred to put their eggs on the petiole than on the leaf. so the infection of pathogen did not affect the oviposition behavior of weevil. 9 biotropia no. 13,1999 experiment 6. monitoring of weevil establishment in situ bagendit, regency of garut, west java generally, the steps for biological control program of weeds are as follows: 1. to determine the conformity of weed species to be controlled biologically. 2. survey of natural enemies. 3. to study the ecology of weeds and their natural enemies. 4. to study the host specificity of natural enemies to secure that they could not become pest in the control area. 5. to introduce/release and establish natural enemies in the control area. 6. evaluation. one of the biological control program steps was to release tv. bruchi. it has been done and it is establishing now. monitoring was carried out after weevils were released to investigate the difference that may occur in the field (kasno 1989). monitoring was done at 2, 4 and 10 months after weevils were released indicating that the feeding symptom was found at distances of ± 45 m, ± 250 m and ± 2 km, respectively, from the initial point of release. however, no symptoms of attack were found on plantation crops surrounding the lake. tables 6 and 7 show the existence of adult weevils, eggs, larvae and pupae at 15 and 16 months after the weevils were released. population of adult weevils at 16 months after weevils were released was higher than at 15 months, i.e. from 0.8 to 1.4 insects per 0.5x0.5 m area. larvae and pupae were not found at 15 months after weevils were released. however, 16 months after the release larvae were also not found, but a small number of pupae were found i.e. 0.4. the number of eggs increased from 5.4 to 9.1. the average of leaf scars per second youngest leaf also increased, i.e. from 13.9 to 17.4. generally, it could be concluded that tv. bruchi weevil was still in the process of establishment, since the number of adult weevils, eggs, larvae, pupae and other parameters increased. 10 integrated use ofneochetina bruchi and altemaria eichhorniae kasno et al. conclusions 1. the integrated use of n. bruchi and a. eichhorniae as a controlling agent ofwater hyacinth caused a better suppressing of water hyacinth and its growth rather than using the agents separately. 2. the infection of a. eichhorniae on water hyacinth leaf did not change the oviposition site of tv. bruchi. 3. n. bruchi prefers to feed on intact than on the infected leaf tissue. 4. the damage severity score from all treatments increased through the time. 5. establishment of n. bruchi in situ bagendit lake, west java is still in progress. acknowledgment the authors would like to express our sincere thanks to the department of higher education, ministry of education and culture for the financial support, and to the plant quarantine department, ministry of agriculture for the release permit of n. bruchi. references andreas, l.a., c.j. da vis, p. harris & a.j. whapshere. 1976. biological control of weeds. in c.b. huffaker & p.s. messenger (eds.). theory and practice of biological control. academic press. new york. bashir, o.m. 1984. the establishment and distribution of natural enemy of water hyacinth released in sudan. tropical pest management 30(3): 320-323. charudattan, r., t.e. freeman & k.e. conway. 1976. progress in the use of plant pathogens as biological control for aquatic weeds. misc. pap. a-77-3. us army engineer waterways exp. stn, vickbury. miss. conway, k.e., t.e. freeman & r. charudattan. 1978. development of cercospora rodmanii as a biological control for eichhorniae crassipes. proc. ewrs 5* symp. on aquat. weeds. po box 14, wageningen, the netherlands, p. 225-230. daigle, d.j. & p.j. cotty. 1991. factors that influence germination and mycoherbicidal activity of alternaria cassiae. weed tech. 5: 82-86. dharmaputra, o.s. 1997. pathogenicity test of altemaria eichhorniae isolated from water hyacinth. seameo biotrop. internal report. galbraith, j.c. 1987. the pathogenicity of an australian isolate of acremonium zonatum to water hyacinth and its relationship with the biological control agent, neochetina eichhorniae. aust. j. agric. res. 38:219-229. ismail, m.a. 1994. biological control efforts towards management of some common weeds in malaysia. proc. symp. biology and management of weeds and 14* tropical weed science society conference. seameo biotrop. bogor. kasno. 1989. pengantar umum pengendalian gulma secara hayati. kumpulan materi kuliah pendidikan dan pelatihan pengelolaan gulma proyek p2tp. direktorat jenderal perkebunan, bogor, 6 nopember 2 desember 1989. seameo biotrop. mangoendihardjo, s. 1995. pengendalian hayati, komponen utama pengelolaan jazad pengganggu (biological control the main component of integrated pest management). pidato pengukuhan guru besar. universitas gadjah mada, 22 maret 1995, yogyakarta. 26 p. 13 biotropia no. 13, 1999 mitchell, j.k. 1988. gibbago trianthema, a recently described hyphomycete with bioherbicide potential for control of horse purslane (trianthema portulacastrum). plant dis. 72: 354-355. nag raj, t.r. & k.m. ponnappa. 1970. blight of water hyacinth by alternaria eichhorniae sp. nov. trans. br. mycol. soc. 55(1): 123-130. sanders, d.r., r.f. theriot & e.a. theriot. 1982. organisms impacting water hyacinth in the panama canal. misc. pap. a-82-1, us army engineer waterways exp. stn, vicksbury, miss. simmons, f.j. 1970. biological control. sompong press, bangkok. 12 p. soerjani, m. el al. 1976. proc. of the southeast asian workshop on aquatic weeds. seameo biotrop. bogor. 45 p. tjitrosoedirdjo, s., kasno & sunjaya. 1993. notes on the management of water hyacinth in rawa pening lake: 1990-1991. in proceedings of integrated pest management control component. biotrop special pub. no. 50: 147-155. 14 integrated use of neochetina bnichi and alternaria eichhorniae kasno et al. appendix 1. analyses of variance on the effect of alternaria eichhorniae spore concentration, tween 80 concentration,type of solvent and their interaction on the damage severity score of water hyacinth. source of variance f value spore concentration (a) tween 80 concentration (b) type of solvent (c) a x b a x c b x c a x b x c 5.18** 9.36** 0.09 0.64 1.06 0.64 0.15 ' = very significantly different at 99% confidence level. appendix 2. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhorniae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination under greenhouse conditions 14 days after weevil release. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 36.50** 173.75** 4.58** 342.33** 2.33 1.56 187.30** 38.95** 4.17** = very significantly different at 99% confidence level appendix 3. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhorniae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination under greenhouse conditions 21 days after weevil release. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 24.58** 239.95** 3.08** 491.60** 8.80** 2.43* 140.70** 50.66** 9.29** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level 15 biotropia no. 13, 1999 appendix 4. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their'interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during dry season at 14 days after weevil release under field conditions. source of variance f value pathogen weevil combination number of weevil pairs (a) spore concentration (b) a x b 1..00 504.27** 1.00 90.53** 1.59 0.18 36.91** 68.57** 8.80** *= very significantly different at 99% confidence level. appendix 5. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during dry season at 21 days after weevil release under field conditions. source of variance pathogen f value weevil combination number of weevil pairs (a)) spore concentration (b) a x b 2.00 874.27** 2.00 104.22** 6.00* 1.11 46.83** 112.32** 14.97** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level appendix 6. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during wet season at 14 days after weevil release under field conditions. source of variance f value pathogen weevil combination number of weevil pairs (a) 2.00 80.73** 75.00** spore concentration (b) 486.00** 9.80** 216.00** a x b 2.00 1.27 5.00** **= very significantly different at 99% confidence level. 16 integrated use of neochelina bruchi and alternaria eichhomiae — kasno et al. appendix 7. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during wet season at 21 days after weevil release under field conditions. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 3.40 80.20** 3.40 176.00** 30.00** 4.40* 55.62** 168.23** 5.77** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level appendix 8. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the change of water hyacinth wet weight. source of variance f value number of weevil pairs (a) 9.41 ** spore concentration (b) 4.31 * a x b 0.48 *= significantly different at 95% confidence level **= very significantly different at 99% confidence level 17 biotropia vol. 29 no. 1, 2022: 7 17 doi: 10.11598/btb.2022.29.1.1443 7 heavy metals contamination level and water quality parameter conditions in jatiluhur reservoir, west java, indonesia gatot prayoga*, bagus amalrullah utomo and hefni effendi environmental research center, institut pertanian bogor, bogor 16680, indonesia received 25 september 2020 / accepted 3 july 2021 abstract waste pollution into the citarum river, the main water source of jatiluhur reservoir, was dominated by the manufacturing industry such as textile, chemical, metal and pharmaceutical. in general, the manufacturing industry is the most common contributor to heavy metal waste, which will cause various health problems. therefore, it is essential to conduct studies on heavy metal contamination and water quality parameters conditions in the jatiluhur reservoir. the study aimed to provide a reference regarding the current condition of the heavy metal contamination level in both sediment and water of the jatiluhur reservoir, as well as to compare the levels of other water quality parameters against the standard of environmental quality. heavy metals contents, such as cu, zn, hg, pb and cd, were determined using x-ray fluorescence (xrf) spectrometry method (for sediment) and atomic absorption spectrometry (aas) method (for water). water quality parameters were analyzed by using methods developed by the indonesian national standard (sni). the data obtained were compared to the canadian sediment quality guidelines (for heavy metal in sediment) and water quality standards from the government of the republic of indonesia regulation number 82 of 2001 (class 3) (for water quality parameters). based on this study, jatiluhur reservoir is divided into three zones i.e., the inlet area, main inundation area and outlet area. within the sedimentary layer, the mercury (hg) was found to be accumulated throughout the jatiluhur reservoir area, exceeding the maximum limit, while cu accumulated in the inlet area, exceeding the minimum limit. the other heavy metals (zn, pb and cd) were found to be exceeding the minimum limit at some locations, but more results were below the minimum limit. the high concentration of heavy metals in the sediment was due to household and/or industrial wastes. although all heavy metals were not detected in water, the presence of heavy metals in sediments could potentially dissolve into the water by means of upwelling. if this happens, the heavy metals can be excessively contained in water, resulting in a harmful habitat for aquatic biota. water containing heavy metals will also be harmful for human. in general, water quality parameters in jatiluhur reservoir meet the standard for water quality. only ammonia, however, was higher than the standard for sensitive fish life due to massive aquaculture activities in this reservoir. considering the conditions of heavy metal contamination levels in both sediment and water, the biota that is most likely to be exposed to heavy metal is benthic organisms, because the organisms live at the bottom of the waters. the priority for further attention and countermeasure in improving the sediment and water quality of the jatiluhur reservoir was toward hg, cu, and pb and ammonia. keywords: heavy metal, reservoir, sediment, water introduction jatiluhur reservoir was the largest reservoir in indonesia having an area of 8,300 ha. jatiluhur reservoir was built in 1957. the reservoir is the first multipurpose reservoir in indonesia with potential available water of 12.9 billion m3/year. the jatiluhur reservoir was built by blocking the citarum river. the river passes through various anthropogenic activities and many cities in the west java province. among functions of the jatiluhur reservoir are as hydropower, as a source of irrigation for 242,000 ha of surrounding rice fields, as a source of drinking water, as a place for inland fisheries (fish farming in floating net cages (kja)), as flood control, and as tourism and water sports facilities. the types of fish commonly being *corresponding author, email: gatotprayoga16@gmail.com biotropia vol. 29 no. 1, 2022 8 cultivated in the floating net cages (kja) in the jatiluhur reservoir include common carp, nile tilapia, red devils and catfish (pangasius spp.) (suprian & salami 2011). in the past few years, fishes in the jatiluhur reservoir seem to have been contaminated and are not suitable for consumption (republika.co.id 2009). likewise, in 2018, a report from anggoro (2018) stated that there was an appeal for not consuming any kinds of fish from this reservoir. the contamination is allegedly due to the influence of various pollutions from the citarum river as the main water source of the jatiluhur reservoir. the contamination may have also occurred due to the wastes of fish feed from the many kjas in the reservoir. industrial activities are the most common contributor of heavy metal waste because heavy metals are used in industrial activities as raw materials, additives and catalysts (hutagalung et al. 1997 in vigers et al. 1996). heavy metal contamination in waters causes various health problems for aquatic biota and humans, such as problems in the nervous system, respiratory system, liver function, kidney function and growth of bones (sanusi 1985). heavy metals are categorized as harmful pollutants because it cannot be destroyed (nondegradable) by living organisms, so they will settle at the bottom of the waters and accumulate (rochyatun & rozak 2007). heavy metals can bond with organic compounds to form complex compounds that eventually settle at the bottom of the water (accumulate in sediments) (marchand et al. 2006). on the other hand, sediments were an inseparable part of aquatic ecosystems that can provide habitat, feeding grounds, spawning grounds, and nurseries for various aquatic organisms. contaminated sediments can reduce or eliminate the aquatic organisms that have important values for ecology, commercial or recreational uses (us epa 2001). fatoki and mathabatha (2001) stated that sediment has a function as a metal container which can release the metal into the water through natural and anthropogenic processes. heavy metals deposited in sediments can cause changes in water quality and transfer toxic chemicals to aquatic organisms (permanawati et al. 2013). physical and chemical water quality parameters such as dissolved oxygen (do), ph, total organic content, temperature and dissolved ion affect the life of aquatic organisms (effendi 2003). water quality parameters is influenced by its catchment area which is related to human activities (wiwoho 2005; asrini 2017). a study conducted by garno (2002) showed that the jatiluhur reservoir was hypertrophic (very nutrient-rich) and phytoplankton bloom can occur any time. the hypertrophic condition was largely influenced by the aquaculture activities in this reservoir and the influx of organic matters from settlements. poor water quality conditions can disrupt aquatic life in the reservoir and reduce the diversity of aquatic biota. a study in cirata reservoir carried out by komarawidjaja et al. (2005) showed that there was a growth disturbance in common carp which was thought to be closely related to water quality, especially with the high concentration of chlorophyll-α and total n in the waters. poor water quality of the jatiluhur reservoir will also have an adverse impact on humans due to the function of the reservoir as a source of drinking water and as tourism and water sports facilities. based on the issues above, the study of heavy metal contamination and water quality parameter conditions in the jatiluhur reservoir should be conducted due to the serious polluted condition in the reservoir. this study aimed to obtain data on current condition of the heavy metal contamination level that has occurred in both sediment and water, as well as to compare the levels of other water quality parameters against the standard of environment quality. materials and methods location and time the research was conducted in jatiluhur reservoir in february 2019. water and sediment samples were taken from 6 (six) locations in the reservoir, consisting of 3 (three) zones i.e., the inlet area, main inundation area, and outlet area (fig. 1). the six locations were: 1. citarum river inlet; 2) jamaras; 3) cilalawi river inlet; 4) kja zone 5; 5) pasir kole and 6) dam (outlet). heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 9 figure 1 map of sampling locations in jatiluhur reservoir methods the water sample was taken using van dorn water sampler, then analyzed in the global qa laboratory using the methods from the indonesian national standard (sni). sediment sampling was conducted by using an ekman grab. the sediment samples obtained were then analyzed in the central nuclear material technology laboratory, batan using the xray fluorescence spectrometry (xrf) method to find out the heavy metals (cu, zn, hg, pb and cd) contents. the xrf spectrometry method is an application of radioisotopes used as an analytical method for detecting heavy metal content, especially in solid substances such as sediment. this method is still not commonly used in aquatic ecological studies that mostly use the aas method. methods used for analyzing the water and sediment samples are presented in table 1. data analyses data of heavy metal contents (in water and sediment) and water quality parameters obtained from this study were compared to the quality standard. the sediment parameters (heavy metal) were compared to the canadian sediment quality guidelines for the protection of aquatic life on freshwater set out by the canadian council of the ministry of environment (ccme). reference to heavy metal quality standards according to ccme standard was presented in table 2. data on water quality parameters were compared to water quality standards from the government regulation of the republic of indonesia number 82 of 2001 (class 3, for the cultivation of freshwater fish, animal husbandry, water to irrigate crops, and or other purposes that require the same water as these uses). inlet area inlet area outlet area main inundation area 1 2 3 4 5 6 biotropia vol. 29 no. 1, 2022 10 table 1 parameters and methods used for analyzing water and sediment samples parameter unit method a. water physical properties: temperaturea °c 5.4/ik/gqa/wq/002 total dissolved solid, tdsa mg/l sni 06-6989.27-2005 total suspended solid, tssa mg/l sni 06-6989.3-2004 chemical properties: pha sni 06-6989.11-2004 biological oxygen demand, bod5a mg/l sni 6989.72:2009 chemical oxygen demand, coda mg/l sni 6989.2:2009 dissolved oxygen, doa mg/l sni 06-6989.14-2004 total phosphate as pa mg/l 5.4-ik-gqa-wq-062 nitrogen, nitrate as n (no3-n)a mg/l 5.4-ik-gqa-wq-043 ammonia, nh3-na mg/l sni 06-6989.30-2005 copper, cua mg/l sni 6989.6:2009 zinc, zna mg/l sni 6989.7-2009 mercury, hga mg/l sni 6989.78:2009 lead, pba mg/l sni 6989.46:2009 cadmium, cda mg/l sni 06-6989.38-2005 b. sediment copper, cu µg/g xrf spectrometry zinc, zn µg/g xrf spectrometry mercury, hg µg/g xrf spectrometry lead, pb µg/g xrf spectrometry cadmium, cd µg/g xrf spectrometry note: a = accredited by the national accreditation committee (kan). table 1 quality standards for heavy metals in sediments (for freshwater) based on ccme (2001) heavy metal concentration (mg/kg dry wt) isqga pelb cu 35.7 197.0 zn 123.0 315.0 hg 0.170 0.486 pb 35.0 91.3 cd 0.6 3.5 notes: a = isqg (interim sediment quality guidelines): the value of the minimum limit, i.e., the limit of metal concentrations that has the low possibility to cause a negative biological effect; b = pel (probable effect level): the value of the maximum limit, i.e., the limit of metal concentrations having a greater possibility to cause a negative biological effect. results and discussion heavy metal contaminations our study showed that all heavy metals (cu, zn, hg, pb, and cd) accumulated in the sediment layer of the jatiluhur reservoir and had exceeded the minimum limit (isqg). mercury (hg) concentration exceeded the maximum limit (pel) throughout the jatiluhur reservoir area. copper (cu) tended to accumulate in the inlet area, with concentration exceeding the minimum limit. the concentration of zinc (zn) and lead (pb) exceeded the minimum limit at some locations (such as sampling point 5), however, the concentration of cadmium was below the detection limit of the measuring tools (table 3). cadmium (cd) seems to be relatively the safest metal found in the sediment samples compared to the other heavy metals. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 11 table 3 results of laboratory analysis for heavy metal in sediment samples station test element (mg/kg) cu zn hg pb cd 1 citarum river inlet #1 39.78 105.16 2.1 29.4 <0.3 #2 40.34 96.32 2.5 29.7 <0.3 #3 35.71 93.51 1.8 28.6 <0.3 2 jamaras #1 36.91 113.59 1 26.8 <0.3 #2 38.42 106.6 <0.7 26.8 <0.3 #3 41.46 133.2 2.5 28.8 <0.3 3 cilalawi river inlet #1 35.79 129.82 <0.7 34.8 <0.3 #2 84.12 103.31 2.4 31.9 <0.3 #3 73.73 92.06 3.2 30.7 <0.8 4 kja zone 5 #1 22.3 114.48 1.1 30.2 <0.8 #2 12.94 104.27 2.5 25.9 <0.3 #3 19.01 113.03 3.8 34 <0.3 5 pasir kole #1 18.69 139.94 2.2 40 <0.3 #2 23.57 143.16 1.5 36.3 <0.3 #3 26.28 147.98 3.2 40.1 <0.3 6 dam (outlet) #1 20.13 89.41 <0.7 35.3 <0.3 #2 74.13 103.39 3.6 32.5 <0.8 #3 27 97.69 <0.7 31 <0.3 quality standarda 35.7 123 0.170 35.0 0.6 197.0 315 0.486 91.3 3.5 notes: a = based on the canadian sediment quality guidelines for the protection of aquatic life on freshwater (ccme 2001). exceed the isqg (minimum limit) exceed the pel (maximum limit) mercury (hg) was among the most toxic metals for aquatic biota. mercury can be released from anthropogenic activities, such as from the antifouling paint for the hull of ships, slimicides used in the lumber and paper industries, pesticides and seed dressings in agriculture and pharmaceuticals (garcia-rico et al. 2006). mercury has many benefits, but it is very toxic and has a high level of bioaccumulation capabilities (garcia-rico et al. 2006; palar 2012). mercury concentration exceeding the safe limit endangers the life of aquatic biota, either directly or indirectly (palar 2012). therefore, the high concentration of mercury in the jatiluhur reservoir should be handled immediately, especially when there is an upwelling process in the reservoir. our study showed that the concentration of cu in sediments was much higher and exceeded the minimum limit in inlet areas, whereas the concentration of zn and pb was high in the main inundation areas. the spatial difference of the heavy metals’ concentrations might be due to the heavy influx from the river into the reservoir, which was already contaminated by various anthropogenic activities. cu is commonly used in insecticides, fungicides, brass alloy materials for household appliances, machine parts, as well as in water purification or as a food additive (palar 2012). household wastes in the form of metabolic waste and corrosion of pipes in residential areas usually also contain cu (connell & miller 2006). the main inundation area has the longest water retention time compared to the other areas and is also much affected by the kja aquaculture activities (marked with black lines in fig. 1). household wastes, water flow from urban areas and phosphate utilization (po4), contributed significantly to the entry of pb metal into the waters (connell & miller 2006; harteman 2011). sources of zn, cu and pb are chemical fertilizers, household wastes such as corrosion of pipes and detergent, as well as industrial wastes such as battery materials, cosmetics, plastics, rubber, soap, paint and ink, television tubes and fluorescent lamps, deodorants and chemicals for wood preservation (connell & miller 2006). biotropia vol. 29 no. 1, 2022 12 table 4 results of laboratory analysis results for heavy metal in water samples station element (mg/l) cu zn hg pb cd 1. citarum river inlet < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 2. jamaras < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 3. cilalawi river inlet < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 4. kja zone 5 < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 5. pasir kole < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 6. dam (outlet) < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 quality standarda 0.020 0.050 0.00200 0.0300 0.01000 notes: a = based on government regulation of the republic of indonesia number 82 of 2001 (class 3). the concentrations of heavy metals in water in this study were very low, below the detection limit of the tool (table 4), which indicated that the concentrations of heavy metals (cu, zn, hg, pb, and cd) in the water of the jatiluhur reservoir were still within the safe limits. this fact was in agreement with a study in jatiluhur reservoir in 2011 conducted by suprian and salami (2011), especially for mercury concentration which was below the quality standard of the government of the republic of indonesia number 82 of 2001 (class 3). despite the results that all heavy metals in the water samples were below the safe limit, however, heavy metals in sediments could potentially dissolve into the water. suprian and salami (2011) stated that although the value of mercury in water is below the safe limit, however, the existence of mercury has to be monitored because it is difficult to eliminate mercury from the water. according to fatoki and mathabatha (2001) and permanawati et al. 2013, sediment can release heavy metal into the water through natural and anthropogenic processes that cause changes in water quality and transfer of toxic chemicals to aquatic organisms. in reservoirs, the most extreme natural process that allows this to happen is upwelling. palar (2012) stated that the 0.01 ppm concentration of cu in water is deadly for phytoplankton, pb concentration of 2.75-49 ppm is deadly for crustaceans, while pb concentration of 188 ppm is deadly for fish, and cd concentration of 0.0028-4.6 ppm is deadly for oligochaeta. mercury concentration of ≥ 0.16 ppm is able to reduce the survival and growth rates of fish caused by the increase of stress and organ damage, whereas mercury concentration of ≥ 3 ppm causes mass mortality in common carp (nirmala et al. 2012; tyas et al. 2013). lethal toxicity test by prayogo et al. (2016) showed that 96 hours mercury exposure with concentration of 0.396 ppm in water had a 50% lethal effect on nilem carp (osteochilus hasselti). if the heavy metals content in the sediment are released into the water, then the concentration of heavy metals can be excessive in the water, causing adverse impact to humans and aquatic biota. considering the conditions of heavy metal contamination levels in both sediment and water, the biota that is most likely to be exposed to heavy metal is benthic organisms, because benthic organisms live at the bottom of the waters (reynolds 2012). heavy metals can cause a decrease in species richness of benthic organisms and a change in species composition of benthic macroinvertebrate communities (qu et al. 2010). cu concentrations of 70-90 mg/kg, zn concentrations of ±350 mg/kg and pb concentrations of 30-40 mg/kg caused the decrease of polychaeta and molluscs biodiversity at the bottom of jakarta bay, especially in industrial areas (takarina & adiwibowo 2011). the same occurrences might happen in the jatiluhur reservoir, especially for cu and pb which concentrations have reached high level of contamination. qu et al. (2010) stated that even though contamination by heavy metals is low in the sampling area, the adverse impact on benthic organisms are significant, suggesting that the chronic effects of long-term exposure to heavy metals in aquatic communities could be serious. thus, it is essential to focus our attention and efforts to handle contamination of hg, cu, and pb in the jatiluhur reservoir. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 13 water quality parameter conditions in general, water quality parameters in our study met the quality standards (qs) based on the government of the republic of indonesia regulation no. 82 of 2001 (class 3). based on direct measurements on-site for all sampling locations, the temperature ranged from 26.5 °c up to 27.1 °c (qs ± 3), dissolved oxygen (do) concentrations ranged from 4.2 mg/l up to 4.5 mg/l (qs min. 3 mg l), and the water ph ranged from 6.37 up to 7.17 (qs 6-9). results of the laboratory analysis on the other parameters of water quality are presented in figure 2. figure 2 values of several water quality parameters measured in the jatiluhur reservoir note: quality standards based on the government of the republic of indonesia regulation no. 82 of 2001 (class 3), except for ammonia which was not available. biotropia vol. 29 no. 1, 2022 14 results of our study showed that tds concentrations ranged from 120 mg/l up to 136 mg/l (qs 1,000 mg/l), tss concentrations ranged from 4 mg/l up to 33 mg/l (qs 400 mg/l), bod concentrations ranged from 0.2 mg/l up to 4.7 mg/l (qs 6 mg/l), cod concentrations ranged from 2 mg/l up to 12 mg/l (qs 50 mg/l), total phosphate concentrations ranged from 0.01 mg/l up to 0.10 mg/l (qs 1 mg/l), nitrate concentrations ranged from 0.01 mg/l up to 0.47 mg/l (qs 20 mg/l) and ammonia concentrations ranged from 0.095 mg/l up to 1.940 mg/l (qs 0.02 mg/l). of the ten observed water quality parameters (not including heavy metals concentrations), only ammonia that did not meet the quality standards of water quality parameters. ammonia is a nitrogen compound that changes to nh4 ions at low ph conditions. ammonia can also come from domestic and industrial wastes (marganof 2007). the high content of ammonia in the reservoir was presumably due to the fish feed and fish fecal wastes as a result of aquaculture activities in the reservoir. fish emit 80-90% ammonia (n-inorganic) through the osmoregulation process, while feces and urine account for 10-20% of total nitrogen (rakocy et al. 1992 in sumoharjo 2010). ammonia in the reservoir can come from organic and inorganic nitrogen sources found in soil and water or from the decomposition of organic matters by microbes and fungi. ammonia also comes from the denitrification process during the decomposition of wastes by microbes under anaerobic conditions (effendi 2003). commonly, the concentration of ammonia in the pond should not exceed 0.05 mg/l. according to sni 6139:2009 (national standardization agency 2009), the ammonia value resulting from the nile tilapia aquaculture activity in calm water ponds should not exceed 0.02 mg/l. ammonia concentrations of 0.020.07 mg/l have been shown to inhibit growth and cause tissue damage in several fish species. the toxicity threshold value for ammonia is highly dependent on the type of species, size, fine solids, surface-active compounds, metals and nitrates (colt 2006). based on anas et al. (2017), the water quality status of the jatiluhur reservoir is classified as moderate. the moderate status was resulted from the storet calculation, stated in the decree of the minister of environment no. 115 of 2003) which was caused by high concentrations of bod, cod and ammonia. the main contributor to the high concentrations of organic matter in the jatiluhur reservoir presumably are the number of operating kja in the reservoir. based on the regent of purwakarta regency decree no. 6 of 2000, the optimal number of kja to operate in the jatiluhur reservoir is 2,100 plots. meanwhile, in 2015 there were 18,038 kja in the reservoir, which exceeded the carrying capacity of the waters (astuti et al. 2016). a recent study conducted by fitri et al. (2016) stated that in 2014 the number of intensive kja in the jatiluhur reservoir was already excessive, amounting to 23,000 cages. according to their study, the optimal number of kja was 19,401 plots. moreover, fitri et al. (2016) stated that the difficulties faced by the jatiluhur reservoir related to the kja problem were caused by multi-parties’ management having different perspectives leading to inconsistent decision making. harmonious perspectives and visions of all managing parties are needed to maximize the productivity of the kja without sacrificing environmental quality. conclusion the jatiluhur reservoir has experienced the accumulation of cu, zn, pb, and cd in the sediment layer with exceeding the minimum limit (isqg) and hg with exceeding the maximum limit (pel). spatially, hg concentration was high in the entire jatiluhur reservoir area, cu was found to accumulate in the inlet area, whereas zn and pb were relatively high in the main inundation areas. cadmium (cd) seemed to be relatively the safest metal in sediment compared to the other heavy metals. heavy metal concentration in the waters of jatiluhur reservoir was below the detection limit of the tool. the other water quality parameters also met the standard of water quality. only ammonia did not meet the quality standards for the life of sensitive fish (such as nile tilapia). the high concentration of heavy metals in the sediment of the reservoir was due to household heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 15 and/or industrial wastes, while the high concentration of ammonia in water was due to fecal materials from the aquaculture activities. benthic organisms may have been affected by the high concentration of heavy metals in the sediment of the reservoir. based on the high level of heavy metal concentrations in the jatiluhur reservoir, the priority for further attention and countermeasure in the reservoir was toward hg, cu, pb and ammonia. further studies are recommended to manage the water quality of the jatiluhur reservoir. acknowledgments this study is part of a research grant from the osaka gas foundation of international cultural exchange (ogfice). the authors are thankful to ogfice for fully funding this study. references anas p, jubaedah l, sudino d. 2017. kualitas air dan beban limbah karamba jaring apung di waduk jatiluhur jawa barat [water quality and wastewater load of floating net cages in jatiluhur reservoir of west java]. jppik 11(1):35-47. anggoro b [internet]. 2018. warga diimbau tak konsumsi ikan dari aliran sungai citarum [residents are advised not to consume fish from the citarum river]. 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[the lethal and sub-lethal toxicity test of mercury chloride (hgcl2) on cyprinus carpio l.]. omni-akuatika 12(17):1-10. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 17 [us epa] u.s. environmental protection agency. 2001. methods for collection, storage and manipulation of sediments for chemical and toxicological analyses: technical manual. epa 823-b-01-002. washington dc (us): u.s. environmental protection agency, office of water. wiwoho. 2005. model identifikasi daya tampung beban cemaran sungai dengan qual2e [model identification of river pollution load carrying capacity with qual2e] [thesis]. retrieved from universitas diponegoro. biotropia no biotropia no. 14, 1999 : 17 35 the occurrence of insects, fungi and organoleptic characteristics in stored coffee beans in lampung okky s. dharmaputra, sunjaya, ina retnowati and muhammad amad seamed biotrop, p.o. box 116, bogor 16001, indonesia cahya ismayadi the indonesian research institute for coffee and cocoa, jl. pb sudirman 90, jember 68118, indonesia abstract a survey on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels was conducted in west lampung and tanggamus regencies of lampung province during harvest time (july 1998). interviews and sampling of coffee beans were carried out during the survey. the number of respondents at farmer, trader and exporter levels was 22, 20 and 4, respectively, while the number of samples collected from each level was 20. all samples were analyzed for moisture content, physical quality, insect and fungal infestation, reducing sugar content, and coffee cupping. the results of the interviews indicated that postharvest handling and technology processing became better from farmers to exporters. moisture contents of coffee beans collected from farmers and traders were higher than the tolerable limit recommended by sni (13%). physical quality of coffee beans collected from exporters was higher than that collected from farmers and traders. insects were found on coffee beans collected from farmers, traders and exporters, but the number of species and the percentage of samples infested by insects from each level were relatively low. the predominant species was liposcelis entomophila. the number of fungal species on coffee beans collected from farmers was higher than that collected from traders and exporters. the predominant species at the three levels was aspergillus niger, but the lowest percentage of beans infected by this fungus was found on coffee beans collected from exporters. the lowest percentage of samples infected by all fungi was also found on coffee beans collected from exporters. reducing sugar content of coffee beans collected from exporters was lower than that from farmers and traders. aroma and flavor values tended to increase from farmers through traders to exporters, while the body decreased. some off-flavors (i.e. earthy, mouldy, fermented and woody) were encountered in a few coffee samples from farmers as well as from traders. there was no off-flavor encountered in the coffee samples from exporters. key words: stored products pests/postharvest handling/technology processing/moisture content physical quality/insect/fungi/reducing sugars/coffee cupping/coffee/ lampung. introduction people all over the world are all facing global competitiveness. with the advent of wider market access and perceived increase in demand for farm products, we face the responsibility of maintaining the sustainability of our resources through appropriate research program in postharvest. it would minimize postharvest losses which in turn should benefit our farmers in terms of higher productivity and incomes. food selfsufficiency can be attained by increasing production and minimizing food losses and quality degradation resulting from irjefficient postharvest handling. 17 biotropia no. 14, 1999 coffee is one of the important export commodities in indonesia. licht (1995) reported that indonesia is the biggest coffee producer in asia. based on data from the central bureau of statistics (1996) between january september 1996; indonesia has exported 253 027 tons of coffee beans. most jf coffee beans production is derived from smallholders. during storage, coffee beans could be infested by insects, mites, microorganisms and rodents. insects are considered the most significant cause of losses. among microorganisms, fungi are the most important cause of deterioration of stored products. the role of insects on fungal infection cannot be disregarded. aside from injuring seeds, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce enough heat and moisture (specially during longterm storage) to allow fungal growth. no intensive study has been carried out on the occurrence of insects and fungi on stored coffee beans in indonesia. the objective of this study was to get information on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels in lampung province. the moisture content, physical quality, pest infestation (insect and fungi), reducing sugar content and coffee cupping (organoleptic test) of coffee beans were also analyzed. materials and methods time and location of surveys a survey was conducted during harvest time (july 1998) in two regencies, i.e. west lampung regency (fajar bulan and sumber jaya villages) and tanggamus regency (data-rajan, air naningan, way harong, gunung megang and tekad villages). these regencies were selected because they produce large quantities of coffee beans. interviews using questionnaires interviews were carried out during the survey to get information on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels. the number of respondents from each level was different depending on the condition in the field during the survey. the number of respondents from farmers, traders and exporters was 22, 20 and 4, respectively. the questionnaires contained questions on postharvest handling and technology processing carried out by farmers, traders and exporters, and problems encountered by them. sampling methods the number of samples taken from each level was 20. not all samples were derived from the respondents. about 1 kg of each sample was taken randomly from 18 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. farmer, trader and exporter levels. samples of coffee (primary samples) from each stack were taken randomly from a certain number of sacks using a sampling spear. insects were separated from each primary sample using graded sieves. each primary sample was divided several times using a sample divider to obtain working samples for analyzing moisture content, physical quality, fungi, reducing sugars, organoleptic test and reserve sample. the presence of insects was estimated within the sacks. moisture content analysis moisture content of coffee beans (wet weight) was determined by the oven method (isc 1992). two replicates were used for each sample. the beans were ground and dried in the oven at 130 ± 2°c for 6 hours (first period), and then they were placed in a desiccator for,a night. they were re-dried in the oven at 130 ± 2°c for 4 hours (second period). the moisture content was determined using the formula: moisture content = p, + where : p, is the first period moisture content (in 100 g of sample) ?2 is the second period moisture content (in 100 g of sample) physical quality analysis physical quality analysis was determined based on sn1 01-2907-1992 (isc 1992). quality level of coffee bean samples was determined based on the number of defective beans from 300 g of sample. quality classification based on defective beans system and determination of the number of defective beans are shown in tables 1 and 2. table 1. quality classification based on defective beans system grade criteria of grade grade 1 maximum number of defective beans 11 grade 2 number of defective beans 12-25 grade 3 number of defective beans 26 44 grade 4-a number of defective beans 45-60 grade 4-b number of defective beans 61-80 grade 5 number of defective beans 81-150 grade 6 number of defective beans 151 225 notes : the number of defective beans was determined based on table 2. 19 biotropia no. 14, 1999 table 2. determination of the number of defective coffee beans no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. kind of defective 1 (one) black bean 1 (one) a half of black bean 1 (one) broken black bean 1 (one) cherry bean 1 (one) brown bean 1 (one) big husk 1 (one) middle husk 1 (one) small husk 1 (one) bean with hull 1 (one) big hull 1 (one) middle hull 1 (one) small hull 1 (one) broken bean 1 (one) immature bean 1 (one) bean with one hole 1 (one) bean with more than one hole 1 (one) big branch, soil, or gravel 1 (one) middle branch,soil, or gravel 1 (one) small branch, soil, or gravel defective value 1 (one) '/2 (a half) '/2 (a half) 1 (one) 1/4 ( a quarter) 1 (one) !/2 ( a half) 1/5 (one fifth) '/2 ( a half) '/2 ( a half) 1/5 (one fifth) 1/10 (one tenth) 1/5 (one fifth) 1/5 (one fifth) 1/10 (one tenth) 1/5 (one fifth) 5 (five) 2 (two) 1 (one) notes : if one bean has more than one kind of defective, the defective value was determined based on the highest defective value. insect and fungal analyses the insects were identified using haines (1991) as the main reference. fungi were isolated using direct plating method on dichloran 18% glycerol agar (dg18) (pitt and hocking 1997). before plating, samples were individually disinfected using 1% sodium hypochlorite for 1 minute. one hundred beans were then plated (10 beans/plate) on the medium and incubated at 25°c for 7 days. the fungi were identified using samson et al. (1996), and pitt and hocking (1997) as the main references. reducing sugars analysis reducing sugars (dry weight) was determined according to somogyi nelson's method (mccready 1970). in alkalic condition, reducing sugars reduce cu 2+ to cu+. alkalies give a specific color if added with arsenicmolibdat and the absorbency of the color was measured by spectrophotometer on wave length 510 nm. reducing sugars was determined using standard curve and glucose as a reference. 20 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. organoleptic test analysis coffee cupping was determined according to lingle's method (1986). coffee bean samples were roasted using probat roaster type pre-2 at 200°c until medium roast level (color degree of # 55 agtron/specialty coffee association of america). roasted coffee was then ground at medium grind degree. one hundred fifty ml boiling water was added to about 10 g of roasted and ground coffee. the test was carried out by 4 experienced panelists on aroma, flavor, body, and defect characteristics. evaluation for each character was determined by numerical system (scoring): 0 = none, 1 = low, 2 = low-medium, 3 = medium, 4 = medium-high, 5 = high, respectively. results and discussion interviews with farmers, traders and exporters a. farmer level the results of the questionnaires are shown in appendix 1. most of the fanners (77.3% respondents) cultivated only robusta coffee, while others (22.7%) cultivated arabica and robusta coffee, especially in west lampung. determination of harvest time was based on visual observation of the color of the berry. the ripeness of coffee berry is indicated by its red color. nevertheless, strip methods of harvesting was carried out when the number of ripe berries was about 80%. the drying process was carried out by farmers. most of the farmers (63.6%) dried the coffee directly without opening the berries, and consequently they stored the coffee in the form of dried coffee berries. the others (36.4%) opened the berries using a machine before drying. the final form obtained was called green coffee. applying this method, coffee beans would dry faster, and thus farmers could sell them faster. the drying facilities used by farmers were varied, i.e. on the ground, on a plastic (polypropylene) sheet and on paved floor. farmers revealed that in general there was no problem with insects as well as fungi in the storage. b. trader level the results of questionnaires are shown in appendix 2. traders bought coffee beans from farmers, although the moisture content was still high (20-30%). in this case, the farmers gave a discount for the undesirable moisture content of the coffee beans. most of traders (85.7%) further sun-dried their coffee beans for less than 1 day or only several hours. 21 biotropia no. 14, 1999 traders dried coffee beans on polypropylene sheet (42.9%) and paved floor (57.1%). in general, traders stored the beans for not more than 2 days by spreading them on the floor in their warehouse/store (65%) and the beans were packed using plastic bags (30%). most of traders (60%) sorted and selected the beans that will be sold to exporters, because exporters classified the price of beans based on moisture content and physical quality (i.e. percentage of extraneous matter, hulls, beans with holes and black beans). most of the traders did not have any problem with insects and fungi. nevertheless, if mouldy beans were found, no action was taken by 65% of the respondents. exporter level the results of questionnaires are shown in appendix 3. exporters in lampung obtained coffee beans from traders in lampung province, java and other provinces in sumatera. determination of price classification was based on moisture content and physical quality of the beans. the beans were packed in jute bags (91.7%) and plastic bags (8.3%) before reprocessing (further dried, sorted and sifted). the duration of storage was usually less than 1 week. further drying was carried out using a mechanical dryer up to moisture content of 12-13%. sorting was conducted using an electronic sorting machine (sortex) to discard extraneous matter and black beans. after sorting, grade quality could be determined. the beans were then packed in jute bags or containers, fumigated, and finally exported. generally, fumigation was conducted by private companies in warehouses or in the containers depending on customer demand. the fumigant used was methyl bromide (87.5% of respondents) or phosphine (12.5%) depending on customer demand and importing countries. all exporters monitored insect pest infestation. most of them (75%) did not have any problem with insect pests, while the others (25%) did. moisture content and physical quality moisture content of coffee beans collected from farmers (16.1 ± 1.3%) and traders (16.5 ± 3.3%) were higher than the tolerable limit recommended by sni (13%). all the samples (100%) collected from farmers has a moisture content of more than 13%. similar moisture content was obtained from 90% samples collected from traders (table 3). based on statistical analysis, moisture content of coffee beans from farmers was not significantly different compared to coffee beans collected from traders, but that from traders was more varied. moisture content of coffee bean samples from exporters (12.6 ± 0.5%) (table 3) was lower compared to samples from farmers and traders, but 20% of the total samples collected from exporters still exceeded the tolerable limit recommended by sni. exporters re-processed the coffee from traders to meet the standard. the 22 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et at. table 3. moisture content and physical quality of coffee beans collected from farmers, traders and exporters source of coffee beans moisture content (%) grade of quality (% samples) number (%) of sam ples with moisture content > 13% farmers 16.1 ± 1.3 a 5 (10), 6 (90) 20 (100) traders 16.5 ± 3.3 a 6 (100) 18 (90) exporters 12.6 + 0.5 b 4-a(10),4-b(15), 5 (20), 6 (55) 4(20) numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level notes : grade 4-a : samples of coffee beans with the number of defective bean 45-60 grade 4-b : samples of coffee beans with the number of defective bean 6 1 — 8 0 grade 5 : samples of coffee beans with the number of defective bean 81 150 grade 6 : samples of coffee beans with the number of defective bean 151 225 process included drying, mixing, grading and packaging. exporters dried coffee beans using mechanical dryers. the temperature of the dryer was set at 200°c or more; consequently, the moisture content of coffee beans from farmers and traders could be decreased from 16% up to about 13% within a short period. based on the number of defective beans, the quality of samples from traders was not better, it even tended to be of lower quality (100% of samples consisted of the lowest grade 6) compared with that from farmers (10% of samples consisted of grade 5 and 90% of samples consisted of grade 6) (table 3). traders bought coffee beans from farmers against the price which is based on moisture content, number of defective beans and extraneous matter. defective beans (black beans, brown beans, immature beans, broken beans and cherries) and the extraneous matter were counted arbitrarily by the traders. most of traders possessed an electronic moisture tester (generally cera tester) which is used to determine the moisture content for pricing. nevertheless, the tester had never been calibrated, thus the result of determination is unreliable. traders mixed the coffee from farmers. as a consequence the mixing of high as well as low quality coffee resulted in downgrading. in general, the physical quality of coffee beans from exporters was better (10% of samples consisted of grade 4-a, 15% of samples grade 4-b, 20% of samples grade 5 and 55% of samples grade 6) than that from farmers and traders (appendix 2). exporters sorted coffee beans obtained from traders in order to reduce the defects especially black beans and beans in cherry. the sorting process was conducted using electronic sorting machine. in addition, the coffee beans were also sieved with screens to get a uniform size and clean beans. 23 blotropia no. 14, 1999 the presence of insects haines (1991) reported that insect species associated with stored coffee beans were araecerus fasciculatus, hetebostrychus brwmeus and prostepanus trunchatus. the number of insect species found in coffee bean samples collected from farmers, traders and exporters, and the percentage of samples infested by the insects were relatively low (table 4). similar results were reported by the respondents. table 4. insect species found on coffee beans collected from farmers, traders and exporters 0% samples infested by insects insect species farmers traders exporters ahasverus advena 0 5 0 araecerus fasciculatus 0 0 5 liposcelis bostrycophila 10 0 30 l. enlomophila 10 10 40 sitophilus zeamais 0 5 0 in this study, insect species found on coffee beans collected from farmers were liposcelis bostrycophila (10% of samples) and l. entomophila (10%) (table 4). this result showed that insect infestation on coffee beans from farmers was relatively low, because farmers did not store the beans for a long period. the results of interviews using questionnaires showed that only 14.3% of farmers stored coffee beans for more than 3 days (appendix 1). haines (1995) reported that l. entomophila was found on 50% of samples of stored coffee beans in java and sumatera. in a tropical country like indonesia, the predominant psocid (insect of genera liposcelis) found were l. bostrycophila and l. entomophila (leong and ho 1991;syarifandhalid 1993). the role of psocids is not well understood, as to whether these insects eat or cause deterioration of stored commodities. psocids are often found in humid warehouses (syarief and halid 1993). according to sidik and cahyana (1992), high populations of liposcelis spp. made the warehouse dirty; consequently working activities were disturbed. recently it was found that liposcelis spp. were real stored product pests. their heavy infestations on stored grains occurred in indonesia, india and australia (roesli et al. 1998). the occurrence of l. bostrycophila and l. entomophila on coffee beans at the farmer level was due to the high moisture content of the beans (16.1 ± 1.3%) and the sanitary conditions of the storage facility which were not managed properly. in indonesia, populations of psocids on rice have increased drastically several months after harvest (santoso et al. 1996). three insect species were found on coffee beans at the trader level, namely ahasverus advena (5% of samples), l. entomophila (10%) and sitophilus zeamais 24 the occurrence of insects, fungi and organoleptic chaiaeteristics okky s. dharmaputra et al. (5%) (table 4). in general, s. zeamais is an insect pest found on milled rice, maize and pulses. the presence of the three insect species on coffee beans was due to migration, because the beans were stored in the stores located at traditional markets which sell milled rice and maize. ahasverus advena is a fungus feeder. its presence was due to high moisture content of coffee beans (10.5-22.7%). according to haines (1991), a. advena often found on damaged and mouldy commodities in storage is an indicator of wet storage facility. three insect species were also found on coffee beans at the exporter level. they were araecerus fasciculatus (5% of samples), liposcelis bostrycophila (30%) and l. entomophila (40%) (table 4). l. bostrycophila and l. entomophila were more often found at the exporter level than at the farmer and trader levels. this was because coffee beans stored by exporters were derived from farmers and traders and infested by those insects, resulting in their presence in greater numbers on the beans at the exporter. besides that, frequent fumigation carried out by exporters affected the population of liposcelis spp. according to roesli et al. (1998), improper fumigation, i.e. shorter exposure period or too low gas concentration, caused an increase in populations of these insects. a. fasciculatus is a major pest of stored coffee beans. its presence caused damage to the beans, thus decreasing the selling value. haines (1991) revealed that the presence of a. fasciculatus on export commodities such as coffee and cocoa beans could decrease the selling value, although the damage might not be heavy. this insect was also found on stored products in lampung and south sumatera (haines 1995). the presence of fungi based on an interview, most of the respondents said that they had no problem with fungi. their opinion was only based on visual observation. nevertheless, based on fungal isolation using dg18, all beans were infected by fungi. fifteen fungal species were isolated on coffee beans collected from farmers. they were aspergillus flavus , a. niger, a. ochraceus, a. penicilloides, a. tamarii, a. versicolor, a. wentii, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, fusarium semitectum, lasiodiplodia theobromae, mucor hiemalis, penicillium citrinum and wallemia sebi. a. niger was the dominant fungus isolated, having been found in 100% of all samples, and 83.5% of all beans examined. a. flavus was the next most common fungus, being present in 90% of the samples, but only 12.3% of all beans examined. p. citrinum was present in only 15% of the samples and infected 18.7% of all beans examined (table 5). ten fungal species were isolated on coffee beans collected from traders. they were a. flavus, a. niger, a. ochraceus, a. penicilloides, a. tamarii, a. versicolor, a. wentii, e. chevalieri, l. theobromae and p. citrinum. a. niger was also the dominant fungus isolated, being found in 100% of the samples, and 94.9% of all beans 25 biotropia no. 14, 1999 26 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. examined. a. flcrvus, a. ochraceus, l. theobromae and p. citrinum were all present in more than 75% of the samples, but their infection on all beans examined were relatively low (14.1, 11.6, 2.3 and 30.7%, respectively) (table 5). eleven fungal species were isolated on coffee beans collected from exporters. they were a. flavus , a. niger, a. ochraceus, a. tamarii, a. wentii, c. cladosporioi-des, e. chevalieri, f. semitectum, l. theobromae, m. hiemalis and p. citrinum. the most commonly occurring species was a. niger, present in 95% of samples, and 60.1% of all beans examined. a. flavus and p. citrinum were found in 100 and 75% of samples, respectively, but infected only 11.9 and 3.7%, respectively, of all beans examined (table 5). according to scott (1994) and ono et al. (1995) a. niger and a. ochraceus could produce ochratoxin a. this toxin caused a swelling of the kidneys (nephropathy) in domestic livestock and poultry, and it has been considered as the causative agent responsible for human endemic nephropathy prevalent in areas of the balkan countries (prelusky et al. 1994) and tunisia (ccfac 1998). among the three levels, the number of fungal species isolated from coffee beans at the farmers level was the highest, because moisture content of all sample was more than 13% (table 3). the dominant fungus isolated at the three levels was the same, i.e. a. niger, but the lowest infection of all beans examined among the three levels was found at exporter level, because the exporters further dried and sorted coffee beans obtained from farmers and traders. moreover, among the three levels the lowest percentage of samples infected by all fungi was obtained from the exporter level (70.7%), while that at farmer and trader levels were not significantly different to each other (99.1 and 99.0%, respectively) (table 6). nakajima et al. (1997) reported that the dominant fungus isolated on coffee beans was a. niger. table 6. percentage of samples infected by all fungi on coffee beans collected from farmers, traders and exporters source of coffee beans samples infected by all fungal species (%) farmers' 99.1 a traders 99.0 a exporters 70.7 b numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level reducing sugar content sugars are important components in the formation of flavor, color/pigment, caramelization and condensation products of roasted coffee. the major free sugar in coffee beans is sucrose, which reaches 6.1% in arabica coffee and 3.4% in robusta 27 biotropia no. 14, 1999 coffee (trugo 1985). besides sucrose, coffee beans also contain simple sugars including reducing ones, but in small amounts. fructose and glucose in arabica coffee constitute 0.030-0.038% and 0.023-0.030%, respectively, while in robusta coffee 0.16-0.18% and 0.19-0.21%, respectively. reducing sugars in arabica and robusta coffees are only 0.1 and 0.5%, respectively (trugo 1985). analysis of reducing sugars in the samples resulted in 2.7 ± 0.3% for coffee from farmers, 3.2 ± 0.6% for coffee from traders, and 2.3 ± 0.2% for coffee from exporters (table 7). those results were higher than the reported data. coffee beans from exporters had the lowest value compared to the coffee from farmers and traders. those lower values were probably due to the coffee that had been further dried at quite high temperature, a condition which makes the sugars caramelized or condensed through the maillard reaction, hence the amount was reduced. according to winarno (1991) caramelization of sucrose occurred when the temperature is higher than the melting point (160°c); the maillard reaction is a reaction among carbohydrates, especially of reducing sugars with primary amine groups yielding pleasant brown compounds. table 7. reducing sugar contents of coffee beans collected from farmers, traders and exporters source of coffee beans reducing sugar content (%) farmers 2.7 ± 0.3 b traders 3.2 ± 0.6 a exporters 2.3 ± 0.2 c numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level coffee cupping (organoleptic test) aroma and flavor characters of all coffee samples were termed as low-medium to medium, while their body character ranged from medium to full (table 8). coffee from lampung is known for its unique taste with full bodied and unclean flavor. the unclean flavor is also frequently found in dry processed coffee beans. some very low off-flavors like earthy, musty, and mouldy shadowing the coffee flavor resulted in a unique character. aroma and flavor values tended to increase for the coffee at farmer (2.4 + 0.4 and 2.5 + 0.5, respectively), trader (2.8 ± 0.3 and 2.6 ± 0.3, respectively), and exporter levels (2.9 ± 0.1 and 2.9 + 0.2, respectively), while the body decreased with values of 4.3 ± 0.5, 3.6 ± 0.2, and 3.6 ± 0.2 for each coffee origin (table 8). the aromatic characters of coffee beans from exporters was the strongest, it was probably due to caramelization during the drying process which further produced specific aroma. 28 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. tables. results of coffee cupping (organoleptic test) on coffee beans collected from farmers, traders and exporters source of coffee beans aroma flavor body off-flavor (% of samples) farmers 2.4 ±0.4 2.5 ±0.5 4.3 ±0.5 earthy : 40 mouldy : 5 fermented : 5 traders 2.8 + 0.3 2.6 + 0.3 3.6 + 0.2 earthy : 35 woody : 5 exporters 2.9 ±0.1 2.9 ±0.2 3.6 + 0.2 score: 0 = none, 1 = low, 2 = low-medium, 3 = medium, 4 = medium-high, 5 = high some off-flavors were encountered from few coffee samples from farmers as well as from traders, i.e. earthy (35% of samples), mouldy (5% of samples), fermented (5%), and woody (5%). there was no off-flavor encountered in the coffee samples from exporters. it was due to the mixing of defective beans with good beans that the off-flavor characters decreased and could not be detected by the panelists. the soil odor was absorbed by fat in the beans causing an earthy flavor. the off-flavor was also caused by fungal infection, since some fungi could produce chemical compounds with earthy flavor, like 2-methylisoborneol and geosmin. the earthy off-flavor was higher in samples collected from farmers, since most farmers dried their coffee beans on the ground with no underlayer or mat used. this made the coffee cherry become dirty and the beans were covered with soil dust after de-hulling. drying of coffee directly on the ground takes a longer time, since the moist ground hampers evaporation. it makes the coffee mouldy and tainted with the earthy and mouldy characters. the off-flavor could be avoided if the drying process was conducted on a sheet or mat, or on patio made of concrete. sanitation along with the drying process should be emphasized, since the dirty coffee would be infected by fungi. mouldy off-flavor is also referred to as musty, which is an odor taint giving the coffee beans a mouldy odor, a result of fats in the coffee beans absorbing organic material from fungi during the drying process. this off-flavor is more pronounced than the earthy off-flavor. the defect may also appear during storage, if the coffee beans had not been dried properly. if the coffee beans from farmers and traders had a high moisture content (± 16%), it could be easily infected by fungi, especially the toxigenic fungi (see fungal analysis). once the taint occurred, it would remain permanently. in order to avoid this defect, it is suggested that the coffee be dried at the very early stage of production (on the farm) properly, and the process be continued until the moisture content reaches below 13%. it is advisable not to store the coffee beans with high moisture contents. 29 biotropia no. 14, 1999 fermented off-flavor is an undesirable taste in the coffee beans that produces a strong unpleasant sour sensation or sting on the tongue. this defect is caused by the coffee cherries that had been kept or heaped for days, thus making sugars in the beans fermented and produce sour substances. fresh cherries could be easily fermented, especially the pulp and mucilage parts. the increase in temperature and microorganisms present in the cherries will hasten the fermentation. the sour substances produced would be absorbed into the beans and taint the coffee. in order to avoid this defect, the fresh cherries had to be processed immediately. the fresh cherries should be dried on the patio soon after harvest. during the drying process, the cherries were frequently turned upside-down to hasten the process and to get more uniformly dried beans. re-wetting by the rain should also be avoided. no off-flavor was encountered in the coffee samples from exporters which showed quality improvement of the coffee beans collected from farmers and traders after being re-processed. the improvement was due to fuijher drying, sorting out of bad beans, and mixing of coffee lots. the mixing of perfect and defective coffee beans could neutralize or dilute the off-flavor which makes the percentage of off-flavor beans present become very low and undetectable by panelists. conclusions most farmers cultivated robusta coffee and they carried out the drying process to obtain commercial coffee beans. postharvest handling and technology processing methods became better from farmers to exporters. physical quality, pest infestation (insect and fungi), moisture content, reducing sugar content and coffee cupping of coffee beans also became better from farmers to exoorters. recommendations extension and dissemination of good handling practices for coffee beans especially at farmer and trader levels are necessary to improve the coffee postharvest handling and technology processing. acknowledgment the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the plantation office province level, lampung and association of indonesian coffee exporters, bandar lampung, for their information and cooperation during the surveys at farmer, trader and exporter levels in lampung. the authors are also grateful to the indonesian research institute for coffee and cocoa, for their collaboration in this study. 30 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. references central bureau of statistics. 1996. summary bulletin of cbs. jakarta. ccfac. 1998. revised position paper on ochratoxin a. paper presented at the 30"' session of the codex committee on food additives and contaminants. the hague, the netherlands, 9 1 3 march 1998. haines, c.p. 1991. insects and arachnids of tropical stored products, their biology and identification (a training manual). natural resources institute, uk. haines, c.p. 1995. insects and arachnids in indonesian food stores-biodiversity in a man-made environment. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p: 95 125. indonesian standardization council. 1992. indonesian national standard. sni 01-2907-1992. coffee. leong, e.c.w. and s.h, ho. 1991. research on liposcelis bostrycophilus and liposcelis entomophilus (psocoptera: liposcelidae). in j.o. naewbanij and a.a. manilay (eds.). proceedings of the 14"' asean seminar on grain postharvest technology, 5-8 november 1991, metro manila, philippines, p: 317-327. licht, p.o. 1995. international coffee yearbook. world coffee production. lingle, t.r. 1986. the coffee cupper's handbook: a systematic guide to sensory evaluation of coffee's flavor. coffee dev. group, washington d.c. mccready, r.m. 1970. monosaccharides. in m.a. joslyn (ed.). methods in food analysis physical, chemical and instrumental methods of analysis, 2"d ed. academic press, new york. p. 475 509. nakajima, m., h. tsubouchi, m. miyabe, and y. ueno. 1997. survey of aflatoxin bi and ochratoxin a in commercial green coffee beans by high-performance liquid chromatography linked with immunoaffinity chromatography. food and agricultural immunology (9): 77 83. ono, h., a. kataoka, m. koakutsu, k. tanaka, s. kawasugi, m. wakazawa, y. ueno and m. manabe. 1995. ochratoxin a producibility by strains of aspergiltus niger group stored in ifo culture collection. mycotoxins (41): 47 — 51. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic and professional publ.,london. prelusky, d.b., b.a. rotter and r.g. rotter. 1994. toxicology of mycotoxins. in j.d. miller and h.l. trenholm (eds.). mycotoxins in grains; compounds other than aflatoxin. eagan press, st. paul, minnesota, p. 359403. roesli, r., r. jones and d.p. rees. 1998. factors affecting outbreaks of liposcelis (psocoptera: liposcelidae) population in grain storage. paper presented at the 7"' international working conference on stored-product protection, beijing, china, 14-19 october 1998. samson, r.a., e.s. hoekstra, j.c. frisvad and o. filtenborg. 1996. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baarn, the netherlands. santoso, t, sunjaya, o.s. dharmaputra, h. halid and r.j. hodges. 1996. pest management of psocids in milled rice stores in humid tropics. international journal of pest management, 42 (3): 189 197. scott, p.m. 1994. penicillium and aspergillus toxins. in j.d. miller and h.l. trenholm (eds.). mycotoxins in grain: compounds other than aflatoxin. eagan press, st. paul. p. 261 -285. sidik, m. and y. cahyana» 1992. storage pest problem and its management in indonesia. biotrop special publication, (45): 75 87. syarif, r. and h. halid. 1993. teknologi penyimpanan pangan. penerbit arcan, jakarta. trugo, l.c. 1985. carbohydrates. in r.j. clarke and r. macrae (eds.). coffee volume 1: chemistry. elsevier applied science publ. london, p: 83 114. winarno, f.g. 1991. kimia pangan dan gizi. pt. gramedia pustaka utama, jakarta. 31 biotropia no. 14, 1999 appendix 1. results of questionnaires on postharvest handing and technology processing of coffee beans at farmer level no. subject % respondents 1. type of coffee cultivated: a. robusta coffee (coffea robusta) 77.3 b. arabica coffee (c. arabica) 0 c. both types of coffee 22.7 2. determination of harvest time was based on: a. color of coffee berry 100 b. size of coffee berry 0 c. others 0 3. harvesting method of coffee berries: a. selected method 9.1 b. strip method 90.9 4. duration of storing coffee berries: a. < 1 day 0.0 b. 1-3 days 35.7 c. > 3 days 14.3 5. storing method of coffee berries: a. in bag 100 b. in another container 0 c. spreading out on the floor 0 6. opening of coffee berry before drying: a. yes 36.4 b. no 63.6 7. drying facilities: a. ground 0.0 b. plastic (polypropylene) 27.3 c. paved floor 22.7 d. other 0 8. duration of drying: a. < 1 week 31.8 b. 1-2 weeks 59.1 c. > 2 weeks 9.1 9. methods for determining dried coffee beans: a. experience/organoleptic 100 b. using equipment for measuring moisture content 0 10. type of stored coffee beans: a. dried coffee berries 63.6 b. green coffee 36.4 11. duration of storing coffee beans: a. < 1 week 95.4 b. 1-2 weeks 4.6 c. > 2 weeks 0 12. type of bag used for storing coffee beans: a. jute bag 4.6 b. plastic bag (polypropylene) 86.4 c. others " 0 d. spreading out on the floor 90 32 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. appendix 1. (continued) no. subject % respondents 13. postharvest problems: a. pest infestation (insects and/fungi) 18.2 b. processing facility 0 c. none 81.8 14. mouldy beans problems: a. yes 27.3 b. no 2.7 15. information on postharvest of coffee: a. yes 77.3 b. no _ ___22.7 number of respondents: 22 appendix 2. results of questionnaires on postharvest handing and technology processing of coffee beans at farmer level 'no. subject %respondents 1. moisture content of coffee beans bought from farmers: a. < 12% 0 b. 12-15% 0 c. 15-20% 0 d. 20-30% 100 e. >30% 0 2. methods for determining moisture content of coffee beans a. using equipment (cera tester) 75.0 b. by estimating (organoleptic) 25.0 3. price classification of coffee beans based on: a. moisture content 40.0 b. moisture content and physical quality . 60.0 c. other criteria 0 d. no classification 0 4. drying method used: a. sun-drying 93.3 b. mechanical drying 6.7 5. drying facilities used: a. ground 0 b. plastic (polypropylene) 42.9 c. paved floor 57.1 6. duration of further drying: a. < 1 day 85.6 b. 1-2 days 7.2 c. > 2 days 7.2 7. storing method of coffee beans: a. packed in jute bag 5.0 b. packed in plastic bag (polypropylene) 30.0 c. spreading the coffee beans on the floor 65.0 33 biotropia no. 14, 1999 appendix 2. (continued) no. subject % respondents 8. duration of storing coffee beans in warehouse/store: a. 1-2 days 70.0 b. 2 days -1 week 30.0 c. > 1 week 0 9. action conducted when mouldy beans were found: a. further dried 35.0 b. no action 65.0 10. pest problems encountered: a. insects infestation 10.0 b. fungal infection 0 c. none 90.0 number of respondents: 20 appendix 3. results of questionnaires on postharverst handing and technology proccesing of coffe beans at fanner level no. subject % respondents 1. price classification of coffee beans based on: a. moisture content 0 b. moisture content and physical quality 100 c. no classification 0 2. further dried coffee beans obtained from traders: a. yes 100 b. no 0 3. drying method used: a. sun-drying 0 b. mechanical dryer (oven/mason dryer) 100 4. moisture content of coffee beans after further dried: a. < 12% 0 b. 12-13% 100 c. > 13% 0 5. sorting coffee beans used: a. manpower/manually 0 b. electronic sorting machine (model sortex) 100 6. type of bag used for storing coffee beans before reprocessing: a. jute bag 91.7 b. plastic bag (polypropylene) 8.3 c. bulk system 0 7. duration of storing coffee beans in warehouse/factory: a. < 1 week 100 b. 1-4 weeks 0 c. > 4 weeks 0 8. pest monitoring: a. yes 100 b. no 0 34 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. appendix 3. (continued) no. subject % respondents 9. fumigation of coffee beans before exported: a. yes 10 b. no 0 10. type of fumigant used: a. phosphine 125 b. methyl bromide 87.5 11. fumigation was carried out by: a. the exporters 0 b. private companies 100 12. fumigation site: a. warehousee 25.0 b. container 75.0 13. pest problems encountered: a. insects infestation * 25.0 b. fungal infection 0 c. rat infestation/others 0 d. none 75.0 14. complaint from importing countries: a. coffee beans were infested by insects 25.0 b. coffee beans were infected by fungi 0 c. physical quality was not good 0 d. coffee taste was not like desired 0 e. none 75.0 number of respondents: 4 35 biotropia no. 8, 1995: 1 10 nutrient transfer in vesicular-arbuscular mycorrhizas: a new model based on the distribution of atpases on fungal and plant membranes*) f.a. smith department of botany, the university of adelaide, south australia, 5005, australia s.e. smith department of soil science, the university of adelaide, south australia, 5005, australia abstract in this paper we review the membrane transport processes that are involved in the transfer of mineral nutrients and organic carbon between the symbiotic partners in mycorrhizas. in particular, we reassess the prevailing hypothesis that transfer in vesicular-arbuscular (va) mycorrhizas occurs simultaneously and bidirectionally across the same interface and that arbuscules are the main sites of transfer. using cytochemical techniques, we and our collaborators have reexamined the distribution of atpases in the arbuscular and intercellular hyphal interfaces in va mycorrhizas formed between roots ofallium cepa (onion) and the fungus glomus intraradices. the results showed that h + -atpases have different localisation on plant and fungal membranes in arbuscular and hyphal interfaces (gianinazzi-pearson et al. 1991). while some arbuscular interfaces had h + -atpase activity on both fungal and plant membranes, in most cases the fungal membrane lacked this activity. in contrast, the plasma membranes of intercellular hyphae always had h + -atpase and the adjacent root cells did not. this suggests that the different interfaces in a va mycorrhiza may have different functions. we propose that passive loss of p from the arbuscules is associated with active uptake by the energised (atpase-bearing) plant membrane and that passive loss of carbohydrate from the root cells is followed by active uptake by the intercellular hyphae. if this model is correct, then variations in "mycorrhizal efficiency" (i.e. the extent to which mycorrhizal plants grow better than non-mycorrhizal controls) might be determined by differences in the numbers of active arbuscules as a proportion of the total fungal biomass within the root. as a first step towards investigating this possibility, we have developed methods for measuring the surface areas of arbuscular and hyphal interfaces in different fungus-host combinations, glomus spp./ allium porrum (leek). we have also measured fluxes of p from fungus to plant and have been able to partition these between the arbuscular and total (arbuscular plus hyphal) interfaces. the implications of this work, and suggestions for future investigations of the molecular mechanisms involved in nutrient transfer in mycorrhizas, are discussed. key words: mycorrhizas/glomus intraradices/atpases/allium cepa. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19-21 april, yogyakarta, indonesia. 1 biotropia no. 8, 1995 smith, s.e., s. dickson, c. morris and f.a. smith. 1994b. transfer of phosphate from fungus to plant in va mycorrhizas: calculation of the area of symbiotic interface and fluxes of p from two different fungi to allium porrum. new phytologist. in press. smith, s.e. and f.a. smith. 1990. structure and function of the interfaces in biotrophic symbioses. new phytologist, 114, 1-38. smith, s.e. and f.a. smith. 1994. membranes in mycorrhizal interfaces: specialised functions in symbiosis. in: membranes specialised functions in plant cells. ed. by m. smallwood and d. bowles, jai press inc. (in press). tester, m. 1990. plant ion channels: whole cell and single channel studies. new phytologist, 114, 305-340. 10 biotropia no. 8, 1995 transfer of nutrients is of central importance in the functioning of the very widespread symbioses between mycorrhizal fungi and their host plants. the host gains mineral nutrients (e.g. phosphate and zinc), and in all mycorrhizas except orchid mycorrhizas the fungus gains carbohydrate. this two-way transfer occurs across interfaces which comprise the membranes of both organisms and an apoplastic region between them. the interfacial apoplast may contain considerable amounts of wall or wall-derived material, which varies according to the type of mycorrhiza and the particular interface (smith and smith 1994). in ectomycorrhizas, the interface is entirely intercellular (extracellular), whereas in some endomycorrhizas (e.g. ericoid mycorrhizas), the interface is virtually entirely intracellular. other endomycorrhizas (e.g. vesicular-arbuscular mycorrhizas) have both an intercellular interface — where the fungus penetrates between cortical cells, and an intracellular interface where the fungus penetrates the cell walls of its host and develops arbuscules. in all cases, the interface has a restricted volume. it is isolated from the external environment, and membrane transport processes in both organisms will control the physico-chemical conditions in the apoplast and hence the overall transport of solutes from one organism to the other. fig. 1 summarises the uptake and transfer of nutrients in all mycorrhizas except orchids. as well as emphasising phosphate (p) and figure 1. transfer of mineral nutrients and carbohydrates (sugars) in mycorrhizas (excluding orchids). with the exception of h + -atpases (see text), the mechanisms of membrane transport are not specified, fpm: fungal plasma membrane; rpm: root plasma membrane; pi: inorganic phosphate. 2 introduction nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith zinc (zn), it shows that other mineral nutrients can be transferred from soil to the host via the fungus. in the case of nh4 + , there is conversion to organic n compounds and subsequent transfer of some of these across the interface. the transfer of sugars to the fungus is shown in fig. 1, and also the possible transfer of "growth factors" i.e. phytohormones, vitamins etc. finally, fig. 1 shows the presence of h + -transporting atpases that are present in the functional plasma membranes of all fungal and plant cells. these "energise" the membranes both electrically, via the generation of electric potential differences (pds), and chemically via the development of ph differences, and so allow selective transport of other solutes. for example, phosphate and sugars are transported into fungal and plant cells by diferrent transport proteins which catalyse co-transport of phosphate or sugar with h + (not shown in fig. 1, for simplicity). other types of transport proteins are "channels" which can open and close in response to various signals at the plasma membrane, including changes in pds and ph. for a general discussion of interfaces and transport in mycorrhizas, see smith and smith (1990, 1994). tester (1990) describes factors that control the operation of channels. membrane transport and atpases in va mycorrhizas for va mycorrhizas, the prevailing hypothesis has been that transfer of nutrients and carbohydrates occurs bidirectionally and simultaneously across the same interface and that the arbuscules, having large surface-to-volume ratio, are important as the main site of transfer. it has been suggested in the past and sometimes even now — thatp transfer can be explained by breakdown (digestion) of arbuscules, releasing p to the host. however, calculations by cox and tinker (1976) showed that this would be far too slow the account for the fluxes of p across the interface. to make these calculations, cox and tinker (1976) measured surface areas of arbuscules in the glomus mosseae/allium cepa (onion) symbiosis using electron microscopy and image analysis, the amount of infection and the total numbers of arbuscules per unit length of root. they also estimated the lifespan of the arbuscules (about 4 days), their p content and the inflow of p i.e. the uptake of p to the host per unit length of root. in this way, they showed that the p that could be released by digestion was about 100 times too small for the actual p flux; hence continuing transfer from living arbuscules is required. this is an important conclusion, and the calculated value for the p flux 13 nmol m (arbuscular surface area) -2s-1 -is also important since it is similar to values for uptake of p into cells by active transport (i.e. by h + -p co-transport), but much larger than ty 3 biotropia no. 8, 1995 pical values for efflux of p. an important question thus becomes: what is the mechanism for release of p from the fungus? this issue is discussed by smith et al. (1994a). the hypothesis of simultaneous two-way transport of p and sugar in va my corrhizas is summarised in fig. 2. details of co-transport and passive transport are shown. the hypothesis has been supported by the cytochemical demonstration of atpases on both fungal and plant plasma membranes in the arbuscular interface (marx et al. 1982), but does not take into account the potential role of the intercellular fungal hyphae in transport processes (smith and dickson 1991). likewise, the possible role of the intercellular interface was not considered by cox and tinker (1976). figure 2. diagrammatic representation of the distribution of h + -atpases and associated transport processes in va mycorrhizas that would result in bidirectional transfer of sugar and phosphate across the same arbuscular interface. based on figure 7 of gianinazzi-pearson et al. (1991). pam: periarbuscular (plasma) membrane of the root cortical cells. 4 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith spatial separation of transport? gianinazzi-pearson et al. (1991) re-examined the distribution of atpases in the arbuscular and intercellular (hyphal) interfaces in va mycorrhizas formed between roots of allium cepa (onion) and glomus intraradices. the cytochemical methods were similar to those used by others: they involved the deposition of lead phosphate at sites of atpase activity and the use of inhibitors (e.g. vanadate and molybdate) to distinguish h + -atpases likely to be involved in active membrane transport from other enzymes with atp-hydrolysing ability. the results showed that h + -atpases have different localisation on plant and fungal membranes in arbuscular and hyphal interfaces (gianinazzi-pearson et al., 1991). while some arbuscular interfaces had h + -atpase activity on both fungal and plant membranes, in most cases the fungal membrane lacked this activity. in contrast, the plasma membranes of intercellular hyphae always had h + -atpase and the adjacent root cells did not. these results suggest that the different interfaces in a va mycorrhiza may have different functions (fig. 3). the arbuscules may indeed play a major role in p transfer. passive p loss from the fungus to the inter facial apoplast would be associated with active uptake (h + -p co-transport) by the energised (atpase-bearing) plant membrane. reabsorption of p from the interfacial apoplast by the fungus may be reduced in the arbuscules, because the fungal membrane frequently appears to lack atpase activity and hence the capacity for active uptake. these figure 3. spatial separation of transfer in va mycorrhizas. a: passive loss of p from the fungus and active uptake (h + -phosphate co-transport) across the energised periarbuscular membrane. b: passive loss of sugar to the intercellular apoplast and active uptake (h + -hexose co-transport) across the energised hyphal plasma membrane. based on figure 7 of gianinazzi-pearson et al. (1991). 5 biotropia no. 8, 1995 processes would result in net transfer of p from fungus to plant. we have shown that transfer of p between the symbionts occurs at rates consistent with high passive efflux from the fungus and active uptake by the plants (smith et al. 1994a & b). gianinazzi-pearson et al. (1991) propose that the intercellular hyphae (but not the arbuscules) may be responsible for carbohydrate transfer from plant to fungus. in this case, passive loss of carbohydrate from the root cortical cells to the intercellular apoplast would be followed by active uptake by the fungal hyphae. as far as we are aware, the proposal that transfers of p and carbohydrate are spatially separated had not been made previously. arbuscules and va mycorrzhizal "efficiency" an attraction of this new model is that variations in mycorrhizal "efficiency" (i.e. the extent to which mycorrhizal plants grow better than non-mycorrhizal controls) might be explained by differences in the numbers or size of metabolically active arbuscules as a proportion of the total fungal biomass within the root. without arbuscules, the fungus would be essentially a parasite, draining carbohydrate from its host but giving no phosphate in exchange. it is well known that mycorrhizal "efficiency" does vary in different fungus/host combinations, or under different environmental conditions, but there has been little attempt to correlate these variations with numbers of arbuscules or their activity. one reason is the difficulty of measuring numbers and surface areas of arbuscules, and length and surface area of intercellular hyphae, rather than just taking the length or percent of the root that contains fungus as a measure of infection. in order to remedy this deficiency, we have developed an updated version of the methods used by cox and tinker (1976), including computer-aided image analysis techniques and the use of nitroblue tetrazolium to distinguish metabolically active fungal tissues from the total fungal biomass (smith and dickson 1991). by means of a size facility in the program, it is possible to distinguish between intercellular hyphae and arbuscules. table 1 shows data for allium porrum infected by glomus sp. 'city beach' (wum16). percentage infection was obtained conventionally and the areas of interface obtained by image-analysis. details for the experiment and methods are given by smith and dickson (1991) and smith et al. (1994b). table 1 shows that the surface area of metabolically active arbuscules per metre of root declined with time, while that of hyphae increased. after 63 days, the hyphae had the larger surface area, illustrating the danger of ignoring them as a site of nutrient transfer, irrespective of what model is adopted. table 2 shows the inflows of p (i.e. amounts of p taken up per metre of root length) into non-mycorrhizal and mycorrhizal plants, with the amount taken into 6 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith the latter via the fungus calculated by subtraction. we are aware that this calculation, which assumes that fungal infection does not affect p uptake by the host, may produce errors, but this possibility has been ignored in our calculations. table 2 shows that inflow of p decreased with time, particularly in non-mycorrhizal plants. table 3 shows the fluxes of p across the interfaces, as obtained by dividing the inflows (table 2) by the average surface areas obtained from table 1. the increasing divergence with time between the values that were obtained assuming that the flux occurred 1) only across the arbuscular interface and 2) across both arbuscules and intercellular hyphae reflects the increasing contribution of the latter to the total surface area. the range of values in table 3 (3.7-12.8 nmol m -2 s1 ) agrees quite well with the single value of nmoll m -2 s 1 that was calculated by cox and tinker (1976). 7 biotropia no. 8, 1995 although these results show that it is possible to make separate calculations for influx of p across the arbuscular interface and the total interface, they do not by themselves answer the question of whether the arbuscular interface alone is responsible for p transfer. to do this, it will be necessary to attempt to correlate p transfer with arbuscular surface area, e.g. in comparison with the total fungal biomass within the root. results that we have obtained so far suggest that where different va mycorrhizal fungi infect the same host species, there is no simple correlation. table 4 shows again the results for allium porrum infected by glomus sp. 'city beach' over 21-42 days of growth and compares them with results for allium porrum/glomus mosseae obtained at the same time and under the same conditions (smith and dickson 1991; smith et al. 1994b). g. mosseae produced a slightly larger % infection but a slightly smaller inflow of p (19.0 compared to 22.3 pmol m 1 s 1 )the areas of interfaces with g. mosseae were more than twice that with g. sp. 'city beach', and the flux of p via mosseae was accordingly much smaller than with g. sp. ' city beach', whether calculated using the area of arbuscular interface or the total interface. these results show that g. sp. 'city beach' loses p faster on a surface area basis. there are several possible explanations including not only differences in numbers of membrane transport proteins at the interface but also different rates of translocation of p through the fungi or different rates of uptake from the soil which could in turn be associated with different lengths of external hyphae. to pursue further the question of the role of the arbuscule as the sole location of p transfer in comparison with that of the intercellular hyphae (or total interface) as the location of exchange of carbohydrate, it will be necessary to do more sophisticated experiments with more fungus/host combinations. the use of va mycorrhizas in which development of arbuscules is suppressed will be particulary valuable. 8 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith conclusions in this paper we have reviewed briefly the mechanisms for transfer of nutrients in mycorrhizas mainly va mycorrhizas and have then proceeded to re-evaluate the sites of nutrient transfer, particularly the question of whether simultaneous bidirectional transfer occurs in arbuscules. we suggest that this may not be the case, and this in turn has led us to quantify the development of the arbuscular interface vis-a-vis the total interface and to attempt to calculate p fluxes across both interfaces. as well as pursuing this approach further, we are starting to attempt to identify the molecular mechanisms for transport at the interface and also to measure the physico-chemical conditions there, including the ionic conditions in the apoplast. in this way we hope that the control of nutrient transfer and its role in determining "mycorrhizal efficiency" will be better understood. acknowledgements we thank our collaborators, particularly vivienne gianinazzipearson, silvio gianinazzi, sandy dickson, christina morris, mark tester and rob reid for all their help in discussions and experiments. funding came from the australian research council, an inra international collaborative research program and the university of adelaide. references cox, g. and p.b. tinker. 1976. translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. i. the arbuscule and phosphorus transfer: a quantitative ultrastructural study. new phytologist, 77, 371-378. gianinazzi-pearson, v., s.e., smith, s. gianinazzi and f.a. smith. 1991. enzymatic studies on the metabolism of vesicular-arbuscular mycorrhizas v. is h + -atpase a component of atp hydro-lysing enzyme activities in plant/fungus interfaces? new phytologist, 117, 61-74. marx, c., j., dexheimer, v. gianinazzi-pearson and s. gianinazzi. 1982. enzymatic studies on the metabolism of vesicular-arbuscular mycorrhizas iv. ultracytoenzymological evidence (at-pase) for active transfer processes in the host-arbuscular interface. new phytologist, 90, 37-43. smith, f.a., s., dickson, c., morris, r.j., reid, m., tester and s.e., smith. 1994a. phosphate transfer in va mycorrhizas: special mechanisms or not? proceedings of the 4th international symposium on the structure and function of roots. in press. smith, s.e. and s. dickson. 1991. quantification of active vesicular-arbuscular mycorrhizal infection using image analysis and other techniques. australian journal of plant physiology, 18, 637-648. 9 1.pdf 10.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biot ropia no. 10, 1997 : 29 -41 the effects of drying and shelling on aspergillus flaws infection and aflatoxin production of maize* o.s. dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia i. retnowati seameo biotrop, p.o. box 116, bogor, indonesia h.k. purwadaria department of agricultural engineering, faculty of agriculture technology, bogor agricultural university, bogor. indonesia h. susilo seameo biotrop, p.o. box 116. bogor, indonesia abstract the effects of drying and shelling on aspergillus fla\-us infection and aflatoxin production of maize stored under laboratory conditions were investigated together with the intactness of grain and change of moisture content during the storage period. fully matured maize var. arjuna and cpi-2 were harvested at 90 and 97 days after planting, respectively, after which they were unhusked and divided into 4 pans. the 1st and the 2nd parts were sun dried up to 20^ moisture content (m.c.) and then shelled and re-dried up to 17 and 14% m.c.. respectively. the 3rd part was sun dried up to 17% m.c. and then shelled but not re-dned. the 4th pan was sun dried up to 17% m.c. and then shelled and re-dried up to 14% m.c. the maize was sun dried by spreading either the cobs or the kernels on the paved floor. the nail-down wood and mechanical sheller were used for shelling the maize. after drying and shelling, maize samples were stored in the jars which were covered with muslin cloth for 3 months under laboratory conditions. a. flavus was isolated using dilution method on aspergillus flavus and parasiticus agar (afpa). the damaged kernel analysis was carried out at the beginning of storage to obtain the percentage of damaged kernel caused by shelling. the m.c. and aflatoxin were determined using oven and high performance liquid chromatography (hplc) methods, respectively. the m.c. decreased at 1 month of storage and then it was almost constant at 2 and 3 mo nths of storage. the percentage of damaged kernels of maize var. cpi -2 was higher than those of var. arjuna. the percentage of damaged kernels of maize shelled at 20% m.c. was higher than that shelled at 17% m.c. the percentage of damaged kernels of maize shelled by mechanical sheller was higher than that shelled by nail-down wood. *paper presented at the sy mposium on pest manag ement for stored food and feed, 5 -7 september 1995 bogor, indonesia. 29 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra population of a. flavus on maize var. arjuna was higher than that of var. cpi-2. the population on maize stored at the initial m.c. of 17% was higher than that of 14%, the population on maize shelled by mechanical sheller was higher than that shelled by nail-down wood, but there was no significant difference. the population increased at 1 and 2 months of storage and then decreased at 3 months of storage. total aflatoxin bi content of maize var. cpi-2 was higher than that of var. arjuna. the content of maize dried up to 17% m.c. and then shelled but not re-dried was the lowest compared with the other methods of drying. the content of maize shelled by nail-down wood was not significantly different than shelled by mechanical sheller. the content increased with the increase of storage duration. key words: drying/shelling/x5perg///mj-/zav«j/aflatoxin introduction before and after harvest, maize (zea mays) could be infected by aspergillus flavus, a fungus that can produce aflatoxin (butler 1974; lillehoj and hesseltine 1977). the most notorious toxin is aflatoxin, as it is an extremely potent carcinogen affecting several animal species. cole et al. (1982) reported that among mycotoxins, aflatoxin was often found in maize. survey conducted by biotrop team in 1992 revealed that 35 maize samples collected from farmers and traders in lampung province contained 23-367 ppb of aflatoxin. thirty samples contained aflatoxin of more than 30 ppb (dharmaputra et al 1993). another survey conducted during 1993/94 showed that 108 maize samples collected from fanners in central lampung and kediri regencies contained 5-291 and 9-283 ppb of aflatoxin bi during dry and wet seasons, respectively. of these, 87 samples contained more than 30 ppb of aflatoxin bl. while 32 maize samples collected from traders in the two regencies contained 10-104 and 21-115 ppb of aflatoxin bl during dry and wet seasons, respectively, 26 of which contained more than 30 ppb (dharmaputra et al. 1994). the high level of aflatoxin content in most maize samples could be caused by the methods of handling that were not carried out properly. limits on the amount of aflatoxins which are permitted in foods vary between countries and products. most european countries have moved towards a limit of below 30 ppb for total aflatoxin concentration in all foods and less than 0.05 ppb of m aflatoxin hi milk (gilbert 1991). in asia, taiwan tolerates up to 50 ppb of aflatoxin bi in cereals and peanuts, while thailand has a tolerance of 20 ppb for b and g aflatoxins hi all foods and japan 10 ppb tolerance for b aflatoxin in all foods. the philippines has a 20 ppb tolerance for aflatoxin bi in coconut and peanut products for export (van egmond 1991). 30 biotropia no. 10, 1997 postharvest handling (among others, drying and shelling) could affect fungal infection. in general, the moisture content of freshly harvested maize is still high, making it a good substrate for fungal growth. shelling could cause mechanical damage, and fungal spores can infect the kernel through the damage. consequently, the kernels should be shelled using a proper tool at certain moisture content to reduce the damage of kernels. according to covanich (1991) drying is particularly a vital operation in gram handling chain, since moisture is the most important factor determining the extent grains are liable to deterioration during storage. dried grains are less susceptible to insect and mold attacks. overdrying of grains in the sun, for example, can cause breakage, loss of viability, etc. in general, the moisture content of freshly harvested maize was between 23-33%. drying of maize was conducted up to 17-20% moisture content (sfcdp 1990). most farmers in lampung and east java shelled cob of maize using mechanical sheller, while in south sulawesi fanners use a nail-down wood (sfcdp 1990). according to purwadaria (1987) the mechanized maize sheller could reduce the quantity of losses as much as 5% and lowered the shelling cost to 67-40% of the manual shelling cost. the objectives of this study were : 1) to determine the effects of some methods of drying and shelling on aspergillus flavus infection and aflatoxin production of maize stored under laboratory conditions; and 2) to determine the effect of drying and shelling on the intacmess of kernel, as well as the effect of storage period on the change of moisture content. materials and methods maize variety two maize varieties (arjuna and cpi-2) were used hi this study. they were grown at the experimental plot of the research institute for food crop biotechnology, bogor, harvested at 90 and 97 days after planting and unhusked immediately after harvest. drying and shelling cobs of maize were divided into 4 parts. the 1st and the 2nd parts were su n dried up to 20% moisture content (m.c.) and then shelled and re -dried up to 17 and 14% m.c., respectively. the 3rd part was sun dried up to 17% m.c. and then shelled but not re-dried. the 4th part was sun dried up to 17% m.c. then shelled and re-dried 31 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra up to 14% m.c. all of the maize samples were sun dried by spreading either the cobs or the kernels on the paved floor. nail-down wood and mechanical sheller type yanmar tf 55-di with cylinder rotation of 500-700 rpm were used for shelling the maize. storing of maize and method of sampling after drying and shelling, 500 g of maize of each treatment were placed in 3.3 l jar covered with muslin cloth, and stored for 1, 2, and 3 months under laboratory conditions. two replicates were used for each treatment. the ambient temperature and relative humidity of the storage were recorded using a wilh. lambrecht thermohygrograph type 252. initial sample was derived from each replicate (jar) at the beginning of storage, and then at 1, 2, and 3 months of storage. this sample was divided twice using a sample divider to obtain working samples for moisture content. damaged kernels, population of a. flavus, and total aflatoxin bi content analyses. moisture content, damaged kernels, population of a. flavus and alfatoxin bi content analyses moisture content (based on wet basis) was determined using oven method at 130° c for 2 hours (bsi 1980). the analyses for damaged kernels were carried out at the beginning of storage to obtain the percentage of damaged kernel caused by shelling. a. flavus was isolated using dilution method on aspergillus flavus and parasi-ticus agar (afpa) (pin and hocking 1985). aflatoxin content was determined using high performance liquid chromatography (hplc) method (rodriquez and mahoney 1994). statistical analysis the data were analyzed using completely randomized factorial design with 4 factors. the 1st, 2nd, 3rd and 4th factors were maize variety, method of drying and shelling, and duration of storage, respectively. results and discussion the effect of maize variety, methods of drying and shelling, and storage period on moisture content and damaged kernel based on statistical analysis, the effects of drying, duration of storage and their interaction gave very significant differences in moisture content, while variety and shelling did not give significant differences. 32 biotropia no. 10, 1997 at the beginning of storage, moisture content on each drying method (i, ii, iii and iv) was 16.84, 14.10, 17.11 and 14.35%, respectively. at 1 month after storage they decreased (13.98, 13.51, 14.05 and 13.75%, respectively), and then they were almost constant at 2 and 3 months after storage (table 1). moisture content of maize at 2 and 3 months after storage would approach the equilibrium moisture content (emc) with relative humidity of the storage. during storage m.c. of grains would move towards the emc. according to hall (1957), henderson and perry (1976), emc was table 1. moisture content of maize treated with different methods of drying during storage numbers followed by the same letter do not differ significantly according to duncan' s multiple range test at 95% confidence level i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shell ed and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re -dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re -dried up to 14% moisture content. reached when the grains did not absorb or release vapor any more. brooker et al. (1974) reported that emc of grains were affected by temperature, humidity, variety and maturity of grains. range of temperature and relative humidity of the storage were 21.75-29.25°c and 47.88-88.25%, respectively. (table 2). the effects of maize variety, drying and shelling gave very significantly differences to damaged kernels, while their interaction was not significantly different. table 2. range of temperature and relative humidity in the storage room 33 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra the percentage of damaged kernels of maize var. cpi -2 (5.0%) was higher than that of var. arjuna (3.6%) (table 3). based on visual observation, the kernels of var. cpi -2 were bigger and less solid than var. arjuna, therefore it could be more easily broken during shelling. table 3. the effect of maize variety, methods of drying and shelling on damaged kernels at the beginning of storages * numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level *damaged kernels analysis was carried out only at the beginning of storage i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re -dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re -dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re -dried up to 14% moisture content. the percentage of damaged kernels of maize shelled at 20% m.c. (drying methods i and ii) (4.7 and 5.4%, respectively) was higher than maize shelled at 17% m.c. (drying methods hi and iv) (3.7 and 3.2%, respectively). according to sfcdp (1990), in general, the percentage of damaged kernels increased if the m.c. was more than 18%. idrc (1988) reported that the maize quality deteriorated more when the shelling process took place directly after harvest at a high moisture level of grain (around 37% wb) compared to the process where maize shelling was done after the initial sun drying which brought the grain moisture content down to aroun d 25% wb. the percentage of damaged kernels of maize shelled by mechanical shelter (5.7%) was higher than that shelled by nail-down wood (2.9%). shelling of each cob of maize using a nail-down wood did not cause friction among the cobs, while shelling usin g mechanical shelter created a friction. moreover, shelling done by manpower 34 biotropia no. 10, 1997 using a nail-down wood can be controlled to reduce the friction between sheller and maize. according to suprayitno (1980), the percentage of damaged kernels of maize shelled using mechanical sheller was high, because there was a friction among intact kernels, between intact kernels and the cylinder of mechanical sheller. the effect of maize variety, methods of drying and shelling, and storage duration on population of aspergillus flavus based on statistical analysis, the effects of maize variety gave significant differences to population of a. flavus; drying and duration of storage gave very significant differences; while shelling and interaction among maize variety, drying, shelling and duration of storage did not give significant differences. population of a. flavus on maize var. arjuna (697.2 colonies/g) was higher than that of var. cpi-2 (306.4 colonies/g) (table 4). wilson el al. (1983) reported that the growth of a. flavus and aflatoxin production were affected by some metabolites (groups of alcohol and aldehyde) produced by maize. population of a. flavus on maize stored at the initial m.c. of 17% (drying methods i and iii) (570.9 and 1224.4 colonies/g) was higher than that of 14% (drying methods ii and iv) (84.2 and 127.7 colonies/g) (table 4). christensen and kaufmann (1974) reported that the minimum m.c. for the growth of a. flavus on maize was 18-18.5%. according to kawashima and kawasugi (1988). grain which was sun dried immediately after shelling on concrete drying floor with m.c. less than or around 15% could be stored with low contamination of a. flavus (1.5-8%) for 56 days in middlemen's storage. if the grains (with m.c. more than 20%) were not dried after shelling, a. flavus contamination prevailed quickly. however, if m.c. was less than 17% the development of a. flavus could be inhibited. population of a. flavus on maize shelled by mechanical sheller (573.4 colonies/g) was higher than shelled by nail-down wood (430.2 colinies/g), but it did not give significant difference (table 4). also the percentage of damaged kernels shelled by mechanical sheller was higher than those shelled by a nail-down wood. the damaged kernels were more easily infected by a. flavus. dharmaputra et al. (1994) reported that there was a positive correlation between the damaged kernels and the percentage of kernels infected by a. flavus of maize obtained by farmers and village traders in central lampung and kediri regencies. population of a. flavus increased at 1 and 2 months of storage, from 48.5 colonies/g to 586.8 and 890.7 colonies/g, respectively, because the m.c. of grain, temperature, relative humidity of storage and nutrition content of grams still supported the fungal growth. garraway and evans (1984) revealed that the growth and development of fungi was very much affected by nutrition contents and substrate. according to 35 the effects of drying and shelling on aspergillus flavus infection o.s. oharmaputra chatterjee et al. (1990) a. flavus growth was highly and linearly correlated with the decrease in the percentage of healthy germ and germinability, root-shoot growth, total carbohydrate and protein of grain. pomeranz (1992) reported that during storage deterioration of grain occurred, consequently nutrients were lost because of changes among others in carbohydrates. however, at 3 months of storage, population of a. flavus (481.1 colonies/g) decreased (table 4). it was assumed that m.c., relative humidity and nutrition contents decreased and there were competitive fungi to a. flavus. according to christensen (1955), mills and abramson (1982), one kernel can be infected by more than one fungal species. in this study, the predominant fungi in addition to a. flavus were acremonium strictum, fusarium moniliforme and penicillium citrinum. zummo and scott (1992) reported that f. moniliforme inhibited a. flavus infection and aflatoxin production on maize. table 4. the effect of maize variety, methods of drying and shelling, and duration of storage on population of aspergillus flavus numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re-dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re-dried up to 14% moisture content. 36 biotropia no. 10, 1997 the effect of maize variety, methods of drying and shelling, and storage duration on total aflatoxin b 1 content according to heathcote and hibbert (1978) production of aflatoxin was affected by strain of fungi, relative humidity, temperature, oxygen, carbohydrate and kind of commodities. in this study, four kinds of aflatoxins were determined, i.e. aflatoxin b 1 , b2, g1 and g2 based on statistical analysis, the effects of maize variety, drying, interaction between drying and shelling and interaction among maize variety, shelling and duration of storage gave significant differences to total aflatoxin b 1 content; duration of storage gave very significant difference, while interaction among maize variety, drying, shelling and duration of storage did not give significant difference. total aflatoxin bi content of maize var. cpi-2 (35.97 ppb) was higher than that of var. arjuna (32.36 ppb) (table 5). this could be attributed to the effect of metabolites produced by maize. total aflatoxin bi content of maize dried up to 17% m.c. then shelled but not re-dried (30.34 ppb) was the lowest compared with the other methods of drying (drying methods i. ii and iv) (36.73, 34.79 and 34.60 ppb, respectively) (table 5), although the population of a. flavus of maize was the highest. according to diener and davis (1969) aflatoxin production depended on the strain of a. flavus. total aflatoxin bi content of maize shelled by nail-down wood (32.78 ppb) was lower and not significantly different with that shelled by mechanical sheller (35.45 ppb) (table 5). it was assumed that the high damaged kernels caused by shelling using mechanical sheller were more easily infected by a. flavus, consequently, aflatoxin producing strains had more chance to infect the kernels. total aflatoxin b 1 content of maize increased with longer storage. at the begin ning of storage, it was 0.49 ppb, while at 1, 2 and 3 months of storage they were 8.19, 50.83 and 76.95 ppb, respectively, because aflatoxin could not be easily decomposed, thus, it accumulated. melting points of aflatoxin b 1 , b2, g1 and g2 were more than 230 c, therefore, they cannot be decomposed by ordinary heating (betina 1989). as a secondary metabolite, aflatoxin production was dependent on stadia of fungal growth, aflatoxin biosynthesis pathways and on factors affecting enzymatic reaction. according to davis and diener (1983) aflatoxin production is as follows: as fungal growth and primary metabolism proceed, little or no aflatoxin was formed initially. eventually, phospha te, nitrogen, or some trace elements become limited and primary growth is retarded. as primary metabolism becomes disorganized, various primary metabolites accumulate. metabolites such as pyruvate, malonate, acetate, various amino acids, etc. trigger the development and stimulate the activity of en 37 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra table 5. the effect of maize variety, methods of drying and shelling, and duration o f storage on total aflatoxin b1 content numbers followed by the same lener do not differ significantly according to duncan's multiple range tea at 95 % confidence level aflatoxin b1. b2. g1 and g2 contents were convened into total aflatoxin b1 i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re-dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re-dried up to 14% moisture content. zymes of secondary metabolism, such as those of the polyketide biosynthetic pathway that in turn promote aflatoxin biosynthesis over fatty acid biosynthesis in toxigenic isolates of a. flavus. according to betina (1984) there were acetyl co-a and malonyl co-a in aflatoxin biosynthesis. there are active ingredients of acetate and malonate which compose lipid. aflatoxin arises naturally when a toxin-producing strain of a. flavus grows on a substrate where environmental conditions are suitable for the development of the fungus. in view of the fact that a. flavus was ubiquitous and capable of development over a wide range of temperature on substrates of high carbohydrate content (heathcote and hibbert 1978). 38 biotropia no. 10, 1997 conclusions 1. moisture contents of maize decreased at 1 month of storage, and then they were almost constant at 2 and 3 months of storage. 2. maize var. cpi-2 was more resistant than var. arjuna to aspergillus flavus infection, although the percentage of damaged kernels of maize var. cpi-2 was higher than those of var. arjuna. 3. in general, the best drying method was as follows: maize was sun dried up to 17% m.c., and then shelled and re-dried up to 14% m.c. 4. a. flavus population of maize shelled by mechanical sheller was insignificantly different with nail -down wood, although the percentage of damaged kernels of maize shelled by mechanical sheller was higher than that shelled by nail-down wood. 5. population of a. flavus increased at 1 and 2 months of storage, and then decreased at 3 months of storage. 6. total aflatoxin bi content 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(ed.). storage of cereal grains and their products. american associations of cereal chemist inc., minnesota, p. 55-141. purwadaria. h.k. 1987. grain post harvest technology system in indonesia: maize. soybean and groundnut in north sumatra. south sumatra and lampung. consultancy report ministry of agriculture, indonesia -fao, undp (df/fao/ins/85/004). rodriquez. s.b. and n.e. mahoney 1994. inhibition of aflatoxin production by surfactants. applied and environmental microbiology 60(1): 106-110. sfcdp report. 1990. studies on postharvest handling on corn, soybean and cassava in lampung. east java and south sulawesi, moa-aid. indonesia. suprayitno. 1980. the effect of some shelling methods on the damaged kernels of maize. faculty of mechanization and agricultural technology, bogor agricultural university. bogor. indonesia (unpublished). van egmond. h.p. 1991. regulator) aspects of mycotoxins in asia and africa. in fungi and mycotoxins in stored products. aciar proceedings no. 36: 194-197. wil so n d.m.. r.c. gue l dne r and r.a. hil l 1983. effect of plant metabolites on the aspergillus flavus group and aflatoxin production. in diener, u . l . , r.l. asquith and j.w. dickens (eds.). aflatoxin and aspergillus flavus in corn. southern cooperative series bulletin 279. p. 13-15. z um m o n and g.e. scot t. 1992. interaction of fusarium moniliforme and aspergillus flavus on kernel infection and aflatoxin contamination in maize ears. plant dis. 76: 771-773. 41 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf biotropia vol. 28 no. 2, 2021: 165 172 doi: 10.11598/btb.2021.28.2.1297 165 karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity ahmad ridwan1 and awaludin2 1school of life sciences and technology, bandung institute of technology, bandung, indonesia 2department of aquaculture, faculty of fisheries and marine science, university of borneo tarakan, indonesia received 4 september 2019/accepted 30 january 2020 abstract white shrimp litopenaeus vannamei is one of the prime shrimp commodities cultivated in indonesia. as such, the discovery of more efficient seed production techniques for this species is deemed necessary. karamunting (melastoma malabathricum) extract contains the cholesterol precursor called lanosterol, a phytosterol which is used by crustaceans to form the animal steroid hormone that is very crucial in their reproduction. hence, this research aimed to determine the ovary development of mature l. vannamei individuals injected with the karamunting ethanol extract. the experiment was carried out in several stages. firstly, injecting the white shrimp at the base of the 5th leg, every 3 days for 15 days with variable control dosage 0 (c), 10 mg/kg bw (t1), 7.5 mg/kg bw (t2), 5 mg/kg bw (t3), 2 mg/kg bw (t4) and 1 mg/kg bw (t5), where bw is body weight. secondly, isolating the white shrimp parent ovary. thirdly, measuring the progesterone level in the ovary using the radioimmunoassay (ria) method. fourthly, observing the histology of white shrimp parent ovary and, finally, analyzing the data. measurements of the increase in progesterone levels showed that the administration of karamunting ethanol extract significantly affected the progesterone production (p˂0.05). histology observations of gonadal development in the control, t5 and t4 showed that the cells developed to previtellogenesis oocytes whereas in treatment t1, t2 and t3 ovary cells developed into endogenous vitellogenesis oocytes and only in t1 did the ovarian cells develop to form exogenous vitellogenesis oocytes. karamunting extract significantly increased the oocyte sizes (p˂0.05). at the start of the experiment, the average oocyte sizes were at 15.57 ± 3.15 µm at the end of the experiment, the control was at 25.29 ± 2.69 µm and the ovarian treatments produced the following oocyte sizes; t1 at 65.65 ± 2.64 µm, t2 at 63.98 ± 3.06 µm, t3 at 39.12 ± 6.01 µm, t4 at 28.08 ± 0.84 µm and t5 at 27.65 ± 0.71 µm. the extract produced oocyte sizes greater than at the beginning of maintenance and control. apparently, the lanosterol in the karamunting extract had increased the hormone progesterone resulting in an accelerated gonadal maturity and enlargement of oocyte sizes in the parent individuals of the white shrimp. keywords: gonadal maturity, histology, melastoma malabathricum, litopenaeus vannamei, progesterone, reproduction introduction one of the important world-wide aquaculture industries is shrimp farming which accounted for the global 2.9 million tons of shrimp farm production in 2016 (fao 2017), of which 75% came from asia-pacific. indonesia contributed about 390 tons. one of the shrimp commodities cultivated in indonesia is the white shrimp (litopenaeus vannamei). for better cultivation, more efficient techniques are needed for white shrimp production. several ways were tried to increase its seed production, among others; eye ablation, hormone injections, and high protein feeding. however, some of these methods have not been able to significantly improve shrimp reproduction. gonad maturity in white shrimp is influenced by two antagonistic hormones, namely gonad inhibitng hormone (gih), which is synthesized in the x sinus organ glands (xo-sg) in the eye and gonad stimulating hormone (gsh) produced by the brain and chest ganglion. moreover, there is the involvement of other hormones in regulating reproduction in crustacean animals, such as the progesterone, follicle stimulating hormone (fsh), and *corresponding author, email: ridwan@sith.itb.ac.id biotropia vol. 28 no. 2, 2021 166 luteinizing hormone (lh). the availability of cholesterol strongly influences the development of crustacean ovaries. in the endocrine system, cholesterol is a precursor of steroid hormones that function for the reproduction and maturation of the gonads (wouters et al. 2001). cholesterol is a sterol compound which is a precursor of steroid hormones and molting hormones (sheen 2000). this is one of the chemical compounds that cannot be synthesized by crustaceans (kanazawa et al. 1988) but is very much needed by the parent shrimp. therefore, to meet the cholesterol requirements, crustaceans obtain it from outside the body through feed intake. cholesterol sources can be obtained from plants that form secondary metabolites. one plant that contains cholesterol is karamunting (melastoma malabathricum) which is widely known for its medicinal uses particularly its secondary metabolic compounds that consist of saponins, tannins, triterpenoids/steroids, flavonoids. karamunting extracts obtained by using ethanol solvents contained lanosterol, the cholesterol commonly found in plants (ridwan et al. 2015). karamunting plants also contained sitosterol α and β amyrin from the hexane fraction (nuresti et al. 2003). mangrove crabs (scylla serrata) supplemented with serotine cholesterol had experienced an accelerated ripening of the parent ovaries (pattiasina et al. 2010). until recently, the use of synthetic cholesterol is still the foundation for accelerating the maturity of shrimp gonads, while the use of natural cholesterol from plants is still very rarely done. therefore, this research was conducted to prove whether or not the administration of karamunting extract can increase the amount or content/quantity of hormone progesterone and accelerate the maturation of white shrimp gonads. materials and methods materials the materials used in this study include karamunting (melastoma malabathricum) ethanol extract, mature individuals of white shrimp (litopenaeus vannamei), feed, formalin, xylol, 100% ethanol, distilled water, paraffin, picric acid, and eosin. experimental design the shrimp broodstock, with shrimps weighing 32 35 g, was placed in a 30 x 30 x 60 cm aquarium. the shrimps were fed with fresh worms and oysters at a dosage of 15% of body weight per day. feeding was carried out 5 times a day at 04.00 h, 07.00 h, 13.00 h, 18.00 h, and 23.00 h. this study was conducted using a completely randomized design (crd) with 6 treatments and 5 repetitions. using a 1 ml tuberculin syringe, the karamunting extract was injected at the base of the fifth leg of the white shrimp following the method of tarsim et al. (2007) at extract dosages of 10 mg/kg bw (t1); 7.5 mg/kg bw (t2); 5 mg/kg bw (t3); 2.5 mg/kg bw (t4); 1 mg/kg bw (t5); and control or without extract (c). the parameters that were used included the increase in the progesterone content by using the radioimmunoassay (ria) method and in the ovary development using the histology staining method. at the end of the 15-day observation period, the shrimp gonads were isolated to measure the level of gonad maturity. all data obtained were statistically analyzed by using anova. results and discussion karamunting ethanol extract on the white shrimp progesterone hormone the injection of estradiol-17β into the female white shrimp had stimulated the development of the gonads to the level of gonad maturity (lgm) i (tarsim et al. 2007). whereas white shrimp with human chorionic gonadotropin, progesterone, or a combination of both in the form of feed and injection, had stimulated the ovaries to lgm iv in the 11-month-old shrimp weighing 80 120 g and lgm ii in the 5.5month-old shrimp weighing 60 80 g (ismail 1991). the progesterone content at the start of the experiment was at 0.03 ± 0 ng/ml. after the 15day experiment the contents were measured as follows:, control (c ) was at 0.03 ± 0 ng/ml, t1 at 0.082 ± 0.050 ng/ml), t2 at 0.058 ± 0.023 ng/ml, t3 at 0.034 ± 0.005 ng/ml, t4 at 0.03 ± 0 ng/ml and t5 at 0.03 ± 0 ng/ml). an increase in the hormone progesterone was observed in karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 167 the shrimp applied with karamunting extract t1 and t2 (fig. 1.). this shows that karamunting extract containing lanosterol had increased the content of the cholesterol hormone, resulting in the acceleration of gonadal maturity of the parent white shrimp. similar results were also observed among the tiger shrimps treated by eye ablation and serotonin hormone injection (wongprasert et al. 2006). histology gonad development was observed not only morphologically but also histologically. histological observation was intended to see the development of oocyte cells in the gonads of the parent white shrimp (l. vannamei) as influenced by the karamunting ethanol extract. the development of oocyte in shrimp were classified as previtellogenic, endogenous vitellogenic oocytes, and exogenous vitellogenic oocytes (wilder et al. 2010). based on observations of oocyte development after exposure to the karamunting ethanol extract, oocyte development results were obtained at the beginning of the observation and the end of the observation (fig. 2). figure 1 the average progesterone content of white shrimp l. vannamei injected with karamunting m. malabathricum ethanol extract notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means with different letters differ significantly from each other at p<0.05. biotropia vol. 28 no. 2, 2021 168 figure 2 histology of litopenaeus vannamei gonads notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means of different letters significantly differ from each other at p<0.05 using anova; o: oogonia; pre: previtellogenic oocytes; en: endogenous vitellogenic oocytes; ex: exogenous vitellogenic oocytes; 20x enlargement. ovarian development among shrimps is characterized by the formation of cortical rods in the oocytes after the accumulation of yolk (clark et al. 1980). at the beginning of the treatment, the gonads were generally not developed yet (fig. 2.a), these gonads were still in the accumulation of oogonia. after the 15 days, the control and all the treatments exhibited gonad cell development. in the control (fig. 2.c), the gonads developed into oocytes at the previtellogenic oocytes stage, and part of the ovary was still oogonia. whereas in t4 (2 mg/kg bw) and t5 (1 mg/kg bw) the development of gonads did not differ from that of the control which was still at the previtellogenic oocytes stage. in ti (10 mg/kg bw) (fig. 2.t1), the oocyte development was observed in the endogenous phase of vitellogenic oocytes, and several parts of the gonad have formed exogenous a o pre t4 pre t5 pre t2 en t1 ex en t3 en pre pre o c karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 169 vitellogenic oocytes and have a larger oocyte size as compared to control (fig. 3). in t2 (7.5 mg/kg bw) (fig. 2.t2), the oocyte development was in the endogenous phase of vitellogenic oocytes as compared with that of the control which was in the previtellogenic oocytes phase. in t3 (5 mg/kg bw) (fig. 2.t3), the ovary development was in the endogenous phase of vitellogenic oocytes and some still have the previtellogenic oocytes as compared with that of the control which was also in the previtellogenic oocytes phase. the mean oocyte size at the beginning of the experiment, at 15.57 ± 3.15 µm, was smaller than that of the control at 25.29 ± 2.69 µm (fig. 3). this indicates that after the 15-day maintenance, the ovary developed in both the control and the treatments. furthermore, the size of the oocytes in t1 at 65.65 ± 2.64 µm, t2 at 63.98 ± 3.06 µm, t3 at 39.12 ± 6.01 µm, t4 at 28.08 ± 0.84 µm and t5 at 27.65 ± 0.71 µm were also significantly bigger than that of the initial size of the oocyte and control. the control had the smallest oocyte at the end of the observation while t1 had the largest. the application of karamunting ethanol extracts significantly increased the oocyte diameter sizes among the treatments (p>0.05) with the largest diameter at t1 and t2 indicating that karamunting has compounds that can accelerate gonad maturity. in another study, the injection of estradiol 17β has also increased the diameter size of oocytes in l. vannamei (tarsim et al. (2007). karamunting ethanol extract on gonad somatic index (gsi) the application of karamunting ethanol extract also increased the gonad somatic index (gsi). the high increase in individual gsi indicated gonadal development manifested by gonads growing bigger (fig. 4). the use of karamunting ethanol extract has shown that it can meet the average gsi requirements in t1 (1.39 ± 0.04), t2 (1.26 ± 0.03) and t3 (1.01 ± 0.08) higher than the control (0.84 ± 0.02). at t4 (0.91 ± 0.03) and t5 (0.83 ± 0.04), the gsi increases were not significantly different from the controls. figure 3 oocyte average diameter size of the parent litopenaeus vannamei injected with melastoma malabathricum ethanol extract notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b, c, d, e = means with different letters significantly differ from each other at p<0.05. biotropia vol. 28 no. 2, 2021 170 figure 4 average somatic gonad index of parent white shrimp litopenaeus vannamei injected with karamunting melastoma malabathricum ethanol extract notes: c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means with different letters significantly differ from each other at p< 0.05. based on the gas chromatography mass spectrometry (gcms) test, the karamunting extract contained high lanosterol α and β amirin levels. in another study, karamunting plants also contain sitosterol α and β amyrin (nuresti et al. 2003). moreover, natural feed such as blood worms and squid had also stimulated the ovary maturation in shrimp (wouter et al. 2011). blood worms and squid are animals that contain high cholesterol (saidin 2000). increases in the gsi values of the treatments were significantly different from the control (p˂0.05). in t1 (10 mg/kg bw) and t2 (7.5 mg/kg bw), the increase in gsi was higher than that of the control (value?) at p <0.05. the t3 (5 mg/kg bw) increase in gsi was not significantly different from that of the control. a study showed that gsi increase was closely related to protein and lipid levels in the gonads, which also reflected high feed levels (rodriguezgonzalez et al. 2009a). the application of cholesterol had also optimized the increase in the ovarian weights of mangrove crabs (pattiasina et al. (2010). furthermore, the injection of serotonin on mature female individuals of the freshwater shrimp macrobrachium rosenbergii had stimulated an increase in gsi (meeratana et al. 2006). increasing the dosage of karamunting plant extract in white shrimp had impacted ovarian development. irrevocably, crustaceans need cholesterol which functions as precursors of steroid hormones in the process of gonadogenesis, maturation, and (wouters et al. 2001). gsi of t4 (2 mg/kg bw) and t5 (1 mg/kg bw) did not significantly differ from that of the controls. this is probably due to the small dosage that was not enough to produce the optimum hormonal increase in the parent white shrimp. the parent shrimp has an optimum level of protein requirements, where increasing the level of protein above the optimum level will not affect the nutritional condition (idris et al. 2011). the recommended protein levels could produce the highest spawning rate of 30% (rodriguez-gonzales et al. 2006a). water quality the optimum qualities for the maintenance medium of the white shrimp is at 28 32 oc, salinity 27 40 ppt, ph 6.5 8.3, and do 4 6 mg/l (farhan 2006). in this study, the water quality parameters were measured every day to monitor and maintain stable environmental conditions (table 1). changes in water quality on broodstock maintenance media will create stress on the shrimp which in turn will disrupt the gonadal maturation process. the growing environment was maintained at optimum conditions for the mature female white shrimp. karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 171 to maintain stability of the water quality in the growing media, certain maintenance activities were carried out every day before feeding. firstly, squeezing the base to remove feces and the rest of the feed; secondly, the water media was replenished every three days at a turnover rate of 30 percent new water. table 1 qualities of the aquatic environment no parameter avarage range morning afternoon evening 1 salinity 33 34 34 2 temperature 28 29 28 3 ph 7.6 7.8 7.8 4 do 6.56 6.01 6.57 conclusion the injection of karamunting melastoma malabathricum ethanol extract on the mature female individuals of white shrimp litopenaeus vannamei had increased its hormone progesterone and accelerated its gonad maturity indicating that the extract can hasten the reproduction time thereby increasing production volume over a shorter period. references clark w h, lynn jw, yudin ai, persyn ho. 1980. morphology of the cortical reaction in the eggs ofpenaeus aztecus. the biological bulletin, 158(2):175-86. doi:10.2307/1540929 farchan m. 2006. teknik budidaya udang vannamei. serang. in: bappl-stp. food and agriculture organization. 2017. globefish highlights. a quaterly update on world seafood markets: (fao) of the united nations. huberman. 2000. shrimp endocrinology: a review. aquaculture 191:191-208. idris m. 2011. energy pathway on two repeated spawning of australian red claw crayfish (cherax quadricarinatus) [dissertation]. bandung (id): institut teknologi bandung ismail a. 1991. pengaruh rangsangan hormon terhadap perkembangan gonad individu betina dan kualitas telur udang windu (penaeus monodon farb). 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[thesis]. bandung (id): institut teknologi bandung. biotropia vol. 29 no. 1, 2022: 18 27 doi: 10.11598/btb.2022.29.1.1568 18 inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi against pathogen pyricularia oryzae witiyasti imaningsih1*, della adventaria2, mariana3 and ahmad budi junaidi4 1microbiology laboratory, biology study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia 2student of biology study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia 3plant protection study program, faculty of agriculture, universitas lambung mangkurat, banjarmasin 70714, indonesia 4chemistry study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia received 22 march 2021 / accepted 4 january 2022 abstract diseases in rice plants (paddy) caused by microorganisms such as pyricularia oryzae lead to a decrease in rice production. therefore, it is essential to find out biological agents for protecting paddy and plants in general, against plant diseases. liquid smoke and endophytic fungi have been known as biological agents to enhance the protection of plants against disease. the purpose of this study was to determine the ability of liquid smoke, endophytic fungi and the concentrations combinations to suppress the growth of p. oryzae. the results showed that liquid smoke concentrations of 0.17% to 1.75% and endophytic fungi filtrate of 2% to 10% showed significant ability against pathogen p. oryzae. however, the combination of liquid smoke and endophytic fungi filtrate at selected concentrations (0.17% liquid smoke combined with 2% endophytic fungi filtrate and 0.34% liquid smoke combined with 2% endophytic fungi filtrate) showed no significant inhibition percentage against p. oryzae compared to control. in conclusion, this study showed that the respective applications of liquid smoke and endophytic fungi filtrate inhibit the growth of p. oryzae. keywords: endophytic fungi, inhibition ability, liquid smoke, p. oryzae introduction rice plants are the most dominating food crop commodity in indonesia (andriani 2008). as many as 95% of indonesians choose rice as a staple food (norsalis 2011). one of the rice plants cultivated in indonesia is gogo rice, which is grown on moorland (a type of habitat with the characteristic of low growing vegetation on acidic soil) or on dry land (hairmansis et al. 2016). “gogo” rice maninjau variety (oryza sativa l. var. maninjau) is one of the indonesian native varieties of brown rice that comes from the area of lake maninjau located in the sumatera barat province of indonesia. this variety is able to live on the dry land of central kalimantan, is resistant to leaf blight disease and able to survive in soil with high iron content (bppp jateng 2014). among microorganisms attacking gogo rice plants are pathogenic fungi such as p. oryzae, rhizoctonia solani, helmithosporium sigoideum and cercospora janseana. p. oryzae is one of the main diseases of rice crops due to its impact on reducing rice productivity (wang et al. 2014; suganda et al. 2016). this pathogen causes blast disease and serious damage to panicles (panicle blast) and leaves (leaf blast) of rice plants, where damage to panicles greatly affects rice productivity (hayashi et al. 2019). plant resistance to pathogens can be improved by utilizing the interaction of microorganisms with endophytic microbes. *corresponding author, email: witiyastiimaningsih@ulm.ac.id inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 19 endophytic fungi are a symbiotic between fungi and plants, which has a role to protect plants from pathogens by using the compounds produced by the symbiosis. symbiosis mutualism of endophytic fungi with plants produces secondary metabolites, such as phytohormones, nutrients and colony formations. some studies state that secondary metabolites produced by endophytic fungi inhibit pathogenic microbes (lalngaihawmi et al. 2019). therefore, endophytic fungi are potential biological agents to inhibit pathogenic fungi. liquid smoke bioactive materials can also be used to prevent disease caused by microorganisms. liquid smoke is a mixture of solution and colloidal dispersion of wood smoke vapor in water obtained from the process of wood pyrolysis or made from a mixture of pure compounds (darmadji 2002; soldera et al. 2008; lee et al. 2011). liquid smoke has good antimicrobial properties because it can inhibit the growth of microbes. this study used liquid smoke from “ulin wood” (eusideroxylon zwageri teijsm. & binn). ulin wood has density characteristics and a tight structure of hardwood, containing complex constituent compounds. several studies reported that one type of hardwood, “alaban” wood (vitex pubescens vahl.), contains phenol, carboxylic acid and carbonyl that are antimicrobial (oramahi & yoshimura 2013). based on literature studies, “ulin” wood liquid smoke may have the ability to inhibit p. oryzae. both endophytic fungi and liquid smoke of “ulin” wood have the ability to inhibit pathogenic microorganisms. in this study, both were tested both independently and by combining the two. as an initial effort to find one of the solutions to overcome diseases that often occur in rice plants “gogo” maninjau varieties. this study was aimed to determine the ability of “ulin” wood liquid smoke, endophytic fungi and their combinations to suppress the growth of p. oryzae. materials and methods sampling samples of “gogo” rice (o. sativa l var. maninjau) were taken from the same stretch of land in jaar village, east dusun subdistrict, east barito district, central kalimantan province. 2°06'53.9" north-south latitudes 115°15'22.1" west-east longitudes. samples consisting of all parts of the gogo rice plants were taken along with the soil around the roots of the gogo rice plants, by using a machete. the samples were stored in polybags and transported to the microbiology laboratory of the faculty of mathematics and natural sciences, universitas lambung mangkurat. isolation, purification and identification of endophytic fungi endophytic fungi were isolated from the roots of the gogo rice plants, by using surface sterilization method. the roots were washed using running water, then cut to a size of 10 cm. the root cuts were surface-sterilized by using 0.05% bleach for 60 sec and rinsed twice using sterile distilled water. each part of the root tip was then slightly cut to totally drain the root tip. the root tip was then planted in a sterile potato dextrose agar (pda) medium and incubated at a temperature of 28 °c for 3-5 days and observed daily. the grown fungi were then purified (manurung et al. 2014; nurzannah et al. 2014). fungi identification was carried out by using morphological observations, consisting of macroscopic and microscopic observations with reference to the identification book titled "illustrated genera of imperfect fungi 4th edition" (barnett & hunter 1998). screening of endophytic fungi pathogenicity and antagonism tests were used for the screening of endophytic fungi. pathogenicity test were carried out by using rice seed refering to waruwu et al. (2016). prior to being used in the pathogenicity test, the surface of the rice seeds (20 grains) were sterilized by soaking the rice seeds in 70% ethanol for 30 sec, followed by soaking in 1% naocl for 60 sec. subsequently, the rice seeds were flushed 3 times in sterile distilled water. after that, the rice seeds were inoculated in pda medium that had been previously overgrown by 7-day pure isolates of endophytic fungi and then incubated for 2 weeks at room temperature (27-29 °c). observation on the growing rice sprouts was carried out at the end of incubation. isolates of endophytic fungi that did not interfere with rice biotropia vol. 29 no. 1, 2022 20 germination were used for further testing. seed germination rate was calculated using the formula (talukdar 2011): germination (%) = number of germinated seed x 100% total number seed a confirmation test for endophytic fungi infection in the roots of rice plants was conducted for germination by using a method of luqman et al. (2015) that has been modified. prior to being used in the test, the roots of rice plants were washed thoroughly in running water, drained, then soaked in 5.25% naclo solution for 5 min, then rinsed using distilled water. subsequently, the roots were soaked in 1% koh solution for 30 min and then rinsed using distilled water. after that, the roots were then pre-soaked in 1% h2o2 solution for 5 min. the coloring stage was started by soaking the roots in 0.5% vinegar solution, followed by being soaked in ink with a ratio of 1:5 for 30 min. then, the roots were rinsed with distilled water. finally, the roots of the rice plants were placed on an object glass and covered by a cover glass, and then observed under a microscope with 40x and 100x magnifications. the antagonism test of endophytic fungi isolates against p. oryzae was conducted by using dual culture method (tomah et al. 2020), which put isolates of pathogenic fungi and endophytic fungi on pda medium in a petri dish that has been divided into two quadrants. each isolate was placed at a distance of 3 cm from the edge of the petri dish and incubated at a temperature of 28 °c for 5-7 days. after incubation, the inhibition percentage of the pathogens was measured using the formula developed by rabha et al. (2014): inhibition (%) = diameter of pathogen control colony x 100% diameter of pathogen treatment colony filtrate harvesting of endophytic fungi filtrate production methods used in this study were modified based on elita et al. (2013) and malinda et al. (2015). endophytic fungi with the highest inhibitory ability obtained in previous tests were inoculated on pda slant media then incubated for 7 days at 28 °c. after being incubated, the filtrate was harvested by adding 9 ml of sterile distilled water. the fungi surface was then gently wiped with a fine brush. subsequently, the water suspension and fungi were transferred to a new test tube, then centrifuged at 3,500 rpm for 20 min. finally, the supernatant was filtered by using a syringe filter with a pore size of 0.45 μm. the filtrate was then used in testing the inhibitory activity of endophytic fungi against pathogens. inhibition test of liquid smokeendophytic fungi to p. oryzae inhibition test of liquid smoke was conducted at various liquid smoke concentrations, i.e., 0.085%, 0.17%, 0.34%, 0.68%, 1.36% and 1.75%, based on a method of malinda et al. (2015) that have been modified. liquid smoke was obtained from condensation during the production process of “ulin” wood charcoal by talasiana charcoal production group, located at tanah laut regency. the liquid smoke was mixed into the potato dextrose agar (pda) medium. the pathogen isolates were then grown on the mixture of liquid smoke and pda for 5-7 days with daily observation. the inhibition percentage was calculated by the formula developed by rabha et al. (2014): inhibition (%) = diameter of pathogen control colony x 100% diameter of pathogen treatment colony the inhibition test for endophytic fungi was carried out by using the serial dilution method. endophytic filtrate with concentrations of 2, 4, 6, 8, and 10% was mixed with pda medium to be used for growing pathogen, and then incubated at 28 oc for 7 days and observed daily. the inhibition percentage was calculated with the same formula as the one used for calculating the inhibition percentage for the liquid smoke. liquid smoke and the endophytic filtrate (ketoconazole) with various concentrations were then combined to be tested for their inhibitory ability against pathogens. testing methods and measurements of inhibition percentage were conducted by using the same method as for the previous tests. results and discussion endophytic fungi of o. sativa l var. maninjau root endophytic fungi obtained from the roots of “gogo” rice var maninjau were coded ap.2, inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 21 ap.3, ap.4, ap.7, ap.8, and ap.9. microscopic observation of the endophytic fungi isolates showed that there are morphological differences. based on barnet & hunter (1998), the six isolates found refers to several species. ap2 is curvularia sp., ap3 and ap7 are penicillium sp., ap 8 is geotrichum sp., ap9 is aspergillus sp., while ap4 has not yet been able to be identified (fig. 1). the four fungi species were also reported by lalngaihawmi et al. (2018) as endophytic fungi in rice. the presence of endophytic fungi in the roots of the gogo rice plants varies based on the various tissues in which they grow. a study conducted by naik et al. (2009) reported that the colonization of endophytic fungi in rice plants happens more dominantly at the roots of rice plants. pathogenicity of endophytic fungi isolated from o. sativa l. var maninjau seeds our study showed that the germination percentage of gogo rice seeds varied (table 1), while the invasions of endophytic fungi against the seed germination were shown in figure 2. each endophytic fungi showed different levels of pathogenicity, both at 7 days after inoculation and 14 days after inoculation. the level of pathogenicity is useful for determining the best isolates to be used for subsequent tests. based on the results of pathogenicity levels of rice sprouts that grew normally, abnormally and did not grow, the best pathogenicity value was provided by geotrichum sp. ap8, followed by penicillium sp. ap7 and curvularia sp. ap 2. figure 1 microscopic characteristics of endophytic fungi isolates obtained from the roots of o. sativa l var. maninjau notes: a. curvularia sp. ap2 (40x); b. penicillium sp. ap3 (40x); c. ap4 (100x); d. penicillium sp. ap7 (40x); e. geotrichum sp. ap8 (40x); f. aspergillus sp. ap9 (40x). table 1 germination rate of the o. sativa l. var. maninjau for the 7 and 14 days after incubation (dai) endophytic fungi germination (%)* 7-dai 14-dai normal abnormal no growth normal abnormal no growth without adding endophytic fungi 60.00 ± 8.16b 0.00 ± 0.00a 36.67 ± 4.71a 63.33 ± 9.43b 0.00 ± 0.00a 33.33 ± 4.71a curvularia sp. ap2 40.00±14.14a 10.00 ± 8.16a 40.00 ±8.16a 20.00 ± 8.16a 36.67 ± 12.47a 33.33 ± 4.71a penicillium sp. ap3 43.33 ± 4.71ab 13.33 ± 4.71a 43.33 ± 9.43a 56.67 ± 4.71ab 16.67 ± 4.71a 26.67 ± 4.71a ap4 0.00 ± 0.00ab 0.00 ± 0.00a 100.00 ± 0.00b 6.67 ± 9.43ab 13.33 ± 12.47a 80.00 ± 14.14b penicillium sp. ap7 43.33 ± 9.43ab 13.33 ± 12.47a 50.00 ± 8.16a 56.67 ± 12.47ab 16.67 ± 9.43a 26.67±4.71a geotrichum sp. ap8 50.00 ± 8.16b 0.00 ± 0.00a 50.00 ± 8.16a 70.00 ± 8.16b 16.67 ± 4.71a 16.67±4.71a aspergillus sp. ap9 43.33 ± 4.71ab 6.67 ± 4.71a 50.00 ± 8.16a 46.67 ± 4.71ab 20.00 ± 0.00a 33.33±4.71a note : * = numbers followed by the same letter are not significantly different based on duncan test at p < 0.05. biotropia vol. 29 no. 1, 2022 22 figure 2 rice germination with various treatments of endophytic fungi notes: a. rice germination without the addition of endophytic fungi; b. rice seeds with the addition of ap4 endophyte fungi (not germinated); c. rice germination with the addition of endophytic fungi geotrichum sp. ap8; d. roots of rice seed undergoing treatment with endophytic fungi geotrichum sp. ap8 (the arrow shows endophytic fungi invading the rice root tissue). the results of the endophytic fungi antagonism test against p. oryzae showed no significant difference between the three selected fungi (p > 0.05) (table 2). although the antagonism test did not show significant differences, the selection of the best isolate for subsequent tests was determined based on the best inhibition percentage and the diameter of the pathogen successfully inhibited. thus, geotrichum sp. ap8 was the chosen isolate. endophytic fungi can inhibit pathogens having metabolite compounds by inhibiting the permeability of the pathogenic cells (ting et al. 2011). white et al. (2019) added that endophytic fungi can use the mechanisms of space and nutrient competition for suppressing pathogen growth. the ability of liquid smoke and endophytic fungi in inhibiting the growth of p. oryzae the results of our study indicated that liquid smoke at all tested concentrations was significantly able to inhibit the growth of pathogens compared to control (fig 3). at liquid smoke concentrations of 0.17% to 1.75%, the inhibition percentages differed significantly (p < 0.05). the selection of the right liquid smoke concentration for subsequent tests is indispensable, given that liquid smoke contains several antimicrobial components that may affect not only the growth of pathogen, but also the growth of endophytic fungi. table 2 diameter of p. oryzae colony and inhibition percentage of endophytic fungi endophytic fungi diameter of p. oryzae (mm)* inhibition percentage (%)* curvularia sp, ap2 41.47±2.92 21.98±5.54 penicillium sp. ap7 32.03±7.84 41.87±14.32 geotrichum sp. ap8 30.21±6.31 41.87±11.71 note: * = not significantly different based on duncan test (p < 0.05). inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 23 figure 3 inhibition percentage of different concentrations of liquid smoke against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on duncan test (p < 0.05). the results also showed that the smallest liquid smoke concentration (0.02%) was able to inhibit the growth of pathogens despite the daily decrease in ability, while the largest liquid smoke concentration (1.75%) was able to inhibit pathogens at an inhibition percentage of 100%. our study also indicated that the 0.17% and 0.34% liquid smoke concentrations were considered the best concentration for inhibiting pathogens compared to other concentrations. the inhibitory curve of those two concentrations had a tendency of increase after passing 4 days of inoculation, in contrast to other concentrations that had a tendency to decrease. the two liquid smoke concentrations also showed inhibiting capabilities against the tested pathogen despite the small concentrations. the inhibiting capabilities of liquid smoke against the growth of microorganisms may have been due to the contents of active compounds originating from the pyrolysis of wood constituents (cellulose, hemicellulose, and lignin). cellulose and hemicellulose produce organic acid compounds such as acetic acid, while lignin produces phenol compounds. the higher the content of the wood constituents, the more complex liquid smoke obtained (pszczola 1995). contents of active compounds in “ulin” liquid smoke are acids, phenolics, alcohol, ketones, ethers and esters, with acetic acid as the main active compounds (71.57% of the total active compounds) (junaidi et al. 2020). liquid smoke of “ulin” wood also contains a total acid of up to 8.88% (junaidi et al. 2019), which has antimicrobial properties. various concentrations of selected isolate endophyte fungi filtrate (geotrichum sp.) significantly inhibited the growth of p. oryzae compared with the control. however, there were no significant differences in inhibition percentages among concentrations (2-10%) (p < 0.05) (fig. 4). biotropia vol. 29 no. 1, 2022 24 figure 4 inhibition percentage of geotrichum sp. ap8 filtrate concentrations against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on the duncan test (p < 0.05). geotrichum sp. ap8 filtrate concentration of 2% was able to inhibit the growth of p. oryzae grown at 1 dai up to 7 dai with inhibition percentages ranging from 91.8 up to 100%. therefore, the 2% concentration of geotrichum sp. ap8 filtrate was chosen for testing the inhibitory synergism with liquid smoke. this result of geotrichum sp. ap8 filtrate is similar to the results of previous research conducted by imaningsih et al. (2021), which used endophytic fungi filtrate of “hiyung” cayenne pepper with a concentration of 2% for inhibiting pathogen colletotrichum capsici at almost 100% inhibition percentage. our study also provides better inhibitory results for the genus geotrichum compared to the study of lalngaihawmi et al. (2019), which tested geotrichum candidum for inhibiting p. oryzae with a 68% inhibition percentage at 7 dai. endophytic fungi inhibit pathogen growth through anti-microbial compounds (schulz & boyle 2005; singh et al. 2021). in our study, the filtrate of endophytic fungi was tested directly to inhibit the growth of p. oryzae and successfully showed high inhibition percentage. results of our study showed that the presence of bioactive substances produced by endophytic fungi has anti-microbial properties against the tested pathogen. singh et al. (2021) stated that bioactive compounds of endophytic fungi can be alkaloid, flavonoid, lignan, saponin, quinone, xanthone and miscellaneous compounds. synergism of liquid smoke and endophytic fungi inhibit p. oryzae growth the growth of pathogenic p. oryzae was inhibited by the combination between liquid smoke concentrations of 0.17% and 0.34% and geotrichum sp. ap.8 endophytic filtrate concentration of 2% with a range of inhibition percentages from 42% up to 100% on 1 dai until 7 dai. however, the inhibition percentages of the combination did not differ significantly among treatment combinations. the inhibition percentages of the treatments were not significantly different when compared to the control (fig. 5). a previous study conducted by imaningsih et al. (2021) showed that the concentrations combinations between liquid smoke of “ulin” wood and endophytic fungi filtrate of “hiyung” chili significantly inhibit the growth of pathogen c. capsici compared to the control. inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 25 figure 5 inhibition percentages of several concentrations combinations between geotrichum sp. ap8 filtrate (ke) and liquid smoke (ac) against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on duncan test (p < 0.05). based on the results of our study, the inhibition percentage achieved when combining liquid smoke and endophytic fungi filtrate was higher compared to the inhibition percentage of only using liquid smoke at the same concentration. the inhibition percentage of combining liquid smoke and endophytic fungi filtrate was lower when compared to the inhibition percentage of only using endophytic filtrate at the same concentration. during the filtration process, there might still be fungi cells carried away, due to the pore size of the filter membrane of 0.45 microns. hyphae fragments and spores of geotrichum sp. ap8 possibly penetrates the filter pores and grows during the inhibition process against the pathogens. meanwhile, sayer et al. (1969) grouped geotrichum sp. into fungi with intermediate spore size, which size is smaller than the usual fungi spores size. it is suspected that the ability of endophytic fungi filtrate decreased due to the presence of the carried-away fungi cells during the filtration process. however, those carriedaway fungi cells died in the presence of the liquid smoke. this was confirmed by a study conducted by oramahi et al. (2011) as well as oramahi and yoshimura (2013) which showed that liquid smoke possesses antifungal properties because it contains phenol, carbonyl and acid compounds. in addition, acetic acid and propionate components are able to neutralize fungi cells and inhibit enzyme activity (karseno et al. 2001). therefore, in addition to inhibiting the growth of pathogenic fungi, liquid smoke is also suspected to inhibit endophytic fungi. conclusion liquid smoke of “ulin” wood and endophytic fungi of “gogo” rice var. maninjau has the ability to inhibit the growth of pathogen p. oryzae. concentrations of 0.17% to 1.75% liquid smoke and 2% to 10% endophytic fungi filtrate showed high inhibition percentage against pathogen p. oryzae. the test of synergism, however, did not show an increase in inhibition percentage. further research on the inhibitory ability and the best concentrations of liquid smoke and endophytic fungi filtrate should be conducted to provide more protection against plant pathogens, especially for rice plants. acknowledgments the authors sincerely thank the ministry of research, technology and higher education of the republic of indonesia who has funded this research (2018-2019), as well as to the talasiana business group, ranggang village, tanah laut regency. our sincere thanks go to mrs. tambuy biotropia vol. 29 no. 1, 2022 26 as an elder of dayak maanyan community, jaar village area, east dusun district, east barito regency, central kalimantan province. references andriani y. 2008. budidaya tanaman padi di indonesia. jakarta (id): sastra hudaya. barnett hl, hunter bb. 1998. illustrated genera of imperfect fungi (4th ed.). st. paul (us): american phytopathological society (aps press). retrieved from https://www.academia. edu/35499449/ illustrated_genera_of_imperfect_fungi_fourth_edi tion_barnett_y_hunter_pdf_pdf. bppp jateng. 2014. kumpulan deskripsi varietas padi. semarang (id): balai penelitian dan pengembangan pertanian. darmadji p. 2002. optimasi pemurnian asap cair dengan metode redistilasi. j teknologi industri pangan 13:267-71. hairmansis a, yullianida y, supartopo s, suwarno s. 2017. rice improvement for upland areas. iptek tanaman pangan 11(2):95-106. retrieved from http://www.ejurnal.litbang.pertanian.go.id/index. php/ippan/article/view/6078. hayashi k, yoshida t, hayano-saito y. 2019. detection of white head symptoms of panicle blast caused by pyricularia oryzae using cut-flower dye. plant methods, 15(1), p. 1-9. imaningsih w, mariana, junaedi ab, rasyidah. 2021. antifungal activities of the combination of ulin wood liquid smoke and hiyung cayenne pepper root endophyte fungi against colletotrichum capsici. agrivita 43(1):69-78. https://doi.org/10.17503/ agrivita.v1i1.2458. junaidi ab, apriyani h, abdullah, santoso ut. 2019. fraksinasi dan karakterisasi asap cair dari kayu ulin (eusideroxylon zwageri teijsm. & binn.) sebagai pelarut kitosan. 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[metabolite endophytic mushrooms of rice plants as an alternative to control pathogenic mushrooms carried by rice seeds]. jurnal fitopatologi indonesia. 12(2):53-61. white jf, kingsley kl, zhang q, verma r, obi n, dvinskikh s, …, kowalski kp. 2019. review: endophytic microbes and their potential applications in crop management. pest manag sci 75(10):2558-65. https://doi.org/10.1002/ps.5527. biotropia vol. 29 no. 3, 2022: 213 224 doi: 10.11598/btb.2022.29.3.1583 213 the molecular approach reveals the relationship among venus clams (meretrix spp.) community in malaysia mohd hanafi idris1, abu hena mustafa kamal1, hadi hamli2*, amy halimah rajaee2,3 and abdulla-al-asif2 1faculty of fisheries and food science, universiti malaysia terengganu, 21300 kuala nerus, terengganu, malaysia 2department of animal science and fishery, faculty of agricultural and forestry sciences, universiti putra malaysia, bintulu sarawak campus, bintulu, sarawak, malaysia 3institut ekosains borneo, universiti putra malaysia, bintulu sarawak campus, bintulu, sarawak, malaysia received 12 april 2021/accepted 2 june 2022 abstract molecular study is important to detect variations and similarities among species from the same genus, in case if they do not encompass any morphological or physiological differences. the study was conducted to differentiate among species of meretrix spp. (meretrix lyrata, m. meretrix, and m. lusoria) obtained from two locations in malaysia through the phylogenetic tree. the adductor muscle tissues were used to extract dna and to perform other procedures; the samples were subjected to analyses using pcr and gel electrophoresis. the multiple sequence comparison was conducted by muscle and the phylogenetic relationships were established using maximum likelihood (ml) statistical methods with mega 6.0 statistical software. m. lyrata samples showed 99% similarity to the three accessions sequence, where m. lyrata indicated 87% similarities, and m. meretrix showed not more than 89% similarities from the deposited sequence. the nucleotide base composition sequences consisted of the mean of thiamine (t) 37.9%, cytosine (c) 15.4%, adenine (a) 27.4%, and guanine (g) 19.4%. maximum likelihood (ml) analysis was conducted using the tamura 3-parameter model to establish five major clades on meretrix spp. and two out-groups clades significantly different from the meretrix spp. these major clades were closely related to each other at the 50% evidence of bootstrap, which grouped as genus meretrix. the present study on meretrix spp. from the sarawak locality was able to differentiate coi sequences between m. lyrata, m. meretrix, and m. lusoria. m. lusoria was close related to m. meretrix with strong bootstrap supporting evidence at 96% scoring. moreover, m. lyrata was inferred as the ancestor to m. meretrix, and m. lusoria from sarawak, malaysia. keywords: bivalve, blast, borneo, mtdna, pcr, phylogenetic analysis introduction genetic variation can be characterized into two groups which are intraspecific and interspecific (arruda et al. 2009; ehlers et al. 2016; layton et al. 2016; thia et al. 2016). intraspecific is the variation within individuals from similar species such as subspecies (stevens et al. 2010). on the other hand, interspecific is the variation within individuals above the species level. genetic variation of natural populations is thought to be governed by the mutual effects of irregular genetic drift, restricted gene flow, and variance of selection pressures. intense natural selection is an important factor influencing variation among, and within groups (endler 1986). the natural selection can be stabilized the species characters such that genetic variation within the population might be reduced. disruptive selection may lead to genetic divergence, and therefore, may eventually increase genetic variation among population (backeljau et al. 2001). mitochondrial dna (mtdna) is one of the dna markers that are popular for dna identification. this marker is the maternal inheritance that supplies information absent in nuclear markers (okumuú & çiftci 2003). *corresponding author, email: hadihamli@upm.edu.my biotropia vol. 29 no. 3, 2022 214 therefore, selecting tissue from the female individual is essential to ensure information regarding the species ancestor. however, the maternal inheritance by mtdna can be restricted by the geographical with no mixing of mtdna haplogroups from the different geological regions (luttikhuizen et al. 2003; mishmar et al. 2003; layton et al. 2016). furthermore, inheritance characteristics on a particular defect gene could be recognized using an mtdna marker (wallace et al. 1999). this is significant, particularly in aquaculture, to ensure cultured species are free from any inherited diseases from the parents. moreover, mtdna could be applied for species identification and phylogeny studies due to the particular inherited gene variance. the typical mitochondria gene used for the phylogenetic study is cytochrome c oxidase subunit i (coi). this mtdna marker has been applied for genetic relationship and phylogenetic analysis for meretrix spp. from japan, korea, and china (yamakawa et al. 2008, chen et al. 2009; torii et al. 2010; kim & yoon 2014; sato et al. 2016). parentage assignment from a similar population but different parents was also successfully implemented for meretrix meretrix culture (lu et al. 2011). due to the intensive mtdna marker application, the universal primer of coi had been created based on the conserve gene sequence across the species to facilitate coi analyzing region on different metazoan species (folmer et al. 1994). the study of mollusca started from the study of morris & purchon (1981), their approach was to enlist and taxonomic study of available species; however, the story of mollusk study goes on, and malaysian scientists and academician studied a different aspect of mollusca; from taxonomy to diversity, along with the genetic aspect of this community (hamli et al. 2012a, b; 2013; 2015; 2017; 2019; 2020a, b, c; foon et al. 2017; al-asif et al. 2020; al-asif et al. 2021; idris et al. 2017a, b). genetic and molecular study of class bivalvia and gastropoda is not new in malaysian territory, including east malaysia (sabah and sarawak provinces). some previous study revealed the genetic diversity of asian green mussel (perna viridis) in the waters of sabah (lau et al. 2018), dna study of neritid (chee & mohd nor 2016), genetic diversity of razor clam in kuala selangor, malaysia (hassan & kanakaraju 2016). there is no genetic approach of venus clam (meretrix spp.) was found in malaysia, and the genetic relationship among the available venus clam species is still unknown. therefore, the present study aims to differentiate among meretrix spp. from different through the phylogenetic tree. materials and methods sample collection a total of 30 samples of meretrix lyrata, m. meretrix, and m. lusoria were collected from two areas, kuching and kabong, sarawak province, malaysia (fig. 1). adductor muscle tissues from the females were separated from the shell and preserved with 95% of ethanol (wang et al. 2010) for further analysis at the parasitology laboratory, department of animal science and fishery, universiti putra malaysia, at the bintulu sarawak campus. dna extraction about 10 to 20 mg of adductor muscle tissue from each sample was used for extraction using a tissue dna extraction kit (vivantis) following the manufacturer's instructions and stored at -20 °c. the extracted dna was then quantified through agarose gel electrophoresis. dna quantification and gel electrophoresis extracted dna was quantified following rengarajan et al. (2002) to ensure the amount of dna within 1 µl. each quantified dna was adjusted to 100 µl to ensure the total amount of dna sample used for amplification was equal. quantification of genomic dna was compared based on lambda hind iii marker (23130 bp). gel agarose concentration used for dna quantification was 0.8% in 1x tae (trisacetate-edta) buffer and the electrophoresis procedure was conducted following lee et al. (2012). a total of 5 µl of extracted genomic sample and 1 µl of dye (ez-vision one dye) were added into the well. after all the samples and markers were loaded, electrophoresis was run for 40 minutes with 120 volts of electricity. the running of electrophoresis was monitored until the red dye was moved 3/4 of the gel length, and the electrophoresis was stopped. molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 215 figure 1 study area of meretrix spp. for genetic study the gel was then removed from the tank and was photographed under a uv light gel imager (redtm, alpha innotech). quantification process based on appeared bands on the gel to be viewed with alpha view software and band intensity from extracted samples was compared with lambda hind iii marker for 100 ng of dna. while 1.5% of gel agarose concentration was used for the polymerase chain reaction (pcr) through 0.3 g of agarose powder into 20 ml of 1x tbe (tris-borate-edta) buffer, the mixture was swirled and heated in the microwave to ensure agarose powder was dissolved. heated mixture was monitored to make sure the mixture was not boiled. after agarose powder dissolved in the buffer, the mixture was left to cool and then was poured slowly into the prepared gel tank while ensuring that no bubble was trapped. immediately, the comb was inserted into the poured mixture, while letting the agarose mixture to be cool and solidify for 30 minutes. after the gel became solid, the comb was removed and the running buffer 1x tba or 1x tbe was poured into the gel tank until all the gel parts submerged. polymerase chain reaction (pcr) amplification amplification of dna was performed in 50 µl of pcr mixture using universal marker cytochrome c oxidase subunit i (coi) with sequence lco1490: 5’-ggtcaacaaatcata aagatattgg-3’ and hco2198: 5’taaacttcagggtgaccaaaaaatca-3’ (folmer et al. 1994). each pcr reaction consisted of 0.1 mm of reverse and forward primers, 0.2 mm of dntp, 2.0 mm mgcl2, 1x buffer, 1.25 units of taq polymerase, 100 ng of template dna, and ultrapure water to complete 50 µl. a total of 50 µl of pcr mixture were amplified in xp thermal cycler block (bioer technology co. ltd) with pre-denaturation at 95 °c for 5 minutes, then followed by 35 cycles of 1 minute at 95 °c for denaturation, 1 minute for annealing at 40 °c, 1 minute 30 second at 72 °c for extension and 72 °c for 7 minutes for final extension (folmer et al. 1994). the last cycle temperature was maintained at 4 °c. the pcr product then underwent electrophoresis. gel electrophoresis gel agarose concentration used was 1.5% in 1x tbe (tris-borate-edta) buffer. the electrophoresis procedure was carried out following lee et al. (2012). a total of 100 ng of pcr product was added into the well with 1 µl of dye (ez-vision one dye) and 1 µl of 100 bp ladder (promega) with 1 µl of dye (ez-vision one dye) at the end of the well. after all samples and markers were loaded, electrophoresis was run for 60 minutes with 80 volts of electricity. the running of biotropia vol. 29 no. 3, 2022 216 electrophoresis was monitored until the red dye was moved 3/4 of the gel length, and the electrophoresis was stopped. the gel was then removed from the tank and was photographed under a uv light gel imager (redtm, alpha innotech). the appeared band was then compared to the ladder to identify the fragment size. purification and sequencing pcr products were purified before sequencing. according to the manufacturer's instruction, purification has been done to remove excess dinucleotide triphosphate (dntp) using themo scientific genejet pcr purification kit. afterward, purified samples were sent to the first base laboratory, malaysia, for dna sequencing. both strands of pcr products and primers were sequenced using the applied biosystems bigdye terminator v3.1 cycle sequencing kit. statistical analysis the obtained sequences were inspected and assembled using bioedit version 7.0. the assembled sequence of m. lyrata, m. meretrix, and m. lusoria were then compared with all species of meretrix spp. in genbank of national center for biotechnology information (ncbi) using basic local alignment system tool (blast) 2.2.31 (national center for biotechnology information, bethesda, md, usa [http://www.ncbi.nlm. nih.gov/blast/]). the highest similarity sequence from the genbank with meretrix spp. was selected for phylogenetic analysis in the present study. the present meretrix spp. sequence, 16 accessions sequence mtdna, coi sequence, and 2 species with coi sequence as an outgroup which mercenaria mercenaria and paphia gallus (table 1) in genbank ncbi were aligned based on multiple sequences comparison by logexpectation using muscle (edgar 2004). phylogenetic relationships were developed using maximum likelihood (ml) statistical methods with mega 6.0 statistical software (tamura et al. 2013). the evolutionary history was inferred using the maximum likelihood method based on the tamura 3-parameter model (tamura 1992). the non-uniformity of evolutionary rates among sites was modeled using discrete gamma distribution (+g) with 5 rate categories. the bootstrap agreement tree inferred from 500 replicates (felsenstein 1985) was taken to represent the evolutionary history of the analyzed taxa (felsenstein 1985). branches equivalent to partitions reproduced in more than 50% bootstrap replicates were illustrated. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test was shown next to the branches (felsenstein 1985). the heuristic search's initial tree was obtained automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach and then selecting the topology with a superior log-likelihood value. evolutionary analyses were conducted in mega6 (tamura et al. 2013). table 1 list of meretrix accession sequences from the genebank (ncbi) meretrix accessions geographical locations genbank acession number citation meretrix lyrate sarawak, malaysia present study present study m. lusoria sarawak, malaysia present study present study m. meretrix sarawak, malaysia present study present study m. lyrata china kc832317.1 wu et al. (2014) m. lyrata china j n898944.1 cheng et al. (2013) m. lyrata china hm124580.1 chen et al. (2011b) m. lusoria china gq903339.1 wang et al. (2010) m. lusoria japan ab853870.1 yamakawa & imai (2013) m. lusoria japan ab853865.1 yamakawa & imai (2013) m. meretrix china gq463598.1 he et al. (2011) m. meretrix china hm124578.1 chen et al. (2011b) m. meretrix china jn043623.1 wang et al. (2011a) m. petechialis china eu145977.1 ren et al. (2009) m. petechialis china hm124582.1 chen et al. (2011b) molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 217 table 1 (continued) m. petechialis china hq703177.1 chen et al. (2011a) m. lamarckii china gu071281.1 wang et al. (2011b) m. lamarckii china hm124579.1 chen et al. (2011b) m. lamarckii japan ab059420.1 hamaguchi et al. (2001) m. casta india jq773441.1 ranjith et al. (2012) mercenaria mercenaria * canada hm884239.1 layton et al. (2014) paphia gallus * china hq703232.1 chen et al. (2011a) note: * = outgroup. results and discussion the coi marker produces a fragment size at 710 bp for m. lyrata, m. meretrix, and m. lusoria (fig. 2). figure 2 fragment size produced by meretrix spp. using coi marker notes: 1 = m. lyrata; 2 = m. lusoria; 3 = m. meretrix. basic local alignment system tool (blast) analysis indicated that the m. lyrata samples from the present study had 99% similarity to the three accessions sequence (kc832317.1, jn898944.1, hm124580.1) of m. lyrata compared to other accessions sequence (table 2). different accession sequences were only showed lower than 87% of similarities with m. lyrata from the sarawak locality. the present study on m. lusoria showed higher similarities with m. lyrata with accessions sequence kc832317.1 and hm124580.1, and m. meretrix with accessions sequence hm124578.1 and jn043623.1. other accessions sequence were less than 89% of similarities with the present study on m. lusoria. there was no similarity of more than 89% within m. meretrix from sarawak province with genebank ncbi accessions sequence of m. meretrix. the higher similarity sequence was represented by the accession sequence hm124580.1 of m. lyrata with 89% similarity. the nucleotide sequence for m. lyrata in the present study showed potential similarity with the accession nucleotide sequence of m. lyrata from the genbank ncbi with more than 90% similarity compared to m. meretrix and m. lusoria. some previous studies conferred the morphology feature on m. lyrata as being the easiest to distinguish from m. meretrix and m. lusoria (hamli et al. 2012b, 2015, 2016, 2017). however, the nucleotide sequence for m. meretrix and m. lusoria showed lower than 90% similarity with the accession nucleotide sequence from the genbank ncbi. hence, the blast biotropia vol. 29 no. 3, 2022 218 analysis could not confirm and distinguish m. meretrix and m. lusoria from the sarawak locality through the coi sequence. current findings were contradictory with results of the morphology and morphometric studies, which distinguished differences between m. meretrix and m. lusoria. however, wang & dunbrack (2004) reported that the application of blast only produces short and conserve fragment alignment. thus, the analysis was frequently unable to identify many relationships, particularly below 40% sequence identity. low sequence identity of fragment creates uncertainty of gaps and insertion, thus compromising confidence in overall modeling of protein related to genome sequence annotation (gan et al. 2002). m. lusoria and m. meretrix were hard to identify by rough morphological inspection due to similar features, particularly the outer shell. however, a detailed examination of the inner shell generated morphological variation of the distinct profile of the pallial sinus scar between m. lusoria and m. meretrix. yet, using the blast application to confirm the differences between the two species appeared to provide uncertain results. rather than depending on sequence identity percentage, phylogenetic relationship perhaps provides more justified evidence on the present genome characteristic of meretrix spp. to be evaluated against the accession sequence in the genbank. the current phylogenetic tree was constructed based on maximum likelihood (ml), which applied an algorithm that calculates the probabilistic approaches to build a relationship tree between compared species based upon the nucleotides or amino acid sequences (guindon & gascuel 2003). meretrix spp. from the sarawak locality indicates the close relationship between m. lusoria and m. meretrix. the variation in the coi sequence of this meretrix spp. can be distinguished, hence, we are able to clarify the intricacy conferred using the blast application. moreover, m. lusoria and m. meretrix had similar ancestors represented by m. lyrata from a similar locality. therefore, coi sequence in m. lyrata had a low level of sequence identity among meretrix spp. this was in agreement with chen et al. (2009) and wu et al. (2014), which suggested that m. lyrata has the level of sequence that portrays as the ancestor to other meretrix spp. by referring to coi and transfer rna (trna), respectively. sequence characteristics a total of 21 nucleotide sequences were analyzed from three meretrix spp. of sarawak locality, 16 accessions sequence of meretrix spp. and two outgroup species represented by mercenaria mercenaria and paphia gallus. codon positions included were 1st + 2nd + 3rd + noncoding. all positions containing gaps and missing data were eliminated. there were a total of 435 positions in the final dataset. the nucleotide base composition sequences were comprised with mean of thiamine (t) 37.9%, cytosine (c) 15.4%, adenine (a) 27.4%, and guanine (g) 19.4% (table 3). table 2 similarity percentage of meretrix spp. in the present study with 16 selected meretrix spp. mtdna accessions sequence from genbank (ncbi) accession sequence genbank species m. lyrata (%) m. lusoria (%) m. meretrix (%) kc832317.1 m. lyrate 99 90 88 jn898944.1 m. lyrata 99 < 83 < 84 hm124580.1 m. lyrata 99 90 89 gq463598.1 m. meretrix 86 86 85 hm124578.1 m. meretrix 87 90 87 jn043623.1 m. meretrix 87 90 87 gq903339.1 m. lusoria < 85 86 86 ab853865.1 m. lusoria 85 87 85 ab853870.1 m. lusoria < 85 86 85 eu145977.1 m. petechialis 86 85 85 hm124582.1 m. petechialis < 85 85 85 hq703177.1 m. petechialis < 85 86 85 gu071281.1 m. lamarckii < 85 < 83 86 ab059420.1 m. lamarckii < 85 < 83 87 hm124579.1 m. lamarckii < 85 < 83 86 jq773441.1 m. casta < 85 88 87 molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 219 table 3 nucleotide base composition for accession sequences species accession t % c % a % g % total meretrix lyrata sarawak 24.8 19.5 42.1 13.6 435.0 m. lusoria sarawak 24.4 20.2 41.8 13.6 435.0 m. meretrix sarawak 23.7 20.7 42.1 13.6 435.0 m. lyrata kc832317.1 41.8 14.0 23.7 20.5 435.0 m. lyrata jn898944.1 42.1 13.8 24.1 20.0 435.0 m. lyrata hm124580.1 42.1 13.8 24.1 20.0 435.0 m. lusoria gq903339.1 42.1 13.6 23.0 21.4 435.0 m. lusoria ab853870.1 23.0 20.5 43.0 13.6 435.0 m. lusoria ab853865.1 22.3 22.1 43.0 12.6 435.0 m. meretrix gq463598.1 42.8 13.1 23.0 21.1 435.0 m. meretrix hm124578.1 41.6 14.3 22.3 21.8 435.0 m. meretrix jn043623.1 42.1 13.8 24.1 20.0 435.0 m. petechialis eu145977.1 42.8 13.1 23.0 21.1 435.0 m. petechialis hm124582.1 41.8 13.8 23.7 20.7 435.0 m. petechialis hq703177.1 42.3 13.3 23.7 20.7 435.0 m. lamarckii gu071281.1 43.7 13.8 19.8 22.8 435.0 m. lamarckii hm124579.1 43.7 13.6 20.7 22.1 435.0 m. lamarckii ab059420.1 42.3 13.8 22.1 21.8 435.0 m. casta jq773441.1 42.3 14.7 23.2 19.8 435.0 mercenaria mercenaria hm884239.1 40.8 14.7 21.1 23.4 436.0 paphia gallus hq703232.1 44.4 12.0 23.0 20.7 435.0 average 37.9 15.4 27.4 19.4 435.0 phylogenetic analysis a discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+g, parameter = 2.0104)). maximum likelihood (ml) analysis conducted using the tamura 3-parameter model has constructed five major clades on meretrix spp. in the present study with designed out-groups mercenaria mercenaria and paphia gallus. these major clades were closely related to each other at the 50% evidence of bootstrap, which grouped as meretrix genus (fig. 3). two monophyly groups were formed, represented by clade 3 and 4, while clade 1, 2, and 5 were formed, paraphyly groups. meretrix spp. from the sarawak locality was clustered in clade 1 alongside two m. lusoria of japan locality with accession sequence number ab853865.1 and ab853870.1. clade 2 comprised two m. meretrix from the china locality with accession sequence number hm124578.1 and jn898944.1. clade 3 consisted of m. lyrata from the china locality with accession sequence number kc832317.1, hm124580.1, jn898944.1, and m. casta from the india locality with accession sequence jq773441.1. meretrix lamarckii from the japan locality (ab059420.1) was clustered with m. lamarckii from the china locality (gu071281.1; hm124579.1) in clade 4. all m. petechialis (hm124582.1, hq703177.1, eu145977.1) from the china locality were clustered with m. meretrix (gq463598.1) and m. lusoria (eu145977.1) from the china locality, respectively, in clade 5. phylogenetic analysis on three meretrix from the sarawak locality is closely related to the m. lusoria from the japan locality. however, the result contrasts with m. lyrata from blast analysis which inferred a high similarity sequence with m. lyrata accession sequence. the application of ml analysis for the phylogenetic tree was calculated using the substitution rate in the sample sequence and accession sequence, which produced different inferences than blast analysis (guindon & gascuel 2003). furthermore, meretrix spp. from the sarawak locality and m. lusoria from the japan locality had a high percentage of cytosine and adenine than that of other meretrix spp. from different localities. meretrix spp. from the sarawak locality only had a relationship with other meretrix spp. localities at more than 50% of bootstrap scoring, which is inferred as under meretrix genus. inconsistent cladistic group between m. meretrix (gq463598.1) m. lusoria (gq903339.1), m. casta (jq773441.1), and m. petechialis (hm124582.1, hq703177.1, eu145977.1) from the present study indicated that the three species had a close relationship following wu et al. (2014) and chen et al. (2009). hence, chen et al. (2009) suggested that m. lusoria and m. petechialis as a junior synonym to m. meretrix. biotropia vol. 29 no. 3, 2022 220 figure 3 phylogenetic tree of meretrix accession sequence inferred from the maximum likelihood analysis using tamura three-parameter model note: only bootstrap score greater than 50% are shown. the present study on meretrix spp. from the sarawak locality was able to differentiate coi sequences among m. lyrata, m. meretrix, and m. lusoria. m. lusoria was closely related to m. meretrix with strong bootstrap supporting evidence at 96% scoring. m. lyrata became the ancestor to m. lusoria and m. meretrix with strong bootstrap evidence at 100% of the scoring. meretrix spp. from the sarawak locality is closely related to the m. lusoria of the japan locality with 100% scoring on bootstrap supporting evidence to form clade 1. meretrix spp. from the sarawak locality was closely associated with other accession sequences for meretrix spp. at bootstrap supporting evidence at 50% scoring. based on ml phylogenetic analysis, all m. lyrata with accession sequence kc832317.1, jn898944.1, and hm124580.1 formed the cladistic group with strong bootstrap evidence at 99%. all m. lamarckii with accession sequence gu071281.1, ab059420.1, and hm124579.1 also formed cladistic at 94% of bootstrap evidence. m. casta (jq773441.1) formed an individual clade which indicated this species distance from other species except for m. lyrata. other meretrix spp. accession sequence, such as m. meretrix (gq463598.1) m. lusoria (gq903339.1), and m. petechialis (hm124582.1, hq703177.1, eu145977.1) formed inconsistent cladistic groups, while m. meretrix with accession sequence hm124578.1 and jn043623.1 formed individual clade apart from other m. meretrix (gq463598.1) at 99% evidence of bootstrap scoring. mitochondria dna is known to have higher transition mutation rates, particularly synonymous sites in the nucleotide sequence that affected the rates of evolution in organisms (brown et al. 1982; caterino et al. 2000; overton & rhoads 2004). this fast rate of evolution is one of the features suitable for phylogenetic study, particularly in higher taxa. moreover, higher taxa less affected by the recombination (guo et al. 2006), uniparental inheritance (passamonti et al. 2003; passamonti & plazzi 2020), heteroplasmy with paternal leakage (bromham et al. 2003; wolff et al. 2013; mastrantonio et al. 2019), and selective sweeps (ballard & rand 2005; james et al. 2016; hill 2019). despite proper application on higher taxa, this is the contrast when nucleotide sequences at the species level take into account those which are more defected. the evolution rate is influenced by thermal adaptation, mitochondrial, nuclear interaction, and infection with wolbachia spp. (ballard & rand 2005). hence, meretrix spp. from the sarawak locality possibly has a different evolution rate in mtdna, which caused divergence from the meretrix spp. from other localities, particularly from the china regions. molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 221 the current phylogenetic analysis is difficult to verify that species meretrix spp. belong to the sarawak locality. bivalves had high variability in size and gene group of mtdna, which vary extensively between species from a similar genus (xu et al. 2012). meretrix spp. from the sarawak locality are separated in terms of geographical distance. the geographical distance affects the mtdna feature as described by mishmar et al. (2003) that maternal inheritance can be restricted by the geographical distance with no mixing of mtdna haplogroups from the different geological region. therefore, other environmental conditions in each region influence the organisms' mutation rate (massey & buckling 2002). ambiguous meretrix spp. identification from the sarawak locality might be possibly related to other several features, such as sample used. the mtdna marker feature is the maternal inheritance which supplies the missing information in nuclear markers (okumuú & çiftci 2003). therefore, the female individual's tissue selection is crucial during the genetic study to ensure reliable information regarding the species ancestor. however, female selections for the genetic research have not been mentioned in each previous study for the genbank accession sequence, causing the doubtness of the reliability of accession sequence in the genbank, leading to the divergence of meretrix spp. from the sarawak locality compared to the previous species sequence. moreover, the accession sequence is well doubted due to the non-intensive morphological identification for each species which only depending on the rough observation. the present study suggested that appliance on a single marker is insufficient to support species identification and species-level phylogenetic analysis. therefore, additional variance types of molecular markers such as allozyme and nuclear markers are required to create substantial evidence on species verification to ensure the intensive morphological study's genetic approachability. depending on the genetic approach, the lack of intensive morphological evidence would create ambiguous species naming in taxonomy. conclusion the molecular approach application was able to distinguish three meretrix spp. from sarawak province, malaysia. moreover, m. lyrata is inferred to as the ancestor of m. meretrix and m. lusoria from sarawak. regardless of ancestor identification and species distinctions, the coi gene in the present study was unable to verify which species meretrix spp. belonged to the sarawak locality. several disadvantages to the coi sequence that influence the phylogenetic analysis involve the consistent accession sequence from genbank as the main reference for the current study. the present study suggested that additional dna markers for species identification and phylogenetic analysis are recommended in supporting the intensive morphological identification. acknowledgments the research team would like to acknowledge the ministry of higher education malaysia f.r.g.s. research grant code, frgs/1/2018/ wab13/upm/02/2, and the department of animal science and fishery, universiti putra malaysia, bintulu sarawak campus, for the technical support. references al-asif a, hamli h, abu hena mk, idris mh, gerusu gj, ismail jb, karim nu. 2020. benthic macrofaunal assemblage in seagrass-mangrove complex and adjacent ecosystems of punang-sari estuary, lawas, sarawak, malaysia. 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(mollusca: veneridae) in the western pacific ocean. pacific sci 62(3): 385-94. biotropia no biotropia no. 16, 2001 : 10 -17 a novel integron in the genome of escherichia coli isolated from indonesian monitor lizard (varanus spp). esti puspitasari2, antonius suwanto1'2 * , amarila malik3, and walter erdelen" 'dept. of biology, faculty of science and mathematics and ivc biotechnology, bogor, agricultural university, bogor, indonesia; 2 south east asian regional center for tropical biology (seameo biotrop), bogor: indonesia 3 dept. of pharmacy, faculty of science and mathematics, university of indonesia, jakarta, indonesia; 'dept. of biology, faculty of science and mathematics, bandung institute of technology, bandung, indonesia abstract the genotype of antibiotic resistance in natural isolates of escherichia coli was determined through integron detection and characterization of the associated antibiotic resistance. e. coli sg2 isolated from varanus salvator of java demonstrated resistance to spectinomycin (50ng/ml) and streptomycin (song/ml). integron detection indicated that eight isolates out of nine e. coli isolates possessed a conserved segment of the integron. amplification of the inserted cassette of the integron in this sg2 isolate yielded a 1-kb dna fragment. sequence analyses indicated that this fragment was homologous with aad gene, which confirmed the resistance to spectinomycin/streptomycin. this is the first report on the presence of integron in the e. coli isolated from the environment. key words: integron / antibiotic resistance / escherichia coli introduction antibiotic is a substance produced by a microorganism as protection from another microorganism (madigan et al. 1997). antibiotics have a specific target site in the cell. for example, in trying to get hold of protein synthesis targeted to 30s ribosom, the presence of tetracycline, spectinomycin and streptomycin results in an invalid codon-anticodon reaction and therefore, the synthesized amino acid is also invalid ( schumm 1992). currently, there is concern on the spread of antibiotic resistance among bacteria. such dissemination is partly a consequence of the antibiotic-resistant genes mobility which reside in mobile genetic elements such as plasmids, transposons, and integrons (francia et al. 1999). integron is a new type of mobile element which has evolved by a site-specific recombination mechanism. integrons consist of two conserved segments of dna separated by a variable region containing one or more genes integrated as cassettes (levesque et al. 1995). the 5'conserved segment contained in the int gene, which encodes a polypeptide of 337 amino acids, has been shown to be homologous to other members of the integrase family and on the opposite strand, a common promoter region p1-p2 is directed toward the site of integration. the 3' conserved segment contains the qace&\, sur genes, and an open reading frame (fig.l). * corresponding author : e-mail address : asuwanto@indo.net.id 10 10 figure 1. integron structure (levesque et al. 1995) genetic changes in bacterial genome could contribute to antibiotic resistance in some bacteria. there are three mechanisms of antibiotic inactivation: (i) modification of antibiotic structure; (ii) prevention of antibiotic to get into the target site; or (iii) change in antibiotic target site (neu 1992). resistance to aminoglycoside antibiotics such as spectinomycin and streptomycin, could be due to the penetration failure, the low affinity of antibiotic to ribosom, or the inactivation of the antibiotic by enzymes such as phosphorylase, adenylase and acetylase produced by the bacteria (gian and gian 1995). desselberger (1998) reported that data on antibiotic-resistant genes in the environment were rare, whereas antibiotic-resistant genes among bacterial strains were increasing at an alarming rate. biawak monitors (varanus spp.) are reptilians lacking venomous gland, although there are some reports on biawak bites resulting in bacterial infection (auffenberg 1981). probably, this infection is caused by toxic substances produced by microbiota living in the oral of varanus spp. auffenberg (1981) identified five bacteria species that could cause infection. yogiara (1998) reported that many of e. coli isolates living in the buccal of varanus spp. are resistant to ampicillin. this study was conducted in order to understand the mechanism of the antibiotic resistance of e. coli in the digestive system of varanus spp. in this experiment, an antibiotic-resistant gene from e. coli was isolated and further characterized by dna sequence analysis. 11 a novel integron in ther genome of escherichia coli – esti puspitasari et al. material and mothods bacterial strains and plasmids the bacterial strains and p;asmids used in this research are shown in table 1. table 1. bacterial strains plasmida growth conditions antibiotic-resistant bacteria were cultured aerobic (100 rpm) at 37°c and kept overnight in luria bertani (lb) medium supplemented with one, or a combination of the following antibiotics: ampicillin (100 u.g/ml), streptomycin (50 ng/ml), spectinomycin (50 ng/ml). dna extraction bacterial isolates were grown in 10 ml lb in the presence of a selective antibiotic at 37°c overnight. alkaline lysis method (sambrook et al. 1989) was used for plasmid dna extraction, and wizard®genomic dna purification kit (promega, madison, wi) was used according to manufacturer protocol for genomic dna extraction. 12 biotropia no. 16, 2001 pcr amplification pcr were performed in gene amp® pcr system 2400 (perkin elmer, branchburg, new jersey). ready to go™ pcr beads (pharmacia biotech, uppsala, sweden) was used for each pcr reaction. each of 25 ul reaction mixtures contained pcr beads, 4 ng/ul of genomic dna, 10 ng/ul of primers (5'cs: ggcatccaagcagca ag, 3'cs: aagcagacttgacctga, qac: atcgcaatagttggcgaagt, sul: gcaaggcggaaacccgcgcc) and distilled water up to 25 ul. pcr condition for the integron was amplified in 35 thermal cycles at 94°c for 1 minute, 55°c for 1 minute, and 72°c for 3 minutes. a final extension step for 7 minutes at 72°c was also included. the primers used were 5'cs and 3'cs that amplify variable regions of integron, qac and sitll primers to amplify the conserved region. pcr product was purified using gene clean kit (bio 101, la jolla, california). cloning and transformation amplified dna was ligated into pas900 (kmr) and pgem-t easy (apr ) vector. the approximate ratio of pas900 vector to insert dna were 1:4 and that of pgem-t easy to insert dna was 1 to 16. ligation reaction consisted of vectorinsert mixture 1 ul of t4ligase, ix ligation buffer, and distilled water up to 20 ul. ligation mixture was incubated at 7°c for 16-18 h. the ligation mixture was transformed into 250 ul of chemically competent e. coli top10, followed by heat shocked at 42°c for 45 seconds. after the addition of 3 ml lb, the culture was incubated with vigorous shaking at 37°c for 90 minutes (sambrook et al. 1989). transformants were then selected on luria bertani agar (lba) supplemented with x-gal (40ug/ml) and appropriate antibiotic/s. dna sequencing ps2t double stranded templates were derived from cloning of sti in pgem-t easy. sequencing reaction consisted of 8 ul big dye-terminator, 360 ng of dna template, 8 ng/ul of primer and distilled water up to 20 ul cycle sequencing was performed using pcr machine with three-step profile for 25 cycles: a 10-second denaturation at 96°c, a 5-second annealing at 50°c, and a 4-minute extension at 60°c. cycle sequencing product was purified by ethanol-sodium acetate precipitation method. a 1.5 ml microcentrifuge tube was filled with 2 ul of sodium acetate (ph 4.6), 50 ul of ethanol 95%, and 20 ul of the cycle sequencing mixture, and then incubated at room temperature for 30 minutes. the mixture was centrifuged at maximum speed for 20 minutes. the supernatant was removed from the tube, and 250 ul of 70% ethanol was added into the tube. the mixture was centrifuged again for 5 minutes. the supernatant was removed and the dna pellet was vacuum-dried at 50 cm hg for 10 minutes. 13 a novel integron in the genome of escherichia coli esti puspitasari et al. the dna was electrophoresed according to sambrook et al. (1989). prior to electrophoresis, dna was denatured with 6 ul of loading buffer containing blue dextran and 25 mm of edta in formamide at 95°c for 2 minutes, and quickly placed on ice. approximately 1.5 ul of dna sample was loaded to each well and ran for 10 hours. the dna was compared with those in the genbank and embl databases. results and discussions integron detection by pcr amplification of dna sequence experiments using qac-sul primers indicated that there were eight out of nine e.coli isolates possessing 3'conserved segment of the integron (table 2). e. coli top 10 containing recombinant plasmid pepio did not show integron amplification, although it carries a 10-kb dna fragment originated from genomic dna of e.coli sg2 isolated from varanus spp. (table 1). we suspected that parts of the integron have been deleted when it was digested by restriction enzymes. pcr product using 5'cs-3'cs primers generated a single amplified dna band of about 1-kb. it was generated from genomic dna of e. coli sg2 isolate, which was designated as sti (table 2 and fig.2 lane 3). at the downstream end of each resistant gene cassette inserted in the variable region of integrons, there is a short imperfect inverted repeat element called the 59base element. each inserted gene has its own version of this element (levesque et al. 1995). the sti was also thought to have a 59-base element, although it was not demonstrated in this study. these 59-base elements are known to be important in the recombination events observed in the evolution of the integron. for example, in plasmid pvsi, which possesses the 5' and 3' conserved segment but no inserted gene between the conserved segments, there is no 59-base element (levesque et al. 1995). the other isolates used in this study were found to be similar to pvsi (table 2). it was reported that pvsi, a plasmid derived from pseudomonas aeruginosa, possessed integron type ino which has an unoccupied integration site and hence may be an ancestor of the more complex integrons (bissonnette and roy 1992). cloning of sti in pas900 (kmr) vector yielded recombinant plasmid called pcras. the purpose of cloning was to detect ampicillin-resistant gene in sti. e. coli top 10 harbouring the recombinant plasmid pcras was sensitive to ampicillin. this result indicated that sti was not an ampicillin-resistant gene, or it was an ampicillin-resistant gene which could not be expressed. expression of antibiotic-resistant genes in the integrated cassettes of integrons depends on cassette position. in all cases, the resistance level was the highest when the gene was present in the first cassette (collis and hall 1995). therefore, there is a need to sequence the sti for gene characterization. 14 biotroopia no. 15, 2001 figure 2. integron pcr product from total dna of pvhal isolate (lane 1), sg2 isolate (lane 3), and kb ladder as marker (lane 2). 15 a novel integron in the genome of escherichia coli — esti puspitasari et al. sequencing cloning of sti in pgem-t easy vector yielded recombinant plasmid designated as ps2t. transformant was isolated, and then sequenced. the result indicated that the sti sequence was homologous to aada gene encoding aminoglycoside adenyltransferase, which was responsible for streptomycin and spectinomycin resistance in e. coli. blast search analysis of the sti sequence indicated that the 700 nucleotides of 3'sti are integron type 3 (int 3) as in the incl/m plasmid from salmonella typhimurium (96% identity) and pnccsol plasmid from enterococcus faecalis (96% identity). the other 600 nucleotides of 5'sti were similar to the pnccsol plasmid from e, faecalis (97% identity) and the r100.1 plasmid from bacteriophage t4 (97% identity). in summary, there were eight isolates out of nine e. coli isolates possessing a conserved segment of integron and only one possessed inserted gene between the conserved segments of the integron. the sequencing analysis indicated that spectinomycinand streptomycin-resistant gene was present in the 1-kb dna fragment. acknowledgments this work was supported by dip project for biotrop 1999 / 2000 to antonius suwanto. we thank yeo chew chieng from department of microbiology, national university of singapore for conducting dna sequencing. references auffenberg,w. 1981.the behavioral ecology of the komodo monitor. university press of florida, florida bissonnette, l. and p.h. roy. 1992. characterization of ino of pseudomonas aeruginosa plasmid pvsl, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. j. bacteriol. 174:1248-1257. collis, c.m. and r.m.hall. 1995. expression of antibiotic resistance genes in the integrated cassettes of integrons. antimicrob. agents. chemother. 39: 155-162. desselberger, u. 1998. resistance to antibiotics and other antimicrobiol agents. sgm quarterly. 25:94-95. francia, mv., j.c. zabala, f. cruz, j.m.g. lobo. 1999. the intl integron integrase preferentially binds single-stranded dna of the attc site. j. bacteriol. 181: 6844-6849. gian, g.s. dan v.h.s.gian. 1995. farmakologi dan terapi: aminoglikosid. ed. ke-4. gaya baru, jakarta. jacoby, g. 1992. exploring new strategies to fight drug-resistant microbes. science. 257:1036-1038. levesque, c., l. piche, c. larose, and p.h. roy. 1995. pcr mapping of integrons reveals several novel combinations of resistant genes. antimicrob. agents. chemother. 39:185-191. 16 biotropia no. 16, 2001 madigan, m.t., j.m. martinko, and j. parker, 1997. biology of microorganisms. 8"1 ed. prentice hall, new jersey. neu, h.c. 1992. the crisis in antibiotic resistance. science. 257:1064-1072. sambrook, j., e.k. frisch, t. maniatis. 1989. molecular cloning, a laboratory manual. ed. ke-2. cold spring harbor laboratory press. cold spring harbor, usa. schumm, d.e. 1992. intisari biokimia. terjemahan moch. sadikin. binarupa aksara, jakarta. yogiara. 1998. keragaman genetik isolat escherichia coli dari rongga mulut biawak (varanus) dan kloning gen penyandi resistensi ampisilin. skripsi. jurusan biologi fmipa ipb, bogor. 17 biotropia vol. 28 no. 2, 2021: 156 164 doi: 10.11598/btb.2021.28.2.1280 156 sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) rasmita adelina1*, irfan suliansyah2, auzar syarif2, and warnita2 1agrotechnology study program, faculty of agriculture, universitas graha nusantara, padangsidimpuan 22715, indonesia 2agriculture science study program, faculty of agriculture, universitas andalas, padang 25136, indonesia received 5 july 2019/accepted 9 january 2020 abstract salacca sumatrana (becc.), known locally as the sidimpuan snake fruit, is one of the specialties prime local commodities of padangsidimpuan city in sumatra. the fruit is known for its sweet, sour and astringent taste which differentiates it from pondoh and balinese snake fruits. recently, the snake fruit farmers have noticed a continuous decrease in production resulting from the failure in its fruit-setting, particularly during the off-season. the use of fertilization and drip irrigation in the off-season had been currently explored as part of the solution. hence, this research investigates the use of these methods in overcoming the fruit setting failure and guaranteeing subsequent production of sidimpuan snake fruit all-year round. specifically, this study aimed to determine the optimal dosage of ammonium sulfate fertilizer and drip irrigation for fruit setting during the offseason. this research used a split-plot design with the main plot for drip irrigation and the subplot for ammonium sulfate. the observed parameters included the number of flower and fruit bunches, fruit set percentage and a nutrient analysis of the leaves. drip irrigation significantly affected the fruit setting percentage and the number of harvested fruit bunches. the best treatment combination was at 400 g ammonium sulfate fertilizer per plant and drip irrigation of 3,000 ml/plant. the fertilization period in july-september produced an off season harvest that was comparable to the fruit set percentage (10.76% difference) and number of fruit bunches (25.65% difference) that were observed in the april-june fertilization for the on-season harvest. this indicated that applying ammonium sulfate with drip irrigation could overcome fruit set failure in sidimpuan snake fruit, particularly, during the off-season. keywords: drip irrigation, off-season, production, snake fruit, sulfate ammonium introduction sidimpuan snake fruit (salacca sumatrana becc.) is one of the specialty products locally produced primary commodities of padang, sidimpuan city. the fruit is known for its uniquely sweet, astringent and sour taste, differing from the pondoh and balinese snake fruits and other types of snake fruits. the species is spread throughout the sub-districts in the southern part of the tapanuli regency, predominantly at the districts of angkola barat, east angkola, south angkola, and marancar. sidimpuan snake fruit production shows high development potential in south tapanuli covering approximately 19,155 ha with a production potential of up to 30 tons/ha (bps 2015). hence, the development of an optimal cultivation technology is necessary. currently, the crop production undergoes high fluctuations between the main harvest season (on season) and harvest outside this season (off season) resulting in a continuously declining production. this decreasing production is due to the fruit-set failure in the off season which is caused by a number of adverse environmental factors, particularly, those which do not support the production process, including low rainfall and few rainy days, and low soil nutrient content resulting in the lack of vital nutrients as indicated by low nitrogen, phosphorus and potassium content in the leaves (rai et al. (2010). *corresponding author, email: rasmita301271@gmail.com sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 157 for the sidimpuan snake fruit to bear well outside the main harvest season, off-season treatments need to be applied. these would include nutrients supplementation through fertilizer application and simple drip irrigation technique. fertilization with ammonium sulfate and potassium chloride and simple drip irrigation were tried to meet the nutritional and water requirements of sidimpuan snake fruit in increasing its growth, fruit formation and production, out of season. under drip irrigation, fruit-setting was at 75.30% while that of no drip irrigation it was at 59.94% (rai et al. (2010). ammonium sulfate fertilizer provides nitrogen and sulfur and potassium chloride fertilizer as the main source of potassium which plays a role in plant growth and development. the availability of nitrogen and sulfur nutrients by fertilizing with ammonium sulfate at a dosage of 300 g had increased suwaru snake fruit production (sudaryono 2005) while the use of simple drip irrigation had efficiently and optimally meet the plants’ water requirements. hence, this study was aimed to obtain the best dosage combination of ammonium sulfate fertilizer and simple drip irrigation techniques to optimize fruit setting and to increase the sidimpuan snake fruit production in the offseason thereby optimally producing fruit throughout the year. materials and methods time and location of research the present study was conducted in april 2018 until september 2018 at the palopat maria village padangsidimpuan hutaimbaru subdistrict, padangsidimpuan city (between 010 28’19’’ 010 18’ 07’’ n and 990 18’ 53’’ 990 20’ 35’’ e). research methodology sixty (60) trees from among the 20 to 30year-old productive snake fruit trees were measured in the study applying the split plot design, consisting of three replications. the main plot is the application technique either, with drip irrigation of 3,000 ml/plant/day (p1) or with no irrigation (p0). the simple drip irrigation installation included infusion bottles, infusion tubes, drippers, plastic hoses and water pumps. the subplots for the fertilization technique consisted of plots without fertilization (p0) and plots with ammonium sulfate fertilizer applied at 250 g/plant + 40 g potassium chloride/plant (p1), 300 g/plant + 40 g potassium chloride/plant (p2), 350 g/plant + 40 g potassium chloride/plant (p3), and, 400 g/plant + 40 g potassium chloride/plant (p4). hence, the study consisted of 10 treatment combinations with 3 replications and 2 plants per plot, totalling to 60 plants. fertilizer applications were carried out in 2 periods; fertilization for the april june 2018 period was carried out on 14 march 2018, while fertilization for the july september 2018 period was carried out on 22 july 2018. previously, fertilization had been done in august 2017 and december 2017. fertilization was carried out by immersing the appropriate amounts of ammonium sulfate and potassium chloride fertilizer according to treatment into a 10 15 cm deep fertilizer groove in the soil that is 50 60 cm from the base of the stem. watering was a simple irrigation system that involved water movement carried out by gravity. drip irrigation equipment included a plastic tube as a water storage container, an infusion hose installation equipped with a dripper at the end that released water at a rate of 250 ml/30 minutes with a watering volume of 3000 ml/day. the drip irrigation was given daily for 6 hours from 10 am 4 pm. however, if it rains very heavily, the drip irrigation was not applied. the observed parameters were the fruit-set percentage, number of flower and fruit bunches, number of harvested fruit bunches, relative water content (rwc) and the nitrogen, phosphorus and potassium content on the leaves. the number of flower bunches and fruits were counted once every two weeks on each sample plant. analysis of leaf nitrogen, phosphorus and potassium content was carried out once every fertilization period. three leaves taken from each sample plant for laboratory analysis were cleaned, ovendried at 70 oc, then blended and sieved using a 0.5 mm grid sieve. the total nitrogen was determined using the kjeldahl semi-micro method, while the dry ashing method was used for determining phosphorus and potassium content. phosphorus concentration was measured with a uv-vis spectrophotometer, while potassium biotropia vol. 28 no. 2, 2021 158 concentration was determined by using a flame photometer. data were analyzed using anova and if differences among treatments were significant, duncan's multiple range test was applied. results and dicussion fruit set percentage (%) during the april-june fertilization period (on season), the highest fruit set percentage was at 76.949% indicating a significant increased caused by using the drip irrigation (table 1). the highest fruit set percentage of 75.615% also occurred at the 300 g/plant za dosage of ammonium sulfate fertilizer. the lowest response was from no irrigation (54.478%) nor fertilization (47.115%). drip irrigation has increased the relative water content of the leaves. hence, the high fruit-set percentage with drip irrigation was a result of the high relative water content (rwc) of leaves which in turn impacted the chlorophyll and potassium content (sunarka 2015). watering the soil then improves its chemical properties encouraging root growth and increasing physiological activity in the snake fruit plants, thereby improving their fruit setting capacity. in the july-september fertilization period (off season), the fruit set percentage (58.863%) was not significantly affected by drip irrigation treatment (68.673%). this was during the wet season, so the additional water was not necessary. the number of bunches formed and flower fall is influenced by environmental factors (adijaya et al. 2013). in the dry months, the flower fall usually increases thereby reducing the number of bunches formed. the use of ammonium sulfate fertilizer also had a statistically significant impact on the fruit setting with the highest response at 400 g per plant (77.792%). in another study, drip irrigation had significanly affected the out of season fruit production in dry conditions showing a fruit set percentage of 75.30% (off-season) and 93.13 % (on-season), compared to 59.94% and 61.67%, respectively, without irrigation (rai et al. 2014). in the july-september fertilization period of this study, the drip irrigation resulted in a significantly higher fruit set percentage. analysis of variance indicated that the effect of the combination of drip irrigation treatment and ammonium sulfate fertilizer dosage on the percentage of fruit formation was not significant (table 2). however, the treatment combination that resulted in the best fruit set percentage in the april-june fertilization period was at a dosage of 350 g fertilizer per plant with drip irrigation (91.667%) (table 2). whereas, in the fertilization period of july-september, the highest percentage of fruit set was with 400 g with drip irrigation (83.997%) (table 2). in another study, the combination of drip irrigation and nitrogen fertilizer had also significantly increased the yield of cotton plants (gossypium hirsutum) (zhong & bai 2013). moreover, under medium irrigation, the ratio of dry matter in nutritional organs to reproductive organs was also increased (zhong & bai 2013). table 1 the average percentage of fruit set, number of flower bunches, fruit and number of harvested fruit bunches with drip irrigation treatment and different dosages of ammonium sulfate fertilizer applied in april june and july september 2018 treatment fruit set (%) number of bunches number of harvested fruit bunches flower fruit apr-jun jul sept apr-jun julsept apr-jun julsept apr-jun julsept without irrigation 54.478a 58.863 19.267b 15.000 10.533 8.467 9.933 12.467a drip irrigation 76.949b 68.673 16.467a 14.267 12.733 9.467 12.067 18.200b without fertilizer 47.115a 52.620a 16.333 16.333 7.667a 7.833a 10.500 13.000 za 250 g 59.708ab 57.800ab 17.833 13.500 0.500ab 7.667a 9.000 14.667 za 300 g 75.615b 68.620ab 18.667 14.833 14.167b 10.000ab 11.000 13.833 za 350 g 74.443b 62.008ab 18.333 14.167 12.833b 8.167ab 10.167 18.000 za 400 g 71.687b 77.792b 18.167 14.333 13.000b 11.167b 14.333 17.167 note: means with different superscripts within a column are significantly different at p≤0.05, with comparisons performed using dmrt. sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 159 the application of ammonium sulfate fertilization and drip irrigation had increased the percentage of fruit set in both fertilization periods. this indicated that these production techniques will be able to minimize the production fluctuations in snake fruit between the harvest season (on season) and small harvests (off season). other study results also showed that both irrigation and nitrogen fertilization promoted cotton growth and yield (zhuan et al. 2017). drip irrigation technique had also increased the fruit set percentage of gula pasir snake fruit during the off season in a dryland ( rai et al. 2014). number of flower bunches during the april june (off season) fertilization period, the flower formation was significantly affected by the drip irrigation as a single factor (table 1). no irrigation treatment resulted in the most numerous flower bunches formed (19.267). ammonium sulfate fertilizer dosage at 300 g/plant also produced the highest average number of flower bunches (18.667) (table 1). during the july september (on season) fertilization period, the drip irrigation nor the fertilization did not significantly influence the flower formation (table 1). however, there was a tendency for more flower bunches with the no-irrigation treatment. this absence of significant difference with the irrigation technique is similar to that found by jose et al. (2013) who used regulated deficit irrigation techniques to improve fruit size. reduced irrigation and fruit thinning were found to affect carbon allocation within the tree by altering a number of interrelated factors (photosynthesis, location and number of competing sinks, storage capacity, and transport) that control the carbon partitioning in fruit trees. other factors affecting flowering could be interspecific differences and different crop seasons which was probably caused by variable atmospheric conditions during vegetation periods (greiner & kohl 2014). no significant relationship exists between fertilizer dosage and flower formation. in a study on sweet orange plants, the application of certain dosages phosphorus and potassium compound fertilizer had only a small effect on the number of flowers formed (ramadhan et al. 2015). other factors that cause unfertilized plants to flower as prolifically as those treated with fertilizer could be the irrigation and less than optimal water management. table 2 average fruit set percentage (%) with the combined drip irrigation and ammonium sulphate fertilization in april-june 2018 and july-september 2018 treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 34.140 44.330 50.437 52.503 67.603 65.157 57.220 60.740 62.990 71.587 54.478 58.863 drip irrigation 60.090 60.910 68.980 63.097 83.627 72.083 91.667 63.277 80.383 83.997 76.949 68.673 average 47.115 52.620 59.708 57.800 75.615 68.620 74.443 62.008 71.687 77.792 notes: * = fertilization in april–june 2018; ** = fertilization in july-september 2018 table 3 average number of flower bunches with ammonium sulfate fertilizer treatment treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 16.667 19.333 19.667 12.000 18.667 16.333 21.667 14.333 19.667 13.000 19.267 15.000 drip irrigation 16.000 13.333 16.000 15.000 18.667 13.333 15.000 14.000 16.667 15.667 16.467 14.267 average 16.333 16.333 17.833 13.500 18.667 14.833 18.333 14.167 18.167 14.333 notes: * = ammonium sulfate fertilization in april-june 2018; ** = ammonium sulfate fertilization in july-september 2018. biotropia vol. 28 no. 2, 2021 160 the combination of drip irrigation and the different ammonium sulfate dosages did not significantly affect the number of flower bunches. similar results were observed when new flower buds formed spontaneously every 1 1.5 months at the base of the leaf midrib but were uninfluenced by the combined irrigation and fertilization treatments (adelina 2017). furthermore, it was then assumed that the two treatments would influence the formation of new flower buds, later, when the fruit bunches were formed and until finally, when the fruit bunches were harvestable (table 3). the application of certain dosages of compound fertilizer phosphorus and potassium had also a minimal effect on the number of flowers in sweet orange plants (ramadhan et al. 2015) number of fruit bunches the number of fruit bunches in the apriljune fertilization period was not significantly affected by the drip irrigation in which the irrigated plants produced an average of 12.733 bunches per plant. in contrast, drip irrigation had a significant effect on both growth traits and fruit production indicating that it was important in zones with water limitations (loewe & delard 2016). the different dosages of ammonium sulfate fertilizer has significantly affected fruit bunch formation but the best dosage appeared to be at 400 g with 13.000 bunches of fruit per plant, and the 250 g at only 10.500 bunches (table 1). in the july-september fertilization period, the drip irrigation had no significant effect on the number of fruit bunches formed but it appeared to give a slightly higher number of fruit bunches (9.467) compared to the no-irrigation technique (8.467) (table 1). during this period, there was sufficient rain so the effect of irrigation in the formation of fruits seemed neligible. the drip irrigation was more effective during the dry season rather than on the rainy season (biswas et al. 2016). the same effect happened in the increased yield of tomato associated with the increasing amount of irrigation water (biswas et al. 2016). the optimal dosage of ammonium sulfate fertilization for fruit bunch formation was at 400 g with 11.167 bunches and the 250 g only yielding 7.667 bunches, showing a statistically significant difference (table 1). the use of nitrogen fertilizer and optimal water application were also recommended for the summer production of cotton (zhuan et al. 2017). in the april-june fertilization period, the combined treatments that resulted in the formation of the largest number of fruit bunches was drip irrigation with the 300 g fertilizer that produced 15.667 bunches (table 4). this interaction between irrigation and fertilization influenced most of the plant physiological functions and growth (wang et al. 2018). the same results indicating better economic production were observed in the use of fertilizer with daily drip irrigation for corn growing on a semi-arid area (chauhdary et al. 2017). number of harvested fruit bunches in the april-june fertilization period, neither drip irrigation nor dosages of ammonium sulfate fertilizer significantly affected the number of harvested fruit bunches. however, drip irrigation with 12.067 bunches and 400 g of fertilizer with 14.333 bunches appeared to give the best fruit harvest compared to 9.000 bunches from the 250 g of fertilized plants (table 1). ammonium sulfate fertilizer, as the main source of nitrogen, is crucial in increasing the crop production process. nitrogen application has increased the seed yield of coriander (coriandrum sativum l.) in a linear manner and the application of 60 kg/ha has improved the yield by 40% (alil et al. (2015). table 4 average number of fruit bunches with combined drip irrigation and ammonium sulfate fertilizer treatment treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 5.667 7.667 10.000 6.333 12.667 11.000 12.000 8.000 12.333 9.333 10.533 8.467 drip irrigation 9.667 8.000 11.000 9.000 15.667 9.000 13.667 8.333 13.667 13.000 12.733 9.467 average 7.667 7.833 10.500 7.667 14.167 10.000 12.833 8.167 13.000 11.167 notes: * = combined treatment in april-june 2018; ** = combined treatment in july-september 2018. sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 161 in the july-september 2018 fertilization period, drip irrigation had resulted in a statistically significant 18.200 harvested fruit bunches while any dosage of the ammonium sulfate fertilizer did not. the plants applied with the drip irrigation also produced a higher number of harvested bunches compared to those plants that were not given (rai et al. 2013). the best yield was at the 350 g fertilizer dosage with 8.000 bunches of harvested fruit compared to 12.467 bunches from the non-irrigated unfertilized plants (table 1). the non-significant effect observed on the combined irrigation and ammonium sulfate fertilizer treatment on the number of harvested fruit bunches implied that no real interactions existed between the two treatments. however, 22.667 fruit bunches resulted from a combination of 400 g fertilizer with drip irrigation and 7.000 bunches from the 250 g with no-drip irrigation (table 5). the lack of influence of ammonium sulfate fertilization suggested that drip irrigation alone can meet certain needs of sidimpuan snake fruit plants. the use of organic fertilization has also increased the fruit set percentage (ability to bear fruit) of snake plants (dewi 2014). the failure of fruit development from the flowers of the gula pasir snake fruit was more likely due to other environmental and plant physiological factors rather than the application of fertilization (rai et al. 2010). the development pattern of snake fruit plant production and distribution is strongly influenced by physiographic environments such as the altitude, land, rainfall, and air temperature (cahyani et al. 2013). these environmental factors include particularly, low rainfall and number of rainy days which lower the relative water content (rwc) of leaves thereby disrupting the metabolic processes. the average rwc of leaves in the july-sept 2018 period was higher than those of leaves in the april-june period (fig. 1). the positive impact resulted in a higher average number of fruit bunches harvested in the july-september period using the drip irrigation. this application of irrigation also resulted in greater fresh biomass, fresh leaf yield, and dry leaf yield in stevia plants (benhmimou et al. 2018). the higher leaf rwc with the drip irrigation treatment showed that irrigation increased the water content of the plant tissues thereby positively affecting the physiological processes as indicated by the increased plant ability to take up nutrients (rai et al. 2014). the nitrogen, phosphorus, potassium content and rwc of leaves in the julyseptember fertilization period was higher than those of the april-june 2018. very high differences were found in the nitrogen content and rwc of leaves (fig. 1). the leaf nitrogen content in the july-september fertilization period was higher (2.194%) than in the apriljune fertilization period (1.384%). nitrogen content and the rwc varied in a similar way to the percentage of fruit set and the highest number of fruit bunches obtained in the julyseptember fertilization period compared to those of the april-june 2018. the low productivity of gula pasir snake fruit was influenced by the low level of nitrogen in the leaves (rai et al. 2010; dewi 2014). this nutrient deficiency has influenced the plant physiological processes resulting in the failure of flower development into fruit due to photosynthate deficiency indicated by sucrose content, total sugar, and reducing sugars in the table 5 average number of harvested fruit bunches with the combined drip irrigation and ammonium sulfate fertilizer treatments in april-june 2018 and in july-september 2018 treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 10.000 11.667 7.000 12.000 9.000 11.000 8.333 16.000 15.333 11.667 9.933 12.467 drip irrigation 11.000 14.333 11.000 17.333 13.000 16.667 12.000 20.000 13.333 22.667 12.067 18.200 average 10.500 13.000 9.000 14.667 11.000 3.833 10.167 18.000 14.333 17.167 notes: * = combined treaments in april-june 2018; ** = combined treatments in july-september 2018. biotropia vol. 28 no. 2, 2021 162 figure 1 average nitrogen, phosphorus, potassium and relative water contents of leaves leaves at low interest due to high competition in fighting photosynthesis. fruit weight and fruit yield have been found to be significantly and positively correlated with nitrogen, phosphorus, potassium, iron, zinc and copper contents of the citrus var. kinnow mandarin leaf (kaul et al. 2014). leaf nutrient content is one indicator of nutrient availability which is critical in plant growth and development (marschner 1986). if the production process is not balanced with the availability of nutrients, in general it will cause a decrease in production. the nitrogen and phosphorus contents of sidimpuan snake fruit leaves were found to be higher than those of pondoh and sumedang leaves but the potassium content was lower (islami 2014). nitrogen status is related to leaf water content which also influences chlorophyll formation (fig. 1). drip irrigation increased the maximum chlorophyll content and photosynthetic nitrogen use efficiency (wang et al. 2018). for chlorophyll production, a schedule combining drip irrigation with 300 kg nitrogen/ha has provided the highest average chlorophyll production at an increase of 62% above non-irrigated levels (perez-ortola et al. 2016). nitrogen when absorbed by plants could play an important role in the chlorophyll formation as indicated by an increase in the green leaf color (pangaribuan et al. 2018). fertilization increases the soil nutrient availability for the plants to absorb. the average nitrogen and potassium levels of the sidimpuan snake fruit leaves were increased after the application of ammonium sulfate fertilizer (adelina et al. 2018). the higher the frequency of fertilization, the more secured is the availability of soil nutrients for plant growth and development (vargas & david 2015). the lack of plant response to fertilizer in this research may have been due to the plants’ need for a continuous supply of nutrients throughout the year. hence, a better result may have been achieved if fertilizers were given more often. for further research, it is recommended to increase the dosage of ammonium sulfate and potassium chloride fertilizers to determine a possible increase in the the fruit set percentage and production of sidimpuan snake fruit. conclussion drip irrigation during the off season in julyseptember has improved both the fruit set percentage and the number of harvested fruit bunches of the sidimpuan snake fruit. the best treatment, as compared to no drip irrigation, was the irrigation with 3000 ml/plant/day. as a single separate factor, the ammonium sulfate fertilizer treatment and the drip irrigation application significantly influenced the fruit set percentage and number of formed fruit bunches. no significant interaction existed between irrigation and fertilization. however, sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 163 based on the average number, the combined treatments that gave the best response was obtained at the ammonium sulfate fertilizer dosage of 400 g/plant. with drip irrigation, the off-season harvest in july-september was comparable to that of the fruit set percentage and number of fruit bunches formed with fertilization, during the main harvest season in april-june. acknowledgements this research was funded by dp2m dikti, through a doctoral dissertation grant in the 2018 implementation year. the researchers, therefore, would like to express their deepest gratitude to the director of dp2m dikti for the funding support. an infinite gratitude from the researchers is also extended for the collaboration of the research team from the department of agrotechnology, faculty of agriculture, graha nusantara padangsidimpuan university, as well as to the analytical staff at the soil department laboratory, faculty of agriculture, andalas university, padang, indonesia. references adelina r, irfan s, auzar s, warnita. 2017. study of sidimpuan salak cultivation (salacca sumatrana becc.). grahatani journal 3(1):434-43. adelina r, irfan s, auzar s, warnita. 2018. evaluation nutrients content in salak sidimpuan leaves (salacca sumatrana becc.). ijair 6(5). adijaya iy, mahaputra nik, rai imy, sukadana im, kertawirawa pa, sugiarta p, priningsih py. 2013. nursery studies, increased productivity and quality of gula pasir salak. final report. bali agricultural technology research institute. 29 p. adijaya in, rai imy. 2014. increasing the salak production of gula pasir variety (salacca edulis) with inovation of cow fertilizer. proceedings of the national seminar on bogor organic agriculture, june 18-19, 2014, bali institute of agricultural technology (bptp), ngurah rai pesanggaran, denpasar. adiwijaya iy, rai imy. 2014. increased production of gula pasir salak (salacca edulis) with fertilization innovations cow manure. bptp. bali. alil h, gohar a, ehsan e, muhammad s, saeed a, nazeer a, 2015. response of coriander (coriandrum sativum l.) to different nitrogen levels and sowing dates. asian j agri biol 3(4): 155-8. arteca rn. 1996. plant growth substance principles and application. pennsylvania (us): springer science business media dordrecht. 332 p. benhmimou a, mohammed i, chaouki a, fatima g, naima s, fatima za, mounira l. 2018. effects of water stress on growth yield quality and physiological responses of two stevia (stevia rebaudiana bertoni) varieties in rabat region morocco. asian j agri biol 6(1):21-34. biswas s.k, akanda ar, rahman ms, hossain ma. 2015. effect of drip irrigation and mulching on yield water-use efficiency and economics of tomato. plant soil environ 61(3):97-102 doi: 10.17221/804/2014-pse bps tapsel. 2015. south tapanuli regency in figures. south tapanuli regency central office of statistic. cahyani nk, suryadi wm, treman iw. 2013. distribution of salak gula pasir (zalacca sp. var. amboinensis) in bebandem district, karangasem regency. (a spatial approach). department of geography education, fis undiksha. 10 p. chauhdar jn, allah b, muhammad a, muhammad m. 2017. effect of different irrigation aand fertigation strategies on corn production under drip irrigation. pak j agri sci 54(4):855-63. doi: 10.21162/pakjas/17.5726 dewi kacj. 2014. application of several types of organic fertilizers and mycorrhizal doses to increase production and quality of salak granulated sugar out of season. 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(abstract). grassland science 62(2). https://doi.org/10.1111/grs.12116 pangaribuan dh, kus h, sheilla re, ade y. 2018. the effect of organic fertilizer and urea fertilizer on growth yield and quality of sweet corn and soil health. asian j agri biol 6(3):335-44. pérez-ortolá m, andre d, jerry wk. 2016. modelling irrigation and fertiliser use for chlorophyll production. grassland science 62(2):102-11. doi:101111/ grs.12116 rai in, semarajaya cga, wiraatmaja iw. 2010a. phenology study of fertilization of salak granulated sugar as an effort to overcome fruitset failure. j hort 20(3):216-22. rai in, wiraatmaja w, semarajaya cga, astiari nka. 2014. application of drip irrigation technology for producing fruit of salak ‘gula pasir’ (salacca zalacca var. gula pasir) off season on dry land. jdmlm 2(1):219-22. doi: 10.15243/ jdmlm. 2014. 021.219. ramadhan ra, baskara wm, suryanto a. 2015. the effect of nitrogen phosphor potassium fertilizer treatment to the fruit set of sweet orange (citrus sinensis osb.) var. pacitan. jurnal produksi tanaman 3(3). salisbury fb, ross cw. 2012. plant physiology. 4th edition. terjemahan lukman dan sumaryono jilid iii. perkembangan tumbuhan dan fisiologi lingkungan. bandung (id): penerbit itb. 343 h. sudaryono t. 2005. the production technology of salak suwaru off season. agricultural sciences. 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[thesis]. denpasar (id): agrotechnology study program, graduate program, universitas udayana. vargas ol, david rb. 2015. growth and fruit production of highbush blueberry fertilized with ammonium sulfate and urea applied by fertigation or as granular fertilizer. hortscience 50(3):479-85. wang z, qingyong b, jinzhu z, bo z. 2018. optimized water and fertilizer management of mature jujube in xinjiang arid area using drip irrigation. water j 10:1467. zhong d, bai d. 2013. effects of water and nitrogen regulation on the yield and water and nitrogen use efficiency of cotton in south xinjiang, northwest china under plastic mulched drip irrigation. chin j appl ecol 24(9):2525-32. zhuan y, yang g, shen xj. 2017. effects of nitrogen and irrigation water application on yield, water and nitrogen utilization and soil nitrate nitrogen accumulation in summer cotton. chin j appl ecol 28(12):3945-54. https://www.actahort.org/books/1015/index.htm https://www.actahort.org/books/1015/index.htm https://doi.org/10.17660/actahortic.2014.1015.37 https://doi.org/10.1111/grs.12116 biotropia vol. 29 no. 1, 2022: 90 94 doi: 10.11598/btb.2022.29.1.1278 90 the range expansion of parachromis managuensis günther, 1867 (perciformes, cichlidae) in java, indonesia maheno sri widodo1, veryl hasan2*, abdul rahem faqih1, maftuch1, r. adharayan islamy1 and felipe polivanov ottoni3 1department of aquatic resources management, universitas brawijaya, malang 65145, indonesia 2department of aquaculture, universitas airlangga, surabaya 60115, indonesia 3laboratório de sistemática e ecologia de organismos aquáticos, centro de ciências agrárias e ambientais, campus universitário, universidade federal do maranhão, br-222, km 04, s/n, boa vista, cep 65500-000, brazil received 28 june 2019 / accepted 24 may 2021 abstract the jaguar cichlid, parachromis managuensis (günther, 1867), is native to central america, with introductions reported from west java and central java provinces of indonesia. on 7-8 january 2019, sixteen specimens of p. managuensis were collected from karangkates, the largest hydropower reservoir in east java province of indonesia. a description of the morphological characters of specimens is provided. keywords: cichlid, distribution, freshwater fish, jaguar guapote introduction parachromis managuensis (günther, 1867), is a cichlid native to costa rica, nicaragua and honduras (conkel 1993), but it has been introduced to several countries in north america (fuller et al. 1999), south america (magalhães & jacobi 2013), and southeast asia (agasen et al. 2006). parachromis managuensis exhibits highly predatory habits and tolerance to new habitats (rosana et al. 2006; agasen et al. 2006), which make p. managuensis potential to become an invasive species (yamamota & annete 2000). parachromis managuensis is generally sold as ornamental fish and has not been cultured openly. p. managuensis in indonesia is firstly found in the freshwaters of west java (dahruddin et al. 2016) and central java (hedianto et al. 2013) provinces of indonesia. the presence of p. managuensis at the karangkates reservoir is considered a new finding because there has been no previous record of exotic fish culture in karangkates reservoir, the largest hydropower reservoir in east java province. materials and methods the fish sampling and description of the study sites sixteen (16) live specimens of p. managuensis were obtained from a local angler during fieldwork conducted on 7 8 january 2019 at the karangkates reservoir (8°11'16"s; 112°27'22"e) (fig. 1). administratively, the karangkates reservoir is located in malang regency, east java province, indonesia. fishing gear used by the angler was a medium hook with a bottom and using worms as bait (stein et al. 2012). *corresponding author, email: veryl.hasan@fpk.unair.ac.id parachromis managuensis in java – maheno sri widodo et al. 91 figure 1 the collecting point of parachromis managuensis at the karangkates reservoir in east java province fish identification the morphological characters of the fish specimens were determined by using the methods employed by kullander and hartel (1997) and bussing (1998). results and discussion specimens collection the sixteen (16) live specimens of p. managuensis had a range of total length between 9.9 mm and 26.6 cm. five (5) of the specimens were preserved in 96% alcohol solution (hasan & taman 2019) and transported to the hydrobiology laboratory, universitas brawijaya, malang, indonesia (voucher no. hb.pm.i.2019). the remaining eleven (11) specimens were kept as livestock at the fish reproduction laboratory, universitas brawijaya, malang, indonesia. the 11 living specimens were transported in oxygen-filled polyethylene bags. diagnosis the morphological characters of the specimens are as follows: a large mouth, projecting lower jaw, prominent enlarged canine teeth, a more or less continuous black stripe between the eye and opercular margin, and another stripe between the eye and the lower angle of the opercle, and a row of black blotches along the middle of the side. the fish can be distinguished from other members of the genus by having the expanded preopercle at the angle. it has silvery or goldengreen to purple body colors and black spots on the fins and body. there are also numerous black spots on the anal and caudal fins. the fish has moss green back, purple iridescence sides, and a whitish or yellowish belly. it also has whitish yellowish, or blue iridescence dorsal interspaces, and a black blotch on the caudal-fin base. all of these characteristics were found in every specimen collected from the karangkates reservoir, east java province, indonesia (fig. 2). biotropia vol. 29 no. 1, 2022 92 figure 2 specimen of parachromis managuensis captured on 8 january 2019 in karangkates reservoir, east java province distribution the discovery of p. managuensis in karangkates reservoir is the first record of this species in east java province. other discoveries occurred at reservoirs in west java and central java provinces. the discovery in karangkates reservoir represents a discovery in the eastern part of java island, which is around 400 km apart from the central java province (fig. 3). this record is an important contribution to understanding the dispersal of alien fish species in indonesia. figure 3 a. distribution of parachromis managuensis in java island notes: red square: west java province; blue square: central java province; green square: east java province). b. location of the karangkates reservoir in east java province note: the green square indicates the new record of p. managuensis. parachromis managuensis in java – maheno sri widodo et al. 93 we speculated that p. managuensis were released into karangkates reservoir in east java province by exotic fish hobbyists without clear purposes. further investigation is warranted to determine the source of p. managuensis in east java province because the reservoir has never been used for any exotic fish culture industry. control and prevention of further introductions are needed to prevent alien fish from disturbing the freshwaters ecosystem (hasan et al. 2020; wijayanti et al. 2021; hasan et al. 2021). conclusion parachromis managuensis is a non-native fish of indonesia. this fish species has been found in freshwater in west java and central java provinces and also in the karangkates reservoir in east java province. the existence of p. managuensis in east java is considered a new finding and added data on the alien fish species distribution in indonesia. acknowledgments the authors sincerely thank the ministry of finance of indonesia for funding this study (grant no. 20160221035555) and the local angler as our field guide. references agasen ev, clemente jp, rosana mr, kawit ns. 2006. biological investigation of jaguar guapote parachromis managuensis (günther, 1867) in taal lake, philippines. j environ sci manag 9(2):20-30. barros lc, santos u, zanuncio jc, dergam ja. 2012. plagioscion squamosissimus (sciaenidae) and parachromis managuensis (cichlidae): a threat to native fishes of the doce river in minas gerais, brazil. plos one 7(6): e39138. https:// doi:10.1371/journal.pone.0039138. bussing wa. 1998. peces de las aguas continentales de costa rica (freshwater fishes of costa rica). 2nd edition. editorial de la universidad de costa rica, san josé. conkel d. 1993. cichlids of north and central americas. new york (us): tfh publications. 191 p. dahruddin h, hutama a, busson f, sauri s, hanner r, keith p, …, hubert n. 2016. revisiting the ichthyo diversity of java and bali through dna barcodes: taxonomic coverage, identification accuracy, cryptic diversity and identification of exotic species. mol ecol resour 17(2):288-99. https://doi:10.1111/1755-0998.12528. fuller pl, nico lg, willians jd. 1999. nonindigenous fish introduced into inland waters of the united states. bethesda (us): american fisheries society. gestring kb, shafland pl. 1997. status and selected life history attributes of the exotic jaguar guapote (cichlasoma managuense) in florida. florida scientist 60(3):137-42. hasan v, tamam mb. 2019. first record of the invasive nile tilapia, oreochromis niloticus (linnaeus, 1758) (perciformes, cichlidae), on bawean island, indonesia. check list 15 (1): 225-227. https:// doi.org/10.15560/15.1.225. hasan v, widodo ms, islamy ra, pebriani daa. 2020. new records of alligator gar, atractosteus spatula (actinopterygii: lepisosteiformes: lepisosteidae) from bali and java, indonesia. acta ichthyologica et piscatoria 50(2): 233-236. https:// doi:10.3750/aiep/02954. hasan v, valen fs, islamy ra, widodo ms, saptadjaja am, islam i. 2021. short communication: presence of the vulnerable freshwater goby sicyopus auxilimentus (gobiidae, sicydiinae) on sangihe island, indonesia. biodiversitas 22: 571-9 https://doi.org/10.13057/biodiv/d220208. hedianto da, purnomo k, warsa a. 2013. interactions of food resources utilization by fish communities in penjalin reservoir, central java. bawal 5(1): 33-40. kullander so, hartel ke. 1997. the systematic status of cichlid genera described by louis agassiz in 1859: amphilophus, baiodon, hypsophrys and parachromis (teleostei: cichlidae). ichthyol explor freshw 7:193-202. kwik jtb, kho zy, quek bs, tan hh, teo dcj. 2013. urban stormwater ponds in singapore: potential pathways for spread of alien freshwater fishes. bioinvasions rec 2(3):239-45. http://dx.doi.org/ 10.3391/bir.2013.2.3.11. magalhães alb, jacobi cm. 2013. invasion risks posed by ornamental freshwater fish trade to southeastern brazilian rivers. neotrop ichthyol 1(3):433-41. https://doi.org/10.1590/s167962252013005000003. page lm, burr bm. 1991. a field guide to freshwater fishes of north america and north of mexico. boston (us): houghton mifflin company. 432 p. rosana mr, agasen ev, villanueva ls, clemente jr jp, kawit ns, de la vega jt. 2006. status and biotropia vol. 29 no. 1, 2022 94 economic impact of parachromis maraguensis in taal lake, philippines. j environ sci manag 9(2):1-19. sampaio wms, belei f, giongo p, dergam ja, orsi ml. 2017. heterotilapia buttikoferi (hubrecht, 1881) (perciformes: cichlidae), an introduced exotic fish in the upper paraná river basin. check list 13(4): 245-250. https://doi.org/10.15560/13.4.245. stein ja, shultz ad, cooke sj, danylchuk aj, hayward k, suski cd. 2012. the influence of hook size, type, and location on hook retention and survival of angled bonefish (albula vulpes). fish res 113:147-52. wijayanti a, hasan v, tamam mb. 2021. range expansion of oreochromis niloticus (linnaeys, 1758) (perciformes, chichlidae) in java sea and first record for masalembo island. iop conference series earth and environmental science 718(1): 012096. yamamoto mn, annete wt. 2000. hawai'i's native and exotic freshwater animals. honolulu (us): mutual publishing. biotropia vol. 29 no. 2, 2022: 95 102 doi: 10.11598/btb.2022.29.2.1625 95 secondary metabolite of sumbawa algae and its potential as a natural preservative baso manguntungi1*, arlinda puspita sari1, ariandi ariandi1, andi masniawati2 leggina rezzy vanggy3, robby erlangga3 and muhammad abdi mahesa3 1department of biological education, universitas sulawesi barat, majene 91412, indonesia 2department of biology, universitas hasanuddin, makassar 90245, indonesia 3department of biotechnology, universitas tekhnologi sumbawa, sumbawa 84371, indonesia received 31 july 2021/accepted 12 november 2021 abstract pathogenic bacterial contamination was a serious matter due to its capability in reducing food quality and health. this study aimed to select various types of algae in luk coast, sumbawa regency that have the potential to produce antibacterial compounds for natural food preservatives. algae on luk coast was identified by means of morphological characters, followed by sample preparation and extraction of secondary metabolites (bioactive compounds). algae extracts were used in antibacterial tests against food spoilage bacteria, such as escherecia coli, staphylococcus aureus, salmonella thypi, enterobacter cloacae and pantoea agglomerans. five types of algae identified were padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose. the five algae have the growth-inhibiting ability toward the tested bacteria. the highest inhibition zone was obtained from the 100% algae extract concentration. keywords: antimicrobial, natural preservative, secondary metabolite, sumbawa algae introduction food spoilage and foodborne infection increasingly become issues in the food industry, due to their serious impact on food quality and safety (vanegas et al. 2017). food spoilage is caused by pathogenic microorganisms. several types of food-spoiling microorganisms include aerobic psychrotrophic gram-negative bacteria, yeasts, molds, heterofermentative lactobacilli, and spore-forming bacteria (rawat 2015). food spoilage due to infection from foodborne microbes is mainly caused by campylobacter spp., salmonella, and eschercia coli (koluman & dikici 2013). contamination from these spoilage pathogenic bacteria, not only decreases food quality but also causes adverse health conditions. in order to inhibit the process of food deterioration, some people use synthetic preservatives such as formaldehyde, benzoic acid, bha (butilated hydroxyanisol), bht (butylated hidroxytoluene) and tbhq (tertier butylated hydroxyanisole), especially for food ingredients having high water content. however, the use of synthetic preservatives is not recommended by badan pengawas obat dan makanan (national agency of drug and food control/bpom). therefore, it is necessary to seek alternative food preservatives from natural ingredients. algae have biological properties that can be used as antibacterial (renhoran et al. 2017; basir et al. 2017). algae have the ability to produce secondary metabolites which are bioactive compounds to protect themselves from unsuitable environmental conditions and to defend themselves from the threat of various diseases and predators. bioactive compounds in algae can also act as antimicrobials, antifeedants, antihelmintic and cytotoxic agents (al-saif et al. 2014). *corresponding author, email: baso.manguntungi@uts.ac.id mscombalicer@up.edu.phveryl.hasan@fpk.unair.ac.id mailto:baso.manguntungi@uts.ac.id biotropia vol. 29 no. 2, 2022 96 several secondary metabolites produced by marine algae include alkaloids, polyketides, cyclic peptides, polysaccharides, phlorotannins, diterpenoids, sterols, quinones, lipids and glycerols (cabrita et al. 2010). al-saif et al. (2014) reported that groups of flavonoid compounds contained in marine algae include rutin, quercetin and kaempferol. these compounds can be used as natural food preservatives (hidayati et al. 2017). algae can be easily found in indonesia, such as on sumbawa island. sumbawa island is located in the nusa tenggara barat (ntb) province. this island has a 3,331.72 km2 water area, rich in various types of algae, including red algae (rhodophyta), brown algae (ocrophyta) and green algae (chlorophyta). cabrita et al. (2010) reported that laurencia is a group of red algae that produce secondary metabolites such as diterpenes, sesquiterpenes, triterpenes and c15-acetogenins. various kinds of compounds derived from phlorotannins (phloroglucinol, eckol, dieckol, etc.) that was beneficial for health were produced by brown algae such as ecklonia cava, ecklonia stolonifera, ecklonia kurome, eisenia bicyclis, ishige okamurae, sargassum thunbergii, hizikia fusiformis, undaria pinnatifida and laminaria japonica (li et al. 2011). bhagavaty et al. (2011) reported that the bioactive compounds of green algae chloroccocum humicola such as lipophilic and phenolic have antimicrobial properties against bacillarius subtilis, staphylococcus aureus, eschericia coli, pseudomonas aeruginosa, salmonella thypimurium, klebsiella pneumoniae, vibrio cholerae. the secondary metabolites of algae might be used in inhibiting the activity of food-spoiling bacteria. however, there have been no studies examining the antimicrobial potentials of algae originating from sumbawa island. this study aimed to conduct a study of antimicrobial compounds extracted from several types of algae originating from sumbawa island, against some food-spoiling bacteria such as salmonella thypi, staphylococcus aureus, escherichia coli, enterobacter cloacae and pantoea agglomerans. materials and methods sampling method samples were taken in the luk coast, sumbawa region. the samples were classified as red algae, green algae and brown algae. the samples were thoroughly cleaned from any debris and substrates by running the samples under tap water. algae identification identification was conducted based on morphological observations and practical guidelines for marine algae identification. samples extraction the collected samples were crushed using a pestle and mortar before being extracted. extraction was carried out using the soxhletation method. this method was carried out to separate bioactive compounds from the samples. the extraction was conducted by using the 1:15 ratio of the sample powder and methanol. the reflux process was carried out for 15 h at 78 oc (methanol's boiling point temperature). filtrates of the reflux process had the basic colors of the algae (red, green and brown). the 150 ml solvents were then separated from the extract by distilling the solvent directly using soxhlet. the purified solvents were subsequently poured into the bottles, followed by repeat heating until there is a sample substance in the flask (fabrowska et al. 2017; liswandari et al. 2018). rejuvenation and manufacture of test bacterial suspensions salmonella thypi, escherechia coli, enterobacter cloacae, pantoea agglomerans and staphylococcus aureus bacteria from pure cultures were taken and inoculated on slanted nutrient agar (na) medium. bacterial cultures were incubated at 37 oc for 24 h. the test bacteria having been rejuvenated for 24 h were taken and suspended into nb media (nutrient broth) then homogenated. liquid cultures were incubated at 37 oc for 24 h. the turbidity of the media indicated the multiplication of bacterial cells. potentials of algae as natural preservative for food – baso manguntungi et al. 97 algae extract preparation in various concentrations the crude extracts were poured into dilution bottles of 200 µl, 400 µl, 600 µl, 800 µl and 1000 µl, respectively, and subsequently added with distilled water until each solution amounted to 1 ml. so, the extracts obtained had respective concentrations of 20%, 40%, 60%, 80% and 100%. the five extract concentrations in the bottles were homogenized. antimicrobial test the antibacterial activity test was carried out in-vitro using the antibacterial sensitivity test with agar diffusion method using disc papers. nine disc papers were immersed in respective crude extract solution and control solution for 30 min. as much as 100 µl of tested bacteria having been cultured for 24 h were leveled using triangular rods on the na medium. the disc papers having been soaked in the crude extract and control solutions were affixed to the surface of the media. the disc papers were arranged as not too close to each other so that the inhibition zones did not intersect with each other. each treatment was conducted with three replications. the petri dishes were then incubated at 37 oc. the inhibition zones were observed at the 16th hour. the diameter of the inhibition zone was determined by subtracting the overall diameter (disc paper + inhibition zone) from the diameter of disc paper (6 mm) and diameter of the inhibition zone of solvent (if the solvent has an inhibition zone) (al-saif et al. 2014; bhagavathy et al. 2011). data analysis the study was carried out using the completely randomized design (crd) method with three replications. data were analyzed using anova (analysis of variance) with a confidence level of 95% by using spss 24.0. results and discussion algae identification there were 5 algae species identified in the luk coast, sumbawa regency. the five algae species were derived from the groups consisting of red algae, brown algae and green algae. identification was carried out based on the morphological characters of each alga (table 1), showing three species of brown algae and one species of red algae and green algae, respectively. one identified type of brown algae was padina sp. this species is also found in several indonesian waters such as in the makassar strait (hamrun et al. 2019), parigi moutong coast, padang (dharmayanti et al. 2019) and belitung lengkuas coast (jannah et al. 2021). according to aulia et al. (2021), padina sp., is fan-shaped with a diameter of 3-4 cm, yellowish-brown or whitish due to calcification, consisting of flexible rhizoids for attaching themselves to a surface. the tip of the blade is thin-widens and forms a lobe. the lobe is segmented and has white circular stripes, which were not visible, so the talus structure consists only of a blade and a holdfast. the holdfast has the slabs form. padina sp. grows on sandstone substrates. besides padina sp. other types of brown algae found were sargassum horneri and sargassum crassifolium. these two types of algae have similar physical characteristics. sargassum crassifolium has a cylindrical or flattened brown thallus with a lush and crossing branch, similar to terrestrial plants. the leaves’ shape is broad, oval, or sword-like. on the edges of the jagged leaves, there are some bubbles called vesicles. the function of these air bubbles is to keep the leaves on the surface of the water. the branching alternates regularly. the leaf has an oval and elongated shape with a thallus length of 13.5 14 cm. the thallus grows closely resembling a stem. the leaves are sparse and table 1 classification of the five algae species in the luk coast algae classification no. class ordo familia genus spesies 1 ocrophyta dyctyotales dyoctaceae padina padina sp. 2 chlorophyta bryopsidales halimedaceae halimeda halimeda opuntia 3 ocrophyta fucales sargassaceae sargassum sargassum horneri 4 ocrophyta fucales sargassaceae sargassum sargassum crassifolium 5 rhodophyta nemaliales galaxauraceae galaxaura galaxaura rugose biotropia vol. 29 no. 2, 2022 98 wavy. the tips are curved or tapered. this type of algae is able to grow on coral substrates in choppy areas (ode 2013). meanwhile, sargassum horneri has a branched and wide thallus, and lush branching. on the leaves, there were serrations and bubbles. sargassum horneri has a slightly flat and smooth thallus, but the main stem is rounded and slightly rough. the length of the pinnatus alternates is 30-50 cm. according to gazali et al. (2018) sargassum sp. contains bioactive compounds such as alkaloids, phenols and triterpenoids. sargassum sp. also contains bioactive compounds such as saponins, tannins, flavonoids, and phenols (pangestuti et al. 2019). these compounds are able to inhibit the growth of s. aureus and e. coli. green algae, namely halimeda opuntia, was also found on luk coast. halimeda opuntia grows on sandy and coral substrates in intertidal to subtidal areas (kepel et al. 2018). halimeda opuntia has the characteristics of a green thallus and is very stiff because it is formed from filaments of branched shiphonus; segmented by tricotome branching; segments form triangles, and segments appear in basal segments. the adhesive device is a filament that comes out of the basal segment and grips the substrate. segments are calcareous, very rigid, and have a three-indented shape. the arrangements are overlapping, irregular and not located in one branching, so the thallus is not located on one side. halimeda opuntia produces several types of bioactive compounds such as alkaloids, flavonoids and triterpenoids (hudaifah et al. 2020). the last type of algae found on luk coast was galaxaura rugose, which belongs to the red algae group. the algae are beneficial because it contains compounds such as sterols, triterpenoids, flavonoids, tannins and coumarins (al-enazi et al. 2018). galaxaura rugose has a thick thallus, stiff, dark red-brown color. branches are cylindrical and have a holdfast to attach to coral reefs. this species is short clumps and reaches 5-7 cm in height. galaxaura rugose grows on every coral reef. antimicrobial activity of algae extracts an antimicrobial activity test is a method to determine the ability of a substance to inhibit bacterial growth. this test was carried out on five types of pathogenic bacteria causing food spoilage, such as salmonella thypi, staphylococcus aureus, escherichia coli, enterobacter cloacae and pantoea agglomerans. the test was carried out on each identified algae extract. the test results indicated that padina sp. has growth-inhibiting ability against all tested pathogenic bacteria (table 2). the antimicrobial activities of padina sp. against p. agglomerans, s. aureus and s. thypi showed significantly different values at each treatment concentration with the highest result obtained by the 100% extract concentration of padina sp. in testing against e. coli, the 60% and 80% concentrations were not significantly different. the highest inhibiting ability was also shown by the 100% extract concentration. the testing against e. cloacae showed that the diameter of the inhibition zone for the 20% and 40% concentrations were not significantly different and the highest inhibiting ability was shown by the 100% extract concentration. the antimicrobial activity of halimeda opuntia extract also showed growth inhibiting ability against all tested bacteria (table 3). the formed inhibition zone showed significantly different results for each treatment concentration against all tested bacteria. the highest inhibition zone for all tested bacteria was obtained by the 100% extract concentration. potentials of algae as natural preservative for food – baso manguntungi et al. 99 table 2 results of testing the antimicrobial activity of padina sp. extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 11.67±0.58 b 9.33±0.58 b 12.33±0.00 b 12.67±0.00 b 13.00±0.00 b 40 15.67±0.58 c 12.00±1.00 c 13.67±0.58 c 13.67±0.58 b 14.67±0.58 c 60 19.33±0.58 d 17.33±0.58 d 16.67±0.58 d 16.33±1.15 c 17.33±1.15 d 80 22.67±0.58 e 18.67±0.58 e 17.67±0.58 d 20.00±0.58 d 20.33±0.58 e 100 25.00±1.00 f 20.33±0.58 f 22.33±0.58 e 22.67±1.15 e 22.67±1.15 f k+ 29.00±1.00 g 28.33±0.58 g 28.67±0.58 f 28.67±0.58 f 28.67±0.58 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a *notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. table 3 results of testing the antimicrobial activity of halimeda opuntia extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 8.33± 0.58 b 8.67±0.58 b 8.67±0.58 b 10.33±0.58 b 10.67±0.58 b 40 11.33±0.58 c 13.67±0.58 c 14.67±0.58 c 14.33±0.58 c 14.67±0.58 c 60 13.00±0.00 d 16.67±0.58 d 18.00±0.00 d 14.67±0.58 c 15.67±0.58 d 80 16.00±0.00 e 18.00±1.00 e 19.33±0.58 e 16.33±0.58 d 18.33±0.58 e 100 21.00±1.00 f 22.33±0.58 f 22.67±0.58 f 20.33±0.58 e 21.67±0.58 f k+ 28.00±0.00 g 28.33±0.58 g 28.33±0.58 g 28.67±0.58 f 29.00±0.00 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. table 4 results of testing the antimicrobial activity of sargassum horneri extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 8.00±0.58b 10.67±0.58b 11.67±0.58b 8.33±0.58b 7.33±0.58b 40 10.75±0.58c 14.33±0.58c 15.33±0.58c 13.00±1.00c 13.33±0.58c 60 12.50±1.15d 16.00±1.00d 17.00±1.00c 16.33±0.58d 16.33±0.58d 80 17.75±0.58e 23.67±0.58e 23.00±1.00d 18.33±0.58e 17.67±0.58e 100 19.00±0.58f 25.67±0.58f 26.00±1.00e 23.67±0.58f 22.67±0. 58f k+ 21.25±0.58g 28.33±0.58g 28.67±0.58e 28.67±0.58g 28.33±0 58g k0±0a 0±0a 0±0a 0±0a 0±0a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. the antimicrobial activity of sargassum horneri extract against p. agglomerans, s. aureus, e. cloacae and s. thypi showed significantly different results at all concentrations (table 4). slightly different results were shown in observations against e. coli. the diameter of the inhibition zone showed no significant difference at concentrations 40% and 60%. the 100% extract concentration of sargassum horneri has the same effectiveness with positive control in inhibiting the activity of e. coli. the sargassum crassifolium extract showed growth inhibiting ability against all tested bacteria (table 5). the diameter of the inhibition zone formed in the four tested bacteria, i.e., s. aureus, e. coli, e. cloacae, and s. thypi showed significantly different results in biotropia vol. 29 no. 2, 2022 100 table 5 results of testing the antimicrobial activity from sargassum crassifolium extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 10.67±0.58b 7.67±0.58b 10.33±0.58b 9.00±1.00b 8.67±0.58b 40 13.67±0.58c 12.00±0.00c 12.00±0.00c 12.67±0.58c 13.33±0.58c 60 18.33±0.58d 14.67±0.58d 14.00±1.00d 16.00±1.00d 16.67±0.58d 80 24.00±0.00e 17.67±0.58e 18.00±0.00e 19.00±0.00e 21.67±0.58e 100 27.00±1.00f 22.33±0.58f 22.33±0.58f 23.67±0.58f 23.67±0.58f k+ 28.67±0.58f 28.33±0.58g 29.00±0.00g 29.00±0.00g 28.33±0.58g k0±0a 0±0a 0±0a 0±0a 0±0a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. each extract concentration with the highest inhibition zone obtained by the 100% extract concentration of sargassum crassifolium. however, there was a slight difference in observations against p. agglomerans test bacteria, showing that the 100% extract concentration was not significantly different from the control treatment. it means that the 100% concentration of sargassum crassifolium extract has the same inhibitory effect against the p. agglomerans test bacteria with the control. the antimicrobial activity test using galaxaura rugose extract against e. coli, e. cloacae, and s. thypi showed significantly different results among extract concentrations with the highest inhibiting ability obtained by 100% galaxaura rugose extract (table 6). the 40% and 60% extract concentrations against the p. agglomerans and s. aureus test bacteria showed a nonsignificant difference effect, with the highest inhibiting ability shown by the 100% extract concentration. the five algae extracts showed the ability to inhibit the growth of all tested bacteria. the largest inhibition zone formed was shown by the 100% extract concentration with an inhibition zone diameter range of 19.00-27.00 mm. according to davis and stout (1971), the bacterial inhibition zone less than 5 mm was categorized as weak, the 5 10 mm zone of inhibition was categorized as moderate, the 10 20 mm zone of inhibition was categorized as strong and more than 20 mm was categorized as very strong. it means that the 100% extract concentration of the five algae was in the very strong category. table 6 results of testing the antimicrobial activity from galaxaura rugose extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 10.67±0.58 b 10.33±0.58 b 8.00±1.00 b 11.67±0.58 b 11.67±0.58 b 40 12.00±0.00 c 13.00±0.00 c 9.67±1.53 c 14.67±0.58 c 14.67±0.58 c 60 12.67±0.58 c 13.67±1.15 c 13.33±0.58 d 17.00±0.00 d 18.00±0.00 d 80 15.33±0.58 d 16.33±0.58 d 15.00±0.00 e 19.33±0.58 e 20.67±0.58 e 100 19.67±0.58 e 20.33±0.58 e 20.00±0.00 f 23.33±0.58 f 24.33±0.58 f k+ 28.00±0.00 f 28.67±0.58 f 28.67±0.58 g 28.67±0.58 g 28.67±0.58 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. potentials of algae as natural preservative for food – baso manguntungi et al. 101 the formation of the inhibition zone indicates that the algae extracts from padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose have the potential as antibacterial agents. the five algae have capabilities to produce bioactive compounds such as steroids, terpenoids and eicosanoid acid. different phytochemical contents in padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose determine the differences in inhibition abilities because each bioactive compound contained in algae extract has a different action mechanism against bacteria. according to dewi (2010), flavonoids are able to inhibit the growth of pathogenic bacteria by damaging the cell wall components so that the cell wall layer is not intact causing cell death. steroids can damage bacterial cell membranes by increasing cell permeability, resulting in cell leakage followed by the release of intracellular material (cowan 1999). alkaloid compounds can inhibit the growth of gram-positive and gram-negative bacteria, wherein alkaloids can cause cell lysis and changes in bacterial morphology (katou 2006). terpenoids have an antibacterial mechanism by reacting with porin (transmembrane protein) on the outer membrane of the bacterial cell wall, forming strong polymer bonds resulting in the breakdown of porin. the damage of porin, i.e., the entrance and exit for compounds, reduces the permeability of the bacterial cell wall resulting in the bacterial cell being deficient in nutrients so that bacterial growth is inhibited or dies (cowan 1999). the ability of the five algae extracts to inhibit the growth of pathogenic bacteria shows that the five algae have the potential to be developed and utilized as natural food preservatives. the bioactive compounds produced through the secondary metabolism of algae were proven by this study to have the ability for inhibiting the growth of food-spoiling pathogenic bacteria. the use of algae as natural food preservatives is expected to replace synthetic food preservatives. food preservatives derived from natural ingredients are relatively safe to consume and do not cause side effects with the same effectiveness as food preservatives as synthetic ones. conclusion the padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose algae were able to inhibit the growth of pathogenic bacteria such as pantoea agglomerans, staphylococcus aureus, escherecia coli, enterobacter cloacae and salmonella thypi. antibacterial activities in the five types of algae have an inhibition zone ability ranging from 19.00 to 27.00 mm at 100% extract concentration with a category of very strong inhibition zone capability. this means that the five types of algae have the potential to be used as natural food preservatives. acknowledgments the authors thank the head of the biotechnology laboratory, universitas teknologi sumbawa. this research was funded by the 2021 research grant of universitas teknologi sumbawa. references al-enazi nm, awaad as, alqasoumi si, alwethairi mf. 2018. biological activities of the red algae galaxaura rugosa and liagora hawaiiana butters. saudi pharm j 26:25-32. al-saif ssa, abdel-raouf n, el-wazanani ha, aref ia. 2014. antibacterial substances from marine algae isolated from jeddah coast of red sea, saudi arabia. saudi j biol sci 21:57-64. aulia a, kurnia sk, mulyana d. 2021. identifikasi morfologi beberapa jenis anggota phaeophyta di pantai palem cibeureum, anyer, banten. 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(code no. r071), and streptomyces sp. (code no. r086) as the consistent and most effective inhibitors. application of the three most promising antagonistic bacteria as seed treatment s howed that they effectively inhibited the growth of the damping-off fungi in the laboratory as exhibited by an increase in percent germination. bacillus subtilis however, was not able to antagonize the effect of p. debaryanum in this particular experiment. seed germination and seedling survival were likewise improved with the application of the three most promising antagonistic bacteria as seed treatment. this was shown after three months under nursery conditions. there were possible mechanisms of control by the antagonistic bacteria against the damping-off fungi. the mycelium and spores of the pathogenic fungus may have been attacked and parasitized by the antagonist when they were simultaneously grown in culture media. there must have been a competitive interaction between the two microorganisms. any essential requirement of microorganisms can serve as potentially possible basis for competition. another was antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds. moreover, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. lysis is often attributed to production of cell wall degrading enzymes like chitinase and gluconase t hat may hydrolyze major constituents of fungal cell walls. 1 biotropia no. 7, 1994 furthermore, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. lysis is often attributed to production of cell wall degrading enzymes like chitinase and glucanase that may hydrolyze major constituents of fungal cell walls. as demonstrated by kapoor and kar (1979), hyphae showed lysis in addition to thickening of hyphae as well as formation of chlamydospores in abundance as an effect of bacillus sp. on f. oxysporum f. sp. lycopersici. inhibitory substance diffused into the media may initiate fungal cell wall lysis. most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. seed treatment gave the antagonist the opportunity to be the first colonizer of the plant roots of the seedlings (elad and chet 1987) thus having an advantage for any competition for nutrients supplied by roots. seed exudates probably occurred in the interaction between the antagonistic bacteria and damping-of f fungi in the root and is responsible at least to a degree for the observed biological control by introduced bacteria. large populations of bacteria established on planting material and roots become a partial sink for nutrients in the rhizophere, thus reducing the amount of carbon and nitrogen available to stimulate sporulation of fungal pathogen or for subsequent colonization of the root (weller 1988). in addition, cell wall degrading enzymes are activated by nutrient deprivation imposed by microbial activity. oospore germination by some damping-off fungi has already been shown to be affected by exogenous nutrients (howell and stipanovic 1980). nutrients supplied by root exudates stimulate oospore germination in the rhizosphere. it therefore, appeared that bacteria may compete with germinating oospore for available carbon or nitrogen sources, and by eliminating these resources, the bacteria reduce the percentage of oospore germination. in addition, elad and chet (1987) stated that there was a significant positive correlation observed between inhibition of oospore germination and disease reduction by bacteria. conclusion the promising antagonistic bacteria therefore, could reduce the incidence of damping-off disease of agoho. its application as biological control agent could be a good substitute for soil fungicides. although soil fumigation is very effective for the control of damping-off pathogens, it is very expensive, impractical and may create problems like environmental pollution and pathogen resistance. 10 biological control of damping-off fungi of agoho f.a. dela pena et al. references dayan, m.p. and v.o. sinohin. 1989. diagnosing forest tree diseases (fungal). ecosystem research and development bureau, college, laguna. 29 p. dela cruz, a. 1986. microbial antagonists of the potato soft-rot pathogen, erwinia carotovora pv. carotovora (jones) bergey, harrison, breed, hammer, and hutton. (unpublished ms thesis, university of the philippines at los banos, college, laguna). dickson, j.g. 1956. diseases of field crops. me. graw hill book, co., new york, 517 p. ela d, y. and i. chet. 1987. possible role of competition for nutrients in biocontrol of pythium damping-off by bacteria. phytopathol. 1: 375-396. garret, s.d. 1965. toward biological control of soil-borne plant pathogens: 4-16. in: k.f. barker and w.c. snyder (eds.), ecology of soil-borne plant pathogens-, prelude to biological control. university of california press, california. henis, y. and i. chet. 1975. microbiological control of plant pathogens. in: d. perlman (ed.) advances in applied microbiology. vol. 19. academic press, new york. 332 p. henis, y. and m. imbar. 1968. effect of bacillus subtilis on growth and sclerotium formation by rhizoctonia solani. phytopathol. new york. howell, c.r. and r.d. stipanovic. 1980. suppression of pythium ultimum induced damping-off of cotton seedlings by pseudomonasfluorescens and antibiotic pyoluteorin. phytopathol. 70: 712 715. jones, r.k. 1985. fungicides for bedding plants. bedding plants inc. news 16(2): 3-4. kapoor, i.j. and b. kar. 1989. antagonism of azotobacter and bacillus to fusarium oxysporum f. sp. lycopersici. indian phytopathol. 42(3): 400-404. larson, r. 1987. growing concerns. pest control: how much is enough. marketletter 2: 5. national research council. 1984. casuarinas: nitrogen fixing trees for adverse sites. national academy press, washington d.c. 118 p. olsen, c.m. and k.f. baker. 1968. selective heat treatment of soil and its effects on the inhibition of rhizoctonia solani by bacillus subtilis. phytopathol. 58: 79-87. raymundo, a.k., e.t. serrano, g.d. reyes and t.o. zulaybar. 1985. isolation and identification of antibiotic-producing bacillus from the soil. phil. agr. 68: 393-402. weller, d.m. 1988. biological control of soilborne plant pathogens in the rhizosphere with bacteria. ann. rev. phythopathol. 26:379-407. 11 biotropia no. 7, 1994 pre-emptive colonization was also a possible mechanism of control. seed treatment gave the antagonist the opportunity to be the first colonizers of the plant roots and moved along the roots of the seedlings thus having an advantage for any competition of nutrients. most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. key words: biological control. fungal diseases, casuarina equisetifolia, antagonistic bacteria introduction agoho (casuarina equisetifolia l.) is a good fuel wood species in scores of tropical countries like the philippines. it has particularly been valuable for stabilizing sand dunes as it withstands salt sprays, thrives/grows in infertile soil and is drought-tolerant (national research council 1984). these indispensable characteristics make it highly recommended for reforestation and afforestation purposes. damping-off disease however, is the major threat in the production of sufficient and quality planting stocks. damping-off is a term applied to any disease that results in the decay of seeds in the soil before seedling emergence, or rapid rotting usually at the soil line of recently emerged seedlings (dayan and sinohin 1989). it is caused by a number of soil-inhabiting fungi that are facultative parasites and not specialized as to host. the ubiguity of damping-off fungi necessitates effective control measures because there are times when seedlings in the entire seedbed are wiped out resulting to shortage of supply for field planting. chemical control is usually not feasible due to the expensive price of fungicides. moreover, they can cause environmental pollution and may even induce pathogen resistance (jones 1985; larson 1987). biological control perhaps offers one of the best alternatives. as defined by garret (1965), biological control is any condition or practice under which survival or activity of a pathogen is reduced through the agency of any organism (except man himself) with the result that there is a reduction in the incidence of the disease caused by the pathogen. one of the ways that biological control may operate to suppress pathogen is introduction of biocontrol agents or antagonists. it is for this purpose that a study on the biological control of damping-off fungi of agoho using antagonistic bacteria was conducted. specifically, the study aimed to determine the efficacy of promising antagonistic bacteria in the laboratory and under nursery conditions. 2 biological control of damping-off fungi of agoho f.a. dela pena et al. materials and methods the antagonistic activity of 100 bacterial isolates consisting of 85 strains of bacillus species and 15 actinomycetes was determined against six fungal pathogens isolated from damped-off agoho. the damping-off fungi were: fusarium oxysporum schet., rhizoctonia solani kuhn., phytophthora parasitica dastur, pythium debaryanutn hesse, and two unidentified pathogens temporarily designated as unk 1 and unk 2. preliminary screening using the agar-plug technique involved simultaneous inoculation of the pathogen (damping-off fungus) and the antagonist (bacterial isolate). bacterial isolates showing good antagonistic activity were then subjected to the agar-diffusion method and further tested in the laboratory and under nursery conditions as seed treatment. agar-plug technique the cell suspension of bacillus species was prepared by growing the organism in tryptone glucose yeast extract agar (tgya) in a flat bottle for 24 -48 hr under room temperature. after incubation, the growth was flooded with sterile distilled water and then aseptically dislodged from the agar. the resulting suspension was adjusted to 0.30 optical density (od) at 540 nm using a spectronic 20 to have the desired population of 10 9 cells/ml (dela cruz 1986). the cell suspension of actinomycete was prepared by growing the organism in yeast-malt extract broth (ymb) for 48 72 hr on a shaker at room temperature. the broth culture was then pooled to make the necessary amount needed and homogenized using an osterizer for 5 min. a 10% suspension was made by diluting 100 ml of the prepared actinomycete suspension into 900 ml sterile distilled water (dela cruz 1986). agar plugs were prepared by initially growing the damping-off fungus on potato dextrose agar (pda) under room temperature for seven days or until sufficient growth was obtained. a sterile 1-cm-diameter cork borer was used to obtain the agar plugs from these culture plates. the agar plugs inoculated on agar plates were initially prepared by plating 1 ml of bacterial cell suspension on tgya for bacillus species and yeast-malt extract agar (yma) for actinomycetes. inoculation of the agar plugs was done in an upside-down position to provide a direct contact between the pathogen and the antagonist. the assay plates were incubated at room temperature for 3 to 5 days, after which growth of the pathogen was observed. this experiment utilized a completely randomized design (crd) with three replications. 3 biotropia no. 7, 1994 agar-diffusion method cultures (24 to 48-hr-old) of the potential antagonistic bacteria were used to inoculate tryptone glucose yeast extract broth (tgyb) for bacillus species and ymb for actinomycetes. these were then incubated at room temperature on a shaker. after 48 hr (for bacillus spp.) and 72 hr (for actinomycetes) incubation, the broth cultures were centrifuged at 14 000 rpm, 0°c, for 15 min. the supernatant was used for the microbial assay. one loopful of growth from 48-hr-old culture of the damping-off fungus was suspended in 2 ml sterile distilled water, mixed and the suspension inoculated into 100 ml pda top agar. this was used to seed the pda plates and then allowed to solidify (raymundo et al. 1985). following the solidification of the top agar, four cup cylinders were positioned on the plate and these were then filled with 0.1 ml of appropriate supernatant from each potential antagonist. after incubation for 48 hr or until sufficient growth was obtained, the assay plates were examined and the diameter of zones of inhibition was measured. this experiment utilized a crd with three replications and three subreplications. significant values were further subjected to duncan's multiple range test (dmrt). antagonistic bacteria showing potential to control the damping-off fungi in the agar-diffusion method were further screened as seed treatment in the laboratory and under nursery conditions. laboratory experiment newly prepared cell suspensions of the antagonistic bacteria were used to coat the agoho seeds by soaking for at least 15 min. before sowing onto sterile germination plates lined with tissue paper. the germination plates were initially inoculated with 5 ml fungal cell suspension prepared by suspending a loopful of growth of a 48-hr-old culture onto 100 ml sterile distilled water. twenty agoho seeds treated with the antagonistic bacteria were sown in each inoculated germination plate. untreated seeds sown in an uninoculated germination plate served as the control. all seeds sown were disease-free and previously surface-sterilized. the plates were incubated at room temperature for 15 days. watering was regulated using sterile distilled water. seed germination and the occurrence of damping-off disease were regularly assessed. the experiment utilized a crd with three replications. 4 biological control of damping-off fungi of agoho f.a. dda pena et al. nursery experiment the promising antagonistic bacteria were further screened as seed treatment under nursery conditions. a newly prepared bacterial cell suspension corresponding to each treatment was used to coat the agoho seeds. this was done by soaking for at least 15 min. before sowing into a pot with naturally infested soil. untreated seeds sown in naturally infested soil served as the control. another treatment made use of untreated seeds sown in a soil medium subjected to sterilization in an autoclave for 1 hr at 15 psi for three consecutive days. there were 20 disease-free and previously surface-sterilized seeds sown in every pot. seed germination and the occurrence of damping-off disease were assessed. the percentage of survival of the seedlings was determined after three months when the study was terminated. the experiment utilized a crd with five replications. significant values were further subjected to dmrt. results and discussion in vitro assay of the antagonistic activity of the 100 bacterial isolates using the agar-plug technique revealed that only 18 inhibited two or more of the damping-off fungi of agoho (table 1). fusarium oxysporum was inhibited by 17 bacterial isolates, r. solani by 8 isolates, p. parasitica by 14 isolates, and p. debaryanum by 15 isolates. the unidentified damping-off fungi unk 1 and unk 2 were inhibited by 13 and 9 isolates, respectively. of those that showed inhibitory effects, 15 belonged to the genus bacillus while three were actinomycetes. further screening using the agar-diffusion method disclosed that 10 were promising antagonists with b. subiilis (code no. r060), bacillus sp. (code no. r071), and streptomyces sp. (code no. r086) as the consistent and most effective inhibitors (table 2). some of the strains listed for inhibitory effects gave values below that of the reference antibiotic mycostatin (200 ug/ml) while others did not produce any inhibition zone, thus there was no antimicrobial activity observed. effect of the three promising antagonistic bacteria on percent germination of agoho seeds inoculated with the damping-off fungi in the laboratory is shown in table 3. the control which was not inoculated with any of the six pathogens gave the highest seed germination of 81.67%. it was however, surpassed though not significantly different by seed treatment with bacillus sp. (code no. r071) against p. parasitica resulting to 83.33%. inoculation with any of the damping-off fungi and with no seed treatment with any antagonist gave the lowest percent germination. bacillussubtilis (code no. r060) however, was not able to antagonize 5 biotropia no. 7, 1994 the effect of p. debaryanum in this particular experiment. application or treatment with b. subtilis may not have been of optimum concentration to affect an increase in percent germination of seeds inoculated with p. debaryanum. this could also be due to the fact that this pathogenic fungus is considered very virulent (dickson 1956). under nursery conditions, the antagonists suppressed the growth of the damping-off fungi (table 4 and fig. 1). seed treatments gave percent germination values of 74, 69 and 78 for b. subtilis, bacillus sp., and streptomyces sp., respectively. seed coating with streptomyces sp. gave a comparable value with untreated seeds sown in sterilized soil (83 %). table 1. inhibitory effects of the bacterial isolates on growth of the damping-off fungi of agoho based on agar-plug technique a code b classification c inhibitory effect d __________ number f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 r013 b + + + + rom b + + + r059 b + + + + r060 b + + + + + + r063 b + + + + + r070 b + + + + r071 b + + + + + + r072 b + + + + r073 b + + + r074 b + + + + r075 b + + + r076 b + + + + r078 b + + r079 b + + r080 b + + + + r086 a + + + + + + r095 a + + + + + + r099 a + + + + total number of active isolates = 18 17 8 14 15 13 9 a results based on three replications b treatments with no antimicrobial activity against any of the six damping-off fungi are not listed c either belonging to the genus bacillus (b) or an actinomycete (a) d positive sign ( + ) means with inhibitory effect: negative sign (-) means no inhibitory effect 6 biological control of damping-off fungi of agoho f.a. dela pena et al. table 2. in vitro assay of the antagonistic activity of the different bacterial isolates against the damping-off fungi of agoho using agar-diffusion method inhibition zone b (mean diameter in mm) f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 control 0 f 0 d 0 d 0 e 0 d 0 e r013 0 f 0 d 0 d 0 e 11.97c 0 e r060 21.65c 29.63a 27.05a 9.31b 19.21a 17.85c r071 29.22b 19.09b 10.06a 11.16a 14.30b 34.67a r072 0 f 0 d 9.89c 0 e 0 d 0 e r074 0 f 10.71c 0 d 0 e 0 d 11.37d r07s 9.73e 11.80c 0 d 0 e 11.20c 10.21d r078 9.62e 0 d 0 d 0 e 0 d 0 a r079 0 f 12.58c 0 d 0 e 0 d 0 e r086 31.42a 27.69a 18.43b 0 e 14.56b 25.26b r095 9.75e 11.96c 9.24c 9.00c 0 d 11.04d m200 d 14.05d 12.64c 9.43c 8.67d 11.84c 11.96d a treatments with no antimicrobial activity against any of the six damping-off fungi are not listed b results based on three replications with three subreplications c means with the same letter within the column are not significantly different at 1 % level using dmrt d reference antibiotic mycostatin (200 ug/ml) table 3. effects of the promising antagonistic bacteria on percent germination of agoho inoculated with damping-off fungi under laboratory conditions treatments percent germination (means) f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 control 91.67a 87.67a 81.67a 81.67a 81.67a 81.67a pathogen alone 18.33d 1.67b 38.33c 0 d 0 e 26.67d pathogen + r060 65.00b 68.33a 66.67b 0 d 71.67b 58.33b pathogen + r071 50.00c 66.67a 83.33a 63.33b 60.00c 41.67c pathogen + r086 75.00ab 76.67a 61.67b 30.33c 51.67d 71.67ab means with the same letter within the column are not significantly different at 1 % level using dmrt furthermore, table 4 and fig. 1 show that percent survival of seedlings was increased by seed treatment with values of 94, 85, and 95 for b. subtilis, bacillus sp. and streptomyces sp., respectively. these were significantly different with the control (62%). seed coating with b. subtilis and streptomyces sp. gave comparable values with untreated seeds sown in sterilized soil (99%). 7 treatments a biotropia no. 7, 1994 figure 1. effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions. bars with the same fill marked with different letter(s) are not significantly different at the 5% level using dmrt 8 biological control of damping-off fungi of agoho f.a. dda pena et al. table 4. effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions treatments germination survival a 57d 62c b 83a 99a c 74bc 94a d 69c 85b e 78ab 95a means followed by a common letter are not significantly different at 5% level by dmrt. legend: a untreated seeds sown in naturally infested soil b untreated seeds sown in sterilized soil c b. subtilis treated seeds sown in naturally infested soil d bacillus sp. treated seeds sown in naturally infested soil e streptomyces sp. treated seeds sown in naturally infested soil results of the assay methods revealed that a considerable number of bacillus sp. and actinomycetes could inhibit the growth of damping-off fungi in culture. the mycelium and spores of the pathogenic fungi may have been attacked and parasitized by the antagonists when they were simultaneously grown in culture media utilizing the agar-plug technique. there must have been a competitive interaction between the two microorganisms as supported by studies of henis and chet (1975). any essential requirement of microorganisms can serve as potentially possible basis for competition. competition for nutrients between the antagonist and germinating spores of the pathogenic fungus could have taken place. olsen and baker (1968) added that b. subtilis which developed along the mycelial wall of r. solani apparently obtained nutrients either from wall components or contents leaking from senescent fungal cells. in the agar-diffusion method, antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds may have likely occurred. the supernatant used in the assay demonstrated the possible role of antibiotics in biological control. moreover, most strains of b. subtilis or any other species of the genus bacillus and actinomycetes produce a wide variety of antibiotics which inhibit filamentous fungi. bacillus subtilis in particular, produces a subtilinlike antibiotic (henis and chet 1975) in addition to bulbiformin, mycosubtilin and bacillomycin (henis and inbar 1968) which have antifungal activity. some of the antibiotics produced by such strains may be extracellular, diffusable in solid agar, and could inhibit the growth of fungal pathogens. 9 1.pdf 10.pdf 11.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no biotropia no. 12,1999 .31-41 vegetative compatibility groups of fusarium oxysporum, the causal organism of vascular wilt on roselle in malaysia k.h. ooi and b. salleh school of biological sciences, universiti sains malaysia, 11800 penang, malaysia abstract forty strains of fusarium oxysporvm isolated from roselle (hibiscus sabdariffa var. sabdariffa) showing vascular wilt symptoms in three states (terengganu, penang and ipoh) in the northern malaysian peninsula were used to investigate the vegetative compatibility. nitrate-nonutilizing (nil) mutants were recovered from all the strains tested and subsequently used to study vegetative compatibility groups (vcg) within the population by nit mutants pairings on minimal medium. thirteen vcgs were found and none were vegetatively compatible with those of other formae speciales (f. spp.) such as asparagi and cubense, and non-pathogenic strains from paddy and oil palm. the results indicate that there is substantial genetic diversity in f. oxysporum that causes vascular wilt disease on roselle as reflected by multiple vcgs, but the distribution of strains into the vcgs is not even as there are 26 representatives in vcg-1001m, two in vcg-1003m and vcg-1013m and only one in the other vcgs. this study may provide new insight into the establishment of a new forma specialis off. oxysporum. key words: vegetative compatibility groups/m/ mutants/fitsarium oxysporwrn/roselle/vascular wilt/ malaysia. introduction fusarium oxysporum schlechtend.: fr. is an asexual, soil-borne fungus found in agricultural soils throughout the world. this species includes many pathogenic strains, known as formae speciales (f. spp.). each forma specialis (f. sp.) is characterized by its ability to cause vascular wilt on a limited taxonomic range of host plants (booth 1971). individual strains of the fungus show a high degree of host specificity which led snyder and hansen (1940) to classify strains of the fungus into f. spp. on the basis of the host plants attacked. for example, f. oxysporum f. sp. melonis attacks muskmelon (cucumis melo l.), whereas f. oxysporum f. sp. vasinfectum is a pathogen of cotton (gossypium spp.). strains of the fungus, whether from the same or different f. spp. are usually morphologically indistinguishable (puhalla 1985). currently, pathogenicity tests are the primary means to distinguish different pathogenic fusarium strains. however, such tests do not indicate whether various strains of a given f. sp. or a physiologic race are genetically related. puhalla (1985) used nitrate-nonutilizing (nit) mutants to show that strains in different f. spp. of f. oxysporum were in distinct vegetative compatibility groups (vcg), based on the ability of complementary nit mutants to anastomose and form wild-type 31 biotropia no. 12,1999 heterokaryons. these nit mutants could be recovered without mutagen treatment from selective media containing potassium chlorate (kc1o3) (puhalla 1985). heterokaryon formation between complementary mutants was indicated by the development of dense aerial growth where mycelia of two thin nit mutant colonies touched and anastomosed when cultured side-by-side on minimal medium with nitrate as a sole nitrogen source (klittich & leslie 1988; puhalla & spieth 1985). mycelia outside the anastomosed region remained thin. diversity within pathogenic strains of f. oxysporum has been evaluated by characterizing strains in terms of vegetative compatibility. strains capable of forming a successful vegetative heterokaryon are referred to as vegetatively compatible. strains that are vegetatively compatible with one another are frequently described as members of the same vegetative compatibility group, or vcg (leslie 1993). strains from different groups would not form heterokaryons with each other. in asexually reproducing fungi such as fusarium, vegetatively compatible strains are much more likely to be genetically similar than vegetatively incompatible strains (correll et al. 1987). puhalla (1985) used nit mutants to test for vegetative compatibility among 21 strains of f. oxysporum. he found a correlation between vcg and f. sp. i.e. members of the same vcg belong to the same f. sp. vegetative compatibility tests, therefore, have been shown to be a powerful tool for studying genetic diversity in f. oxysporum (correll et al. 1987). most of the studies revealed a limited number of distinct vcgs within a f. sp. (correll et al. 1986a; jacobson & gordon 1988; and larkin et al. 1988). in contrast, 97 strains of f. o. f. sp. asparagi were found to include a minimum of 42 vcgs (elmer & stephens 1989), and 110 strains of f. oxysporum isolated from celery roots but nonpathogenic on celery included a minimum of 14 vcgs (correll et al. 1986b). vascular wilt caused by f. oxysporum is the most important soil-borne disease on roselle in malaysia (ooi et al. 1998). roselle (hibiscus sabdariffa var. sabdariffa) has recently been domesticated and is being planted in a large scale for growing juice, jam and confectionary industries in malaysia (mat isa et al. 1985; tan & said 1994). the seeds contain 17% oil similar in properties to cotton seed oil. roselle is suitable to be planted on mineral or bris soils (chin 1986). however, the cultivation of this newly domesticated crop in malaysia has been disrupted due to various diseases mainly vascular wilt caused by a soil borne fungus, f. oxysporum. the pathogenicity of f. oxysporum isolated from the roots and stems of roselle showing vascular wilt symptoms in the field was proven in the greenhouse (ooi et al. 1998). in the present study, forty strains of f. oxysporum isolated from roselle showing vascular wilt symptoms in the northern malaysian peninsula were used to investigate the vegetative compatibility among the fungal strains. 32 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh materials and methods strains forty strains of f. oxysporum isolated from roselle in three states of the malaysian peninsula were examined (table 1). the strains were each grown on peptone pentachloronitrobenzene (peptone-pcnb) agar (nash & snyder 1962) for 57 days. conidia from these strains were transferred to 3% water agar (wa), and individual uninucleate microconidia were isolated using a micromanipulator and transferred to potato sucrose agar (psa). colonies from these single-spore cultures were produced on psa under standard incubation conditions and the resultant colonies were stored in liquid nitrogen (salleh & sulaiman 1984; salleh & strange 1988). these parent cultures were used as the starting inoculum for all subsequent tests. table 1, complementation reactions between nitrate-nonutilizing (nit) mutants off. oxysporum1 nitl nit3 nitm nit i o r ± ± o r + nit3 ± o r + nitm + + + ' + = complementation occurs readily, = no complementation occurs, ± = weak and/or slow complementation occurs. media the basal medium (bm) was described by correll et al. (1987). minimal medium (mm) was made by adding 2.0 g of sodium nitrate (nano3) to 1 l of the basal medium. nit mutants were generated on minimal agar medium with chlorate (mmc) as described by correll et al. (1987). psa were made as described by booth (1971). peptone-pcnb medium was prepared by adding the following to 1 l of distilled water: difco peptone, 15.0 g; kh2pcv 1.0 g; mgso4, 0.5 g; pentachloronitrobenzene (pcnb 75 wp), 1.0 g; agar, 20.0 g; and streptomycin, 300 ppm (nash & snyder 1962). recovery of nitrate-nonutilizing mutants (nit mutants) nit mutants were generated on mmc at 1.5 % concentration of chlorate. mycelial transfers (approximately 2-mm3 psa blocks) of the fungus were placed on three equidistant places of three plates of mmc and incubated under standard incubation conditions (salleh & sulaiman 1984) and examined periodically (3, 5 and 33 biotropia no. 12,1999 7 days after incubation) for the appearance of fast-growing fan-shaped sectors from the initially restricted colony. all sectors were transferred to mm and those that grew as thin expansive colonies with no aerial mycelium were considered nit mutants. all nit mutants were resistant to chlorate and showed wild-type growth on psa. nit mutant phenotypes the physiological phenotypes of nit mutants recovered were distinguished by their growth on mm amended with different compounds as the sole nitrogen source (correll et al. 1987; klittich & leslie 1988). the plates were incubated as described above, and colony morphology was scored relative to the wild-type parent after four days. complementation tests vegetative compatibility was determined by observing heterokaryon formation between complementing nit mutant on mm. pairings were made by placing mycelia from each nit mutant 1-3 cm apart on mm. pairings were incubated as described above for 7-10 days and then scored for complementation. phenotypically distinct nit mutants, a nitl and a nitm, were obtained from 10 strains. these nit mutants were paired in all possible combinations to establish which strains were vegetatively compatible. nitm from one isolate in each of the vcgs thus identified was selected to serve as a tester for each group. two nitls were obtained from each of the remaining 30 strains and paired with a nitm from each of the established vcgs. from within the group of strains that did not pair with any of the testers, a second group of testers was obtained and the procedure repeated. this process was continued until all strains were assigned to a vcg. each pairing was repeated at least once (gordon & okamoto 1991). all of the nit mutants recovered from the same parent were paired with at least one nitl, one nit3, and one nitm mutant from that parent to test for self-compatibility. vegetative compatibility tests with testers of other f. spp. and non-pathogenic strains of f. oxysporum two nit mutants (one nitl and one nitm) of at least one isolate from each of the established vcgs were paired on mm with four nit testers of the following two f. spp.: asparagi (two testers) and cubense (two testers) ; and four nit testers of nonpathogenic strains of f. oxysporum from paddy (oryza saliva) (one tester) and oil palm (elaeis guineensis) (three testers). 34 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh results nit mutant isolation spontaneous chlorate resistant sectors were readily recovered from all 40 strains of f. oxysporum when cultured on mmc. the majority of the chlorate resistant sectors recovered were unable to utilize nitrate as a sole nitrogen source and consequently grew as thin expansive colonies with no aerial mycelium on mm. these sectors were designated nit mutants. the rate of radial expansion of these nit mutants is compatible to that of the wild types. successive subcultures of the nit mutants onto mm continue to show this thin growth habit. most of the nit mutants were stable, but a few (2-4%) of them developed small patches of heavy growth after prolonged incubation on mm. nil mutants phenotype identification recovered nit mutants could be divided into three distinct phenotypic classes by their growth and colony morphology on media containing one of five different nitrogen sources. the classes presumably reflect mutations at a nitrate reductase structural locus (nitl), a nitrate assimilation pathway-specific regulatory locus (nit3) and loci (at least five) that affect the assembly of a molybdenum-containing cofactor necessary for nitrate reductase activity (nitm). the majority of nit mutants recovered on mmc were nitl. several sectors recovered from some of the strains were resistant to chlorate but had wild-type colony morphology on mm. correll et al. (1987) have observed this type of chlorate resistant nitrate utilizing sectors in f. oxysporum. they examined in more detail such sectors to determine if these sectors resulted from a mutation in a single locus or from the heterokaryotic growth of complementary mutations in two or more different nuclei. the analysis of microconidia from these sectors indicated that the chlorate-resistant nitrate utilizing sectors could be heterokaryotic or homokaryotic. microconidia from homokaryotic sectors had a wild-type morphology on mm and presumably were mutants that were both chlorate-resistant and able to utilize nitrate (cm mutants). microconidia recovered from heterokaryotic sectors were often a mixture of nit mutants conidia, wild-type conidia, and/or crn mutant conidia. complementation tests complementation occurred between nit mutants with different phenotypes. complementation occurred more rapidly and growth of the resulting heterokaryon was more robust in pairings of nitm with nitl or nit3 mutants than in pairings of nitl with nit3 mutants (table 1). when nitl and nit3 mutants were paired, 35 biotropia no. 12, 1999 complementation reaction was weak (very little aerial mycelium). to confirm that weak reactions could not result from cross feeding without anastomosis, 10 pairs of weakly reacting strains were tested with sterilized cellophane separating the two nit mutants following a method devised by puhalla (1985). where cellophane prevented hyphal contact, there was no visible reaction. outside the cellophane barrier, where hyphae of the two nit mutants came into direct contact, a weak reaction was observed. this result was consistent with a requirement for hyphal anastomosis to produce weak reactions. in our delineation of compatibility groupings, an isolate was placed in a vcg only if it reacted strongly and developed dense aerial growth where the mycelia of the nit mutant colonies came in contact and anastomosed to form a heterokaryon, with at least one other isolate in that group. on this basis, 40 strains were assigned to a total of 13 vcgs (table 2). the largest of these included 26 strains, whereas 10 vcgs were represented by only a single strain while two vcgs were represented by 2 strains. in no case was a strain vegetatively compatible with strains from two different vcgs. table 2. strains of f. oxysporum classified by vegetative compatibility and their source strain1 source (naturally wilted roselle) l location vcg 1001m 72558% root mengabang bakung, 7erengganu t2560% root mengabang bakung, 7erengganu t2561% root kuala berang, 7erengganu t2563% root rhu 7apai, 7erengganu t2564% root rhu 7apai, 7erengganu t2566% root rhu 7apai, 7erengganu t2568% root rhu 7apai, terengganu t2577% root kuala berang, 7erengganu t2605% nonsterile seed kuala 7erengganu, terengganu 12607% rotten fruit mengabang bakung, 7erengganu 72608% rotten stem mengabang bakung, 7erengganu 72609% rotten leaf kampung pasir nering, 7erengganu 72610% rotten leaf kampung pasir nering, 7erengganu 72623% stem mengabang bakung, terengganu 72625% root kuala berang, 7erengganu 72633% rotten stem mengabang bakung, terengganu p2883% stem usm, penang p2884% stem usm, penang p2885% root usm, penang p2886% root usm, penang p2887% root usm, penang p2888% stem usm, penang p2889% rotten root usm, penang p2890% rotten root usm, penang p2893% stem usm, penang p2894% stem usm, penang 36 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh table 2. continued strain' source (naturally wilted roselle)2 location vcg 1002m t2567% root rhu 7apai, 7erengganu vcg 1003m 12573% root marang, 7erengganu 12618% stem kampung pasir nering, 7erengganu vcg 1004m t2575% root marang, 7erengganu vcg 1005m 72620% leaf kampung pasir nering, 7erengganu vcg 1006m t2621% leaf kampung pasir nering, 7erengganu vcg 1007m 72622% stem mengabang bakung, 7erengganu vcg 1008m 72628% root mengabang bakung, 7erengganu vcg 1009m 72629% root rhu 7apai, 7erengganu vcg 1010m 72637% healthy root rhu 7apai, 7erengganu vcg 1011m a2891% fruit ipoh, perak vcg 1012m a2882% fruit ipoh, perak vcg 1013m a2892% fruit ipoh, perak a2898% healthy root ipoh, perak 't= strains from terengganu a= strains from perak p= strains form penang '%= strains from roselle !parts of roselle where the strains were isolated heterokaryon self-incompatibility no complementation occurred between any nit mutants of eight strains tested, even after repeated attempts. furthermore, no complementation was observed when the nitl or nitm mutants were paired among themselves. the lack of complementation between phenotypically distinct nit mutants recovered from these strains lead us to designate these strains as heterokaryon self-incompatible (table 3). vegetative compatibility tests with testers of other f. spp. and non-pathogenic strains of f. oxysporum two nit mutants (one nitl and one nitm) from each of the established vcgs were paired with four nit testers from two f. spp.: asparagi and cubense', and four nit 37 biotropia no. 12, 1999 table 3. source of self-incompatible strains of f. oxysporum strain1 source2 location 12566% root rhu tapai, terengganu t2575% root marang, terengganu t2605% nonsterile seed kuala terengganu, terengganu t2608% rotten stem mengabang bakung, terengganu t2609% rotten leaf kampung pasir nering, terengganu t2637% healthy root rhu tapai, terengganu a2882% fruit ipoh, perak p2883% stem usm, penang t = strains from terengganu a= strains from perak p= strains from penang '%= strains from roselle 2parts of roselle where the strains were isolated testers from non-pathogenic strains. no heterokaryon formed (no complementation) between the testers from each vcg with any of the other f. spp. and the nonpathogenic strains. discussion fusaria are often considered to be genetically unstable because they frequently produce sectors in culture which differ from the original colony in morphology, virulence, or other characteristics (bumett 1984; puhalla 1981). a moderate degree of genetic instability could have a selective advantage for a plant-pathogenic fungus such as fusarium, allowing rapid adaptation to environmental stress such as fungicides or to the introduction of resistant genes into the hosts (sapumohotti & salleh 1992). a source of genetic variability is essential particularly for organisms such as f. oxysporum, that depend entirely on asexual reproduction. genetic instability could generate variants that are fungicide resistant, able to overcome host resistance, or tolerant to toxic wastes in the soil. a high mutation rate could be an important means of generating variability if sexual or parasexual recombination is rare (klittich & leslie 1988). heterokaryosis and other types of mycelial interactions have been recognized in the genus fusarium for at least 100 years (page 1961; stover 1959). a heterokaryon is a multinucleate cell containing genetically distinct nuclei in a common cytoplasm. such cells are often created by hyphal fusions between vegetatively compatible strains. heterokaryosis provides an opportunity for genetic recombination via the parasexual cycle in sterile, homothallic, or imperfect fungi such as fusarium (adams etal. 1987). in this experiment, spontaneous nit mutants were readily recovered without mutagenic treatment. because these mutations probably affect at least seven loci with three distinct phenotypes, the nit mutants can be used as forcing markers in the 38 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh formation of heterokaryon to test fungal strains for vegetative compatibility (correll et al. 1986a, 1987; gordon et al. 1986; sidhu 1986; jacobson & gordon 1988; elmer 1991). studies of fungal populations using vcgs as a means to measure diversity have become widespread in recent years. vcgs serve as a natural means to subdivide fungal populations. the vie loci and alleles that define vcgs are presumed to be selectively neutral with respect "to traits such as pathogenicity and vegetative viability. different strains are vegetatively compatible (i.e., capable of forming a heterokaryon) only if alleles at all vegetative compatibility loci are identical. vegetative compatibility groups (vcgs) are ideal markers for population studies because they occur naturally and are easy to score using spontaneous nit mutants. strains which belong to the same vcg can form heterokaryons in which cytoplasms may mix, mitotic recombination may occur, and deleterious cytoplasmic agents, such as mycoviruses, can be exchanged. vcgs, which are analogous to anastomosis groups, have been correlated with pathogenicity in many fungal species including fusarium spp. (bosland & williams 1987; correll et al. 1986a; correll et al. 1987; jacobson & gordon 1988; ploetz & correll 1988; puhalla 1985). results obtained from this study suggest that 26 (65%) of the 40 strains of f. oxysporum tested belonged to a dominant vcg designated as 1001m, but only 2 strains (5%) were grouped in vcg 1003m and 1013m, respectively. vcg 1002m and 1004m-1012m each consist of only one strain (0.025%). this result suggests that anastomosis occurs infrequently among strains in this population and implies that the wilt pathogen of roselle in malaysia is genetically diversified. but the distribution of different genotypes into vcgs among the strains is not even. there are some closely related strains and grouped into one vcg, while some others which showed no relatedness (vegetatively incompatible) with other strains were grouped into several vcgs separately as isolated groups. at least one strain from each of the 13 vcgs was tested and the results showed that they were vegetatively incompatible with the wilt pathogens, f. oxysporum f. sp. cubense and f. sp. asparagi; and also incompatible with the non-pathogenic strains from paddy and oil palm. thus, the wilt pathogen of roselle in malaysia likely appears to constitute a distinct genetic population within the f. oxysporum complex. generally, mutants deficient in the molybdenum-containing cofactor (nitm) are infrequently isolated but readily form complementing heterokaryons, making them the most suitable choices for vegetative compatibility tests. on the other hand, nit i and nit3 mutants may form weak, slow-growing heterokaryons, giving ambiguous results. we also found that the strength of a heterokaryon varied depending on the combination of the nit mutants used. the ideal combinations were my7-nitm anc nitm-nitm which form more stable heterokaryons. our study demonstrates the importance of characterizing heterokaryosis in f oxysporum. once the heteroploids are better characterized, they should be useful ir studying the effects of genetic interactions on the expression of economical!} 39 biotropia no. 12, 1999 important traits such as virulence and the production of secondary metabolites such as mycotoxins. this study may also provide new insight into the establishment of a new forma specialis of f. oxysporum, causing wilt of roselle. acknowledgments this research is supported by the government of malaysia (1rpa grant 01-0205-6003). we thank our research assistants mr. kamarudin mohd. maidin and miss wan faridah maydin for their capable assistance, the department of agriculture (state of terengganu) and the owners of the roselle sampling plots throughout the northern malaysian peninsula. references adams, o., n. johnson, j.f. leslie and l.p. hart. 1987. heterokaryons of gibberella zeae formed following hyphal anastomosis or protoplast fusion. experimental mycology 11: 339-353. booth, c. 1971. the genus fusarium. commonwealth mycological institute, london, p. 21-22. bosuand, p.w. and p.h. williams. 1987. an evaluation of fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility and geographical origin. canadian journal of botany 65: 2067-2073. burnett, j.h. 1984. aspects of fusarium genetics. in: the applied mycology of fusarium, edited by m.o. moss and j.e. smith, cambridge university press, cambridge, p. 39-69. chin, h.f. 1986. the hibiscus, queen of tropical flowers. tropical press, kuala lumpur. 151p. correll, j.c., c.j.r. klittich and j.f. leslie. 1987. nitrate non-utilizing mutants of fusarium oxysporum and their use in vegetative compatibility tests. phytopathology 77: 1640-1646. correll, j.c., j.e. puhalla and r.w. schneider. 1986a. identification of fusarium oxysporum f. sp. apii on the basis of colony size, virulence and vegetative compatibility. phytopathology 76: 396 400. correll, j.c., j.e. puhalla and r.w. schneider. 1986b. vegetative compatibility groups among nonpathogenic root-colonizing strains of fusarium oxysporum. canadian journal of botany 64: 2358-2361. elmer, w.h. 1991. vegetative compatibility groups of fusarium proliferatum from asparagus and comparisons of virulence, growth rates and colonization of asparagus residues among groups. phytopathology 81: 852-857. elmer, w.h. and c.t. stephens. 1989. classification of fusarium oxysporum f. sp. asparagi into vegetatively compatible groups. phytopathology 79: 88-93. gordon, t.r., j.c. correll and a.h. mccain. 1986. host specificity and vegetative compatibility in verticillium albo-atrum. phytopathology 76: 1111 (abstract). gordon, t.r. and d. okamoto. 1991. vegetative compatibility groupings in a local population of fusarium oxysporum. canadian journal of botany 69: 168-172. jacobson, d.j. and t.r. gordon. 1988. vegetative compatibility and self-incompatibility within fusarium oxysporum f. sp. melonis. phytopathology 78: 668-672. klittich, c.j.r. and j.f. leslie. 1988. nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi). genetics 118: 417-423. 40 vegetative compatibility groups of fusarium oxysporum k..h. ooi and b. salleh larkin, r.p., d.l. hopkins and f.n. martin. 1988. differentiation of strains and pathogenic races of fusarium oxysporum f. sp. niveum based on vegetative compatibility. phytopathology 78: 1542 (abstract). leslie, j.f. 1993. fungal vegetative compatibility. annual review of phytopathology 31. 127-151. mat isa, a., p.m. md. isa and a.r. aziz. 1985. chemical analysis and process of roselle (hibiscus sabdariffa l,). mardi research bulletin 13: 68-74. nash, s.m. and w.c. snyder. 1962. quantitative estimations by plate counts of propagules of the bean root rot fusarium in field soil. phytopathology 52: 567-572. ooi, k.h., b. salleh and m.h. hafiza. m.h. 1998. vascular wilt caused by fusarium oxysporum on roselle in malaysia. journal of plant protection in the tropics (submitted). page, o.t. 1961. variation in the banana-wilt pathogen fusarium oxysporum f. sp. cubense canadian journal of botany 39: 545-557. ploetz, r.c. and j.c. correll. 1988. vegetative compatibility among races of fusarium oxysporum f. sp. cubense. plant disease 72: 325-328. puhalla, j.e. 1981. genetic considerations of the genus fusarium. in: fusarium: diseases, biology and taxonomy, edited by p.e. nelson, t.a. toussoun and r.j. cook, pennsylvania state university press, university park. p. 291-305. puhalla, j.e. 1985. classification of strains of fusarium oxysporum on the basis of vegetative compatibility. canadian journal of botany 63: 179-183. puhalla, j.e. and p.t. spieth. 1985. a comparison of heterokaryosis and vegetative compatibility among varieties of gibberella fujikuroi (fusarium moniliforme). experimental mycology 9: 39 47. salleh, b. and r.n. strange. 1988. toxigenicity of some fusaria associated with plant and human diseases in malaysian peninsula. journal of general microbiology 134: 841-847. salleh, b and b. sulaiman. 1984. fusaria associated with naturally diseased plants in penang. journal of plant protection in the tropics 1: 47-53. sapumohotti, w.p. and b. salleh. 1992. nitrate reduction mutants of fusarium nygamai, the causal organism of reddish brown rot on asparagus. journal of plant protection in the tropics 9: 187-194. sidhu, g.s. 1986. genetics of gibberella fujikuroi. viii. vegetative compatibility groups. canadian journal of botany 64: 117-121. snyder, w.c. and h.n. hansen. 1940. the species concept in fusarium. american journal of botany 27: 64-67. stover, r.h. 1959. studies on fusarium wilt of banana. iv. clonal differentiation among wild type isolates of fusarium oxysporum f. sp. cubense. canadian journal of botany 37: 245-255. tan, h.h. and n.m.s. said. 1994. cultivation of roselle (hibiscus sabdariffa l.). department of agriculture, terengganu, malaysia. 18 p. 41 biotropla vol. 28 no. 3,2021: 221 230 doi: 10.1 1598/btb.2021.28.3.1339 diversity of endophytic fungi associated with fruits a n d leaves of tamarind (tamarindus indica l.) based o n its ribosomal d n a sequences nurul asyiqin mohd zaini', nurul huwaidah md nizam, dayang fatin zafira awg zainal abidin, nor izanis azni mohd nazri and nur ain izzati mohd zainudin* department $biology, factllo $science, universiti putra mala_ysia, 43400 serdang, selangor, malysia received 18 february 2020 /accepted 2 may 2020 abstract plant-associated microbes are among essential natural resources that abundantly exist in a natural environment, such as endophyuc fungi. studies o n endophytic fungi in medicinal plants have allowed the discovery of numerous fungi species and their hidden potentials. therefore, this study focused o n the isolation and identification of endophyuc fungi from several plant parts of tamarind (t. indica), such as leaves and fruits. a total of 69 fungal cultures were successfully isolated and identified into 31 distinct species from 15 genera based on morphological characteristics and internal transcribed spacer (its) sequence analysis using a maximum likelihood method. a high diversity of endophytic fungi associated with t . indica were observed by shannon wiener index h' (3.083). there were six different species obtained from the genus colletotrichzlm (c. aenigma, c. brevisporum, c. cobbittiense, c. fmcticola, c. gloeosporiaides and c. siamense), and diaporthe (d. arecae, d. ceratoxamiae, d. phaseolorzrm, d. pseudomangzjirae, d. pseudooctlii and d. pseudophoenicicala), four species of aspergilzlzls (a. aczlleatzrs, a. carbonarizls, a.flautls and a. tzlbingensis), two species of czlrvzrlank/cochliobolzrs (c. geniculatzrs and c. lunata) and lv&rospora (n. lacticolonia and n. oryxae), two species of lasiod$lodia (l. psetldotheobromae and l theobmmae) and penicillm (p. m@ii and p. verruczllos~m). other fungal species that were also identified are botyosphaeria mamane, fusaritlm solani, tmncospora tephropora, ph_yllostictafallopiae, sarcostroma bisettllatnm, tiichodema asperelhm and xylarid j2ejeensi.r. keywords: endophyuc fungi, internal transcribed spacer (its), phylogenetic tree, tamarind introduction endophytic fungi are microorganisms inhabiting plant tissues in a part of their life without showing any harm toward the host plants. the species of endophytic fungi are expected in over a million species, which arisen from the natural surroundings w s h r a e t al. 2018). they are widely dstributed, which have been found in many plant species that can grow in natural environments such as terrestrial plant communities (nisa e t al. 2015). endophytic fungi such as aspel.gllus, colletotm'cbum, fusam'um, penicillm and tm'cbodema may colonize several parts of plants, includmg fruits and leaves *corresponding author, email: ainizzati@upm.edu.my (hanada e t al. 2010). there are many research studes reported the abundance of fungi associated with plants, however, there is a lack of study in the endophytic fungi associated with t. indica. bourou e t al (2010) reported, three genera of arbuscular mycorrhzal fungi (acaulospora, glomzls and scutellospora) were associated with t. indim. tamarind tree has been reported to be infected by some wood decay fungi such as daldinid concentm'ca, scbixopbylh commune, flavodon jlavus, @ex bydnoides, and pbellinus fastuosus (nnagadesi & arya 2015). in previous reports, aspep!lius n&eq rhixopus stolonifeq ulocladium cban'drum, penicillium cb ysogenum, p. citm'num and pbomopsis liquidambaris were associated with infected-tamarind fruit biotropia vol. 28 no. 3,2021 (danggomen e t al. 2013; peter & patrick 2017). isolation, purification and preservation of penin'ilium cblysgenum and p. n'tm'nam are microfungi confirmed pathogens and caused spoilage in all plant samples were washed in running tap fruits (peter & patrick 2017). recently, water for 30 rnin to remove any debris or soil a.pergiiius nniger has been proven as a pathogen before being processed. the leaves were cut into that causes black pod of tamarind (meena e t al. segments of 5 x 5 mm. then, the surface of the 2018). leaves and fruits was surface sterilized by due to the regarbg e n d o ~ h ~ c following the described by ravindran fungal diversity associated with l indica is et al (2012) by immersing in 700,0 ethanol lacking, this study will provide important (5 sec), 4% sodium hypochlorite (naoci) information regarding the diversity of fungal (90 set., with sterile distilled water e n d o p h ~ e s associated with l ifldica. this study (30 sec) and blotted dry with sterile filter paper. was aimed to determine the ~ulturable all of the segments were placed (3 segments endophytic fungal diversity associated with each plate) on potato dextrose agar (pda) l indicd using molecular phylogenetic analysis of supplemented with streptomycin (0.05 g/rnl) its rdna sequences. and neomycin (0.01 g/l) using sterilized forceps. the culture plate was incubated at room temperature (27 f 2oc) for 5 to 7 days or materials and methods until there was an appearance of mycelium or colony from the sample fragments. plant samples the fungal mycelia grown from the parts of the sample were streaked on 4% water agar collection of leaves and fruits samples of f a ) for purification. the wa plate was t. was com~leted in 2018 and 2019 at incubated for another 24 hours. then, the single jalan asam jaws, universiti tip of hyphae was cut and transferred onto a serdang selangor located at 3"00709.0"n n, pda plate and incubated at 2 7 & 2 0 ~ for 101"42'34.gne (fig. 1). the fruits and leaves seven days. the pure isolated fungi were samples were collected using fruit picker from preliminarily identified by examining their 20 l indicd trees with 2 m apart. all samples morphological characteristics. all isolates were were further placed in paper bags, properly maintained and preserved at -20 "c using a labeled, and brought to the mycology modified filter paper method for working and laboratory, department of biology for fungal stock cultures with slight modifications (fang e t isolation. al. 2000). figure 1 samples of fruits (a) and leaves (b) of t. indica were collected in persiaran asam jawa, universiti putra malaysia endophytic fungi from tamarinds mohd zaini eta/ dna extraction, pcr amplification and sequencing all isolates were cultured on pda and incubated for 5 days. dna of the isolates was extracted using ultracleanb microbial dna isolation igt (mo bio, carlsbad, ca, usa) according to manufacturer's instruction. amplification of the its regions was conducted using polymerase chain reaction (pcr) machine (hercuvan lab systems, california, usa) involved primers its1 (5' tccgtaggtgaacctgcgg-3') and its4 (5'-tcctccgcttattgatatgc-3') (xkte e t al. 1990). the pcr master mix was prepared from 4 pl of 5xpcr buffer, 2 pl of 2 mm dntp, 2 pl of 25 mm mgclz, 1 pl of 10 mm for each primer, 0.1 pl of taq dna polymerase with concentration 5 u pl, 6.9 pl of nuclease free water and 3 pl of dna in a total volume of 20 pl. the pcr protocol with initial denaturation step was done for 30 sec at 95 oc, followed by 35 cycles of denaturation (95 oc for 10 sec), annealing (59 oc for 15 sec) and extension (72 oc for 30 sec), and was completed by final extension step at 72 oc for 5 min. then, the pcr product was prepared for gel electrophoresis or stored at -20 oc. the pcr products were gel-electrophoresed using 1.5% agarose gel. the mixture of 2.5 yl of 6x loading dye (blue/orange) and 2.5 yl of 100 bp dna marker were used as a ladder. the dna and ladder were pipetted with 5 yl in volume into the holes using a micropipette and electrophoresed. the amplicon size was visualized under a uv trans-illuminator. the pcr products were purified using a qiaquick gel extraction kit (qiagen, usa), following the manufacturer's instructions. the purified pcr products were sequenced by using an applied biosystem 3730x1 dna analyzer (mytacg bioscience company, my). phylogenetic analysis evolutionary analyses of its sequences were conducted in molecular evolutionary genetics analysis @ega) 6.0 software to obtain alignment sequences (tamura e t al. 2013). homologous sequences were obtained from the genbank database ncbi (http://blast. ncbi.nlm.nih.gov/) using blastn search (https://blast.ncbi.nlm.nih.gov/blas t.cgi? page-type=blastsearch) of the its sequences. the phylogenetic analysis was conducted using the maximum likelihood method based on the tamura-nei model with 1000 bootstrap test (tamura & nei 1993) in mega version 6.0. saccharomyces cerevisde cbs 1171 (ab018043) was used as an outgroup (fig. 3). the genbank accession number of new sequences were listed in table 1. species diversity the species diversity was calculated by using the shannon-weiner index (spellerberg 2008) as formula below: where: h' = value of shannon wiener's diversity index pi = proportion of species s = number of species in community 1 = number of indviduals in species results and discussion a total of 69 isolates of fungi were obtained from 20 fruit and leaf samples of t. indica, and were identified based on their morphological characteristics (fig. 2) and its sequence analysis (table 1 and fig. 2). thirty-two species belong to 15 genera were found in the present study including aspergillas (4 species), botyospbaerid (a single species), colletotm'chmn (6 species), cochliobolzls/ czawalarid (2 species), diaporthe (6 species), fzuam'am (a single species), lasiod$lodia (2 species), nigrospoa (2 species), penin'iiium (2 species), tmncospora (a single species), pbyllostictd (a single species), sarcostroma (a single species), tm'cboderma (a single species), and xylarid (a single species) (table 1). biotropia vol. 28 no. 3,2021 e l i 1;igut-e 2 1:ungal morphological retrieved in the culture media isolation procedure. f,ndoph\-tic fungi were isolated from fruits and leaves o f t ilzdi~u. i u n g i a-ere cultivated i n pda m e d i u m a t 27 o c foi7 da1.s based o n phylogenetic analysis of its basidiomycota). clade a was divided into 2 sub sequences of the 69 endophytic fungi isolated clades; clade a1 represents isolates of from tamarind fruits and leaves, two major a.pe?gi/h.r, bot!yoshaerid, co//etot~icbzm, i>iapof-t/3e, clades ( a and b) were generated (fig. 3). the fi~samum, nigrospa, ,.i'arcostroma, tncboderma, first clade (clade a) comprises isolates of fungi p~e/?ici//iunz, ph~~lhsticta, and x y l a t a , whereas clade under phylum ascomycota and clade b a2 represents isolates of lasiod$lodia and contains tru~zcospora tephropora b3 148 (phylum cu~-~j~t/aria/ cocbliobolus. endophyuc fungi from tamarinds m o h d zaini e t al. table 1 its sequences genbank accession number o f deposited fungal isolates from fruits and leaves of t. indicd no. isolates s~ecies plant oart genbank accession number aspeqi/lus aculeatus a . carbonarizls a . javus a. tubingensis a. tubingensis boflyosphaeria mamane colletotrichnm aenigma c. brevispom c. cobbittiense c.ficticola c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gheosporioides c. siamense c. siamense c. siamense c. siamense c. sidmense c. siamense c. siamense c. siamense cumlaria lunata c. lnnata cochliobolus geniculatns c. lunata diqorfhe arecae d. ceratozamiae d. phaseolorzlm d. phaseolom d. phaseolom d. phaseolomm d. phaseolomm d. phaseolorcrm d. psendomang$rae d. pseudooculi d. psendoocnli d. psendophoenicicola d. psendophoenicicala fusarium solani k o d i p l o d i a psendo fheobmae l theobromae l theobromae nigrospora lacticolonia n. lacticolonia n . lactiolonia n . olyzae n. olyzae n . oryxae n. ovyxae penicillinm roifsii p. ro&ii p. rofsii p. rofsii p. ro&si; p. uemculosum ph_yllostictaf.ll@iae sarcostmma bisetnlatum trichodemza asperellurn t . asperellurn t . asperellurn fruit fmit fmit fruit leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf fruit fruit fruit fruit leaf leaf leaf leaf leaf leaf leaf leaf fruit leaf fruit leaf leaf leaf leaf leaf fruit leaf leaf leaf leaf fruit leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf fmit fruit fruit leaf leaf fruit leaf leaf fruit fmit fmit leaf leaf biotropia vol. 28 no. 3,2021 figure 3 phylogenetic tree generated from the maximum likelihood method based on the its sequences of 69 fungal endophytes sequences associated with t. indim. the tree generated using tamura-nei model with 1000 bootstrap replications. all bootstrap scores with less than 50% are not shown in the tree mt043776 c gloeosporrodes 83154 mt043780 c glaeosponades 83158 mt043796 c glaeosponades 83186 mt043799 c gloeosporiodes 83169 the shannon index (h' = 3.083) indicated most diverse fungal genera isolated from that the tamarind fungal community possesses a tamarind leaves was colletot?ichzlm and diaporthe vast diversity of endophyuc fungi (table 2). the (fig. 3, table 1). 58 mt043784 c glaeosponardedes 83162 mt043786 c cobbiffiense 83170 mt043776 c glaeosponardedes 83154 mt043781 c glaeosporiardes 83154 mt043792 c glaeosporiaides 83182 mk204297 c. siamense 82885 mk204295 c siamense 82892 mt043774 c g1aeospo"oides 83152 mt043801 c glaeosporioides 83191 mk204289 c froct,cola 82961 mk204314 c. aengma 82881 93 mk204292 c. siamense 82907 mk204294 c. siamense 82921 ~ l i a x 69 mt043769 c. brev,sparum 83145 mk204285 f salani82964 mk204288 t asperellum 82963 mt043803 n aryrae 83193 mt043779 n lact,calonra 83157 mt043782 n lact,calonra 83160 mk204313 n a w e 82889 mt043788 s biswfulatum 83172 mt043785 x feejeensis 83163 5 3 mt043765 d phaseolomm 83141 a l i a 1 76 -1g mt043763 d phaseolomm 83161 mt043777 d phaseolomm 83155 mt043770 d phaseolomm 83147 mt043800 d. phaseolomm 83190 mk204303 d. phaseolomm 82940 mk204305 d pseudomangtferae 82928 mt043798 d pseudooculr 83168 mt043772 d ceatozamiae 83150 mt043773 d pseodophoenicicola 83151 mt043790 d pseudoculr 83180 mt043793 d pseodophoenicicola 83183 71 mk204301 d. arecae 82952 mk204300 p venuculasum 82958 89 a l i b 99 mk204304 a aculeatus 82931 52 mk204306 p mlfw, 82925 mk204308 p mlfsir 82919 mk204310 p mlfw, 82899 mk204309 p mlfw, 82894 mt043804 p falloprae 83194 a l i i 9 3 mt043767 b mamane 83143 mt043789 l theobmmae 83179 mt043794 l pseudotheobmmae 83184 mk204311 a tubrngensrs 82916 mk204302 a carbonanus 82948 a 2 mk204299 a lvus 82959 b mk204312 c lunsta 82902 mt043766 c lunata 83144 mt043771 t tephmpora 83148 nr 111007 sacchammyces cerevrsrae endophyuc fungi from tamarinds mohd zaini e t al. table 2 endophyuc fungal percentage and shannon-wiener index obtained from culture media isolation using fruits and leaves of t. indicd no. species number of isolate c. breuisporm c. cobbittiease c. fmcticola c. &eosporioides c. siamense c. lunata cochliobolm geniculatu~ diaporthe arecae fusarium solani lasiodiplodia theobromae l pseudotheobromae nigrospora lactz'colonia n . olyxae penicillum ro@ii tmncospora tephropora ph_yllostictafallqbiae sarcostroma bisetulatum tiicboderma asperelhi xylaria feejeensis percentage (o/o) 1.45 1.45 1.45 2.90 1.45 1.45 1.45 1.45 1.45 14.49 11.59 4.35 1.45 1.45 1.45 8.70 1.45 2.90 2.90 1.45 2.90 1.45 4.34 5.79 7.25 1.45 1.45 1.45 1.45 4.34 1.45 shannon-wiener index (h') total 69 100 3.083 in this study, the most abundant fungal (26 isolates) species obtained from t. indicd leaves was from genus colletotm'chum where 10 isolates were identified as c. gloeosporioides with 14.49% (h' = 0.280). endophyuc c. fructicola and c. siamense have been recovered from healthy cymbopogon citratgs (manamgoda e t al. 201 3). weir et al. (2012) stated that c. siamense is geographically diverse with a varied host range and is a common saprobe or endophyte. colletotm'chum species can be found abundantly forming its association with temperate plants and they are widely distributed in the tropical and subtropical areas (cannon e t al. 2012), but no report on associations with t. indim. a study by boddy (2016) also reported that colletotm'chum species could be existed w i h n plant tissues without causing any harm while it is in an inactive state. these studies showed that members of colletotm'chum exhibit a multiple life styles. six isolates of endophytic diaporthe phaseolomm have been isolates from healthy fruits and leaves of t. indica. diapon'he spp. are known to be existed symbiotically alongside plants as saprobic, endophytic or phytopathogenic (udayanga e t al 201 1; tan e t al. 201 3; gomzhina & gannibal 201 8). according to gonzalez and tello (2011), endophytic diapon'he species are commonly isolated from several hosts in the temperate and tropical region. research on diapon'he species by gomes e t al. (2013) collected several species of diaporthe from vaccinium growing regions in europe i n c l u h g d. phaseolomm and d . arecae. diaporthe pseudomangiferae has been reported cause inflorescence rot, rachis, canker, and flower abortion of mango (serrato-diaz e t al. 2014). biotropia vol. 28 no. 3,2021 endophytic c. lanata and cochliobolas pbyllostica species have been known to form genicalata; (telemorph of c. genicalata) have been isolated from leaves of t. indim. two distinct species from genus lasidioplodid that were isolated from the leaves of tamarind were lasidioplodia theobromae and lsidioplodia pseadotheobromae with a simdarity percentage of 99% and 97% respectively. similar to colletotn'cham species, carvalaria/cochliobolas and ldsiodiplodia are well-known plant pathogens and can also be endophytes. in t h s study, aspe@ills tabengensis was found associated with the t. indicd leaves. this species was found to form an association with many plant species such as the mangrove plant, sonora desert plant (nadumane et al. 201 6), and strawberry (palmer et al. 2019). previously, other species of aspe@llas which is aspe~illas niger was isolated from diseased-fruits of t. indica and caused black pod (meena et al. 2018). two species of penicillm, p. ro@ii and p. vemcdosam have been isolated from healthy fruits and leaves of t. indim. penicillizlm spp. are common pathogens and caused spoilage in fruits (peter & patrick 2017). the assemblage of endophytic fungi in healthy tissue of t. indica may indicate that some of the fun@ are possible latent pathogens and some may saprophytic. the other genus dominated the t. indica leaves was nigrospora sp. wang et al. (2017) claimed that nigrospora sp. is a common in forming symbiosis with plants as pathogens, endophytes or saprophytes. nigroqora sphaevica (synonym of n. oy~ae) was found inhabiting numerous hosts such as the zea, andropogon and cymbopogon as reported by wang et al. (2017). supaphon and preedanon (2019) also claimed, the species was isolated from hehntbas annas as an endophyte. botyo~haerid mamane was only one isolate obtained from this genus. according to phdlips et al. (2013), this species that belonged to the botryosphaeriaceae is existed diversely in nature as pathogenic, endophytic or saprobic with more preferable to woody plants. a study by li et al. (201 8), also recorded the discovery of species of botryosphaeriaceae from plantation trees including canninghamina lanceolata, dimocaqas longan, melastoma sanguineam and phoenix hanceana, whch were growing adjacent to eaca4pta-r. their association with plants widely and can be either pathogens or endophytes. in this study, one isolate of phyllostictd fallopide with a 100% percentage of similarity with the established sequence in the genbank database. the morphology of the isolate characterized as p. fallpiae also fit the description of this species by zhang et al. (2013). one isolate was identified as xylarid feejeensis which was isolated from healthy leaves samples with 98.90% similarity to the genbank sequences. according to chen et al. (201 3) xylariaceous fungi are dominantly associated with the dendrobiam species of class orchdaceae. this f i n h g had supported the existence of xyb& sp. as an endophyte. trancospora tephropora (synonym of perenniporid tephropora) was the only basidiomycete found associated with healthy t. indicd leaves with similarity percentage of 99.84% from the sequence from genbank database. conclusion ' i h s study revealed that various endophytic fungi were isolated from the fruits and leaves of tamarind. the 31 species that have been successfully identified were a. acaleatas, a . carbonakas, a . flavas, a . tabingensis, b. mamane, c. aenigma, c. brevispomm, c. cobbittiense, c. fmcticola, c. gloeo~;pokoides, c. sidmense, c. genicalatas, c. lanata, d. arecae, d. ceratoxamiae, d. phaseoloram, d. pseadomangiferae, d. pseadoocali, d. pseadophoenicicoh, f. solani, l. psezldotheobromae, l. theobromae, n. lacticolonia, n. ogqae, p. ro@ii, p. vemcalosam, t. tephropora , p. fallopiae, s. bisetalatzlm, 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protocols: a guide methods and applications. san diego: academic press, 315-22. xia y sahib mr, amna a, opiyo so, zhao z, gao yg. 2019. culturable endophytic fungal communities associated with plants in organic and conventional farming systems and their effects on plant growth. sci rep 9:1669. zhang i<, su w, cai l. 2013. morphological and phylogenetic characterisation of two new species of phyllosticta from china. mycol prog 12:547-56. biotropia vol. 30 no. 2, 2023: 116 127 doi: 10.11598/btb.2023.30.2.1038 116 removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater using aquatic macrophytes nguyen tan chung* and nguyen van ha department of environmental biotechnology, faculty of biological sciences, nong lam university ho chi minh city, vietnam received 23 march 2018 /revised february 26, 2023 /accepted 3 march 2023 abstract wastes from breeding farms have globally increased greenhouse gases and caused a serious pollution to aquatic environments. biogas treatment polymer bags could significantly reduce organic compounds; however, they could not effectively treat other pollutants in animal wastewater. the objective of this study was to assess removal efficiency of salinity and pollutants associated with pig wastewater using aquatic macrophytes. four macrophytes namely acrotichum aureum, eleocharis dulcis, typha domengensis, and limnophyton obtusifolium and a soil control without vegetation were randomly assigned into fifteen mesocosms (1.2 x 0.7 x 0.6m) with 3 replicates for each treatment. pig wastewater was filled continuously into input chambers of mesocosms in every three day with 5 liters. water samples were collected from output chambers with 60 and 120 days after treatment while soil and vegetation samples were collected at the beginning and the end of the experiment. the results showed that e. dulcis, t. domengensis, and l. obtusifolium were dominant in removal of n, p, cu and zn and suspended solids as well; e. dulcis and t. domengensis significantly increased the dissolved oxygen; whereas the treatment of l. obtusifolium species showed the best efficiency in salt-ion removal. pollutants of n, p, cu and zn tend to accumulate more in the macrophyte roots than in their leaves. accumulation of n, p, cu and zn in the l. obtusifolium’s biomass is the highest compared with other treatments. from findings, it is suggested that a combination of three aquatic macrophytes including e. dulcis, t. domengensis, and l. obtusifolium could establish a constructed wetland system to directly treat pollutants of livestock wastewaters keywords: aquatic macrophytes, nitrogen, phosphorus, salinity, swine wastewater treatment introduction rapid growth of poultry and livestock sectors have caused seriously adverse effects on environmental quality and natural ecosystems and threatened human health (mawdsley et al. 1995, honda et al. 2000, nguyen 2010). wastes from poultry and livestock activities annually contribute to a large number of greenhouse gases which result in the global warming (fao 2014). throughout the vietnam, there are more than 10 million different households and farms with approximately 27 million pigs that consume annually about 18 million tons of industrial feed (nguyen 2009). the livestock sector discharges about 73 million tons of wastes per year, in which wastes of pig farms count for 33.4% (xuan 2009). this could not only reducing the quality of products but also increasing pollutants and heavy metals in wastes. major pollutants associating with the swine wastes include organic matters, nitrogen, phosphorus, copper, and zinc and other trace metals (cang et al. 2004, truong 2010, dang et al. 2014). according to the surveyed results about status of waste treatment of pig farms from eight different regions throughout vietnam, nguyen (2010) showed that 74% of pig farms have applied biogas plants to treat pig wastes while about 26% of the households and farms have been directly discharged the wastes into the environment. although treatment polymer bags have effectively been removed organic carbon but they could not reduce pollution of nitrogen (n), phosphorus (p) and other trace metals. nguyen & pham (2012) who evaluated the treatment effectiveness of pig wastewater removal by biogas treatment polymer bags at a *corresponding author, email: ntchung@hcmuaf.edu.vn removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 117 level of households showed that biogas plants could reduce up to 85% cod (chemical oxygen demand) and 76% bod5 (biological oxygen demand in five days) but only 12% and 7% for total nitrogen (tn) and total phosphorus (tp). other study which assessed the wastewater treatment effectiveness by biogas plants at 12 pig farms in the provinces of red river delta in the northern part of vietnam, vu et al. (2008) pointed out that biogas reduced 6570% cod, 75-81% bod5, and 10-28% tn. meanwhile, biogas treatment polymer bags did not impact on reducing heavy metals in pig wastewater. in recent decades, the use of constructed wetlands to treat different polluted sources has achieved a very high efficiency (vymazal 2011, shelef et al. 2013). four major factors determining a successful application of constructed wetlands include type of the aquatic plants, retention time of wastewater staying on the treatment system, the depth of the mud, and the depth of water column (usepa 2000). cattail and eleocharis species are two species of aquatic plants that have a wide distribution throughout the world and have a high performance in pollutant treatment (marois et al. 2015, mitsch et al. 2015). particularly, eleocharis species is recently used to remove n and p because of its high treatment efficiency in a short time (pham 2013). suhendrayatna et al. (2012) used cattails (typha sp) and reed species (phragmites communis) to treat municipal wastewater and showed that cattailspecies were more resilience to the wastewater and removed more bod, cod and tss (total suspended solid) higher reed species. in the other research, lam & ngo (2013) pointed out that cattail species was capable of absorbing 0,17g n and 0,09g p/m2/day in wastewater treatment of intensive catfish ponds with closed recirculation, whereas absortion ability of heavy metals was 456 1549 mg/kg (fe) and 1.2 to 7.6 mg/kg (pb) (anning et al. 2013). the main goal of this research was to identify some species of aquatic plants that peform a high efficiency in handling salinity and majority pollutants in pig wastewater. to achieve the research objectives, a series of examinations were conducted : (i) assessing capability to remove bod, cod, n, p, cu, zn and salinity in pig wastewater between treatments, (ii) determining the ability to absorb and accumulate n, p, cu and zn in soil and biomass of plant species. research results will provide a scientific basis for a suitable choice of aquatic plant species in handling organic pollutants, salinity and heavy metals for wastewater in general, particularly pig wastewater. materials and methods experimental design four plant species consisting of typha domengensis, eleocharis dulcis, acrostichum aureum, limnophyton obtusifolium and one control without plant were assigned a completely random into 15 composite barrels with 1.2m (length) x 0.6m (width) x 0.7m (depth) with 3 replications for each treatment (figure 1). a layer of soil was placed at the bottom of each barrel with a depth of 30cm. plants were grown with an initial density of 6 plants/experimental barrels, exception to the control. water level in each treatment was gradually raised to plant height after plantation, and water column-maintained depth of 30 cm from the mud surface by an effluent system with pvc ф-34 pipes. the composite barrels were buried into the soil with a depth of 30 cm equal to the soil depth inside each barrel. raw wastewater collected from pig farms was contained in a wastewater tank and filled into the barrels with frequency of every 3 days and 5 liters per time at an inlet zone of each treatment through the influent wastewater system as described in figure 1. based on the volume of water column, the surface area of barrel and the amount of wastewater entered (adelaja et al. 2014; dong et al. 2022), a retention time of wastewater stayed in each treatment system was estimated approximately 14 days. determination of soil and water temperature in order to examine if temperature affects efficacy of pollutant removal, hoboware device was used to measure temperature in water column and soil of treatments during experimental time. fifteen hoboware gauges (figure 2a) were deployed into the middle of soil layer and water column within each treatment and established to automatically recorded temperature in every 30 minutes. the gauges were mounted on pvc ф-27 pipes and fasten by biotropia vol. 30 no. 2, 2023 118 figure 1 a) experimental design with a treatment system; b) plant growth after 3-months handling plastic tubes. every two gauges were arranged on the same pvc pipe and aligned in the middle of water column and soil layer of each treatment. measurement of salinity, do, and ph to measure salinity among experimental treatments, electrical conductivity (ec) was measured, and then be converted ec to salt concentration (rhoades et al. 1999, nrw service centre 2007). dissolved oxygen (do), ec, and ph in effluent were measured at the outlet zone of each effluent system in every 2 weeks from 9:00 am to 10:00 am using ysi650 mds machine (figure 2b). ysi’s readers were deployed into water column with a depth of 10cm at the outlet zone to measure do, ec and ph. the readers were set up stabilizing in 60 seconds before recording and displaying results. to ensure qa/qc, the machine was calibrated in one hour before conducting the measurement and checked right after measurement by following the user manual’s standard operating procedures (ysi incorporated 2009). figure 2 a) hoboware; b) ysi650 mds machine effluent system wastewater tank influent system e. dulcis t. domengensis l. obtusifolium a. aureum a b a b removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 119 collection and analysis of effluent samples to evaluate effectiveness of pollutant removal among experimental treatments, samples of effluent water were collected after two and four months. collection of effluent samples was conducted at the outlet zone of effluent system in the morning by using a mini pump and collected in one day before putting the wastewater. effluent water was directly pumped into a 2-liter plastic bottle and kept inside an ice cooler before sending to nlu’s research institute of biotechnology and environment on the same collection day for analyzing bod5, cod, n, p, zn, and cu. methods applied to analyze for these components are presented in table 1. table 1 methods used to analyze samples sample type criteria analytical methods water bod5 tcvn 6001:2008 cod sweww 5220c:2012 tn tcvn 6638:2008 tp tcvn 6020:2008 cu2+, zn2+ aciar-aas 007-2007 soil tn tcvn 6498 – 1999 tp tcvn 8940 – 2011 cu, zn aciar-aas 019-2007 plant tn 10tcn 451 – 2001 tp 10tcn 453 – 2001 cu, zn aciar-aas 0192007 accumulation of pollutants in soil and plant soil and vegetation samples were collected in one day before wastewater was initially filled into barrels corresponded with one month after planting, and at the end of experiment to determine contents of n, p, cu and zn in soil and plant biomass. soil samples were taken at a depth of 5cm from soil surface by a soil core. in each treatment barrel, soil was randomly collected at three different positions, mixed them together, then placed it into a ziplock bag and kept it in an ice cooler before sending to laboratory for analysis. similarly to soil sampling, plant sample was collected from three plants in three different locations of each barrel. the sample was distinctly divided into leaves and roots. these samples were cut into 2-cm pieces, arranged neatly in paper bags, and dried in oven at 65oc for 7 days before sending for analysis. analytical methods of n, p, cu, and zn applied for plant and soil are presented in table 1. an increasing accumulative rate of pollutants in biomass computed by the following equation: h (%) = 100 x (cb-final– cb-initial)/cb-final where: cb-initial = concentration of pollutants in plant biomass (mg/kg) collected in one day before putting wastewater cb-final = concentration of pollutants in plant biomass (mg/kg) collected in one day before putting wastewater at the end of the experiment. n:p ratio = tn/tp, where concentrations of n and p accumulated in the plant biomass (mg/kg). statistics and anova analysis method this one-factor experiment was completely randomized factor with three replications for each treatment. software of minitab 17 was used for anova analysis to evaluate significantly differences among treatments, and for tukey’s multiple comparision method (α = 0.05). charts and figures were created by using the software of sigma plot 12. results and discussion concentrations of influent pollutants table 2 shows that concentrations of untreated pollutants in raw pig wastewater were very high. bod5 and cod were 2687.0 and 31516.0 mg o2/l, whereas concentrations of total nitrogen (tn), total phosphorus (tp), zn2+ and cu2+ were 2207.0; 1335.0; 23.4; 15.4 mg/l respectively. especially the pig wastewater contained a high electrical conductivity (ec) of 8.3 ms/cm, but a very low do content of 1.8 mg o2/l. concentrations of these pollutants were much higher than those of previous studies (chang & liu 2002, chung et al. 2004, nguyen & pham 2012). the reason for that was because wastewater samples were taken from tunnels of pigsty which directly received waste from pig manures and pigsty washing, and have not been decomposed with a condensed concentration. based on concentration of the untreated pollutants in the raw wastewater, loading rate established for each treatment, and retention time of wastewater in each treatment, initial influent concentrations of cod, bod5, tn, tp, zn and cu were 806.1; 9455.0; 662.2; 400.6; 7.0; and 4.6 mg/l respectively. biotropia vol. 30 no. 2, 2023 120 table 2 concentration of influent pollutants parameter unit raw wastewater initial average influent* ph 7.2 ec ms/cm 8.3 do mg/l 1.8 bod5 mg/l 2687.0 806.1 cod mg/l 31516.0 9455.0 tn mg/l 2207.0 662.2 tp mg/l 1335.0 400.6 zn2+ mg/l 23.4 7.0 cu2+ mg/l 15.4 4.6 * concentration estimated by basing on the raw wastewater concentration and retention time of the wastewater temperature variation in soil and water column temperature is an important ecological factor and directly affects respiration of microorganisms in a treatment system. variation of daily average temperature in soil and water among treatments were showed in figure 3. in general, the temperature in soil and water fluctuated from 24 – 31oc. in the first stage of development, there was a temperature difference. there was also a temperature difference among treatments in soil due to young plants, but no difference of those in water. in the final growth period, there was no different temperatures among the treatments in both soil and water. temperatures of t. domengenis and l. obtusifolium showed lower than those of other treatments. variations of temperatures among treatments could impact on removal efficiency of pollutants (ab-halim et al. 2016; alisawi, 2020; shatat and al-najar, 2011). figure 3 temperature fluctuation of treatments following the treated time: a) soil; b) water a b 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 121 do, ec and removal efficacy of salt dissolved oxygen (do) in water is dependent on photosynthesis processes of aquatic plants and a microbial respiration in water column and soil. in the respiration, microorganisms consume a large amount of do to decompose organic compounds. an amount of do used for the microbial respiration are often supplemented by photosynthesis of aquatic vegetation. the low level of dissolved oxygen in water is not only a sign of contamination and is but also an important factor in determining water quality and pollution (bozorg-haddad, 2021; kulkarni, 2016). in freshwater ecosystems, most of aquatic animals require amount of dissolved oxygen higher than or equal to 3 mg o2/l for survival. for example, fish cannot survive for long in water with dissolved oxygen less than 5 mg/l (bozorg-haddad, 2021). table 2 shows that the pig wastewater contained a large amount of organic compounds with a low do of 1.8 mgo2/l. althought do concentrations of each treatment fluctuated in over the experimental periods, two treatments of t. domengenis and e. dulcis generally improved the do (figure 4). do concentration of these two treatments was generally ranged from 5 to 9 mg o2/l. most aquatic plants and animals require oxygen to survive; fish, for instance, cannot survive for long in water with dissolved oxygen less than 5 mg/l. the low level of dissolved oxygen in water is a sign of contamination and is an important factor in determining water quality, pollution control and treatment process. the do in a saturated solution varies with the water temperature and elevation. figure 4 changes of do, ec and salt ion contents following the treated time among treatments biotropia vol. 30 no. 2, 2023 122 electrical conductivity (ec) shows concentration of ions which exist in water is usually derived from the soluble salt ions such as na+, k+, cl-, so4 2-. a high content of soluble ions would negatively influence and inhibit a growth of aquatic organisms. salinity in fresh water normally ranges from 0.01 to 0.50‰. the pig wastewater contained a large amount of soluble salt ions with ec of 8.3 ms/cm (table 2) which corresponded to salinity content of 4.6‰, ten times higher than the normal range of salinity in natural freshwater ecosystems. due to no vegetation, ec in control treatment accumulatively increased following the treated time resulted in a gradual accumulation of the salt content during the experiment (figure 4). species of t. domengenis, l. obtusifolium, and e. dulcis performed a high absorption of salt ions which resulted in a very low ec and salinity. salinity of these treatments was always lower than 0.50‰ over 14 weeks of the treated experiment (figure 4). exception for e. dulcis, since this species grew very quickly and had its shorter growth cycle than those of others, about 10 weeks, when this species died and returned its turnovers into water, and decomposed turnovers quickly (marois et al. 2015, mitsch et al. 2015). this resulted in an increasing of ec and salinity for this treatment in the last stage of the experiment. removal efficacy of bod5, cod, n, p, zn and cu as showed in table 2, the pig wastewater contained a high concentration of organic matters with cod of 31,516 mg o2/l and biochemical oxygen demand (bod5) of 2,687 mg o2/l. based on the input load, the retention time and the total volume of each treatment system, initial influent concentrations of cod and bod5 in all treatments were 9455.0 and 806.1 mg o2/l respectively (table 2). sixty days after treated, three treatments of l. obtusifolium, e. dulcis, and t. domengensis removed a large amount of organic matters, and as a result, the cod contents of these treatments reduced to 55.5, 36.0, and 176.0 mg o2/l respectively (figure 5). but in 120 days after treated, the cod content of l. obtusifolium treatment continued dropping to 46 mg o2/l, whereas the cod of e. dulcis treatment went up to 560 mg o2/ l, which was explained by due to the 10-month growth cycle of e. dulcis species. similar to effectiveness of cod handling, the treatments of l. obtusifolium, and t. domengensis showed the best efficacy of bod5. pig wastewater was polluted with high n and p, where the concentrations of tn and tp are respectively 2207.0 and 1335.0 mg/l, while their influent concentrations at each treatment were 662.0 mg/l (tn) and 400.6 mg/l (tp) (table 2). decomposition of organic pollutants by microorganisms associated with treatments of l. obtusifolium, e. dulcis, and t. domengensis reduced the bod and cod. this resulted in decomposition and transformation of organic n and p compounds into inorganic compounds for plant uptakes. these three treatments showed the best performance of n and p removal after 60 days treated. the tn contents of l. obtusifolium, e. dulcis, and t. domengensis were respectively 2.4, 10, and 3.3 mg/l. but after treated in 120 days, tn contents of all treatments increased more than doubled in comparison with those at the treated time of 60 days. similarly to total n, the content of total p (tp) was also removed significantly after 60 days and increased after 120 days, except for two treatments of l. obtusifolium and t. domengensis (figure 5). cu and zn ussualy supplements to food of livestocks, especially in pig foods, but pollution of cu and zn in pig wastewater is paid less attention. concentrations of these two heavy metals in the pig wastewater are so high, approximately 23.4 and 15.4 mg/l for cu and zn. based on input load and retention time on the treatment system, the initial average concentration of cu and zn were 4.6 and 7.0 mg/l (figure 5). removal capabilities of cu and zn are very different among treatments. the treatment of l. obtusifolium removed completely cu and zn due to plant uptake. as a consequence, concentrations of cu and zn in this treatment was nearly equal to zero (non detection) after 60 days treated. in addition to the l. obtusifolium treatment, the a. aureum also showed a higher efficiency of cu and zn removal than others. removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 123 figure 5 changes of bod5, cod, n, p, zn and cu following the treated time among treatments accumulation of n, p, zn, cu in soil according to results of anova analysis shown in table 3, the initial n and p contents in soil were not different from the treatments (p = 0.186 for tn, p = 0.669 for tp). concentration of initial soil tn ranged from 284 to 323 mg n/kg dried soil, whereas the initial tp concentration in soil ranged from 62 to 67 mg p/kg dried soil. baseline concentrations of tn and tp used for the experiment were very low. however, both n and p concentrations in the soil were increased for all treatments. due to without vegetation, the control treatment accumulated the lower than those of three other treatments because of its ability of low n transformation and decomposition in water, while the treatment of l. obtusifolium had a the highest accumulation of tn and tp contents in soil with 504 mg n/kg and 210 mg p/kg, respectively. this result were corresponded with the lowest tn and tp concentrations of l. obtusifolium treatment in water. initially baseline cu and zn concentrations in the soil utilized for the experiment were very low and did not differ among treatments with the cu concentration under non-detection zero and zn concentration ranged from 3.1 to 3.9 mg/kg (p = 0.822) (table 3). at the end of the experiment, although accumulation of cu content in soil increased in all treatments, approximately 6 mg cu/kg, there was no significant difference (p = 0.157). in contrast to cu accumulation in soil, zn concentrations were significantly different from the treatments (p < 0.000). the treatment of l. obtusifolium showed the highest zn accumulation with 11.4 mg zn/kg. this is resulted from the high zn removal efficiency of l. obtusifolium treatment in water which assisted by organic matter breakdown associated with microorganisms in this treatment. treated time (day) biotropia vol. 30 no. 2, 2023 124 table 3 accumulation of n, p, cu and zn in soil/sediment parameter treated time (day) concentration of treatments (mg/kg dry soil weight) (mean ± se) t. domengensis e. dulcis a. aureum l. obtusifolium control tn 0 313 ± 22 301 ± 5 323 ± 3 284 ± 31 304 ± 15 120 467 ± 21 409 ± 13 386 ± 48 504 ± 9 636 ± 16 tp 0 65 ± 8 63 ± 2 67 ± 1 62 ± 1 63 ± 5 120 164 ± 8 123 ± 24 140 ± 2 210 ± 19 267 ± 8 cu 0 nd nd nd nd nd 120 6.0 ± 0.5 6.0 ± 1.0 6.2 ± 0.7 6.1 ± 0.8 7.4 ± 0.5 zn 0 3.8 ± 1.9 3.9 ± 0.9 3.3 ± 0.2 3.1 ± 0.3 3.4 ± 0.5 120 5.2 ± 2.2 4.1 ± 0.8 4.3 ± 1.0 11.4 ± 2.6 14.6 ± 1.4 nd: none detection contents of n, p, cu and zn in plant biomass since the initial n baseline content in foliages and roots are significantly different among treatments (table 4). the treatment of l. obtusifolium contained the highest n concentrations in root (0.97% ± 0.15) and foliage (1.90% ± 0.10). this difference could be explained that because in first month after planting (not input the wastewater to treatments), the l. obtusifolium species grew faster than others treatments resulting in higher accumulation of n content in its root and foliage. this corresponded with the lowest initial n content of l. obtusifolium treatment in the soil (table 3). after treated in 120 days, root and foliage n concentrations of all treatments significantly increased in comparison with their initial baseline concentrations (table 4). the increasing percentage of n absorption rate by vegetation treatments within root and foliage were presented in figure 6. all treatments showed a high n absorption rate in roots with greater than 50%, but there was no a significant difference of the rate among the treatments. the highest n absorption rate in roots reached 67% for the a. aureum treatment while the lowest n absorption rate was 50% for the l. obtusifolium treatment. on the other hand, the l. obtusifolium treatment showed the highest foliage n absorption with the 28.9%, followed by the treatment of e. dulcis (19.6%), from which the rates of two these treatments were significantly different from two other remaining treatments. if combined with the root and foliage absorbed n contents, the treatment of l. obtusifolium performed the highest capacity to accumulate nitrogen in its biomass. generally, under a normal condition, all treatments accumulated lower n in roots than in foliages, but with the addition of n increasing amount from pig wastewater, n tended to be accumulated more in roots than in foliage in order to create a balance of n content in their biomass. table 4 concentrations of n, p, cu and zn in plant biomass treatment bio-mass type one day before treated 120 days after treated tn (%) tp (%) zn (mg/kg) cu (mg/kg) tn (%) tp (%) zn (mg/kg) cu (mg/kg) t. domengensis root 0.75 ± 0.05 0.182 ± 0.005 77.1 ± 20.4 19.9 ± 4.7 2.27 ± 0.30 0.907 ± 0.113 154 ± 15.0 19.5 ± 10.5 leaf 1.65 ± 0.09 0.240 ± 0.025 nd nd 1.71 ± 0.02 0.263 ± 0.015 nd nd e. dulcis root 0.55 ± 0.05 0.254 ± 0.048 19.5 ± 5.0 13.7 ± 7.9 1.54 ± 0.24 0.607 ± 0.014 93.5 ± 13.5 26.0 ± 1.0 leaf 1.25 ± 0.15 0.314 ± 0.003 nd nd 1.59 ± 0.41 0.477 ± 0.021 nd nd a. aureum root 0.95 ± 0.05 0.213 ± 0.061 32.1 ± 13.5 26.8 ± 11.4 2.09 ± 0.17 0.689 ± 0.031 45.0 ± 17.0 34.0 ± 16.6 leaf 1.85 ± 0.06 0.105 ± 0.005 nd nd 1.97 ± 0.18 0.272 ± 0.037 nd nd l. obtusifolium root 0.97 ± 0.15 0.329 ± 0.004 73.2 ± 1.2 5.3 ± 0.4 2.00 ± 0.26 1.078 ± 0.017 154 ± 43.8 80.0 ± 6.0 leaf 1.90 ± 0.10 0.339 ± 0.094 nd nd 2.68 ± 0.17 0.486 ± 0.017 nd nd nd: none detection removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 125 similar to n status accumulated in biomass, the initial root and foliage p concentrations were very different among treatments. the treatment of l. obtusifolium always performed the best ability to uptake p into its biomass, from which the initial p baseline concentration were 0.329% ± 0.004 in root and 0.339% ± 0.094 in foliage and that the p concentration accumulated in its roots and foliage after 120 days were respectively 1.078% ± 0.017 and 0.486% ± 0.017 (table 4). in contrast to n absorption results, phosphorus mainly accumulated in roots of treatment’s plants after the 4-month handling period. if it takes into account of absorbed p rate in biomass, the treatment of t. domengensis showed the best performance of p absorption rate in its biomass, about 68% in root and 62% in foliage (figure 6). the n/p ratio in plant biomass is an ecological indicator, which is used to assess a level of excess or deficiency of n or p content provided for growth of trees in different ecosystems. n/p ratio of 14-15 reflects a balance between n and p contents in an ecosystem. the n/p ratios in biomass of all treatments in this study were rather low, approximately 3 in root and 8 in foliage for treatments (figure 6). these results indicated that the pig wastewater was more n polluted than p in a term of n/p ratio for plant uptake. according to the analyzed results presented in table 4, both cu and zn were only accumulated in roots of treatments. cu and zn concentrations in foliage were very low and even under detectable in some treatments. at the same rate of initial zn and cu influent loading, two treatments of t. domengensis and l. obtusifolium accumulated the highest zn content in their roots, 154.0 ± 15.0 and 154.3 mg/kg ± 43.8 respectively (table 4), where only the treatment of l. obtusifolium showed the highest cu absorption in its roots with cu concentration of 80.0 mg/kg ± 6. figure 6 n and p absorption rate and n:p ratio in plant biomass among treatments root foliage biotropia vol. 30 no. 2, 2023 126 conclusion three species of t. domengenis, l. obtusifolium, and e. dulcis performed a high removal efficacy of pollutants accociated with the pig wastewater including salinity, cod, bod, n, p, cu and zn. with its ecological 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6-series multiparameter water quality sondes. biotropia vol. 29 no. 2, 2022: 103 111 doi: 10.11598/btb.2022.29.2.1634 103 mercury chloride (hgcl2) exposure changes the histopathological figure of eye and brain of tilapia fish (oreochromis mossambicus) dwinna aliza1*, nazaruddin1, aya sofia abda2 and annisa asri2 1pathology laboratory, faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia 2veterinary medicine study program, faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia received 8 august 2021/accepted 26 january 2022 abstract mercury pollution brings harmful effects to aquatic animals, the environment and eventually to human health. mercury accumulates in the liver, kidney, eye lens and brain of fish, resulting in organ damage. this study aimed to determine the effect of hgcl2 exposure on anatomical pathology and histopathology of tilapia fish eye and brain. a total of 36 male tilapia fish were allotted into 4 treatment groups with 3 replications. fish were exposed to 0.00, 0.25, 0.50, and 0.75 ppm of hgcl2 for 10, 20, and 30 days. subsequently, the anatomical pathology was observed followed by histopathological examination. anatomical pathology examination of fish eye on day 30 showed white membrane on the eye lens surface, pupil diminution, and sunken eyes. the brain demonstrated hemorrhage, necrosis, discolorations, and granulated area. the retina showed necrosis, retina pigmentation flexiform layer widened, and cone cell atrophy. brain depicted structural and cellular damage such as degeneration necrosis and vacuolation. hgcl2 exposure changes the anatomical pathology and histopathology of tilapia fish eye and brain. keywords: cellular damage, fish brain, fish eye lens, histopathology, mercury chloride introduction heavy metals polluted the waters of the sea and rivers. among heavy metals pollutants is mercury (hg). mercury has volatile properties, takes the form of liquid at a certain temperature, capable of dissolving various metals and very toxic element to all living things (gworek et al. 2020; wang et al. 2017). mercury in nature comes in two forms, namely inorganic mercury such as mercury chloride (hgcl2) and mercury oxide (hgo), and organic/organomercury consisting of methylmercury (ch3hg+) and ethylmercury (c2h5hg+). both types are toxic, however the natural and anthropogenic emissions occur as inorganic form (zulaikhah et al. 2020; macirella et al. 2016). mercury chloride in sediments on the seabed and rivers can be converted by microorganisms into organic metal mercury (r-o-hg) compounds, which remain soluble in water and accumulate in sediments. high hg concentrations can cause death in fish, whereas sublethal concentrations lead to disruption of organ function (saturday 2018; rauf et al. 2018). mercury accumulated in the body of aquatic animals will damage or stimulate the enzymatic system, which resulting in the decreased adaptability of the animal concerned to the polluted environment (mehouel et al. 2019; lee & wendy 2017). mercury enters the tissues of living organisms through several ways, namely the respiratory, digestive, and penetrating through the skin (dogan & canli 2018; ismail & mahboub 2016). mercury that enters the body of aquatic organisms cannot be digested, however, it can be dissolved in fat. the fat-soluble metal is able to penetrate the cell membrane, so that eventually the mercury metal ions will accumulate in the cells and other organs. the highest accumulation is usually in the detoxification organs (liver) and excretory *corresponding author, email: dwinna.aliza@unsyiah.ac.id biotropia vol. 29 no. 2, 2022 104 organs (kidneys) (macirella et al. 2016; jyotsna et al. 2014; moustafa et al. 2016). in fish, the organs that accumulate the most heavy metal of mercury are the kidneys, liver, brain, and lens of the eye (sobhanardakani et al. 2018; gupta & kumar 2006; pereira et al. 2014; kimakova et al. 2018). severe sublethal toxicity affects the morphology of tissue histology, physiological conditions such as metabolism, growth and development of fish morphology, biochemistry (blood chemistry and enzyme activity), behavior (neurophysiology) and fish breeding (korbas et al. 2017; nazaruddin et al. 2020). according to morcillo et al. (2017), exposure to hg can produce highly variable effects which depend on the magnitude and duration of exposure. this phenomenon was proven by nazaruddin et al. (2020) which found the most severe histopathological condition on male gonad organs of tilapia fish exposed to the longest time period of hgcl2. korbas et al. (2017) stated that fish eye has the potential for hg absorption since it has a wide surface of exposure and continuously contact with external media and thus, becomes an effective absorption route of hg. high accumulation of mercury in the eye lens causes ocular anomalies such as cataracts or opacity, as previously reported in humans affected by hg (lemire et al. 2010). pereira et al. (2014) also stated that eye is the often-neglected organ in accumulation of heavy metals mercury studies. a thoroughly histopathological study on changes of fish eyes exposed to hg has never been reported. in the brain, mercury will accumulate in the cerebrum cortex and cerebellum where it will be oxidized to a form of mercuric (hg++). mercury ions will bind to sulfhydryl from enzyme and cellular proteins that interfere with enzyme function and cell transport (morcillo et al. 2017), leading to degeneration of nerve cells in cerebellum of the brain (berntssen et al. 2003). although there is a considerable amount of information available on toxicity of mercury to fish tissues and cells, which is among the most sensitive biological responses due to mercury exposure. furthermore, this study focusing on eye and brain which proved to be the most impacted organs physiologically affected by mercury pollution. materials and methods fish and experimental design this study used 36 male tilapia (oreochromis mossambicus) fish within similar sexual maturity stage and body weight of 250-300 g. fish were obtained from farmer’s ponds at cadek village, banda aceh, indonesia. fish were apparently healthy and free from skin lesions or external parasites. the fish were maintained in glass aquaria (80 x 60 x 40 cm capacity) having 100 l of dechlorinated tap water. each aquarium was equipped with aerator. fish were acclimatized for 7 days to the laboratory environment and fed three times daily. this experimental study implemented a completely randomized design. fish were allotted into 4 groups (n = 9) with 3 replications. group 1 (k1) was negative control, while group 2, 3, and 4 (k2, k3 and k4) were treated with hgcl2 0.25, 0.5 and 0.75 ppm. the treatments were exposed to hgcl2 for 10, 20, and 30 days. histopathological examination the fish were dissected to collect the eye and brain then subsequently fixed with davidson solution for 24 h. the fish tissues were then dehydrated in an ascending series of alcohol, cleared in xylene and embedded in paraffin wax. section of 5 µm thick were cut, processed and stained with haematoxylin and eosin (h&e). the observations of the slides were done using olympus light microscope and the histopathological changes of the tissues were documented using the photo microscope. results and discussion the anatomical pathology of tilapia fish eye the observation of the anatomical pathology of tilapia fish showed that fish eye in the control group (k1) was normal indicated by clear, fresh, and intact eye ball and lens (fig. 1). the observation did not show any changes of eye performance of the fish exposed to hgcl2 on day 10 and 20, however on day 30 fish in group k2 and k3 demonstrated soft and transparence white layer on the surface of eye ball. this layer became thicker and wider, which almost cover mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 105 all surface of the eye ball, thus, looks like cataracts. furthermore, the diminution of the eye pupil and sunken eyes was also clearly observed (fig. 1). normal fish eye is bright, intact, transparent, and no mucos layer on the eye ball surface. fish eye, although modified a lot, is basically similar to other vertebrates, consisting of the cornea, corneal epithelium, lens, retina, choroid, optic nerve and iris (takashima & hibiya 1995). fish eye anatomy is rather horizontal in the anterior part, with the intention that the convex lens almost touches the cornea which is an important transparent part of the eye ball scleroid coat. the lens has a layer that resembles a layer of onions, the layer on the lens is transparent, nonnucleated and inelastic. not like the lenses in mammals which are elastic and can be modified by the contraction of the eye muscles, the lenses in fish are not elastic, thus they must be pulled inward toward the retina with the retractor lentic muscles to accommodate changes in vision (azab et al. 2017). anatomical pathology changes occured in the form of white membranes in the eye ball surface might be caused by physical and systemic factors. physical factors occur due to direct contact between hgcl2 and the surface of the eye mucosa, while systemic factors occur through inhalation or ingestion of toxic compounds which are then carried by blood circulation and reaches the eye organ resulted in nerves and balance disorders such as blindness and swim abnormally. this happens since the eyes are connected to the optic lobe of the brain through the optic nerve (rajeshkumar et al. 2017). furthermore, korbas et al. (2013) stated that fish eyes have wide surface which continuously contact with external media leading to an effective absorption route of hg. in addition, fish eyes appeared sunken, indicated high accumulation of hgcl2 in the eye tissue which lead to contraction and sunken of the eye ball. according to pereira et al. (2013), sunken eyes are caused by physiological reaction and fish resistance to exposure of pollutants, bacterial infections or viruses which changes the morphology of fish eyes such as sunken and cloudy. in this study cataracts were also found due to hgcl2 exposure. figure 1 anatomical pathology of tilapia fish exposed to hgcl2 on day 30 notes: k1 = control group; k2 = 0.25 ppm hgcl2; k3 = 0.50 ppm hgcl2; k4 = 0.75 ppm hgcl2; a = eye pupil; b = white layer on eye ball surface; c = sunken eye. k3 k1 k4 k2 biotropia vol. 29 no. 2, 2022 106 histopatology of tilapia fish eye lens and retina histopathology of tilapia eye lens in control group (k1) showed lens layer was transparent and pink in color. similar results were observed on tilapia fish lens in group k2 on observation days 10 and 20. in contrast, on day 30, k2 showed a change in the form of hemorrhage in the iris but the lens was still intact. k3 treatment showed a severe necrosis area in the lens of the eye as well as in the k4 treatment. moreover, in k4 groups a change in the color of the lens that was pink turned into purple in the middle of the lens and became granulated were observed (fig. 2). normal tilapia retina were shown in the control group (k1). the structure of the tissue showed very clear layers and there were no changes of the structure, color and size. the fundamental layers of the retina were still complete and remain unchanged. the structure of the retina layers from inner to outer layer consists of the optic nerve layer, the ganglion layer, the inner flexiform layer, the inner nucleus layer, the outer flexiform layer, the outer nucleus layer, cone cells and stem cells, the retinal pigment cell layer, and choroid (genten et al. 2009) (fig. 3). the histopathological examination of tilapia fish eye retina exposed by hgcl2 revealed necrosis in inner and outer layer of nucleus in k2 and k3 on day 10 observation, while k4 showed cell necrosis in outer layer of nucleus, cone cell and rod cell atrophy which resulted in unarranged of both cell structure and widened inner flexiform layer. moreover, on day 20, fish eye retina in k2 showed cell necrosis in the inner and outer layers of nucleus, while in k3 cell necrosis of inner nucleus layer and cone cell necrosis leading to widened rod cell was revealed. fish in k4 group showed cell necrosis in inner and outer nucleus layer and cone cell atrophy, furthermore, retina pigment cell started to widened (fig. 3). severe damages of the eye retina tissue were observed on day 30. k2 demonstrated inner nucleus cell necrosis, cone cell and rod cell atrophy, and widened retina pigment. fish in k3 groups showed cell necrosis in almost all layers, thus the borders between layers disappeared. severe condition was shown in k4 group, eye retina was damaged which resulted in total changes of all layers. all cells were necrosis causing the borderless condition between the layers and became one-piece tissue. in additon, hyperplasia of the cells also found in k2, k3, and k4. the highest hgcl2 concentration and the longest exposure time caused cells degeneration and lost of their function which end with cell death. this observed in day 20 and 30 observation, which cell damages were clearly seen in each component of retina layers. figure 2 histopathology of tilapia fish eye lens exposed to hgcl2 on day 30 notes: k1 = control group; k2 = 0.25 ppm hgcl2; k3 = 0.50 ppm hgcl2; k4 = 0.75 ppm hgcl2; a = eye lens; b = necrotic area; c = haemorrhage. (he 40x). k1 k2 k3 k4 mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 107 histologically, eye lens is composed of collagen produced by the epithelium cells of the eyepiece, forming a layer that resembles a layer of onions, transparent, thin, has no core, and not elastic (mumford et al. 2007). results of this study indicated that exposure to hgcl2 for 30 days has the potential to cause damage to eye tissue and high concentration of hgcl2 also show obvious histopathological damage. this is in accordance with the statement of mela et al. (2013) that the longer exposure and the higher concentration of hg can produce severe and varied forms of effects. for example, purple discoloration of fish eye lens was caused by a chronic hgcl2 exposure since the discoloration was only observed on the 30th day of observation. other lesion found was hemorrhage that occurred in the iris of fish eye due to the accumulation of hgcl2 in the blood leading to blood vessels to rupture. when hemorrhaging occurs, the intake of nutrients and other substances to the eye will stop, thus the cells will experience the lack of nutrients which eventually cause cell necrosis followed by lost in its function (pereira et al. 2013). in this study most necrosis was found in the retina layer which was marked by the loss of cell parts and appeared empty (vacuole). this is in accordance with takashima & hibiya (1995) that necrosis describes a condition where a decrease in tissue activity is characterized by the loss of several parts of the cell, one by one from one tissue, so that in a short time the cell will be dead. jeevanaraj et al. (2016) also reported that there was necrosis in retinal tissue due to chronic mercury exposure in h. malabaricus traira fish. the observations also showed hyperplasia of retina cells as a response of cell adaptation to the stimulation of toxic compounds. it was found that cellular proliferation caused the retina layer to undergo hyperplasia and caused the boundary layer components to be irregular. this can affect the function of the retina which is very sensitive to light (azab et al. 2017). similar mechanism of proliferation occurred to the brain cerebellum tissue causing the lost of layers boundaries of the tissue. atrophy occurred in the cone and rod cells of fish eye in group k2, k3, and k4 on day 10, 20 and 30. athrophy is suspected to be caused by inhibited vascularization activity caused blood intake to the eye is inadequate. cone cells and rod cells are receptor cells in the retina that are very sensitive to light. fish tend to use their vision to adapt to light when looking for food, because according to hunt et al. (2015) cone cells have the ability in terms of sensitivity to light and sharpness of vision. the damage of the cone cells or rod cells will affect the behavior of fish related to the sharpness of vision, axis of vision, and maximum visibility in fish (pereira et al. 2014). histopathology of the brain microscopic examination of the brain cerebellum of tilapia fish in control group demonstrated that the fish brain cerebellum consists of two parts namely cortex (gray matter) dan medulla (white matter). cortex consists of three layers, the outer layer called molecular layer, middle layer (ganglioner), and inner layer called granular layer (periventriculare). every layer has specific cells. satelite cell, neuroglia cell and basket cell were found in molecular layer, while purkinje cell was found in ganglioner layer, and granuler cells and golgy cells were found in granuler layer. cerebellum tissue of tilapia fish in group k1, k2, and k3 showed relatively similar figure between 10, 20, and 30 days of treatment that the molecular layer was widened, while the ganglioner layer was thinner compared to control group (fig. 4). the highest the mercury concentration was exposed the widest the molecular layer observed. the widening of molecular and granular layers of brain cerebellum was caused by cell proliferation of one layer to the others. in contrast, ganglioner layer was thinner along with the higher dosage of hgcl2 exposure. granular layer showed different patterns compared to the two previous layers. in the low dose of hgcl2 exposure the granular layer was thinner than normal tissue, however, in higher dose of exposure the granular layer became thicker. the ganglioner and granulare layer disappeared after being exposed to hgcl2 for 30 days. other than structural damage of cerebellum tissue caused by cell proliferation, cellular changes were also observed such as cloudy swelling degeneration, cell swelling, necrosis and vacuolation. biotropia vol. 29 no. 2, 2022 108 figure 3 histopathology of tilapia eye retina exposed to hgcl2 on day 30. notes: a = cell necrosis; b = cone cell atrophy; c = widened of eye retina pigment; d = widened of inner fleksiform layer. (he 400x). the tilapia fish brain cerebellum exposed to hgcl2 also showed the presence of vacuolation in all treatment groups. similar results were reported by berntssen et al. (2003) in atlantic salmon (salmo salar) fish exposed to chronic dietary mercury. vacuolysis has characteristics such as a round, hollow hole that occurs because of the accumulation of fat. the contributing factors of vacuolations are the accumulation of toxic substances, oxygen damage and excess fat consumption (chamarthi et al. 2013). vacuolysis will lead to disruption of cell metabolism process which eventually resulted in cell lysis (lakshmaiah 2017). these vacuoles are seen more in brain samples with the highest dose of mercury exposure. this is consistent with the statement of rajeshkumar et al. (2017), that the degree of pathological damage is highly dependent on the dose and duration of exposure. under conditions of higher mercury exposure the vacuole turned into expanding empty spaces. similar results are reported by lakshmaiah (2017), in catfish exposed by various concentrations of glyphosate herbicides which is neurotoxic, characterized by severe degeneration, edema and vacuole changes into large empty spaces. the vacuolation that occurred in this study might share similar effect as the herbicide since mercury is also neurotoxic. cell swelling or vacuole degeneration is reversible. thus, cell returns to normal when the exposure to toxic substances is terminated. bose et al. (2013) reported that increasing the dose of heavy metal exposure causes vacuolation in the molecular layer of cerebellum. k4 k3 k1 k2 mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 109 figure 4 histopathological feature of tilapia fish brain cerebellum notes: a-b = control group; c-d = k3 group; a = medula; b = cortex; c = molecular layer; d = ganglioner layer; e = granulare layer; f = proliferative purkinje cell; g = proliferative neuroglia cell; h = proliferative granulare cell. (he 40x, 400x). cell changes mainly occurred in neuroglia cells in the molecular layer, purkinje cells in the ganglioner layer and granulare cells in the granular layer. it is clearly seen that necrosis were observed on purkinje cells and granulare cells. the cell swelling was shown by purkinje cells as well as neuroglia cells. swelling of purkinje cells was reported by chamarthi et al. (2014) in the cerebellum of the teleost catfish induced by benzene petrochemical. the neuroglial cell swelling was reported by erhunmwunse et al. (2014) in tilapia exposed to glyphosate herbicide. conclusion hgcl2 exposure changes anatomical pathology and histopathological of tilapia fish eye and brain. the higher the hgcl2 concentration and the longest time of exposure, the most severe the changes of tilapia fish eye and brain tissues. acknowledgments the authors thank mrs. denny irmawati hasan from the department of pathology, universitas syiah kuala for her tremendous support in histopathological and staining method processes. this study was a laboratory grant project supported by the faculty of veterinary medicine, universitas syiah kuala. references azab am, shoman hm, el-deeb rm, abdelhafez hm, samei sea. 2017. comparative studies on the histology of eye retina in some nile fishes with different dial activities. ejhm 68:815-23. berntssen mhg, aatland a, handy rd. 2003. chronic dietary mercury exposure causes oxidative stress, brain lesions and altered behavior in atlantic salmon (salmo salar) parr. aquat toxicol 65(1): 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histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 111 planktonic biomass of the river ravi, pakistan. trjfas 19(10): 857-64. saturday a. 2018. mercury and its associated impacts on environment and human health: a review. j environ health sci 4(2):37-43. sobhanardakani s, hosseini sv, tayebi l. 2018. heavy metals contamination of canned fish and related health implications in iran. trjfas 18:951-7. takashima f, hibiya t. 1995. an atlas of fish histology. normal and pathological features. 2nd edition. tokyo (jp): kondansha ltd. wang lk, chen jp, hung yt, shammas nk. 2017. heavy metals in the environment. new york (us): crc press. zulaikhah, st, wahyuwibowo j, pratama aa. 2020. mercury and its effects on human health: a review of the literature. ijphs 9(2):103-14. biotropia no. 9,1996: 38-52 the genus mimosa with special reference tom. quadrivalvis l. var. leptocarpa (d.c.) earnedy, a new species record for the weed flora in malaysia *) baki, b. bakar, h. noorma wati and m.a. haji mohamed botany dept., university of malaya, 59100 kuala lumpur, malaysia abstract an exploratory floristic survey of the genus mimosa was conducted in 1993 to ascertain species diversity and their spatial patterns of distribution in peninsular malaysia. a new species record of uncertain indigene, m.. quadrivalvis was recorded for the first time in restricted localities along the roadsides in pekan darat and bertam, seberang perai, gurun and bedong in kedah in addition to widely distributed and seemingly ubiquitous presence of m. invisa mart. ex. colla and m. pudica l. the latter two species were mostly found in open, disturbed and derelict habitats, agricultural areas and ex-mining lands. both species exhibited largely contagious and overdispersed distribution patterns with positive peaks in pattern intensity values although regularity or underdispersed distribution patterns do manifest in certain localities. the giant mimosa, m. pigra inhabited in clustered thickets, large pockets of lands in the urban and sub-urban localities in the states of penang, perak, kelantan, kuala lumpur and negeri sembilan. in other states, m. pigra was confined to a few localities in smaller patches. except for m. quadrivalvis, the other species of mimosa are serious weeds in the agricultural, recreational and residential and derelict areas. a key to the mimosa species is constructed along with brief descriptions on their morphology and ecology. key words: malaysia/weed ecology/mimosa invisa/mimosa pigra/mimosa pudica/mimosa quadrivalvis/ weed distribution/weed anatomy and morphology. introduction the genus mimosa, in the family mimosaceae (syn. leguminosae or fabaceae) is large and cosmopolitan with 300-500 species worldwide (holm et al. 1977; nielsen 1981, 1983, 1992). the intricacy of this genus has made it a subject of continued research interest and taxonomic revisions (e.g. ridley 1925; corner 1952; henderson 1959; burkill 1966; metcalfe & chalk 1972; nielsen 1992). according to index kewensis (1991), there are well over 1800 binomials of mimosa worldwide with heavy concentrations in tropical america. in malesia, there are three introduced and naturalised species. recent studies by nielsen (1992) gave extensive accounts of the genus *) paper presented at the symposium on biology and management of weeds and fourth tropical weed science conference, 22 24 november 1994, bogor, indonesia 38 38 the genus mimosa baki, b. bakar et al. on the world scale. in malaysia, the genus mimosa is known for its weedy elements viz. m. invisa, m. pigra and m. pudica infesting large areas of agricultural, residential, recreational, roadsides and derelict areas and ex-mining lands. ridley (1925) gave the first account of the family in the malay peninsula while burkill (1966) described a brief history of the various species that has been introduced into the malay peninsula. waterhouse (1993) enlisted the three weedy species of mimosa with the 15 to 19 ratings among the 140 most important weed species as appropriate targets for biological control in southeast asia in general and in malaysia in particular. this paper reports briefly on the morphology and distribution patterns of mimosa spp. with emphasis on m. quadrivalvis l. var. leptocarpa (dc.) barneby. materials and methods an exploratory floristic survey was conducted in 1993 in peninsular malaysia to ascertain species diversity and spatial distribution patterns of the genus mimosa. for such purpose, samplings were made at random in selected vegetable gardens, derelict areas, immature rubber and oil palm estates, ex-mining lands and roadsides. in addition, the list count quadrat (1m x 1m) method of kim & woody (1983) was also used. in the case of m. pigra, the line transect method was used with quadrat size of 2m x 2m. only adjacent and accessible areas close to the roads were sampled. a total of 72 quadrat samples for each species were obtained (table 1). the spatial distribution date for each species were subjected to pattern analysis (allinson 1981) with pattern intensity values used as indices to measure the departure from randomness. detailed morphological comparisons were made between species. table 1. plot size,number of transects and quadrats transects employed in pattern analysis of plant population of mimosa species 39 biotropia no. 9, 1996 results and discussion pattern of distribution figure 1 illustrates the extent of infestation of the four species of mimosa in peninsular malaysia. generally, heavy concentrations of m. pigra are found in ex-mining lands, ex-padi farms and derelict areas both in rural and urban localities in the states of penang, perak, kelantan, kuala lumpur and negeri sembilan, often in clustered thickets and large pockets. mimosa invisa and m. pudica are widely distributed and seemingly ubiquitous mostly in open, disturbed and derelict habitats, agricultural areas and ex-mining lands. the soils range from rather wet of the inseptisol, vertisol and entisol types or the relatively dry soils of the oxisol or ultisol types. soil samples taken in areas where rampant infestations of mimosa spp. occur registered ph values ranging from 4.0 5.0. mimosa invisa and m. pudica are also found on the bris soil habitat in terengganu, pahang, kelantan and pantai remis areas of perak. clump sizes of adult populations vary considerably according to the species of mimosa. in the case of m. pigra, it ranges from 1.0 m to 6 m in diameter while for m. pudica and m. invisa these were about 0.25 m 2.0 m. mimosa quadrivalvis has a relatively smaller clump size of about 0.2 1.0 m (table 2). genet populations range from 0.2 1.0 plant/m 2 for m. pigra; 0.5 4.0 plants/m 2 (m. invisa and m. pudica) to 1.0 5.02 plants/m 2 for m. quadrivalvis, thereby emphasizing different morphology and growth habits of the different species of mimosa (table 2). table 2. clump size and genet population density of mimosa spp. in peninsular malaysia species clump size (m) genet population density (per m 2 ) m. invisa 0.25 2.0 0.5 4.0 m. quadrivalvis 0.20-1.0 1.0-5.0 m. pigra 1.0 -6.0 0.2-1.0 m. pudica 0.25 2.0 0.5 4.0 the spatial distribution of the four species of mimosa differed accordingly, showing pattern at different block sizes in different regions of survey. in the case of m. pigra population in the northern states of penang, perak and kedah (region a), pattern was observed with peak pattern intensity values at block size 12. similar clusterings were observed among m. pigra populations in malacca, negeri sembilan, johore (region c) and kelantan (region d). mimosa pigra populations in the federal territory, 40 the genus mimosa baki, b. bakar el al figure 1. the distribution of (a) mimosa invisa, (b) m. quadrivalvis, (c) m. pigra, and (d) m. pudica in peninsular malaysia. each symbol represents at least one record within 5 m radius. 41 biotropia no. 9, 1996 selangor (region b) and southern perak showed pattern at block sizes 8 and 16. a tendency towards regular undispersed spatial distribution was observed among m. pigra populations in region b at block size 1 (figure 2a). individual genets among m. invisa population for all regions showed regularity for block sizes 1, 2 and 3. contagious overdispersed individuals were observed at block size 6 for region c and block size 8 for regions a, b and d (figure 2b). regularity in spatial distribution of individuals of m. pudica was recorded at block sizes 1, 2 and 3 for all regions saved region b. positive peaks of pattern intensity values were recorded at block size 6 for regions a, b and d and block sizes 6 and 16 for region c (figure 2c). the spatial distribution of individual genets of m. quadrivalvis showed pattern at block size 6 but exhibited regularity at block sizes 1, 2 and 3 (figure 2d). no concrete arguments can be forwarded to explain the spatial heterogeneities exhibited by different populations of mimosa pigra, m. invisa, m. pudica and m. quadrivalvis. differential growth rates and differences in morphology among the species studied are too simple a notion to help illustrate such differences. further analyses on the possible roles of environmental variables such as soil, ph, nutrient status, soil types, etc. in explaining the spatial heterogeneities and distribution patterns of the different mimosa species are needed. morphology and species description morphological illustrations and habit of m. quadrivalvis are shown in figures 3 -6. comparative morphological traits and characteristics of the species are shown in tables 3 and 4. 1. mimosa invisa auct. non mart, ex colla: backer & bakh. f., fl. java 1 (1963) 561; burkill, diet. econ. prod. mal. penins. 2 (1966) 1498; nielsen, fl. camb., laos, vietnam 19 (1983) 34, pi. 5: 1-7; nielsen, fl. malesiana ser. 1, 2, 1 (1992) 184-185. a procumbent perennial shrub, growing up to 3 m high. stem conspicuously 4-angled, up to 4 m long, densely distributed with prickles. leaves bipinnate, primary leaflets 4 to 9 pairs, leaflets opposite sessile 12 to 30 pairs, lanceolate, 6 to 12 mm long, 1.5 mm wide. inflorescence a globular head, axillary, flowers pinkish violet, corolla 4-lobed with 8 pinkish violet exserted stamens. fruit softly spiny, 3to 4-seeded borne as clustered axillary, flattened pods, 1.0 to 3.5 cm long, 6 to 10 mm wide, splitting along the valves into 1-seeded segments when mature. seeds flattened, ovate light brown, rather glossy, 2 to 3 mm long. 42 the genus mimosa baki, b. bakar et al figure 2. pattern intensity values of spatial distribution of (a) m. pigra, (b) m. invisa, (c) m. quadrivalvis, and (d) m. pudica plants at different block sizes. lower case letters on the curve denote regions of occurrence (see text for details). 43 biotropia no. 9, 1996 figure 3. mimosa quadrivalvis. 44 the genus mimosa baki, b. bakar et al. figure 4. mimosa quadrivdvis. (a) stamens, (b) axillary inflorescence, (c) opened flowers. 45 biotr0pia no. 9, 1996 figure 5. mimosa quadrivalvis. (a) mature pod densely covered with long, fine prickles, (b) opened pod, (c) young pod, (d) seeds. 46 the genus mimosa baki, b. bakar et al. figure 6. mimosa quadrivalvis. (a) seeds, (b & c) emergence of hypocotyl, (d & e) hypocotyl with cotyledons, (f) emergence of once-pinnate, first true-leaf, (g) young plant. 47 biotropia no. 9, 1996 48 the genus mimosa baki, b. bakar et al. 49 biotropia no. 9, 1996 2 mimosa quadrivalvis l. var. leptocarpa (dc.) barneby. a straggling shrub. mature stem angular, prickles sparsely distributed; younger stem slightly rounded. leaves evenly bipinnate, primary leaflets 1 2 pairs, 0.5 1 cm apart from each other, leaflets opposite, 9 1 6 pairs, almost sessile, 2 8 mm long, 0.5 -2.0 mm wide, margin entire, pubescent, oblanceolate-oblong, both abaxial and adaxial surfaces sparsely distributed with glands and sparsely hairy but abaxial surface of outermost leaflet usually more hairy compared to abaxial surface of the inner leaflets. secondary veins obscure, apex mucronate-truncate, base truncate. secondary rachilla covered with short fine hairs, sparsely distributed. inflorescence a globular head, axillary, 1 8 2 0 flowers per inflorescence. stamen numerous, pinkish, 1.0 1.5 mm long, arranged in several rows forming a globular head. bracts present, hairy. fruits in axillary flattened pods, 1 3 in a group, usually the third one abortive, 4.0 9.5 cm long with prominent raised ridge along the edges, apex sharply pointed, tapered to a point 1.0 1.5 cm long, almost linear, moderately to densely covered with long, fine prickles, pedicel 0.8 1.1 cm long, hairy; mature fruits dehisce along sutures. seed ovate to rhomboid in shape when dry, brownish-black, about 3 mm long. 3. mimosa pigra l., backer & bakh. f., fl. java 1 (1963) 561; nielsen, fl. camb. laos, vietnam 19 (1981) 34, pi. 5: 8-14; nielsen, fl. malesiana ser. 1, 2,1 (1992) 185. mimosa sepiaria auct. non benth.: ridley, fl. mal. pen. 1 (1922) 656, p.p.; burkill, diet. econ. prod. mal. pen. 2 (1966) 1499. an erect, perennial woody shrub, growing up to 5 m high. stem woody, up to 3 m long, sparsely distributed with prickles. leaves bipinnate, primary leaflets about 15 pairs, leaflets sessile, lanceolate. inflorescence a globular head, axillary, flowers pink or mauve, corolla 4-lobed with 8 pink stamens. fruits densely hairy, 20to 25-seeded axillary, borne as clustered flattened pods, 6.5 to 7.5 cm long, 7 to 10 mm wide, breaking into 1-seeded segments when mature. seed brown or olive green, oblong, flattened, 4 to 6 mm long, 2 m wide. 4. mimosa pudica l., backer & bakh. f., fl. java 1 (1963) 561; nielsen, fl. camb. laos vietnam 19 (1981) 35, pi.; nielsen, fl. malesiana ser. 1, 2, 1 (1992) 185-186. a procumbent, perennial, creeping shrub growing up to 50 cm high. stem rounded, stiff, up to 150 cm long, prickles scattered along the internodes. leaves bipinnate, primary leaflets 1 to 2 pairs, leaflets opposite, 12 to 25 pairs, oblong or linear, 9 to 12 mm long, 1.5 mm wide, margins hairy. inflorescence an ovoid or a globular head, borne on prickly axillary stalks, flowers purplish pink, corolla 4-lobed, stamens 4, purplish pink. fruits oblong, flattened 1to 5-seeded pod, 1 to 2 cm long, 3 to 6 mm wide, edge with prickles, borne in cluster on leaf axils, breaking into 1-seeded segments 50 the genus mimosa baki, b. bakar et al. when mature. seeds light brown, flattened with a finely granular surface, 2.5 to 3 mm long. key to mimosa species in peninsular malaysia 1. primary leaflets 12 pairs 2. leaflets almost sessile, pubescent, 2 8 mm long, 0.5 2.0 mm wide. stem angular. flowers pink, in globular heads, stamens pink. pods 4.0 9.5 cm long, apex sharply pointed tapered to a point 1.0 1.5 cm long, covered with long, fine prickles ..................................................................................................... m. quadrivalvis 2. leaflets sessile, 9 1 2 mm long, 0.5 mm wide, margin hairy. stem rounded. flowers purplish pink, in ovoid or globular head, stamens purplish pink. pods 1 2 cm long, edge with prickles ............................................................................ m. pudica 1. primary leaflets more than 2 pairs 3. primary leaflets 4 9 pairs, leaflets lanceolate. stem conspicuously 4-angled with numerous prickles. flowers pinkish violet. pods 1.0 3.5 cm long, 3 to 4-seeded. seed light brown, glossy, ovate, 2 3 mm long ................................................ m. invisa 3. primary leaflets about 15 pairs, leaflets lanceolate. stem woody, prickles sparsely distributed. flowers pink or mauve. pods 6.5 7.5 cm long, 20to 25-seeded. seeds brown or olive green, oblong, 4 6 mm long .......................... m. pigra acknowledgements the authors are extremely thankful to dr. g.p. lewis of kew botanical gardens, u.k. for identifying the species and mr. anuar pandak and mr. zainal mustafa for their technical assistance. thanks are also due to mr. tan lip hong for typing the manuscript. references allinson, j.m. 1981. numerical and experimental analysis of species relationships in grassland. phd thesis, university of wales. backer, c.a. and r.c. bakhueen van den brink. 1963. flora of java. vol, 1. rijksherbarium. leyden. 547-565. 51 biotropia no. 9, 1996 burkill, i. h. 1966. a dictionary of the economic products of the malay peninsula. 2nd ed. the crown agent for the colonies, london. corner, e.j.h. 1952. wayside trees of malaya. 2nd ed. govt. print. off. singapore, p 413. henderson, m.r. 1959. malayan wild flowers. dicotyledons. malayan nature society, p 100. holm, l.g., d.l., plucknett, j.v. pancho and j.p. herberger. 1977. the world's worst weeds. distribution and biology. univ. press of hawaii, honolulu. index kewensis 1991. oxford university press. kim, s.c and k. woody. 1983. comparison of some methodologies for vegetation analysis in transplanted rice. korean journal of crop science 28(3): 310-318. metcalfe, c.r. and l. chalk. 1972. anatomy of the dicotyledons. vol. 1. oxford, clarendon press, p 487. nielsen, i. 1981-83. flora of cambodia, laos and vietnam, vol. 19. nielsen, i.c. 1992. mimosaceae (leguminosae mimosoideae). flora malesiana ser. 1, vol. 2, prt. 1. ridley, h.n. 1922. flora of malay peninsula vol. 1. reeve and co. london . waterhouse, d.f. 1993. the major arthropod pests and weeds of agriculture in southeast asia: distribution, importance and origin. aciar, canberra. 141 p. 52 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf biotropia no biotropia no. 11, 1998: 22 41 nutrient stocks in four stages of a lowland rain forest at pasirmayang, jambi, central sumatra, indonesia pamuji lestari faculty of agriculture, university of bengkulu , bengkulu, sumatra, indonesia abstract studies of nutrient cycling of tropical forests should differentiate between dynamic stages of the forest. we studied the nutrient concentration (n, p, k, ca and mg) and phytomass of aboveground (living and non living parts) and belowground compartments (soil) in four dynamic stages, namely building (bl and b2), and mature (ml and m2) stages, for a lowland rain forest. nutrient concentrations in various compartments differed between the dynamic stages. bark contains higher nutrient concentration than wood parts, both in stems and branches. leaves contain higher nutrient concentration than wood parts, both in litterfall and litter. the concentration of k in throughfall is the highest, ca and mg perform similar value. throughfall exhibits lower nutrient concentration than open area rain water. the nutrient concentration of 10-20 cm is higher than in the 0-10 cm soil depth. the phytomass values are highly variable among tree parts, diameter classes and dynamic stages. the phytomass is generally the highest in m2 and significantly different from b l , ml and b2. the phytomass of leaves in litterfall and litter is higher than wood parts. more litterfall and litter are accumulated in mature than building phases. key words : nutrient / phytomass / dynamics / rain / forest introduction understanding the cycling of the original forest ecosystem mineral nutrients is essential in evaluating consequences of forest replacement to agriculture, tree plantations, or best methods by which forests can be managed for sustained timber production. reviews of overall patterns of mineral cycling in the tropics (jordan 1985; vitousek and sanford 1986; proctor 1987; bruijnzeel 1989) clearly show a wide diversity. the tropical rain forests are very rich in species with large interspecific differences in chemical composition (grubb and edwards 1982; hobie 1992), and they differ in species competition and structure over sometimes short distances (trichon 1996). moreover, the tropical forests are very dynamic systems, mosaic of regenerating and mature "phases" (halle et al. 1978; whitmore 1984). this invariably rises the question of the representativeness of field samples that are always small for obvious logistic reasons, when extrapolating the data to the whole ecosystem. the present study is part of a wider research program on nutrient patterns and variability in tropical forested lowland landscapes, both accross physiographical and 22 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari successional gradients in jambi province, sumatra. it was initiated not only because there is so little information available on productivity (soerianegara 1966; schaik and mirmanto 1985) and nutrient cycling (soedjito 1988; strigel et al. 1993) of rain forest in indonesia in general, but also because the understanding of the variability of nutrient availability in such forested landscape is needed for forest land management. this study was conducted to determine whether nutrient stocks and phytomass significantly vary among dynamic stages and how the nutrient maintains the rain forest ecosystems. site description the pasirmayang permanent plot in the jambi province of sumatra (1°5's latitude and 102°10'e longitude, north muarabungo city) offers an excellent opportunity to study lowland rain forest function and dynamics since topography is very gentle, meaning that there are no or few interaction between dynamics and slopes. average altitude varies between 70 and 100 in and the plot's physiography is gentle with slopes ranging from 2 to 8% for drops of 5 to 30 m. the underlying geology is that of vast tertiary deposition basins mainly formed in sedimentary rock. these geological formations consist of layers several kilometers thick and mainly include sandstones, calcareous sandstone and marls. the soils encountered in the plot are mainly oxisols. the a horizon, located at a depth of 2 to 10 cm, contains a superficial fine root layer, and its low organic content is a characteristic feature (c/n from 8 to 15). clay content increases with depth (a-b located between 10 and 40 cm). kaolinite sensu stricto accounts for at least 80% of the clay fraction and the low loam/clay ratio is characteristic. in the brown or yellow-brown b horizon, soil acidity remains relatively constant (ph between 4 and 5). the cation exchange capacity is very low, less than 15 meq/loog of clay. these soils rank amongst the least fertile and are extremely fragile once forest has been cleared (erosion on slopes, compacting by heavy vehicles, insolation and rapid loss of surface minerals). few changes in soil characteristics are observed between the lower slopes and the top of the ridges in the plot. the litter is mostly very thin and covers a humus horizon of 2 to 3 cm thick, sometimes 15 cm deep from place to place. these thicker layers are related to dominance of certain species like dipterocarpus crinitus or d. lowii whose leaves apparently decompose very slowly. the humus horizon is extremely acid in these instances (ph 3.8 to 4). 23 biotropia no. 11, 1998 in general, the climate of the area shows an annual rainfall between 200 and 2500 mm/year. monthly mean rainfall can drop below 100 mm for one month at most, and the yearly average of rainy days varies from 120 to 150, with a mean temperature of the coldest month always above 20°c. however, as indicated by laumonier (1995) climatic variability is high and rather long dry periods (2 to 3 months) can be experienced for some years. the forest is a typical lowland dipterocarp forest as described for the eastern sumatra plains by laumonier (1995). materials and methods a. field sampling a.l. sampling unit selection in relation to forest dynamics following previous research on forest dynamics in that area by torquebiau (1986) and laumonier (1991), several criteria were taken into account for the selection of what can be seen as representative of mature and "old" building forest phases. for the particular purpose of the study a former classification of the forest dynamics made by laumonier (op.cit), was combined with whitmore (1984) tree diameter classes for mature, building and gap phases, and refined using vertical and horizontal structure criteria e.g : frequency histogram for diameters and heights, scatter and profile diagrams, crown projection, canopy cover, crown overlapping, treefall gaps and added with a concept by halle et al. (1978) that classifies trees according to their architecture: 1) trees of the "future" which are represented by the first stage of development when either conform to their original model or are regenerated by reiteration, 2) trees of the "present" are trees that survive from earlier phase of development, show no regeneration in an architectural sense as a whole and have established a new energetic balance, and 3) trees of the "past" are trees that show no particular response to the outside energy, facing an architectural chaos from disturbance and are gradually eliminated. a total of eight plots assigned in four dynamic stages with two replications in each dynamic stage were used to evaluate those vertical and structural criteria. 24 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari a.2. vegetation and soil sampling a. nutrient concentration in each plot (20 m x 20 m), aboveground compartments as living parts (leaves, stems, branches and twigs), non living parts (litterfall, litter, and throughfall) and belowground (soil) compartments were sampled for the nutrients that are most likely to limit primary production and other ecosystem function (n, p, k, ca and mg). a.l. aboveground compartments living parts (trees) trees were sampled for three stem diameter classes 30 cm and up (all diameter higher than 30 cm which vary among plots), 010-29.99 cm and 0 3-9.99 cm. a composite of leaves, stems (wood, bark), branches (wood, bark), and twigs from five trees in each of the three diameter class was collected for each plot of various dynamic stages obtained from the structural analysis. an approximate of 0.5-1 kg of leaves, branches and twigs were obtained by cutting, whereas samples of stems were obtained by slashing the main stems to reduce tree damage. non living parts litterfall two semi-permanent circular litter traps (diameter 1 m) were used for litterfall collections. they were equipped with plastic mesh 1.25 m above the ground to allow proper water drainage. at emptying, the contents of each trap were put in a paper bag and sorted into leaves and woody parts. there is a total of 32 samples (8 plots; two replications in each stage with two litterfall traps in each plot and two different parts; leaves and wood). litter two litter samples (one square meter each) were taken from every plot. a total of 32 samples both for leaves and woody parts were analyzed. throughfall a total of seventeen water samples were collected for nutrient analysis. two plastic drums with a polyethylene funnel of diameter 22.5 cm in every plot were 25 biotropiano. 11, 1998 used for throughfall collection. an additional drum was put in the open for sampling bulk precipitation. a, 2 belowground (soil) a metal ring of 8.5 cm diameter and 10 cm height was used for soil collection. a total of 32 soil samples (two samples in every plot at two soil depths: 010 cm and 10-20 cm) were used for nutrient analysis. roots were not included in the present study due to logistic reason. b. phytomass living parts (trees) the phytomass of living parts in the three diameter classes has been estimated from allometric equations developed by yamakura et al. (1986) who studied plant biomass with reference to forest dynamics in east kalimantan (table 1) the phytomass calculation was based on the number of trees in each plot according to diameter classes. the results of 8 plots were then averaged and presented in each dynamic stage. table 1. regression equation for calculating biomass of tree parts developed by yamakura (1986) tree regression equation stem biomass (sb) 2.903 x 10"2(d2h)° 98'3 branch biomass (bb) i 259 leaf biomass (ib) ' -, 07266 9.146xlo"(sb+bb)° 266 sb, bb, ib, in kg d (diameter) in cm h (height) in m non living parts litterfall the equipments used are the ones used for sampling the litterfall for nutrient concentration study. the litterfall phytomass obtained is the g/m2 of samples dry weight and extrapolated into ton/ha. 26 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lcstari litter the litter phytomass was obtained from the dry weight inside a square meter subplot (g/m ), then it was extrapolated into ton/ha. b. laboratory analysis prior to nutrient analysis, above ground and below ground materials were oven dried at 105°c for 48 hours or until constant weight. after determining oven dry weight, the samples were ground and nutrient analyzed. total nitrogen was determined using kjeldahl digestion and distillation method and phosphorus was analyzed using spectrophotometry. an atomic absorption spectrophotometer was used for potassium and calcium analysis, whereas magnesium was determined by using flame photometer. c. data analysis the nutrient concentration of different parts of trees, litterfall, litter and soil in various dynamics were compared using least significant difference (lsd) of the statistix program package version 4.0 (siegel 1992). results and discussion a. structural and dynamic classification of the plots the height of the forest canopy varies from 30 to 40 m with emergent trees rising to heights of 45 to 55 m. mature phase of the forest may have up to four distinct foliage mass layer respectively, situated at the following heights: 5 to 10 (15), 15 20(25), 25 30(35), and 35 40(45) m (laumonier 1995). a total of eight plots were selected representing mature phases and building phases (figure la b) following the former classification by laumonier (1991). eventhough it is important to study the early stages of succession i.e. treefall gaps and young aggrading phases to complete the comparison of each dynamic stage, they were excluded from the study due to logistic reasons. 27 biotropia no. 11, 1998 as shown in table 2, further analysis of the horizontal structure of these plots (number of trees, diameter, height, basal area, abundance, dominance and canopy cover), allows to group them into 4 classes corresponding to four dynamical status situation. building 1 (bl), building 2 (b2), mature 1 (ml), and mature 2 (m2). the number of trees in the four different classes are not significantly different except for b2 where a high tree density is observed. in all plots, the density is always higher for trees of the future, with the exception of ml where abundance of trees of the present is conspicuous. the trees of the past are the most abundant in m2. total, covered and overlap crown surface are the highest in bl followed by m2, ml and b2, whereas gap surface is the highest for b2 followed by ml, m2 and bl. in term of coverage, the horizontal structure is always dominated by "the trees of the present" (m1>m2>b2>b1) followed by "trees of the future" (b2>b1>m2>m1) and "past trees" (b1>m1>b2>m2). average basal area of the four different classes is similar, but more variations are observed in the vertical structure and the respective arrangement of the tree classes (table 2). b. nutrient concentration the complete results of nutrient concentration in various compartments (tree, litterfall, litter, throughfall and soil) are presented in figure 2a d. b.i. aboveground a. living parts (trees) the general pattern of nutrient concentration in trees observed in this forest is n > k > ca > mg > p. mostly n concentration in leaves is the highest compared to other parts this condition is mainly due to the physiological role of the leaves in photosynthetic activity. other nutrient concentrations are varied among parts. barks which play considerable role in transferring nutrients from trees into soil are always present in higher nutrient concentration than sap wood, both in stems and branches. b. non living parts b. 1. litterfall most nutrient concentration is varied between parts and among dynamics. the variable nutrient concentration in litterfall may be mainly the result of water penetration versus leaching from rainfall, influenced by the position of leaves in the trees, and also by the species composition. the higher nutrient concentration of 30 biotropia no. 11, 1998 leaves and wood parts in building phase may be due to the condition that in this stage plants are very active in uptaking and storing nutrients from soil. b.2. litter all nutrients in both parts are not significantly different among dynamics. the concentration of n, k and ca of leaves is significantly different from wood parts. similar to litterfall, the nutrient concentration of leaves is higher than wood parts, litter concentrates higher nutrients in building than mature stages. the fallen leaves are accumulated as forest litter. thus, the nutrient concentration of the litter may mainly come from the concentration of the forming materials. therefore, the higher nutrient concentration of the litterfall may result in the higher nutrients in the litter. moreover, in the process of decomposition more nutrients will become available, and so the concentration of nutrients in litter may be increased. by comparing aboveground living parts, it shows that the nutrient contents in leaves > litterfall > litter. the average percentage of four dynamic stages in nutrient withdrawal before litterfall is the highest for k (68%) followed by p (46%), ca (42%), n (24%) and mg (12%). the poorer quality of litter compared to litterfall may suggest that the process of decomposition is very slow. b.3. throughfall in the four phases, k exhibits the highest concentration both as input (open area) and throughfall in the forest ecosystem, whereas ca and mg show similar values. this result agrees to that of manokaran (1980) that the nutrient concentration increased as precipitation passed through the canopy as throughfall due to leaching at the canopy with conspicuous amount for k, relatively less for mg and ca. the average nutrient concentration of throughfall is higher in building than mature stage. this may be caused by the higher surface covered in building than mature stage that allows more water penetration after reaching the canopy. the values of ca and mg in the pasirmayang forest are lower than that obtained in open area. this is in contrast with strigel et al. (1993) who studied the nutrient concentration of throughfall in two forest sites in east kalimantan (table 3 ). 36 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari table 3. the nutrient concentration (mg/1) of throughfall in pasirmayang, sumatra vs bukit suharto and lempake, east kalimantan throughfall open k ca mg k ca mg pasirmayang (bph) bukit suharto 1.21 1.80 0.30 0.40 0.30 0.05 1.33 0.10 0.41 0.20 0.30 0.02 lempake 1.70 0.60 0.08 0.30 0.20 0.02 in pasirmayang, the nutrient concentration of open area is higher than in throughfall, although they are not significantly different. this condition may be due to the water sample collectors'position in the open area of pasirmayang (20 m x 20 m plot size) that may still be affected by leaching of nearby canopy, i.e. increasing the nutrient concentration. in bukit suharto and lempake, the nutrient concentration of throughfall is higher than in the open area. this phenomenon may be the result of the water sampling position that is far from the forest area (100 m apart), therefore the effect of adjacent trees could be avoided. b.2. bcltm ground (soil) the concentration of p and ca is not significantly different between depths in all stages. other nutrients are varied. all nutrient concentrations are not significantly different among dynamics. at 0-10 cm soil depth, the pattern of nutrient concentration in old building (b2) and mature phase (m2) is similar with n> ca> k> p or mg. however, some differences in nutrient concentration of top soil among dynamics were observed among the plots. at 10-20 cm depth, the pattern of nutrient concentration is similar to that at 0-10 cm with n> ca> k> p or mg. the p in both soil depths are generally limited because of the small mass circulation in most forests, small input from atmosphere to compensate losses from available pools and fixation by fe and al oxides in highly weathered acid soil. the concentration of nutrients is higher in top than in the lower layer. this may be the result of higher litter decomposition process above the top soil that releases many nutrients to the soil. apparently, the concentration of nutrients in the soil of the building phase was higher than that of the mature phase. knowing, however, the extremely high variability of soil nutrient availability in the rain forest environment, it may be difficult to draw conclusions. the relationship between forest dynamics with soil 37 biotropia no. 11, 1998 variation pattern is a very complex phenomenon which is difficult to assert within the present study. c. phytomass c.l. living parts (trees) the general aboveground phytomass for the entire pasirmayang plot varies between 320 and 400 ton of dry matter per hectare (laumonier 1991) for trees with a diameter > 10 cm. undergrowth samples provide a further estimate of 15 to 20 ton/ha for individuals with a diameter ranging from 3 to 10 cm. at an average of 30 to 40 kg/m2 (extremes being 0 to 80 kg/m2), epigeal phytomass varies from 2.65 to 30.7 ton/400 m2. in the selected plots, the phytomass pattern of trees in different diameter classes (0 > 30 cm, 10 < 0 < 30 cm and 3 < 0 < 10 cm) and dynamic stages is shown in figure 3a c. among the plots, the general phytomass pattern for living parts is m2> bl> ml> b2. m2 stage exhibits the highest phytomass and differs significantly from ml, b2 and bl. for the diameter class 10 < 0 < 30 cm alone, bl > ml > b2 > m2. bl is the type of dynamic stage where fierce competition exists between a large number of "future" trees reaching the canopy. the phytomass in the 3 < 0 < 10 cm diameter class performs similar to that of pole trees with bl exhibiting the highest followed by ml, m2 and b2. c.2. non living parts a. litter/all as shown in fig. 4a, in all stages, leaves litterfall phytomass is higher than wood litterfall. the pattern of total litterfall and leaves litterfall is ml > b2 > bl > m2, whereas for wood litterfall, the pattern is slightly different with bl > b2 > ml > m2. b litter regarding utter accumulated on the ground, the pattern of leaves and total litter is the highest in m2 followed by b2, ml or bl (fig. 4b). in woody litter, the phytomass of mature is higher than in building stages. this phenomenon seems to correspond to a natural succcssional pattern of forest dynamics from building to mature phase, the "older" the phase, the more litter accumulated 38 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari references bruunzeel, l.a. 1989. moist tropical forest nutrient cycling : the hydrological framework. in proctor, j. (ed). mineral nutrienu in tropical forest and savana ecosystems, p. 383-416. grubb, p.j and p.j. edwards. 1982. studies of mineral cycling in a montane rain forest in new guinea. iii. the distribution of mineral elements in the aboveground material. journal of ecology 70:623-648. halle, f.r., a.a. oldeman and p.b. tomlinson 1978. tropical trees and forests. springer-verlag, heidelberg. hobbie, s.e. 1992. effects of plant species on nutrient cycling. tree7(10):336-339. jordan, c.f. 1985. nutrient cycling in tropical forest ecosystems. wiley. london. laumonffir, y. 1991. vegetation de sumatra, indonesia . ecologie, flore, phytogeographie. th doct. etat, ups toulouse, france. 337p. laumonier, y. 1995. the vegetation and physiography of sumatra. geobotany 22. kliiwer academic, dordrecht, 234 p. manokaran, n. 1980. the nurient contents of precipitation, throughfall and stemfiow in a lowland tropical rain forest in peninsular malaysia. the malaysian forester 43(3):266-289. proctor, j. 1987. nutrient cycling in primary and old secondary forest. applied geography 7:135-152. schadc, c.p. van and e. mkmanto. 1985. spatial variation in the structure and litter production in a sumatran rain forest. biotropica 17:196-205. slegel, j. 1992. statistix version 4. analytical software. borland international inc. and fleming software. minnesota, usa. soerianegara, i. 1966. the primary productivity of selected forests in indonesia. rimba indon. 10(4):246-256. soediito, h. 1988. spatial pattern, biomass, and nutrient concentrations of root systems in primary and secondary forest trees of a tropical rainforest in kalimantan, indonesia. indonesian institute of sciences (l1pi), indonesian national mab commitee, 41-59. str1gel, g.d, ruhiyat, d. prayitno and s. sarmina. 1993. nutrient input by rainfall into secondary forests in east kalimantan, indonesia. j. trop. ecol. 10(2):285-288. torquebiau, e.f. 1986. mosaic pattern in dipterocarps forest in sumatra. agroforestry systems 2(2):103-128. trichon, v. 1996. heterogeneite spatiale des structures en foret naturelle de basse a sumatra, indonesie. doctoral de 1'universite paul sabatier. toulouse, france. vitousek, p.m and r.l. sanford, jr. 1986. nutrient cycling in moist tropical forest. ann; rev. ecol. syst. 17:137-167. whitmore, t.c. 1984. tropical rain forests of the far east. clarendon press, oxford. yamakura, t, a. hagihara, s. sukardjo, and h. ogawa 1986. aboveground biomass of tropical rain forest stands in indonesian borneo. vegetation 68:71-82 41 biotropia no. 7, 1994: 18-29 . the possibility of controlling sclerotium rolfsii on soybean (glycine max) using trichoderma and tebuconazole*) okky s. dharmaputra department of biology, faculty of mathematics and natural sciences, bogor agricultural university; and seameo biotrop, p.o. box 116, bogor, indonesia and ina retnowati seameo biotrop, p.o. box 116, bogor, indonesia abstract the possibility of controlling s. rolfsii on soybean (glycine max) var. rinjani using t. aureoviride and tebuconazole under field conditions was studied. the experiment was conducted at the experimental plot of seameo biotrop. the pathogen was mixed with the soil (2 kg/plot) 4 days before the inoculation of the antagonist (2.25 kg/plot). the measurement of each plot was 2.5 x 6 m 2 . n, p and k (120 kg/ha) were applied at the same day with the inoculation of the pathogen. soybean seeds were planted 7 days after the inoculation of the antagonist. the distance between plants and between plots were 20 and 40 cm, respectively. the fungicide at concentration of 100 g/ha (in vitro concentration) and 210 g/ha (field or recommended concentration) were applied using 2 methods, i.e. 1) spraying on the planting hole at the same day as the planting of soybean seeds, and 2) spraying on the soil surrounding the plants 7 days after planting. soils that were neither inoculated with the antagonist nor the fungicide were used as controls. three replications (3 plots) were used for each treatment (including the control). the results showed that the inoculation of the antagonist, the concentrations of the fungicide, and time of application gave very significant differences in the percentages of the plants infected by the pathogen and significant differences in seed production; while the interaction between the inoculation of the antagonist and the concentrations of the fungicide, between the concentrations of the fungicide and the time of application, and between the inoculation of the antagonist, the concentrations of the fungicide and the time of application did not give significant differences either in the percentages of the plants infected by the pathogen or seed production. the percentage of plants infected by the pathogen was lower on soil inoculated with the antagonist (31.6%) than on soil not inoculated with the antagonist (52.9%). the percentage of plants infected by the pathogen was lower on soil treated with the fungicide either at in vitro concentration (37.5%) or at field concentration (37.4%) than on the soil not treated *) a part of research funded by the government of indonesia fy 1991/1992 18 18 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati with the fungicide (61.5%). nevertheless, based on statistical analysis, the fungicide at in vitro concentration was not significantly different from that at field concentration. the percentage of plants infected by the pathogen on the soil sprayed with the fungicide at the same day of seed planting was lower (30.5%) than sprayed 7 days after planting (44.4%). the seed production on the soil inoculated with the antagonist (1893.3 kg/ha) was higher than on the soil not inoculated with the antagonist (1465.7 kg/ha). the production on the soil sprayed with the fungicide either at in vitro (1758.0 kg/ha) or at field concentration (1817.1 kg/ha) was higher than on the soil not sprayed with the fungicide (1247.2 kg/ha). the production on the soil sprayed with the fungicide at the same day of seed planting (2010.9 kg/ha) was higher than sprayed 7 days after planting (1564.2 kg/ha). the combination between the inoculation of the antagonist and the fungicide application at in vitro concentration at the same day of seed planting gave higher seed production (2391.2 kg/ha) than the inoculation of the antagonist (1711.7 kg/ha) or the fungicide application either at in vitro concentration (1771.9 kg/ha) or at field concentration (1939.1 kg/ha) at the same day of seed planting. however, based on statistical analysis, the interaction among the three treatments (the antagonist, the concentrations of the fungicide, and the time of application) was not significantly different. keywords: sclerotium rolfsii, glycine max, trichoderma, tebuconazole, antagonist introduction sclerotium rolfsii is a soil-borne fungal pathogen that can cause root rot and damping-off of crops, among others, soybean (glycine max) (agrios 1988). one of the control methods of the pathogen is by using antagonistic fungi (cook and baker 1983). some soil fungi especially trichoderma have been reported to be potential biocontrol agents of soil-borne fungal pathogens. according to upadhyay and mukhopadhyay (1986) t. harzianum was able to control the disease of sugarbeet seedlings as high as 88% under greenhouse conditions. under field conditions, integration of pcnb (pentachloronitro-benzene) and t. harzianum significantly reduced the incidence of sclerotium root rot (76% disease control) and increased the root, green foliage and sucrose yield per ha. cole and zvenyika (1988) reported that t. harzianum integrated with triadimenol fungicide enhanced disease control in tobacco caused by other soil-borne fungal pathogens, i.e. rhizoctonia solani and fusarium solani. under greenhouse conditions, four strains of t. harzianum suppressed damping-off of snapbean caused by s. rolfsii (papavizas and lewis 1989). tebuconazole (folicur 250 ec) is a fungicide that can control the pathogen (bayer 1990). the objective of the study is to determine the effect of trichoderma combined with tebuconazole to control s. rolfsii on soybean. 19 biotropia no. 7, 1994 materials and methods this research was conducted at the experimental plot of biotrop, bogor, indonesia. the soil type is latosol (ph ± 5). soybean variety, isolates of s. rolfsii and trichodernna, and fungicide s. rolfsii isolate bio-1 and soybean variety rinjani which was the most susceptible variety to the pathogen, were used in this study (dharmaputra and retnowati 1992). t. aureoviride bio-5 was used as antagonist because it caused the highest percentage of inhibition to the pathogen on potato dextrose agar (pda) of ph 5 (dharmaputra and retnowati 1992). tebuconazole was used as a fungicide to control the pathogen (bayer 1990). preparation of pathogen and antagonist inocula inoculum of the pathogen was prepared based on riker and riker (1936), while the inoculum of the antagonist was based on dharmaputra and suwandi (1989). for the preparation of the inoculum of the pathogen, a mixture of sand : corn : water ( 2 : 2 : 3 ) was put in plastic bags (2.5 kg/bag), sterilized in an autoclave for 1 h, and then incubated at room temperature for one night. for the preparation of the inoculum of the antagonist, a mixture of sand : husk : water ( 2 : 4 : 5 ) was treated in the same manner as the preparation of the pathogen's inoculum. five pieces of the pure cultures (5 mm in diameter each) of the pathogen and the antagonist (3 days old on pda) were grown on each medium. they were then incubated at 28 °c for 7 days. inoculation of the pathogen and the antagonist, application of the fungicide, and planting of soybean the pathogen was inoculated 4 days before the inoculation of the antagonist. it was mixed homogeneously on the surface of the soil in each plot (2.5 x 6 m 2 ). the inocula of the pathogen and the antagonist were 2 and 2.25 kg/plot, respectively. n, p and k (120 kg/ha) were given at the same day as the inoculation of the pathogen. soybean seeds were planted 7 days after the inoculation of the antagonist (aia). the distance between plants and between plots were 20 and 40 cm, respectively. 20 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati tebuconazole at concentrations of 100 g/ha (in vitro concentration) and 210 g/ha (field concentration) were applied using 2 methods: a) spraying in the planting hole at the same day as planting of soybean seeds b) spraying on the soil surrounding the plants (7 days after planting). soils that were neither inoculated with the antagonist nor the fungicide were used as controls. three replications (3 plots) were used for each treatment (including the control). observation of the percentage of infected plants was carried out 14 days after planting (dap), and of the soybean production at 90 dap. factorial in randomized completely block design (rcbd) was used in this study consisting of 3 factors: a) the antagonist (not inoculated and inoculated with t. aureoviride bio-5), b) the concentration of tebuconazole (0, 100 and 210 g/ha), and c) the time of tebuconazole application. results and discussion percentage of plants infected by the pathogen analysis of variance showed that the inoculation of the antagonist and the concentrations of the fungicide gave very significant differences in the percentage of plants infected by the pathogen; the time of fungicide application gave significant difference. the interaction between the inoculation of the antagonist and the concentrations of the fungicide; the concentrations of the fungicide and the time of fungicide application; among the inoculations of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 1). infected and non-infected plants at 14 dap are presented in figure 1. the percentage of plants infected by the pathogen was lower (34.1%) and significantly different on the soils inoculated with the antagonist than on the soils not inoculated with the antagonist (56.8%) (table 2). the percentage of plants infected by the pathogen was lower on the soil sprayed with the fungicide either in vitro (37.5%) or field (37.4%) concentration compared to the soil not sprayed with the fungicide (61.5%). nevertheless, the fungicide application at in vitro concentration did not differ significantly from field concentration. the percentage of plants infected by the pathogen was lower and significantly different when the fungicide was applied at the same day as the planting of soybean 21 biotropia no. 7, 1994 figure 1. non-infected (a) and infected (b) plants by sclerotium rolfsii 14 dap 22 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati table 1. analysis of variance on the effect of trichoderma aureoviride bio-5, tebuconazole and the time of fungicide application on the percentage of plants infected by sclerotium rolfsii table 2. the effect of the inoculation of trichoderma aureoviride bio-5, the concentrations of tebuconazole and the time of fungicide application on the percentage of plants infected by sclerotium rolfsii 23 biotropia no. 7, 1994 seeds (40.8%) than when the fungicide was applied 7 dap (50.1%) (table 2). it was assumed that the plants were already infected by the pathogen before spraying. it is interesting to note that the percentage of infected plants on soil sprayed with the fungicide at in vitro concentration combined with the inoculation of the antagonist (sft1a1 = 20%) was lower than on soil sprayed with the fungicide at field concentration (sf2t0a = 35.3%) (table 3), but based on statistical analysis the interaction among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 1). figure 2 shows the plants on soil inoculated with the pathogen 14 dap; while figure 3 shows the plants inoculated with the pathogen and sprayed with the fungicide at field concentration (210 g/ha) at the same day of seed planting. the plants on soil inoculated with the pathogen, sprayed with the fungicide at concentration of 100 g/ha (in vitro concentration) at the same day of seed planting, and inoculated with the antagonist is presented in figure 4. according to upadhyay and mukhodhyay (1986), a combination of pcnb with t. harzianum was able to control basal stem rot in sugar beet caused by s. rolfsii up to 76%. seed production analysis of variance showed that the inoculation of the antagonist and the concentrations of the fungicide gave very significant differences in seed production; the time of fungicide application gave significant difference. the interaction between the inoculation of the antagonist and the concentrations of the fungicide; the concentrations of the fungicide and the time of fungicide application, and among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant differences (table 4). the seed production of the soil inoculated with the antagonist was higher and significantly different (1863.3 kg/ha) than that of the soil not inoculated with the antagonist (1351.8 kg/ha) (table 5). according to elad et al. (1980) t. harzianum was able to control the disease on bean, cotton or tomato caused by s. rolfsii and rhizoctonia solani. the antagonist was also able to increase the production of bean. the soybean production of the soil sprayed with the fungicide either at in vitro concentration (1758.0 kg/ha) or at field concentration (1817.1 kg/ha) was significantly different from that not sprayed with the fungicide (1247.2 kg/ha) (table 5). nevertheless, the soybean production of the soil sprayed with fungicide at field concentration was higher than that of the soil sprayed with the fungicide at in vitro concentration. 24 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati table 3. percentage of soybean plants infected by sclerotium rolfsii with different treatments table 4. analysis of variance on the effect of trichoderma aureoviride bio-5, tebuconazole and the time of fungicide application on the production of soybean seeds 25 biotropia no. 7, 1994 figure 2. soybean plants on soil inoculated with sclerotium rolfsii, 14 dap figure 3. soybean plants (14 days after planting) on soil inoculated with sclerotium rolfsii and sprayed with tebuconazole at concentration of 210 g/ha at the same day of seed planting 26 figure 4. soybean plants (14 dap) on soil inoculated with sclerotium rolfsii and trichoderma aure-oviride bio-5, and sprayed with tebuconazole at concentration of 100 g/ha at the same day of seed planting the soybean production of the soil sprayed with the fungicide at the same day with the planting of seeds was higher and significantly different (1756.3 kg/ha) than that of the soil sprayed with the fungicide 7 dap (1458.5 kg/ha) (table 5). the combination of inoculation of the antagonist, and fungicide application at in vitro concentration at the same day as the planting of soybean seeds (s1f1t1a = 2391.2 kg/ha) gave higher soybean production than inoculation of the antagonist (s1f0t1 = 1711.7 kg/ha) or the fungicide application either at in vitro concentration (s1f1t0a = 1771.9 kg/ha) or at field concentration (s1f2t0a = 1939.1 kg/ha) (table 6), but based on statistical analysis the interaction among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 4). 27 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati biotropia no. 7, 1994 table 5. the effect of the inoculation of trichoderma aureoviride bio-5, the concentrations of tebuconazole and the time of fungicide application on the production of soybean seeds table 6. the production of soybean seeds with different treatments 28 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati conclusions t. aureoviride bio-5 or tebuconazole could be used to control s. rolfsii. they also could increase seed production. the fungicide was more effective when applied at the same day of seed planting than at 7 dap. the use of the fungicide at in vitro concentration combined with the use of the antagonist was more effective in decreasing the percentage of infected plants and increasing the seed production than the use of the fungicide at field concentration at the same day of seed planting. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the technicians of the laboratory of plant pathology, seameo biotrop. references agrios, g.n. 1988. plant pathology. academic press, new york. bayer. 1990. folicur; systemisches fungizid technische information. cole, j.s. and z. zvenyika. 1988. integrated control of rhizoctonia solani and fusarium solani in tobacco transplants with trichoderma harzianum and triadimenol. plant pathology 37: 271-277. cook, r. j. and k.f. baker. 1983. the nature and practice of biological control of plant pathogens. the american phytopathological society, st. paul, minnesota. dharmaputra, o.s. and w.p. suwandi. 1989. substrat untuk produksi besar-besaran trichoderma. (substrate for mass production of trichoderma). annual report. research collaboration between marihat research centre and biotrop. biotrop/tagr/89/736: 44-52. dharmaputra, o.s. and i. retnowati. 1992. the possibility of controlling sclerotium rolfsii on soybean (glycine max) using trichoderma, gliocladium and tebuconazole. seameo biotrop. internal report. elad, y., i. chet and j. katan. 1980. trichoderma harzianum: a biocontrol agent effective against sclerotium rolfsii and rhizoctonia solani. phytopathology 70: 119-121. papavizas, g.c. and j.a. lewis. 1989. effect of gliocladium and trichoderma on damping-off and blight of snapbean caused by sclerotium rolfsii in the greenhouse. plant pathology 38: 277-286. riker, a.j. and r.s. riker. 1936. introduction to research and plant diseases. john swift and co., st. louis. mo. sinaga, m.s. 1986. biological control of some soil-borne fungal pathogens of soybean (glycine max (l.) merr.) with gliocladium spp. ph.d. thesis. university of the philippines at los banos. sivan, a., y. elad and i. chet. 1984. biological control effects of a new isolate of trichoderma harzianum on pythium aphanidermatum. phytopathology 74: 498-501. upadhyay, j.p. and a.n. mukhopadhyay. 1986. biological control of sclerotium rolfsii by trichoderma harzianum in sugar beet. trop. pest management 32: 215-220. 29 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 5 phytochemical (elfahmi).cdr biotropia vol. 18 no. 1, 2011: 42 49 phytochemical study of cell culture jatropha curcas elfahmi , artri , komar ruslan belongs to the euphorbiaceae family which has a high economic value as a source of biofuel.. this plant has been reported containing toxic compounds such as curcin and phorbol ester and its derivatives which may become a problem if is processed to biofuel. in order to investigate the chemical constituents of this plant, a research on phytochemical and initiation of cell and organ culture has been carried out. collected from different regions in indonesia showed containing relatively the same profile of chemical contents. dominant compounds that were detected by gcms are hydrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as octadecanoate acid, ethyl linoleate, ethyl stearate, hexadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. no phorbol ester and its derivatives have been detected yet by the gcms method. callus and suspension cultures of have been established using murashige and skoog medium supplemented with 4 % (w/w) sucrose, solidified with 0.9% agar and growth hormon naa 2 mg/l : bap 0,5 mg/l. , stigmasterol, fucosterol, sitosterol, phorbol ester, fatty acid 1* 1 1 1 pharmaceutical biology research group, school of pharmacy, itb, bandung, indonesia jatropha curcas j. curcas jatropha curcas j. curcas jatropha curcas abstract introduction key words: jatropha curcas j. curcas j. curcas j. curcas belongs to the family euporbiaceae which has a multipurpose use such as medicinal plant and source of biofuel. as an alternative source of biofuel, has a high economic value. the development of this plant as source of alternative energy has been supported by researches and government regulations. since this plant is very potential, it has been cultivated in many regions in indonesia. several aspects have to be considered to develop this plant not only from the view point of production capacity but also the social condition of the population. aside from a potential source of biofuel, contains secondary metabolite compounds which have pharmacological and also toxicity effects. several compounds isolated from have been reported to be toxic, they are toxalbumin, cursin and phorbol ester. toxic effects of this plant caused skin irritation, vomiting, diarhea, etc. these * corresponding author : elfahmi@fa.itb.ac.id 42 toxicities are closely related to its chemical contents. several other secondary metabolites from have been reported such as terpenoid, coumarine, flavonoid, etc. the level of these chemical contents of cause the different toxicity levels of several strains of (gadir 2003, rug m & ruppel 2000). high phorbol ester content has been proved to be responsible for the toxicity of . this toxicity effect could not be eliminated using high temperature, since phorbol ester is very stable in high temperature. chemical reactions can reduce phorbol ester content of (aregheore 2003). these toxic compounds can be a problem in processing to biofuel. attempts to detoxificate toxic compounds of have been done in order to reduce the toxic effects (haas 2000). in addition to physical and chemical detoxification, biotechnological approaches could be applied. the toxic compounds may also be eliminated or reduced by regulation of their biosynthetic pathway. several genes which are responsible for synthesis of toxic compounds have been cloned, furthermore the expression and properties of the enzyme have been studied (qin . 2005). identification of genes which are responsible for cursin production give the challenges to regulate their inhibition activities. several tools to study the biosynthesis of toxic compounds of are needed. cell, tissue and organ culture are tools that can be used to regulate the production of secondary metabolites from plants. furthermore, these tools can be used for study at genetic level. to understand the secondary metabolites in relation to its toxicity and side effects, a research concerning phytochemical study of collected from wild types and cultivated regions have been done. in addition, cell, organ and supension cultures have been initiated. the aims of this study were to investigate the phytochemical contents of and to establish the cell culture of which can be used for further study. plant materials were collected from different regions in indonesia such as padang, lampung, garut, ciamis, tasikmalaya, bantul and gunung kidul. plant organs ie. leaves, fruits and stems were separated all materials were dried at 50 c and powdered. to isolate secondary metabolites from , each plant material from different organs and collection regions was extracted using soxhlet apparatus and solvent with increasing polarity such as n-hexane, ethyl acetate and methanol. extracts were checked by thin layer chromatography (tlc). chromatogram profile was used as the first indication to choose the best system for further isolation. n-hexane extract of leaves, stems and fruits were further fractionated using vacuum liquid chromatography using silica gel 60 h as adsorbents, the eluents were the mixtures of n-hexane and dichloromethane. the compositions of the eluents consisted of n-hexane 100 %, n-hexane dichloromethane (9:1) till 1:9, and dichloromethane 100%, yielded 12 fractions (fraction 1-12). the fractions were checked by tlc. several j. curcas j. curcas j. curcas et al. j. curcas j. curcas et al. j. curcas j. curcas et al. et al j. curcas j. curcas j. curcas j. curcas j. curcas materials and method 0 extraction, isolation and identification of secondary metabolites 43 phytochemical study of cell culture elfahmi .jatropha curcas et al fractions contained the dominant compounds. identification of isolated compounds and selected fractions were conducted by gcms. spectrums were analyzed and compared with available library. the explants used in this experiment were leaves and seeds. the sterile leaves or seeds were cut into slices and callus induction was obtained using media with different compositions. these media were modifications of the murashige and skoog (murashige & skoog 1962) or the gamborg's b5 medium (gamborg 1968). medium contained the combination of macro and micro-nutrient, carbon sources and other nutritions as well as growth hormones is needed. all media are supplemented with 4 % (w/w) sucrose and solidified with 0.9% agar. the callus cultures were grown under an l/d regime (16/8 h: 3000 lux) at 26°c. the compositions of growth hormones were as follows : 1) naa 2 mg/l : bap 0.5 mg/l, 2) naa 2.22 mg/l : bap 0.5 mg/l, 3) naa 2.5 mg/l : bap 0.5 mg/l, 4) naa 2.22 mg/l:bap 0.4 mg/l. cell suspension cultures were initiated by transferring callus clumps into 100 ml sterile conical flasks with 50 ml of liquid medium of the same composition as described above but without agar. cultures were incubated on a rotary shaker (100 rpm) at 26°c under an l/d regime (16/8 h: 3,000 lux, daylight l 36w/10, osram, germany). after 1 month, 50 ml of cell suspension cultures were transferred into a 500 ml conical flask, with fresh medium yielding a total volume of 300 ml. subcultures were prepared every 3 weeks by adding 100 ml of a full-grown cell suspension culture to 200 ml of fresh medium. phytochemical screening showed that contains alkaloid, flavonoid, tanin chatecate, saponin and steroid/triterpenoid (table 1). screening results were summarized in table 1. samples from other collection regions gave the same result as shown in the table. based on the screening results, the extraction process was used as previously mentioned in materials and method section. tlc analysis resulted that chromatogram profile of n-hexane, ethyl acetate and methanol extracts from collected from different places showed relatively the same, respectively. from gcms data of fraction and isolated compounds, several spectrums showed containing hydrocarbon, fatty acid and steroid compounds. however, phorbol ester and its derivatives reported having toxic effects, could not be detected. it was probably due to low content of phorbol ester. to confirm whether these compounds did not exist in from selected regions, another gcms measurement and or using different columns and conditions should be done. from these confirmations, no phorbol esters contents were detected. based on gcms, compound 1 has molecular weight of 412 with m/z: 83, 159, 255, and 300. these fragmentation patterns of 284 with m/z 73, 129 and 241 which were similar to octadecanoic acid (fig. 2). compound 3 has molecular weight of 152 with m/z 81 which were similar to 2,4 decadienal (fig. 3). dominant callus, cell, and suspension culture of j.curcas et al. j. curcas j. curcas j. curcas results and discussion were similar to β-stigmasterol (fig. 1). compound 2 has molecular weight biotropia vol. 18 no. 1, 2011 44 crude sample alkaloid flavonoid tancin quinon saponin steroid/ terpenoidchatecate gallat lea ves ciamis garut tasik + + + + + + + + + + + + + + + stem ciamis garut tasik + + + + + + + + + + + + + + + notes : + = contain secondary metabolites = did not contain secondary metabolites table. 1. phytochemical screening of collected from ciamis, garut and tasikjatropha curcas figure 1. gcms spectrum and fragmentation pattern of a) isolated compound of jatropha curcas and b). βstigmasterol compounds detected by gcms were hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as octadecanoate acid, ethyl linoleate, ethyl stearat, hexadecanoate acid etc and steroid such as stigmasterol, fucosterol, sitosterol. using leaf as the explant, callus can grow within 22 days, callus growth started with the folding of the green leaves, then callus slowly proliferated. callus was soft, compact and yellow green. the optimum callus formation was found at 82 days after inoculation. at day 90, callus started to be brownies and then died for more than 100 days. then callus were subcultured every month (fig. 4). while using seeds as the explant, callus can grow in a week after inoculation. callus was easily proliferated. formed callus was soft, compact, pale green. the optimum callus formations of seeds were found at 63 days after inoculation (fig. 5). using similar medium without agarose, cell suspension culture were grown, yielded cell suspension culture of seed 45 phytochemical study of cell culture elfahmi .jatropha curcas et al figure 2. gcms spectrum and fragmentation pattern of a) isolated compound of and b). octadecanoic acidjatropha curcas figure 3. gcms spectrum and fragmentation pattern of a) isolated compound of and b). 2,4-decadienal jatropha curcas 46 biotropia vol. 18 no. 1, 2011 a b c d e f g h figure 4. callus culture of leaf in growth phase (a) 22 days, (b) 29 days, (c) 45 days, (d) 52 days, (e) 59 days, (f) 65 days, (g) 82 days and (h) 90 days, incubation in ms medium added with naa 2 mg/l : bap 0.5 2mg/ml. j. curcas a b c d e f g h figure 5. callus culture of seeds in growth phase (a) 7 days, (b) 14 days, (c) 17 days, (d) 21 days, (e) 28 days, (f) 35 days, (g) 42 days, (h) 63 days, incubation in ms medium added with naa 2 mg/l : bap 0.5 2mg/ml. j. curcas a b c d figure 6. cell suspension culture of seeds in growth phase (a) 14 days, (b) 60 days, (c) 70 days, and (d) 80 days, , incubation in ms medium added with naa 2 mg/l : bap 0.5 2 mg/ml without agarose j. curcas 47 phytochemical study of cell culture elfahmi .jatropha curcas et al a b c d figure 7. cell suspension culture of leaf in growth phase (a) 14 days, (b) 25 days, (c) 40 days, and (d) 60 days, , incubation in ms medium added with naa 2 mg/l : bap 0.5 2 mg/ml without agarose j. curcas and leaf. but the amounts of cell suspension culture were not enough for further experiment with elicitor. scale up production of cell suspension culture is in progress (fig. 6 and 7) long time is needed to optimize callus formation (approximately 2 months for the seeds and almost 3 months for the leaves). therefore, the cell suspension cultures were not enough for the elicitor treatment. the secondary metabolites of original plant (seed, leaf) have different profiles compared to the callus and cell suspension. it could be concluded that the callus and cell suspension produced additional compounds. however, this research should be continued to determine the produced compounds of cell suspension and callus culture. some bands of tlc chromatogram of original plants were not found in the callus and cell suspension culture. analysis of n-hexane, ethyl acetate and methanol of leaves, stems and fruits of collected from different location in indonesia (sumatera barat, lampung, jawa barat dan yogyakarta) showed a similar profile (qualitatively). based on the gcms analysis of fractions and isolates of showed that the dominant compounds were hydrocarbon, fatty acid and steroid. gcms data did not show any phorbol ester compound and its derivaties which has a toxic effect. the growth of callus and suspension cultures of leaves and seeds of could be optimized in ms medium added with naa 2 mg/l : bap 0,5 mg/l. secondary metabolites content of callus and suspension culture showed different profiles. it can be concluded that the induction of callus and cell suspension can influence the production of secondary metabolites of plants. the authors would like to express their sincere thanks to the department of national education, directorate general of higher education, project of development of tropical biology, indonesia 2007, for the financial support. thanks are also due to seameo biotrop management and staff for the opportunity given to conduct this research, and to the school of pharmacy itb for providing all instruments needed for this research. conclusions acknowledgements j. curcas j. curcas j. curcas 48 biotropia vol. 18 no. 1, 2011 references aregheore em, becker k, makkar hps, 2003. detoxification of a toxic variety of jatropha curcas using heat and chemical treatments, and pr liminary nutritional evaluation with rats. s. pac. j. nat sci, 21, 50-56 gadir wsa, onsa to, ali wem, el badwi sma, adam sei, 2003. comparative toxicity of croton macrostachys, jatropha curcas and piper abyssinica seeds in nubian goats. small ruminant research 48, 61-67 gamborg ol, miller ra, oijama v. 1968. nutrient requirements of suspension cultures of soybean root cells. experimental cell research, 50, 151-158 haas w, mittelbach m. 2000. detoxification experiments with the seed oil from jatropha curcas l. industrial crops and products, 12, 111-118 murashige t, skoog f. 1962. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiologia plantarum, 15, 473-497 qin w, ming-xing h, ying x, xin-shen z, fang c. 2005. expression of a ribosome inactivating protein (curcin 2). j. biosci. 351-357. 49 phytochemical study of cell culture elfahmi .jatropha curcas et al biotropia no. 8, 1995: 39-44 effects of various soil environmental stresses on the occurrence, distribution and effectiveness of va mycorrhizae a.g. khan department of biological sciences, faculty of business and technology, university of western sydney macarthur, p.o. box 555, campbelltown n.s. w. 2560, australia introduction the vesicular arbuscular (va) mycorrhizal fungi are geographically ubiquitous soil inhabitants and form universal symbiotic relationship with plants from every phylum. these fungi link host plants with host soils and their biota in the mycorrhizosphere and play an important role in plant health, productivity and soil structure. although va mycorrhizal fungi do not show any host specificity, there is increasing evidence that various climatic and edaphic environmental factors such as land use and management practices, physical, chemical and biological properties of host soils and host plant characteristics influence their occurrence, taxonomic distribution and effectiveness. the interaction of these factors with vesicular-arbuscular mycorrhizae (vam) is poorly understood except in a few cases. it is now very clear that va mycorrhizal associations are ecologically significant factors that require more attention than previously accorded. this paper discusses the occurrence, distribution and significance of vam in environmentally stressed soil conditions that limit plant growth such as drought, waterlogging and salinity. key words: mycorrhizas/environmental factors/waterlogging/soil salinity/growth development stages/ inoculum. vam and drought evidence is accumulating which supports the contention that va mycorrhizal fungi are present in arid regions in xerophytes (khan 1974) and improved resistance of plants to water stress in controlled environments (bethlanfalavay et al. 1988), as well as in the field (sylvia et al. 1993), primarily due to improved nutritional status of plants. vam are known to occur in plants growing naturally in pioneer arid habitats of sand dunes, industrial waste lands and disturbed sites such as coal tips and 39 biotrop1a no. 8, 1995 strip-mined land (khan 1978). evidence has also been accumulating, linking the presence of vam infection to survival and improved plant growth in mine soil (khan 1981). vam has great potentials in the reclamation of drastically disturbed land and arid lands for agriculture. our findings, that different vam endophytes had quantitatively different effects on growth of onions in unsterilized coal waste (khan 1981) and the extent of colonization of onion roots was dependant upon inoculum density (khan 1988), may have practical implications for the establishment of a particular endophyte in arid conditions and the extent of plant response to the infection. vam symbionts can assist in the accelerated rehabilitation of unsightly industrial waste lands. increased growth of grasses and other indigenous flora on the dump sites may be obtained by either increasing the population of ecologically adapted va endophyte strain(s) and or transplanting plants pre-inoculated with suitable strain(s) instead of applying and fertilising top soil cover. commercial quantities of vam inoculum are now available for revegetation efforts of mined or cleared land. nurseries selling plants for revegetation or transplants into fumigated soils are the immediate beneficiaries of the vam biotechnology. also, the determination of abiotic factors governing efficiency of an isolate must be made before we can realistically select superior vam fungal isolates for use in the colonization of disturbed sites. vam and waterlogging vam occur over a wide range of soil water contents (khan 1974). vam colonization has been found in plants growing in frequently waterlogged soils (khan 1993a), as well as in free floating and submerged aquatic plants (khan & belik 1994). the vam fungi are aerobic, so soil water and aeration influence their distributions and effectiveness in soils and vam spore numbers along with vam infection are positively correlated to redox potential values (khan 1993b). a relationship between characteristics of vam infection and soil moisture gradient was found in a study of a casuarina cunninghamiana-transect on a creek embankment (khan 1993a). typical vesicles and arbuscules were found in roots from drier soil. roots from relatively wet soils lacked arbuscules but contained large liquid filled intracellular vesicles. typical vesicles and arbuscules were absent in flooded creek beds where roots were associated with coenocytic intracellular hyphae with abundant lipid contents. vam roots of submerged aquatic plants also harboured vesicles and/or coenocytic hyphae only, however, arbuscules were rare or absent. it is possible that the endobiont may be existing even in the aquatic plants recorded as non-mycorrhizal in the form of mycelia, not considered by the researchers as vam infection. vam fungi become non-functional in these habitats when the soil is saturated for extended 40 effects of various soil environmental stresses a.g. khan periods of time (liberia et al. 1983). however, when sites become seasonally dry, a functional symbiosis redevelops (khan 1993c). the variations between functional and non-functional associations may be common in wet and waterlogged habitats where conditions fluctuate on a seasonal or annual basis to favour or hinder mycorrhizal formation. the nature of the periodic non-functional associations, whether benign or a mild form of parasitism, is unknown and requires further study, as suggested by anderson et al. (1994). these fluctuations may be due to a change in redox potential from reducing to oxidising by the lateral liberation of oxygen from plant roots (kludge et al. 1993). the reports of the presence of vam under waterlogged conditions may be related to the (1) internal oxygen transport from the stem to the root due to presence of aerenchyma, (2) the oxygen release from the roots of large trees to support vam fungi, (3) strains of vam fungi in the sediments that can withstand highly reduced habitats, or (4) a combination of all these factors. but these have not been demonstrated and further studies are needed. the ability of vam to tolerate submersion became evident from various studies on the commercial production of inoculum by using aerated nutrient solutions in hydroponics or aeroponics. many global studies have established that vam fungi can infect permanently submerged plants under natural condition (khan and belik 1994). however, the process of infection and various biotic and abiotic factors in the aquatic environment affecting the process are unknown and further research is needed to understand the ecological significance of vam in these habitats. an understanding of the adaptions and mechanisms of plants living in or on water is valuable in explaining the interaction between plants, vam fungi and sediment/water including its biota as a dynamic system. mycorrhizal infection of roots causes significant physiological changes in the host plant which make the mycorrhizal plant grow and respond to environmental stresses differently from a non-mycorrhizal plant. although the occurrence of mycorrhizal infection in waterlogged and submerged aquatic plants has been known for some time, very few workers have attempted to identify species involved in these associations (khan and belik 1991). various global studies suggest that environmental factors such as soil moisture, soil type and soil gases influence the taxonomic distribution of vam fungi. spores of gigaspora margaritawere reported from the mycorrhizospheres of various wet and submerged plants (khan and belik 1994), which suggests that this species may be adapted to grow in these conditions of low oxygen availability and high p,n, and phytotoxin contents of the reduced sediment. although the presence of spores in aquatic sediments suggests that they probably enter the aquatic sediments through run-off from terrestrial ecosystems, information is accumulating that support the 41 biotropia no. 8, 1995 contention that there is significant genetic and physiological diversity in population of vam fungi from dissimilar environments (bethlenfalvay 1992). further research is needed to demonstrate this 'ecological specificity'. studies are also needed to separate the effects of flooding stress on the vam fungus from those on the host root. vam and soil salinity va-mycorrhizal fungi occur naturally in saline environment (khan 1974; khan and belik 1994) and several researchers who investigated the relationship between soil salinity and occurrence of mycorrhizae on halophytes have indicated that the number of vam spores or infectivity of vam fungi based on germination and subsequent hyphal growth decreased with increasing salt (juniper and abbott 1993). saline soils pose physiological stresses such as nacl toxicity and low osmotic potentials to plants growing in them. these stresses effect the growth of plants fungus or both. nacl (750 mg nacl per kg soil) reduced the number of vesicles and arbuscules, but not the growth of hyphae in the guayule roots inoculated with glomus intraradices (pfeiffer and bloss 1998). under higher nacl concentrations, endosymbionts may be reduced to coenocytic hyphae only as under waterlogged conditions and act almost as benign parasite. there are few studies indicating that mycorrhizal fungi can increase growth of plants growing in saline habitats (ojala et al. 1983; pond et al. 1984). va-mycorrhizal fungi may have the ability to protect plants from salt stress (hirrell and gerdemann 1980; rosendahl and rosendahl 1991), but the mechanism is not fully understood. the few data available at present suggest that fungi do have a potential to enhance plant growth but further studies are needed with additional plants, fungi under variable saline conditions. va-mycorrhizal fungi most commonly observed in saline soils are glomus spp. (juniper and abbott 1993) which suggest that this may be adapted to grow in saline conditions, but ecological specificity has not been demonstrated. there is evidence that vam species distribution is markedly changed with increased salinity (stahl and williams 1986). soil salinity and waterlogging often co-occur and interact in low-lying areas but no attempts have been made to study their collective effects on vam. va-mycorrhizal infection and vamycorrhizal fungal propagules have been observed in inundated salt marshes (khan and belik 1994). a comparative study of the effects of salinity and the co-related stress of low water potential on host plant and vam fungus is desirable before plant endophyte combinations are selected for. also, it is necessary to separate na effects from other salt effects. 42 effects of various soil environmental stresses a.g. khan aforementioned studies show that flood tolerant plants and va endobiont fungi exist together. whether these fungi are adapted to such conditions play any role in alleviating plant stress brought on by flooding under aquatic environments had yet to be elucidated. some recent studies in rice-growing areas such as india, thailand and usa and our preliminary investigations in australia suggest that va mycorrhizal endobiont would benefit both wetland and upland rice. but there are many biological principles which need to be explained, ie. how the infection occurs under flooded conditions and what initiates it, how the nutrients are obtained by the vam fungi under waterlogged conditions, what vam fungi are adapted to aquatic conditions, what conditions favour the symbiosis, etc. close attention should be given to the effects of edaphic, abiotic (physical and chemical) and biotic (soil microflora and fauna, including pathogens) factors on the mycorrhizal symbiosis while researching this area. references anderson, r.c., a.c. liberta and l.a. dickman. 1984. interaction of vascular plants and vesiculararbuscular mycorrhizal fungi across a soil moisture gradient. oecologia, 64: 111 -117. bethlenfalvay, g.j. 1992. mycorrhizae and crop production. in bethlenfalvay, g.j. and lindermann, r.g. (eds). mycorrhizae in substantial agriculture. asa spec. pub. 54, am. soc. agron. madison, wisconsin, p. 1-27. bethlenfalvay, g.j., r.s. thomas, s. dakessian, m.s. brown, and r.n. ames. 1988. mycorrhizae in stressed environments: effects on plant growth, endophyte development, soil stability and soil water. in whitehead, e.e. arid lands, today and tomorrow. westview press, boulder, co. p. 1015-1029. juniper, s. and l. abbott. 1993. vesicular and arbuscular mycorrhizae and soil salinity. mycorrhizae, 4: 45-57. khan, a.g. 1974. the occurrence of mycorrhizae in halophytes, hydrophytes and xerophytes, and of endogone spores in adjacent soils. j. gen. microbiol., 81: 7-14. khan, a.g. 1978. vesicular-arbuscular mycorrhizas in plants colonising black wastes from bituminous coal mining in the illawarra region of new south wales. new phytol., 81: 53-63. khan, a.g. 1981. growth responses of endomycorrhizal onions in unsterillised coal waste. new phytol., 87: 363-367. khan, a.g. 1988. inoculum density of glomus mosseae and growth of onion plants in unsterilized bituminous coal spoil. soil biol. biochem., 20 (5): 749-753. khan, a.g. 1993a. the occurrence and importance of mycorrhizae in aquatic trees of new south wales, australia. mycorrhizae. 3: 31-38. khan, a.g. 1993b. the influence of redox potential of formation of mycorrhizae in trees from wetland and water-logged areas of new south wales, australia. abst. 9th north am. conf. on mycorrhizae, aug. 812, 1993, university of guelph, guelph, ontario, p. 18. 43 biotropia no. 8, 1995 khan, a.g. 1993c. vesicular-arbuscular mycorrhizae (vam) in aquatic trees of new south wales, australia, and their importance at land-water interface. in gopal, b., hillbricht-ilkowska, a. and wetzel, r.g. (eds). wetland and ecotones: studies on land-water interaction, national institute of ecology, new delhi, p 173-180. khan, a.g. and m. belik. 1994. occurrence and ecological significance of mycorrhizal symbiosis in aquatic plants. in: varma a, hock b (eds). mycorrhiza: function, molecular biology and biotechnology. springer-verlag, heidelberg, germany, 1994 (in press). kludge, h.k., r.d. delaune and w.h. patrick jr. 1993. aerenchyma formation and methane and oxygen exchange in rice. soil sci.soc. am. j., 57: 386-391. liberta, a.e., r.c. anderson and l.a. dickman. 1983. vesicular-arbuscular mycorrhizae fragments as a means of endophyte indentification at hydrophytic sites. mycologia, 75: 169-171. ojala, j.c., w.m. jarrell, j.a. menoe and e.l.v. johnson. 1983. influence of mycorrhizal fungi on the mineral nutrition and yield of onion in saline soil. agron. j., 75: 255-259. pfeiffer, c.m. and h.e. bloss. 1988. growth and nutrition of guayale (parthenium argentatum) in a saline soil as influenced by vesicular-arbuscular mycorrhizae and phosphorous fertilisation. new phytol., 108: 315-321. pond, e.c., j.a. menoe and w.m. jarell. 1984. improved growth of tomatoes in salinized soil by vesicular-arbuscular mycorrhizal fungi collectors from saline soils. mycologia, 76: 74-84. rosendahl, c.n. and s. rosendahl. 1991. influence of vesicular-arbuscular mycorrhizal fungi (glomus spp.) on the response of cucumber (cucumis sativus l.) to salt stress. environ. exp. bot, 31: 313-318. stahl, p.o. and s.e. williams. 1986. oil shale process water affects activity of vesicular-arbuscular fungi and rhizobium four years after application to soil. soil biol. biochem. 18: 451-455. sylvia, d.m., l.c. hammond, j.m. bennett, j.h. haas and s.b. linda. 1993. field response of maize to a vam fungus and water management. agron. j. 85. 44 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf biotropia vol. 28 no. 2, 2021: 141 148 doi: 10.11598/btb.2021.28.2.1274 141 biocontrol potential of endophytic aspergillus spp. against fusarium verticillioides ron patrick cuagdan campos* and james kennard sanz jacob department of biological sciences, college of arts and sciences, isabela state university, echague 3309, isabela, philippines received 15 june 2019/accepted 8 september 2019 abstract the soil-borne fungus fusarium verticillioides is the causal agent of ear, stalk and root rot of maize resulting in severe reduction in yields and quality of infected products. endophytic fungi have been reported as potential candidates in controlling pathogens since they are considered strong plant mutualists that confer disease resilience to their host. the present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic aspergillus spp. associated with plectranthus amboinicus leaves against f. verticillioides. three fungal endophytes from the genus aspergillus, namely; a. tamarii, a. terreus and a. niger were isolated and identified from the leaves of p. amboinicus,. the fungal isolates were tested for their antagonism against f. verticillioides in dual culture plates. results indicated that the aspergillus endophytes can restrict the growth of f. verticillioides through varying mechanisms of antagonism. a. niger inhibited f. verticillioides by 47.37%, followed by a. tamarii (41.02%) and a. terreus (27.91%). dual culture observations revealed that a. tamarii and a. niger antagonized the growth of f. verticillioides via overgrowth mechanism while a. terreus employed antibiosis to restrict the pathogen. these varying degrees of antagonism by the aspergillus endophytes exhibited their potential as biocontrol agents and source of bioactive compounds. keywords: aspergillus, biocontrol, endophytic fungi, fusarium, rot introduction fusarium verticillioides is one of the most commonly reported soil-borne fungal pathogens infecting maize (abbas et al. 1998; bacon & hinton 1996). it is the causal agent of ear, stalk and root rot of maize. this fungus also produces secondary metabolites such as fumonisins that accumulate in maize kernels, consequently causing severe reductions in yields and quality of the products (leyva-madrigal et al. 2017; chu & li 1994). in order to maintain the abundance and quality of agricultural products world-wide, these plant pathogens need to be controlled and managed appropriately (pal & gardener 2006). currently, chemical fungicides are the most effective agents in preventing the infection of f. verticillioides. however, efforts to control postharvest diseases employing synthetic chemical control agents pose danger to the environment as they affect soil microorganism diversity (cardoso et al. 2010). endophytic fungi are microorganisms that inhabit internal plant tissues, usually involving metabolic interactions without apparent symptoms (bacon & white 2000; petrini 1991; wilson 1995). growing evidences from several studies indicate that endophytes are found in all plants and are extremely abundant and diverse (arnold et al. 2000). endophytic fungi are believed to be strong plant mutualists which can produce increased resilience against pests and plant pathogens (carroll 1988). since these microorganisms are systemically distributed throughout the host via metabolic translocation, they are noteworthy candidates for biological control (rai et al. 2007). studies have been done on the antagonistic mechanisms and actions, particularly the efficacy, of many endophytic fungi for their biocontrol potential against different plant pathogens (rahman et al. 2009). furthermore, considering their long-lasting effects and not requiring repeated periodic *corresponding author, email: rpcampos023@gmail.com biotropia vol. 28 no. 2, 2021 142 application, the use of fungi for biocontrol has more advantages than chemical fungicides (okigbo & ikediugwu 2000). several studies recorded that aspergillus species acted as biological control agents against fungal pathogens (fusarium oxysporum, pythium spp. and sclerotinia sclerotiorum) through competition, mycelial lysis, mycoparasitism and antibiosis via the synthesis of volatile and/or non-volatile metabolites (gomathi & ambikapathy 2011; daami-remadi et al. 2006; bhattacharyya & jha 2011). hence, the present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic aspergillus spp. associated with plectranthus. amboinicus against f. verticillioides. materials and methods collection of plant material mature and healthy leaf samples of plectranthus amboinicus were collected in may 2018 from echague, isabela (16.6701° n, 121.7171° e). the plant materials were authenticated based on taxonomic characters through the assistance of an experienced botanist. samples were transported in sterile polypropylene bags and processed within 6 hours of collection. isolation of endophytic fungi plant samples were surface sterilized and the endophytic fungi were isolated using the method described by kusari et al. (2009) with minor modifications. leaves of p. amboinicus were washed and rinsed with running tap water and cut into 10 mm (length) by 5 mm (width) segments. each segment was then surface sterilized by sequential immersion in 75% ethanol for 2 minutes, 1% sodium hypochlorite (naocl) for 3 minutes, and then once again in 75% ethanol for 1 minute. the leaf segments were finally rinsed three times in sterile distilled water to remove excess sterilant and blot dried in sterile filter paper. the leaf segments were later inoculated onto potato dextrose agar (pda) plates supplemented with streptomycin (1 ml/l) to suppress bacterial growth. four (4) leaf segments were equidistantly placed on each amended pda plate. the plates were then sealed with parafilm and incubated at 28 °c until the growth of endophytic fungi was detected. the hyphal tip of each endophytic fungi growing out from the leaf segments were separately transferred into new amended pda plates and routinely maintained. identification of endophytic fungi the endophytic fungi were identified according to their macroscopic and microscopic characteristics, particulalrly the fruiting structures and spore. colony morphology of the endophytes was observed on coconut water agar (cwa), potato dextrose agar (pda) and malt extract agar (mea). to ascertain the identification of species, a microscopic examination of morphological structures was conducted using the agar block technique. the identification of fungi was done using the dichotomous keys and descriptions provided by quimio and hanlin (1999) and samson et al. (2014). source of test fungus pure cultures of the pathogenic fungus f. verticillioides were obtained from the maintained cultures of mycology laboratory, college of arts and sciences, isu-echague, isabela. the cultures were transferred into sterilized potato dextrose agar (pda) plates and incubated at ±30 °c to allow growth of the mycelia for seven (7) days. in vitro antagonistic activity the in vitro biocontrol potential and antagonistic activity of the endophytic fungi were tested against the pathogenic fungus f. verticillioides using the dual culture method described by matroudi et al. (2009) and john et al. (2010). agar plugs of each endophytic fungi and pathogen were obtained from the edge of 7-day-old pure cultures using sterile cork borer. the plugs were aseptically transferred 20 mm apart respectively on the center of 90 mm mea plates. control plates were inoculated with the pathogenic fungi alone. plates were incubated at 37 °c for 15 days. the interactions exhibited by the co-cultures were monitored daily and the diameter growth of both endophyte and pathogen were recorded at 5, 10 and 15 days, respectively. all control and test plates were biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 143 conducted in triplicates. percentage inhibition was calculated as compared to control (gaspar et al. 2004). the growth inhibition was calculated by using the formula: statistical analysis each of the tests was carried out using completely randomized design (crd) with three replicates for each treatment. all means were treated statistically using one-way analysis of variance (anova) and compared by tukey’s honest significant difference test at p < 0.05 using ibm™ spss v25. results and discussion identification and characterization of endophytic fungi three fungal isolates belonging to aspergillus species were selected and their morphology and growth characteristics on cwa, pda and mea were recorded for the determination of their biocontrol potential (table 1). the colony color of a. tamarii endophytes ranged from yellow to olive green which turned dark green with age (fig. 1a). the margin and form are both filamentous and elevation is slightly raised. production of colorless exudates and brown-black sclerotia was observed in various plates after extended incubation. the colonies of a. terreus showed cinnamon-brown color with floccose white mycelia which eventually turns brown to yellow-brown consisting of a dense felt of conidiophores (fig. 1b). reverse morphology was brownish to orange in color, indicating the secretion of metabolites into the medium. sclerotia was absent. meanwhile, a. niger has a distinct blackbrown colony (fig. 1c). it was both filamentous on margin and form and has umbonate elevation. the colonies initially grow with feltlike yellow to white hyphae, turning black with the formation of conidia. formation of black sclerotia and black exudate beads was also observed. the microscopic structures of the endophytes were observed in a microscope through the agar block method. the vesicles of a. tamarii have sub-globose shape, while a. terreus and a. niger have pyriform and globose shaped vesicles, respectively (fig. 1d-1f). the asexual conidia of a. terreus are smooth and hyaline, while a. tamarii conidia are finely roughened compared to the conidia of a. niger which are echinulate (fig. 1g-1i). the endophytic fungi also exhibited some common morphological structures, such as biseriate conidial heads and septate hyaline hyphae. the obtained microscopic descriptions of the aspergillus endophytes coincide with the keys and descriptions provided by samson et al. (2002). table 1 macroscopic and microscopic characteristics of the aspergillus endophytes macroscopic characteristics microscopic characteristics endophytic fungi culture media colony color reverse color colony density shape of vesicle texture of conidia seriation a. tamarii cwa browngreen white abundant subglobose smooth/finely roughened biseriate pda parrot green white luxuriant mea yellow light yellow abundant a. terreus cwa beige tan sparse pyriform smooth biseriate pda cinnamon brown sparse mea cream yellow yellow orange abundant a. niger cwa grey white luxuriant globose echinulate biseriate pda black cream abundant mea brownblack black luxuriant biotropia vol. 28 no. 2, 2021 144 figure 1 colony morphology of (a) a. tamarii; (b) a. terreus; and (c) a. niger; conidiophore of (d) a. tamarii; (e) a. terreus; and (f) a. niger; conidia of (g) a. tamarii; (h) a. terreus; and (i) a. niger in vitro antagonistic activity of endophytic fungi results of this study showed that the endophytes can variably restrict the growth of f. verticillioides (table 2). among the three fungi, a. niger produced the largest inhibition on the mycelial growth of f. verticillioides by 47.37%, followed by a. tamarii (41.02%) and a. terreus (27.91%). f. verticillioides recorded a mean radial growth diameter of 69.91 mm on the control plate. in comparison, the pathogenic fungi produced radial growth of 49.85 mm, 60.92 mm and 44.48 mm in the dual culture with a. tamarii, a. terreus and a. niger, respectively. the smaller radial growth on the dual culture plates indicated the presence of antagonism between the pathogen and the endophytes. c b a d e f g h i biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 145 table 2 radial mycelial growth and percent inhibition of f. verticillioides by three endophytic strains of aspergillus after 14 days of incubation fungal antagonists f. verticillioides radial mycelial growth (mm) percent inhibition (%) a. tamarii 49.85±10.64a 28.69% a. terreus 60.92±11.51b 12.86% a. niger 44.48±4.39a 36.38% control 69.91±16.31b notes: values are means of three replications. means in the same column with different superscript indicate significant difference at p ≤ 0.05. all the fungal antagonists inhibited the mycelial growth of f. verticillioides through different antagonistic mechanisms (fig. 2). observations on the dual culture plates of a. tamarii and f. verticillioides indicated that the endophyte antagonized the pathogen through overgrowth mechanism. the overgrowth is achieved when a fungus exhibits a higher growth rate, tolerance against metabolites produced, and a higher capacity of antibiotic production (mathiavanan et al. 2000). a noticeable change in the morphology of a. tamarii in all co-culture plates was also observed wherein the isolates became highly floccose and conidia were rarely present (fig. 2a). this suggested that the a. tamarii isolates were adapting and responding to the presence of f. verticillioides. aspergillus species are known to grow via the formation of a floccose mycelium, producing aerial hyphae that are capable of enhanced oxygen absorption and increased rates of respiration (rahardjo et al. 2005). isolates of a. niger outgrew those of f. verticillioides implying that the antagonism involved is overgrowth mechanism. macroscopic observation also revealed that the a. niger has a mutually intermingled growth with f. verticillioides without any zone of inhibition, indicating the failure of the production of antibiotics either by the pathogen or by the antagonist. a. terreus zone of inhibition was clearly observed in which there was a conspicuous space between the antagonist and the test fungus (fig. 2e). the inhibition zone is observed in all co-culture plates of a. terreus and f. verticillioides and mycelial growth of both fungi is either stunted or severely decreased in the region. the formation of a zone of inhibition is an indication of the production of antibiotic substances either by the pathogen against antagonistic fungi or vice versa (gomathi & ambikapathy 2011). the capacity of aspergillus species to inhibit fusarium isolates has been reported by several authors. a. niger initiated lysis of f. oxysporum mycelium through antibiosis (patibandn & sen 2007; dwivedi & enespa 2013). a. niger, a. tamarii and a. terreus successfully controled the growth of fusarium sambucinum and phytophthora erythroseptica (abdallah et al. 2015). a. niger was also reported as one of the best antagonists for several soil-borne, seed-borne and foliar plant pathogens (kamil et al. 2009; ahmed & upadhyay 2009). moreover, certain atoxigenic strains of aspergillus have been reported to competitively exclude aflatoxin-producing strains during crop infection and thereby reduce aflatoxin contamination. one of these, af36, has been registered as a biological control for the competitive exclusion of aflatoxin producing fungi from cottonseed (cotty 2018). aspergillus species have diverse adaptations and responses to cellular stress which allows them to be resilient in the presence of other organisms (abdallah et al. 2015). these include the deployment of biophysically diverse compatible solutes and functionally diverse protein‐stabilization proteins; hyperaccumulation of melanin in the cell wall; oxidative stress responses; ability to resist high temperatures; production of extracellular polymeric substances (eps) and formation of biofilms; and the ability to compete with other microbes. biotropia vol. 28 no. 2, 2021 146 figure 2 antagonism of f. verticillioides with a. tamarii (a, d), a. terreus (b, e) and a. niger (c, f); microscopic hyphal interactions of a. tamarii and f. verticillioides (g) and a. niger (h) with f. verticillioides conclusion the three aspergillus endophytes, namely a. tamarii, a. terreus and a niger isolated from the foliar segments of plectranthus amboinicus restricted the growth of fusarium verticillioides and the mechanisms involved overgrowth and antibiosis. the capacity of these endophytes to restrict the growth of f. verticillioides implied that these organisms can be exploited as possible alternatives to chemical control agents. acknowledgements the authors acknowledge the provision of materials and facilities by the department of biological sciences of isabela state university, echague, philippines. we also gratefully acknowledge dr helen c. ramos and dr florenda b. temanel for their assistance in checking this manuscript. c b a f e d endophyte endophyte endophyte endophyte endophyte endophyte pathogen pathogen pathogen pathogen pathogen pathogen g h f. verticillioides hyphae a. tamarii hyphae a. niger hyphae f. verticillioides hyphae biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 147 references abbas hk, mirocha cj, meronuk ra, pokorny jd, gould sl, kommedahl t. 1988. mycotoxins and fusarium spp. associated with infected ears of corn in minnesota. appl environ microbiol 54:1930-3. abdallah r, jabnoun-khiareddine h, mejdoub-trabelsi b, daami-remadi m. 2015. soil-borne and compostborne aspergillus species for biologically controlling post-harvest diseases of potatoes incited by fusarium sambucinum and phytophthora erythroseptica. j plant pathol microbiol 6:313. ahmed m, upadhyay rs. 2009. rhizosphere fungi of tomato: their effect on seed germination, seedling growth and the causal agent of tomato wilt. j mycopathol res 47:99-110. arnold ae, maynard z, gilbert gs, coley pd, kursar ta. 2000. are tropical fungal endophytes hyperdiverse? ecol lett 3:267-74. bacon cw, hinton dm. 1996. symptomless endophytic colonization of maize by fusarium moniliforme. can j bot 74:1195-202. bacon cw, white jf. 2000. microbial endophytes. new york (us): marcel decker inc. bhattacharyya pn, jha dk. 2011. optimization of cultural conditions affecting growth and improved bioactive metabolite production by a subsurface aspergillus strain tsf 146. intl j app biol pharmacol tech 2:133-43. cardoso ra, pires lta, zucchi td, zucchi fd, zucchi tmad. 2010. mitotic crossing-over induced by two commercial herbicides in diploid strains of the fungus aspergillus nidulans. gen mol res 9:231-8. carroll gc. 1988. fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. ecology 69:2-9. chu fs, li gy. 1994. simultaneous occurrence of fumonisin b1 and other mycotoxins in moldy maize collected from people’s republic of china in regions with high incidences of esophageal cancer. appl environ microbiol 60:847-52. cotty pj. 1994. influence of field application of an atoxigenic strain of aspergillus flavus on the populations of a. flavus infecting cotton bolls and on the aflatoxin content of cottonseed. phytopathology. 84:1270-7. daami-remadi m, jabnoun-khiareddine h, ayed f, hibar k, znaïdi iea. 2006. in vitro and in vivo evaluation of individually compost fungi for potato fusarium dry rot biocontrol. j biol sci 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37(4):277-85. rai r, dash pk, prasanna bm, singh a. 2007. endophytic bacterial flora in the stem tissue of a tropical maize (zea mays l.) genotype: isolation, identification and enumeration. world j microb biot 23:853-8. samson ra, visagie cm, houbraken j, hong sb, hubka v, klaassen chw, ... , frisvad jc. 2014. phylogeny, identification and nomenclature of the genus aspergillus. stud mycol 78:141-73. wilson d. 1995. endophyte-the evolution of a term, and clarification of its use and definition. oikos 73:274-6. biotropia no biotropia no. 16, 2001 : 1 9 in vitro propagation of angiopteris evecta using spores damien cupitt, poonam bhatia* and nanjappa ashwath primary industries research centre, school of biological and environmental sciences, central queensland university, rockhampton, qld 4702, australia abstract techniques of establishing angiopleris evecta plants in vitro were studied. soaking of a. evecta spores in water for 24 hours markedly reduced spore contamination. soaking of the spores in 1 -2 % of sodium hypochlorite for less than 5 minutes allowed satisfactory disinfestation without affecting spore viability. lower concentration of minerals (1/4 ms), presence of charcoal in the medium and exposure of the spores to light were crucial for spore germination and gainetophytc development of a. evecta. keywords: angiopleris evecta i k in g fern / spore / ms medium / bleach / light / tissue culture / sporophyte / gamctophytc abbrevations ba benzylaminopurine; bb bold's basal medium; pcpa para-chlorophenoxyacetic acid; 2,4-d 2,4-dichlorophenoxyacetic acid; iaa indoleacetic acid; iba indolebutyric acid; kinetin -6-furfurylaminopurine; mm moss's mineral medium; ms murashige & skoog media; naa naphthaleneacetic acid; noa naphthoxyacetic acid. introduction the fern and fern allies are the surviving members of the earliest lineages of vascular plants. none of these primitive plants produce either seeds or flowers; instead they reproduce via single-celled spores. ferns are usually found in moist terrestrial or aquatic environments and their size may vary from a few centimeters to many meters. angiopteris evecta is known as king fern or giant fern and it belongs to the family marattiaceae. it is a magnificent fern, the fronds are reputedly largest in the world, and are about 5 m long, arching, semi-weeping, bi-pinnate, and glossy (figure la). lower pinnules have earlike lobes at the base. sporangia always occur `* corresponding author : e-mail address : p.bhatia@cqu.edu.au fax: 61-7-4930 9255 1 biotropia no. 16 , 2001 figure 1. a. mature sporophyte with well developed fronds; b. pinnule showing sori; c. sporangia in dense clusters; d&e. developing gametophytes in ms vi c medium; f. germinating sporophyte from gametophyte. 2 in vitro propagation of angiopteris evecta using spores damien cupitt et al. in dense clusters of five to eight opposite pairs (figure ib & ic). the king fern is found in australia (queensland and new south wales), malaysia, polynesia and southern and northeastern parts of india. it also occurs in new guinea and indonesia (s. marsterson; department of e.p.a., rockhampton, pers. com.). in northeastern parts of india, the massive stem is cooked and eaten by the tribes. an intoxicating drink called 'ruchshi' is also made out of it. the plant yields aromatic oil that is used for perfuming coconut oil in south sea islands (manickam and irudayaraj 1992). conventionally, king ferns are almost impossible to propagate from spores but may be reproduced vegetatively from the fleshy earlike projections which are commonly known as auricles. plant development is very slow and it may take 12 months or more before a small frond and sufficient roots develop to support the plant (blomberry and maloney 1994). tissue culture can prove to be the fastest and the most effective method of multiplication of the king fern. studies on in vitro spore propagation and culture media requirements of different ferns have been conducted by bernabe et al. (1999); amoroso and amoroso (1998); fernandez et al. (1997); goller and rybczynski (1995) and dong and su (1993). since spores remain the only source of fern propagation, considerable importance has been placed on spore disinfestation and culture media selection for different ferns. fernandez et al. (1997) observed optimal growth of blechnum spicant gametophytes in ms (murashige and skoog 1962) liquid medium spread over a 2% agar and exposed to 16 hours of light. borelli et al. (1990) used both calcium and sodium hypochlorite solution for disinfesting spores and they obtained the best results with 2% sodium hypochlorite solution. borelli et al. (1990) also observed better germination of the ferns cyathea schanschin and dicksonia sellowiana on john and knop's media after 4-8 weeks, with the prothalli developing 30-40 days later. in a similar study, goller and rybczynski (1995) disinfested spores of cyathea australis using 3% chloramine with tween. they reported better germination of spores on anderson's medium supplemented with 80 mg t1 of adenine sulfate solidified by 0.8% agar. no studies are currently available for a. evecta either for spore disinfestation or for selecting suitable culture medium. the present study was carried out to optimize spore disinfestation procedures and to determine suitable culture medium and growth conditions for establishment of a. evecta in tissue culture. materials and methods collection and storage of spores recently, matured pinnules containing sori were collected fresh from the rockhampton city council botanical garden, rockhampton, australia. after a close microscopic examination, closed sori containing spores were isolated from the 3 biotropia no. 16, 2001 fronds for disinfestation. another set of pinnules was stored in a brown paper bag to allow drying without fungal infection. effect of soaking spores on contamination the sori were separated from the pinnulus by scraping with a scalpel. the released sporangia were then collected onto a piece of paper. these sporangia were exposed to three treatments. in treatment 1, sporangia were filtered through a 106 um sieve using demineralised water and the filtrate was collected in a glass jar. the sporangia were allowed to soak in water for approximately 24 hours. once soaked, the spore solution was microfuged (x 10,000 rpm) for five minutes to concentrate the spores prior to sterilisation (sieved and soaked). the spore solution (1 ml) was inoculated onto agar plates to test for contamination and germination. in treatment 2, the unsieved sporangia were used, and they were soaked in sterile water for 24 hours before microfuging as in treatment 1 (not sieved but soaked). in treatment 3, plant debris were removed by sieving and the spores were not soaked in water (sieved but not soaked). the rest of the operations were the same as for treatment 1. in treatment 4, spores were neither sieved nor soaked (neither sieved nor soaked). spore disinfestation two concentrations (1% and 2%) of sodium hypochlorite were used for disinfestation, with the exposure times of 3, 5, 7 and 9 minutes. a known volume (1 ml) of solution containing sporangia was placed in an eppendorf tube and 1.5 ml of 1% or 2% naocl was added. after 30 seconds of soaking, the eppendorf tube was centrifuged for 3 minutes. one milliliter solution was removed from the top of the eppendorf tube and a milliliter of sterile water was added. the contents were centrifuged again for 2 minutes. the washing with sterile water was repeated 4 times, to remove all traces of naocl prior to inoculation. inoculation using aseptic bench top tunnel technique, culture tubes (50 ml capacity, plastic) were inoculated with two drops of spore solution and the tubes sealed before being placed in a controlled environment room. media media with low nutrient content were selected, because the spores, like seeds, contain all required nutrients for early growth. wide ranges of media were used. these include ms, msc (ms media + 1 g/l charcoal), ms%c (ms medium with v* nutrients +'/« sugar + 1 g/l charcoal), ms low (ms medium + low hormones*), ms'/z low (ms medium with vi nutrients + low hormones*), ms% low (ms medium 4 in vitro propagation of angiopteris evecta using spores damien cupitt et al. with 1/4 nutrients + low hormones*), fn (fern normal), bold's basal (bischoff and bold 1963) and moss's mineral media. *here de fossard's (1981) broad spectrum low hormones were used. low hormones consisted of auxins (iaa+iba+naa+noa+2,4-d+pcpa; 0.1 um each) and cytokinins (bap+kinetin; 0.1 um each). incubation one set of inoculated tubes was randomly stacked in a clear plastic bag and incubated in a controlled environment room (cer) at 30 °c with a light intensity of 100 uem~2s~'. the cer was maintained at a photoperiod of 12 hours light and 12 hours dark. the second set of tubes was sealed within a black plastic bag to simulate dark environment that is often found in dense rainforest floor where mature ferns are usually found. both sets of tubes were placed side by side in the cer to ensure that they were exposed to similar climatic conditions except for light. results effect of soaking on contamination and spore germination pre-soaking of spores for 24 hours before disinfestation reduced contamination. the contamination rate could be reduced to as low as 3% with pre-soaking treatment alone. most of this contamination was caused by fungi (penicillium sp. and aspergillus sp.) and rarely by bacteria. contamination was as high as 75% when the spores were neither sieved nor soaked. the sieving and soaking reduced the contamination rate to 13% (table 1). sieving had very little effect on contamination, but the soaking markedly reduced the contamination rate. table 1. effect of pre-soaking treatments on contamination and spore germination of a. evecta 5 biotropia no. 16, 2001 response of a. evecta spores to naocl the degree of contamination varied from 0% to 38%, with 1% naocl and from 0% to 9% at 2% naocl (table 2). exposure of king fern spores to 1 or 2% naocl for either 7 or 9 minutes resulted in complete disinfestation. however, none of these spores germinated when the exposure time exceeded 5 minutes. thus, the treatment of king fern spores with 2% naocl solution for less than 5 minutes appear to provide the best results. the tubes that were inoculated with untreated spores were fully contaminated suggesting that disinfection is a must for king fern spores. table 2. contamination status of a. evecta spores in response to naoci concentration and time of exposure (number of tubes used per treatment varies from 8 to 38) effect of culture media on spore germination the highest germination (69%) was observed in msvic medium followed by moss's mineral medium (25%), hold's basal medium (17%), ms 'alow (4%) and ms'/tlow (0%) (table 3). spores failed to germinate on full strength ms medium, either with or without hormones and charcoal. reduced concentration of ms medium and the presence of charcoal maximized king fern spore germination (figure l d & le). table 3. effect of culture media on spore germination of a. evecta (please see methods for description of acronyms) 6 in vitro propagation of angiopteris evecta using spores damien cupitt et al. role of light in germination of a. evecta spores light played a major role in 'the germination of a. evecta spores. irrespective of soaking treatment, the length of exposure to naocl, or the type of medium used, almost all the spores failed to germinate in the absence of light (table 4). gametophytes that grew well in tissue culture conditions produced sporophytes within 6 months (figure if). the sporophytes were then successfully transplanted and raised in a potting mix. table 4 the role of light on spore germination of a. evecta light dark number of tubes inoculated 170 49 number of tubes showing spore germination 15 2 percentage of tubes showing spore germination 9 2* * tubes germinated only after the cultures were exposed to light discussion pre-disinfestation treatments are usually beneficial in reducing contamination, to a certain level, without harming the explant. this experiment gave a very useful clue for the use of pre-disinfestation treatment for decontaminating spores of angiopteris evecta. contamination percentage was reduced to as low as 3% when sporangia were soaked in water for 24 hours. it is likely that the bacteria and fungi that were adsorbed onto the fern spores become more susceptible to naocl treatment if they are soaked in water prior to disinfestation. prior to soaking of sporangia, the whole material was passed through 106 urn sieve (treatment 1) with the aim of reducing the quantity of material to be disinfested, as it included a considerable amount of sori cases and dried leaf pieces. sieving did not seem to make any difference to contamination rates both in soaked and unsoaked treatments. our results revealed that 2% naocl is highly effective in disinfesting a. evecta spores at both 3 and 5 minutes. these results are in agreement with those of borelli et al. (1990) who obtained best results with 2% sodium hypochlorite for cyathea schanschin and dicksonia sellowiana spores. in contrast, goller and rybczynski (1995) obtained better results when they disinfested intact leaves containing the sori of cyathea australis with 3% chloramine and tween for 30 minutes. the length of exposure of a. evecta spores to sodium hypochlorite also had a considerable bearing on the success of germination. our results showed that three minutes exposure to sodium hypochlorite reduced contamination to a considerable 7 biotropia no. 16, 2001 extent whereas, 7 and 9 minutes exposure proved to be lethal as the spores failed to germinate. based on these results, we recommend the exposure of a. evecta spores to high concentration (1-2%) of naoclfor short duration (<5 minutes). the pattern of spore germination in various media compositions appears to reflect the need for low concentrations of mineral nutrients and the presence of charcoal for a. evecta spore germination. this observation is in consistence with the findings of khoo and thomas (1980), who found that high mineral salt concentrations tended to retard spore germination and sporophyte formation of adiantum raddianum cv tassel. however, the current findings contrast with those of fernandez et al. (1997) who reported that ms full strength liquid media being optimal for growth of blechnum spicant gametophyte. apart from ms media, andersen's media for cyathea australis (goller and rybczynski 1995), jone's media and knop's solution, respectively, for cyathea schanschin and dicksbnia solviana (borelli et al. 1990) and knudson's media for cyathea spinulosa (agrawal et al. 1993) have also been advocated. similar to our findings, wardle et al. (1998) recommended the use of charcoal for the spore germination. spores are analogous to seeds and they contain all the required nutrients for early growth, therefore it can be justified to use low nutrient media during initial stages of germination. light is an important factor and it plays a significant role in the germination of a. evecta spores. the results of this study concur with the field observations (s. marsterson; department of e.p.a., rockhampton, pers. com.) where the king fern seedlings usually occur in patches of rainforests that have been exposed to light possibly due to fire or other sources of damage to canopy. light has been found to govern the development stage of whole gametophyte (turnwald et al. 1999). kiss and kiss (1998) reported that light frequency in the red region is found to promote spore germination more than in far-red region. light not only regulates the growth mechanism in haploid gametophyte, but it also affects development of sporophyte due to involvement of a photoreceptor phytochrome (christensen et al. 1998). presence of light has been shown to increase nuclear dna and this increase is known to have an influence on fern spore germination (raghavan 1993). this study has demonstrated the importance of soaking of a. evecta spores to minimize contamination, sensitivity of king fern spores to high mineral concentration of the media and the need for light and charcoal for its spore germination. these data highlight the desirable conditions for disinfestation and germination of king fern spores. references agrawal dc, ss pawar, af mascarenhas. 1993. cryopreservation of spores of cyathea spinulosa wall, ex. hook. f. an endangered tree fern. j plant physiol 142: 124-126. amoroso cb, vb amoroso. 1998. spore culture studies on some economic ferns of mindanao, philippines. acta horticulturae 461: 231-235. 8 in vitro propagation of angiopteris evecta using spores — damien cupitt et al. bernabe n, g williams-linera, m palacios-rios. 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.study on the spore propagation of the ostrich fern, matteuccia struthiopteris todaro. acta horticul.turae sinica 20: 274-278. fernandez h, am bertrand, i feito, r sancheztames. 1997. gametophyte culture in vitro and antheridiogen activity in blechnum spicant. plant cell tissue and organ culture 50: 71-74. goller k, jj rybczynski. 1995. in vitro culture used for woody fern cyathea australis (r.br.) domin vegetative propagation. acta societatis botanicorum poloniae 64: 13-17. khoo si, mb thomas. 1980. studies on the germination pf fern spores. the plant propagator 26: 11-15. kiss hg, jz kiss. 1998. spore germination in populations of schizaea pusilla from new jersey and nova scotia. international journal of plant sciences 159: 848-852. manickam vs, v irudayaraj. 1992. pteridophyte flora of the western ghats south india b. i. publications, new delhi p. 56-57. murashige t, f skoog. 1962. a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum 15: 473-497 raghavan v .1993. chloroplast activities of dark-imbibed and photo induced spores of the fern onoclea-sensibilis. protoplasma 175: 75-84. turnwald s, r scheuerlein, m furuya .1999. phytochrome-dependent modulation and re-induction of growth of the first rhizoid in dryopteris paleacea sw. planta. 208: 98-106. wardle da, o zackrisson, mc nilsson. 1998. the charcoal effect in boreal forests: mechanisms and ecological consequences. oecologia 115: 419-426. 9 biotropia no biotropia no. 12, 1999 : 1 18 the occurrence of insects and moulds in stored cocoa beans at south sulawesi okky s. dharmaputra seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia sunjaya, muhammad amad and ina retnowati seameo biotrop, p.o. box 116, bogor 16001, indonesia teguh wahyudi the indonesian research institute for coffee and cocoa, jl. pb sudirman 90, jember 68118, indonesia abstract surveys on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels in south sulawesi were conducted together with investigations on moisture content, pest infestation (insect and mould) and quality characteristics in terms of reducing sugar, free amino acid and free fatty acid content. surveys were conducted during dry (july 1997) and wet seasons (february 1998) in three regencies (pinrang, polewali-mamasa and luwu) and ujung pandang, south sulawesi province. interviews were carried out during surveys in the dry season. number of respondents from farmers, trailers and exporters was 38, 15 and 5, respectively. in each season, number of samples taken from farmers, traders and exporters was 9, 21 and 15, respectively. in general, farmers, traders and exporters did not carry out postharvest handling and technology processing properly. moisture content of cocoa beans collected from farmers, traders and exporters were higher than the tolerable limit recommended by sni (7.5%). moisture content of cocoa beans collected during the wet season was higher than in the dry season. insects were found on cocoa beans collected from traders and exporters. species composition and the presence of each insect species were varied among the two seasons, but the predominant species was tribolium castaneum. at trader level the percentage of insect-damaged beans during the wet season was higher than that during the dry season, while at exporter level it was lower. during the two seasons the percentage of mouldy beans at farmer level was lower than the tolerable limit recommended by sni (4%), while those from some samples at trader and exporter levels were higher than 4%, but based on the direct plating method, all of the samples at trader and exporter levels were mouldy. species composition and the percentage of beans infected by each mould species at farmer, trader and exporter levels during the two seasons were varied. the percentage of mouldy beans increased at trader and exporter levels. the predominant moulds were aspergillus flaws, eurotium amstelodami, e. chevalieri and penicillium citrinum. the predominant mould at farmer level during wet season was saccharomyces cerevisiae (yeast). reducing sugar and free amino acid content of cocoa beans collected during the dry season was higher than those collected during the wet season, either at farmer, trader or exporter levels. free fatty acid content of cocoa beans tends to be higher during the wet season than the dry season at the three levels. keywords: stored products pests/postharvest handling/technology processing/moisture content/insect/mould/reducing sugars/free amino acids/free fatty acids/cocoa/south sulawesi. 1 biotropia no. 12, 1999 introduction indonesia ranks third among the cocoa producing countries in the world after ivory coast and ghana (ico 1996). in south and southeast sulawesi cocoa production was dominated by smallholders, and generally the quality was low, i.e. high moisture content, low fat content, less flavour, and inconsistency in the quality of beans (wahyudi 1994). in relation to health and food safety to consumers due to insect infestation and mould infection, the quality of cocoa beans in indonesia is considered low (siswoputranto 1994). zaenudin and wahyudi (1996) also reported that the problem of exported cocoa beans was due to insect infestation and mould infection, so that it was subjected to automatic detention, and thus should be refumigated. the quality of cocoa beans can be increased by improving the method of postharvest handling from farmer to exporter levels. aziz (1996) assumed that the low quality of cocoa beans was attributed to farmers and traders in indonesia who did not conduct postharvest handling and technology processing properly. the objective of this study was to get information on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels in south sulawesi province. the moisture content, pest infestation (insect and mould) and quality characteristics (reducing sugar, free amino acid and free fatty acid content) of cocoa beans were also analyzed. materials and methods time and location of surveys surveys were conducted during the dry (july 1997) and wet seasons (february 1998) in three regencies (pinrang, polmas and luwu) and ujung pandang, south sulawesi province. these districts were selected because they produce large quantities of cocoa beans, while ujung pandang was the main port for exporting of cocoa beans. range of the number of rainy days and rainfalls from those regencies during dry and wet seasons is presented in table 1. interviews using questionnaires with farmers, traders and exporters, on the spot observation at survey locations, and random sampling of cocoa beans derived from farmers, traders and exporters in selected areas were carried out during the surveys. interviews using questionnaires interviews were carried out during the dry season to get information on postharvest handling and technology processing of cocoa beans at fanner, trader and exporter levels. data were collected randomly from interviews with respondents. number of respondents from each level was different depending on the condition in 2 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. table 1. range and average number of rainy days and rainfall on location of the surveys during dry and wet seasons dry season wet season june 1997 july 1997 january 1998 february 1998 rf rd rf rd rf rd rf rd range 49-155 3-11 68-106 4-14 85-175 8-10 138-264 8-10 average 91.3 6.0 82.7 8.7 135.4 8.7 207.3 9.3 rf = rainfall (mm) rd= rainy days (day) the field during the surveys. number of respondents at farmers, traders and exporters was 38, 15 and 5, respectively. the questionnaires contain questions on postharvest handling and technology processing carried out by farmers, traders and exporters. sampling methods about 1 kg of each sample was taken randomly from farmer, trader and exporter levels. in each season, number of samples taken from farmers, traders and exporters were 9, 21 and 15, respectively. samples of cocoa (primary samples) from each stack were taken randomly from a certain number of sacks using a sampling spear. insects were separated from each primary sample using graded sieves. each primary sample was divided several times using a sample divider to obtain working samples for analyzing moisture content, insect-damage, mould and quality characteristics in terms of reducing sugars, free fatty acids and free amino acids. the presence of insects was estimated on sack surface as well as within the sacks. moisture content, insect, mould and quality characteristics analyses moisture content (wet weight) was determined based on sni 01-2323 (isc 1995). the insects were identified using the publication of haines (1991) as the main reference. insect-damaged beans were determined according to sni 01-2323 (isc 1995). mouldy beans were determined based on 2 methods, namely cut test (isc 1995) and direct plating methods on dichloran 18% glycerol agar (dg 18) (pitt and hocking 1985). the moulds were identified using the publications of pitt and hocking (1985), samson et al. (1996) as the main references. reducing sugars, free amino acids and free fatty acids were determined according to somogyi (mccready 1970), spectrometry method (lillevik 1970) and sni 01-2323 (isc 1995), respectively. 3 biotropia no. 12, 1999 results and discussion interviews with farmers, traders and exporters the results of questionnaires are shown in table 2. commonly, bulk cocoa was cultivated by smallholders in pinrang, polmas and luwu regencies (99.4% of table 2. result of questionnaires on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels * = generally, ex fertilizer bag ** = generally, ex sugar bag 4 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al respondents). immediately after harvesting, the pods were opened using a knife or a wooden billet. nevertheless, some farmers delayed opening the pods for several days (23.7%).there were only 44.7% of farmers who fermented their cocoa beans properly before drying. usually, the beans were fermented using traditional fermenting boxes. most of the traders bought wet cocoa beans from farmers (75% of respondents), consequently farmers did not dry their cocoa beans up to recommended moisture content (7.5%). method of drying used by farmers was usually sun-drying for less than 3 days (57.9%), or 3-5 days (42.1%). they spread the beans on various drying facilities, namely fish net, jute bag, polypropylene bag, wooven bamboo and pandanus mat. traders redried their cocoa beans for less than 3 days (80%), consequently the moisture content was still high and the beans could easily be infected by mould. in this condition, cocoa beans could not be stored for a long time. the common bag type used for storing cocoa beans at trader level was jute bag (33.3%) ex fertilizer and sugar bags. exporters also redried their cocoa beans by the sun-drying method, but during the wet season they used a mechanical dryer until 7.5% moisture content was attained. jute bags were usually used to store the beans. some exporters did not inspect the beans for insects and mould infestations in the warehouse. moisture content a. a. farmer level moisture content (m.c.) of cocoa beans collected from farmers during dry and wet seasons were 7.9-11.7% (mean 9.7%) and 9.3-14.0% (mean 12.3%), respectively (table 3). moisture content of the samples were higher than the tolerable limit recommended by sni (7.5%). most of the farmers (75%) sold cocoa beans in wet condition to the traders (table 2). if the cocoa beans were not dried immediately and properly by the farmers, they could readily be infected by moulds. the farmers did not pay too much attention to the m.c. of cocoa beans sold, because they want to get money quickly. their main source of living comes from cocoa beans. table3. moisture content, insect-damage, and mouldy cocoa beans collected from farmers during dry and wet seasons dry season wet season parameter mean range mean range moisture content (%) insectdamage beans (%) mouldy beans (%) cut test direct plating method 9.7 b 0 0.2 c 18.4 e 7.9-11.7 0-2 7-76 12.3 a 0 0.7 c 32.1 d 9.3-14.0 0-2 14-53 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level 5 b1otrop1a no. 12, 1999 , based on statistical analysis, there was a significant difference between dry and wet seasons on m.c. of cocoa beans (table 3). aside from economical factors, the rainfall and the number of rainy days affected the duration of drying, so that the m.c. of cocoa beans during the wet season (12.3%) was higher than during the dry season (9.7%). differences in rainfall and number of rainy days between wet and dry seasons are shown in table 1. b. trader level moisture content of beans collected from traders during dry and wet seasons were 7.3-11.8% (mean 9.4%) and 7.3-12.9% (mean 9.6%), respectively (table 4). m.c. of cocoa beans during the wet season was higher than the dry season, but based on statistical analyses the m.c. was not significantly different for the two conditions (table 4). m.c. of almost all the samples collected (95.4% samples) during dry and wet seasons were higher than 7.5% (tolerable m.c. for storing of cocoa beans). the traders redried the cocoa beans for less than 3 days, consequently their m.c. was still high, so that they could be infected by moulds. table 4. moisture content, insect-damage, and mouldy cocoa beans collected from traders during dry and wet seasons parameter dry season wet season mean range mean range moisture content (%) insect-damage beans (%) mouldy beans (%) cut test direct plating method 9.4 a 0.2 b 1.1 d 26.7 f 7.3-11.8 0-1 0-8 5-100 9.6 a 0.5 b 2.2 c 47.6 e 7.3-12.9 ,0-2 0-7 3-100 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level c. exporter level moisture content of cocoa beans collected from exporters during dry and wet seasons were 7.2-9.7% (mean 8.2%) and 7.5-11.5% (mean 9.4%), respectively (table 5). statistical analysis revealed that m.c of cocoa beans during the wet season was significantly higher than the dry season (table 5). m.c. of twelve samples (80%) collected during the dry season and all samples collected during the wet season were higher than 7.5%. the high m.c. of cocoa beans collected from exporters during the wet season was due to the high m.c. of cocoa beans bought from traders. however, m.c. of cocoa beans always change with the change of relative humidity in storage. m.c. of 7.5% is in equilibrium with the relative humidity of storage of 75% (wood 1985). 6 the occurrence of\jnsects and moulds in stored cocoa beans okky s. dharmaputra el al. table s. moisture content, insect-damage, and mouldy cocoa beans collected from exporters during dry and wet seasons parameter dry season wet season mean range mean range moisture content (%) insect-damage beans (%) mouldy beans (%) cut test direct plating method 8.2 b 0.7 c 3.7 d 67.4 e 7.2-9.7 0-3 0-6 8-100 9.4 a 0.5 c 1.7 d 73.4 e 7.5-11.5 0-2 0-3 41-100 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level the presence of insects and insect-damaged beans cocoa beans could be damaged by insects during storage, mainly of the orders lepidoptera and coleoptera. kalshoven (1981) and wood (1985) reported that insect species associated with stored cocoa beans were araecerus fasciculatus (coffee bean weevil), ephestia cautella (tropical warehouse moth), and tribolium castaneum (rust red flour beetle). insect pests of cocoa beans feed directly on grain constituents. their feeding action causes loss of weight of the commodity, thus reducing its value, commercially and nutritionally. according to sni 01-2323, insect-damaged beans should not be higher than 4%. however, the quality of exported beans must be free from dead or live insects. a. farmer level there were no insect-damaged beans and live insects found on cocoa beans collected from farmers during dry and wet seasons, because most of the farmers did not store the beans for a long time. usually, soon after drying or storing for only less than 1 week, the beans were sold. b. trader level eight insect species found during the dry season were anisopteromalus calandrae, araecerus fasciculatus, callosobruchus chinensis, carpophilus sp., cryptolestes ferrugineus, liposcelis entomophila, sitophilus zeamais and tribolium castaneum. ten insect species found during the wet season were ahasverus advena, anisopteromalus calandrae, araecerus fasciculatus, c. chinensis, carpophilus sp., l. entomophila, oryzaephilus surinamensis, palorus sp., s. zeamais and t. castaneum. haines (1991) reported that these insects were major species infesting grain during storage, except a. calandrae which is a parasitic insect. tribolium castaneum was the most frequently found insect. the percentages of samples infested by t. castaneum during dry and wet seasons were 66.7 and 71.4, respectively (table 6). 7 biotropia no. 12, 1999 table 6. the number and percentage of cocoa bean samples infested by each insect species at trader level during dry and wet seasons n insect species dry season wet season number of infested samples percent of infested samples number of infested samples percent of infested samples 1 ahasverus advena 0 2 9.5 2 anisoptermalus calandrae 1 4.8 1 4.8 3 araecerus fasciculatus 5 23.8 3 14.3 4 callosobruchus chinensis 1 4.8 1 4.8 5 carpophilus sp. 1 4.8 2 9.5 6 cryptolesles fernigineus 1 4.8 0 7 liposcelis entomophila 1 4.8 1 4.8 8 oryzaephilus surinamensis 0 1 4.8 9 palorus sp. 0 2 9.5 10 sitophilus zeamais 3 14.3 4 19.0 11 tribolium castaneum 14 66.7 15 71.4 most of the traders sold various commodities in their shops which are located at traditional markets, consequently insect species of other commodities were also found on cocoa beans. sitophilus zeamais and c. chinensis are the major insects associated with maize and mung beans. their presence on stored cocoa beans was due to migration. the difference among variability of insect species between dry and wet seasons was caused by the difference in physical conditions, such as temperature, relative humidity and moisture content. haines (1991) reported that the development and behaviour of some insects could be affected by the physical condition of the environment. at high moisture content, competition between mould and other microorganisms could decrease the number of some stored insects which are replaced by fungus-feeder insects. in this study, this is shown by the presence of a. advena on 2 samples collected during the wet season. percentage of insect-damaged beans in samples collected during the wet season (0.5%) was higher than the dry season (0.2%), but they were not significantly different (table 4). c. exporter level seven insect species were found on cocoa beans collected during the dry season, they were a. fasciculatus, bracon hebetor, carpophilus sp., corcyra cephalonica, ephestia cautella, necrobia rufipes and tribolium castaneum, while during the wet season 10 insect species were found, i.e. ahasverus advena, araecerus fasciculatus, b. hebetor, carpophilus sp., corcyra cephalonica, cryptolestes ferrugineus, e. cautella, liposcelis entomophila, oryzaephilus surinamensis and t. castaneum. most of these insects were grain stored pest, except b. hebetor which is a parasitic insect of corcyra cephalonica and e. cautella. necrobia rufipes is a common insect associated with copra (haines 1997), therefore 8 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. its presence on stored cocoa beans was due to migration, because cocoa beans and copra were stored in the same warehouse. variability of insect species at exporter level during the wet season was higher than the dry season. tribolium castaneum was the most frequently found insect. the percentages of samples infested by t. castaneum during dry and wet seasons were 93.3 and 80.0, respectively (table 7). table 7. the number and percentage of cocoa bean samples infested by each insect species at exporter level during dry and wet seasons n insect species dry season wet season number of infested samples percent of infested samples number of infested samples percent of infested samples 1 ahasverus advena 0 5 33.3 2 araecerus fasciculatus 12 80.0 4 26.7 3 bracon hebetor 3 20.0 3 20.0 4 carpophilus sp. 1 6.7 2 13.3 5 corcyra cephalonica 1 6.7 2 13.3 6 cryptolestes fermgineus 0 2 13.3 7 ephestia cautella 8 53.3 1 6.7 8 liposcelis entomophila 0 1 6.7 9 oryzaephilus surinamensis 0 5 33.3 1 necrobia rufipes 1 6.7 0 1 tribolium castaneum 14 93.3 12 80.0 the variability of insects found on cocoa beans at trader level, both during dry and wet seasons was different than at the exporter level. it might be due to the difference in environmental conditions. the structures and sanitary conditions of exporter's warehouses were better than that of trader's warehouse. exporters usually stored only cocoa beans in their warehouse, while traders did not. according to sidik (1997) the presence of pests in stored grain was usually affected by the physical structure and sanitary conditions of the storage facility, and characteristic of the stored grain. the percentage of insect-damaged beans during the wet season (0.5%) was lower than the dry season (0.7%), but they were not significantly different (table 5). mouldy beans the difference between the two analyzing methods (cut test/snl and direct plating method) gave different results on mouldy beans. the percentage of mouldy beans using the cut test was lower than for the direct plating method, because the mould was invisible before it produces mycelium or spore mass inside the beans. nonmouldy beans observed using the cut test might be already infected by mould, but their intensity was very low. visual observation as well as examination of the beans using a stereoscopic microscope at low magnification also did not reveal the 9 biotropia no. 12, 1999 differences in the appearance of the beans that might be correlated with levels of mould infection. niles (1981) reported that on invisible mouldy beans which had been further redried, the mould count was 2.0 x 10s cfu/g beans. nevertheless, hanson and welty (1971) stated that the presence of microorganisms on non-mouldy beans (visual observation) has no correlation with bean quality. a. farmer level in general, farmers did not store the beans for a long period of time, therefore, the intensity of mould infection was still low. statistical analysis showed that percentage of mouldy beans by the cut test was not significantly different between dry and wet seasons, while for the direct plating method it was significantly different (table 3). the percentage of mouldy beans by the cut method during the wet season (32.1%) was higher than the dry season (18.4%). mouldy beans obtained using the cut test during dry and wet seasons were between 0-2%, respectively, while the direct plating method gave 7-76% and 14-53%, respectively (table 3). cut tests showed that 1 sample (11%) collected during the dry season and 5 samples (11%) collected during the wet season had mouldy beans. examination using the direct plating method showed that all of the samples had mouldy beans, during the two seasons. it suggests that mould infection has occurred at the farmer level, although its intensity was low. thirteen species of moulds were isolated from cocoa beans collected during the dry season, i.e. aspergillus flavus, a. niger, a. ochraceus, a. restrictus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, mucor piriformis, nigrospora oryzae, penicillium citrinum and saccharomyces cerevisiae (yeast). the predominant species were aspergillus flavus and a. niger. table 8 shows that the average number of beans infected by both table 8. the percentage of cocoa beans infected by each mould species at farmer level during dry and wet seasons n mould species infected beans (%) dry season wet season mean range mean range 1 aspergillus flavus 10.4 0-67 8.4 2-26 2 a. niger 10.4 0-59 6.2 0-14 3 a. ochraceus 0.1 0-1 0 0 4 a, reslriclus 0.3 0-2 0 0 5 a. tamarii 0.5 0-3 0.2 0-1 6 a. wentii 7.3 0-64 0 0 7 botryodiplodia theobromae 1.7 0-7 4.2 0-30 8 cladosporium cladosporioides 1.8 0-6 0 0 9 eurotium amstelodami 1.9 0-9 5.1 0-20 1 e. chevalier! 0 2.6 0-13 1 mucor piriformis 0.3 0-3 0 0 1 nigrospora oryzae 0.2 0-2 0 0 1 penicillium citrinum 5.6 0-19 0.2 0-2 1 saccharomyces cerevisiae 0.6 0-3 34.4 0-100 10 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al. species was 10.4%. the other most frequently isolated moulds were a. \ventii (7.3%) and penicillium citrinum (5.6%). eight species of moulds were isolated from cocoa beans during the wet season, i.e. a. flavus, a. niger, a. tamarii, b. theobromae, e. amstelodami, e. chevalieri, p. citrinum and saccharomyces cerevisiae. the percentage of the beans infected by each mould species is presented in table 8. the predominant moulds were a. flavus (8.4%) and a. niger (6.2%). on some samples, yeast was most frequently isolated. however, yeast did not give negative effects, because infection was only on the shell of cocoa beans due to improper washing and drying of the beans. botryodiplodia theobromae was isolated from cocoa beans collected from farmers. the presence of this mould indicated that the pods were infected by pod rot. according to wood (1985) b. theobromae caused diplodia pod rot and infected the beans before harvest. b. trader level based on statistical analysis, the percentage of mouldy beans collected during the wet season was higher than that during the dry season and significantly different either using the cut test or the direct plating method (table 4). based on the cut test, mouldy beans obtained during the dry and wet seasons were 0-8% and 0-7%, respectively. the percentage of mouldy beans using the direct plating method was higher than the cut test. the direct plating method yielded 5-100% (mean 26.7%) mouldy beans during dry season and 3-100% (mean 47.6%) during wet season (table 4). based on the cut test, 2 samples (0.1%) collected during the dry season and 4 samples (0.2%) collected during the wet season had low quality, because the percentage of mouldy beans was more than 4%. the direct plating method showed that all samples collected either during dry or wet seasons were mouldy. the percentage of mouldy beans collected from the traders were higher than that from the farmers, because the traders did not carry out sorting properly. they also stored the beans with high moisture content for long time. nineteen species of moulds were isolated from cocoa beans collected during the dry season, i.e. aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. restrictus, a. tamarii, a. •wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, fusarium graminearum, f. proliferatum, libertella faginea, mucor piriformis, nigrospora oryzae, penicillium citrinum, rhizopus oryzae, and saccharomyces cerevisiae. the predominant moulds were a. niger (10.0%), followed by p. citrinum (7.6%) and a. flavus (5.6%). fourteen species of moulds were isolated from cocoa beans collected during the wet season, i.e. aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, libertella faginea, mucor piriformis, nigrospora oryzae, penicillium citrinum and 5. cerevisiae. the predominant moulds were e. amstelodami (15.9%) 11 biotropia no. 12, 1999 followed by a. flavus (12.6%). the percentage of cocoa beans infected by each mould species is presented in table 9. table 9. the percentage of cocoa beans infected by each mould species at trader level during dry and wet seasons c. exporter level statistical analysis showed that there was no significant difference on the percentage of mouldy beans collected during dry and wet seasons, either using the cut test or the direct plating method (table 5). the percentage of mouldy beans using the cut test and collected during dry and wet seasons were 0-16% (mean 3.7%) and 0-3% (mean 1.7%), respectively, while those using the direct plating method were 8-100% (mean 67.4%) and 41-100% (mean 73.4%), respectively (table 5). based on the cut test all samples collected during the wet season were still within the tolerable quality recommended by sni 01-2323 (less than 4%), except 1 sample collected during the dry season was higher than 4%. based on the direct plating method, all samples collected had mouldy beans in excess of 4%, either during dry or wet seasons. the percentage of mouldy beans collected from the exporters were higher than that from the traders. it was assumed that the exporters did not carry out sorting properly, or they stored the beans with high moisture contents, consequently the beans were more easily infected by mould. sixteen species of moulds were isolated 12 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al. from cocoa beans during the dry season, i.e. aspergillus candidus, a. jlavus, a. niger, a. ochraceus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, e. repens, libertella faginea, mucor piriformis, penicillium citrinum, rhizopus oryzae and saccharomyces cerevisiae (table 10). the predominant mould was p. citrinum (37%). other moulds frequently isolated were eurotium amstelodami (31.5%) and a. niger (13.3%). the domination by p. citrinum and e. amstelodami was correlated with low moisture content of beans (about 8%). eleven species of moulds were isolated from cocoa beans during the wet season, i.e. aspergillus flavus, a. niger, a. tamarii, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, libertella faginea, mucor piriformis, m. racemosum, penicillium citrinum and rhizopus oryzae (table 10). the predominant moulds were a. flavus (20.5%) and e. chevalieri (20.9%). table 10. the percentage of cocoa beans infected by each mould species at exporter level during dry and wet seasons quality characteristics the quality requirements for both marketing and manufacturing stages can be divided into factors. firstly, the yield-determining factors are bean size, shell content, fat content, and foreign matter. secondly, the quality-determining factors are flavour, purity or wholesomeness, consistency, and cocoa butter characteristics. quality analysis of cocoa bean samples in this study were conducted in terms of reducing sugars and free amino acids, whereas cocoa butter quality was represented by free fatty acids analysis. 13 biotropia no. 12,1999 reducing sugars reducing sugars (fructose and glucose), amino acids and peptides are flavour precursors (biehl et al. 1985). reducing sugars are important flavour precursors. the role of this sugar is to perform as a degrading agent of amino acids during flavour development (rohan and stewart 1967a). hastori et al (1987) reported that reducing sugar content of cocoa beans correlated with the duration of fermentation. the results show that during the dry season they tend to be higher than the wet season, especially at farmer and trader levels (table 11). these results imply that postharvest handling of cocoa beans should be conducted more carefully at farmer and trader levels, especially during the wet season, in order to produce higher reducing sugar content. table 11. reducing sugar content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons reducing sugar content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 1.32 a 1.34 a 1.27 a 1.05-1.51 0.96-1.63 0.96-1.51 1.15 b 0.93 b 1. 19 a 1.04-1.34 0. 53-1.22 1.03-1.47 numbers followed by the same letter in the same row do not differ significantly according to dmrt a 95% confidence level reducing sugar content of cocoa beans collected from farmers, traders an< exporters during the dry season were 1.05-1.51% (mean 1.32%), 0.96-1.63% (mea 1.34%) and 0.96-1.51% (mean 1.27%), respectively, while during the wet seaso 1.04-1.34% (mean 1.15%), 0.53-1.22% (mean 0.93%), and 1.03-1.47% (mea 1.19%), respectively (table 11). free amino acids as mentioned above, free amino acids constitute a flavour precursor. roh and stewart (1967b) revealed that flavour development of cocoa beans correlai with amino acid synthesis during fermentation. hastori et al. (1987) reported t amino acid content could be affected by duration of fermentation. therefr fermentation must be stopped when the condition of free amino acid content reached a maximum. under-fermentation and over-fermentation cause loss of s< precursors of flavour development. the results of free amino acid analysis also show that during the dry se; they tend to be present at higher levels than the wet season. statistical analysis sh that there was a significant difference between free amino acid content of c beans collected from farmers during dry and wet seasons, but neither from tn nor exporters (table 12). 14 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. free amino acid content of cocoa beans collected from farmers, traders, and exporters during the dry season were 0.47-1.04% (mean 0.64%), 0.15-1.26% (mean 0.66%), and 0.44-1.50% (mean 0.76%), respectively, while during the wet season 0.31-0.71% (mean 0.49%), 0.41-0.84% (mean 0.59%), and 0.42-1.14% (mean 0.68%), respectively (table 12). table 12. free amino acid content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons free amino acid content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 0.64 a 0.66 a 0.76 a 0.47-1.04 0.151.26 0.44-1.50 0.49 b 0.59 a 0.68 a 0.31-0.71 0.41-0.84 0.42-1.14 numbers followed by the same letter in the same row do not differ significantly according to dmrt at 95% confidence level. free fatty acids the measurement of free fatty acids (ffa) provides an indication of the quality of cocoa beans to produce cocoa butter. cocoa butter with high level of free fatty acids tend to be soft, have poor crystallization properties, contain off-flavour, and have a poor shelflife (nickless 1994). the ffa level must be less than 1.0%, so that ffa in the cocoa butter produced from these beans will be less than 1.75%. this is the legal limit for cocoa butter within the eec and the limit proposed by codex (ccca 1984). the free fatty acid contents of cocoa beans collected from farmers, traders and exporters were higher during the wet season than the dry season (table 13). this might be due to incomplete or delayed drying, long period of storage under humid conditions (ccca 1984), or accidental wetting during storage, transport or shipment (wood 1985). based on statistical analysis, there were significant differences between ffa content of cocoa beans collected during the dry and wet seasons at farmer and exporter levels. these results also justified the need of a better postharvest handling technique at farmer, trader and exporter levels. free fatty acid content of cocoa beans collected from farmers, traders and exporters during the dry season were 0.25-2.19% (mean 0.90%), 0.43-2.86% (mean 0.96%), and 0.52-1.89% (mean 1.10%), respectively, while during the wet season 0.29-1.83% (mean 1.45%), 0.53-3.81% (mean 1.25%), and 0.83-2.04% (mean 1.39%), respectively (table 13). the free fatty acid contents of cocoa beans collected from farmers, traders and exporters were higher during the wet season than the dry season. this might be due to incomplete or delayed drying, long period of storage under humid condition (ccca 1984), or accidental wetting during storage, transport or shipment (wood 1985). 15 biotropla no. 12,1999 table 13. free fatty acid content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons free fatty acid content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 0.90 a 0.96 a 1.10 • 0.25-2.19 0.43.2.86 0.52-1.89 1.45 b 1.25 a 1.39 b 0.29-1.83 0.53-3.81 0.83-2.04 numbers followed by the same letter in the same row do not differ significantly according to dmrt at 95% confidence level. conclusions in general, farmers, traders and exporters did not carry out postharvest handling and technology processing of cocoa beans properly. moisture content of cocoa beans collected from farmers, traders and exporters were higher than the tolerable limit recommended by sni (7.5%). m.c. of cocoa beans during the wet season was higher than the dry season. insects were found on cocoa beans from traders and exporters. species composition and presence of each insect species were varied during the two seasons, but the presence of tribolium castaneum occurred at higher frequency. at trader level the percentage of insect-damage beans was higher during the wet season than the dry season, while at exporter level it was lower. based on the cut test during the two seasons the percentage of mouldy beans at farmer level was lower than the tolerable limit recommended by sni (4%), while those from some samples collected at trader and exporter levels were higher than 4%, but based on the direct plating method all of the samples collected at the two levels were mouldy. species composition and the percentage of beans infected by each mould species at farmer, trader and exporter levels during the two seasons were varied. the percentage of mouldy beans increased at trader and exporter levels. aspergillus flavus, eurotium amstelodami, e. chevalieri and penicillium citrinum were the predominant moulds. the predominant mould isolated from cocoa beans collected from farmers during the wet season was saccharomyces cerevisiae (yeast). reducing sugar and free amino acid content of cocoa beans collected during the dry season were higher than those collected during the wet season, either at farmer, trader or exporter levels. free fatty acid content of cocoa beans tends to be higher during the wet season than the dry season at the three levels. postharvest handling of cocoa beans should be conducted more carefully at farmer, trader and exporter levels, especially during the wet season, in order to produce good quality cocoa beans. 16 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the plantation office province level, south sulawesi; plant quarantine service, ujung pandang; and dpd askindo south sulawesi for their information and cooperation during the surveys at farmer, trader and exporter levels in south sulawesi. the authors are also grateful to the indonesian research institute for coffee and cacao, for their collaboration in this study. references aziz, m.f. 1996. upaya peningkatan mutu biji kakao rakyat melalui sentralisasi pengolahan. wart a puslit kopi dan kakao 12(1): 12-17. biehl, b., e. brunner, d. passern, v.c. quesnel and d. adomako. 1985. acidification, proteolysis, and flavour potential in fermenting cocoa beans. j. sci. fd. agric. 36 : 583-598. ccca (the cocoa, chocolate and confectionery alliance). 1984. cocoa beans: chocolates manufacturer's quality requirements. 11 green street, london, 20p. haines, c.p. 1991. insects and arachnids of tropical stored products: their biology and identification (a training manual). natural resources institute, u.k. haines, c.p. 1997. insects and arachnids in indonesian food stores-biodiversity in a man-made environment. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p. 95-125. hansom, a.p. and r.e. welty. 1971. microflora of raw cocoa beans. mycopath. mycol. appl. 44 : 309 316. hastori, b., djatmiko and t. wahyudi. 1987. pengaruh perlakuan pendahuluan pada fermentasi kakao (theobroma cacao l.) terhadap mutu biji keringnya. bul. teknol. industri pertanian 1 (i) : 23-40. indonesian standardization council. 1995. indonesian national standard, sni 01-2323 1995, rev. 1994: cocoa beans. international cocoa organization. 1996. production of cocoa beans by country, 1986/87 1995/96. quart, bull, of cocoa statistics 22 (3): 6. international cocoa organization, london. kalshoven, l.g.e. 1981. the pests of crops in indonesia. revised by p.a. van der laan. p.t. ichtiar baru-van hoeve, jakarta. lillevik, h.a. 1970. the analytical chemistry of the protein, peptides and amino acids. in methods in food analysis: physical, chemical and instrumental methods of analysis (ed. by joslyn, m.a.), 2nd ed. academic press, new york. p. 617-700. me cready, r.m. 1970. monosaccharides. in methods in food analysis: physical, chemical and instrumental methods of analysis (ed. by joslyn, m.a.), 2nd ed. academic press, new york. p. 475-509. nickless, h. 1994. cocoa butter quality. in proceeding of the malaysian international cocoa conference (ed. by j. selamat, b.c. lian, t.k. lai, w.r.w. ishak and m. mansor). malaysian cocoa board : 322-336. nlles.e.v. 1981. microflora of imported cocoa beans. j. stored prod. res. 17:147-150. pirr, j. i and a.d. hocking. 1985. fungi and food spoilage. academic press, sydney. roman, t.a. and t. stew art. 1967a. the precursors of chocolate aroma : production of reducing sugars during fermentation of cocoa beans. j. food sci. 32:399-402. _____. 1967b. the precursors of chocolate aroma : productions of free amino acid during fermentation of cocoa beans. j. food. sci. 32: 395-398. samson, r.a, e.s. hoekstra, j.c. frisvad and o. filtenborg. 19%. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baam, the netherlands. 17 biotropia no. 12,1999 sidik, m. 1997. state-of-the-art of storage management. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p. 11-23. siswoputranto, p.s. 1994. prospek perkakaoan dunia dan beberapa masalah yang perlu digarap. makalah disampaikan dalam gelar teknologi pascapanen kakao rakyat, samarinda, 13-14 september 1994. wahyudi, t. 1994. teknologi pengolahan untuk menghasilkan kakao bermutu baik dan usaha diversifikasi produk. makalah disampaikan dalam gelar teknologi pascapanen kakao rakyat. samarinda, 13-14 september 1994. p. 1-17. wood, g.a.r. 1985. from harvest to store. in cocoa. (ed. by wood, g.a.r and r.a. lass). longmann, london, pp.444-504. zaenudin and t. wahyudi. 1996. laporan kunjungan tim askindo ke amerika serikat dalam upaya meniadakan automatic detention terhadap kakao indonesia. warta puslit. kopi dan kakao 12(1): 44-47. 18 biotropia no. 8, 1995: 30-38 arbuscular mycorrhizal fungi from the rfflzospheres of soybean crops in lampung and west java*) k. kramadibrata research and development centre for biology indonesian institute of sciences, bogor, indonesia e.i. riyanti and r.d.m. simanungkalit central research institute for food crops, agency for agricultural research and development, bogor, indonesia abstract the occurrence of arbuscular mycorrhizal (am) fungi in the rhizospheres of field-grown soybean crops in the provinces of lampung and west java was examined. nineteen taxa of am fungi were identified as follows: acaulospora delicata, a. foveata, a. rehmii, a. scrobiculata and a. tuberculata; gigaspora cf. gigantea and gigaspora sp. 1; glomus clavisporum; glomus cf. fasciculatum, glomus micro-aggregatum, glomus sp. 1, glomus sp. 2, glomus sp. 3 and glomus sp. 4; scutellospora cf. heterogama, scutellospora cf. pellucida, scutellospora sp. 1. scutellospora sp.2. and scutellospora sp. 3. key words: mycorrhizas/soybean/rhizosphere fungi/identification. introduction there are only a few reports on arbuscular mycorrhizal (am) fungi derived from the rhizospheres of food crops in indonesia. widiastuti and kramadibrata (1992) reported on some am fungi from the acid soils of west java, among others from the rhizospheres of corn. soybean is one of the main staple foods in indonesia. it is often grown on uplands as well as on lowlands after rice in the dry season. however, the information on am species and its distribution has never been reported. schenck and smith (1982) investigated three am fungi which occurred in soybean in florida, u.s.a., i.e. glomus claroideum, g. tortuosum and gigaspora albida. the purpose of this paper is to report on species and distribution of am fungi from the rhizospheres of field-grown soybeans in the provinces of lampung and west java. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom 111), 19-21 april 1994, yogyakarta, indonesia. 30 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit materials and methods the soybean-grown upland areas in lampung (central lampung) and west java (garut and bogor) have been selected for soil sampling. soil samples were collected from ten locations (four in central lampung, two in garut and four in bogor). some soil characteristics and number of am propagules of each location are described in table 1. ph of the soils measured in water suspension ranged from 4.78 to 6.94. p availability by bray-2 ranged from 2.32 to 55.59 mg/100 g soil. infective am propagules were determined by the 'most probable number' method as described by sieverding (1991). the number of am propagules ranged from 383 to 12 266 g air dried soil. a total of three to five subsamples weighing about one kg were systematically taken from each location at a depth of 0-20 cm. subsamples were then mixed 31 biotropia no. 8, 1995 thoroughly and air dried. from each sample 100 g of soil were used for spore examinations. spores were extracted by wet sieving and decanting methods in combination with 48% sucrose (walker et al. 1982), mounted on slides in polyvinyl alcohol-lactic acid-glycerol (pvlg) and then examined under a compound microscope for spore details (100looox). spores were identified by using the species description of walker (1983, 1986), walker and koske (1987) and walker and sanders (1986). melzer's reagent was used to determine spore wall reaction when necessary. spore preparations were coded krs (abbreviations of family names of the authors) and deposited partly at the central research institute for food crops and partly at the "herbarium bogoriense", research and development centre for biology-indonesian institute of sciences; both are located in bogor. results and discussion all soil samples collected from lampung and west java contained am fungal spores. however, the species were not distributed evenly as shown in table 2. thirteen were identified from the rhizospheres of soybean in central lampung, three in garut, nine in bogor. the sampling locations new sukadana and suryamataram yielded six am species (highest) each; the experimental farm cikeumeuh i and cikeumeuh ii had five species each; gantiwarno had four species; sidobinangun, experimental farm muara i and muara ii had three species each; karang pawitan and cihuni (both located in garut) had only two am species each. some spores could be identified well because they bore very specific characteristics. some identified species showed very similar characteristics with known species like gigaspora cf. gigantea, glomus cf. fasciculatum, g. cf. microaggregatum, scutellospora cf. heterogama and scutellospora cf. pellucida. the species of some spores could not be identified due to some problems such as poor condition and the limited number. species identification will be done after pot cultures of the related soil samples are established. the soil samples from sukadana contained extramatrical cells of scutellospora, while that from gantiwarno contained extra-matrical cells of gigaspora. however, the three species reported by schenck and smith (1982) on am fungi from the rhizospheres of soybean in florida were not found in soil samples collected from lampung and west java. 32 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit the am fungi from the rhizospheres of soybean in lampung and west java are described as follows: 1. acaulospora delicata walker, pfeiffer & trappe the spores were globose to subglobose, yellow, 94116 x 96101 µm. the spore surface was smooth. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#41, krs#42, krs#43, krs#44 and krs#53, gantiwarno village, pekalongan subdistrict, central lampung district; krs#82, experimental farm cikeumeuh i, bogor; krs#83, experimental farm muara i, bogor; krs#94 and krs#98, experimental farm muara ii, bogor. 33 biotropia no. 8, 1995 2. acaulospora foveata trappe & janos the spore was pale yellow to greenish yellow, globose, 200 x 200 µm. the spore surface was covered with round to oblong sometimes irregular depressions with curved bottom, separated by ridges. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#60, cihuni village, wanaraja subdistrict, garut district. 3. acaulospora rehmii sieverding & toro the spores were yellow, globose to subglobose, 180 190 x 190 – 200 µm. the spore surface had labyrinthi form folds with ridges and depressions between ridges. a sporiferous saccule was not observed in any collection made during the study. collections examined: krs#66 and krs#68, experimental farm cikeumeuh i, bogor. 4. acaulospora scrobiculata trappe the spores were pale yellow to yellow, globose to subglobose, 96-125 x 96 135µm. the surface was uniformly pitted with depressions, separated by ridges with a circular, linear or y-shaped pattern. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#23, suryamataram village, sukadana subdistrict, central lampung district; krs#55, sidobinangun village, seputih banyak sub-district, central lampung district; krs#65 and krs#69, experimental farm cikeumeuh i, bogor; krs#87, experimental farm muara i, bogor. 5. acaulospora tuberculata janos & trappe the spores were light yellowish brown to light brown, globose to subglobose 220-240 x 210-240 µm. the surface was covered by tubercules. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#31, suryamataram village, sukadana subdistrict, central lampung district; krs#70, experimental farm cikeumeuh i, bogor; krs#91, krs#92, krs#93 and krs#96, experimental farm muara ii, bogor. 34 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit 6. gigaspora cf. gigantea (nicol. & gerd.) gerd. & trappe the spores were pale yellow, globose to subglobose, 288 403 x 384 403 µm. the surface was smooth. a bulbous suspensor ± 40 µm diameter was found on every spore. collections examined: krs#11, krs#13 and krs# 18, new sukadana village, sukadana subdistrict, central lampung district; krs#25 and krs#27, surya-mataram village, sukadana subdistrict, central lampung district. 7. glomus clavisporum (trappe) almeida & schenck sporocarps were brown to dark brown, globose to subglobose, 300-400 x 300-380 µm. spores were brown to dark brown 100120 x 20-40 µrn, clavate to subcylindric, tapering to a cylindric subtending hypha. collections examined: krs#20, krs#21, krs#22, krs#28, krs#29 and krs#30, suryamataram village, sukadana subdistrict, central lampung district. 8. glomus cf. fasciculatum (thaxter sensu gerd.) gerd. & trappe emend. walker & koske the spores were pale yellow to pale yellow brown, globose to subglobose, 90 110 x 100110 µm. the surface was smooth. collections examined: krs#2, krs#3 and krs#19, new sukadana village, sukadana subdistrict, central lampung district; krs#63, krs#64 and krs#71, experimental farm cikeumeuh i, bogor; krs#78, krs#79 and krs#81; experimental farm cikeumeuh ii, bogor; krs#84, krs#86 and krs#88, experimental farm muara i, bogor. 9. glomus cf. microaggregatum koske, gemma & olexia the spores were found as loose aggregate. they were yellow, globose to sub-globose, 39-54 x 35-43 µm. the surface was smooth. collections examined: krs#1, new sukadana village, sukadana subdistrict, central lampung district. 10. scutellospora cf. heterogama (nicol. & gerd.) walker & sanders the spores were brown to dark brown, globose to subglobose, 200-300 x 200 — 300 µm. the surface was ornamented or covered by spines. a suspensor-like cell was found on spore, it was pale brown, ±40 µm. 35 biotropia no. 8, 1995 collections examined: krs#73 and krs#74, experimental farm cikeumeuh ii, bogor. 11. scutellospora cf. pellucida (nicol. & schenck) walker the spores were hyaline to pale yellow, pear-shaped, 160 204 x 80 – 102 µm. the surface was smooth. a suspensor-like cell was found on spore, it was hyaline, ± 40 µm collections examined: krs#76 and krs#77, experimental farm cikeumeuh ii, bogor. collections which could not be identified due to poor conditions and the small number of spores are listed below: 12. gigaspora sp.l. spores were globose to subglobose, brown to dark brown, 172.8-480 x 211.2-480 µm. the spore had one group of wall consisting of a thin unit wall, < 1 µm, hyaline and a brown laminated wall, 10-48 µm thick. the muronym is a(ul). one specimen had a germ tube. collections examined: krs#4, krs#7, and krs#17, new sukadana village, sukadana subdistrict, central lampung district; krs#45, krs#46, krs#47, and krs#52, gantiwarno village, pekalongan subdistrict, central lampung district. 13. glomus sp.l. spores were globose or sometimes subglobose, yellow, ±97µm, in groups. however, the spores of the specimens were deflated so that spore forms were not distinct, irregular; wall arrangement could not be examined. 14. glomus sp.2. spore forms could not be determined because it was deflated; it was pale yellow and in loose aggregate. collections examined: krs#49, gantiwarno village, pekalongan subdistrict, central lampung district. 15. glomus sp.3. spores were globose to subglobose, yellow; hyphae were hyaline to pale yellow. spore sizes were 100115 x 90104 µm. wall arrangements could not be analyzed. 36 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit collections examined: krs#58, central seed institute karang pawitan, karang pawitan subdistrict, garut district. 16. glomus sp.4. spores were globose, pale yellow, 172 x 172 µm; wall was 2 µm thick. wall arrangements could not be analyzed. collections examined: krs#62 and krs#67, experimental farm cikeumeuh i, bogor. 17. scutellospora sp.l. spores were ellipsoid, brownish yellow, 288 x 355 µm. spore wall possibly consisted of two wall groups, a and b. wall group a consisted of two unit walls, wall 1 was thinner than wall 2, both were brownish yellow. wall group b had one membranous wall, hyaline and very thin ( < 1 µm). the muronym is temporarily a (uu) b(m). collections examined: krs#12, new sukadana village, sukadana subdistrict, central lampung district. 18. scutellospora sp.2. spores were globose or subglobose, pale brown 220.8 280 x 201.6 288µm. the wall may have been structured in two wall groups, a and b. wall group a consisted of wall 1 and 2. wall 1 was a unit wall and wall 2 was thicker than wall 1; both were hyaline. wall 2 appreared to be laminated. wall group b had one membranous wall. the muronym is a (uu) b(m). some specimens clearly had a germ shield and an initial germ tube. collections examined: krs#5, krs#6, krs#14, krs#15 and krs#16, new sukadana village, sukadana subdistrict, central lampung district; krs#57, sidobinangun village, seputih banyak subdistrict, central lampung district; krs059, karang pawitan central seed institute, garut district; krs#61, cihuni village, wanaraja subdistrict, garut district; krs#75, experimental farm cikeumeuh ii, bogor. 37 biotropia no. 8, 1995 19. scutellospora sp.3. spores were globose, brownish yellow, 326 x 326 µm. it has two wall groups, a and b. wall group a consisted of wall 1, laminated and brown. wall group b had one membranous wall. the muronym is a(l) b(m). collections examined: krs#56, sidobinangun village, seputih banyak sub-district, central lampung district. references schenck, n.c. & g.s. smith. 1982. additional new and reported species of mycorrhizal fungi (endogonaceae) from florida. mycologia 74(1): 771-92. sieverding, e. 1991. vesicular-arbuscular mycorrhiza management in tropical agrosystems. technical cooperation, federal republic of germany, eschborn. walker, c. 1983. taxonomic concepts in the endogonaceae: spore wall characteristics in species description. mycotaxon 18: 443-445. walker, c. 1986. taxomonic concepts in the endogonaceae: ii a fifth morphological wall type in endogonaceous spores. mycotaxon 25: 95-99. walker, c. & r.e. koske. 1987. taxonomic concepts in the endogonaceae: iv glomus fasciculatum redescribed. mycotaxon 30: 253-262. walker, c., c.w. maize and h.s. mcnabb jr. 1982. populations of endogonaceous fungi at two locations in central iowa. canadian journal of botany 60: 2518-2529. walker, c. & f.e. sanders. 1986. taxonomic concepts in the endogonaceae: iii the separation of scutellospora gen. nov. from gigaspora gerd. & trappe. mycotaxon 27: 169-182. widiastuti, h. & k. kramadibrata. 1992. jamur mikoriza bervesikula arbuskula di beberapa tanah masam dari jawa bar at. menara perkebunan 60: 73-77. 38 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf biotropia vol. 28 no. 2, 2021: 109 116 doi: 10.11598/btb.2021.28.2.1162 109 interleukin levels in the zingiber cassumunartreated mice nurkhasanah*, nanik sulistyani, yuni arum handayani, qanita kamila and annisa candra nur isnaini faculty of pharmacy, universitas ahmad dahlan, yogyakarta 55164, indonesia received 6 december 2018/accepted 12 january 2020 abstract the protein compound, cytokine, is responsible for the body’s immune system. several cytokines acting as key regulators of infection include il-10, il-12, and il-14. the chemical content of zingiber cassumunar shows potential immunomodulatory effects. this study aimed to determine the effect of the zingiber cassumunar ethanol extract (eezc) on the expressions of il-10, il-12, and il-14. the test animals, balb/c mice which were treated for 21 days, were divided into five groups, i.e., normal group (untreated), negative control group (treated with 10% of tween 80), and three treatment groups that respectively received 1.25 mg, 2.5mg, and 5mg/20g bw of eezc. on the 22nd day, the mice were induced with lipopolysaccharide (lps) intraperitoneally (except for the normal group). the interleukin expression was observed by immunohistochemistry using specific antibodies, and the expressed cells were counted under a microscope. the 21-day administration of eezc at doses of 1.25 mg, 2.5mg, and 5mg/20g bw significantly increased the expression of il-10, il-12, and il-14 in proportion to the dose thereby suggesting the potency of the extract to induce both innate and adaptive immunity. this activity may be attributable to curcumin as the active compound of the extract. keywords: curcumin, immunomodulator, interleukin, zingiber cassumunar introduction the immune system which is responsible for protecting the host from various pathogenic microorganisms also controls the immune responses and prevents over-reaction of the body’s own cells (saraiva & o’garra 2010). a decrease in the immune system can affect the body's strength to fight infections or other diseases. therefore, the presence of immunomodulator compound that can improve the immune response to diseases or infections is a vital component of the immune system. immunomodulator is a substance, which can stimulate, suppress or modulate any of the components of the immune system including both the specific and nonspecific immune system (das et al. 2014). modulation of the immune system is marked by induction, expression, amplification or inhibition of certain parts in the immune signaling and response mechanism. thus, the immunomodulator substance is used as immune stimulant for its effect on the immune system. the anti-inflammatory cytokine interleukin10 (il-10) plays a crucial role in preventing inflammatory and autoimmune pathogens. it can both impede pathogen clearance and ameliorate the immunopathology process. several types of cells can produce il-10, with the major source of il-10 varying in different tissues or during acute or chronic stages of the same infection (couper et al. 2008). il-10 plays a central role during infection by limiting the immune response to pathogens and thereby preventing damage to the host. the production of il-10 was associated with the regulatory t (treg) cells. (saraiva & o’garra 2010). interleukin-12 cytokine, produced mainly by the antigen-presenting cells (apc) which include the macrophages and dendritic cells that respond to microbes, is also vital in the immunoregulation process (abbas et al. 2017). *corresponding author, email: nurkhas@gmail.com biotropia vol. 28 no. 2, 2021 110 during the immune response, il-12 is produced as a reaction to stimuli of various compounds (including lipopolysaccharide/lps). interleukin-14 (il-14) cytokines, produced by the immune system, particularly by the activated b cells and t cells, regulates the b-cell proliferation (leca et al. 2008). il-14 can increase antibody responses to vaccinations causing autoimmunity and contribute to b-cell lymphoma formation (shen et al. 2006). zingiber cassumunar of the zingiberaceae family, known locally as bengle (javanese, indonesia), has been traditionally used for treating various diseases. z. cassumunar contains terpenoids, essential oil, and curcuminoids. several studies on z. cassumunar include its performance as anticancer (varalakshmi et al. 2008), antioxidant (vankar et al. 2006) (bua-in & paisooksantivatana 2009) and immunomodulator (nurkhasanah et al. 2017; rahmawati 2013). this plant exhibited an immunomodulatory activity by increasing phagocytic activity in vitro (chairul et al. 2009), increasing nitric oxide (no) and reactive oxygen species (ros) (nurkhasanah et al. 2017) and decreasing malondialdehyde products in plasmodium berghei-infected mice (nurmasari et al. 2014). this study documents the activity of zingiber cassumunar ethanol extract (eezc) in stimulating the immune response in vivo as observed from its effect on interleukin-10, -12 and -14. it focuses on the immune responses of these cytokines on both the innate and adaptive body immunity. the expression il-10 and il-14 are closely related to activation of adaptive immunity, while the il-12 is important in cell communication between the macrophage and t cells. this study was conducted for 28 days through oral administration on balb/c male mice. this in vivo experiment provides evidence for the higher effectiveness of eezc in increasing the immune responses, a very vital information for the development of z. cassumunar as an immunomodulatory product. materials and methods materials the zingiber cassumunar rhizome, collected from a local yogyakarta market, was identified at the biology laboratory, universitas ahmad dahlan. the test animal was obtained from the animal house of the integrated research and testing laboratory, universitas gadjah mada (lppt ugm) yogyakarta, indonesia. extraction the rhizome was selected, washed, sliced and finally oven-dried at a temperature of 50 oc. the dried rhizome was then blended or ground into powder. the extraction was carried out by maceration method using 96% ethanol as the solvent. the maceration lasted for 24 hours, and the yield were evaporated in a vacuum rotary evaporator to obtain the concentrated extract (eezc) which was used as the treatment. thin-layer chromatography (tlc) analysis of the extract the thin-layer chromatography (tlc) analysis was used to identify the active eezc compound. a total of 100.0 mg of eezc was dissolved in 10.0 ml of absolute ethanol. this procedure used curcuminoids (sigma) that was dissolved in ethanol as a standard. each 2 μl of the extract and curcuminoid were applied on silica gel gf 254 as the stationary phase and eluted with the mobile phase of chloroform : ethanol : glacial acetic acid (94 : 5 : 1). the detection of eezc was done under daylight and uv 254 nm. animal treatment the procedure of the study and the use of test animal were ethically approved by the research ethics committee of ahmad dahlan university on february 9, 2016, with reference no. 011601011. the test animals, 8 week-old balb/c mice, were acclimatized for a week before the treatment. the mice were divided into five (5) groups, namely; the normal group, negative control group which were treated with the solvent tween 80 at 10% concentration, and the 3 treatment groups (1.25 mg/20g bw; 2.5 mg/20g bw; 5 mg/20g bw; bw is abbreviation of body weight). the administration of eezc was carried out once a day, orally for 21 days (3 weeks). on the 22nd day, the mice were sacrificed using co2 gas. following sacrificing, lipopolysachcharide (lps) (sigma) with dose of interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 111 0.01 mg/20 g bw was injected into the peritoneal cavity area. after 1 hour, the macrophage was isolated and the expressions of interleukin-10, -12 and -14 were observed using the immunohistochemistry method with the specific antibodies of il-10, il-12 and il-14. macrophage isolation following the 21-day treatment, the mice were injected with lps in the intraperitoneal cavity. the mice were then dissected by opening the skin in the peritoneal area. as much as 10 ml of roswell park memorial institute (rpmi) (sigma) medium was injected into the stomach. the stomach was massaged, then the rpmi medium was drawn again. the medium was centrifuged for 10 minutes, and the supernatant was removed. the macrophage was washed with the medium and incubated for 24 hours. after overnight incubation, the macrophage was harvested. immunohistochemistry assay the immunohistochemistry assay was based on the method reported in nurkhasanah (2015), an indirect method using specific primary antibody that was conjugated with secondary antibody and chromogen. the expressed browncolored interleukin was the product of dimethyl amino benzidine (dab) chromogen detected under the light microscope. the cultured macrophage, which was previously fixed with 1 ml of methanol that was later removed, was washed in pbs (phosphate buffer saline) for 5 minutes. the fixed macrophage was then immersed in peroxidase blocking solution at room temperature for 10 minutes. the macrophage was then washed with running water and then re-washed with pbs. a total of 50 µl of blocking serum was added to the preparation which was then incubated in a humid temperature for 10-15 minutes. the 100 µl (with dilution 1:100) of specific antibodies (antiil-10, anti-il-12, and anti-il-14, murine recombinant, biovision) was then added to the preparation and incubated on a moist tray at room temperature for 1 hour. after the incubation, the preparations were washed with 1 ml of pbs. a total of 50 µl (with dilution 1 : 100) of anti-mouse biotin secondary antibody (biovision) was added to each preparation, which was incubated at a humid temperature for 20 minutes and then re-washed with pbs. the preparations were incubated with 50 μl of the streptavidin-peroxidase enzyme for 10 minutes, washed with pbs, and re-incubated with 50 µl of dimethyl amino benzidine (dab) chromogen (peroxidase substrate solution). the preparations were then washed with pbs and incubated with mayer’s hematoxylin as the counterstain and then rewashed with pbs in preparation for the microscopic observation at 400x magnification. the macrophages that expressed interleukin manifested brown stains. the observation was carried out from several fields of view (fov) of the microscope. the number of expressed positive cell was compared with the total number of observed cell and presented as percentage value. the results of the treated groups were statistically compared with that of the control group to analyze the effect of treatment. statistical analysis the quantitative percentages of il-10, il-12 and il-14 expressions were analyzed statistically for normality and homogeneity and then further analyzed using anova and followed by lsd analysis among the treated group. results and discussion extraction the zingiber cassumunar ethanol extract (eezc) was dark brown with a specific odor, exhibited thick consistency, and has slightly bitter taste. the extraction process produced 25.55% yield which has met the standard of the indonesian herbal pharmacopoeia (depkes ri 2008). curcumin was found to be the major content in the eezc extract, h however, the other curcumin derivates (i.e., demethoxycurcumin and bisdemethoxycurcumin) were not detected (fig. 1). curcumin reportedly showed immunomodulatory activities (varalakhmi et al. 2008). after the treatment, significant increases of il12 levels were observed among the curcumintreated animals on day 10 and 20. curcumin was biotropia vol. 28 no. 2, 2021 112 also found to induce generation of reactive oxygen species (ros) which are important in the immune responses (varalakhmi et al. 2008). curcuminoids (cassumunin a and cassumunin b) isolated from z. cassumunar were observed to have a protective effect on living cells suffering from oxidative stress (nagano et al. 1997). besides curcumin, essential oil was also reported as one main compound in zingiber cassumunar rhizome. the high essential oil content was responsible for the specific odor of z. cassumunar rhizome and extract. several studies on the phytochemical compounds and biological activities of z. cassumunar roxb had reported the main component of z. cassumunar rhizome essential oil as triquinacene 1,4-bis (methoxy), (z)-ocimene and terpinen-4-ol (buain & paisooksantivatana 2009). previous studies on its rhizome also found several phenylbutenoid compounds, curcuminoid, and sesquiterpene (zerumbon) (nakamura et al. 2009). expression of il-10 indirect immunocytochemistry was used to detect the interleukin expression in the macrophage (fig. 2). the specific antibody of il-10 interacted with interleukin-10 in the cells and attached itself to the secondary antibody. during the detection process, the secondary antibody attached itself to dimethyl amino benzidine (dab) as the chromogen, and the expression appeared as brown stains on the cytoplasm area, while the cells with negative expression appeared as blue stains as the result of counterstaining. the percentage of the il-10 expression is shown in table 1. figure 1 the tlc profile of curcuminoids standard (a) and zingiber cassumunar ethanolic extract (b), detected in daylight (a) and uv 254 nm (b). a b interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 113 figure 2 immunocytochemistry of il-10 expression in macrophage cells after beign treated with ethanol extract of zingiber cassumunar which was observed with 400x magnification: (a) macrophage with no expression of interleukin; and (b) macrophage with positive expression of interleukin table 1 percentage of il-10 expression on the macrophage cells of balb/c mice treated with ethanol extract of zingiber cassumunar groups mean ± sd normal negative control treatment dose of 1.25mg/20gbw treatment dose of 2.5mg/20gbw treatment dose of 5mg/20gbw 45.65 ± 1.92%* 51.86 ± 1.42% 55.91 ± 3.07% 63.68 ± 2.93%* 68.65 ± 4.42%* notes: * = significant difference with negative control (p<0.05); bw = body weight. the treatment of zingiber cassumunar ethanol extract (eezc) has increased the expression of il-10, affirming its potential as an immunomodulator. il-10 was expressed by macrophages and other dendritic cells (dc) as a response to microbial infection. the increase of il-10 expression may be attributable to the activation of extracellular signal-regulated kinase 1 (erk1) and erk2 (saraiva & o’garra 2010). such an increase will activate the specific response of the immune system and inhibit the nonspecific response. il-10 has been identified as an inhibitor of the synthesis of inflammatory mediators and pro-inflammatory cytokines that play a role in modulating fever and sickness (harden et al. 2013). the present study also found that the expression of il-10 in the negative control group significantly increased as compared to the normal group, which could be caused by the tween 80 effect. another study also reported that polysorbate (tween) 80 could increase the immune response (maggio 2012). the increased expression of il-10 was proportional to the administered dose. the higher the treatment dose, the higher the expression of il-10. however, an extremely high level of il-10 can inhibit chemokine production and prevent its role in directing lymphocytes to the lymph nodes, as manifested in mycobacterial infection, resulting in a failure to recruit and induce th1 cell differentiation (couper et al. 2008). therefore, il-10 has both immunosuppressive and immunostimulatory properties (acuner-ozbabacan et al. 2014). regulation of the il-10 expression involved the enhancement or silencing of il10 transcription and is performed by certain transcription factors activated by discrete signaltransduction pathways. following transcription, the post-transcriptional mechanisms existed and involved many of the molecular events leading to il-10 expression (saraiva & o’garra 2010). some molecules of zingiber cassumunar were involved and had affected the transcriptional biotropia vol. 28 no. 2, 2021 114 process of il-10 and resulted in the increasing il-10 levels. expression of interleukin-12 interleukin-12 (il-12) is a pro-inflammatory cytokine that induces the production of interferon-γ (ifn-γ), leading to the differentiation of t helper 1 (th1) cells and connecting the link between innate and adaptive immunity. dendritic cells (dcs) and macrophages produce il-12 in response to pathogens and infection (trinchieri 2003). production of il-12 is strongly regulated by positive and negative regulatory mechanisms. microorganism products including bacteria, intracellular parasites, fungi, double-stranded rna, bacterial dna and oligonucleotides are strong inducers of il-12 production by macrophages, monocytes, neutrophils and dcs. in this study, the lps was used to activate the macrophage production of the il-12 expression after the administration of eezc and was analyzed quantitatively (table 2). the study found out that the il-12 expression in the negative control group was not significantly different from the normal group, indicating that the solvent (tween 80) did not affect the immune response. tween 80 can stimulate the immunogenicity (maggio 2012) as also shown by the increased il-10 levels in the present study. however, the dosage applied in this study was not enough to increase the il-12 expression. eezc treatment at a dose of 1.25mg/20g bw resulted in an il-12 expression lower than the negative control. hence, the lower the dose, the less effective is the eezc active compound in increasing the il-12 expression. when the dose was increased, the il-12 expression was also heightened. the eezc immunomodulatory effects might have been due to the presence of curcumin, the active compound known to increase the immune response of the cells (nagano et al. 1997; nurkhasanah et al. 2017). curcumin treatment also elevated the il-12 expressions in mice (varalakhmi et al. 2008). the increasing level of il-12 in the treatment was caused by the capacity of curcumin in increasing reactive oxygen species (ros) and nitric oxide (no) (nurmasari et al. 2014; rahmawati 2013). ros is known to regulate the il-12 generation. curcumin was also found to stimulate the t cells, b cells, neutrophil, nk cell, and dendritic cell (nurmasari et al. 2014). the essential oil, which emitted a special odor, was also identified in the eezc (bhuiyan et al. 2008). these eezc essential oils were also reported to boost the body immune response, including the phagocytic activity of macrophages (chairul et al. 2009; nakamura et al. 2009). the active compounds from the volatile oil are the phenilbutenoids which were successfully identified as immunomodulatory (chairul et al. 2009). the eezc treatment increased the il-12 expression, thereby activating the t cells and stimulating the production of ifn-γ, which led to macrophage activation and secretion of reactive oxygen species (ros) that eliminate infections (abbas et al. 2017). furthermore, this treatment has intensified the phagocytic activity of macrophages (nurkhasanah et al. 2017). expression of interleukin-14 eezc treatment also increased the il-14 expressions after lps induction (table 3). il-14 was the first known high-molecular-weight bcell growth factor, originally identified as a b cell growth factor (shen et al. 2006). as produced by t cells and b-cells, the il-14 binds and signals through a 90-kda receptor that promotes b-cell proliferation (akdis et al. 2016). high levels of il-14 can enhance b-cell proliferation and can expand a subpopulation of memory b cells (leca et al. 2008), and if followed by the secretion of antibody, it can also eliminate the invader. table 2 il-12 expression in the macrophage cells of balb/c mice treated with ethanol extract of zingiber cassumunar (eezc) groups mean ± sd normal 64.63% ± 9.763 negative control 66.39% ± 1.603 treatment dose of 1.25mg/20g bw 51.56% ± 4.528* treatment dose of 2.5mg/20g bw 70.62% ± 3.469 treatment dose of 5mg/20g bw 77.00% ± 5.110* note: * = showed significant difference from the negative control (p<0.05). interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 115 table 3 interleukin-14 expressions in mice treated with ethanol extract of zingiber cassumunar groups expressions (x ± sd) normal 59.19 ± 3.07% negative control 61.24 ± 1.51% treatment dose of 1.25 mg/20g bw 57.02 ± 1.94%* treatment dose of 2.5 mg/20g bw 67.41 ± 6.60% treatment dose of 5 mg/20g bw 71.07 ± 1.30%* note: * = showed significant difference with the negative control (p<0.05). previous researches studied the increase of il-14 expression by using some medicinal herbal extracts (nurkhasanah 2015). the treatment of anthocyanin-rich rosella extract increased both the il-10 and il-14 expressions in vitro. the present research also found that the treatment of eezc increased the il-10, il-12, and il-14 expressions after lps induction. this induction stimulated the immune response as lps was recognized as an endotoxin, consisted of a lipid and a polysaccaride, found on the outer membrane of gram-negative bacteria. eezc’s active role in increasing the il-10, il12 and il-14 exhibited its potency in inducing both the innate and adaptive body immunity. studies on zingiberaceae family, including curcuma mangga, kaempferia angustifolia, and zingiber cassumunar recorded that zingiber cassumunar displayed the highest immunomodulatory activity (chairul et al. 2009). this is probably due to the active curcumin compound and its essential oil which has the phenyl butanoic substance. furthermore, a toxicity study confirmed that z. cassumunar extract has no observable adverse effect and it is well-tolerated for both acute and chronic toxicity studies (koontongkaew et al. 2014). conclusion the zingiber cassumunar ethanol extract (eezc) exhibited immunomodulatory activities by increasing the levels of il-10, il-12 and il14 cytokines in the treated mice. this study also suggested the extract’s potency to induce both the innate and adaptive body immunity. acknowledgment the 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the family dipterocarpaceae from pasir mayang, jambi, sumatera hilman affandi, arif nuryadin, susilo b. prayoga seameo b1otrop, jl. raya tajur km. 6 bogor. indonesia and roger w. read department of organic chemistry the new south wales university, p.o. box / kensington, n.s.w. 2033, australia abstract screening of 12 species of shorea and 2 species of anisoptera from the forest of pasir mayang, jambi with brine shrimp (anemia salina) lethality bioassay showed that shorea gibosa and anisoptera marginata have sufficient activity for further investigation. bioassay-guided fractionation of the active extract of s. gibosa led to the isolation of stigmasterol and the shoreaphenol. bioassay-guided fractionation of the active extract of a. marginata resulted in the isolation of lupenone and 3-methoxy-4-hydroxybenzoic acid o-p-c glucopyranoside as the bioactive compound. key words: plant natural products/sftorea gibosalanisoptera marginatalactive extracts/bioassay/pasir mayang. introduction dipterocarp timbers are well known to resist biological attack from many sources. shorea robusta was shown to be highly resistant to the termites microcerotermes beesoni and heterotermes indicola (sen-sarma 1963). particle boards constructed from shorea species are also protected against cryptotermes cynocephalus (moi 1980). fresh resin of anisoptera thurifera appears to protect beenests from termites (messer 1984). dipterocarp woods cause substantial mortality to insects feeding on them. over a three-month test period, termites feeding on shorea species suffered up to 99% mortality, while termites feeding on the nondipterocarp dyera costolata showed only a 13% death rate (moi 1980). chemical factors in either resins and bark may also protect dipterocarps from microbial attack. untreated dipterocarp timbers are reported to be highly resistant to fungal invasion (bakshi et al. 1967), and volatile components of hopea papuana were shown to inhibit fungal growth (messer 1984). bacterial growth inhibitors of dipterocarp origin include essential oils of valeria indica (bhagarva and chauan 1968) and stemnoporol and alpha-copalliferol from sri lankan dipterocarps (sootheeswaran et al. 1983). 42 screening for natural products ofshorea spp. and anisoplera spp. hilman affandi el al. the research described here attempts to identify the active compounds that are responsible for their toxicities against brine shrimp (artemia salina). the crude extracts of the stem bark of twelve species of shorea and two species of anisoptera collected from pasir mayang, jambi, sumatera were screened with brine shrimp lethality bioassay. bioassay-guided fractionation of the active extracts led to the isolation of the active substances reported in this paper. methodology general 'h n.m.r spectra were recorded at 500 mhz and 13c n.m.r spectra were measured at 125.6 mhz on a bruker am 500 spectrometer. mass spectra were recorded at 70 ev with an a.e.i. ms 12 spectrometer. all measurements were performed at the school of chemistry, university of new south wales, australia. initial column chromatographic separations were made on merck 60 silica gel. merck kieselgel 60 f254 was employed for preparative tlc with a 1 mm layer of adsorbent on 20 x 20 cm glass plates and 200 mg of material applied to each plate. analytical tlc was performed on commercial aluminium-packed merck kieselgel 60 f254 art. 5554 plates, visualised under uv 254 nm light or by charring after applying a 4% h2so4/meoh spray. plant material the stem bark of twelve species ofshorea i.e. s. opalis, s. guiso, s. selanica, s. seminis, s. pinanga, s. stenoptera forma, s. stenoptera (burck)., 5. mecistopteryx, s. leprosula, s. palembanica, s. multiflora, s. gibosa and two species of anisoptera namely a. marginata, and a.costata were collected from pasir mayang, jambi, sumatera. extraction and isolation an amount of 20 g of air dried plant materials from each species ofshorea and anisoptera was extracted with methanol at room temperature. the crude extracts were evaporated under reduced pressure. sea water was poured into a small tank and shrimp eggs were added to the tank provided with an aerator. after two days the shrimp eggs hatched and mature as naupili. each crude extract was weighed (20 mg) and dissolved in dichloromethane (2 ml). from this solution 500, 50, 5 ul were transferred to small petri dishes corresponding to 1000, 100, 10 ug/ml (ppm), respectively. the solvent was evaporated by standing at ambient temperature overnight. after two days (when shrimp larvae are ready), sea water was added to each petri dish, inoculated with 10 shrimp per dish (30 shrimp per dilution), and the 43 biotropla no. 12, 1999 volume was adjusted with sea water to 5 ml/dish. the number of surviving shrimps were recorded after 24 hours. the data were analysed with a finney computer program to determine lc50 values and 95% confidence intervals (hostettmann 1991). each crude extract of the sample was tested in three replicates against a single control. the controls were treated in a similar manner without the presence of crude extracts. the results of the initial screening are compiled in table 1. table 1. relative toxicity of the crude extracts against brine shrimp at 1000, 100, and 10 ppm no species lc50 (nq/ml) (c.i. 95%)* 1 lshorea opalis 513 2 anisoptera marginata 293 3 s. guiso 1114 4 s. selanica 270 5 s. seminis 392.4 6 s. pinanga 717 7 s.stenoptera forma 691 8 s. mec/sfopterix 394.9 9 s. leprosula 259 10 s. stenopfera burck. -ve 11 anisoptera costafa -ve 12 s. palembanica 882 13 s. multiflora 346 14 s. gibosa 205 c.i. 95% = confidence intervals 95% -ve = not toxic at a concentration of more than 1000 ppm the result of the screening indicated that the crude extracts of shorea gibosa, s leprosula, and s. selanica with toxicity level (lc50) at 205, 259, and 270, respectively, showed significant activity against brine shrimp. meanwhile among anisoptera spp., a.marginata showed sufficient toxicity (lc50 = 293). in this preliminary work, however only s. gibosa and a. marginata were chosen for further investigation. therefore, large-scale extraction and fractionation of both species were carried out in the next phase of the research. extraction and fractionation of shorea gibosa. the air-dried plant material of s. gibosa was extracted with methanol. the crude extracts (100.2 g) were extracted with hexane to remove the hexane-soluble fraction (19.8 g), and the residues were chromatographed by column chromatography (silica gel 60). the initial solvent system used in the column was hexane. the polarity of the solvent was gradually increased by the addition of chloroform into the solvent system until the mixture of hexane and chloroform became 60% in hexane. the solvent system was then changed, initially by adding 5% of methanol into the chloroform until the con 44 screening for natural products ofshorea spp. and anisoptera spp. hilman affandi et al. centration of methanol became 30% in chloroform. the fractions were grouped and monitored by analytical thin layer chromatography (tlc) to obtain 14 fractions. all fractions, including the hexane fraction, were screened with the brine shrimp lethality bioassay (table 2). table 2. relative toxicity of the fractions against brine shrimp fractions lcso (ng/ml) (c.i.95%) fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7 fraction 8 fraction 9 fraction 10 fraction 1 1 fraction 12 fraction 13 fraction 14 -ve -ve 823 882 226 240 132 127 c.i. 95% = confidence intervals 95% -ve = not resistant at a concentration of more than the results showed fraction 9 to be the most toxic fraction, followed by fraction 14, 10, 8, and 7. however, due to time limitation and budgetary constraints only fraction 9 was subjected to further investigation. therefore, fraction 9 (1.15 g) was purified by preparative tlc, eluted initially with hexane, and later with the mixture of hexane and chloroform ( 3 : 7) to yield three fractions. fraction 1 (0.46 g) consisted of a mixture of 4 5 compounds as shown by analytical tlc. fraction 2 (0.31 g) was dissolved in a mixture of chloroform and methanol. the mixture was allowed to stand overnight and precipitation occurred. the precipitation was filtered to give solid material as a white amorphous crystal of stigmasterol (0.13 g), m.p. 169°c. meanwhile solid material present in fraction 3 (0.22 g) was filtered and recrystallised with chloroform-methanol, afforded shoreaphenol as orange crystalline compounds (13.27 mg; m.p. 240°c). extraction and fractionation of anisoptera marginata. air-dried stem bark of a. marginata (1.7 kg) were extracted with methanol. after removing the solvent under reduced pressure the crude extract (121.4 g) was extracted with hexane, to remove the hexane-soluble fraction (19.02 g). the remaining crude extract (102.38 g) was subjected to column chromatography (silica gel 60), initially eluted with 45 biotrop1a no. 12,1999 chloroform, and later with methanol by increasing the polarity at 30%, 60%, 80% and 100% levels. the fractions were pulled and monitored by analytical tlc to yield 18 fractions. fraction 1 to 10 were combined, since the components of those fractions were relatively similar judging from analytical tlc. all fractions, including the hexane fraction, were screened with the brine shrimp lethality bioassay (table 3 ). table 3. relative toxicity of the fractions against brine shrimp fractions lcjo (ng/ml) (c.i.95%) hexane fraction fraction 1 1 fraction 12 fraction 13 fraction 14 fraction 15 fraction 16 fraction 17 -ve 300 114 65 44 72 121 c.i 95% = confidence intervals 95% -ve = not resistant at a concentration of more than 1000 ppm the results indicated that fraction 14 was most toxic. fraction 14 (1.43 g) was subjected to preparative tlc, eluted twice initially with hexane followed by 30% chloroform in hexane, to yield fraction 1 (0.87 g), fraction 2 (0.13 g), fraction 3 (0.03 g); and fraction 4 (0.49 g). solid material present in fraction 1 was filtered, followed by recrystalisation in chloroform to yield the triterpenoid lupenone as a white crystalline amorf (0.21 g), m.p 160°. fraction 4 was dissolved in methanol and a crystalline solid was produced upon addition of a few drops of methanol. the solid residue was filtered and recrystallised with a mixture of chloroform and methanol to yield a colorless crystal (0.16 g), m.p. 145°. all fractions, including two compounds isolated from fraction 1 and 2, were tested with the brine shrimp lethality bioassay (table 4). table 4. relative toxicity of the fractions against brine shrimp fractions lcw (tig/ml) (c.i. 95%) lupenone fraction 2 fraction 3 glue. comp. -ve -ve -ve 7 c.i 95% = confidence intervals 95% -ve = not toxic at a concentration of more than 1000 ppm 46 screening for natural products ofshorea spp. and anisoptera spp. hilman affandi et al. results and discussion methanolic extract of the stem bark of s. gibosa, followed by fractionation by column chromatography and monitored by tlc resulted in 14 fractions. the fractions were screened with brine shrimp lethality bioassay and purification of the active fractions led to the isolation of non-biologically active compound stigmasterol. the structure of stigmasterol was established by 'h and i3c n.m.r. spectroscopy (fig. 1). the 'h n.m.r. spectrum showed two methyl singlets, 8 0.57, 1.01; three methyl doublets, 8 0.79, 0.84, 1.03; and a methyl triplet, 8 0.80. also three proton multiplets corresponding to two internally coupled, trans olefinic protons 8 5.03 (dd, j 15.1, 8.4 hz, and 5.15 (dd, j 15.1, 8.4 hz) and an isolated olefinic proton (8 5.18, m) with long range coupling were present, but no other signals occurred at higher chemical shift than 2.5 ppm. the presence of a 1,2disubstituted and a 1,1,2-trisubstituted olefin was supported by the presence of signals at 8 117.0 (ch), 129.6 (ch), 138.1 (ch) and 139.5 (c) in the "c n.m.r. figure 1. structure anisopte 1. stigmasterol 3. lupenone 4. 3-m acid of chemical constituents isolated from the ba ra marginata (3 and 4). 2. shoreaphenol ethoxy-4-hydroxybenzoic -o-p-c glucopyranoside rk of shorea gibosa (1 and 2) and 47 biotropia no. 12,1999 spectrum. the presence of a quaternary signal at 8 212.0 also confirmed the occurrence of a carbonyl group in the compound. the structure of stigmasterol was also confirmed by mass spectrometry at mlz 367 (m-c3h7), 298 (m-c8h,6), 271 (mc,0h,9), 269 (m-c10h2,), and comparative melting point analysis. stigmasterol is widely distributed in the plant kingdom. application of this compound as therapeutic agents is very limited, although extensive exploratory activities in this area have been underway during recent years (mahato'ef a/. 1992). the solid material produced from fraction 4 of the active fraction was filtered, followed by repeated crystallisation to give an orange crystalline solid of shoreaphenol. the 'h n.m.r. spectroscopy (table 5) data revealed the presence of signals at 6 8.61 (2h, br d, j 8.7hz, h-ll and h-15), 7.84 (2h, br d, j 8,7hz, h12 and h-14), 7.74 (2h, br d, j 8.8hz, h-24 and h-28), and 7.44 (2h, br d, j 8.8hz, h-25 and h-27). these signals clearly indicated the presence of two pdisubstituted benzene nuclei. the 'h n.m.r. spectrum also established signals for four m-coupled proton at 6 8.42 (1h, d, j 2.1hz, h-3), 8.11 (1h, d, j 2.1hz, h5), 7.58 (1h, d, j 2.5hz, h-19) dan 7.33 (1h, d, j 2.5hz, h-21). in addition to these signals, five table 5. 'h n.m.r. spectral data of compounds (1) (4) (ppm in cdc13 cd3od, and acetone d6) screening for natural products ofshorea spp. and anisoptera spp. hilman affandi el al. phenolic hydroxyl protons were also discernible in the 'h n.m.r. spectrum in the region 7.92 to 7.96. the i3c n.m.r. spectra (table 6) were characteristic of substituted a and (3carbons of the benzofuran nucleus. furthermore, the appearance of one proton at 5.98 (8c 54.97, d) clearly suggested the presence of-ch-coin the molecule of shoreaphenol. the mass spectrometry data are consistent with the assigned structure m/z 466. comparison of the 'h and 13c n.m.r. spectral data and its melting point of shoreaphenol with those reported (saraswathy et al. 1992) suggested close similarity. the methanolic extract of the bark of the tree of anisoptera marginata was fractionated by column chromatography using silica gel, and monitored with tlc to give 9 fractions. brine shrimp bioassay of those fractions showed the fraction eluted with chloroform-methanol (8 : 2) to be the most active fraction. the active fraction was subjected by preparative tlc, and the precipitation that occurred in fraction 1 biotropia no. 12, 1999 was filtered and crystallised with chloroform to yield the terpenoid lupenone as a white crystalline amorf (0.21 g), m.p 160°. the structure of lupenone was established from 'h and 13c n.m.r. (table 5 and 6). the 'h n.m.r. spectral data indicated the presence of five singlet resonances at 8 0.89, 0.94, 0.97, 1.07 and 1.19, arising from six methyl groups attached to quaternary carbons. there also appeared one isolated singlet at 8 1.68 due to a vinylogous methyl group, and the olefmic proton signals at 8 4.56 (dd, j 2.2, 1.4 hz) and 4.68 (d, j 2.2 hz) were consistent with a 1,1-disubstituted alkene, a propenyl group was likely. low field signals resonance at 8 3.18 (dd, j 10, 9, 5.3 hz), 2.38 (dt, j 5.7, 11.1 hz) and 1.91 (m) occurred, but there were no other olefinic resonances. the n.m.r. signal at 8 3.18 was suggestive of an axial proton adjacent to a quaternary centre and attached to a carbon bearing hydroxyl group. the 13c n.m.r. spectrum (table 5) supported the structural assignment of lupenone. in particular there appeared seven methyl signals, only two olefinic carbon signals 8 109.3 (ch2) and 151.0 (c), and a low field methine resonance ( 8 78.9) for c3. the identity of compound (1) as lupenone was confirmed by melting point and mixed melting point with an authentic sample (affandi et al. 1998). the solid material produced in fraction 4 was filtered, followed by recrystallisation in methanol to give 3-methoxy-4-hydroxybenzoic acid o-p-c glucopyranoside as a colorless crystalline amorf (0.98 g), m.p 145°. the 'h and 13c n.m.r spectral data of 3-methoxy-4-hydroxybenzoic acid-o-p-c-glucopyranoside are shown in table 5 and 6. the infrared (ir) spectrum of the compound disclosed the broad absorption at 3600 2500 cm"1 and the carbonyl stretch at 1700 cm'1. this is in agreement with the compound being an acid. the 'h n.m.r. spectrum of the compoundshowed the signals of the following protons (acetone-d6, 500 mhz): proton in 3-methoxy-4hydroxybenzoic acid, 8 3.79 (3h,s), 7.01(1h, s), 8.56 (1h, br s), 9.30 (1h, br s). proton in a glucopyranoside ring, 8 4.11 (1h, dd), 3.77 (1h, m), 3.44 (1h, dt), 3.84 (1h, m), 3.62 and 4.07 (each 1h, ddt), proton in a hydrogen bonded hydroxyl group, 8 4.87 (1h, dd), 5.43 (1h, d), 5.62 (1h, d). the 13c n.m.r, spectrum of the assigned structure indicated the presence of six quaternary carbon signals at 8 109.0, 115.7, 118.7, 139.9, 148.3, 150.9. primary carbon signals were obtained at 8 70.5, 72.3, 74.1, 79.6, 81.6, and 109.0. signals for a carbon methoxy group at 8 59.6, carbon methylene at 8 61.3 and carbon acid at 8 163.1 also were obtained. mass spectroscopy confirmed m/z 328 of the molecular formula c14h16o4. furthermore, the location of the c-glucoside ring was confirmed by the two dimensional noe spectroscopy (noesy) spectrum as described in figure 2. the long-range c-h correlation spectra with proton detection (hmbc) as described in figure 3 permits any coupling between oh protons and the carbons to which they are attached, to be detected by the appearance of cross peaks. brine shrimp lethality bioassay of this compound showed to be highly toxic against brine shrimp (lc 50 = 7), it means that the compound caused the mortality of 50 biotropia no. 12, 1999 brine shrimp more than 50% at concentration 7 ppm (jig/ml). future investigations might reveal medicinal or other uses of the compound. references affandi, h., a, nuryadin, and r.w., read 1998. studies on natural products of albizia sp., b1otropia, 11, 1 -8. bhargarvca, a.k., and c.s., chauan. 1968. antibacterial activity of some essential oils., indian j. pharm. 30:151-152. bakshi, b.k., y.n., puri, and s., singh. 1967. natural decay resistance of indian timbers. i. introduction and method. ii. resistance of sal (shorea robusta gaertn.) and teak (tectona grandifloris l.f), indian for., 93 : 305-328. hostettman, k., ed., 1991. method in plant biochemistry, vol. 6: assays for bioactivity, pergamon press, new york. mahato, s.b., a.k., nandy, and g., roy. 1992. triterpenoids, phytochemistry, 31(7), 2199-2249. messer, a.c., 1984. chalicodoma pluto : the world's largest bee rediscovered living communally in termite nests (hymenoptera: megachilidae), j. kans. entomol, soc., 57 : 165-168. moi, l.c., 1980. a new laboratory method for testing the resistance of particle boards to the drywood termite cryptotermes cynocephalus. malaysian for., 43:350-355. saraswathy, a., k.k., purushothaman, a., patra, a.k., dey, and a.b., kundu. 1992. shoreaphenol, a polyphenol from shorea robusta. phytochemistry, 31(7), 2561-2562. sen-sarma, p.k.. 1963. studies on the natural resistance of timbers to termites, i. observations on the longevity of the test termite heterotermes indicola wasm. in the saw dust from forty common indian timbers. indian for. bull. (n.s.) entomol, 220 : 1-3. sootheeswaran, s., m.v.s., sultanbawa, s., surendrakumar, and p., blandon. 1983. poly phenols from a dipterocarp species: copalliferol and stemnoporol. i. chem. soc. perkin trans. i, 699-702. 52 2 development og gene.cdr biotropia vol. 18 no. 1, 2011: 13 23 13 comparison of three different techniques of gene transfer in humpback grouper ( ) cromileptes altivelis slamet subyakto , alimuddin , rustidja , m. sasmito jati , irvan faizal , ratu siti aliah , gemi triastutik , and komar sumantadinata humpback grouper is one of the most cultured fishes in asia, including indonesia. the main problem faced by humpback culture is its slow growth rate. one of the methods that will be more effective and efficient to solve the problem is using transgenic technique. this study was conducted to determine the effectiveness of transfection, microinjection and electroporation techniques on gene transfer in humpback grouper. transfection was performed by incubating sperm to the foreign dna (pktbp-ktgh gene construct)-transfectant complex solution, while was by injecting those complex solution into testis of mature males. microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign dna solution, and duration of injection was 1, 2 and 3 seconds. electroporation by 50 v, 30 ms of pulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using three dna concentration of 5, 10 and 20 μg/ml. the incorporation of foreign dna in sperm and embryos were analyzed using pcr method. based on pcr analysis, an optimum dna concentration for electroporation was 10 μg/ml. limited number of embryos could be microinjected during 20-30 min to reach 2-4 cell stage. microinjection for 1 second showed higher survival rate of embryos, although none or very low number of larvae was hatched. transfast was an effective dna delivery reagent for humpback grouper sperm. foreign dna could be detected in sperm from two out of ten transfected fish at least 36 hours post transfection (hpt). by transfection, foreign dna was detected in sperm at 48 hpt 25 c incubation temperature. our study revealed that transfection, microinjection as well as electroporation could be used as transgenesis methods in humpback grouper. by means of simplicity and efficacy, however, electroporation was an appropriate gene transfer method. transfection, microinjection, electroporation, transgenesis, humpback grouper 1 2 3 4 5 5 1 2 o * 1 2 3 4 5 brackishwater aquaculture development center, situbondo, ministry of marine affairs and fisheries, indonesia department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, indonesia faculty of fisheries and marine science, university of brawijaya, malang, indonesia faculty of mathematics and natural sciences, university of brawijaya, malang, indonesia agency for the assessment and application of technology, jakarta, indonesia in vitro in vivo in vivo in vitro abstract key words: * corresponding author : alimuddin_alsani@yahoo.com biotropia vol. 18 no. 1, 2011 14 introduction materials and method in the last two decades, a variety of transgenic aquatic organisms have been successfully produced by researchers around the world. these transgenic organisms have been produced by introducing gene/transgene encoding protein using various transgenesis methods, that is microinjection, electroporation, transfection and particle gun bombardment (chen . 1998; sarmasik 2001). transgene can be integrated and expressed in descendants of transgenic fish. furthermore, transgenic organisms have produced the expected phenotypes. until now, most of the generated transgenic aquatic organisms are freshwater fish because these fish are relatively easy to maintain and spawn under laboratory condition. in contrast, marine fish receive very little attention despite the fact that marine fish have a high potential economic value in aquaculture. humpback grouper is one of the most cultured fishes in asia, including indonesia. the main problem faced in humpback culture is its slow growth rate. for instance, to reach consumption size of about 0.5 kg 1.0 kg, this species needs 8 up to 24 months (tucker 1991; teitelbaum 2007). its slow growth makes the operational cost of this fish aquaculture increases drastically. therefore, developing a proper method that will be more effective and efficient to solve the problem is urgently needed. application of transgenesis technique is expected to solve the problem for humpback grouper and also other marine fish, though the use of transgenic organisms in aquaculture remains controversial. several methods of transgenesis have been successfully applied to create transgenic fish. devlin (1994) utilized microinjection method to generate transgenic fish. pacific salmon, alimuddin (2005) succeeded in zebrafish. electroporation method has also been successfully used by sin . (1993) and symons . (1994) on production of transgenic salmon, while patil and khoo (1996) and rambubu (2005) succeeded in zebrafish. the transfection method was successfully applied for gene transfer in silver snapper (lu 2002) and white shrimp (sun 2005). this study was conducted to determine the effectiveness of transfection, microinjection and electroporation methods in gene transfer for humpback grouper. transfection method is one of gene transfer methods using a specific reagent that is capable of binding to and delivering foreign dna to enter the cell. foreign dna used in this study was all-humpback grouper gene construct, pktbp-ktgh, which has been constructed by rusnas (national strategic prime research) program of ministry of research and technology, republic indonesia) for genetic improvement of hump et al et al. et al. et al. et al et al et al. et al. et al. transfection back grouper research team in 2007. pktbp-ktgh contains growth hormone gene (ktgh) and β-actin promoter (ktbp) from humpback grouper. 15 gene transfer method in humpback grouper alimuddin .et al a. transfection b. transfection microinjection electroporation dna extraction preliminary experiment was conducted by two transfectanst, i.e. transfast (promega) and jetpei (polyplus) to determine their efficacy to deliver pktbp-ktgh into sperm. in this experiment, 100 μl of grouper sperm was exposed to 360 μl of physiological solution containing 10 μl pktbp-ktgh and either 30 μl transfast or jetpei, at 17ºc and 25ºc incubation temperature for 24, 48, and 72 hours transfection. plasmid pktbp-ktgh was injected into humpback grouper testes prior to spawning according to the method of lu . (2002) with slight modifications. foreign dna-transfast complex solution at the solution was injected into the right and left testes of mature broodstock, respectively. at 12, 36, 48, 60 and 72 hours post injection, dna from the transfected sperm was extracted and used as template of pcr amplification to determine the success of transfection method. microinjection was conducted as described previously (alimuddin . 2005) microinjection was carried out to fertilized grouper eggs at 2-4 cell stage. a microinjection plate that consists of ten grooves of 3% agarose gel was used to support embryos when penetrated by microinjection needle. humpback grouper embryos were gently transferred onto the groove of microinjection plate and blastodisc should face the direction of the microinjection needle. dna solution at concentration of 25 μg/ml was slowly injected into blastodisc. the volume of injected dna solution was about one-fifth of the blastodisc volume (ath-thar 2007). number of embryos that can be injected by one microinjector in each spawning time was 60-100 embryos. three durations of injection (1, 2 and 3 seconds) into each embryo were examined to obtain length of injection time resulting to higher survival number of injected embryos. electroporation is a gene transfer method that utilizes a series of electric shock to help foreign dna to enter the cell. electroporation was carried out using gene pulser xcell electroporation system (biorad) by square wave method with parameters of 50 volt, 30 ms of pulse length, 5 pulse numbers, 0.1 s of pulse interval, and using cuvettes of 0.2 and 0.4 cm gap size. these electroporation parameters were obtained from the preliminary study. concentrations of dna used in this study were 5, 10 and 20 μg/ml, respectively. each dna concentration was mixed with 25 μl grouper sperm and sodium chloride solution to final volume of 260 μl. an amount of 100 microliters of electroporated sperm was used for dna analysis using pcr method. transfected and electroporated sperm solution was subjected to centrifugation for 1 min at 3000 rpm to remove remaining dna in solvent and transfection reagent. sperm was washed two times with 200 μl physiological salt solution before performing in vitro in vivo et al et al ratio of 1:2 (1 μg of dna: 6 μl transfast) was dissolved in physiological solution to final volume of 500 μl, and then half of 16 dna extraction. sperm and the injected embryos were lyzed with 500 μl of lysis buffer for 10 minutes at 95ºc. dna was precipitated using 400 μl of ethanol 96% and then diluted with 50 μl depc water. dna solution was stored at -20ºc. pcr analysis was performed in 10 μl of 1 microgram dna extracted from sperm and embryos, 1 μl primer forward and reverse (10 pmol), 1 μl dntps mix, 1 μl ex buffer, 0.05 μl ex polymerase (takara bio) and sdw to reach the final volume of 10 μl. forward and reverse primers designed and used in this study were located at 3' terminus of ktbp promoter (fbp2) and ktgh (rgh1) sequences, respectively. the sequence of those primers was 5' ttcatccagctgatgatt gccagatgtaac-3' and 5'-agttggcttca-ggagagagtcgacattt ag-3'. a total of 35 cycles of denaturation for 30 s at 94ºc, annealing at 62ºc for 30 s, and extension at 72ºc for 1 min were conducted. two microliters of pcr product was electrophoretically separated using 1.5% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. sperms survived when transfection was conducted using transfast reagent in both incubation temperatures, while contrarily jetpei-transfected sperm died (table 1). furthermore, the results of pcr analysis showed that transfection using transfast reagent for 48 hours at 25 c incubation temperature allowed foreign dna to incorporate into sperm (fig. 1 lane 2, table 1). the forward primer located at 5' terminus of ktbp (fbp1) and ap2 reverse (5'-ctatagggcacgcgtggt-3') was used to ensure that the amplified dna product was the foreign dna. pcr product using this set primer was about 2 kb in size (fig. 1 lane 1). the result suggested that foreign dna was incorporated in sperm. pcr amplification transfection taq taq results and discussion in vitro o biotropia vol. 18 no. 1, 2011 table 1. survival and incorporation of foreign dna into transfected sperms using transfast and jetpei reagents at 17 c and 25 c incubation temperatures. in vitro o o treatments duration of transfection (hours) 24 48 72 incubation temp. 25 oc transfast sperm survived, did not carry the foreign gene sperm survived and carry the foreign gene sperm survived, did not carry the foreign gene jetpei sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene control sperm survived, no carry the foreign gene incubation temp. 17 oc transfast sperm survived, did not carry the f oreign gene sperm survived, did not carry the foreign gene sperm survived, did not carry the foreign gene jetpei sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene control sperm survived,did not carry the foreign gene 17 in vivo transfection in vivo transfection was carried out using ten mature male fish by injecting dnatransfast complex to testis through a canulation tube. this technique was developed, since fish could die when directly injected by syringe into testis through urogenital pore was conducted. two out of ten broodstocks contained sperm carrying foreign gene at 36 hours and 60 hours after transfection, respectively. the size of dna band of pcr product of dna from sperm containing foreign gene was similar with that of pktba-ktgh as template (fig. 2, indicated by arrow head). kb 10,0 2,0 1,0 0,5 0,1 m 1 2 figure 1. results of pcr analysis with dna template extracted from transfected sperm using transfast. lane 1: pcr amplification product using primers fbp-1 (5'-gtgwgtgacgcyggaccaatc-3') and ap2 (5'-ctatagggcacgcgtggt-3'), lane 2: pcr amplification product using primers as described in materials and methods. arrow head indicates pcr product of foreign gh gene. transfection was carried out for 48 hours at 25ºc. m is 2-log ladder dna marker (biolabs, inc., new england). the amplified fragment is ~700 bp in size. kb 10,0 3,0 1,2 1,0 0,7 0,5 0,3 0,1 m 1 2 3 4 5 6 m figure 2. results of pcr amplification with dna that have been extracted from transfected sperm at 24 and 60 hours post transfection (hpt). lane 1 and 2: pcr product of dna from broodstock no. 2919 at 36 hpt and 60 hpt, respectively. lane 3 and 4: pcr product of dna from broodstock no. 2935 at 36 hpt and 60 hpt, respectively. lane 5: pcr product using pktba-ktgh as template, lane 6: product of pcr with no dna template. m is 2-log ladder dna marker (biolabs, inc., new england). in vivo gene transfer method in humpback grouper alimuddin .et al 18 biotropia vol. 18 no. 1, 2011 microinjection high survival rate of uninjected embryos (92%) indicated that a high quality of fertilized eggs was used for microinjection (table 2). percentage of survived microinjected embryos 14 hours after injection decreased with the increase in duration of dna injection into blastodisc (table 2). increasing the duration of injection into blastodisc increased dna volume entering the cytoplasm. thus, high dna volume in cytoplasm of blastodisc probably caused embryos to die. similar result has been obtained for catfish (at-thar 2007). in addition, embryo may also die when injection is too deep into yolksac (at-thar 2007). this might also contribute to the decrease in survival rate of injected humpback grouper embryos. as blastodisc of humpback grouper is very thin, it is highly possible that the microinjection needle enters the yolksac. furthermore, twenty microinjected embryos for each treatment at 14 hours post injection were pooled into a tube for dna analysis. pcr amplification product showed that all microinjected embryos group contained foreign dna (fig. 3). this suggested that foreign dna was transferred into blastodisc. table 2. number and percentage of developed embryos after injection of 25 μg/ml at 1, 2 and 3 seconds. treatment repetition no. developed embryos undeveloped embryos percentage developed embryos 1 second 1 42 2 95,5 2 41 14 74,5 mean 85.0±14.8 2 second 1 1 18 5,3 2 3 42 6,7 mean 6.1±1.0 3 second 1 0 31 0,0 2 0 105 0,0 mean control 508 39 0.0±0.0 92,9 electroporation in this study, application of electroporation method for transferring the gene revealed that high percentage of electroporated sperm was motile (table 3). there was no difference in sperm motility of treated and untreated dna i.e. during 5-10 minutes in water. this result suggested that electroporated sperm could fertilize the eggs as in untreated sperm. percentage of motile slight difference of water quantity in dna solution might take account to reduce percentage of motile ml dna concentration. furthermore, droop value when using 0.2 cm cuvette was slightly higher (6-12%) compared with 0.4 cm cuvette (0-6%). thus, droop seems to be affected by the size of cuvette used, although no effect on electroporated sperm motility (table 1). droop electroporated spermatozoa using dna concentration of 5 μg/ml was slightly lower (90%) compared to that of the two other treatments (100%). sperm in 5 μg/ 19 is a function of voltage reduction at the end of electric shock (v v ) from a starting voltage v (anonymous 2006). 0 t 0 table 3. actual voltage, droop and percentage of motile sperm in electroporation using different dna concentration and cuvette gap size dna concentration (μg/ml) repetition setting voltage (volt) cuvette gap (cm) actual voltage (volt) droop (%) sperm motility (%) 1 50 0.4 37 6 90 5 2 50 0.4 37 0 90 3 50 0.2 37 12 90 1 50 0.4 37 6 100 10 2 50 0.4 37 6 100 3 50 0.4 37 0 100 1 50 0.2 37 12 100 20 2 50 0.2 37 12 100 3 50 0.2 37 6 100 m 1 2 3 4 5 6 7 8 kb 10,0 3,0 1,0 0,7 0,5 0,3 0,1 figure 3. pcr amplification product of dna extracted from microinjected embryos at 14 hours post injection. lanes 1-2: 1 second injection, lane 3-4: 2 seconds injection, lane 5-6: 3 seconds injection, lane 7: pcr product with no dna template, and lane 8 is pcr product with plasmid pktbp-ktgh as template. m is 2-log ladder dna marker (biolabs, inc., new england). pcr analysis showed that electroporated sperm in all treatments contained foreign dna (fig. 4). thus, parameters of electroporation used in this study could deliver foreign dna to enter spermatozoa. in addition, semi-quantitative pcr was applied to determine whether increase of dna concentration used in electroporation could improve number of sperm carrying foreign gene. as shown in figure 4, the thickness of dna band increased by increasing concentration of foreign dna used in this electroporation method. volume of semen (25 μl, equal to about 175 million spermatozoa) utilized in electroporation and concentrations of the extracted gene transfer method in humpback grouper alimuddin .et al biotropia vol. 18 no. 1, 2011 20 dna from electroporated spermatozoa used as template in pcr amplification were similar among treatments. thus, most likely increasing of dna concentra-tion in this study increased copy number of foreign dna in spermatozoa. furthermore, higher copy number of foreign gene entering spermatozoa may raise the possibility of foreign gene to integrate to the host genome. through fertilization, those spermatozoa would then contribute to enhance the number of embryo carrying foreign gene. n 1m kb 3.0 1.0 0.5 2 3 4 5 6 7 8 9 p figure 4. pcr amplification product of dna extracted from electroporated sperm. lanes 1-3: 20 μg/ml plasmid dna concentration, lanes 4-6: 10 μg/ml plasmid dna concentration, lanes 7-9: 5 μg/ml plasmid dna concentration. m is 2-log ladder dna marker (biolabs, inc., new england). n is pcr product without dna template. p is pcr product with plasmid pktbp-ktgh as template. efficacy of sperm-mediated gene transfer (smgt) technique to produce a transgenic marine species has been proved by lu . (2002) and sun (2004). smgt method has also been applied to introduce foreign gene into humpback grouper sperm. three smgt methods (electroporation, and transfection) were examined in this study. transfection using jetpei reagent succeeds to be used in white shrimp (sun . 2005). in this study, humpback grouper sperm died when transfection was performed using jetpei reagent. any compounds of jetpei may be toxic for humpback grouper sperm. in contrast, transfection using transfast reagent allowed humpback grouper sperm to survive. transfast transfected-sperm also carried foreign gene, although this was only found when transfection was conducted for 48 hours at 25 c incubation. furthermore, transfection by injecting dna-transfast complex into testis could also deliver foreign gene to sperm. foreign gene could be detected in transfected sperm at least 36 hours post injection. transgenic sea bream could be obtained by transfection for at least 48 hous prior to spawning (lu . 2002). in the same way with sea bream, transgenic humpback grouper may also be produced. in this study, however, only two out of ten injected broodstocks carried foreign gene in their sperm. this might be due to the difference in testis maturity. in addition, by applying the spawning system for humpback grouper, screening of founder transgenic fish generated by transfection method is costly, labor-extensive and time-consuming. microinjection is generally applied to produce transgenic fish. this method has also been used to introduce foreign gene through fertilized eggs towards generation of transgenic humpback groper. as shown in figure 3, injected embryos carried the foreign gene at least until 14 hours post injection. however, in this study, none or very low number of injected embryos hatched. as in other marine finfish, eggs of et al et al. in vitro in vivo in vitro et al in vitro in vivo in vivo et al in vivo o 21 humpback grouper float in water, small in size and has unclear blastodisc. these conditions hamper microinjection precisely. in addition, the time to reach 2-cell stage of humpback grouper embryos is about 20-30 min post fertilization. so the number of embryos which could be injected by one microinjector is very limited (60100 embryos). hatching rate of uninjected embryos is high, but the survival rate of larvae is lower i.e. 5-10% in average. based on the results in zebrafish transgenic research, the number of germline transgenic f0 is 2-4% of survived fish (alimuddin . 2005; alimuddin . 2008). if we assume that similar number of germline transmitted f0 in zebrafish can be achieved in humpback grouper, the number of embryos injected will be at least 1000 embryos to obtain 2 transgenic f0. this means that microinjection should be conducted 10 times, or we need more microinjectors and technicians. thus, the use of microinjection technique to produce humpback grouper requires facilities, and is labor-extensive as well as time-consuming electroporation has been reported to be a simple and mass transgenic production method. this technique also has similar efficiency with transfection method to produce transgenic sea bream (lu . 2002). voltage (50 v), pulse length (30 μs) and number of pulse (5 pulses) were also lower compared to those of sea bream. electroporation in sea bream is conducted using 600-2000 v, 40 μs pulse length, and up to 8 pulses. this suggests that optimum level of electroporation parameters may be species specific. finally, compared to the three other methods examined for humpback grouper as discussed above, electroporation to sperm is a fast, simple, and efficient transgenic method for humpback grouper. production of transgenic humpback grouper using electroporation technique is in progress in our laboratory. electr production of transgenic grouper carrying genes regulating the important traits for aquaculture holds exciting possibilities for the future, though the cultivation of transgenic organisms remains controversial. . we thank the research team for genetic quality improvement of humpback grouper broodstock of rusnas (national strategic prime research) program of ministry of research and technology republic indonesia for providing the pktbpet al et al . in vivo et al et al . 2002). as shown in figure 4, all treatments allowed the sperm to carry the foreign gene. based on the sperm motility and results of pcr analysis, 10 μg/ml was considered as the optimum dna concentration for electroporation of humpback grouper. this dna concentration was lower than that used for sea bream, 25 μg/ml (lu oporation to sperm was considered as an appropriate approach by means of efficacy and simplicity, to generate transgenic humpback grouper. optimum dna concentration for electroporation was 10 μg/ml. conclusions acknowledgements gene transfer method in humpback grouper alimuddin .et al 22 biotropia vol. 18 no. 1, 2011 ktgh plasmid; head of the department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university for supporting laboratory facilities; our thanks are also due to the staffs of situbondo brachishwater aquaculture center, especially the grouper parent team for their cooperation and technical support in broodstock handling. references alimuddin, nugrahani w, aliah rs, sumantadinata k, faizal i, carman o and g. yoshizaki. . jurnal riset akuakultur, 2: 199-209. (in indonesian). alimuddin, yoshizaki g, carman o. and k. sumantadinata. 2003. application of gene transfer technology in aquaculture. jurnal akuakultur indonesia, 2: 41-50. (in indonesian). alimuddin, yoshizaki g, kiron v, satoh s and t. transgenic research, 14: 159 165. alimuddin, kiron v, satoh s, takeuchi t and g. yoshizaki. 2008. cloning and over-expression of a masu salmon ( ) fatty acid elongase-like gene in zebrafish. aquaculture, 282: 13-18. anonymous, 2006. instruction manual: gene pulser xcell electroporation system, biorad catalog number 74, biorad office. ath-thar mf. 2007. effectiveness of oryzias latipes using hrgfp (humanized renilla reniformis green fluorescent protein) gene as a marker in catfish clarias sp f0 generation. undergraduate thesis. department of aquacuture, faculty of fisheries and marine sciences, bogor agricultural university. (in indonesian). chen tt, kight k, lin cm, powers da, hayat m, chatakondi n, ramboux ac, duncan pl and ra dunham. 1993. expression and inheritance of rsvltr-rtgh1 complemetary dna in the transgenic common carp, . molecular biology and biotechnology, 2:88-95. devlin rh, yesaki ty, donaldson em, du sj and cl hew. 1995. production of germline transgenic pacific salmonids with dramatically increased growth performance. canadian journal of fisheries aquatic sciences, 52: 1376 1384. lu jk, fu bh, wu jl and tt chen. 2002. production of transgenic silver sea bream ( ) by different gene transfer methods. marine biotechnology, 4: 328-337. patil jg and khoo hw. 1996. nuclear internalization of foreign dna by zebrafish spermatozoa and its enhancement by electroporation. journal of experimental zoology, 274: 121-129. rambubu km, rao shn and nm rao. 2005. efficient expression of transgene in adult zebrafish by electroporation. bmc biotechnology, 5:29. rusnas of humpback grouper broodstock research team. 2007. final report on genetic improvement of humpback grouper broodstock. national strategic primer research program of the ministry of research and technology, republic indonesia. 78 p. sarmasik a, jang ik, chung cz, lu jk and tt chen. 2001. transgenic live-bearing fish and crustacean produced by transforming immature gonads with replication-defective pantropic retroviral vector , 3: 470-477. sin fyt, bartley al, walker sp, sin il, symonds je, hawke l and cl hopkins. 1993. gene transfer in chinook salmon ( ) by electroporating sperm in the presence of prsv-laz dna. aquaculture, 111:57-69. sin fyt, walker sp, symonds je and il sin. 1994. sperm-mediated gene transfer in chinook salmon. online symposium paper, international congress of fish biology. vancouver-canada, 360-365. http://www.biologybrowser.org/bb/subject/ …/index251.shtml. august 14, 2009. 2007. isolation and characterization of β-actin promoter from humpback grouper takeuchi. 2005. enhancement of epa and dha biosynthesis by over-expression of masu salmon δ6-desaturase-like gene in zebrafish. β-actin promoter from japanese medaka oncorhynchus masou cyprinus carpio sparus sarba . marine biotechnology oncorhynchus tshawytsca 23 sun ps, venzonnc, calderon fro and esaki dm. 2005. evaluation of methods for dna delivery into shrimp zygotes of ( ) . , 243:19-26. symon je, walker sp, sin fyt and l. sin. 1994. development of a mass gene transfer method in chinook salmon: optimization of gene transfer by electroporated sperm. molecular marine biology and biotechnology 3:104-111. teitelbaum a. 2007. pacific islanders gain specific knowhhow on grouper hatchery techniques.http:// www.spc.int/coastfish/news/fish_news/ 121/teitelbaum_ 121.pdf. spc fisheries newsletter #121 april/june 2007. tsai hj, lai ch and hs yang. 1997. sperm as carrier to introduce an exogenous dna fragment into the oocyte of japanesse abalone ( ). transgenic research, 6:85-95. tucker jr. and w john. 1999. species profile grouper aquaculture. srac publication no. 721. http://www.ca.uky.edu/wkrec/ grouperaquaculture.pdf. penaeus litopenaeus vannamei aquaculture , haliotis deversicolor supertexta gene transfer method in humpback grouper alimuddin .et al biotropia vol. 28 no. 2, 2021: 102 108 doi: 10.11598/btb.2021.28.2.1079 102 true shallot (allium cepa var ascalonicum) seed production during off season ramadhani eka putra1,2 *, d. beta ramadan1, adriyanita adin3, ida kinasih4 and indah oktaviani2 1school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia 2department of biology, institut teknologi sumatera, jalan terusan ryacudu, lampung 35365, indonesia 3pt east-west seeds indonesia, benteng, kecamatan campaka, purwakarta 41181, indonesia 4department of biology, universitas islam negeri sunan gunung djati bandung, cibiru, bandung 40615, indonesia received 8 june 2018/accepted 10 february 2020 abstract seed cultivation for true shallot is an alternative for the more common cultivation practice in which 30% of the harvested tubers are used for cultivation purposes. seed production of this temperate tuber in the tropical region, however, is quite challenging due to its low flowers and seed formation. several studies have shown that vernalization (cold induction) and application of benzil amino purin (bap) had improved the flowering and seed production of shallot. however, such studies were conducted during the best cultivation period for about 3 months and thus, limit the production period of seeds during the rainy season. this study was conducted to observe the effect of both methods outside cultivation periods on the flower and capsule numbers, fruit set, and weight of 100 seeds when compared with commonly practiced cultivation during the dry season. the onion bulbs vernalized at 10 oc for 30 days were subjected to synthetic hormone (bap) prior to planting. the shallot group treated with bap had the lowest values for all observed parameters, i.e., 1,552.67 number of flowers; 312.11 number of capsules; 22.5% seed set; and 0.2244 g weight of 100 seeds, compared to those in the vernalization treated group, i.e., 1,592.44 number of flowers; 623 number of capsules; 30.5% seed set; 0.2261 g weight of 100 seeds and control group 6,774.67 number of flowers; 3,898.44 number of capsules; 57.06% seed set; 0.3304 g weight of 100 seeds. in conclusion, the commonly practiced cultivation of sowing bulbs directly without vernalization and plant growth regulator treatment is probably the better method to produce shallot seeds during the offseason, the rainy season. keywords: benzil amino purin, true shallot seeds, vernalization introduction shallot (allium ascalonicum) is one of the most important tubers in indonesia, which is commonly used as food ingredients and traditional medicine. market demand for the certain commodity has increased annually with 5.30% average consumption growth (kementerian pertanian 2015). the shallot farmers in indonesia usually cultivate this commodity by its vegetative form. however, this method has several challenges, namely 1. short storage period of planting stock (suwandi & himan 1995); 2. variable quality (balai penelitian dan pengembangan pertanian 1995); 3. high susceptibility to disease spread (wibowo et al. 2016); 4. high production cost (gina & rofik 2010); and 5. significant amount of unsold harvested tuber (permadi & putrasamedja 1991; basuki 2009). these factors have prevented the true shallot total production to fulfill its market demand. to meet its market demand, the improvement of national shallot production through the use of botanical seeds or true shallot seed (tss) in shallot cultivation is necessary. seeds have longer storage time, up to six times their vegetative form, and eliminate the need for large storage room (basuki 2009) thereby also reducing the production cost (permadi & putrasamedja 1991; basuki 2009). *corresponding author: ramadhani@sith.itb.ac.id true shallot (allium ascalonicum) seed production during off season – putra et al. 103 however, most of the local growers have not applied this method due to the very limited amount of available true shallot seeds (rosliani 2013). environmental factors, such as average temperature, photoperiod, average humidity, are believed to be the limiting factors for seed production in indonesia (fahrianty 2013; wu et al. 2015). this biennial plant produces bulb as an overwintering stage of the life cycle and produces flowers in the spring after a period of winter or vernalization (brewster 2008). furthermore, true shallot also requires long photoperiod (> 12 hours) to ensure flowering and seed production (kamenetsky & rabinowich 2001). vernalization of bulb before planting ensures early flowering of seed crop (brewster 1994) and enables the seeds to produce a heavier yield (jones & mann 1963; mollah et al. 2005; ami et al. 2013) as the result of increasing gibberellin endogen and auxin production (dinarti et al. 2011). therefore, most true shallot seed producers in indonesia apply low-temperature shock treatment to the mother bulb and establish the plantation in higher elevation. moreover, improving the production application of plant growth regulators (pgr) is one of the common practices. a small number of plant regulators can be applied to adjust plant physiology, such as plant growth and development (yamaguchi & kamiya 2000). control of flowering and seed formation are among the most important practical aspects of seed production. among all plant regulators, cytokinin plays an important role from seed germination to delaying the onset of senescence (chatsudthipong & muanprasat 2009). furthermore, synthetic cytokinin is available, such as benzil amino purin (bap). some studies showed the beneficial effect of bap on flower numbers, flower size, flower longevity, and the number of seed produced (youngkoo et al. 2006; roslian et al. 2012; el-kinany et al. 2019). in indonesia, the true shallot seeds have been produced by applying either vernalization or bap to the mother bulb and planting in the highland during the dry seasons (rosliani et al. 2012). the present study, however, explored the possibility and limitation of the application of the vernalization technique and bap on true shallot seeds during the rainy season, with the intent to improve its production by providing viable seeds all year round. materials and methods study site the study was conducted at the field greenhouse of pt east west seed indonesia research station in lembang and school of life sciences and technology, institut teknologi bandung, indonesia. shallot for seed production was cultivated from october 2016 to april 2017 at the greenhouse, while the quality of seed produced was assessed at the institute. the field experiment was conducted during the rainy season at average temperature of 19 oc to 23 oc which is considered as off-season for true shallot seed production. vernalization of the bima mother bulbs bima variety, which is widely planted in center of shallot production and released by the balai penelitian tanaman sayuran was used as mother bulbs in this study. one hundred and twenty bima mother bulbs were put in white cotton cloth bags and vernalized in a refrigerator at a calibrated temperature of 10 °c for 30 days. after bulbs vernalization, a total of 60 bulbs were subjected to synthetic hormone treatment by dipping them in the bap solution, while another 60 bulbs were stored under controlled temperature (21±3 °c) and serve as the untreated control. to prevent the mother bulbs from fungi attack, all of the mother bulbs were dipped into fungicide solution before planting. land preparation the land was thoroughly prepared by plowing and cross plowing followed by laddering. the subsequent operations were done with harrow, spade, hammer, and other tools. weeds and stubbles were collected and removed from the field. irrigation and drainage channels were made around the plots with the corners trimmed by the spade. plant spacing the planting distances between rows and between bulbs were 25 cm and 20 cm, biotropia vol. 28 no. 2, 2021 104 respectively. each of the 3 plots contained four rows with 15 bulb seeds sown in each row, amounting to 180 bulbs sown at 7 cm depth. application of fertilizer and cultivation practices the true shallot planting stock was fertilized with the recommended doses of n:p:k 16:16:16 and dolomite. watering, weeding, and fungicide applications were conducted once a week during the cultivation period (115 130 days). during the flowering period, the plants were protected under plastic sheets, harbored above them to protect them from rainfall damage, while pollination was conducted by hand. harvesting and processing the cultivation period lasted for 115 130 days. when the seeds inside the capsules turned black and more than 25% black seeds were exposed on the umbel, each umbel was cut at 5 cm of the flower stalk. harvesting was conducted on days 116, 123, and 130 and the umbels were then sun-dried. threshing was done by light beating and hand rubbing of the umbels. the seeds were cleaned and sun-dried up to 7 days until seed moisture was reduced to below 8%. each of the harvested groups of seeds were processed separately and contained in separate paper bags and preserved for later use (mollah et al. 2015). seed weight and germination one of the methods to determine the quality of seeds is by measuring the total weight and germination rate of 100 seeds. one hundred seeds were randomly selected from each harvest and weighed (in gram, g) on an electric balance, placed in a plastic tray, and allowed to germinate. the plastic tray was filled with a paper towel previously dipped in liquid fertilizer. germination test was carried out according to the international rules for seed testing (ista 1996). the number of normal seedlings, abnormal seedlings, dead seeds, and ungerminated seeds were recorded for two weeks. then the germination percentage was determined by the following formula. germination = [(number of seedlings/number of seeds tested)] x 100% (1) data analysis the normality of data was analyzed by the one-sample kolmogorov-smirnov method. the differences among treatments on flowering initiation period, flower numbers, capsule numbers, fruit set, seed numbers, weight of 100 seeds, and seeding rates were analyzed using one-way anova with a significant level of p< 0.05. tukey analysis was conducted as the post hoc test when anova showed significant differences. all analyses were conducted using spss 16.0. results and discussion flowering initiation the time required to produce flowers in the untreated shallot plants (control group, with temperature of 21±3 °c) was significantly longer than those in the other groups, while those in the v+bap and v groups were relatively similar (fig. 1). these results conformed with previous studies that vernalization treatment on shallot bulbs required a shorter time to produce flowers (satjadiputra 1990; yan et al. 2003; islam et al. 2010; andres & coupland 2012; fahrianty 2013; ream et al. 2013; wu et al. 2015). the results showed the importance of vernalization treatment to initiate the flowering, which might be related to the temperate origin of true shallot. vernalization blocked the flowering repressor and induced the expression of genes responsible for the flowering (florigen) (lee et al. 2013). vernalization could also promote the upregulation of some key cytokinin signaling regulators which induced the flowering (wen et al. 2017). the application of bap, a cytokinin synthetic, might have induced gibberellin signaling that reduced the flowering initiation time (tarkowska 2012; wong et al. 2013). number of flowers, capsules, and seeds produced the control groups produced significantly more flowers than the other groups. on the other hand, the number of flowers produced by both v+bap and v groups were relatively similar (fig. 2). true shallot (allium ascalonicum) seed production during off season – putra et al. 105 figure 1 time required for producing flowers among all treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization, control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. figure 2 number of true shallot flowers among treatments. notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. vernalization positively affected flower initiation and the number of flowers produced by shallot (fahrianty 2013). however, the information was based on the study conducted in dry season, the best season for true shallot seed production. the lower number of flowers produced by vernalization groups in the current study could be related to insufficient photoperiod. the true shallot is a long day plant that required 12 hours light period (currah & proctor 1990). shorter and epileptic light conditions during the rainy season may negate the positive effect of vernalization on flower production due to less optimal photoperiod (dennish & peacock 2009; wu et al. 2015). the lower number of flowers resulted in a fewer number of capsules (fig. 3a) and seeds (fig. 3b) of the vernalized shallot groups. most of the seed losses were caused by flower abortion and infection by fungi on the flowers of vernalized groups. the results indicated that plants of the vernalized groups had lower resistance to diseases probably due to the high humidity. vernalization reduced the vegetative period which benefited the seed production because when plants were growing in optimum condition, most of the plant energy was fully used in seed production. however, under suboptimal conditions, the plants have to overcome environmental stress and allocated less energy to seed production. on the other hand, the longer growing period of the control group could increase seed yield (farghali 1995), probably due to more available energy for seed production. seeds quality the weight of 100-seeds of the control groups was significantly higher than those of other groups, while the v+bap group produced slightly heavier seeds than the v group (fig. 4). biotropia vol. 28 no. 2, 2021 106 figure 3 (a) number of true shallot capsules and (b) seeds per plant among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization, control = no treatment (with temperature of 21±3 °c); * = significant at p < 0.05. figure 4 weight of 100-seeds among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. figure 5 germination rate of seeds produced among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); (*) = significant at p < 0.05. the seed weights of the vernalization groups were in contrast with those in previous studies that indicated the positive effect of vernalization on seed weight (mollah et al. 2005; ami et al. 2013). the high humidity of the rainy season might have caused significant damage to the seed resulting in its reduced weight (ku et al. 2008). the addition of bap after vernalization has improved the seed weight as it could have induced cell growth and tissue differentiation (rosliani 2013). based on this current study, vernalization probably induced only flower production, while the seed quality depends on different mechanisms such as the effect of vegetative propagation, flower numbers and the availability of the pollinator (krontal et al. 2000). therefore, further study is suggested to test this hypothesis. true shallot (allium ascalonicum) seed production during off season – putra et al. 107 germination rate of seeds produced by the control group was significantly higher than those of other groups, followed by v+bap group and v group (fig. 5). germination rate of seed highly depends on seed weight which explained the low germination rate of the vernalization groups (gamiely et al. 1990; mollah et al. 2005). germination rate in this study was also much lower than those produced in optimal season which indicated the importance of planting date (mollah et al. 2005; el-helaly & karam 2012). conclusion vernalization is required to induce the flowering of true shallot. however, the planting date plays an important role in the volume of seed production and quality. during the rainy season, the offseason, the commonly practiced cultivation technique is still the recommended method for the optimum production of true shallot seeds. reducing the detrimental effect of the rainy season to the true shallot plant induced with vernalization, is also suggested, such as maintaining seed production inside a closed system with a controlled environment, which is obviously a key factor in the sustainable production of true shallot seed in indonesia and other areas with similar climate. acknowledgements the authors would like to thank pt east weast seed indonesia – lembang for providing the shallot seeds and the research facilities. this research was partly funded by the hibah penelitian unggulan terapan perguruan tinggi and the arisa grant granted to the corresponding authors. references ami ej, islam t md, farooque am. 2013. effect of vernalization on seed production of onion. agric for fish 2(6):212-7. andrés f, coupland g. 2012. the genetic basis of flowering responses to seasonal cues. nat rev genet 13(9):627-39. basuki rs. 2009. analisis kelayakan teknis dan ekonomis teknologi budidaya bawang merah dengan benih biji botani dan benih umbi tradisional. 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abstract analyses of landsat tm and spot multispectral data were performed with a very detailed description of the vegetation cover in the field to get a relevancy and consistency of digital image classification in a semi-automatic approach. three main vegetation types, i.e. primary forest, logged-over forest and secondary forest after clear cut were analyzed and the microclimatic parameters were also measured to describe the ecological condition of the vegetation. spectral and textural analysis of data obtained from field measurements and spectral reflectance values of the remote sensing data are the main topic of this report as one aspect of study on the digital method of detection and monitoring on forest ecosystem change using high resolution satellite data funded by the indonesian national research council. this study shows that spectral reflectance values alone cannot differentiate the logged-over forest from the primary forest, but it is very sharply distinguished from the secondary forest. as for the texture analysis, it is possible to distinguish the logged-over forest from the primary forest, as shown by different values of degree of entropy, although spatially, it is still doubtful. key words: indonesia/jambi/tropical rain forests/lowland areas/remote sensing/vegetation analysis/ logged-over forests/primary forests/secondary forests. introduction analysis of remotely sensed images is commonly done by calculating the multispectral reflectance values of the objects or themes, regardless of the spectral reflectance of the neighboring pixels. this has affected the mean values and variances, which is why different objects can have the same spectral reflectance or the same objects give different spectral reflectance values. digital data are principally dedicated to quickly evaluate and monitor the change and dynamics of the earth features such as land cover, land use, vegetation cover, development areas, etc. especially for the vegetation cover, the change usually occurs in a slightly dynamic phenomenon and in the form of a mosaic of gaps. the conventional photo interpretation of analog satellite imageries by using the elements of interpretation such as tone or color, size, shape, texture, pattern, shadow, site, association, becomes limited to evaluate and to monitor the change, especially if the degradation occurs in a mosaic pattern. although this conventional interpretation contains texture and pattern, it is considered only qualitatively, and is therefore sometimes inconsistent. at present, the problems of deforestation and forest degradation are becoming a concern for many people and need to be solved before forests totally disappear. in this study, we tried to detect and indicate the significant degradation from the primary forest into 18 biotropia no. 13, 1999 logged-over forest, secondary forest and finally to grassland alang-alang by using spectral and textural analyses of high resolution satellite spot and landsat tm data. in remote sensing analysis, the mosaic of degradation or perturbation of the vegetation cover can be translated into the form of textural pattern of pixels. however, not so many studies have been performed to analyze the textural phenomena of the vegetation status. most studies convert the textural phenomena of the mosaic into structural information of spectral reflectance values and consider the average/mean values and variances as a major information to be used in the classification process. to be able to understand how the texture of pixels in remote sensing data significantly correlates with ground truth, a very detailed information on the vegetation characteristics obtained from field observations and measurements are provided accordingly. study area field observations and measurements are mainly conducted inside the biotrop-barito permanent plot, in pasirmayang, muarabungo, jambi province, located between 1°1'35" 1°5'55" latitude south and 102°4'35" 102°6'45" longitude east. the observation plot for the primary forest is about 10 ha (data analyzed only from 6 ha), logged-over forest plot, 6 ha, secondary forest plot, one (1) ha and alang-alang plot varied according to the period of abandoned fallow (fig. 1). the logged-over forest plot was selectively cut in 1979/1980 and 1983/1984, whereas the secondary forest was formerly devoted to transmigration area and was figure 1. location of study site at biotrop-barito permanent plot. 19 spectral and textural characteristics upik r. wasrin et al. totally cut (clear cut) in 1978/1979. as for grassland alang-alang, its occurrence is closely related to shifting cultivation practices, therefore, the size and distribution is varied from one place to another. all these vegetation types are located inside the 2700 ha research area near the forest plantation of acacia mangium in pasirmayang. the landscape of the plot is moderately undulating with an altitude between 50 150 m above sea level. the forest is mainly considered as tropical lowland rain forest dominated by dipterocarpaceae, with a total height of the emergent tree up to about 50 m and usually composed of multilayers. laumonier (1992) described that the forest inside the biotrop observation plot consists of four dynamic phases, while torquebiau (1988) considered the mosaic of the forest is built up by four different eco-units following the dynamic phases of reorganizing, aggrading, steady state and degrading eco-units. many research activities have been done in the area, providing a detailed information on the vegetation community, structure, floristic and ecological factors such as soil and microclimate. it is,for this reason that the plots were chosen to support the study on spectral and textural behavior of the tropical lowland rain forest and its main derived vegetation types. methods vegetation measurements the characterization of vegetation based on the parameters measured in the field is done spatially to get a synopsis of mosaic or texture. the field parameters used to highlight the textural phenomena are biomass/phytomass, leaf area index (lai), tree density, structure (height) and crown projection. the biomass/phytomass and lai were obtained by using the allometric equations of ogawa et al. (1989) as follows: for primary forest for secondary forest stem biomass: ws = 0.0396 (d2h) 09326 stem biomass : ws = 0.0396 (d2h) 09326 branch biomass: wb = 0.00602 (d2h)' °27 branch biomass: wb = 0.003487 (d2h) 1027 ws ws leaves biomass: wi =————————— leaves biomass: wi = —————————— 13.75 + 0.025 ws 22.5 +0.025 ws total biomass/tree = ws + wb + wi total biomass = ws + wb + wi ws ws leaf area index lai = ——————————— leaf area index lai = —————————— 0.907 + 0.00205 ws 2.385 + 0.00465 ws the position of each tree and its crown projection are drawn inside the observation transect of 200 x 300 m2, and measurements of total height, clear bole height at the first branch, diameter at breast height are done for each single tree with total amount of 672 trees/ha or 4032 trees/6 ha in each forest type (primary forest and logged-over forest). as for the secondary forest, the observation plot size is 1 ha 20 biotropia no. 13, 1999 with a total number of 702 trees/ha. calculation was done for data gathered from the 6 ha primary forest and 6 ha logged-over forest following the size of a unit of spectral reflectance in remote sensing data, i.e. 20 x 20 m as imitating spot pixel with a total unit of 150 parcels, and 30 x 30 m according to landsat tm pixel size with a total unit of 65 parcels. the calculated vegetation parameters will further be correlated with the spectral reflectance values and will be spatially analyzed to get the textural pattern of the dynamic phenomena. spectral analysis the remote sensing data used in this study are spot cct data k/j = 272/353, july 1986; k/j = 278/354, july 1986 and k/j = 272/352, april 1990 and landsat tm data path/row = 126/061, april 1994; path/row 121/060, april 1994; and aerial photographs taken in october 1992, with scale 1 : 20 000 and its enlargement of 1:10 000 and 1:5000. a significant number of training areas (ta) were extracted from spot and landsat tm data, covering the five themes selected i.e. primary forest, logged-over forest, secondary forest, thickets and grassland alang-alang found inside the study areas. a simple statistical calculation of all ta is executed to obtain the mean value and standard deviation of each theme according to different spectral bands (spot, xs1-xs2-xs3; landsat tm, tm2-tm3-tm4). to have a good figure on the representativeness of the themes, a geographical repetition is performed by using ta of other regions of west kalimantan for landsat tm and south sumatera for spot data. a total of 179 training areas (ta) consisting of 122 536 pixels were taken from five (5) different vegetation types such as hill and lowland primary forest, loggedover forest, secondary forest, plantation/estate, mosaic of grassland and thickets, were analysed. those spectral reflectances were extracted from 3 scenes of spot and 2 scenes of landsat tm data available and tested using tukey student range procedure to know the difference between the mean spectral values of the vegetation types. textural analysis an almost representative sub-sample of training areas (ta) are taken for textural analysis. six ha field observation plots were geographically plotted in remote sensing data of landsat tm path/row = 126/061, april 1994 and spot k/j = 272/352, april 1990 (figures 2 and 3). spectral reflectance values are extracted from each band xs1-xs2-xs3 for spot and tm2-tm3-tm4 for landsat tm and presented in the form of spectral values distribution as given in the example of tm-2 landsat (table 1) and spot xs-1 (table 2). various regressions are used to get the relationships between these spectral reflectances with the field parameters especially biomass and lai which are taken as exactly as possible from the same unit/pixel. while a general feature of the vegetation mosaic is obtained from aerial photo's interpretation on scale 1 : 5769 21 spectral and textural characteristics upik r. wasrin et al. figure 2. position of primary forest plot (yellow) and logged-over forest plot (red) on landsat tm false color composite, with an enlargement of 9 times. figure 3. position of primary forest plot (yellow) and logged-over forest plot (red) on spot false color composite, with an enlargement of 7 times. 22 biotropiano. 13, 1999 table 1. spectral reflectance values of band tm2 landsat from primary forest 61 58 29 13 13 11 11 31 28 19 13 11 11 14 15 13 16 12 11 11 11 13 13 12 12 10 10 12 13 12 13 13 10 11 10 13 12 12 12 12 13 12 12 11 13 12 12 11 11 13 11 10 13 11 12 11 11 12 12 12 11 12 12 12 10 11 13 12 13 12 table 2. spectral reflectance values of band xs1 spot from primary forest 25 24 25 24 24 23 23 23 25 26 25 25 25 24 25 25 24 24 27 25 25 24 24 24 25 26 24 25 26 25 25 23 25 25 25 24 24 25 24 26 25 25 25 27 25 25 24 24 25 25 25 26 28 26 25 25 26 23 24 26 27 26 28 25 25 25 25 24 25 25 24 25 26 25 25 24 23 22 24 24 24 24 25 24 25 25 25 24 23 24 24 24 24 23 24 24 23 25 25 26 23 25 25 24 23 24 25 24 25 24 23 23 25 24 24 24 25 23 24 25 23 24 24 26 25 25 24 24 24 23 25 24 24 24 24 24 23 24 25 24 24 23 24 25 24 24 24 25 27 24 with 95% accuracy for the primary forest and 96% accuracy for the logged-over forest. in the aerial photos, the vegetation covers were divided into 4 layers i.e. the emergent frees with height from 45 55 (60) m, the canopy layer from 30 40 m, the under layer (sub-canopy) layer from 15 25 m and the ground cover/gaps from 2 10 m above the forest floor. to get the indication of textural pattern, a simple statistical approach is used to calculate the level of entropy (h') by using the equation as follows: m h' = 3.3219 (logio n 1/n ∑ ni iog10 ni) i=1 23 spectral and textural characteristics upik r. wasrin et al. where : h' = entropy level n = total number of pixels/parcels ni = number of pixels/parcels of class 1 = 1,2, 3,... m although this index is not an absolute value, it still can give a general idea on the differences between one texture of a certain object compared to the texture of the other objects. to make the value of entropy level (h') more significant, it has to be complemented with a spatial arrangement of the pixel's values. results vegetation characteristics the quantitative description of the forest according to diameter and height distributions of the trees gives an indication that the two forest types under study are considered to have a normal distribution, following j inverse for diameter distribution and cloche-like form for height distribution. therefore, there is no significant difference between these statistical and quantitative approaches. figure 4 gives a general view of the primary forest and logged-over forest from aerial photos at scale 1 : 20 000. these figures show a very different synopsis of the two forest types, where the mosaic of primary forest appears to be more rigorous than those of logged-over forest. the primary forest is still densely covered by the canopy layer and the emergent trees, while in the logged-over forest the canopy layer and the emergent trees are randomly scattered throughout the observation plot (6 ha). from aerial photographs, it is certainly possible to distinguish between the figure 4. general view of primary forest (yellow) and logged-over forest (red) on stereo-pair of aerial photos (scale ± 1 : 20 000). 24 biotropia no. 13, 1999 primary forest and the logged-over forest by looking at the ratio of percentage of the vegetation layers and other parameters such as tree density, volume and tree distribution. a similar procedure is done in the analysis of field parameters such as lai (figures 5,6, 7 & 8) and biomass (figures 9, 10, 11 & 12) of the primary forest and logged-over forest. to enhance the pattern of textural phenomena, the data are regrouped into 8 classes according to pixel sizes of landsat (30x30 m) and spot (20x20 m) for both vegetation types, primary forest and logged-over forest. 25 26 27 28 biotropia no. 13, 1999 spectral analysis the mean spectral values obtained from 3 different sites of spot images and 2 sites on landsat tm images are still varied, especially for thickets and alang-alang on spot and almost for every vegetation type on landsat tm image (table 3). although the landsat tm resolution is nearly as high as spot resolution, a relatively different mean and standard deviation or variance are recorded from the same vegetation types. it can be explained by the fact that radiometric resolution of landsat tm is not at the optimum range of radiation wavelength particularly for the vegetation response. to have a proper analysis on the mean spectral values of various vegetation types according to spectral band and images used, a procedure of tukey student range is applied to give a confidential range as given in tables 4 and 5. the tstudent range is very high in all spectral band of spot and landsat tm, indicating that statistically the mean spectral values are not significantly different from one to another. table 5 also shows no significant difference among the mean spectral values, except between primary forest and thickets and between logged-over forest and thickets, in spectral band of landsat tm-3. based on the spectral reflectance alone, it is hard to distinguish the difference between the primary forest and the logged-over forest. however, the physiognomy has a great contribution in differentiating the primary forest with the other degraded types such as secondary forest, thickets and alang-alang, and plantation or estate, both in spot and landsat tm data. the tukey student test for analyzing the mean spectral values supports this result, where primary forest and logged-over forest almost have no difference in spectral reflectance values. a quite important variability of spectral reflectance values (mean and variance) obtained in landsat tm data is probably due to inhomogeneity and different geographical location of the training areas extracted from the images (jambi and west kalimantan). figures 13 and 14 show the distribution of the spectral reflectance from different training areas (ta) taken from two different geographical location using landsat tm and spot data. no clear pattern of spectral distribution classes is shown in these graphs. therefore, this spectral analysis should eventually be complemented with textural analysis, in order to distinguish the pattern of the vegetation mosaic according to dynamic phenomena. correlation between spectral reflectance and vegetation parameters tests of correlations between spectral reflectance values and vegetation parameters such as lai and biomass are performed to know the relevancy of data gathered from vegetation measurement in the field with respect to the spectral relectance response. from tables 6 & 7, it is obvious that all parameters tested are highly correlated, especially between bands tm3 and tm4 with biomass for the primary forest as well 29 spectral and textural characteristics upik r. wasrin et al. table 3. spectral reflectance values extracted i om spot and landsat tm images spot data spot xs-1 spot xs-2 spot xs-3 no. vegetation types no. of pixels ta mean sd mean sd mean sd 1 lowland primary forest 1413 15284 1. 2. 3. 17.00 16.68 0.70 0.78 45.00 31.42 1.10 1.04 64.00 74.40 3.90 6.08 average 16.84 0.74 38.21 1.07 69.20 4.99 2 logged-over forest 6245 6726 3252 1. 2. 3. 22.50 18.20 19.00 0.80 1.54 0.70 44.10 32.80 33.00 1.24 1.28 0.70 75.40 68.50 72.00 5.40 6.01 2.80 average 19.90 1.01 36.63 1.07 71.97 4.74 3 secondary forest 1960 4801 2041 1. 2. 3. 24.00 18.00 20.00 0.90 0.67 0.80 46.00 32.60 36.00 1.00 0.85 0.90 84.00 9.60 75.00 4.20 4.17 2.60 average 20.67 0.79 38.20 0.92 79.53 3.66 4 thickets & alangalang 1004 2258 1066 1. 2. 3. 25.00 18.20 24.00 0.70 0.76 1.00 48.00 29.20 39.00 0.90 0.28 1.30 98.00 95.60 74.00 2.80 4.70 4.10 average 22.40 0.82 38.73 0.83 89.20 3.87 5 plantation/ estate 714 2730 1. 2. 3. 18.20 18.00 0.75 0.80 31.80 32.00 0.90 1.00 82.50 77.00 2.48 2.90 " average 18.10 0.78 31.90 0.95 79.75 2.69 landsat tm tm-1 tm-2 tm-3 no. vegetation types no. of pixels ta mean sd mean sd mean sd 1 low primary forest 4994 2598 1. 2. 23.30 21.60 0.91 0.93 23.06 17.50 1.08 1.06 67.20 67.60 7.09 7.01 average 22.45 0.92 20.28 1.07 67.40 7.05 2 logged-over forest 3905 6763 1. 2. 20.40 21.95 1.26 0.93 20.40 17.88 1.54 1.18 60.30 65.28 6.96 6.41 average 21.18 1.10 19.14 1.36 62.79 6.69 3 secondary fore'st 3391 1046 1. 2. 24.70 23.60 0.81 0.88 24.60 19.10 1.08 0.93 76.70 79.90 4.00 5.75 average 24.15 0.85 21.85 1.01 78.30 4.88 4 thickets & alangalang 1955 1414 -1. 2. 23.73 27.30 1.34 1.31 26.50 21.70 1.44 1.20 90.50 99.40 6.93 8.15 average 25.52 1.33 24.10 1.32 94.95 7.54 5 plantation/ estate 2128 2760 1. 2. 23.80 26.33 0.84 0.89 23.90 22.84 1.05 1.00 77.50 89.84 3.88 4.90 average 25.07 0.87 23.37 1.03 83.67 4.39 30 spectral and textural characteristics upik r. wasrin et al. table 4. tukey student range (w) values to compare mean spectral value of each spectral band spot landsat tm xs-1 18.28 tm-1 5.27 xs-2 38.92 tm-2 9.96 xs-3 75.53 tm-3 16.98 table 5. tukey student range for mean spectral values with confidential level 95% as for the logged-over forest. the other vegetation parameters like structure of tree height, tree density and crown projection are not significantly correlated. the same analysis for the correlation between the spectral reflectance of spot data with the vegetation parameters showed similar results where biomass and lai have a good correlation with spot spectral values, while no significant correlationn was found between spectral reflectance and tree height, tree density and crown projection (tables 8 and 9). however, franklin and mcdermid (1993) found that there is a significant correlation between green band (xs-2) with mean diameter at breast height, height, age and volume of the pinus forest stand, while with the infrared band (xs-3) these stand parameters showed a poor correlation. as for landsat tm data, trotter et al. (1997) examined the relationship between wood volume of the coniferous stand and landsat tm data and gave the conclusion that landsat tm only provides an acceptable information for estimating the wood volume in plantation forest of a minimum 40 ha areas. while cohen and spies (1992) obtained a correlation coefficient between 0.45 and 0.88 for the relation between hrv panchromatic texture with stand attributes such as tree bole diameter, tree height, tree density, age and a structural complexity index; while for tm wetness the coefficient correlation is between 0.51 and 0.90. he also concluded that the lowest correlation is for tree 31 biotropia no. 13, 1999 figure 13. distribution of spectral reflectance. values of the treaining areas obtained from spot scene, k/j : 272/352 and 278/354 figure 13. distribution of spectral reflectance. values of the treaining areas obtained from landsat-tm scene, path/row : 126/061 and 121/060. 32 33 33 34 34 spectral and textural characteristics upik r. wasrin et at. height. this confirms the hypothesis that structural parameter is less important to support the study on spectral reflectance behavior of the vegetation. therefore, measurement of the spectral reflectance values directly above the forest canopy is highly recommended to get a significant correlation between spectral reflectance of high resolution satellite data with the spectral reflectance obtained from field measurement and the vegetation parameters. textural analysis textural analysis was done referring to spectral reflectance values which are randomly distributed on landsat tm and spot data. likewise, the vegetation parameters, especially lai and biomass were calculated inside each parcel as imitating the pixel of landsat tm and spot spatial resolution. table 10 shows the value of entropy level as a measure of desegregation of parcels or pixels inside the 6 ha observation plot in the primary forest and logged-over forest. both in the landsat tm and spot data, the entropy level of the logged-over forest is in general greater than those of the primary forest. it means that the perturbation occurring in the logged-over forest which appears in the form of mosaic of gaps (rigorousness or smoothness of the pixels/parcels) can be expressed by the value of "entropy level". the same aspect is equally found for lai and biomass where the entropy values are greater in logged-over forest compared to those of the primary forest, indicating the heterogeneity level of the parcels analyzed. this is supported by the findings of cohen and spies (1992) where tree bole diameter, tree density, age and structural complexity index have contributed to the texture of the stand but were not significant for the tree height. table 10. level of entropy of spectral reflectance values and vegetation parameters according to the pixel size of landsat tm and spot parcel/pixel parameters primary forest logged-over forest 35 blotropia no. 13, 1999 conclusions analysis of the spectral and textural patterns on landsat tm and spot high resolution data to differentiate the logged-over forest from the primary forest mainly from the point of view of vegetation parameters and its response to radiation wavelength gave interesting results. from this study, it is obvious that the spectral reflectance alone cannot distinguish the logged-over forest from the primary forest; but in complement with the textural pattern analysis, it is possible to separate this logged-over forest from the primary forest. for the practical implementation of this findings, further study is needed to translate this formula into image classifier hi the image processing module. acknowledgement the authors would like to express their sincere gratitude to the peer-reviewers and to the indonesian national research council for providing funds which made the conduct of this research possible from 1994 until 1996. cordial appreciation is also addressed to the barito pacific timber group who has kindly provided facilities during the field work. references cohen, w.b. and t.a. spies. 1992. estimating structural attributes of douglas-fir western hemlock forest stands from landsat and spot imagery. remote sensing of environment, 41(1): 1-17. franklin, s.e. and g.j. mcdermid, 1993. empirical relation between digital spot hrv and casi spectral response and logepole pine (pinus concorta) forest stand parameters. international journal of remote sensing, 14(12): 2331-2348. laumonier, y. 1992. the vegetation of sumatera. seameo biotrop spec. pub. bogor. ogawa h., t. kira, k. yoda and k. ogino, 1989. comparative ecological studies on three main types of forest vegetation in thailand. ii. plant biomass. in: t. kira and i. iwata (eds). nature and life in sea. fauna and flora res. society, kyoto, japan. torquebiau, e.f. 1988. photosynthetically active radiation environment, patch dynamics and architecture in a tropical rainforest in sumatera. aust. j. plant physiol., 15. trotter, c.m., j.r. dymond and c.j. gotjlding. 1997. estimation of timber volume in a coniferous plantation forest using landsat tm. international journal of remote sensing, 18(10): 2209-2223. 36 biotropia no biotropia no. 16, 2001 : 28 38 characterization of malaysian wild bananas based on anthocyanins muhammad asif javed*, mak. chai and rofina yasmin othman division of genetics, institute of biological sciences, university of malaya 50603 kuala lumpur, malaysia abstract the male buds of 16 musa species (musaceae) populations were investigated by hplc for the occurrence of anthocyanins. the investigation was based on the presence of 6 anthocyanins. the 16 musa samples could be classified into three distinct species i.e. musa acuminata, musa violascens and musa balbisiana. musa acuminata could be divided into two subspecies : malaccensis (lowland) and tmncata (highland) according to their constituents and content of major anthocyanins. no variation was observed in the composition of the anthocyanins of kedah type ssp. siamea and selangor types ssp. malaccensis. the classification of m. acuminata into two subspecies based on anthocyanin data further supported the current taxonomic grouping of the species. key words: musa acuminata/musa violascens/musa balbisiana/musaceae /hplc /chemotaxonomy introduction the classification of the genus musa as proposed by cheesman (1947) is the only one which accords satisfactorily with the known taxonomic and cytogenetic facts. of the four named sections, eumusa is the best known partly because the plants are hardy and fairly easy to grow whereas the callimusa is the least known section. section eumusa is the most important as the progenitor of all cultivated bananas. musa acuminata (aa) and musa balbisiana (bb) belong to this section. among the cultivated bananas, different cultivars are classified according to their genomic constitution and ploidy level. malaysia is one of the centers of diversity of wild and cultivated bananas. simmonds (1955) reported four species from the malaysian peninsula. the two species musa violascens and musa gracilis are ornamental wild bananas whereas musa acuminata and musa balbisiana are the two most important species of the genus musa. musa acuminata is the most variable species of the two in malaysia being the progenitors of several local diploid and triploid banana cultivars. three different subspecies of musa acuminata suggested (simmonds 1955) were ssp. malaccensis (selangor), truncata (cameron highland) and siamea (kedah). this classification was questioned by hari (1968) and then later on shepherd (1988) based on chromosomal translocations. therefore, the nomenclature of local wild musa species has been complicated by taxonomic revisions. recent renewed interest * corresponding author: e-mail. j95asit@umcsd.um.edu.my rocketmail. asif_javed@rocketmail.com 28 biotropia no. 16, 2001 in the utilization of wild banana sources had further elucidated the need to characterize different wild musa species in malaysia. anthocyanins constitute the most important group of plant pigments. in 70 species of 33 families of angiosperms, they were found in membrane-bounded anthocyanoplasts located within the main cell vacuole. apart from contributing cyanic color to flower and fruits, the pigments also play an important role during pollination, and are of great economic and genetic importance. previous surveys of anthocyanins in the bracts of banana have been reviewed (simmonds 1954 b,c; horry and jay 1988 a,b). the distribution of various anthocyanidins among different banana cultivars gave good support to the taxonomic ordering within the genus (horry and jay 1988a,b). the present study described the relationship between different wild bananas of malaysian origin based on the distribution of anthocyanins. materials and methods sample collection wild musa species samples were collected from different parts of peninsular malaysia. the types and number of samples collected and their locations are shown in table 1. fourteen populations of wild musa acuminata were sampled. samples of musa violascens and musa balbisiana were also selected for comparative studies. ten suckers each of musa acuminata, musa violascens and musa balbisiana were collected from different mother plants and these were planted in the university farm for evaluation. suckers were planted in holes of 30-40 cm deep, spaced at 2 m x 2 m. at planting, organic compost was applied in the holes. weeding was done manually. anthocyanin extraction variation in anthocyanin pigments was studied from male buds collected from 16 samples (table 1). each banana sample consisted of three male buds as three replicates. the outermost male bracts were used for anthocyanin extraction by grinding 4-5 g of bracts to powder form in liquid nitrogen. anthocyanin pigments were then extracted by boiling the powder in 40 ml of meoh etoh (1:1) for 40 minutes. crude extracts were concentrated to 5 ml, and filtered through glass wool (horry and jay 1988a). filtrates were stored at -70°c till used for thin layer chromatography (tlc) and high pressure liquid chromatography (hplc). tlc and hplc analysis thin layer chromatography (tlc) was carried out on 0.1 mm cellulose plates (merck. 1.05716. 20x20 cm). development was accomplished with solvent mixtures prepared from analytical grades of hydrochloric acid, formic acid and deionized dis 29 characterization of malaysian wild bananas – muhammad asif javed et al table 1. samples of wild musa species ``tilled water. two solvent systems used for tlc were : a. system i. cone. hc1 formic acid water (24.9:23.7: 51.4), b. system ii. cone. hc1 formic acid water (7.1:31.4:61.5). developing tanks were lined with heavy grade filter paper and allowed to equilibrate overnight prior to use. tlc plates were kept at 80°c overnight. a base line was drawn on the loading end and a 1-2 ul of concentrated anthocyanin solutions were loaded. there were 10 samples loaded on each plate. rf values taken for each pigment according to their mobility on the tlc plate were calculated as follows; distance of migration substance rf = distance of migration of solvent front hplc analysis of anthocyanin pigments was carried out on a millipore waters™ liquid chromatograph equipped with a variable wavelength waters™ 486 detector, waters™ 600 controller and waters™ 717 plus auto-sampler was used. anthocyanin pigments separation was carried out using reverse phase c 18, inertsil ods -2 (4.6 x 1.6 mm) 5 urn column (horry and jay 1988a). hplc was run according to horry and jay (1988a), with slight modifications. two solvents used were 5% formic acid and absolute methanol (5:95, v/v). concentrated anthocyanin extracts were diluted ten times with 0.1n hc1, and filtered just before injection. flow rate was maintained at 0.80 ml/min with an injection volume of 20 ul. detection was observed at 514 nm or 240 to 600 nm with scanning detector for spectral identification of the peaks. the aglycone moiety of the 30 biotropia no. 16, 2001 anthocyanin for each hplc peak was identified according to the maxima of absorbance in the visible range (compounds 3, 6, 8: x max vis = 530, 535, 535 nm; compounds 4, 5, 7: a. max vjs = 520, 522, 522 nm) and to their retention times whereas glycosidic pattern was determined by tlc. the composition of the two solvents, flow rate and time are given in table 2. table 2. method used for anthocyanin separation by hplc system (horry and jay 1988a) the peaks showing different anthocyanin pigments (delphinidin, derivative of cyanidin, cyanidin, petunidin, peonidin and malvidin) were used for sample comparison. chemometric analysis hplc data from 16 musa samples were subjected to cluster analysis and principal component analysis (pca) using spss statistic program (spss, inc.). relative amounts of the anthocyanins expressed by percentages were used as an input matrix. results anthocyanins were extracted from the outer most bracts tested through thin layer chromatography (tlc) followed by high pressure liquid chromatography (hplc). different pigments separated by hplc were determined according to increasing retention times, and only six compounds were identified. the absence of a shoulder around 330 nm indicated the absence of acylation. the presence of a shoulder in the visible spectrum at 440 nm and the rf values obtained on tlc indicated the presence of 3rutinosides as also reported by horry and jay (1988a). thin layer chromatography on 0.1 mm cellulose layer plates clearly showed the separation of different anthocyanin pigments. rf values obtained on tlc indicated the presence of 3rutinosides namely 3-o-rutinosyldelphinidin, 3-o-rutinosylcyanidin, 3-o-rutinosylpeonidin, and 3-o-rutinosylmalvidin (table 3). high pressure liquid chromatography (hplc) of anthocyanins resolved musa samples into a maximum of nine peaks. the peaks appearing between 10 to 21 minutes were the six peaks representing the six anthocyanins according to their retention times (horry and jay 1988a,b). 31 characterization of malaysian wild bananas muhammad asif javed et al the same sample injected three times did not show any difference in the chromatograms showing the stability of the system and the anthocyanin pigments. similarly, anthocyanin components extracted from the three outermost bracts from the same male bud did not show any difference in the number of anthocyanin components. the color of the bract in the male inflorescence progressively vanishes from the outside bract (bract 1, the oldest) to the inner bracts. it was observed that the relative distribution of the anthocyanin pattern is stable from the outer bracts to the inner bracts. extracts from musa acuminata accessions, bc2 and gala were analyzed and the results are presented in table 4. six previously reported anthocyanins were observed to be piesent in different combinations and percentages among all the samples studied except flava (table 4). table 3. anthocyanin of banana bracts separated through tlc by using two different solvent systems 32 biotropia no. 16,2001 the data matrix of relative amounts of floral anthocyanins expressed by percentages for musa samples (table 4) was then subjected to cluster and principal component analysis (pca). cluster analysis was conducted by using ward's method with squared euclidean distance (fig 1). four principal components with eigenvalues more than 1.0 were obtained, which could explain 99.89% for the total variation in the data matrix. thus all samples were grouped into four based on cluster and principal component analysis (table 5). rescaled distance cluster combined figure 1. cluster analysis of musa accessions based on anthocyanins using ward's method. all musa acuminata accessions were grouped into two major clusters except for the sample flava. the highland banana bc3 was well separated from other musa acuminata accessions. similarly, gala (musa balbisiana) and bc2 (musa violascens) were also well separated. 33 characterization of malaysian wild bananas muhammad asif javed et al gala was completely separated from the musa acuminata and musa violascens by larger values in the score for third pc (table 5), and is characterized by the larger accumulation of cyanidin (84.63%) followed by cyanidin derivative (table 4). factor loadings of pigments cyanidin and cyanidin derivative for the third pc were also great (table 6). highland musa acuminata sample (bc3) was completely separated from other samples of the species gaining the highest score for the second pc (table 5), for which factor loadings of pigment petunidin were great (table 6), whereas the cyanidin derivative was also accumulated in the highest percentage compared to other accessions of m acuminata samples. however, this sample showed the lowest peonidin compared to other samples of the same species. table 5. principal component scores of musa samples table 6. factor loadings of respective pigments exiting in musa species to principal components 34 biotropia no. 16, 2001 musa violascens (bc2) showed a very different chemotype and a number of peaks produced varied from other two species samples greatly. the anthocyanins detected in the other two species were present in a very small percentage. similarly, musa acuminata sample flava was observed with a very small percentage of the six anthocyanins detected. twelve samples were classified into first group gaining the largest scores for first principal component (table 5), for which factor loadings of peonidin, cyanidin and malvidin were great (table 6), whose anthocyanin composition of the bracts seems to be the standard of the musa acuminata samples. the only variation observed among these samples was the ratio of peonidin and malvidin. different from the highland banana samples with a large accumulation of peonidin whereas, cyanidin derivative and petunidin were present in a very low percentage (table 4). as shown in fig (1) and table (5), three groups of musa acuminata were readily distinguishable. flava diverges from other two groups of samples by'the absence of anthocyanins, as it had yellow color bracts i.e. acyanic bracts. highland banana sample bc3, had a very distinct deep chocolate brown color of the male bracts, and was characterized with accumulation of the simple anthocyanins in this subspecies, whose total composition could be described as 3 rutinosides of cyanidin derivative (cyd), 3 rutinosides of petunidin (pt) (table 4). these anthocyanins were mainly present in this sample. however, a low percentage of peonidin was detected. the remaining musa acuminata accessions belonging to lowlands of malaysia showed similar anthocyanins and their bract color ranged from red and purple. similarly, all musa acuminata accessions suggested a very high ability of methylation of anthocyanidins compared to gala (m. balbisiana). discussions many cytotaxonomists have argued that a unique chemical feature should merit the same importance as an unusual leaf shape or an odd chromosome number in determining plant relationships. they have shown that chemical characters correlate in genetic surveys satisfactorily with one or more biological characters. harborne (1967) made a survey of flavonoid pigments in 45% of the genera in the family gesneriaceae against the classification proposed based on morphological grounds. the differences at the subfamily level indicated a correlation between anthocyanin chemistry and plant geography. anthocyanin components were found to be very stable and the chromatograms obtained were highly reproducible under the same conditions. although the yield of anthyocyanins in musa accessions may vary, the qualitative aspect of it remains relatively consistent, thus making them good taxonomic characters. more than nine peaks were observed in most of the samples, indicating that at least similar number of components were present. however, in the present study only six components were recognized following horry and jay (1988 a,b). from the chromatograms, all the musa species can be identified. musa acuminata accessions were characterized 35 characterization of malaysiar, wild bananas muhammad asif javed el al with partially methylated anthocyanins based on cyanidin delphinidin mixtures i.e. mixtures of cyanidin, peonidin, delphinidin, petunidin and malvidin in different proportions. flava, a yellow bracted accession, did not show significant amount of anthocyanins. simmonds (1954) reported that musajlava ridl. from malaysia was merely a yellow bracted form of musa acuminata and the yellowness was due a single recessive gene and flava was a good example of acyanic bracts (bracts without anthocyanins). musa violascens was observed with a very different chromatogram than the other two and it showed a very little amount of six anthocyanins as observed in remaining species. the anthocyanin variation observed in this species could also be related to the light pink bract color compared to the red purple color observed in other species. anthocyanin variation among these species was further suggested by their different pollinators. musa violascens were reported to be bird pollinated whereas m. acuminata and m. balbisiana flowers were pollinated by bats (simmonds 1962; argent 1976). horry and jay (1988 a, b) described a schematic sequence for anthocyanin biosynthesis in musa. they described that cyanidin is the first and simplest element in a sequence of hydroxylations whereas methylations ending at malvidin. cyanidin or peonidin always occur, suggesting that cyanidin is an obligatory intermediate. we have observed high levels of cyanidin and peonidin in all musa acuminata accessions except for the highland banana form bc3. bc3 was observed to have a very low level of peonidin whereas a new compound cyd (cyanidin derivative) was deposited in high percentage compared to all other accessions. this compound was also reported by horry and jay (1988a, b) in musa balbisiana and suggested a different biochemical pathway. it is found to be an interesting marker for highland banana forms, therefore, it merits further characterization. a similar situation was observed for petunidin, which was accumulated in a very high percentage in bc3. horry and jay (1988b) suggested that a low level of petunidin and the fact that it never accumulates without malvidin, leads to the assumption of o-methylations. these two compounds were observed in high percentage in all lowland banana samples except in bc3. in terms of evolutionary significance of anthocyanins, there is a trend for delphinidin to be replaced by cyanidin, which is considered to be the most primitive pigment. therefore, o-methylation represents an advanced feature in comparison with simple hydroxylation (harborne 1977). all lowland musa acuminata accessions were observed with low levels of petunidin compared to the high percentage of malvidin, suggesting an advanced feature of anthocyanins whereas it was different in highland banana accession showing a high percentage of petunidin. simmonds (1962); horry and jay (1988a,b) suggested that chemotype of musa balbisiana is fairly unique and possesses no "advanced" anthocyanin forms and that environmental constraints were not so extreme that specialized biochemical pathways were needed to be developed in the evolution of this species. on the other hand, musa acuminata was found to possess a broad range of chemotypes, indicating the presence of a more diversified metabolism. this suggests that musa acuminata has undergone more evolutionary changes than musa balbisiana. 36 biotropia no. 16, 2001 cluster analysis and principal component analysis (pca) produced five groupings in musa acuminata accessions based on anthocyanins. the highland banana bc3 showed completely different anthocyanin patterns and was well separated from the remaining accessions. it was also observed to be morphologically distinct. all lowland musa acuminata accessions showed variation mainly in the proportion of the peonidin and malvidin. malvidin was predominant in ri, iptj, bd1, bc1, segun, sintok, rangis, and perak whereas peonidin in kra, bd2, ppc and j-4, respectively. the discrimination found among accessions, therefore, was mainly due to the ratios of these two anthocyanins whereas overall anthocyanin distribution was found similar among these accessions and the mean chemotype proceeded from a simple readjustment of the metabolic balance between methylated anthocyanins (horry and jay 1988a). the relationship revealed by the anthocyanin fingerprints could be compared with the geographical distribution and origin of these accessions. musa acuminata accessions were collected from lowlands and highlands. a large variation in the anthocyanin composition was observed for the highland banana form (ssp. truncatd) compared to lowland samples. northern (ssp. siamea) and central malayan (ssp. malaccensis) region samples were observed with similar anthocyanin compositions. therefore, the geographical origin of lowland accessions had little effect upon the anthocyanins metabolism. whereas the highland banana was different in ecology and geography and also showed distinct anthocyanin pattern. anthocyanin data further supported the two groupings of the musa acuminata i.e. lowland (ssp. malaccensis) and highland (ssp. truncatd) as suggested by hari (1968) and shepherd (1988). acknowledgements the work was supported by a grant from the ministry of science and technology and environment, malaysia. we also would like to thank mr. gopal krishnen and mr. asokan for their kind help in running the hplc system analysis. references anderson, o. m. and g. w. francis. 1985. simultaneous analysis of anthocyanins and anthocyanidins on cellulose thin layers. j. chromatography. 318,450-454. argent, g. c. g. 1976. the wild bananas of papua new guineae. notes of royal botanical garden, edinburgh 35: 77-114. cheesman, e. e. 1947. classification of the bananas. kew bull. 3, 11-28. hari, p. c. 1968. bract imbrication as a taxonomic character in musa acuminata. trop. agric. trinidad. vol. 45, no. 2,99-108. harborne, j.b. 1967. comparative biochemistry of the flavonoids. academic press. london. harborne, j. b. 1977. flavonoids and the evolution of angiosperms. biochem. sys. ecol. 5, 5-22 37 characterization of malaysian wild bananas muhammad asif javed et al horry, j. p. and m. jay. 1988a. distribution of anthocyanins in wild and cultivated banana varieties. phytochemistry. 27, 2667-2672. horry, j. p. and m. jay. 1988b. an evolutionary background of bananas as deduced from flavonoids diversification. in: (jarret, r. l. ed.). identification of genetic diversity in the genus musa, inibap. montpellier, france, p. 158-165. shepherd, k. 1988. observations on musa taxonomy. in: (jarret, r. l. ed.). identification of genetic diversity in the genus musa, inibap. montpellier, france, p. 158-165. simmonds, n. w. 1954b. anthocyanins in bananas. nature, 173,402. simmonds, n. w. 1954c. anthocyanins in banana. ann. bot. lond. 18, 471-82. simmonds, n. w. 1955. wild bananas in malaya. malayan nature journal. 10, 1-8. simmonds, n. w. 1962. the evolution of the bananas. london. uk. longman. 38 biotropia vol. 28 no. 1,2021: 46 53 doi: 10.1 1598/btb.2021.28.1.1051 determining an appropriate age for estimating site index of acacia hybrid plantations i n southeastern vietnam tran thi ngoanl and nguyen tan chung2* 'vietnam national university of foresty, dong n a i province, viet n a m ' f a c ~ ~ l g dfbiological sciences, nong lam uniuersio, ho chi minh ciiy, ho chi minh, viet nam received 5 april 2018/accepted 21 january 2020 abstract this article documents the research results of a site index classification for acacia hybrid plantations in dong nai province. the objectives of this study were to 1) determine a baseline age for acacia hybrid plantations to establish their site indices and 2) develop site index curves for acacia hybrid plantations. three standard plots were established for each age g o u p of 1-10 years with 111 trees per plot of which 108 trees were measured for the determination of tree growth criteria. the other three trees were cut for the tree truncation estimate, and were excluded in the computation of tree growth criteria. i n this study, the site index (si) for acacia hybrid plantations was divided into three levels based o n the mean total heights of the dominant trees; levels i (24 m), i1 (20 m), and i11 (16 m). the heights of the 108 trees were used t o build the si functions, and the three truncated trees were used to examine the possibilities of the si functions. research results showed that the appropriate baseline age of acacia hybrid plantations at dong nai province is 8 years. moreover, in order to improve the effectiveness of acacia hybrid plantation businesses, the owners should focus on growing plantations at site index levels of i or ii. keywords: acacia hybrid plantations, curve of site index, dominant trees, site index introduction climate change has negatively affected human and environmental health, particularly forest ecosystem health (ipcc 2000). tropical rainforest ecosystems are one of the most important carbon sinks on the earth, playing a very critical role in balancing the global carbon cycle and co2 concentration (chaiyo et al. 2011). as such, on forest biomass and carbon sequestration researches are very relevant in forest management and planning, as well as biomass energy use (brown 2000 & 2002; zianis et al 2005). moreover, biomass changes are associated with increases in forest ecosystem growth and rates of carbon absorption and emission (fa0 2009). a forest population ratio of biomass to carbon intensity depends on an age and site index (nguyen & tran 2016). an *corresponding author, email: ntchung@hcmuaf.edu.vn accurate estimation of forest biomass is an important factor in assessing the global carbon cycle (chavt et al. 2005). several studes are interested in determining the mechanisms for worldwide carbon sequestration in different environments (bouman e t al. 1999). it is therefore, necessary to develop appropriate methods for surveying and evaluating forest biomass and carbon stocks (chambers et al. 2001; brown 2002, chavi et al: 2005). several studes also developed mathematical models for the estimation of forest productivity (dong 1974; nguyen & dao 1999; vu 2005 & 2012) and biomass functions (vien 2008: bao 2010; vu & vo 201 1; nguyen 2012; dang 2014; nguyen & tran 2016) for timber trees and different types of natural forests. although a few studies have focused on the site index curves of acacia mangizlm wdld (knsnawati etal. 2010; lumbres e t al. 2018), very limited information are available on biomass estimation of acacia hybrid determining an appropriate age for estimating site index of amcia &bed ... t h i ngoan and t a n chung plantations at various tree ages and soil site some researchers have studied the planting indices. therefore, it is essential to predict acacia hybrid plantation biomass to determine some functions based on different site indices. the forest soil site index or soil productivity is a criterion for evaluating the site suitability for forest productivity (nguyen & dao 1999). soil site quality (or productivity of a soil site) reflects the productivity capacity of a forest, which might change due to the effects of over exploitation, fire, ferulizer application and/or soil erosion (clutter e t al. 1983). site quality is determined through forest growth and yield, mean annual increment (mai) and periodic annual increment (pai) by site index (si) and growth intercept (gi) methods (clutter e t al. 1983; larsen 1999). si can be estimated dlrectly based on the average height of dominant trees (ho) associated with a baseline age (ao) (monserud 1984). si values also depend on tree species and is determined as functions of ho or g i (monserud 1984; larsen 1999; vu 2005). the selection of a baseline age for a tree species is dependent on the life cycle (choosing a period at which population growth does not depend on species density) or business cycle (monserud 1984; larsen 1999). the site index is determined by three different methods (monserud 1984; larsen 1999). the first method involves the construction of a site index population curve, which has been commonly used since the 1940s. this method is based on the related function of h o = f(a0) to construct a site index curve. the second method is based on selecting pairs of h o and age and defining the function ho = f(a), where a is the age of a forest. therefore, site index curves have different shapes by applying different functions. the third method involves truncation of individual trees, using site index curves based on results of tree truncation. this method was created in the 1980s and has been widely applied ever since. in general, each method gives a unique interpretation result (monserud 1984; larsen 1999; vu 2005 & 2012). the si curve converts h o at a b a s e h e age (ao) to ho at a matured age of trees (a). si curves are usually constructed as tree growth functions related to mature ages; in other words, they are based on data pairs of ho/a. a suitable si curve must be fitted by mathematical, statistical methods and must be chosen based on statistical tests (larsen 1999; vu, 2005 & 2012). and breeding of acacid hybrid species in vietnam (le 2000). the mature age of acacia hybrid plantations in southern vietnam is eight years (nguyen e t al 2006). nguyen et al. (2020) records the total area of acacid hybrid plantations in dong nai province as approximately 23,557 ha. plantations are mainly distributed in the dstricts of vinh cuu, xuan loc, and dinh quan. as of writing this manuscript, very limited information is avdable on the classification of site indices for acacia hybrid plantations in dong nai province. the objectives of h s study, therefore were; (i) to determine a baseline age of acacia hybrid plantations to establish their site indices; and (ii) to develop site index curves for acacid hybrid plantations in dong nai province. dong nai province was selected for the study site because many acacia hybrid plantations have been planted in this area with different climatic conditions, topography and soil types. currently, the total area of acacid hybrid plantations in dong nai province is 23,000 ha (institute for forest ecology and environment 2017). the results of this study will provide the scientific basis for the application of silvicultural methods to effectively manage and use acacia hybrid plantations. materials and methods description of the study site this research was conducted on one to 10 year old acacia hybrid (mention the hybrid type or parental species) plantations from in the districts of vinh cuu, dlnh quan, xuan loc, tan phu, long thanh, and bien hoa in dong nai province. these sites have three main soil types formed on basalt, shale and silt soil foundations in dong nai and all located between 1 o0 30' 03"1 1 ' 34' 57" in the northern latitudes and 106' 45' 30"107' 35' 00" in the eastern longitudes, near the equator at 50-350 m above sea level. the rainy season occurs in may to october, and the dry season occurs in november to april. the average temperature, rainfall, and humidity are 22 "c, 2100 mm, and 80°/o, respectively. biotropla vol. 28 no. 1,2021 experimental design and sample collection the site index (si) for acacia hybrid (acacia aaticahyormis or acacia mangk) plantations was determined based on the dominant tree height (ho) at a baseline age (ao). based on a preliminary survey of the age dstribution of acacia hybrid plantations in the study locations, standard sampling plots were established to measure growth criteria for each age group. based on secondary data, three standard plots were established for each of the acacia hybrid plantation age group, and each of those plots represented a typical kind of soil, as mentioned above. the areas of each standard plot varied due to plantation density at each study location; however, the number of trees per plot was consistently 39. within each plot, three sample trees representing different growth status (vietnam standard 11 567-1:2016, 201 6) were cut for tree truncation; one tree of good growing status, one of medum growing status, and one of poor growing status. the remaining thirty-six trees were used to measure tree growth criteria. thus, a total of 30 standard plots were established, in which 108 trees were measured to estimate the growth criteria and nine trees were used to measure truncation for each acacid hybrid plantation age group. the truncated trees were not included in the estimation of tree growth criteria. an average height (ho) for each age group (from 1 to 10 years) was determined by the tree truncation method. the trees used for truncation were cut at 10 cm above the ground, and the diameter at breast height (dbh or d) and stem height (ho) were determined before carrying out the tree truncation steps. each trunk was cut into segments 1.0 m in length, except for the top trunk section, which was 0.5 m. annual rings were counted at 0.0 m, 1.0 m, 1.3 m, 2.0 m, 3.0 m, 4.0 m and so on. at each truncated dameter of the tree, annual rings of the tree were counted to determine a tree age corresponding to the tree height reached within the truncated section. the rings within each tree were assembled and then labelled accrodingly for each truncated tree. the statistical characteristics described for ho, such as the mean height (ho), standard deviation (sd), coefficient of variation (cv), minimum height (ho, -), maximum height (ho, ,,,) and dfference of ho,,,, ho,-, were calculated by using results of statistical analyses. determination of a site index function the site index for acacia hybrid plantations was constructed based on functions described by schumacher (1 939). the observed empirical data of the 108 sample trees in each age group were used to fit the functions. the consistency of each fitted function (equation (3)) was evaluated by considering statistical factors, such as coefficient of determination (r2), standard deviation (sd), mean absolute error (mae) and mean absolute percent error (mape). ho = a*exp(-b/aac) (1) or ln(h0) = ln(a) b/aac (2) ln(h0) = b o + b l / a a c (3) ho = exp(bo + bl/aac) (4) bo = ln(h0) bl/aac (5) where: ln(a) = bo and b = b~ from equations (4) and (5), a function of site index, si = f (a), was defined by equation (6), and parameters of si function, such as boy were defined by equation (7). ln(si) = bo + bl/aoac (6) bo = ln(si) bl/aoac (7) by substituting equation (7) into equation (3), equations (8-10) were obtained. equation (10) is described as the function of site index, with ho at a certain baseline age (ao). ln(ho) = ln(s1) bl/aoac + bl/aac (8) ln(s1) = ln(h0) bl/aoac + bl/aac (9) si = exp(ln(ho) bl(1 /aac -1 /aoac)) (10) determination of a baseline age (&) and parameters of site index curve an appropriate baseline age (ao) was chosen at the time when si was used to convert h o at a0 to ho at a certain age (a) with the smallest regression sum of squares (ssr-). the appropriate baseline age was tested for only acacia hybrid plantations with age groups of 6 10 years (a = 6-10 years). the si value was defined based on the fluctuation of h o at ao (ho,~,, ho,,,~,). the slope (parameter bl) remained the same across the site index functions. to obtain an si curve, a value of bl from equation (4) was first calculated and then substituted into equation (10) along with the value of ho at ao. the si curves were validated by using empirical data measured from the truncated trees. determining an appropriate age for estimating site index of amcia &bed ... thi ngoan and tan chung results and discussion of acacid hybrid plantations for age groups from 1 to 10 years, was defined (equation 11). statistical characteristics of acacia hybrid plantation the average ho values of the dominant trees varied from 2.6 m (year 1) to 22.4 m (year 10) (table 1). the different ranges between ho, ,,, and ho, ,,, were from 2.1 to 3.3 m (year 1) and from 15.7 to 29.0 m (year 10). coefficients of variation (cv) fluctuated from the highest 23.9% (year 2) to the lowest 14.7% (year 10). in general, ho values exhibited large variations with age and site con&tions. therefore, a categorization of acacia hybrid plantations in dong nai province into different site levels is necessary. selection of a site index function ho = exp (3,65344 2,76734/aa0,70746) (1 1) with 2 = 83.4%; mae = 2.2; mape = 16.2%. ho values were fitted by substituting the values of age groups into equation 10 (table 2). the annual periodc growth of height (zho) increased gradually for the first year (2.4 m/ year), reached a peak at 4.7 m/year in year 2, and then gradually decreased unul age 10 (0.9 m/year). the annual average height growth (aho) also increased gradually starting at year 1 (2.4 m/year), reached the highest at year 3 (3.6 m/year), and then gradually decreased unul age 10 (2.2 m/year). height growth rate (pho) decreased rapidly from 100% in year 1 to 14.1% based on the result of the regression and in year 5 and decreased again to 4.1% in year 10. correlation analyses, the function ho = f (a) that thus, at age 2 the acacia hybrid plantations expresses a relationship between height and age transitioned from rapid to slow growth. table 1 statistical characteristics of the tree heights in different age groups (n = 108) a (year) ho, observed (m) cv ("/o) ho, min (m) ho, max (m) ho, max ho, min (m) 1 2.6 15.4 2.1 3.3 1.2 2 6.7 23.9 4.0 9.1 5.1 3 11.0 17.3 7.1 14.2 7.1 4 14.0 17.1 9.4 17.9 8.5 5 15.9 18.2 10.5 20.4 9.9 6 18.0 15.0 12.2 22.7 10.5 7 18.5 20.0 12.3 24.4 12.1 8 20.3 18.7 13.6 26.8 13.2 9 21.9 15.1 14.8 27.6 12.8 10 22.4 14.7 15.7 29.0 13.3 notes: a = age; ho = height of dominant tree; cv = coefficient of variation; ho, fin = minimum height; and ho, ,,, = maximum height. table 2 fitted values of periodic annual and average annual height growths and growth rate notes: zho = periodic annual height growth; a h 0 = average annual height growth; and pho = height growth rate. biotropia vol. 28 no. 1,2021 zho (m) -----ah0 (m) figure 1 periodic annual and average annual height growth of acacia hybrid plantation by age baseline ages (&) of acacia hybrid plantations baseline ages (ao) of the acacia hybrid plantations were chosen at a year when the functions si = f(a) were fitted with the smallest regression sum of scpares (ssr,i,). in this study, the ages of acacia hybrid plantations ranged from 1 to 10 years; therefore, determination of an appropriate baseline age was tested at ages 6, 7, 8, 9 and 10 years. predicted ho values based on equation (10) and differences between observed ho and predicted ho values are presented in table 3. the highest and lowest values of ssr were 2.87 and 0.86 at the ages of 7 and 8, respectively. thus, the appropriate baseline age used for constructing si curves was 8 years. age 8 is consistent with the mature age for acacia hybrid plantations in southern vietnam (nguyen e t al. 2006). table 3 predicted ho at ao and ssr associated with ho for ages 6-10 predicted ho (m) at a. (year) ssr values associated with the predicted ho total sum of ssr 1.14 2.87 0.86 1.84 0.98 determining an appropriate age for estimating site index of amcia &bed ... thi ngoan and tan chung levels of site index at the selected baseline age the dfference between ho, ,, and ho, ,,in (ho, ,,, ho, mi, = 26.8 m 13.6 m) at the selected baseline age of 8 is 13.2 m (rounded to 13.0 m) (table 1). at the age of 8, the average height ho value for acacia hybrid plantations was 20.4 m (rounded to 20.0 m). the measured error of height was usually from k0.5 to kl.o m. if the ho value at the age of 8 (13.0 m) is divided into three levels, a range of each level is equal to 4.3 m (rounded to 4.0 m). the &stance between two levels of adjacent site indices (4.0 m) is four to eight times higher than the measured error of height. therefore, the acacia hybrid plantation was divided into three levels of site in&ces (i, 11, 111) based on the ho values in which the distance between two levels of adjacent site indices was 4.0 m. the si values of the three levels are; 24 m (level i), 20 m (level 11) and 16 m (level 111) at the baseline age of 8. the si values midway between levels i and i1 and midway between levels i1 and i11 were 22 m and 18 m, respectively. similarly, the si value at the lower margin of level 111 was 14 m, while the upper margin of level i was 26 m. table 4 functions of site index (si) at selected levels. selected site index level curves in this study, the slope @i) of si curves was the same for all three si levels. the results of the regression analysis showed that the slope value (bl) was 2.76734 and l/aoac was equal to 1/8"0.70746 = 0.22967. the values of these parameters were substituted into equation 9, and the si curves were fitted (table 4). there were no significant differences in the growth intercepts between functions of si = f (a) among the three site index levels and functions of ho = f (a), with p-values of 0.239, 0.285 and 0.261 for levels i, i1 and 111, respectively. similarly, the slopes of the site index functions were not sigmficantly different from those of the functions of ho (p-value = 0.570 for level i, p-value = 0.61 1 for level i1 and p-value = 0.380 for level 111). these results proved that functions 11 to 17 can be used to construct site index curves for acacia hybrid plantations as represented by the predicted values of site indices for each age group (table 5) and the fitted site index curves of height values by years of acacid hybrid plantations at levels of iupp,,, i, 1-11, 11, 11-111, 111, and 111~0,,, (fig. 2). si levels fitted functions of si iupper si = exp (ln (26) 2.76734*(1/aa0.70746 0.22967)) i si = exp (ln (24) 2.76734*(1 /aa0.70746 0.22967)) i1 i si = exp (ln (22) 2.76734*(1 /aa0.70746 0.22967)) i1 si = exp q n (20) 2.76734*(1 /aa0.70746 0.22967)) i1 i11 si = exp (ln (18) 2.76734*(1 /aa0.70746 0.22967)) i11 si = exp (ln (1 6) 2.76734*(1 /aa0.70746 0.22967)) iiilowcr si = exp (ln (14) 2.76734*(1 /aa0.70746 0.22967)) table 5 predicted values of site index levels for age groups from 1 to 10 years predicted ho (m) at different site index levels a (year) i,,,, i i i1 i1 i1 i11 i11 iiilo,,, biotropia vol. 28 no. 1,2021 si curve 30 5 6 a (year) figure 2 curves of siteindices associated with ho at different age groups conclusion brown s. 2002. measuring carbon in forests: current status and future challenges. journal of environmental pollution 116:363-72. acacid hybrid plantations in dong nai province could be divided into three site index chaiyo u, garivait s, wanthongchai k. 2011. carbon storage in above-ground biomass of tropical levels namely; level 1 (24 m height), i1 (20 m) deciduous forest in ratchaburi province, thailand. and i11 (16 m). for these three site index levels world academy of science, engineering and the appropriate baseline age of the plantations technology 5(10):495-500. was at age 8. the site index curves for each level chambers jq, santos js, ribeko rj, hlguchi n. 2001. of the acacia hybrid plantation sites were tree damage, dometric relationships, and above established according to the baseline age. hence, ground net primary production in central amazon to improve the efficiency of plantation forest. forest ecology and management 152:73-84. industries. the owners need to focus on chavc j, andalo c, brown s, cairns ma, chambers jq, developing acaka hybrid plantations with site eamus d, folster h, fromard f, higuchi n, kira t, lescure jp, nelson bw, ogawa h, puig h, index levels of i and 11. ricra b, yamakura t. 2005. tree allometry and references bao h. 2010. study on methodologies for estimation of the carbon stock on natural forests as a basis for calculating coz emissions from degradation and deforestation in vietnam. vietnamese journal of agriculture and rural development 1 : 1 -1 0. bournan bam, plant raj, nieuwenhuyse a. 1999. quantifying economic and biophysical sustainability tradeoffs in tropical pastures. journal of ecological model 120:31-46. improved estimation of carbon stocks and balance in tropical forests. ecosystem ecology, oecologia 14597-99. clutter jl, fortson jc, pienaar lv, brister gh, bailey rl. 1983. tmber management: a quantitative approach. new york (us): john wiley & sons, inc. 333 p. dong sh. 1974. volume curves of forest plants in vietnam. hanoi (vn): forest science institute of vietnam. 200 p. fao. 2009. land. assessment of the status of the development of the standards for the terrestrial essential climate variables. t9 f a 0 report. rome determining an appropriate age for estimating site index of acacia &bed ... thi ngoan and tan chung (it: food and agriculture organization of the united nations. institute for forest ecology and environment. 2017. results of forest inventory in dong nai province in 2016. available at website: http://ifee.edu.vn/ vi/news/du-an-trong-diem/ket-qua-kiem-ke-rung tinh-dong-nai-nam-2016-64.hunl. ipcc (intergovernmental panel on climate change). 2000. a special report of the ipcc. land use, landuse change, and forestry. cambridge (ukj: cambridge university press. ig-isnawati h, wang y , ades pi<. 2010. generalized height-diameter models for acacia mangium willd. plantations in south sumatra. indonesian journal of forestry research 7(1):1-19. larsen dr. 1999. site index, natural resource biometrics, construction of site index equations for pinu yluestnis l. using permanent plot data in sweden. columbia (us): the school of natural resources, university of missouri-columbia. le di<. 2000. acacia hybrid species and its soil improvement ability. vietnamese journal offoresty, 6. lumbres ric, seo yo, son ym, doyog nd, lee yj. 2018. height-age model and site index curves for acacia mangizm and euca~ptus pellita in indonesia. forest science and technology 14(2): 91-6. monserud ra. 1984. height growth and site index curves for inland doughlas-fu based on stem analysis data and forest habitat type. journal of forest science 30: 943-65. nguyen hs, nguyen vt, bui th, nguyen tm, phan ms. 2006. study on growth characteristics of acacia hybrid plantations and its matured age in the southern vietnam. vietnamese journal of forest sciences 4. nguyen vl. 2012. biomass estimation for calculating carbon stocks and coz absorption in yok don national park, central highlands, vietnam, using remote sensing technology. journal of vietnam environment 3:14-8. nguyen nl, dao ci<. 1999. study on growth and productivity of plantations (pinus k y i a royle ex gordon) in vietnam. hanoi (vn): vietnam publishing house of agriculture. 207 p. nguyen vt, tran tn. 2016. functions of biomass and adjusted coefficients for pinus kysba royle ex gordon o n site index level i in duc trong district, lam dong province. vietnamese science journal of agriculture and forestry 2:57-65. nguyen vp, tran qb, la nk. 2020. the economic and social efficiency of production acacia &brid plantations (acacid hybrid) in dong nai province. vietnamese journal of forest technology and sciences 3:105-12. onyekwelu jc. 2003. choosing appropriate index age for estimating site index of gmeha arborea timber plantations in the oluwa forest reserve. journal of food, agriculture & environment 1(3&4):286-90. vien nn. 2003. study on biomass and primary productivity of auicennia alba in can gio biosphere, ho chi minh city. hanoi (vn): forest science institute of vietnam. 172 p. vietnam standard (tcvn) 11 567-1 :2016. 201 6. lantation large timber plantation transformated from small wood -part 1: acacia hybrid (a.mangitmd auriccuhzrmis). available at the website: http://luattrongtay.vn/viewfulltext/id/664433ff -f408-4367-9258-05b2eob146bd. vu th. 2005. forest productivity. hanoi (vn): vietnam publishing house of agriculture. vu th. 2012. volume chart of standing trees. hanoi (vn): vietnam publishing house of agriculture. 212 p. vu tp, v dh. 2011. biomass structure of pine plantations in lam dong province. vietnamese joural of forest science 2:1813-27. zianis d, muukkonen p, makipaa r, mencuccini m. 2005. biomass and stem volume equations for tree species in europe. monographs 4. 63 p. biotropia no. 10,1996:1 -13 influence of moisture content and length of storage on fungal invasion of paddy rice *) danilo e. paderes college of agriculture, department of crop protection central luzon state university, nueva ecija, philippines t.w. mew department of plant pathology, international rice research institute college, laguna, philippines lina l. ilag college of agriculture, department of plant pathology university of the philippines, college, laguna, philippines abstract the relationship of moisture content and storage period to fungal population, seed germination, grain whiteness and translucency was determined. various fungal species predominated at different moisture conditions and storage periods. the fungi observed belong to the groups of aspergillus flavus-oryzae, a. glaucus, a. mdulans. a. candidus, a. versicolor, a. terreus and a. niger and an unidentified species of penicillium, trichoconiella, curvularia, fusarium, syncephalastrum and verticillium. the predominant storage fungi were a. fla\iis-oryzae and a. candidus whereas, the predominant field fungi were trichoconiella sp., cun-ularia sp. and syncepfialastrwn sp. a decrease in the number of field fungi and an increase in the number of storage fungi with storage time were observed. storage fungi were noted as early as five weeks after storage at moisture contents from 9.3 to 18.33%. the percentage germination of paddy remained high when stored at moisture contents of 9.3 to 14% but decreased with storage time at 14.5 to 18.33% moisture content. the percentage germination of paddy reached a peak at 10-15 weeks of storage. a significant negative correlation between percent germination and moisture content was observed. at 14.5-18.33% moisture content, the germination of stored paddy decreased with a marked increase of storage molds. changes in grain whiteness was not affected by moisture content. however, a decrease in percent whiteness and translucency was noted after 25 weeks of storage. keywords: stored products pests / rice / moisture content / storage / time / fungi / aspergillus sp. / penicillium sp. / trichoconiella sp. / curvularia sp. / fusarium sp./ syncephalastrum sp. / verticillium sp. *) paper presented at the sy mposiu m on pest man agement for stored food and feed, 5 -7 september 1995, bogor, indonesia 1 biotropia no. 10, 1997 morphological and cultural characteristics of the different groups of aspergillus a. flavus-oryzae. colonies on czapek's solution agar grew rapidly, reaching a diameter of 5.5-6.0 cm after 10 days incubation at room temperature. vegetative mycelium was largely submerged with loose texture, surface growth of long-stalked conidial structures and intermixed aerial mycelium. at first, conidial heads appeared white and later developed to a pale greenish yellow to deep yellow-green or olive-brown when mature. globose black sclerotia were produced with" age. conidial heads were predominantly large arising from a long colorless conidiophore. vesicles are typically subglobose bearing either uniseriate or biseriate sterigmata consisting of subglobose conidia. a. glaucus. colonies on czapek's solution agar reached a diameter of 1-1.5 cm after 10 days of incubation at room temperature. conidial heads were typically bluish-green. smooth-walled and brown colored conidiophore terminated to a domelike vesicle. conidia arising from a uniseriate sterigmata were typically globose to subglobose in chains. cleistothecia, yellow and subglobose were present. a, nidulans. growth in czapek's solution agar reached a diameter of 5-6 cm in 10 days at room temperature. conidial heads were usually dark green, short columnar. conidiophores were smooth and light brown in color which terminated to hemispherical vesicle bearing a biseriate sterigmata. the primary and secondary sterigmata were about equal in length. conidia were globose and echinulate. a. candidus. this organism was easily detected by having white condial heads when young and becoming yellowish cream in age. colonies in czapek's solution agar had a diameter of 2.5-3.0 cm after 10 days incubation at room temperature. conidiophores were smooth colorless bearing globose to subglobose vesicle with biseriate sterigmata. a. versicolor. the size of the colonies on czapek's solution agar reached a diameter of 1.5 cm in 10 days at room temperature. this organism has radiate to loosely columnar conidial heads. the brown conidiophore terminated to an ovate or elliptical vesicle. the echinulate conidia are attached to a strictly biseriate sterigmata. a. terreus. colonies on czapek's solution agar obtained a diameter of 4-5 cm. it can easily be identified because of its brighter color in buff, cinnamon, to orange brown compactly columnar conidial heads. the vesicles were hemispherical attached to biseriate sterigmata where globose, smooth conidia were borne. a. niger. this fungus is similar to a. flavus-oryzae by its rapid and vigorous growth on czapek's solution agar which reached a diameter of 2.5-4.0 cm after 10 days at room temperature. conidial heads were in black dark brown shades. conidiophores coming from submerged mycelia terminated to a globose vesicle. the vesicles consisted of biseriate sterigmata where subglobose, elliptical conidia were borne. 10 influence of moisture content danilo e. paderes el. al discussion moisture content is known to be the primary contributing factor in determining the kinds of fungi that invade stored seed and the degree to which they invade it (coleman and fellow 1925; and koehler 1938). in the present study, the predominant fungal species tended to vary at different moisture levels. the variation in the percent fungal incidence found either on or in the seed may probably be due to the competition among organisms. there are fast and slow invading molds (neegaard and adib saad 1962). some fungi may overgrow and obscure other species, thus preventing their identification. overgrowth of one species by another may result from an inherent growth rate difference between species (schroeder and sorenson 1961). expectedly, high percentages mold infection were observed in paddy stored at high moisture content levels. the predominant fungi found in grains with high moisture content were a. flavusoryzae, a. glaucus and a. candidus. these findings agree with those of tuite and christensen (1957), bottomley et al. (1952) and del prado and christensen (1952) on stored cereal seeds, corn and rice, respectively. the decrease in the germination of rice may be attributed to the ability of the fungi to grow on rice grains. at 14.5-18.33% moisture content, the rice grains showed a marked increase in mold infection. similar findings were reported by del prado and christensen (1952), tuite and christensen (1957) and christensen and lopez (1965) in stored rice seeds. christensen and kaufmann (1965) stated that different species of storage fungi require certain limit of moisture content for growth which determines the range over which they will predominate. they further claimed that in the starchy cereal seeds, the lower limits of moisture content that permit invasion by the common storage fungi were: aspergillus halophilicus, 13.0 to 13.2%; a. restrictus, 13.2 to 13.5%; a. amstelodami, a. chevalleri, a. repens and a. ruber, 14.0 to 14.2%; a. candidus and a. ochraceus, 15.0 to 15.2%; a. flavus, 17.5 to 18.0%. christensen and sauer (1982) also reported the different lower limits of fungi for growth in starchy cereal seeds as follows: a. halophilicus 13.0 to 14.0%; a. restrictus, a. glaucus, a. candidus and a. ochraceus, 14.5 to 16.0%; a. flavus and penicillium spp., 16.0 to 20.0%. likewise, christensen and lopez (1965) reported the invasion of storage fungi on rough rice took over at grain moisture contents of 13.4-13.8%. in the present study, storage fungi such as a. flavus-oryzae, a. candidus and a. glaucus were detected in rice grains stored at very low moisture contents (9.3-11.3%). these moisture levels are much lower than the lower limits for growth reported by other workers specially for a. candidus (christensen and kaufmann 1965; and christensen and sauer 1982). 11 biotropia no. 10, 1997 the succession of field fungi by storage fungi during storage observed in the present study agrees with the results of quitco (1982) and those at irri (1977). the percent whiteness and translucency of milled rice showed varying results. thus, the relationship of moisture content and mold count on grain whiteness and translucency needs further studies. conclusion fungi associated with stored rice were a. flavus-orywe, a. glaucus, a. nidulans, a. candidus, a. versicolor, a. terreus, a. niger, and species of penicillium, trichoconiella, curvularia, fusarium, syncephalastrum and verticillium. a high fungal population of the species of aspergillus were noted in rice paddy stored at high moisture content level. seed germination of rice remained high in rice stored at moisture contents from 9.3 to 14.0% and markedly decreased from 14.5 to 18.33% moisture content. likewise, an increase of storage fungi and decrease of field fungi was observed with increased storage period. the high mold count obtained from grain with high moisture levels favorable for growth of various species of aspergillus has contributed to decrease in seed germination. references beuchat, l.r. 1984. survival of aspergillus fla\us conidiospores and other fungi on cowpeas during long term storage under various environmental conditions. j. stored prod. res. 21: 47-52. bottomley. r.a.. c.m. christensen, and w.f. geddes. 1952. grain storage studies. x. the influence of aeration, time and moisture content on fat acidity, non-reducing sugars, and mold flora on stored yellow gram. cereal chem. 29: 53-64. christensen. c.m. 1967. germinability of seeds free of and invaded by storage fungi. proc. assoc. seed analysts?: 141-143. christensen. c.m. and h.h. kaufmann. 1965. deterioration of stored grains by fungi. ann. rev. phytopathol. 3: 69-84. christensen. c.m. and l.c. lopez. 1965. relation of moisture content and length of storage to changes in the microflora and germination percentage of rough rice. phytopathology 55: 953-956. christensen, c.m. and d.b. sauer. 1982. microflora. in storage of cereal grains and their products. c.m. christensen (ed). american association of cereal chemist inc. st. paul minnesota, p. 219240. coleman. d.a. and h.c. fellows. 1925. hygroscopic moisture of cereal grains and flaxseed exposed to atmospheres of different relative humidity. cereal chem. 2: 275-287. david. m.w., e. jay, and r.a. hill. 1985. microflora changes in peanuts (groundnuts) stored under modified atmospheres. j. stored prod. res. 21: 47-52. 12 influence of moisture content danilo e. paderes et. al del prado, f.a. and c.m. christensen. 1952. grain storage studies. xii. the fungus flora of stored rice seeds. cereal chem. 29: 456462. fandialan, i.m. and l.l. ilag. 1973. aflatoxin production ofaspergillusflavus link, isolates from rough rice, corn, sorghum, peanut and copra. phill. agric. 57: 254-263. international rice research institute. 1977. annual report. college, laguna, philippines, p. 177-178. international seed testing associations. 1976. international seed testing association rules. seed sci. and technol. 4:51-177. koehler, b. 1938. fungus growth in shelled corn as affected by moisture. j. agr. res. 56: 291-307. mallick, a.k. and b. nandl 1982. deterioration of rough rice 3. volatile compounds in short term preservation of grains. seed sci. and technol. 10: 315-320. neergaard, p. and adib-saad. 1962. some seedborne diseases and their control. indian council agr. res. 44 p. quasem. s.a. and c.m. christensen. 1958. influence of moisture content, temperature and time on the deterioration of stored corn by fungi. phytopathology 48: 544-549. qurrco, r.t. 1982. paddy deterioration from procurement to storage. naphire tech. bul. no. 2, ftt complex, mm, philippines. qurrco. r.t. and l.l. ilag. 1982. fungi as a cause of yellowing and other discolorations of rice. proc. 5th workshop grains postharvest technology, p. 118-123. raper, k.b. and d.i. fennel. 1977. the genus aspergillus. robert e. krieger pub. co., huntington, new york. 686 p. schroeder, h.w. and j.w. sorenson jr. 1961. mold development in rough rice as affected by aeration during storage. rice journal 64: 21-23. tuite. j.f. and c.m. christensen. 1957. moisture content of wheat seed in reladon to invasion of the seed by species of the aspergillus glaucus group and effect of invasion upon germination of the seed. phytopathology 47: 323-327. vaidehi, b.k. and s. waghray. 1982. storage fungi of paddy and their role in seed deterioration. national seminar on seed pathology. dept. of plant pathology. tamil nadu agricultural university. india, p. 17-19. 13 biotropia no. 10,1997 introduction rice (oryza sativa linn.) is one of the worlds major food crops. in rice producing regions where the relative humidity often ranges from 60 to 90% and the prevailing temperature is high, it has been found that these conditions are conducive for microbial growth. field and storage fungi rapidly multiply, thus affecting the quality of stored rice seeds. the dominant and often the only fungi present on stored rice paddy are species of the genus aspergillus (mallick and nandi 1982; and vaidehi and waghray 1982). like seedborne pathogens, aspergillus spp. are also responsible for the decrease in percentage of seed germination (christensen 1967) and enhance damage or discoloration of glumes (quitco and hag 1982) and aflatoxin formation (fandialan and hag 1973). studies have been conducted on the effect of physical factors on stored corn (quasem and christensen 1958x wheat (tuite and christensen 1957), peanuts (david et al. 1985) and cowpeas (beuchat 1984) but there are few studies focusing on stored rice. since most of the researches on storage fungi have been conducted under temperate and arid tropical conditions, this study identified the storage fungi affecting rice grains and determined the relation of moisture content and length of storage to the predominant mycoflora and on grain deterioration. materials and methods ir60 paddy rice seeds were used in the study. the paddy were sundried to moisture contents ranging to 9.33% and 10.56%. five 150 g paddy rice samples inside small sacks were placed in each desiccator containing super saturated solution of reagent salts, namely: potassium nitrate, potassium chloride and sodium chloride. constant temperature of 22°c was maintained by an incubator which can accommodate 12 desiccators. at every five weeks interval, the desiccators were opened and one sack in each treatment replicated three times including the control was taken and the moisture content, percent seed germination, percent whiteness, translucency and presence of fungi were determined. moisture content the high constant temperature oven method of determining seed moisture content based on the international seed testing association rules (1976) was used. twenty grams of seeds were taken in each treatment including the control and placed inside a brown paper envelope. the envelopes containing the seeds were dried inside the circulating air oven for three hours maintained at 13°c. the envelopes were cooled in a 2 influence of moisture content danilo e. paderes et al desiccator above silica gel for 30 minutes and weighed. the seed moisture content was calculated on the wet weight basis. the experimental layout consisted of a split plot design with three replications. the different salts and the control were assigned to the main plot and storage period to the subplots. percentage germination the percentage germination test was adapted from christensen and lopez (1965) with some modifications. that is, 400 instead of 100 seeds taken from each treatment including the control were used. the seeds were surface sterilized with 20% of 5.25% sodium hypochlorite for 10 min. and spaced on blotter moistened in sterile water with 100 seeds in each petri dish. the seeds were incubated at room temperature for 7 days. a seed which produced a root or coleoptile was considered to have germinated. the relationship of moisture content and percentage germination was determined. percent whiteness a kelt whiteness meter was used to determine the percent whiteness of milled rice. the whiteness meter was conditioned for 30 min. before a sample was placed inside the apparatus. approximately 35 g of milled rice from each treatment and the control were placed inside the whiteness meter. each sample was read three times in all the treatments and the control. translucency a rice meter was used to calibrate the translucency of rice grain. the rice meter was conditioned for 30 min. before a sample was placed inside the loading disk. a standard value of 83.0% was used to calibrate the sample. twenty five grams of milled rice from each treatment and the control were placed inside the loading disk. a corresponding value of the translucency of the samples was registered and recorded. each sample was read three time. a split plot design with moisture contents assigned to the main plot and storage period to the subplot was followed. storage fungi four hundred seeds from each of the treatments and the control were disinfected with 5.25% sodium hypochlorite for 1 min. and spaced in malt salt agar, usually 25 seeds in each petri dish and incubated at 25°c for 7 days. the kinds and numbers of storage 3 biotr opia no . 10, 199 7 fungi were observed using a stereobinocular microscope. fungi showing identical cultural characteristics were grouped together to determine the number of fungi associated on the seeds. a series of transfers were made to obtain a pure culture of the fungus. czapek's solution agar was used as the medium for identifying the isolates belonging to the genus aspergillus. three point inoculation was used as one of the basis of identification of the genus aspergillus (raper and fennel 1977). spores from the purified slants were suspended in melted agar at approximately 45°c. using a cork borer small amounts of the inoculated gelled medium were planted on desired spots on the previously plated czapek's solution agar. the plates were incubated for 10 days at 25°c. the numbers of storage molds, as affected by levels of moisture content and storage period, were analyzed using split split plot design. the levels of moisture content were assigned to the main plot, storage period to the subplot and the fungal genera to the subsubplot. results rice paddy stored in different salt solutions as well as storage period had different moisture contents (fig. 1). the equilibrium moisture content provided by the different figure 1. mean moisture contents of paddy stored in different salt solution and storage periods. bars having the same letter in each salt solution are not significantly different at 5% by dmrt. 4 moisture content (%) kcl nacl salt control influence of moisture content danilo e. paderes et. al salt treatments was highest in potassium nitrate, followed by potassium chloride, then with sodium chloride and the control after 25 weeks storage. the moisture content of the paddy stored in potassium chloride and the control varied with sampling time. however, an increasing trend of moisture content with storage period was noted in grains stored in potassium nitrate. there was no significant differences in moisture content of paddy stored in sodium chloride. effect of moisture contents and length of storage on seed germination the germination of stored paddy was affected by moisture content. at 14.5-18.3% moisture content, the percentage germination increased at 10 and 15 weeks of storage but decreased with further storage (fig. 2). rice seeds stored after 5 weeks still remained figure 2. mean percent germination of paddy stored at different time intervals. bars having the same letter at each moisture content level are not significantly different at 5% by dmrt. dormant, hence, no significant differences were observed in the percentage germination of rice seeds at all levels of moisture content. the percentage germination did not vary significantly at 15, 20 and 25 weeks of storage at moisture content level from 9.3 to 14%. however, at higher moisture levels (14.5-18.33%), the percentage germination significantly decreased at 20 and 25 weeks of storage (fig. 3). 5 biotropia no. 10, 1997 figure 3. mean percent germination of paddy stored at different moisture conditions. bars having the same letter in each storage period are not significantly different at 5% by dmrt. there was a negative correlation between moisture content and percentage germination with a correlation coefficient (r), of 0.61 (fig. 4). figure 4. relationship between percent germination and moisture contents of stored rice 6 influence of moisture content danilo e. paderes et. al effect of moisture content and length of storage on grain whiteness and translucency figure 5 illustrates the effect of moisture content and length of storage on the percent whiteness of milled rice. the percent whiteness did not vary significantly at the different moisture content levels. however, there was a decrease in percent whiteness of milled rice at 25 weeks of storage. figure 5. mean percent whiteness of milled rice stored in different moisture conditions and storage period. vertical bar (1) indicates 1 sd (0.05). the effect of moisture contents and length of storage on the translucency of grains is presented in fig. 6. there were fluctuations in the translucency of the paddy stored at moisture contents 9.3-11.3% and 12.0-14.0% with time. no significant differences were noted on the translucency of paddy at 14.5-18.33% moisture content. from 11.6 to 12.3% moisture content, the translucency of paddy taken after 5 weeks was significantly higher than the succeeding sampling periods 7 biotropia no. 10, 1997 figure 6. mean translucency of milled rice stored in different moisture conditions and storage period. bars having the same letter at each moisture content level are not significantly different at 5% by dmrt. fungi associated with rice stored in different moisture contents the majority of the fungal genera that were consistently associated with stored rice belong to the genus aspergillus. the vegetative mycelium consisted of septate branching hyphae. conidial apparatus developed as conidiophore and head from specialized, enlarged, thick walled hyphal cells. conidiophore originated from a foot cell and enlarging upward and broadening into fertile vesicles. the vesicles bore fertile cells or sterigmata producing spores or conidia borne successively from the tips of the sterigmata. further characterization of the aspergillus into group level was revealed by the different reactions of 3 point inoculation on czapek's solution agar. the cultural and morphological characteristics of the different genera were revealed by agar block technique. the isolated fungal genera were aspergillus flavus-oryzae, a. glaucus, a. nidulans, a. candidus, a. versicolor, a. terreus, a. niger, penicillium, trichoconiella, curvularia, fusarium, syncephalastrwn and verticillium. the percentage fungal incidence varied with moisture levels. there was a shift in the number of different fungal species with storage period at different moisture levels. at 5 weeks of storage, field fungi (curvularia, trichoconiella and fusarium) had higher fungal incidence and these markedly decreased with prolonged storage at all moisture levels (figs. 7 and 8). the storage fungi (a. flavus-oryzae, a. glaucus and a. candidus) slowly increased with storage period until they became the predominant fungi after 25 weeks of storage. 8 influence of moisture content danilo e. paderes et. al figure 7. fungal incidence in rice paddy stored for 25 weeks at 9.3 -11.3% and 11.6-12.35% moisture contents figure 8. fungal incidence in rice paddy stored for 25 weeks at 12.014.0% and 14.4 -18.3% moisture contents 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia vol. 28 no. 1,2021: 74 83 doi: 10.11598/btb.2021.28.1.1092 land suitability assessment for patchouli (pogostemon cablin) development a n d essential oil production ad1 setiawanl?'*, deffi armita', aldila putlu rahayu' and nunun barunawati' 'department ofagronomy, faczrlq ofagni.nltzlre, universio ofbrawzjqa, jalan veteran, malang 65145, indonesia 21nstzt~te ofatxim', universitas brawzjqa, jalan veteran, malang 65145, indonesia received 3 july 2018/accepted 4 january 2020 patchouli (pogostemon cablin) is one of the important crop species in indonesia, since over 80% of patchouli oil global market is produced in indonesia. pacthouli oil is the key ingredients for fragrance and aromatherapy products. patchouli oil is extracted from the stems and leaves of pathouli plants. therefore, it is important to improve patchouli plant productivity and increase resources for sustainable patchouli cultivation. the suitability of abiotic factors in the growing environment of crops remarkably determines the success of crop production. this study aimed t o assess and evaluate land suitability for plant growth and development of patchouli (pogostemon cablin) in dilem wilis, bendungan district, trenggalek regency, indonesia. initially, a survey was conducted and then an analysis was done to classify the land suitability for crops cultivation. the research was conducted on 3 locations from may t o july 2017 for land suitability and from july to november 2017 for patchouli crops cultivation experiment. the results indicated that location 1 had a land suitability of n class, implying that this location was not suitable due to its limiting factor of low i g o content (0.08 me/100 g). meanwhile, both location 2 and location 3 showed similar land suitability class of s3s, tc, f, n signifying as less appropriate. the results of this study also indicated the influence of land suitability classes on plant growth however, the different classification (in this case s3 compared to n) did not demonstrate a correlation between land classes and oil yield and patchouli alcohol, where the element potassium was the limiting factor. keywords: abiotic factor, land suitability, patchouli alcohol, potassium introduction patchouli (pogostemon cabhz) is one of the important crop species in indonesia, since indonesia is one of top producers of patchouli oil. patchouli oil is used as key ingredients for fragrance as well as for cosmetics and aromatherapy products (ramya et al. 2013; nugraha et al. 2019). indonesia is the world's largest producer of patchouli oil, accounting for 80% of the global market (i<usumaningrum et al. 2017). patchouli oil is difficult to synthesize or substitute. therefore, patchouli oil produced by natural patchouli plant remains the only source of the oil (van beek & jouhan 2018). as patchouli oil is extracted from the stems and leaves of patchouli plants, it is important to improve patchouli plant productivity and resource use efficiency through suitable cultivation. land suitability is known to significantly affect the growth and the quantity and quality of crop yields, especially in essential oils. differences in land suitability classes is expected to be directly proportional to plant growth and yield. however, the essential oil produced from secondary metabolites, is strongly influenced by stress con&tions (baher et al. 2002; sangwan et al 2001; simon et al. 1992; taarit et al. 2009). land suitability determines the success of crop production, especially if the target output involves primary metabolites such as carbohydrates, proteins and fats. however, little is known on production targets that include metabolic secretions such as flavonoids, 'corresponding author, e-mail: adisetiawan@ub.ac.id alkaloids and terpenoids. therefore, the assessing and evaluating land suitability in the development of patchouli effect o n patchouli essential oil setiawan e t al. assessment of land suitability for essential oil producing plants is necessary particularly for the patchouli (pogostemon cablin) plant. patchouli plants are considerably easy to grow like other herbaceous plants. however, an optimum ecological condition is deemed necessary to obtain the maximum production. patchouli can grow and develop in the lowlands up to a plateau at 1,200 m above sea level (m asl), although its best growth is at altitudes between 50 400 m asl. the oil content of patchouli on the lowlands is higher but the alcohol content is lower (low plateau patchouli plant would generate low oil content and high alcohol levels). environmental factors greatly affect patchouli alcohol (pa) levels (singh et al. 2002). for the optimal growth, patchouli plants require hot and humid temperatures; with annual rainfall ranging from 2,000&2,500 mm/year, evenly distributed throughout the year; optimum temperature of 24 28 "c; moisture of more than 75%, and radiation intensity ranging between 75 100%. in certain protected areas, patchouli can still grow well with lower oil content. patchouli planted under shady location will grow better, with wider, thinner and greener leaves, but with low oil content. patchouli plants grown in the open area are considerably less dense with smaller plant habitus, small and thick leaves that are yellowish and slightly red, yet having hgher oil content. therefore, the addition of little shade at the beginning of growth was suggested because patchouli is vulnerable to stress drought (rltung et al. 2007). patchouli plants grow best on fertile and loose soil, which is rich in humus and properly drained. the most suitable soil types are those that have a crumb texture, such as andosol or latosol. for clays, a more intensive processing is required to obtain optimal conditions. in soil with less humus, the application of manure is highly recommended to improve soil fertility and humidity (rltung et al! 2007). a problem arises when the plant is cultivated in suitable condition, yet producing low essential oil content. thus, to increase its essential oil production level, the plants require relatively stress-free environmental condtions. several factors could affect the growth and yield of patchouli plants (both quality and quantity) however, this study focused only on the assessment of land suitability for the growth of patchouli and its oil production. materials a n d method study site this study involved two steps, namely: 1. land suitability assessment conducted in may 2017 to july 2017 and 2. patchouli crop production conducted in july to november 2017 at dilem wilis plantation, dompyong village, bendungan district, trenggalek regency, indonesia (figs. 1 & 2). laboratory test was conducted at the chemistry laboratory of soils department, faculty of agriculture, universitas brawijaya. data were then analyzed at the environmental resources laboratory, department of agronomy, universitas brawijaya. steam distillation process was performed to extract the oil and to analyze the patchouli alcohol content (pa) at the institute of atsiri, universitas brawijaya, indonesia. land suitability assessments the experiment involved the use of several tools, such as ruler to measure the depth of the soil, hoe to obtain soil samples from the different locations, clmometer to measure the percent slope of the land, gps (global positioning system) to find the location of coordinates and altitude of the place. laboratory procedures and facilities were also used to analyze c-organic, cec, i g 0 , and ph, as well as computer for map workmanship and data analysis. the soil texture and drainage were also analyzed. the first step in the evaluation of land suitability involved the ground check for the location of patchouli plantation. soil samples were then collected at some locations for observation of land physiography. the composite soil samples were obtained from each location at a depth of 10 20 cm. these samples were analyzed for soil texture. meanwhile, laboratory procedures were done to examine the organic matter content, k20, ph, and cec level. observations of land physiography included recording of coordinate locations, altitude, and slope of the land. the land suitability assessment method in this study was based on dengiz (2013). figure 1 study site i dilem willis plantation trenggalek regency i settlements location street footpath boundary 0 40 80 160 240 320 li meter source : 1. google earth 2. dem srtm 3. rbi bakosurtanal map figure 2 location of assessment plot the data analysis was initiated by establishing the data base to cluster the land suitability by using the matching method adjusted to each parameter as growth requirement of patchouli plant (fa0 1976; djaenudin e t al. 2003; ritung e t al. 2007; safuan e t al. 2013). the land suitability classification was determined by the most constraining limiting factor (rossiter & van wambeke 1997; djaenudin 2008; wirosoedarmo e t al. 2011; jayanti e t ak. 2013). this study applied such basis to investigate the effect of land suitability on the agronomic aspects of plants. the assessment was performed by matching the requirement of patchouli plant growth and the characteristics of the land which were assessing and evaluating land suitability in the development of patchouli effect o n patchouli essential oil setiawan e t al. categorized into 3 groups based on: topography, patchouli growth and result evaluation soil and climate p t u n g e t al 2007). the land suitability assessment was used to reveal the limiting factor of patchouli cultivation, as reference to improve the patchouli cultivation area. the land suitability classification based on f a 0 (1976) was consisted of four categories, namely: a. order: indicating the general state of the land suitability; b. class: indicating the order level of conformity; c. subclass: inhcating the status of the class level based on the type of constraint or improvement as required in the class; and d. unit: indicating the level in the subclass based on minor differences that affect the management. three locations were identified to obtain the com~osite soil sam~les and the land after conducting land suitability analysis, the locations were classified based on suitability. to evaluate the effect of land suitabfity on growth and yields, the patchouli was planted without adding improvements to the soil conditions or to the existing topography. it is suspected that there is a positive correlation between patchouli yield and land suitabfity evaluation. the one-month old sidikalang variety of patchouli seedlings were planted at a spacing distance of 50 x 100 cm. the soil was cultivated applying minimum ullage with weed control every 2 weeks. soil was irrigated every 2 days to adjust the soil conditions with the same dosage equally given for each location without addition char~ctenstics, inclu&& the required ph, of so^ n~trients/fedizer. this study utilized a c-organic, n, p, i<, na, ca, mg, and cec. randomized block design with 3 replications at the slope, altitude, temperature, drainage and each location, each replication consisting of 27 texture. plants with 3 plants for each replication. table 1 land suitability classification of patchouli plants land suitability classes category s1 s2 s3 n land position (s) slope (yo) altitude (m asl) temperature (tc) average temperature pc) 24 26 22 24 or 26 28 2 0 2 2 0 1 2 8 3 3 1 8 2 0 o r > 3 3 water availability (wa) rainfalls (mm) 2300 3000 1750 2300 or 30001200 1750 or > <i200 or >5000 3500 3500 root media (rc) texture drainage soil depth (cm) sandy clay, sandy clay and other sandy clay, clay quartz clay other other very good good bad very bad > 100 cm 75 100 cm 50 75 cm < 50 cm nutrient retention (f) acidity (ph) acidic (5.5 7) acidic to neutral acidic to very very acidic to (5.5 5) acidic (4,5-5) alkaline (< 4.5 or > 7.5) c-organic (yo) 2 3 3 5 < 2 cec (me/100 g) > 17 5.6 < 5 nutrition (n) i<2o (me/100 g) pzos (ppm) source: djaenudin e t al. (2003). biotropia vol. 28 no. 1,2021 the plant growth was assessed based on the branch height and canopy diameter of the plant at 2, 4 and 6 months after planting. results were obtained by destructive sampling based on wetlfresh weight and curing dry weight (t20% water content). the oil yield component was obtained by the 8-hour steam distillation method to obtain patchouli alcohol (pa) level as an indicator of the quahty of essential oils (idris e t al 2014; bur6 & selher 2004). the obtained 768 m asl. the average altitude for optimum patchouli cultivation was at 780 m asl. average air temperatures at each location based on the formula of braak (1928) were: location 1 at 21 oc, location 2 at 21 oc, and location 3 at 22 oc. the air temperature got cooler as the altitude went higher. based on the formula of braak (1928), the temperature registered a steady decline by 0.61 oc with an increasing altitude of 1 m. growth and yield data were tested by using the soil texture at the study site was analysis of variance (f-test) at significance level dominated by clay and dust. at location 1, the p < 0.05). furthermore, to test the significant soil texture was sandy clay, while at location 2 differences between treatments, the tukey test and location 3 it was clay. drainage at location performance by r statistics was applied. 1 is considerably good as water moves quickly or seeps into the soil through the infiltration process, supported by sandy clay texture having results a n d discussion a larger pore than the sand fraction. meanwhile, land suitability the differences among location 1, location 2 and location 3, were based on the land suitabhty class (location 1 is n as not appropriate, locations 2 and 3 are less appropriate (table 2). the slopes of mount wilis is considered hilly to mountainous reliefs. location 1 has a slope of 8%, location 2 has 10°/o, and location 3 has 12%. at the observation point the altitude was about 600 850 m above sea level (m asl). location 1 lies at an altitude of 792 m asl, location 2 at an altitude of 809 m asl, and location 3 at an altitude of both location 2 and location 3 have a good or medium drainage class. soil acidity (ph) at location 1 is 4.6, at location 2 is 4.9 and at location 3 is 4.5 (where the soil ph at the observation point is acidic). based on laboratory test, c-organic at the observation site was 1.63% at location 1, 1.50% at location 2 and 0.71% at location 3. the cation exchange capacity (cec) at location 1 is 18.75 me/100 g, at location 2 is 5.38 me/100g, and at location 3 is 18.45 me/100 g. the availability of i<20 macro elements at location 1 is 0.08 me/100 g, at location 2 is 0.46 me/100 g, and at location 3 is 0.25 me/100 g. table 2 land suitabhty classification of location 1 , 2 and 3 at dilem wilis plantation, trenggalek regency, indonesia land characteristics data category location 1 location 2 location 3 land position (s) slope (o/o) 8% 10% 12% altitude (m asl) 792 809 768 temperature (tc) average temperature ("c) 21 2 1 22 water availability (wa) rainfalls (mm) 1,428 1,428 1,428 root media (rc) texture clay dust clay clay drainage good good very bad soil ~ e p t h (cm) > 50 > 50 >-15 nutrient retention (f) acidity (ph) 4.6 4.9 4.5 c-organic (o/o) 1.63 1.50 0.71 cec (me/100 g) 18.75 5.38 18.45 nutrition (n) iqo (me/ 1 00 g) 0.08 0.46 0.25 -. p205 (ppm) land suitability class n n s3s, tc, f, n s3s, tc, f, n source: primary data (2017) assessing and evaluating land suitability in the development of patchouli effect on patchouli essential oil setiawan e t al. crops production evaluation the number of branches and canopy diameter . did not differ sigmficantly. correiation between cmp production yield and ~ h , dfferent land classes produced higher suitabikp biomass in plant fresh/wet weight and curing the results showed that the different land dry weight, indicating the effect of land classes significantly affected the plant height at 6 suitabiliq on plant growth (fig. 6). months after planting (figs. 3, 4, 5). however, month after planting figure 3 number of branches notes: a = 2 months; b = 4 months; c = 6 months after planting. month after planting figure 4 plant height notes: a = 2 months; b = 4 months; c = 6 months after planting; different letters in the figures indicate significant difference at 0.05 level. biotropia vol. 28 no. 1,2021 month after planting figure 5 canopy diameter among location (l) 1,2, and 3 on the 2, 4 and 6 months after planting location location figure 6 fresh weight (g per plant) notes: a = curing dry weight (g per plant); b = harvested 6 months after planting; different letters in the figures indicate significant differences at 0.05 level. although location 1 has the lowest yield value, the average oil yield and patchouli alcohol (pa) quality in each location were not significantly affected by the limiting factors (table 3). table 3 average oil yield and quality pa (patchouli alcohol) of patchouli location yield (?/o) pa (yo) location 1 2.36 ns 23.40 ns location 2 2.42 ns 22.82 ns location 3 2.49 ns 24.14 ns note: ns = no significant difference. have s3 class, tc, f, n indicating that the cultivation area was less suitable because of the limiting effect of the slope, altitude, temperature, ph, c-organic, and igo. potassium (i<) plays a key role in plant metabolism affecting the synthesis and accumulation of nutrients and secondary metabolites (bihter e t a l 2016). limiting factors that have significant effects included the altitude and the s l o ~ e s . the cultivation area was located on the southern slopes of mount wilis which is hilly to mountainous with an altitude of 600 850 m asl. the slope of the land varies. the land on slopes patchouli cultivation and land suitability were to erosion due to the lack of soil reinforcement plants and the absence of soil matching the land characteristics with the protection from splashing rainwater. the land growth requirement of patchouli resulted in on the slopes will be eroded if there is no location 1 having a land suitability class "n", improvement on it. in the highlands with low indicating that the cultivated land was not temperatures, soil f e r a t y wdl be preserved, but suitable because of the limiting effect of the very steep slopes and unstable lands wdl lead to low igo content. location 2 and location 3 landslides making it more suitable for trees assessing and evaluating land suitability in the development ( ~f patchouli effect o n patchouli essential oil setiawan eta/ (djaenudin 2008). the elevation of the observed location is in the range of > 700 m asl. patchouli plants can grow and produce well at an altitude of 10 400 m above sea levels with air temperature ranging from 24 to 28 oc (pujiharti et al. 2008). the other limiting factor was the acidic soil ph which has a value ranging from 4.5 to 4.9. the optimum growth and production of patchouli plants requires optimum soil ph value of 5.5 to 7 which is quite acid to neutral. nutrients and microorganisms are affected by the soil ph as nutrients are only available at certain ph level. the soil ph is influenced by the manner of land utiluation. the c-organic value, representing the organic matter percentage in the soil, at all the locations were also low (< 2%), as compared to the growth requirement of patchouli plants whch is about 2 3%. i<20 content at all three locations was also low (< 0.6 me/100 g), lower than the optimal growth requirement of patchouli (> 10 me/100 g of g o ) . low i<20 content is due to several factors such as lack of fertihzation. potassium is an alkaline cation, which balances charges of organic and inorganic anions activating more than 50 enzymes (nurzynska wierdak et al. 201 1). agronomic factor land suitability increased the plant growth despite the insignificantly different oil quantities (figs. 3, 4, 5, 6; table 3). the current study showed that the effect of limiting factors (potassium) and land suitability does not affect patchouli oil and alcohol yields, similar results with singh (201 4). however, distinguishable differences were observed in the potassium content among location 1, 2 and 3 (table 2). potassium is considered a plant essential mineral which has h g h concentration in the meristematic tissues and in the phloem. however, i< uptake by the plant roots is accomplished by at least two distinct hnetic systems such as the h g h and low affinity i<+ transporters (hafsi et al. 2014). potassium serves as an important element in plant metabolism, promoting carbohydrates, fats and protein synthesis, increasing crop yield and improving fresh produce quality. moreover, i< enables plants efficacy to resist pests and diseases as well as acting as enzymes co-factor, including enzymes related to the essential oil synthesis (hafsi et al. 2014). the application of i< has also affected the growth and essential oil yield of lemongrass (cymbopogon flxaoszls), dittany (origdnum dictamnas), basil (ocimm basilicam) and rosemary (rosmrinus ofiknalis) (economakis 1993; puttanna et al. 2010). improvement of land characteristics and cultivation the improvement of land characteristic was aimed at optimizing the land condition for patchouli cultivation by using the limiting factors as a reference. the slope of the land is unchangeable, yet such condition could still be addressed by constructing terraces to reduce surface runoff that leads to erosion (wkunan et al. 1985); and the position of the porch slope affects the reception of light (auslander et al. 2003). fertilization is another alternative to improve the soil ph value, i<20 content, and c organic percentage, including appropriate fertilization system such as: fertilization time and method, and fertilizer type selection. the application of organic fertilizer is intended to supply the nutrients that could not be provided by the chemical fertilizer, and also to improve the physical and biological properties of the soil (abdurachman e t al. 2008). liming is another method applied to increase soil acidity values (ph) to be more neutral during land cultivation. potassium (i<) affects the growth and essential oil synthesis in aromatic plants as it is required by plants to build abundant organic compounds such as amino acids, proteins, enzymes and nucleic acids. these minerals affect the function and levels of enzymes involved in the terpenoides biosynthesis (hafsi et al. 2014). the low potassium content at location 1 is a h t i n g factor. however, certain strategies could be applied to ensure the quantity of essential oil yields, where the main target of the harvest is secondary metabolites. it is necessary to synchronize the suitabihty of the growth conditions for secondary metabolite-producing plants in as much as both the growth and yield targets also implied the achievement of good quality essential oils. the planting patterns are also considered as a solution to increase stress as the harvesting period approaches (sacks et al. 2010). biotropia vol. 28 no. 1,2021 conclusion the land suitability analysis of the three sampling locations (location 1, 2, 3) revealed that location 1 is not suitable for the growing of patchouli because of its low ig0 content at 0.08 me/100 g. both location 2 and location 3 showed similar land suitability class of s3s, tc, f, which is less appropriate, because of the limiting effects of land, altitude, rainfall, air temperature, c-organic, soil ph (acidity), and i<zo. however, the dfferent locations did not significantly demonstrate any inhibiting effects on the growth and yleld of the patchouli plants. although the land suitability test indicated differences in growth as well as quality and quantity of yields, h s correlation between land suitability and growth becomes less relevant to patchouli oil yield and alcohol level (indication of the quality of oil obtained). differences in locations under certain conditions, in this case s3 compared to n, did not affect the patchouli alcohol (pa) content of the essential oil. lastly, potassium was a limiting element on the growth of the essential oil-producing plant patchod, pogostemon cablin. acknowledgement the authors would like to acknowledge and thank the director of the institute of atsiri for providing the research fund and the dilem wilis plantation company of trenggalek as the research area. references abdurachman a, dariah a, mulyani a. 2008. strategi dan teknologi pengelolaan lahan kering mendukung pengadaan pangan nasional p h e strategy and technology of dry land management support national food procurement]. jurnal litbang pertanian 27(2):43-9. auslander m, nevo e, inbar m. 2003. the effects of slope orientation o n plant growth, developmental instability and susceptibility t o herbivores. journal of arid environments 55(3):405-16. baher zf, mirza m, ghorbanli m, bagher rm. 2002. the influence of water stress on plant height, herbal and essential oil yield and composition in satureja hortensis l. flavour and fragrance journal 17(4):275-7. bihter ce, bintug 0 , ozgiir c, dilek a. 2016 sweet basil (ocimzlm basiliczlm l.) and potassium fertilization, journal of plant nutrition 39(1):35-44. braak c. 1928. the climate of the netherlands indies. proc royal mogn meteor observ batavia nr. 14. pp.192. bur6 cm, sellier nm. 2004. analysis of the essential oil of indonesian patchouli pogostemon cabin benth.) using gc/ms (ei/ci). journal of essential oil research 16(1):17-9. dengiz 0 . 2013. land suitability assessment for rice cultivation based o n g i s modeling. turhsh journal of agriculture and forestry 37(3):326-34. djaenudin d , manvan, subagyo h, hidayat a. 2003. petunjuk teknis evaluasi lahan untuk komoditas pertanian l a n d evaluation technical guidelines for agricultural commodities]. first edition. bogor (id): lembaga penelitian tanah. djaenudin d. 2008. perkembangan penelitian sumberdaya lahan dan kontribusinya untuk mengatasi kebutuhan lahan pertanian di indonesia p h e development of research on land resources and their contribution to overcome the problem of agricultural land in indonesia]. jurnal litbang pertanian 27(4):137-45. economakis cd. 1993. effect of potassium o n growth and yield of origanm dictamnzl~ in growth culture. acta hort. 331 :339-44. f a 0 [food and agriculture organization]. 1976. a framework for land evaluation. f a 0 soil bulletin no. 32 rome 013: soil resources management and conservation service land and water development division. fao-uno. hafsi c, debez a, abdelly c. 2014. potassium deficiency in plants: effects and signaling cascades. acta physiol plant 36(5):1055-70. idris a, minarni r, said i. 2014. analisis kualitas rninyak nilam (pogostemon cablin benth) produksi icabupaten buol [quality analysis of patchouli oil (pogostemon cablin benth) production buol district]. j akad igm 3(2):79-85. jayanti ds, g o e n a d s, hadi p. 2013. evaluasi kesesuaian lahan dan optimasi penggunaan lahan untuk pengembangan tanaman kaliao (theobroma cacao l.) (studi kasus di icecamatan batee dan icecamatan padang tiji igbupaten pidie propinsi aceh) [land suitability evaluation and land use optimization for cacao (theobroma cacao l.) development (case study in batee district and padang tiji district, pidie sub-province, aceh province)]. agritech 33(20):208-18. i<usumaningrum hp, purbajanti ed, icusdiyantini e. 2017. enhancement of patchouli oils quality using traditional distillation methods from batang, indonesia by plant improvement. advanced science letter 23(3):2450-3. assessing and evaluating land suitability in the development of patchouli effect o n patchouli essential oil setiawan et al. nugraha at, prayitno g, maulidi c, jahra sl, limantara lm. 2019. patchouli plant development in trenggalek regency. international journal 16(53):95-100. nurzynska-wierdak r, rozek e, dzida i<, borowski b. 201 1. growth response to nitrogen and potassium fertilization of common basil (ocimum basilicum l.) plants. acta sci-pol hortoru 11:275-88. pujiharti y, rumbaina m, slameto d . 2008. teknolog budidaya nilam patchouli cultivation technology]. bogor (id): center for assessment and development of agricultural technology. agency for agricultural research and development bogor. puttanna i<, rao evsp, singh r, ramesh s. 2010. influence of nitrogen and potassium fertilization o n yield and quality of rosemary in relation to harvest number. commun soil sci plant anal 41:190-8. ramya hg, palanimuthu v, rachna s. 2013. an introduction to patchouli (pogostemon cablin benth): a medical and aromatic plant: it's importance to mankind. agricultural engineering international. cgiar journal 15(2):243-50. ritung s, agus f, hidayat h. 2007. guidelines for the evaluation of land suitability with the example of land use map of aceh barat district. soil research institute and world agroforestry center 39. rossiter dg, van wambeke ar. 1997. automated land evaluation system ales version 4.65 user's manual. management 6(1):7-20. sacks wj, deryng d , foley ja, ramankutty n. 2010. crop planting dates: an analysis of global patterns. glob ecol biogeogr 19(5):607-20. safuan lo, kandari am, natsir m. 2013 land suitability evaluation of cocoa (theobroma cacao l.) crop based o n climate data analysis using geographic information systems applications j agroteknos 3(2):80-5. sangwan ns, farooqi aha, shabih f, sangwan rs. 2001. regulation of essential oil production in plants. j plant growth regul34(1):3-21. simon j e , reiss-bubenheim d , joly rj, charles dj. 1992. water stress-induced alterations in essential oil content and composition of sweet basil. j essent oil res 4(1):71-5. singh m, sharma s, ramesh s. 2002. herbage, oil yield and oil quality of patchouli [pogostemon cab& (blanco) benth.] influenced by irrigation, organic mulch and nitrogen application in semi-arid tropical climate. ind crops prod 16(2):101-7. singh m. 2014. effect of potassium o n growth and yield of patchouli [pogostemon cablin (blanco) benth.]. josac 23(1):76-9. taarit mb, msaada i<, hosni k, hammami m, i<chouk me, marzouk b. 2009. plant growth, essential oil yield and composition of sage (saluia o$cinaalir l.) fruits cultivated under salt stress conditions. ind crops prod 30(3):333-7. van beek ta, joulain d . 2018. the essential oil of patchouli, pogostemon cab&: a review. flavour and fragrance journal 33(1):6-51. whitman ce, hatfield jl, reginato rj. 1985. effect of slope position o n the microclimate, growth, and yield of barley. agron j 77(5):663-9. wirosoedarmo r, sutanhaji at, icurniati e, wijayanti r. 2011. evaluasi kesesuaian lahan untuk tanaman jagung menggunakan metode analisis spasial f a n d suitability assessment of corn (zea mqs l.) using spatial analysis method]. agritech 31(1):71-8. biotropia no. 10, 1997 : 63-74 influence of media gelling agents on root biomass and in vitro va-mycorrfflzal symbiosis of carrot with gigaspora margarita alok adholeya*, anjali verma and naveen pal bhatia tata energy research institute, durban seth block, habitat place, lodi road, new delhi 110 003, india abstract an in vitro study with ri-tdna transformed roots of carrot (daucus carota) was carried out to evaluate the role of macro-elements contributed as impurities in the gelling agent (phytagel) over an d above those present in the minimal (m) medium. production of root biomass was taken as a measure to quantify the influence of macro -elements added to the minimal medium. the levels of phosphorus when adjusted to 1.19 mg/1 and 1.09 mg/l, lead to dry root biomass production at par with the control. attempts made to lower the amount of impurities in phytagel by de-ionization using different alkalies, proved naoh to give the best results in terms of relatively high amount of root biomass. in an in vitro dual culture system with carrot as host and gigaspora margarita as the vesicular-arbuscular mycorrhizal fungus, phytagel impurities helped to produce maximum number of infection units and auxiliary cells when phytagel was added to the minimal medium. key words: agrobacterium rhiiogenesfdaucus caro/a/gelling agents/diaspora margarita/macro elements/vesicular-arbuscular mycorrhiza/transformed roots. introduction production of vam fungi under axenic conditions continues to be one of the most challenging goals of modem biology (becard and piche 1992). mosse and hepper (1975) were the first to report a simplified in vitro system for the study of vam development wherein they used excised roots instead of whole plants. mugnier and mosse (1987) modified the technique further by using ri-tdna transformed hairy roots as the host tissue. plants with such tumoral roots are potentialy valuable for this kind of study as they not only have exceptionally low nutritional requirements but also allow cultivation on synthetic media axenically (nuutila et al. 1995). amending an appropriate medium for co-cultivating the two components, plant roots and the fungus are the most important factor for a successful vesicular-arbuscular mycorrhizal formation in root organ culture (becard and fortin 1988). after the first *corresponding author 63 biotropia no. 63-74 report about the m medium (becard and fortin 1988), the agar component in this media was replaced by other gelling agents, such as gellen gum (gel gro, icn biochemicals, usa) or phytagel (sigma chemical co., usa). these gelling agents made the media more transparent, which facilitated observation under the light microscope. diop et al. (1992) reported that these gelling agents are polysaccharides with very few impurities. however when analyzed, they were found to contain a number of elemental ions, which significantly increased the concentration of many major and minor ions in the medium. taking these observations into account, the present study was undertaken to optimize the level of some of the major elements, which are known to help in root growth and eventually improve in vitro culture conditions towards a more stronger symbiosis hi the dual culture system. materials and methods transformation of carrot (daucus carota l.) agrobacterium rhizogenes strain (a4 wild type atcc 43057) was used to induce transformation (mugnier and mosse 1987). a loopfull of overnight grown culture was suspended in ms broth (murashige and skoog 1962) (optical density adjusted between 0.6 and 0.7). the tap root of carrot was obtained from agricultural fields and was surface sterilized for 10 min. in 30% haoi solution and rinsed three tunes hi sterile, distilled water. the cut ends of roots were then dipped in bacterial suspension for 15 min., filter soaked and put on ms medium supplemented with 500 mg/1 cefotaxime. after about 3-4 weeks, roots emerging from the cut ends were excised and put on mw medium (becard and fortin 1988). healthy white root tips with numerous laterals showing negative geotropism (unlike the non-transformed root cultures) were routinely maintained on m 0.2% medium (w/v) (becard and fortin 1988). transformation of cucumber (cucumis sativus l.) cucumber seeds were surface sterilized as described above and germinated in the dark at 27°c on moist, sterile filter paper. when radicles were approximately 2 cm long, the seedlings were transferred to ms medium slants and kept in growth room at 25°c with 16/8 h light/dark photoperiod and a relative humidity of 65±5%. once the plants became healthy, sections and slanting incisions were made on stems and leaves. cut ends were then subjected to transformation. the roots that emerged (showing negative geotropism) were subsequently maintained on m 0.2% medium (w/v). 64 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. non-transformed root cultures seeds of tomato (lycopersicon esculentum mill.), were surface sterilized and the seedlings were put on ms slants. after establishment, healthy roots were excised and placed on mw medium. after 3-4 weeks, they were transferred onto m 0.2% medium (w/v) and maintained subsequently on the same medium. testing host for colonization fungal inoculum azygospores of vam fungus, gigaspora margarita becker and hall (daom 194757, deposited at the biosystematic research center, ottawa, canada), grown in pots in a greenhouse, were collected by wet sieving (gerdmann and nicolson 1963) and purified by density gradient centrifugation in 60% urografm (furlan et al. 1980). spores thus obtained were then surface sterilized with 2% chloramine t and rinsed in antibiotic solution which consisted of 1% streptomycin and 0.5% gentamycin (becard and fortin 1988). they were then stored on 1 % sterile water agar at 4°c. viability of the spores was ascertained visually by their light colour and globule filling. only viable candidates were picked up and inserted into 1 % sterile water agar and the plates were incubated in a co2 incubator (biocenter 2001, salvis, switzerland) with 2% co2 at 26°c. later on, germinated spores were transferred to m medium prepared with 0.4% (w/v) phytagel. selection of host for selection of a suitable host for subsequent studies, various root cultures of carrot, tomato, and cucumber were examined for their affinity to in vitro symbiosis with g. margarita. many of these cultures were developed in our laboratory (except for errc-i which was obtained from g. becard, errc, philadephia. usa) and classified on the basis of rate of root growth, (table 1) as a=fast (root growth > 2 cm/day), b=moderate (root growth 1-2 cm/day), and c=slow (root growth < 1 cm/day). gp-i root culture proved to be one of the fastest growing and besides being myc+ was selected for subsequent studies. table 1. host species tested for colo nization study growth rate species a b c c arrot errc-i gp-i gp-ii gp-iii tomato tom cucu mber cu-i cu-ii cu iii 65 biotropia no. 63-74 establishment of dual culture in vitro dual culture was initiated from a single spore of g. margarita and a single healthy root tip. different root cultures of carrot, tomato and cucumber were used to establish the experimental unit. the petri dishes were kept for incubation in the dark at 26°c in order to facilitate the growth of the germ tube (becard and piche 1989). the experiment was terminated at the end of 4 weeks. the medium was dissolved and roots were recovered using 10 mm sodium citrate buffer, ph 6 (doner and becard 1991). roots were carefully picked, cleared and stained (phillips and hay man 1970). stained roots were mounted on glass slides and observed at x 200 magnification under compound microscope attached to video camera and image analyser system (leica, switzerland) loaded with quantiment 500 + software (leica, cambridge, uk) having colour option. to get the number of infectious propagule per unit length of root, the number of entry points were divided by total root length. root growth and rate limiting factors in order to identify the role of certain major nutrient elements such as nitrogen, phosphorus, calcium, magnesium, and sulphur, the basic m medium was modified in different ways (table 2). the level of each ion was altered while keeping the levels of j other ions constant (table 3). both lower and higher concentrations than what is prescribed were tested to study the role of deficiency, alternatively excess availability of the ion selected. root tip length of carrot gp-i at start of an experimental unit was 2.5 cm in various treatments. at the end of four weeks, the root system was harvested according to doner and becard (1991) and dry weight of the total root system was recorded. the experiment was laid in completely randomised block design and data! were subjected to the analysis of variance (anova) and means were separated using duncan's multiple range test (p<0.01). table 2. elemental (ionic) composition of m medium 66 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. table 3. levels of selected ions attained with the basic m medium phytagel de-ionization and root growth phytagel was de-ionized by stirring it in distilled and de-ionized water in an erlenmeyer flask at 60°c (doner and douds 1995). the requisite amount of mixed bed resin, tmd-8 (sigma chemical co., usa) was then added and the mixture was incubated on a shaker at 60°c for four hours. it was then filtered through a clean cheesecloth. to the resultant turbid filtrate 200 mm koh solution was added till it became clear (ph>8.0). this clear filtrate was then poured into pre-chilled iso-propanol and the flakes were recovered by re-filtering it through a cheesecloth and were dried overnight in an oven at 50 ± 5°c. the dried de -ionized phytagel was then subjected to icp-aes (inductiviry coupled plasma-atomic emission spectroscopy) analysis to determine the concentration of ions (table 4) and to decide subsequently the extent of changes to be made in the ionic composition of elements selected for the present study. table 4. icp-aes analysis of various gelling agents 67 biotropia no. 63-74 test of de-ionized phytagel with different alkalies was done with an aim to improve rooting. in the routine procedure of phytagel de-ionization 200 mm koh is added. this addition increases the k content of the medium from 135.35 mg/1 to 188.14 mg/1. since the k level goes even higher than the impurity level of ordinary phytagel, different alternative alkalies, namely 200 mm naoh, 200 mm nahco3, 200 mm trizma and 5% nh4oh, were all tested to raise the ph of the solution. young, actively growing root tips of carrot (gp-i, 2.5 cm long) were placed in petri plates, sealed and incubated in the dark at 27°c. subsequently, at the end of four weeks, roots were recovered by the method of doner and becard (1991) and were dried in a hot air oven at 55 ± 5°c till a constant weight was achieved. test of different gelling agents and root colonization because phytagel contains significant amount of elemental impurities (table 4), various gelling agents namely extra pure (ep) agar 0.7% (w/v) (hi media, india), phytagel 0.4% (w/v), de-ionized phytagel 0.5% (w/v) and agarose 0.4% (w/v) were used to solidify the m medium. attempts were made to keep the physical state identical by varying the amount of different gelling agents in the media. in vitro dual cultures were established on these medium using a single spore of g. margarita and carrot gp-i root tip. auxiliary cells formed after 2 weeks were counted and the experiment was terminated at the end of 4 weeks. after recovering (doner and becard 1991) and staining the roots (phillips and hay man 1970), the total number of infection units formed under various treatments were counted. results and discussion amended recipes and root biomass an evaluation in terms of dry root weight production (g) for various amended recipes of nitrogen (fig. la) shows that when the basic m medium recipe was modified, there was a reduction in dry mean root weight of carrot. the percentage reduction over control was highest (50.82) in hi compared to remaining two recipies (l140.86 mg/1 and l235.86 mg/1). as none of the set concentrations performed at par with the control, it is likely that either the reduced levels of nitrogen fail to meet the biochemical and physiological demands of the roots or the disturbances in the no3 :nh4+-n ratio in the medium lowered the recovery of dry root biomass. in contrast to the basic m medium recipe, amongst all the five lower levels of phosphorus (p) tested, l1 and l2 recipes were at par with the control while l3, l4 and l5 recipes produced significantly lower root biomass (fig. 1b). of all the 68 influence of gelling agents on root biomass and in vitro va-myconhizal symbiosis alok adholeya et al. nutrients, insufficiency of p affects vam symbiosis the most (habte and aziz 1991). it is also well established that high p content is detrimental to vam establishment and decreases mycorrhizal infection (abbott and robson 1977, 1979; menge et al. 1978) and probably, reduces the uptake of some minor elements namely zn, fe, and cu. the m recipe was therefore, modified to incorporate lower levels of p as, subsequently, there is a need to optimise the p concentration in the medium for achieving better colonization. similarly, out of the four amended recipes for calcium (table 3) root growth deteriorated to a great extent with h1 and h2 recipes (fig. 1c). between the other two recipes (l1 and l2) a considerably higher dry root weight was obtained with l1 (percentage reduction over control being 39.96) and a remarkably lower recovery of root biomass was observed in case of l2. calcium is known to increase the rigidity of plant cell walls by cross-linking protein components by the formation of calcium pectate. ca ++ however, could adversely affect the biochemical and physiological processes within the root tissues as calcium at higher concentrations tends to shorten lateral branching in roots (hirrel 1981) resulting in the low recovery of root biomass and subsequently reducing the uptake of cations like mg. lower recovery at l1 and l2 levels could be because of insufficient calcium, which leads to the disorganization of the plasma and vacuolar cell membranes, resulting in increased permeability and subsequent loss of certain vital nutrient ions (burstrom 1968; simon 1978). amendments carried out in minimal (m) media recipe to achieve two of the higher levels of magnesium, namely h1 and h2 produced significantly lower values of dry root biomass (fig. 1d) when compared with the control. out of the three different recipes made by amending sul phur in the basic (m) recipe, h1 and li recipes produced significantly lower root biomass and were at par while h2 recorded 77.05% reduction over control (fig. le). therefore, the original levels of the two elements mg (87.35 mg/1) and s (97 mg/1) (in m medium and those added as impurities through phytagel) are optimal to meet the me tabolic requirements of young carrot roots in vitro. the reduction in root biomass at higher levels of magnesium may be due to the ionic interactions such as ca ++ and k + , resulting in lower availability of this element. colonization of roots maximum infection units and auxiliary cells were produced on ordinary phytagel, followed by ep agar, perhaps because certain very important elements present in the phytagel added significantly towards better root growth and symbiosis. a minimum of one infection unit and three auxiliary cells were formed on the medium prepared with de-ionized phytagel, while agarose plates produced the least number of auxiliary cells and infection unit (table 5). 69 biotropia no 63-74 fig 1. effect of m medium recipes, amended for various nutrients, on in vitro root biomass. a-e, various recipes amended for nitrogen, phosphorus, calcium, magnesium and sulphur, respectively those bars followed by the same letter do not differ significantly by duncan’s multipke range test at p<0.01 70 influence of gelling agents on root biomus and in vitro va-mycorrhizal symbiosis alok adhoteya et al. table 5. test of various gelling agents for colonization of gigaspora margarita icp analysis of ep agar indicates very high sulphur and iron contents compared to phytagel. this could be detrimental to the root growth and colonization. in de-ionized phytagel the level of k rises significantly, affecting the symbiosis negatively. agarose (0.4%) is devoid of impurities and its water-holding capacity is not as high as that of other gelling agents. probably, due to this, root growth ceases after some time and affects drastically the colonization process. root biomass production in phytagel de-ionized with different alkalies the dry root biomass values obtained with phytagel de-ionized by naoh and trizma were highly significant and were at par, while the remaining alkalies tested for de-ionization produced significantly lower root biomass values (fig. 2). though sodium is one of the critical components of the medium and the extent of its uptake in plants is sometimes related to the activity of vam fungi, the level of sodium present in the medium however is quite low (1.96 mg/1), therefore, root growth was not adversely affected. moreover significantly low recovery of root biomass in case of nahco3 may be due to increase in level of na above the critical level. de-ionization of phytagel with koh definitely increases the k level in the resultant product. k + uptake by any plant is strongly influenced by the form of nitrogen available (whether no 3 or nh4 + ) as well as by the cations, particularly na + . it might also be expected to be influenced by the metabolism and polymerization of phosphate (harley and smith 1983). increased k + concentration in the m medium drastically decreases percent colonization of vam fungus (becard and fortin 1988) and high k + content was also found to be detrimental to root growth (harley and smith 1983). de-ionization of phytagel with nh4oh also failed to support the root growth. there was no significant difference between nh4oh and nahco3 treatments. the significant reduction may be due to the fact that excess ammonium 71 biotropia no. 63-74 fig 2. test of different alkalies for phytagel de-ionization those bars followed by the same letter do not differ significantly by duncan's multiple range test (p<0.01) ions in m medium result in a rapid drop in the ph of the culture medium and is detrimental to root growth (becard and piche 1992). in summary, concentration of major and minor elements, ph, temperature and the biochemical or physiological demands of the tissues of the test species play a critical role in the root growth. a balance must be reached between the requirements of actively growing roots, which need a complex medium, and those of the extra-radical phase of the vam fungus, which normally grows in a rhizosphere (i.e. in a relatively nutrient-poor medium). the present study puts forth a major challenge in the task of identifying appropriate nutritional and physiological demands of root cultures and vam fungus to develop a strong symbiosis. the impurities identified hi "phytagel" makes the task of identifying an optimal defined medium much more complex. therefore, there is a need to look for an alternative while defining the requirement for vam symbiosis. 72 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. acknowledgements thanks are due to mr. y.p. kalra for carrying out icp-aes analysis of various gelling agents and ms. s. krishna sundari for root transformation work. dr. david douds and dr. sadhna alstrom made critical comments on an earlier draft of the manuscript, their efforts are thankfully acknowledged. thanks are due to director teri for providing infrastructural support and the department of biotechnology govt. of india, for sponsoring the work carried out under this programme. references abbott, l.k. and a.d. robson. 1977. growth stimulation of subterranean clover with vesicular-arbuscular mycorrhizas. aust. j. agric. res. 28: 639-649. abbott, l.k. and a.d. robson. 1979. a quantitative study of the spores and anatomy of mycorrhizas formed by a species of glomus, with references to its taxonomy. aus. j. bot. 27: 363 -375. bayley, j.m., j. king and o.l. gamborg. 1972a. the effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures. planta 105: 15-24. bayley. j.m., j. king and o.l. gamborg. 1972b. the ability of amino compounds and conditioned medium to alleviate the reduced nitrogen. planta 105: 25-32. becard. g. and j.a. fortin. 1988. early events of vesicular-arbuscular mycorrhiza formation on ri-tdna transformed roots. new phytol. 108: 211-218. becard, g. and y. piche. 1989. new aspects on the acquisition of biotrophic status by a vesicular-arbuscular mycorrhizal fungus. gigaspora margarita. new phytol. 112: 77-83. becard. g. and y. piche. 1992. establishment of vesiculararbuscular mycorrhizae in root organ culture: review and proposed methodology. in: methods in microbiology: experiments with mycorrhizae. varma a.. j.a. norris, d.j. read (eds). academic press. new york. p. 89-108. burstrom, h. 1968. calcium and plant growth. biol. rev. 43: 287316. diop, t.a., g. becard and y. piche. 1992. long term in vitro culture of an endomycorrhizal fungus, gigaspora margarita, on ri t-dna transformed roots of carrot. symbiosis 12: 249-259. doner, l.w. and g. becard. 1991. solubilization of gellan gels by chelation of cations. biotech. tech. 5(1): 25-28. doner, l.w. and d.d. douds. 1995. purification of commercial gellan to monovalent cation salts results in acute modification of solution and gel forming properties. carbohydrate res. 273: 225-233. furlan, v., h. bartschi and j.a. fortin. 1980. media for density gradient extraction of endomycorrhizal spores. trans. br. mycol. soc. 75: 336-338. gerdemann, j.w. and t.h. nicolson. 1963. spores of mycorrhizal endogon species extracted from soil by wet sieving and decanting. trans. br. mycol. soc. 46: 235-244. habte, m. and t. aziz. 1991. relative importance of ca, n, and p in enhancing mycorrhizal activity in leucaena leucocephala grown in an oxisol subjected to stimulated erosion. j. plant nutr. 14(5): 429-442. 73 biotropia no. 63-74 harley, j.l. and s.e. smith. 1983. mycorrhizal symbiosis. academic press inc., london. hirrel, m.c. 1981. the effect of sodium and chloride salts on the germination of gigaspora margarita. mycologia 73: 610-617. menge, j.a., d. sterile, d.j. bagyaraj, e.l.v. johnson and r.t. leonard. 1978. phosphorus concentrations in plants responsible for inhibition of mycorrhizal infection. new phytol. 80: 575 -578. mosse, b. and c.m. hepper . 1975. vesicular-arbuscular mycorrhizal infections in root or gan cultures. physiol. pi. path. 5: 215-223. mugnier, j. and b. mosse. 1987. vesicular-arbuscular mycorrhizal infection in transformed root inducing t-dna roots grown axenically. phytopathology 77: 1045-1050. murashige, t. and f. skoog. 1962. a revised medium for rapid growth and biomass assays with tobacco tissue cultures. physiol. plant. 15: 473-497. nuutila, a.m., m. vestberg and v. kauppinen. 1995. infection of hairy roots of strawberry (fragaria x ananassa duch.) with arbuscular-mycorrhizal fungus. pi. cell rep. 14: 505-509. phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. br. my col. soc. 55: 158-161. simon, e.w. 1978. the symptoms of calcium deficiency in plants. new phytol. 80: 1 -15. 74 63.pdf 64.pdf 65.pdf 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf biotropia vol. 29 no. 2, 2022: 124 133 doi: 10.11598/btb.2022.29.2.1656 124 developmental morpho-anatomy and germination of the seeds of pterocarpus indicus f. echinatus willd. variants kate c. capilitan, lerma sj. maldia, marilyn o. quimado, crusty e. tinio and marilyn s. combalicer* department of forest biological sciences, college of forestry and natural resources (cfnr), university of the philippines los baños (uplb), college, laguna 4031, philippines received 8 september 2021/accepted 10 december 2021 abstract previous studies on the embryo structure of legumes species had resulted in the division of the fabaceae family into two great subfamilies based on embryo axis curvature. research on seed morphology and anatomy adds to the knowledge of taxonomy, evolution and ecology. this study determined the seed developmental anatomy, pod and seed morphology as well as germination characteristics of the observed variants (t1 small prickles; t2 medium prickles; t3 long prickles) of pterocarpus indicus willd. f. echinatus locally known as prickly narra in the mount makiling forest reserve (mmfr). based on the anatomy of the root (radicle) and shoot apex, the formation of the leaf primordium in t2 seeds after radicle protrusion was more progressive. it was observed that the germination rate and the percentage were the highest in t2, where the apical dome was welldeveloped. the germination, pod and seed morphological characters as well as seed anatomical characters were proven to be systematically informative by showing significant differences among the variants. keywords: fabaceae, faboidae, meristem, pod, radicle introduction the fabaceae family consisting of 686 genera with more than 18,000 species is the third largest flowering plant family after asteraceae and orchidaceae (mabberley 1997). the study of de candolle (1825), resulted in the division of fabaceae into two great subfamilies based on embryo axis curvature (i.e., curvembriae and rectembriae). the first subfamily contains the faboideae, while the second one contains the caesalpinioideae and mimosoideae. even though embryo axis curvature is currently not considered as the best character for primary divisions in the family, it may be one of the seed characters (especially hilar characters) used to divide the faboideae from the other subfamilies (oliveira & paiva 2005). faboideae is the largest of the fabaceae subfamilies, with about 440 genera and 12,000 species (polhill 1981). certain seed characteristics are very helpful for faboid generic identifications i.e., aril, endosperm, radicle concealment by the cotyledons, cotyledon lobes over the radicle, overall radicle shape, radicle tip shape, and radicle length relative to that of the cotyledons (kirkbride et al. 2003). capitaine (1912) stated the importance of legume seed morphology in legume classification and identification at the tribal, generic and specific levels. in the last 30 years, there has been a revival of interest in seed morphology and about 225 publications on the subject have been documented (kirkbride et al. 2003). research on seed morphology and anatomy adds to the knowledge of taxonomy, evolution, and ecology of angiospermae species (cortez & carmello-guerreiro 2008). however, it is essential to emphasize that seed morphology generally presents little phenotypic plasticity. conversely, embryological characters, typically *corresponding author, email: mscombalicer@up.edu.ph developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 125 constant in the genera, serve as an important indicator of taxonomic affinity (von teichman & van wyk 1991). according to oliveira and paiva (2005), there are only few descriptive and ontogenetic studies on seed structure causing difficulties in building the hypothesis of evolutive trends affecting seeds. knowledge on fabaceous seeds is mainly related to hard seeds (baskin & baskin 1998; baskin et al. 2000) while studies on seeds are limited, with the majority of such studies concentrating on species of agricultural interest, mainly soybean and bean (qutob et al. 2008). basic knowledge about seed characterization has proven its importance in dealing with problems in the field of seed technology and is essential in activities designed to maintain biodiversity and germplasm conservation. external morphology characterization of seeds has been used by various authors in species identification (youngberg et al. 1998; cappers & bekker 2013). characters mostly used include shape, weight, diameter, color and texture. populations of pterocarpus indicus or narra over the natural range of distribution has declined over the years due to unselective cutting and overall habitat loss. therefore, this species has been categorized under endangered category (iucn v. 2021-1) while being categorized as critically endangered under the philippine national red list for plants (denr-dao 2017-11). to conserve the remaining population, intensive research on the species have been conducted (gazal et al. 2004; krishnapillay et al. 1994; xu et al. 2016). there are two recognized forms of narra (p. indicus) namely smooth narra (pterocarpus indicus forma indicus) and prickly narra (p. indicus f. echinatus). however, some authors have recognized the presence of intermediate forms (jøker 2000; duke 1983). this may be attributed to the high degree of cross-pollination in p. indicus. there have been a few studies on morphology and field germination of the species, but there remains a dearth of information on the anatomy of the seeds, especially of p. indicus f. echinatus. this study determined the developmental anatomy, pod and seed morphology as well as germination characteristics of the observed variants of p. indicus f. echinatus collected from mmfr to assess the extent of variation in this form. materials and methods study site and seed collection seeds were collected in the mt. makiling forest reserve (mmfr) (14o8’ n and 121o12’ e) in 2018. flowering of p. indicus f. echinatus starts in january and peaks around april and may. regular fruiting season starts from january to july or september to november. selection of mother trees was based on characters that distinguish p. indicus f. echinatus trees from the smooth form; leaflets distinctly more obovate with acuminate apex and inner bark relatively whitish to yellowish. however, the observed variation in prickles length was confirmed during pod maturity, usually four months after fruit bud formation. pods were collected and air-dried. seeds were extracted from the pod using scissors and forceps. pods are disc-shaped, flat and have winged margins or samara. unlike most legumes, the pterocarpus pod is indehiscent and is wind dispersed. the pod also floats in water and can be water dispersed. around 5 cm across, the pod has a central woody corky bulge containing 1 3 seeds. the seeds have very thin seed coats (orwa et al. 2009). morphological measurement pod and seed characters were described and compared among the three variants. for the pod morphology, different variants of p. indicus f. echinatus were categorized not only by the length of the prickles but also by the shape and size of the pods, number of seeds per pod, and number of prickles. for the seed morphology, seed length and width were determined using a ruler (mm). the mean for every characteristic was calculated. anatomical measurement permanent sections of the germinated seeds were obtained using a modified histological paraffin technique by johansen (1940). the seeds were fixed using formalin: acetic acid: alcohol (faa) solution for two weeks, dehydrated using different percentages of alcohol and infiltrated with paraffin wax (tables 1 and 2). the seeds were then embedded in paraffin wax and mounted on wooden blocks measuring 2 x 2 x 2 cm. the samples were sectioned using a rotary microtome with a biotropia vol. 29 no. 2, 2022 126 thickness of 10 to 15 µm. the resulting paraffin ribbons were then mounted on glass slides, decerated, stained with safranin and counterstained with fast green. drops of entellan were added over the stained sections prior to the addition of cover slips and were then air-dried. photomicrographs of the seed’s embryo were obtained using optika microscope under 400x magnification. the thickness of different tissues (root apical meristem, shoot apical meristem, procambium, ground meristem, protoderm and leaf primordium) were measured using the optika software. the development of the embryo was observed starting from the ungerminated seeds until the radicle protrusion and elongation of the embryo. haupt (1953), fahn (1967), bell (2008) and shipunov (2020) were followed to describe the developmental anatomy of the seed. table 1 paraffin schedule (fixation, dehydration, infiltration and embedding) solution procedure per day duration in solution (hours) fixation (faa-a and faa-b mixture) one week 50% etoh day 1 1st dehydration 1 hour 50% etoh day 1 2nd dehydration 1 hour 50% etoh day 1 3rd dehydration 1 hour 50% etoh day 1 4th dehydration 1 hour j1 day 1 2 hours j2 day 1 overnight j3 day 2 2 hours j4 day 2 2 hours j5 day 2 2 hours j6 day 2 (in warm place; vial uncorked) overnight j6 day 3 (3 tba changes every 2 hours) 6 hours tba (1) day 3 (uncorked at room temperature) (fumehood) 1 4 hours tba + paraffin pellets day 3 uncorked at 65 oc) 3 to 4 hours table 2 staining schedule for pterocarpus indicus samples solution time (minutes) xylene 15 tba 15 absolute ethanol 15 95% ethanol 15 50% ethanol 15 h2o 3 safranin 30 h2o 3 to 4 rinses 50% ethanol 15 95% ethanol 15 fast green 5 95% ethanol 15 absolute ethanol 15 tba 30 xylene 30 developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 127 germination viability test was conducted to p. indicus f. echinatus seeds via flotation method (dayan & reaviles 1995). viable seeds were washed with running water and were arranged in sterilized petri dishes containing filter paper with water. the set-up was done in the microtechnique laboratory of the department of forest biological sciences, college of forestry and natural resources, university of the philippines los baños (dfbs, cfnr, uplb). the seeds are considered germinated when visible protrusions of plumule is observed. for the germination percentage (equation 1) and germination rate (equation 2) (awasthi et al. 2016), the following formulae are used: germination percentage (%) = number of total germinated seeds x 100 (1) total number of seeds tested germination rate = number of germinated seeds + + number of germinated seeds (2) day of first count day of final count experimental design and analysis the germination experiments used a simple complete randomized design (crd) with three treatments having four replicates of 50 seeds each. the experiment included three variants (as treatments) of p. indicus f. echinatus, namely: t1 = small prickles; t2 = medium prickles; and t3 = long prickles. the one-way analysis of variance (anova) and duncan’s multiple range test (dmrt) were used to test for significance of the mean differences in terms of pod and seed morphological characters (pod diameter, length of prickles, number of prickles, seed length and seed width) and germination characteristics of p. indicus f. echinatus. analyses were performed using r studio version 4.1 (r studio team 2020). results and discussion morphological features certain characters (pod diameter, length of prickles and number of prickles) displayed variation among variants and were found to be potentially informative, whereas other characters (pod shape, presence of prickles and seed shape) were observed to be similar in all variants studied. all p. indicus f. echinatus pods found in this study were thin, papery-winged and disc-shaped. generally, the pods had bulge at the center containing the seeds. pod usually has a diameter of 5 8 cm, but all have their own unique characteristics. pods of t1 variant contained up to four (4) seeds, while only 1 3 seeds were found in t2 and t3 variants. on the other hand, the pod diameter had nearly significant variations among the three variants (p = 0.0535), while both the length of prickles (p = 0.000) and the average number of prickles (p = 0.000) were significantly differentiated among the variants. t2 had numerous prickles (95.095±0.461) per pod compared to those of t1 and t3. lastly, t3 had soft and the longest prickles (8.764±0.027) while t2 and t1 had hard prickles and shorter prickles (table 3). table 3 pod morphological characteristics of the pterocarpus indicus f. echinatus variants collected from mmfr morphological characteristic variant t1 t2 t3 pod shape disc-shaped disc-shaped disc-shaped pod diameter (cm) 6.450±0.136b 6.746±0.071a 6.524±0.047ab no. of seeds 1 4 1 3 1 3 length of prickles (mm) 6.248±0.026c 7.230±0.028b 8.764±0.027a number of prickles 62.095±0.809b 95.095±0.461a 55.524±0.562c stiffness of prickles hard hard soft notes: t1 = small prickles; t2 = medium prickles; t3 = long prickles. biotropia vol. 29 no. 2, 2022 128 table 4 seed morphological characteristics of the pterocarpus indicus f. echinatus variants collected from mmfr morphological character variant t1 t2 t3 seed shape falcate falcate falcate seed color orange-brown orange-brown to reddish-brown reddish-brown to brown seed width (mm) 5.167±0. 122ns 4.905±0. 127ns 5.095±0. 159ns seed length (mm) 13.571±0.316ns 13.333±0.349ns 12.905±0.325ns note: ns = no significant difference among the variants at p < 0.05. seed color of variants varied from orange brown to reddish-brown. seed width (p = 0.380) and seed length (p = 0.354) showed no significant variations among the three variants (table 4). in this study, the seeds of p. indicus f. echinatus were flat, falcate-shaped and had almost similar width and length ranging from 4.905 to 5.167 mm and 12.905 to 13.571 mm, respectively. the pod description of p. indicus f. echinatus in this study is consistent with the reports of orwa et al. (2009), thomson (2006), francis (2002) and flores et al. (2021) having indehiscent discshaped and flat pod with winged margins. about 5 cm across, it has a central woody-corky bulge containing several seeds. dayan and reaviles (1995) reported p. indicus f. echinatus pod length of 5 6 cm including the wing (1.5 2.5 cm), while rojo (1977) and duke (1983) reported 4 7 cm and 4 6 cm pod diameter, respectively. in this study, 5 8 cm pod diameter was observed. flores et al. (2021) confirmed that p. indicus f. echinatus has pod size ranging from 5 8 cm. light environment and soil moisture influence fruit quality including fruit size and color (kozlowski & pallardy 1997; raina 2003). different literatures reported various number of seeds per pod, such as francis (2002) reported 1 4 seeds per pod, orwa et al. (2009) reported 1 3 seeds per pod, while jøker (2000) and duke (1983) reported 1 2 seeds per pod. in this study, the t2 and t3 variants of p. indicus f. echinatus contained 1 3 seeds per pod while t1 has 1 4 seeds per pod. according to kelly (1984), the number of seeds per seeding plant is one of the basic parameters necessary for a description of the population dynamics of species which does not reproduce vegetatively. moreover, it was emphasized that in situations where the number of seeds per fruit was found to vary within years or among treatments, there seemed to be a stable relationship between fruits per plant and seed numbers. in terms of prickles, t2 was found to have numerous hard prickles, while t1 and t3 were found to have a lesser number of prickles (table 3), which is in agreement with the study of flores et al. (2021). kellogg et al. (2011) defined prickles as outgrowths of epidermal tissues and can provide a simple developmental system for the study of the control of cell proliferation and growth. prickles constitute one of the many types of plant defense against vertebrate herbivore (janzen & martin 1982; cooper & owen-smith 1986; milewski et al. 1991 as cited by ronel & lev-yadun 2012). for seed morphology, seed width and seed length in this study were not significantly different among p. indicus f. echinatus variants. jøker (2000) and thomson (2006) reported 6 8 mm seed length for p. indicus with brown papery testa. according to harper (1977) and silvertown (1989) as cited by chacon et al. (1998), seed size is a life history trait that may affect the fitness of the parent’s plants and the population regeneration process. large seeds tend to have a positive effect on germination. in this study, t2 had a significantly similar germination percentage to that of t1, but different from that of t3 (table 5). the morphology distinction of angiosperm seeds and the relative consistency of seed structures in narrow taxonomic units allow the use of seed characteristics in taxonomic research (esau 1977). the most significant seed morphological characters are shape, size, testa surface, the position of hilum and the presence or absence of specialized structures such as aril, caruncle or elaiosomes. the differences in bristle-like prickles or spicules in terms of length and number as well as number of seeds per pod as observed in p. indicus f. echinatus may support the argument of jøker (2000) and duke (1983) that intermediate forms may occur. developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 129 anatomical features in the root apex, the root cap had become more developed after protrusion, consisting of 7 10 layers of cells. for the germinated seeds, the root tip of t2 was more round compared to the root tips of t1 and t3 (fig. 1). the procambium became more evident after radicle protrusion and was the thickest in t3. the ground meristem also increased in thickness after radicle protrusion. the shoot apical meristem (sam) of the embryo was domeshaped which was observed in the embryo of all p. indicus f. echinatus variants. well-developed leaf primordium was observed in all of the embryos after radicle protrusion. on the other hand, the formation of leaf primordium in t2 seeds after radicle protrusion was more progressive (fig. 2). sam is essentially a dome-shaped structure with undifferentiated cells at the tip, surrounded by a differentiating peripheral zone that participates in leaf formation. a well-developed apical dome is directly related to high germination frequency (corredoira et al. 2002). a less developed apical dome would mean lesser germination frequency. in this study, welldeveloped leaf primordium can be observed in all of the embryos after radicle protrusion (fig. 2). figure 1 longitudinal section of the root apex of the variants of pterocarpus indicus willd. f. echinatus collected from mmfr notes: a-c = before protrusion; d-f = after protrusion. a and d = t1 root apex; b and e = t2 root apex; c and f = t3 root apex. rc = root cap; ram = root apical meristem; pc = procambium; pd = protoderm; gm = ground meristem. scale bar = 200 µm. figure 2 longitudinal section of the shoot apex of the variants of pterocarpus indicus willd. f. echinatus collected from mmfr notes: a-c = before protrusion; d-f = after protrusion. a and d = t1 root apex; b and e = t2 root apex; c and f = t3 root apex. sam = shoot apical meristem; pc = procambium; pd = protoderm; lp = leaf promordium. scale bar = 200 µm. biotropia vol. 29 no. 2, 2022 130 seed anatomical characters showed value in verifying taxonomic relationships (esau 1977). p. indicus f. echinatus was found to have a root tip cap of 7 10 layers of cells. in this study, the root tip cap of t2 variant was found to be round, while those of t1 and t3 were pointed. roue et al. (2020) proved in their study that root tip cap structure affects apex penetration and reorientation, in which rectangular-shaped root tip cap showed enhanced penetration abilities compared to the pointed root tip cap. kumpf and nowack (2015) mentioned that modern plant biology has unraveled that many of the functions that darwin attributed to the root tip are actually accomplished by the root tip cap, which is a multi-layered dome of spindleshaped parenchyma cells that overlies the growing root tip (iijima et al. 2008). the root tip cap surrounds and protects the meristematic stem cells at the growing root tip. in addition, the root tip cap shows a rapid turnover of shortlived cells regulated by an intricate balance of cell generation, differentiation and degeneration. in arabidopsis thaliana, the root tip cap cells are actively killed and degraded on the root surface, while a limited amount of short-lived ‘borderlike’ cells are released into the rhizosphere (durand et al. 2009; fendrych et al. 2014). based on the classification of kumpf and nowack (2015), p. indicus f. echinatus can be considered to have a closed meristem structures which form their cell lineages from specific stem cells, including the root tip cap lineage, which shows a defined root cap stem cells and a layered root cap structure. while new root cap cells are constantly produced by root cap stem cells in an indeterminate fashion, the size and cell number of the root cap are determinate (barlow 2003). the procambium in this study became more evident after radicle protrusion and was the thickest in t3. the ground meristem also increased in thickness after radicle protrusion. the procambium provides the basis for the differential modulation of long-distance transport capacities and plant body stability (jouannet et al. 2015). germination results of germination percentage and germination rate showed significant differences for the three variants. germination percentage and germination rate were both the highest in t2 with 82.67% and 42.86, respectively (table 5; fig. 3). table 5 mean germination percentage and rate ± se of the pterocarpus indicus f. echinatus variants collected from mmfr parameter variant t1 t2 t3 germination percentage (%) 60±1.15ab 82.67±3.53a 50.667±0.667b germination rate (no. of seeds per nth day) 16.73±1.15b 42.86±1.95a 23.46±1.79b note: numbers followed by the same letter are not significantly different based on duncan test at p < 0.05. figure 3 seeds showing germination up to radicle protrusion notes: a = t1; b = t2; c = t3. day 0 to day 3 from left to right. scale bar = 1 cm. developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 131 in this study, seeds began to germinate 3 4 days after sowing, which is similar to the observation of thomson (2006). germination usually commences with the uptake of water by the dry seed through imbibition and is completed when the radicle extends to penetrate the structures that surround it (bewley 1997). in the study of de sedas et al. (2019) the osmotic effect due to salinity was the main inhibitory factor that reduced germination of inland neotropical tree species such as minquartia guainensis, apeiba menbranacea, ormosia ccoccinea and ochroma pyramidale. high germination percentage and germination rate in t2 (table 5; fig. 3) can be attributed to various factors. in the study of vozzo (2003) as cited by luna et al. (2014), tropical species that benefit from reagent, such as hydrogen peroxide, included albizia species and camphor tree seeds. valio and scarpa (2001) proved that the seeds germination percentage and rate of seven tropical pioneer species in brazil (cecropia hololeuca, c. pachystachya, c. glazioui, solanum gracillimum, s. granuloso-leprosum, s. tabacifolium and miconia chamissois) were significantly higher in the irradiated condition than in shaded condition. on the other hand, seed germination rate of peltophorum dubium varied with water potential treatment (daibes & cardoso 2020). conclusion the germination, pod and seed morphological characters and seed anatomical characters proved to be informative by distinguishing significant differences among the observed variants of p. indicus f. echinatus. the variants can be differentiated by the average number of prickles, length of prickles, stiffness of prickles, pod diameter, number of seeds per pod and seed color. the formation of the leaf primordium in t2 seeds after radicle protrusion was more progressive. the observed variants did not differ in their germination characteristics. differences of shoot apical meristem can be observed based on the development of the apical dome of each variant, which is related to the germination percentage of each variant. this study on p. indicus f. echinatus is limited to mmfr only and examines seed only and does not include other parts of the tree. also, this study does not include parameters such as seed coat and fruit anatomy. it is therefore recommended that further research, such as molecular and dna analyses, be conducted in the future to shed light on the observed variation in this study. characters such as seed coat anatomy can add more knowledge on the separation of the variants being studied. acknowledgments the authors wish to thank the department of forest biological sciences and the makiling center for mountain ecosystems (mcme), cfnr-uplb for allowing the use of their facilities to conduct this study. this study is partially supported by the dost-pcaarrdfunded project “germplasm conservation of selected indigenous forest trees in mmfr”. references abud hy, goncalves nr, pereira mds, pereira dss, reis rdge, bezerra ame. 2012. germination and morphological characterization of the fruits, seeds and seedling of pilosocereus gounellei. rev braz bot 35(1):11-6. awasthi p, karki h, vibhuti bk, bargali ss. 2016. germination and seedling growth of pulse crop (vigna spp.) as affected by soil salt stress. curr agric res j 4(2):159-70. barlow pw. 2003. the root cap: cell dynamics, cell differentiation and cap function. j plant growth regul 21:261-86. baskin cc, baskin jm. 1998. seeds: ecology, biogeography and evolution of dormancy and germination. san diego (us): academic press. 666 p. baskin jm, baskin cc, li x. 2000. taxonomy, anatomy and evolution of physical dormancy in seeds. plant species biol 15:139-52. benedict jc, smith sy, collinson me, skornickova jl, specht cd, marone f, xiao x, parkinson dy. 2015. seed morphology and anatomy and its utility in recognizing subfamilies and tribes of zingiberaceae. am j bot 102(11):1814-41. cappers rtj, bekker rm. 2013. a manual for the identification of plant seeds and fruits. gröningen (nl): barkhuis & university of gröningen library. 272 p. biotropia vol. 29 no. 2, 2022 132 chacon p, bustamante ro, henriquea c. 1998. the effect of seed size on germination and seedling growth of cryptocarya alba (lauraceae) in chile. rev chil hist nat 71(2):189-97. cervantes e, martin jj, saadaoui e. 2016. updated methods for seed shape analysis. scientifica [internet]. 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[cited 2019 may 31]; 10: 706. doi: 10.3389/ fpls.2019.00706 xu cx, zeng j, cui tc, chen qd, ma yp. 2016. introduction, growth performance and ecological adaptability of hongmu tree species (pterocarpus spp.) in china. j trop for sci 23(3):260-7. youngberg h, hannaway d, mosley a. 1998. 4-h plant and seed identification and crop judging. lecture guide. corvallis (us): oregon state university extension service. 20 p. biotropia no. 10, 1997 : 42-62 the effects of milling degree and type of bag on fungal infection and some chemical contents of stored milled rice okky setyawati dharmaputra seameobiotrop, p.o. box 116, bogor, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the effects of milling degree and type of bag on fungal infection of stored milled rice were investigated together with some chemical contents (glucose, amylose, p rotein and total lipid contents), and changes in moisture content. rice var. ir 64 with different milling degrees (85, 90, 95 and 100%) packed in jute and polypropylene bags (1 kg of milled rice/bag) were stored under laboratory conditions with temperature between 24.3-27.3 c and relative humidity 52.6-81.9% for 3 months. the initial moisture content (m.c.) of milled rice was ± 14%. three replications (3 bags) were used for each treatment. each bag was put individually and was arranged randomly on a wooden pallet. the results showed that in general, the increase of milling degree and duration of storage decreased the m.c. type of bag did not give significant differences on the m.c. twenty eight fungal species were isolated from rice with different milling de gree and bag type during storage. the predominant species was aspergillus candidas. total fungal population decreased with the increase of milling degree and duration of storage. bag type did not give significant differences on total fungal population. in general, the increase of milling degree increased glucose content. glucose content in milled rice packed in jute bag was higher than that in polypropylene bag. glucose content tended to decrease with the increase of storage duration. the increase of milling degree increased amylose content in milled rice. amylose content of milled rice packed in jute bag was lower than that in polypropylene bag. the increase of storage duration decreased amylose content in milled rice. in general, protein content decreased with the increase of milling degree and duration of storage. 1 type did not give significant differences on protein content. total lipid content decreased with the increase of milling degree and duration of storage. total lipid content of milled rice packed in jute bag was lower than that in polypropylene bag, but based on chemical analysis the difference was not significant. based on statistical analyses, correlation between the m.c. and total lipid content with total fungal population was positive. there was no correlation between glucose, amylose and protein contents with total fungal population. rice with high milling degree can be stored safely for long period, but it has low chemical (nutritional) contents. key words: milling degree/bag type/fungal infection/chemical content/milled rice. introduction rice (oryza saliva l.) is the primary foodcrop in indonesia. the national logistics agency (bulog) buys and stores a large amount of milled rice as national stock to maintain price stability. 42 the effects of milling degree and type of bag on fungal infection okky setyawad dhannaputra during storage rice could be infested by insects, mites, microorganisms and rodents. among the microorganisms, fungi are the most important cause of deterioration of stored products (christensen and kaufmann 1974). the tropical climate of indonesia provides a favourable condition for fungal growth. according to garraway and evans (1984) the development of fungi was affected by the nutritional content of the substrate. the degree of milling is one of the physical characters of rice that may influence the fungal development. another factor that may affect the fungal development is the type of bag used to store milled rice. the objective of the study is to get information on the effects of milling degrees and bag types on moisture content, fungal population, and some chemical contents (glucose, amylose, protein and total lipid) of milled rice during 3 months of storage. materials and methods rice variety, milling degree, bag type, and storing of milled rice rice var. ir 64 was used in this study, because it is widely cultivated in indonesia. the paddy was obtained from food technology research and training centre (balai penelitian teknologi pangan) bulog, tambun, and it was stored for 2 weeks from the harvest time. the paddy was milled with different milling degrees, i.e. 85, 90, 95 and 100%. rice with 85% milling degree was enclosed by 15% of bran layer and embryo, while rice with 100% milling degree was die rice that had neither bran layer nor embryo (juliano 1972; bulog 1994). two types of bag were used, i.e. jute and polypropylene bags. rice with milling degree of 85, 90, 95 and 100% were stored in jute and polypropylene bags (1 kg/bag) under laboratory conditions for 3 months. the initial moisture content of milled rice was ± 1 4 % . before storage rice was fumigated with phos-phine at dosage rates 2 g/tonne of rice for 6 days. three replications (3 bags) were used for each treatment. each bag was arranged randomly on a wooden pallet (figure 1). during storage the temperature and the relative humidity of the storage were recorded using a thermohygrograph. obtaining working samples initial samples were obtained from each replication (bag) of treatment at the beginning of storage, and then at 1, 2 and 3 months of storage. a sample from each bag was divided using a sample divider into 4 sub samples for (1) moisture content analysis, (2) fungal analysis, (3) some chemical content analyses, and (4) reserve sample. 43 figure 1. milled rice packed in jute (a) and polypropylene (b) bags, and arranged randomly on a wooden pallet. moisture content, fungal and chemical content analyses moisture content was determined using an oven method (bsi 1980). fungi were isolated using dilution method on dichloran 18% glycerol agar (dg 18) (pitt and hocking 1985). the glucose, amylose, protein and total lipid contents were determined using modified anthrone, williams, kjeldahl and soxhlet extraction methods, respectively (yoshida et al. 1976). identification of the fungi fungal identification was carried out according to samson et al. (1984), pitt and hocking (1985). experimental data were analysed using completely randomized factorial design with 3 factors. the 1st, 2nd, and 3rd factors were milling degree, type of bag, and duration of storage, respectively. results and discussions moisture content moisture content (m.c.) is the most important factor in determining the development of microorganism, insect and chemical reaction hi stored products (muir 1973; sinha 1973). 44 biotropia no. 10, 1997 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra based on statistical analysis, milling degree, duration of storage, interaction be tween bag types and duration of storage, interaction among milling degree, bag types and duration of storage gave very significant differences on the m.c., while interaction between milling degree and bag type was significantly different. type of bag and interaction between milling degree and duration of storage were not significantly different. the increase of milling degree decreases the m.c. (table 1). the m.c of rice with 85, 90, 95, and 100% milling degree were 14.18, 14.04, 13.91 and 13.73%, respectively. table 1. the effect of milling degree, duration of storage, interaction between milling degree and bag type, interaction between bag type and duration of storage, and interaction among milling degree, bag type, and duration of storage on moisture content of rice according to dharmaputra et al. (1993) the increase in milling degree increased the m.c. of milled rice that was stored under laboratory conditions for 3 months with temperature of 25 ± 2 c and relative humidity (rh) of 80 ± 5%. in this study the result was not the same; the increase of milling degree decreased the m.c. it was assumed that the milled rice was stored at higher temperature (24.3-27.3 c) and lower rh (52.6-81.9%) (table 2). 45 biotropia no. 10, 1997 table 2. range of temperature and relative humidity at storage condition the endosperm and the embryo of milled rice were enclosed by bran layer. the layer consists of pericarp, legmen and aleuron (figure 2). the three layers contain more lipid and protein than the endosperm, while the latter contains more carbohydrate. the higher milling degree causes the higher loss of the three layers. water could be easily bound by carbohydrate compared to lipid or protein. it was assumed that the high temperature and the low rh during storage caused the evaporation of water in the grain. although the pores of polypropylene bag was smaller than jute bag, type of bag did not give a significant difference on the m.c. it was assumed that only one bag (with 1 kg of rice) was put on the wooden pallet, so that air circulation did not affect the m.c. of rice. figure 2. structure of the rice grain (cited from grist, 1969). 46 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra during storage the m.c. of rice decreased, although the m.c. of rice at 2 months of storage was not significantly different from that at 3 months of storage (table 1). this was probably because of the higher temperature and the lower rh during storage. the lowest m.c. was found in rice with milling degree of 100% packed in polypropylene bag at 2 months of storage (13.39%); while the highest was in rice with milling degree of 85% packed in jute and polypropylene bags at the beginning of storage (14.59% respectively). species and fungal population twenty eight species of fungi were isolated from rice with different milling degrees and bag types during storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. niger, a. oryiae, a. penicilloides, a. sydowii, a. terreus, a. versicolor, a. wentii, cladosporium cladosporioides, curvularia lunala, eurotium chevalieri, e. repens, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, paecilomyces varotii, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. corylo-philum, p. herquei, p. islandicum, p. raistrickii, rhizopus microsporus, synce-palastrum racemosum and wallemia sebi. the predominant fungus was a. candidus. it was assumed that the m.c. of milled rice during storage was suitable for its growth, or the fungus was able to compete with other fungal species in colonizing the substrate. among the above species, there were some species of field fungi i.e. cladosporium cladosporioides, curvularia lunata, f. moniliforme, and n. oryzae. it was assumed that the fungi infected the grain before harvest and they survived during storage. according to christensen and kaufmann (1969) field fungi need m.c. in equilibrium with the high rh (90%) or more for their growth. the presence of each fungal species on rice with different milling degree during storage is presented in table 3. a. candidus, c. cladosporioides and p.. citrinum were always isolated, while a. flavus and e. chevalieri were often isolated. most of the fungi isolated belong to genera aspergillus, eurotium, and penicillium. according to christensen and kaufmann (1974) fungi that belong to genera aspergillus and penicillium caused deterioration in grain during storage. pitt and hocking (1985) reported that eurotium is a teleomorph stage of aspergillus which is a xerophilic fungus. based on statistical analysis, milling degree, duration of storage, and interaction between milling degree and duration of storage gave very significant differences on total fungal population, while bag type, interaction between milling degree and bag type, interaction between bag type and duration of storage, and interaction among milling degree, bag type, and duration of storage were not significantly different. 47 biotropia no. 10, 1997 table 3. the presence of each fungal species on rice with different milling degree during storage total fungal population (transformed into log x) on rice with milling degree of 85, 90, 95 and 100% during 3 months of storage were 2.94, 2.50, 2.08 and 1,73 colonies/g, respectively (table 4). total fungal population decreased with the increase of milling degree. the total fungal population was related to the nutritional content of the rice. protein, lipid, some organic acids, and vitamins decreased with the increase of milling degree. they are micro and macro-elements needed for fungal growth. according to juliano (1972) the nutritional content of the rice with high milling degree was low because the outer layer of the rice was removed during milling, consequently a part of the nutrition found in this layer was lost. 48 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 4. the effect of milling degree, bag type, duration of storage, and interaction between milling degree and duration of storage on total fungal population of nee type of bag did not give a significant difference on the total fungal population (table 4). the m.c. is the environmental factor in determining the fungal growth and type of bag did not give a significant difference on it, so that the bag type did not also give a significant difference on total fungal population. total fungal population decreased with the increase of storage duration (table 4). the total fungal population (transformed into log x) at the beginning of storage was significantly different from that at 1, 2 and 3 months of storage, while after 2 and 3 months of storage they were not significantly different, although their population tended to decrease. the decrease of total fungal population was presumably related to the decrease of nutritional and moisture contents in rice. the total fungal population (not transformed into log x) at the beginning of storage, and at 1, 2 and 3 months of storage were 650.48, 410.79, 390.15 and 183.81 colonies/g, respectively. the lowest total fungal population was found in rice with milling degree of 100% after 3 months of storage (32.17 colonies/g), while the highest was in rice with milling degree of 85% at the beginning of storage (1505.92 colonies/g). fungi in rice with milling degree of 85% bulog (1994) determined that rice with 85% milling degree was enclosed by 15% of bran layer and embryo. twenty four fungal species were isolated from rice with 85% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. niger, a. oryzae, a. penicilloides, a. sydowii, a. versicolor, a. wentii, cladosporium dadosporioides, curvularia lunata, eurotium 49 biotropia no. 10, 1997 chevalieri, e. repens, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. herquei, p. raistrickii, rhizopus microsporus, syncephalastrum racemosum and wallemia sebi. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidus with the population of 1474.33, 947.67, 1045.59 and 372.09 colonies/g, respectively. fungi in rice with milling degree of 90% bulog (1994) determined that rice with 90% milling degree was enclosed by 10% of bran layer and embryo. twenty one fungal species were isolated from rice with 90% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. penicilloides, a. sydowii, a. terreus, a. ver-sicolor, cladosporium dadosporioides, curvularia lunata, eurotium chevalieri, e. re-pens, endomyces fibuliger, nigrospora oryzae, paecilomyces varotii, penicillium sp, nr. adametzioides, p. chrysogenum, p. citrinum, p. islandicum, p. raistrickii, rhizopus microsporus and syncephalastrum racemosum. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidus with the population of 656.08, 332.41, 355.83 and 84.67 colonies/g, respectively. dharmaputra (1994) reported that milled rice with the same variety (ir 64) and milling degree (90%) stored for 3 months under laboratory conditions (in jars), was infected by eleven fungal species i.e. a. candidus, a. flavus, a. niger, a. penicilloides, a. versicolor, a. wentii, c. dadosporioides, e. chevalieri, e. repens, p. citrinum and p. herquei. the predominant species was a. flavus. the difference in fungal species that infected milled rice was presumably due to the difference of location where the paddy was cultivated. fungi in rice with milling degree of 95% bulog (1994) determined that rice with 95% milling degree was enclosed by 5% of bran layer and embryo. twenty two fungal species were isolated from rice with 95% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. oryzae, a. penicilloides, a. sydowii, a. terreus, 50 the effects of milting degree and type of bag on fungal infection okky setyawati dharmapuera 51 biotropia no. 10, 1997 a. versicolor, cladosporium cladosporioides, curvularia lunata, eurotium chevalieri, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, paecilomyces varotii, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. islandicum, p. raistrickii, rhizopus microsporus and syncephalastrum racemosum. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidas with the population of 211.50, 95.17, 125.00 and 50.25 colonies/g, respectively. fungi in rice with milling degree of 100% rice with 100% milling degree had neither bran layer nor embryo. it had only an endosperm (juliano 1972; bulog 1994). eighteen fungal species were isolated from rice with 100% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidas, a. flavus, a. fumigatus, a. niger, a. oryzae, a. penicilloides, a. sydowii, a, versicolor, a. wentii, cladosporium cladosporioides, curvularia lunata, eurotium chevalieri, fusarium moniliforme, nigrospora oryzae, penicillium sp. nr. adametzioides, p. citrinum, p. corylophilum, and p. raistrickii, population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidas with the population of 84.17, 41.34, 35.75 and 19.34 colonies/g, respectively. glucose content most of fungi use glucose (d-glucose) for their growth (moore-landecker 1982). glucose is a form of monosaccharide with six carbon atoms (hexose). mono -saccharide is considered as carbohydrate which cannot be hydrolized into a simple form and has a very simple bond compared to polysaccharide. consequently, their chemical bond can be easily separated by enzymes compared to amylose, a polysaccharide which has a more complex chemical bond than monosaccharide (mayes 1981). based on statistical analysis, milling degree, bag type, duration of storage, interacti on between milling degree and bag type, and interaction among milling degree, bag type and duration of storage gave very significant differences on the glucose content, while interaction between milling degree and duration of storage was no! significantly different. interaction between bag type and duration of storage was noi significantly different. 52 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra glucose content increased with the increase of milling degree (table 6). the higher milling degree caused the higher loss of the bran layer and embryo. therefore, the percentage (w/w) of glucose content in rice with high milling degree was higher than that of low milling degree. glucose content on rice with milling degree of 85, 90, 95 and 100% were 2.99, 3.50, 3.27 and 3.41%, respectively. milled rice packed in jute bag had glucose content higher than packed in polypropylene bag. it was assumed that total fungal population in milled rice packed in jute bag was lower than in polypropylene bag, although based on statistical analysis it was not significantly different (table 4). glucose content tended to decrease with the increase of storage duration. it was attributed that the glucose was used for the fungal growth (moore-landecker 1982) and as energy source for grain respiration (pomeranz 1992). glucose content at the beginning and one month of storage were significantly different from that at 2 and 3 months of storage. the glucose content of milled rice at the beginning of storage, and at 1, 2, and 3 months of storage were 3.74, 3.86, 2.84 and 2.73%, respectively. table 6. the effect of milling degree, bag type, duration of storage, interaction between milling degree and bag type, interaction between nulling degree and duration of storage, and interaction among milling degree, bag type and duration of storage on glucose content of rice. milling bag degree ( % ) type glucose content ( % ) duration of storage (month) 0 1 2 3 x 85 jute 3.29uvw 3.85 stu 2.57 wx 2.56 wx 3.07 ij polypropylene 3.29 uvw 3.66 stu 2.23 x 2.47 z 2.91 j 3.29 mno 3.76 kiln 2.40 r 2.52 qr 2.99 a 90 jute 3.78 stu 3.79 stu 4.06 st 3.43 tuv 3.78 g polypropylene 3.78 stu 3.68 stu 2.84 vwx 2.61 wx 3.23 ij 3.78 kl 3.74 klm 3.45 lmn 3.02 nop 3.50 b 95 jute 3.94 stu 3.43 tuv 2.47 x 2.78 vwx 3.15ij polypropylene 3.94 stu 4.30 st 2.69 wx 2.61 wx 3. 38 hi 3.94 kl 3.86kl 2.58 pqr 2.69 pqr 3.27 ab 100 jute 3.94 stu 4.89s 2.94 vwx 2.87 vwx 3.66 gh polypropylene 3.94 stu 3.24 uvw 2.93 vwx 2.50 x 2.15ij 3.94 kl 4.06k 2.93 opq 2.68 pqr 3.41 b y jute * * * * 3.41c polypropylene * * * * 3.17d 3.74 e 3.86e 2.84 f 2.73 f — numbers followed by the same letter do not differ significantly according to tukey multiple range test at 95% confidence level *not significantly different 53 biotropia no. 10, 1997 the highest glucose content was found in rice with milling degree of 100% packed in jute bag at one month of storage (4.89%), while the lowest was in rice with milling degree of 85% packed in polypropylene bag at 2 months of storage (2.23%). although a sugar other than glucose may give maximum growth of a fungus, it appears that the more closely the configuration of a sugar resembles glucose, the more fungi use it. this might be due to the ability of a fungus to use a particular sugar depending on how easy it can be converted into a phosphorylated derivative of glucose which can enter the respiratory pathways (moore-landecker 1982). amylose content most of the carbohydrate found in rice is in the form of starch (90% of dry weight) and only a very small part is in the form of hemicellulose, cellulose and some free sugars such as sucrose, raffinose, glucose and fructose (juliano 1972). starch is a polysaccharide that can be separated into two fractions depending on their ability to dissolve hi water, that is amylose (± 20%) and amylopectin (± 80%) (mayes 1981). juliano (1972) reported that based on dry weight, the amylose content of milled rice was between 7-33% depending on rice variety. based on statistical analysis, milling degree and duration of storage gave very significant differences on the amylose content, while bag type gave a significant difference. interaction between milling degree and bag type, interaction between milling degree and duration of storage, interaction between bag type and duration of storage, and interaction among milling degree, bag type and duration of storage did not give significant differences. the increase of milling degree increased amylose content (w/w) in milled rice. the amylose content of milled rice with milling degree of 85, 90, 95 and 100% were 23.84, 23.99. 24.98 and 25.50%, respectively (table 7). amylose content of milled rice packed in jute bag (24.09%) was lower than that in polypropylene bag (25.07%). it was assumed that the pores of jute bag was bigger than that of polypropylene bag, so that the air surrounding jute bag could make easier to hydrolize the amylose to form maltose or glucose. the increase of storage duration decreased amylose content in milled rice. the amylose content at the beginning of storage, and at 1, 2 and 3 months of storage were 26.72, 25.40, 24.29 and 21.90%, respectively. presumably the phenomena was due to the activities of (3-amylase and fungal enzymes which infected the molecule a-glucans and supported the hydrolization of amylose to disaccharide and monosaccharide. 54 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 7. the effect of milling degree, bag type, and duration of storage on amylose content of rice amylose content ( % ) milling bag duration of storage (month) degree (%) type 0 1 2 3 x 85 * * * * 23.84 a 90 * * * * 23.99 ab 95 * * * * 24.98 be 100 * * * * 25.50 c y jute * * * * 24.09 d polypropylene * * * * 25.07 e 26.72 f 25.40 g 24.29 h 21.90i numbers followed by the same letter do not differ significantly according to tukey multiple range test a t 95% confidence level * not significantly different protein content rice is one of the cereals which contains a higher percentage of lysine (± 4%) than other cereals. lysine is an essential amino acid which is found in very small quantities in other food stuff (juliano et al. 1973). brown rice contained ± 8% protein, 75% carbohydrate and a small amount of lipid, fibre and ash, while milled rice contained ±7% protein (juliano 1976). based on statistical analysis, milling degree and duration of storage gave very significant differences on the protein content, while interaction between milling degree and duration of storage was significantly different. bag type, interaction between milling degree and bag type, interaction between bag type and duration of storage, and in teraction among milling degree, bag type and duration of storage did not give significant differences. the protein content tended to decrease with the increase of milling degree and duration of storage (table 8). the protein content in rice with milling degree of 85 and 100% during 3 months of storage were significantly different. they were 9.14 and 8.84%, respectively. according to juliano (1972) the decrease of milling degree decreased the nutritional content (including protein). dharmaputra et al. (1993) reported that total protein in milled rice var. cisadane decreased with the increase of milling degree. at the beginning of storage the protein content in milled rice was 9.44%, and then decreased to 9.18% at 3 months of storage. dharmaputra et al. (1993) reported that the increase of storage duration decreased the protein content in milled rice. 55 biotropia no. 10, 1997 table 8. the effect of milling degree, duration of storage, and interaction between milling degree and duration of storage on protein content of rice according to sinha and muir (1973), the decrease of protein content may also be caused by degradation of amino acid and non-protein nitrogen such as vitamin b and phospholipid. . the lowest protein content was found in rice with milling degree of 100% after 1 month of storage (8.28%), while the highest was in rice with milling degree of 90% at the beginning of storage (9.77%). in general, the increase of milling degree and duration of storage decreased the protein content hi milled rice. total lipid content according to juliano (1976), brown rice contained ±1.9% lipid, while milled rice contained ± 0.5% lipid. pomeranz (1974) reported that lipid was convened into free fatty acid and glycerol with the aid of lipase enzyme during storage. t his con version was accelerated with the existence of storage fungi because the fungi have high ly poly tic activity. based on statistical analysis, milling degree, duration of storage, and interaction between milling degree and duration of storage gave very significant differences on the total lipid content, while bag type and interaction among milling degree, bag type and duration of storage gave significant differences. interaction between milling degree and bag type, and interaction between bag type and duration of storage did not give significant differences. total lipid content decreased with the increase of milling degree and duration of storage (table 9). total lipid content in milled rice with milling degree of 85, 90, 95 and 100% were 0.26, 0.23, 0.20 and 0.15%, respectively. juliano (1972) reported; 56 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 9. the effect of milling degree, bag type, duration of storage, interaction between milling degree and duration of stor age, and interaction among milling degree, bag type and duration of storage on total lipid content of nee that milling removes the embryo and the bran layer, therefore total lipid found in this pan will decrease. total lipid content of milled rice packed in jute bag (0.20%) was lower than packed in polypropylene bag (0.21 %), but chemical analysis showed no significant difference. total lipid content in milled rice at the beginning of storage, and at 1, 2 and 3 months of storage were 0.28, 0.25, 0.17 and 0.14%, respectively. dharmaputra et al. (1993) reported that the increase of milling degree and du ration of storage, decreased total lipid content. according to pomeranz (1992) degra dation of fat or oil in grains can be affected by several factors, such as 1) oxydative process which gives rancid odour and taste, and 2) hydrolytic process which results to free fatty acid and glycerol. the lowest total lipid content was found in rice with milling degree of 100%, packed in jute bag at 3 months of storage (0.10%), while the highest was in rice with milling degree of 85%, packed in jute bag and polypropylene bags at the beginning of storage (0.34% respectively). 57 biotropia no. 10, 1997 correlation between moisture content and total fungal population based on statistical analysis, the m.c. and total fungal population were positively related with coefficient correlation (r) = 0.52. the increase of m.c. increased total fungal population (figure 3). figure 3. correlation between moisture content and total fungal population on milled rice during storage water is required for the growth of fungi just as for other organisms. fungi usually require a film of water around their cells through which nutrients and enzyme diffuse (garraway and evans 1984). ramakrishna et al. (1993) reported that spore germination and initial growth of fungi that infected the grain were affected by water activity and temperature of storage. correlation between glucose content and total fungal population statistical analysis showed no correlation between glucose content and total fungal population. it was assumed that glucose was not only used for the fungal nutrition, but also for respiration process of grains (pomeranz 1992). 58 the effects of milling degree and type of bag on fungal infection okky setyawati dbarmaputra according to garraway and evans (1984) glucose is a good carbon source for 4-5 fungal species. most of the fungal species also used oligosaccharide and polysaccharide as carbon source. correlation between amylose content and total fungal population based on statistical analysis, there was no correlation between amylose content and total fungal population. it was assumed that amylose was hydrolized into disaccharide by fungi-infected milled rice during storage. in this study twenty eight fungal species were isolated (table 3). according to garraway and evans (1984) each fungal species may have different enzyme to hydrolize carbon sources, such as amylose. 1& correlation between protein content and total fungal population statistical analysis showed no correlation between protein content and total fungal population. it was assumed that fungi require other nitrogen source for their growth and development. according to garraway and evans (1984) most fungi are able to utilize simple inorganic nitrogen sources as well as organic sources such as amino acids. correlation between total lipid content and total fungal population based on statistical analysis, the correlation between total lipid content and total fungal population was positive with r = 0.64. the increase of total lipid content increased total fungal population (figure 4). fungi contain a specific enzyme that could hydrolize lipid from the substrate for their development and growth (patterson 1989). according to garraway and evans (1984), at first fungi hydrolized lipid with lipase enzyme, then changed them into glycerol through (3-oxidation pathway. conclusions in this study the increase of milling degree and duration of storage, decreased the m.c. of rice. the type of bag did not give a significant difference on the m.c. twenty eight fungal species were isolated from rice with different milling degree and bag type during storage. most of the fungi isolated belong to genera aspergillus, eurotium and penicillium. the predominant species was a. candidus. 59 biotropia no. 10, 1997 figure 4. correlation between total lipid content and total fungal population on milled rice during storage. total fungal population decreased with the increase of milling degree and duration of storage. type of bag did not give a significant difference on the total fungal population. glucose content of milled rice increased with the increase of milling degree. when packed in jute bag the glucose content was higher than packed in polypropylene bag, but it decreased with the increase of storage duration. amylose content of milled rice increased with the increase of milling degree. when packed in jute bag the amylose content was lower than packed in polypropylene bag, but it decreased with the increase of storage duration. protein content in milled rice tended to decrease with the increase of milling degree and duration of storage, but it was not affected by bag type. total lipid content of milled rice decreased with the increase of milling degree and duration of storage. when packed in jute bag the lipid content differed significantly than packed in polypropylene bag, but chemical analysis showed no significant difference. 60 the effects of milling degree and type of bag on fungal infection okky setyawati dharmiputn rice with low milling degree has high nutritional content, but it cannot be stored for long period. on the other hand rice with high milling degree has low nutritional content, but it can be stored for long period. to get more information on the effect of bag type on fungal infection and chemical contents of stored milled rice, a research using stack of rice is needed. acknowledgement the author gratefully acknowledges the financial support of the government of indonesia. thanks are due to dr. haryanto susilo, dr. hariyadi halid, dr. mulyo sidik, mr. amad and mrs. asmarina s.r. putri for their valuable assistance, and to dr. a.d. hocking from csiro, australia, for the confirmation of fungal identification. references bsi. 1980. methods of test for cereals and pulses. pan 3. determination of moisture content of cereals and cereal products (routine method). british standards institution. isbn 0 058 114333. bul og. 1994. tatacara teknis pemenksaan kualitas gabah, beras dan karung goni/plastik dalam rangka pengadaan pangan dalam negeri. badan urusan logistik. jakarta. (in indonesian). christe nse n. c.m. and h.h. kaufman n. 1969. grain storage: the role of fungi in quality l oss. university of minnesota, minneapolis. ___. 1974. microflora. in c.m. chrstensen (ed.). storage of cereal grains and their product. american association of cereal chemist., inc. minnesota, pp. 158-192. dharmaputra, o.s. 1994. kapang pada beras yang berasal dari beberapa varietas padi. hayati 1(2): 37-41. ___. h. halid, h. susilo and a.s.r. putri. 1993. the effects of milling degree on fungal infection, protein and total lipid content in milled rice. proceeding 16th asean seminar on grain postharvest technology, phuket, thailand. 24-26 august 1993. oarraway. m.o. and r.c. evans. 1984. fungal nutrition and physiology. john willey & sons inc., new york. grist. d.h. 1959. rice 3 rd ed. longmans, london. juliano, b.o. 1972. rice caryopsis and its composition. in d.f. houston (ed). rice chemistry and technology. american association of cereal chemist., minnesota. ___. 1976. the chemical basis of rice grain quality. proceedings of the workshop on chemical aspect of rice grain quality. irr1. los banos, philippines. __. a.a. ant onio and b.v. esmama 1973. effects of protein content on the distribution and properties or rice protein. j. sci. food agr. 24: 295-306. maye s. p.a. 1981. carbohydrate. in d.w. martin, p.a. mayes and v.w. rodw ell (ed}. 18th edition. harper's review of biochemistry. lange medical publication, los altos, california. moore landecker, e. 1982. fundamentals of the fungi. 2rd edition. prentice-hall, inc., new jersey. muir. w.e. 1973. temperature and moisture in grain storage. in r.n. sinha and w.e. muir (ed). grain storage: part a system. the avi publishing company, connecticut. 61 biotropia no. 10, 1997 patterson, h.b.w. 1989. handling and storage of oilseeds, oils, fats and meal. elsevier applied science, new york. pitt, j.i. and a.d. hocking. fungi and food spoilage. academic press, sydney. pomer anz. y. 1992. biochemical, functional and nutritive change during storage. in sauer, d.b. (ed). storage of cereal grains and their products. american association of cereal chemist., minnesota, pp. 55-141. ramakrishna, n., j. lacey and j.e. smith. 1993. effects of water activity and temperature on the growth of fungi interacting on barley grain. mycol. res. 97(11): 1393-1402. samson, r.a., e.s. hoekstra and c.a.n. van oorschot. 1984. introduction to foodborne fungi. centraalbureau voor schimmelcultures, baam, the netherlands. sinha. r.n. 1973. interrelation of physical, chemical and biological variables in the deterioration of stored grains. in r.n. sinha and w.e. muir («/). grain storage: part a system. the avi publishing co. yoshida, s., d.a. forno. j.h. cook and k.a. gomez. 1976. laboratory manual for physiological studies of rice. irri, los banos, philippines. 62 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf biotropia vol. 29 no. 2, 2022: 134 141 doi: 10.11598/btb.2022.29.2.1664 134 macrozoobenthic community structures in seaweed culture ponds in muara gembong estuary, bekasi, west java province, indonesia raden indarjani* and siti nurhayati 1department of biology, faculty of science and technology, universitas islam as-syafiiyah, jakarta 17411, indonesia received 13 september 2021/accepted 24 march 2022 abstract the study of macrozoobenthic in seaweed culture in muara gembong estuary, bekasi district, west java province, was conducted in may to july 2018. the study was aimed at understanding the roles of macrozoobenthic organisms as ecosystem engineering in seaweed habitat by identifying macrozoobenthic community structures using various biological indices. sampling sites were conducted at three selected intertidal ponds used for seaweed culture at different distances and perpendicular to the coastline. samples of macrozoobenthic organisms were collected using an ekman grab during low tide periods. the study results showed that the macrozoobenthic community from the three ponds were consisted of 9 major benthic families and 14 genera with a total of 139 individuals. the results also showed that gastropod of the genus cerithiidae was the dominant taxa found in every pond which contributed to 42.45% of the total macrozoobenthic found in the three ponds and became the main contributing taxa to the macrozoobenthic community structure. in addition, genus platynereis of the polychaeta class was found to be another important taxon which contributed to 14.39% of the total macrozoobenthic found in the three ponds. the genus platynereis were mostly found in the second pond with muddy coarse sandy sediment substrate containing more silt compared to the other two ponds. the rare taxon was the genus lithophaga from family mytilidae represented by 1 individual. our study concluded that the macrozoobenthic community structure in the three ponds was categorized as poorly diverse indicating that the pond system was unstable. the shannon-wiener diversity index (h’) was only 0.87 on average with the highest diversity index (h’=1.47) was found in the third pond located at the farthest area of the coastline. meanwhile, the average of evenness index was 0.34 indicating that the distribution of the taxa was uneven with a tendency of being dominated by certain taxa. keywords: biological indices, community structure, macrozoobenthic, seaweed aquaculture introduction indonesia is the among the largest global producers of seaweed and supplies around twothird of the global demand (deswati & luhur 2014). gracilaria sp. and eucheuma cottonii are the two popular species of seaweed out of the 50 cultured seaweeds species in indonesia (deswati & luhur 2014). west java provincial authorities claimed that seaweed is one of the primadonna of the aquaculture export commodities with bekasi district contributing 10,000 tonnes of dried seaweed, in which 7,000 tonnes was produced from the muara gembong seaweed culture area (mujiyanto et al. 2014). seaweed is often cultivated in polyculture system with milkfish (chanos chanos) and/or tiger shrimp (penaeus monodon). these practices cause multiplying effects to the environment, economy growth and food security, if this practice is done properly. a case study in the gulf of castelamarre, italy reported valuable polyculture practices as a reliable economic tool through increasing fish production and sustainable environmental management (zenone & sara 2007). as an ecological engineering practice, seaweed polyculture can reduce the impact of wastes from fish cultivation through recycling of particulate and dissolved matters and enhancing the total productivity for both commodities (troell et al. 2013). *corresponding author, email: indarjani61@gmail.com mailto:indarjani61@gmail.com macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 135 the growth of gracilaria sp. mostly depends on the availability of nutrients as this species need nutrient uptake of around 0.0082 -0.0149 ppm/day which in turn can eliminate the excessive fish food (komarawidjaja & kurniawan 2008). thus, water quality parameters such as the level of nutrient content, suspended organic matter as well as weedsand diseases-free environment are important requirements for culturing gracilaria sp. to attain maximum growth (reksono et al. 2012; mujiyanto et al. 2014). in aquatic ecosystem, nutrients availability was maintained by macrozoobenthic organisms. the macrozoobenthic community has a complex structure consisting of a wide range of invertebrate animals inhabiting the bottom of aquatic ecosystem. macrozoobenthic have critical roles in maintaining energy flow and mineralization cycles ensuring that the aquatic ecosystem is functioning at an optimum level (indarjani 2003). macrozoobenthic are mostly found as sessile animals, crawling, burrowing and digging the sediment, permanently or temporarily. macrozoobenthic’s live is associated with substrates. macrozoobenthic is favorable as bioindicator organisms due to its capacity to record the environmental changes. the distribution of macrozoobenthic is determined by physical, chemical and biological parameters which will influence the abundance and composition of the macrozoobenthic as well as the producers, herbivores and predators (snelgrove 1998; indarjani 2003). seaweed and macrozoobenthic perform a mutualistic relationship referring to nutrientcycling and food web, even though the magnitude and mechanism of this relationship is still limited. thomsen et al. (2013) reported that the increase biomass of seaweed gracilaria vermiculophylla is associated with the abundance of benthic invertebrates because the root system of the seaweed facilitated a creation of a 3d mosaic structures and interstitial spaces that provide spaces for different sizes of invertebrates to live and grow. in addition, cummin et al. (2004) suggested that seaweed enteromorpha intestinalis influences the structure of macrobenthos communities compared with unvegetated plot in an australian estuary. the thickness of algal and oxygen concentration at the bottom layer explained the best structure of macrozoobenthic community and enhanced the species richness (lauringson & kotta 2006). muara gembong is located at the north of bekasi district adjacent to jakarta bay. in 2008, the gracilaria sp. polyculture system was introduced and became one of the potential local commodities for the livelihood of the local community and strengthening the food security in that area. the muara gembong estuary is around 13,205,702 ha and has been developed intensively causing impact that may influence the macrozoobenthic communities (pirzan & pong-masak 2008). looking at the potential of increasing the productivity of seaweed culture, this study aimed to gain understanding on: 1) the roles of macrozoobenthic organisms as ecosystem engineering in the habitat of gracilaria sp. by identifying macrozoobenthic community structure using several biological indices and 2) the effect of physicochemical variables on the types of macrozoobenthic community. materials and methods study site and time the study was conducted from may to july 2018, in an urban estuarine called muara gembong, that located under the administration of muara gembong subdistrict which lies at latitude 6º10’53”-6º30’6” s and 106º48’28”107º27’29” e. sampling site determination the location of sampling site was pantai sederhana village, one of the six villages under the administration of muara gembong subdistrict (fig. 1). in this location, seaweed culture was carried out in intertidal ponds with varying substrates of the pond bottom. the ponds have separate inlet and discharge sluice gates and water canals surrounded by various densities of mangrove trees, i.e., avicennia sp., sonneratia sp., rhizophora spp. and bruguiera sp. biotropia vol. 29 no. 2, 2022 136 figure 1 sampling sites located in pantai sederhana village, muara gembong estuary, bekasi district, west java province, indonesia the first pond was located at 100 m from the coastline and has an area of approximately 1.3 ha surrounded by low density of mangrove tress. at this location, the gracilaria seaweed seemed to be well maintained. visually, the substrate type of the pond bottom was fine sandy sediment. the second pond was at 250 m from the coastline with an area of 3.7 ha. the gracilaria sp. seaweed seemed to be poorly grown. the substrate type of the pond bottom was muddy coarse sandy sediment. mangrove trees with low density were planted around the second pond. the third pond of around 4.0 ha was located at 500 m from the coastline and was close to the fishermen’s residential area. the gracilaria sp. seaweed in the third pond seemed to be better maintained when compared with the seaweed in the second pond. due to the closeness of the third pond’s location with the fishermen’s residential area, household wastes may overflow to the third pond during high tide. the third pond may also receive less seawater compared to the other two previous ponds because of its distance from the coastline. the substrate of the third pond bottom was fine sandy sediment. a high density of mangrove tress was found around the third pond. sample collections samples of macrozoobenthic organisms were collected from each seaweed culture pond during low tide. five replicates of macrozoobenthic samples were taken at each pond (a total of 15 samples) by using the ekman grab sampler having a diameter of 152 x 152 x 152 mm, which was suitable for fine sediment (putro 2011). the samples were then stored in airtight plastic bags and preserved in 10% buffered formalin before being transported to the laboratory. in the laboratory, the samples west java administrative areas city regency capital west java map (source: www.google.com) map of muara gembong, bekasi (source: www.google.com) pantai sederhana village (source: www.google.com) macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 137 were washed through a 0.5 mm mesh sieve. the subsequent identification of macrozoobenthic organisms was carried out up to the genus level, referring to indarjani (2003) and bremmer (2005). after the identification process, the macrozoobenthic samples were preserved in 70% alcohol. water quality parameters such as ph, dissolved oxygen, water temperature, water clarity and salinity were recorded in situ. ph was measured using ph meter, salinity using refractometer, water clarity using secchi disk, dissolved oxygen using do meter. the water sample collection were also done in five replications. data analysis data analysis was conducted using descriptive statistics to describe and summarize the data. the data analysis may show certain patterns of macrozoobenthic distribution. to examine ecological function of macrozoobenthic community, several biological indices were calculated, as suggested by pakpahan et al. (2013) such as: 1) shannonwiener diversity index (h’) to calculate the taxa diversity (odum 1971); 2) evenness index (e) to examine the distribution of taxa (elliott 1971); 3) simpson dominance index (d) to observe the dominance of taxa related to the macrozoobenthic community (odum 1971); and 4) abundance relative index (r) to examine the population degree of certain taxa that support to the total abundance of macrozoobenthic community. the total abundance (n) was then standardized by dividing the total of biota of each pond to densities per m2 resulted in a value in number of abundances of each taxon per m2 (cox 1995). results and discussion water quallity parameters one of the most important factors in marine culture fisheries is the quality of seawater which act as growth medium for benthic organisms, both flora and fauna. results of water quality paramaters measurement are presented in table 1. the results showed that water quality among the culture ponds was quite varied. data of temperature, salinity and ph were considered to be suitable for culturing graciliaria sp., even though water clarity and dissolved oxygen was found not suitable (mujiyanto et al. 2014). data of water clarity in all ponds were lower than the suitable range. the low water transparency may have been caused by the period of low tide when the measurement was carried out. during the measurement, the secchi disk may have been covered by seaweed plants, while the pond water may have been murky due to high suspended organic matter from the fish food wastes, fertilizers, floating debris from the surrounding mangrove trees and household wastes. the lower dissolved oxygen value could be explained as a result the shallowness of the pond, high decomposition process, disturbed water, residue of organisms’ respiration during the night, and relatively high temperature during the day when water sampling was conducted. data of abiotic water quality parameters showed that temperature was around 31.6-31.8 oc, which nearly reached the highest temperature for suitable range of seaweed culture. the depth of each culture pond was around one meter due to dry season when the sample collection was conducted. in addition, mujiyanto et al. (2014) stated that the muara gembong estuary was classified as having shallow waters. however, in regard to the existence of macrozoobenthic communities, the water quality parameters were within the acceptable range (marpaung et al. 2014; syamsurisal 2011). macrozoobenthic compositions our study found 4 phyla, 5 classes, 6 ordos, 9 families and 14 genera of macrozoobenthic with a total number of 139 individuals in muara gembong seaweed culture ponds (table 2). previous research conducted by wijaya and indriatmoko (2019) reported that 44 genera of benthic organisms was identified from the muara gembong waters and dominated by the class polychaeta that made up 52.50% of macrozoobenthic community. the high yield of macrozoobenthic organisms obtained presumably due to large area of sampling sites, while in this study the macrozoobenthic organisms were collected from three selected seaweed culture ponds. in addition, the abundance of macrozoobenthic organisms and taxa richness were relatively different among the three ponds which could influence the macrozoobenthic community structures in this area. biotropia vol. 29 no. 2, 2022 138 table 1 average values of water quality parameters at each seaweed culture pond no abiotic water quality parameters first pond second pond third pond suitability range of water quality parameters for seaweed culture 1 temperature (oc) 31.8 31.6 31.6 29-32 2 secchi disk transparency (cm) 16.3 17.7 16.7 75-153 3 salinity (‰) 20.8 20.6 19.4 16-25 4 ph 7.6 7.5 7.8 7.5-8.5 5 do (mg/l) 3.20 3.20 3.25 6.5-8.5 table 2 composition of macrozoobenthic assemblages collected during the study no genus first pond (individuals) second pond (individuals) third pond (individuals) total (individuals) 1 telescopium 2 0 0 2 2 cerithiidae 43 2 14 59 3 cerithideopsilla 0 1 1 2 4 cerithium 0 1 5 6 5 tarebia 0 0 9 9 6 sermyla 0 1 5 6 7 terebralia 1 1 4 6 8 melanoides 0 0 4 4 9 hydrobia 0 0 5 5 10 lithophaga 0 1 0 1 11 platynereis 5 9 6 20 12 notomastus 0 0 2 2 13 sipuncula 0 0 1 1 14 litopenaeus 1 3 12 16 total (individuals) 52 19 68 139 results of our study indicated that the existing macrozoobenthic communities in seaweed culture ponds were mostly dominated by marine gastropods cerithiidae (59 individuals or 42.45%) which was extremely abundant in the first pond. the second dominant taxon was platynereis of the class polychaeta (20 individuals or 14% of the total macrozoobenthic found in this study) which was abundant in the second pond. in addition, marine bivalve of lithophaga and unsegmented marine worm sipuncula could be reckoned as rare taxa. only one lithophaga was found in the second pond and only one sipuncula was found in the third pond. the second pond inhabited by 19 individuals of 8 taxa or 14% of the total macrozoobenthic organisms seemed to be an unfavorable habitat for macrozoobenthic compared with the other two ponds. conversely, the platynereis was found quite many in the second pond where the seaweed culture was poorly maintained. in contrast, the third pond inhabited by 68 individuals of 12 taxa or 49% of the total macrozoobenthic found in this study was considered as a favorable habitat for macrozoobenthic where the seaweed culture was well maintained and surrounded by dense mangrove trees. meanwhile, the first pond which was situated adjacent to the coastline had the lowest taxa richness (52 individuals of 5 taxa) with gastropods cerithiidae as the most abundant macrozoobenthic organisms (82%) in the first pond. different compositions of macrozoobenthic communities may have been caused by the types of substrates and the microenvironment factors where the macrozoobenthic organisms live. based on visual (qualitative) observations, the first pond has the fine sandy sediment substrate which is relatively similar to the type of substrate in the third pond. the second pond has the muddy coarse sandy sediment substrate. wiyaningtiyah et al. (2014) stated that fine sandy sediment has a better capability in accumulating organic materials compared with coarse sandy sediment. the different types of substrates influence abundance and types of macrozoobenthic community structures (hadiyanto 2018). community structure of macrozoobenthic assemblages the macrozoobenthic community structure was assessed using various biological indices, i.e., the shannon-wiener diversity index, the evenness index and the dominancy index. the calculated indices indicated that the macrozoobenthic community structure was categorized as poorly diverse which suggested that the community system was unstable (fig. 2). macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 139 figure 2 macrozoobenthic community structure showing irregular trends the average of shannon-wiener diversity index (h’) was 0.87 with the highest index of 1.47 was found in the third pond which was the farthest pond from the coastline. snelgrove (1998) predicted that species diversity is mainly controlled by the fluctuations of environmental factors and tends to be low in physically controlled environment, such as in seaweed culture pond. meanwhile, the evenness index value ranged from 0.25 to 0.55 with an average of 0.34. the low value of evenness index indicated the uneven distribution of the taxa in which the community tends to be dominated by certain taxa. in our study, the cerithiidae dominated the macrozoobenthic community structure in the first pond, located adjacently to the coastline. the existence of cerithiidae contributed significantly to the macrozoobenthic community structure in the muara gembong seaweed culture ponds. this finding is in agreement with the work of ludwig and reynolds (1988) which indicated that the evenness index should be maximum when all species in the samples were equally abundant. the evenness index can drop to zero when the relative density of species diverges away from the evenness. referring to the values of the calculated biological indices, the third pond was considered as the favorable habitat for macrozoobenthic organisms, having the highest score of the shannon-wiener diversity index (h’ = 1.46) and the evenness index (e = 0.55). the low dominancy index (c = 0.31) indicated that macrozoobenthic organisms had relatively similar opportunity to grow and the population was distributed accordingly. the third pond occupied by seaweeds and surrounded by dense mangrove trees with fine sandy sediment substrate may have been contributed to the richness of macrozoobenthic taxa. seaweed root system might facilitate a creation of a 3d mosaic structure and interstitial spaces of substrate so that different sizes of invertebrates organisms could live and grow (thomsen et al. 2013). the curve linearity of the macrozoobenthic species living in a densely algal environment has higher increasing slope compared with macrozoobenthic assemblages in a poorly vegetated area (lauringson & jonne 2006). in addition, mangrove debris around the pond might become potential food resources for the macrozoobenthic functioning as wastes decomposer (palanisamy & khan 2013). dominant taxa examinations the first pond in this study was dominated by gastropods cerithiidae and had density of 8 individuals per meter square with relative abundance of 82%. the gastropods cerithiidae is considered as the characterized macrozoobenthic assemblages of seaweed culture in muara gembong. previous research reported by ardli et al. (2015) and kamaluddin (2017) showed that the distribution and density of cerithiidae were influenced by types of substrates and other various supporting factors such as dietary factors, water conditions, predators and competition. those factors also influenced the abundance of cerithiidae in our study which were varied among the three ponds. thus, in this case, the cerithiidae can be reckoned as a cosmopolitan taxon and has a good adapting capability to live and grow in the fine biotropia vol. 29 no. 2, 2022 140 and muddy sandy sediment substrates. the fine sandy sediment may be favorable for the digging and burrowing macrozoobenthic organisms, like gastropods cerethiidae (schirjvers et al. 1998). besides, the fine sandy sediment substrate is effective in trapping organic materials that stimulate the optimum growth of macrozoobenthic organisms. another important taxon in our study that contributed to the macrozoobenthic community structure was platynereis of the polychaeta class. this organism was found in every pond with the highest relative abundance of 47.37% and became the main contributing taxa to the macrozoobenthic community structure in the second pond. the type of substrate in the second pond may have been more suitable for platynereis than for other taxa. taqwa et al. (2014) confirmed that class polychaeta can easily be found in muddy as well as fine sandy substrates, which is relatively similar with the second pond’s substrate type. according to cambi et al. (2000) platynereis dumerii as mezograzer species prefers the brown seaweed sphacelaria. our study showed that the density of platynereis in every pond were not much different indicating that the environment of the three ponds was suitable for platynereis to grow. conclusion the study of macrozoobenthic community inhabiting seaweed culture ponds in muara gembong estuary showed that the macrozoobenthic community structure was characterized with low diversity, low evenness and high dominance indices, which indicated that the environmental condition was unstable and the macrozoobenthic community structure was dominated by certain taxa. marine gastropod cerithiidae seemed to be the main contributing taxa to the macrozoobenthic community structure as this organism was found in every pond. the second abundant macrozoobenthic organisms was platynereis. this study also showed that the third pond located at the farthest distance from the coastline seemed to be the favorable habitat for macrozoobenthic organisms. seaweed in this pond was grown luxuriant and surrounded by dense mangrove trees, with a higher diversity index, high taxa richness, and high evenness index, indicating that organisms in this pond have similar opportunities to grow. the first pond located adjacent to the coastline was considered favorable for gastropode genus cerithiidae, even though this pond had the lowest taxa richness, lower evenness index and high dominance index values. the second pond can be seen as the “transition” pond between the first and the third ponds considering its biological indices values were in between. references ardli er, widyastuti a, yani e. 2015. study of bioecological changes in mangrove ecosystem restoration in segara anakan. cilacap, central java province [kajian perubahan bioekologi pada restorasi ekosistem mangrove di segara anakan, cilacap]. biosfera 32(1): 19. bremmer j. 2005. assessing ecological functioning in marine benthic communities. 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[connectivity of macrozoobenthos community structure along gradient mangrove, seagrass and reef crest habitats in pulau kelapa dua, kepulauan seribu, jakarta]. bonoworo wetlands 4(1): 37-48 doi:10 1 3057/bonorowo/w040103 zenone a, sara g. 2007. polyculture as a tool to increase the economic income: a study case in the gulf of castellamare. j animal sci 6 (suppl 1):837-48. https://doi.org/10.1007/s10750-005-1009-4 https://doi.org/10.1007/s10750-005-1009-4 http://dx.doi.org/10.1186/2046-9063-9-15 https://ejournal.undip.ac.id/index.php/sm/article/view/7912 https://ejournal.undip.ac.id/index.php/sm/article/view/7912 https://doi.org/10.14710/marj.v3i1.4429 https://doi.org/10.14710/marj.v3i1.4429 biotropia vol. 29 no. 2, 2022 142 biotropia no. 6, 1992/1993: 55-65 shading effects on growth and partitioning of plant biomass in paspalum conjugatum berg. i.b. ipor universiti pertanian malaysia, kampus bintulu, p.o. box 396, 97008, bintulu, sarawak, malaysia and c.e. price imperial college, silwood park, ascot, berkshire sls 7py, england abstract glasshouse studies were carried out to determine the effect of shading on the growth and partitioning of plant biomass in paspalum conjugatum berg. the invidual leaf rate expansion, final leaf are, specific leaf area, and the whole plant vegetative growth pattern, dry-matter production, leaf area as well as biomass partitioning were significantly influenced by shading. at the 75% level of shading, p. conjugatum produced the highest values of leaf weight ratio, specific leaf area and leaf area ratio. individual leaf assessment revealed that shading significantly increased the final leaf area, duration of leaf expansion and specific leaf area. introduction weeds are often considered as very competitive plants. weeds competing for light, water and nutrient have adverse effects on crop growth and yield. the availability of these resources influences the physiological, morphological and competitive responses of both weeds and crops. the effects of light on weed growth vary between species. generally, these effects can be distinguished on the basis of their physiological characteristics. among the common response of plants upon shading in general, are the alteration of leaf size (bjorkman and holmgren 1966), internode extension, number of branching (vince-pruce 1977) and stem elongation rate (morgan and smith 1981). paspalum conjugatum berg, is an important, stoloniferous creeping perennial weed in malaysia and in many regions of southeast asia. it grows well in the moist, fertile soils of agricultural fields, roadsides and wastelands. p. conjugatum grows profusely in most estates such as rubber, oil palm, cocoa and coconut (holm et al. 1977). it can tolerate, survive and grow under shade condition (beetle 1974; ward and woolhouse 1986). p. conjugatum possesses several important characteristics 55 biotropia no. 6, 1992/1993 that enable it to interfere with crop growth: rapid growth with extensive stolon formation, early and profuse tillering, heavy seed production and readily rooting at most nodes whenever in contact with the soil surface. however, little information has been reported on the morphological and physiological response of p. conjugatum when exposed to different environmental conditions. the present study was conducted to determine the effects of shade on the growth of p. conjugatum in different shade levels. materials and methods plant materials and growing conditions uniformed size seedlings of two leaf stage were transplanted into 10-cm diameter pots filled with john innes no. 1 compost mixture. watering was carried out daily and 15 ml of nutrient solution ( 7 : 7 : 7 npk foliar fertilizer) was given twice a month. the temperature of the glasshouse was maintained at 30±40°c during the day and 12±3°c at night. the plants were exposed to sunlight and supplemented with 12 hours of fluorescent white light each day. these plants were grown under three shade levels: 0% (control), 50% and 75%. single and double layers of green nettings (netlon, uk) were used to cover the cages to obtain 50% and 75% shade, respectively. the light intensities of the shading regimes were regularly checked with skye light meter (skye instrument limited, uk). harvesting and growth analysis ten plants from each light regimes were used to determine weekly increments in plant height or length, leaf number, internode length, stolon and node numbers. the other group of plants were harvested at 35 and 42 days after transplanting to determine the leaf area and total dry weight (65°c, oven-dried). in addition, dry weights of plant parts were also determined on the 42nd-day of harvest. dry-matter production and biomass allocation patterns of the three shade levels were compared using mathematical growth analysis techniques (kvet et al. 1971; patterson et al. 1979). data from 42nd-day harvest were used to calculate leaf weight ratio, shoot weight ratio, specific leaf area and leaf area ratio. calculation of dry-matter production, net assimilation rate and leaf area duration were based on 35th-day and 42nd-day harvests. all experiments were repeated twice and data were subjected to analysis of variance. duncan's multiple range or least significant difference (lsd) test was used to determine significant differences among the mean values for each treatment. 56 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price growth of individual leaf the leaf shape was traced on a white paper and cut out with scissors. the area traced was taken as the leaf area and determined with an optomax tracer. leaf measurements were made on alternate leaves, starting from the second leaf till the sixteenth leaf. measurements were made at the time of leaf appearance, i.e. when the leaf was sufficiently distinct from the apical bud so that it could be measured without damaging the bud. the measurement was continued until the area remained constant after three successive readings. an example of the measurements on one plant is given in figure 1. the individual leaf was finally harvested and oven dried at 65°c for determination of dry weight. two sets of experiments were arranged in completely randomized blocks with ten replicates. final leaf size, duration of leaf expansion, weighed mean growth rate and specific leaf area of each individual leaf were calculated and subjected to variance analysis. means were compared using the lsd test. figure 1. example of measurement of leaf area of individual leaves of p. conjugatum. each number indicates leaf position. 57 12 16 20 24 28 32 36 40 44 days after transplanting 48 52 56 60 biotropia no. 6, 1992/1993 results and discussion the number of leaves recorded for p. conjugatum was significantly influenced by light intensity (figure 2). thus, plants at 0% shade had significantly more leaves than those provided with 50% and 75% shade. however, height of plants at 75% shade was significantly greater than those at 0% and 50% shade, 35 days after transplanting (figure 3). the shortest plant height were all recorded at 0% shade. reducing light intensity to 75% had significantly decreased the number of stolons (figure 4). plants at 0% shade produced more stolons as compared with those at 50% shade, 35 days after transplanting. bjorkman (1968) and ishimine etal. (1985) noted that reduced light levels to a certain degree would result in increased stem extension of solidago virgaurea l. and height of vaseygrass (paspalum urvillei steud.), respectively. however, further reduction of light suppressed the plant growth. this pattern of growth was also reported by soerjani (1970) and moosavi and dore (1979) in their work on imperata cylindrica. decreasing the light intensity tends to increase the total dry weight of p. conjugatum (table 1). plants grown under 0% shade produced the highest total dry weight, followed by 50% and 75% figure 2. effect of shading on leaf number of p. conjugatum. 58 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price figure 3. effect of shading on the height of p. conjugatum. figure 4. effect of shading on stolon number of p. conjugatum. 59 biotropia no. 6, 1992/1993 table 1. effect of shading on vegetative growth, leaf area production and biomass allocation in p. conjugatum (42nd day harvest). shade levels. total dry matter of three populations of cogon grass [imperata cylindrica (l.) beauv.] decreased three fold at 56% of full sunlight and 20 fold at 11% of full sunlight (patterson 1980b). imperata cylindrica that grew with 11% of full sunlight had a leaf area ratio 2.5 times greater than those grown in full sunlight. the leaf area of the p. conjugatum increased correspondingly with increase in shade levels (table 1). the highest leaf area was 433 cm 2 from 50% shade, followed by 344 cm 2 from 75% and 274 cm 2 from 0% shade. p. conjugatum is a c4 grass native to shaded habitats or shade tolerant species (ward and woolhouse 1986) which normally produced linear thin leaves. the highest value of leaf areas of plants from 50% shade was possibly due to a sufficient number of large leaves as compared with those from 75% shade. leaves at 0% shade were reduced in size although the number exceeded from those in the other light regimes. the reduction in total leaf area with 75% shade may have resulted from a reduction in the leaves. partitioning of plant biomass into roots and leaves differed significantly among the plants under the three shade levels. at 0% and 50% shade levels, the plants partitioned more biomass into roots and stems than the other two light regimes (table 1). rwr (root weight ratio) and swr (shoot weight ratio) were highest at 0% and 50% shade. plants at 75% shade, partitioned most biomass as leaf area was also greatest in 75% shade, as indicated by their high specific leaf area and leaf area ratio values. the values differed significantly among the three shade levels. plants at 75% shade had the highest leaf area ratio followed by those at 50% shade. leaves produced under shade conditions were generally thinner than those produced in unshaded treatment (blackman and black 1959). the concomitant increases in specific leaf area and leaf weight ratio resulted in substantial increases in the leaf area ratio which means that the amount of leaf area per unit of plant 60 shading effects on growth and partitioning of plant biomass .i.e. ipor & c.e. price weight was increased by shading. morgan and smith (1978) reported that the lar increased by 38%, after 32 days in the shade treatment (15% sunlight) of cyperus rotundus grown for 30 days compared with full sunlight conditions. leaf area ratio and specific leaf area of veronica chamaedrys, v. montana and v. offlcinalis were also increased at low radiance (dale and causton 1992). increase in leaf area ratio with shading was an adaptation to low light at the whole-plant level because it reflected a greater allocation of plant biomass to photosynthetic tissue and a greater distribution of this tissue as light-intercepting structure in the form of the leaf area (patterson 1980b). analysis of the components of dry-matter production, net assimilation rate and leaf area duration at the three shade levels indicated that dry-matter production and net assimilation ratio showed the highest value at 0% and 50% shade for leaf area duration (table 2). leaf area duration significantly differed among the three shade levels. table 2. effect of shading on dry-matter production, net assimilation rates, leaf area duration of p. conjugatum during the 35th-to-42nd-day interval. shade dmp nar lad level (g) (mg.cm 2 .day) (cm 2 . day) 0% 3.74a 0.70a 5443c 50% 2.88ab 0.32b 8546a 75% 1.42b 0.21b 6701 b within each column, values sharing the same letter are not significantly different at 5% level, according to duncan's multiple range test. in general, decreasing the light intensity tends to increase the final leaf areas (figure 5). the largest size of individual leaves was always found at 75% shade. newton (1963) observed a similar trend as reduction in light significantly reduced the final areas of individual leaves of cucumber. the final areas of individual leaves in all treatments showed a characteristic variation with leaf position with an increase in size up to about leaf six. final leaf sizes of different leaves across shading treatment were significantly different except for leaf 12 and 14 under 0% and 50% shade. leaf areas were maximum at leaf 6 of plants at 75% and 50% shade while this was at least 4 of plants at 0% shade. dennet et al. (1979) observed that the final leaf area increased with leaf position (numbered from the base) up to the 8th leaf of faba beans. a similar general pattern of variation in final leaf area with leaf position had been reported for sunflower (yegappan et al. 1982), maize (thiagarajah and hunt 1982) and sugar beet (milford et al. 1985). 61 biotropia no. 6, 1992/1993 figure 5. effect of shading on final leaf area of p. conjugatum. the duration of expansion increased significantly with increasing shade and decreased with increasing leaf number (figure 6). leaves at both 50% and 75% shade took a significantly longer period to reach their maximum size. initially, the duration of expansion was increased with increasing leaf number but decreased after leaf 6 for 50% and 75% shade. the results revealed that leaves from higher shading particularly at the earlier growth stage needed longer time to reach their maturity. much of the variations in final leaf area with leaf position or shade level were associated with changes in the weighed mean growth rate rather than with changes in the duration of expansion. weighed mean growth tended to increase with decrease in light intensity (figure 7). at 75% shade, the weighed mean growth rate was significantly higher than at 0% and 50% shade. weighed mean growth rate reached a maximum of 1.3 cmvday for leaf 6 at 75% shade. the specific leaf area was significantly increased by increase in shade (figure 8). for example, specific leaf area of leaves under 75% shade differed greatly compared with other shade treatments. maximum specific leaf areas were 299, 318 and 334 for 0%, 50% and 75% shade, respectively. it was also observed that the 62 shading effects on growth and panitioning of plant biomass i.e. ipor & c.e. price figure 6. effect of shading on the duration of expansion of p. conjugatum leaves figure 7. effect of shading on weighed mean growth rate of p. conjugatum leaves 63 biotropia no. 6, 1992/1993 figure 8. effect of shading on specific leaf area p. conjugatum increase in a specific area depended on the leaf position that tended to decrease as the leaf number increased. plants grown in shade typically have lower maximum photosynthetic rates than plants grown in full light condition. patterson (1980a) showed that shaded plants normally have lower rates of dark respiration which result in conservation of the photosynthates produced. as a result of the lower respiration rates, shade-grown plants typically have a lower light compensation point, the light level at which respiratory carbon dioxide loss equals photosynthetic carbon dioxide uptake and no net carbon dioxide exchange occurs. references bjorkman, o. 1968. further studies on differentiation of photosynthetic properties in sun and shade ecotypes of solidago virgaures l. physiol. plant, 21: 84-89. bjorkman, o., and p. holmgren. 1966. adaption to light intensity in plants native to shaded and exposed habitats. physiol. plant, 19: 854-914. 64 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price blackman, g.e. and j.n. black. 1959. physiological and ecological studies in the analysis of plant environment. xi. a further assessment of the influence of shading on the growth of different species in the vegetative phase. ann. bot. 23: 51-63. dale, m.p., and d.r. causton, 1992. the ecophysiology of veronica chamaedrys, v. montana and v. officinalis. ii. the interaction of irridiance and water regime. journal of ecology 8 0(3): 493 504. dennet, m.d., j., elston and j.r. milford. 1979. the effects of temperature on the growth of individual leaves of vicia faba l. in the field. annals of botany, 43: 197-208. holm, l.g., d.l. plucknett, j.v. pancho, and j.p. herberoer, 1977. the world's worst weed. distribution and biology., university press of hawaii, p. 609. ishimine, y.k., k. miyazato, and s. matsumoto. 1985. physiological and ecological characteristics of weeds of sugarcane fields in the ryukus islands. 3. effects of shading on growth and seed production of paspalum urvellei steud. weed res. (japan) 30: 148-150. kvet, j., j.p. ondok, j. necas and p.o. jarvis. 1971. method of growth analysis in plant photo-synthetic production : manual of methods, eds. z. sestak, j. catsky and p.o. jarvis, w. junk. n/v publ. the hague: 343-391. milford, g.f.j., t.o. pocock, j. riley and a.b. messem, 1985. an analysis of leaf growth in sugarbeet. leaf expansion in field crops. annals of applied biology 106: 187-203. moosavi-nia, h. and j. dore. 1979. factors affecting glyphosate activity in imperata cylindrica l. beauv. and cyperus rotundus l. ii. effect of shade. weed res. 19: 321-327. morgan, d.c. and h. smith. 1978. linear relationship between phytochrome photoequilibrium and development in light grown chenopodium album l. planta 142: 187-193. morgan, d.c. and h. smith. 1981. control of light. the effect of light quantity (total influence rate) and light quality (red : far-red ratio) new phytologist 88: 239-248. newton, p. 1963. studies on the expansion of the leaf surface. ii. the influence of light intensity and photoperiod. j. exp. bot. 14: 458-482. patterson, d.t. 1980a. light and temperature adaption p. 205-235. in predicting photosynthesis for ecosystem model eds. j.d. hesketh and j.w. jones, vol. 1 crc press, inc. boca raton, f.l. patterson, d.t. 1980b. shading effects on growth and partitioning of plant biomass on cogon grass (imperata cylindrica) from shaded and exposed habitat. weed sci. 28: 735-740. patterson, d.t., c.r. meyer, e.p. flint and p.c. quimby, 1979. temperature response and potential distribution of itchgrass (rottboellia exaltata) in the united states. weed sci. 27: 77 82. soerjani, m. 1970. alang-alang, imperata cylindrica (l.) beauv. (1812). patterns of growth as related to its problem of control. biotrop bull. no. 1. thiagarajah, m.r. and l.a. hunt, 1982. effect of temperature on leaf growth in corn (zea mays). canadian j. of botany 60: 1647-1652. vince-pruce, d. 1977. photo control of stem elongation in light growth plants of fuchsia hybrids. planta 133: 149-56. ward, d.a. and h.w. woolhouse, 1986. comparative effects of light during growth on the photosyntetic properties of nadp-me type c4 grasses from open and shaded habitats. gas exchange, leaf, anatomy and ultra structure. plant, cell and environment 9: 261 -270. yegappan, t.m., d.m., patton, c.t. gates and w.j. muller, 1982. water stress in sunflower (helianthus annuus l.) ii. effect of leaf cells and leaf area. annals of botany 49: 63 -68. 65 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf 65.pdf microsoft word 62 biotropia no. 24, 2005 : 62 68 molecular phylogenetic analysis of monascus fungi based on internal transcribed spacer region n. suharna1, y. kikuchi2'3 and t. fukatsu3 'research center for biology, indonesian institute of sciences, bogor, indonesia 2natural history laboratory, faculty of science, ibaraki university, mito 310-8512, japan 3national institute of biological functions and resources, advanced industrial science and technology (aist), tsukuba 305-8566, japan abstract a molecular analysis of internal transcribed spacer region has been carried out to reveal the relationship among 16 strains of monascus spp. a primer set comprised primer its1 and its4 was used to amplify this region in which they were cloned and scqucnccd. we also compared the sequence result with m. purpureus af458473, m.ruber af458470, m. kaoliang af451859, m. araneous af458471 and m. pilosus af451856 and one outgroup species thermoascus crustaceus u18353. the result showed that 16 monascus spp. were divided into two large clades while m. ruber af458470 was basically separated from all those monascus. one of the two large clades included the seven m. purpureus strains, m. purpureus af458473, m. araneosus af458471 and m. kaoliang af451859. another large cladc included the six monascus sp. strains which typically have whitish colonies, the three m. ruber strains and m.pilosus af451856. however, even outstanding morphological differences possessed by several white monascus and one whitish m. purpureus strain, all monascus strains were suggested to be very closely related with similarity >99% almost 100%. although this its analysis could not discriminate cultural and morphological differentiation of monascus strains studied, yet there is still little genetic variation within these strains. key words : molecular genetics/monascus spp./fungi introduction hawksworth and pitt (1983) cited that monascus species (monascaceae) are important for producing asian fermented foods particularly red rice (ang-kak), rice wine and kaoliang brandy, soy bean cheese and food colorants; for their antibacterial properties; production of mycotoxin; and a major component of silage mycofloras. lakrodi et al. (200) cited that one of these world wide distributed fungi, m. purpureus is known as the red rice fungus that has been used for over a thousand years by the chinese as a traditional herbal medicine. however, this fungus was firstly isolated from chinese red rice (ang-kak) in java. in indonesia, we isolated and collected m. purpureus from ang-kak in java and sumatra and from other several monascus species isolated from deteriorated invertebrate specimens. based on morphological observation on growth in agar media, we found some interesting not ordinary properties in m. purpureus such as two m. purpureus isolates one of which has unique character such as bigger ascomata and the other one has white colony. while the other four monascus 62 biotropia no. 24, 2005 isolates have very remarkable properties such as they resist ethanol at very extreme concentration and their morphological characters are also unique. so, it is of interest to know the genetic relationship rather than morphological relationship among those isolates within monascus species. actually, dna sequence analysis based on the d1/d2 regions of lsu rrna genes of monascus species have been conducted by park and jong (2003). they suggested that m. lunispora, m. floridanus, and m pallens were separated in different clades. however, five monascus species such as m pilosus, m. purpureus, m. ruber, m. eremophilus and m. sanguineus were reflected in monophyletic relationship (park and jong 2003). these five species were definitely different species based on morphological characters on agar media by hawksworth and pitt (1983) (m. pilosus, m. purpureus, m. ruber), hocking and pitt (1988) (m eremophilus) and cannon et al. (1995) (m sanguineus). as park and jong (2003) have suggested no separation among the five species of monascus, we intended to do analysis on internal transcribed spacer region of monascus. this region is known for its various nucleotides sequences so it might be more perspective rather than on lsu rrna genes. this similar work was not only to reveal genetic diversity of monascus in indonesia, but also to understand the relationship among monascus species and direction of mutation by analysis materials and methods monascus strains a number of 16 monascus strains used in this study were isolated and identified by suharna in 2002 and 2003 (table 1). molecular phylogenctic analysis of monascus fungi — n. suharna et al. cultivation and purification of monascus strains all fungi were cultivated on ym agar plate and for purification of cultures water agar 2% was used. incubation was carried out at room temperature (25°c) for three days. a little amount of mycelial mass of each fungus was picked up using toothpick then transferred into centrifuge tube containing 10 ml of ym broth medium and incubated at 30°c for three days. the mycelial mass of each fungus was then harvested for dna extraction. dna extraction the harvested mycelial mass of each fungus was put onto paper to remove excess of medium then subsequently put onto mortar before freezing by pouring liquid nitrogen and grounded by mortar and pestle. the following step of dna extraction was carried out using qiaamp tissue kit (qiagen). the yield of dna samples was quantified and qualified by both spectrophotometer and gel electrophoresis. pcr, cloning and sequencing of its region its region was amplified by pcr with specific primer set, its1 (5'tccgtaggtgaacctgcgg-3') and its4 (5'-tcctccgcttattgatatgc-3') (white et al. 1990). pcr was conducted using amplitaq dna polymerase (roche, basel, switzerland) and supplemented with buffer system under a temperature profile of 94 °c for 4 min. followed by 30 cycles of 94 °c for 30 sec, 55 °c for 1 min. and 72 °c for 1 min. the pcr products were directly cloned with ta-cloning vector pt7blue (takara) and escherichia coli dh5a competent cells (takara) using ampicillin and x-gal blue-white selection system. to check the length of the inserted dna fragment, white colonies expected to contain inserted plasmid were directly subjected to pcr using the primers univl9 (5'-gttttcccagtcacgacgt-3') and rev20(5'agctatgaccatgattacgc -3'). when a pcr product of expected size (600 bp) was obtained, two clones of which were cultured overnight in 3 ml of lb medium containing ampicillin, and subjected to plasmid extraction using qiaprep-spin miniprep kit (qiagen). the purified plasmids were eluted with 50 ul distilled-water and used for sequencing. dye terminator-labelled cycle sequencing reaction was conducted with dna sequencing kit fs (perkin elmer) and four sequencing primers univl9 and rev20 under a temperature profile of 94 °c for 4 min. followed by 30 cycles of 94 °c for 30 sec, 50 °c for 1 min. and 60 °c for 4 min. the products were analyzed by abi prism 377 dna sequencer (perkin elmer). 64 biotropia no. 24, 2005 molecular phylogenetic analysis the its region sequences determined were subjected to molecular phylogenetic analysis together with those retrieved from the ddbj nucleotide sequence database. a multiple alignment of the its region sequences was generated by the program package clustal w (thompson et al. 1994). phylogenetic trees were constructed by the neighbor-joining method using clustal w (thompson et al. 1994). bootstrap tests (felsenstein 1981) were performed with 1000 replications. results figure 1 shows that amplification by primer set comprised its1 and its4 were successful for all monascus strain tested. the size of amplified dna products of all monascus isolates by the primer set was 700 base pair each as shown in figure 1. molecular phytogeny figure 2 shows that thermoascus crustaceus u18353 used as outgroup was significantly separated from all monascus species that integrated in one main clade. this monascus clade consists of very similar two large clades, while m. ruber af458470 was basically separated from all those monascus. one of the two large 65 molecular phylogenetic analysis of monascus fungi n. suhama el al. clades includes the seven m. purpureus strains, m. purpureus af458473, a araneosus af458471 and m. kaoliang af451859. another large clade includes th six monascus sp. strains which typically have whitish colonies, the three m. rube strains and m.pilosus af451856. the similarities within the seven m. purpurei> strains and m. purpureus af458473, m. araneosus af458471 and m. kaolian af451859 are very high more than 99% or almost 100%. however, m. purpure^ pkb5 shows a little more different. the six monascus sp. strains, the three m. rube and m. pilosus af451856 also showed very high similarity. but lesser difference i shown by monascus sp. ka30.1. discussions with regard to the level of very high similarity more than 99% almost nearl; 100% among all monascus fungi we analyzed, indicated that there is little mutatioi on its region of monascus species. concerning genetic diversity it seemed tha there is a diverse mutation tendency as several monascus strains such a 66 biotropia no. 24, 2005 m.purpureus pkb1, m.purpureus prba, m.purpureus jms, m.purpureus af458473, m. rubber cka1 and monascus sp. ka30.1 appeared more different though these differences were so slight. therefore, this phylogenetic analysis revealed very close relationship among monascus strains so we suggested that all m.purpureus were identical to each other as shown by m.purpureus ngk-j, m.purpureus jmba, m.purpureus pkb1, m.purpureus pkb5, m.purpureus srba, m.purpureus af458473 and m. araneosus af458471 in the same clade. in another clade, all indonesian m. ruber strains and m. pilosus were identical to each other. however, m. ruber af458470 was separated in another clade. this work also confirmed that m. araneosus af458471 and m. kaoliang af451859 are the same species with m. purpureus. previously, hawkswoth and pitt (1983) have included m. araneosus and m. kaoliang as m. purpureus synonym based on morphological characteristics. it is also of interest to note that m. purpureus srba has a uniqueness of its morphological features such as having bigger ascomata (generally 90 um in diameter) than "normal" m. purpureus. the normal m. purpureus has ascomata up to 70 um in diameter. the other white isolates such as monascus sp. mm, monascus sp. coel, monascus sp. ktb, monascus sp. myom and monascus sp. myot have different morphological characters. these white isolates at least have two different morphological characters compared to m. ruber such as shorter ascospores, cleisthothecium wall transparent. moreover, monascus sp. myom and monascus sp. myot have fusiform aleurispores, while m. ruber has no this spore form(suharna 1999). besides d1/d2 regions of lsu rrna genes, phylogenetic analysis on its region was also made by park and jong (2003) to know the relationship among monascus strains accessed from genebank. the phylogenetic tree constructed showed similar result with our analysis on its sequence of monascus where m. ruber and m. pilosus were very closely related and all m. purpureus strains were included together in the same clade. though it is still suggested that the two clades were very closely related, interestingly, all indonesian m. ruber strains were in the same clade with m. pilosus af451856. while m. ruber with accession number af458470 retrieved from ddbj was separated in another clade. this indicated that three m. ruber isolates and six white monascus isolates showed little difference from m. ruber af458470, but identical to m. pilosus af451856 except for monascus sp. ka30.1 and m. ruber cka3 with genetically little difference. however, despite of this finding, it is still much surprising in regard with the outstanding morphological differences of several strains as mainly possessed by whitish strains such as monascus sp. ka30.1, monascus sp. myom, monascus sp. ktb, monascus sp. myot, monascus sp. mm, monascus sp. coel. park and jong (2003) also reported similar result as their comparison of two very distinct species based on morphological observation such as m. ruber and m. pilosus. therefore, we are in concordance with park and jo (2003) that it is needed to reveal in more details about molecular phytogeny to elucidate molecular taxonomy included in the identification of monascus species. 67 molecular phylogenctic analysis of monascus fungi — n. suharna et al. acknowledgments this research was funded by the general exchange program, japan societ the promotion of science and conducted in early 2004 at the national institu biological functions and resources, advanced industrial science and techno (aist), tsukuba 305-8566, japan. references cannon, p.p. , s.k. abdullah and b.a. abbas. 1995. two new species of monascus from iraq, \ key to known species of the genus. mycol. res. 99: 659-662 fclsenstcin, j. 1981. evolutionary trees from dna sequences : a maximum likchood approach. j. evol 17: 368-376 hawskworth, d.l. & j.i. pitt. 1983. a new taxonomy for monascus based on cultural and micros characters. aust. j. bot. 31: 51-61 lakrodi, k., c. chaisrisook, b. yongsmith and d.z. skinner. 2000. rapd analysis of genetic vari within a collection of monascus spp. isolated from red rice (ang-kak) and sofu. mycol. res (4): 403-408 park, hg., jong, s-c. 2003. molecular characterization of monascus strains based on the d1/d2 re of lsu rrna genes. mycoscicnce 44: 25-32 thompson, j.d., d.g. higgins, and j.j. gibson. 1994. clustal w: improving the sensitivity of progrc multiple alignment through sequence weighting, position-specific gap penalties and weight it choice. nucleic acids res. 22:4673-4680 white, t. j., t. bruns, s. lee, and j. w. taylor. 1990. amplification and direct sequencing of fi ribosomal rna genes for phylogcnctics. pp. 315-322 in: pcr protocols: a guide to method: applications, eds. innis, m. a., d. h. gelfand, j. j. sninsky, and t. j. white. academic press, new york. suharna, n. 1999. a study on the fungal occurrences in several animal specimens preserved in alcohc gakuryoku 5 (3): 139-144 68 62.pdf 63.pdf 64.pdf 65.pdf 66.pdf 67.pdf 68.pdf biotropia vol. 29 no. 3, 2022: 193 202 doi: 10.11598/btb.2022.29.3.1551 193 heavy metal bioaccumulation in ducks and possible risks to human health r. susanti* and karima widiyastuti department of biology, faculty of mathematics and natural sciences, universitas negeri semarang 50229, indonesia received 12 february 2021/accepted 27 june 2022 abstract meat is part of duck carcass mostly consumed by humans compared to other parts. this study aimed to analyze the heavy metal bioaccumulation factor (baf) of duck meat and its possible risks to human health. a total of 25 duck samples with their drinking water and feed were taken from five intensive duck farms in central java province, i.e., semarang (a), temanggung (b), magelang (c), pati (d) and salatiga (e). heavy metals concentration (as, cd, hg, and pb) were measured following the icp-oes method. heavy metals concentration data obtained were then compared with the quality standard. the baf value was calculated and the risks to human health were assessed. our study found that drinking water provided for ducks in all farms contained heavy metals (as, cd, hg, and pb) concentrations exceeding the quality standards. hg concentration of 0.089 5.01 ppm in duck feed exceeded the quality standard. concentrations of cd (0.0713 0.075 ppm) and hg (3.1 4.84 ppm) in duck meat exceeded the quality standard. the average of bafduck meat values was in the range of 0.443 0.955. the edi value of heavy metals (as, cd, hg, and pb) for adults and children through consumption of duck meat in the central java region was lower than rfd. this study showed that the health risk parameters (edi, hq, and hi) were within safe limits. exposure to heavy metals through duck meat consumption both in adults and children was unlikely to cause adverse health effects. keywords: bioaccumulation, duck, estimated daily intake, heavy metals, human health introduction indonesia is an agricultural country where most of the population work as farmers and breeders. ducks are among the leading livestock commodities in indonesia. the duck population in indonesia ranks third in the world after china and vietnam (ismoyowati & sumarmono 2019). in 2018, indonesia's duck population was recorded to reach 51,239,185 (department of livestock and animal health 2019). the duck population in central java province ranks fourth after east java province, with a population of more than 5 million (the central statistics agency of central java province 2018). in the last decade, duck meat has been favored by many people. duck meat also determines the agricultural economy. based on the total production of duck meat in the world, asian countries contributed 84.2%. the muscle fiber content of duck breast is higher than chicken meat, so it is often referred to as red meat (biswas et al. 2019). duck meat contains essential amino acids, vitamins, and minerals (reis et al. 2010) and it tastes very delicious and is favored by most people. based on those reasons there has been quite an increase in duck farming. in general, however, the quality of the foodstuff is not only determined by its nutritional content, but also by the safety of the food itself. foodstuff must be free from harmful substances. the public’s concern about the importance of food safety has increased. some cases related to food safety occur due to contamination of microorganisms, pesticides, hormones, antibiotics, and heavy metals. the consumption of foodstuffs is considered to be one of the main routes of heavy metal exposure to the human population. heavy metal contamination is a major problem because it affects the structural and functional integrity of the ecosystem. heavy metal contamination of *corresponding author, email: basanatha8@mail.unnes.ac.id biotropia vol. 29 no. 3, 2022 194 meat, vegetables, and fish is a serious problem for food safety and a threat to human health (bortey-sam et al. 2015) as most of these metals are toxic even at very low concentration levels. dietary intake accounts for more than 90% of heavy metal exposure compared to other routes such as inhalation and dermal absorption (loufty et al. 2006). likewise, the source of heavy metal contamination in duck meat is most likely from its feed and drinking water. abdulkhaliq et al. (2012) suggested that the accumulation of heavy metal in ducks is caused by contamination at every point of duck production, sources of nutrition, drinking water, and the environment. faten et al. (2014) also suggested that the heavy metal accumulation in ducks' bodies can come from water, feed, air, and the environment contaminated with these metals. meat is the part of duck carcass that is most consumed by humans compared to other parts such as the brain, lungs, liver, kidney, etc. therefore, this study aimed to analyze the levels of heavy metal bioaccumulation factor (baf) in duck meat and to assess the possible risks to human health. materials and methods this research is an exploratory observational study. a total of 25 duck samples were taken from five intensive duck farms in central java province, i.e., semarang (a), temanggung (b), magelang (c), pati (d), and salatiga (e). the criteria for selecting duck farms were laying ducks with an intensive care system (ducks were kept in cages, not released from the cage), 18 months years old, and the total number of ducks in a cage was 200 500 ducks. samples of ducks, drinking water, and feed were taken from each duck farm to be analyzed for heavy metal contents and concentrations. sample preparations meat samples five ducks, duck feed, and duck drinking water were randomly taken from each intensive duck farm. each duck was slaughtered. the thigh and breast meat were cut into sizes of 2 x 2 x 3 cm. furthermore, the meat was weighed and dried in an oven for > 72 hours at 70 °c. a total of 6 g of dry meat was added with 2 ml of 65% hno3 (merck) and then ashed using a furnace (nabertherm l3/c6) at 500 °c for 2 hours (demirel et al. 2008). subsequently, the ashed meat was dissolved with distilled water (merck) to a volume of 50 ml, to be analyzed for the heavy metal contents and concentrations. drinking water samples as much as 100 ml of duck drinking water was acidified with 65% hno3 until the water ph became 2 (demirel et al. 2008). after that, distilled water was added to the sample until the volume reached 200 ml, to be analyzed for the heavy metal contents and concentrations. feed samples as much as 1,500 g of duck feed was dried in an oven at 70 °c for > 48 hours. after being dried, the feed sample was mashed in a blender. a total of 6 g of feed sample was added with 2 ml 65% hno3 (merck) and mixed until homogeneous. then, feed samples were ashed using a furnace (nabertherm l3/c6) at 500 °c for 2 hours (demirel et al. 2008). the ashed feed was dissolved in 50 ml of distilled water (merck), to be analyzed for heavy metal contents and concentrations. quantification of heavy metals each sample of drinking water, feed, and duck meat was analyzed for heavy metal contents and concentrations following the icpoes method (perkin elmer optima 8300) following the procedure of naschan et al. (2017). sample solutions and standard solutions of as, cd, hg, and pb were prepared and injected into the icp-oes equipment. the analytical wavelengths of investigated heavy metals were 546.074 nm for hg, 193.696 nm for as, 220.353 nm for pb, and 226.506 nm for cd. each heavy metal measurement of each sample was repeated 3 times. data of as, cd, hg, and pb contents in the samples of drinking water, feed, and duck meat were compared with the determined quality standards (goi 2001; sni 2009; fao 2017). bioaccumulation factor (baf) bioaccumulation factor (baf) was calculated according to wang et al. (2017), with the following equation: heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 195 baf = concentration in organism (ci) ………….. (1), concentration in ingested food and/or water (c0) where: c0 = concentration of heavy metals in drinking water and or feed; ci = concentration of heavy metals in duck meat. thus, there were 3 types of baf calculated in this study, i.e., bafwater, baffeed, and bafduck meat. baf values were categorized according to arnot and gobas (2006), i.e., 1) baf < 1,000: no possibility of accumulation; 2) 1,000 < baf < 5,000: bioaccumulative; and 3) baf > 5, 000: highly bioaccumulative. potential health risk assessment non-carcinogenic risk to humans in the central java province in relation to the consumption of duck meat contaminated with heavy metals was assessed following the usepa (2000) method. heavy metal (as, cd, hg, and pb) accumulation in duck meat can cause adverse effects on human health. the estimated daily intake (edi) value through consumption of duck meat was calculated from the average concentration of heavy metals observed in duck meat samples and the average daily consumption of duck meat in indonesia. the edi (mg/kg/day) was estimated using the following equation (usepa 1989): edi = cm x adc ………………………….. (2), bw where: adc = average daily consumption of duck meat in indonesia, determined to be 0.0225 kg/person/day (ministry of trade of the republic of indonesia 2014); bw = body weight, determined to be 70 kg for adults (usepa 1989) and 30 kg for children (bortey-sam et al. 2015); cm = average heavy metal concentrations in duck samples (mg/kg). the non-carcinogenic risk due to the consumption of duck meat containing heavy metals by the population of central java province was called hq (hazard quotient). the hq is calculated using the following formula (usepa 1989): hq = cm x ef x adc x ed x 10-3 ….……….. (3), rfd x bw x at x 1000 where: cm = average heavy metal concentrations in duck samples (mg/kg); ef = exposure frequency (365 days/year); adc = average daily consumption of duck meat in indonesia, determined to be 0.0225 kg/person/day (ministry of trade of the republic of indonesia 2014); ed = exposure duration (70 years); rfd = an estimate of the number of contaminants per day that a human can tolerate over their lifetime. the rfd (oral) values used in this assessment were 3.00e-04 (for as), 5.00e-04 (for cd), 3.50e-03 (for pb), and 3.00e-04 (for hg) (tay et al. 2019); bw = body weight, determined to be 70 kg for adults (usepa 1989) and 30 kg for children (bortey-sam et al. 2015); at = average exposure time (ed x 356 days). if the hq value is less than one (< 1), pollutant exposure to the population does not cause adverse effects on human health. duck meat contained more than one type of heavy metal. therefore, the level of adverse effects on the exposed population is called hazard index (hi), which is the sum of all calculated hqs of all heavy metals found in the sample (usepa 1989): hi = hqas + hqcd + hqhg + hqpb ……… (4) hi is considered to be a potential risk estimated from exposure to some heavy metals (usepa 1989). data analysis heavy metal concentration data, baf, edi, hq, and hi values were analyzed descriptively. biotropia vol. 29 no. 3, 2022 196 results and discussion heavy metal toxicity can cause teratogenic, mutagenic, and carcinogenic effects in biological organisms including poultry (hashmi et al. 2013). in ecosystems, metal contamination can also affect the habitat, food chain, and community structure of organisms in an environment (kim & oh 2013). drinking water for ducks in all sampled intensive farms in this study contains heavy metals of as, cd, hg, and pb which exceeded the determined quality standards (goi 2001; sni 2009; fao 2017). the concentrations of heavy metals in duck drinking water were 0.0513 0.081 ppm (for as), 0.07 0.08 ppm (for cd), 1.74 2.4 ppm (for hg), and 0.06 0.07 ppm (for pb). the order of heavy metal concentrations in the drinking water of ducks from the highest to the lowest in temanggung, magelang, and salatiga was hg > cd > as > pb. on the other hand, the order of heavy metal concentrations in the drinking water of ducks from the highest to the lowest in semarang was hg > cd > pb > as; while in pati was hg > as > cd > pb (table 1). table 1 heavy metal content in drinking water and feed for ducks, and duck meat heavy metals water feed duck meat as standard fao 2017 0.01 0.5 0.5 sni 2009 0.05 0.5 0.5 pp no 82 year 2001 0.01 0.25 0.25 samples from: semarang (a) 0.0513abc 0.0750 0.0700 temanggung (b) 0.0773abc 0.0800 0.0770 magelang (c) 0.0797abc 0.1097 0.0770 pati (d) 0.0810abc 0.0987 0.0770 salatiga (e) 0.0710abc 0.1003 0.0700 cd standard fao 2017 0.003 0.5 0.5 sni 2009 0.003 0.1 0.3 pp no 82 year 2001 0.003 0.1 0.05 samples from: semarang (a) 0.0700abc 0.0760 0.0740abc temanggung (b) 0.0800abc 0.0750 0.0713abc magelang (c) 0.0800abc 0.0787 0.0750abc pati (d) 0.0800abc 0.0783 0.0740abc salatiga (e) 0.0800abc 0.0783 0.0740abc hg standard fao 2017 0.001 0.1 0.1 sni 2009 0.001 0.05 0.03 pp no 82 year 2001 0.001 0.06 0.03 samples from: semarang (a) 1.740abc 2.000abc 6.000abc temanggung (b) 2.400abc 0.089abc 3.100abc magelang (c) 2.380abc 4.420abc 4.840abc pati (d) 2.260abc 5.010abc 4.220abc salatiga (e) 2.380abc 4.560abc 4.400abc pb standard fao 2017 0.01 0.3 0.1 sni 2009 0.01 0.3 0.3 pp no 82 year 2001 0.01 0.2 0.5 samples from: semarang (a) 0.0600abc 0.0740 0.0640 temanggung (b) 0.0700abc 0.0730 0.0690 magelang (c) 0.0700abc 0.0780 0.0670 pati (d) 0.0700abc 0.0730 0.0460 salatiga (e) 0.0700abc 0.0650 0.0650 notes: a = the data exceed the quality standards set by fao 2017 (fao 2017); b = the data exceeds the quality standards set by indonesian national standard year 2009 (sni 2009); c = the data exceeds the quality standards set by indonesian government regulation no 82 year 2001/pp no. 82 year 2001 (goi 2001). heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 197 the source of drinking water for the sampled intensive duck farms in semarang, pati, and salatiga is groundwater, while in temanggung and magelang, the source is ponds. the high concentration of heavy metals in drinking water for ducks in this study was probably related to the timing of conducting the water sampling, which was during the dry season (may june). the concentration of chemicals in groundwater is greatly influenced by the season. the seasonal analysis report from obasi and akudinobi (2020) showed a relative decrease in chemical concentration in groundwater during the rainy season compared to that during the dry season. although heavy metal levels in drinking water for ducks increased relatively during the dry season, analysis of heavy metal concentrations in groundwater in various regions of central java province and indonesia is still required, both during dry and rainy seasons. the analysis is essential because groundwater is a source of drinking water for most livestock and humans. accumulation of heavy metals in the body of animals and humans will cause an adverse impact on health, especially arsenic, cadmium, lead, and mercury which are reported of having high toxicity. exposure to heavy metals in humans can increase the risk of cancer and other health problems such as anemia, liver and kidney damage (malik & khan 2016), decreased intelligence, decreased function of various organs, and even death (kulkarni & kaware 2013). the variation of heavy metal concentrations in water is influenced by water ph, temperature, dissolved oxygen, and water flow rate (li et al. 2013). the lower value of water ph causes higher metal solubility. water ph can convert carbonate (co3) into hydroxide (oh) forming bonds with particles on the water surface (palar et al. 2008). heavy metal solubility of zn, cu, cd, cr, and pb is greater at low ph conditions (4 7) than that at high ph conditions (8 10) (li et al. 2013). the amount of cd released from polluted sediment will decrease with increasing ph value (zhang et al. 2018). in this study, the results of water and soil ph measurements in the five samples of intensive duck farms were in the range of 6 7 and 6.9 7, respectively. these ph values were within the ph range of 6 9 which is a safe ph value based on indonesian government regulation no. 82 of 2001 (goi 2001). temperature also has an important effect on heavy metal solubility. at higher temperatures (30 35 °c), the release rate of metals (zn, cu, cd, cr, and pb) becomes faster than that at low temperatures (li et al. 2013). the temperature at each location of the sampled intensive duck farms in this study was in the range of 25.6 34.6 oc. this temperature was in the range of 27 35 °c which is a safe temperature per determination from the indonesian government regulation no. 82 of 2001 (goi 2001). in duck feed, hg was found in the concentration range of 0.089 5.01 ppm. this hg concentration exceeded the determined quality standards (goi 2001; sni 2009; fao 2017). meanwhile, the levels of other heavy metals (as, cd, and pb) were recorded as still under the determined quality standard. the range of as concentration detected in duck feed was 0.075 0.1097 ppm. meanwhile, the concentration range of cd, hg, and pb were 0.075 0.0787 ppm, 0.089 5.01 ppm, and 0.065 0.078 ppm, respectively (table 1). a study by ukpe and chokor (2018) also showed that there was heavy metal contamination of ni (1.71 g/kg), co (0.39 g/kg), pb (1.17 g/kg), cr (0.529 g/kg) and cd (0.031 g/kg) sourced from poultry feed. poultry feed has the potential to pollute the environment through the fecal matter of cultivated poultry. a large-scale duck farm produces large amounts of fecal matter. compost made from poultry fecal matter may still contain a lot of heavy metals (alvarenga et al. 2015). apart from being used as compost, fecal matter can be digested by maggots and the harvested maggots can be used as animal feed (wang et al. 2017). heavy metals in manure will contaminate the soil, then from the soil, heavy metals can be transferred to plants and animals. in turn, humans can also be exposed to heavy metals if they consume the contaminated plants and animals. heavy metals can contaminate various organisms in the ecosystem, through the food chain cycle. poultry feed is made from plant material, thus, if plants are contaminated, the feed will also be contaminated. the raw materials of duck feed in this study varied, i.e., concentrate feed (commercial), rice bran, leftover vegetables, dry rice, trash fish, shrimp waste, and fish waste. concentrate feed may also biotropia vol. 29 no. 3, 2022 198 contribute to heavy metal contamination in duck feed. a study by okoye et al (2011) showed that some concentrate feeds in nigeria contained 50.575 170.075 mg/kg of iron, 6.52 14.20 mg/kg of copper, 1.10 7.85 mg/kg of lead, and 0.038 0.463 mg/kg of cadmium. wang et al. (2013) also reported that several animal feeds in china contained several heavy metals with an average of 15.9 2,041.8 mg/kg of zn, 0 392.1 mg/kg of cu, and < 10 mg/kg of hg, as, pb, cd, and cr. the concentrations of cd (0.0713 0.075 ppm) and hg (3.1 4.84 ppm) in duck meat were identified to exceed the determined quality standards (goi 2001; sni 2009; fao 2017). meanwhile, as and pb concentrations were still under the determined quality standards. analysis of heavy metals in mallard ducks (anas platyrhynchos) in iran showed that the lowest concentrations of pb, cd, and zn metals were found in muscle (meat) and the highest concentration was found in the liver (alipour et al. 2016). some previous studies also revealed heavy metal contamination in the meat of ducks and other livestock. a study by widayanti and widwiastuti (2018) showed that duck meat in malang city contained 0.37 ppm of cadmium (cd). poultry meat in egypt contained 0.086 ppm of cd, 6.092 ppm of cu, 0.136 ppm of sn, and 1.280 ppm of zn (ali et al. 2017). heavy metal cd was detected at 0.003 ppm in poultry meat in ghana (nesta et al. 2015). heavy metals of as, cd, and hg were also detected in duck meat products in argentina (hyun et al. 2018). study results of islam (2018) showed that beef, goat, chicken, and duck meat in bangladesh contained 0.533 6.55 mg/kg of chromium (cr), 0.005 7.70 mg/kg of nickel (ni), 0.581 15.99 mg/kg of copper (cu), 0.080 11.34 mg/kg of arsenic (as), 0.001 0.22 mg/kg of cadmium (cd), and 0.061 13.52 mg/kg of lead (pb). bioaccumulation is the absorption process of chemicals by an organism through all exposure routes, such as those that occur in the natural environment, i.e., food sources, drinks, and surrounding environment. the bioaccumulation factor (baf) is a value to measure the bioaccumulation rate. baf is calculated based on the ratio of metal concentration in an organism to metal concentration in water and feed. baf value is influenced by several factors such as sex, reproductive status, age, body size, and lipid content of an organism. organisms having a lot of lipids have a greater capacity to store hydrophobic organic chemicals, so their baf values are higher (arnot & gobas 2006). in this study, the baf value in duck meat was analyzed based on the accumulation of water, feed, and the total of water and feed. the baf value of all heavy metals in duck meat was in the range of 0.065 5.01. according to arnot and gobas (2006), a baf value < 1,000 means that accumulation is not possible. the average values of bafwater for as and pb were greater than those of baffeed. on the other hand, the average values of baffeed for cd and hg were greater than those of bafwater (table 2). the bafwater value of as, cd, hg, and pb in duck meat in semarang was the highest compared to the values calculated in other locations. meanwhile, the highest baffeed value of as was found in temanggung (0.962) followed by semarang (0.933) and pati (0.78). the highest baffeed value of cd was found in semarang (0.973) followed by magelang (0.952) and temanggung (0.95). the highest baffeed value of hg was found in pati (5.01) followed by magelang (4.42) and temanggung (3.48). the highest baffeed value of pb was found in temanggung (0.945) followed by semarang (0.864) and magelang (0.858) (table 2). the average of bafduck meat values was in the range of 0.443 0.955, with the details of 0.443 for pb; 0.457 for as; 0.475 for cd, and 0.955 for hg. the highest bafduck meat values of as, cd, and hg were found in semarang. meanwhile, the highest bafduck meat value of pb was found in temanggung. the lowest bafduck meat values of as, cd, hg, and pb were found in magelang, temanggung, and pati, respectively (table 2). heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 199 table 2 baf values of heavy metals in drinking water and feed for ducks, and duck meat heavy metals location of duck farm average a b c d e as water 1.364 0.966 0.966 0.950 0.985 1.046 feed 0.933 0.962 0.701 0.780 0.697 0.815 duck meat 0.554 0.490 0.407 0.428 0.409 0.457 cd water 1.054 0.891 0.937 0.925 0.925 0.946 feed 0.973 0.950 0.952 0.945 0.945 0.953 duck meat 0.507 0.460 0.473 0.467 0.467 0.475 hg water 3.448 1.291 2.034 1.870 1.849 2.098 feed 3.000 3.483 4.420 5.010 0.964 3.375 duck meat 1.604 1.245 0.712 0.580 0.634 0.955 pb water 1.066 0.985 0.957 0.657 0.928 0.919 feed 0.864 0.945 0.858 0.630 0.065 0.672 duck meat 0.478 0.483 0.453 0.322 0.481 0.443 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. there have not been many studies related to baf values of heavy metals in terrestrial animals. research results by wang et al. (2017) showed that younger maggot larvae had a higher baf value of heavy metal than older larvae. the baf value of cd in 3-day-old larvae was 1.20, while the baf value of cd in 4-day-old larvae was 1.10. this comparison showed that as the maggots grow, heavy metals are released from the maggots' bodies. a study conducted in gadani shipbreaking pakistan to find out heavy metal baf values in the gills and muscles of seven fish species showed that the baf values found were in the order of mn > cd > ni > pb (kakar et al. 2020). estimated daily intake (edi) values of heavy metals (as, cd, hg, and pb) for adults and children through the consumption of duck meat in the central java province were calculated and presented in table 3. our study showed that children are more susceptible to the acute and chronic effects of a chemical contaminant because children consume more food per unit of body weight compared to adults (enhis 2007). therefore, toxic contamination by means of food consumption is higher in children than that in adults, leading to higher edi scores in children compared to that in adults for all heavy metals. the edi values calculated for as, cd, and pb in our study were lower than the oral reference dose (rfd) of each metal. however, the edi value for hg metal was higher than the oral rfd. this result may be caused by the high concentrations of hg in water, feed, and duck meat, which were above the determined quality standards (goi 2001; sni 2009; fao 2017). high concentrations of hg had also been reported in water at the ports of mayangan, kenjeran, and gresik, east java, indonesia (hertika et al. 2018). table 3 estimated daily intake (mg/kg/day) values of heavy metals (as, cd, hg, and pb) for adults and children through duck meat consumption heavy metals location of duck farm average a b c d e as adults 2.25e-05 2.47e-05 2.47e-05 2.47e-05 2.25e-05 2.38e-05 children 5.25e-05 5.77e-05 5.77e-05 5.77e-05 5.25e-05 5.56e-05 cd adults 2.38e-05 2.29e-05 2.41e-05 2.38e-05 2.38e-05 2.37e-05 children 5.55e-05 5.35e-05 5.63e-05 5.55e-05 5.55e-05 5.53e-05 hg adults 1.93e-03 0.99e-03 1.56e-03 1.36e-03 1.41e-03 1.45e-03 children 4.50e-03 2.33e-03 3.63e-03 3.12e-03 3.30e-03 3.38e-03 pb adults 2.06e-05 2.22e-05 2.15e-05 1.48e-05 2.09e-05 2.00e-05 children 4.80e-05 5.18e-05 5.03e-05 3.45e-05 4.88e-05 4.67e-05 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. biotropia vol. 29 no. 3, 2022 200 table 4 hazard quotient (hq) values hazard quotient location of duck farm average a b c d e as adults 7.50e-05 8.25e-05 8.25e-05 8.25e-05 7.50e-05 7.95e-05 children 1.75e-04 1.92e-04 1.92e-04 1.92e-04 1.75e-04 1.85e-05 cd adults 4.76e-06 4.58e-05 4.82e-05 4.75e-05 4.75e-05 4.73e-05 children 1.11e-04 1.07e-04 1.12e-04 1.11e-04 1.11e-04 1.10e-05 hg adults 6.43e-03 3.32e-03 5.19e-03 4.52e-03 4.71e-03 4.83e-03 children 15.00e-03 7.75e-03 12.10e-03 10.55e-03 11.00e-03 11.28e-03 pb adults 5.88e-06 6.34e-06 6.15e-06 4.22e-06 5.97e-06 5.71e-05 children 1.37e-05 1.48e-05 1.44e-05 0.98e-05 1.39e-05 1.33e-05 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. the non-carcinogenic risk of heavy metals through the consumption of duck meat was evaluated based on the value of the hazard quotient (hq). the estimated hq value of all heavy metals in this study was < 1 (table 4). the hq values indicate that the people in central java province who consume duck meat are unlikely to suffer adverse health effects due to heavy metal contamination in the duck meat. the highest average of hq values for adults and children were recorded at hg intake (4.83e-03 and 11.28e-03, respectively) followed by as (7.95e-05 and 1.85e-05, respectively). the lowest averages of hq values were recorded at cd intake, i.e. 4.73e-05 in adults and 1.10e-05 in children. in this study, the order of hq values from the highest was hg > as > pb > cd. hazard index (hi) is a parameter to assess the cumulative risk due to exposure to several heavy metals. the average of hi values of our study showed a number of < 1 for both adults (4.97e-03) and children (9.41e-03) (table 5), which indicated that there was no significant health risk due to cumulative exposure to heavy metals (as, cd, hg, and pb) in duck meat. a study by kakar et al. (2020) showed that the hq values of seven fish species in gadani shipbreaking pakistan were safe with regard to pb and mn (hq < 1), but may cause potential risk with regard to cd and ni (hq > 1). estimated values of hq and hi of heavy metals (cd, cr, cu, fe, ni, pb, and zn) through consumption of baladi chickens were < 1, indicating that chicken consumption did not have the potential to cause health risks for consumers in the jazan region of saudi arabia (al bratty et al. 2018). table 5 hazard index (hi) values ducks farm location adults children semarang (a) 6.56e-03 15.29e-03 temanggung (b) 3.46e-03 8.06e-03 magelang (c) 5.32e-03 12.42e-03 pati (d) 4.66e-03 10.86e-03 salatiga (e) 4.84e-03 11.29e-03 average 4.97e-03 9.41e-03 all of the risk parameters (edi, hq, and hi) calculated in this study were within safe limits. exposure to heavy metals through duck meat consumption, both in adults and children in central java province, was not potential to present an adverse health risk. however, exposure to heavy metals contained in duck meat cannot be underestimated because it will accumulate in the human body. exposure to heavy metals from other foodstuffs, dermal absorption, urban air inhalation, etc, which were not studied in this study, can increase the collective risk of heavy metal contamination in the human body. conclusion drinking water for ducks in all sampled intensive duck farms contained heavy metals of as, cd, hg, and pb which exceeded the determined quality standards. all health risk parameters (edi, hq, and hi) were within safe limits. exposure to heavy metals through duck meat consumption, both in adults and children, was unlikely to cause adverse health effects. acknowledgments this research was partially funded by the directorate general of research and development, strengthening the ministry of heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 201 research, technology, and higher education through the national competitive fundamental research grant number 192/sp2h/lt/ drpm/2019, 11 march 2019. references abdulkhaliq a, swaileh km, hussein rm, matani m. 2012. levels of metals (cd, pb, cu, and fe) in cow’s milk, dairy products, and hen’s eggs from the west bank, palestine. int j food res 19(3): 1089-94. al bratty m, alhazmi ha, ogdi sj, otaif ja, al-rajab aj, alam mf, javed sa. 2018. determination of heavy metals in various tissues of locally reared (baladi) chicken in jazan region of saudi arabia: assessment of potential health risks. pak j zool 50(4): 1509-17. ali ma, dalia mh, nagwa te. 2017. evaluation of some heavy metals residues in batteries and deep liter rearing systems in japanese quail meat and offal in egypt. vet world 10: 2231-0916. alipour h, solgi e, majnouni f. 2016. concentrations of heavy metals in tissues of the mallard anas platyrhynchos in kanibarazan, northwestern iran. podoces 11(2): 35-42. alvarenga p, mourinha c, farto m, santos t, palna p, sengo j, ..., cunha-queda c. 2015. sewage sludge, compost and other representative organic wastes as agricultural soil amendments: benefits versus limiting factors. j waste manag 40: 44-52. arnot ja, gobas fa. 2006. a review of bioconcentration factor (bcf) and bioaccumulation factor (baf) assessments for organic chemicals in aquatic organisms. environ rev. 14: 257-97. biswas s, banerjee r, bhattacharyya d, patra g, das ak, das sk. 2019. technological investigation into duck meat and its products: a potential alternative to chicken. worlds poult sci j 75(4): 609-20. bortey-sam n, nakayama smm, ikenaka y, akoto o, baidoo e, yohannes yb, ..., ishizuka m. 2015. human health risks from metals and metalloids via consumption of food animals near gold mines in tarkwa, ghana: estimation of the daily intakes and target hazard quotients (thqs). ecotoxicol environ saf 111: 160-7. demirel s, tuzen m, saracoglu s, soylak m. 2008. evaluation of various digestion for trace element contents of some food materials. j hazard mater 152: 1020-6. department of animal husbandry and animal health [dinas peternakan dan kesehatan hewan]. 2019. animal husbandry and animal health statistics [statistik peternakan dan kesehatan hewan]. https://ditjenpkh.pertanian.go.id. 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seniati]. itn malang, 3 february 2018. p: 361-4. zhang y, zhang h, zhang z, liu c, sun c, zhang w, marhaba t. 2018. ph effect on heavy metal release from polluted sediment. j chem 2018 (id 7597640): 7 pages. doi.10.1155/2018/7597640 biotropia vol. 30 no. 2, 2023: 137 146 doi: 10.11598/btb.2023.30.2.1678 137 optimization of talinum paniculatum gaertn. root induction and the effect of phosphate concentrations and ammonium:nitrate ratio on biomass of adventitious roots in in vitro culture palupi dasawulan lestari, syifa fajrisani, putri gehasti, sugiharto and yosephine sri wulan manuhara* department of biology, faculty of science and technology, universitas airlangga, surabaya 60115, indonesia received 11 october 2021 / revised 15 march 2023 / accepted 21 march 2023 abstract java ginseng (talinum paniculatum gaertn.) is a medicinal plant, the roots of which are commonly used in traditional medicine. in its natural habitat, the roots grow very slowly, requiring two to three years to produce 100 g of roots per plant. plant tissue culture could therefore provide an alternative means of accelerating root growth. this research aimed to optimize root induction and determine the effect of phosphate (kh2po4) concentration and the ratio between ammonium and nitrate (ammonium:nitrate) on the biomass of java ginseng adventitious roots in in vitro culture. stem and leaf were used as explants and various combinations and concentrations of iba and bap, kinetin, and tdz were used as growth regulators. leaf explants were grown in murashige and skoog (ms) media supplemented with iba 2 mg/l and various concentrations of phosphate (170; 212.5; 255; 297.5; 340; 382.5; 425; 467.5; 510 mg/l) and various ammonium:nitrate ratios (21:19 mm as the control, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, 30:0 mm). cultures were maintained for 6 weeks. the observed parameters were fresh weight, dry weight, the duration of root formation, and the number and length of adventitious roots. the data were analyzed using analysis of variance. the results showed that the concentration of phosphate and the ammonium:nitrate ratio significantly influenced the amount, length, fresh weight, and dry weight of java ginseng adventitious root. the highest fresh weight (37.47 mg) and dry weight (5.53 mg) were achieved in the treatment of double phosphate concentration (kh2po4 340 mg/l), while an ammonium:nitrate ratio of 10:20 mm was the optimum treatment to produce the highest biomass (fresh weight 73.6 mg and dry weight 8.2 mg). keywords: adventitious roots, ammonium, nitrate, phosphate, talinum paniculatum gaertn. introduction plants provide an incredible source of new medicinal product inventions for development. one of the medicinal plants often used by the community is java ginseng (talinum paniculatum gaertn.) from the portulacaceae family. java ginseng is widely used as a substitute for korean ginseng, which continues to be imported, because it is relatively cheap, easy to obtain, and easy to cultivate (widiyani, 2006). the plant’s chemical content comprises saponins, triterpenes, polyphenols, and essential oils (komatsu, 1982). the root of the java ginseng plant is the part that can be used as a medicinal ingredient. the most important and dominant component in the chemical content of java ginseng root is saponins. talinum paniculatum gaertn. has very slow root growth in its natural habitat, requiring around two to three years to produce 100 g of roots per plant (manuhara et al., 2015). therefore, in vitro culture techniques offer potential as an alternative means of accelerating the root growth of this plant. root growth can be increased by manipulation in culture media with the addition of nutrients (manuhara, 2014). as such, in this study, an increase in root biomass was achieved with the addition of phosphate and nitrogen sources. phosphate has an important function in *corresponding author, email: yosephine-s-w-m@fst.unair.ac.id biotropia vol. 30 no. 2, 2023 138 plant growth due to its role in transferring energy molecules such as adp and atp, nad and nadp, as well as genetic information system compounds such as dna and rna (barker and pilbeam, 2007). in plant tissue culture, the concentration of phosphate in the medium can be a major factor influencing plant growth. according to dormatey et al. (2021), phosphite supply in the murashige and skoog (ms) medium influenced root morphological characteristics and fresh biomass in five genotypes of potatoes. furthermore, pavlov et al. (2000) stated that lavandula vera biomass and rosmarinic acid was maximally produced with the addition of a two-time phosphate concentration in ms media. apart from phosphate, nitrogen sources also affect cell growth and the formation of secondary metabolites. nitrogen functions as a component of amino acids, proteins, and nucleic acids in plants (wiedenhoeft, 2006). ammonium (nh4+) and nitrate (no3-) are used as the main sources of nutrition in plant cell and tissue culture (zhang et al. 1996, in kim et al. 2005). when both nitrogen sources were administered simultaneously, growth and yield were significantly increased compared to ammonium or nitrate alone (zhang et al. 2011). research by yin et al. (2013) on pseudostellaria heterophylla plants showed that the highest adventitious root biomass, namely 9.11 g fresh weight and 0.54 g dry weight, was obtained with an ammonium and nitrate ratio of 20:40. furthermore, panda et al. (1992) reported that the only received information on ammonium was used as not only the nitrogen source. therefore, it is very important to determine the optimal ratio of ammonium to nitrate. no study has reported the effect of various concentrations of phosphate and the ammonium:nitrate ratio on the biomass production of javanese ginseng plants. this study therefore aims to investigate their effect in culture media on the adventitious root biomass of javanese ginseng. materials and methods adventitious root induction from leaf and stem explants the adventitious roots of java ginseng were induced from stem and leaf explants grown in solid ms medium supplemented with 30 g/l sucrose, 6 g/l agar, and various combinations of iba growth regulator 2 mg/l with three types of cytokinins, namely bap (0.1, 0.3, and 0.5 mg/l), kinetin (0.1, 0.3, and 0.5 mg/l), and tdz (0.1, 0.3, and 0.5 mg/l). the leaf samples (1 cm2) were taken from the second and third leaves of the shoots from intact plant. all explants were immersed in detergent solution for 3 minutes and rinsed with water. the explant surfaces were sterilized by immersing them in 10% clorox solution for 10 minutes. the explants were then washed 3 times using sterile distilled water. following this, they were maintained in an incubation room with an average temperature of 25oc in dark conditions. after 28 days, the adventitious roots were harvested and the fresh weight, dry weight, number of roots, and duration of adventitious root formation were measured. phosphate concentration treatments the explants used the second and third leaves from the shoots. solid ms medium was supplemented with a combination of iba 2 mg/l and tdz 0.1 mg/l, agar 8 gr, and sucrose 30 gr/l. different concentrations of kh2po4 (0; 42.5; 85; 127.5; 170; 215.5; 255; 297.5; and 340 mg/l) were added to the medium. the leaf explants were grown in culture bottles containing the medium and incubated at 25oc in the dark for 6 weeks. after 6 weeks the fresh weight, dry weight, root formation time, number of roots, and root length were measured. adventitious root growth was first recorded and used as the initial observational data for the first time that root growth occurred. after 6 weeks of cultivation, the number of roots, fresh weight, and dry weight were measured. ammonium: nitrate ratio treatments the explants used the second and third leaves from the shoots. the ms medium was supplemented with a combination of iba 2 mg/l and tdz 0.1 mg/l, agar 8 gr, and sucrose 30 gr/l. different ammonium:nitrate ratios (21:19 mm as the control, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30: 0 mm) were added to the medium. the leaf explants (1 cm2) talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 139 were grown in culture bottles containing the medium and incubated at 25oc in the dark for 6 weeks. after 6 weeks, the fresh weight, dry weight, root formation time, number of roots, and root length were measured. adventitious root growth was first recorded and used as the initial observational data for the first time root growth occurred. after 6 weeks of cultivation, the number of roots, fresh weight, and dry weight were measured. root fresh weight was measured after the roots had been rinsed with distilled water and drained. dry weight was measured after drying in an oven at 50oc for 5 days to obtain a constant dry weight. data analysis the data obtained, including the fresh weight, dry weight, number of adventitious roots, and length of adventitious roots, were analyzed using anova (analysis of variance) at a significance level of 5%. results and discussion adventitious root induction the reactions of t. paniculatum adventitious root induction to the addition of an iba growth regulator with various types of cytokinins in the third week can be seen in figure 1 (stem explants) and figure 2 (leaf explants). figure 1 root induction of talinum paniculatum stem explants in various combinations of iba and ba, kinetin, and tdz during a 3-week culture. a) i2b0.1; b) i2b0.3; c) i2b0.5; d) i2k0.3; e) i2k0.5; f) i2t0.1; g) i2t0.3; h) i2t0.5. (bar scale = 1cm). i: iba, b: ba, k: kinetin, t: tdz. the number after the letter indicates the concentration of the growth regulator in mg/l biotropia vol. 30 no. 2, 2023 140 figure 2 t. paniculatum adventitious root induction using leaf explants at the third week. a) i2b0.1; b) i2b0.3; c) i2b0.5; d) i2k0.3; e) i2k0.5; f) i2t0.1; g) i2t0.3; h) i2t0.5 (bar scale = 1cm) the stem and leaf explants induced from various combinations of growth regulator iba 2 mg/l with the different types of cytokinins (bap, kinetin, and tdz) at separate concentrations (0.1, 0.3, and 0.5 mg/l) produced varied responses, as seen in tables 1 and 2. table 1 the average duration of root formation, number of roots, root length, fresh weight, and dry weight of t. paniculatum gaertn. adventitious roots with various combinations of concentrations of iba 2 mg / l and bap, kinetin, and tdz (0.1, 0.3, 0.5 mg / l) on stem explants treatment duration of root formation (days ) number of roots root length (mm) fresh weight (mg) dry weight (mg) i2b0.1 9.33 ± 4.04 8.33 ± 1.52 11.44 ± 5.58 2.86 ± 1.30 1.00 ± 0.20 i2b0.3 14.00 ± 0.00 4.00 ± 2.64 14.68 ± 5.77 4.10 ± 1.94 0.93 ± 0.28 i2b0.5 14.00 ± 0.00 1.33 ± 0.57 8.50 ± 2.50 1.23 ± 0.05 0.53 ± 0.28 i2k0.1 4.67 ± 8.08 1.67 ± 2.88 2.60 ± 4.50 0.12 ± 0.21 0.03 ± 0.05 i2k0.3 11.67 ± 4.04 8.67 ± 6.11 11.81 ± 2.50 11.63 ± 9.60 6.67 ± 7.23 i2k0.5 16.33 ± 4.04 4.00 ± 0.00 7.08 ± 1.52 4.67 ± 3.78 1.06 ± 0.90 i2t0.1 0 0 0 0 0 i2t0.3 16.33 ± 4.04 2.00 ± 1.00 13.33 ± 3.78 3.83 ± 2.41 1.43 ± 1.00 i2t0.5 21.00 ± 7.00 1.33 ± 0.57 8.83 ± 2.36 4.63 ± 1.05 1.63 ± 0.15 talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 141 table 2 the average duration of root formation, number of roots, root length, fresh weight, and dry weight of t. paniculatum gaertn. adventitious roots with various combinations of concentrations of iba 2 mg/l and bap, kinetin, and tdz (0.1, 0.3, 0.5 mg/l) on leaf explants treatment duration of root formation (days to) number of roots root length (mm) fresh weight (mg) dry weight (mg) i2b0.1 16.33 ± 4.04 5.33 ± 1.15 13.3 ± 5.67 1.93 ± 1.04 1.16 ± 0.23 i2b0.3 21.00 ± 7.00 3.33 ± 3.21 10.9 ± 2.85 0.93 ± 0.23 0.10 ± 0.11 i2b0.5 11.67 ± 10.69 1.00 ± 1.00 3.33 ± 3.05 2.53 ± 2.96 0.83 ± 1.27 i2k0.1 0 0 0 0 0 i2k0.3 11.67 ± 10.69 2.00 ± 2.00 3.66 ± 3.32 0.63 ± 0.92 0.50 ± 0.86 i2k0.5 16.33 ± 4.04 2.00 ± 1.00 9.00 ± 5.29 2.40 ± 0.85 1.06 ± 0.90 i2t0.1 21.33 ± 9.01 7.33 ± 1.15 21.33 ± 9.01 4.53 ± 1.76 1.33 ± 0.64 i2t0.3 18.67 ± 8.08 4.33 ± 2.30 8.15 ± 2.58 3.23 ± 1.88 1.03 ± 0.95 i2t0.5 14.00 ± 0.00 1.67 ± 1.15 8.22 ± 5.17 4.83 ± 2.43 1.10 ± 0.90 the fastest mean root formation time was obtained from the induction of stem explants with the addition of a combination of growth regulator iba 2 mg/l + kinetin 0.1 mg/l, namely in 4.67 days. meanwhile, for the leaf explants, the fastest root formation time, at 11.67 days, was obtained by adding the combinations of growth regulator iba 2 mg/l + kinetin 0.3 mg/l and iba 2 mg/l + bap 0.5 mg/l. whereas the addition of a combination of growth regulator iba 2 mg/l + tdz 0.5 mg/l for stem explants and iba 2 mg/l + tdz 0.1 mg/l for leaf explants resulted in the longest mean times to root formation, namely 21 and 21.33 days, respectively. based on table 1, the highest average fresh weight, dry weight, and number of roots for the stem explants were obtained in the iba treatment of 2 mg/l + kinetin 0.3 mg/l. while the highest average root length of stem explants, namely 13.33 cm, was obtained in the iba treatment of 2 mg/l + 0.3 mg/l tdz. in table 2, the greatest average root fresh weight was obtained from the results of leaf explant induction with the addition of a combination of growth regulator iba 2 mg/l + tdz 0.1 mg/l, namely 4.53 mg. meanwhile, the highest dry weight, number of roots, and root length were obtained from the induction of leaf explants with the addition of a combination of growth regulator iba 2 mg/l + tdz 0.1 mg/l. the lowest average dry weight was obtained with the combination of iba 2 mg/l + bap 0.3 mg/l, namely 0.1 mg. adventitious roots can be induced from various explants including leaves, stems, roots, and various other factors such as auxins (baque et al., 2010). the selection of the types, concentrations, and combinations of growth regulators is very important. based on previous research, adventitious roots have been successfully induced from javan ginseng leaf explants on a solid ms medium with the addition of iba 2 mg/l, which produced the highest root mass of 5.929 g (solim et al. 2017). meanwhile, erin et al. (2020) reported that the treatment of iba 2 mg/l + ethephon 1 mg/l produced the highest average number of roots, namely 7.33, compared to other treatments. bap and kinetin hormones are chemical compounds that are included in the cytokinin group and play a role in shoot growth. this time, however, they were combined with auxin iba to stimulate adventitious root growth on t. paniculatum stem and leaf explants. in the research of isda and fatonah (2014), the highest number of roots was found at a bap concentration of 0.5 mg/l + 1.0 mg/l naa, namely 5.00 fruit on the explants of grammatophyllum scriptum orchid shoots. however, in the results of this study on stem and leaf explants, the combination of iba 2 mg/l + bap 0.5 mg/l did not provide optimal results in all parameters of the observation. it is thus evident that the effect of these growth regulators depends on the type of plant and the dosage concentration of growth regulator combination that is suitable; as such, for t. paniculatum this is not the optimal concentration of auxin and cytokinin combination to produce the most roots. apart from bap, kinetin is also often combined with the auxin hormone in its use in vitro, as in the research of mahadi et al. (2013) where the highest average number of dragon fruit explant roots was found in the n0.4 k4 treatment, namely 5.25 roots. however, the lowest average number of roots was also found in the n0.4 k4 treatment. this is presumably because high kinetin administration can produce stunted explant growth; wahidah (2011) stated biotropia vol. 30 no. 2, 2023 142 that kinetin hormone can affect the process of plant development at low concentrations while inhibiting growth at high concentrations. tdz can play a role in stimulating endogenous cytokinin production. therefore, tdz can increase the action of other cytokinins, both exogenous and endogenous cytokinins (guol et al., 2011). the administration of tdz at a low concentration induces callus faster than at a high concentration; for callus regeneration, it is better to combine tdz and naa at low concentrations than tdz alone (oláh et al., 2003). this is corroborated by the findings of this study, where tdz with a concentration of 0.1 mg/l induced the formation of a large number of adventitious roots to produce a large fresh weight and dry weight compared to concentrations of 0.3 mg/l and 0.5 mg/l. the formation of adventitious roots is a type of positive synergy between tdz and iba as the best adventitious root-forming hormone. effect of phosphate concentration (kh2po4) on adventitious root growth the best treatment for inducing root growth (a combination of iba 2 mg/l and tdz 0.1 mg/l) was used to determine the effect of phosphate concentration and the ammonium:nitrate ratio on adventitious root growth. the average fresh weight, dry weight, root growth, number of roots, and adventitious root length of t. paniculatum in various phosphate concentration treatments of ms medium are listed in table 3. the highest average values for fresh weight, dry weight, and the number of roots were obtained for the p5 treatment (phosphate concentration 340 mg/l). meanwhile, the fastest root growth rate of 7.3 days was achieved with the p7 treatment (phosphate concentration 425 mg/l), and the longest average root is shown for the p8 treatment (phosphate concentration 467.5 mg/l). the highest average fresh weight and dry weight values were identified for the p5 treatment. this is consistent with research conducted by curtis et al. (1991), which stated that the growth of opium poppy in cell suspension culture increased by 50% in media with two times the concentration of phosphate added. this occurred since phosphate plays an essential role in the transfer of energy for cell metabolism, the constituents of cell membranes, and nucleic acids. the lowest fresh weight was found on the media with the p9 treatment (510 mg/l) due to the very high phosphate concentration. the plant cells in these explants were stressed due to the very high salt concentration. the increase in the plant’s dry weight is attributable to the nutrients that were absorbed by the root and the accretion of protoplasm due to the increase and size of the cell count (khristyana et al. 2005). the fastest average duration of root formation was 7.3 days (phosphate concentration 425 mg/l) based on the application of the control treatment (phosphate concentration 425 mg/l) for 8 days. in this study, the p9 treatment (phosphate concentration 510 mg/l) showed the longest average duration of root formation (11.6 days) because media with high phosphate concentrations can suppress growth (george et al. 2007). phosphate can bind with calcium and other microelements to reduce the absorption of other elements below the maximum level (buddh, 2014). table 3 average fresh weight, dry weight, duration of root formation, number of roots, and root length of t. paniculatum adventitious roots in various phosphate concentrations of ms medium treatment code kh2po4 concentratio n (mg/l) fresh weight (mg) dry weight (mg) duration of root formation (days to) number of roots root length (cm) p1 170 18.77 ± 9.96 b 3.23± 0.60 b 8.00 ± 2.64 3.67 ±2.52 a 2.56 ±1.76 ab p2 212.5 2.67 ± 2.56 a 1.10 ± 0.60 a 9.00 ± 1.73 3.00 ± 1.00 a 1.79 ± 0.31a p3 255 8.63 ± 5.55 a 2.07 ± 0.81 ab 10.67 ± 1.15 1.33 ± 0.58 a 2.85 ±1.52 ab p4 297.5 16.30± 5.03 ab 3.30 ± 0.09 b 8.33 ± 3.21 4.00 ±1.73 a 1.39±0.22 a p5 340 37.43 ± 5.09 c 5.53 ± 1.03 c 7.67 ± 0.57 5.33 ±0.58 ab 1.70 ±0.39 a p6 382.5 8.8 ± 6.61 ab 1.77 ± 1.22 ab 11.33 ± 1.52 4.00 ± 1.00 ab 1.55 ± 0.96 a p7 425 19.13 ±14.89 b 3.17 ± 1.56 b 7.33 ± 0.57 4.67 ± 1.15 ab 2.15 ± 0.46 a p8 467.5 19.17 ± 9.03 b 3.27 ± 0.50 b 8.00 ± 1.73 1.67 ±0.58 b 4.20 ±0.75 b p9 510 2.30± 1.15 a 1.13 ± 0.67 a 11.67 ± 2.51 2.00 ±1.00 ab 1.56±0.66 a note: numbers followed by different letters show real differences according to duncan’s test. the number in bold shows the highest value for each treatment parameter. talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 143 figure 3 the number of adventitious roots formed from explants of javanese ginseng leaves (talinum paniculatum gaertn.) after a 6-week incubation period, namely (a) p1 (kh2po4 170 mg/l / control), (b) p2 (kh2po4 212,5 mg/l ), (c) p3 (kh2po4 255 mg/l), (d) p4 (kh2po4 297,5 mg/l), (e) p5 (kh2po4340 mg/l), (f) p6 (kh2po4 382,5 mg/l), (g) p7 (kh2po4425 mg/l), (h) p8 (kh2po4 467,5 mg/l), and (i) p9 (kh2po4 510 mg/l) (bar scale = 1cm) the number of roots produced in each explant is different (figure 3). the highest mean number of roots was found in the explants treated with p5 (kh2po4 340 mg/l). the treatment in this study was controlled by p1 (170 mg/l of kh2po4). the number of roots stood at only 2.56; thus, while both were from the second and third leaves of the shoots, they were from different plants. auxin plays a role in cell elongation, cell division, and adventitious root formation (george, 2007). therefore, different endogenous auxins lead to differences in root formation, the number of adventitious roots, and the length of adventitious roots. effect of ammonium:nitrate ratio on adventitious root growth the average fresh weight, dry weight, root growth, number of roots, and the adventitious root length of t. paniculatum in various ammonium:nitrate ratios on ms medium are listed in table 4. the highest average fresh weight, dry weight, and number of roots were obtained for the ammonium:nitrate ratio of 10:20. meanwhile, the fastest average time to root formation was 10 days and the longest root length was 2.3 cm. however, the data showed no significant difference between all treatments, meaning the data did not affect the adventitious root biomass of java ginseng. figure 4 contains pictures of 6 different ammonium:nitrate ratios, namely 21:19 mm as the control, and 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30: 0 mm for the adventitious roots of java ginseng at 6 weeks. biotropia vol. 30 no. 2, 2023 144 table 4 average fresh weight, dry weight, duration of root formation, number of roots, and root length of t. paniculatum adventitious roots in various ammonium:nitrate ratios (21:19 mm, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30:0 mm) ammonium:nitrate ratio fresh weight (mg) dry weight (mg) duration of root formation (days to) number of roots root length (cm) a21 : n19 (normal ms) 47.9 ± 14.9a 5.0 ± 1.7a 10.2 ± 3.7a 5.6 ± 5.3ab 1.9 ± 0.8a a0 : n30 17.5 ± 9.5b 2.1 ± 0.8b 13.0 ± 4.3a 3.0 ± 2.3a 2.0 ± 0.9a a10 : n20 73.6 ± 32.5a 8.2 ± 3.2a 10.2 ± 3.0a 8.8 ± 2.9b 1.8 ± 0.6a a15 : n15 23.3 ± 17.6b 3.4 ± 2.5b 10.0 ± 1.9a 7.0 ± 4.6ab 1.4 ± 1.0a a20 : n10 19.6 ± 14.9b 2.5 ± 1.5b 11.0 ± 1.9a 5.6 ± 4.7ab 1.0 ± 0.3a a30 : n0 21.3 ± 9.6b 3.2 ± 1.2b 11.4 ± 3.4a 5.8 ± 3.1ab 2.3 ± 1.0a note: numbers followed by different letters show real differences according to duncan’s test. numbers in bold show the highest value for each treatment parameter. a = ammonium; n = nitrate. figure 4 the adventitious roots (shown by arrows) of 6-week-old t. paniculatum were cultured in vitro with 6 types of treatment and ammonium:nitrate ratios, namely (a) 21:19 mm (control); (b) 0:30 mm; (c) 10:20 mm; (d) 15:15 mm; (e) 20:10 mm, and (f) 30: 0 mm adventitious roots (i.e., roots that form from non-root tissue) can arise in various tissue locations from groups of mature cells that renew their cell division activity (taiz and zeiger, 2003). in the treatment ratio ammonium:nitrate 10:20, nh4cl has good potential to replace nh4no3 in control as a nitrogen source to increase adventitious root biomass production. this is because nitrogen is used for protein synthesis, both structural and enzymatic, and is thus needed for cell and organ growth, including for the production of plant biomass (lawlor et al. 2001). in addition, the form and amount of nitrogen in the in vitro media have a significant effect on the rate of cell growth, differentiation, and cell totipotency (kirkby and mengel, 1987). the average time to root formation in this study ranged from 10 to 13 days (table 4). this aligns with the findings of research by palestine (2008) on pule pandak (raufolvia serpentine, l.) showing that the addition of iba with a concentration of 2 to 4 mg/l can initiate root growth faster than other treatments, namely in 15 days. the highest number of roots was found in the ammonium:nitrate ratio of 10:20, while the lowest number of roots was found in the ammonium:nitrate ratio of 0:30. efforts to increase the number of roots include the addition of auxin growth regulators that can stimulate root induction. wattimena (1988) explained that auxin is a plant hormone essential talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 145 for cell division and root formation. root emergence is influenced by the number of roots and correlates with the absorption of nutrients present in the culture medium. several studies have shown that nitrogen compounds and the ratio between ammonium and nitrate can affect the differentiation, dedifferentiation, growth, and development of explants, as well as organ formation (preece 1995). the average root lengths of the treatments in this study are shown in table 4; they range from 1 cm to 3 cm with no influence between one treatment and another. the increase in plant size reflects the increase in protoplasm that occurs due to the increase in cell size and number (khristyana et al. 2005). conclusion the combination of iba 2 mg/l + kinetin 0.3 mg/l is the optimal concentration to produce the highest mean number of roots, fresh weight, and dry weight of talinum paniculatum adventitious roots in stem explants. meanwhile, for leaf explants, the best results were obtained with the combination of iba 2 mg/l + thidiazuron 0.1 mg/l. the highest fresh weight (37.47 mg) and dry weight (5.53 mg) were obtained in the phosphate concentration (kh2po4) 340 mg/l treatment. meanwhile, the ammonium:nitrate ratio of 10:20 was the best treatment to produce the highest biomass (fresh weight 73.6 mg and dry weight 8.2 mg). acknowledgments this research was funded by the directorate general of higher education, research and technology no. 852/un3.14/pt/2020 in the master’s thesis research scheme. references baque ma, hahn ej, paek ky. 2010. induction of adventitious root from leaf explants of morinda citrifolia affected by auxin and light quality. in vitro cell dev biol plant 46: 71-80. barker av, pilbeam dj. 2007. 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agriculture bogor]. widiyani t. 2006. efek antifertilitas ekstrak akar som jawa (talinum paniculatumgaertn.) pada mencit (mus musculus l.) jantan [the effect of antifertility of talinum paniculatum gaertn. root extract in male mice (mus musculus l.)]. bul. penel. kesehatan 34(3): 119-28. wiedenhoeft ac. 2006. the green world plant nutrition. new york: infobase publishing. yin s, liang y, gao w, wang j, jing s, zhang y, li h. 2013. influence of medium salt strength and nitrogen source on biomass and metabolite accumulation in adventitious root cultures of pseudostellariaheterophylla. acta physiologiae plantarum 35(8): 2623-28. zhang yh, gao wy, wang j, li xl, xiao pg. 2011. improvement of growth and periplocin yield of periploca sepium adventitious root cultures by altering nitrogen sources supply. chin herb med. 3: 226-31. biotropia vol. 29 no. 3, 2022: 203 212 doi: 10.11598/btb.2022.29.3.1569 203 diversity and status of butterflies in awasian water forest reserve, mt. hilong-hilong, philippines mary cris g. abao1, kevin c. quiňonez1, lea jane g. elegio1, mark freddie d. suarez1, alma b. mohagan3,4 and arturo g. gracia jr.2 1college of teacher education, surigao del sur state university-main campus, tandag city, surigao del sur 8300, philippines 2department of natural sciences and mathematics, college of arts and sciences, surigao del sur state university-main campus, tandag city, surigao del sur 8300, philippines 3department of biology, college of arts and sciences, central mindanao university, bukidnon 8310, philippines 4zoological section, university museum, central mindanao university, musuan, bukidnon 8310, philippines received 22 march 2021/accepted 14 october 2021 abstract butterflies are deemed as an essential faunal group in the ecosystem due to their ecological services. however, continuous habitat loss leads to the decline of its population. thus, this study was conducted to assess its diversity and status in one of the watersheds of mt. hilong-hilong. sweep netting (336 man-hours) and butterfly trapping (200 trap days) were carried out to document the species. fifty-seven species of butterflies were recorded with the family nymphalidae as the most represented group (n = 30). species diversity (h') was relatively higher in dipterocarp (h' = 1.49) forest than agroecosystem (h' = 1.39), a result primarily influenced by favorable ecological support like food availability. endemicity was 31%, which comprised mostly of rare species. noteworthy findings are the listing of globally and nationally rare species but locally assessed as common. based on the results, the area harbors an array of butterfly species and various rare species that requires an effective management plan to conserve the organisms. keywords: butterflies, diversity, lepidoptera, mt. hilong-hilong, richness introduction lepidoptera is one of the largest families in the insects' realm, where butterflies well represent most of the species. its occurrence and diversity are considered good indicators for any terrestrial biotopes (kunte 2000; aluri & rao 2002; thomas 2005; arya & dayakrishna 2014), which also denote environmental quality changes and served a vital role in agricultural landscapes (munyuli 2012; nacua et al. 2015a). lepidoptera are excellent pollinators that ensure the reproduction and survival of plants used by other organisms as sources of food, reproductive areas, and medicine (mohagan & treadaway 2010). despite the high diversity, sociological and ecological functions of butterflies, the taxa are still not spared from gradual extinction due to overexploitation, illegal trading, and habitat loss which are the manifestations of uncontrollable anthropogenic activities (myers et al. 2000; brook et al. 2008; serengil et al. 2010; parria et al. 2017). owing to habitat destruction for developmental activities in an urban environment and unscientific management of natural resources, most of our native butterflies are fast disappearing. at present, their survival is under threat (nair et al. 2014) and these threats are highly observable in the philippines, making the country one of the hottest hotspots (bisson et al. 2003). hence, determining the diversity, level of endemism, and distribution of species is necessary for it serves as bases and guidelines for the formulation of conservation measures (ehrlich & hanski 2004; pyke & ehrlich 2010). awasian water forest reserve is one of the watersheds of mt. hilong-hilong, a key *corresponding author, email address: artzgracia@gmail.com mailto:artzgracia@gmail.com biotropia vol. 29 no. 3, 2022 204 biodiversity area (kba) in the philippines located in the northeastern mindanao. mt. hilong-hilong itself has been considered as an ideal abode for various forms of flora and fauna (pef 2008). however, ecological information is observed to be fragmented and limited, with scientific studies mainly focused on vertebrates and the western part of the ecosystem (agusan provinces). in contrast, invertebrates, especially butterflies and the eastern part (surigao provinces), are relatively poorly known, with only one accessible data set on butterflies from the study of ramirez and mohagan (2012). the area is also considered a vulnerable habitat under the criterion of very high due to highly observed anthropogenic pressures (birdlife international 2020), especially on the aspect of the rapid growth of human population in the uplands, mining, agricultural expansion, and road expansion and development (haribon 2017). for these reasons, the study was conducted to address the scarcity of ecological information on butterflies in the area and provide information that can be utilized for effective environmental management planning. the study generally aimed to assess the diversity through the determination of species composition and richness of butterflies across habitat types, as well as the evaluation of its status in comparison with the global and national assessments. materials and methods duration and description of the study area the study was conducted at awasian water forest reserve, mt. hilong-hilong in tandag city surigao del sur, philippines located at 9.07579 n and 126.14006 e (fig. 1). the study was carried out on 1 9 october 2017, covering nine days of sampling. the site's topography is generally plain, rolling, and gently sloping. the area can be reached for approximately two hours by walking from the nearest human settlements. the climatic condition in the area falls under the type ii climate condition of the philippines. it has rainfall distributed throughout the year, with a negligible short dry season (mpdo 2004). the area has two vegetations, the agroecosystem and dipterocarp forests. figure 1 location and spot map of the study site (birdlife international 2020) butterflies of awasian water forest reserve – abao et al. 205 establishment of study stations and habitat assessment two transect lines with a length of 1,000 m were established in the study area with an aerial distance of 200 m from each transect. the reason for such aerial distance is to avoid or lessen possible bias in documenting the species. the first transect line was laid across the human trail in the agroecosystem and labeled as transect 1 (t1), while the second transect line was laid across the dipterocarp forest and labeled as transect 2 (t2). habitat assessment was carried out through the documentation of the plant community and composition, canopy cover, nearness of water bodies, distance from the nearest human settlements, and temperature. sampling techniques collection and capturing the butterflies was carried out primarily through sweep netting. the activity was actively performed from 9:00 am to 3:00 pm, for these are the hours the butterflies are highly active. a total of 336 man-hours of sampling effort was spent for the entire duration of the study. wherein 168 man-hours were spent per transect or habitat. as for this technique, the researchers wore brightly colored clothes to attract butterflies. also, 25 pieces of classical butterfly traps containing muscovado sugar solutions were deployed along the first 500 m of the transect lines. each trap was placed with a 20 m interval from each other. the traps were hung in the place which was convenient for butterflies feeding like open fields. identification, preservation, and data analysis preliminary identification of butterflies was carried out using taxonomic keys. other references, such as books, journals, and photographs (mohagan & treadways 2010; treadway 2012; ramirez & mohagan 2012) of the previously identified specimens, were also used. after the initial identification, the samples were sent to the zoological section of the university museum of central mindanao university (cmu), a state-governed research university for verification. the collected species of butterflies were pinned and preserved using naphthalene balls and powder. data analyses that include the computation of species rarefaction and diversity indices were analyzed using biodiversity professional (biopro) software version 2.0 (mcaleece 1997). the butterfly's global status assessment was based on the international union conservation for nature (iucn 2020), while the established national and local assessments by treadaway (1995) as well as mohagan and treadaway (2010) were adopted. results and discussion taxonomic composition and overall richness fifty-seven (57) species of butterflies were recorded in the study area. these species were classified into 43 genera belonging to 5 families. among the five families, family hesperiidae was the least represented with three species observed, followed by family papilionidae (n = 5), pieridae (n = 8), lycaenidae (n = 11), and nymphalidae (n = 30) (table 1). the low representation of species under hesperiidae was attributed to a generally thicker canopy in the area. the thick canopy makes the habitat shadier, which is not favorable for the hesperiidae, which species used to inhabit an open place and near the shrubs (braby 2016). hesperiids also prefer to feed on various weedy plants, including pigweeds and lamb's quarter (hilty 2013), which is not present in any vegetation types of the current sampling area. biotropia vol. 29 no. 3, 2022 206 table 1 species list, endemicity, and status of observed butterflies in awasian water forest reserve, mt. hilong-hilong taxon assessment endemism local national global family hesperiidae 1 hasora moestissima moestissima r c ne 2 tagiades japetus titus r c c ne 3 tagiades trebellius martinus r c c pe family lycaenidae 4 allotinus pallax apsecus c c ne 5 arhopala abseus abseus c c ne 6 caleta angola angola r c ne 7 cheritra orpheus orpheus r c pe 8 eooxylides neduna neduna c 9 hypolycaena sipylus tharrytas c c ne 10 jamides alecto manillana c r pe 11 jamides celeno lydanus c r r ne 12 jamides philatu osias c r ne 13 nacaduba borenice leei c 14 prosotas nora semperi r c ne family nymphalidae 15 acrophtalmia albofasciata r r me 16 acrophtalmia leto ochine c c ne 18 amathusia phidippus pollicaris r c c ne 19 cirrochroa tyche tyche c c ne 20 cyrestis maenalis c c ne 21 danaus melanippus c c ne 22 elymnias beza beza r c me 23 euploea amulciber mindanensis c c ne 24 euploea euniceleucogaris r 25 faunis phaon leuces c c ne 26 junonia hedonia ida c c c ne 27 lassipa pata semperi r r ne 28 lexias panopus miscus r c ne 29 milanitis boisduvalia r r pe 30 mycalesis micromede micromede c r ne 31 mycalesis federi federi r r pe 32 mycalesis mineus philippina c c r ne 33 mycalesis tagala semiraza c r ne 34 neptis mindorana pseudosoma c pe 35 neptis pampanga boholica r r c ne 36 pantaporia dama commixta c c c pe 37 pantoporia cyrilla cyrilla r c pe 17 pantoporia sp. r 38 phaedyma columella messogai c c ne 39 phalantha phalantha phalantha c c ne 40 ptychandra schadenbergi r r pe 41 ragadia melindena mindeninse c r pe 42 tanaecia leucotaenia acquamarina r c ne 43 tarattia cosmia cosmia c pe 44 ypthima sempera chaboras r r pe family papilionidae 45 atrophaneura semperi r r r pe 46 graphium argamemnon argamemnon r c ne 47 melenaides deiphobus rumanzovia r c ne 48 melenaides helenus hystaspes c c c me 49 pachliopta mariae mariae c c pe family pieridae 50 appias nepheleelis r r ne 51 cepora aspasia orantia r c ne 52 eurema blanda valli volans c c c ne 53 eurema hecabeta miathis c c ne 54 eurema sarilalas arilata c r r pe 55 gandaca harina mindanensis c c ne 56 leptosia nina terantia r c ne 57 pareronia boebera trinobantes c c ne total number of families 5 total number of genera 43 total number of species 57 notes: c = common; r = rare; ne = non-endemic; pe = philippine endemic; me = mindanao endemic. butterflies of awasian water forest reserve – abao et al. 207 the family nymphalidae was observed to be the most represented group. this finding is attributed to the study site's general characteristics as a forest. nymphalids are perceived to be dominant in a forested area, particularly in tropical regions (sarkar 2011; harsh et al. 2015). its abundance is attributed to the availability of food resources from the variety of host plants and favorable microclimate conditions (widhiono 2015). at the course of the conduct, various plants were flowering and fruiting. among these plants are the dominant species in the area like shorea spp. and other trees like lansium dosmesticum, artocarpus odoratissimus, and durio zibithenus, which make the condition suited to the requirements of the butterfly group for their feeding behavior. this observation agrees with opler et al. (2017) claim that nymphalids' feeding behavior depends on the nectar, sap flows, and rotting fruit, wherein the food availability from one vegetation type influences the butterfly composition (toledo & mohagan 2011). the total richness observed in this study is comparatively higher compared to some of the faunistic studies conducted in the philippines. zapanta et al. (2016) at bulusan, bulakan, only recorded 21 species, whereas the lepidopteran assessment carried out in lipa, batangas documented only 25 species (nacua et al. 2017). the same observation was noted for the studies of toledo and mohagan (2011) at mt. hibokhibok, camiguin (n = 41) and sumagaysay and sumagaysay (2012) at mt. nebo, bukidnon (n = 31). as compared to the global findings, the study surpasses the records of arya and dayakrishna (2014) in naital, uttarakhand, india (n = 27); haroon et al. (2020) in tanga, charsadda, khyber pakhunkhwa, pakistan (n = 22); castro and espinosa (2015) in arenillas ecological reserve, ecuador (n = 22); and koneri et al. (2016) at manembo-nembo wildlife reserve, north sulawesi, indonesia (n = 44). findings of our study suggested that the study area is an ideal abode for butterflies due to its capability for supporting larger communities. our study also indicated that the habitat has a better support system coming from the butterflies, especially on the aspect of pollination. on the other note, the results of our study are comparatively lower compared with the records in mts. apo, kitanglad, musuan, and timpoong in the philippines with 104, 148, 114, and 79 species, respectively (mohagan et al. 2011). the reports of nacua et al. (2015b) at san fernando la union botanical garden, mohagan et al. (2018) at mt. pinamantawan, bukidnon, as well as mohagan and treadway (2010) at mt. hamiguitan, davao oriental, philippines were also noted to have higher richness with 104, 118, 142 species, respectively. even in comparison with the study results of ramirez and mohagan (2012) at maitum village, tandag city which is an area adjacent to the sampling site of this study, recorded a total of 104 species. the discrepancy between the results is attributed to various factors ranging from sampling effort to study duration. unlike in different studies, the participation of a wellversed taxonomist maximizes the observation since visually observed species are added to the list, like the case of the abovementioned studies. in contrast, our study only represents the verified captured samples. the influence of sampling duration could also be another factor due to more time provided for further documenting the faunal group. the concept conforms to the elaborated observation in the faunistic study of lepidoptera in one of the wildlife sanctuaries in misamis oriental (guadaluiver et al. 2019) and mt. hamiguitan (mohagan & treadway 2010). as shown in figure 2, the species rarefaction entails that sampling effort is still unachieved. thus, observing additional species is still feasible by doing reassessment in the field and could lead to an increase in the overall butterfly richness. not to mention that various uncaught morphologically distinct individuals were observed during the fieldwork that could mean a different species as well. biotropia vol. 29 no. 3, 2022 208 figure 2 butterfly species rarefaction plot in awasian water forest reserve ecological profile of butterflies across habitats in this study, 155 individuals of butterflies were captured. butterfly abundance was higher in the dipterocarp forest, with 83 (53%) individuals than in the agroecosystem with 72 (47%) individuals. in terms of species richness, dipterocarp forest had higher species-richness with 42 species while the agroecosystem had only 34 species. diversity index (h') and species evenness (j') were relatively higher in dipterocarp forest (h'=1.49; j'=0.93) compared to those in agroecosystem (h' = 1.39; j' = 0.90) (table 2). these consistent results suggest that the dipterocarp forest has better ecological support for the survival of the butterflies in the area. the dipterocarp forest was observed to have numerous flowering and fruiting plants during the sampling. this environmental set-up could have resulted in wider ecological support concerning food preference, unlike in the agroecosystem, where limited resources were observed. the result conforms with the report at mt. malindang (ballentes 2006) and in the lowland forest at maitum, mt. hilong-hilong (ramirez & mohagan 2012). the findings supported the idea that butterfly assemblage was more diverse in the dipterocarp forest than that in the agroecosystem. this finding could be attributed to the diversity and abundance of butterflies which are highly correlated with the availability of food plants and assemblage of floral species in the surroundings (kunte 2000; stefenascu 2004; ansari 2015). this common ground of findings is linked to the butterflies' voracious eating behavior, particularly in their larval stage, to meet the demand for nutrients in their fast development through the process of metamorphosis. moreover, schneider (2003) reported that the habitat characteristics and landscape structure influenced species abundance and richness, thus, supporting the variation of the result in this study. table 2 ecological data of butterflies representing species richness, abundance, evenness, and diversity index in the two study areas habitat ecological profile richness abundance shannon-wiener diversity index (h’) evenness (j’)  agroecosystem 34 72 1.49 0.93  dipterocarp forest 42 83 1.39 0.90  overall 57 155 1.58 0.90 rarefaction plot e s (n ) 0 10 20 30 40 50 60 0 50 100 150 200 butterflies of awasian water forest reserve – abao et al. 209 the lesser diversity in the agroecosystem is also attributed to the influence of human disturbance. the habitat was more vulnerable to anthropogenic activities compared to the situation in the dipterocarp forest since the agroecosystem is nearer to human settlements. the butterflies are profoundly affected and endangered by land use and forest cultivation (avigliano et al. 2019) because those activities affect the butterflies’ continual survival by delimiting the needed resources, such as food and good habitat (ozden et al. 2008). hence, forest cultivated areas have comparatively low butterfly diversity than any other habitats (malagrino et al. 2008; laghude et al. 2019). among the documented species, acrophtalmia leto ochine and euploea amulciber mindanensis (nymphalidae) and pachliopta mariae mariae (papilionidae) were the most abundant with 15, 10, and 12 individuals, respectively. representatives of a. leto ochine were mostly seen in the agroecosystem, particularly in the open fields and grasslands. in contrast, p. mariae mariae mainly were seen in the dipterocarp forest in an area with at least 50 70% canopy coverage and near the water systems. as for the e. amulciber mindanensis, samples of its population were equally observed in both habitats. species assessment out of the 57 species recorded, only 21% (n = 12) of the butterfly species have international union for conservation of nature assessment status (iucn 2020), consisting of 5 (9%) common non-endemic species (cnes), 2 (4%) rare non-endemic species (rnes), 2 (4%) common philippine endemic species (cpes), 2 (4%) rare philippine endemic species (rpes), and 1 (2%) common mindanao endemic species (cmes). the philippines national assessment levels were also carried out following treadaway (1995) for 52 (91%) butterfly species. these were categorized into cnes (n = 28; 53%), rnes (n = 7; 13%), cpes (n = 5; 9%), rpes (n = 8; 15%), cmes (n = 2; 4%), and rare mindanao endemic species (rmes) (n = 1; 2%). it is noticeable that most of the species are unassessed globally and even some species nationally, thus, signifying the importance of the findings to the global and national platforms for the eventual global and national synopsis, especially on the local and national levels, because species assessment is considered important for any management planning on any forest reserves and protected areas (haribon 2017; pef 2008). as for the local assessment, 21 (37%) species were evaluated as cnes, 14 (25%) as rnes, 7 (12%) as cpes, 8 (14%) as rpes, 1 (2%) as cmes, and 2 (4%) as rmes (fig. 3). other noteworthy findings are the observation of the rare species. most importantly, the listing of the globally rare but observed to be locally common species such as jamides celeno lydanus (lycaenid), mycalesis mineus philippina (nymphalid), and eurema sarilalas arilata (pierid). the same pattern was observed for the following: jamides alecto manillana, jamides philatu osias, j. celeno lydanus, mycalesis micromede micromede, mycalesis tagala semiraza, ragadia melindena mindeninse, and eurema sarilalas arilata. these species were abundantly observed in the area but nationally assessed as a rare species. this observation entails that habitat has different dynamics, and it varies from one another and could support species in the various ecological spectrum. thus, indicating every ecosystem is unique and requires different conservation measures. biotropia vol. 29 no. 3, 2022 210 figure 3 distribution of butterfly status based on global, national, and local level of assessments notes: cnes = common non-endemic species; rnes = rare non-endemic species; cpes = common philippine endemic species; rpes = rare philippine endemic species; cmes = common mindanao endemic species; rmes = rare mindanao endemic species. the overall percentage of endemism was 31%. the finding is comparatively higher compared to the records of martinez and mohagan (2012) at the adjacent forest of the study site with 22% endemicity. the same observation was noted compared to the findings of nacua et al. (2015b) at la union and mohagan et al. (2018) at mt. pinamantawan with a percentage of endemism difference of 9 to 10%. as compared with the major forest reserves in the philippines such as mt. malindang (ballentes et al. 2006), mt. hamiguitan (mohagan & treadway 2010), and mimbilisan protected landscape (guadelquiver et al. 2019), the findings were observed to be closed with only 1 to 3% difference on its endemicity. this infers that the area is a relatively preferable habitat for endemic species and is comparable with other pristine environments. conclusion based on the findings, awasian water forest reserve is home to various butterfly species and has good ecological support. these supports span from multiple factors, especially on the perspective of plant community structure, which is vital to the survival of the organisms. endemism was relatively high as compared with other ecosystems and showed to be comparable with other pristine habitats. in the context of rarity, the habitat houses various globally and nationally rare species that require conservation attention. acknowledgments the authors are grateful to the department of environment and natural resources (denr) for the issuance of the gratuitous permit (gp). to tandag water district (twd) and the local government unit (lgu) for the clearance and logistic support. to mr. gerry bernadas, mr. aldrin dua, jhony boy, and venz joy verano for the support during the conduct of this study. sincerest gratitude is also extended to all the people who contributed to the success of the study. references aluri jsr, rao sp. 2002. psychophily and evolution consideration of cadaba fructicosa (capparaceae). j bombay nat hist soc 99(1): 59-63. 50 45 40 35 30 25 20 15 10 5 0 cnes rnes cpes rpes cmes rmes unassessed local national global butterflies of awasian water forest reserve – abao et al. 211 ansari na, ram j, nawab a. 2015. structure and composition of butterfly (lepidoptera: rhopalocera) fauna in surajpur wetland, national capital region, india. asian j 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asian j biodiv 3(1): 142-55. thomas ja. 2005. monitoring change in the abundance and distribution of insects using butterflies and their indicator groups. philos trans r soc lond b biol sci 360: 339-57. toledo jms, mohagan ab. 2011. diversity and status of butterflies in mt. timpoong and mt. hibok-hibok, camiguin island, philippines. jpair multidisciplinary journal 6: 103-16. treadaway cg. 1995. checklist of the butterflies of the philippine islands (lepidoptera: rhopalocera). nachr ent ver apollo 14: 7-118. treadaway cg. 2012. revised checklist of the butterflies of the philippine islands (lepidoptera: rhopalocera). nachr entomol apollo suppl 2012 (20): 1-64. widhiono i. 2015. diversity of butterflies in four different forest types in mount slamet, central java, indonesia. biodiversitas 16(2): 196-204. zapanta mrg, victoria jv, del rosario mpn, empasis mgd, gasat vjp, bonoan jm, ..., nacua ae. 2016. diversity of butterflies (rhopalocera) in bulusukan (san ildelfonso, bulacan, philippines). international journal of advanced engineering, management and science (ijaems) 2(9): 1477-83. biotropia no. 4, 1990/1991: 1-8 growth and rooting system of acacia mangium obtained by tissue culture iwan setiawan, m.i. umboh and supriyanto seameo biotrop, p.o. box 17, bogor, indonesia introduction since 1980/1981, the government of indonesia through the ministry of forestry has started to reforest logged-over, alang-alang, unproductive areas and to convert them to forest industry plantation. the target is 300 000 ha per year. it means, 750 million seedlings should be provided per year (planting distance 2 m x 2 m). the tree species to be planted in forest industry plantation should have shorter life cycle (8 10 years), good stem-form, good rooting system, and should be fast growing. acacia mangium has been selected as one of the important tree species for forest industry plantation due to its growth, quality of fiber wood (pulp and paper industry) and rooting system (produce a lot of secondary root and nitrogen fixater) (soebardjo 1986). the reforestation of logged-over dipterocarp forests in malaysia with a. mangium has also been considered (appanah and weinland 1989). generally, reforestation with a. mangium is done with seedlings obtained by seed germination. a. mangium produce a lot of seeds but its production is still limited by the season, while the conventional method of vegetative propagation through cuttings gave very low percentage of rooted-cuttings (1%) (umboh and syamsul yani 1989). the micropropagation of a. mangium through tissue culture is a promising method. the production of a. mangium plantlets through that method has been done at the forest genetic laboratory, tropical forest biology, seameo biotrop (situmorang 1988, umboh 1988, umboh et al. 1989, 1990). these rooted-plantlets (plantlings) were first put in the green house (acclimatization) before planting in the field. field tests of some agricultural plants have been done but information on forest trees species is still lacking because the production of plantlings through tissue culture is still limited as there are still problems of their rooting. in fact, the progress of reproducing woody plants by tissue culture has been much slower than with herbaceous plants. the major reason for this limited success with forest trees appears to be due to lack of efforts and because it takes longer time than herbaceous plants (durzan and campbell 1974). generally, rooted-cuttings or rooted-plantlets do not produce tap root, but secondary roots only. the most frequent origin of adventitious roots are the cambium, phloem and pericycle. it is less frequent for 1 biotropia no. 4, 1990/1991 the cortex, pith and xylem (haissig 1974). the time and place of initiation of induced root primordia vary greatly among species (kramer and kozlowski 1979). the rooting system will influence the growth pattern of the rooted-cuttings or plantlings. the objective of this study is to observe the growth and rooting system of a. mangium obtained by tissue culture in the field. the information gathered will be used for sylviculture and genetic study of a. mangium plantlings. materials and method the plantlings of a. mangium were obtained by tissue culture method described previously by umboh and syamsul yani (1990). during the acclimatization period, the plantlings were planted in mixed soil-sand medium (1:1) for one month and green house conditions. after 4 months in the green house, the plantlings were transferred to the field (figure 1). forty plantlings (50-60 cm high) were planted in the field at 3 m x 3 m planting distance. to avoid strong direct sunlight, simple shading with alang-alang leaves was implemented. figure 1. four-month old a. mangium plantlings before planting in the field. the experimental site is located at the forest research and development experimental garden, cikarawang, darmaga, bogor. the area was dominated by imperata cylindrica (alang-alang), open area, 244 m a.s.l. and 3552 mm/year rainfall. the soil type is red brown latosolic and is very fertile up to 1 m soil depth. 2 growth and rooting system of acacia mangium-i. setiawan, m.i. umboh & supriyanto the height and diameter growth rate were measured once a month. the clear-bole height was also recorded. to confirm its rooting system, two trees were dug and compared with other a. mangium trees obtained from seed germination, of the same age, at 2 m x 2 m planting distance, situated 150 meters from the field test and planted by the forest research and development center, gunung batu, bogor. this plantation is designated for provenance trials of a. mangium. results and discussion the height and diameter growth rate of a. mangium plantlings are shown in figures 2 and 3. during the 2.5 years of field test, some important developments were observed: during the first six months, the plantlings were trying to adapt to field conditions. the mean height growth was slow (0.5 m whithin 6 months) and diameter increment was very low. during the second six months the stems were more vigorous and some branches were formed. the mean increase in height was 1.5 m within 6 months. it was figure 2. height growth of a. mangium tree obtained by tissue culture. a : growth during adaptation period b : growth after adaptation period c : growth during maturation period 3 biotropia no. 4, 1990/1991 figure 3. diameter growth of a. mangium tree obtained by tissue culture. faster than during the first six months and the mean diameter increment was more evident (2.5 cm within 6 month). after one year of planting, the mean height increased remarkably (3.5 m within 6 months) and 4.5 cm within 6 months for mean diameter growth rate. many branches at the lowest level broke off naturally (natural pruning). the natural pruning of branches can reach up to 3/4 of the tree height among seed germinated trees because the planting distance is very close (figures 4a and 4b). since the planting distance among the a. mangium plantlings was greater, lower natural pruning (1/2 of tree height) was observed together with more branching (figure 5a). the mean height, diameter and height clear-bole of a. mangium plantlings after 2.5 years of field test were 10.3 m, 15 cm and 5 m, respectively. in the philippines, the height of 3-year old a. mangium (from seed germination) reached up to 8.3 m and 9.3 cm in diameter, without mentioning its soil type. while in bangladesh, a. mangium (from seed germination) planted in good soil condition grew up to 8 m in height and 15 cm in diameter (soebardjo 1986). the height and diameter growth of a. mangium obtained by tissue culture were better than those obtained from seed germination. this might be caused by its rooting system and soil fertility. a tree has a good rooting system if the secondary roots develop well and has some vertical roots. the volume of soil occupied by the roots is an important factor in determining the amount of minerals and water available to trees. roots of forest trees tend to 4 growth and rooting system of acacia mangiumi . setiawan, m.i. umboh & supriyanto figure 4a. plantation of a. mangium, 2.5 years old, 2 x 2 m planting distance, obtained from seed germination. provenance trial at the research institute of forestry cikarawang, bogor. figure 4b. the rooting system of a. mangium obtained from seed germination, 2.5 years old, planting distance 2 x 2 m. 5 figure 5a. the plantation of a. mangium produced by tissue culture, 2.5 years old, 3 x 3 m planting distance. form a dense mat on the surface of soil intercepting minerals released by decaying litter (russel 1973). based on the observations of the root formation and development, a. mangium trees obtained by tissue culture produce a lot of lateral roots (secondary roots) but no tap root. three to four adventitious roots initiated in vitro developed as main roots and some vertical roots were also formed (figure 5b). the number of secondary roots (bigger than 0.5 cm diameter) was 20-30. in the nursery, the formation of secondary roots can be stimulated by cutting the tap root (abod 1984). a. mangium trees obtained from seed germination formed tap roots and less lateral roots. since the tap root is still being formed, the formation of lateral roots was inhibited. consequently, the mineral absorbtion in this system is less efficient than in trees obtained by tissue culture. sylviculturally, this field test gives better understanding of the growth pattern, rooting system and ecological adaptation of a. mangium produced by tissue culture. since its growth (height and diameter) and rooting system are very good, seedlings production through tissue culture should be used in reforestation programme specially for forest industry plantation. 6 biotropia no. 4, 1990/1991 figure 5b. the rooting system of a. mangium obtained by tissue culture, 2.5 years old, planting distance 3 x 3 m. these results should be supplemented either by cytological or electrophoretic studies before proceeding to the clone trees in bigger quantity because most seedlings obtained by tissue culture (plantlings), specially callus culture, produce high genetic variability (d'amato 1975; berljak 1984). from the wood technology point of view, the wood qualities of a. mangium that need to be examined include hardness and the fiber length. since tissue culture of forest trees is relatively new, there is little information on wood quality of trees produced by tissue culture. the main constraint is the fact that forest trees take longer time to grow than agricultural plants (fruit trees). fortunately, the life cycle of a. mangium is very short compared to other forest trees (6 — 8 years for albizia falcataria, 35 year for dipterocarps, 6-100 years for tectona grandis). since a. mangium is used also for pulp and paper industry, the examination of fiber quality is also needed, i.e. possible improvement of fiber length. conclusions 1. 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(3/4): 179-184. umboh, m.i.j., syamsul a. yani and j. situmorang. 1990. pengadaan bahan tanam acacia mangium dan eucalyptus urophylla dari beberapa pohon pilihan di hutan tanaman subanjeriji. in: seminar on biotechnology for forestry, held by pau, university of gadjah mada, 12-13 february 1990, wanagama, yogyakarta. 8 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf biotropia vol. 29 no. 1, 2022: 28 38 doi: 10.11598/btb.2022.29.1.1584 28 characterization and polyphasic identification of novel rhizobacteria strain isolated from sand dunes ecosystem ketut arte widane1, annisaa widyasari2 and endah retnaningrum3* 1undergraduate student, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2graduate student, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 3microbiology laboratory, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia received 9 april 2021 / accepted 3 december 2021 abstract the coastal sand dune ecosystem at the parangtritis coast of yogyakarta, indonesia has unique characteristics such as low moisture sandy soil, high salinity and low nutrient content. fimbristylis cymosa is one of the plant species having the capability to survive in that unique ecosystem. in this study, rhizobacteria isolated from the rhizosphere of f. cymosa were isolated to be further analyzed on their phosphate solubilizing and antagonistic properties against fusarium oxysporum which cause the wilt disease. the isolates of phosphate solubilizing rhizobacteria (psr) having the most potential capabilities were then polyphasically identified based on phenotypic and genotypic characters followed by 16s rdna sequencing. the results showed that four psr isolates (i8, i11, i12 and i24) have high phosphate dissolution indices. the highest indices were observed in isolates i11 (3.08) and i12 (3.44), respectively. analysis of the dual plate experiments for psr i11 and psr i12 isolates against the growth of f. oxysporum also showed quite high inhibitory activities, i.e., isolate psr i11 was 42.40%, while isolate psr i12 was 42.08%. the two isolates were polyphasically identified as burkholderia dolosa. this study clearly showed that psr i11 and psr i12 isolates are very potential and prospective to be used as marginal land inoculants and as providers of phosphorus. this study also showed that the isolates are useful as biocontrol agents against f. oxysporum in plants. keywords: inhibitory activity, phosphate dissolution index, phosphorus, polyphasic identification, sandy soil introduction coastal sand dunes are aeolian landforms commonly found in coastal areas at all latitudes in the world. indonesia has coastal sand dunes located in the southern part of java island, extending from the southern coast of west java province to yogyakarta province. the most significant sand dunes formation occurs in the parangtritis coastal area, located on the southern coast of yogyakarta province. the sand dunes ecosystem has a high temperature, relatively little vegetation, strong winds, high salt content and very low groundwater content, making it a dry and infertile area (mahdavi & bergmeier 2016; campos et al. 2020). in general, plants are difficult to grow in dry and less fertile areas. however, there are several groups of plants that can survive in the sand dunes habitat. among plants that survived the sand dunes habitat on yogyakarta’s southern coast is fimbristylis cymosa, a family of cyperaceae, which grows well and is widespread along with the coastal sand dunes habitat. the growth and development of f. cymosa in the coastal sand dunes habitat are strongly influenced by the availability of essential soil macronutrients. one of the main macronutrients supporting the growth and development of f. cymosa is phosphorus. phosphorus is among minerals needed at 0.2% dry weight of f. cymosa, as a component of nucleic acids (dna and rna) for conserving energy. plants absorb phosphorus in the form of phosphate anions (po4 or po4 2-) which are dissolved in groundwater. however, phosphate anions have *corresponding author, email: endahr@ugm.ac.id novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 29 a high tendency to bind with calcium, aluminum, and iron in the soil, which makes them insoluble and easily settled so that they cannot be absorbed by plants, including f. cymosa (yang et al. 2021). undissolved phosphorus in the soil can be dissolved by the phosphate solubilizing bacteria (psb) group. a study conducted by liu et al. (2015) in calcareous soil found psb genera bacillus, pseudomonas, rhizobium and acinetobacter. pastore et al. (2020) reported psb genera burkholderia, bacillus, and pseudomonas in forest soils. in the mangrove forest rhizosphere, teymouri et al. (2016) observed the existence of psb genera bacillus, pseudomonas and acinetobacter. rasul et al. (2019) obtained psb genera pseudomonas and bacillus from rhizospheric paddy field soil, while the psb genus paenibacillus was found from wheat rhizosphere (cherchali et al. 2019). in soil environments, there are also soilborne pathogens causing plants infection. one species of a soil-borne pathogen is fusarium oxysporum causing the fusarium wilt disease in various types of agricultural crops within the solanaceae family, such as tomatoes, potatoes, and chilies (ávila et al. 2019; srinivas et al. 2019; chowdhury et al. 2020). the wilting phenomenon that occurred in those crops due to fusarium wilt disease resulted in a significant reduction in crop yields and has been a major problem for agriculture worldwide. the use of chemical compounds for treating the wilt disease has adverse effects on non-target organisms. therefore, it is pertinent to find biocontrol agents which is considered safer for the environment. in soil environments, phosphate solubilizing bacteria (psb) are reported to be more dominantly existing in the rhizosphere. in addition, psb from the rhizosphere is known to produce active metabolites compared to other sources and is able to increase plant growth and development. psb originating from extreme environments such as sand dunes, might have specific characteristics as a biocontrol agent against f. oxysporum (ahluwalia et al. 2021; rasool et al. 2021) therefore, this study aimed to: 1. isolate and characterize phosphate solubilizing bacteria (psb) from the rhizosphere of f. cymosa living in sand dunes habitat; and 2. screen the capability of isolated psb as a biocontrol agent against f. oxysporum. materials and methods rhizosphere soil samples and isolate of f. oxysporum rhizosphere soil samples from the planting area of f. cymosa were taken and collected from 8 different sampling points along with the coastal sand dune habitat in the parangtritis coastal area, yogyakarta. the roots of the f. cymosa were carefully extracted with a shovel. soil attached to the roots was collected in sterile plastic containers as rhizosphere soil samples. the samples were then stored at 4 oc for further isolation of phosphate solubilizing bacteria (psb). the isolate of fusarium oxysporum was obtained from the culture collection of the microbiology laboratory, faculty of biology, universitas gadjah mada, yogyakarta, indonesia and was grown in potato dextrose agar (pda) medium at 30 oc for 10 days. psb isolation and phosphate dissolution index analysis ten (10) g of rhizosphere soil samples were poured into a 50 ml erlenmeyer and suspended in 90 ml of sterile distilled water, then shaken using a vortex at a speed of 200 rpm for 30 min. the sample suspension was diluted in a series of up to 10-4 dilutions. a total of 0.1 ml of the respective 10-3 and 10-4 diluted sample suspensions was inoculated on the national botanical research institute's phosphate (nbrip) agar medium by means of the spread plate method, then incubated at 30 oc for 48 72 hours. the nbrip medium consists of 1% glucose, 0.5% ca3(po4)2, 0.5% mgcl2, 0.01% (nh4)2so4, 0.0.5% mgso4.7h2o, and 0.02% kcl (nautiyal 1999). psb isolates having a clear zone around the colony were then re-isolated as psr, and purified on an nbrip medium. in order to measure the phosphate dissolution index, the psr isolates were recultured in nbrip liquid medium and incubated at 30 oc for 48 h. forty (40) μl of the incubated culture was diluted until reaching a colony having cell number 108 cfu/ml. the liquid culture was then poured into a petri dish containing nbrip agar medium by means of the pour plate method and incubated at 30 oc for 48 h. biotropia vol. 29 no. 1, 2022 30 the formed clear zone and the size of the bacterial colony were calculated. the phosphate dissolution index was determined with the following formula developed by haile et al. (2016): psi = (hzd + cd) cd where: psi = phosphate solubilization index hzd = halo zone diameter (mm) cd = colony diameter (mm) antagonistic activity of psr against f. oxysporum in vitro test of psr antagonist activity against f. oxysporum was carried out by using the dual plate experiments method according to zhang et al. (2017) with minor modifications. potato dextrose agar (pda) medium was prepared on a 9 cm diameter petri dish, followed by making a 5-mm diameter well in the pda medium at the center of the petri dish by using a cork borer. the other four 5-mm diameter wells were made at a distance of 2 cm from the well located at the center of the petri dish and from each other. f. oxysporum mycelium with a diameter of 5 mm was taken from the outermost part of the 10day-old fungal colony and was inoculated into the well at the center of the petri dish containing pda medium by using an inoculation needle. a total of 8 µl of psr isolate suspension with a cell number of 109 cfu/ml was inoculated into the other four wells in the same petri dish and then incubated at 30 °c for 10 days. the control agar plates were then prepared with the same agar medium without inoculating the psr isolates suspension. at the end of the incubation time, the diameter of f. oxysporum colonies was measured diagonally, vertically and horizontally. the inhibition percentage of psr isolates against f. oxysporum was determined using the following formula of royse and ries (1978): ip = (r1 r2) x 100% r1 where: ip = inhibition percentage r1 = diameter of f. oxysporum colonies on the control plate r2 = diameter of f. oxysporum colonies on the treated plate polyphasic identification of psr isolates polyphasic identification of psr isolates was determined based on a combination of phenotypic and genotypic characters. phenotypic characters of the observed isolates, including the morphological and biochemical characters, were determined by referring to bergey's manual of systematic bacteriology (brenner et al. 2005). the morphological characters were observed based on cell and colony morphologies. the cell morphology of psb isolates was observed based on the shape and characteristics resulting from the gram staining procedure. meanwhile, colony morphology of psb isolates was observed based on the shape, color, margins, elevation, internal structure and optical features of the colony. the biochemical characters were determined based on several tests, such as the motility, catalase, oxidase tests, glucose, sucrose, lactose fermentation tests, urease and voges-proskauer (vp) tests. the isolates were then characterized genotypically based on the 16s rdna nucleotide sequence. total genomic dna of psb isolates was extracted using the quickdnatm fungal/bacterial miniprep kit (zymo research, usa). pcr amplification of the 16s rdna gene of the psb isolates was analyzed by using 27f primers (5’-agagtttgatcct ggctcag-3’) and 1492r (5’ggttacctt gttacgactt-3’) (hossain et al. 2020). the pcr amplification was performed using a dna thermal cycler (bio-rad, usa) with the following program: pre-denaturation at 95 °c for 30 min, followed by 30 cycles consisting of denaturation at 95 °c for 30 sec, annealing at 57 °c for 1 min, extension at 72 °c for 1 min, and final extension at 72 °c for 10 min. the pcr products were purified and sequenced. the nucleotide sequences were then compared with dna sequence databases (genbank) through the blast program (blastn) at the national biotechnology information center (ncbi). a phylogenetic tree was constructed through the maximum likelihood method using molecular evolutionary genetics analysis x (mega version x). statistical analysis data were statistically analyzed using the oneway variance analysis (anova) followed by the duncan multiple range test (dmrt) approach for testing the mean differences. at p < 0.05, novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 31 the differences were considered significant. results are expressed as means of three replicates ± standard deviation. the software used was ibm spss statistics version 21. results and discussion psr isolate and phosphate solubilization index a total of 26 bacteria were isolated from f. cymosa rhizosphere soil samples in the sand dunes habitat of parangtritis coastal area, yogyakarta, indonesia. of the 26 rhizobacterial isolates, 4 rhizobacterial isolates were selected as psr based on the clear halo formation around the colony in the nbrip medium containing tricalcium phosphate. the clear halo was formed due to the production of organic acids or polysaccharides as a result of the phosphatase enzyme activity of the psr isolate (paul & sinha 2013; behera et al. 2017). the phosphate solubilization indices of each psr isolate after 10 days of incubation are presented in table 1 (widane et al. 2018). the highest phosphate solubilization index of 3.44 was obtained from psb i12. meanwhile, the lowest phosphate solubilization index of 3.00 was obtained from psb i24. a previous study by teymouri et al. (2016) reported the phosphate solubilization index of 3.5 which was obtained from bacillus sp. found in the rhizosphere of mangrove forest. mardad et al. (2013) reported the phosphate solubilization index of 3.5 which was obtained from enterobacter hormaechei found in the phosphate rock deposits. in contrast, several other researchers reported a lower-than-three phosphate solubilization index, such as those obtained from pseudomonas (2.6) and acinetobacter (2.0) found in the rhizosphere of forest (teymouri et al. 2016). the paenibacillus genus from the wheat rhizosphere was reported to have a very low phosphate solubilization index of 1.32 (cherchali et al. 2019). pseudomonas and serratia obtained from the rhizosphere of allium cepa l. were also reported to have low phosphate solubilization indices of 2.0 and 2.1, respectively (blanco-vargas et al. 2020). table 1 phosphate solubilization index of psr isolates obtained from the f. cymosa rhizosphere after 10 days of incubation no psr isolate phosphate solubilization index 1 i8 3.04 ± 0.51bcd 2 i11 3.08 ± 0.38cd 3 i12 3.44 ± 0.19d 4 i24 3.00 ± 0.43abcd notes: the index is presented as mean ± sd; numbers followed by the same letter in a column do not show a significant difference according to the duncan test (dmrt) at p < 0.05. different values of phosphate solubilization index among psr isolates are closely related to organic acids production, causing a ph decrease in bacterial cells and their environment, leading to proton substitution in the phosphate mineral and p release (ludueña et al. 2018; rasul et al. 2021). organic acids produced by several psrs include gluconic, glycolic, oxalic, malonic and succinic acids. gluconic acid is an organic acid having a role as a phosphate solvent (joe et al. 2018; santos-torres et al. 2021), which is useful in providing p minerals needed for plant growth (valetti et al. 2018; sahandi et al. 2019). psr i11 and psr i12 isolates have a high ability to dissolve phosphate in vitro. therefore, these two isolates are the potential to be developed as agents for providing p for plants. antagonistic activity of psr isolates against f. oxysporum psr i8, psr i11, psr i12, and psr i24 isolates were tested in-vitro for their antagonistic activity against f. oxysporum by using the dual plate technique. the results showed a significant inhibitory effect on the growth of f. oxysporum mycelium (fig. 1), which was clearly seen by comparing the inhibitory effect of the four isolates with the control against the growth of f. oxysporum. the four psr isolates clearly caused a smaller colony size of f. oxysporum compared to that in the control treatment. the colony diameter of f. oxysporum, however, was different among the four psr isolates, i.e., 44.75 mm was observed in psr i11 isolate, 45.00 mm in psr i12, 53.00 mm in psr i24 and 62.50 mm in psr i8 (widane et al. 2018). biotropia vol. 29 no. 1, 2022 32 a b c d figure 1 antagonistic activity of psr isolates against f. oxysporum observed in-vitro after 14 days incubation period notes: a = psr i8; b = psr i12; c = psr i11; d = psr i24. in addition, our study showed that psr isolates also caused thinner f. oxysporum mycelium growth compared to that in the control treatment. this explains the inability of f. oxysporum to grow well in the presence of psr isolates. the graph of the inhibition percentage of psr isolates against f. oxysporum is presented in figure 2 (widane et al. 2018). the dual culture method applied in this study allows direct in-vitro interactions between psr isolates and f. oxysporum. these interactions can be in the form of competition for space or for nutrients from the growth medium, leading to growth inhibition of f. oxysporum. growth inhibition can also be caused by metabolite compounds produced by psr isolates. several metabolite compounds are reported to have the most effective antagonistic activity in inhibiting the growth of plant pathogens in the form of antibiotics, siderophores and bacteriocins (khedhera et al. 2021). bacteria can produce hydrolytic enzymes such as protease, lipase, chitinase and glucanase which can lyse fungal cells so that they have antagonistic activity against plant pathogenic fungi (cui et al. 2019; lau et al. 2020). our study also showed that the psr i11 isolate was the most effective at inhibiting the growth of f. oxysporum with an inhibition percentage of 42.40%, while the psr i12 isolate had an inhibition percentage of 42.08% which is not significantly different from that of psr i11. lower inhibition percentages were observed for psr i24 and psr i8, i.e., 31.82% and 19.58%, respectively (fig. 2). figure 2 inhibition percentage value of psr isolates against f. oxysporum after 14 days of incubation period novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 33 several studies have also reported that bacteria isolated from the rhizosphere are able to protect plants from fungal pathogens (babu et al. 2015; panda et al. 2016). bacillus amyloliquefaciens from the rhizosphere of apple trees were reported to have inhibition capability against f. oxysporum with an inhibition percentage of 35% (guleria et al. 2016). meanwhile, research conducted by jangir et al. (2018) showed that bacillus sp. from the rhizosphere of tomato plants had antagonistic activity against f. oxysporum with higher inhibition percentage (70%) and therefore, is potential as a biocontrol agent against fusarium wilt disease in tomato plants. karthika et al. (2020) also isolated bacillus cereus from the rhizosphere of a tomato plant, which has an inhibition percentage of 66%. our study further showed that psr i11 and psr i12 isolates have the capability of phosphate solvent and have high antagonistic activity against f. oxysporum. polyphasic identification of psr isolates observation of the morphological characters of psr i11 and psr i12 showed different colony morphology forms, i.e., circular and irregular, respectively (table 2). however, the two isolates have the same colony color, elevation, internal structure, optical features and shape, such as cream, entire, convex, smooth, translucent and rod-shaped (table 2). biochemical properties of the two isolates, psr i11 and psr i12 showed similarities in terms of motility, production of catalase and oxidase enzymes and positive reaction toward the voges-proskauer test. different reactions occurred when being tested in a medium containing urea, showing that psr i11 isolate is able to produce urease, while psr i12 isolate is not able to produce urease (table 3). comparing the results from the identification of the morphological and biochemical characters with the characters described in bergey’s manual of determinative bacteriology, it is concluded that psr i11 and psr i12 isolates belong to the genus burkholderia (brenner et al. 2005). table 2 morphological characters of psr isolates from rhizosphere of f. cymosa no morphological characters psr isolate i11 i12 1. colony morphology shape circular irregular color cream cream margin entire entire elevation convex convex internal structure smooth smooth optical feature translucent translucent 2. cell morphology shape rod-shaped rod-shaped gram properties negative negative tabel 3 biochemical characters of two psr isolates from rhizosphere of f. cymosa psr isolate no biochemical characters i11 i12 1 motility test + + 2 catalase test + + 3 oxidase test + + 4 glucose fermentation 5 sucrose fermentation 6 lactose fermentation 7 urease + 8 voges-proskauer (vp) test + + biotropia vol. 29 no. 1, 2022 34 burkholderia spp. is reported of having the ability to dissolve phosphate and at the same time has antagonistic activity against f. oxysporum. simonetti et al. (2018) isolated burkholderia ambifaria from the rhizosphere of barley having a role as phosphate solvents and antagonistic properties against f. oxysporum. burkholderia cepacia and burkholderia contaminans with similar capabilities were isolated from the rhizosphere of maize (zhao et al. 2014; tagele et al. 2018). burkholderia sp. was also obtained from the rhizosphere of juçara palm (euterpe edulis mart) (de castilho et al. 2020). in addition to the plant rhizosphere, burkholderia spp. with the same properties is also obtained from the root nodules of fenugreek (trigonella foenumgraecum l.) (kumar et al. 2017). b. contaminans were also existed at the nodule of the common bean (phaseolus vulgaris) (tapia-garcía et al. 2020). apart from dissolving phosphate and having antagonistic properties against f. oxysporum, b. contaminans also has nitrogenase properties (silva et al. 2012). both psr isolates were also genotypically identified using the 16s rdna genetic marker. based on the 16s rdna gene sequencing, the psr i11 and psr i12 isolates had similarities with burkholderia dolosa strain lmg 18943b with a similarity index of 99.51% and 99.44%, respectively (table 4). the two psr isolates were categorized in the same species because the similarity percentage in the 16s rrna gene sequences was both ≥ 99% (schlaberg et al. 2012). phylogenetic trees based on 16s rdna sequences of psr i11 and psr i12 isolates were reconstructed by using comparative sequences with the in-group and out-group categories obtained from ncbi (table 4). some of the relatively close sequences of burkholderia spp. included burkholderia dolosa strain lmg 18943 (yarza et al. 2013), burkholderia latens strain r-563 (vanlaere et al. 2008), burkholderia multivorans strain struelens (bauernfeind et al. 1999), burkholderia vietnamiensis strain lmg 10929 (lipuma et al. 1999), burkholderia vietnamiensis strain tvv75 (viallard et al. 1998), burkholderia metallica strain r-16017 (vanlaere et al. 2008), burkholderia territorii strain lmg 28158 (de smet et al. 2015) and burkholderia seminalis strain r-2419 (vanlaere et al. 2008) as the in-group. meanwhile, alcaligenes faecalis subsp. parafaecalis strain g 16s was used as the out-group because the species is a classlevel classification group betaproteobacteria with burkholderia spp. in addition, alcaligenes sp. has the ability to dissolve phosphate and antibiosis properties against f. oxysporum (rasool et al. 2021). reconstruction of the phylogenetic tree was carried out using the maximum likelihood (ml) method with a substitution model (tamura-nei 93, g: gamma-distributed) and a bootstrap of 1,000 replications (fig. 3). this phylogenetic tree shows that both strains form clusters with burkholderia dolosa strain lmg 18943 in 83% bootstrap replications; therefore, reconstruction of these phylogenic trees can be trusted (hillis & bull 1993). the phylogenetic tree reconstruction of the two isolates was also in accordance with the blast results, which indicated that the two isolates could be identified as burkholderia dolosa. in addition, we already submitted the 16s rdna sequences of both strains (b. dolosa i11 and b. dolosa i12) to the genbank, with accession numbers of ok083732 and ok083731, respectively https://www.ncbi.nlm.nih.gov). tabel 4 identify sequences of psr isolates from the rhizosphere of f. cymosa based on the genbank data by using blast isolate accession number species of psr homolog identity query cover reference psr i11 nr_104973.1 burkholderia dolosa strain lmg 18943 99.51 % 99% yarza et al. (2013) nr_042632.1 burkholderia latens strain r-5630 99.30 % 99% vanlaere et al. (2008) nr_029358.1 burkholderia multivorans strain struelens 99.09 % 99% bauernfeind et al. (1999) nr_041720.1 burkholderia vietnamiensis strain lmg 10929 99.09 % 99% lipuma et al. (1999) nr_118872.1 burkholderia vietnamiensis strain tvv75 99.22% 98% viallard et al. (1998) nr_042636.1 burkholderia metallica strain r-16017 98.95% 99% vanlaere et al. (2008) nr_136496.1 burkholderia territorii strain lmg 28158 98.95% 99% de smet et al. (2015) nr_042635.1 burkholderia seminalis strain r-2419 98.74% 99% vanlaere et al. (2008) psr i12 nr_104973.1 burkholderia dolosa strain lmg 18943 99.44 % 99% yarza et al. (2013) nr_042632.1 burkholderia latens strain r-5630 99.16 % 99% vanlaere et al. (2008) nr_029358.1 burkholderia multivorans strain struelens 99.09 % 99% bauernfeind et al. (1999) nr_041720.1 burkholderia vietnamiensis strain lmg 10929 99.09 % 99% lipuma et al. (1999) nr_118872.1 burkholderia vietnamiensis strain tvv75 99.43% 98% viallard et al. (1998) nr_042636.1 burkholderia metallica strain r-16017 98.95% 99% vanlaere et al. (2008) nr_136496.1 burkholderia territorii strain lmg 28158 98.88% 99% de smet et al. (2015) nr_042635.1 burkholderia seminalis strain r-2419 98.81% 99% vanlaere et al. (2008) novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 35 figure 3 phylogenetic tree based on 16s rdna sequences constructed by the maximum likelihood method note: numerals at the nodes indicate the bootstrap value (%) derived from 1,000 replication. conclusion psr i11 and psr i12 bacterial isolates obtained from the rhizosphere of f. cymosa in the sand dunes habitat of parangtritis, yogyakarta, indonesia showed the capability to dissolve phosphate. the two isolates also showed an inhibition percentage of more than 40%, and therefore, can be used as biocontrol agents against f. oxysporum. psr i11 and psr i12 isolates were polyphasically identified as burkholderia dolosa. since the two isolates were obtained from f. cymosa living in sand dunes habitat, which is an extreme environment, this study can be further developed into determining the possibility of obtaining these two isolates in other marginal lands, to be used as phosphate solvent and 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[the capability of phosphate solubilizing bacteria and antipathogen fusarium oxysporum isolated from the rhizosphere of fimbristylis cymosa r.br. obtained in sand dunes habitat in parangtritis coastal area of yogyakarta]. final research project of undergraduate student. yogyakarta (id): universitas gadjah mada. https://pubmed.ncbi.nlm.nih.gov/?term=tapia-garc%c3%ada+ey&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=hern%c3%a1ndez-trejo+v&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=guevara-luna+j&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=rojas-rojas+fu&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=arroyo-herrera+i&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=meza-radilla+g&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=estrada-de+los+santos+p&cauthor_id=32585580 biotropia vol. 29 no. 1, 2022 38 yang f, sui l, tang c, li j, cheng k, xue q. 2021. sustainable advances on phosphorus utilization in soil via addition of biochar and humic substances. sci total environ 768:145106. yarza p, sproer c, swiderski j, mrotzek n, spring s, tindall bj, …, rossello-mora r. 2013. sequencing orphan species initiative (sos): filling the gaps in the 16s rrna gene sequence database for all species with validly published names. syst appl microbiol 36(1):69-73. zhang x, yingying zhou y, yan li y, fu x, wang q. 2017. screening and characterization of endophytic bacillus for biocontrol of grapevine downy mildew. crop prot 96:173-9. zhao k, penttinen p, zhang x, ao x, liu m, yu x, chen q. 2014. maize rhizosphere in sichuan, china, hosts plant growth-promoting burkholderia cepacia with phosphate solubilizing and antifungal abilities. microbiol res169:76-82. biotropia no biotropia no. 18, 2002 : 1 20 preliminary study on the palm flora of the lore lindu national park, central sulawesi, indonesia johanis p. mogea herbarium bogoriense, research center for biology, lipi, bogor. indonesia abstract the population size, structure, and composition of the palm flora in a 1350 m by 20 m rectangular plot in gunung potong and a 1500 m by 20 m rectangular plot in tongoa were measured. the total palm species from both plots numbered 33 represented by 8 genera. eight species, namely calamus omatus var. celebicus, pinanga caesia, arenga pinnata, daemonorops sp.3. calamus didymocarpus. calamus sp.4 (rapid spines), caryota mitis, andareca vestiaria have relatively high frequency values ranging from 5.46% to 10.66%. in addition, palm specimens previously collected from the park were examined at herbarium bogoriense to set up a preliminary checklist. so far, the national park is recorded as having 48 palm species represented by 11 genera which give figures of about 68% species and 58% genera of the total native sulawesi palm flora. though the number of endemic palms in sulawesi is high (72%), namely 51 out of total native 71 species, only two species are locally endemic to central sulawesi namely gronophyllum sarasinorum and pinanga sp. nov. 1 (longirachilla). so far only the latter species is endemic to the national park. key words: palm diversity/lore lindu national park/sulawesi/indonesia/endemic species introduction with emphasis on the sustainable use of biological diversity and its conservation, the indonesian-german research program had initiated a comprehensive interdisciplinary research funded by the german research council (dfg) for a duration of 15 years beginning in 2000. the program will be evaluated every three years. the research partners are two universities from germany namely, gottingen and kassel, and two from indonesia, bogor agricultural university (ipb, bogor) and university of tadulako (untad, palu). the overall goal is to identify factors which cause changes in forest margin areas. therefore, the program is known as "stability of rainforest margins" abbreviated as storma. in addition to the research program, a development of related sciences, manpower and laboratory equipment are also provided. the field of research consists of social and economic dynamics, water and nutrient turnover, biological diversity, and land use systems. the location used for the research is in and around the lore lindu national park, central sulawesi, indonesia (fig. 1). within this framework, an exploration of knowledge on palm diversity in and around the lore lindu national park is being conducted. the present paper reports a preliminary study, based mainly on the field observations made during the two weeklong field trips to the national park in november 2000 and march 2001, as well as examination of earlier herbarium collections at herbarium bogoriense. 1 biotropia no. 18, 2002 figure 1. valley study sites map in and around the lore lindu national park, central sulawesi 1. palolo, 2. sopu, 3. napu, 4. besoa, 5. bada, 6. kulawi, and 7. lindu. mountains and others: n: gunung nokilalaki, m: gunung malemo, b: boundary of the national park,f: foot path, r: road. location: km: kamarora, ll: lore lindu national park, pl: palu, pp: gunung potong plot, ra : rahmat, ro : rompo, sa : sadaunta, si : sintuwu, to : tongoa,tp : tongoa plot, tr: torire, ts: transmigration sites, ws: wuasa. 2 preliminary study on the palm flora of the lore lindu national park johanis p. mogea the lore lindu national park covers 217 000 ha of lowland and montane rainforest. approximately 70% of the park lies between elevations of 1000 to 1500 m, and less than 10% is below 1000 m. the highest place located south of tongoa is the summit of gunung (mount) nokilalaki at elevation 2355 m, the other summit is located south of the park namely gunung malemo at 2263 m (mogea & suhardjono 1981). three big rivers drain the park, namely lariang, gumbasa, and sopu. the headwaters of lariang drain at the eastern part of the park, then flow to the south and round to the west then to the north of the park. near gimpu, the river divides into two branches, one to the west ending in the west coast in south sulawesi, the other branch to the north of the park joins the gumbasa river and ends at the coast near palu. the upper gumbasa river is the outlet of lake lindu. it flows to the north around kamarora and sintuwu then unified with lariang. the lake is located at west of gunung nokilalaki. at the eastern north of the park flows sopu river, its lower reaches join at the gumbasa river. two enclaves in the park are excluded from the national park, namely lindu and besoa valleys. five other valleys are located around the park, namely palolo, sopu, napu, bada, and kulawi. there are two moderate roads available for large and public vehicles. the west is a route from palu to gimpu through kulawi valley outside the park. the east road is a route from palu to doda. the road segments near tongoa to wuasa, and torire to doda are through the eastern part of the park. the road from wuasa to rompo in november 2000 was badly damaged, therefore it can only be transversed by a four-wheel drive vehicle (fig. 1). further basic information of the park is presented in the indonesian program information sheet of the nature conservancy (tnc 2000). materials and methods the sites selected for the study were at tongoa, gunung potong, rompo, moa, gimpu, and sadaunta (fig. 1). a national park topographical map, 12xl garmin gps, and altimeter were used to locate the site and to measure the elevation. paln\ diversity was analyzed in each site using a modified mueller-dombois & ellenberg (1974) rectangular plot transect. the transects were established from the lowest elevation on each site to the highest evaluation preferably following an existing foot path. for these two trips, palm diversity was studied in two sites. the first site was in a new cruising transect of gunung potong from 850 m to 1250 m altitude, on the plot of 1350 x 20 m; and the second site was in tongoa on a foot path to the summit of gunung nokilalaki, at an average altitude of 750 m, on the plot of 1500 x 20 m. each plot consisted of many rectangular subplots of 50 x 20 m for observation of either solitary or clustered young and mature palms. for the sapling's study, each subplot consisted of one sapling square plot of 5 x 5 m and one seedling square plot of 1 x 1 m for observation of seedling coverage. through this study, species density, frequency, cover dominance, important value, structure, composition, and palm 3 biotropia no. 18, 2002 population dynamics can be calculated. during the field observation, voucher specimens were made. herbarium specimens, ripe fruits, and living seedlings were collected when necessary, particularly for proper identification and ex. situ conservation. herbarium duplicates are deposited in herbarium celebense (ceb) at the university of tadulako in palu, herbarium bogoriense (bo), netherlands national herbarium leiden branch (l), and herbarium of the royal botanic gardens kew (k). palm collecting followed standard methods; for rattans a method introduced by dransfield (1974) was followed. palm classification is in accordance with the genera palmarum which was introduced by uhl & dransfield (1987). temporary herbarium specimen preservation followed a modified schweinfurth technique (steenis 1950). identification was done in the field as well as at bo and ceb. to facilitate remembering unfamiliar or unidentified taxon in the field, a temporary nickname based on its significant morphological character was applied, hence in this paper a name like calamus sp.8 (tuberosus) or calamus sp.2 (ligule) is used. in addition, sketch drawings and digital photographs were made. field observations were supported by examination of 61 herbarium specimens at bo. new taxa will be described and published in separate papers. results and discussions during identification of palms from the study sites, it was found that 15 taxa of calamus and 4 taxa of daemonorops (table 2) need further identification particularly due to either incomplete herbarium specimens or lack of specimen types at bo, or a need for taxonomic revision. hence, naming these palms required three ways. the first is the taxon's name based on its morphological similarity to a related species, hence the epithet "aff." is used, such as calamus aff. c. zollingeri. the second is an expression of its significant character, such as when the taxon typically has long ligule, then the taxon is called calamus sp.2 (ligule). another example is where there is no spine on its knee (fig. 7-9), the taxon is called calamus sp. 1 (knee no spine). the third is when the epithet required long sentence to define the taxon, then it is called for example daemonorops sp.3 (table 2). some taxa are definitely new species, but their description is not published yet, hence the name is informal, such as calamus sp. nov.l (ahlidurii). the latter case is found as well as in caryota, gronophyllum, gulubia, and pinanga (tables 2 and 3). the number of species from both the gunung potong and tongoa plots is 33 which belong to 8 genera (table 1). calamus is represented by 20 species, among them are the well-known commercial rattans namely 'batang' (c. zollingeri), 'lambang' (c. ornatus var. celebicus), 'tohiti' (c. inops), and 'ombol' (c. symphysipus)', daemonorops represented by five species, among them is an edible young sweetest cabbage 'uwe manis' (d. macropterus); pigafetta represented by p. elata, an ornamental tree palm known as 'wanga' (fig. 6). this palm, for about three decades, was misidentified as p.filaris (dransfield 1998). 4 preliminary study on the palm flora of the lore lindu national park johanis p. mogea table 1. absolute (fa) and relative frequency (fr) of palms from 27 subplots in gunung potong (p) and 30 subplots in tongoa (t). subplot size is rectangular 50 m by 20 m 5 biotropia no. 18, 2002 6 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 7 biotropia no. 18, 2002 8 preliminary study on the palm flora of the lore lindu national park johanis p. mogea while the number of species in gunung potong and tongoa is the same (23 in 7 genera), the species composition and population sizes are different (table 1). pigafetta is only found in gunung potong and korthalsia only in tongoa. thirteen species were found in both gunung potong and tongoa, among them 8 species have high absolute and relative frequency values (table 1), meaning that those species occur in most of the 57 subplots (namely 27 subplots in gunung potong and 30 subplots in tongoa). calamus ornatus var. celebicus occurred in 41 subplots (10.66%), pinanga caesia in 40 subplots (10.40%, fig. 3 5), arenga pinnata in 33 subplots (8.58%), daemonorops sp.3 in 32 subplots (8.32%), calamus didymocar-pus in 29 subplots (7.54%), calamus sp.4 (rapid spines) in 29 subplots (7.54%), caryota mitis in 28 subplots (7.28%), and areca vestiaria in 21 subplots (5.46%). of the 10 species found only in gunung potong, calamus aff. c. paspalanthus is the most significant occurring in 9 subplots (2.34%). likewise 10 species were only found in tongoa, 4 species had much higher absolute and relative frequency values namely calamus aff. c. reinwardtii occurred in 18 subplots (4.68%), arenga undu-latifolia in 14 subplots (3.64%), daemonorops sp.l (didymophylla) in 12 subplots (3.12%), and korthalsia celebica in 10 subplots (2.60%). these figures on palm diversity may change as'a result of further identification and further study as more plots are established. outside the gunung potong and tongoa plots, other species of palms were found such as calamus macrosphaerion var. macrosphaerion and c. orthostachyus in gunung malemo and moa (kramadibrata & dransfield 1992); 'ronti' (calamus leiocaulis) and 'togisi' (c. leptostachys) in moa (siebert 1997). a study on the role of rattan resources in the moa and au in the southern part of the park has been done by siebert (1997). it revealed that 'togisi' (calamus leptostachys), 'ronti' (c. leiocaulis), 'lambang '(c. ornatus var. celebicus), 'batang' (c. zollingeri), and 'noko' (daemonorops robusta) are the important rattans which produced a quite good rural cash income. other palms outside the two above mentioned plots are caryota sp. nov.l (angustifolia) in gunung malemo (zumaidar 2001), this species is solitary as c. rumphiana (fig. 2); licuala celebica and livistona rotundifolia were common and widespread throughout the park (personal observation); pinanga sp. nov.l (longirachilla), pinanga sp. nov.2 (rubiginosa), and pinanga sp. nov.3 (tenuirachis) were found in and around gimpu (sinaga 2000). the rachillae of p. caesia and pinanga sp. nov.2 (rubiginosa) are set up in many planes, the leaf sheath is dull green to bluish deep green in p. caesia (fig. 2 4), while it is reddish orange in pinanga sp. nov.2 (rubiginosa). the rachillae of pinanga sp. nov.l (longirachilla) and pinanga sp. nov.3 (tenuirachis) are set up in one plane, the leaf sheath is yellowish green in pinanga sp. nov.l (longirachilla) and it is yellow in pinanga sp. nov.3 (tenuirachis). pinanga celebica was not listed in the current sulawesi palms (table 3) due to uncertainty of its existence. the story of this taxon started in 1871, when scheffer described pinanga patula var. celebica based on the specimen of riedel s.n. the label was annotated with the plant coming from gorontalo north celebes, but in 9 biotropia no. 18, 2002 2. solitary caryota rumphiana, stem 6 m long 3. solitary pinanga caesia, stem 7 m long, 12 cm in diameter 4. solitary pinanga caesia (living collection of mogea 7467), stem 10m long, 16 cm in diameter 5. top portion of pinanga caesia (living collection of mogea 7467) shows top portion of the stem, infructescences, and leaf sheath 80 cm long, 10 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 6. at the center are three tree palms of pigafetta data, the tallest is about 25 m 7. calamus sp.l (knee no spine; living collection of mogea 7466), leaf rachis 140 cm long, cirrus 100 cm long, white triangle indicates the rachillae bearing reddish brown fruit 8. top portion of infructescence of calamus sp. 1 (knee no spine, mogea 7466) bearing one ellipsoid reddish brown almost ripe fruit of 15 mm long and 7 mm in diameter 9. base portion of infructescence of calamus sp. 1 (knee no spine, mogea 7466) bearing many very young fruits, leaf sheath 15 mm in diameter 1876 he changed its status to pinanga celebica. he wrote that the plant clusters, had whitish leaves, a long inflorescence rachis with numerous rachillae, and obovoid fruits. this character diagnosis, however, is inadequate for identification to the species level as they are too general. moreover the clustered habit is not found in any of the east malesian pinanga (sinaga 2000). 11 biotropia no. 18, 2002 betel nut palm or 'pinang sirih' areca catechu and coconut palm or 'kelapa' cocos nucifera is often planted in a village garden, the former usually used for a living fence to mark boundaries. sugar palm or 'aren' or 'saguer' arenga pinnata is often grown in gardens around the park, and are often found on the forest border or in disturbed primary lowland and montane forest. hence, including these last mentioned palms, the number of species or taxa so far known to occur in and around the park is 48 represented by 11 genera (table 2) namely areca 2 species, arenga 2 species, calamus 26 species, caryota 3 species, cocos 1 species, daemonorops 5 species, korthalsia 1 species, licuala 1 species, livistona 1 species, pigafetta 1 species, and pinanga 4 species. including here are some other of unidentified genera (table 2 and appendix 1). as shown in appendix 1, many of the herbarium specimens are sterile, though it is still important to figure out the diversity but to describe or to identify up to the genus level is often inadequate. calamus are often difficult to separate from daemonorops when they are all still in the vegetative stage. on the other hand, the morphology of seedling, sapling, juvenile, and mature in the palm species is often distinct. therefore, adequate observation in the field, fertile and correct collection for the herbarium specimens is very necessary. if this field study is successfully done, it is believed that there might be some new taxa particularly of calamus and daemonorops, but also of pinanga, licuala and caryota. most calami in and around the park are clustering rattans but few of them are solitary such as in calamus inops, c. leptostachys, and c. symphysipus. stem diameter varies from 6-40 mm, with the leaf sheath diameter varying from 8 4 5 mm. the smallest cane is c. leiocaulis and the largest is c. zollingeri. petioles are mostly green, but are reddish brown in calamus sp.14 (mogea 7465, fig. 13). the leaf sheath and the petiole are covered by scarcely small spines such as in c. ornatus var. celebicus; but calamus sp.4 (rapid spines; mogea 7461) are covered by slender, rapid, greenish, large spines (fig. 10); in c. macrosphaerion var. macrosphaerion, by rapid, flat, triangular, blackish brown spines (fig. 12); and in calamus sp.14 (mogea 7465) by scarcely brown, flat, triangular comblike spines (fig. 13). the specimens of mogea 746.5 might be calamus sp. nov.l (ahlidurii) or daemonorops macropterus. when already matured, the identity of the specimen will be much easier to determine as the difference between calamus and daemonorops can only be seen after the flowering stage, namely the peduncular bract in calamus is tubular and attached to the rachis of the inflorescence while it is leafy-like and deciduous in daemonorops. calamus sp.2 (ligule; mogea 7456) has the only conspicuous ocrea or ligule (fig. 11). calamus in sulawesi are mostly cerriate, hence from this point the vegetative characters of calamus are not different from daemonorops. however, sometimes the species identity can be found based on the number of the leaflets on either side of the rachis, mostly daemonorops has regular (rarely in groups) and very narrow leaflets compared to calamus. the number of leaflets in daemonorops is mostly on the average between 40 to 80 leaflets, while in a small diameter cane of 12 preliminary study on the palm flora of the lore lindu national park johanis p. mogea calamus, the leaflets are very much less in number, usually 6 to 16 such as in calamus minahassae which has only 12 leaflets set up in 6 groups, each group consisting of one or two leaflets. when ripe, the fruit colour is pale greenish-white such as in calamus macrosphaerion (fig. 14) or glossy white (fig. 15) as in calamus sp. 1 (knee no spine) or black as in calamus zollingeri. native palms in sulawesi are represented by 71 species in 19 genera (table 3) based on observations from about 20 sites all over sulawesi (mogea 2000) updated by recent current examination of the specimens and recent field work. pending further identification, the number of genera and palm species in and around the park represents about 58% and 68% of genera and species of sulawesi's palm flora, respectively. metroxylon sagu was probably planted long ago for their edible carbohydrate and is considered here. as an introduced species. its original distribution is in moluccas and new guinea. sagu baruk (arenga microcarpa) has apparently been cultivated in district sangir talaud for 80 years already. its original distribution is the same as for the sago palm. sihombing et al. (1980) incorrectly identified it as a. obtusifolia. the sagu baruk covers about 19 890 ha or about 30% of the total agricultural area in the district (sihombing et al. 1980). 'salak pangu', salacca zalacca var. amboinense, was cultivated for some decades in pangu village, district minahasa, north sulawesi . the salak pangu now is popular as an edible fresh fruit in north sulawesi. it was said that the cultivation started in 1961. now salak pangu plantations cover about 654 ha with about 2 million individual plants (dannie 1999). other introduced palms are mostly indoor or outdoor ornamental plants. so far known, the number of the introduced palms in sulawesi is 20 (table 4), including the palm for plantation namely 'sawit' or oil palm elaeis guineensis. based on their origin, the introduced palms consist of 12 groups. these introduced palms were found in palu and on the way to the park. 13 biotropia no. 18, 2002 10. base portion of young calamus sp.4(rapid spines, living collection of mogea 7461) shows 50 cm long petioles and leaf sheaths covered by rapid slender long greenish white spines 11. top portion of young 1.5 m tall calamus sp.2 (ligule, living collection of mogea 7456) stows top of leaf sheaths bearing conspicuous young entire and old tattered ocrea 12. middle portion of calamus macrosphaerion var macrosphaerion (living collection of mogea 7470) shows leaf sheaths of 4 cm in diameter and petioles covered by rapid flat triangular blackish brown spines 13. base portion of young calamus sp. (living collection of mogea 7465) shows reddish brown petioles covered by scarcely black flat triangular combed-like spines 14 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 14. nearly top portion of infructescence of calamus macrosphaerion (living collection of mogea 7470) bearing pale greenish white almost ripe ellipsoid fruits of 10 mm in diameter 15. middle portion of infructescence of calamus sp. 1 (knee no spine, living collection of mogea 7487) bearing white ellipsoid ripe fruits 8 mm in diameter table 4. checklist of introduced palms in sulawesi and their origin 15 biotropia no. 18, 2002 1) actinorhytis calapparia (blume) h. wendl. & drude ex scheti. it is listed that native palms of sulawesi consist of 71 species and can be divided into 22 groups based on local and regional plant geographical distribution, namely tropical asia, malesia, borneo; north, central, south, and southeast sulawesi; unknown locality in sulawesi, and moluccas (table 3). the number of palm species endemic to sulawesi is moderately high (70.83%) namely 51 out of total native 71 species. however, only two species namely, gronophyllum sarasi-norum and pinanga sp. nov.l (longirachilla), are endemic to central sulawesi (table 3, group 7). the latter species is so far known only from gimpu (fig. 1) the south portion of the park (table 3 and appendix 1). conclusion this preliminary study on palms in and around the lore lindu national park, including observations on 1350 m by 20 m rectangular plot sites in gunung potong, a 1500 m by 20 m rectangular plot in tongoa, herbarium specimens collected from gunung nokilalaki, sopu valley, moa, and gunung malemo revealed that there are 48 species belonging to 11 genera. calamus and daemonorops are very dominant as shrubs and climbers. the largest genus in the park-is calamus with 25 species, followed by daemonorops (6 species) and pinanga (4 species). however, more 16 preliminary study on the palm flora of the lore lindu national park johanis p. mogea fertile collections of these genera are still required. it is therefore expected that some undescribed and locally endemic palms may still be found as calamus, daemonorops, licuala, caryota and pinanga. acknowledgments the author would like to express his sincere gratitude to storma for providing funds which made the conduct of this study possible. cordial appreciation is also addressed to the officers of lore lindu national park who have kindly provided facilities during the field work and the indonesian-german research team, particularly to dr. paul kesler. thanks are also due to dr. sri s. tjitrosoedirdjo of seameo biotrop for her suggestions to improve the manuscript, and dr. arie budiman, head of the research centre for biology, lipi, bogor, for allowing the author to conduct the study. references dannie, a.t.e. 1999. salak pangu "imigran" asal bali trubus 30 9358): 69. dransfield, j. 1974. a short guide to rattans. biotrop (seameo regional centre for tropical biology). bogor. mimeographed. 69 p. dransfield, j. 1998. pigafetta. principes 42 (1): 34 40. kramadibrata, p. & j. dransfield. 1992. calamus inops (palmae: calamoideae) and its relatives. kew bulletin 47 (4): 581-593. mogea, j.p. & suhardjono. 1981. struktur dan komposisi pepohonan di gunung malemo sulawesi tengah. paper presented at the national biological seminar xv.semarang indonesia, june 26 28. mimeographed. 14 p. mogea, j.p. 2000. status konservasi palem indonesia, khususnya sulawesi. paper presented at the national biological seminar xvi, bandung, july 25 27, 2000. mimeographed. 15 p. mueller-dombois, d. & h. ellenberg. 1974. aims and methods of vegetation ecology. new york, ca 160 p. siebert, s.f. 1997. economically important rattans of central sulawesi, indonesia. principes 41 (1): 42 -46. sinaga, n.i. 2000. pinanga blume (arecaceae) in east malesia. m.sc. thesis. bogor agriculture university, darmaga, bogor indonesia, 66 p. steenis, c.g.g.j., van. 1950. the technique of plant collection and preservation in the tropics. flora malesiana i (1): xlv ixix. 17 biotropia no. 18, 2002 the nature conservancy. 2000. lore lindu national park. indonesia program information sheet sppi. jakarta. leaflet 2 p. uhl, n.w. & j. dransfield. 1987. the genera palmarum. alien press. lawrence kansas. 610 p. zumaidar. 2001. taxonomy and species relationship of indonesian caryota (palmae). m.sc. thesis. bogor agriculture university, darmaga, bogor indonesia, 44 p. 18 preliminary study on the palm flora of the lore lindu national park johanis p. mogea appendix 1. specimens examined: note: plant name: locality, elevation, phenological state (st: sterile, ffl: female flower, mfl: male flower, fl: hermaphrodite flower, fr: fruit), date, collection number, deposited hersbarium, with the exclamation point means that the author has seen the specimens) areca vestiaria: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 in, fr.02-03-01, mogea 7473 (bo!, ceb!) arenga undulatifolia: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 in, st.02-03-01, mogea 7474 (bo!; ceb!) calamus didymocarpus : mt. nokilalaki, toro, fr.23-04-75, meijer 9465 (bo!). calamus inops: kulawi, moa, mt. malemo, mfl.24-10-77, mogea 1474 (bo!); ditto., mogea 1475 (bo!); ditto., mogea 1476 (bo!); ditto., mogea 1477 (bo!, k). tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, ffl.05-03-01, mogea 7490 (bo!; ceb!). calamus macrosphaerion var. macrosphaerion: kulawi, mt. malemo, fr.20-10-77, mogea 1347 (bo!). calamus minahassae: sopu valley, fr.05-05-79, de vogel 5212 (bo!, l). calamus omatus var. celebicus : sopu valley, fr.26-04-79, de vogel 5054 (bo!, l); ditto., 02-05-79, de vogel 5172 (bo!); kulawi, moa, fr.22-10-77, mogea 1411 (bo!). calamus orthostachyus: kulawi, moa, mt. malemo, fr.18-10-77, mogea 1329 (bo!, k, l), ditto., mogea 1330, mfl. (bo!). calamus symphysipus: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, st.02-03-01, mogea 7472 (bo!, ceb!). undeterminate calamus from tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m: calamus aff. c omatus, st.03-03-01, mogea 7478 (bo!, ceb!), ditto, mfl.03-03-01, mogea 7479 (bo!, ceb!). calamus aff. c. reinwardtii: path to the st.02-03-01, mogea 7476 (bo!, ceb!); calamus sp.4 (rapid spines), st.18-11-00, mogea 7442 (bo!, ceb!) = mogea 7461 (see at gunung potong alt. 950 m); calamus sp.7 (tattered ligule), st.03-03-01, mogea 7477 (bo!, ceb!); calamus sp.8 (tuberosus), fr.03-0301, mogea 7486 (bo!, ceb!); calamus sp.9 mfl.05-03-01, mogea 7488 (bo!, ceb!). undeterminate calamus from gunung potong, alt. 850 m: calamus sp.8 (tuberosus), st.26-02-01, mogea 7455 (bo!, ceb!). calamus sp.2 (ligule), st.27-02-01, mogea 7456 (bo!, ceb!). calamus sp.6 (soft white spine), mfl.01-03-01, mogea 7471 (bo!, ceb!). undeterminate calamus from gunung potong, alt. 950 m: calamus sp.4 (rapid spines), st.27-02-01, mogea 7461 (bo!, ceb!) = mogea 7442 (see at tongoa ); ditto., alt. 1200 m, mfl.01-03-01, mogea 7468 (bo!, ceb!). undeterminate calamus from mt. roroka, timbu: calamus sp.10, st.20-05-79, de vogel 5482 (bo!); ditto., fr. 13-05-79, de vogel 5334 (bo!). undeterminate calamus from sopu valley: calamus sp.ll, st.05-05-79, de vogel 5209 (bo!); st.05-05-79, de vogel 5218 (bo\). undeterminate calamus from mt. nokilalaki: calamus sp.12, st.22-04-75, meijer 9438 (bo!); ditto., st. 0405-75, meijer 10021 (bo!). undeterminate calamus from kulawi, moa: calamus sp.13, st.14-10-77, mogea 1290 (bo!); ditto., st. 1810-77, mogea 1324 (bo!), ditto., mogea 1325 (bo!); ditto., mogea 1326 (bo!); ditto., mogea 1328 (bo!); ditto., st.22-10-77, mogea 1433 (bo!) 19 biotropia no. 18, 2002 caryota sp. nov.l (angustifolia); kulawi, moa, mt. malemo, alt. 1200 m, fr.23-10-77, mogea 1441 (bo!); ditto., fl.+fr.24-10-77, mogea 1473 (bo!) daemonoropx macropterus: sopu valley, fr.22-05-79, de vogel 5326 (bo!); tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, st.05-03-01, mogea 7491 (bo!; ceb!) korthalsia celebica: kulawi, moa, mt. malemo, st.,23-10-77, mogea 1462 (bo!); ditto., mogea 1463 (bo!) pinanga caesia blume: palu: sopu valley, alt. 1000 m, fr. 12-04-79, de vogel 5064 (bo!); gunung potong, alt. 950 m, fr.28-02-01, mogea 7467 (bo!, ceb!); parigi, alt. 800 m, fr.17-04-75, meijer 9378 (bo!); kulawi, gimpu, moa, alt. 1200 m, st.19-10-77, mogea 1343 (bo!); ditto., fr.l 1-10-77, mogea 1271 (bo!); ditto., fr.l 1-10-77, mogea 1274 (bo!); ditto., mogea 1275 (bo!): mt. malemo, fr.23-10-77, mogea 1443 (bo!). pinanga sp. nov.l (longirachilla): kulawi, gimpu, alt. 600 m, f r . l 1-10-77, mogea 1275 (bo!); mt. malemo, alt. 1000 m, fr.l7-04-79, mogea 1443 (bo!). pinanga sp nov.2 (rubiginosa): east of tongoa, alt. 720 m, fr.02-03-81, johannxxal, nybom & riehe 126 (bo!). pinanga sp. nov.3 (tenuirachis): kulawi, alt. 600 m, fr. 11-10-77, mogea 1271 bo!). 20 biotropia no. 4, 1990/1991: 41-48 the role of haltica sp. (coleoptera: halticidae) as biological control agent of polygonum chinense kasno faculty of forestry bogor agricultural university, bogor, indonesia s. tjitrosemito and sun jay a tropical agricultural pest biology programme seameo biotrop, bogor, indonesia abstract the role of haltica sp. (coleoptera: halticidae) with emphasis on host specificity and damage potential in controlling polygonum chinense was evaluated under laboratory condition. starvation test of the weevil on 33 weeds and 14 crop plant species indicated that only 6 weed species were attacked: polygonum chinense, p. nepalense, p. barbatum, p. longisetum, ludwigia octovalvis and l. parennis with p. chinense as the most preferred host plant. preliminary damage potential test indicated that a population of 0, 1,2 and 3 pairs of adult weevil reduced the percentage of fresh weight increment of p. chinense by 0; 46.2; 74.7 and 75.5% respectively. field observations indicated that the larvae as well as adult weevils are potential biological control agents of p. chinense. further studies are, however, on the host-range of this weevil. introduction polygonum chinense l. is one of the weeds growing amongst tea plantations in west java but it has never been reported as a serious problem in the area. locally it is known as "titiwuan" (backer & slooten 1924) and in thailand it is called "phayaadong" (harada et al. 1987). it was reported to be distributed also from india eastward to japan and it grows mostly on highland or open forest, coffee and tea plantations (harada et al. 1987). although west java is classified as a populated province, the utilization of labour to control weeds manually in large tea plantations is considered costly. chemical control with herbicides is considered cheaper and more practical. on the other hand there is a strong demand to save the environment from any pollutant for better living. therefore, it is necessary to develop other control methods in line with minimizing the side effects of herbicides and lowering the cost. in the tea producing areas of west java, there are many kinds of insects associated with p. chinense. haltica sp. (coleoptera: halticidae) seems to be one of the most common and it causes considerable damage to the weed. a study on the potential role of the insect as biological control agent is needed. 41 biotropia no. 4, 1990/1991 materials and methods haltica sp. was collected from a tea plantation of ptp xii, gunung mas located about 30 km on the way from bogor to bandung. they were brought to biotrop laboratory at bogor for rearing and further observations on its life cycle, host specificity and damage potential. some behavioural aspects of haltica sp. were casually observed in the field. the insects were reared in petri dishes and fed with fresh cut leaves of polygonum chinense. the room temperature and relative humidity of the laboratory varied from 24-31°c and 48-82% respectively. starvation test of newly hatched larvae and newly emerged weevil of haltica sp. was done against 14 species of crop plants and 33 species of weeds (appendix 1). five heads of larvae and adult weevils in separate petri dishes were reared with a single fresh cut leaf of test plants. replacement of fresh cut leaves of the test plants was done every day during the test. p. chinense was used as the control. the response parameter of the insect on the test plants was mainly the presence of feeding scars. observation was done every 24 hours till most of the test insects died. when the test insects produced feeding scars, the test was continued till the sixth day to make sure that they can survive by consuming the cut leaves of the test plants. the positive test plants were tested further for food preference. preferential test using larvae of the insect was carried out against six species of weeds namely polygonum chinense, p. longisetum, p. barbatum, p. nepalense, ludwigia octovalvis and l. parennis. six heads of haltica larvae were released in petri dish containing six arranged pieces of the weed species. the inner space of each petri dish was equally divided into six radial sectors (figure 1) and each piece of fresh cut leaves of test plants was put into each sector. six heads of test weevil were released at the center of the petri dish to allow them to select freely the test leaves. the response parameter of the preferential test was the level of feeding scars on each test leaf. preliminary damage potential test of the weevils against p. chinense was carried out by releasing various numbers of adult weevils on 20 day-old plants. the average fresh weight of newly cut p. chinense before being transplanted into plastic pots was 14.30±2.27 gram and the average leaf number was 11.85 ± 1.59 pieces. the pots were 15 cm in height, 10 cm and 15 cm in bottom and upper diameters, respectively. the pots were fully filled with light soil. the potted plants were allowed to grow for 20 days. watering was done daily with tap water. to avoid attack of any insect, the potted plants were covered singly with screen cage. various numbers of weevils e.g. 0, 1, 2 and 3 pairs were released on a single pot plant and kept for ten days. the test was run following complete randomized design with 42 figure 1. six radial sectors of the petri dish used for preferential test. 5 (five) replications. the response parameter of the test was mainly based on the percentage of reduction of fresh weight compared with the control at the end of the test. results and discussion under laboratory conditions of 24 31 °c temperature and 48 82% relative humidity, respectively, the life cycle of the insect varied from 26 to 28 days. the incubation, larval and pupal periods were 5-6, 13-15 and 7 days, respectively. during the larval period, it molted three times, i.e. it had four larval instars. other biological data such as adult longevity and egg production of an adult female have not been precisely observed. however, it seemed that the adult longevity was more than a month and egg production of a female was more than a hundred. the adult females laid their eggs in groups of ten on the abaxial surface of the leaves of p. chinense. usually the early larvae feed on the outer tissues of the leaf where the adult female oviposited but when they have developed bigger, they feed on most of the leaf tissues. 43 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya biotropia no. 4, 1990/1991 the adult weevils feed mostly on leaves and soft stem tissues of p. chinense. the adults feed randomly but the larvae produced local spot symptom. both larvae and adults caused defoliation of p. chinense. starvation test of both larvae and weevils of haltica sp. against 14 crop species and 33 weed species showed that the insects feed on six of the test weeds: p. chinense, p. barbatum, p. nepalense, p. longisetum, ludwigia octovalvis and l. parennis. p. chinense was the most preferred by the larvae. the rank of preference of the larvae is presented in appendix 2. it seemed that the insect is polygophagous but feeds especially on polygonum spp. it is strongly suspected that p. chinense, p. barbatum, p. nepalense and p. longisetum contain the key feeding stimulant for haltica sp. biological control using insects has a risk of changing from control agent to pest of crop plants in the future. luckily in indonesia, there is no known crop plant belonging to polygonum sp. and ludwigia sp. there are some closely related insects to haltica sp. namely h. caerulea oliv, h. cyanea (weber) and h. caerulea which were reported as promising biological control agents of water primrose (ludwigia spp.). a total of 123 plant species have been tested by cibc (sankaran et al. 1967 cited by mangoendihardjo et al. 1977) which reported that these insects feed on nicotiana tabacum under laboratory condition (rao et al. 1977). h. cyanea has also been reported to feed on leaves of some forest trees such as ammonia baccifera, a. rotundifolia, terminalia myriocarpa in india (beeson 1941), and some food crops in indonesia (kalshoven 1981). haltica sp. that was observed in this study was formerly suspected as h. caerulea but due to the supporting data on its host range, the authors of this paper name it haltica sp. for the moment. the correct name of the insect will be confirmed later. preliminary damage potential test using weevils on p. chinense grown in plastic pots indicated that the insect produced serious damage on the weed. populations of 1, 2 and 3 pairs of adult weevil caused a reduction on the fresh weight increment of 46.19, 74.33 and 75.53%, respectively within the last 10 days of the 30-day experiment. conclusion and recommendations conclusion haltica sp. (coleoptera: halticidae) is a promising candidate as biological control agent of polygonum chinense. 44 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya recommendations 1. further studies are needed especially on its host range before deciding to recommend it as a biological control agent of p. chinense. 2. confirmation of the correct identification of the insect is necessary. references backer, c.a. and d.f.v. slooten. geillustreerd handboek der javaansche theeonkruiden en hunne beteekenis voor de cultuur. drukkerijen ruygrok & co. jakarta. beeson, c.f.c. 1941. the ecology and control of the forest insects of india and the neighbouring countries. dept. of agriculture. dehra dun. harada, j., y. paisooksantivatana and s. zungsontiporn. 1987. weeds in the highlands of northern thailand. project manual no. 3 nat. weed science res. inst. project. bangkok. kalshoven, l.g. 1981. the pests of crops in indonesia. revised edition by p.a. van der laan. pt ichtiar baru van hoeve. jakarta. mangoendihardjo, s., o. setyawati, r.a. syed and s. sosromarsono. 1977. insects and fungi associated with some aquatic weeds in indonesia. proc. sixth asian pacific weed science society conference. jakarta. rao, v.p., m. a. gani and t. sankaran. 1971. a review of the biological control of insects and other pests in south east asia and the pacific regions. techn. communication no. 6. cibc, trinidad. 45 biotropia no. 4, 1990/1991 appendix 1. response of the larvae and adults of ha/tica sp. on several test plants during starvation test no. plant species response amaranthaceae 1. amaranthus spinosus l. asteraceae 2. ageratum conyzoides l. 3. bidens pilosa l. 4. crassocephalum crepidioides (benth.) s. moore 5. chromolaena odorata (l.) r.m. king & h. robinson 6. eupatorium riparium reg. 7. eleutheranthera ruderalis poit. 8. mikania micrantha h.b.k. 9. tridax procumbens l. balsaminaceae 10. impatiens platypetala lindl. carparaceae 11. cleome rutidosperma dc cruciferaceae 12. nasturtium heterophyllum bl. cyperaceae 13. cyperus rotundus uphorbiaceae 14. phyllanthus niruri l. gramineae 15. imperata cylindrica (l.) raeuschel 16. paspalum conjugatum berg. 17. sporobulus berteroanus trin. melastomataceae 18. oxalis barrelieri l. 19. oxalis corniculata l. onagraceae 20. ludwigia octovalvis (jacq.) raven + 21. ludwigia perennis l. + 22. ludwigia peruviana (l.) polygonaceae 23. polygonum barbatum l. + 24. polygonum chinense l. + + + + 25. polygonum longisetum de br. + + 26. polygonum nepalense meissn. + + + 46 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya appendix 1. (continued). no. plant species response rubiaceae 27. borreria data (aubl) dc 28. borreria laevis griseb. 29. diodia sarmentosa swartz. verbenaceae 30. lantana camara l. 31. stachytarpheta indica (l.) crop species amaryllidaceae 1. a ilium cepa l. 2. allium fistulosum l. araceae 3. colocasia esculenta (l.) brassicaceae 4. brassica oleraceae l. convolvulaceae 5. ipomoea batatas poir. gramineae 6. oryza sativa l. 7. zea mays l. leguminosae 8. arachys hypogea l. 9. glycine max (l.) 10. phaseolus vulgaris l. musaceae 11. musa paradisiaca l. solanaceae 12. capsicum annuum l. 13. solarium lycopersicum l. theaceae 14. cammelia sinensis (l.) o.k. note: — no feeding scars + feeding scars present. 47 biotropia no. 4, 1990/1991 appendix 2. response of larvae of haltica sp. on several weed species during preferential test no. weed species response 1. polygonum chinense + + + + 2. p. nepalense l. + + + 3. p. longisetum de br. + + 4. p. barbatum l. + 5. ludwigia octovalvis (jacq.) raven + 6. l. perennis l. + 48 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf microsoft word 1 biotropia no. 21, 2003 : 1 10 isolation and characterization of a novel benzoate utilizing serratia marcescens dwi suryanto1 and antonius suwanto2'3* 'department of biology, faculty of mathematics and natural sciences, north sumatra university, medan, indonesia . department of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia . 3seameo-biotrop, p.o. box. 116, bogor, indonesia abstract a new benzoate-utilizing strain, serratia marcescens ds-8, isolated from the environment was characterized. the strain was enterobacilli, gram negative, mesophilic, non halophilic, and aerobic bacterium that showed motile ovalerod shaped cells. the isolate produced extracellular chitinase, protease, and prodigiosin (a red pigment produced by several serratia strains yielding bright red or pink colonies). a physiological assay using microbact* test showed that the strain was closely related to klebsiella ozaenae (49.85%) and serratia liquefaciens (24.42%), respectively. however, 16s rrna sequence analysis indicated that the strain was closely related to s. marcescens dsm 30121 with similarity level of 98%. ds-8 strain was able to synthesize its own vitamins. optimum growth in benzoate was obtained at ph between 7-8.5 and nacl concentration of 1-1.5% (w/v). the isolate could grow in benzoate-containing medium up to 10 mm. other carbon sources that could support the growth of ds-8 were casamino acid, glutamate, glucose, acetate, potato starch, and ethanol. keywords: serratia marcescens/aromatic degradation/168 rrna sequence introduction a large amount of monocyclic hydrocarbon aromatic and its derivatives, as well as polycyclic hydrocarbon aromatic, have been deliberately introduced to the environment. many of these compounds, particularly the chlorinated derivatives, are toxic and carcinogenic even at low concentrations. the metabolism of these hydrocarbon aromatic compounds in nature depends on the ability of catabolic reaction of particular microorganisms (semple and cain 1996). of these microorganisms, bacteria of the genera such as alcaligenes, bacillus, pseudotnonas, bulkholderia, and rhodococcus (guerin and boyd 1995; lenke et al. 1992; mars et al. 1996; shen and wang 1995) are the main degrading microbes. several other microorganisms such as fungus (gurujeyalakshmi and oreil 1989), and algae (semple and cain 1996) have also been reported to aerobically catabolize aromatic hydrocarbons. one of the important monocyclic aromatics introduced to the environment is benzoate. it has been introduced through some herbicides application or other industrial 'corresponding author, e-mail: asuwanto@indo.net.id biotropia no. 21, 2003 practices (werwath et al. 1998). hence, it is also found as one of the important intermediate in metabolic pathway of many aromatic compounds (powlowski and shingler 1994). the increasing need of this aromatic compound for industrial purposes and its application should make us aware of its environmental potential hazard. so far, a study on aerobic benzoatc and its derivative degradation has been done in acinetobacter sp. strain 4cb1 (adriaens et al. 1989), amycolatopsis and streptomyces spp. (grund et al. 1990), and pseudomonas (altenschmidt et al. 1993). in the group of enterobacteriaceae, klebsiella pneumonia was reported to degrade 3and 4-hydroxy-benzoate (suarez et al. 1991), and salmonella typhimurium was able to metabolize m-hydroxybenzoate and gentisate (goetz et al. 1992). diaz et al. (2001) noted the involvement of escherichia coli in the metabolism of phenylacetic acid, 3and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3hydroxyphcnylpropionic acid, and 3-hydroxycinnamic acid and amines (phenylethylamine, tyramine, and dopamine). in this study, another member of enterobacteriaceae, serratia marcescens strain ds-8 was found capable of using benzoate as its sole c-source. we would like to examine further the ability of s. marcescens ds-8 to utilize benzoate and to characterize its physiological properties as well as its taxonomic identity. materials and methods bacterial isolate and culture condition the isolate was previously isolated from household sewage water in bogor, west java, indonesia. screening was carried out by growing the isolate in modified salt medium (dong et al. 1992) supplemented with 5 mm na-benzoate as sole carbon source. the isolate was maintained in 12.5% glycerol at -70° c. benzoate and other c source utilization test benzoate utilization was determined by growing the isolate in a modified salt medium supplemented with or without vitamins with 5 mm na-benzoatc as a carbon source. escherichia coli top 10 was used as a control. to determine the degrading ability in different conditions, the isolate was grown in modified salt medium with different initial ph, nacl concentration, and benzoate concentration. growth in other c sources was performed in modified salt medium supplemented with vitamins with either 5 mm succinate, 5 mm glucose, 1% casamino, 5 mm citrate, 5 mm glutamate, 5 mm acetate, 3% ethanol, or 1% potato starch as carbon sources. cell, density in other c source utilization test and benzoate utilization test were measured turbidimetrically at 660 nm after 72 hours and 120 hours of incubation time, respectively. benzoate-utilizing serratia marcescens — dwi suryanto & antonius suwanto growth conditions, measurement of growth, and quantification of benzoate utilization all liquid cultures were cultivated aerobically in 250 ml erlenmeyer. cultures were grown in 200 rpm at 30°c. growth was measured turbidimetrically at 660 nm. benzoate concentration was measured at its absorption maximum of 276 nm using hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) following the establishment of standard curve relating benzoate concentration to uv absorbance (shoreit and shabeb 1994). for all inoculations, the seed cultures were taken from 2-day-old culture of modified salt medium with 5 mm na-benzoate as carbon source. the cultures were grown with the initial cell concentration of 5xl06 cell/ml. unless otherwise indicated, all media were adjusted to ph 7.2. benzoate solution was filter-sterilized. examination of cell morphology and physiological properties cell shape, motility, and gram staining were evaluated using a nikon ys2-t microscope. physiological characteristics were analyzed using microbact kit test (medvet science pty. ltd., adelaide, australia). test of production of extracellular protease and chitinase was monitored on salt medium agar supplemented with colloidal chitin and skim milk. emb agar was used for preliminary screening for enterobacteriaceae isolates. the appearance of the colonies was observed in luria bertani (lb) agar supplemented with either 50 (-ig/ml ampicillin, 50 ng/ml spectinomycin/streptomycin, 10 ng/ml trimethoprim, 5 mm benzoate, 5 mm salycilate, 5 mm gentisate, or 5 mm phenol. a test of growth in different temperatures was monitored on lb agar. amplification and sequencing of part of 16s rrna gene the 16s-rrna genes were pcr-amplified using specific primers of 63f and 1387r from genomic dna (200 ng) using ready-to-go pcr beads (pharmacia-biotech). modified phenolchloroform-isoamylalcohol treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. the total volume of pcr reactions (25 \i\) consisted of 1.5 u taq dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 \m of each dntps, and stabilizer including bovine serum albumin. the reaction was incubated in a gene amp pcr system 2,400 thermocycler (perkin-elmer cetus, norwalk, conn). part of the genes for 16s-rrna were sequenced to infer the closest related organism from ribosomal database project (rdp) maintained in the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit and purification with ethanol biotropia no. 21,2003 sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn.). construction of phylogenic tree cluster analysis of 16s-rrna gene was done using the computer program from european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of department of biochemistry, university of antwerp) was used to determine the relatedness in phylogenic tree based on the nucleotide sequences. results and discussion the new benzoate-utilizing bacteria, ds-8, described in this study was a motile, ovale-rod shaped, gram negative, mesophilic, non halophilic, and aerobic bacterium. instead of microscope observation, the motility could be seen by its swarming activity. on lb solidified with 1.2% agar, the strain swarm on the agar surface, colonized the entire plate with expansion rate of c.a. 19 mm/hours. eberl et al. (1999) also observed a similar behavior in s. liquefaciens mg1. an auto-induction phenomenon might be involved in this swarming activity (eberl et al. 1999;lindumefa/. 1998). based upon microbact test (table 1), ds-8 was closely related to k. ozaenae (49.85%) and s. liquefaciens (24.42%), respectively. however, the test might have no significant match to the available characteristics in microbact data of bacterial species. this might occur since the test is designed for hospital purposes. any other specific characters for isolates might not be incorporated in this detection kit. growth on emb media indicated that the isolate was a member of enterobacteriaceae. partial sequencing (c.a. 500 bp of the 5'-end) of 16s rrna gene of ds-8 showed that the isolate might likely be one species of s. marcescens dsm 30121 (98% of similarity) (figure 1). the isolate showed lower similarity (94%) to strain k. ozaenae. complete sequencing of the 16s rrna gene, however, should give more definitive information about the taxonomic position of the isolate. the isolate produced a red or pink pigmentation on lb agar, or on lb agar supplemented with ampicillin, and streptomycin and spectinomycin, trimethoprim, tetracyclin, skim milk, or chitin (table 2). on the former antibiotic-containing media, the pink color appeared later in the growth stage. no red or pink color was produced when grown in media containing 1 mm phenol, 5 mm salycilate, 5 mm gentisate, or in lb agar supplemented with gentamicin. the red pigment might be prodigiosin, a red pigment produced by the genus of serratia. we did not examine whether the media affected the prodigiosin production, or a quorum sensing might take an effect. other interesting physiological traits were that the isolate produced extracellular chitinase and protease. a study on genetic and cloning of gene encoding chitinase of s. marcescens has been done by watanabe et al. (1997). benzoate-utilizing serratia marcescens — dwi suryanto & antonius suwanto the ability of the isolate to grow on structurally similar aromatic compounds like benzoate, gentisate, salycilate, and phenol, can be explained as it might have similar cell membrane transport system or similar catabolic pathway after ring fission (shimp and pfaender 1987). the different ability to grow in benzoate, gentisate, salycilate, and phenol (table 2) might be due to different uptake efficiency (altenschmidt et al. 1993). ds-8 was able to grow aerobically in 5 mm benzoate with or without vitamin supplement (figure 2). however, different responses of growth in these media were significant. bacterial growth in media supplemented with vitamins was faster than they were without vitamins. ds-8 clearly required vitamins for optimal growth in benzoate. the generation time in media with vitamins and media without vitamins were 11.4 hours and 16.8 hours, respectively. consequently, benzoate degradation also occurred much faster in media supplemented with vitamins. in media with benzoate-utilizingserrarta marcescens dwi suryanto & antonius suwanto vitamin supplementation, benzoate utilization rate was 0.16 mm/hour compared to 0.04 mm/hour in media without vitamin supplement. the ability to grow in the aromatic compound without vitamins indicated that the isolate was capable of synthesizing its own vitamins. relatively low cell density (absorbance at 660 nm) in this study might be a result of using poor media ( figure 3a). minimal media for this purpose are usually supplemented with 0.01% yeast extract or other common c sources (adriaens et al. 1989; gurujeyalakshmi and oriel 1989; shimao et al. 1989). as shown in figure 3, optimum ph and nacl concentration for bacterial growth was between 7-8.5 and 1-1.5%, respectively. the optimum range of ph and nacl concentrations showed that the isolate was not acidophilic or halophilic. optimum growth temperature was in the range of 20-35°c. this isolate could not grow at 40°c. at 37°c, no red pigment appeared. andreeva and ogorodnikova (1999) observed that prodigiosin was not accumulated by growing s. marcescens at this temperature. ds-8 grew very poorly in media with benzoate concentration exceeding 10 mm. a common concentration for benzoate degradation test was up to 5 mm (altenschmidl et al. 1993; bundy et al. 1998). an increase in cell tolerance against toxic substrates was crucial to improve the degradation capabilities. alteration of cm to trans-fatty acid of cell membrane might improve cell tolerance to toxic substrates (heipieper et al. 1992). relatively poor growth of the isolate was also observed in 5 mm benzoate without vitamin supplement. hence, vitamins were needed for optimum growth. benzoate utilization might indirectly depend on ph, nacl concentration, as well as benzoate concentration, since the cell growth was affected by initial ph, nacl concentration, and benzoate concentration (figure 3). the experiment with other organic compounds showed that the isolate grew well in media with casamino acid and glutamate (figure 4). the isolate grew poorly in glucose, acetate, potato starch, and ethanol and failed to grow in succinate or citrate. it seemed that the availability of organic nitrogen in casamino acid and glutamate supported the growth of ds-8. eberl et al. (1999) showed that the doubling time of s. liquefaciens mg1 was significantly increased even in only 0.01% casamino acid. the inability of ds-8 to utilize succinate and citrate might be due to the lack of special transport system. for example, an inducible transport system for malate, succinate, and fumarate was present in rhodobacter (rhodop-seudomonas) sphaeroides (gibson 1975). acknowledgements this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor, indonesia.   benzoate-utilizing serratia marcescensdwi suryanto & antonius suwanto references adriaens, p., kohler, h-pe., kohler-staub, d., and d.d. focht. 1989. bacterial dehalogenation of chlorobenzoate and coculture biodegradation of 4,4'-dichlorobiphenyl. appl. environ. microbiol., 55, 887-892. andreeva, i.n. and t.i. ogorodnikova. 1999. the effect of the cultivation conditions on the growth and pigmentation on serratia marcescens. zh. mikrobiol. epidemiol. immunobiol., 3,16-20. (abstract). altenschmidt, u., oswald, b., steiner, e., herrmann, h., and g. fuchs. 1993. new aerobic benzoate oxidation pathway via benzoyl-coenzyme a and 3-hydroxybenzoyl-coenzyme a in a denitrifying pseudomonas sp. j. bacteriol. 175,48514858. bundy, b.m., campbell, a.l., and e.l. neidle. 1998. similarities between the ont4sc-encoded anthranilate dioxygenase and the ie«^5c-encoded benzoate dioxygenase of acinetobacter sp. strain adp1. j. bacteriol. 180, 4466-4474. eduardo diaz, e., ferrandez, a., prieto, m.a., and j.l. garcia. 2001. biodegradation of aromatic compounds by escherichia coli. microbiol. mol. biol. rev. 65, 523-569. dong, f., wang, l., wang, c., cheng, j., he, z.,.sheng, z., and r. shen. 1992. molecular cloning and mapping of phenol degradation genes from bacillus stearothermophilus fdtp-3 and their expression in escherichia coli. appl. environ. microbiol. 58,2531-2535. eberl, l., molin, s., and m. givkov. 1999. surface motility of serratia liquefaciens mg1. j. bacteriol. 181,1703-1712. gibson, j. 1975. uptake of c4 dicarboxylates and pyruvate by rhodopseudomonas sphaeroides. j. bacteriol. 123,471480. grund. e., knorr, c., and r. eichenlaub. 1990. catabolism of benzoate and monohydroxylated benzoates by amycolatopsis and streptomyces spp. appl. environ. microbiol. 56,1459-1464. biotropia no. 21,2003 guerin, w.f. and s.a. boyd. 1995. maintenance and induction of naphthalene degradation activity in pseudomonas putida and an alcaligenes sp. under diiferent culture conditions. appl. environ. microbiol. 6, 4061-4068. gurujeyalakshmi, g. and p. oriel. 1989. isolation of phenol-degrading bacillus stearothermophi/us and partial characterization of the phenol hydroxylase. appl. environ. microbiol. 55,500-502. hiepieper, h.j., diefenbach, r., and h. kuweloh. 1992. conversion of cis-unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading pseudomonas putida p8 from substrate toxicity. appl. environ. microbiol. 58,18471852. lenke, h., pieper, d.h., bruhn, c., and h. knackmuss. 1992. degradation of 2,4-dinitrophenol by two rhodococcus erythropolis strains, hl 24-1 andhl 24-2. appl. environ. microbiol. 58,2928-2932. lindum, p.w., anthoni, u., christofferson, c., eberl, l., molin, s., and m. givskov. 1998. af-acyl-l-homoserine lactone autoinducers control production of an extracellular surface-active lipopeptide required for swarming motility ofsetratia liquefaciens mg1. j. bacteriol. 180,6384-6388. mars, a., houwing, j., dolfmg, j., and d.b. janssen. 1996. degradation of toluene and trichloroethylene by bulkholderia cepacia g4 in growth-limited fed-batch culture. appl. environ. microbiol. 62, 886-891. powlowski, j. and v. shingler. 1994. genetics and biochemistry of phenol degradation by pseudomonas sp. cf600. biodegrad. 5,219-236 semple, k.t. and r.b. cain. 1996. biodegradation of phenols by the alga ochromonas danica. appl. environ. microbiol. 62,1264-1273. shen, h and y. wang. 1995. simultaneous chromium reduction and phenol degradation in a coculture of escherichia coli atcc 33456 and pseudomonas putida dmp-1. appl. environ. microbiol. 61, 2754-2758. shimao, m., onishi, s.; mizumori, s., kato, n., and c. sakazawa. 1989. degradation of 4-chlorobenzoate by facultatively alkalophilic arthrobacter sp. strain sb8. appl. environ. microbiol. 55,478^182. shoreit, a.a.m. and m.s.a. shaheb. 1994. utilization of aromatic compounds by phototrophic purple nonsulfur bacteria. biodegrad. 5,71-76. suarez, m., gibello, a., allende, j.l., martin, m., ferrer, e., and a. girrido-pertierre. 1991. degradation of 3and 4hydroxybenzoate by k/ebsiellapneumoniae. appl. microbiol. biotechnol. 34,677-682. shimp, r.j. and f.k. pfaender. 1987. effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities. appl. environ. microbiol. 53,1496-1499. watanabe, t., kimura, k., sumiya, t., nikaidou, n., suzuki, k., suzuki, m., taiyoji, m., ferrer, s., and m. regue. 1997. genetic analysis of the chitinase system of serratia marcescens 2170. j. bacteriol. 179, 7111-7117. werwath, j., arfrnann, h., pieper, d.h., timrnis, k.n., and r. wittich. 1998. biochemical and genetic characterization of a gentisate 1,2-dioxygenase from sphingomonas sp. strain rw5. j. bacteriol. 180,4171-4176. 10 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 1 dietary expo (okky).cdr biotropia vol. 18 no. 1, 2011: 1 12 1 dietary exposure assessment for aflatoxin b from processed peanut products in municipality of bogor 1 santi ambarwati , okky setyawati dharmaputra and ina retnowati a research on dietary exposure assessment for aflatoxin b (afb1) from processed peanut products in municipality of bogor was carried out. the objectives of this study were to determine the contents of afb1 in processed peanut products at retail levels, and to obtain information whether there is a risk to public health caused by the consumption of processed peanut products contaminated by afb1. survey of processed peanut product consumption was carried out by interviewing each respondent using a questionnaire of weekly processed peanut product consumption. sampling of processed peanut products was conducted at the locations where the respondents obtained processed peanut products. the number of roasted peanuts with skin pods, flour-coated peanuts and or sauces samples was 33, respectively, while the number of and sauces samples was 18 and 12, respectively. the total number of processed peanut product samples was 129. afb1 content was determined using thin layer chromatography method. estimation of the dietary exposure assessment was determined using the actual survey data consisting of afb1 content, consumption data and body weight. the highest contaminated sample percentage and mean of afb1 content was found in roasted peanuts with skin pods i.e. 42% of 33 samples and 43.2 μg/kg, respectively, followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). mean of estimated dietary exposure for afb1 found in children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while in adults 9.0 ng kg bw day and 95 percentile exposure was 27.0 ng kg bw day . the excess cancer risk of afb1 exposure in bogor from this study on children and adults was calculated as 193 and 115 cancers/year, respectively dietary exposure assessment, aflatoxin b , processed peanut products 1 1,2 1 -1 -1 th -1 -1 -1 -1 th -1 -1 * 1 2 seameo biotrop, bogor, indonesia department of biology, faculty of mathematics and natural sciences, pecel gado-gado siomay satai pecel gado-gado bogor agricultural university, darmaga campus, bogor, indonesia abstract 1 1 . key words: * corresponding author : ambarwati@biotrop.org biotropia vol. 18 no. 1, 2011 2 introduction peanuts are next to rice, maize and soybean as the most important secondary crop in indonesia. since indonesia has a humid tropical climate, peanuts can easily be infected by fungi during the drying phase in the field, or under poor storage conditions. according to sauer . (1992) fungal infection can cause a decrease in physical quality of kernels and nutritional content, rancidity, discoloration, and production of mycotoxin, among others aflatoxin. the toxin has been recognized as human and domestic animals carcinogen, and is produced following the infection of peanuts among others by certain strains of . in general, aflatoxins found in foodstuffs and their processed products are aflatoxins b , b , g and g . the most dangerous aflatoxin is b (afb1). the adverse health effects of aflatoxins can be categorized as either acute or chronic. acute aflatoxicosis occurs when moderate to high levels of the toxins are consumed and may result in hemorrhage, acute liver damage, rapid progressive jaundice, edema of the limbs, alteration in digestion, absorption and/or metabolism of nutrients, high fever, vomiting, swollen livers and possibly death (fung & clark 2004). hepatocelullar carcinoma (hcc), or liver cancer, is the third leading cause of cancer deaths worldwide, with roughly 550 000-600 000 new hcc cases globally each year (who 2008). it has been known for several decades that aflatoxin causes liver cancer in humans, however, the exact burden of aflatoxin-related hcc worldwide was unknown. liu and wu (2010) conducted a quantitave cancer risk assessment i.e. using global data on food-borne aflatoxin levels, consumption of aflatoxincontaminated foods, and hepatitis b virus (hbv) prevalence. aflatoxins have been classified as group 1 human carcinogen by the international agency for research on cancer (iarc) and demonstrated carcinogenic effects on many animal species, including some rodents, non human primates, and fish (international programme on chemical safety 1998). groopman . (2008) reported that specific p450 enzyme in the liver metabolize aflatoxin into a reactive oxygen species (aflatoxin-8,9-epoxide), which may then bind to proteins causing acute toxicity (aflatoxicoses) or to dna causing lesions that over time increase the risk of hepatocelullar carcinoma (hcc) or liver cancer. for cancer risk assessment, it is traditionally assumed that there is no threshold of exposure to a carcinogen below which there is no observable adverse effect. national research council (2008) stated that cancer potency factors are estimated from the slope of the dose response relationship, which is assumed to be linear, between doses of the carcinogen and cancer incidence in a population. according to ipsc/who (1998), aflatoxin risk assessment selected two different cancer potency factors for aflatoxin : 0.01 cases/ 100 000/year/nanogram/kilogram body weight per day aflatoxin exposure for individuals without chronic hbv infection, and 0.30 corresponding cases for individuals with chronic hbv infection. kirk . (2005) and ok . (2007) reported that several epidemiological studies confirm that aflatoxin's cancer potency is about 30 times greater among hbv-positive than among hbv-negative individuals. et al aspergillus flavus et al et al et al 1 2 1 2 1 . . . 3 dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al acute outbreaks of aflatoxicosis have been reported from kenya (ngindu 1982, cdc 2004), india (krishnamachari 1975) and malaysia (chao 1991, lye 1995). chronic aflatoxicosis results from ingestion of low to moderate levels of aflatoxins and the effects are impaired food convertion (shane 1993), slower rates of growth (gong 2002, 2004), and a decrease in various micronutrient levels (pimpukdee 2004). many countries have determined maximum tolerable levels of aflatoxins in peanuts and their processed products. maximum tolerable levels of aflatoxins b , b , g and g in peanuts and their processed products in australia, canada, philippines and singapore were 15, 15, 20 and 5 μg/kg, respectively (fao 2004). based on sni (2009) in indonesia, maximum tolerable limit of afb1 and total aflatoxins in peanuts and their processed products were 15 and 20 μg/kg, respectively. researches on infection and aflatoxin contamination in raw peanut kernels collected from farmers, collectors, wholesalers, retailers at traditional markets have been conducted by dharmaputra . (2005, 2007a). the results indicated that in general the highest aflatoxin contamination of raw peanut kernels was at retailers in traditional markets. dharmaputra . (2007b) reported that the high afb1 contents in raw kernels were due to among others by damaged kernels (discoloured, cracked and broken kernels). lilieanny . (2005) stated that the highest aflatoxin content was found in compared to in roasted peanuts with skin pods, roasted peanuts without skin pods, flour-coated peanuts, , and . data on aflatoxin level in processed peanut products and their consumptions are needed to prepare the dietary exposure assessment for aflatoxin. most asean countries (including indonesia) have some data on aflatoxin content in foods, however, no formal risk assessment on aflatoxin has been conducted for the region. this may be due to the lack of technical and financial resources to develop the necessary data and information needed to support or to conduct risk assessment. more data on aflatoxin contents in processed peanut products are needed. in addition, a survey of individual processed peanut product consumption should be conducted to determine aflatoxin exposure assessment (sparringa 2008). dietary assessment is a part of risk assessment, i.e. the process of estimating potential exposure of a population to food chemicals (among others aflatoxin) from the diet and comparing the potential exposure against a reference health standard for risk characterisation purpose. aflatoxin exposure assessment could be used to estimate the potential exposure/intake of the toxin, to assess the potential risk of health for a population group, and to maintain safe food supply. the objectives of this study were to determine the contents of afb1 in processed peanut products at retail levels, and to obtain information whether there is a risk to public health caused by the consumption of processed peanut products contaminated by afb1. et al. et al. et al. et al. et al. et al. a. flavus et al et al et al bumbu pecel bumbu pecel enting-enting gepuk 1 2 1 2 4 materials and method pre-survey survey of processed peanut product consumption pre-survey consisted of : a. determination of data consumption survey location of processed peanut products : the location of data consumption survey was carried out at (subdistrict) bogor tengah covering 11 (lowest local government), i.e. babakan, babakan pasar, cibogor, ciwaringin, gudang, kebon kelapa, pabaton, paledang, panaragan, sempur, and tegallega. bogor tengah has the highest population density (13 445 inhabitants/km ) and was selected to conduct the survey and sampling. in addition the government center including business activities are also found in this (bps kota bogor 2008). b. determination of respondents : the data of bogor tengah office showed that the population at bogor tengah until march 2009 was 116 686 people. the number of respondents was determined based on the square root of population at bogor tengah, i.e. 342 respondents. in each , the number of respondents was determined proportionally based on the number of inhabitants. the respondents were grouped into two categories, i.e. children (6-15 years old, 169 respondents) and adults (16-44 years old, 173 respondents). it was assumed, that most children of 6 years and older like to eat processed peanut products, while the adults are more sensitive to hepatitic. survey of processed peanut product consumption was carried out by interviewing each respondent using a questionnaire of weekly processed peanut product consumption concerning : the kinds of processed peanut products ( or , , , , , , , or , , , and ) consumed by each respondent during the last one week the frequency of processed peanut products to be consumed by each respondent during the last week portion or product number consumed by each respondent during the last week the location where each respondent bought the processed peanut products the body weight of each respondent in addition to interviewing each respondent, observation was also conducted to obtain information about the number of processed peanut product sellers found in the surrounding of respondent domiciles. the information was used at the stage of sampling. during survey in each , the research team was accompanied by one or two staff of the who are familiar with the sites. kecamatan kelurahan kelurahan kecamatan kecamatan kecamatan kecamatan kecamatan kelurahan bumbu pecel gado-gado bumbu karedok bumbu siomay bumbu batagor bumbu satai bumbu ketoprak oncom hitam kacang garing kacang kulit kacang atom kacang telur kacang bawang kelurahan kelurahan 2 . . biotropia vol. 18 no. 1, 2011 5 sampling method of processed peanut products determination of aflatoxin b content estimation of the dietary exposure assessment for afb1 sampling of processed peanut products was conducted at the locations where the respondents obtained processed peanut products, i.e. from or , and vendors , and small shops. the number of samples of each processed peanut product was determined based on the number of processed peanut product sellers found in the surroundings of the respondent domiciles. the number of roasted peanuts with skin pods, flour-coated peanuts and or sauces samples were 33, respectively, while the number of dan sauces samples were 18 and 12, respectively. the total number of processed peanut products samples was 129. each sample consisted of 5 portions of processed peanut products in the form of sauce ( or , , and sauces) and 2 kg (= 100 small packs, weight @ 20 g) of other processed peanut products (roasted peanuts with skin pods and flour-coated peanuts). at the time of purchase, the main materials of or , and were packed separately from their peanut sauces. one portion of , and contained 75, 50 and 60 g peanut sauces, respectively. each sample was mixed manually and homogenously, and then divided into two parts to obtain working samples for afb1 content determination and a reserve sample. afb1 content was only determined in peanut sauces. afb1 content was determined using thin layer chromatography (tlc) method (aoac 2005). two replicates were used from each sample. afb1 was extracted from ground processed peanut products using methanol-h o. the filtrate was diluted using nacl solution and defatted using hexane. afb1 was partitioned into chloroform, then it was removed through evaporation, and quantitated using tlc on silicagel plate by visual estimation, i.e. by comparing the spot of standard and sample. exposure assessment involves estimating the intensity, frequency, and duration of human exposures to a toxic agent. dietary exposure to aflatoxin b in bogor tengah was estimated using the afb1 concentration data, food consumption data and mean body weight. afb1 exposure was calculated on the group of average consuming (mean) and high consuming (95% percentile). consumption data were obtained from food frequency questionnaire. the estimation of dietary exposure assessment for afb1was determined based on the following formula (who 2008): warung pecel gado-gado siomay satai pecel gado-gado siomay satai pecel gado-gado siomay satai pecel gado-gado siomay satai gadogado siomay satai kecamatan 2 1 1 food consumption (kg/day) × b1 aflatoxin concentration (μg/kg) body weight (kg) ×dietary exposure = (ng/kg bw/day) 1,000 dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al 6 results and discussion pattern of processed peanut product consumption aflatoxin b content based on five big ranks of consumers, roasted peanuts with skin pods (63% of the total respondent number) ranks first among 11 kinds of consumed processed peanut products in bogor tengah, followed by flour-coated peanuts (54.5% of the respondents ), sauce (54% of the respondents), or sauce (49% of respondent number) and sauce (34% of the respondents) (table 1). in general, either child or adult respondents, bought roasted peanuts, flour-coated peanuts, or , and from , or and vendors. in general child respondents bought from vendors, while adult respondents bought it from . the mean highest number of processed peanut product consumption of child and adult respondents was 0.0110 kg/day and 0.0149 kg/day, respectively, for or (table 2). the mean consumption number was obtained from the mean consumption frequency in one day multiplied by the mean consumption portion. afb1 contents were determined in five most consumed processed peanut products i.e. roasted peanuts, flour-coated peanuts, or sauce, and sauces. the highest contaminated sample percentage and mean of afb1 conteent was found in roasted peanuts with skin pods (42% of 33 samples and 43.2 μg/kg), followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). (table 3). kecamatan siomay pecel gado-gado satai pecel gado-gado siomay warung warung pecel gado-gado satai warung satai pecel gado-gado pecel gado-gado siomay satai pecel gado-gado 1 respondent gender number of respondents kind of processed peanut products male female 1 2 3 4 5 6 7 8 9 10 11 childs 73 96 169 70 (21%) 12 (4%) 109 (32%) 84 (25% ) 59 (17%) 25 (7%) 11 (3%) 125 (37%) 109 (32%) 23 (7%) 9 (3% ) adults 33 140 173 97 (28%) 28 (8%) 75 (22%) 25 (7%) 58 (17%) 30 (9%) 48 (14% ) 90 (26%) 77 (22.5%) 23 (7%) 19 (6% ) total 106 236 342 167 (49 % ) 40 (12% ) 184 (54% ) 109 (32% ) 117 (34 % ) 55 (16%) 59 (17% ) 215 (63 % ) 186 (54.5 % ) 46 (13.5% ) 28 (9%) rank of the products consumed 4 3 5 1 2 note : 1 = 7 = (black oncom) 2 = 8 = (roasted peanuts with skin pods) 3 = 9 = (flour-coated peanuts) 4 = 10 = (egg-coated peanuts) 5 = 11 = (garlic peanuts) 6 = pecel/gado-gado oncom hitam karedok kacang kulit siomay kacang atom batagor kacang telur sate kacang bawang ketoprak table 1. number of respondents who consumed processed peanut products in (subdistrict) of bogor tengah kecamatan st up to 5thfrom 1 biotropia vol. 18 no. 1, 2011 7 table 2. frequency, portion and consumption number of processed peanut products in bogor tengahkecamatan respondent kind of product place of purchase * ) mean of consumption frequency per day mean of portion (kg) mean of consumption number (kg/day) children roasted peanuts with skin pods warung (small shop) 0.40 0.0204 0.0082 flour-coated peanuts warung 0.31 0.0096 0.003 pecel/gado -gado sauce warung pecel/gado -gado 0.24 0.0460 0.0110 siomay sauce vendor 0.31 0.0164 0.00 satai sauce vendor 0.28 0.0224 0.00 adults roasted peanuts with skin pods warung 0.34 0.0290 0.009 flour-coated peanuts warung 0.32 0.0090 0.00 pecel/gado -gado sauce warung pecel/gado -gado 0.26 0.0572 0.014 siomay sauce vendor 0.30 0.0263 0.007 satai sauce warung satai 0.21 0.0305 0.0064 51 63 29 0 9 9 9 * small part of the respondents bought processed peanut products in traditionaland supermarkets or they prepared the products by themselves table 3. aflatoxin b content of processed peanut products at bogor tengah and maximum tolerable limit (mtl) of afb1 based on sni (2009) ) 1 kecamatan kind of processed peanut products no. of samples number (%) of samples contaminated by afb 1 number (%) of samples contaminated by afb 1 > 15 μg/kg roasted peanuts with skin pods 33 14 (42%) 14 (42%) flour-coated peanuts 33 10 (30%) 10 (30%) pecel/gado -gado sauce 33 9 (27%) 7 (21%) siomay sauce 18 2 (11%) 2 (11%) sat ai sauce 12 2 (17%) 2 (17%) mtl (sni 20 09) range and mean of afb 0 0 0 0 0 – – – – – content (μg/kg) 316.80 (43 160.00 (34.28) 197.80 (17.11) 39.90 (4.41) 198.58 (23.17) 15 .21) 1 the best way to control the presence of aflatoxins in foods and feeds is through good agricultural and manufacturing practices which could prevent fungal growth. aflatoxins are thermostable compounds and, once formed, they can persist in animal feeds and food. the usual methods of processing peanuts to make peanut butter and processing nuts for confectionery may appreciably reduce aflatoxin contamination. effective means of reducing aflatoxin contamination include removing undersized nuts, removing nuts that resist splitting and blanching, and removing discoloured nuts by hands or electric sorting (cole 1989). percentage of samples contaminated by afb1 and afb1 content of or sauce was relatively high. it was probably due to the low quality of peanuts used to prepare the sauces or containers used to store peanut kernels and to prepare the sauces were not clean. percentage of samples contaminated by afb1 and the mean of afb1 content in sauce were relatively low (11% of 18 samples and 4.4 μg/kg). the percentage of sauce samples contaminated by afb1 was also relatively low, i.e. 17% of 12 pecel gadogado siomay sate dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al samples, but the mean of their afb1 contents was relatively high, i.e. 23.2 μg/kg (table 3). in indonesia the maximum tolerable limit (mtl) of afb1 for peanuts and their processed products is 15 μg/kg (sni 2009). percentage of roasted peanuts with skin pods, flour-coated peanuts, or sauce, sauce and sauce samples contaminated by afb1 exceeded 15 g/kg i.e. 42% of 33 samples, 30% of 33 samples, 21% of 33 samples, 11% of 18 samples, and 17% of 12 samples, respectively (table 3). based on the mean of afb1 content, among the five processed peanut products, sauce has a mean afb1 content lower than the mtl (4.41 g/kg). two out of 18 samples were contaminated by afb1 exceeding 15 g/kg. the other four processed peanut products contained mean of afb1 contents higher than mtl. consequently, risk management of the four products is suggested to be applied. lilieanny . (2005) stated that total aflatoxin contents of roasted peanuts with skin pods (47 samples), flour-coated peanuts (22 samples), (12 samples) and (4 samples) collected from several factories, supermarkets, and traditional markets in bogor, malang, pati and yogyakarta from january up to august 2002, were 1.8, 5.2, 41.6 and 20.8 μg/kg, respectively. aflatoxin was not detected in roasted peanuts without skin pods (3 samples). mean of estimated dietary exposure for afb1 in children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while in adults 9.0 ng kg bw day and 95 percentile exposure 27.0 ng kg bw day (table 4). the major contributing foods for afb1 in children and adults was roasted peanuts with skin pods, followed by sauce, sauce, flour-coated peanuts and sauce, while the highest contaminated level was roasted peanuts with skin pods, followed by flourcoated peanuts, sauce, sauce and sauce. the estimated highest exposures to afb1 by consuming roasted peanuts with skin pods were 44.5% (adult respondents) and 43% (child respondents) of total exposure to afb1 (table 4). further researches on the risk management of roasted peanuts with skin pods are needed. the mean dietary intake of aflatoxin for australian and swedes were 0.15 and 0.8 ng kg bw day , respectively (thuvander . 2001), for americans 0.26 ng kg bw day (jecfa 1998), for french adults (>15 years old ) was 0.1 ng kg bw day , while for children (ages 3-14 ) was 0.3 ng kg bw day. it was assumed, that bogor community consumed processed peanut products containing higher content of afb1 compared to other countries. however, li (2001) reported that in guangxi, china, the probably daily intake of processed peanut products was estimated 3,680 ng kg bw day . from the five processed peanut products, the lowest dietary exposure for afb1 was sauce, either on children or adult respondents. dietary exposure for afb1 of the five products on children respondents was higher than that of adult respondents (table 4). this was due to the body weight of children which was lower than that of adults. the lower the body weight of the consumer, the higher the dietary exposure of afb1. pecel gado-gado siomay satai siomay et al bumbu pecel enting-enting gepuk pecel/gado-gado satai siomay satai gado-gado siomay et al et al. siomay estimation of the dietary exposure for aflatoxin b1 -1 -1 th -1 -1 -1 -1 th -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 biotropia vol. 18 no. 1, 2011 8 9 respondent kind of product mean of consumption number (kg/day) mean of afb1 content (μg/kg) mean of body weight (kg) dietary exposure for afb1 (n g/kg body weight /day ) percentage of total daily intake childs roasted p eanuts with skin pods 0.0082 43.21 33 10.6 43.0 flour-coated peanuts 0.0030 34.28 32 3.4 13.7 pecel/gado-gado sauce 0.0110 17.11 32 5.6 22.8 siomay sauce 0.0051 4.41 32 0.7 2.8 satai sauce 0.0063 23.17 31 4.4 17.7 adults roasted peanuts with skin pods 0.0099 43.21 56 7.4 44.5 flour-coated peanuts 0.0029 34.28 57 1.8 10.7 pecel/gado -gado sauce 0.0149 17.11 56 4.4 26.3 siomay sauce 0.0079 4.41 56 0.6 3.5 satai sauce 0.0064 23.17 57 2.5 15.0 table 4. dietary exposure assessment for aflatoxin b from processed peanut products at bogor tengah 1 kecamatan risk assessment to evaluate the potential health risk of bogor community to afb1, a risk assessment of afb1 should be conducted by comparing the estimation of dietary intake and provisional maximum tolerable daily intakes (pmtdi). as afb1 is a genotoxic carcinogen, the safety factors used for non-genotoxic carcinogens cannot be applied. therefore, most agencies, including jecfa and us fda, have not determined yet a tolerable daily intake for afb1. jecfa proposed the potency value of 0.3 cancer cases per year per 100 000 population per ng aflatoxin per kg body weight for hepatitis b positive individuals, while the potency value for the non-hepatitis b population was 0.01 cancer cases per year per 100 000 population per ng aflatoxin per kg body weight (jecfa 1997). based on the potency value of 0.3 and 0.01 cancers per year per 100 000 population per ng aflatoxin per kg body weight, prevalence of hepatitis b in bogor was 1.4% (departemen kesehatan republik indonesia 2008), while the population of bogor end 2007 was 905 132 (bps kota bogor 2008). consequently, the results of this study showed that the cancer risk of afb1 exposure in bogor on children and adults was 193 and 115 cancer cases/year, respectively. therefore, cancer risk could increase due to the consumption of highly afb1 contaminated processed peanut products. afb1 is an unavoidable food contaminant. to evaluate the potential health risk of afb1 caused by food consumption, it is important to determine the natural occurrence of afb1 in food and to estimate the risk for liver cancer through dietary exposure to afb1. ok (2007) reported that the level of afb1 contamination in 28 of the 32 food products in south korea was less than 10 kg , which is the legal tolerance limit in korea. from data on daily food consumption, the exposure dose of afb1 was estimated to be 6.42 x 10 mg kg body weight day . the risk of liver cancer et al. -1 -7 -1 -1 . . . . dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al 10 for those exposed to afb1 through food intake was estimated to be 5.78 x 10 for hepatitis b-negative individuals and 1.48 x 10 for hepatitis b-positive individuals. the highest contaminated sample percentage and mean of afb1 content was found in roasted peanuts with skin pods (42% of 33 samples and 43.2 μg/kg), followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). the percentage of sauce samples contaminated by afb1 was also relatively low, i.e. 17% of 12 samples, but the mean of their afb1 contents was relatively high, i.e. 23.2 μg/kg. among the five processed peanut products, sauce has a mean afb1 content lower than the mtl (4.41 g/kg). the other four processed peanut products contained afb1 higher than mtl. consequently, risk management of the four products is suggested to be applied. in addition, aflatoxin contamination can be minimized by using good practices, from farm up to table. mean of estimated dietary exposure for afb1 on children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while on adults 9.0 ng kg bw day and 95 percentile exposure was 27.0 ng kg bw day . the cancer risk of afb1 exposure in bogor from this study on children and adults showed 193 and 115 cancer cases /year, respectively. therefore, cancer risk could increase due to the consumption of highly afb1 contaminated processed peanut products. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the office of for the permission in conducting the survey, to the office of and 11 offices of which belong to for the cooperation during the survey; to mr. edi suryadi, ms. amanda windyarini, mr. rahadian pratama, mr. putra hidayat nasution, ms. rena yulia vernanda, and to the technicians of the laboratory of food analysis, seameo biotrop, for their assistance. we would like also to express our appreciation to the reviewers of this manuscript. -6 -4 -1 -1 th -1 -1 -1 -1 th -1 -1 conclusions acknowledgements references pecel gado-gado sate siomay kesatuan bangsa dan perlindungan masyarakat (kesbanglinmas) kecamatan bogor tengah kelurahan kecamatan bogor tengah aoac. 2005. natural toxins. in : horwitz w, editor. official methods of analysis of aoac international. association of official analytical chemist, gaithersburg. 18 ed. ch.49. p 11 bps kota bogor. 2008. kota bogor dalam angka 2008. badan pusat statistik kota bogor, bogor. center for disease control and prevention (cdc). 2004. outbreak of aflatoxin poisoning easter and central provinces, kenya. morbid mortal weekly report 53: 790 793 th biotropia vol. 18 no. 1, 2011 chao tc, maxwell sm, 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( ). paper presented at seminar pra-2 widyakarya nasional pangan dan gizi ke-ix 2008, kelompok kerja mutu dan keamanan pangan. jakarta, 9 june 2008 thuvander a, moller t, barbieri he, janson a, salomonsson aca, olsen m. 2001. dietary intake of some important mycotoxin by swedish population. food additives and contaminant 18 : 696-706 world health organization of united nation (who), 2004. worldwide regulations for mycotoxins in food and feed in 2003; fao food and nutrition paper 81. food and agriculture organization of the united nations, rome who. 2008. the global burden of disease : 2004 update. geneva : world health organization. available at http://www.who. int/healthinfo/global_burden_disease/ 2004_reportupdate/en/index.html) who. 2008. dietary exposure assessment of chemicals in food. report of a joint fao/who consultation. annapolis, maryland, usa, 2-6 may 2005. who press, geneva yan liu, felicia wu. 2010. global burden of aflatoxin-indiced hepatocellular carcinoma : a risk assessment. environ health perspectives 118 (6): 818-24 1 batas maksimum kandungan mikotoksin dalam pangan studi diet total dan kajian paparan bahan kimia dalam pangan th . biotropia vol. 18 no. 1, 2011 12 microsoft word 30 biotropia no. 24, 2005 : 30 45 analysis of the temperature dependence of co2 assimilation rate (study case: glycine maxl. merr) tania june biotrop-jcsea, seameo biotrop, btic building, jl. raya tajur km. 6 bogor, indonesia; email: taniajune@biotrop.org; laboratory o/agrometeorology, bogor agricultural university, bogor abstract the maximum rate of carboxylation (kcmax) and maximum rate of regeneration of ribulose bisphosphate (rubp) (controlled by the rate of electron transport, jmax) arc two processes governing the photosynthetic capacity of plants. both processes are affected by temperature. this paper examines how the response of these two photosynthetic capacities to temperature determines the temperature response curve of the co2-assimilation rate for plants grown at different temperatures, by using the concept of the farquhar €3 photosynthesis model. the goal is to use photosynthetic parameters from co2 and light curves to predict the temperature dependence of the co2-assimilation rate (a) of soybean and to estimate the preferred growth temperature. analysis shows that the optimum temperature of the assimilation rate changes with the changing temperature dependence of carboxylation and regeneration of rubp. key words : temperature dependence/soybean/modeling photosynthesis/preferred growth temperature. introduction photosynthesis is strongly affected by temperature. during gas exchange measurements, the short-term temperature dependence of photosynthesis is strongly affected by other environmental factors such as light intensity and intercellular coa concentration (berry & bjorkman 1980). some of these short-term effects can be modeled by c3 photosynthesis models of farquhar et al. (1980). however, the response varies not only among species but even within an individual species subjected to changing growth temperature regimes during their developments (long-term effects) (berry & bjorkman 1980). in many species, the optimum temperature at which maximum short-term photosynthesis is obtained, shifts upwards when plants are grown at higher temperatures (lange et al. 1974; slatyer 1977; berry & bjorkman 1980; ferrar, slatyer & vranjic 1989), which is commonly known as acclimation or adaptation. the long-term temperature acclimation during growth may affect both the maximum photosynthetic rate per unit leaf area and the shape of the photosynthetic temperature response curve. how this growth temperature (long-term effect) affects the short-term temperature response of photosynthesis is still unclear. it can be attributed at the enzyme level or at the gene action-dna level. to understand the mechanism of this change in temperature dependence, it is important to know the limiting factor to photosynthesis at each measured temperature. 30 biotropia no. 24, 2005 according to the farquhar cj photosynthesis model (farquhar et al. 1980), carboxylation and regeneration of rubp are two processes governing photosynthesis. in the model, the photosynthetic rate is limited either by the capacity of rubp carboxylase (rubisco) to consume ribulose bisphosphate (rubp), denoted as fcmax, or by the capacity for rubp regeneration, denoted as jmax/4. these capacities have different temperature dependencies, in the original model and as confirmed, for example, by kirschbaum & farquhar (1984) and later by june (2002). for a fixed temperature dependence of the electron transport rate (j), farquhar & von caemmerer (1982) showed that increasing the ratio of the capacity of rubisco to consume rubp to that of rubp regeneration could change the optimum temperature (ro) by changing the relative amounts of the two components, so that the optimum temperature would be higher when the ratio is increased. june (2002) has shown that this ratio decreased with increasing temperature. however, june (2002) also showed that temperature dependence of electron transport rate is not fixed but changed, both at short-term and long-term time scales. the net effect is on the change in the ratio jmm/vcmm with temperature. in the farquhar & von caemmerer (1982) paper, the ratio of rubp consumption to rubp regeneration increased with increasing growth temperature. in june (2002), the jmm/vcmax ratio decreased as the short-term temperature measurement increased, for each of the growth condition. this paper examines how the changes in these two photosynthetic capacities with temperature affect the temperature response curve of the co2-assimilation rate for plants grown at different temperatures, using the concept of the farquhar et al. (1980) c3 photosynthesis model. the goal is to use parameters from cc>2 and light curves from june (2002) (vcmm and jmax temperature dependences) to predict the temperature dependence of the co2-assimilation rate (a) and then to test the model with independent measurements of the temperature dependence of the cc>2-assimilation rate. the plants used for testing the model were grown under the same conditions as the plants used for the parametrisation of the model in june (2002). materials and methods seeds of indeterminate soybean (glyclne max [l.] merr. cv stephen) were sown in 12 liter plastic pots containing a mixture of sand and vermiculite (1:1, v/v) and plants were thinned to one plant per pot after germination. plants were grown in a controlled environment chamber with a 14hour photoperiod of around 700 umol quanta m"2 s"', 60/70 % relative humidity day/night and three different temperature regimes: 20/15, 25/20, 32/27 day/night °c under ambient [co2], 350 umol mol'1. the lowest and highest temperature regimes were repeated with atmospheric [ccy enrichment to 700 umol mol"1. the source of light used in the growth chamber was a metalarc lamp (general electric lighting), mvr 1000/u. plants were well spaced (30 cm apart at sowing) to avoid mutual shading. rhizobial inoculation was not provided for the plants. each 31 analysis of the temperature dependence of co2 assimilation rate tania june pot was flushed every second day with full-strength herridge's solution (0.50 mm mgso4, 0.25 mm cacl2, 0.25 mm kc1, 0.125 mm kh2po4, 0.125 mm k2hpo4, 25 um ferric monosodium salt of edta, 12 um h3bo3, 3.6 um mncl2, 77 um zncl2, 76 nm cucl2, 25 nm namoo4) (herridge 1977) and watered twice daily on days when nutrients were not given. to obtain a range of nitrogen levels in the plant leaves, three different concentrations of kno3 were added to the nutrient solution (2, 5 and 16 mm). the nutrient solutions were added to each pot until they drained at the base (2.5 to 3.0 liters per pot). for gas exchange measurement, several temperature response curves of assimilation rate were measured at a light intensity of 1200 umol m~2 s"1 with co2 concentrations of 350 umol mol" 1 using a photosynthetic gas exchange system developed in the environmental biology group, research school of biological sciences, australian national university. two pots of plants were used for replication. the vapour pressure difference was kept constant at around 12.5 mbar in most cases and was less than 17 mbar in all cases. for each leaf, the co2-assimilation rate was measured at six temperatures from 15°c to 40°c, holding for 15 to 20 minutes at each temperature measurement to reach a steady state condition. data obtained from measurements are then compared to simulation results where parameters are obtained from measurements by june (2002). results and discussion temperature dependence of co2 assimilation rate figure 1 shows the temperature dependence of the absolute value of the co2-assimilation rate (a) measured at 350 umol mol"1 [co2] and irradiance of 1200 umol m" 2 s"1, for plants grown under 350 umol mol'1 [co2]. as expected, increasing the temperature from 15°c to 25°c increases the assimilation rate: 18 % for plants grown at 20/15°c (day/night temperature), 88 % for plants grown at 25/20°c and 98 % for plants grown at 32/27°c. increasing the measurement temperature further resulted in a reaching a maximum at the optimum temperature and then decreasing. after reaching the optimum temperature, the photosynthesis rate dropped with further increasing temperature; by 40°c the drop in a was as much as 10 % for plants grown at 20/15°c, 24 % for plants grown at 25/20°c and 10 % for plants grown at 32/27°c. it is clear that the temperature dependence of the co2-assimilation rate is different depending on the growth temperature. 32          the photosynthetic processes in soybean exhibit a capacity for temperature acclimation. plants  maintained at different day/night temperatures had different optimum photosynthetic temperatures (t0).  plants grown at 32/27°c have an optimum temperature for the co2‐assimilation rate of 32.7±0.2°c. when plants  were grown at 25/20°c, the optimum temperature was 30.1±0.8°c and when plants were grown at 20/15°c, the  optimum temperature was reduced to 28.5 ±0.9°c (table 1).  table  1.  optimum  temperature  for  coz  assimilation  rate,  7"0,  and  assimilation  rates,  a,  at  optimum  temperature and at various other temperatures for plants growing at different temperatures,+  s.c. plants were grown at the temperatures  indicated  in the table, co2 concentration of 350  (imol mol"1 and nitrogen concentration of 16 mm. gas exchange measurements were conducted  at [co2]=350 (imol mol"' and light intensity, 7=1200 ̂ mol m" 2 s"'.        analysis of the temperature dependence of co2 assimilation rate tania june in a similar study with festuca arundinaceae grown at 10 and 25°c, treharne & nelson (1975) found that at measurement temperatures above 15°c, net photosynthesis rates per unit area were higher in plants grown at 25°c. the authors concluded that greater photorespiration rates were important in accounting for the low net photosynthetic rates of the 10°c plants at higher temperature measurements. however, in the photosynthesis model used in this manuscript, photorespiration is already taken into account in the form of f * (co2 compensation partial pressure in the absence of dark respiration) and c\ (co2 concentration in the intercellular air spaces). treharne & nelson (1975) further concluded that the decrease in photosynthesis above 30°c seen in both groups of plants was due primarily to a decrease in stomatal conductance, and hence c\, the decrease in c\ was not observed in my results (as seen in figure. 2) with increasing temperature. for net cc>2 uptake for plants grown at day/night temperature of 10/10°c was 12°c for agave americana and 15°c for a. deserti. when the growth temperature was raised to 30/30°c, the optimum temperature shifted upward by 7°c for a. americana and 3°c for a. deserti. shifting a. americana to the higher growth temperature caused the maximum rate of net cc>2 34 biotropia no. 24, 2005 uptake at the optimal temperature to increase, whereas the same shifting of a. deserti caused it to decrease. the results showed that a(t0), i.e. the optimum temperature for net assimilation rate or rate of photosynthesis, increase with increasing growth temperature. plants grown under 700 ^mol mol"' [co2] exhibited a similar pattern as those grown under ambient [co2], with t0 = 28.0 ± 0.4 and 32.2 + 0.7°c and a(t0) = 20.7 + 2.0 and 21.8 ± 0.7 tamol m' 2 s'j for plants grown at 20/15 and 32/27°c, respectively. it is important to note that a change in c\ (the intercellular cc>2 concentration) can cause a shift in the optimum temperature of photosynthesis, with t0 increasing with ci (farquhar & von caemmerer 1982; kirschbaum & farquhar 1984). there is a linear correlation between growth temperature and the optimum temperature for co2 assimilation rate (figure 3). the linear regression from table 1 can be solved to give a "preferred" temperature for the cc>2 assimilation rate of 33°c. this concept of "preferred" temperature was initially suggested by slatyer & ferrar (1977), and is the intersection of the linear regression line and the 45° line. the "preferred temperature" was also used in june (2002) to descibe the acclimation of the electron transport rate (j) to growth temperature.   analysis of the temperature dependence of co2 assimilation rate ‐ tania june  what controls the reduction in co2assimilation rate after reaching                                     optimum  temperature?  it has been reported that high temperature limits co2 availability, because c the physiological  responses of leaves which result in increased resistance to the ga diffusion (mukohata et al. 1971;  monson et al. 1982). high temperature also alter the substrate specificity of rubisco (jordan &  ogren  1984;  brooks  &  farquha  1985)  and  its  activity  (weis  1981;  santarius  et  al.  1991).  working  with  neriun  oleander,  badger  et  al.  (1982)  showed  that  plants  grown  at  low  temperature ha< higher activity of several photosynthetic enzymes at low temperatures but had  lowe heat stability relative to plants grown at high temperatures. temperature acclimatioi in the  electron transport system could also be the reason for the change in the co: assimilation rate at  high temperature. several studies have shown that the temperature dependence of electron  transport capacity changes with growtr temperature (armond et al. 1978; badger et al. 1982;  mitchell  &  barber  1986)  such  changes  were  also  observed  in  june  (2002),  where  plants  grown at highei temperature had a lower electron transport rate.  modeling the temperature dependence of the co2‐assiiniiation rate  the basic assumption underlying the modelling is that the rate of photosynthesis at any  temperature is controlled by the activity of enzyme rubp carboxylase‐oxygenase (rubisco),  denoted as vcmm, and the potential rate of regeneration of rubp, denoted as jmax/4. therefore,  at a given temperature, the net co2‐assimilation rate, a, is taken as being either the rubisco‐limited  rate, av, or the estimated rubp‐regeneration‐limited rate of photosynthesis, aj, whichever is  smaller (units of umol m"2 s"1).  where c\ = partial pressure of co2 in the leaf, f* = co2 compensation partial pressure in the  absence of dark respiration, /?d  = dark respiration by the  leaf which continues in the light, o =  ambient  partial  pressure  of  oxygen,  and  atc  and  k0  are  the  michaelis‐menten  constants  for  carboxylation and oxygenation by rubisco, respectively. temperature dependence of c\ follows the  equations from figure 2. r^ follows this equation:  36              biotropia no. 24, 2005 rd =a l +a 2 (t-25)+a,(t-25)2 (3) where t i s leaf temperature (°c) and ,4], a2andai are the fitted parameters of the rf temperature relationship (table 2), which was obtained from june (2002). the temperature dependence of kc and k0 follows an arrhenius function as where r is the universal gas constant, 8.3144 j mol"1 k"'. ef and e0 are the apparent activation energies with the 25 subscript representing the value at 25°c. the effect of temperature on the cc>2 compensation point of photosynthesis in the absence of mitochondrial (dark) respiration follows the equation of von caemmerer et al. (1994), assuming infinite wall conductance. the temperature dependence ofj and fcmax is given by the following equations june (2002): where j(t0), t0, q, c\, c2 and c3 are the fitting parameters, specific for each set of growth conditions (table 2). the capacity of the electron transport rate can be inferred from where the light dependence of electron transport, j, follows the equation by .farquhar & wong (1984): 37 analysis of the temperature dependence of co2 assimilation rate tania june •/max is the maximum (light-saturated) rate of electron transport capacity of the leaf, 0 is the curvature factor of the light response curve of eq. (10) and a-i is the quantum yield of electron transport. / is the amount of light intensity incident on the leaf surface. /2 in eq. (9) is equal to 021. the temperature dependence of © follows the equation of june (2002): where t\= 0.93, t2 = 0.0145, and t-> = -8.13 the w 4' these curvature factor parameters were obtained from june (2002), where measurement of the light curves was done with light incident on both the upper and lower surface of the leaf. biotropia no. 24,2005 simulated co2 assimilation rate the simulation results, based on table 2, at measurement temperatures of 15 to 40°c and / = 1200 umol m"2 s"1, are shown in figure 4. figure 5 shows the results for jmax, converted from j using eqs. (9) to (11). fcmax is constantly increasing with temperature, in relatively good agreement with wang et al. (1996) and other estimates (wullschleger 1993). however, ferrar et al. (1989) who investigated several species of eucalyptus grown at contrasting temperatures found that in leaves grown at high temperature, f"cmax increased with short-term temperature measurement, but in leaves grown at low temperature, kcmax did not increase as measurement temperature increased. they speculated that rubisco may be inactivated or damaged at measurement temperatures higher than the growth temperature. in my experiment, although fcmax increases with short-term temperature measurements for all growth conditions, plants grown at higher temperature have a slightly lower fcmax than plants grown at lower temperature. june (2002) showed that there is an acclimation in the electron transport rate which favours the lower growth temperature (20/15 °c). hence, if there is no stomatal effect due to different growth temperatures, then the co2-assimilation rate of plants grown at 20/15 °c will be higher than that of plants grown at 32/27 °c in the model (as seen in figure 4). the lower observed co2-assimilation rate of plants grown at 20/15°c (figure 1), and their lower assimilation rate at t0, make these plants a poor representation of this simulation (compare figure 1 and figure 4). the standard measurements of those particular leaves (standard measurement was done at light intensity of 1200 umol irfv, vpd =12.5 mbar, [ccy = 350 umol mol"', and temperature = 25°c), before starting each measurement were lower on the first day the plants were taken out from the growth chamber (the day when those measurements were done). they were increased by 19.5 % the next day. if the data (of the 20/15°c plants) were corrected with this percentage, plus taking into account the reduced ci( then the co2-assimilation rate at t0 of plants grown at 20/15°c would be higher than the other two growth temperature of plants as shown by the simulation result in figure 4. the simulation shows that differences in the temperature dependence of a are due to the differences in the processes limiting^. for example, a for 20/15 °c plants is limited by rubp regeneration below 23°c and is limited by rubisco activity at temperatures higher than 23°c, while a for 32/27°c plants is limited by rubp regeneration below 30°c and is limited by rubisco activity at temperature higher than 30°c. for plants grown at 25/20°c, a was limited by rubp regeneration below 28°c and limited by rubisco activity at temperature higher than 28°c. a co-limitation of these two capacities occurs at the optimum temperature (where av and aj lines cross in figure 4) and only one of them would limit photosynthetic rate at other temperatures, with the penalty of excess investment in the other capacity. therefore, when growth temperatures vary, changes in the organization of the photosynthetic apparatus are necessary, and this can be shown by the changed optimal ratio of jmaxs / vmaxs (farquhar & caemmerer 1982). 39 analysis of the temperature dependence of co2 assimilation rate tania june the simulated ratio of jmaxs / vcmaxs figures 4 and 5 show that between 15 and 35°c, the relative slope of the increase in jmax is higher than it is in fcmax; then the opposite happens with further increase in temperature. as the slope of increase in jmax with temperature was higher than that of fcmax within the range of 15 30°c (figures 4 and 5), the simulation predicts that the ratio of jmax/vcmax would increase with temperature within this range. figure 5 indicates that plants change the allocation of their photosynthetic resources between these two capacities as growth temperature changes. june (2002) showed that ./max/fcmax did have a lower value when plants were grown at 32/27°c compared to plants grown at lower temperatures, which is supported here by the simulation. 40 biotropia no. 24, 2005 farquhar & von caemmerer (1982) and recently hikosaka (1997) predicted that the photosynthetic rate should be co-limited by fcmax and jmax at the growth temperature for efficient nitrogen utilization of photosynthesis. figure 4 shows that the temperature where the two processes co-limit photosynthesis in the simulation (i.e. where av and aj lines cross) was close to the growth temperature. it was around 23°c for plants grown at 20/15°c, 27 °c for plants grown at 25/20°c and 30°c for plants grown at 32/27°c. the slight differences are probably due to the different light intensity used in the measurement (1200 iimol m"2 s"') compared to that in the growth chamber (600-700 umol m~2 s"'). under lower light, the electron transport rate would be lower and it might co-limit at a lower temperature than the ones from figure 4, which would improve the agreement for plants grown at the lower two growth temperatures. when j is converted to jmax using eq. (9), the value depends on which 0 value is used (as shown in figure 5). using 0 = 0.7, the estimated jmax was higher as tem 41 analysis of the temperature dependence of co2 assimilation rate tania june perature increased than the estimated jmm calculated using a θ that increased with temperature. this difference affects the ratio of jmm/ycmm, with a higher ratio obtained when θ was held constant at 0.7. now, the temperature where co-limitation occurs changes from that in figure 4. for plants grown at 20/15 and at 25/20°c, it is almost the same at 33 -35°c. plants grown at 32/27°c, were always limited by a-y figure 6 also shows that for conditions in which electron transport becomes limiting (that is, growth at high temperature), the electron transport rate will dominate the behaviour of the co2-assimilation rate, so the optimum temperature of a should shift towards the optimum temperature of jmm. conclusions the temperature response characteristics of the photosynthetic process are not fixed but depend on the prevailing conditions during growth. the present study shows how the temperature dependence of the photosynthetic rate differs between plants grown at different temperatures. the factors responsible for the difference include the change in temperature dependences of the two processes controlling photosynthesis, carboxylation and regeneration of rubp. the change in the proportion of photosynthetic resources into these two capacities can be shown by the change of the jmax/ vcmaxs ratio with growth temperature. 42 biotropia no. 24, 2005 acclimation to growth temperature occurred as shown by the changing optimum temperature with growth temperature. this acclimation is important for plants in optimising their photosynthesis rate in the environment they are exposed to in terms of the most economical way of using photosynthetic resources. for the soybean plants used in this experiment, the optimum temperature for electron transport (j) was a few degrees higher than the preferred temperature for co2-assimilation rate (33°c). hence an increase in temperature during the day to more than 33°c will still increase the electron transport rate, although not the co2-assimilation rate. for the purpose of modelling plant growth or modelling the capacity of plants in absorbing co2 in relation to the changing environmental conditions (atmospheric co2 concentration, temperature, light intensity, nitrogen, and water availability effect on supply of co2), the most useful models will be those which incorporate acclimation processes at all levels of organization within the plants. these models will be the most precise and the most responsive to changes in the physical and biological factors which control photosynthesis. however, the knowledge of the mechanisms of temperature dependence of photosynthesis is still limited and so the combination of an empirical approach with the mechanical ones in these areas where the mechanism is already established would be 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37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf biotropia vol. 29 no. 2, 2022: 112 123 doi: 10.11598/btb.2022.29.2.1637 112 dragonflies diversity and land cover changes in the batubolong river, west lombok district muhammad zulhariadi1*, raden dedi irawan2, aulia zulfaeda3, nurul hidayani4 and frendi irawan5 1,2,3biology education department, faculty of tarbiyah and teaching, universitas islam negeri mataram, mataram 83116, indonesia 4remote sensing and gis department, vocational college, universitas gadjah mada, yogyakarta 55281, indonesia 5indonesia dragonfly society, yogyakarta 55272, indonesia received 19 august 2021/accepted 13 october 2021 abstract west lombok district is the second largest district in lombok islands. the diversity of dragonflies (order odonata) as a bioindicator of environmental quality has not been widely studied in the west lombok region. this study aimed to determine the species diversity of dragonflies (order odonata) found in batubolong river, west lombok district and its relation to the occurring land cover changes. this study was carried out by using a descriptive explorative method, where the sampling technique was done by means of line transects. maps of study and sampling locations as well as land cover changes were made using arcgis 10.4.1 software based on primary and secondary data. the results showed that there were 11 species of dragonflies with a shannon-wiener diversity index value (h') of 2.212 (medium diversity), a population density index (c) of 0.126 (low dominance), and a species evenness index (e) of 0.923 (high uniformity). our study also found two rare species of dragonfly in lombok island i.e., 3 individuals of nososticta emphyla (lieftinck, 1936) with 9% relative abundance and 1 individual of drepanosticta berlandi (lieftinck, 1939) with 3% relative abundance. the discovery of d. berlandi in lombok is the third time after the findings in 1896 and the 19th century. analysis of satellite data around the sampling area within the period 2013-2020 showed that there has been an increase in land cover of 6,149.29 m2. the increase in land cover changes may have caused the disappearance of several odonata species in the sampling location. keywords: batubolong river, diversity, dragonfly, land cover, odonata introduction west lombok district is among districts in west nusa tenggara province, indonesia, located at 115°49.12'04”-116°20'15.62”e and 8°24'33.82"-8°55'19"s and is directly adjacent to mataram city, central lombok and north lombok districts and the indian ocean (bps statistics lobar 2021). the west lombok district has a large area with relatively dense population and settlements in the west nusa tenggara province (ntb). biodiversity in this district is similar to that in other cities/districts in the province. the west lombok district has several forests and extensive beaches, rivers and a nature tourism park (taman wisata alam/twa) managed by the provincial office of the natural resources conservation center of the ntb (rahmawati et al. 2019). several tree species as birds’ habitat as well as rice fields and plantations in several areas of the district still exist. there are also various types of dragonfly species in various areas of west lombok district. dragonfly (odonata) is a good bioindicator of water quality (dolný et al. 2013; koneri et al. 2020). the damselfly (suborder zygoptera) and the common dragonfly (suborder anisoptera) are members of the order odonata of the phylum arthropoda. various types of dragonflies are identified in various spots in west lombok district. the habitats of the damselfly and the dragonfly are scattered at various points, ranging from forests, rice fields, areas close to housings to areas far from s ettlements, hot and open areas to shady and closed areas. several study *corresponding author, email: zulhariadi@uinmataram.ac.id dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 113 locations regarding dragonflies have been reported in the west lombok area, namely the batubolong river and pusuk area (kosterin 2014), kerandangan twa (rahmawati et al. 2019) and suranadi twa (ilhamdi et al. 2021). changes in land cover and forest land conversions contribute to the decreasing diversity of various fauna species, especially several types of dragonflies that are vulnerable to environmental changes. several types of dragonflies inhabit several areas of west lombok regency which are still beautiful because dragonflies choose to live in a safe and comfortable place for their reproductive activities, especially in the life of their nymphs (ansori 2009; dolný et al. 2011). the batubolong river is a river that has its headwaters in lembah sari village, batulayar district and passes through batubolong hamlet, west batulayar village and has an estuary on the batubolong beach, batulayar district. this study aimed to observe the diversity of dragonflies (order odonata) in the batubolong river, west lombok district and to determine the impact of the recent changes in land cover around the river area toward dragonfly diversity. the results of this study will be compared with previous studies that have taken data in the batubolong river, west lombok. materials and methods this study was carried out by using descriptive explorative method for identifying the types of dragonflies (suborder anisoptera) and damselfly (suborder zygoptera) in the batubolong river. sampling was carried out for 4 months from april 2021 (rainy season) up to july 2021 (dry season) in the middle part of the batubolong river (fig. 1). the purposive sampling technique was used with line transects. environmental parameters observed were water ph and temperature, total dissolved solids (tds), electrical conductivity (ec), altitude, wind speed, air temperature and humidity. documentation of dragonflies was carried out by using a camera, while the identification of dragonflies was conducted by using references from irawan & rahadi (2018), kosterin (2014), steinmann (1997) and gbif.org. the relative species abundance, species diversity index, population density index and species evenness index were then calculated based on the data obtained from the field. maps of study and sampling locations as well as land cover changes were developod by using arcgis 10.4.1 software based on primary and secondary data. figure 1 map of the study location in the batubolong river, batulayar barat village, batulayar subdistrict, west lombok district biotropia vol. 29 no. 2, 2022 114 the relative species abundance was calculated using the formula: rsa = ni x 100% n where: rsa = relative species abundance ni = number of individual dragonflies i n = number of individuals of all (total) types of dragonflies the shannon-wiener diversity index (h') was calculated using the formula: h' = σ (pi ln pi) where: h' = shannon-wiener diversity index pi = ratio of the number of individuals of one species to the total number of individuals in the sample within the plot (n/n) diversity index criteria: h' < 1 = low diversity 1 < h' < 3 = medium diversity h' > 3 = high diversity population density/dominance index (c) was calculated using the formula: where: c = dominance index ni = number of individuals of one species n = total individuals of all species environmental community criteria based on dominance index: 0.00 ≤ c ≤ 0.30 = low dominance 0.30 ≤ c ≤ 0.60 = medium dominance 0.60 ≤ c ≤ 1,00 = high dominance the evenness index of species at the sampling point was calculated using the formula: e = h' ln s where: e = evenness index h' = shannon-wiener diversity index s = number of species environmental community criteria based on evenness index: 0.00 < e < 0.50 = small uniformity/depressed community 0.05 < e < 0.75 = moderate uniformity/ unstable community 0.75 < e < 1.00 = high uniformity/stable community results and discussion landscape and canopy in the batubolong river sampling area batubolong river is a type of river with small to large granite rocks adorning the river (fig. 2). various types of canopy plants over the riverbanks providing shady and beautiful ambiance are dominated by various types of bamboo, ivory mahogany (dysoxylum gaudichaudianum), cembirit/kumbi (tabernaemontana sphaerocarpa), pacific walnut (dracontomelon dao), tamarind (tamarandus indicus), mahogany (swietenia mahagoni), acacia (acacia sp.), kapok (ceiba pentandra), mango (mangifera indica), sugar palm (arenga pinnata), cashew (anacardium occidentale), black rosewood (dalbergia latifolia), and sengon (paraserianthes falcataria). several types of shrubs and medium-sized trees growing along the riverbanks include lantana (lantana camara), siam weed (chromolaena odorata), quickstick (gliricidia sepium), largeleaf rosemallow (hibiscus macrophyllus), various types of bananas (musa sp.), bandicoot berry (leea aequata l.) sparrow mango (buchanania arborescens), taro (araceae), grasses (graminaceae), vines such as spurred butterfly pea (centrosema virginianum), sweet leaf (sauropus androgynous), papaya (carica papaya), ferns (pteridophyta), and turmeric (curcuma sp.). dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 115 figure 2 landscape area at the dragonfly observation point on the batubolong river notes: a & b = river view during rainy season (4 april 2021); c & d = river view during dry season (6 july 2021). source: personal data (2021). most of the batubolong river stream is far from the residential area. only the downstream is close to the residents' villages. the canopy cover area on the batubolong river is in the class i-iv category (class i: 0-25% canopy cover, class ii: 26-50% canopy cover, class iii: 51-75% canopy cover, and grade iv: 76-100% canopy cover) (buckley et al. 2018; hendriks 2020). topographical and water physicalchemical parameters in the batubolong river table 1 presents the results of topographical and water physical-chemical parameters measurement in the batubolong river. in the topographical aspect, the batubolong river which passes through the area around the batulayar barat village is still at a low altitude (20 40 masl) with the highest air temperature of 36.1 oc and the lowest is 25.1 oc at the sampling time. the neutral water ph (6.5) combined with low tds (tds = 66 ppm), and low ec values (ec= 136 µs/cm) indicate that river water quality was still within the range of standard water quality (khairunnas & gusman 2018). the river water is also free of total dissolved solids and low in electrical conductivity; thus, it is safe as drinking water (afrianita et al. 2017; gasim et al. 2015). the standard of total dissolved solids for drinking water is below 1,000 ppm (who 1996). the water temperature (25 28 oc) of the batubolong river is still in the moderate category which is very good for the development of nymph larvae of several types of dragonflies. in the water bodies, medium to small fish and small crabs are found which can be a threat to dragonfly nymphs. biotropia vol. 29 no. 2, 2022 116 table 1 measurement of topographical and water physical-chemical parameters in the batubolong river no. topographical and water physicalchemical parameters measurement results observation date 1. altitude (masl) 20 40 6 july 2021 2. air temperature (oc) 30.4 36.1 25.1 28.9 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 3. humidity (%) 74 86 57 65 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 4. water ph 6.5 4 april 2021 and 7 june 2021 5. soil ph 5.8 4 april 2021 6. total dissolved solid (tds) (ppm) 66 7 june 2021 7. electrical conductivity (ec) (µs/cm) 136 7 june 2021 8. water temperature (oc) 27 28 25 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 9. wind speed (knot) 0.0 1.6 0.0 0.5 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) dragonfly (odonata) diversity in batubolong river the observed dragonflies (odonata) at various points on the batubolong river is presented in table 2. the distribution map of dragonflies (order odonata) found at the sampling location on the batubolong river is presented in figure 3. table 2 dragonflies (odonata) distribution in the batubolong river no. suborder and species name total observation time coordinate point perch and brightness altitude (masl) air temperature (oc) / humidity(%) a. suborder zygoptera 1. euphaea lara lombokensis (mclachlan, 1898) 5 10.50 wita, 04/04/2021 and 06/07/2021 -8.497485, 116.065951 on a rock, on a small branch 200-300 lux (shady) 20 30 / 86 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 5 10.50 wita, 04/04/2021 and 06/07/2021 -8.497485, 116.065951 on a rock, on a small branch 200-300 lux (shady) 20 30 / 86 3. nososticta emphyla (lieftinck, 1936) 3 13.53 wita, 04/04/2021 and 06/07/2021 -8.497350, 116.066083 -8.496795, 116.067063 on the leaves and twigs of the lantana (lantana camara) and kirinyuh (chromolaena odorata) trees 200-1500 lux (shady bright) 40 36.1 / 74 4. drepanosticta berlandi (lieftinck, 1939) 1 11.01 wita, 06/07/2021 -8.496795, 116.067063 under the leaves and stems of trees on the river 200 lux (shady) 40 28.7 / 66 dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 117 table 2 (continued) b. suborder anisoptera 5. orthetrum testaceum soembanum (forster, 1903) 5 11.11 wita, 04/04/2021 and 06/07/2021 -8.497503, 116.065891 on a wooden branch < 2,000 lux (hot) 20 31.5 / 81 6. neurothemis ramburii (brauer, 1866) 3 11.32 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on a wooden branch < 2,000 lux (hot) 20 30.4 / 85 7. trithemis festiva (rambur, 1842) 3 11.35 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on the rock < 2,000 lux (hot) 20 30.4 / 85 8. orthetrum glaucum (brauer, 1865) 2 11.45 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on the rock < 2,000 lux (hot) 20 30.4 / 85 9. agrionoptera insignis insignis (rambur, 1842) 1 11.39 wita, 06/07/2021 -8.495512, 116.068244 on the taro leaves 2,000 lux 20 28.9 / 57 10. orthetrum sabina (drury, 1773) 1 11.04 wita, 06/07/2021 -8.496763, 116.067334 on the branches of the bush < 2,000 lux (hot) 20 34.5 / 45 11. diplacodes trivialis (rambur, 1842) 2 11.41 wita, 06/07/2021 -8.495515, 116.068239 on stones and twigs < 2,000 lux (hot) 20 28.9 / 57 figure 3 distribution map of dragonfly (order odonata) at sampling locations on the batubolong river note: several species have the same location point. biotropia vol. 29 no. 2, 2022 118 sampling was carried out in the rainy (april) and dry (july) seasons. more dragonfly species were found in april (rainy season) than in july (dry season). dragonfly species found both in the rainy and dry seasons were: euphaea lara lombokensis (mclachlan, 1898), pseudagrion pilidorsum declaratum (lieftinck, 1936), nososticta emphyla (lieftinck, 1936), orthetrum testaceum soembanum (forster, 1903), neurothemis ramburii (brauer, 1866), trithemis festiva (rambur, 1842), and orthetrum glaucum (brauer, 1865). species of dragonflies (order odonata) that were only found during sampling in july (dry season) were drepanosticta berlandi (lieftinck, 1939), agrionoptera insignis insignis (rambur, 1842), orthetrum sabina (drury, 1773), and diplacodes trivialis (rambur, 1842). during the rainy season, the river water is abundant so that dragonflies have more opportunities to reproduce because water is a medium for dragonflies to lay eggs and develop larvae. the canopy and the light intensity also determine the types of dragonfly species existing around the river. dragonflies which prefer hot areas in the batubolong river are: orthetrum sabina (drury, 1773), diplacodes trivialis (rambur, 1842), orthetrum testaceum soembanum (forster, 1903), neurothemis ramburii (brauer, 1866), trithemis festiva (ramburi, 1866). 1842), and orthetrum glaucum (brauer, 1865). this findings is in agreement with the work of buchori et al. (2019). on the other hand, dragonflies which prefer shaded to moderate light areas in the batubolong river are: euphaea lara lombokensis (mclachlan, 1898), pseudagrion pilidorsum declaratum (lieftinck, 1936), nososticta emphyla (lieftinck, 1936), drepanosticta berlandi (lieftinck, 1939), and agrionoptera insignis insignis (rambur, 1842). relative species abundance, species diversity, population density and species evenness indices table 3 presents the calculation results of relative species abundance, species diversity, population density and evenness indices. euphaea lara lombokensis had the highest relative abundance of 21%, followed by pseudagrion pilidorsum declaratum and orthetrum testaceum soembanum of 15%, respectively, indicating that these species were able to breed and adapt well to the batubolong river. these three species are easy to find in rivers or streams having clean water with water ph close to neutral. the calculation results on the shannonwiener diversity index (h') of 2.212 (medium diversity category) indicated that the dragonfly species found in the sampling area were relatively few. the medium diversity may be caused by the crowded activity of the population around the river, especially in the downstream. at several points in the sampling area, there were logged trees and cowsheds owned by residents. therefore, it is necessary to expand the sampling area, especially in the upstream part which is still in the form of primary forest, considering that some dragonflies are very sensitive to community activities and environmental changes. table 3 calculation of relative species abundance, species diversity, population density and evenness indices no. species name total pi rsa ln pi h' c e 1. euphaea lara lombokensis (mclachlan, 1898) 7 0.212 21% 1.551 0.329 0.045 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 5 0.152 15% 1.887 0.286 0.023 3. nososticta emphyla (lieftinck, 1936) 3 0.091 9% 2.398 0.218 0.008 4. drepanosticta berlandi (lieftinck, 1939) 1 0.030 3% 3.497 0.106 0.001 5. orthetrum testaceum soembanum (forster, 1903) 5 0.152 15% 1.887 0.286 0.023 6. neurothemis ramburii (brauer, 1866) 3 0.091 9% 2.398 0.218 0.008 7. trithemis festiva (rambur, 1842) 3 0.091 9% 2.398 0.218 0.008 8. orthetrum glaucum (brauer, 1865) 2 0.061 6% 2.803 0.170 0.004 9. agrionoptera insignis insignis (rambur, 1842) 1 0.030 3% 3.497 0.106 0.001 10. orthetrum sabina (drury, 1773) 1 0.030 3% 3.497 0.106 0.001 11. diplacodes trivialis (rambur, 1842) 2 0.061 6% 2.803 0.170 0.004 total 33 1 100% 28.615 2.212 0.126 0.923 notes: pi = ratio between the number of individuals of a species with the number of individuals of all species (n/n); rsa = relative abundance; h' = shannon-wiener diversity index; c = dominance index; e = evenness index. dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 119 nososticta emphyla is a beautiful and rare dragonfly with only 3 species found in the sampling location (rsa = 9%). this dragonfly has a characteristic of having a metallic purple combined with black colors on the thorax. the last 3 segments at the end of the abdomen have a bright purple color. lieftinck (1953) described an adult male of n. emphyla dragonfly found on flores island has a deep reddish-purple color on the head. kosterin (2014) described n. emphyla dragonfly as having purple color gradually changing to citron-yellow on the thorax on the abdominal segments 1-3 with purple and brilliant cobalt blue on segments 8-10. this dragonfly is classified as rare on lombok island and is only found in the batubolong river area. according to the iucn redlist, n. emphyla is in the data deficient (dd) category and its presence is only recorded in the nusa tenggara islands, namely lombok island, sumbawa island and flores island (dow 2020). n. emphyla is found in the batubolong river at coordinates -8.497350, 116.066083 and -8.496795, 116.067063. when found, this dragonfly were flying across the riverbank and then perched on the nearby lantana (lantana camara), kirinyuh (chromolaena odorata) and bamboo trees. these dragonflies tend to be shy and will fly up into taller trees when caught. drepanosticta berlandi (lieftinck, 1939) was the rarest needle dragonfly found at the sampling point because only one male was found in immature condition. according to the iucn redlist, this species is endemic to the island of lombok and is recorded 2 times, namely in 1896 at an altitude of 2,000 masl as many as 2 males and in the 19th century as many as 1 male (kalkman 2009). the latest data indicated that in 2016, d. berlandi was also found on sumba island, east nusa tenggara in forest areas in the manupeu tanah daru and laiwangi wanggameti national parks, with high humidity, wet vegetation and dense canopy (irawan & rahadi 2018). when found in the batubolong river, d. berlandi was perching on the leaves under a shady tree. d. berlandi is classified as a long needle dragonfly with a body length of 5.96 cm. d. berlandi is categorized as dd (data deficient) in the iucn red list or the distribution and population data are still lacking so that the risk of extinction of this dragonfly is still unclear (kalkman 2009). several quite rare dragonfly species of the suborder anisoptera (large dragonflies) were found in the batubolong river. dragonfly orthetrum testaceum subsp. soembanun (foerster, 1903) is one of the large dragonflies that is quite sensitive to environmental changes and is only found in rivers or streams that are still clean. according to a report from gbif.org (20122019), the dragonfly orthetrum testaceum subsp. soembanun is only recorded in the islands of nusa tenggara (lombok island, sumbawa island, flores island, alor island and rote island). in addition, the dragonfly orthetrum testaceum subsp. soembanun is also reported to be in the batubolong river on lombok island (kosterin 2014) and in the manupeu tanah daru and laiwangi wanggameti national parks on sumba island (irawan & rahadi 2018). this dragonfly mating behavior can be seen during copulation and oviposition. at the time of copulation, the male and female dragonflies form a tandem formation, namely the female attaches the end of the abdomen (appendage) to the male secondary genitalia (segments 1–2 on the ventral abdomen), while oviposition is when the female lays her eggs on the substrate with an ovipositor (loiola & de marco 2011). the uniqueness of oviposition behavior in dragonflies orthetrum testaceum subsp. soembanun, happens when the female lays her eggs in the water, the males fly over them to guard them (irawan & rahadi 2018). at the sampling time of our study in the batubolong river, the male orthetrum testaceum subsp. soembanun dragonflies were at the river, while the female orthetrum testaceum subsp. soembanun perched on tree branches at the top of the river. another rare large dragonfly found in the batubolong river is agrionoptera insignis subsp. insignis (rambur 1842). according to gbif.org data between 1997-2020, this dragonfly is only found in 3 locations in indonesia, namely java island, kalimantan island and papua island. another report states that this dragonfly has been found in the batubolong river (lombok island) (kosterin 2014) and in the manupeu tanah daru and laiwangi wanggameti national parks on sumba island (ntt) (irawan & rahadi 2018). at young age, this dragonfly is similar to lathrecista asiatica, especially in terms of the biotropia vol. 29 no. 2, 2022 120 pattern on the thorax and abdomen. if you look closely, the two species have different thorax patterns. agrionoptera insignis insignis has a thinner (flat) abdomen with a slightly thicker septum than l. asiatica. at the end of agrionoptera insignis insignis abdomen there are 3 black segments, while in l. asiatica only the last 2 segments are black. land cover changes around the sampling locations on the batubolong river the analysis results of land cover changes using satellite imagery data for the period 20132020 around the dragonfly sampling locations on the batubolong river is presented in figure 4, figure 5 and table 4. figure 4 map of land cover changes around the sampling locations for the period 2013-2020 dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 121 figure 5 land cover changes in area surrounding the sampling locations notes: spatial analysis using gis applications obtained from: 1) high resolution satellite map of ntb province from lapan (2021); 2) map of ntb provincial rtrw administrative data from pupr ntb province. table 4 land cover area around the batubolong river no. year land cover area (m2) 1 2013-2015 24,532.95 2 2016 25,064.20 3 2017 25,689.16 4 2018 25,689.16 5 2019 26,340.81 6 2020 30,682.24 notes: spatial analysis using gis applications obtained from: 1) high resolution satellite map of ntb province from lapan (2021); 2) map of ntb provincial rtrw administrative data from pupr ntb province the largest land cover of 30,682.24 m2 occurred in 2020 (table 4), or an increase in land cover area of 6,149.29 in the period of 2013-2020. the occurring land cover changes may cause changes in the existing biodiversity, especially in the dragonfly population (kosterin 2014). previous studies of kosterin (2014) reported several dragonflies species found in the middle of the batubolong river in february 2014. the research data regarding dragonflies obtained by kosterin (2014) is quite diverse. there are differences in the dragonfly species data previously obtained by kosterin (2014) compared to the data in our study in 2021. the data difference is regarding the dragonflies diversity on the batubolong river. the data comparison is presented in table 5. table 5 data comparison of odonata research on the batubolong river (middle part) between 2014 and 2021 no. aspect kosterin (2014) our study (2021) 1. month of research februari april – juli 2. number of odonata species found 11 11 3. the odonata species found by kosterin (2014) and in our study (2021) 1. euphaea lara lombokensis (mclachlan, 1898) 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 3. nososticta emphyla (lieftinck, 1936) 4. orthetrum testaceum soembanum (forster, 1903) 5. trithemis festiva (rambur, 1842) 6. orthetrum glaucum (brauer, 1865) 7. agrionoptera insignis insignis (rambur, 1842) 8. orthetrum sabina (drury, 1773) 4. the odonata species found by kosterin (2014), but not found in our study (2021) 1. pantala flavescens (fabricius, 1798) 2. trithemis lilacina (förster, 1899) 3. rhyothemis phyllis (sulzer, 1776) 5. odonata species found in our study (2021), but not found by kosterin (2014) 1. drepanosticta berlandi (lieftinck, 1939) 2. diplacodes trivialis (rambur, 1842) 3. neurothemis ramburii (brauer, 1866) biotropia vol. 29 no. 2, 2022 122 the numbers of odonata dragonflies found in 2014 and 2021 are the same, but of different species. the difference may have been caused by: 1) the difference in study period and 2) the land cover changes occurring around the middle of the batubolong river. in 2014, it rained almost every month, except in september. in february 2014 the rainy season started with 111 mm of rainfall and 11 rainy days (bps-statistics lobar 2015). meanwhile, april 2021 was the rainy season, where river water was overflowing. in july 2021, it was the beginning of the dry season which dried up the batubolong river. in the middle of august 2021 it rained intensively. in 2021, as much as 34.8% of indonesia's territory experienced above normal dry conditions (wetter dry season, namely the dry season rainfall is higher than the climatological average) (bmkg 2021). the weather changes indicates that there has been a climate change that occurring between 2014 and 2021 which can affect the diversity of various kinds of invertebrates, including the order odonata (brook et al. 2006; clausnitzer et al. 2009). changes in the environment around the forest due to human disturbances can cause changes in the diversity of the order odonata (buchori et al. 2019; dolný et al., 2013; & šigutová et al., 2019). species drepanosticta berlandi (lieftinck, 1939) was found at the beginning of the dry season, at an altitude of 40 masl. according to harabiš & dolný (2010), the distribution of dragonfly is determined by several ecological factors, one of which is the altitude range. d. berlandi is a rare dragonfly recorded only on the lombok island and is a new species found on sumba island (irawan & rahadi 2018). meanwhile, trithemis lilacina was first discovered on sumbawa island by förster in 1899, by lieftinck in 1936 and 1953 on lombok island (steinmann 1997). based on data from gbif.org, t. lilacina was recorded only on islands of lombok, sumbawa, flores and timor (bánki et al. 2021). conclusion there are differences in dragonfly research data between those obtained by kosterin (2014) and the ones found in our study in 2021, in terms of different species found. our study discovered a rare needle dragonfly, namely drepanosticta berlandi (lieftinck, 1939) which has never been reported before in the batubolong river. satellite data showed that there has been a change in land cover in the period 2013-2020 around the batubolong river. the government and residents are expected to continually preserve the environment around the batubolong river by wisely utilizing the natural resources to support the sustainability of the existing rare dragonfly species. acknowledgments the authors sincerely thank the faculty of tarbiyah and teaching, universitas islam negeri mataram for funding this study. special gratitudes are also extended to the students for their assistance in collecting field data and to my beloved wife for developing the map of land cover change. references afrianita r, edwin t, alawiyah a. 2017. analisis intrusi air laut dengan pengukuran total dissolved solids (tds) air sumur gali di kecamatan padang utara. 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7(1):16-25. doi: 10.33394/ bjib.v7 i1.2381 šigutová h, šipoš j, dolný a. 2019. a novel approach involving the use of odonata as indicators of tropical forest degradation: when family matters. ecol indic 104(7):229-36. doi: 10.1016/ j.ecolind. 2019.05.001 steinmann h. 1997. world catalogue of odonata i. in world catalogue of odonata i. doi: 10.1515/9783110824438 [who] world health organization & international programme on chemical safety. 1996. guidelines for drinking-water quality. vol. 2. health criteria and other supporting information, 2nd edition. world health organization. https:// apps.who.int/iris/handle/10665/38551. biotropia vol. 29 no. 3, 2022: 244 253 doi: 10.11598/btb.2022.29.3.1711 244 role of mycorrhiza helper bacteria on mycorrhizal colonization and nematode pratylenchus coffeae infection reginawanti hindersah1*, elena f. l. lilipaly2, imam mudakir2, iis nur asyiah2 and rita harni3 1faculty of agriculture, universitas padjadjaran, sumedang 45363, indonesia 2biological education study program, faculty of teacher training and education, universitas jember, jember 68121, indonesia 3indonesian centre for estate crop research and development, sukabumi 43357, indonesia received 21 december 2021/accepted 3 august 2022 abstract the coffee nursery is susceptible to endoparasitic pratylenchus coffeae. application of biological method in the nursery is suggested to control the nematode population and maintain the seedling health. the objectives of this study were to observe the ability of arbuscular mycorrhiza fungi (amf) glomus spp. and liquid inoculant of mycorrhiza helper bacteria (mhb) consortium pseudomonas diminuta and bacillus subtilis for increasing amf colonization and reducing the infection p. coffeae in arabica coffee seedling and their growth. a pot experiment was conducted using a completely randomized block design with four treatments and five replications. the treatments were glomus spp. spore inoculation without and with two concentrations of mhb. the control treatment did not receive glomus spp. and mhb. the seedlings were growing in the greenhouse for three months. the results indicated that glomus spp. and mhb consortium significantly reduced the nematode total number in soil and roots by approximately 30%; and infection degree of p. coffeae by 50%. the application of glomus spp. significantly increased root colonization by mycorrhizal fungi, but mhb inoculation did not affect the mycorrhizal colonization. seedlings treated with mhb had higher shoot length compared to the plant without mhb and control; but the leaves number and shoot dry weight of seedlings were not affected by all treatments. even though the root fresh weight was reduced after mhb treatment, the lateral roots growth of mhb-treated seedling visually was improved. the experiment demonstrated that mhb was efficient to reduce p. coffeae infection of arabica seedling. keywords: glomus spp., infection degree, nematode population, root colonization, seedling growth introduction arabica coffee is the valuable agriculture commodity in global market. brazil is still the first coffee producer worldwide while indonesia is the 4th largest producer and exporters of coffee beans. in 2020, the coffee beans production in indonesia was 12.0 million of 60-kg bags. in growing coffee many challenges occur and have to be dealt with endoparasitic pratylenchus coffeae which well adapted to warm tropical and subtropical agroecosystem (thiep et al. 2018; tarno et al. 2021). pratylenchus are root lesion nematode; mainly infecting the cortical parenchyma which inhibit the water and nutrients absorption and thereby, cause serious root damage and limit plant growth (yu et al. 2012). in general, this problem was resolved by applying chemical and soil disinfectant as well as shading. nowadays, the use of arbuscular mycorrhizal fungi (amf) is suggested in order to reduce the harmful effect of chemicals on the agricultural environment. the amf is well known as biofertilizer, which inoculation become a prominent way to save phosphorous fertilizer and increase coffee tree growth in tropical agroecosystem (sewnet & tuju 2013; hernández-acosta et al. 2018). to date, researcher have reported that other advantage of mycorrhizal formation in roots is to reduce nematodes in coffee tree seedlings as *corresponding author, email: reginawanti@unpad.ac.id role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 245 well as replanted coffee fields (asyiah et al. 2015; pham et al. 2020). moreover, biocontrol strategy using amf may protect the plant form nematode pathogen and induce plant resistance against this pathogen (veresoglou & rillig 2012; poveda et al. 2020). among the amf species, the natural occurrence of glomus spp. in soil and rhizosphere of coffee tree are reported (prates júnior et al. 2019). the effect of amf on coffee growth were recorded. introducing amf on coffee nursery resulted in increment of height, foliar area and root volume arabica coffee seedling (hernandez-acosta et al. 2018). coffee tree inoculated by amf spores combined with chemical fertilizer showed better mycorrhizal colonization and tree growth (daras et al. 2015). certain soil bacteria have been reported to develop positive interaction with amf, which bacteria are known as mycorrhiza helper bacteria (frey-klett et al. 2007). the bacteria increase fungal spore germination and mycelium growth in soil and root-mycelium recognition (deveau & labbé 2016). the ability of gramnegative proteobacteria pseudomonas and a grampositive bacillus to help mycorrhizal establishment has been reviewed (frey-klett et al. 2007; labbé et al. 2014). both bacteria are well known plant growth promoting rhizobacteria (pgpr) which induce plant growth by the mechanisms of phytohormones production, biocontrol of soilborne diseases, arbuscular mycorrhiza stimulation and acquired systemic resistance induction (jankiewicz & kołtonowicz 2012; patel & saraf 2017; hashem et al. 2019). we have developed mycorrhiza helper bacteria (mhb) liquid inoculant of bacillus as well as pseudomonas. in previous research, single application of b. subtilis and p. diminuta suppressed the population of nematode p. coffeae in coffee seedlings by 71.3% and 64.2%, respectively compared to the untreated seedlings (asyiah et al. 2015). nonetheless, the ability of mixed cultures of both bacteria to induce mycorrhizal formation and repress p. coffeae infection have not been studied. controlling coffee pest in the nursery is an important step to enhance tree growth and coffee production in the field. improving the quality of coffee seedlings is essential to support the sustainability of coffee plantation. the objectives of this study were to determine the ability of mycorrhiza glomus spp. and the liquid formula of mhb consortium to enhance amf colonization and reduce p. coffeae infection, as well as to observe the effect of glomus spp and liquid formula of mhb consortium on the growth of coffee seedlings. materials and methods the experiment was carried out in the greenhouse of coffee and cocoa research institute (ccri) at jember, east java province, indonesia which is located in the tropical area at 60 m asl. the range of annual temperature and humidity in year 2019 were 23.0 29.9 oc and 67 86%, respectively. preparation of mhb and p. coffeae inoculants was conducted at the microbiology laboratory of universitas jember. the spores of glomus spp. (zygomycetous fungi) were provided by laboratory of mycology at the faculty of agriculture, universitas gadjah mada, while the arabica coffee seedlings were obtained from ccri. table 1 major essential nutrient composition of soil before experiment parameters methodsa value phh2o potentiometry 5.6 organic c walkey and black 2.39% total n kjeldahl 0.24% c to n ratio calculation 9.95 available p spectrophotometry 14.65 mg/kg available k spectrophotometry 79.82 mg/kg note: a = based on aoac proximate analysis method (2012). the top soil of inceptisols with loam texture (prastowo et al. 2013) was collected from the agricultural field at ccri. the soil properties showed that the soil was average in organic carbon (c) and total nitrogen (n), low in available phosphor (p) and high in potassium (k) (table 1). the c to n ratio of soil was low indicating the nitrogen was available for plants. preparation of glomus spp. and mhb inoculants arbuscular mycorrhizal fungi propagation was carried out in zeolite-based media with corn as host plant. a 150-g sterilized zeolite were poured into 250-ml transparent cup and biotropia vol. 29 no. 3, 2022 246 saturated with 0.5% nacl solution. then 100 glomus spp. spores were spread evenly on the surface of zeolite prior to sow two corn seeds and covered with 50 g of zeolite. substrate humidity was maintained by adding 30 ml water per day. the first fertilization was carried out a week after sowing by applying 30 ml of liquid mixed fertilizer at a concentration of 1 g/l; similar fertilization was done twice a week. two months later watering was reduced gradually in order to induce spore formation. the corn plants were eliminated and the growth substrates containing glomus spp. spores were used as amf inoculant. the production of bacterial liquid inoculant was carried out using molasses-based broth. molasses containing 19.58 c/n ratio, 0.18% p2o5, and 0.39% k2o was obtained from probolinggo sugar factory. each mhb species was grown separately in 2% molasses-based broth for 2 days at a temperature of 30 oc, then two volumes of b. subtilis were mixed with three volumes of p. diminuta. the liquid bacterial consortium was then put on a shaker at room temperature for three days. the final concentration of mixed mhb liquid inoculant was 109 colony forming unit (cfu)/ml. experimental design and set up the pot experiment was conducted in completely randomized block design composed of four treatments and six replications. the arabica coffee seedlings were treated with glomus spp. inoculant without mhb consortium (mhb-0) and with 108 cfu/ml mhb inoculant (mhb-1) and 109 cfu/ml mhb inoculant (mhb-2). control plants received neither glomus spp. nor mhb inoculant. the mature p. coffeae was applied to all treatments. plant substrate consisting of air-dried soil, sand and commercial compost in balanced composition was sterilized at 115 oc for 30 min and placed overnight at room temperature. as much as 5 kg plant substrate was put into opaque polyethylene pot with 5 drainage holes on its bottom and stored at the greenhouse. an 8-cm depth planting hole with 5 cm diameter was made in each pot prior to growing 3-month old coffee seedlings. the average shoot height and leaves number of seedlings at planting time were 11.2±0.08 cm and 4.2±0.05, respectively. soon after transplanting, 1 g of urea fertilizer was applied into 2-cm depth hole with a distance of 5 cm away from the stem seedlings. then, the holes were covered with the growth media. a week after transplanting, 50 mature p. coffeae was spread on 4-cm depth circular band with a distance of 5 cm from plant stem and then covered with thin layer of substrate. subsequently, 9.1 g glomus spp. inoculant containing 100 spores were spread over similar circular band and covered with substrates. inoculation of mixed mhb were carried out by pouring the liquid inoculant evenly around the seedling stem. the mhb-treated plants were inoculated with 1 ml of bacterial inoculant which was diluted in 99 ml distilled water, while control plants were watered with 100 ml distilled water. all seedlings on potted soil were grown in the greenhouse for 3 months. parameters measurement and statistical analysis plant height and leaves number were measured once a month until 3 months after treatment. fresh weight of shoot and root as well as shoot dry weight were measured at the end of the experiment. the root dry weight was not analyzed since the roots were used for nematode enumeration. the shoot dry weight was determined after being oven-dried at 70 oc for two days. after 3 months of treatment, the number of nematodes in root and soil were counted by extracting the nematodes from 10 g of soil sample and 100 ml of root extract following the method of baermann funnel (van bezooijen 2006). the soil and roots extracts samples were passed through a different set of filters with an opening size of 40 mesh and followed by the 325 mesh before counting the nematodes under a light microscope at 100x magnification. determination of root colonization degree (rc) by amf was carried out following the method of kormanik dan mc graw (1982). the roots were cut into 2-cm length root segments and subsequently soaked in 2% koh at 80 oc, followed by staining the roots with acid fuchsin. a total of 10 stained root segments were placed in object glass and covered with cover glass. the cover glass then pressed manually for flatting the roots and removing excess liquid. the number of infected roots was observed using a light microscope at 400 x magnification. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 247 the infected roots were determined by the hypha, vesicle and arbuscule occurrence in root segments. the rc (%) was calculated by dividing the number of infected root segments by number of observed root segments. at the end of experiment, the infection degree (id) for estimating disease severity due to p. coffeae was calculated using a scale of five classes (table 2) by using the townsendheuberger formula described by scalzo et al. (2012): id (%) = σ1i (ni.vi) ............................................ (1) (n.v) where: vi = infection class; ni = number of seedlings in one class; n = total seedling number; v = the highest class; i = the number of classes. table 2 the classes for scaling p. coffeae infection based on leaves and roots conditions class description 0 healthy seedling, the roots are normal without color alteration; leaves are green without yellow spots. 1 less than half leaves had a yellow spot but the leaves number was similar to healthy seedling; roots had altered their color to yellow and brown. 2 at least half of leaves number showed yellowing symptom and seedlings had some fallen leaves; roots are not healthy with rottenlateral roots. 3 the seedling is dominated by yellow leaves and the number of intact leaves were less than half of healthy seedling; their root system was not fully developed and rotted. 4 seedling had 1-2 yellow leaves and their roots were badly damaged indicated by brown in color and rotten-lateral roots. all data were subjected to analysis of variance (p ≤ 0.05). if the effect of treatment on the parameter was significant then the least significant difference (lsd) test was carried out at p ≤ 0.05. statistical analysis was performed using spss 16 statistical program. results and discussion root colonization and infection degree without glomus spp. inoculation, the roots did not show any root colonization (rc) by amf (fig. 1a), while inoculation of glomus spp. resulted in rc higher than 80%. the result verified that seedlings without mhb showed similar rc compared to seedlings treated with lower concentration of mhb inoculant (mhb-1). furthermore, seedlings inoculated with higher concentration of mhb (mhb-2) caused significant rc reduction up to 12.4%. low nematode infection degree (id) was demonstrated by seedlings grown with the application of glomus spp. inoculation compared to control (fig. 1b). the application of mhb combined with glomus spp. inoculation enabled to decrease id up to 50% compared to the control. seedlings inoculated with mhb containing 108 cfu/ml (mhb-1) demonstrated lower id even though it was not significantly different from the results showed by the inoculation of mhb-2 (109 cfu/ml) based on lsd test. microscopic observation revealed that nematode p. coffeae infected roots of arabica coffee at 3 months after treatment (fig. 2). moreover, mycorrhizal colonization in the root cell was clearly showed by arbuscular and vesicular formations. figure 1 effect of mycorrhiza helper bacteria consortium on (a) root colonization by amf and (b) infection degree caused by p. coffeae on coffee seedlings treated with glomus spp. at 3 months after treatment note: control = without glomus spp. and mhb inoculations. c c a b a a 120 100 80 60 40 20 0 120 100 80 60 40 20 0 c b b a biotropia vol. 29 no. 3, 2022 248 figure 2 mature nematode in coffee root cell of coffee seedling infected by (a) p. coffeae and (b) amf colonization resulted in vesicular formation in the root cells note: photo source: elena f.l.lilipaly, using olympus digital camera. the glomus spp. inoculation with and without mhb lowered the nematode population compared to the control even though the differences were not statistically significant (table 3). regardless of statistical analysis, lower nematode population in roots was shown by seedlings with glomus spp. and mhb. the lowest nematode population in soil was shown by seedlings treated with low concentration of mhb inoculant even though this decline was not statistically different based on lsd test. in general, the significant reduction of total nematode count was performed by seedlings treated with 108 cfu/ml and 109 cfu/ml of mhb liquid inoculants. results of this experiment found that glomus spp. inoculation with or without mhb increased root colonization by amf. the soil was sterilized before experiment and considered free from indigenous amf. therefore, only exogenous glomus spp. colonized the roots. this result verified that mhb did not induce mycorrhizal formation in roots and mhb slightly reduced root infection. microscopic observation showed that the presence of external hyphae, arbuscular and vesicular were only detected in glomus-treated roots. the lower rc in mhb-treated seedling shown in this experiment disagrees with the induction of amf spore germination, mycelium growth, and rootmycelium recognition proposed by deveau & labbé (2016). the failure of mbh to increase rc might be related to the inability of mhb to perform those roles. the arabica coffee seedlings were responsive to glomus spp. inoculation which relates to soil acidity and available p in soil. before experiment the soil acidity was 5.6. in general, soil fungi showed optimal mycelial growth at ph of 4.5 to 5.5. certain glomus spp. species produce the highest number of spores in soil ph of 6.5 (costa et al. 2013). the acidic and slightly acidic soil condition was suitable for the growth of glomus spp. and hence, their colonization in roots (rohyadi et al. 2004). table 3 effect of mycorrhiza glomus spp. and mycorrhiza helper bacteria consortium on p. coffeae population in roots and soils at 3 months after treatment treatments nematode number nematode total reduction (%)a root soil total control 221±29.6a 214±54.1a 435±72.0a glomus spp. without mhb 281±60.8a 104.4±4.4a 385.4±66.0a 11.4 glomus spp. + mhb-1 200.6±77.5a 74.4±12.5a 275±77.6a 36.7 glomus spp. + mhb-2 181.8±27.9a 112.2±18.1a 294.2±37.6a 32.4 note: values followed by the same letter in the same column are not significantly different according to lsd test at p ≤ 0.05. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 249 amf colonization in roots treated with glomus spp. is promoted by low available p content in soil. before experiment, the soil contained as low as 14.65 mg/kg of available p. the growth media composed of soil-sandcompost without inorganic p fertilizer. high p in soil and high rate of p fertilizer can inhibit am colonization (linderman & davis 2004). in contrast, adding only moderate inorganic p fertilizer enhanced the root colonization and diversity of amf communities (higo et al. 2020). the experiment found that inoculation of glomus spp. combined with mhb resulted in the significant reduction of nematode population up to 32 36% and id by 43 56%. both mhb produce phytohormone that play an essential role to activate the mechanisms of plant defence response against unfavourable condition (egamberdieva et al., 2014). chitinase is probably produced by mhb pseudomonas and bacillus as described by some researchers (zhong et al. 2015; amar et al. 2016). this chemical substance might limit nematode pathogen development. in the root knot of nematode, chitinase produced by paenibacillus illinoisensis was capable to lysis nematode eggshell and inhibit egg hatching (jung et al. 2002). however, the mechanism of chitinase to repress p. coffeae infection is remained unclear. increased root colonization by amf in glomus-treated seedlings might be related to low id of p. coffeae. mycorrhiza can suppress the population of p. coffeae through symbiont competition on space, carbon and nitrogen (bell et al. 2021) and induce the lignification of root endodermic cells that possibly decreased nematode pathogen infection (linderman 1994; elsen et al. 2001). the decrease of nematode population in our experiment agrees with the 68% of pratylenchus population reduction in soil of apple seedling after glomus intraradices inoculation (ceustermans et al. 2018). plant growth glomus spp. inoculation with or without any concentration of mhb liquid inoculant did not affect plant height and number of leaves at one and two weeks based on lsd test (fig. 3a & 3b). the application of glomus spp. and mhb enhanced plant height at 3 months after treatment, with decreasing number of leaves compared to control plants. this experiment showed that mixed inoculation of glomus spp. and mhb significantly increased plant height up to 22% compared to seedlings inoculated with glomus spp. without mhb (fig. 3a). the highest number of leaves was demonstrated by seedlings inoculated by glomus spp. without mhb (fig. 3b). the fresh and dry weight of shoots had not altered due to mhb inoculation (table 4). in contrast, growing coffee seedlings with glomus spp. but without mhb inoculation resulted in higher root weight over mhb-treated seedlings. inoculation of mhb clearly increased plant height at 3 months after treatment (fig. 3) but their dry weight was not different. figure 3 effect of mycorrhiza helper bacteria inoculation on plant height and number of leaves of coffee seedlings treated with glomus spp. at 1, 2 and 3 months after treatment note: control = without glomus spp. and mhb inoculations. a b a a a b b a b a 30 25 20 15 10 5 0 16 14 12 10 8 6 4 2 0 biotropia vol. 29 no. 3, 2022 250 table 4 effect of mycorrhiza glomus spp. and mycorrhiza helper bacteria inoculation on weight of shoot and root of coffee seedlings at 3 months after treatment treatments shoot weight (g) root fresh weighta dry weight fresh weight control 0.44 ± 0.25a 1.60 ± 1.09a 0.43 ± 0.28b glomus + without mhb 0.49 ± 0.26a 1.93 ± 1.11a 0.36 ± 0.26b glomus + mhb-1 0.44 ± 0.05a 1.64 ± 0.60a 0.11 ± 0.04a glomus + mhb-2 0.42 ± 0.08a 1.49 ± 0.36a 0.14 ± 0.08a note: values followed by the same letter in the same column are not significantly different according to lsd test at p ≤ 0.05. based on visual observation, the performance of seedlings treated with glomus spp. was better than that of the control plant (fig. 4). despite lower root fresh weight, seedlings inoculated with glomus spp. combined with mhb liquid in general had more rigorous lateral roots than plants without being inoculated with glomus spp. and mhb (fig. 5). moreover, the tap root of control seedlings and seedlings without mhb are likely thicker than roots of seedlings inoculated with mbh. significant increase in shoot height was observed in the seedlings inoculated with glomus spp. combined with mhb (b. subtilis and p. diminuta) at 3 months after treatment. shoot elongation is determined by several growth factors including the regulation of cell division and enlargement that affected by sufficient nutrient and plant growth substances. in the photosynthate partitioning, the stems are the sink that demands the energy (sugar) to elongate, while leaves are the source that produces the starch through photosynthesis. the sugar allocation from the leaves and essential ion taken by the roots were not suffice for shoot development. a plant growth hormone called auxin influences and integrates in plant growth (sachs 2005). figure 4 shoot performance of arabica coffee after being infected by p. coffeae and inoculation by glomus spp. and mhb note: control = without glomus spp. and mhb inoculations. figure 5 root performance of arabica coffee after being infected by p. coffeae and inoculated with glomus spp. and mhb note: control = without glomus spp. and mhb inoculations. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 251 plants produce auxin in the shoot tissues and has an essential role in initiating root growth and inhibiting primary root development (sachs 2005; alarcón et al. 2019). visual observation showed better growth of the seedlings’ lateral roots growth inoculated with glomus spp. combined with mhb compared to roots without glomus spp. and mhb inoculations. (fig. 3). mhb liquid inoculant of p. diminuta and b. subtilis consortium contained 4.01 mg/l of iaa, a group of auxin hormone that is capable of improving lateral root growth and subsequently facilitating better nutrient uptake and shoot growth. the results of this experiment agree with a study showing an increase in cucumber rooting after the application of low concentrations of exogenous auxin (balliu & sallaku 2017). the intensive growth of lateral roots in mhb-treated seedlings might cause low fresh weight of root because the primary roots become thin. in relation to root colonization by glomus spp., better root growth provides more physical contact establishments between fungi and roots. the mycorrhizal fungi are obligate symbiont characterized by forming arbuscular and/or vesicular in root cells. both particular and functional structure are found in glomus-treated coffee seedlings. this experiment found that glomus spp. are compatible with coffee seedlings as host plant and possibly build a positive interaction with the community of indigenous amf. both pseudomonas and bacillus in this experiment are not endophytic bacteria, so they do not compete with amf for infection sites. the precise mechanisms by which p. diminuta and b. subtilis decrease the infection degree of p. coffeae and repress nematode population in the presence of amf have to be studied. conclusion mixed liquid formula of mycorrhiza helper bacteria (mhb) consisted of p. diminuta and b. subtilis combined with spore of arbuscular mycorrhiza fungi glomus spp. significantly reduced the total number of p. coffeae in roots and soil by 30%. both microbial inoculations decrease the infection degree (id) of p. coffeae. introducing 108 cfu/ml and 109 cfu/ml mhb cells to the arabica coffee seedlings increased the id up to 50%. mhb has no significant role on the enhancement of root colonization (rc) by glomus spores in root seedlings inoculated with glomus spp. slightly but significant reduction of rc was shown in roots treated with mhb. a combination of mhb and glomus spp. increased shoot height at 3 months after treatments without affecting its dry and fresh weight. the decrease of root weight was observed in seedlings inoculated with mhb and glomus spp., with thinner primary roots and more intensive lateral roots. we suggest to introduce the inoculation of glomus spp. spores and mhb at the coffee nursery in order to minimize p. coffeae infection. acknowledgment the research was funded by the indonesian agricultural research and development agency of the ministry of agriculture. we would like to thank the director of the coffee and cocoa research institute for providing greenhouse and coffee seedlings. references andrade cc, young ki, johnson wl, villa me, buraczyk ca, messer wb, hanley ka. 2016. rise and fall of vector infectivity during sequential strain displacements by mosquito-borne dengue virus. j evol biol 29: 2205-18. alarcón mv, salguero j, lloret pg. 2019. auxin modulated initiation of lateral roots is linked to pericycle cell length in maize. front plant sci 10: 11. amar b. taha c, zaghloul i, el-mahdy ar, el-massry mh. 2016. effect of ph and temperature on bacillus sp. r2 chitinase activity and stability. proc technol 22: 471-7. asyiah in, harni r, wiryadiputra s, fauzi i. 2015. populasi pratylenchus coffeae (z.) dan pertumbuhan bibit kopi arabika akibat inokulasi pseudomonas diminuta l. dan bacillus subtilis (c.). [the population of pratylenchus coffeae (z.) and growth of arabica coffee seedling inoculated by pseudomonas diminuta l. and bacillus subtilis (c.)]. pelita perkebunan 31(1): 30-40. balliu a. sallaku g. 2017. exogenous auxin improves root morphology and restores growth of grafted cucumber seedlings. hort sci 44(2): 82–90. bell ca, magkourilou e, urwin pe, field kj. 2021. the influence of competing root symbionts on belowground plant resource allocation. ecol evol 11: 2997-3003. biotropia vol. 29 no. 3, 2022 252 ceustermans a, van hemelrijck w, van campenhout j, bylemans d. 2018. effect of arbuscular mycorrhizal fungi on pratylenchus penetrans infestation in apple seedlings under greenhouse conditions. pathogens 7(4): 76. costa fa, haddad lsm, kasuya mcm, oton wc, costa md, borges ac. 2013. in-vitro culture of gigaspora decipiens and glomus clarum in transformed roots of carrot: the influence of temperature and ph. acta scientiarum. agronomy maringá 35(3): 315-23. daras u, sobari i, trisilawati o, towaha j. 2015. pengaruh mikoriza dan pupuk npkmg terhadap pertumbuhan dan produksi kopi arabika [effect of mycorrhiza and npkmg fertilizers on growth and production of arabica coffee]. jurnal tanaman industri dan penyegar 2(2): 91-8. deveau a, labbé j. 2016. mycorrhiza helper bacteria. in: martin f, editor. molecular mycorrhizal symbiosis. new york (us): john wiley & sons, inc. p. 437-540. egamberdieva d, wirth sj, alqarawi aa, abd_allah ef, hashem a. 2017. phytohormones and beneficial microbes: essential components for plants to balance stress and fitness. front microbiol 8: 2104. frey-klett p, garbaye j, tarkka m. 2007. the mycorrhiza helper bacteria revisited. new phytol 176(1): 22-36. hashem a, tabassum b, abd-allah ef. 2019. bacillus subtilis: a plant-growth promoting rhizobacterium that also impacts biotic stress. saudi j biol sci 26(6): 1291-7. hernández-acosta e, trejo-agular d, ferrera-cerrato r, rivera-fernández a, gonzález-chávez mc. 2018. arbuscular mychorrhizal fungi in coffee growth (coffea arabica l.) varieties garnica, catimor, caturra and catuaí. agroproductividad 11(4): 61-7. higo m, azuma m, kamiyoshihara y, kanda a, tatewaki y, isobe k. 2020. impact of phosphorus fertilization on tomato growth and arbuscular mycorrhizal fungal communities. microorganisms 8(2): 178. jankiewicz u., kołtonowicz m. 2012. the involvement of pseudomonas bacteria in induced systemic resistance in plants. appl biochem microbiol 48(3): 244-9. jung w-j, jung s-j, an k-n, jin y-l, park r-d, kim ky, ..., kim t.h. 2002. effect of chitinaseproducing paenibacillus illinoisensis kja-424 on egg hatching of root-knot nematode (meloidogyne incognita). j microbiol biotechnol 12(6): 865-71. kormanik pp, mc graw ac. 1982. quantification of vesicular arbuscular mycorrhizae in plant roots. in: schenck nc, editor. methods and principles of mycorrhizal research. st paul (us): american phytopathological society. p. 37-45. labbé jl, weston dj, dunkirk n, pelletier 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an inceptisol of a cocoa plantation. j agric sci technol a 3: 878-85. prates jr p, moreira bc, da silva mdcs, veloso tgr, stürmer sl, fernandes rba, ..., kasuya mcm. 2019. agroecological coffee management increases arbuscular mycorrhizal fungi diversity. plos one 14: e0209093. rohyadi a, smith f, murray r, smith se. 2004. effects of ph on mycorrhizal colonization and nutrient uptake in cowpea under conditions that minimize confounding effects of elevated available aluminium. pl soil 260: 283-90. sachs t. 2005. auxin’s role as an example of the mechanisms of shoot/root relations. pl soil 268: 13-9. scalzo r, fibiani m, pietromarchi p, mandalà c, la torre a. 2012. effects of different fungicide treatments on grape, must and wine quality. comm appl biol sci 77(3): 151-61. sewnet tc, tuju fa. 2013. arbuscular mycorrhizal fungi associated with shade trees and coffea arabica l. in a coffee-based agroforestry system in bonga, southwestern ethiopia. af focus 26(2):111-31. tarno h, marsudi ew, widjayanti t, setiawan y. 2021. nematodes associated with robusta coffee plantations in malang district, east java, indonesia. biodiversitas 22(8):3306-12. thiep nv, soytong k. oanh ntk, hung pm. 2019. study on nematodes (pratylenchus spp.) on arabica coffee in the northwestern vietnam. int j agric technol 15(4): 675-84. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 253 van bezooijen j. 2006. methods and techniques for nematology. [cited 2019 june 6]. available from http://www.nematologia.com.br/files/tematicos/ 5.pdf veresoglou sd, rillig mc. 2012. suppression of fungal and nematode plant pathogens through arbuscular mycorrhizal fungi. biol let 8: 214-7. yu y-t, liu h-l, zhu a-g, zhang g, zeng l-b, xue sd. 2012. a review of root lesion nematode: identification and plant resistance. adv microbiol 2012(2): 411-6. zhong w, ding s, guo h. 2015. the chitinase c gene pschic from pseudomonas sp. and its synergistic effects on larvicidal activity. genet mol biol 38(3): 366-72. biotropiano. 5, 1991/1992: 10-14 the performance of upland rice established on alang-alang dominated area after various techniques of alang-alang control s. tjitrosemito seameo biotrop, p.o. box 17, bogor, indonesia and a. purwanto pt. monagro kimia, wisma kosgoro jl. thamrin, llth floor, jakarta, indonesia abstract pot experiment to investigate the performance of upland rice in a previously alang-alang dominated area was conducted under greenhouse condition at biotrop, bogor, indonesia from november 1986 to may 1989. the treatments were factorially combined, replicated 5 times and randomized completely. the first factor was alang-alang control consisting of 5 different techniques, i.e. (1) glyphosate applied at 2.2 kg a.e./ha; (2) imazapyr applied at 1.5 kg a.e./ha; (3) dalapon applied twice at 7.4 + 7.4 kg a.i./ha; (4) slashing followed by soil cultivation; (5) slashing of alang-alang only; while the second factor was nitrogen fertilizer at 4 different levels, i.e. (1) 0 kg n/ha, (2) 60 kg n/ha, (3) 120 kg n/ha given twice, 60 kg n/ha at planting time and 60 kg n/ha at 38 dap, (4) 180 kg n/ha given twice, 90 kg n/ha at planting and 90 kg n/ha at 38 dap. plant height (cm), tiller number/pot, productive tiller (%), panicle length (cm), spikelets/panicle, empty spikelet (%), weight 1000 grains (g) and grain yield (ton/ha) were observed. upland rice grown with zero tillage technique using glyphosate (2.2 kg a.i./ha) or dalapon (14.8 kg a.i./ha) performed as good as or even better than manual cultivation. imazapyr at 1.5 kg a.e./ha was phytotoxic to rice planted 1 month after spraying. the application of n fertilizer lower than 60 kg n/ha was not sufficient, but more than 60 kg n/ha was too high; it stimulated the production of too many tillers, with high percentage of unproductive tillers and empty grains. introduction areas under alang-alang (imperata cylindrica (l.) beauv.) have recently been utilized for transmigration schemes in indonesia. the transmigrants from java and bali used to cultivate their land before planting upland rice. this practice, however, is very tedious, especially on areas dominated by alang-alang. they are usually able to cultivate only 0.4-0.7 ha of land manually in this alang-alang dominated area. 10 the performance of upland rice established s. tjitrosemito & a. purwanto the area under alang-alang is known to be infertile, producing a very low rice yield, insufficient to support the transmigrant's family. it forces them to find additional income, and when the area is close to the forest, they join the group of illegal loggers. it is imperative to find ways to raise the transmigrant's income to improve their condition. in this context pot experiments were carried out under greenhouse condition to investigate the growth and yield of upland rice in alang-alang dominated areas. material and methods pot experiments were conducted under greenhouse condition at biotrop, bogor, indonesia from november 1986 to may 1989 to investigate the performance of upland rice grown in a previously alang-alang dominated area established by various techniques at different levels of nitrogen fertilizer. the alang-alang plants were first established in plastic pots of 20 cm diameter and 21 cm height by 5 single-node cuttings/pot for approximately 9 months. for the experiment, 100 pots were utilized. the treatments were factorially combined, replicated 5x and randomized completely. the first factor was alang-alang control consisting of 5 different techniques, i.e. (1) glyphosate applied at 2.2 kg a.e./ha; (2) imazapyr applied at 1.5 kg a.e./ha; (3) dalapon applied twice at 7.4 + 7.4 kg a.i./ha; (4) slashing followed by soil cultivation; (5) slashing of alang-alang only; while the second factor was nitrogen fertilizer at 4 different levels, i.e., (1) 0 kg n/ha, (2) 60 kg n/ha, (3) 120 kg n/ha given twice, 60 kg n/ha at planting time and 60 kg n/ha at 38 days after planting, (4) 180 kg n/ha given twice, 90 kg n/ha at planting and 90 kg n/ha at 38 days after planting. dalapon was sprayed twice at 7.4 kg a.i./ha each time at intervals of 3 weeks; the other herbicide treatments were done on the same days as the second spraying of dalapon, while slashing and cultivation treatments were carried out on the same day of seed planting. the sprayings were carried out using cp20 at high pressure with yellow nozzle calibrated to deliver an equivalent amount of 600 1/ha. upland rice (oryza sativa c.v. sentanu) seeds were selected for uniformity and planted one month after the last herbicide spraying, by dibbling 5 seeds/pot at about 2 cm deep. the plants were later thinned to 3 plants/pot. the basal fertilizer consisting of kc1 and triplesuper-phosphate (tsp) at 60 kg k2o/ha and 60 kg p2o5/ha, respectively, were given to all pots by dibbling about 5 cm deep at a distance of 5 cm from the rice hill. the nitrogen fertilizer in the form of urea was also given in the same manner. 11 biotropia no. 5, 1991/1992 during the experiment, the soil was kept moist and plants were prevented from pests by spraying with sevin 855 and from pathogens by spraying with dithane m-45. the data collected were plant height at harvest time, number of tillers, number of panicles, length of panicle, number of spikelets/panicle, percentage of empty spikelets, the yield of grain/pot at 14% moisture content, weight of 1000 grains and yield/ha equivalent. the data were analysed statistically using f-test and the means were compared with dmrt. results and discussion upland rice plants grown on pots previously treated with imazapyr at 1.5 kg a.e./ha were killed. it seems that 1.5 kg a.e./ha of imazapyr even 1 month after application was still too toxic for upland rice in this pot condition. similar results were also reported by tjitrosemito & tentamia (1986) with soybean; but it was contrary to the results of field experiments on soybean reported by tjitrosemito & suwinarno (1988), where application of imazapyr at 2.0 kg a.e./ha to alang-alang fields when planted with soybean 1 month later showed no symptoms of phyto-toxicity. it is important, therefore, to study further the behaviour of imazapyr molecules in the soil and to specify what conditions may increase or decrease its availability to plants to become toxic. the performance of upland rice is summarized in tables 1 and 2. table 1. the performance of upland rice established in pots previously occupied by alang-alang after various control techniques variables rice establishment after alang-alang control glyphosate + zero tillage dalapon + zero tillage imazapyr + zero tillage soil cultivation slashed + zero tillage plant height (cm) 85.2 c 76.9 b 0.0 a 78.1 b 69.7 b tiller number/pot 5.5 c 4.9 b 0.0 a 5.6 c 4.9 b productive tiller (%) 76.4 c 69.4 be 0.0 a 67.8 be 63.3 b panicle length (cm) 19.1 c 18.8 c 0.0 a 17.0 be 16.0 b spikelets/panicle 76.5 d 65.3 c 0.0 a 61. 8 c 55.4 b empty spikelet (%) 18.7 b 18.6 b 0.0 a 16.6 b 16.6 b weight 1000 grain (g) 23.6 b 22.9 b 0.0 a 23.0 b 23.2 b grain yield (ton/ha) 0.81 d 0.58 c 0.0 a 0.67 cd 0.39 b nb: numbers in a column followed by the same letter do not differ significantly at 5% level 12 the performance of upland rice established s. tjitrosemlto & a. purwanto table 2. the effect of n-fertilizer on the performance of upland rice established on land previously dominated by alang-alang variables rate of nitrogen fertilizer (kg n/ha) 0 60 120 180 plant height (cm) 38.5 a 53.0 b 57.2 b 58.0 b tiller number/pot 3.5 a 4.3 b 6.5 c 6.6 c productive tiller (%) 51.4 a 55.8 b 50.8 a 48.5 a panicle length (cm) 9.7 a 12.3 b 13.0 bc 13.5 c spikelets/panicle 33.1 a 64.6 b 79.5 c 81.7 c empty spikelet (%) 13.3 b 9.6 a 10.9 a 10.7 a weight 1000 grain (g) 14.3 a 15.3 c 14.4 ab 14.7 bc grain yield (ton/ha) 0.16 a 0.44 b 0.57 bc 0.65 c this local upland rice variety produces only a small number of tillers. its performance was quite well when established by zero tillage after alang-alang had been cleared using glyphosate at (2.2 kg a.e./ha). it produced 0.81 ton/ha of grain which was as good as that produced when the rice planted after alang-alang was slashed followed by manual soil cultivation. under dalapon treatment it also showed quite a good yield i.e. 0.58 ton/ha which was not different from that under manual cultivation. when alang-alang was only slashed before dibbling the seed, the rice performed badly. it produced only 0.39 ton/ha equivalent. this was expected because it had to compete against regrowth of alangalang from rhizomes. the tillering was somehow very limited under dalapon treatment. there were only 4.9 tillers/pot which was the same as that under slashing, while under glyphosate it was 5.5 tillers/pot. under glyphosate treatment, this upland rice also showed the highest percentage of productive tillering (76%) and the highest number of spikelets/ panicle. it was not surprising to see that the performance of this upland rice with grain yield of 0.81 ton/ha equivalent was the best under glyphosate treatment in this experimental condition. the effect of nitrogen fertilizer is shown in table 2. the application of nitrogen fertilizer (60-180 kg n/ha) increased the performance of upland rice. when unfertilized, rice grew short (38.5 cm) with a small number of tillers, a small part of which became productive. the panicle was also short with a few spikelets and the panicle had a high percentage of empty spikelets. morever, the grain was light, therefore, the total grain yield was the lowest among the treatments. it is clear that n-fertilizer is badly needed for this rice production. the application of 60-180 kg n/ha increased the height and the tiller number' considerably (p < 0.01); however not all of these increased tillers developed into productive tillers. when this upland rice was fertilized with 120 and 180 kg n/ha, 13 biotropia no. 5, 1991/1992 apparently too many tillers were produced to develop further into productive tillers. in fact, under 180 kg n/ha, less than 50% of the tillers produced developed into productive ones, which is worse than rice not treated with fertilizers. nitrogen fertilizer application at 60 kg n/ha increased the tiller number and the proportion of tillers developing into productive ones. n-fertilizer rate at 60 kg n/ha is probably the appropriate rate. nitrogen fertilizer application increased the number of panicles. treatments with 120 and 180 kg n/ha increased the spikelet number/panicles more than double, i.e. 79.5 and 81.7 respectively. but, again, this increase was too much to fill up the spikelet which gave lighter grain as assessed by the weight of 1000 grains. nitrogen fertilizer application at 60 kg n/ha showed the heaviest grain among the treatments, which supports further the conclusion that the appropriate n-fertilizer application is 60 kg n/ha in this condition. with the available results, it is obvious that food crops can actually be established in alang-alang dominated area using zero tillage technique, provided that alang-alang is controlled with appropriate herbicides and an appropriate rate of fertilizer is applied. references tjitrosemito, s. and tentamia. 1986. the growth performance of soybean under the influence of herbicide residue. biotrop internal report. _____, and d. suwinarno. 1988. the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides. biotropia 2: 12-17. 14 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf biotropia no. 5, 1991/1992: 41-52 histopathology of the telencephalon and diencephalon of t1lapia nilotica exposed to sublethal dose of malathion s-[1,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate] edna a. amparado institute of biology, university of the philippines, diliman, quezon city, philippines abstract a 35-day exposure of tilapia nilotica embryos to sublethal doses of 3.0 ppm and 0.3 ppm malathion, s-[l,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate], commercial grade, ec 57, produces cellular and ultrastructure changes in the brain. a number of nuclear centers of the treated animals are markedly larger than those of the control. aberrant features observed in day-45 embryos are the neoplastic masses and increased vascularization. ultrastructure defects include the presence of nuclear blebs, cytoplasmic vacuolations and increased lysosomal bodies. introduction the reliance of agriculture on pesticides to increase crop yield has resulted in the extensive and intensive use of these chemicals. since the ban on the use of the organochlorine, organophosphates have been used increasingly to control pest infestation. though neither as deadly nor as persistent as the organochlorines, organophosphates are potent inhibitors of acetylcholinesterase (rao et al. 1984; anthony 1986; antwi 1987). a number of studies have shown that these chemicals cause, too, morphopathologic effects. among these are vertebral malformations, parrot beak syndrome in fowl (van leewen et al. 1986), curved body, axis, blister formation and delayed melanogenesis in frog (pawar et al. 1983), proliferation of type ii pneumocytes, proliferation of interstitial cells and delayed septal and capillary development in lungs (koizumi et al. 1988), atresia of follicles (ansari et al. 1986), and demyelination and degeneration of axon (hoffman et al. 1984), disruption of oogenesis in pontius conchonus (kumar and pant 1988) and vacuolation of tubules and degeneration of glomeruli in glorias batrachus (mandal 1987). this study focuses on the histopathology of the telencephalon and diencephalon of tilapia nilotica, exposed to commercial grade malathion. 41 biotropia no. 5, 1991/1992 materials and methods a. chemical malathion s-[l,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate], commercial grade, 57 ec, used in the study for this concentration is utilized in agriculture, poultry and livestock spray. it is a deep brow~n to yellow liquid, slightly soluble in water (145-147 ppm), mixable with many organic solvents including alcohol, esters ketones, ethers, aromatic and alkylated hydrocarbons and vegetable oils. it has a limited solubility in certain paraffin hydrocarbons. it has a boiling point of 156 157°c; melting point of 2.9°c; density of 1.23 g/cc. it is hydrolyzed at ph > 7.0 or < 5.0 but is stable in aqueous solution buffered to ph 5.26 (merck index 1983). b. collection and rearing of fry tilapia nilotica eggs, approximately 1000, were obtained from the bureau of fisheries and aquatic resources in tanay, rizal and allowed to hatch in finger bowls. the hatchlings were divided into control and experimental groups, each consisting of 300-350 larvae and reared in 1.0 x 1.0 x 1.5 m concrete ponds in dechlorinated tap water until day 45, post-fertilization. malathion, commercial grade, ec 57, diluted to sublethal doses of 0.3 ppm and 3.0 ppm served as pond water for the experimental group. pond water was changed every week, taking care to maintain the dose concentration in the experimental pond. histological studies a. paraffin series five fry from each group were harvested every three days until day 45, fixed in zenker's solution, dehydrated in alcohol series, sectioned serially at the brain region and stained in delafied's hematoxylin-eosin for light microscopic examination. the sizes of nuclear centers in each brain region at a particular developmental stage were measured using an ocular micrometer (fig. 1a and 1b). statistical analyses of the data were done by the use of two-way anova. 42 figure 1a. paraffin transverse section of themedial olfactory area of untreated, day-19, tilapia nilotica. the olfacto-somatic area (os) consisted of small neurons. x 400 figure 1b. paraffin transverse section of the thalamus of untreated, day-45. tilapia nilotica. the nucleus rotondus (nr) is a large nuclear center in the lateral region of the thalamus. x 400 b. resin sections specimens were decapitated and heads were fixed overnight at 20°c in 2.5% glutaraldehyde in sodium phosphate buffer, ph 7.2. brains were dissected and washed in millonig's buffer for 45 minutes and fixed for one hour at 20°c in 1% osmium tetroxide. after a 45-minute buffer wash, they were dehydrated through an acetone series, embedded in araldit (luft's araldit formula) at 40-50°c for 24 h each and at 60°c for 3 days. sections for light microscopy were cut at 0.5 um using the lkb ultramicrotome and stained with 1% toluidine blue. for electron microscopy, ultrathin sections, 100 um, were made with the same ultramicrotome, stained with 1% uranyl acetate, counterstained with lead citrate, and examined with joel, jem 100u electron microscope. 43 histopathology of the telencephalon and diencephalon edna a. amparado biotropia no. 5, 1991/1992 results light microscopy a. telencephalon the early histologic aberration which occurred among the free-swimming larvae, days 16-19, was the early differentiation of the olfacto-somatic area. unlike in the control group, this nuclear center of embryos exposed to 0.3 ppm malathion was composed of large polyploid neurons (fig. 2). at this stage, too, the olfactory bulb and a number of nuclear centers had larger sizes than those of the control group which size differences persisted until day 35 (table 1). thereafter, the nuclei of treated embryos showed two types of changes: i) increased areas of olfactory bulb and olfacto-somatic areas and ii) decreased areas of nucleus olfactorius pars bulbaris, nucleus pars commissuralis, preoptic nuclei and somatic area (table 1). day 45 specimens showed a number of histopathologic effects.the medial olfactory area of 3.0 ppm-dosed embryos showed hypertrophy and increased vascularization (fig. 3). table 1. comparative sizes (um) of the nuclear centers in the telencephalon of tilapia nilotica at specific developmental stage, post-fertilization 44 biotropia no. 5, 1991/1992 table 1. continued nuclear center 36-45 days a b c nucleus olfactorius 3505.30+138.80 2202.58+370.88 3341.79+462.10 pars bulbaris nucleus olfactorius 1428.92+29.10 1560.00+39.30 1992.25+ 58.40 pars commisuralis nucleus preopticus 375.63+26.40 333.29+38.40 258.56+27.50 olfacto-somatic area 190.85+17.50 121.59+11.10 181.25+269.00 somatic area 20927.40+591.40 19836.80+820.90 19396.40+835.80 nucleus taenia 843.60+77.80 618.87+40.60 819.26+67.30 table 2. comparative sizes (um2) of selected nuclear centers in the diencephalon of tilapia nilotica at specific developmental stage, post-fertilization 45 histopathology of the telencephalon and diencephalon edna a. amparado table 2. continued b. diencephalon larger nuclear centers than those of the control group were observed as early as day 16 and the disparity in sizes persisted until day 45, post-fertilization. table 2 shows the sizes (um) of selected diencephalic nuclei at specified developmental stage. during the late free-swimming stage, day 16-19, the epithalamic centers were larger than those of the control while other nuclear centers were smaller. on days 21-28, nuclear masses were much larger than those of the control. on days 36-45, a number of nuclear centers exhibited regression in sizes with the notable exception of hypophysis and nucleus geniculatus lateralis which showed steady increase in sizes. in the dorsal thalamus, the region lateral to the nucleus ventrolateralis thalami showed hypertrophy (fig. 4). 46 figure 2. paraffin transverse section of the medial olfactory area of day-19 tilapia nilotica exposed to 0.3 ppm malathion since day 10, post fertilization. the olfacto-somatic area (os) consisted of large polyploid neurons. x 400 figure 3. paraffin transverse section of the medial olfactory area of day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. the brain showed hypertrophy (arrow) and increased vascularization (arrowhead). x 400 figure 4. paraffin transverse section of the thalamus of day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. the lateral region showed hypertrophy (arrow). x400 47 histopathology of the telencephalon and diencephalon edna a. amparado biotropia no. 5, 1991/1992 electron microscopy ultrastructure defects were observed in the telencephalic and diencephalic neurons of pesticide-exposed fishes. the electromicrograph of normal neurons of t. nilotica in day-45 is shown in fig. 5. in both 0.3 and 3.0 ppm malathion exposed fish, the nucleus showed blebbing, ruffling and nucleolar disintegration (fig. 6). extensive vacuolations and lysosomal bodies were observed in the cytoplasm (fig. 7 and 8). discussion the present study describes the impact of chronic exposure to commercial grade malathion on the neurons of tilapia nilotica embryos. membrane deformations are similar to the necrosis in the brain of pacific herring larvae, clupea harenguipallasi, exposed to crude oil (cameron and smith 1980), larvae hatched from eggs that have been incubated in varying concentrations of zinc (somasundaram et al. 1984) and in paraclithys lethosigma exposed to mercury (trump et al. 1975) and mouse brain exposed to the neurotoxicant trimethyltin (chang et al. 1982). necrosis is attributed to the binding of the toxicant to the sh group in the neuronal membrane (trump et al. 1978), failure of the neuronal plasma membrane atpase system (chang et al. 1982) and interaction of the lipophilic pesticide with the cell membrane (triebskorn and kunast 1990), and increase in acetylcholine release that induced increased membrane permeability to sodium, potassium, and calcium ions (kabayashi et al. 1988). these authors contend that changes in fluid and electrolyte balance resulted in membrane damage. while these researchers categorized nuclear damage as effects caused by lethal concentrations, in this study, nuclear blebs are induced by sublethal concentrations. interestingly, these cytopathologic conditions are observed only on older embryos, day 45 to day 60 specimens. chronic exposure may have exhausted the defense mechanisms such as the depletion of energy resources needed to initiate protective processes (recio et al. 1988). other histopathologic effects include hypertrophy of a number of nuclear centers and increased vascularization. such aberrations were observed in the gill epithelium of teleosts exposed to acute doses of heavy metals such as zinc (khangarat 1982), cadmium (norgren et al. 1988), copper, silver and lead (simula 1988). similarly, thickening of the basement membrane of the digestive tract of the garden slug resulted from exposure to lethal dose of carbamate molluscide chloethocarb (triebskorn and kunast 1990). the observations that hypertrophy and increased vascularization were visible in day 45 embryos may indicate that prolonged exposure to sublethal concentrations eventually produce histologic aberrations induced by lethal exposure. these pathomorphologic changes may be due to prolonged 48 histopathology of the telencephalon and diencephalon edna a. amparado figure 5. electromicrograph of normal neurons in day-45 tilapia nilotica. the nuclear membrane is intact and the cytoplasm does not exhibit vacuolization. x 6000 49 biotropia no. 5, 1991/1992 figure 6. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. neurons exhibited nuclear blebs (arrow), x 6000 figure 7. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. cytoplasmic vacuolations were extensive in both the telencephalon and diencephalon. x 6000 figure 8. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. lysosomal bodies were rampant in the cytoplasm, x 6000 50 histopathology of the telencephalon and diencephalon edna a. amparado deprivation of acethylcholinesterase which is required for cellular interactions in the maintenance of the nervous system structure (chase and kankel 1988). the findings that pesticides cause numerous neuronal pathology have repercussions on the reproductive functions and life span of tilapia nilotica. that a very low dose is capable of producing various neuron aberrations, evaluation on the intensive and extensive use of pesticides is an urgent necessity. references anthony, j., e. banister and p.c. oloffi. 1986. effects of sublethal levels of diazinon: histopathology of liver. bull. environ. contain. toxicol. 37: 501-507. antwi, l.a.k. 1987. fish head acetylcholinesterase after aerial application of tenephos in two rivers in burkina faso, west africa. bull. environ. contain. toxicol. 37: 501-507. chase, b.a. and d.r. kankel. 1988. on the role of normal acetylcholine metabolism in the formation and maintenance of the drosophila nervous system. dev. biol. 125: 361-380. chang, l.w., t.m. triemeyer, g.r. wenger, p.e. mcmillan, k.r. reuhl. 1982. neuropathology of trimethyltin intoxication. ii. electron microscopy study on the hippocampus. env. res. 29: 445-458. chang, l.w. 1983. neuropathology of trimethyltin intoxication iii. changes in the brain stem neurons. env. res. 30: 399-411. delpire, e., c. duchene, g. goessens and r. gilles. 1985. effects of osmotic shocks on the ultra structure of different tissues and cell types. ex. cell. res. 160: 106-116. khangarot, b.s. 1982. histopathological changes in the branchial apparatus of pontius sophore (hamilton) subjected to toxic doses of zinc. arch. hydrobiol. 93: 352-358. kumar, s. and s.c. pant. 1988. comparative sublethal ovarian pathology of some pesticides in the teleost. pontius conchonius hamilton. bull. environ. contain. toxicol. 41: 227-231. mandal, p.k. and a.k. kulshrestha. 1980. histopathological changes induced by the sublethal smithion in glorias batrachus l. indian j. exp. biol. 18: 547-552. norgren, l.k., p. runn, c. haux and l. forlin. 1985. cadmium induced changes in the gill morphology of zebrafish, brachydamio nerio (hamilton-buchanan) and rainbow trout, salmo gairdneri (richardson). j. fish biol. 27: 81-95. pant, j.c. and t. singh. 1983. inducement of metabolic dysfunction by carbamate and organophos phorous compounds in a fish, pontius conchonius. pestic. biochem. physiol. 20: 294-298. rao, d.m. and a.s. murthy. 1980. toxicity, biotransfortmation and elimination of endosulfan to the indian major carp, catla catla with special reference to some biochemical changes induced by the pesticide. pestic. biochem. physiol. 33: 220-229. recio, a., a. marjgomez, e. angulo and j. moya. 1988. zinc treatment of the digestive gland of the slug arion ater l. 2. sublethal effects at the histological level. bull. environ. contain. toxicol. 41: 865-871. sunila, i. 1988. acute histological responses of the gill of mussel, mytilus edulis to exposure by environmental pollutants. j. inverteb. pathol. 52: 137-141. 51 biotropia no. 5, 1991/1992 triebskorn, r. and c. kunast. 1990. ultrastructural changes in the digestive system of deroceras reticulatum (mollusca: gastropoda) induced by lethal and sublethal concentrations of the carbamate molluscicude cloethocarb. malacologia 32: 87-104. van leeuwen. c. j., t. helder and w. seinen. 1986. aquatic lexicological aspects of dithiocarbamates and related compounds. iv. teratogenicity and histopathology in rainbow trout (salmo gairdnerf). aq. toxicol. 9: 247-259. wester, p.w. and j.h. canton. 1986. histopathological study of oryzias latipes after long-term b-hexachlorocyclohexane exposure. aq. toxicol. 9: 21-45. 52 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf biotropia vol. 29 no. 3, 2022: 234 243 doi: 10.11598/btb.2022.29.3.1705 234 physical and chemical qualities of corn with different moisture levels supplemented with mold inhibitor catootjie l. nalle*, max a. j. supit, angga m. akbar, angriana so’o and emiliana langodai animal husbandry department, politeknik pertanian negeri kupang, kupang-85228, indonesia received 2 december 2021/accepted 7 july 2022 abstract corn grain is used as the main energy source in poultry diet formulation. the quality of corn is easy to deteriorate during storage because of insect, fungal, and mycotoxin contamination. efforts should be made to maintain the quality of corn during storage. the present study aimed to evaluate the physical and chemical qualities of different moisture levels of corn supplemented by a mold inhibitor. a total of 750 kg of corn grains was used in the present study. a commercial mold inhibitor was used with a dose of 0.045%. the experimental design used was a 3 x 2 factorial complete randomized design. the first main factor was the different moisture levels (ml) of corn (≤ 10%, 10.0-10.9%; 11.0-11.9%), while the second main factor was mold inhibitor (mi, or +). thus, there were six treatment combinations, and each treatment comprised five replications. the results showed that ml, mi, and ml x mi interaction significantly (p < 0.05 to 0.001) affected the percentage of grain damage and fungal grain but not (p > 0.05) the moisture level of corn during 90 days of storage. except for crude protein content, the ml did not affect (p > 0.05) the proximate composition (pc) and gross energy (ge) content of corn. except for dry matter (dm), the pc and ge content of corn were not affected (p > 0.05) by mi. ml x mi interaction did not affect (p > 0.05) the pc and ge content. the aflatoxin b1 (afb1) content was similar (p > 0.05) among all treatments. except for histidine and lysine contents, the amino acid contents of corn were not affected by ml, mi, or ml x mi combination. in conclusion, the supplementation of mi in corn with different ml improved the physical quality, dm, ash, and ge content of corn grain during the storage; mi maintained the dm content but did not reduce the afb1 content of corn. except for histidine and lysine, the supplementation of mi in corn with different ml did not affect the amino acid content of corn. keywords: corn, moisture levels, mold inhibitor, quality introduction corn as a feed ingredient is the dry seeds of zea mays l that have been removed and cleaned from the cobs (national standardization agency 2013). furthermore, it was explained that based on the color, corn kernels are classified into two types, namely white corn, and yellow corn. both yellow and white corn is the main energy source in poultry diet formulation in indonesia. the proportion of corn used in poultry and monogastric diets ranges from 50 to 60%. the quality of corn as a feed ingredient is determined based on its nutrient content and the presence or absence of unwanted materials (national standardization agency 2013). corn is categorized into two levels of quality. the first and second qualities of corn contain maximum 14 and 16% moisture content, respectively (national standardization agency 2013). corn grain having high moisture content is often associated with aflatoxin content. in indonesia, freshly harvested corn usually has a high moisture content, approximately 31.28%, so that if it is not immediately and properly dried, it will very quickly become contaminated with various fungi including aspergillus flavus and fusarium (hausufa & rusae 2018; mukkun et al. 2018). the growth of aspergillus flavus is influenced by various factors such as initial moisture content, relative humidity, temperature, *corresponding author, email: catootjienalle@gmail.com physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 235 atmospheric gases, light, oxygen, carbon dioxide, ph, mechanical damage, contamination, and competitive effects of other molds (kumar et al. 2021; muga et al. 2019; mukkun et al. 2018). aspergillus flavus can live well at a temperature range of 20 35 oc, ph 4-6, relative humidity 80 to 90%, aerobic atmosphere conditions, and 18% water content (talanca & mas'ud 2009; muga et al. 2019). daou et al. (2021) reported that the optimal temperatures for aspergillus flavus to grow and to produce toxins were 35 and 33 oc, respectively. the pathogenic fungal species infesting corn during the storage will utilize corn nutrients for their growth and development, leading to a decrease in physical and chemical qualities of corn (elsamra et al. 2012; talanca & mas'ud 2009). the decrease in the physical quality of corn can be identified through odor, color, and texture (talanca & mas'ud 2009). the decrease in chemical quality of corn was identified through the decrease in nutrient content and the presence of aflatoxin. aflatoxins, which are the most potent fungal toxins (mycotoxins), are carcinogenic and teratogenic. the aflatoxins are produced during the infection and growth of aspergillus flavus and aspergillus parasiticus in several food/feed ingredients, such as maize and beans (fountain et al. 2015). aflatoxins also adversely affect the growth of livestock and humans. in terms of livestock, the toxicity of aflatoxin depends on the level of aflatoxin in the feed and the impact is different for each type of livestock. chickens are the most resistant to acute aflatoxicosis compared to other poultry (monson et al. 2015). when poultry is exposed to aflatoxins it does not cause mortality or morbidity, but considerable losses are experienced by the poultry industry because of hepatotoxicity. strategies to suppress the growth and development of pathogenic fungi is a major concern, which should lead to serious efforts in maintaining the physical and chemical quality of corn during storage. the efforts should also suppress the production of fungal toxins produced by corn so that the danger of aflatoxicosis can be reduced or even eliminated. several strategies can be applied, such as immediately drying the harvested corn, the use of antagonistic microbes such as neurospora sp. and rhizopus sp., the use of chemicals (mold inhibitors) such as ammonia and propionic acid, and natural materials such as clove powder (elsamra et al. 2012; talanca & mas'ud 2009; wang et al. 2019; oliveira et al. 2020). the results of research by elsamra et al. (2012) showed that the use of clove powder as a natural fungal inhibitor can suppress the growth of aspergillus flavus and also reduce the crude fat content of corn. the same research also proved that the use of fix-a-tox is also effective in suppressing the growth of aspergillus flavus and reducing the content of some amino acids in corn. the adverse effects of aflatoxin and strategies for preventing and eliminating aflatoxins are still a global issue. indonesia's tropical environmental conditions strongly support the growth of pathogenic fungi, such as aspergillus spp. according to weinberg et al. (2008), the harvested grains are vulnerable to molding leading to rapid decline of quality under humid and warm conditions. therefore, it is very important to find the appropriate strategy to prevent the growth activity of these pathogenic fungi. based on these considerations, a study has been conducted to evaluate the physical and chemical qualities of corn grains having different moisture contents supplemented by commercial mold inhibitor during storage. materials and methods feed ingredients the yellow corn grains having different moisture content (< 10%, 10.0 10.9%, and 11.0 11.0%) and a commercial mold inhibitor was used in this study. the yellow corn grains were obtained from farmers in south central timor regency. the mold inhibitor product was provided by a feed producer. the product contains 57% propionic acid, 30 mg/kg lead, and 0.54 mg/kg arsenic. the dose used in this study was 450 g/ton of feed. experimental design this study was designed using a factorial completely randomized design with a 3 x 2 factorial pattern with 3 levels of corn moisture content (< 10%, 10-10.9%, and 11.0-11.9%) and 2 levels of fungal inhibitors (-, +), resulting to six treatment combinations altogether. each biotropia vol. 29 no. 3, 2022 236 treatment consisted of five replications (25 kg of corn per replication). the treatments were: corn (<10% moisture content) corn (<10% moisture content) + mold inhibitor (0.045%) corn (10.0-10.9% moisture content) corn (10.0-10.9% moisture content) + mold inhibitor (0.045%) corn (11.0-11.9% moisture content) corn (11.0-11.9% moisture content) + mold inhibitor (0.045%) experimental procedure the initial moisture content of corn was measured with a grain moisture meter. then, the corn grains with different moisture contents (< 10%, 10.0-10.9%, 11.0-11.9%) without mold inhibitors were put into polyethylene bags (5 bags per treatment; 25 kg corn per bag). meanwhile, the treatments with mold inhibitor were conducted as follows: corn grains were added with mold inhibitor 0.045%, mixed, and then put into a polyethylene bag (25 kg/bag). all treatment bags were then placed on pallets and stored for three months in a feed storage room which had been cleaned and sanitized. a thermo-hygrometer was placed on the wall to control the temperature and humidity. on day 90, the moisture content of the corn grains was measured using a grain moisture meter. the sampling of corn was carried out using the cone and quartering method (campos-m & camposc 2017) and was followed by sample reduction using a seed sampler to obtain laboratory samples. laboratory samples were packed in sealed plastic bags, labeled, and sent to the laboratory for chemical analysis. chemical analysis the dry matter, crude protein, crude fat, and ash contents were determined using the aoac official method (aoac 2005). gross energy (ge) level was determined using an automatic bomb calorimeter (ika c2000). the analysis of aflatoxin (b1, b2, g1, and g2) content of corn grains was conducted at the food and feed laboratory of seameo biotrop in bogor using thin layer chromatography (tlc) (bainton et al. 1980). the limit of aflatoxin detection with tlc was 3.01 ppb for afb1, 3.50 ppb for afb2, 0.54 for afg1, and 1.0 ppb for afg2. the amino acid content of corn samples was analyzed using high-performance liquid chromatography (hplc, ici instrument/ shimadzu scl-10a/shimadzu cbm 20a) with four main steps, namely the manufacture of protein hydrolyzate, drying, derivatization, and injection into hplc. the analysis procedure was as follows: corn sample was hydrolyzed with 10 ml of 6 n hcl at 100 oc for 24 hours. the results of the hydrolysis were transferred to the evaporator flask and rinsed with 2 ml of 0.01 n hcl. this process is done 2 3 times. then the sample was dried using a rotary evaporator for 15 30 minutes to convert cysteine into cystine. the dried sample was added with 5 ml of 0.01 n hcl, then filtered. the derivatization solution was prepared by adding potassium borate buffer ph 10.4 to the sample in a ratio of 1:1. a total of 50 ml of the sample was put into an empty vial and added with 250 ml of orthoflaaldehyde, left for one min, and then filtered. subsequently, 5 ml of the sample was injected into the hplc and then made a standard chromatogram using ready-to-use amino acids that underwent the same treatment as the sample. measurements 1. insect-damaged seed (%): corn grains from each plastic bag was sampled and reduced several times to get to 1.5 kg of samples by using the cone and quartering method (campos-m & campos-c 2017). then, the insect-damaged seeds were taken from the reduced sample and weighed. the percentage of insect-damaged seeds was then calculated using the following formula (nyarko et al. 2021): % insect-damaged seeds = insect-damaged seeds (g) x 100% total weight of corn sample (g) 2. moldy seeds (%): the sampling procedure to quantify the moldy seeds was similar to the sampling method for insect-damaged seeds. the moldy seeds were characterized by color change (shahbazi & shahbazi 2018). the percentage of moldy seeds was calculated by the formula: % moldy seeds = moldy seeds (g) x 100% total weight of corn sample (g) physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 237 statistical analysis the data obtained were analyzed by using the two-way analysis of variance (anova) following the general linear model procedure of sas (sas ondemand of the sas system). significance was determined at p < 0.05 and the duncan test was then conducted to determine the significant differences between mean values. results and discussions effect of mold inhibitor on physical quality of corn physical and chemical damages to corn kernels during storage can be caused by various factors, such as the initial moisture content of corn during storage, the temperature, humidity, corn variety, and warehouse pests (mutungi et al. 2019; mukkun et al. 2018; muga et al. 2018; li et al. 2014; suleiman et al. 2013). table 1 shows the effect of treatments on the physical quality and moisture content of corn stored for 90 days. the results showed that a significant (p < 0.05) interaction was found between the moisture level (ml) and mold inhibitor (mi) in the percentage of insect-damaged seeds and moldy seeds. on the other hand, no significant interaction (p > 0.05) between the moisture level (ml) and mold inhibitor (mi) was observed in the moisture content of corn harvested on day 90. table 1 shows that corn with different moisture contents (10.1 10.9% and 11.0 11.9%) supplemented with mold inhibitor had a significant lower percentage of damaged seeds and moldy seeds (p < 0.05) compared to the group of corn with the same moisture contents without mold inhibitor. the reduction of the damaged seeds and moldy seeds ranged from 1.46% to 77.9% and 16.9% to 80.5%, respectively (table 1). the percentage of reduction increased in the group of corn with a higher moisture content supplemented by mold inhibitor. this phenomenon indicates that the mold inhibitor works more effectively to prevent physical damage to corn with high moisture content (figs. 1 & 2). table 1 the effect of treatments on the physical quality and the moisture content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) insect-damaged seed (%) moldy grain (%) moisture content (%) < 10.0% 2.73c 1.24b 10.38 + 2.69c 1.03b 10.42 10.0-10.9% 8.86b 2.92a 10.42 + 5.05c 1.58b 10.48 11.0-11.9% 19.25a 3.94a 10.38 + 4.25c 0.77b 10.30 sem 0.828 0.457 0.533 main factors moisture level (ml) ≤ 10.0% 2.71c 1.14c 10.40b 10.0-10.9% 6.96b 2.25b 10.45a 11.0-11.9% 11.75a 2.35a 10.34c sem 0.585 0.323 0.037 mold inhibitor (mi) 10.28a 2.70a 10.39 + 3.99b 1.13b 10.40 sem 0.478 0.264 0.307 probability p > f ml *** * ns mi *** *** ns ml × mi *** * ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. biotropia vol. 29 no. 3, 2022 238 figure 1 stored corn grains without mold inhibitor figure 2 stored corn grains with mold inhibitor results of the present study was in agreement with those conducted by elsamra et al. (2012) who reported that the addition of mold inhibitor reduced the percentage of damaged seeds (80%). damaged corn is generally characterized by the appearance of holes and maize weevil (sitophilus zeamais). cannepele et al. (2003) stated that the damage of cereal grains due to sitophilus zeamais has an impact on weight loss, decreased physical and chemical qualities of seeds, and reduced germination. bhusal and khanal (2019) reported that the presence of maize weevil leads to the increase of aspergillus flavus infestation in corn. mukkun et al. (2018) reported from their experiment that aspergillus flavus, a. niger, a. fumigatus, fusarium spp., penicillium spp., rhizophus spp., and mucor spp. were the fungal species identified in corn during the storage. the mold inhibitor used in the present study contains some active compounds, namely propionic acid (≥ 57%), lead (≤ 30 mg/kg), arsenic (≤ 0.54 mg/kg) which can inhibit fungal growth. choojun and yoonprayong (2011) stated that propionic acid is able to inhibit the growth of fungi (having antifungal activity), such as aspergillus spp., rhizopus sp., penicillium sp., and zygosaccharomyces rouxii. telaumbanua (2019) in his literature review stated that propionic acid can inhibit the respiration process of grains and the metabolic activity of grain microorganisms. yun and lee (2016) in their literature review explained that the mechanism of propionic acid (ch3ch2cooh) in killing fungi is through mitochondrial apoptosis (programmed cell death). regarding the lead (pb) compound, amari et al. (2017) reported that lead (pb) binds into the cell wall or cell membrane of fungi causing damages to the plasticity of the cell wall of fungal cell membrane so that the mitotic activity of fungal cells is reduced. meanwhile, the arsenic (as) compound changes the ph of the corn medium to acid so that fungi cannot grow (ceci et al. 2020). results of the present study are in agreement with those carried out by nahm (1991) who reported that mold inhibitor was effective in preventing the growth of fungi and reducing the percentage of moldy seeds. the insignificant differences in moisture content of corn can be attributed to the temperature and humidity factors during the 90-day storage remains stable. these findings agreed with those conducted by telaumbanua et al. (2019). effect of mold inhibitor on proximate composition and gross energy content of corn the effect of treatments on the proximate composition and gross energy content of corn grains stored for 90 days is presented in table 2. the results showed that the interaction between moisture level (ml) and mold inhibitor (mi) did not affect (p > 0.05) the proximate composition and gross energy of corn during the experiment. however, the content of dry matter, ash, and gross energy of corn tended to be lower in a group of corn without a mold inhibitor supplementation. on the other hand, the crude protein and crude lipid contents tended to decrease in a group of corn added with mold inhibitor. physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 239 table 2 the effect of treatments on the proximate composition and gross energy content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) dry matter crude protein crude lipid ash gross energy ……………..% (as fed)………………. (kcal/kg dm) <10.0% 89.02 7.01 4.96 0.922 2914 + 89.70 6.94 4.74 0.935 3004 10.0-10.9% 89.10 7.71 4.93 1.082 2971 + 89.77 7.35 4.69 1.250 2994 11.0-11.9% 89.33 7.38 4.74 1.067 3002 + 90.05 7.04 4.91 1.250 3014 sem 0.166 0.195 0.103 0.137 995 main effects moisture level (ml) <10.0% 89.36 6.97b 4.85 0.929 2959 10.0-10.9% 89.43 7.52a 4.81 1.166 2983 11.0-11.9% 89.67 7.21a 4.82 1.159 3008 sem 0.117 0.138 0.072 0.097 703 mold inhibitor (mi) 89.15b 7.36 4.87 1.024 2962 + 89.83a 7.11 4.78 1.145 3004 sem 0.095 0.112 0.059 0.079 574.2 probability p> f ml ns * ns ns ns mi *** ns ns ns ns ml × mi ns ns ns ns ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. except for crude protein content, the first main effect of moisture level (ml) did not affect (p > 0.05) the proximate composition and gross energy content of corn over the 90-day storage period. significant differences (p < 0.05) in crude protein (cp) content were observed between the 10% ml and the other two moisture content (10.0 10.9% and 11.0 11.9%). the low cp content of corn with ml < 10% was an unexpected result. the second main effect of mold inhibitor was that the mold inhibitor affected (p < 0.001) the dry matter (dm) content, but it had no significant effect (p > 0.05) on the content of crude protein, crude fat, ash, and gross energy of corn during the 90-day storage period. corn supplemented with mold inhibitor had a higher dm content (p < 0.05) than those without mold inhibitor supplementation. the present results indicated that mold inhibitor supplementation was effective in inhibiting the growth of fungi and maize weevil (sitophillus zeamais) so that the dry matter was not used by these living organisms to propagate. the insignificant difference in crude protein content was in agreement with the finding of telaumbanua et al. (2019). however, the insignificant effect of crude lipid, ash, and gross energy did not agree with those found by telaumbanua et al. (2019). the difference was probably due to the difference in the method applied, especially the duration of the experiment and the type and dose of mold inhibitor used. effect of mold inhibitor on aflatoxin content of corn according to negash (2018), there are six types of aflatoxin, involving aflatoxin b1, b2, g1, and g2, m1 (a metabolite of b1), and m2. among all types of aflatoxin, aflatoxin b1 (afb1) is the most dangerous type of aflatoxin, which can cause several harmful effects in humans and animals, such as enlarged liver, liver cancer, and hepatitis b virus infection (nalle et al. 2021; benkerroum 2020). in addition, nalle et al. (2021) also reported that even at the low level, the afb1 reduced the fat digestibility and feed efficiency, and changed the liver color. thus, it is important to minimize the adverse effect of afb1 in poultry feed ingredients. biotropia vol. 29 no. 3, 2022 240 table 3 the effect of treatments on the aflatoxin content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) afb1* afb2** afg1*** afg2**** …..……….……………..ppb……….……………....... ≤10.0% 1.72 nd nd nd + 1.15 nd nd nd 10.0-10.9% 1.72 nd nd nd + 1.72 nd nd nd 11.0-11.9% 1.15 nd nd nd + 1.15 nd nd nd sem 1.462 main effects moisture level (ml) ≤10.0% 1.43 nd nd nd 10.0-10.9% 1.72 nd nd nd 11.0-11.9% 1.15 nd nd nd sem 1.033 mold inhibitor (mi) 1.52 nd nd nd + 1.34 nd nd nd sem 0.844 probability p> f ml ns ns ns ns mi ns ns ns ns ml × mi ns ns ns ns notes: ns = not significantly different (p > 0.05); sem = standard error of mean; * = the limit of detection of afb1 was 3.01 ppb; ** = the limit of detection of afb2 was 3.50 ppb; *** = the limit of detection of afg1 was 0.54 ppb; **** = the limit of detection of afg2 was 1.0 ppb. table 3 depicts the effect of treatments on the content of aflatoxins b1, b2, g1, and g2 in corn stored for 90 days. the results proved that the interaction between the level of moisture (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the aflatoxin content (b1, b2, g1, and g2) of corn during the trial period. the inefficacy of mold inhibitor in reducing the aflatoxin content of corn presumably because the aflatoxin level in corn was too low. however, it seems that the addition of mold inhibitor in corn grain with < 10% ml reduced the afb1 level during the storage. the main effect of moisture level (ml) or mold inhibitor (mi) had no significant effect (p > 0.05) on the content of aflatoxins (b1, b2, g1, and g2). however, the group of corn supplemented with mold inhibitor had lower afb1 concentration (1.34 ppb) compared to those that were not added with a mold inhibitor (1.54 ppb). the numerical reduction in aflatoxin level was in agreement with the findings of telaumbanua et al. (2019) who found that the aflatoxin level in the group of corn grains supplemented with mold inhibitor (propionic acid) was lower than that of control treatment. effect of treatments on the amino acid content of corn amino acid is the building block of protein and plays an important role in protein cell synthesis in animal and human beings. table 4 describes the effect of treatments on the indispensable amino acid content of corn stored for 90 days. the results showed that except for histidine and lysine, the interaction of moisture level (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the indispensable amino acid content of corn during the 90-day storage period. the comparison was difficult to be made due to the difficulties in finding the references which conducted similar research. physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 241 table 4 the effect of treatments on the indispensable amino acid content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) arginine histidine isoleucine leucine lysine methionine phenylalanine threonine valine ………..……………….…………………% as fed…………………………………………………. <10.0% 0.710 0.545 0.480 1.200 0.510 0.300 0.595 0.245 0.265 + 0.710 0.565 0.485 1.210 0.560 0.365 0.580 0.300 0.370 10.0-10.9% 0.690 0.510 0.440 1.190 0.510 0.355 0.575 0.235 0.300 + 0.710 0.530 0.495 1.200 0.505 0.295 0.575 1.065 0.265 11.0-11.9% 0.710 0.620 0.445 1.190 0.535 0.345 0.590 0.205 0.310 + 0.680 0.525 0.455 1.195 0.520 0.345 0.615 0.240 0.255 sem 0.008 0.013 0.023 0.014 0.006 0.002 0.020 0.338 0.043 main effects moisture level (ml) <10.0% 0.710 0.555a 0.482 1.205 0.535a 0.332 0.587 0.273 0.317 10.0-10.9% 0.700 0.520b 0.467 1.195 0.507b 0.325 0.575 0.650 0.282 11.0-11.9% 0.695 0.572a 0.450 1.192 0.527a 0.345 0.602 0.222 0.282 sem 0.006 0.009 0.016 0.010 0.004 0.015 0.014 0.239 0.031 mold inhibitor (mi) 0.703 0.558 0.455 1.193 0.518 0.333 0.587 0.228 0.292 + 0.700 0.540 0.478 1.202 0.528 0.335 0.590 0.535 0.297 sem 0.005 0.007 0.013 0.008 0.004 0.012 0.011 0.195 0.025 probability p> f ml ns * ns ns * ns ns ns ns mi ns ns ns ns ns ns ns ns ns ml × mi ns *** ns ns *** ns ns ns ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. the main effect of moisture level (ml) significantly affected (p < 0.05) the content of histidine and lysine, but it did not affect (p > 0.05) the other indispensable amino acid content of corn during the trial period. the histidine and lysine content of corn with 10.1 10.9% ml was lower (p < 0.05) than the histidine and lysine content of two other treatments. the main effect of mold inhibitor (mi) did not affect (p > 0.05) all indispensable amino acid content of corn during the experimental period. this proves that the use of mold inhibitor with a dose of 0.045% did not have an adverse impact on the indispensable amino acid content of corn. table 5 shows the effect of treatment on the dispensable amino acid content of yellow shelled corn stored for 90 days. the results of the analysis of diversity showed that the interaction of moisture level (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the essential amino acid content of dry shelled yellow corn which was stored for 90 days. table 5 the effect of treatments on the dispensable amino acid content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) alanine aspartic acid cysteine glycine glutamic acid proline serine tyrosine ………………………..………..…........% as fed..…...………………….………………... ≤10.0% 0.825 0.625 0.205 0.805 1.900 0.200 0.585 0.290 + 0.845 0.620 0.205 0.815 1.900 0.205 0.630 0.350 10.0-10.9% 0.835 0.590 0.215 0.835 1.535 0.205 0.595 0.295 + 0.890 1.120 0.200 0.820 1.855 0.215 0.605 0.290 11.0-11.9% 0.855 0.605 0.200 0.805 1.920 0.230 0.620 0.300 + 0.855 0.620 0.195 0.805 1.865 0.230 0.615 0.305 sem 0.023 0.217 0.010 0.012 0.150 0.019 0.010 0.018 main effects moisture level (ml) <10.0% 0.835 0.622 0.205 0.810 1.900 0.225 0.607 0.320 10.0-10.9% 0.862 0.855 0.207 0.827 1.695 0.210 0.600 0.292 11.0-11.9% 0.855 0.612 0.197 0.805 1.892 0.230 0.617 0.302 sem 0.016 0.154 0.007 0.008 0.106 0.013 0.008 0.013 mold inhibitor (mi) 0.838 0.606 0.207 0.815 1.785 0.232 0.600 0.295 + 0.863 0.787 0.200 0.813 1.873 0.212 0.617 0.315 sem 0.013 0.125 0.006 0.007 0.087 0.010 0.006 0.010 probability p> f ml ns ns ns ns ns ns ns ns mi ns ns ns ns ns ns ns ns ml × mi ns ns ns ns ns ns ns ns biotropia vol. 29 no. 3, 2022 242 conclusion the supplementation of mold inhibitor (mi) in corn with different moisture levels (ml) is effective to maintain the physical quality, the dry matter (dm), ash and energy content of corn grains during the 90-day storage period. the concentration of crude protein and crude lipid tended to decrease in the group of corn with different moisture level supplemented with mold inhibitor. the addition of mold inhibitor maintained the dm content but was not effective to reduce the afb1 content of corn during the storage. except for histidine and lysine, the supplementation of mi in corn with different ml did not affect the amino acid content of corn. further research is needed to evaluate the mold inhibitor-treated corn on the growth performance of broilers and other poultry. acknowledgments the author would like to thank the state polytechnic of agriculture kupang that has provided funding for this research (contract number: 03/p3m/dipa.023.18.2.677616/ 2021). we are grateful of the in-kind contribution from pt japfa comfeed tbk in the form of mintai feed anti-mold product. the assistance provided during the research by maria wonda, geti pahnael, and suhartini salih was highly appreciated. references abass a, fischler m, schneider k, daudi s, gasper a, rüst j, ..., kabula e. 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control the toxigenic fungal microbiome and major mycotoxins during storage of maize. front microbiol 10: 1643. doi: 10.3389/ fmicb.2019.01643 weinberg zg, yan y, chena y, finkelmana s, ashbella, g, navarro s. 2008. the effect of moisture level on high-moisture maize (zea mays l.) under hermetic storage conditions: in vitro studies. j stored prod res 44: 136-44. yun j, lee dg. 2016. a novel fungal killing mechanism of propionic acid. fems yeast res 16(7): 1-8. doi: 10.1093/femsyr/fow089 microsoft word 19 biotropia no. 21, 2003 : 19 31 antagonistic effect of three fungal isolates to aflatoxin-producing^spergiy/hs/javhs okky setyawati dharmaputra seamed biotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia asmarina s.r. putri, ina retnowati and santiambarwati seameo biotrop, p.o. box 116, bogor, indonesia abstract aflatoxin contamination in preharvest peanuts can be controlled among others by using antagonistic fimgi to aflatoxin-producing fungi. aspergillus flavus is one of the fungal species where certain strains can produce aflatoxin. informations regarding the type of interactions between antagonistic fungi and toxigenic a. flavus, and the effects of culture filtrates of the test fungi on the growth and aflatoxin production of toxigenic a. flavus are necessary, before antagonistic fungi could be used as biocontrol agent. three fungal isolates (nontoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130) were tested for their antagonistic properties against toxigenic a. flavus bio 2132 using direct and indirect confrontation methods. on direct confrontation method, four kinds of agar media were used, i.e pda (potato dextrose agar), mea 1% (malt extract agar 1%), smkya (sucrose 200 g, mgso47h2o 0.5 g, kno3 3 g, yeast extract 7 g, and block agar aa 20 g), and the mixture of mea 1 % + smkya (1:1). the results indicated that the type of interactions between toxigenic a. flavus either with nontoxigenic a. flavus or with t. harzianum was b type. in this type of interaction, the growth of both toxigenic a. flavus and the test fungi inhibited each other (mutual inhibition) with the zone of inhibition < 2 mm. type of interaction between toxigenic a. flavus and a. niger depended on the kind of media. on smkya and mea 1% + smkya media, the interaction was b type, while on pda and mea 1% media it was d type. in this d type of interaction, toxigenic a. flavus and a. niger inhibited each other (mutual inhibition) at a distance > 2 mm. culture filtrates derived from nontoxigenic a. flavus and a. niger grown on me 1%, smky and me 1% + smky inhibited the growth (based on dry weight) of toxigenic a. flavus, except culture filtrates derived from t. harzianum grown on smky and me 1% + smky media stimulated the growth of toxigenic a. flavus. culture filtrates of nontoxigenic a. flavus, a. niger and t. harzianum inhibited aflatoxin b\ production of toxigenic a. flavus. culture filtrates of a. niger and t. harzianum with conidial concentrations of ixlo6, 2xl06 and 3xl06 per ml inhibited aflatoxin b, production up to 100%. the percentage of inhibition of aflatoxin bi production increased with the increase of conidial concentrations of nontoxigenic a. flavus. the highest percentage of inhibition of aflatoxin bi production (62.5%) was obtained from conidial concentration of 3xl06 per ml. aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic a. flavus, either on agar media or on culture filtrates of test fungi. culture filtrate of a. niger was also the most potential filtrate in inhibiting aflatoxin bi production of toxigenic a. flavus. keywords: antagonistic effect / aspergillus flavus i aspergillus niger i trichoderma harzianum i aflatoxin 19 biotropia no. 21,2003 introduction aflatoxin contamination in peanuts occurs when kernels become infected by aspergillus flavus, a. parasiticus and a. nomius, under drought stress before harvest, during the drying phase in the field, or under unsuitable storage conditions. pitt et al. (1991) reported that a. flavus (and a. parasiticus) are able to grow as commensals in developing peanut plants, and from there the fungi can invade developing peanuts. levels of 1000 ppb of aflatoxin could cause acute liver damage in man and animals. lower levels of aflatoxin in peanut products if consumed could cause liver cancer and premature death in humans, as well as reducing productivity of livestock (pitt and hocking 1996). according to pitt and hocking (1996), 45% of 215 peanut samples collected from retailers in bogor and yogyakarta contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22% exceeding 1000 ppb. in foodstuff, the limit of aflatoxin content determined by u.s. food and drug administration was 20 ppb (park 1993). codex alimentarius commision adopted the maximum level of total aflatoxin content in peanuts intended for further processing at 50 ppb (fao and who 1999). during the past 20 years, a number of approaches have been advocated and tested for reduction of aflatoxins in peanuts: resistant cultivars, thickened shells, waxy testa, improved farm management techniques and postharvest procedures involving drying and storage. some of these approaches have merit, but despite the expenditure of large sums of research funding aflatoxin in peanuts remains a serious commercial problem. other approaches such as using antagonistic fungi to aflatoxin-producing fungi are still needed (pitt 1999). one of the more promising method is the concept of biocontrol by competitive exclusion (pitt 1999). this involves the use of competitive fungi to reduce the possibility of toxigenic fungi present in the soil entering developing peanuts and then producing aflatoxins in them. according to dorner et al. (1992), nontoxigenic a. parasiticus can be used as biocompetitive agent to control aflatoxin contamination in peanuts before harvest. dharmaputra et al. (2001) reported that among fungi isolated from the soil of peanut farms at wonogiri regency (central java), nontoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130 were antagonistic to toxigenic a. flavus bio 2128. the objectives of this study were to get information on the type of interactions between each of the three fungal isolates (nontoxigenic a. flavus, a. niger and t. harzianum) and toxigenic a. flavus; and to investigate the effects of culture filtrates of the test fungi on the growth and aflatoxin production of toxigenic a. flavus. materials and methods fungal isolates fungal isolates used were toxigenic a. flavus bio 2132, isolated from peanut farms in pati regency, central java, in january 2002; nontoxigenic a. flavus bio 20 antagonistic effect of three fungal isolates okky s. dharmaputra et al. 2127, a. niger bio 2129 and t. harzianum bio 19130, isolated from soils of peanut farms at wonogiri regency, central java, in may 2000 (dharmaputra et al 2001). the four fungal isolates belong to the plant pathology laboratory culture collection, seameo biotrop. each fungal isolate was subcultured on potato dextrose agar (pda) medium and incubated at room temperature (28 ± 2°c) for 7 days. test of antagonism between three fungal isolates and toxigenic a. flavus 1. direct confrontation between fungal colonies nontoxigenic a. flavus, a. niger and t. harzianum isolates were tested for their antagonistic property against toxigenic a. flavus using direct opposition method (dennis and webster 1971) on four agar media, i.e. pda, me a 1% (malt extract agar 1%), smkya (sucrose 200 g, mgso4.7h o 0.5 g, kno3 3 g, yeast extract 7 g, block agar aa 20g) and the mixture of mea 1% + smkya (1:1). toxigenic a. flavus (4 mm in diam) was placed on the four media in a petri dish (diam 9 cm). at the same time at a distance of 3 cm from toxigenic a. flavus inoculum, each test fungus was inoculated on the same dish. the plates were incubated at room temperature. three replications were used for each treatment. observations were carried out seven days after incubation on the inhibition of mycelial growth of toxigenic a. flavus by the test fungi using the formula of fokkema (1973): r i r 2 1= x l o o % r1 where i = percentage of inhibition f] = radius of toxigenic a. flavus away from the test fungi r2 = radius of toxigenic a. flavus towards the test fungi the type of interactions between toxigenic a. flavus and each of the test fungus was determined based on wheeler and hocking (1993), adapted from magan and lacey (1984), as presented in table 1. 2. indirect confrontation 2.1. preparation of cultural filtrates of three test fungal isolates isolates of nontoxigenic a. flavus, a. niger and 7". harzianum were grown respectively on pda medium in petri dishes (9 cm in diam) and incubated at room temperature for 7 days. fungal conidia were suspended in sterile distilled water. two ml of each test fungal isolate suspension with three conidial concentrations (ixlo6, 2xl06 and 3xl06 conidia/ml) were'grown on 100 ml me 1%, 100 ml smky, and 100 ml of the mixture of me 1% + smky (1:1) liquid media in 250 ml erlenmeyer flask, respectively, and then incubated at room 21 biotropia no .21,2003 antagonistic effect of three fungal isolates okky s. dharmaputra et al. temperature for 14 days. the concentrations of conidia were determined by microscopically counting the number of conidia using a haemacytometer. after incubation, the culture filtrates of the fungi were separated from their colonies using sterile filter paper no. 1, followed by sterilization of the culture filtrates using millipore (0.45 jam pore size). 2.2. effect of culture filtrates of test fungi on the growth and aflatoxin production of toxigenic a. flavus one ml of conidial suspension of toxigenic a. flavus ( i x l o 6 conidia/ml) was inoculated into 50 ml of sterilized culture filtrates derived from each treatment combination in a glass bottle volume 100 ml. they were then incubated at room temperature for 10 days. as control, toxigenic a. flavus was grown on me 1%, smky and in the mixture of me 1% + smky liquid media. three replications were used for each treatment (including the control). observations were made on the dry weight of mycelia of toxigenic a. flavus and aflatoxin production. the dry weight of mycelia was determined by drying the fungal colonies in an oven at 95°c until a constant weight was attained (gourama and bullerman 1995). aflatoxins were analyzed using a thin layer chromatography method (bainton et al. 1980). in this case, aflatoxin in culture filtrates was extracted using chloroform and identified by two dimensional tlc using standard comparison. aflatoxin content produced by the most potential test fungal isolate was confirmed using a high performance liquid chromatography method (rodriguez and mahoney 1994). statistical analysis the data were analyzed using a completely randomized factorial design with 2 factors. the first and the second factors were the isolates of test fungi (nontoxigenic a. flavus, a. niger, and t. harzianum) and conidial concentration of test fungi (1x106, 2x106 and 3xl06 conidia/ml), respectively. results and discussion type of interaction and percentage of growth inhibition between toxigenic a. flavus and test fungi on various agar media two types of interactions (b and d types) were found between each of the three test fungi and toxigenic a. flavus after 7 days of incubation (table 2). on the type of interaction b, the test fungi and the toxigenic a. flavus inhibited each other's growth with a zone of inhibition < 2mm. this type of interaction was found on the interaction between the toxigenic and the nontoxigenic a.flavus on pda, mea 1%, smky a, and mea 1% + smkya media; between the toxigenic a. flavus and 23 biotropia no. 21,2003 a.niger on smkya and mea 1% + smkya media; between the toxigenic a. flavus and t. harzianum on pda, mea 1%, smkya, and mea 1% + smkya media. on the d type of interaction, the test fungi and the toxigenic a. flavus inhibited each other's growth with a zone of inhibition > 2 mm. this type of interaction was found on the interaction between the toxigenic a. flavus and a. niger on pda and mea 1% media. on the two media a zone of inhibition was found between the colony of a. niger and toxigenic a. flavus. it was assumed that a. niger produced antibiotic. according to jeffries and young (1994), production of extracellular metabolites (such as antibiotics and lytic enzymes) was one of the mechanisms of antagonism between two fungal isolates. dharmaputra et al. (2001) reported that the type of interaction between toxigenic a. flavus isolate 557 and t. harzianum, and between a. flavus isolate 102 and t. harzianum were a and b types, respectively; between toxigenic a. flavus isolate 23j and nontoxigenic a. flavus isolate 18t was b type; between toxigenic a. flavus isolate 102 and a. niger was d type. the kind of media did not give significant differences on the percentage of growth inhibition (based on mycelial growth) of toxigenic a. flavus by the three test fungal isolates. nevertheless, the highest percentage of growth inhibition was found on smkya medium (43.77%) (table 3). the test fungal isolates gave very significant differences on the percentage of growth inhibition of toxigenic a. flavus on smkya medium. trichoderma harzianum caused the highest growth inhibition (49.09%) compared to toxigenic a. flavus on smkya medium (table 4). 24 antagonistic effect of three fungal isolates okky s. dharmaputra et al the effect of culture filtrate of test fungi on the growth and aflatoxin production of toxigenic a. flavus the effect of culture filtrates of nontoxigenic a. flavus and a. niger on the percentage of growth inhibition of toxigenic a. flavus (based on the dry weight of mycelia) was not significantly different. nevertheless, the highest percentage of growth inhibition (69.44%) of toxigenic a. flavus caused by the culture filtrate of nontoxigenic a. flavus was on me 1%, while that of the culture filtrate of a. niger (72.51%) was on smky (table 5). it indicated that nontoxigenic a. flavus was more competitive to toxigenic a. flavus on me 1% medium compared with those of smky and me 1% + smky media, while a. niger was more competitive on smky medium compared with those on me 1% and me 1% + smky media. culture filtrate derived from t. harzianum grown only on me 1% medium inhibited the growth of toxigenic a. flavus (63.64%), while on smky and me 1% + smky media the filtrate stimulated the growth of toxigenic a. flavus (38.0 and 31.58%, respectively) (table 6). it indicated that culture filtrate of t. harzianum was only effective in inhibiting the growth of toxigenic a. flavus on me 1% medium. using tlc method, only aflatoxin b, was detected by toxigenic a. flavus. as aflatoxin was not produced on me 1 % medium, consequently only smky and me 25 biotropia no. 21, 2003 1% + smky media were used to study their effects on the growth (based on dry weight of mycelia) and aflatoxin production of toxigenic a. flavus. the kind of culture filtrates of test fungi on the growth of toxigenic a. flavus on smky media was significantly different. conidial concentration of test fungi and the interaction between the kind of the culture filtrates and conidial concentrations were not significantly different. the percentage of growth inhibition of toxigenic a. flavus caused by culture filtrate of a. niger (72.51%) was higher than that of nontoxigenic a. flavus (45.62%). on me 1% + smky medium, culture filtrates and conidial concentration of test fungi, and their interaction were not significantly different. nevertheless, the percentage of growth inhibition of toxigenic a. flavus caused by culture filtrate of a. niger (68.68%) was higher than that of nontoxigenic a. flavus (64.0%). antagonistic effect of three fungal isolates okky s. dharmaputra et al. the inhibition percentage of aflatoxin b, production caused by culture filtrate derived from nontoxigenic a, flavus grown on smky medium (41.67%) was not significantly different with that of me 1% + smky medium (58.33%). culture filtrate derived from a. niger either grown on smky or me 1% + smky media completely inhibited aflatoxin production effectively (100%) (table 7). culture filtrate of t. harzianum grown on the two media also completely inhibited aflatoxin bi production up to 100% (table 7), but also stimulated the growth of toxigenic a. flavus (table 6). the kind of culture filtrates derived from test fungi and conidial concentration of test fungi and their interaction gave significantly different effects on the percent inhibition of aflatoxin production using smky medium. on me 1% + smky medium, the kind of culture filtrates of the test fungi gave very significantly different effect on aflatoxin b) production, while conidial concentrations and the interaction between kind of culture filtrates and conidial concentrations were not significantly different. culture filtrates derived from a. niger with the concentrations of 1x106, 2xl06 and 3xl06 conidia/ml grown on smky medium inibited aflatoxin bt production up to 100% (table 8). the percent inhibition of aflatoxin b, production increased with the increase of conidial concentrations of nontoxigenic a. flavus. the culture filtrate of nontoxigenic a. flavus with the concentration of 1x106 conidia/ml was not significantly different with that of 2x106 conidia/ml against the inhibition percentage of aflatoxin bj production, while that of 3xl06 conidia/ml was significantly different. the culture filtrate derived from a niger grown on me 1% + smky medium inhibited aflatoxin b] production higher (100.00%) than that of nontoxigenic a. flavus (56.25%). aflatoxins produced by the most potential test fungal isolate were confirmed by using high performance liquid chromatography (hplc). two kinds of aflatoxins (bi and g|) were found using this method. 27 biotropia no. 21,2003 the results of test of antagonism using direct confrontation showed that smkya medium was the best medium for antagonism study between toxigenic a. flavus either with nontoxigenic a. flavus, a. niger or t. harzianum. this is because the highest percentage of growth inhibition of toxigenic 'a. flavus caused by the three test fungal isolates occurred on smkya medium (table 3). on smkya medium the highest percentage of growth inhibition of toxigenic a. flavus was caused by t. harzianum followed by a. niger, while the lowest was caused by nontoxigenic a. flavus (table 4). however, the results of test of antagonism study using indirect confrontation showed that the culture filtrate derived from t. harzianum grown on smky medium stimulated the growth of toxigenic a. flavus (table 6). therefore, a. niger was the most potential test fungus in inhibiting the growth and aflatoxin b, production of toxigenic a. flavus, either on agar media or on culture filtrates of the test fungi. confirmation of aflatoxin contents produced by toxigenic a. flavus grown on culture filtrate of a. niger cultivated on smky and me 1% + smky is presented in table 9. the results showed that there were differences in aflatoxin bi contents analyzed using tlc and hplc methods, because different methods could have different results. nevertheless, based on the two methods used, culture filtrates derived from a. niger cultivated either on smky or me 1% + smky liquid media with different conidial concentrations completely inhibited aflatoxin b] production of toxigenic a. flavus. 28 antagonistic effect of three fungal isolates okky s. dharmaputra et al. table 9. aflatoxin bj contents produced by toxigenic a. flavus grown on culture filtrate derived from a. niger cultivated on smky and me 1% + smky liquid media with various conidial concentrations. aflatoxin bl content (ppb) conidial concen tration of a. niger (conidia/ml) media tlc method hplc method 1 x 106 culture filtrate (using smky) 0 0 control (smky) 160 97.57 culture filtrate (using me 1% + smky) 0 0 control (me 1% + smky) 120 60.79 2x10" culture filtrate (using smky) 0 0 control (smky) 160 98.89 culture filtrate (using me 1% + smky) 0 0 control (me 1% + smky) 120 69.37 3x 10' culture filtrate (using smky) 0 0 control (smky) 160 103.58 culture filtrate (using me 1 % + smky) 0 0 control (me 1% + smky) 120 73.68 tlc = thin layer chromatography; hplc = high performance liquid chromatography conclusions the type of interactions between toxigenic a. flavus either with nontoxigenic a. flavus or with t. harzianum was b type, respectively. in this type of interaction, mutual inhibition on contact or space between the test fungi and toxigenic a. flavus was small (< 2 mm). the type of interactions between toxigenic a. flavus and a. niger depended on the kind of media. on smkya and mea 1% + smkya media, the interactions were b type, while on pda and mea 1% media, it was d type. in this d type of interaction, toxigenic a. flavus and a. niger inhibited each other (mutual inhibition) at a distance > 2mm. culture filtrates derived from nontoxigenic a. flavus and a. niger grown on me 1%, smky and me 1% + smky inhibited mycelial growth (based on dry weight) of toxigenic a. flavus, except those derived from t. harzianum grown on smky and me 1% + smky media which stimulated the growth of toxigenic a. flavus. 29 biotropia no. 21,2003 the culture filtrates of nontoxigenic a. flavus, a. niger and t. harzianum inhibited aflatoxin bi production of toxigenic a. flavus. the culture filtrates of a. niger and t. harzianum with the conidial concentrations of 1x106, 2xl06 and 3xl06 per ml could inhibit aflatoxin b] production up to 100%. the percent inhibition of aflatoxin b, production increased with an increase of conidial concentrations of nontoxigenic a. flavus. the highest percent inhibition of aflatoxin b\ production (62.5%) was obtained from the conidial concentration of 3x106 per ml. aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic a. flavus, either on agar media or on culture filtrates of the test fungi. the culture filtrate of a. niger was also the most potential filtrate in inhibiting aflatoxin bi production of toxigenic a. flavus. the results of this study gave important informations on antagonistic effect of three fungal isolates (nontoxigenic a. flavus, a. niger and t. harzianum) before they could be used to control aflatoxin-producing a. flavus in peanuts grown under green-house and field conditions. acknowledgements the authors gratefully acknowledge the financial support of the government of indonesia. we also thank the technicians of the plant pathology laboratory, seameo biotrop, who have in one way or another contributed to this research. references bainton, s.j., r.d. coker, b.d. jones, e.m. morley, m.j. nagler, r.l. turner. 1980. mycotoxin training manual. tropical products institut (tpi), london. dennis, c and j. webster. 1971. antagonistic properties of species groups of trichoderma. iii. hyphal interaction. trans. brit. mucol. soc. 57: 363 369. dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. soil mycobiota of peanut fields at wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropia no. 17: 30 59. dorner, j.w., r.j. cole and p.d. blankenship. 1992. use of a biocompetitive agent to control preharvest aflatoxin in drought stressed peanuts. journal of food protection 55 (11): 888 892. fao and who. 1999. codex aliraentarius commision. food standard programme. report of the 23'd session, rome, 28 june 3 july 1999. food agriculture organization and world health organization, rome, italy. fokkema, n.j. 1973. the role of saprophytic fungi in antagonism against dreschslera sorokiniana (helminthosporium sativum) on agar plates and on rye leaves with pollen. physiological plant pathology 3: 195-205. gourama, h. and l.b. bullerman. 1995. inhibiton of growth and aflatoxin production of aspergillus flavus by lactobacillus sp. j. food protection 58 (11) 1249 1256. 30 antagonistic effect of three fungal isolates okky s. dharmaputra et al. jeffries, p. and t.w.k. young. 1994. interfungal parasitic relationship. cab international, wallingford. magan, n. and j. lacey. 1984. the effect of water activity, temperature and substrate on interaction between field and storage fungi. trans. br. mycol. soc. 92: 83 93. park, d.l. 1993. controlling aflatoxin in food and feed. food technology 47 (10): 92 96. pitt, j.i. 1999. controlling aflatoxins in peanuts by competitive exclusion of toxigenic fungi. in: dietzgen, r.g. (ed). elimination of aflatoxin contamination in peanut, pp. 21 -22. aciar proceedings no. 89, canberra. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia. in: highley, e. and g.i. johnson (eds). mycotoxin contamination in grains, pp. 5 -10. aciar technical reports 37, canberra. pitt, j.i., s.k. dyer and s. mccammon. 1991. systemic invasion of developing peanut plants by aspergillusflavus. letters in applied microbiology 13: 16 20. rodriguez, s.b and n.e. mahoney. 1994. inhibition of aflatoxin production by surfactants. applied and environmental microbiology 60 (1): 106 110. wheeler, k.a. and a.d. hocking. 1993. interactions among xerophilic fungi associated with dried salted fish. j. appl. bacteriology 74: 164 169. 31 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf biotropia vol. 29 no. 2, 2022: 150 160 doi: 10.11598/btb.2022.29.2.1687 150 pregnant and lactating macaca nigra: behavior and food selection eka arismayanti1, james o. waterman2, andre pasetha1, indira nurul qomariah1, dyah perwitasari-farajallah1,4,5*, and dewi apri astuti3 1animal biosciences study program, institut pertanian bogor, bogor 16680, indonesia 2biological and environment sciences, liverpool john moores university, united kingdom 3animal nutrition and feed technology study program, institut pertanian bogor, bogor 16680, indonesia 4primate research center, institut pertanian bogor, bogor 16680, indonesia 5macaca nigra project, north sulawesi 95535, indonesia received 3 november 2021/accepted 6 march 2022 abstract pregnancy and lactation are reproductive phases that require large amounts of energy. females in the reproductive period need good quality and quantity of food to provide nutrition for the fetus, milk production and child care. the mother will adapt to changes in behavior patterns and food type to meet these needs. the influence of parity and environmental conditions can affect the behavior patterns of females. during pregnancy, the macaca nigra is known to have different proportion of activities in each period, while the behavior during the lactation phase in each mester is unknown. therefore, this study aimed to analyze the behavior patterns in each mester and the food selection of macaca nigra during the pregnancy and lactation phases, as well as the influence of female parity and environmental toward the behavior patterns. there were 39 females macaca nigra observed from two groups from august 2018 to july 2019. an instantaneous focal sampling method was performed to observe females’ daily activities, continuous focal sampling to monitor food types and a selectivity index to analyze food type preferences. the results showed that the female macaca nigra pattern was influenced by the reproductive phase, female parity and environmental conditions. females at the end of the pregnancy and lactation phases had a high proportion of feeding and eat more arthropods. primiparous females mostly performed resting activities. food preference was influenced by reproductive factors and food availability. the choice of fruit could be affected by fruit availability, and their favorite food was d. mangiferum and euginia sp. keywords: daily activity, fruits availability, fruits preferences, macaca nigra, reproductive phase introduction sulawesi black crested macaque (macaca nigra) is one of the seven macaques species endemic to sulawesi island. female m. nigra has three phases of reproduction, namely oestrous, pregnant and lactation. several studies have shown that the reproductive phase requires a large energy cost (pond 1977; altmann & samuels 1992; garber & leigh 1997). pregnancy and lactation are essential stages for females, especially lactation, which is the reproductive phase with the highest energy costs (pond 1977; garber & leigh 1997). during lactation, females need foods with higher protein and calories for milk production and baby care, such as infant transport and thermoregulation (altmann & samuels 1992; guedes et al. 2008). the gestation period of m. nigra lasted about 180 days (engelhardt & perwitasari-farajallah 2008) and was characterized by discoloration around the urogenital area from red to dark purple (gholib 2017). the lactation period was characterized by a discoloration of the urogenital skin from dark red-purple to pale pink (gholib 2017). the lactation lasts approximately one year (pasetha 2018, unpublished data). females allocate time to optimize reproductive success (mccabe & fedigan 2007). the reproductive phase affects the behavior and proportion of female daily activities. females change feeding strategies to meet nutritional *corresponding author, email: witafar@apps.ipb.ac.id behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 151 needs (dunbar & dunbar 2002). in addition to internal factors such as reproduction and birth experience (parity), external factors such as environmental conditions (availability of food and seasons) can affect individual nutritional intake, thus triggering changes in activity and selection of food types (murray et al. 2006; cui et al. 2019). selected foods are generally high quality, easy to process and eaten more often than expected based on their estimated availability (leighton 1993). the study of food preference focused on the type of fruit eaten by m. nigra, because m. nigra is a frugivorous monkey. fruits are expected to be the food resource that most restricts the feeding places of this species. sulawesi black crested monkeys spend 59% of their time for locomotion and eating, while the other 41% are for resting and socializing (o'brien & kinnaird 1997). during the reproductive phase, females of m. nigra have a different proportion of activity, i.e., higher feeding activity during the pregnancy and higher resting during the lactation phase (pasetha et al. 2019). the activity changes in each lactation period are not known, as well as the fruit preference during the pregnancy and lactation phases. the purpose of this study was to investigate the daily proportion of female m. nigra behavior in pregnancy (b1, b2, b3) and lactation (l1, l2, l3) phases, as well as their fruit preferences during those two phases. analysis of the influence of female parity and environmental conditions was also carried out on the behavior and food preferences of female m. nigra. materials and methods research subjects and locations two groups of habituated m. nigra in pantai batu 1b (pb1b) and rambo (r1) were observed in their natural habitats. sixteen individuals in the pregnancy phase and 23 individuals in the lactation phases of m. nigra were observed from august 2018 to july 2019. data collection was conducted in the macaca nigra project (mnp) research station, at the conservation forest management unit (kphk) area of tangkoko, north sulawesi, indonesia (10 32'39''n, 1250 12'42''e). this area covers 8,867 ha and consists of primary and secondary lowland rainforests with an altitude of 1,350 meters above sea level (o'brien & kinnaird 1997). research tools data were collected using the 10-inch odys tablet, samsung smartphone j3 (2016) with the cyber tracker application, counters, cameras, books, pencils, markers, ziplock plastics, garmin gps, binocular, obrometer, thermometer maximum-minimum and portable scales. demographics and daily behavior data collection the group was followed from 05:00 to 17:00, five days a week. demographic data collected were females' reproductive status and the day of parturition (as an indicator of the end of the gestation phase). the oestrus cycle was observed qualitatively with visual observations. the observation was based on the size of swelling and skin color around the urogenital area (0 = deflating, 1 = inflating, 1-2 = medium swelling, 2 = maximum swelling) (higham et al. 2012). pregnancy was divided into three periods, namely b1, b2, and b3 (one period equals to two months). lactation was divided into three periods, namely l1, l2, and l3 (one period equals to four months). behavioral data were collected with focal animal sampling and instantaneous sampling for 30 min with a oneminute interval (altmann 1974). food intake was recorded by continuous focal sampling, while data on the consumed food species were recorded (rothman et al. 2011). daily activities recorded were feeding, foraging, locomotion, resting and socializing. observation time was divided into three periods: morning (06:0009:00), noon (10:00-13:00), and afternoon (14:00-17:00) (pasetha et al. 2019). identification of fruit species fruit species identification was carried out based on mnp database. unknown fruit species were sampled, made into herbarium specimens and identified in the biology research center, indonesian science institute (lipi). collected fruits were stored in ziplock plastic bags and labeled with a local name, scientific name, date, location and part-eaten (rothman et al. 2011). biotropia vol. 29 no. 2, 2022 152 environmental data collection environmental data taken were the air temperature and the rainfall around the mnp research station area. the air temperature and rainfall were measured every morning during the study period at 07:00 or 08:00 micro temperature (around the mnp research station), and the numbers on the obrometer boundary were recorded. the data were tabulated monthly. food availability index fruit availability was calculated by observing the parts of the trees eaten by m. nigra, from which the food availability index (fai) was then calculated. measurements were taken on the second week of every month. the calculation of food availability index was carried out by making 20 plots sized 100 x 100 m covering 6.9% of the total group area. all the tree species were recorded and identified. the number of tree parts counted, such as leaves (young leaves, old leaves and leaf buds), flowers (flowers and flower buds) and fruit (raw fruit, ripe fruit and old fruit). we used two types of measurements. the 1st type was logarithmic scale for measuring leaves: 0 = 0-9 leaves; 1 = 10-99; 2 = 100-999 and so on. the 2nd type was rank scale for measuring fruits: 0 = almost nothing; 1 = multiples; 2 = many but not full; 3 = full or almost full. the diameter (dbh) of tree trunks was measured for all the trees with dbh of more than 10 cm and propagated more than 5 cm (sari 2013). the data was analyzed by using the food availability index (fai) formula (1). fai =  n (aixdi) …….……….…………. (1) i where: fai = total food availability index for species n ai = average value of food availability for species i (logarithmic scale) di = average density of species i per hectare (based on 20 plots). behavioral data analysis data on daily activities were tabulated using pivot tables. the daily behavior proportions were grouped and calculated based on pregnancy phase differences (b1, b2, and b3) and lactation (l1, l2 and l3) phase differences. the frequency of daily activities was calculated by using formula (2): activity frequency i (%) = activity i x 100 ..….. (2) total all activity analysis of food type frequency and fruit preference all types of food eaten were tabulated using pivot tables. the frequency of food and fruit types was analyzed to see the relationship between reproductive conditions and seasons. the frequency of food types was calculated by using formula (3): food i frequency (%) = food i x 100 …… (3) total all food food preferences were calculated by food selection index method (krebs 1999; manly et al. 2002). the fai values were used to determine a proportion of fruit availability in habitats. each type of eaten food was calculated as the ratio of food selection. the resulting value can be interpreted as the probability of food desired choice by primates. the null hypothesis test using the g-test indicates that an animal randomly chooses food resources based on food availability at a confidence interval of 95%. the food selection index was calculated by using formula (4): w = oi …………………….………..……… (4) pi where: w = forage ratio oi = proportion of fruit species eaten pi = proportion of fruit species i in the habitat (fai). statistical analysis statistical analyses were run using a nonparametric multivariate analyses. shappiro-wilk test for normality test and kruskal-wallis for testing the significance of reproductive phase differences were performed. g-test was used for selectivity analysis of selected fruit. pairwisewilcoxon test was performed to know which variables influence the behavior. the statistical analyses were run using program r alpha level 0.05. behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 153 results and discussion female behavior during reproduction reproductive phase is an important phase for females, which affects changes in daily activities and types of food eaten (muruthi et al. 1991; white et al. 2007). energy required by female mammals during pregnancy and lactation phases are generally higher than those of nonproductive females (lee 1997). energy costs for reproduction in primates can be achieved by implementing several strategies such as increasing energy intake, reducing energy excretion, using up body tissue reserves and reducing energy output from physical activity (bercovitch 1993; dufour & sauther 2002; national research council 2003). based on the observations, females during reproductive phase had higher feeding and social activities compared to other activities. locomotion was the least activity performed by pregnant and lactating females. lactating females had feeding (31%, p = 0.01) and foraging (16%, p = 0.21) activities higher than pregnant females. other activities such as resting (19%, p = 0.7), locomotion (12%, p = 0.81) and social (25%, p = 0.6) were performed more by pregnant females than lactating females (fig. 1). figure 1 the behavior frequency of female m. nigra during pregnancy and lactation phases notes: observed behaviors were feeding (fe); foraging (fo); resting (re); locomotion (lo) and socializing (so). based on this study, females in the pregnant and lactating phases had significantly higher feeding activity, especially the lactating females. the increased energy intake shown by the increasing feeding frequency is an adaptation done by females to meet the energy needs (dufour & sauther 2002). social activities were also highly performed by the pregnant and lactating females, although activity was not significantly different between pregnant and lactating females. these results align with a study done on macaca thibetana, where females with infants receive more grooming and affection from other members than females without infants. this phenomenon can be explained by the biological market theory, which states that grooming is a form of a commodity that can be exchanged for other individuals' infant caring (jiang 2019). females in the pregnancy period generally had high feeding activity at b1 (28%) and b3 (29%) period with p = 0.35 (fig. 2) because at that time, the female needed more nutrition for fetal development and energy reserves after parturition. foraging activity decreased as the gestation period progressed (p = 0.57), similar to locomotion behavior (p = 0.07) which also reduced toward the end of pregnancy. in contrast to foraging behavior, females in the final period had the highest resting percentage (21%, p = 0.046) which was significantly different compared to other periods. social activity of pregnant females was more performed on the b1 and b2 levels of pregnancy (p = 0.5) than b3 in the last pregnancy period. in general, the social activity among levels of pregnancy were not significantly different. ruivo et al. (2017) and gould et al. (2011) stated that due to the high energy and nutritional needs during pregnancy, females in the early gestation period will spend a lot of time eating. this behavior aims to compensate for the nutrition shared with the fetus. the behavior was also shown by a study conducted by pasetha et al. (2019) that m. nigra females had the highest feeding activity during the pregnancy period. this pattern is also shown in baboon (papio cynocephalus), which compensate for their energy needs by having longer mealtime (altmann & altmann 1970; dunbar 1977). this result is in contrast with a study on green monkeys (cercopithecus sabaeus). green monkeys adapt their dietary patterns by eating better quality foods but shorter meal time to save energy (harrison 1983). our study showed that the foraging, social and locomotion activities of m. nigra females decreased with the gestation period progressed, while the resting activity was significantly rising. pregnant females biotropia vol. 29 no. 2, 2022 154 specifically reduce their activity to save energy lost, including social activities, especially before parturition. this behavior is also done to avoid other members' aggression (pusey et al. 2008; murray 2009). females in lactation period had the highest feeding activity compared to other activities (fig. 2). feeding (34%, p = 0.002) and foraging (23%, p < 2.2e-16) activities were higher as the lactation period progressed. along with the lactation period, the infants will be more independent, so that the movement of females is more flexible to eat and prepare for the next reproductive cycle. resting activity was most often performed by females in l1 (25%, p = 1.5e-14) and continues to decrease by females in l2 and l3, similar to locomotion activity (15%, p = 5.98e-11). in contrast, social activity was stable around lactation period (23%, p = 0.19). our finding indicated a similar result to previous studies done in other primate species. in baboons and vervet monkeys, the feeding activity of lactating females are less efficient when the infant is still dependent on the mother's body. at the beginning of infant age, female often spends longer time foraging than other females (whitten 1982; koenig et al. 1997; sauther 1994). as the infants get older, the dependence of infants on their mother will be reduced so that the female can increase the proportion of forage for subsequent reproductive needs (koenig 1997; dunbar & dunbar 2002). usually, lactating females raise food intake (tarnaud 2006), forage efficiently (maccabe & fedigan 2007), or eat nutrient-rich foods such as protein and carbohydrates (herrera & heymann 2004; maccabe & fedigan 2007; muruthi et al. 1991; serio-silva 1999). resting activity and locomotion in lactating females m. nigra significantly decreased along with the age of infants. the results of our study were in contrast to a study conducted by pasetha et al. (2019) showing that lactating female m. nigra had the highest resting behavior a few days after parturition based on only taking data on lactating females in the early period during the postpartum period. female m. nigra increased their social activity at the end of the lactation period. lactating females spend a lot of time socializing with other females as a strategy to share the burden of infant care, grooming access and foraging access (henzi & barrett 2002; jiang et al. 2019). effect of parity on female behavior we observed 13 primiparous females and 24 multiparous females of m. nigra. multiparous pregnant females performed more feeding (28%, p = 0.65), social (26%, p = 0.3) and locomotion (12%, p = 0.93) activities compared to primiparous females. besides, foraging activity (17%, p = 0.47) and resting (22%, p = 0.029) activities performed by primiparous females were higher than multiparous females (fig. 3). only resting activity showed a significantly different proportion between females in two parity types. multiparous females in lactation phase had feeding (31%, p = 0.69), foraging (15%, p = 0.51) and locomotion (12%, p = 0.52) activities higher than primiparous females (fig. 3). primiparous females performed more resting (21%, p = 0.03) and social (23%, p = 0.30) activities. figure 2 the behavior frequency of pregnant and lactating females m. nigra notes: b1, b2 and b3 indicate the pregnancy period; l1, l2, and l3 indicate the lactation period. activity budget of pregnant m. nigra activity budget of lactating m. nigra behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 155 figure 3 the behavior of females m. nigra with different parity types in pregnancy and lactation phases parity or experience in having children can be one of the individuals' characteristics that can influence their behavior patterns. a part from resting, there were no significant differences between primiparous and multiparous female behavior. primiparous females tend to increase alertness and resting activity, in exchange, they decrease feeding activity to compensate for time (barrett et al. 2006). in contrast to muruthi et al. (1991), primiparous females need more time to eat because primiparous females are not fully mature. they still need more energy to increase body weight and adequate calorie intake for reproduction. environmental influence on female behavior based on the phenological analysis, the abundance of fruit was directly proportional to rainfall. from october to february, the rainfall was high with low micro-temperatures (fig. 4). feeding activities were directly proportional to the high rainfall. pregnant females did a lot of social (p = 0.31) and resting (p = 0.02) activities when the rainfall was high, but the feeding activity was relatively not much different each month (p = 0.31). locomotion activity was increased during dry season (p = 1.25e-06). they walked more often during the low rainfall season from august to october to search for more food. during the lactation period, females had the highest feeding activity when the rainfall was high (p = 0.002). resting activity was also higher in the rainy season (p < 2.2e-16) (fig. 5). figure 4 phenology of fruit trees and environmental conditions (rainfall and temperature) in kphk tangkoko effect of parity on activity budget of pregnant m. nigra effect of parity on activity budget of lactating m. nigra biotropia vol. 29 no. 2, 2022 156 figure 5 the effect of different seasons on female daily activity the intensity of rainfall was high from october 2018 to february 2019. our study showed that during the rainy season, the feeding activity of female m. nigra rose. in lactation period, females had significantly higher feeding (p = 0.002) and resting (p < 2.2e-16) activities during the peak of rainy season. the proportion of these activities fluctuated until it increased in july 2018 when the rainfall was low. locomotion activities were significantly higher during the dry season. the increasing proportion of locomotion activity also occurs in baboon (papio cynocephalus), which during the dry season and less food availability, baboons walk more and look for alternative food (altmann & samuel 1992). frequency and preference of female food during reproduction reproductive period can affect the females on selecting food (mccabe & fedigan 2007). due to high energy needs, the females are required to consume food that suits their needs (serio-silva et al. 1999; herrera & heymann 2004). based on the observations in our study, females m. nigra during reproduction period ate more arthropods such as ants, termites, grasshoppers, crickets, spiders and larvae. arthropods contain many proteins and lipids that are important for females during the reproductive phase (rothman et al. 2014). pregnant and lactating females ate arthropods up to 45% and 40% (p = 0.003), respectively. the proportion of fruit eaten by pregnant and lactating females was 20% and 13% (p = 0.36), respectively (fig. 6). in addition, females also ate mushroom (1%, p = 0.34), stems (1.1%, p = 0.43), leaves (1-2%, p = 0.25), buds (0.07%, p = 0.26), sap (0.008%, p = 0.57), and flowers (0.1%, p = 0.08). calorie consumption and crude protein are critical during pregnancy and lactation phases so that females will choose certain foods during that period (lee 1997; dufour & sauther 2002). sulawesi black crested macaques are fruit-eating monkeys. the main foods eaten are fruit and insects as companion food (o'brien & kinnaird 1997; sari 2013). our study indicated that arthropods and fruits are the dominant food for the pregnant and lactating females. effect of season on activity budget of pregnant m. nigra effect of season on activity budget of lactating m. nigra behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 157 figure 6 frequency of the type of food eaten by females m. nigra in contrast to a previous study that stated m. nigra in the pregnant and lactating phase eats more fruit (pasetha et al. 2019) because m. nigra is a frugivore macaque that needs glucose as the primary source of energy. arthropods are a widely available food source containing high concentration of fat and protein in nature. protein and fat are essential nutrients for females during the reproductive phase (gould 2011). besides, lactating females significantly ate more insects and have more variety of food types than pregnant females. we recorded 71 fruit species eaten by females during their reproductive period. the four most-eaten fruit species were i.e., koosiodendron pinnatum (p = 0.17), ficus variegate (p = 0.26), dracontomelon dao (p = 0.8) and morinda citrifolia (p = 0.22). based on the period of lactation, female of l1, l2, and l3 mostly consumed k. pinnatum, f. variegata and d. dao, respectively. based on the period of pregnancy, females of b1, b2, and b3 mostly consumed m. citrifolia, f. variegata and k. pinnatum, respectively. the differences in fruit types eaten by pregnant and lactating females in different periods did not show statistical significance. these fruits were consumed due to the abundant availability throughout the season except for k. pinnatum. m. citrifolia and d. rao has carbohydrate content of 93.23% and 93.12%, respectively, which is quite high. fruits are essential sources of nutrition for m. nigra, especially carbohydrates, vitamins and minerals (national research council 2003). based on previous research by o'brien and kinnaird (1997), there are 145 types of fruit eaten by m. nigra. according to pasetha et al. (2019), there are 34 types of fruit eaten by females during reproduction. similar to our findings, these previous studies stated that dracontomelon dao, spondias sp., and ficus sp. are the most eaten species (o'brien & kinnaird 1997; pasetha et al. 2019). those fruits have a high carbohydrate, fiber and protein content among fruits they consume, respectively. the vegetation analysis results support this finding because both fruit types have the highest important value index (ivi) (pasetha et al. 2019, unpublished). pregnant and lactating female diet preferences a selectivity index was conducted to indicate which food types are the favorite choice of animal species. the results showed that dracontomelon mangiferum (35.13%) and eugenia sp. (42.38%) was the most preferred fruit types by females during reproduction period. based on the proximate analysis, d. mangiferum has a relatively high carbohydrate content (93.28%), and eugenia sp. has a high-fat content (5.4%) compared to other fruit types. based on fruits availability analysis, arenga pinnata, ficus variegate, caryota mytis, garcinia tetranda, morinda citrifolia, morinda bracheata and piper aduncum are species that bear fruit throughout the year and are included in fruit species having a high consumed frequency. environmental condition is a limiting factor causing variations in the primate's diet (agetsuma 1995; agetsuma & nakagawa 1998; jaman & huffman 2008; yeager et al. 1996). the availability of fruit increases in the months during high rainfall, resulting in the increased of feeding activity. based on the g-test, which rejected the hypothesis, pregnant and lactating m. nigra did not randomly select the food type. effect of female parity with food type based on their parity, pregnant females ate more arthropods (fig. 7). primiparous female ate fruits (25%) more than multiparous females (23%, p = 0.48). on the contrary, arthropods consumption was mostly done by multiparous females (38%, p = 0.47). lactating females also ate more arthropods. multiparous lactating females consumed more insects (49%) than primiparous females (43%, p = 0.07). type of food eaten by females m. nigra biotropia vol. 29 no. 2, 2022 158 fruit consumption in primiparous and multiparous lactation females was quite similar (p = 0.56). species of fruit eaten by pregnant primiparous and multiparous were k. pinnatum (p = 0.86), f. variegate (p = 0.006) d. dao (p = 0.28), t. cattapa (p = 0.16) and m. citrifolia (p = 0.37). species of fruit eaten by primiparous and multiparous lactating females were k. pinnatum (p = 0.82), f. variegate (p = 0.63) d. dao (p = 0.03), t. cattapa (p = 0.0005) and m. citrifolia (p = 0.5). figure 7 effect of parity on the frequency of food types based on parity, both primiparous and multiparous females in the reproductive period ate more arthropods. primiparous females consumed more arthropods such as crickets, grasshoppers, spiders and larvae than multiparous females. arthropods contain high protein and fat concentrations compared to other types of food such as fruits and other plants part. in small quantities, arthropods can also be a supplement for primates due to their content of vitamins and fatty acids (cluttonbrock et al. 1989; rothman et al. 2014). those nutrients are essential during pregnancy and lactation periods. our finding has a similar result to previous studies. female orangutans, titi monkeys and capuchins tend to increase arthropods' consumption during pregnancy and lactation (fox et al. 2004; herrera & heymann 2004; mccabe & fedigan 2007). fruit consumption in primiparous and multiparous lactating females had a similar proportion. most fruit eaten by pregnant and lactating females was k. pinnatum, f. variegata, d. dao, t. cattapa and m. citrifolia. intraspecific variability in primate feeding behavior is closely related to several factors, e.g., reproduction conditions and favorite foods (post et al. 1982). conclusion the behavior pattern of female macaca nigra can be influenced by the reproductive phase, female parity and environmental conditions. females in the 3rd period of pregnancy and lactation had high percentage of feeding. resting had significant changes in behavior proportion. feeding activity was done more often by multiparous females compared to primiparous female. food preference was influenced by the female reproductive phase and food availability. the selectivity in choosing food was not random. during the pregnancy and lactation periods, females preferred to eat arthropods and fruits. their favorite fruits were d. mangiferum and euginia sp. more comprehensive and in-depth research on behavioral and feeding ecology needs to be done. research with more individual samples of female in the reproduction phase is recommended, as they may have different strategies to adapt to ecological changes in the future. acknowledgments we would like to thank james o. waterman for the funding and support that has been given during the research. special gratitudes are also delivered to antje engelhardt and macaca nigra project, who supported and provided invaluable assistance. many thanks to the natural resources conservation agency of north sulawesi (bksda sulut) which has granted research permission in kphk tangkoko. effect of parity on pregnant m. nigra food types effect of parity on lactating m. nigra food types behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 159 references agetsuma n. 1995. dietary selection by yakushima macaques (macaca fuscata yakui): the influence of food availability and temperature. int. j. primatol 16(4):611-27. doi:10.1007/bf02735284 agetsuma n, nakagawa n. 1998. effects of habitat 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influence of infant age and plant phenology. am j primatol 68:966-77. white fj, wood kd. 2007. female feeding priority in bonobos, pan paniscus, and the question of female dominance. am j primatol 69:837-50. doi:10.1002/ajp.20387 whitten t. 1987. the ecology of sulawesi. yogyakarta (id): ugm pr. yeager cp. 1996. feeding ecology of the long-tailed macaque (macaca fascicularis) in kalimantan tengah, indonesia. int j primatol 17:51-62. doi:10.1007/ bf02696158 microsoft word 40 biotropia no. 23, 2004 : 40 46 preservation of garut rams spermatozoon as a source of male germ plasm arief boediono'*, herdisu, and muhammad rizal' 'faculty of veterinary medicine, bogor agricultural university, bogor 16680, indonesia 2 center for the assessment and application of farming technology, agency for the assessment and application of technology, jakarta, indonesia abstract this study was conducted to examine the quality of ejaculated sperm by garut rams to be used for artificial insemination (ai) and viability of sperm that were collected from preserved cauda epididymis (4°c up to 12 days) for assisted reproductive technology. the semen was collected by artificial vagina, with the sperm motility, live sperm, acrosomal intact, and intact plasma membrane observed. sperm motility was 75%, while for the live sperm, intact plasma membrane and sperm abnormality were 91.5%, 90.0%, and 1.8%, respectively. in the other study, sperm was collected from cauda epididymis by aspiration method and diluted in different media: 1) brackett oliphant (bo) media and 2) modified phosphate buffer saline (mpbs). evaluation of sperm motility and intact plasma membrane were conducted after washing, counting and dilution of the sperm. the results of this study showed that the sperm motility and intact plasma membrane could be maintained better in bo rather than pbs medium although they were not statistically different (p>0.05). at day 12 of preservation, the motility and intact plasma membrane of sperm collected from cauda epididymis were 0.7% and 1.33% for motility and plasma membrane intact, respectively. these findings showed that the garut rams semen was qualified for ai and frozen processing; in vitro embryo production by introducing the assisted reproductive technology such as intracytoplasmic sperm injection (icsi) could be applied by using the sperm collected from preserved cauda epididymis until 12 days of preservation at 4°c. keywords : reproduction/spcrmatogenesis/inscmination/garut rams/small ruminant introduction indonesia is a country with tremendous amounts of genetic resources and biodiversity. in indonesia, the percentage of meat and wool produced by sheep as a small ruminant in west java province is approximately 46.2% (anonymous 1999). one of the ruminants is garut sheep, endemic to indonesia, and potential for meat production. moreover, they have a good performance for sheep contest. the population of garut sheep decreased from 7.6 million in 1997 to 7.2 million in 2001. in an attempt to increase the garut sheep population, several efforts should be done to maintain the population. application of reproduction biotechnology such as artificial insemination (ai) and in vitro embryo production are the solutions. artificial insemination has been routinely done in large ruminants such as cattle and * correspondence: arief boediono, lab. of embryology, dept. of anatomy, fac. of veterinary medicine bogor agriculturaluniversity, jalan agatis, ipb campus darmaga, bogor 16680, indonesia. e-mail: abl@cbn.net.id 40 biotropia no. 23, 2004 buffaloes. however, in small ruminants like sheep and goat this technique should be modified according to the small size of the reproductive tract. sperm usually was collected from ejaculation for artificial insemination procedure. collected sperms were then diluted for fresh sperm insemination, sperm preservation or sperm cryopreservation. in the case of dead ram, sperm was collected from cauda epididymis. epididymal sperm may be the last chance to ensure preservation of genetic materials after injury or death of a valuable male. studies have been conducted to determine if epididymal sperm can be used to produce viable embryos and offsprings. iwamatsu and chang (1971) have reported successful fertilization of mouse oocytes using epididymal sperm. fuller and whittingham (1996) reported the production of normal mouse fetuses after ivf using epididymal sperm that had been cooled at 4°c. sperm collected from the caudal epididymides has been used for both ivf and intracytoplasmic sperm injection (icsi) in domestic cats (pope et al. 1998). caprine epididymal sperm has also been reported for use in the production of ivf embryos to the blastocyst stage (song and iritani 1988), and production of an ivf goat offspring born from artificial insemination (slash et al. 2000). from recent studies, viable epididymal sperm can be harvested post-mortem for possible use in assisted reproductive technologies (art). most of the species evaluated so far, produce more viable sperm when the testicles are stored at 4°c, prior to harvesting the epididymal sperm. the objective of this study was to identify an alternative to collect ovine ejaculate and epididymal sperm for subsequent fertilization. materials and methods sperm collection ejaculated sperm. sperm ejaculate was collected from 5 healthy garut rams by using artificial vagina. the artificial vagina consists of a hose 20 to 25 cm in length and 5 to 7 cm in diameter, with a rubber liner. the temperature of artificial vagina was set between 42 to 46°c, and it is necessary to lubricate it with k-y jelly. at the time of the ram mounts to the ewe, his penis was gently guided inside the artificial vagina connected with a warm tube (37°c) to avoid the cold shock. after ejaculation, the tube containing the semen was removed and placed in a water bath at 30°c. testicle preservation ovine testicles were collected as pairs from mature rams from a slaughterhouse. one pair of testicle (control testicle) was processed shortly after the rams were slaughtered (day-0), while the other testicle of the pair was processed and stored at 4°c up to 12 days of preservation. collected sperms were evaluated daily. 41 preservation of garut rams spermatozoon arief boediono and muhammad rizal epididymal sperm. after storage at 4°c, each testicle was allowed to warm at room temperature (25°c) for not more than 30 minutes. during this warming period, the testicle was dissected away from the connecting tissues. sperm dilution media (bracket! and oliphant, bo and modified phosphate buffer saline, mpbs) were warmed in water bath to 37°c prior to exposure to the epididymal sperm. sperm was collected from the cauda epididymis region by aspiration method. briefly, sperm was collected by aspiration using 21g needle connected to 3 ml spuit already filled with 1 ml warmed dilution media. collected sperm was then washed by centri-fugation at 500g for 3 minutes to create sperm pellet. the supernatant was discarded and the pellet was resuspended in sperm dilution media. evaluation the volume of ejaculated sperm was recorded in each collection. microscopic evaluation of the sperm was done including the sperm concentration, motility, percentage of live sperm, intact plasma membrane, and morphology. for epididymal sperm, analysis was done on percentage of sperm motility and intact plasma membrane. an evaluation of live and dead sperm was done by eosin-negrosin staining. the appearing red sperms were considered dead, otherwise they are alive. the sperm which had an intact plasma membrane was shown by coiled sperm tail after incubation in hypo-osmotic solution at 37°c for 30 min (correa and zavos 1994). hypo-osmotic solution contained 0.032 m nacl in destilled water. data were analyzed using the simple one-way anova to make comparisons between testicle pairs and duncan's test to identify the difference in means between the treatment groups. results and discussion ejaculated sperm was successfully collected from 5 rams. the mean number of garut ram ejaculated sperm was 3463 x 106 sperm rnl"', with the average volume of semen per ejaculation of 0.7 ml (table 1). by comparison, the volume of semen per ejaculation was 1.7 ml in st. croix sheep (feradis 1999), and 1.05 ml in suffolk sheep (boland et.al. 1985). the difference had correlation with individual factors such as age, body weight, and breed (hafez and hafez 2000). the motility and percentage of live sperm of garut ram ejaculation were 75.0% and 91.5%, respectively. these-results were higher than that reported previously in st. croix (64.0% and 76.0% for motility and percentage of live sperm, respectively) (sirman and situmorang 1987). 42 biotropia no. 23, 2004 the percentage of intact plasma membrane of garut ram ejaculated sperm was 90.0%. this was higher than the percentage of intact plasma membrane of st. croix sheep ejaculated sperm i.e. 86.3% (feradis 1999). when sperm was exposed to hypo-osmotic solution, the spermatozoa that had intact plasma membrane would be swollen. if the sperm have intact plasma membrane, the water that influx into the cell could not come out from the cell. the percentage of intact plasma membrane had positive correlation with sperm motility. no single test accurately predicted fertility of a sperm sample; however, examining various physical characteristics of semen could determine greater fertility potential. processing semen for artificial insemination or sperm freezing depends on semen quality. analysis of ram semen involves obtaining maximum information about the physiology status of testicular and epididymis functions by examining one or several ejaculations. in general, the minimal requirements for semen processing (fresh or frozen semen) would include: at least 65% motility, concentration 700 x 106 sperm ml"', and less than 20% morphological abnormalities (hafez and hafez 2000). motility and intact plasma membrane of sperm collected from cauda epididymis could be found up to 24 hours of incubation in vitro both in mpbs or bo medium. at the time of collection sperm motility was 58.0% in mpbs medium and 55.7% in bo medium. they decreased gradually during the in vitro incubation up to 24 hours (0.3% in mpbs and 0.6% in bo medium) (figure 1). intact plasma membrane of sperm collected from cauda epididymis was 60.0% in mpbs and 56.7% in bo medium at the time of sperm collection. the intact plasma membrane in both media during in vitro incubation exhibited a declining pattern, as with sperm motility. however, the intact plasma membrane was higher than sperms motility after 18 hours of incubation. this was shown by some of the immotile sperm that had intact plasma membrane. in this case, spermatozoa may be used for in vitro fertilization by injection of single sperm directly into the oocyte cytoplasm called intracytoplasmic sperm injection (icsi). 43 preservation of garut rams spermatozoon — aricf boediono and muhammad rizal 1812 incubation incubation period (hours) 60.0 ■ mot mpbs q mot bo □q mpi □mpbs q figure 1. motility and intact plasma membrane of sperm collected from cauda epididymis after incubation in vitro in different medium. mot mpbs: sperm motility in mpbs medium, mot bo: sperm motility in bo medium; mpi mpbs: intact plasma membrane of sperm in mpbs medium, mpi bo: intact plasma membrane in bo medium. the sperm motility and intact plasma membrane of sperm collected from cauda epididymis decreased significantly after 2 days of preservation at 4°c. however, 0.7% motile sperm and 1.3% sperm with intact plasma membrane were found after 12 days of preservation (figure 2). according to motility and live sperm, rizal et al. (2004) reported that sperm collected from testicles after 3 days of preservation at 5°c could be used for artificial insemination or in vitro embryo production of sheep. epididymal sperm was used in a goat in vitro embryo production that resulted in cleavage and development of blastocyst, although none of the blastocysts were transfered to the recipients (song and iritani 1988). further work is needed to optimize the in vitro embryo production, through micro-fertilization procedure. the valuable sperm has the ability to fertilize the oocytes and, therefore, could increase the chances of propagating valuable genetics. intracytoplasmic sperm injection (icsi) could initiate the early embryo development to cleavage in goat oocyte (boediono 2001). moreover, cochran et al. (1998) reported that live ivf offsprings from oocyte donor mares have been produced by using icsi procedure. although there has been some success using assisted reproductive technologies, more researches are needed to improve the efficiency of the procedures. 44 conclusions these results indicate that garut rams semen was qualified for ai and frozen processing, and motile sperm can still be collected from cauda epididymis after being stored at 4°c up to 12 days. sperm cooled in the testicle at 4°c could be used in assisted reproductive technologies (intracytoplasmic sperm injection) for the untimely death of a valuable sheep and an effective tool to conserve important genetic resources. this study investigated the standard parameter of sperm analysis. it would stand to reason that further investigations with particular emphasis on chromatin damage and/or fertility are suggested. acknowledgements we would like to thank lesan putra breeding farm, bogor for providing the garut rams. this research was partly financed by seameo-biotrop fy 2003, no.l05/res/iii/2003. references anonymus. 1999. buku statistik petcmakan. direktorat jenderal pcternakan. departemen pertanian republik indonesia dan asosiasi obat hcwan indonesia. jakarta. blash s., mcllican d, and w. gavin. 2000. cryopreservation of cpididymal sperm obtained at necropsy from goats. theriogenology, 54:899-905. 45 preservation of garut rams spermatozoon arief boediono and muhammad rizal boediono a. 2001. sperm immobilization prior to intracytoplasmic sperm injection (icsi) and oocytc activation improve early development of microfertilizcd goat oocytcs. reprotech, 1:29-34. boland m.p., a.a. al-kamali, t.f. crosby, n.b. haynes 1, c.m. howlcs 1, d.l. kelleher and i gordon. 1985. the influence of breed, season and photoperiod on semen characteristics, testicular size, libido and plasma hormone concentrations in rams. animal reproduction science, 9: 241-252. cochran r, meintjes m, rcggio b, hylan d, carter j, pinto c, paccamonti d, and r.a. godke. 1998. live foals produced from sperm-injected oocytcs derived from pregnant mares. j equine vet sci, 18:736-740. correa j.r. and p.m. zavos. 1994. the hypo-osmotic swelling test : its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. theriogenology, 42:351-360. feradis. 1999. penggunaan antioksidan dalam pcngcnccr semen beku dan metodc sinkronisasi estrus pada program inseminasi buatan domba st. croix. disertasi doktor. program pascasarjana institut pcrtanian bogor. bogor. fuller sj and d.g. whittingham . 1996. effect of cooling mouse spermatozoa to 4°c on fertilization and embryonic development. j. rcprod. fcrtil., 108:139-145. hafez e.s. e, and b. hafez 2000. reproduction on farm animals. 7th ed. lippincott williams & wilkins. philadelphia. iwamatsu y. and m. c. chang. 1971 factors involved in the fertilization of mouse eggs in vitro. j. reprod. fcrtil., 26:197-208. pope c. e., johnson c.a. , mcrae m.a. , kellcr g.l. and b.l. dresser. 1998. development of embryos produce by intracytoplasmic sperm injection of cat oocytes. anim rcprod sci, 53:221-236. rizal m, herdis and a. boediono. 2004. viability of rams epididymal sperm after preservation in low temperature (5°c). animal reproduction 6(l):30-36. sirman p and p. situmorang. 1987. evaluasi semen domba cair. ilmu dan peternakan, 3:1-3. song h.b and a. iritani. 1988. in vitro fertilization of goat follicular oocytes with epididymal spermatozoa. korean j anim sci, 30:636-642. 46 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf microsoft word 36 biotropia no. 20, 2003: 36 48 heterologous expression of a chitinase gene from aeromonas caviaein pseudomonas fluorescens amarila mahku'+, antonius suwanto1'2'", budi tjahjono3, rob harling4 'southeast asian regional center for tropical biology (seameo biotrop), bogor, 2 department of biology, faculty of mathematics and sciences, 3 department of plant pests and diseases, faculty of agriculture, bogor agricultural university, bogor-16144, indonesia: ' 'department of crop science. scottish agricultural college, edinburgh eh9 3jg, uk. abstract a transcriptional fusion for an aeromonas caviae chitinase gene was constructed under the control of a constitutive promoter of the kanaraycin resistance gene (pkmr). the construct was inserted into a medium copy number broad host range plasmid vector to yield recombinant plasmid pam340, which harbored transcriptional fusion pkmrchi. another transcriptional fusion, ptac-chi, in a recombinant plasmid pam630, was conducted as comparison. triparental mating of e. coli carrying the recombinant plasmids with pseudomotws fluorescens 5100, a phyllosphere bacterium, was performed. pseudomonas fluorescens 5100 exconjugants were examined for constitutive expression of chitinase employing a spectrophotometric assay; they showed stronger chitin degradation activity than escherichia coli transformants. using a fungal antagonism plate assay, this chitinolytic p. fluorescens, however, could not inhibit selected phytopathogenic fungi. keywords: aeromonas caviae/ chitinase gene/transcriptional fusion/pkm'v vtac-chilpseudomonas fluorescens introduction chitinase is an enzyme that catalyzes the hydrolysis of chitin to oligomers. chitin is an insoluble linear p-1, 4-linked unbranched polymer of 7v-acetylgluco-samine, and is a major constituent of fungal cell wall. chitinase is found widely distributed amongst many organisms, but its physiological role is different among them. in chitinase-producing plants, this enzyme is used for self-defense against plant pathogens and pests, i.e. to hydrolyse chitin in the cell wall of certain fungal pathogens and cuticle of pests (boiler et al. 1983; hedrick et a\. 1988; oppenheim and chet 1992). it has been suggested that chitinase production in plants is induced by microbial infections or other injuries (hedrick et al. 1988). in chitinase-producing bacteria, extracellular chitinases are produced to digest chitin and utilize * author to whom all correspondence and reprint requests should be addressed : tel : +62 (251) 625965; fax:+62 (251) 315107 ; e-mail : asuwanto@indo.net.id + present address : department of pharmacy, faculty of mathematics and natural sciences, university oflndonesia. biotropia no. 20,2003 it primarily as a carbon and energy source (gooday 1990). the exploration and utilization of bacterial chitinase in plant protection is centred around its use to protect against pathogenic fungi as a biocontrol mechanism. this potential has been a major concern in constructing biocontrol agents against fungi. several studies regarding this have been reported (inbar and chet 1991; koby et al. 1994). we have cloned and sequenced a heterogenous chitinase gene (chi) from a soilborne aeromonas caviae isolated from a blackpepper plantation in bangka island, indonesia in our previous study ( malik et al. in press). this gene has 97% identity with the chitinase gene chia from a. caviae (uo9139) (sitrit et al. 1995). in this present study, we intended to utilize this gene under control of a constitutive promoter, i.e. the kanamycin resistance gene from tn903, in a suitable vector for expression in a phyllosphere bacterium, as a means of producing a chitinolytic biocontrol agent. we cloned this chi gene into a relatively small size and medium copy number broad host range plasmid vector, pbbrlmcs-2 (kovach et al. 1994), rather than into a high copy number plasmid as reported by koby et al. (1994). this strategy was employed to avoid accumulation of defective escherichia coll mutants during high expression of chitinase (koby et al. 1994). therefore, the aim of this work was to construct a chitinolytic p. fluorescens biocontrol strain through the introduction of a chitinase gene under the control of the kmr promoter as a transcriptional fusion (pkmr-c/j/). materials and methods bacterial strains, plasmids, and growth conditions bacterial strains and plasmids used or constructed during this study are described in table 1. e. coli strains which were used as hosts for cloning experiments throughout this study, and their derivatives also, were routinely grown in luria bertani (lb) medium (broth or solidified with 1.5% agar) at 37°c for 18 hours. ampicillin (100 ng/ml), kanamycin (25 μg/ml), trimethoprim (100 μg/ml) and gentamycin (10 μg/ml) were added to the growth media when needed. pseudomonas fluorescens strains were grown routinely in king's b (kb) 10% agar (king et al. 1954) at 25°c for 18-20 hours. spontaneous rifampicin resistant mutants (rii ) of p. fluorescens, which was done according to the method described by eisenstadt et al. (1994), were isolated by plating a 25ul aliquot of a 10-fold concentration of a stationary phase culture onto plates containing a gradient concentration from 10 to 100 ug/ml of rifampicin. dna manipulation plasmid dnas were isolated either by alkaline lysis (sambrook et al. 1989), or hv dna nurification kits (promeea wis.. or biorad, richmond, calif.). restriction heterologous expression of a chitinase gene from aeromonas caviae amarila malik el al table 1. bacterial strains and piasmids bacterial strains and plasm relevant characteristics reference or source e. coli sambrook et dh5a f", for a-complementation, general host for cloning al. 1989 top10 f", for a -complementation, general host for cloning invitrogen, inc. (carlsbad, calif.) hb101(prk2013) mod" res", general helper strain for bacterial conjugation dittae/fl/1980 top10(pam340) top10 carries fusion transcription pkmr-c/i; this study top10(pam630) top10 carries fusion transcription ptac-chi this study p.jluorescens 5100 phyllosphere biosurfactant* strain, wildtype, derived from campbell et al brassica oleracea var italica 1995 5100 rif spontaneous rifampicin resistance mutant pfsloo this study 5100(pam340) pf 5100 (rif) carries fusion transcription pkmr-c/i; this study 5100(pam630) pf 5100 (rif) carries fusion transcription ptac-chi this study aeromonas caviae ws7b chitinolytic, wildtype malik etal in press plasmids puc19 lacz. apr cloning vector sambrook et al. 1989 pas385 mcs from psl301(£cori-.eo/i) cloned into puc19 suwanto & kaplan (smal-ecorl), apr 1992 pas396 source of trimethoprim resistance gene (tpr) in pas385 suwanto & kaplan 1992 pbbrlmcs-2 broad-host range, medium copy number, kmr kovache/a/. 1994 puc4k pkmr source, kmr gene from 1n903 oknetal 1981 p34s-gm source of gentamycin resistance gene (gmr) dennis & zylstra 1998 pws506 pas385 carrying 2.9 kb xho\ + ffmdlu chitinase gene malik et al in fragment from a. caviae ws7b press pam201 c/i/-tpr dna fragment as sail cassette in pas385 this study pam330 pknf-chi in pbbrlmcs-2, tpr this study pam340 pkmr-c/i/ in pbbrlmcs-2, tp", gmr this study pam630 ptac-chi in pbbrlmcs-2, gmr this study fragments were purified from agarose gels with the gene clean kit (biolol inc., la jolla, calif.). dna filled-in reaction, utilizing t4 dna polymerase (boehringer mannheim biochemicals, indianapolis, ind.), dna fragment ligations, recombinant dna transformation, and other accessory techniques were carried out as described by sambrook et al (1989). escherichia coli transformants harboring the chitinase recombinant were selected on lb-chitin agar plates, supplemented with the appro biotropia no. 20,2003 ing pkmr, as well as ptac, were selected by a blue-white assay on lb agar plates, containing iptg (0.5mm), and x-gal (5-bromo-4-chloro-3-indolyl-b-d-thiogalacto-pyranoside) 40 mg/ml. plasmids were maintained by selection in the presence of appropriate antibiotics. bacterial conjugation recombinant plasmids pkmr-c/» and ptac-chi were introduced into p. fluorescens 5100 (campbell et al. 1995) by bacterial conjugation as described by ditta et al (1980). triparental mating was performed, employing e. coli hb101 (prk2013) (ditta et al. 1980) as helper, e. coli top10 (pam340), as well as e. coli top10 (pam630), as donor, and p. fluorescens 5100 rif as recipient. the cells of all bacteria employed in this mating were collected from mid-exponential phase cultures, harvested by centrifiiging recipient, donor, and helper cultures in a ratio of 8:1:1, washing the pellet twice in sterile saline solution, and resuspending in a small volume of lb broth (30 ul). all cell suspensions were mixed, spotted onto lb agar plates without any antibiotic supplements, and incubated at 25°c. polymerase chain reactions standard pcr reactions were performed to verify the presence of cloned chi in p. fluorescens exconjugants, followed by electrophoretic analysis. an approximately 800-bp fragment of chi (accession number aj431785) was amplified in a geneamp 2400 thermal cycler (perkin elmer). single colonies of exconjugants were resus-pended into a reaction mixture prepared with taqdna polymerase (finnzyme). the thermocycling program used in this study consisted of denaturation at 94 °c for 60 s, annealing at 54°°c for 60 s, and elongation at 72 °°c for 90 s, for 25 cycles, as a modification of the method described in chernin et al. (1997). a set of chi internal primers, which were designed based on a. caviae chitinase dna sequence (accession number aj431785), were used, i.e. forward primer 5 ' g t g a a g a a c t a c c a g g c -3', and reverse primer 5 ' g g c a g a t c a g t t g c a g c t c g -3' (genset biotech, singapore). chitinase plate assay and semimicro-quantitative assay an assay for chitinase was carried out on lb agar supplemented with colloidal chitin 1% (hsu and lockwood 1975) for e. coli derivatives, whilst for p. fluorescens derivatives this was carried out on 10% kb agar supplemented with colloidal chitin at the same amount. p. fluorescens exconjugants were selected on 10% kb agar supplemented with rifampicin (50 ug/ml) and gentamycin after introducing transcriptional fusion recombinant plasmids by bacterial conjugation. semimicro-quantitative assay was performed as described previously (malik et a/. in press). cultures of the recombinant bacteria were collected after growing for 18-20 hours both for e. coli and p. fluorescens, whilst cultures of a. caviae were heterologous expression of a chitinase gene fromaeromonas caviae amarila malik et al. collected from 15 hours. enzyme activity was measured in both extracellular and intracellular fractions, after sonicated the cell. the protein concentration was measured according to the method as described by bradford (1976). chitinase activity was assayed by a modification of methods as described using colloidal chitin azure as substrate (wirth and wolf 1990; evrall et al. 1990; hood 1990). the amount of dye released from this chromogenic substrate, remazol brilliant blue, was measured at 590 nm. a standard curve was constructed using a chitinase standard from streptomyces griseus (sigma chemical, st. louis, mo) against colloidal chitin azure as substrate, in citrate-phosphate buffer ph 6.0 as described by hood (1990), and incubation temperature 37°c for 2 hours. the enzymatic activity was expressed as units of chitinase/mg of protein. assay for antagonism to fungi an antagonism assay was performed as described by chernin et al. (1995): test bacteria were grown for 24 and 48 h for a. caviae ws7b (malik et al. in press) and p. fluorescens strains, respectively, in luria or nutrient broth at 30°c with aeration. the suspension of cells was streaked in a line at the centre of a pda plate and incubated at 30 °c for 24 and 48 h for ws7b and p. fluorescens strains, respectively. after placing 3-mm diameter agar disks of an actively growing fungal culture of either botrytis cinerea tom98ld and fusarium solani f. sp. pisi, at 3 to 4 cm away from each side of the bacterial growth area, the plates were incubated for 3 to 25 days, until mycelium growing from the two sides on a control plate came into contact. results construction of sail-cassette ofchi constitutive promoter of kanamycin resistance gene (pkmr) from tn903 used in this study possesses a unique restriction site, xhol (oka et al. 1981). this xhol site was employed to construct a transcriptional fusion of a. caviae ws7b chitinase gene chi (accession number aj431785) under the control of pkmr (fig.l). the chi gene in pws506 was cloned without its indigenous promoter (malik et al. in press). plasmid pam201-a carrying a sail cassette of chi was constructed through several intermediate-cloning steps (fig. 1). the first step was the insertion of trimethoprim resistance gene (tpr) isolated from pass 96 (suwanto and kaplan 1992), as a marker downstream ofchi fragment at hpal site in pws506. the chi-tpr fragment was then isolated subsequently employing hindhl+ecorv double digestion. the protruding end of hinalll was made blunt by means of t4 dna polymerase to obtain blunt end chi-tpr fragment, which was then ligated with blunt-end hpal of vector pas385 (suwanto and kaplan 1992), generated recombinant plasmid pam201 (fig 1). plasmid pam201-a carries c/z/-tor figure 1. schematic structure of sail cassette construction of chi. the promoter of kanamycin resistance gene (pkmr) from tn903 in puc4k is located upstream of the xhol site (oka et a/,1981) (a). a trimethoprim resistance gene (tpr) fragment, isolated from pas396 (suwanto & kaplan 1992), was inserted downstream of the chi fragment in pws506 (malik et al. in press) as marker at hpal site in polyl inker site (b). the c/i/-tpr fragment was isolated employing hindlll+ ecorv double digestion. the protruding end of ifiruh.ll was made blunt, which was then ligated with blunt-end hpal end of linearized vector pas385 (suwanto & kaplon 1992), resulting in recombinant plasmids pam201-a (chi+) and pam201-b (chi-) that carry sail cassette of c/i;-tpr (c). heterologous expression of a chitinase gene from aeromonas caviae amarila malik et al. construction of pkmr-chi transcriptional fusion in pbbrlmcs-2 a medium copy number broadhost-range plasmid vector, pbbrlmcs-2 (kovach et al. 1994), was utilized as cloning vehicle to introduce pkmr-chi transcriptional fusion into pseudomonas fluorescens 5100. the c/»/-tpr sail cassette from pam201-a was inserted into xhol site of pbbrlmcs-2 under pkmr to generate pam330, in which c/»'-tpr will be transcribed under control of kmr promoter (fig 2). bgli figure 2. restriction map of recombinant plasmid pam330 carrying pkmr-c/i; transcriptional fusion in pbbrlmcs-2. the additional gmr marker was inserted at ///«diii site downstream of tpr, which generated pam340 in addition to tpr marker, a gmr cassette isolated from p34s-gm (dennis and zylstra 1998) was used as an alternative marker by inserting the cassette downstream of tpr resulting in pam340 (fig 2), since tpr marker was not sufficient to select for pf exconjugants after mobilizing pam330 from the e. coli transformant. construction of ptac-chi transcriptional fusion in pbbrlmcs-2 a bamhl fragment of ptac isolated from pkk223-3 was inserted into bamhl site in pbbrlmcs-2, generating plasmid pam601. chitinase gene chi, which was isolated from pam202 as hindlll-ecorl c/i/-gmr fragment, was inserted subsequently into this plasmid vector, resulting in recombinant transcriptional fusion plasmid harboring ptac-chi, designated as pam630 (fig 3). plasmid pam202 was generated from pam201-b by replacing tpr with gmr. biotropia no. 20. 2003 ecori hind iii. abbreviations : km" = kanamycin resistance gene; pkmr = promotor of kanamycin resistance gene; apr = ampicillin resistance gene; tp" = trimethoprim resistance gene; gmr = gentamycin resistance gene; chi = chitinase gene figure 3. restriction map of recombinant plasmid pam630 carrying ftac—chi transcriptional fusion in pbbrlmcs-2 chitinase expression of transcriptional fusions in e. coli and p. fluorescens. screening of e. coli transformants harboring pam340, or pam630 on chitin agar plate showed only weak clearing zones observed after 7 and 11 days of incubation, respectively (table 2). on the other hand, e. coli harboring pws506 showed strong clearing zones after only 3 days of incubation. table 2. chitinolytic expression of chi on chitin agar plates. strains expression chitinolytic activity control as clearance zone* incubation time (days) e. co//top10(pws506) plac +++ 3 e. co//top10(pam340) pkmr + 7 e. co//top10(pam630) ptoc + 11 p. fluorescens 5100 (pam340) pkmr ++ 7 p. fluorescens 5100 (pam630) ptoc ++ 11 * relative chitin degradation ability was indicated as +, ++ and +++ for slightly, moderate, and very clear zone, respectively. plasmid pam340 and pam630 were mobilized into spontaneous rifampicin resistance (rif) mutants of strain pf5100. screen ino fnr pf phitmr.krf;/ heterologous expression of a chitinase gene from aeromonas caviae — amarila malik et al. selection, in conjunction with pcr techniques using chi primers as described above. approximately 800 bp bands were observed on agarose gel after electrophoresis of exconjugants (data not shown). the latter was a rapid and easy method to verify the exconjugants after isolating single colonies. constitutive chitinase expression of pkmr-chi the modified chitin azure assay used in this study could demonstrate the constitutive expression of the cloned chitinase gene by growing the e. coli recombinant and pf exconjugant without chitin. the colloidal form of chitin azure, which consists of oligomers and monomers, was fairly easy to degrade by chitinolytic activity compared to the flake form of this compound. we also constructed a transcriptional fusion of the same chitinase gene under the tac promoter (fig. 3), in order to compare the promoter activity. expression assays of both transcriptional fusions were carried out under uninduced growing conditions. the result showed that constitutive expression of chi under pkmr was slightly stronger (tables 2 and 3). by comparing chitinolytic activity of these two p. fluorescens 5100 exconjugants to the wild type bacterium a. caviae ws7b (the origin of chi gene in this study), it could be demonstrated that constitutive chitinolytic activity of these two strains of p. fluorescens were less active. this is consistent with the result of our previous study on e. coli recombinant harboring chi of ws7b in a puc vector that ws7b presumably carries more than one chitinase gene, and has a complex regulation of chitinase gene expression. table 3. chitinase activity assay chitinase assay (unit/mg protein) strains extracellular intracellular a. caviae ws7b 7.70" nd e. co//top10 0.29 0.41 e. co//top10(pws506) 2.31 4.17 eco/itop10(pam340) 0.93 1.12 e. co/;top10(pam630) 0.91 1.05 p. fluorescens 5100 rif" 0.77 0.84 p. fluorescens (pbbrlmcs-2) 0.78 0.83 p. fluorescens (pam340) 1.91 3.27 p. fluorescens (pam630) 0.97 2.53 #as positive control,collected from90 hrs of growth incubation time nd = not determined biotropia no. 20,2003 antifungal plate assay the antagonism assay of p. fluorescens strains harboring pam340, and pam630, was conducted with a. caviae ws7b as positive control for chitinolytic activity. p. fluorescens 5100 rif and p. fluorescens (pbbr!mcs2) were used as negative control strains. the inhibition zones between the pathogenic fungus botrytis cinerea tom98ld and the tested strains were observed after 3 to 24 days of incubation. the positive control, ws7b, showed an inhibition zone up to 17 mm. however, all bacteria strains tested did not show any fungal growth inhibition against fusarium solani f. sp. pisi. discussion a transcriptional fusion of a. caviae chitinase gene (chi) under kanamycin resistance gene promoter (pkmr) has been constructed. kmr gene is known to be expressed constitutively. by inserting chi under this promoter, we assume this will cause simple constitutive regulation of chi. the expression of other chitinase genes has been reported under nonindigenous promoter, ptac (koby et al. 1994; downing and thomson 2000). however, the expression of chitinase gene under the control of pkmr has previously not been reported. in this study, we demonstrated the constitutive expression of chitinase under this promoter by a simple semi micro-quantitative chromogenic assay using colloidal chitin azure as substrate. the enzyme activity was determined by measuring the amount of remazol brilliant blue dye released employing spectrophotometer. the results from the expression of chitinase transcriptional fusion assay demonstrated that both transcriptional fusions were expressed in e. coli, and were able to degrade chitin in the medium. longer incubation time for chitinolytic activity of pam340 and pam630 compared to pws506, might be due to the influence of plasmid copy number in this expression (table 1). plasmid pam340 and pam630 were constructed on a medium copy number plasmid vector pbbrlmcs-2, while pws506 was constructed on a high copy number plasmid vector pucl 9. the strategy to isolate and insert the promoters into plasmid vector pbbrlmcs-2 before fusion with chi, rather then subcloned chi under pkmr directly in puc4k, as well as under ptac in pkk223-3, was carried out to yield a broad host-range plasmid vector that carry a constitutive promoter pkmr, as well as the strong promoter ptac, which will be useful in future studies of other heterologous gene expressions. the result of the semimicro-quantitative of chitinase activity assay indicated that chi was expressed constitutively both in e. coli or p. fluorescens 5100 recombinants (table 3). however, chitinase activity of intracellular fractions were higher than the extracellular fractions which can be assumed that the chitinase product might not be well secreted in e. coli, as we have discussed in our previous study, as well as in p. fluorescens (malik et al. in press). 45 heterologous expression of a chitinase gene from aeromonas caviae amarila malik et al. the inability of transformed p. fluorescens strains to slow fungal growth is more likely caused by secretion problem, as well as low expression of chitinase product in p. fluorescens based on the results obtained from chitinolytic expression on agar plates and semiquantitative chitinase assays as shown in tables 2 and 3. the gene was isolated from a. caviae, and was cloned and expressed in e. coli and p. fluorescens, which are not indigenous hosts. nevertheless, the fact that the protein expression machinery between these three microorganisms are not the same, could explain this low chitinolytic activity. it could be also suggested that this chitinase gene (chi) in its source bacteria a. caviae ws7b, did not act alone to slow fungal growth. it might also require other antifungal mechanisms as reported previously in jones et al. (1986) and sundheim et al. (1988). conclusions transcriptional fusion of the chi gene under the control of kanamycin resistance gene promoter generated constitutive expression of chitinase in both e. coli and p. fluorescens 5100, which has previously not been reported. this chitinase gene expression might be useful to be developed further for construction of a biocontrol strain to prevent the growth of phytopathogenic fungi. acknowledgment this work was supported by graduate team research grant (urge) grant 029/htpp/ii/ urge/96 to b.t., and a higher education link award from the department for international development (dfid)/british council to r.h. and a.s. references boiler, t., gehri, a., mauch, f. and u. vogeli. 1983. chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. planta 157,22-31. bradford, m.m. 1976. a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72,248-254. campbell, l.j., marling, r., chard, j. and m. sutton. 1995. calabrese: factors controlling symptom development in bacterial spear rot. final report, project fv104b, horticultural development council, bradbourne house, east mailing, kent me 19 6dz, uk. cherain, l., z. ismailov, s.haran, and i. chet. 1995. chitinolytic enterobacter agglomerans antagonistic to fungal plant pathogens. appl. environ. microbiol. 61,1720-1726. chernin, l.s., de la fuente, l., sobolev, v., haran, s., vorgias, c.e., oppenheim, a.b. and i. chet. 1997. molecular cloning, structural analysis, and expression in escherichia coli of a chitinase gene from enterobacter agglomerans. appl. environ. microbiol. 63, 834 -839. 46 biotropia no. 20,2003 dennis, j.j. and g.j. zylstra. 1998. plasposon: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. appl. environ. microbiol. 64,2710-2715. ditta, g., stanfield, s., corbin, d. and d.r. helsinski . 1980. broad-host range dna cloning system for gramnegative bacteria: construction of gene bank of rhizobium melihti. proc. natl. acad. sci. 77,7347-7451. downing, k.j. and j.a. thomson. 2000. introduction of the serralia marcescensa chia gene into an endophytic pseudomonas jluorescens for the biocontrol of phytopathogenic fungi. can j microbiol 46, 363-369. eisenstadt, e., b. c. carlton and b. j. brown. 1994. gene mutation. in: gerhardt, p., r. g. e. murray, w. a. wood, n.r. krieg (eds). methods for general and molecular bacteriology. american society for microbiology, washington, d.c.: 297-315. evrall, c.c., atwell, r.w., and c.a. smith. 1990. a semi-micro quantitative assay for determination of chitinolytic activity in microorganisms. j. microbiol. methods 12,183-187. gooday, g.w. 1990. physiology of microbial degradation of chitin and chitosan. biodegradation 1, 177-190. hedrick, s.a., bell, j.n.., boiler, t. and c.j. lamb. 1988. chitinase cdna cloning and mrna induction by fungal elicitor, wounding, and infection. plant physiol. 86,182-186. hood, m.a. 1990. comparison of four methods for measuring chitinase activity and the application of the 4-muf assay in aquatic environments. j. microbiol. methods 13, 51-160. hsu, s.c. and j.l. lockwood. .1975. powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. appl. microbiol. 29, 422-426 inbar, j and i. chet. 1991. evidence that chitinase produced by aeromonas caviae is involved in the biological control of soil-borne plant pathogens by this bacterium. soil biol. biochem. 23, 973-978. jones, j.d.g., k.l.grady, t.v.suslow, and j.r.bedbrook. 1986. isolation and characterization of genes encoding two chitinase enzymes from serratia marcescens. embo j. 5: 467-473. king, e.o., ward, m.k. and d.e. raney. 1954. two simple media for the demonstration of pyocyanin and fluoreescin. j. labor. clin. med. 44, 301-307. koby, s., schickler, h., chet, i. and a.b. oppenheim. 1994. the chitinase encoding tn7-based chia gene endows pseudomonas jluorescens with the capacity to control plant pathogens in soil. gene 147,81-83. kovach, m.e., phillips, r.w., elzer, p.h., roop ii, r.m. and k.m. peterson. 1994. pbbrlmcs: a broad-host range cloning vector. biotechniques 16, 800-802. malik, a., wenuganen, s., suwanto, a. and b. tjahjono. cloning, dna sequence and expression of aeromonas caviae chitinase gene. molec. biotech. (in press) oka, a., sugisaki, h. and m. takanami. 1981. nucleotide sequence of the kanamycin resistance transposon tn905. mol. biol 147: 217-226. oppenheim, a. b. and i. chet. 1992. cloned chitinases in fungal plant pathogen control strategies. trends biotechnol. 10,392-394. sambrook, j., fritsch, e.f. and t. maniatis. 1989. molecular cloning. cold spring harbor laboratory press. cold spring harbor. new york. usa 47 heterologous expression of a chitinase gene from aeromonas caviae amarila malik el al. sitrit, y., vorgias, c.e., chet, i. and a.b. oppenherm. 1995. cloning and primary structure of the chia gene from aeromonas caviae. j. bacteriol. 177, 4187-4189 sundheim, l., a.r. poplawsky and a.h. ellingboe. 1988. molecular cloning of two chitinase genes from serratia marcescens and their expression in pseudomonas species. physiol. mol. plant. pathol. 33: 483-491. suwanto, a. and s. kaplan. 1992. a self-transmissable narrow-host range endogenous plasmid of rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer function. j. bacteriol. 174, 1124-113 wirth, s.j. and wolf, g.a. (1990. dye-labelled substrates for the assay and detection of chitinase and lysozyme activity. j microbiol. methods 12,197-205. 48 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf biotropia vol. 29 no. 1, 2022: 56 68 doi: 10.11598/btb.2022.29.1.1626 56 incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) cake extract to improve bioplastic characteristic rini umiyati1*, chusnul hidayat2, ria millati2 and teguh ariyanto3 1department of food technology, faculty of engineering, universitas pgri semarang, semarang 50125, indonesia 2department of food and agricultural product technology, faculty of agriculture technology, universitas gadjah mada, yogyakarta 55281, indonesia 3department of chemical engineering, faculty of engineering, universitas gadjah mada, yogyakarta 55281, indonesia received 1 september 2021 / accepted 25 february 2022 abstract the cross-linking agent plays an important role in bioplastic mechanical properties. this study aimed to determine the effect of sodium hyaluronate (soha) as a synthetic cross-linking agent and the addition of nyamplung cake extract (nce) as an antimicrobial agent in the manufacture of bioplastic with hydroxypropyl starch (hps) as based ingredient using the thermo-compression method. the novelty of the study was thiocyanate (scn) formation in zone 3 (2,161.66-2,162.02/cm) and cyanate (c-n=o) in zone 6 (1,411.57 1,412.61/cm) of (1, 2 and 3%) soha bioplastic and cyanate formation in zone 6 and 7 (1,411.37-1,558.59/cm) of (1, 2 and 3%) soha-20% nce combined bioplastic originating from acetanilide group in soha and amide group in nce. the formation of scn and c-n=o in 2 and 3% soha bioplastic improved its sensitivity against gram-positive bacteria (staphylococcus aureus) indicated by 0.6 mm and 0.45 mm inhibition zone, respectively. c-n=o formed in (1, 2 and 3%) soha-20% nce combined with bioplastic had 3.25 mm average inhibition zone against gram-positive bacteria (s. aureus), 2.75 mm against gram-negative bacteria (escherichia coli), and 0.71 mm against fungi (aspergillus niger). the analysis of mechanical properties showed that an addition of 3% soha was able to increase tensile strength and modulus of elasticity while reducing elongation, water solubility and water vapor permeability. addition of (1, 2 and 3%) soha-20% nce resulted in a reverse effect. keywords: acetanilide, amide, cross-linking agent, cyanate, thiocyanate introduction natural polymers, such as starch, have various potential applications to replace commercial market-dominant petroleum-based plastics due to their abundant availability, degradability, renewability and low price (samsudin & hani 2019). the most critical property of plastic is its ability to be adjusted for specific purposes. the increasing utilization of starch for commercial use, such as to produce biodegradable thermoplastic, is expected to reduce problems caused by the petroleum-based polymer in solid waste disposal (manoi & rizvi 2010). as a renewable polymer source with filmforming ability, starch fulfills several requirements as an alternative for plastic, such as having abundant availability, high extraction yield, cheap, biodegradable and biocompatible. starch-based bioplastic is also odorless, tasteless, colorless, non-toxic, and semi-permeable to carbondioxide, temperature, oxygen, as well as fat and aroma components (shah et al. 2016). starch-based bioplastic as an environmentally friendly package has various disadvantages related to the hydrophilic and hygroscopic properties of starch. in this study, hydroxypropyl starch (hps) as a modified starch was used as the main material, because hps is more feasible to be processed into bioplastic due to its lower solubility temperature, with more transparent and flexible plastic properties (woggum et al. 2015). therefore, the mechanical properties of bioplastic can be improved and *corresponding author, email: riniumiyati@upgris.ac.id incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 57 adjusted by adding a plasticizer or cross-linking agent. a plasticizer is a group of small molecules that are able to blend and interact among polymer chains by intermolecular force, thus reduces glass transition temperature of a material to improve malleability, flexibility, extension and to soften the texture (wypych 2017). on the other hand, cross-linking mechanism works by formating bridges of immediate intermolecular bond by chemicals known as cross-linking agent. cross-linking means polymer molecules are interconnected by bond (canisag 2015). cross-linking agent is substances that are able to form permanent chemical bond among polymer chains, to increase material rigidity, firmness, strength, less flexible and increase glass transition temperature (tg). cross-linking agent might affect permeability property, thermal stability, glass transition temperature and the rate of wear (frost et al. 2013) due to formation of covalent or ionic bond connecting a polymer chain to the others which enhance polymer strength. cross-linking process is critical to modify starch properties by enhancing the starch’s intra and inter-molecular bonds in random patterns. the bonds tend to inhibit starch interaction to water while bringing structural integrity on starch-based biodegradable materials during hydraulic pressure and high humidity. crosslinking process can be done by starch treatment, both in semi-solid or pulp form, using reagent with ability to form ether or esther bond among hydroxyl (-oh) in starch molecule (manoi & rizvi 2010). cross-linking agents are able to form cross bond that enhances matrix density and tensile strength, and even permanent chemical bond among polymer chains, thus generate more rigid, strong, less flexible material and increase glass transition temperature (tg). polyfunctional chemicals such as phosphor oxychloride (pocl3), sodium trimetaphosphate (stmp), sodium tripolyphosphate (stpp), epiclorohydrine (epi), a mixture of adipic, acetate anhydride, and mixture of succinic anhydride and vinyl acetate are some of the common cross-linking agents for starch. in this study, we used sodium hyaluronate (soha) as a cross-linking agent. soha is a polymer of disaccharides, consisting of d-glucuronic acid and d-n-acetyglucosamine, linked by β-1, 4 and β-1, 3 bonded by glycosidicbonds, into polymer compounds that do not have uv absorbing chromophores (ruckmani et al. 2013). hyaluronate supports the structure of connective tissue by acting as a magnet to maintain fluidity and form viscous liquids with lubrication properties. soha has low toxicity and no report of negative effect on human (spec-chem ind. 2014). soha is also suitable as cross-linking agent and encapsulates various active ingredients (contipro 2018). this study used nyamplung cake extract (nce) as antimicrobial agent because of the ability of nce against gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli). bioplastic is more sensitive against gram-positive bacteria compared to gram-negative bacteria. bioplastic has the largest inhibition zone of 30 mm against staphylococcus aureus and that of 23 mm against escherichia coli. the abiltiy of bioplastic as antimicrobial agent might be related to the content of the bioplastic extract which serves as natural cross-linking and antimicrobial agents (umiyati et al. 2019). the purpose of this study was to determine the effect of soha as synthetic cross-linking agent and nyamplung cake extract (nce) as antimicrobial agent in the manufacture of bioplastic with hps as basic ingredient using thermo-compression method. this study demonstrated that the features of bioplastic produced showed better mechanical properties, having antimicrobial ability compared to the conventional bioplastic. materials and methods materials nyamplung cake (calophyllum inophyllum l.) was obtained from biodiesel industry in purworejo, central java, indonesia. nyamplung cakes were sorted and dried until reaching moisture content below 10-15%. the dried cakes were finely ground and sifted using a 120mesh sieve to obtain nyamplung cake powder. subsequently, 20 g of the nyampung cake powder was extracted by using 120 ml of 96% ethanol and placed on a hot plate stirrer at 80 °c for 1 h. the mixture was filtered using whatman filter paper no. 1. based on the optimal method biotropia vol. 29 no. 1, 2022 58 by chana-thaworn et al. (2011), the extracted liquid was then evaporated and ovendried at 50 °c for 24 h. other materials used in this study were hydroxypropil starch (hps), sodium hyaluronate (soha), glycerol 20%, etanol 96% and distilled water. instruments thermo-compression machine, ftir (thermo scientific nicolet is10), scanning electron microscopy (jeol jsm 6510) and texture analyzer (brookfield usa) were used in this study. bioplastic preparation various concentrations of soha (1%, 2% and 3% b/b) were mixed with 10 g of hps, 2 g of nyamplung cake extract, 1.5 g of glycerol and 1 g of distilled water, then mixed at 21,000 rpm for 45 min until homogen. the mixture was then made into bioplastic using thermocompression mold. the mixture was then placed in the middle of a 0.5 mm-thick aluminum frame sized 10 × 10 cm that was put between the two previously heated aluminum plates, molded at 140 °c and 250 kg/m2 pressure for 6 min in heated hydrolic pressure machine (rasheed et al. 2015). fourier transform infrared spectroscopy (ftirs) analysis is a method to identify structural changes in starch chains due to interaction among extract, glycerol, naha, and hps molecules (bilal et al. 2015). ftir spectra of bioplastic was measured by thermo scientific nicolet is10, equipped with smart atr diamond at an area of 650-4,000/cm using 32 scanning and resolution of 8/cm. scanning electron microscopy (sem) was used to analyze microplastic structure of the bioplastic. sample of bioplastic was stored in a desiccator with p2o5 absorbent for two weeks to ensure no moisture left in the bioplastic sample. for cross-section observation, the bioplastic sample was frozen in n2 liquid for cryofracture preservation (espinel et al. 2014). all samples were placed in bronze stub and covered with gold sheets before imaging. micrograph of the surface of bioplastic and fracture was obtained using sem (jsm-6510la) at 10-300,000x magnification and 1-10 nm resolution. characterization of bioplastic physical properties mechanical properties such as tensile strength (σ), elongation (ε), modulus of elasticity (y), water solubility (ws) and water vapor permeability (wvp) were measured based on the methods in astm d638. prior to mechanical properties analysis, the bioplastic sample was conditioned at rh of 50 ± 5 and 23 °c for 48 h in humid chamber. tensile strength at room temperature was measured using brookfiled usa analyzer texture. bioplastic specimen was cut according to the method by astm d1708 at a constant pull rate of 5 mm/minute. solubility measurement of bioplastic sample in water was conducted by drying the bioplastic sample at 105 °c and then weighed as m3. subsequenty, the bioplastic sample was soaked in 50 ml distilled water at 24 °c for 6 h, then ovendried at 105 °c for 24 h and weighed as m4 (hassannia-kolaee et al. 2016; kumari et al. 2017), before calculation using formula below (1): ws = m3 – m4 x 100 ..….……………….. (1) m4 where: m3 = weight of bioplastic sample before drying m4 = weight of bioplastic sample after drying water vapor permeability (wvp) was analyzed using standard method in astm (1996) e96. the bioplastic sample was placed as a cover on circle permeation cells sized 4 cm in diameter containing silica gel (0% rh) and placed in a desiccator which was previously filled with saturated sodium chloride liquid (75% rh), then stored at 30 °c. water vapor permeability rate was measured by permeation cell weighing at a frequency of every 30 min to obtain several points. the wvp was counted using a formula (2) developed by wirawan et al. (2012): wvp = wptr x m ….…………….. (2) ps (rh1 – rh2) where: wvp = water vapor permeability (g.mm/ kpa.s.m2) wvtr = mass increase (g) ps = pressure of saturated water vapor (pa) incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 59 rh1 = relative humidity in desiccator rh2 = relative humidity in permeation cell m = average thickness of bioplastic layer (m) characterization of bioplastic antimicrobial properties disk agar method was used for analyzing the antimicrobial activity of bioplastic sample using distilled water as solvent. as much as 0.1 ml of 10% bacteria and fungi suspension was inoculated on mueller hinton agar and on potato dextrose agar media, respectively. bioplastic sample of 1 cm diameter was placed on a sterile disk sized 6 mm diameter and let to dry. subsequently, the bioplastic sample was placed on the surface of culture media in a petri dish that was previously inoculated with tested microorganisms, then incubated at 37 °c for 1-2 days for bacteria and at 30 °c for 2-3 days for fungi, based on optimum condition reported by chanwitheesuk et al. (2007). results and discussion bioplastic characteristics mechanical properties comparison of mechanical properties in the form of tensile strength (σ), elongation at break (ε) and modulus of elasticity (y) of bioplastic prepared with (1, 2 and 3%) soha, combination of (1, 2 and 3%) soha-20% nce compared to that prepared by using 20% nce and control is presented in figure 1. those mechanical parameters depend on microstructure characteristics (aguirre et al. 2013). bioplastic thickness ranged from 0.24 to 0.32 mm (data not shown). results of this study showed that the control bioplastic had significant increase of tensile strength (σ) compared to 3% soha bioplastic and 20% nce bioplastic, by 23.7% and 50.8%, respectively, while that of (1, 2 and 3%) soha20% nce bioplastic had significant reduction by an average of 62.4% (fig 1). in terms of elongation at break (ε), the (2 and 3%) soha bioplastic showed significant reductions compared to the control bioplastic by an average of 32.4%, while the (1, 2 and 3%) soha-20% nce bioplastic had a significant increase of 224.2%. for modulus of elasticity (y), there was significant difference between the control and (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce combination, and 20% nce. this indicated that soha addition to 3% could function as effective cross-linking agent to improve bioplastic mechanical properties. soha works to facilitate esterification of starch oh groups in overcoming the poor polymer compatibility by incorporating compatible substances (ortega-toro et al. 2014). figure 1 effect of soha addition and combination of soha-nce on the mechanical properties of hps bio-plastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). biotropia vol. 29 no. 1, 2022 60 soha is a widely distributed natural mucopolysaccharide polymer, which in mammals is known as connective tissue. crosslinking process is done by soha to increase the in vivo retention time, so that the hydroxyl groups within the polymer chains bind to one another. the cross-linking reaction in soha is carried out in a homogeneous aqueous alkaline solution which can enhance anti-biodegradation ability by increasing the degree of cross-linking and extending the in vivo retention time (xuejun et al. 2015). utilization of the modified hps starch as basic material may also serve to improve bioplastic mechanical properties due to its high amylose content of modified starch. therefore, hps is feasible as bioplastic raw material, has mechanical strength and functions as good oxygen inhibitors (woggum et al. 2015). concentration of cross-linking agent, ph, treatment temperature and storage period might determine cross-linking bond. reaction condition can be varied according to the type of cross-linking agent (canisag 2015). utilization of soha-nce combination reduced σ and y. higher concentration of soha on nce resulted in a lower σ. these effects were probably due to the loss of amide group in nce bioplastic (20% extract) by the combination. on the other hand, soha-nce combination increased ε, which probably due to h2o formation from reactions involving soha and hydroxyl groups from hps starch, glycerolsoha and amide groups of nce-soha. c-n=o is an intermediate product of the first stage of thiocyanate hydrolysis, which was then hydrolyzed into ammonia and bicarbonate (doble & kumar 2005). stronger interaction of hps-soha-glycerol reduces y and maximum tension as well as tension-when-separate resulted in a more flexible bioplastic, and therefore, showing the function of soha as cross-linking agent (contipro 2018). modified starch was reportedly a more stable raw material for making bioplastic compared to native starch (gutiérrez et al. 2015). bioplastic preparation might also play a role as thermo-compression utilizes high temperature and hydroulic pressure. there was also the possibility of maillard reaction which can form more compact networks (leceta et al. 2013). hps as raw material might affect bioplastic mechanical properties due to the effect of hydroxypropylation that interfere the interand intra-molecular hydrogen bond of the starch chain. the effect can enervate starch granule while increasing starch chain movement in an amorph area and enhancing hps swelling power compared to native starch with increasing molar substitusion (woggum et al. 2015). soha addition brought significant increase on σ at ≥ 3%, but nce addition in soha bioplastic did otherwise. water solubility (ws) soha and soha-nce combination affected bioplastic water solubility (ws) in terms of bioplastic integrity in watery media. high solubility of bioplastic indicates low water resistency (gutiérrez et al. 2015). ws is defined as the ratio of dry matter in water, such as bioplastic soaked in distilled water (hassanniakolaee et al. 2016). results of this study showed that ws of bioplastic made by using soha, combination of soha-nce and nce were significantly lower than that of control (fig. 2). soha bioplastic when compared to control had an average of lower ws values by 6.6%, while bioplastic made by using soha-nce combination had an average of lower values by 15.9%, then bioplastic made by using nce had an average of lower values by 20.95%. insignificant ws decrease was observed in 2% soha compared to 3% soha bioplastics and vice versa, as well as in 1% soha-20% nce combination compared to nce bioplastics (fig. 2). this showed that nce addition affected the molecular structure of soha bio-plastic. during bioplastic preparation, phosphor of stmp (as a cross-linking agent in making modified starch) reacted with hydroxyl (-oh) group of starch to form phosphate distarch (cross-bond) and other phosphate derivates as indicated by the ft-ir results. cross-linking process enhances granule structure starch and inhibit both water absorption and starch solubility, thus limit the mobility of starch chain in amorph area (manoi & rizvi 2010). bioplastic prepared by hps-soha had lower solubility compared to control, with increasing concentration of cross-linking agent. this incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 61 figure 2 effect of soha addition and combination of soha-nce on the water solubility of hps bio-plastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). finding was comparable to previous study conducted by (manoi & rizvi 2010) which indicated that the formation of phosphate distarch act as cross-linking agent might limit swelling and hydration of starch granules. hyaluronate is known as a hydrophilic polymer derivative polysaccharide that has ability as an enhancer of percutaneous penetration by changing composition of cells of tightly arranged materials into more tenuous so that permeability is increased. permeability has an inverse relationship with ws so that the addition of soha can decrease ws of bioplastics (djajadisastra et al. 2014). ws is an important property of bioplastic for food prevention application, particularly in high water activity, or when bioplastic must be in contact with water, such as during food processing. generally, high solubility indicates lower water endurance, though high solubility might be advantageous for some applications (chana-thaworn et al. 2011). according to (spec-chem ind. 2014), soha can function as a lubricant and film maker, where soha is a high molecular weight polymer which is a strong lubricant and film maker. soha as cross-linking agent can be observed from its ability to reduce the ws of bioplastic. it was contrary to the results obtained from soha-nce that increased ws due to water formation from reaction between hps, soha and nce, showing the hydrophilic property of bioplastic. water vapor permeability (wvp) wvp is a bioplastic ability to withold penetrating water vapor and provides information on water vapor transmission through bioplastic as a critical property of food packaging (leceta et al. 2013). bioplastic permeability is determined by the difference of water vapor concentration between one-side to the other side of bioplastic, with higher difference indicating faster mass transfer and also affected by bioplastic thickness. the results of wvp analysis showed that the control bioplastic had significant difference compared to bioplastic prepared by using 3% soha, sohance combination and nce. increasing concentration of 3% soha and 20% nce bioplastic was able to decrease wvp by an average of 3.66% and 3.89%, respectively, while (1, 2 and 3%) soha-20% nce combination on the contrary increased wvp by an average of 8.25% (fig. 3). biotropia vol. 29 no. 1, 2022 62 figure 3 effect of soha addition and combination soha-nce on the wvp of hps bioplastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). the results of wvp analysis showed that the control bioplastic had significant difference compared to bioplastic prepared by using 3% soha, soha-nce combination and nce. increasing concentration of 3% soha and 20% nce bioplastic was able to decrease wvp by an average of 3.66% and 3.89%, respectively, while (1, 2 and 3%) soha-20% nce combination on the contrary increased wvp by an average of 8.25%. previous study conducted by gutiérrez et al. (2015) reported a tendency of increasing wvp and ws in bioplastic containing native starch, which indicated higher rate of hydrophilic property. another possibility for increasing wvp in (1, 2 and 3%) soha-20% nce bioplastic was by implementing higher hydrophilic property in the (1, 2 and 3%) soha20% nce bioplastic compared to (1, 2 and 3%) soha and 20% nce bioplastic. water holding capacity of soha is very high compared to other moisturizers. the constanta of soha moisture evaporation rate is lower than that of other moisturizers. this shows that soha has strong water retention properties, with moisture evaporation rate constanta of 8.0 ± 0.1x100-2/min (spec-chem ind. 2014). the main function of bioplastic or edible film is to inhibit vapor transfer from the surrounding environment to the food covered by the bioplastic, or between two different components of food products. therefore, wvp value needs to be as low as possible (chana-thaworn et al. 2011). wvp is also determined by bioplastic thickness as well as by the glycerol and starch concentrations. thickness of hydrophilic film might determine wvp, with higher thickness resulted in higher resistance against mass transfer and increasing partial water pressure in equilibrium of film inner surface, thus enhance water vapor permeability of film hydrophilic property with higher thickness (gutiérrez et al. 2015). the enhancing wvp is caused by the changes of partial water vapor pressure of the exposed inner surface of the film. separate utilization of soha and 20% nce was able to reduce wvp, indicating their function as cross-linking agent due to their ability to inhibit water vapor penetrating the bioplastic. on the other hand, different results occurred in bioplastic made from a combination of (1, 2 and 3%) soha-20% nce due to deteriorated cross-linked bond from water formation which subsequently caused bioplastic became hydrophilic. fourier transform infrared spectroscopy (ftir) analysis the recent development of material technique has resulted in demand increase for fast, reliable and non-destructive analytical methods to control preparation process and physicochemical characterization. ftir provides general information on chemistry of the surface and overall characters as well as their functions in polymer (ricci et al. 2015). ftir spectra of control, (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastic was presented to measure the results of reaction involved in bioplastic preparation made from hps with soha as cross-linking agent, as well as combination of soha-nce (fig. 4). incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 63 figure 4 ftir spectra of control, (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastics differences in several peaks of bioplastic made by using 20% nce compared to (1, 2, and 3%) soha and combination of (1, 2 and 3%) soha-20% nce (fig 4; table 1). zone 1 representing bioplastic made by using soha at 3,285.91-3,288.09/cm; soha-nce at 3,287.46 3,297.68/cm; and nce at 3,282.13/cm showed oh bond vibration, related to bound, free, interand intra-molecular hydroxyl group (bilal et al. 2015). zone 2 representing bioplastic made by using soha at 2,926.41-2,927.94/cm; sohance at 2,927.28-2,927.61/cm; and control at 2,924.49/cm indicated stretching vibration of ketones (ch3-co). zone 3 representing bioplastic made by using soha at 2,161.66 2,162.02/cm and 1% soha-20% nce showed stretching vibration of scn (thyocianates). scn can be derived from cyanide-rich plants, such as cassava, sweet potato, corn, sugar cane, sorghum and linseed (chandler & day 2012). scn presence in (1, 2, and 3%) soha bioplastic was probably derived from corn starch as hps raw material. scn functions as host defense and as cyanide detoxification product. scn is a preferable substrate for lipid peroxidation (lpo), for catalytic reduction caused by hydrogen peroxyde (h2o2) into hypocyanate acid (hoscn) (chandler & day 2012). eighty percent (80%) scn in the body comes from cyanide absorbed from food which transformed into scn in metabolism (simeonova & fishbein 2004). in addition, the presence of scn vibrations may originate from reaction between acetanilide group in soha and sulfate compounds from hps. zone 4 representing (2% and 3%) soha and (1, 2, and 3%) soha-20% nce bioplastic showed oh-p=o bond area, which arguably indicated cross-linking activity through transformation of starch granule by bifunctional or multifunctional reagents which were able to form ether or ester bond with hydroxyl group of starch (gui-jie et al. 2006). in zone 8 (1,411.37 1,558.59/cm) of (1, 2 and 3%) soha-20% nce bioplastic and zone 9 (1,411.571,412.61/cm) of (1, 2 and 3%) soha bioplastic, cyanate (c-n=o) was formed. whereas, in the 20% nce bioplastic at 1,704.17/cm, amide group was present. this indicated that nce combined with (2 and 3%) soha removed scn vibration. besides hydroxyl group and glycerol, amide group from nce and acetanilide from soha also played important roles in determining bioplastic mechanical properties as well as cyanate (c-n=o) formation observed in peak (1,558.46-1,558.59) (fig. 4; table 1). there was a higher presence of c-n=o in (1, 2 and 3%) soha-20% nce compared to that in (1, 2 and 3%) soha bioplastic which probably caused by nce addition, in dosedependent manner with the decreasing cyanide concentration due to oxidation. cyanide can be photocatalytic oxidized into c-n=o, biotropia vol. 29 no. 1, 2022 64 20% nce bioplastics incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 65 particularly in alkali media (destanoğlu & gümüş-yılmaz 2016). the photocatalytic oxidation was shown by increasing the rate of c-n=o formed in (1, 2 and 3%) soha-20% nce bioplastic due to nce addition, which low ph was caused by hydrolyzed tannin dominated by gallic acid. in zone 10, 13 and 14, both soha and soha-nce bioplastics, ether group was formed as an indication of crosslinking process between hydroxyl group of hps and gallic acid or amide from hydrolyzed tannin. moreover, peaks dominated by ether and aromatic groups also found at peaks 15, 16, 18 and 19. utilization of (1, 2 and 3%) soha in hps bioplastic preparation caused scn and c-n=o formation, while 20% nce addition caused increasing c-n=o concentration that arguably determined bioplastic mechanical properties and antimicrobia activity. scanning electron microscopy analysis micrograph resulted from the scanning electron microscopy (sem) analysis was used to study the tensile surface fracture of the eight bioplastic samples and microstructure changes caused by the addition of (1, 2 and 3%) soha and combination (1, 2 and 3%) soha-20% nce. the control bioplastic had homogenous surface microstructure with cracks in several parts. the microcracks formation was possibly caused by the absence of cross-linking agent in bioplastic (ortega-toro et al. 2014). addition of cross-linking agent can be detected by white spots of agglomeration on bioplastic surface made by applying addition of (2 and 3%) soha. the spots appeared as a side effect of uneven mixing or lack of solvent to dissolve crosslinking agent. different to bioplastic microstucture made by using (1, 2 and 3%) soha, those made by using (1, 2 and 3%) soha-20% nce combination had a very significantly different result compared to control, (1, 2 and 3%) soha and 20% nce. addition of 20% nce resulted in aggregated formation on bioplastic surface, hence more flexible compared to other bioplastic samples. the flexibility was as also indicated by higher ε of bioplastic made by using (1, 2 and 3%) soha-20% nce compared to the other bioplastic samples (fig. 5). sem micrograph of the eight bioplastic samples showed significantly different results, with (2 and 3%) soha bioplastic had agglomeration as indication of cross-linking agent addition, whereas (1, 2 and 3%) soha bioplastic had more flexible texture from water formation because the final reaction product rendered hydrophilic property of bioplastic. nce contains organic acid having plasticizing effect that contributes to inhibiting starch partial recrystalization (ortega-toro et al. 2014). control 1%soha 2%soha 3%soha 1%soha 20%nce 2%soha 20%nce 3%soha 20%nce 20%nce figure 5 sem micrographs of the control, (1, 2, and 3%) soha, (1, 2, and 3%) soha-20% nce and 20% nce bioplastics biotropia vol. 29 no. 1, 2022 66 antimicrobial analysis antimicrobial capabilities from control bioplastic was compared to that of (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastics by determining the inhibition zone diameter against gram-positive (s. aureus) and gram-negative (e. coli) bacteria, as well as fungi (a. niger). the results showed that control and 1% soha had no antimicrobial activity, although the ftir spectrum analysis showed that there is a cyanate suspected as an antimicrobial agent. the lack of antimicrobial activity was probably due to the small concentration of the cyanate. the 2 and 3% soha had inhibition zone indicating antibacteria activity against grampositive bacteria (s. aureus) with diameter of 0.6 mm and 0.45 mm, respectively (table 2). the presence of antimicrobial activity was probably caused by the presence of scn and c-n=o in the soha addition during bioplastic preparation, or in the soha addition within the hps which generated scn and c-n=o groups. scn can be generated from catalysis of cyanide (fawell et al. 2007). other study conducted by (destanoğlu & gümüş-yılmaz 2016) mentioned that scn is a chronic exposure of low dose of cyanide. besides, scn is utilized in various industrial process, such as photofinishing, production of herbicide and insecticide, colorant, production of acrylic fiber, thio-urea production, metal separation and coating, as well as soil sterilization and corrosion inhibition (doble & kumar 2005). nce addition to (1, 2 and 3%) soha-20% nce bio-plastic, was able to improve antimicrobial properties against gram-positive (s. aureus) and gram-negative (e. coli) bacteria as well as fungi (a. niger). antimicrobial activity of (1, 2 and 3%) soha20% nce bioplastic was considered better than the control, (1, 2 and 3%) soha and 20% nce, which probably due to the presence of c-n=o that was predictably increased with the increasing nce. the existence of c-n=o is probably derived from acetanilide group found in soha and amide group contained in nce. acetanilide is the first derivative of aniline having analgesic and antipyretic properties (khan et al. 2016). c-n=o concentration can be gradually increased with time, in line with the reduction of cyanide due to oxidation (destanoğlu & gümüş-yılmaz 2016). the analysis results showed that (1, 2 and 3%) soha-20% nce bioplastic was more sensitive against s. aureus than e. coli or a. niger (table 2). this indicated that s. aureus was more sensitive to c-n=o in (1, 2 and 3%) soha-20% nce bioplastic. different results were obtained by 20% nce bioplastic containing amide, toward which e. coli had higher sensitivity than s. aureus, with no antimicrobial activity against a. niger. results of this analysis also indicated that gram-positive bacteria (s. aureus) were more sensitive toward scn or c-n=o in both 3% soha and (1, 2 and 3%) soha-20% nce bioplastics compared to gram-negative bacteria (e. coli) and fungi (a. niger). however, c-n=o compounds provided antimicrobial activity for (1, 2 and 3%) soha-20% nce bioplastic against s. aureus, e. coli and a. niger. on the other hand, amide content in 20% nce showed higher sensitivity against e. coli than s. aureus, with no antimicrobial activity against fungi (a. niger). table 2 inhibition zone obtained in the antimicrobial analysis no sample inhibition zone against e. coli s. aureus a. niger 1 hps-0 0a 0a 0a 2 hps-0-soha-1 0a 0a 0a 3 hps-0-soha-2 0a 0a 0a 4 hps-0-soha-3 0a 0.45 ± 0.26b 0a 5 hps-20-soha-1 3.17 ± 0.29b 3.57 ± 0.55c 0.77 ± 0.12b 6 hps-20-soha-2 2.67 ± 0.06c 3.23 ± 0.31c 0.80 ± 0.00b 7 hps-20-soha-3 2.40 ± 0.17d 2.97 ± 0.42d 0.57 ± 0.06c 8 hps-20 5.07 ± 0.20e 4.32 ± 0.25e 0a incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 67 conclusion addition of (1, 2, and 3%) soha as crosslinking agent and combination of (1, 2, and 3%) soha-20% nce had significant effects on mechanical properties and antimicrobial activity of hps bioplastic. soha at 2 and 3% in hps bioplastic improved mechanical properties in form of σ and y increase while reducing ɛ, ws and wvp. the reduction was caused by the presence of scn and c-n=o which probably derived from acetanilide group found in soha causing antimicrobial activity against grampositive bacteria (s. aureus). on the contrary, combination of (1, 2 and 3%) soha-20% nce degraded mechanical properties by reducing σ and y followed by ɛ, ws and wvp decrease, which probably due to water formation from reactions involving hps, 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hydroxypropylated rice starch based biodegradable films. food hydrocoll 50: 54-64. https://doi.org/ 10.1016/ j.foodhyd.2015.04.010 wypych g. 2017. typical methods of quality control of plasticizers. a handbook of plasticizers (3rd edition). toronto (ca): chemtec publishing. pp.85-109. https://doi.org/10.1016/b978-1895198-97-3.50003-2 xuejun c, xiaobin z, zou, y. 2015. cross-linked sodium hyaluronate gel for tissue filler for plastic surgery and preparation method thereof 1(19): 1-4. united states: 14/432 (a patent). biotropia vol. 30 no. 2, 2023: 128 136 doi: 10.11598/btb.2023.30.2.1654 128 elasticity analysis of the grazing and detrital pathways in a shallow philippine seagrass meadow michael a. clores partido state university – caramoan campus, caramoan, camarines sur, 04429 philippines received 6 september 2021 / revised 27 may 2023 / accepted 31 may 2023 abstract ecotrophic efficiency (ee) is an estimate of the proportion of production that is utilized by the next trophic level through direct predation or fishing or exported out of the ecosystem. in seagrass systems, analysis of ee provides crucial information on how biomass, when used or lost in biological functioning, affects the higher trophic levels via death or grazing relative to the energy lost via decomposition (i.e., flow to the detritus, ftd) and exports to another ecosystem (i.e., sum of all exports, sae). in this study, projections on the effect of change in the ee of functional groups in seagrass systems due to the alteration of biomass were established heuristically using elasticity analysis. using a previously constructed ecopath model for a shallow philippine seagrass meadow, the simulations of altering the biomass of seagrasses and their grazers were done to determine the change in ee, ftd, and sae, thereby generating information on the dynamics of the grazing and detrital pathways in the seagrass ecosystem. results showed the effects of biomass increase and decrease of grazers (herbivorous gastropods, tripneustes gratilla, and polychaetes). if the grazers’ biomass increases, their ee tends to decrease, and biomass accumulation tends to increase. this implies that a fraction of their production used in the system is reduced even if their predators' density and feeding rate are still constant. in addition, the ee of seagrasses tends to increase, leading to a decrease in biomass accumulation at the primary producers’ trophic level. lastly, the ee of detritus decreased because the ftd and sae of its major contributors (the seagrasses) had also decreased. the findings contribute to the ongoing analysis of the role of herbivores versus detritivores in the energetics of seagrass habitats. keywords: biomass elasticity, ecotrophic efficiency, flow to detritus, grazing, seagrass, trophic introduction the two major food chains in any ecosystem, namely, grazing and detrital or decomposer food chains, vary primarily in terms of energy source: autotrophs and living plant materials for the former and the detritus or dead organic matter for the latter. furthermore, the directional flow of net primary production characterizes the grazing pathway (attayde and ripa, 2008). in contrast with the grazing pathway, the detrital pathway has a unidirectional flow that dominates, wherein the recycling of detritus eventually results in inputs at the food chain (smith and smith, 2009). odum (1956) first presented the y-shaped model in which the energy flow in the grazing and detritus food chain are sharply separated. lindeman and lindeman (2007), in a comprehensive trophic and energy flow model of ecosystems, presented an integrated ecological interaction of the grazing and detrital pathways. in seagrasses, the detrital food chain relies on input from waste materials and decaying matter from the grazing food chain. the two food chains are also linked via the process of predation. aside from the breaking down of dead organic matter, decomposer organisms are also food for numerous other animals (hearne et al. 2019; johnson et al. 2019; breithaupt et al. 2019; boncagni et al. 2019; johnson et al. 2020). quantifying the flux of energy through the seagrass ecosystem requires evaluating the consumption, ingestion, assimilation, respiration, and production at each trophic level *corresponding author, email: michael.clores@parsu.edu.ph; mclores@gbox.adnu.edu.ph elasticity analysis of the seagrass model – clores 129 (du et al. 2020). within the food webs in the seagrass system, the trophic level represents seagrasses and their consumers' position. seagrass biomass enters the second trophic level by either secondary production through grazing, recycling biomass by the detrital loops, and feeding on phytoplankton by certain species as early-stage larvae. biomass outputs happen at all trophic levels involving predation and mortalities (blomberg et al. 2014; gloeckner and luczkovich, 2008). following lindeman's (1942), and lindeman and lindeman (2007) tropho-dynamics concept, this study aims to examine the trophic levels of the seagrasses and their consumers and how they are leveraged by the various scenarios of increasing and decreasing biomass. using a previously constructed model by clores and cuesta (2019), such a model was subjected to elasticity analysis by focusing on the seagrasses (producers) and the benthic invertebrates (consumers). the model's scope reflects the total size of the ecosystem, thereby reflecting the properties of the system, particularly the extent of the trophic structure (gascuel 2005; heyman et al. 2014). the modeling outputs are the ecotrophic efficiency (ee) and flow to detritus. the model subjected to elasticity analysis concentrated on the grazing effects on the seagrasses by the invertebrates and almost trifling recreational fisheries. materials and methods the study site is sabitang-laya island located at maqueda channel, caramoan peninsula, philippines (13051’56.22” n, 123057’35.20” e) (fig 1). figure 1 study area in maqueda channel, caramoan peninsula, philippines biotropia vol. 30 no. 2, 2023 130 a trophic model for the seagrass meadow of this island was constructed using ecopath with ecosim (ewe) version 6.6.3 (christensen, waters, and pauly, 2000) and was already reported by clores and cuesta, 2019. the model used an estimate of 22 functional groups' biomass to construct a food web model that described the trophic structure and linkages, and energy flows in the seagrass system (clores and cuesta, 2019) (table 1 and figure 2). ewe required the following: biomass (b) of functional groups, ratios of productivity: biomass (p/b), consumption: biomass (q/b), proportion of unconsumed food, or other collective variables (e.g., gross food conversion efficiency (ge as pb/qb) (christensen et al. 2000). ecotrophic efficiency (ee), estimated with ewe, is the proportion of any of the functional groups’ production assimilated inside the, and is the proportion of production taken by the predators or are transported out from the system (ullah et al. 2012). in this study, the ee was subjected to elasticity analysis. table 1 basic inputs and estimated outputs (bold) of the sabitang-laya is., maqueda channel) seagrass system model group name tl b (t km-2) p/b (yr-1) q/b (yr-1) ee p/q predat mort other mort flow to detritus 1 carnivorous gastropods 3.04 2.35 2.80 5.60 0.96 0.50 2.69 0.12 1.45 2 p. nodosus 3.02 12.89 0.52 2.60 0.31 0.20 0.16 0.36 5.67 3 crustaceans 2.96 1.85 8.40 28.00 0.99 0.30 8.37 0.03 5.21 4 diadema spp. 2.86 0.99 7.51 25.00 0.95 0.30 7.13 0.38 2.67 5 sand dollar 2.60 0.27 10.00 50.00 0.87 0.20 8.86 1.14 1.50 6 nematodes 2.47 2.94 10.00 50.00 0.79 0.20 7.85 2.15 17.86 7 ophiuroids 2.46 0.80 8.63 34.52 0.97 0.25 8.36 0.27 2.87 8 pelecypods 2.26 25.03 2.06 6.86 0.99 0.30 2.04 0.02 17.46 9 polychaetes 2.33 4.20 1.63 12.46 0.54 0.13 0.89 0.74 6.80 10 s. maculata 2.22 2.09 4.45 22.25 0.55 0.20 2.44 2.02 6.76 11 other holothuria 2.22 0.30 4.45 22.25 0.74 0.20 3.30 1.15 0.84 12 zooplankton 2.11 2.87 67.00 192.0 0.68 0.35 45.69 21.31 85.68 13 herbivorous gastropods 2.00 3.11 2.80 5.60 0.90 0.50 2.51 0.29 2.19 14 t. gratilla 2.00 3.77 4.47 25.00 0.33 0.18 1.50 2.98 15.03 15 s. isoetifolium 1.00 178.5 8.43 --0.07 --0.06 8.37 747.1 16 h. uninervis 1.00 262.3 8.43 --0.01 --0.05 8.38 1099 17 h. minor 1.00 10.24 8.43 --0.15 --1.30 7.13 36.51 18 c. serrulata 1.00 1424 8.43 --0.01 --0.03 8.40 5983 19 c. rotundata 1.00 269.6 8.43 --0.01 --0.08 8.35 1125 20 e. acoroides 1.00 5501 8.43 --0.01 --0.00 8.43 23181 21 phytoplankton 1.00 48.00 30.42 --0.38 --11.79 18.63 447.0 22 detritus 1.00 19.80 ----0.01 -------- notes: tl = trophic level; b = biomass (t wet weight (ww) km-2); p/b = production/biomass ratio or the instantaneous rate of total mortality (z) (yr-1); q/b = consumption/biomass ratio or consumption rate (yr-1); ee = ecotrophic efficiency, p/q = production/consumption ratio or gross efficiency; predat = predator; mort = mortality; flow to detritusis in t ww km-2 yr-1. elasticity analysis of the seagrass model – clores 131 elasticity analysis (caswell, 2001) of the ecopath model, a form of sensitivity analysis, was done to determine the effects of biomass alterations of functional groups in seagrass systems. the analysis allowed the simulation of various forms of disturbances by using small proportional changes in the parameter values. elasticity is defined by barbeau and caswell (1999) as: ep (%) 100 = xp – xo xo where: ep (%) = elasticity of the yield to % rise of parameter p xo = yield of the original model xp = yield of the model with a change in parameter p results and discussion the sabitang-laya seagrass model identified three functional groups in the seagrass system to be subjected to elasticity analysis: important grazers, tripneustes gratilla, herbivorous gastropods, and polychaetes. the seagrass system's impacts were explored by conducting an elasticity analysis that entailed altering (i.e., increasing and decreasing) their biomass. the results of the elasticity analysis done on the seagrass model showed that much of the flows to detritus were contributed by the seagrasses (figure 2). but in general, if the biomass of grazers increased, the flow to detritus also decreased, suggesting that much of the seagrass material goes to the grazing pathway. an overall result of elasticity analysis was shown in figure 3. if the density and biomass of grazers: t. gratilla, herbivorous gastropods, and polychaetes increased, their ee decreased. this observation indicates that a fraction of their production used in the system reduces, this is despite that their predators' density and feeding rate are still constant. also, there is increased biomass accumulation at the grazers' trophic level. when grazing is intense, the ee of seagrasses increased because of the high consumption of their production in the system due to grazing. this eventually decreases the biomass accumulation at the primary producers’ trophic level. furthermore, since much of the seagrass materials goes to the grazing pathway, the ftd from them decreased. lastly, the ee of detritus decreased because the flow into the detrital pool of its major contributors (the seagrasses) had also decreased. figure 2 simplified representation of the grazing scenarios effects of biomass increase and decrease of grazers (herbivorous gastropods, tripneustes gratilla and polychaetes) on a shallow philippine seagrass systems biotropia vol. 30 no. 2, 2023 132 projections showed that if the biomass of grazers (polychaetes, herbivorous gastroprods, tripneustes gratilla) increased under steady-state assumptions, their ecotrophic efficiency (ee) decreased, and flow to detritus (ftd) increased. for example, if the biomass of t. gratilla decreased by 99%%, their ee increased by almost 30% while their ‘flow to detritus’ (ftd) increased to about 60 tons/km2/year this finding means that when grazers become abundant in the seagrass system, their biomass accumulates and enters the detrital pool when they die (figures 3). furthermore, if the biomass of grazers (polychaetes, herbivorous gastropods, and t. gratilla) in the seagrass system increased, the ee of seagrasses had relatively increased also, suggesting that a fraction of the production of seagrass was used in the system or passed up the food web (fig 4). however, the ftd from the seagrasses almost remains unchanged, indicating that their biomass goes to the grazing pathway (fig 5). grazers also tend to consume the seagrasses cymodocea serrulata, syringodium isoetifolium, halodule uninervis and c. rotundata, suggesting intense utilization of these seagrasses through grazing. the low ee of e. acoroides, despite the increasing biomass of grazers, suggests that this seagrass group was underexploited or not favored by the grazers; hence their supply exceeded demand, and much of this excess production went to detritus (fig 4). figure 3 projected ecotrophic efficiency (ee) and ‘flow to detritus’ (ftd, ton/km2/year) of the seagrass grazers as their biomass decrease or increase elasticity analysis of the seagrass model – clores 133 figure 4 projected change in the ecotrophic efficiency (ee) of seagrasses as the biomass of their grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) is decreased or increased figure 5 projected change in the flow to the detritus (ftd, %) of seagrasses as the biomass of their grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) is decreased or increased biotropia vol. 30 no. 2, 2023 134 it was also revealed that, as the biomass of grazers increased, the ee of phytoplankton slightly increased, and their ftd remain unchanged. for instance, an increase in the biomass of grazers by 90% resulted in an increase of their ee by 0.20 (figure 6). the ‘sum of all exports’ (sae) decreased as the biomasses of grazers increased. this means that an increase in grazer biomass leads to the consumption of all biomass that otherwise will be exported from the seagrass systems (figure 7). figure 6 projected change in the ecotrophic efficiency (ee) of phytoplankton and detritus as the biomass of seagrass grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) decreased or increased figure 7 projected change in the ‘sum of all exports’ (t/km2/year) and ‘sum of all flows into detritus’ (t/km2/year) if the biomass of grazers is increased in the seagrass system. elasticity analysis of the seagrass model – clores 135 elasticity analysis of the seagrass model in sabitang-laya island, maqueda channel, caramoan peninsula, a shallow seagrass meadow in the philippines, was useful in predicting the impacts on the seagrass systems' energy flows. fluctuations of the ee fractions and the ftd from the seagrasses as their grazers' biomass change was used to make projections. the projections showed that the seagrasses' energy/biomass proceeds to either the grazing or detrital pathways; it is expected that the energy/biomass of seagrasses will proceed to either the grazing or detrital pathways. however, if there are possible disturbances brought by overgrazing or overharvesting of grazers, the result is a restraint of these routes, or rerouting of the energy/biomass in the system. the overall flows in the system remain constant. it is expected that if an increase in the biomass of grazers (e.g., t. gratilla) happens, a large amount of energy/biomass from the seagrasses flows directly into the grazing pathway. this data indicates that grazers have a critical role in consuming seagrass material and biomass disposal due to grazing, which ultimately ends up in detrital food webs. projections also revealed that the seagrass that was discarded or not consumed by grazing might be exported from the seagrass systems. this could be through wave action and water currents. this export of biomass/energy to other ecosystems means that grazing on seagrass systems has an important role in seagrass beds' connectivity with other habitats like coral reefs and mangrove forests. elasticity analysis of the seagrass model in bageing bay, sabitang-laya island showed increasing the biomass of seagrass grazers t. gratilla, polychaetes, and herbivorous gastropods in shallow philippine seagrass meadows could lead to significant overexploitation for resources of low trophic levels. the model became imbalanced as grazers’ biomass decreased, hence the collapse of biomass affecting the functional biodiversity that may have substantial consequences, particularly on ecosystem resilience. in effect, the model simulations provided relevant theoretical bases to explain the distribution of biomass per trophic level and the impact of grazing on biomass distribution. consequently, real observations should be compared with the model results to validate virtual ecological modeled ecosystems. the interactions of the essential components of the shallow philippine seagrass systems, as shown by this seagrass meadow at maqueda channel, caramoan peninsula, and their influence in regulating the ecosystem are manifested by the steady-state models and elaborated by the elasticity analysis done in this study. the links from the seagrasses as producers to the invertebrates as intermediate consumers portrayed by the results contribute to a better understanding of how the ecosystem functions. the results of the elasticity analysis proved to be a practical tool for diagnosing and forecasting the impacts of overgrazing in seagrass systems commonly reported in the literature (burnel et al. 2013; yeager and layman, 2011; fourqurean et al. 2010) and the mechanistic interactions between predators and prey in seagrass meadows (clores and cuesta, 2019; archer, stoner, and layman, 2015; clores, conde and perez, 2020). conclusion the findings of the current study contribute to the ongoing reevaluation of the role of herbivores and detritivores in the energetics of seagrass habitats. clearly, when in steady-state conditions, as shown by the shallow philippine seagrass meadow in this study, seagrass systems significantly contribute their seagrass production to the detrital pool. this result of the elasticity analysis of the seagrass trophic model analysis showed that when grazers become abundant, much of this production proceeds to the herbivory pathway. this research provides robust elasticity models of the dynamics of a seagrass system that offer relevant insights into the seagrass structure's energy dynamics. analysis of this study's data showed that interactions are strong between the functional groups at the lower trophic levels in seagrass systems. in the context of conservation programs for seagrasses, top-down (detrital pathway) and bottom-up (grazing pathway) factors that control seagrass ecosystem structure and function should be considered. for instance, when grazing rates could surpass the seagrass growth rates (i.e., overgrazing), the biotropia vol. 30 no. 2, 2023 136 possible detrimental threats on the seagrassassociated ecosystem services should be mitigated. acknowledgments this research was supported by a research grant from partido state university’s research, extension, and knowledge management office. references archer, s.k., stoner, e.w., layman, c.a. 2015. a complex interaction between a sponge (halichondria melanadocia) and a seagrass (thalassia testudinum) in a subtropical coastal ecosystem. j exp mar bio ecol, 465: 33-40. attayde, j. l., & ripa, j. 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1department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia 2reseacrh group of metabolomics and chemical ecology, universitas indonesia, depok 16424, indonesia 3research group of wildlife biology and sustainable landscape, universitas indonesia, depok 16424, indonesia received 15 june 2022/accepted 24 september 2022 abstract air pollution is a common environmental problem. planting trees can minimize the adverse effects of air pollution. plants can absorb and accumulate air pollutants through stomata. biochemical changes in the leaves will appear as a physiological response of plants to air pollution that can be known by calculating the apti ( air pollution tolerance index) value. this study aimed to analyze the differences in physiological responses of five tree species in taman margasatwa ragunan (tmr) south jakarta and universitas indonesia (ui) depok campus as well as to find out the proper tree species planted in areas with high levels of air pollution. the leaves of five species (hevea brasiliensis, manilkara kauki, artocarpus heterophyllus, ficus septica, and mangifera indica) were used to examine the effect of air pollution. biochemical parameters (relative water content, leaf extract ph, total chlorophyll content, and ascorbic acid content) were observed from each species. the value of each parameter was calculated into the apti equation. h. brasiliensis, f. septica, and m. indica were categorized as moderately tolerant plants, m. kauki were included as intermediate plants, and a. heterophyllus was a sensitive plant to air pollution in both locations. the highest apti values were observed in m. indica in both locations. thus, the recommended species planted in a polluted area was m. indica. keywords: apti, ascorbic acid content, leaf extract ph, relative water content, total chlorophyll content introduction air pollution is one of the environmental problems faced by many countries in the world (shaddick et al. 2020), especially in developing countries (mannucci & franchini 2017). air pollution comes from natural factors and anthropogenic factors (humans) (susanto 2020). the use of motor vehicles is the largest contributor to pollution in the air. the use of low-quality fuel can worsen air quality in the environment (uka et al. 2019). pollutant gases produced include carbon monoxide (co), sulfur dioxide (so2), nitrogen oxides (nox), hydrocarbons, volatile organic compounds (vocs), and particulate matter (pm) (uka et al. 2019). developing countries account for 50 80% of the no2 and co gases in the air from motor vehicles (adeyanju 2018). poor air quality can cause diseases in humans, such as cough, asthma, respiratory diseases, lung cancer, to cardiovascular disease (manisalidis et al. 2020). plants can absorb and accumulate various air pollutants by absorption through stomata on the leaves (uka et al. 2019). based on research by roshintha and mangkoedihardjo (2016) mentioned that samanea saman can absorb co2 of 3,252.1 g/hour, swietenia macrophylla 3,112.43 g/hour followed by bauhinia purpurea 1,331.38 g/hour, alstonia scholaris 1,319.35 g/hour, and ficus benjamina 1,146.51 g/hour. this proves that plants can absorb and accumulate air pollutants, such as co2. plants exposed to air pollutants will show morphological, anatomical, biochemical, and physiological responses (uka et al. 2019). *corresponding author, email: ratnayuniati@sci.ui.ac.id trees physiological responses to air pollution – hamid et al. 255 changes in biochemical content in the leaves, namely rwc (relative water content), leaf extract ph, total chlorophyll content, and ascorbic acid content will occur when pollutant gases enter leaf tissue (uka et al. 2019). this happens in response to plant physiology to air pollution. the level of plants tolerance to air pollution can be determined by calculating the value of the air pollution tolerance index (apti) based on changes in biochemical content that happens in the leaves (zhang et al. 2016). plants having a good tolerance level can play a role in controlling air pollutants in the environment (ogunkunle et al. 2015). taman margasatwa ragunan (tmr) south jakarta and ui depok campus are green areas located in urban areas (taman margasatwa ragunan 2021; universitas indonesia 2021) with a high level of air pollution. however, conditions in both locations are assumed to be different. the intensity of vehicles in the ui depok campus is higher than that in the tmr. hence, the study aimed to: 1) analyze the difference in physiological responses of five tree species in tmr and ui depok campus based on the tolerance index and 2) find out the right tree species to plant in areas with high levels of air pollution. materials and methods study area the study was conducted in november 2021 located at taman margasatwa ragunan (tmr) south jakarta and ui depok campus (intersection area near the rectorate building). sampling points were depicted in figure 1. five tree species observed in this study were hevea brasiliensis, manilkara kauki, artocarpus heterophyllus, ficus septica, and mangifera indica. the coordinate points of the five tree plant species in both locations were recorded based on gps (global positioning system) (table 1). figure 1 location of sampling points in taman margasatwa ragunan (left) and ui depok campus (right) (intersection area near the rectorate building) notes: plants shown are: 1 = h. brasiliensis, 2 = m. kauki, 3 = a. heterophyllus, 4 = f. septica, and 5 = m. indica. table 1 coordinate points of five tree species at tmr and campus ui depok no. species tmr ui 1. hevea brasiliensis 06°18.385' s 106°49.165' e 06°20.923' s 106°49.609' e 2. manilkara kauki 06°18.492' s 106°49.206' e 06°21.998' s 106°49.571' e 3. artocarpus heterophyllus 06°18.602' s 106°49.158' e 06°22.017' s 106°49.628' e 4. ficus septica 06°18.658' s 106°49.142' e 06°21.996' s 106°49.580' e 5. mangifera indica 06°18.699' s 106°49.239' e 06°21.993' s 106°49.567' e biotropia vol. 29 no. 3, 2022 256 environmental parameters soil temperature, relative humidity, and soil ph were measured in november 2021 at both study locations. temperature and relative humidity were measured over the 4 weeks at both study locations starting from the first sampling activity. monthly average rainfall data were obtained from bps kota jakarta selatan (2021) for south jakarta and from the study conducted by said and widayat (2014) for depok area. soil samples were collected from 3 different points at each location. the measurement of soil ph was based on a method described by nadgórska-socha et al. (2017) using a digital ph meter with a ratio of soil and distilled water weight of 1 : 2.5. sampling and preparation leaf samples came from five species, i.e., h. brasiliensis, m. kauki, a. heterophyllus, f. septica, and m. indica. the samples came from a tree with a height of at least 1.5 m (kaur & nagpal 2017) which faced toward sunlight and roads (zhang et al. 2016). samples were collected three times from the same individual. the samples were taken to the laboratory for cleaning, and then weighed according to the required leaf weight for each test. subsequently, the samples were stored in a freezer having temperature of -20 oc until the sample were ready to be tested (zhang et al. 2016). relative water content (rwc) rwc measurement was carried out following the method described by ghafari et al. (2020) with modifications. as much as 5 g of leaves samples were soaked in distilled water for 24 hours. then, the samples were re-weighed and recorded as turgid weight. subsequently, the samples were oven-dried at 50 oc until reaching a constant weight and the weight was recorded as dry weight. the rwc values were expressed in percent and calculated based on the formula: rcw (%) = fw – dw x 100 tw – dw where: fw = fresh weight (g); tw = turgid weight (g); dw = dry weight (g). leaf extract ph the ph measurement of leaf extract was conducted following the method described by kaur and nagpal (2017) with modifications. a total of 5 g of leaves samples were extracted with 50 ml of distilled water. the extract was filtered and measured using a digital ph meter. total chlorophyll content measurement of total chlorophyll content was carried out following the method described by manjunath and reddy (2019). a total of 0.5 g of leaves samples were extracted and homogenized with 10 ml of 80% acetone. the extract was centrifuged at 2,500 rpm for 3 minutes. supernatant volumes were measured and absorbed at wavelengths of 663 nm and 645 nm using uv-visible spectrophotometers. the total chlorophyll content was calculated based on formula as follows (bharti et al. 2018): chlorophyll a (mg/g) = 12.7 (a663) 2.69 (a645) x v x w 1,000 chlorophyll b (mg/g) = 22.9 (a645) 4.68 (a663) x v x w 1,000 total chlorophyll (mg/g) = chlorophyll a + chlorophyll b where: a663 = absorbance at wavelength 663 nm; a645 = absorbance at wavelength 645 nm; v = supernatant volume (ml); w = sample weight (g). ascorbic acid content measurement of ascorbic acid content were carried out following the method described by patel and kumar (2018) using titration. the leaves samples solution was extracted with 100 ml of 4% oxalic acid. the extract was centrifuged at 2,500 rpm for 3 minutes. the sample solution and blanko were titrated using a dye solution until the color turned pink. the ascorbic acid content was calculated based on the formula: ascorbic acid (mg/100 g sample) = 0.5 mg x v2 ml x 100 ml x 100 v1 ml 5 ml w where: w = sample weight (g); v1 = volume of blanko solution; v2 = volume of sample solution. trees physiological responses to air pollution – hamid et al. 257 air pollution tolerance index (apti) the value of each parameter is calculated into an equation described by zhang et al. (2016): apti = a (t + p) + r 10 where: a = ascorbic acid content (mg/100g sample); t = total chlorophyll content (mg/g); p = leaf extract ph; r = relative water content (rwc). the values obtained are categorized based on the category of plant responses described by sahu et al. (2020): a) apti < mean apti – sd : sensitive (s) b) mean apti – sd < apti < mean apti : intermediate (i) c) mean apti < apti < mean apti + sd : moderately tolerant (mt) d) apti > mean apti + sd : tolerant (t) air pollution data collection the daily average of air pollutants (pm2.5, pm10, co, hc, no2, o3, and so2) in both study locations (tmr and ui depok campus) was obtained from the ispunet klhk ver 1.4.5 application. the data were recorded in the morning at 7 am, noon at 12 pm, and in the afternoon at 5 pm. data were collected from early october to the end of november 2021. the established range of ispu (air pollutant standard index) values is as follows: a) good: 1 50, b) medium: 51 100, c) unhealthy: 101 200, d) very unhealthy: 201 300, e) dangerous: > 301 (kementerian lingkungan hidup dan kehutanan republik indonesia 2020). data analysis data were analyzed descriptively. environmental and biochemical parameters as well as apti values were calculated for the average values. the average values of those parameters at the two study locations were then compared. data were presented in the form of tables and bar charts. results and discussion the average air temperature in ui depok campus was relatively higher than that in tmr (table 2). both study locations are green areas that have similar environmental conditions. higher air temperatures can reduce the humidity (utami et al. 2020). our study found out that the average air temperature in tmr was lower with higher relative humidity than that in ui depok campus. within the period of our study, the average rainfall in south jakarta was 235.96 mm per month (bps kota jakarta selatan 2021) and in depok was 278 mm per month (said & widayat 2014). table 2 comparison of environmental parameters in tmr and ui depok campus in november 2021 no. environmental parameters tmr ui depok campus 1. average temperature (c) 30.350.53 30.750.64 2. average relative humidity (%) 57.503.51 55.503.11 3. average soil ph 7.450.77 7.210.88 based on the ispu data, the range of apti value for all types of pollutants was 0.00 80.72 (fig. 2). the lowest index was observed on hc in depok (0.00) and the highest on pm2.5 in jakarta (80.72). both were observed in october 2021. in october 2021, the pm2.5, co, hc, no2, and o3 indices were observed to be higher in jakarta, while so2 was higher in depok. meanwhile, similar indices in both regions were observed in pm10. furthermore, in november 2021, the pm2.5, pm10, no2, o3, and so2 indices were observed higher in depok, while co and hc remained high in jakarta. the high so2 and pm2.5 indices in depok was presumably due to the high level of vehicle traffic on the highway, especially during peak hours. visibility will decrease along with the increased levels of pm2.5 and pm10 in the air (kementerian lingkungan hidup dan kehutanan republik indonesia 2021) because the air is filled with fine dust that can be inhaled by the respiratory system. the main sources of pm2.5 and pm10 pollutants come from combustion activities, such as the use of vehicles, construction activities, up to coal-fired power plants (haryanto et al. 2016). biotropia vol. 29 no. 3, 2022 258 figure 2 average air pollutant index in jakarta and depok in october and november 2021 h. brasiliensis, m. kauki, f. septica, and m. indica are the three tree species which have higher scores of rwc in the ui depok campus than that in tmr (fig. 3a). meanwhile, the rwc value of a. heterophyllus in both locations had the similar lowest rwc values. water plays a role in maintaining the physiological balance of plants under environmental stress. humidity and temperature in the environment affect the rwc values of the leaves. rwc values tend to decrease in plants that are exposed to high temperature and drought environment (rowshanaie et al. 2014; zhang et al. 2016). meanwhile, tolerant plants usually have a high rwc value (bahadoran et al. 2019). rwc values are related to plant tolerance levels to air pollution. the high rwc value of a species indicates that plants have a good tolerance to air pollution (zhang et al. 2016; kaur & nagpal 2017). based on rwc values in our study, m. indica, f. septica, and h. brasiliensis are more tolerant to air pollution. meanwhile, the lowest rwc values were observed in a. heterophyllus at both locations. this is presumably because a. heterophyllus cannot adapt well to an environment that tends to be dry (centre for agriculture and bioscience international 2019). leaf extract ph of the five species in ui depok campus was higher than that in tmr (fig. 3b). leaf extract ph is related to the sensitivity of plants to air pollution (kaur & nagpal 2017). plants exposed to air pollutants, especially so2, tend to produce a large amount of h+ cellular fluid. the h+ will react with so2 to form h2so4 which cause a decrease in leaf extract ph. the high leaf extract ph indicates that the plant can well absorb so2 and nox (zhang et al. 2016). trees having low leaf extract ph values indicate that the trees are more sensitive to air pollution compared to those having high leaf extract ph, which are more tolerant to air pollution (bahadoran et al. 2019; uka et al. 2019). f. septica and h. brasiliensis in our study have high leaf extract ph value compared to other species and are thought to be more tolerant to air pollution. the total chlorophyll content of three species namely h. brasiliensis, f. septica, and m. indica was observed to be higher in tmr than in ui depok campus. on the other hand, m. kauki and a. heterophyllus have higher total chlorophyll in ui depok campus than that in tmr (fig. 3c). chlorophyll is the main component of green coloring in plants (kaur & nagpal 2017). air pollutants entering the leaf tissue through the stomata cause chlorophyll to degrade. so2 in the air affects the total chlorophyll of the leaves. high concentration of so2 cause a decrease in total chlorophyll content (zhang et al. 2016). total chlorophyll in the leaves is also affected by high temperatures, dry environments, salt stress, and light intensity (zhang et al. 2016). other factors, such as plant species and leaf age, also affect total chlorophyll content (kaur & nagpal 2017). the observed higher so2 index value in depok area is suspected to be the cause of the low total chlorophyll content in tree species in ui depok campus. the higher average air temperature, trees position, and high light intensity are also suspected to be the cause of the low total chlorophyll content. meanwhile, m. kauki and a. heterophyllus showed lower total chlorophyll content in tmr, which is attributed to the location of trees that are more exposed to sunlight, thus having high light intensity. october 2021 november 2021 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 trees physiological responses to air pollution – hamid et al. 259 the ascorbic acid content of h. brasiliensis, m. kauki, and a. heterophyllus was higher in tmr. meanwhile, f. septica and m. indica in ui depok campus had a higher ascorbic acid content than that in tmr (fig. 3d). ascorbic acid is an antioxidant that plays an important role in maintaining the stability of cell division and cell membranes while in environmental stress. the ascorbic acid plays an important role in the synthesis of the cell wall. ascorbic acid in plants is related to plants responses to environmental stress such as air pollution, heavy metals, drought, and high temperatures (gallie 2013; zhang et al. 2016). high ascorbic acid content indicates a good tolerance to so2 in the air (uka et al. 2019). plants having high ascorbic acid content are more tolerant to air pollution compared to those having low ascorbic acid content (zhang et al. 2016). m. indica and f. septica in ui depok campus had a higher ascorbic acid content than those in tmr, which indicated that both species had a good tolerance to so2 gas that was observed to be higher in depok. h. brasiliensis in tmr was also observed to have the highest ascorbic acid content compared to other species, which indicated that the three tree species (m. indica, f. septica, and h. brasiliensis) were more tolerant plants. air pollution tolerance index (apti) values illustrate the level of plants tolerance to air pollution. based on our study, the apti values of h. brasiliensis, m. kauki, f. septica, and m. indica in ui depok campus were higher than that in tmr. meanwhile, apti value of a. heterophyllus were higher in tmr compared to that in ui depok campus. based on the categories of plant responses to air pollution described by sahu et al. (2020), each species has the same responses in both locations (table 3). the higher the apti value, the higher the tolerance of plant to air pollution. plants that are sensitive to air pollution tend to have low values of rwc, leaf extract ph, and ascorbic acid content. meanwhile, plants that are more tolerant of air pollution tend to have high values of rwc, leaf extract ph, and ascorbic acid content. there is a link between the leaf extract ph and ascorbic acid content. the high leaf extract ph increases the efficiency of converting hexose sugar into ascorbic acid which leads to an increase in the ascorbic acid content (bakiyaraj & ayyappan 2014). conversely, the low leaf extract ph decreases the efficiency of converting hexose sugar to ascorbic acid, so the ascorbic acid content tends to be low (kaur & nagpal 2017). figure 3 biochemical parameters of five tree species in tmr and ui depok campus notes: a = relative water content; b = leaf extract ph; c = total chlorophyll; and d = ascorbic acid. a c d b biotropia vol. 29 no. 3, 2022 260 table 3 biochemical parameters, apti values, and response categories of five tree species in tmr and ui depok campus species rwc (%) leaf extract ph total chlorophyll (mg/g) ascorbic acid (mg/100g sample) apti response categories tmr ui tmr ui tmr ui tmr ui tmr ui tmr ui hevea brasiliensis 93.450.58 98.130.57 7.040.41 7.100.32 1.380.03 1.290.04 0.290.00 0.100.00 9.590.50 9.890.57 mt mt manilkara kauki 85.111.33 88.612.68 5.800.20 5.840.07 1.000.09 1.070.14 0.170.03 0.100.00 8.620.15 8.930.27 i i artocarpus heterophyllus 72.664.35 72.224.12 6.300.29 6.420.18 1.360.01 1.400.02 0.120.03 0.100.00 7.350.43 7.300.41 s s ficus septica 95.521.10 96.380.38 7.310.00 7.330.01 1.120.04 1.100.07 0.080.03 0.100.00 9.620.12 9.720.04 mt mt mangifera indica 97.510.61 98.390.27 5.860.18 5.920.15 1.130.02 0.970.05 0.230.02 0.260.03 9.910.06 10.020.02 mt mt notes: mean (n = 3); s = sensitive, i = intermediate, mt = moderately tolerant, t = tolerant. plants tolerance levels studied in tmr can be sequenced as follows: m. indica > f. septica > h. brasiliensis > m. kauki > a. heterophyllus. meanwhile, the sequence of tolerance level in ui depok campus is as follows: m. indica > h. brasiliensis > f. septica > m. kauki > a. heterophyllus. in addition, the study results showed that apti values in tmr and ui depok campus had similar values, which was presumably due to the current condition of the covid-19 pandemic. during the covid-19 pandemi, the number of vehicles passing through the ui depok campus area become fewer, so the pollutants produced are also less compared to the conditions before the covid-19 pandemic. the environmental conditions in both locations tend to be the same. plants having low apti values can act as environmental bioindicators, as they are more sensitive to air pollution. meanwhile, plants having higher apti values can be used as bioaccumulators of air pollution because they have a good ability to absorb and accumulate various pollutants scattered in the air through leaf stomata. plants which are tolerant and moderately tolerant to air pollution can be grown in environments having high levels of air pollution, such as in urban areas, areas with heavy traffic, and industrial areas (kaur & nagpal 2017; uka et al. 2019). a. heterophyllus has the lowest apti values in both locations and is categorized as a sensitive plant to air pollution. the plant has a high total chlorophyll content and low leaf extract ph as well as ascorbic acid content. meanwhile, m. indica has the highest apti values in both locations. the total chlorophyll content and leaf extract ph in m. indica is quite low when compared to other species. however, m. indica has a high ascorbic acid content and the highest rwc value compared to other species. therefore, m. indica is the best plant to be grown in areas with high levels of air pollution. other than that, m. indica has a large tree stature, a large and wide canopy, and a large number of leaves (febrianti & sulistyantara 2020). m. indica is also a native plant of indonesia (centre for agriculture and bioscience international 2022) and resistant to strong winds, so it is not easily uprooted (plants for a future 2022). f. septica and h. brasiliensis can be the alternative choices because these two tree species have moderate level of tolerance as m. indica to air pollution. conclusion h. brasiliensis, m. kauki, a. heterophyllus, f. septica, and m. indica had the same responses in taman margasatwa ragunan south jakarta and ui depok campus. h. brasiliensis, f. septica, and m. indica are categorized as moderately tolerant, m. kauki includes intermediate plant, and a. heterophyllus is a sensitive plant to air pollution based on air pollution tolerant index (apti) values. based on apti values and physical characteristics of the tree, the right tree species to plant in an area with high levels of air pollution 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2016. pollution resistance assessment of existing landscape plants on beijing streests based on air pollution tolerance index method. ecotoxicol environ saf 132: 212-23. microsoft word 40 biotropia no. 22, 2004: 40 58 medicinal herbs of pasir mayang, jambi: ethnopharmacy and toxicity screening hilman affandi*, arif nuryadin* and susilo b. prayogo* *seameo biotrop, jl. raya tajur km. 6 bogor, indonesia abstract this article presents the results of an investigation concerning the use of herbal medicinal plants by the people of pasir mayang, sub-district (municipality) of vii koto, district of tebo, jambi province, sumatera. the data collection was based on interviews with the healers and other villagers of pasir mayang who possess knowledge of the different plants and their medicinal uses. the study recorded 57 species of medicinal plants used in pasir mayang. the detailed uses of the 57 medicinal plants are given. all plant species were subjected to phytochemical analysis and toxicity tests, and the outcome of the analysis on the presence of alkaloids, saponins, steroids/terpenoids, and the level of toxicity against brine shrimp (anemia salina) are presented. a comparison with other studies reported in the literature seems to indicate that a high frequency of the use of leaves in therapy may be a part of a larger cultural phenomenon among the tropical forest tribes of southeast asia. keywords : indonesia/jarabi/pasir mayang/medicinal plants/ethuophannacy/toxicity screening introduction pasir mayang is the name of an area in the district of tebo in the province of jambi. in the beginning, the name of pasir mayang was dusun tuo. however, since the establishment of pt industries et forest asiatiques (ifa), it has used the name pasir mayang. in line with the increasing population and then later, with the influx of settlers from neighboring communities, pasir mayang developed to become several villages which were administratively included in the sub-district of vii koto. pasir mayang can be reached by road from the district of muara bungo, traversing about 20 km, then turning right at the simpang sawmill for about another 20 km on the way to the transmigration area of rimbo bujang to the village of dusun tuo. from there, the sub-district vii koto can be reached by continuing 8 km more. the access to the village of sungai karang can be reached from dusun tuo through unpaved roads traversing 3 km to batanghari river, then crossing the batanghari river by boat and taking another 15 km more of unpaved roads to the village of sungai karang. the sub-district vii koto covers an area of 112,700 ha bordering the province of west sumatera in the northern side. the southern part of the area is bounded by the sub-district of tebo ulu, and the western side by the district of bungo. meanwhile, the eastern side is bounded by the province of riau. 40 biotropia no. 22, 2004 the population of the sub-district vii koto, based on a census conducted by the office of subdistrict vii koto on april 2001, is composed of 19,249 individuals which consisted of 9,595 men, and 9,654 women, comprising over 11 villages. forest is no longer a source of income for the people of pasir mayang. the forest in the area has been used for collecting a wide range of forest fruits and medicinal plants, not to mention building materials and firewood. occasionally, people collect rattan from the forest, but even this is hard to find, because lots of rattan had been collected for a long time for commercial purposes. while the people of pasii mayang are not "primitive" forest-dwellers, they have retained a relatively simple agricultural way of life. therefore, agriculture is the main occupation of the people of pasir mayang. traditional farming (ladang) usually takes place far away from the villages and this usually involves one-quarter to one-half hectare of forest which are cleared and burned and planted with a wide variety of crops. most of their harvest, however, are adequate only to supply their own needs. the cultivation of "pohon karet" or rubber tree (hevea brasiliensis) is important in pasir mayang as one of the main sources of income in this otherwise moneyless society. rubber is planted in the forest close to the villages. at certain ages of the rubber trees, farmers tap and collect the latex, and sell them to the collectors in the district of vii koto or tebo. they also plant coffee in the forest for their own use or sell to the market. the health center, more widely known as puskesmas, is located in the sub-district of vii koto, headed by a medical doctor, assisted by another medical doctor, one dentist and eight paramedical personnel. health services are provided free of charge for those who could provide proof that they are not able to pay. a bidan (nurse) is available in each village. nurses are trained to diagnose basic illnesses and diseases and can provide a number of common medicines. these common generic medicines which are subsidized by the government with generic names, are supplied every three months from the health center in the sub-district of vii koto and are given free of charge. pembantu puskesmas (health sub-centers) exist in each village in the sub-district of vii koto, headed by a nurse and some paramedical personnel. the most prevalent illnesses are diarrhea, cholera and malaria. meanwhile, other communicable diseases include leprosy, framboesia, tetanus neonatorum, diphtheria, whooping cough, and filiariasis. no smallpox cases have been registered. due to the proximity of the area, and also on account of scarcity of medicine, doctors could not conduct regular visits to villages, which are far from the sub-district of vii koto. only when an epidemic occurs or the situation becomes really threatening medical assistance will be sent. the distance between the district and the health centers, as well as the high expenses which travel incurs are the main obstacles preventing the population from making use of the health facilities provided by the government for them. people generally accept the modern medicines that provide wide therapeutic measures for illnesses and diseases that people suffer from, except for mental illness. 41 medicinal herbs of pasir mayang, jambi hilman affandi et al. for this group of diseases, people will then ask for help directly from the traditional healers. in much the same way, if health problems persist and cannot be solved by the health center, they will then turn to traditional healers who combine herbal remedies with mystical ceremonies which, according to those interviewed, counter the influence of what was generally believed to be "evil spirits" which are responsible for the illnesses, diseases, or even accidents the citizens experience. almost all cultures have a long history of folk or traditional medicine, which includes the use of medicinal plants or herbs prescribed by local folk or traditional doctors in the neighborhood. even in ancient cultures, people methodically and systematically collected information on herbs and developed well-defined herbal pharmacopoeias. this must have also occurred to the ancient people of pasir mayang, for there are still quite a number of traditional healers who do their ritual practices in the communities. this study provides an outline of the contemporary knowledge on the use of herbal medicinal plants by the community around pasir mayang with the purpose of preserving this aspect of traditional knowledge for the generations to come. specifically, this research aims to make an inventory of herbal medicinal plants available in pasir mayang and how these are used by the community residents. it further aims to describe the herbal plants used for medicinal treatment by the indigenous people paying particular attention to the methods of preparation prior to its use, identification of the specific illness or disease that can be treated by each plant, and the effect of the treatment. phytochemical analysis and toxicity tests had also been conducted on the medicinal plants collected in the area, to determine the toxic level of the methanolic plant extracts against brine shrimp (artemia salina) and to trace the presence of specific chemical substances such as alkaloids, steroids, saponins, and flavonoids. methodology data collection of pasir mayang medicinal plants information on the traditional medicinal plants and herbs in pasir mayang was collected by a team of four workers in a duration of two months in the year 2003, in april and october. information was obtained for the series of one-hectare plots. in each case, one-strip transects (25 x 100m) were made within each plot marked out by coloured plastic ropes at both ends and in the centre. inputs were collected in the presence of local healers or other villagers by walking along the centre line. the villagers were asked to identify the local names and uses of each plant occurring within the series of transects. for every plant with indication of traditional use(s), every information was recorded and herbarium specimens of each species were collected by pressing between newsprints, stacked and placed in heavy plastic bags, treated with denatured alcohol and packed, opened only upon arrival at the seameo biotrop laboratory in bogor. small amounts of plant materials from 42 biotropia no. 22, 2004 each plant were taken to the seameo biotrop natural products laboratory for phytochemical analysis carried out at a later date. toxicity test (brine shrimp lethality bioassay) twenty grams of air-dried plant materials from each species were extracted with methanol at room temperature. the crude extracts were evaporated under reduced pressure until dried. sea water was poured into a small tank and shrimp eggs were added to the tank provided with aerator. after two days the shrimp eggs hatched and mature as naupili. each crude extract was weighed (20 mg) and dissolved with dichloromethane (2 ml). from this solution, 500, 50, 5ul were transferred to small petri dish corresponding to 1000, 100, 10 (j.g/ml (ppm), respectively (meyer et al. 1982). the solvent was evaporated by standing overnight at ambient temperature. after two days, when the shrimp larvae were ready, sea water was added to each petri dish containing 10 shrimps per dish (30 shrimps per dilution), and adjusted the volume with sea water to 5 ml/dish. the number of shrimps that survived were counted and recorded after 24 hours. the data were analyzed with finney computer program to determine lc50 values at 95% confidence intervals (hostettmann 1991). each crude extract of the sample was tested in three replicates with one control each. the controls were treated in a similar manner without the presence of the crude extracts. phytochemical analysis alkaloid testing was conducted according to procedures described by culvenor and fitzgerald (1963) : two to four leaves, fruits and/or bark of each plant of the collected species were preserved in ethanol, after being dried in the laboratory, grounded with clean sands and added with 10 ml chloroform. ammoniacal chloroform (10 ml, 0.05 m) was added, stirred and filtered, then shaken with aqueous sulphuric acid (10 drops, 2m). then the aqueous layer was tested with mayer's reagent. a dense, heavy precipitate was designated as 3+, strong precipate as 2+ and weak precipate as 1+. terpenoids/steroids and saponins were screened on the basis of liebermann-burchard and froth test, respectively, as generally described by simes et al. (1959). an amount of 2-4 g dry leaves were cut into small pieces and boiled in ethanol (25 ml, 15 min.), filtered while still hot and evaporated to dryness. the extract was triturated with ether and the insoluble ether shaken with water (ca. 5 ml) in a vi x 5-inch test tube. a foam more than 3 cm and lasting for more than 15 minutes was designated as 3+, between 2-3 cm was designated as 2+ and foam of 1-2 cm was designated as 1+. the ether soluble fraction was subjected to the liebermann-burchard test using acetic anhydride and sulphuric acid. the formation of a bright purple, red or pink coloration was considered 3+ while moderate and weak colora 43 medicinal herbs of pasir mayang, jambi hilman affandi et at. tion were designated 2+ and 1+, respectively. in the case of doubtful coloration produced, the ethereal fraction was passed through a pasteur pipette filled with active charcoal just enough to absorb chlorophyll and treated as above. results and discussion a total of 57 herbs were collected and noted according to their uses for various purposes by the healers and villagers of pasir mayang. of these, the medicinal uses which were presumed magical or superstitious in nature were considered of no interest for the present study and have been omitted for further discussions. many of these comprise common weedy species and a few cultivated ones, all of which were easily available, in those areas where this study was conducted, as well as in most other villages in pasir mayang (affandi et al. 1996). many of the plants, however, were of forest origin, growing in the secondary and little-disturbed forests in the surrounding areas. in the list, species were arranged alphabetically by family/followed by common name / plant part(s) used / and a description of their uses. these were summarized in appendix 1. medicinal uses the process to cure common maladies and illnesses among the people of pasir mayang involves accessing both traditional and modern methods. generally, the people of pasir mayang rely mostly on traditional medicines to treat minor ailments or are used as first aid towards a cure. they seek " modern " medicine only when the traditional methods have been exhausted. the most frequently utilized plant part is the leaf (table 1). this preference for leaves is apparently derived from the fact that therein, many species store high concentrations of bioactive compounds (moore 1994). in addition to efficacy, other factors that may contribute to the preference for leaves as medicine include the ease with which they may be collected, stored, and transported and the ease with which bioactive compounds may be extracted. the most frequently cited modes of preparation (table 2) were: poultice (mashed, crushed, or chopped plant part), decoctions (boiling of plant parts), and used directly from the plants (none). the most frequently encountered modes of administration are oral and topical (table 3) which may be preferred because they are believed to be the most effective means for delivering bioactive compounds into the body. 44 phytochemical analysis and toxicity tests the phytochemical analysis is a preliminary test to trace the presence of specific chemical substances such as alkaloids, steroids/triterpenes, and saponins without resulting to a biological screening. a total of 57 species from 30 families were phytochemically tested in the laboratory of natural products at seameo biotrop. the results of the tests are summarized in table 4. 45     biotropia no. 22, 2002 medicinal herbs of pasir mayang, jambi hilman affandi et al.  alkaloids  of the 57 plant species, three species gave strong positive reaction (3+ 4+) to alkaloid. those species were fibraurea sp , zizyphus sp., and uncaria sp. (table 5).        saponins  of the 57 species tested for positive reaction to saponins, 12 species gave a strong positive reaction (3+ 4+) (table 6). saponin is particularly prevalent in bridelia glauca, b. minutiflora, macaranga triloba, callophylum mucumense, litsea aurea, barringtonia racemosa, magnolia mackottii, pternandra coerulescens, ficus variegata, eugenia sp., arenga pinnata, calamus sp., miletia sp., mucuna gigantea, xanthophyllum sp., uncaria sp., euphoria malaiensis, payena acuminata, lygodium circinatum, trema orientalis, and lantana camara.   steroids/triterpenes almost all plant species collected indicated positive response to steroids/ terpenes. nine plant species showed strong reaction with 3+ or 4+ rating (table 7). le compounds are particularly prevalent in mallotus paninlatus; clochidion •borescens; litsea monopetala; dictyopteris irregularis; selaginella hieron.           toxicity tests brine shrimp (artemia salind) lethality test is a simple, fast and inexpensive oassay in the search for bioactive compounds from plant extracts. the value of ore than 1000 ppm is considered non-lethal and hence shown as negative (-ve). a tal of more than 57 samples were tested and only ten showed toxicity against brine rimps. of the ten plant species tested, three species (dictiopteris irregularis pr., intana camara linn, and gleichenia imearis clarke) indicated some potential xicity which will require further investigation (table 8).   medicinal herbs of pasir mayang, jambi hilmau affandi et al. references affandi, h., nuryadin, a., susilo.b.p and supriatno. 1996. phytochemical survey of the forest tree species of pasir mayang, jambi, sumatera, seamed biotrop, research report. culvenor, c.c.j. arid j.s. fitzgerald. 1963. a field method for alkaloid screening of plants. j. pharm. sci. 52:303304. hostettmann, k., ed. 1991. method in plant biochemistry, vol 6 : assays for bioactivity, pergamon press, new york. meyerb.n., n.r. ferrigni, j.e. putman, l.b.jacobsen, d.e. nichol and j.l. mclaughli. 1982 brine shrimp: a convenient general bioassay for active plant constituents. planta medica, 45:31-34 moore, p.o. 1994. 'trials in bad taste." nature 370:410-411. simes, j.j.h., j.g. tracey, lj. webb and dj. dunstan. 1959. australian phytochemical survey ih, saponins in eastern australian flowering plants. bulletin no. 281 c.s.i.r.o., australia, melbourne. 52             40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf biotropia vol. 30 no. 2, 2023: 171 182 doi: 10.11598/btb.2023.30.2.1780 171 spatial distribution of invasive plants in bandung, west java, indonesia rahmawati and dian rosleine* department of biology, school of life sciences and technology, institut teknologi bandung, bandung, west java, 40132, indonesia received 15 july 2022/ revised 10 march 2023 /accepted 10 march 2023 abstract the urban area is a source of invasive plants that enter through human activities such as agriculture and land-use conversion. studying the invasive plant in urban areas is essential to understanding the city’s ecosystem health condition. therefore, this study aims to inventory invasive plants, map their distribution, and explain the relationship between land use with the community diversity and species richness of invasive plants in bandung. the vegetation analysis was performed using line-transect in 22 study sites distributed using a systematic random sampling method in bandung to observe the plant species composition. the study plots were placed based on the land-use type. the species name, individual number, frequency, and sampling site locations were noted and analyzed to calculate the important value index (ivi) and the invasive species distribution pattern using the principal component analysis (pca). the dominant invasive species was spatially mapped. six types of land use were used in this study, i.e., settlements, street green lanes, gardens, paddy fields, urban parks, and urban forests. there were 187 species found in bandung, which can be categorized into alien invasive species (39%), invasive native plants (25%), non-invasive alien species (18%), non-invasive native species (15%), and unidentified plants (3%). the most common invasive plants found were eleusine indica (ivi=10.50%), trimezia martinicensis (ivi=7.22%), and cyperus rotundus (ivi=6.74%). based on the plant community similarity index, the study area with the highest similarities were paddy fields with gardens (50.5%), settlements with road lanes (44.4%), urban parks with road lanes (26.2%), and urban forests with road lane (17.5%). pca showed swietenia macrophylla as the most common invasive plant found in urban forests, urban parks, and road lanes, with air humidity as the most influencing environmental factor. trimezia martinicensis is the most common species in the settlement area affected by high air humidity. bidens pilosa is an invasive plant commonly found on paddy fields, gardens, settlements, road lanes, and urban park edges. this species can easily and rapidly reproduce with a high survival rate. the many invasive plants found in bandung must be managed to maintain the urban ecosystem’s health. keywords: bandung, interpolation, invasive species, species mapping, urban area introduction alien species are brought or accidentally brought into an ecosystem unnaturally. invasive species are native or alien species that can widely impact their habitat, causing environmental damage, economic loss, or harm to humans (tjitrosoedirdjo 2017). dominating their habitat is the main characteristic of invasive species. they can cause a decrease in biodiversity through the loss of native species and disturbance in the ecosystem functioning (sunaryo 2015). urban areas create multiple habitats that accommodate plant species diversity, and invasive species can often develop in such habitats (štajerová et al. 2017). mainly, anthropogenic disturbances introduce invasive species into the new habitat, such as the landuse change to establish agricultural areas of paddy fields and gardens or newly built settlements. settlement areas can be a focal point of the species’ invasive movement from spreading to the surrounding landscape (chytrý et al. 2005). the diverse land use in urban areas caused the difference in the invasive species composition in each land-use type. bandung is one of indonesia’s major cities with vastly developed and diverse land use. so far, studies *corresponding author, email: drosleine@gmail.com biotropia vol. 30 no. 2, 2023 172 on invasive species have mainly been done in conservation areas with limited knowledge in urban areas. many alien plant species were introduced to urban areas to provide, augment or restore specific ecosystem services. however, some species negatively impact existing ecosystem services and create novel ecosystem disservices within urban areas (potgieter et al. 2017). for example, the alien invasive acacia mangium found in the highway green lanes and urban parks was first introduced as street tree shade. besides, this plant was also planted on eroded soil to repair the soil structure due to its robust, extensive rooting system (environmental management agency 2014). on the other hand, a. mangium can change the soil composition through nitrogen fixation, competing on water and light resources with surrounding native species due to its deep rooting system and dense shade, producing allelopathic substances that can hamper the germination of surrounding plant seeds (datiles & rodriguez 2017). a study of invasive species in urban areas must be carried out to manage invasive species to reduce the negative impact on ecosystem services and prevent their spread to the natural areas. therefore, this study aims to inventory invasive species and map their distribution. data gathered (invasive species number and composition on each land use type, relations between invasive species with the land use, and distribution map of the ten most dominant invasive species in bandung) can serve as early detection of the invasiveness of each species. it can also provide information for policymakers to determine further steps. materials and methods study area this study was done in 22 sampling sites (figure 1) placed with systematic random sampling in bandung (6°50′20″-6°58′3″ sl, 107°32′44″-107°44′15″ el). the sampling sites were determined by 22 coordinates systematically set in the thematic map of bandung on arcgis 10.4.1. these selected coordinates were then exported from arcgis to the gps essential application for the location survey. monitoring plots were randomly assigned based on the land use in those coordinate points. figure 1 location of the study site spatial distribution of invasive plants in bandung – rahmawati and rosleine 173 land-use paddy fields were found in six areas, i.e., sukaati, buahbatu, rancasari, mekarmulya, sukapura, and kopo. gardens were found in five sampling areas, i.e., isola, cigondewah, nagrog, cisurupan, and jatihandap. the street green lanes were found at five points, i.e., cigondewah, mekar mulya, cisaranten wetan, kiaracondong, and pahlawan. there were nine points of settlements, i.e., geger kalong, babakan jeruk, rancasari, buahbatu, sukaati, panyileukan, dago, antapani tengah, and pahlawan. last, urban parks were found at tegalega and bandung wetan. meanwhile, the urban forest was only found in the area point of babakan siliwangi. vegetation survey the vegetation survey was done from april 2021 to january 2022. depending on the environmental situation, a twenty-eight-line transect was installed for 100 or 150 meters. the plant species’ name with their life form (i.e., tree, shrub, liana, or herb) found in the line transect area and their number was written in the monitoring data sheet. the plants were directly identified in the field. unidentified plants were sampled from the field to be further identified using the identification book main weeds of rice in asia. identified plants were then grouped into four types, i.e., invasive alien species, invasive native, non-invasive alien, and non-invasive native, based on the guidebook provided by biotrop, convention of biological diversity (cbd), center for agriculture biosciences international (cabi) and guide book to invasive species in indonesia, forest in southeast asia-indonesia program (forisindonesia). measurement of environmental climatic condition environmental and climatic conditions were measured to support the making of invasive species distribution prediction maps. measured data were temperature and air humidity using a thermo hygrometer. light intensity was also measured using a light meter. measurements were done triplicate on each transect at the transect’s start, middle, and endpoint, 50 to 100 cm above the ground. calculation of importance value index (ivi), diversity and habitat similarity the importance value index (ivi) was calculated for each species by summing up the relative density (1) and relative frequency (2) as follows: density (d) = species individual number length of line transect relative density (rd) = d of species x 100% (1) total d of all species found frequency (f) = number of plots where species was found total plot number relative frequency (rf) = f of species x 100% (2) total f of all species found the ivi was calculated twice. the first was done for each land-use type for community analysis, where each land-use type was assumed to represent a specific vegetation community, totaling six vegetation communities. the second calculation was the ivi of the overall study sites. the calculation of shannon wiener diversity index h’ (3) on each community was done with the following formula, h’ = -∑ pi ln (pi ) (3) with pi = proportion of species’ individual number to overall species. the sorensen similarity index (4) was calculated for every combination pair of land use to find the most similar vegetation communities. the calculation of the sorensen similarity index used the following formula, s = 2c x 100% (4) a+b with a = species number in community a b = species number in community b c = species number in communities a and b statistical analysis density values from 20 plant species with high ivi scores (>2.06%), average temperature, average air humidity, and average light intensity in each community were analyzed with pca. pca is used to extract meaningful information from a multivariate data table and to express this biotropia vol. 30 no. 2, 2023 174 information as a set of a few new variables called principal components. these new variables correspond to a linear combination of the originals (kassambra 2017). the number of principal components is less than or equal to the number of original variables. using r studio version 4.2.0, package factominer, only 15 plant species with the highest contribution of the principal component were shown on the pca plot. graphs from the analysis were visualized using the package factoextra. pca analysis was used to choose ten invasive species in making the invasive species distribution prediction map. invasive species distribution mapping ten species with the highest score were mapped for their distribution in the bandung area. the score indicates that those species highly influence study sites (kassambara 2017). the plant distribution map in bandung was based on the data of the individual number of each species and the mean climatic factor measured on each coordinate point (study sites). climatic data were adjusted from the pca results, where only one to two climatic data influence that species. the distribution map was made using the interpolation method on the application arcgis 10.4.1. spatial interpolation is a procedure for estimating the variable value in the field that is not included in study samples and located inside an area of the sampling location (aswant 2016). results and discussion vegetation composition in bandung the total number of species found in all study sites was 187, comprising 47 tree species, 21 shrub species, 3 liana species, and 116 herb species. invasive species, whether alien or native, have a higher proportion than noninvasive species, with a total ratio of 64% (figure 2). the presence of invasive species is supported by disturbance and human activities. these two phenomena are pervasive in urban areas. thus, the presence and frequency of invasive species are relatively high in urban areas (gulezian & nyberg 2010). the proportion of alien invasive species found was higher than the native ones. most alien invasive species were initially brought to the urban areas to provide, add, or recover a specific ecosystem service. however, besides giving ecosystem services needed by humans, the alien invasive species may negatively impact the available ecosystem services (potgieter et al. 2017). figure 2 the proportion of invasive and non-invasive species in bandung spatial distribution of invasive plants in bandung – rahmawati and rosleine 175 table 1 ten species with the highest importance value index (ivi) on all study sites nr species name native family ivi (%) 1 axonopus compressus tropical america poaceae 11.46 2 eleusine indica india poaceae 10.50 3 asystasia gangetica india, ceylon acanthaceae 7.87 4 alternanthera philoxeroides tropical america asteraceae 7.46 5 trimezia martinicensis mexico iridaceae 7.22 6 cyperus rotundus india, africa cyperaceae 6.74 7 bidens pilosa south africa asteraceae 5.42 8 cynodon dactylon africa poaceae 5.12 9 swietenia macrophylla south america, central america meliaceae 4.36 10 synedrella nodiflora south america, central america asteraceae 4.23 the calculation of ivi in table 1 shows that ten plants with the highest ivi were invasive species. invasive species tend to have a high ivi due to their characteristics of the high tolerance range, enabling them to adapt well to various environments. axonopus compressus is the most abundant species in street green lanes, settlements, and urban parks. annual plant a. compressus can grow vegetatively well with stolon and produce many seeds. e. indica was abundant in paddy fields and found in gardens, street green lanes, and settlements. e. indica can grow well in high light intensity and has a high adaptation level (setyawati et al. 2015). meanwhile, a. gangetica was abundant in gardens and found in paddy fields, streets, and urban forests. a. gangetica is a climber plant that can form a highly dense population (sandoval & rodriguez 2012). vegetation composition in six communities ivi calculation in each community (table 2) shows three species with the highest value in communities’ settlements, street green lanes, and urban parks, i.e., a. compressus, rivina humilis, and syngonium podophyllum. the three species are dominant and co-dominant in the urban forest community. dominant species are defined as species with a higher ability to utilize their environment more efficiently than other species (smith 1977). invasive species tend to have a high ivi due to their wide tolerance range. thus, they can adapt very well to various environments. table 2 three species with the highest importance value index (ivi) were found on six land-use types/communities land-use species name family ivi (%) paddy fields eleusine indica poaceae 29.66 cynodon dactylon poaceae 17.22 bidens pilosa asteraceae 13.90 gardens asystasia gangetica acanthaceae 19.71 alternanthera philoxeroides amaranthaceae 14.94 galinsoga parviflora asteraceae 12.76 street green lanes axonopus compressus poaceae 32.63 trimezia martinicensis iridaceae 25.49 swietenia macrophylla meliaceae 11.57 settlements axonopus compressus poaceae 15.20 trimezia martinicensis iridaceae 9.03 arachis pintoi fabaceae 8.55 urban parks axonopus compressus poaceae 37.56 arachis pintoi fabaceae 18.47 hymenocalis speciosa amaryllidaceae 14.51 urban forests rivina humilis phytolaccaceae 29.54 syngonium podophyllum araceae 27.37 xanthosoma violaceum araceae 13.89 biotropia vol. 30 no. 2, 2023 176 table 3 mean climatic factors measured in six communities land-use light intensity (lux) temperature (°c) air humidity (%) urban forests 191 23 90 settlements 17 074 30 84 gardens 58 037 30 78 paddy fields 54 687 30 79 street green lanes 7 030 29 78 urban parks 2 030 27 85 dominating species in the paddy field community were mainly plants from the family poaceae, with leaf characteristics resembling ribbon and fibrous root and reproducing vegetatively with stolon. grasses tend to grow optimally in areas exposed to sunlight. the paddy field community has a relatively high light intensity (table 3), allowing this plant group to grow optimally and reproduce well or widely spread. the garden community was dominated by three invasive species, i.e., a. gangetica, a. philoxeroides, and g. parviflora. the habitat suitability of invasive species developing in the garden community is affected by several factors, e.g., species commodity planted and spatial variations such as microclimate, soil character, and human presence (wang & wan 2020). five garden communities in this study comprised two cauliflower gardens, a bok choy garden, and two mixed cassava and sweet potato gardens. various commodities planted in those five study sites also influenced the invasive species found. r. humilis and s. podophyllum were two invasive species dominating the urban forest community. r. humilis is a tropical plant commonly found in forests, scrubs, street edges, and disturbed sites at a wide range of altitudes from 0 to 1700 masl. this species proliferates, mainly under shade. due to those characteristics, this species can significantly change its habitat and harm the native vegetation (parker 2013). meanwhile, s. podophyllum is a climber that can rapidly grow, invading the forest’s floor into canopies, covering huge trees to understory vegetation underneath (pacific islands ecosystems at risk 2012). species a. compressus dominated the settlements, street green lanes, and urban parks, while t. martinicensis was a co-dominant species in the street lanes and settlements. the third dominant plant in the green street lanes community was s. macrophylla. the ornamental plant a. compressus is mainly used in domestic gardens and parks. this species adapts to humid and warm environments and is also adequately tolerant to shade, even though it can grow well in areas exposed to sunlight (cabi 2019). climatic conditions in settlements, urban parks, and street lanes support the optimal growth of a. compressus. light intensity in settlements, urban parks, and street lanes was lower than in paddy fields and gardens. even though the settlement temperature was the same as those in the garden and paddy fields, the settlement had higher humidity. t. martinicensis is also intentionally introduced as an ornamental plant. almost 40% of invasive plants in the united states were initially introduced as ornamental plants (lehan et al. 2013). ornamental plants can quickly grow and resist pests and pathogens (guo et al. 2019). these characteristics support most ornamental plants developing into invasive species. s. macrophylla is a fast-growing tree with a high tolerance to low light intensity. a study by norghauer et al. (2011) suggests that the abundance of s. macrophylla is negatively correlated to the abundance of plants under their shade. when s. macrophylla grows as the landscape canopy, a dense shade forms, limiting sunlight penetration to understory plants under its shade. therefore, the germination of understory plants can be disturbed, and the mature individual does not grow optimally. the highest invasive species richness (table 4) was found in the community of settlements, followed by gardens, paddy fields, street green lanes, and urban parks. decker et al. (2012) also report a similar result, mentioning that the richness of invasive species positively correlates to the percentage of public area use, human population, and agricultural area use. the settlement vegetation community has the highest diversity and richness of invasive and spatial distribution of invasive plants in bandung – rahmawati and rosleine 177 table 4 the shannon wiener diversity index (h’) calculated in six communities land-use h’ invasive species richness non-invasive species richness settlements 3.71 60 55 street green lanes 2.6 39 17 gardens 3.03 45 3 paddy fields 2.74 39 4 urban parks 2.4 18 10 urban forests 2.31 10 13 non-invasive species. the presence of humans is the most influencing factor in invasive species introduction. the settlement area has the highest human population of the other five land uses. factors influencing the high diversity and abundance of invasive species in settlement areas are human population, trading activity, nutrition source, warm and protected microclimate, and the potential of herbivore or competitor absence (francis & chadwick 2015). the vegetation community in settlements, street green lanes, and urban parks, including urban and suburban areas, had more invasive species than non-invasive ones (table 4). the settlementsgreen street lanes and urban parksstreet green lanes communities had a high similarity of vegetation composition (table 5). human-made ecosystems have often supported the establishment and development of alien species (hulme, 2003). besides the high similarity of vegetation composition, those four communities mentioned (i.e., settlements, urban parks and forests, and green street lanes) also belong to the same cluster in the pca results (figure 3). these four communities are located in quadrant 1 with a. compressus, t. martinicensis, s. macrophylla, and mangifera indica, with a particular characteristic of climatic factor being air humidity. the four species had a high presence in the community with high humidity, indicating that high humidity is a suitable climatic condition for the growth of the four species. the intensity of human existence is high in this land use compared to gardens and paddy fields. species found in quadrant 1 are invasive species, mainly introduced with the intention of ornamental plants, food sources, and street shade trees. the paddy fields and garden communities had a high similarity index (50.5%). both of these communities are included in the suburban area. meanwhile, the urban forest community had a species composition that tends to be unique, indicated by the low value of the similarity index with other communities. in addition, the urban forest also had different climatic conditions from other communities, such as low light intensity and low temperature with high humidity. other invasive species found in these four communities were tagetes erecta and cosmos sulphureus as ornamental plants, artocarpus heterophyllus and psidium guajava as consumable plants, delonix regia and albizia saman as shade trees. the presence of these species depends on human preference. human plays a vital role in managing invasive species. people often tend these species by pruning or splitting them, directing their growth without disturbing their aesthetics. thus, it can be concluded that anthropogenic factor has a more considerable influence than climatic factors on determining the presence of invasive species in urban areas. humans, through their preferences, control the presence and dominance of invasive species in urban areas without reducing the number of invasive species. table 5 sorensen index calculated in six paired communities paddy fields gardens street green lanes settlements urban parks urban forests paddy fields 50.5 30.3 22.8 16.9 6.0 gardens 32.7 25.8 7.9 5.6 street green lanes 44.4 26.2 17.5 settlements 21.0 14.4 urban parks 11.5 urban forests biotropia vol. 30 no. 2, 2023 178 figure 3 pca results on the contrary, findings in the urban forests show that although non-invasive species had a higher species number, this community had the lowest diversity index of others. apart from discussing species’ invasiveness in urban forests, species richness was lower than species richness in other communities. several factors affect small-scale species richness, including geographic factors such as the regional species pool, dispersal distance and ease of dispersal, biological factors such as competition, facilitation, and predation, as well as environmental factors such as resource availability, environmental heterogeneity, and disturbance frequency and intensity (brown et al. 2016). urban forest’s climatic conditions differ from other communities, such as low light intensity, temperature, and high humidity. this condition causes limited species that can survive in urban forests, and the low diversity of species in the urban forest can be caused by management areas related to its function as a recreation facility. when viewed from an ecological perspective, it is also included as an ecosystem disturbance that causes a decrease in species richness. based on the pca results, paddy fields and gardens belong to different quadrants, even though they share similarities in their vegetation composition. gardens were placed in quadrant 2, while paddy fields at quadrant 3. both quadrants were influenced by the same climatic factors, i.e., light intensity and air temperature. it indicates that an abundance of amaranthus spinosus, ageratum conyzoides, cleome rutidosperma, cyperus rotundus, g. parviflora, a. gangetica, a. philoxeroides, eclipta prostrata, e. indica, c. dactylon, and b. pilosa is influenced by light intensity and air temperature, whereas the abundance of a. compressus, t. martinicensis, s. macrophylla, and m. indica is influenced by humidity. quadrant 2 is occupied by species, i.e., a. spinosus, a. conyzoides, c.rutidosperma, c. rotundus, g. parviflora, a. gangetica, and a. philoxeroides. these species were initially introduced as contaminants to propagules planted in the garden and managed to “escape” to the surrounding areas. these species were then able to adapt and invade the surrounding garden areas. most species can survive in intense light exposure and grow optimally in warm temperatures. for example, a. gangetica is dispersed by seeds and rhizomes. the seeds are dispersed from explosive capsules, but long-distance dispersal is affected by humans. the risk of introducing rhizome material as a contaminant of soil and compost remains high in those countries where the plant is well established (sandoval & rodríguez 2012). spatial distribution of invasive plants in bandung – rahmawati and rosleine 179 meanwhile, quadrant 3 is occupied by eclipta prostrata, e. indica, c. dactylon, and b. pilosa. in the paddy field community, in our observation, species such as c. rotundus, a. gangetica, and a. philoxeroides had relatively high frequency but low density. due to the inter-species interaction, their density was lower than e. indica, c. dactylon, and b. pilosa. interspecific interactions, including competition, interference, and facilitation, determine the natural community’s composition, distribution, and species abundance (belote & weltzin 2006). invasive species distribution in bandung the distribution pattern of e. prostrata shares similarities with c. dactylon (figure 4). c. dactylon belongs to the c4 plant group that can adapt well and grow optimally in high light intensity. although occupying the same pca quadrant, b. pilosa has a different distribution pattern from e. prostrata and c. dactylon. instead, b. pilosa distribution tends to be similar to a. philoxeroides and a. conyzoides. the three species (i.e., b. pilosa, a. philoxeroides, and a. conyzoides) are abundant in paddy fields, gardens, and settlements. they produce abundant seeds with a wide dispersal range, allowing them to be present in various land-use types. on average, seeds of a. conyzoides are 3.4 mm in length and 0.33 mm in width, equipped with pappus that enables them to attach to garments or animal body parts. this species is widely considered a weed in agricultural and anthropogenic areas and its natural habitat (united states department of agriculture 2019). meanwhile, b. pilosa can produce up to 6000 seeds per year that can easily be dispersed by attaching to animals, birds, human clothes, wind, and water. their propagules can remain viable for 5-6 years (sandoval 2018). biotropia vol. 30 no. 2, 2023 180 figure 4 distribution map of invasive species in bandung: a. e. prostrata; b. c. dactylon; c. b. pilosa; d. a. philoxeroides; e. a. conyzoides; f. a. spinosus; g. c. rutidosperma; h. a. gangetica; i. t. martinicensis; j. s. macrophylla a. conyzoides and a. spinosus are frequently found in gardens. there is no specific distribution pattern of a. gangetica, but it can be commonly found in paddy fields, settlements, and green street lanes. this species propagates through rhizomes and dehiscent capsules. furthermore, the dehiscent capsule explodes and disperses the seeds in the surrounding areas. humans can assist in its long-distance dispersal. the rhizome of this species can contaminate the soil, and it can spread further when present in compost used as planting media (sandoval & rodríguez 2012). two species, i.e., s. macrophylla and t. martinicensis, are introduced in urban areas such as parks, settlements, and streets with an initial specific purpose. the shape of s. macrophylla resembles a huge umbrella. thus, it can provide broad shade areas and decorate the road due to its pleasant form (environmental management agency 2014). therefore, it is unsurprising that this species is widely found in urban parks, forests, and green street lanes. on the contrary, t. martinicensis is abundant in settlements since it is widely planted as ornamental. conclusion there were 187 species found in bandung that can be grouped into invasive alien species (39%), invasive native plants (25%), noninvasive alien species (18%), non-invasive native species (15%), and unidentified plants (3%). species with the highest individual found on the edge of urban areas and were absent in the city’s center were eclipta prostrata, c. dactylon, b. pilosa, a. philoxeroides, a. conyzoides, a. spinosus, c. rutidosperma, and a. gangetica. meanwhile, s. macrophylla and t. martinicensis had the highest number of individuals in the city center. paddy fields and gardens have similar vegetation composition but differ in dominant and co-dominant species. invasive species found in paddy fields and gardens were agricultural weeds. meanwhile, ornamental plants were invasive in urban parks, street green lanes, and settlements. forest had the lowest number of invasive species, corresponding with its function to maintain biodiversity. further research on the distribution map of invasive species in bandung can focus only on one or several species by analyzing the relationship between plant populations and distance to the city center, distance to main roads, edaphic factors, and human population. this information is vital for determining how to control invasive species properly. experimental research can be used to determine interactions between invasive species in one community. in addition, it is essential to carry out a risk analysis to study further the risks posed and the spatial distribution of invasive plants in bandung – rahmawati and rosleine 181 management of each species. thus, the subsequent steps can be more focused. acknowledgments this research is supported by research, community services, and innovation program (ppmi) – itb 2020 for ecology research group. we also thank nadiya syafia, desi sari, diah frisda, resti lutfiani, and rahayu merdekawati, who helped execute this study. references aswant, i a. 2016. analisis perbandingan metode interpolasi untuk pemetaan ph air pada sumur bor di kabupaten aceh besar berbasis gis 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[cited 2022 apr 23]. avaialble from: www.botany.hawaii.edu. wang, cj, and wan, jz. 2020. assessing the habitat suitability of 10 serious weed species in global croplands. global ecology and conservation, 23: e01142. biotropia vol. 29 no. 3, 2022: 272 282 doi: 10.11598/btb.2022.29.3.1792 272 macrofungal diversity in different vegetation compositions in teghari community forest, kailali, west nepal kausalya joshi1, hari sharan adhikari1,*, hari prasad aryal2 and laxmi joshi shrestha1 1department of botany, amrit science college (tribhuvan university), kathmandu 44600, nepal 2central department of botany, tribhuvan university, kathmandu 44600, nepal received 1 august 2022/accepted 18 october 2022 abstract macrofungi are high-value forest resources that have functionally significant roles in the forest ecosystem. the macrofungal community of three different vegetation compositions, i.e., sal (shorea robusta) forest, tropical deciduous riverine forest, and tropical evergreen forest of teghari community forest were investigated. systematic random sampling was made where 60 plots (10 x 10 m) were laid in all different forest types (20 plots in each). a total of 102 macrofungi species were reported belonging to 36 families. polyporaceae (17 species) was the largest family followed by tricholomataceae (13 species) and saprophytic fungi were more frequent than mycorrhizal and parasitic fungi. the tropical evergreen forest was rich in macrofungi (59 species) followed by sal forest (40 species) and tropical deciduous riverine forest (38 species). macrofungal diversity was directly related to surrounding host species. similarly, increased soil moisture and canopy cover intensified the abundance of saprophytic fungi. the species richness was increased with increasing organic carbon, canopy, moisture, ph, and litter cover. however, soil nitrogen, phosphorus, and potassium were less significant in affecting species richness. also, the disturbance was negatively correlated with the species richness of macrofungi. this study highlights the hidden diversity which is necessary for the conservation of macrofungi, to optimize forest ecosystem integrity and resilience against biotic and abiotic agents. keywords: macrofungal diversity, sal forest, species richness, tropical evergreen forest, tropical riverine forest introduction biodiversity is simply defined as the presence of the total organism of a particular group at a particular time in a particular area. conservation of these natural resources is the priority for ecosystem functioning as well as human welfare. fungi are an enormous usly diverse group of organisms ranging from microscopic to macroscopic forms that grow mostly in the dead and decaying substrate. they appear in all seasons, mostly rainy season, wherever nutrient organic matters or decomposed products are easily available (jha & tripathi 2012). macrofungi are a group of higher fungi that produce mature spore-bearing fruiting bodies, which are visible to the naked eye (chang & miles 1992). they are known to inhabit diverse kinds of habitats varying in the composition of their tree species and substrates. based on ecology, they are parasitic or saprophytic or may show some mycorrhizal associations with vascular plants (kumar & sharma 2011). however, some macrofungi are neutral to the abundance of dominant tree species, in particular, habitat type (zhang & zak 1998). the relationship between the tree and fungal communities is reflected in host trees affecting fungal specialization and providing unique habitat availability and different resource quality. the composition and structure of aboveground vegetation are responsible for diverse macrofungi communities (buee et al. 2011). *corresponding author, email: aharisharan@gmail.com macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 273 generally, macroscopic fruiting bodies of the fungi is called mushroom which can be epigeous or hypogeous and vary in different shape and sizes. they are fleshy, sub-fleshy, or sometimes leathery and woody and bear their fertile surface either on lamellae or lining the tubes, opening out through pores. the most suitable condition for the growth of carpophores depends upon the high humidity, nutritionally rich substrate, and warm atmospheric temperature (dickinson & lucas 1979). similarly, other environmental conditions such as geographic location, light, and surrounding vegetation types also play a major role in the distribution of the macrofungi (sibounnavong et al. 2008). diversity-related studies are carried out in different forests but their relationship with higher plants was poorly explored except these studies such as pradhan (2013), baral et al. (2015), and bhandari and jha (2017). this study aimed to to optimize forest ecosystem integrity and resilience against biotic and abiotic agents, by looking at the effect of different vegetation characteristics and environmental factors on macrofungal species composition and richness in the tropical region of western nepal. materials and methods study sites the study was carried out in three different vegetation patches within teghari community forest in the tropical riverine belt of kailali district, west nepal (fig. 1). the study area lies between latitudes from 28°50'45" n to 28°51'01" n and longitude 80°33 ̍ 3" e to 80°33'13" e, covering an area of 340 ha. the altitude range of the study area is 155 254 masl. meteorological data of the dhangadi airport in the year 2019 was obtained from the department of hydrology and meteorology, government of nepal which revealed that the study area is represented by a tropical climate and receives an average of 1,406.6 mm annual rainfall with the highest monthly rainfall happens in july (466.9 mm) and the lowest in may (5 mm). the highest monthly mean temperature happens in may (40.41°c) and the lowest in january (6.77 °c). figure 1 map of the study area biotropia vol. 29 no. 3, 2022 274 study design the teghari community forest was selected for the field study as it has three different forest types at the same elevation, i.e., sal forest, tropical riverine deciduous forest, and tropical evergreen forest. shorea robusta (sal) is the dominant tree species in the sal forest which forms magnificent forest stand on the edges of the godawari river. the tropical deciduous riverine forest is also located similarly and is mainly dominated by acacia catechu and dalbergia sissoo along with bombax ceiba, syzygium cumini, adina cordifolia, hollarrhena pubescens, murraya koenginii, aegle marmelos, and semicarpus anacardium. the tropical evergreen forest lies on the northwest side of mahakali highway and is dominated by terminalia alata, lagerstroemia parviflora, terminalia bellerica, ficus religiosa, schleichera oleosa, aegle marmelos, and cassia fistula. mallotus phlippensis is present all over the study area. in each forest type, rectangular plots of 10 × 10 m were established. the number of plots to be sampled were determined based on the spatial area of each forest. field sampling detailed sampling of macrofungi diversity was made by applying a systematic random method within the period of june october 2019, where plots were laid in each forest type. a total of 20 plots were laid in each forest type along with the two transects for maintaining an inter-plot distance of at least 20 m (baral et al. 2015). presence or absence data of macrofungal species were recorded in each plot. biophysical variables, such as tree canopy cover, litter cover, and anthropogenic disturbances (trampling, fire grazing, non-degradable waste, etc.) were also recorded in each plot. tree canopy cover and litter cover (in percentage) was estimated visually. for tree canopy cover, observation was made from the middle of each plot. soil samples were collected at a depth of 15 cm from four corners and at the middle of each plot using a soil digger. the soil samples from each plot were mixed thoroughly. from the mixed soil sample, about 200 g of soil sample was taken and put in a zipper polythene bag. the soil samples were air-dried in shade for a week and stored in airtight plastic bags until laboratory analysis. the physiochemical parameters of soil, such as soil ph, moisture, organic carbon, nitrogen, potassium, and phosphorus were assessed using a standard soil analysis manual (zobel et al. 1987). macrofungal specimens were collected, preserved (dry), and taken to national herbarium (kath) in lalitpur, nepal. collected specimens were studied based on their morphological characters and ecology with the help of several websites, such as https://www.mushroomexpert.com and http://www.indexfungorum.org. finally, the identification of specimens was confirmed using relevant literature (pacioni & lincoff 1981; adhikari 2014; laessoe 2013) along with identification conducted by macrofungi expert. all of the collected macrofungal specimens were deposited in ascol herbarium, amrit science college, kathmandu, nepal. data analysis all data were entered in microsoft excel 2010 for further analysis. pearson correlation method was used to know the effect of a different environmental variable on macrofungal diversity. simpson’s diversity index (simpson 1949) and shannon-wiener index (shannon & weaver 1963) were also calculated. regression analysis was performed using spss version 20 and microsoft excel version 2010. species composition of different macrofungi species along with different environmental components were evaluated by canonical correspondence analysis (cca). results and discussion macrofungal diversity in different vegetation composition a total of 102 macrofungi consisting of ascomycetes-5 and basidiomycetes-97 species were documented, in which 100 species were identified up to species level and 2 species were identified up to genus level. out of the 36 families, 17 species belonged to the polyporaceae family, 13 species to tricholomataceae, 11 species to marasmiaceae, 9 species to agaricaceae, 8 species to coprinaceae, 4 species each to russulaceae and xylariaceae, 2 species representing each of the cortinariaceae, ento macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 275 lomataceae, fomitopsidaceae, ganodermataceae, hydnangiaceae, podoscyphaceae, and suillaceae family and the rest of the family was represented by single species only (fig. 2). tropical evergreen forest harbored the highest macrofungal diversity (59) in all three different substrates (fig. 3), followed by sal forest (40) and tropical deciduous riverine forest (38). the maximum numbers of macrofungi were found growing on the soil, followed by wooden logs. the least number of macrofungal species were found growing on litters in all forest types. the present study relates to a study conducted in india where macrofungi were reported in various habitats, like wood, litter, and moist soil, among others (nagaraju et al. 2014). as compared to litter and wood, the soil was the most important substrate for maintaining macrofungal diversity in all three forest types studied. in our study, higher number of macrofungi were grown on moist soil compared to those on litter and decaying wood. these findings resemble the previous findings of a study conducted by bhandari and jha (2017). figure 2 number of species with their respective family figure 3 distribution of macrofungi based on their habitat in different forests biotropia vol. 29 no. 3, 2022 276 based on the ecology of macrofungi, the maximum number of macrofungi were consisted of saprophytes, followed by mycorrhizal and parasitic macrofungi, while the least number belonged to termitophilous macrofungi. the mycorrhizal fungi serve as an extension of the plant root system, exploring soil far beyond the roots and transporting water and nutrients to the roots (tapwal et al. 2013). the flourishing of carpophores is enhanced by litter accumulation and decomposition as well as the presence of extracellular microbial enzymes (pushpa & purushothama 2012). the rapid change in the weather and high response of mycelia were among the main factors for the increasing number of saprotrophic fungi (pradhan et al. 2012). a similar result was obtained in the research of topwal et al. (2013) and dey et al. (2016). higher species diversity in basidiomycota compared to ascomycota is probably contributed by a higher number of mycorrhizal species found on the soil as studies have shown that soil moisture and decaying litter facilitate many diverse macrofungi (muller & schmit 2007). simpson’s diversity index (table 1) was found to be the highest in tropical deciduous riverine forest and tropical evergreen forest (0.91) in comparison to sal forest (0.88). similarly, shannon-wiener diversity index was also found to be the highest in tropical evergreen forest (2.91) followed by tropical deciduous riverine forest (2.77) and sal forest (2.53). the presence of diverse kinds of macrofungi communities is specifically related to the dominant tree species of the forest has been confirmed by many other studies (straatsma & krisai-greilhuber 2003; gates et al. 2011; o’hanlon & harrington 2011; bhandari & jha 2017; collado et al. 2021; kutszegi et al. 2021). such variation may be attributed to microclimate conditions (santos-silva et al. 2011) and forest management practice (kouki & salo 2020). the high macrofungal diversity in the tropical evergreen forest is mainly related to high soil moisture and greater cover of tree species. the high diversity may be also due to suitable habitat, such as soil moisture, litter, and canopy cover which help to maintain sufficient moisture (trudell & edmonds 2004). a tropical deciduous riverine forest located on the water edges has a more open canopy and less humidity in the soil which creates a less suitable habitat for the growth of macrofungi. also, thinning of trees caused a decrease in fruit-body production of the delicate and fragile macrofungi, but this effect varied greatly depending on the season, the macrofungi fruiting pattern and the levels of trees thinning (luoma et al. 2004). therefore, thinning and pruning, which are common silvicultural activities in the community forests of nepal (shrestha et al. 2010), might also affect the composition and abundance of macrofungi. similarly, sal forest has a magnificent stand of tall trees and has a more open canopy in comparison to tropical evergreen forest. the growth of macrofungal species like pycnoporus cinnarius and scleroderma cepa was specifically recorded in sal forest. a similar finding was also reported by prasad & pokhrel (2017) at amrite community forest, kapilvastu district (central nepal), which might be due to the host specificity of macrofungi with particular plant species. the presence of specific macrofungi communities in the present study may be due to host preferences which were related to the findings by ding et al. (2011) and lang et al. (2011). species richness of macrofungi and different environmental variables the canonical correspondence analysis (cca) revealed the relationship between macrofungi species composition and environmental gradient (table 2). the analysis results indicated the effective separation of species along the main gradient (table 3). table 1 diversity indices of macrofungi in different vegetation stands forest stands simpson’s index simpson’s diversity index shannon-wiener diversity index sal forest 0.12 0.88 2.53 tropical deciduous riverine forest 0.09 0.91 2.77 tropical evergreen forest 0.09 0.91 2.91 macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 277 table 2 summary of the results of canonical correspondence analysis axes 1 2 3 4 total inertia eigenvalues 0.369 0.34 0.279 0.208 11.381 species-environment correlations 0.886 0.903 0.945 0.88 cumulative % variance of species data 3.2 6.2 8.7 10.5 cumulative % variance of species-environment data 18.7 35.9 50 60.5 sum of all canonical eigenvalues 1.978 table 3 relative importance of environmental variables and their significance (p value) on macrofungal species composition derived by using the monte carlo permutation test from the canonical corrrespondence analysis with 9999 replications environmental variable abbreviation f p organic carbon orgcarb 0.914 0.682 ph ph 1.086 0.227 moisture moist 1.249 0.11 nitrogen nitro 1.057 0.347 phosphorus phosp 1.176 0.182 potassium potas 1.4 0.162 litter litter 1.33 0.046 canopy cover canop 1.033 0.36 disturbances distrb 1.209 0.052 our study showed that environmental variables, such as moisture, ph, canopy, organic carbon, nitrogen, phosphorus, potassium, and anthropogenic disturbances had significant effect on the distribution and composition of macrofungi. organic carbon, moisture, ph, canopy, litter, and macrofungal species richness were positively correlated and was comparable with the study of bhandari and jha (2017). this finding indicated that these environmental variables play a vital role in shaping macrofungi communities. however, other soil properties such as nitrogen, phosphorus, and potassium were negatively correlated with the species richness. there were no significant effects on the increasing disturbances with the abundance of macrofungi (fig. 4). -0.6 1.0 -0 .8 0 .8 gea_lexpod_des sch_une mar_eus ter_pus rus_ceastr_tussui_tus pod_ata col_nis ple_ensscl_epa xyl_lon xyl_phagan_tum spo_lor hex_ida lac_ata mar_cus mic_pus pyc_nus hyg_lus tri_ora myc_ptaama_iae lep_ria pol_ius ale_tia leu_onipsi_ata omp_era aga_sis hel_ata aur_ata len_aju lep_pes pan_vus mar_lus cya_tus mar_lislac_nda ear_osa ent_num cla_ata can_ius mac_des lep_ata hyg_ica lae_eus rus_ria leu_tusdae_ina cre_lis aga_tus xyl_ila lyc_eumcop_tus gan_dum cop_ans oud_atalac_ina tra_ens cop_pus ter_zus dal_ica mar_ula rus_ans ent_tum rus_sps inf_bba cal_osa cre_lis psa_ata mar_ans pol_nus tra_lor con_ens mac_nii lep_nea pan_tus col_ila cli_mis cop_tus ple_ius mar_dus arm_ens ant_ina tra_ans ram_cta hex_uis fom_ius ter_nus hyg_eus sui_dus psa_mis psa_pes cli_sps pan_cus pos_ica cop_lis len_ina mic_pes ph orgcarbo moist nitrogen phosph potas canop litter distrb figure 4 cca biplot representing the effect of environmental variables on macrofungal species composition biotropia vol. 29 no. 3, 2022 278 macrofungi species, like pycnoporus cinnarius (pyc_ius), scleroderma cepa (scl_epa), agaricus arvensis (aga_sis), tricholomopsis decora (tri_ora), termitomyces microcarpus (ter_pus), aleuria aurantia (ale_tia) and lentinus sajorcaju (len_aju), etc. showed strong presence in the sal forest and were more resistant to disturbances. tropical deciduous riverine forest was dominated by the macrofungi species, like ramaria stricta (ram_cta), antrodia juniperina (ant_ina), fomes fomentarius (fom_ius), hexagonia tenuis (hex_uis), trametes elegans (tra_ans), clitocybe infundibuliformis (cli_mis), lenzites betulina (len-ina), panus fasciatus (pan_tus), coltricia perennis (col_nis) and microporus xanthopus (mic_pus), etc. which favored more open canopy. similarly, tropical evergreen forest harbored macrofungi species, like macrolepiota rickenii (mac_nii), marasmius haematocephalus (mar_lus), daldinia concentrica (dal_ica), marasmius androsaceus (mar_eus), lepiota clypeolaria (lep_ria), macrolepiota rhacodes (mac_des), lacrymaria lacrymabunda (lac_nda), lepiota clypeolaria (lep_ria), lycoperdon subcretaceum (lyc_eum), cyathus striatus (cya_tus) microporus xanthopus (mic_pus) and podoscypha multizonata (pod_ata). our present study also showed that the species found in the tropical deciduous riverine forest and tropical evergreen forests were more similar than those in the sal forest. however, some macrofungi species, like schizophyllum commune (sch_une), polyporus arcularius (pol_ius), termitomyces tylerianus (ter_nus), microporus xanthopus (mic_pus), and geastrum triplex (gea_lex) were found in all three forest types. the cca biplot showed environmental variables and aboveground vegetation were the major components for determining macrofungi composition (fig. 4). the overlapping of macrofungi species was due to a similar ecological niche. as we found in our present study, most of the soil fungi such as geastrum triplex (gea_lex), podocypha petaloides (pod_des), suillus granulatus (sui_tus), and russula species occurred toward the moisture. our study also showed that the presence of thin canopy cover and low soil moisture seemed to enhance the growth of wood-inhabiting fungi, such as spongipollis unicolar (spo_lar), crepodotus mollis (cre_lis), xylaria sp., trametes elegans (tra_ans), antrodia juniperina (ant_ina), fomes fomentarius (fom_ius), hexagonia tenuis (hex_uis), micrporus vernicipes (mic_pes) and lenzites betulina (len_ina), which were mostly presented opposite direction to the moisture and ph. organic carbon was also one of the major components which control the distribution pattern of soil fungi. macrofungi species, like termitomyces sp., rusulla sp., macrolepiota rhacodes (mac_des), omphalina umbellifera (omp_era), and marasmiellus ramealis (mar_lis) were found predominantly toward the direction of organic carbon. in our present study, disturbances seemed to have a poor impact on macrofungi species composition. most of the fleshy, soft, and gilled macrofungi, like clitocybe sp., psathyrella obtusata (psa_ata), agaricus augustus (aga_tus), lepiota clypeolaria (lep_ria) and coprinus disseminates (cop_tes) were favored by the higher soil ph. species richness of macrofungi increased with the increasing soil organic carbon, moisture, ph, litter coverage, and canopy coverage. soil ph and organic carbon ranged from 4.06 to 7.07 and 0.79 to 5.67, respectively. similarly, soil moisture, litter cover, and canopy cover ranged from 7.5 to 45.46%, 11 to 49%, and 15 to 95%, respectively. among all environmental variables, organic carbon, soil moisture, soil ph, litter cover and canopy cover had the most significant positive relationship with macrofungi species richness (fig. 5). also, the species richness of macrofungi showed a weak positive relationship with disturbances. soil nitrogen, phosphorus, and potassium were negatively correlated with macrofungi species richness but the result was statistically insignificant (table 4). species diversity of macrofungi depends on their particular habitat. geographic location, elevation, temperature, the humidity of air and soil, light, surrounding flora, and anthropogenic activity greatly influence the growth and reproduction of macrofungi (zervakis & venturella 2007; topwal et al. 2013). soil moisture is one of the most important environmental factors responsible for affecting the growth of the macrofungi (kropp & albee 2002). present findings also showed increasing species richness is affected by the increasing moisture content of the soil. this finding was similar to the research of bhandari and jha (2017). the fungal diversity studies in greece and sicily (venturella & zervakis 2000; zervakis & venturella 2002) confirmed that fungi require a certain level of moisture; rainfalls, macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 279 figure 5 correlation between macrofungi species richness and organic carbon, moisture, soil ph, canopy cover, and litter cover notes: each point in each figure represents a sampling plot; total number of sample plots = 60. less number of points in the figure may be due to the overlapping of the data among the plots. the fitted line is based on the linear regression model for sampling plots. table 4 pearson correlations between environmental variables and species richness of macrofungi soil ph organic carbon moisture n p k canopy cover litter cover disturbances species richness 0.506** 0.519** 0.580** -0.074 -0.183 -0.14 0.513** 0.612** 0.034 notes: * = correlation is significant at p < 0.05 level; ** = correlation is significant at p < 0.01 level. air humidity, and soil moisture, which are all significant factors. canopy cover and litter cover had also provided positive influence on macrofungal species richness in the studied area and a similar result was found by baral et al. (2015). the result unveiled that canopy cover plays a vital role in increasing macrofungal diversity in forests which was also reinforced by other previous findings (dighton et al. 1986; bonet et al. 2004; sysouphanthong et al. 2010; santos-silva et al. 2011). the reason is likely to be the presence of more substrate on the forest floor and high humidity which favor the growth of more fungal species (lodge et al. 2004). litter is an important component of every ecosystem and it constitutes the major source of organic matter in the soil. the removal of litter directly affects the diversity and growth of macrofungi (eaton et al. 2004; sayer 2006). when the forest floor is covered with layers of well-decomposed biotropia vol. 29 no. 3, 2022 280 leaves, saprotrophic fungi are favored by this organic resource which maintains the temperature and moisture of the surrounding area (fernandez-toiran et al. 2006). also, the abundance of macrofungal species is closely correlated with soil organic matter and other soil parameters (zamora-martinez & de pascual-pola 1995; engola et al. 2007). the growth of saprophytic fungi was enhanced at ph 7 or 8; while the ectomycorrhizal species showed the peak growth at ph 5 or 6 (yamanaka 2003). soil ph was also a major abiotic component responsible for changing macrofungi communities and was found to be positively correlated with macrofungi species richness. the ph range of 5 to 6 favors the growth of soil fungi (bhandari & jha 2017; pavithra et al. 2016; zhang et al. 2016). in this study, it was observed that there were no direct relationships between macrofungi richness and other soil parameters, such as nitrogen, phosphorus, and potassium with macrofungi diversity. however, the importance of forest soil chemistry parameters in fungal species distributions has also been reported by hansen (1988) and ruhling & tyler (1990). conclusion the study area is rich in macrofungal diversity with species’ richest families being the polyporaceae followed by tricholomataceae, marasmiaceae, agaricaceae, and coprinaceae. the presence of diverse kinds of vascular plants and different environmental conditions in different forest types have created unique habitat for the growth and development of a wide variety of macrofungi species. species diversity is higher in moist and dense canopy forests such as tropical evergreen forests and sal forest compared to that in the open and dry tropical deciduous riverine forests. soil moisture, organic carbon, soil ph, litter cover, and tree canopy cover are the most important variables affecting macrofungal diversity. acknowledgments the authors are grateful to the forest users groups of teghari community forest and mr. yagya raj bhatta for their support and cooperation in field. we are also thankful to the national herbarium and plant laboratories (kath) for the specimen identification which had been crucial for this study. finally, we thank anonymous reviewers and editors for their valuable 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tokyo, tokyo, japan abstract this experiment was carried out to confirm the role of water level and water temperature in inducing the spawning of tropical walking catfish. mature males and females reared under 23 25 °c, were paired and induced to spawn by controlling water level and water temperature. decreasing water level and returning it to its original level resulted in a low spawning rate (less than 6.7 %) at 23 °c. decreasing water level with simultaneous increase in water temperature, followed by returning the respective levels to their originals gave high spawning rates (41.7 — 50 %); whereas the same treatment but without any water temperature decreased when the water level was returned to the initial level, gave a low spawning rate (16.7 %). increasing water level only, failed to induce spawning. a high spawning rate was obtained also when changes in water level were carried out under high temperature of 28 °c. no fish spawned in the absence of the environmental stimulation. from the results, it is confirmed that water level and temperature play important roles in inducing spawning of tropical walking catfish. changes in water temperature probably increase the sensitivity of fish to the change in water level. prolonged exposure to high water temperature could also improve the sensitivity of fish. key words: walking catfish / spawning / water level / water temperature introduction in the previous reports (zairin et al. 1992a,b), it has been suggested that under long term rearing and constant warm water temperature, female catfish maintained conditions of maturity for an extended period, and no spontaneous spawning has been seen to occur. on the other hand, from the practical point of view, one of the constraints in employing environmental factors in inducing the fish to spawn in the pond is a low spawning rate. therefore, it is necessary to investigate the ability of this fish to respond to environmental stimulation for the induction of spawning. • corresponding author: e-mail address: zairinm@indo.net.id; fax: 62-251-622541 18 18 biotropia no. 16, 2001 in nature, both environmental and physiological factors are considered as important cues in triggering final oocyte maturation, ovulation and spawning in teleosts. in tropical areas, natural water temperature is relatively high, and water level amplitude is large in association with the occurrence of the rainy and dry seasons. it has been reported that peaks of spawning activity of tropical species are often associated with rainfall and flood (lam 1983; lam and munro 1987; munro 1990). several species have been reported to spawn in relation to the change in water level, for example weakly electric fish (eigenmannia virescens) (kirschbaum 1979) and the african catfish (glorias gariepinus) (bruton 1979). moreover, mimicking environmental phenomena in nature, e.g. by regulating water level, has been commonly employed in aquaculture in southeast asian countries to induce tropical walking catfish to spawn (areerat 1987; tarnchalanukit 1987; knud-hansen et al. 1990). however, little is known on the combinatory effects of water level and water temperature in the action of inducing spawning. therefore, experiments were conducted to study the effects of water temperature and water level in the induction of spawning in walking catfish materials and methods experimental fish two month-old walking catfish were transported from bogor, indonesia to japan, and reared in an indoor concrete pond (width x length x depth = 1.5 x 3 x 0.5 m) supplied with running freshwater of deep-well origin at the fisheries laboratory, the university of tokyo, maisaka, shizuoka prefecture. during this rearing period, fish were subjected to a photoperiodic cycle of 12l(light):12d(dark), and relatively constant water temperature (23-25°c) throughout the year. fish were fed twice per day with commercially available trout dry pellets no. 5 (40% protein, chubu shiryo, co.) at a daily ration of 2-3% of body weight. under the above stocking conditions, fish began to mature at the age of 9 months and thereafter; conditions of maturity were maintained without undergoing natural spawning. two experiments (i and ii) were carried out. a week before the start of the experiment i, 15 pairs of fish were selected from the stock pond. males weighing 325-400 g and females weighing 375-500 g were used. these fish were paired and placed in each compartment. at the end of the experiment i, all fish were replaced by a new group for experiment ii. for this experiment, males weighing 250-375 g and females weighing 300-425 g were selected and paired. experimental condition five indoor concrete ponds 1 x 3 x 1 m in size were used. each pond was divided transversely into three compartments by framed plastic nets, so that every 19 induction of spawning in the tropical walking catfish m. zairin jr. et al. compartment was 1 x 1 x 1 m (fig. ib). each compartment was equipped with one spawning box, which was placed in the compartment only during the observation period of spawning (day 14-24, 31-35, 45-55 in experiment i, day 11-25, 35-45 in experiment ii). the spawning boxes 30 x 35 x 42 cm, were painted dark brown, with a hole of 12 cm in diameter (fig. la). half of the upper portion was made removable for checking for deposited eggs. an artificial plastic spawning nest made of soft plastic fibres was placed inside each box. the box was fixed in the corner of every compartment, in which 2-3 cm of its upper portion rose above that of water level. photoperiod was maintained at constant 12l:12d throughout the experiment, but water temperature and depth were varied according to the type of treatment. limitation in sample numbers did not allow statistical analysis. such experiments are very difficult to be carried out for a large number offish. treatment treatment a and a'. a sudden decrease in water level from 70 cm to 20 cm (in one hour) for 13 days (treatment a) or to 35 cm (in one hour) for 7-10 days (treatment a'), followed by an increase back to the original level of 70 cm (in one hour). water temperature was maintained at 23 ± 0.5°c during the experiments. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10 days (treatment a) and for 4-10 days (treatment a'). for more details, refer to fig. 2. figure 1. (a) spawning box and its placement relative to water surface. (b) pond compartments and box position in each compatmentent. 20 biotropia no. 16, 2001 21 induction of spawning in the tropical walking catfish m. zairin jr. et al. treatment b. a sudden drop in water depth from 70 cm to 35 cm (in one hour), and an increase in temperature from 23 °c to 28°c (in six hours) were simultaneously carried out for 7-10 days, and after that, both water level and temperature were returned to their originals. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 4-10 days (fig. 2). treatment c. similar to treatment b, a sudden drop in water level from 70 cm to 35 cm (in one hour), and an increase in water temperature from 23°c to 28°c (in six hours) for 10 days were simultaneously carried out, but the temperature was maintained at 28°c while the water level was returned to its original (in one hour) after 10 days. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 14 days (fig. 3). treatment d and d'. only an increase in water temperature from 23°c to 28°c (in six hours) for 10 days (treatment d), and for 24 days (treatment d'), without accompanying water level manipulation. the duration of observation was 10 days. on days 11 and 35, a spawning box was placed in each compartment. the boxes were checked everyday for deposited egg for 14 days (treatment d) and for 10 days (treatment d'). for more details, refer to fig. 3. treatment e. a sudden drop in water level from 70 cm to 35 cm (in one hour) at 23°c for 10 days and then return to the original water level at 28°c. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10 days (fig. 3). treatment f. no manipulation. this treatment is regarded as controls for all experiments. water temperature and water level were kept at 23°c and 70 cm, respectively. at days 11 and 35, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10-14 days (fig. 3). results and discussions results experiment i the results are presented in fig. 2. during experiment la, the fish were subjected to treatment a. out of 15 pairs employed, only one pair spawned (6.7%). during experiment ib, fish were subjected to treatment a' and b. from 9 pairs of fish employed in treatment a', no fish spawned. in treatment b, however, the combination of water level and temperature manipulation gave a high spawning rate (50%). out of six pairs employed, three pairs spawned or ovulated. during experiment ic, fish were subjected also to treatment a' and b. the result was still in agreement with those of experiment ib. no fish spawned in treatment a'. treatment b again gave a high spawning rate (41.7%). out of 12 pairs of fish employed, five pairs spawned. 22 biotropia no. 16, 2001 figure 3. spawning success after water level and water temperature manipulation for treatment c to f (see text for details). closed line: water level; broken line: water temperature. each of the open and closed star represents an ovulated or spawned fish, respectively. ha and lib represent the experimental period of each treatment. 23 induction of spawning in the tropical walking catfish m. zairin jr. et al. experiment ii the results are presented in fig. 3. during experiment ha, the fish were subjected to treatment c, d and f. treatment c, which was similar to treatment b, but without dropping water temperature upon increasing water level, gave a spawning rate of 16.7%. out of six pairs employed, one pair spawned. on the other hand, increasing only water temperature (treatment d) or no manipulation in water level and temperature (treatment f) could not induce spawning. during experiment lib, the fish were subjected to treatment d', e and f. manipulation of water level under 28°c (treatment e) gave a high spawning rate (50%). out of six pairs employed, three pairs spawned or ovulated. no fish spawned in treatment d' or f. in our experimental conditions, at least three days (after the placement of spawning box) were needed to reach the ovulation or spawning. the results of all treatments are summarized in table 1. table 1. spawning success after water level and water temperature manipulation 24 biotropia no. 16, 2001 discussions mimicking the environmental phenomenon to induce spawning in fish has been a common practice in aquaculture in southeast asian countries, including the use of the water level manipulation (areerat 1987; tarnchalanukit 1987; knud-hansen et al. 1990). unfortunately, no scientific effort has been made to substantiate this interesting practice. understanding this problem will add value to the development of the domestication of tropical wild edible river fish. considering the typical physical conditions in the tropic and to simplify the problem, water temperature and water level have been chosen as variables. both factors were chosen because changes in water levels of tropical river were usually accompanied by changes in water temperature. the possibility of photoperiod as an environmental cue has been omitted since our previous results showed that these fish were found mature throughout the year in japan, regardless of photoperiod (zairin et al. 1992a,b). environmental stimulation usually induces spawning by a long process through the hypothalamus-hypophysial-gonadal axis (blazquez et al. 1998). and as expected, time elapses are needed between the stimulation and the initiation of spawning. overall in this experiment, at least three days were needed for walking catfish to spawn. in the african catfish clarias gariepinus, in which fish were kept in earthen ponds, a shorter time interval was sufficient (bruton 1979). this difference may be due to the more complex physico-chemical conditions in the earthen pond. the possibility of more males needed to induce courtship behaviour has been disregarded since this fish is not a mass spawner. changes in water levels alone (treatment a) actually could induce spawning, although the spawning success was low (6.7%). this means that the fish may be responsive to the fluctuation in water level after a long time exposure to a constant temperature and photoperiod condition. a low spawning success reflects a low sensitivity of fish to water level fluctuation. this low responsiveness may be due to relatively low water temperature (23°c) employed in this experiment. this is supported by the fact that in the tropics such as indonesia, the average water temperature in places where the fish are commonly cultured, is usually higher than 23°c. from the daily observation on the performance of fish in experiment la, we considered that lowering the water level to 20 cm gave insufficient space for fish to maneuver because the depth was less than the average length of fish employed. thereafter, the minimum depth was increased to 35 cm (treatment a'). although treatment a' gave no spawned fish, we used 35 cm as the minimum depth. the results from treatment b, in which more fish spawned (41.7-50%) when the change in water level was accompanied by the change in water temperature indicate the importance of water temperature amplitude. furthermore, the results suggest that fluctuations in temperature could increase the sensitivity of fish to the water level manipulation. further supporting evidence came from treatment c and 25 induction of spawning in the tropical walking catfish m. zairin jr. etal. d. treatment c was similar to experiment b but without water temperature decrease upon the increase in water level; the spawning rate in treatment c (16.7%) was lower than treatment b (41.7-50%). no spawning occurred under treatment d and d' which indicated that increasing only the water level gave no stimulation on the fish to spawn. treatment d and d' were similar, and differed only in the duration of exposure to high water temperature. exposure to treatment d' was longer than that of treatment d. additionally, when changes in water level were carried out at a higher temperature of 28°c (treatment e), the spawning success was almost the same as those obtained when water fluctuation was accompanied by water temperature changes. these results suggest that changes in temperature could be replaced by prolonged exposure to higher temperature. furthermore, this phenomenon indicates that temperature still plays an important role in the spawning of the tropical walking catfish. from the practical point of view, the results from the best treatment of our experiments give a higher spawning success compared with those of fish farms. calculation based on figures provided by knud-hansen (1990) indicates only less than 15 % of spawning success; and in practice, 10% spawning success is regarded as an acceptable rate. therefore, for practical purposes, the role of these factors should get more attention. furthermore, we believe that combination of environmental factors with simple hormonal treatment could improve the spawning success further. research in this area should receive more attention. acknowledgment we express our thanks to all members of the fisheries laboratory of the university of tokyo, maisaka, shizuoka prefecture, for the facility for field experiments. we thank m. n. wilder for reviewing the manuscript. this study was partially funded by a grant-in-aid for scientific research from the ministry of education, science and culture, japan. references areerat, s. 1987. clarias culture in thailand. aquaculture, 63, 355-362. blazquez, m.p., bosnia, p.t., eraser, e.j. van look, k.j.w., and v.l trudeau, 1998. fish as a model for the neuroendocrine regulation of reproduction and growth. comp. biochem. physiol. partc, 199, 345364. bruton, m.n. 1979. the breeding biology and early development of clarias gariepinus (pisces: clariidae) in lake sibaya, south africa, with a review of breeding in species of the subgenus clarias. trans. zool. soc. london. 35, 1-45. 26 biotropia no. 16, 2001 kirschbaum, f. 1979. reproduction of the weakly electric fish eigenmannia virescens (rhamphichtydae, teleostei) in captivity. behav. ecol. sociobiol., 4, 331-355. knud-hansen, c.f., batterson, t.r., mcnabb, c. d., hadiroseyani, y., dana, d., and h.m eidman,. 1990. hatchery techniques for egg and fry production of clarias batrachus (linnaeus). aquaculture, 89, 919. lam, t. j. 1983. environmental influences in gonadal activity in fish. in: w.s. hoar, d.j. randall, and e.m. donaldson (eds.), fish physiology, vol. ix, part b, p. 65-116, academic press, new york. lam, t.j., and a.d. munro, 1987. environmental control of reproduction in teleost: an overview. proc. of the third int. symp. on reprod. physiol. of fish. st. john's, newfoundland, canada, p. 279-288. munro, a.d. 1990. tropical freshwater fish. in: a.d. munro, a.p. scott, and t.j. lam. reproductive seasonality in teleost: environmental influences, cec press, boca raton, florida, p. 145-239 tamchalanukit, w. 1987. induced spawning of walking catfish, clarias batrachus, by water level jegulation. proc. of the third int. symp. on reprod. physiol. of fish. st. john's, newfoundland, canada, p. 138. zairin, m., jr., furukawa, k.., and k. aida, 1992a. induction of ovulation by hcg injection in the tropical walking catfish clarias batrachus reared under 23-25°c. nippon suisan gakkaishi. 58, 1681-1685. zairin, m., jr., furukawa, k., and k. aida, 1992b. changes in ovarian maturity in the tropical walking catfish, clarias batrachus reared under 23-25°c. nippon suisan gakkaishi. 58, 2033-2037. 27 microsoft word 49 biotropia vol. 13 no. 1, 2006 : 49 55 localization of gfdd4-1 expressed protein in physcomitrella patens cells diah ratnadewi faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the expression of a new dehydration-related gene of physcomitrella patens, gfdd4-i, was traced for its localization in the plant cells. this revelation is useful to predict the possible roles of the protein in plant tolerance to environmental stress. this gene was fused to gfp marker gene and transfected into the plant protoplasts. under a confocal laser microscope, it was detected that the gfdd4-1 protein associated with the off started to generate at the cell periphery and developed more intensively inwards to cytoplasm, forming vesicles and cystemal structures or network. the protein might be membrane protein which may involve directly in membrane maintenance or cellular protection against stress conditions. key words : protoplast transformation, protein localization, dehydration-related gene, gfp, physcomitrella patens introduction abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. diverse environmental stresses often activate a number of cell signalling pathways and cellular responses, such as production of stress proteins, up-regulation of antioxidants and accumulation of compatible solutes (vierling and kimpel 1992). in plant species which is tolerant to a stressing condition, the metabolic responses activated in response to the stress may contribute to the mechanism of tolerance. the production of heat-shock proteins (hsps) and chaperons (ingram and bartels 1996; bray et al. 2000), osmoprotectants and free-radical scavengers (bohnert and sheveleva 1998) are among the examples of substances that may involve in the protection of cell membranes and proteins. physcomitrella patens is a bryophyte which was reported to be highly tolerant to various abiotic stresses, in particular to salt, osmotic, and dehydration stresses (frank et al. 2005). genes ppshpl and ppshp2 in p. patens which demonstrate homology to rci2a and rci2b , respectively, of arabidopsis thaliana, encoding highly conserved small hydrophobic proteins, have been confirmed to be up-regulated by desiccation, salt, sorbitol, cold, and abscisic acid (kroemer et al. 2004), while an original dehydration related gene of p. patens, gfdd4j(genefishmg differential display clone 4-1), demonstrated to be activated additionally by cold and aba (ratnadewi and frank 2005). this indicated that p. patens has a number 49 corresponding author: dratnadewi@yahoo.com biotrop1a vol. 13 no. 1, 2006 of genes regulated by those diverse stress conditions conferring its high level ot tolerance, and whose pathways are overlapping and complementary one to the other. under light stress condition, sepl and sep2 encoded proteins (stress-enhanced proteins, seps) in a. thaliana have been detected to localize in thylakoid membrane of chloroplasts; they might function in one or another way as plant protector to high-intensity light and are not likely light harvesting (heddad and adamska 2000). by attaching the open reading frame of the gene gfdd4-1 to gfp marker gene, we attempted to investigate the localization of the encoded protein in p patens cells, the protein site may indicate its possible function in plant tolerance tc environmental stress. materials and methods isolation of cdnas this research employed copy dna of the gfdd4-1 gene. searching of cdism clones was carried out in the ppest database (basf-albert ludwigs university o: freiburg) (rensing el al 2002) using gfdd4-1 gene sequence as query. the cdnj* cloned in bacterial cultures have been retrieved as glycerol stocks from the clom depository. the clones were subject to pcr reactions, sequencing and selection foi the full length one. plasmid reconstruction the coding region of the selected cdna clone was amplified througl pcr reactions. the primers used were supplemented with bamhi at the 5 prime and with kpnl at the 3' prime: the forward primer wa 5'-ggatccatgaattccgagggtctt-3' and the reverse primer wa 5'ggtaccatgaccaccacgactattc-3' to obtain the expected dw fragment size of about 600 bp. the pcr product was subsequently loaded on ai agarose gel (1.0% wv"1) and the appropriate band was isolated using qiaexi purification kit for gel extraction (qiagen). the cloning of the dna fragment wa performed in pcr*4-topo* vector (invitrogen), and the dnas were then harvestei and purified through mini-preparation procedure (qiagen). when the concerne< fragment would be used as insert, digestion by bamhi and kpnl followed by dm; isolation and purification using qiaexii kit were executed again. plasmid pmav4 contains gfp gene and was selected as vector to fuse the dn/ fragment to the reporter gene. it was linearized at the bamhl and kpnl sites whei the dna fragment would be inserted (figure l).the ligation was executed with t-dna ligase (mbi fermentas). 35s promoter drove the expression of these tandei genes. 50 bacterial transformation escherichia coli competent cells clone xl1 blue mrf" (100 ul) were proceeded for transformation by inoculating the new pmav4 plasmid construct (2 to 8 ul). the bacterial mixture was incubated on ice (30 seconds), in water bath at 42°c (90 seconds) and on ice again (2 min). one millilitre of lb medium was added to the mixture prior to incubation in 37°c shaking water-bath for one hour. an amount of 100 ul of the transformed bacteria was plated on lb+amphycilin agar medium (sambrook and russel 2001). the rest was spinned down at 5000 rpm for 3 min; most of the supernatant was removed and the more concentrated solution was spread over a fresh medium of the same composition. bacterial colonies were expected to grow after an overnight incubation at 37°c. the appropriate ligated plasmid was isolated through dna maxi-preparation (qiagen) from a single colony derived cell suspension. protoplasts isolation and transformation moss plants at protonema stage were harvested from 200 ml bioreactor liquid culture, which corresponds to about 10 mg dry weight. aseptically the moss was proceeded for protoplasts isolation according to the protocol described by rother et al. (1994) with slight modification. in a glass tube, 100 ul of dna solution (0.5 ug ul~' in ca(no3)2) was added to 250 ul of protoplast solution (1.2 x 10* protoplasts ml"1); 350 ul of peg 4000 solution (40%) was then incorporated into the mixture. it was mixed gently by rolling the tube between fingers. the subsequent procedure followed exactly the method cited in hohe et al. (2003). the transformed protoplasts were re-suspended in 3 ml of regeneration medium. it comprised 0.25 gl"1 kh2po4, 0.25 el'1 kc1, 0.25 gr1 mgs04-7h20, 1 gr 1 ca(n03)2, 12.5 mgl' 1 feso4-7h20, 50 gp and 30 gl' 1 mannitol. the ph of the medium was adjusted to 5.8 with koh and its osmolarity to 51 biotropia vol. 13 no. 1, 2006 approximately 540 mos using mannitol. the protoplast solution (1.5 ml) was transferred into 3 cm-well of culture plate. the protoplast cultures were incubated under dim-light at 25°c. observations were carried out at 1,2, 4, and 8 days after transfection. transgenic cells were detected visually under a uv light microscope. when the expected green fluorescent intact cells were visible, the images were taken in more detail under a confocal laser scanning microscope. results and discussion cdna isolation and plasmid reconstruction from searching in the ppest database with gfdd4-1 gene sequence as query, three hits came out from there, e.g. clones no. ppoo1088038, pp004071329 and pp004083128. the three cdna clones were retrieved from the depository to proceed to pcr for amplification using standard primers ml3-20 and m13-rev. dna sequencing revealed that the clone pp004071329 is the full-length cdna and was used further in this work. by using bamhl and kpnl, insert of 600 bp dna fragment and linearized pmav4 were obtained prior to their ligation to a new construct. figure 2 demonstrates the pcr products resulted from these digestions. gfdd4-l/gfproteins in transformed cells one day after the transfection, few green protoplasts were already seen under uv-light microscope, and they looked multiplying in number and intensity over the days of observation. the green fluorescence was assumed to be the protein expressed by the gf'p gene that was closely associated to the gfdd4-1. it has been proven that the intrinsic fluorescence of gfp allows for non-invasive and monitoring to track the expression and location of proteins and other structures within cells or organism without killing or destroying the biological samples. this makes gfp efficient as a transformation marker. at the first till the fourth day, the green structures scattered unevenly along the periphery of the cells (figure 3). chloroplasts are represented by the red auto fluorescence; it was obvious that the protein was not generated in the plastid membrane. when a barrier filter was used to block the red fluorescence of chlorophyll, typical green fluorescence appeared more clearly. at the fourth and eighth day, the protein was more abundant, extending into the cytoplasm, forming vesicles and cysternal structures in some cases, and in some other cells it formed a network. 52 abiotic stresses such as drought, salinity, cold, or heat are often interconnected and cause disruption of osmotic and ionic homeostasis as well as damage of functional and structural proteins and membranes (wang et al. 2003). plant responses to abiotic stress are quite complex due to the fact that, in one hand, it involves many genes and biochemical-molecular mechanisms, on the other hand, different stressing stimuli can induce only a single gene. in their response to environmental stresses, plants activate a large set of genes leading to the accumulation of specific stress-associated proteins. the product of stress-related genes can be classified into two major categories: 1). those that directly involve in the cellular protection against environmental stresses, such as hsp and lea proteins and chaperons, various osmo-protectants and detoxification enzymes, 2). those that play roles in signalling cascades and in transcriptional control, such as transcription factors and protein kinase (seki el al. 2003; wang ef a/. 2003). upon water, salinity, and/or extreme temperature stress, plants produce predominantly hsps and lea proteins. hsp70s are found in several cellular compartments such as in cytoplasm, in the lumen of endoplasmic reticulum (er), in the matrix of mitochondria, as well as in chloroplasts. they have been shown to act as molecular chaperons that function in the stabilization of proteins and membranes, and in assisting protein refolding under stress conditions (vierling 1991). accordingly, in this preliminary work, the gfdd4-1 protein associated to the gfp was observed spreading intensively over the cell. the protein in the peripheral area and the structures of vesicles and cysternae exhibited that the protein might be membrane protein which may involve in membrane maintenance or cellular protection against stress conditions. conclusions the gfdd4-1, a dehydration-related protein in physcomitrella patens, locates at cell membranous system. it may function directly or indirectly in cellular protection against environmental stresses. 54 localization of gfdd4-1 expressed protein d. ratnadewi acknowledgement the author would like to gratefully acknowledge the german academic exchange service (daad) for the great opportunity given to do a part of her research in germany. high appreciation also goes to prof. dr. ralf reski and dr. wolfgang frank who permitted me to join their research group at the institute of plant biotechnology, albert-ludwigs university of freiburg. references bohnert, h.j. and e. sheveleva. 1998. plant stress adaptations making metabolism move. curr. opin. plant biol., 1:267274. bray, e.a., j. bailey-serres and e. weretilnyk. 2000. responses to abiotic stresses. in: gruissem w., buchanan b., jones r. (ed). biochemistry and molecular biology of plants. rockville md: amer. soc. plant physiologists, p. 11581249. frank, w., d. ratnadewi and r. reski. 2005. physcomilrella patens is highly tolerant against drought, salt and osmotic stress. planta, 220:384-394. heddad, m. and i. adamska. 2000. light stress-regulated two-helix proteins in arahidopsis thaliana related to the chlorophyll a/2>-binding gene family. pnas 97(7):3741-3746. hohe, a., t. egener, j.m. lucht, h. holtorf, c. reinhard, g. schween and r. reski. 2003. an improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, physcomitrella patens. curr. gen., 44:339-347. ingram, j. and d. bartels. 1996. the molecular basis of dehydration tolerance in plants. annu. rev. plant biol., 47:377-403. kroemer, k., r. reski and w. frank. 2004. abiotic stress response in the moss physcomitrella patens: evidence for an evolutionary alteration in signaling pathways inland plants. plant cell rep., 22:864-870. ratnadewi, d. and w. frank. 2005. ekspresi gen gfdd4-1 pada physcomitrella patens dan gen homolog pada arahidopsis thaliana dalam responsnya terhadap cekaman abiotik. hayati, 12(4):127-130. rensing, s.a., s. rombauts, y. van de peer and r. reski. 2002. moss transcriptome and beyond. trends plant sci., 7:535538. rother, s., b. hadeler, j.m. orsini, w.o. abel and r. reski. 1994. fate of a mutant macrochloroplasts in somatic hybrids. j. plant physiol., 143:72-77. sambrook, j. and d.w. russel. 2001. molecular cloning: a laboratory manual 3"' ed. cold spring harbor: cold spring harbor laboratory press. scki, m., a. kamei, k. yamaguchi-shinozaki and k. shinozaki. 2003. molecular responses to drought, salinity and frost: common and different paths for plant protection. curr. opin. biotech., 14:194-199. vierling,e. 1991. the roles of heat-shock proteins in plants. annu. rev. plant biol., 42:579-620. vierling, e and j.a. kimpel. 1992. plant responses to environmental stress. curr. opin. biotech., 3:164-170. wang, w., b. vinocur and a. altaian. 2003. plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. planta, 218:1-14. 55 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf biotropia no biotropia no. 17,2001 : 9 17 characterization of three benzoate degrading anoxygenic photosynthetic bacteria isolated from the environment dwi suryanto1, antonius suwanto2'3*, and anja meryandini3 ; dept. of biology, faculty of science and mathematics, north sumatra university, medan, indonesia 2 south east asian regional center for tropical biology (seameo-biotrop), bogor, indonesia 3 dept. of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract three anoxygenic photosynthetic bacteria, ds-1, ds-4 and cas-13, have been examinated for their morphological and physiological properties. all strains were rod-shape cells with a swollen terminal end, gram negative, motile, non-halophilic, non-alkalophilic and non-acidophilic, and capable of utilizing benzoate aerobically and photo-anaerobically. sequence analysis of part of 16s rrna genes showed that ds1 and cas-13 were closely related to rhodopseudomonas palustris strain 7 with a similarity of 97%, whereas ds-4 may not be closely related to the former two strains with a similarity of 78% based on the constructed phylogenic tree. spectral analysis indicated that the three bacteria had bacteriochlorophyl a and normal spirilloxanthin series. growth in medium enriched with vitamin and supplemented with benzoate as their sole c-sources was better than in medium without vitamin. benzoate degradation in medium with vitamin was accelerated. the ability to grow on benzoate without added vitamins indicated that the bacteria were able to synthesize their own vitamins. key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16s rrna gene. introduction some toxic compounds are slowly degraded in polluted aerobic zones and, therefore, may leach into anaerobic subsurface environments (kohring et al. 1989). anaerobic degradation of such compounds may play an important role in eliminating toxic substances. anaerobic bioremediation has been proposed as an inexpensive method for in situ removal of organic contaminants in the environment (kuo and genthner 1996). recent concern about the environmental fate of industrially produced organic compounds has prompted a resurgence of interest in the anaerobic degradation of aromatic compounds (harwood and gibson 1988). anoxygenic photosynthetic bacteria (apb) are nutritionally versatile in their ability to utilize diverse sources of carbon ranging from simple aliphatic organic acids to complex polysaccharides (hiraishi et al. 1995). the occurrence of these bacteria as common inhabitants in aquatic and some terrestrial habitats in nature ' corresponding author: e-mail address : asuwanto@indo.net.id 9 biotropia no. 17, 2001 (hiraishi et al. 1995) might be explained partly by the fact that these bacteria survive on various modes of energy-generating systems (hiraishi et al. 1995). the ability of phototrophic purple non-sulfur bacteria to grow aerobically and anaerobically in the presence of diverse aromatic compounds makes these good candidates for potential biodegradation of harmful compounds (hanvood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). commercialization of these bacteria for purification treatment plants was initiated about 15 years ago (kobayashi and kobayashi 1995). not many phototrophic purple non-sulfur bacteria species are known to degrade aromatic compounds. rhodopseudomonas palustris (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994), rhodomicrobium vannielii (wright and madigan 1991), rhodobacter capsulatus (blasco and castillo 1992; shoreit and shaheb 1994), rs. blastica, and rhodospirilium rubrum (shoreit and shaheb 1994) are able to degrade a variety of monocyclic aromatic compounds with or without other c-sources. benzoate and its derivatives are among the common aromatic compounds that can be completely mineralized (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). however, blasco and castillo (1992) noted that rhodobacter capsulatus e1f1 is able to degrade mononitrophenol and dinitrophenol with acetate as its carbon source. rs. palustris utilized several phenolic compounds, hydroxylated and methoxylated aromatic acids, aromatic aldehydes, and hydroaromatic acids (harwood and gibson 1988). other diverse aromatic compounds have also been reported to be utilized by this group (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). degradation of aromatic compound by phototrophic purple non-sulfur bacteria in general was emphasized in this investigation based on their ability to utilize diverse aromatic compounds. to date, this group of bacteria has not been subjected to intensive examination (harwood and gibson 1988). this study focused on the ability of three new isolates of apb to utilize benzoate. identification of the bacterial isolates based on their 16s-rrna genes as well as their morphological and physiological properties was also carried out. materials and methods bacterial cultures and cultivation three isolates of apb, designated as ds-1, ds-4, and cas-13 were studied. the two former strains were isolated from java, and the last was isolated from moluccas. all isolates were maintained on modified sistrom medium with benzoate as the sole carbon source. to determine the ability of the bacteria to utilize and degrade aromatic compounds, the isolates were grown in modified sistrom by omitting all carbon sources, including nitrilo-triacetic acid, supplemented with 5 mm benzoate as the c 10 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. source with or without vitamins in 100 ml completely filled screw-capped tubes. isolates also were grown in modified sistrom supplemented with vitamins and 5 mm succinate, or 5 mm acetate as their carbon sources to serve as a means of comparison. for cultures grown on benzoate, the inocula were obtained from 3-day old cultures of bacteria grown in modified sistrom with benzoate with an initial cell number of 5xl06 cells/ml. for cultures to test other c-sources, inocula were taken from 2-day old cultures grown in sistrom media with succinate as c-source. unless mentioned otherwise, all media were adjusted to ph 7.2. growth condition, measurement of growth, and quantitation of benzoate cultures were illuminated with a 40 w tungsten bulb at a distance of 30 cm. growth rates were determined by measuring turbidity at 660 nm every 24 hours. for cultures grown in 5 mm succinate and 5 mm acetate, growth rates were measured every 12 hours. for cultures grown in benzoate, residual benzoate levels were measured at 120 hours of incubation. benzoate concentration was measured at 276 nm using a hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) according to shoreit and shabeb (1994). cell density of the isolates grown in other c-sources was measured after 72 hours of inoculation. spectral analysis cells were harvested from 7-day old anaerob-phototrophic cultures grown on modified sistrom supplemented with vitamins and 5 mm succinate as sole carbon source, and suspended in icm buffer (10 mm phosphate buffer ph 7.0 and 1 mm naedta ph 7.0). sonication was carried out using soniprep 150 (msb, uk) at an amplitude of 2 u. for 2 minutes, three times with a time interval of 1 minute. cell extracts were centrifuged at 3000 rpm for 30 minutes at 4°c. protein concentration was determined according to the pierce bca* protein assay kit (rockford, iii, usa). spectral analysis was performed using hitachi u-2010 in protein concentration of ± 100 (ig/ml. amplification and sequencing of part of 16s rrna genes to sequence part of the 16s-rrna genes, the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna (200 ng) on readyto-go pcr beads (pharmacia-biotech, uppsala, sweden). phenol-chloroformisoamylalcohol (25:24:1) treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. total volume of the pcr reaction (25 ul) consisted of 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in a gene amp pcr system 2400 thermocycler (perkin-elmer cetus, norwalk, conn.). 11 biotropia no. 17, 2001 part of 16s-rrna gene was sequenced to infer the closest related organism from the ribosomal database project (rdp) maintained at the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purified by ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (pe applied biosystems, foster city, ca.). construction of phylogenetic trees for the construction of phylogenetic trees, cluster analysis of 16s-rrna gene was done by a computer program from the european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of the department of biochemistry, university of antwerp) was used to determine their phylogenetic relatedness based on their nucleotide sequences and mflp profiles obtained from pulsed-field gel electrophoresis. results and discussion the three new isolates described in this study were similar in their morphological properties. all were gram negative, non-halophilic, non-alkali or acidophilic, aerobic and anaerob-phototrophs that have motile rod-shaped cells with terminal swellings. under anaerobic photothrophic growth conditions, all new isolates produced brick-red and pink cultures in succinate and benzoate, respectively. spectral analysis of cell free extracts (figure 1) of photosynthetic pigments showed absorption maxima for ds-1 at 374, 493, 587, 803 and 852 nm, ds-4 at 371, 501, 585, 803 and 848 nm, and cas-13 at 373, 502, 585, 802 and 871 nm indicating the presence of bacteriochlorophyl a and normal spirilloxanthin series including lycopene and rhodopin (imhoff 1995). in this respect, the three isolates were nearly identical to each other and might be considered as one group. however, these isolates clearly differed from rhodobacter sphaeroides 2.4.1., which has different absorption maxima (376, 451, 453, 477, 507, 587, 746, 799, and 850 nm), with yellow-brown colonies grown anaerobically in succinate. the results from the analysis of partial sequencing of 16s-rrna (c.a. 500 bp) of ds-4, ds-1, and cas-13 demonstrated that ds-4 and cas-13 were closely related (figure 2). partial sequencing of the first 500 bp of 16s rrna gene could lead to a main line of descent (stackebrandt and rainey 1995). comparison with the rdp database of the university of illinois indicated that both ds-1 and cas-13 demonstrated 97% similarity with rs. palustris strain 7, and of 83% and 84% with rb. sphaeroides il106, respectively (table 1). similarity of those of ds-4 to rs. palustris strain 7 was 78%. these results suggested that ds-1 and cas13 are likely to be one species, rs. palustris. ds-4, however, is significantly different from ds-1 and cas-13 by 22%, and from rb. sphaeroides il106 by 30%. complete sequence analysis is needed to give more definitive information about the taxonomic position of these three isolates. 12 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 1. absorption spectrum of cell extracts ds-1, ds-4, cas-13, and rb. sphaeroides 2.4.1. figure 2. phylogenetic tree 16s rrna gene sequences. the number at the tree lines represented bootstrap values. 13 biotropia no. 17, 2001 table 1. percent similarity of the ds-1, ds-4, and cas-13 with other relative members of anoxygenic photosynthetic bacteria. unlike rb. sphaeroides 2.4.1, all of the three isolates were able to metabolize benzoate (figure 3). among the members of apb, rs. palustris is the most common species capable of utilizing benzoate (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994). the ability to grow in the presence of aromatic compounds such as benzoate without vitamins (figure 3) may suggest that the organisms were able to synthesize their own vitamins. however, supplemented vitamins could increase their potential in metabolizing benzoate. the rate of benzoate utilization for ds-1, ds-4, and cas-13 were 0.024 mm/hour, 0.02 mm/hour, and 0.03 mm/hour in media with vitamin supplements compared to 0.017 mm/hour, 0.012 mm/hour, and 0.018 mm/hour in media without vitamins. a relatively similar pattern of extended lag phase was observed in growth of the cultures. time was needed in preparation of producing a number of enzymes. carbon availability might affect cell growth. cell density was observed to be higher in media with benzoate (c7) (figure 3), followed by succinate (c4) and acetate (c2) (figure 4). relatively low cell density of all isolates was shown in 5 mm benzoate with no vitamins. vitamins were certainly necessary for their metabolic activity. 14 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 3. growht of ds-01, ds-4 and cas-13 in 5 mm benzoate with vitamins (above) and without vitamins (below). rb. sphaeroides 2.4.1. was used as negative control. 15 biotropia no. 17, 2001 figure4. growth of ds-1, ds-4, cas-4, cas-13, and r6. sphaeroides 2.4.1. in 5 mm succinate (above) and 5 mm acetate (below) with vitamins. 16 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. acnowledgement this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor indonesia. references blasco, r. and f. castillo. 1992. light-dependent degradation of nitrophenols by the phototrophic bacterium rhodobacter capsulatus e1f1. appl. environ. microbiol. 58: 690-695. gibson, k. j and j. gibson. 1992. potential early intermediates in anaerobic benzoate degradation by rhodopseudomonaspalustris. appl. environ. microbiol. 58: 696-698. harwood, c. s. and j. gibson. 1988. anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium rhodopseudomonas palustris. appl. environ. microbiol. 54: 712-717. hiraishi, a., k. muramatsu, and k. urata. 1995. characterization of new denitrifying rhodobacter strains isolated from photosynthetic sludge for wastewater treatment. j. fermen. bioengine. 79: 39-44. imhoff, j. f. 1995. taxonomy, phylogeny and general ecology of anoxygenic phototrophic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1-15. kobayashi, m. and m. kobayashi. 1995. waste remediation and treatment using anoxygenic photosynthetic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1269-1282. kohring, g., x. zang, and j. wiegel. 1989. anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate. appl. environ. microbiol. 55: 2735-2737. kuo, c. and b.r.s. genthner. 1996. effect of added heavy metal ions on biotransformation and biodegradation of 2chlorophenol and 3-chlorobenzoate in anaerobic bacterial consortia. appl. environ. microbiol. 62: 23172323. shoreit, a. a. m. and m. s. a. shaheb. 1994. utilization of aromatic compounds by phototrophic purple nonsulfur bacteria. biodegrad. 5: 71-76. stackebrandt, e. and f.a. rainey. 1995. partial and complete 16s rdna sequences, their use in generation of 16s rdna phylogenetic trees and their implications in molecular ecological studies. in molecular microbial ecology manual. ed. akkermans, a.d.l., j.d. van elsas, and f.j. de bruijn. pp. mmem -3.1.1/1-17. wright, g. e. and m. t. madigan. 1991. photocatabolism of aromatic compounds by the phototrophic purple bacterium rhodomicrobium vannielli. appl. environ. microbiol. 57: 2069-2073. 17 isolation and production of extracellular enzyme from mannanolytic thermophilic bacterium originally from lampung isolation and characterization of mannanolytic thermophilic bacteria from palm oil shell and their mannanase enzyme production properties biotropia no. 25, 2005 : 1 – 10 sumardi1, antonius suwanto2,, maggy thenawidjaja3, and tresnawati purwadaria4 1department of biology, faculty of science and mathematics, university of lampung, jl. s. brojonegoro no. 1, bandar lampung 35145, indonesia 2department of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor 16144, indonesia 3department of food science and human nutrition, bogor agricultural university, darmaga, 16680, bogor, indonesia 4indonesian research institute for animal production, p.o. box 221, bogor 16002, indonesia abstract a mannanolytic thermophilic bacterium (l-07) was isolated from palm oil shell after 2 days of enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source. sequence analysis of 16s-rrna indicated that l-07 was similar (98%) to geobacillus stearothermophilus, a species of thermophilic aerobic bacteria. we found that g. stearothermophilus l-07 produced extracellular β-1,4-mannanases, but no β-manosidase and α-galactosidase activities. the growth of l-07 reached its maximum (3.0 x 106 cell/ml) at 12-20 hours, while the highest β-mannanase activity (0.52 u/ml) was observed in culture medium after 36 hours of cultivation at 60oc. the medium containing locust bean gum was the best for producing extracellular β-1,4-mannanases compared with kolang kaling, konjak, and palm kernel meal. sds-page and zymogram analysis demonstrated that crude mannanase complex of l-07 from locust bean gum containing medium comprised three active bands with molecular weight of 85, 73 and 50 kda. keywords : extracellular enzyme/mannanase/geobacillus stearothermophilus introduction hemicelluloses are the second most abundant polysaccharide in nature after cellulose. the major constituents of hemicellulose are the hetero-1,4-β-d-xylans and hetero-1,4-β-d-mannans (galactoglucomannan, galactomannan, and glucomannan). the heteroxylans are found mainly in grasses, cereals, and hardwoods (angiosperms). the mannans are more abundant in copra, palm, coffee, and locust bean endosperms (araujo and ward 1990). mannanolytic microbes were found in soil, compost, and animal rumen (zakaria et al. 1998). biodegradation of ß-mannans is caused by β-mannanase (1,4-β-d mannan manohidrolase [ec 3.2.1.78]) produced from bacteria and fungi. the enzyme hydrolyses the ß-(1,4) linkages in backbone of mannan polymer, ∗corresponding author : asuwanto@indo.net.id 1 producing short chain mannoligosaccharides. then, these compounds can be further degraded by the action of β-mannosidase (β-d-mannosidase [ec 3.2.1.25]) and a αgalactosidase (ec 3.2.1.22) (duffaud et al. 1997). mannan degradation from glucomannan and galactomannan produces manno-oligosaccharide, mannobiose, and mannose. mannan-degrading enzymes can be used for numerous applications in food, feed, pulp, and paper industries. biotropia no. 25, 2005 in the palm oil factory, during the composting process of palm shells containing hemicellulose the temperature rises to 65oc. at such temperature, many thermophilic bacteria are able to develop special properties to survive and prosper in the habitat. the purposes of this study were to isolate the mannanolytic thermophilic bacterium from palm shells, a solid waste of palm oil factory in lampung, and to study its ß-mannanase production. materials and methods materials palm shell as source of thermophilic bacteria was obtained from palm oil industry in natar, lampung at 65oc. locust bean gum (galactomannan) was obtained from sigma chemical company. other chemicals were analytical grade from merck industry. isolation of thermophilic mannanolytic strains one milligram of palm shells was enriched in a medium containing 0.2% yeast extract; 0.2% trypton, 1.0% palm kernel meal, 0.02% mgso4, 0.14% kh2po4, and 0.1% (nh4)2so4 at 70 oc for two days with agitation (120 rpm). one hundred microliters of each enrichment culture was spread aerobically on the same medium onto agar plates containing 0.3% locust bean gum instead of palm kernel meal. thermophilic mannanolytic isolates showed a clear zone around the colony after staining with a solution of 0.1% congo red for 15 minutes and destaining through repeated washing with 1 m nacl. characterization and identification of isolate morphological properties and taxonomic characteristics of the best isolate were studied according to the methods as described in “bergey’s manual of systematic bacteriology” (holt et al. 1994). identification of the isolate was determined through 16s rrna sequence analysis. genomic dna extraction was performed using the ctab method. pcrmediated amplification of the 16s rrna was performed as described by marchesi et al. (1998) employing geneamp pcr system 2400 (perkin elmer). pcr product was purified and sequenced. cluster analysis was conducted according to a program 2 provided by european bioinformatics institute (http://www. ebi.ac.uk) and phylogenetic tree was constructed using treecon software (peer and watcher 1993). isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. extracellular enzyme production the best bacterial isolate was grown in the medium for mannanase production which contained 0.35% yeast extract, 0.35% trypton, 0.035% mgso4, 0.245% kh2po4, 0.175% (nh4)2so4, 0.2% nacl, and 0.65% locust bean gum (ph 7.0). locust bean gum was replaced with other carbon sources (kolang kaling, konjak, palm kernel meal) when the effect of carbon sources on the enzyme production was examined. a 250-ml flask containing 50 ml of the medium was inoculated with a loopful of cells taken from a stock slant and was precultured at 60oc on shaker (120 rpm) for 6-8 hours. the same volume of medium and flasks for enzyme production were inoculated with 2 ml of this culture and cultivated at 60oc for 48 h. aliquots of the culture medium were sampled at 4 h intervals to determine β-mannanase activity and viable bacterium number. intracellular enzyme extraction g. stearothermophilus l-07 cells were concentrated from 50 ml liquid medium by centrifugation at 3,200 x g for 10 min. at 4oc. the cell pellets were resuspended in 1 ml of ice-cold condition containing 50 mm phosphat buffer. the cell lysis process used sonication with a soniprep 150 (usa). the sonicator was set to 16 micron amplitude for 5 minutes. intracellular enzymes were detected specifically βmannanase, β-mannosidase, and α-galactosidase. enzyme assay β-mannosidase and α-galactosidase activities were determined by monitoring the release of p-nitrophenol from p-nirophenyl b-d-mannopyranoside or pnitrophenyl α-galactophyranoside (sigma chemical co., usa), respectively. for each assay, 0.9 ml aliquots of 1 mm substrate in 50 mm sodium phosphate buffer (ph 7.0) and 0.1 ml of enzyme were mixed and incubated at 80oc for 30 minutes and the reaction stopped by the addition of 0.1 ml solution of 0.4 m na2co3. the release of p-nitrophenyl (pnp) was measured spectrophotometrically by monitoring the changes in absorbance at 405 nm. control was prepared with the addition of enzyme after na2co3 solution. one unit of β-mannosidase or α-galactosidase activity was defined as the amount of enzyme releasing 1 μmol pnp per min. under the specified assay condition. β-mannanase activity was determined by monitoring the release of reducing sugars. the reaction mixture, containing 0.5% locust bean gum, 50 mm sodium phosphate buffer (ph 7.0), suitably diluted enzyme solution in a total volume of 1 ml, was incubated at 80oc for 30 minutes. the reducing sugar content was determined by dinitrosalisylic acid method. one unit of enzyme was defined as the 3 amount of enzyme producing 1 μmol of mannose per minute under the given assay condition. biotropia no. 25, 2005 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and zymogram sds-page was performed in 8% polyacrylamide gels. protein bands were visualized after staining with silver nitrate. zymogram was prepared using 0.1% locust bean gum co-polymerized with polyacrylamide. after separation, sds was removed by method of spindler and rapp (1997). then, the gel was equilibrated through several washings and finally immersed in 50 mm sodium acetate buffer (ph 6.0), followed by incubation at 65oc for 35 minutes, staining with 0.1% congo red for 15 minutes, and destaining in 1 m nacl. results and discussion we found 15 bacterial isolates from palm oil shell. then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oc with ph 7.0. only isolate l-07 showed mannanase activity. the isolate l-07 could grow at 55-70oc and showed mannanase activity as high as 3.1 u/mg. a. b. figure 1. bacterial isolate l-07. a. gram-positive rod-shaped cells were cultured at 65oc for 1 day. b. cells produced endospore in terminal position after 5 days of incubation. table 1 shows the morphological and physiological characteristics of l-07. l07 was gram-positive (figure 1.a), aerobic, catalase-positive, and endosporeforming bacterium (figure 1.b). it was non-motile and rod-shaped (1-1.2 x 5-5.7 μm). based on these characteristics l-07 belonged to the genus bacillus. 4 table 1. morphological and physiological characteristics of l07 isolate isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. characteristics strain l-07 shape rod endospora shape oval/cylindrical endospora position terminal gram stain positive catalase positive motility negative growth at 37oc negative growth 55 – 75oc optimum growth temperature 60-70oc cell size (μm) 1,2x5,7 nitrate reduction positive indole production negative urease positive voges proskauer test negative utilization as sole carbon source of glucose positive mannose positive xylose positive sucrose positive arabinose positive ramnose negative citrate negative lactose negative mannitol negative sorbitol negative adonitol negative rafinose negative hydrolysis of mannan positive hydrolysis of carboxymethyl cellulose positive hydrolysis of starch negative hydrolysis of xylan negative then, the total sequence of 1315 bp of 16s rrna gene of l-07 showed 98% similarity to that of geobacillus stearothermophilus. the constructed phylogenetic tree for l-07 is shown in figure 2. g. stearothermophilus (formerly bacillus stearothermophilus) was a highly resistant thermophilic organism. major strains of the genus geobacillus live in geothermal areas, such as the oil field subsurface and hydrothermal vents (nazina et al. 2001; and euzeby 2005). at this time, no study has reported on the presence of mannanolytic thermophilic bacteria from palm shell. as comparison aurora et al (2003) showed that bacillus pumilus dyp2, a mannanolytic mesophilic bacteria, was isolated from west sumatra copra soil sample, indonesia. another bacterium, b. stearothermophilus atcc 12016 has been previously reported to produce thermostable β-mannanase (ethier et al. 1998). 5 figure 2. phylogenetic tree construction for l-07 isolate. bootstrap values are shown at each node tree. when g. stearothermophilus l-07 produced thermophilic mannanase, the enzyme was observed primarily in culture media. the other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were not detected in extracellular enzyme preparation (table 2). these enzymes might act to provide simple sugar to geobacillus stearothermophilus l-07 growing on locust bean gum as base media. regarding the cellular localization of these three activities, we speculate that the mannan backbone was cleaved by endo-acting β-1,4 mannanase prior to the transport of smaller galactomannans to the site of the other two enzymes. the other two enzymes, exo-acting α-1,6 galactosidase and β-1,4 mannosidase, are found in the cell membrane. as comparison, duffaud et al. (1997) showed that extracellular β-1,4 mannanase of thermotoga neopalitana 5068 was sevenfold greater than the cell extract. the other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were found in the cell the extract. table 2. the specific activity of extracellular and intracellular mannanolytic enzymes from geobacillus stearothermophilus l-07 cultured at 60oc for 36 hours specific activity (u/mg) no kind of enzyme intracellular extracellular 1 β-1,4-mananase 0.02 ± 0.002 3.10 ± 0.050 2 β-1,4-manosidase 0.03 ± 0.003 0.00 ± 0.000 3 α-1,6-galactosidase 24.1 ± 0.060 0.00 ± 0.000 72 93 92 100 100 pyrococcus furiosus subsp. woesei alicyclobacillus acidocaldarius mih321 g. stearothermophylus bgsc 9a21 l-07 10% biotropia no. 25, 2005 e. coli thermus aquaticus yt-1 c. botulinun kyto-f l. delbrueckii subsp. bulgaricus atcc11842 b.subtilis wl-7 100 50 b. thermoleovorans ccr11 85 6 we also observed the growth and extracellular β-mannanase activity of g. stearothermophilus l-07 as shown in figure 3. after 12-20 hours of observation strain l-07 reached its maximum growth, while the highest β-1,4-mannanase activity was found in culture medium after 36 hours of cultivation. β-1,4-mannanase may be associated with the cell membrane. so, after g. stearothermophilus cells died β-1,4-mannanase leaked out from membrane and increased the activities. besides for mannan degradation to produce manno-oligosaccharide, this thermostable enzyme can be used in the bleaching process (ethier et al. 1998). the other β-mannanase from bacillus sp. was active at 70oc. the enzyme was stable when incubated for 1 h at ≤ 60oc (ooi and kikuchi 1995). while the βmannanase from rhodothermus marinus retained 87% of its initial activity after 1 h at 90oc (politz et al. 2000). 4,5 5 5,5 6 6,5 7 0 4 8 12 16 20 24 28 32 36 40 44 48 time (h) l og c el l n um be r/ m l 0 0,1 0,2 0,3 0,4 0,5 0,6 m an na na se a ct iv ity (u /m l) figure 3. growth curve and extracellular mannanase activity of geobacillus stearothermophilus l-07. strain l-07 was cultured at 60oc for 48 hours in the enzyme production medium. -■ logarithmic viable cell number and -οmannanase activity. we studied the effect on the kind of carbon source on β-1,4 mannanase production. the bacteria produced active β-1,4 mannanase when they were grown in a kind of carbon source as medium (table 3). table 3. effect of carbon sources on the mannanase production. g. stearothermophilus l-07 was cultured at 60oc for 36 hours. no carbon sources relative activity (%) 1 none 2 ± 0.01 locust bean gum 2 100 ± 0.04 kolang kaling (endosperm from arenga pinata) 46 ± 0.06 3 konjak 4 13 ± 0.05 palm kernel meal 0 ± 0.00 5 isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. 7 the enzyme activity in the medium containing locust bean gum was the highest, followed by that in kolang kaling, and konjak. no activity was observed in palm kernel meal. thus, the best induction medium consisted of locust bean gum. it may be pointed out that the mannanase activity is preferably induced by polysaccharides containing mannose or galactose as monomeric unit, and the induction is strongly increased by heterogeneous polysaccharides containing mannose and galactose. locust bean gum consists of 88% galactomannan (whistler and bemiller 1973). the composition may support the increase of mannanase production. so far, there was no accurate information on mannan composition in kolang kaling and palm kernel meal. however, they are found in palm seeds where galactomannans are major part of their dry weight. biotropia no. 25, 2005 konjak mannan consist of 50-60% glucomannan containing mannose and glucose. as comparison, torrie et al. (1990) reported that the highest mannanase production was found from mold of trichoderma harzianum e58 in the medium containing locust bean gum (26.7 u/mg), followed by konjak (glucomannan) (7.5 u/mg). zakaria et al. (1998) showed that the enzyme activity from flavobacterium sp. in the medium containing guar gum was the highest, followed by locust bean gum. in another research, hossain et al. (1996), showed that bacillus sp. kk01 in coconut meal resulted in mannanase activity of 0.04 u/mg. in non-mannan medium, 2% relative activity of mannanase was still found. in spite of non-mannan carbon source addition, the base medium might contain small amount of mannan from yeast extract. the mannan from yeast extract could induce mannanase production. there was no mannanase activity in media containing mannan from palm kernel meal. when g. stearothermophilus l-07 was isolated by enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source, the bacterium might survive due to synergism with other bacteria. furthermore, g. stearothermophilus l-07 could not survive alone if it was cultured in palm kernel meal medium. according to sumardi (2005), the palm kernel meal produced inhibiting substance after sterilization using an autoclave. thus, g. stearothermophilus l-07 could neither grow nor produce β-mannanase. the enzyme from g. stearothermophilus l-07 in the locust bean gum was further run on sds-page and analyzed by zymogram assay towards locust bean gum. three bands were observed at molecular mass of approximately 85, 73, and 50 kda, respectively (figure 4). the presence of three isozymes indicated that at least three mannanase encoded genes are present in l-07. other g. stearothermophilus strains have been previously reported to produce mannanase with molecular mass of 76 kda and active at 70oc (ethier et al. 1998). 8 m cr z isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. kda 85 kda 73 kda 50 kda 94 67 43 30 20 14 figure 4. zymogram of crude mannanase from g. stearothermophilus l-07. lane m indicates the molecular size standard used in this study. lane cr indicates crude enzyme mannanase separated in sds-page. lane z indicates bands of mannanase activity detected by zymogram. conclusions we isolated g. stearothermophilus l-07 from palm shell, a solid waste of palm oil factory in lampung. the bacterium produced extracellular β-1,4mannanase in locust bean gum medium, while β-mannosidase and α-galactosidase were not detected. the optimum enzyme activity was observed after 36 hours of incubation at 60oc. the mannanase comprised three active bands with molecular weight of 85, 73, and 50 kda. acknowledgements this research was financially supported by bpps fellowship from national education department and central microbial diversity of bogor agriculture university. references araujo, a & o.p. ward. 1990. extracellular mannanases and galactanases from selected fungi. j. indust. microbiology. 6: 171-178 aurora d d, y. lestari, & a. meryandini. 2003. identifikasi bakteri penghasil mananase serta karakterisasi enzimnya. j. mikrobiol. indon. 8(1) : 31-33 duffaud,g.d, c.m. mccutchen, p. leduc, k.n. parker, & r.m. kelly. 1997. purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and α-galactosidase from 9 the hyperthermophilic eubacterium thermotoga neopalitana 5068. appl. environ. microbiol. 63: 169-177. biotropia no. 25, 2005 euzeby. 2005. list of prokaryotic names with standing in nomenclature. last full update september 14, 2005. url: http://www.bacterio.net. ethier n, g. talbot, & j. sygusch. 1998. gene cloning, dna sequencing, and expression of thermostable β-mannanase from bacillus stearothermophilus. appl. environ. microbiol. 64: 4428-4432. holt jg, nr krieg, pha sneath, jt staley, & st williams. 1994. bergey,s manual of determinative bacteriology. ninth edition. baltimore : william and wilkins. hossain, h.z., j. abe, & s. hizukuri. 1996. multiple forms of β-mannanase from bacillus sp. kk01. enzyme microb. technol. 18 :95-98. marchesi, j.r.,t. sato, a.j. weightman, t.a. martin, j.c. fry, s.j. hiom, d. dymock, & w.g. wade. 1998. design and evolution of useful bacterium spesific pcr primers that amplify genes coding for bacterial 16s-rrna, j. bacteriol. 64: 795-799. nazina t n, t p tourova, a b poltaraus, e v novikova, a a grigoryan, a e ivanova, a m lysenko, v v petrunyaka, g a osipov, s s belyaev, & m v ivanov. 2001. taxonomyc study of aerobic thermophilic bacilli : description of geobacillus subterraneus gen. nov., sp. nov. and geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of bacillus stearothermophilus, bacillus thermocatenulatus, bacillus thermoleovorans, bacillus kaustophilus, bacillus thermoglucosidasius, and bacillus thermodenitrificans to geobacillus as the new combinations g. stearothermophilus, g. thermocatenulatus, g. thermoleovorans, g. kaustophilus, g. thermoglucosidasius, and g. thermodenitrificans. int. j. syst. evol. microbiol. 51: 433-446. ooi t & d. kikuchi. 1995. purification and some properties of β-mannanase from bacillus sp. world j. microbiol. biotechnol. 11: 310-314. peer & watcher. 1993. treecon: a software package for the construction and drawing of evolutionary trees. comput. applic. biosci, 9: 177-182 . politz o, m krah, kk thomsen, & r borriss. 2000. a highly thermostable endo-(1,4)β-mannanase from the marine bacterium rhodothermus marinus. appl. microbiol. biotechnol. 53: 715-721. spindler, k.d. & c. rapp. 1997. detection of chitin degrading enzymes on gels in: chitin handbook by eds. r.a.a. muzzarelli & m.g. peter. european chitin society. sumardi. 2005. isolasi, karakterisasi, dan produksi β-mananase ekstraseluler dari geobacillus stearothermophilus l-07 [disertasi]. bogor: sekolah pascasarjana, institut pertanian bogor . torrie, j.p., d.j. senior, & j.n. saddler. 1990. production of β-mannanases by trichoderma harzianum e58. appl. microbiol. biotechnol. 34: 303-307. whistler, r.l. & j.n. bemiller. 1973. industrial gum: polysaccharides and their derivates. academic press. new york. zakaria, m.m., m. ashiuchi, s. yamamoto, & t. yagi. 1998. optimization for β-mannanase production of a psychrophilic bacterium, flavobacterium sp. biosci. biotechnol. biochem., 62: 655-660. 10 http://www.bacterio.net/ introduction isolation of thermophilic mannanolytic strains characterization and identification of isolate extracellular enzyme production enzyme assay results and discussion we found 15 bacterial isolates from palm oil shell. then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oc with ph 7.0. only isolate l-07 showed mannanase activity. the isolate l-07 could grow at 55-70oc and showed mannanase activity as high as 3.1 u/mg. biotropia vol. 28 no. 1,2021: 54 63 doi: 10.1 1598/btb.2021.28.1 .i055 ethnobotanical study of digestive systems disorders in baduy ethnic, indonesia rida oictorida igiastini',', indria wahyuni' and irma saraswat13 'dqartment o f biology education, faculq o f teacher training and education, universitas sultan ageng tirttdyasa, kota serang, banten 4 2 1 17, indonesia 'center ofexcellencefor food sectin$ (icefory), universitas sultan ageng tirtqasa, serang, banten 42163, indonesia 'department ofelectrical engineering, faculo ofengineering, universitas sultan ageng tirttdyasa, ciltgon, banten 42435, indonesid received 20 april 2018/accepted 20 december 2019 abstract digestive disorders rank among the most commonly faced problems in indonesia, particularly for the baduy people in banten province. the baduy population lives along water-rich areas, yet their lack of sanitation facilities and unawareness of disease prevention methods have resulted in high morbidity and mortality rates in their communities, largely due to digestive system disorders that they continue to treat with medicinal plants. using quantitative ethnobotanical approaches this survey was undertaken to document the use of indigenous medicinal plants to treat and prevent different types of digestive system disorders among the baduy communities. ethnic knowledge o n their medicinal plants were collected from 30 informants from the baduy people. quantitative approaches were used to determine the use value and informant consensus factor values of the collected data. the baduy population currently uses 54 medicinal plant species belonging to 30 families, in treating digestive system disorders. additional research is, however required, to validate the function of the medicinal plants and identity of their active compounds. keywords: baduy people, digestive disorders, medicinal plants introduction indonesia is a mega-&verse country with its flora composition particularly important in traditional medicine and agriculture and whose identity is deeply rooted among the indigenous groups throughout the indonesian archipelago. more than 1,800 plant species inhabit several forest formations across indonesia, 940 of which are used in herbal medicine by indigenous communities, yet only 300 species are used by the indonesian pharmaceutical industry (lip1 2014). indigenous knowledge is a product of generations upon generations of experience and constitutes a connection between indigenous communities and local natural resources (davies & kassler 2015). among these resources are plants that indigenous community members gather for food, medicines, religious rituals, and *corresponqng author, email: rida.khastini@untirta.ac.id other cultural activities. the indigenous baduy communities in banten province, indonesia, form part of the immense cultural diversity of humanity, and their collective knowledge, passed down from generation to generation, constitutes a source of immeasurable cultural wealth. the baduy people have traditionally used medicinal plants in the treatment of various diseases. the communities continued use of ethno medicine stems from its cost effectiveness, social acceptability, minimal side effects, and accessibihty (chawla e t al. 2013). since the baduy typically reside in mountainous areas where access to public healthcare systems remains limited, they have accumulated profound experience with treating and preventing diseases with medicinal plants, whose diversity in their environment is exceptionally high. digestive system &sorders have recently gained considerable attention among scholars in ethno medicine. such disorders are a major ethnobotany of digestive system disorder among the baduy people in indonesia khastini e t al. cause of morbidity both in indonesia and around the world, particularly among indigenous people, includmg the baduy, whose inadequate access to hygienic levels of sanitation exacerbates the transmission and prevalence of various digestive diseases. only 34% of people in low-income countries have access to adequate sanitation, and that figure is roughly 2.6 blllion people worldwide (mars e t al 2010). researches on local knowledge about medicinal plants is becoming increasingly important in defining strategies for the conservation management of biological resources (jeruto e t al 2008). fortunately, through extension activities, these ethnobotanical studes have become increasingly useful in developing healthcare and conservation programs (vandebroek e t al 2010). given those trends, valuable information about the importance of mekcinal plants and indigenous knowledge disseminated orally warrants its collection, documentation and quantitative evaluation in order to guide future researches and prevent knowledge loss and erosion during its transmission from generation to generation. although some ethnobotanical surveys have been conducted among the baduy (iskandar & iskandar 2017), there was no comprehensive report on the use of medicinal plants to specifically treat digestive system disorders. hence, this paper aims to assess the traditional uses of medcinal plants among the baduy and to compile profiles of the plants by applying quantitative methods. the information that would be generated could not only expand ethno medicinal knowledge but also support general awareness on conserving indigenous medicinal plants in indonesia. materials a n d methods the workflow contained the following specific steps: data collection and data processing analysis (fig. 1). study site the study was conducted at inner baduy, cibeo hamlets, i<anekes village, and leuwidamar sub district, in lebak, banten province, indonesia (figure 2). geographically, the d a g e is located at longtude 6°27'27'1 6"30'07' s and latitude 108°3'911 106°4'5" e at an altitude of 300 600 m asl. the region is located roughly at 172 krn west of jakarta, indonesia. the region experiences an average temperature of 20 oc, and the average rainfall is 4,000 mm/year. generally, three types of soil are available in the region: dark latosol, brown alluvial, and andosol. ethnographically, the baduy community inhabits 5,101.85 ha, of which 2,101.85 ha constitutes the residential area and roughly 3,000 ha of which are protected forest areas. the baduy speak their ethnic language sunda, and as indigenous people, they depend on immediate natural resources in their vicinity for their livelihood, which they supplement with production from the primary sectors such as agriculture, horticulture, and livestock. data collection data processing analysis plants for treating and figure 1 flow chart of ethnobotanical study in the baduy ethnic community biotropia vol. 28 no. 1,2021 legends g v r n g k s n d a g . . . . . . . . . . . village boundary lnner baduy e!33l study site i outer baduy footpath inner baduy hamlets 7 river outer baduy hamlets okm figure 2 map of the study site. baduy area of the ignekes village, leuwidamar subdistrict, lebak, banten province (iskandar & iskandar, 2017) data collection prior to documenting ethno pharmacological data, semi-structured interviews and focus group discussions, personal conversations with locals, and field surveys (martin 1995) were conducted in september 2016 until july 2017. a total of 30 household heads from the cibeo hamlet with age ranging from 20 to 40 years with an average age of 35 years, participated in the study as informants. all informants were ethnic baduy and were born and raised in their respective villages. the names and ages of the informants, the local names of plants and plant parts used, the method of preparation, and the medicinal uses, were recorded. prior to the field survey, several textbooks of plant diversity were consulted for identification purposes including the flora malesiana series (van steenis 1972) and flora of java (backer & van den brink 1965). medicinal plants were photographed and made into herbarium specimen for complete identification by the botanist at the laboratory of biology education, faculty of teacher training and education, university of sultan ageng tirtayasa, indonesia. the observed plant morphology included the characters of the roots, stems, leaves, flowers, seeds and fruit. analysis the use values (uv) of the medcinal plant species were analyzed based on galeano e t al. (2000) and according to the equation uv = u / v, in whch u is the number of mentions per species, and n is the number of informants. uv is a quantitative parameter that indicates the relative importance of species known by the locals. the informant consensus factor (icf), initially developed by trotter and logan (1986), was used to identify the relative importance of medicinal plants in ailment categories in a particular culture. icf was calculated according to the equation icf = nur n t / nur 1, in which nur is the number of uses mentioned in ethnobotany o f digestive system disorder among the baduy people in indonesia iulastini e t al. each category, and nt is the number of species indicated in each category. icf values range from 0 to 1; values closer to 1 indicate a higher proportion of informant consensus on plant species used against a disease category, whereas values closer to 0 indicate the opposite, a lower informant consensus on plant species used as a cure. results a n d discussion a total of 54 species belonging to 47 genera and 30 families were recorded for the treatment of dgestive system disorders (table 1). for each species, the scientific name with voucher number, family, vernacular name in sundanese, ailments treated, parts used, forms of preparation, and uses were documented. the reported plant families included arecaceae, fabaceae, zingiberaceae (four species each), acanthaceae, euphorbiaceae, moraceae, pipera ceae (three species each), apocynaceae, convolvulaceae, melastomataceae, menisperma ceae, poaceae, rubiaceae, solanaceae (two species each), amaranthaceae, begoniaceae, compositae, dilleniaceae, gnetaceae, lamia ceae, liliaceae, magnoliophyta, malvaceae, meliaceae, mimosaceae, myristicaceae, myrtaceae, palmae, phyllanthaceae, and verbenaceae (one species each). all the reported plants were commonly used in treating digestive system disorders by the baduy community because they are readily cultivated or grow as wild plants that can be harvested from the nearby forest where the baduy live. table 1 medicinal plants used t o treat digestive system disorders in baduy communities ailments treated or parts forms of local name use value family species cultipreparation (sundanese) vated plant uses used and use (uv) acanthaceae strobilanthes n'spus b1. pecah beling c hemorrhoids leaf decoction 0.50 staurogyne elongata pi.) o.i< reundeu w stomach ache leaf edible 0.47 andrographispaniculata purm.f.) s a d o t o w diarrhea leaf decoction 0.83 wall. ex nees amaranthaceae celosia azentea l. gamet w appetite stimulant leaf crushed 0.43 apocynaceae qlophora n'ssioides b1. areuy peujit w stomach ache latex infusion 0.23 i<otok alstonia scholaris r. br. lame w intestinal worm latex infusion 0.17 infection arecaceae areca catechu l. jebug w toothache root crushed 0.60 ageratum cony~oides l. babadotan w flatulence leaf decoction 0.53 blumea balsamifera (l.) dc. capeu w appetite stimulant leaf decoction 0.33 . . cocos nucifera l. var. uiridis l<alapa hejo c flatulence bark peel decoction 0.87 begoniaceae begonia isoptera dryand areuy w stomach ache leaf decoction 0.20 bingbiringan compositae mikania cordata (burm.f.) b.l. areuy w jaunchce, stomach whole decoction 0.27 rob. caputuher ache plant convolvulaceae lepistemon binectan$mm (wall.) areuy w stomach ache leaf, resin decoction 0.40 i<untze plumpung merremiapeltdta (l.) merr. areuy w flatulence leaf decoction 0.17 palungpung dilleniaceae tetracera indica merr. areuy asahan w stomach ache bark juice drink 0.10 euphorbiaceae acabpha caturus b1. i<i lauk w appetite stimulant juvenile edible 0.53 leaf macaranga tanarius (l.) muell.ai-g. mara w gastric ulcer leaf decoction 0.13 eubhorbia hirta l. patikan kebo, w mouth ulcer latex rubbed 0.60 nanangkaan fabaceae eytbrina litho.pemza miq. dadap w stomach ache leaf crushed 0.47 abmsprecatorius l. saga w mouth ulcer leaf, root crushed 0.60 pterocapus indicus wdld. angsana w toothache resin crushed 0.77 mimosapudica duchass. & walp gehgeran, w mouth ulcer leaf crushed 0.70 putri malu gnetaceae gnetum neglecturn b1. ihsungka c intestinal worm stem juice decoction 0.67 infection larniaceae orthosiphon starnineus (blume) i<umis ucing c flatulence leaf decoction 0.80 miq. ldtaceae cordilnefisticosa (l.) a.chev. hanjuang c hemorrhoids rhizome, decoction 0.53 leaf biotropia vol. 28 no. 1,2021 table 1 (continued) ailments treated or parts forms of local name culti use value family species preparation (sundanese) vated plant uses used and use (uv) magnoliophyta villebrzmea mbescens (bl.) b1 nangsi w toothache stem juice edible 0.63 malvaceae durio +bethinus murr. icadu c toothache nectar edible 0.43 melastomataceae melastoma mlabathricum auct. harendong w diarrhea, leaf crushed 0.60 toothache, mouth ulcer bellucia amnanthera triana harendong w stomach ache fruit edible 0.43 leuweun~ meliaceae sandoricum koe$ape (burm.f.) icecapi c diarrhea leaf, bark edible 0.43 merr. menispermaceae arcangeliisaflava merr. areuy ki w diarrhea, jaundice root, bark decoction 0.20 koneng tinospora nnspa (l.) diels. martawali w jaunqce, intestinal bark, leaf decoction 0.57 worm infection mimosaceae pitbecelobim lobatum benth. jengkol c appetite stimulant juvenile edible 0.77 leaf moraceae ficus hispida l. f. bisoro w diarrhea, stomach juvenile edible 0.27 ache, jaundice leaf, fruit ficus sagittutu vahl. areuy h baok w mouth ulcer, latex decoction 0.27 stomach ache, jaundice ficzls racemosa roxb. walen w stomach ache juvenile edible 0.63 leaf myristicaceae horsfzeldiaglabra warb. i(i beo, c toothache fruit, leaf edible 0.77 icalapa ciung mvrtaceae psiditm pl~aiava l. tambu batu c diarrhea fruit. leaf edible 0.27 palmae arengapinnata (wurmb) merr. icaung w constipation root decoction 0.83 phvllanthaceae phvllanthus acidus skeels. cereme w a ~ ~ e t i t e stimulant fruit edible 0.87 piperaceae piper betle l. sereh c toothache, mouth leaf crushed 0.83 ulcer piper sulcatum b1. areuy rinu w flatulence fruit edible 0.27 pipar aduncum l. gedebong w nausea seed crushed 0.63 poaceae imperata ylindrical (i,.) p.beauv. eurih w mouth ulcer, root decoction 0.50 pharyngitis andropogon nardus l. sereh c appetite stimulant leaf decoction 0.57 rubiaceae morinda citrz@lia l. mengkudu c jaundice fruit edible 0.67 nauclea orientalis l. gempol w nausea root decoction 0.67 solanaceae physalis minima l. cecenet w intestinal worm root decoction 0.80 pbsalir angulata l. infection cecendet w mouth ulcer, gastric fruit edible 0.67 ulcer, stomach ache verbenaceae clerodendmm serratum (l.) moon singugu w intestinal worm leaf decoction 0.67 infection zingberaceae zingiber oficinale roscoe jahe c flatulence root decoction 0.93 zingiber xemmbet (l.) roscoe ex lempuyang c appetite stimulant root decoction 0.67 sm. zingiber amaricans b1. lampuyang c stomach ache leaf decoction 0.67 costus speciosus smith pacing c jaunqce stem juice decoction 0.83 different plant parts were used for the the uvs of the plants ranged from 0.1 to treatment of various digestive system disorders 0.733. the interview results revealed that the (fig. 3). the kaves were the most commonb three most selected species were zingiber oflknale used (430/0), the (150i0), fruit (0.73) that had the reatest uv among all plants, (13%), resin (loo/o), bark (~oo), stem substance followed by cocos nac@ra l. var vir;;dis and (5%), whole plant (2%), nectar (2%), and seed pblantas kdas skeels (table 1 ) . a high u v value (2%). in most cases, the medicinal plants were prepared in the forms of juice, decoction, for a particular plant indcated a high number of infusion, paste, and powder. uses reported by informants. ethnobotany of digestive system disorder among the baduy people in indonesia khastini e t a l q o l leaf root fruit i43: resin 4 bark ' stem substance i whole plant nectar seed figure 3 use patterns of the documented medicinal plant parts table 2 category of ailment treated and informant consensus factor (icf) ailments treated or plant uses number of taxa number of use report icf constipation 2 26 0.96 gastric ulcer 2 12 0.91 nausea 2 11 0.90 hemorrhoid 2 8 0.86 diarrhea 6 28 0.81 intestinal worm infection 5 21 0.80 flatulence 6 25 0.79 appetite stimulant 7 25 0.75 mouth ulcer 6 18 0.71 toothache 6 16 0.67 pharyngitis 2 4 0.67 stomach ache 13 20 0.37 jaundice 7 10 0.33 icf was determined for 13 ailments or plant uses: appetite stimulant, constipation, diarrhea, flatulence, gastric ulcer, hemorrhoids, intestinal worm infection, jaundice, mouth ulcer, stomach ache, toothache, pharyngitis, and nausea. icfs ranged from 0.33 to 0.96, with a mean value of 0.733 (table 2). the ailment treated with the most use reports was constipation (0.96). the icf values for disease treatment depended upon the availability of the medicinal plant species in the area of the studied baduy community. increasing access to medicinal plants in indonesia, a country with one of the world's most highly diversified plant species, has greatly benefited the locals, especially members of indonesia's indigenous communities. many studies conducted among indigenous communities elsewhere in indonesia. for instance, among the dayak communities in west kalirnantan @iba e t al. 2013) and sundanese communities in west java (roosita 2008), have documented not only the plant parts used (e.g., leaves, wood, fruits, roots, and flowers) and their processing techniques but also their beneficial effects. this current study documented that information on the uses of 54 plant species in treating 13 different aliments among the baduy people were collected from reliable informants and were based on their personal experience. the commonly used plants that the baduy have a b s t e r e d to treat and prevent digestive disorders were readily available and had healing properties. biotropia vol. 28 no. 1,2021 to obtain extracts used for medicine, the ginger has the greatest uv, its rhizomes are informants collected the fresh or dried plant materials from the entire plants or their parts. of all the plant parts used, the leaves were the most commonly prepared part as medicines (43%). leaves contain a myriad of secondary metabolites or phytochemicals that play a vital role in the treatment and prevention of various digestive disorders (i<umar et al. 2009). the baduy typically use fresh leaves by boiling these in water for decoctions, as they also d d with other plant parts used for medicinal materials. such preparation usually involved reducing the water volume by a third. studies conducted in other indigenous communities around the world have shown that leaves were also the most used plant part for medicine at rates of 51.39 o/o in the turgo hamlet of yogjakarta, indonesia (nahdi et al. 2016), 47.65% in argentina (teves et al. 2015), and 34% in the limpopo &strict of south africa (semenya & maroni 2012). the current results also supported the findings of handayani (2015), that leaves were the most frequently used parts in plant-based medcines. leaves and other plant materials, including rhizomes, were also prepared for medicines by pounding and squeezing. according to the informants, the baduy also mixed some plants with other plants depending on their intended uses. single and mixed medicinal plant preparations may have different effects, for a mixed-medicinal plant formula may contain several hundred times the natural constituents that numerous single-medicinal plants contained. quantitative analysis was performed to authenticate and project the relative importance of medicinal plant species in their traditional use. plants with high uv were also used among other indigenous communities throughout indonesia (nulfitriani et al 2013). the plants with the first three largest uv among the medcinal plants documented in baduy area were; ginger (zingiber o@cinale roscoe), coconut (cocos nucfera l.), and pkyllanthus a d u s skeels. used not only as a component in medical treatments but also as an additive in numerous foods and daily beverages (e.g., bandrek and b.jigur) of the baduy people (figure 4). due to its aromatic compounds, ginger is a valuable rhizome that releases a spicy, pungent, pleasant smell. the effectiveness of using ginger in the preparation of reme&es against various digestive problems, including loss of appetite, flatulence, indgestion, diarrhea, and dysentery, was also reported by sidhu & pannu (201 0). coconut (cocos nucifera l.) registered the second-greatest uv among the baduy who cultivate the plant and use all of its parts in one way or another for diverse purposes in their daily lives. the parts of the fruit, including the coconut kernel and tender coconut water, have numerous medicinal properties, including antibacterial, antifungal, antiviral, antiparasitic, and immunostimulant properties, that are effective against digestive system disorders (debmandal & mandal, 2011). the medcinal properties of coconut emanating from its constituents, include vitamin b, nicotinic acid (b3, 0.64 pg/ml), pantothenic acid (b5, 0.52 pg/ml), biotin (0.02 pg/ml), riboflavin (b2, <0.01 ng/ml), folic acid (0.003 pg/ml), and trace quantities of vitamins b1, b6, and c, as well as pyridoxine, thiamine, amino acids, l arginine, plant hormones (auxin, 1,3 diphenylurea, and cytokinin), enzymes (acid phosphatase, catalase, dehydrogenase, diastase, peroxidase, and rna polymerases), and other growth-promoting factors (solangih & iqbal, 2011). other studies conducted in several countries have also reported the use of coconut in treating digestive system disorders. people in brazil have used the extract from the husk fiber of c. nucifera to treat diarrhea (akinyele et al. 201 i), which people in ghana have treated with coconut milk (lima et al. 201 5). ethnobotany of digestive system disorder among the baduy people in indonesia khastini e t al. figure 4 the third largest uv medicinal plants used by baduy ethnic. a. zingiber oflcinale roscoe. b. cocos nxn9m l. var. viridis. c . pbylzannthxs acidas skeels. plylkantbzts acidus skeels has the third largest uv in this study. this species is not only functioned as edible fruit, but also is known to cure wide spectrum of diseases in asia, the carribean region, as well as central and south america (tan et al. 2020). several studies showed that this plant is abundant in a number of phytochemicals, such as flavonoids, alkaloids, lignans, terpenes, steroids, and essential oil (chakraborty e t al 2012; nisar et ak201 8), which have remarkable health benefits. among the study plants, tetrdcera indim, has the lowest uv. most informants were unfamiliar with the medicinal plant species and had little knowledge about its ethnobotanical uses. species in the genus twrscera have also been used as medicinal plants by the ehotile people near aby lagoon in c6te d'ivoire, africa. however, these plants containing the sought-after exudate are found only in old marshlands that are scarce and difficult to access (malan et al. 2015). according to one key informant, most of the raw materials of these medicinal plants were difficult to find along the village periphery, as the original habitats of the cultivated plants were in the wild and were located in the forest. based some areas in the forest are considered to be sacred, and entry is forbidden. forests and other types of habitat provide a wide range of medicinal plant resource-base for primary healthcare, especially in rural areas (lindborg e t al.2012). this study has identified a fairly large number of plants used to treat various ailments, particularly among the baduy community. the high diversity of species reported to be readily available for medicinal purposes could indicate not only a dependency on plants in the treatment of ailments among the baduy but also an immense amount of local knowledge about herbal medicines in the community. despite the widespread use of those plants, however, no pharmacological studies have been conducted to determine their efficacy and safe use. hence, the identity of the medicinal plants used by the baduy provided a platform for formulating further preclinical and clinical studies to determine the efficacy and safe use of herbal preparations in indonesia and elsewhere. conclusions on vegetation structure, the baduy forest lands can be divided into two categories-numa which in summary, the showed that the is developed from fields that have long been b a d u ~ people banten indonesia, abandoned, and are covered with dominant use immediate natural resources namely, 54 plants growing on the land, and leuwet/ng kokot as plant species, both as a whole plant and in part protected areas, forbidden from entry or use as a and as fresh and dried materials, and that they field. both of these contain a high diversity of possess indigenous knowledge to treat digestive plant sources for medicinal purposes. however, system disorders in their community. biotropia vol. 28 no. 1,2021 acknowledgements the study was conducted with financial support from the islamic development bank arranged by the universitas sultan ageng tirtayasa (no. 356/un43.9/pl/2017). the authors would like to express their gratitudes to the baduy community for participating in this study as well as for sharing their indigenous knowledge and hospitality. the authors would also like to acknowledge all of the students of biology education at the universitas sultan ageng tirtayasa who contributed to this study in various ways. references akinyele ta, okoh 0 0 , akinpelu da, okoh ai. 2011. in vitro antibacterial properties of crude aqueous and n-hexane extracts of the husk of cocos nncifera. molecules 16(3):2135-45. backer a, van den brink b. 1965. flora of java (spermatophytes only). volume i. noordhoff (nl): n.v.p. groningen. international conference of masyarakat biodiversitas indonesia, bandung. 1 (6):1425-32. indonesian institute of sciences. 2014. present status of indonesian biodiversity. jakarta (id): indonesian 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pp.91-112. vandebroek i, balick mj, vandebroek a, igonenberg f, yukes j, wade c, castillo d. 2010. the importance of botellas and other plant mixtures in dominican traditional medicine. journal of ethnopharmacology 128(1):20-41. van steenis cggj. 1972. the mountain flora of java. leiden (nl): e.j. brill. world health organization and united nations children's fund joint monitoring programme for water supply and sanitation (jmp). 2008. progress on drinktng water and sanitation: special focus on sanitation. unicef. new york and who, geneva. microsoft word 1 biotropia no. 22, 2004: 1-14 morphological variation of the ecotypes of echinochloa crus-galli var crus-galli (l). beauv (barnyard grass: poaceae) in malaysia and indonesia arifin tasrif' . abdul shukor juraimi*)', jugah kadir', suhaimi napis2 and soet1kno s. sastroutomo3 1 faculty of agriculture, univerxiti putra malaysia, 43400 serdang,selangor, malaysia 2faculty of food science and biotechnology, univerinti putra malaysia, 43400 serdang, selangor, malaysia 3cab international, se asia regional centre, 43400 serdang, selangor, malaysia abstract greenhouse experiments were conducted to examine the morphological traits of barnyard grass ecotypes from diverse geographic origin. seeds (caryopsis) were collected from 17 locations of rice fields throughout malaysia (11 states) and indonesia (six provinces) and were grown in pots each containing 10 kg of paddy field soil. the experiments were arranged using completely randomized design (crd) with five replicates. mean separation was calculated using duncan multiple range test at 5% probability level. unweighted pair-group method of arithmetic averages (upgma) was performed to determine the individual relationship within ecotypes of barnyard grass. twelve morphological traits such as culm, panicle, leaf, and spikelet traits were measured. the growth characters such as emergence date, heading time, and growth duration were also evaluated. the average of emergence date, heading time, and growth duration of barnyard grass collected from perils, kedah, penang, and johor were relatively earlier than other ecotypes. six groups were classified based on the cluster analysis of malaysian ecotypes of barnyard grass. principal component indicated that group six was found to be highly variable compared to others. while three groups were identified in indonesian ecotypes of barnyard grass. group one was observed to be highly variable. results demonstrated that morphological variation among ecotypes of barnyard grass showing differences between the two regions illustrate the role of geographic variation. key words : variation / ecotypes / paddy field weeds / barnyard grass. introduction the barnyard grass (echinochloa crus-galli var crus-galli l. beauv.) is an annual weed native to asia and presently can be found throughout the world (holm et at. 1977). the most widespread and economically important member of the genus is the annual e. crus-galli, which can be found within 50°n latitude and latitude 40°s. (holm at al. 1977; maun and barret 1985). barnyard grass is a noxious weed in rice field and have been shown to reduce rice yield by 40% in direct seeded rice in malaysia (azmi 1988 ) and about 21 % annually in indonesia (tjitrosemito 1994). *) correspondence to: abdul shukor juraimi, department of crop science, faculty of agriculture, universiti putra malaysia, 43400 serdang, selangor de, malaysia. e-mail a_shukor@ agric.upm.edu.my biotropia no. 22, 2004 this weed belongs to the tribe paniceae, family gramineae. the genus may include 20 to 50 species that are widely represented in tropical and warm temperate regions of the world (clayton and ronvoize 1986; martines et al. 1999; michael 1983). with an increase in direct seeding rice practices, barnyard grass is becoming a greater problem in rice fields (azmi and baki 2002; loux and barry 1991 ). in addition, continuous chemical control of barnyard grass in rice plant has caused herbicide resistance to barnyard grass (martines et al. 1999; rutledge et al. 2000). klingaman and oliver (1996) defined ecotypes as plants genetically adapted to the habitat they colonized and biotypes as plants showing a random genetic variant within an ecotype. individual plants of barnyard grass can produce thousands of seeds, but seed germination declines rapidly over time, and sometimes they have long dormancy (martinkova 1989). barnyard grass typically grows 1 to 1.5 m tall and is capable of producing a large number of seeds (azmi and baki 1995; itoh 1991). in certain conditions, it may grow up to 2 m tall (anonim 2001). in addition, the plant can grow in diverse environment (yabuno 1983; yamasue 1997), and its growth habit is strongly affected by environmental conditions. studies have shown that soil type, fertility level, and cultural regimes affect barnyard grass of some morphological traits. the production of a large number of easily dispersed seeds and the ability to flower under a wide range of photo periods contribute to the success of this weed (holm et al. 1977). phenotypic and genetic variation among barnyard grass ecotypes would be divergent resulting from the selection pressure imposed by agricultural practices, crop characteristic, geographic origin, and herbicide application. studies have shown that soil type and fertility level affect morphological characters of barnyard grass in rice field (martines et al. 1999; yamasue 1997). recent evidence has shown that substantial variation exists among seed size within plant ecotypes (marshal et al. 1986) and between geographical regions (ransom et al. 1998; sterling et al. 2000). phenotypic variability has been observed in numerous weed species, comprising half of the species reported as having biotypes or ecotypes. weeds with biotypes differing in growth and morphological characteristics include canada thistle (circium arvense l. scop) (hodgson 1964), field bindweed (convolvulus arvensis l.) (degennaro and weller 1984, quack grass (eltrygia repens l.) (westra and wyse 1981), johnson grass (sorghum helepense l.) (mcwhorter and jordan 1976), yellow nutsedge (cyperus esculentus l.) (holt 1994), leafy spurge (euphorbia esula l.) (harvey et al. 1988), and hemp dogbane (apocynum cannabinum l.) (ransom et al. 1998). differences between weed population can influence the competitive nature of weed species and may affect response to chemical or cultural control strategies. most biotype studies previously reported have been conducted under different environment. it is very important to study variation in morphological characters among ecotypes to determine how plant genotype and diverse environmental conditions could influence the plant morphology of barnyard grass under uniform conditions. thus, the objective of this morphological variation of echinochloa crus-gaili var. crus-galli l. beauv. ecotypes arifin tasrif et al. study is to determine if barnyard grass seeds collected from geographically divergent ecotypes exhibit morphological variation when grown in uniform environmental glass house conditions. materials and methods from december 2000 to march 2001, barnyard grass seeds were collected from 11 rice field locations across peninsular malaysia (100 119° e and 7°n) and 6 locations across indonesia (94 141° band 6° 8' 11° 5' s) (figure 1, table 1). at each location, seeds from a single plant were randomly collected from different sites in1 rice growing areas, so that the collected seeds would be genetically identical. seed collection biotropia no. 22, 2004 the seeds were chosen to represent the populations from which they were collected. since seeds were collected from geographically divergent locations, they can be described as ecotypes (klingaman and oliver 1996). at each location, five sampling sites were randomly selected. from each of the five sampling sites, one individual ecotype per site, but more than one ecotype were collected when morphologically different types were present at each site using the nearest-neighbor techniques (solbrig 1960) and the distance between sampling site of each location was more than 10 km (barret 1982). propagation of the ecotypes a total of 85 ecotypes of e. cruss-galli collected from the 17 locations were grown under uniform glass house conditions. before planting, seeds were soaked in tap water for 24 hours at room temperature. seeds were grown in each pot size containing 10 kg of paddy soil. basal fertilizer (0.4 g n/pot, 0.4 g p/pot, and 0.2g k/pot or equal to 40 kg n/ha, 45 kg p/ha, and 30 kg k/ha) were applied directly in each pot, while one third of nitrogen were applied at five to six weeks after seedling emergence (pane and mansor 1997). two weeks after emergence, seedlings were thinned to one plant per pot, leaving the tallest emergence individual in all replications. the pots were flooded shortly after thinning and five cm of water level of the soil surface were maintained throughout the study. environmental glasshouse morphological variation of echinochloa crus-galli var. crus-galli l. beauv. ecotypes arifin tasrif et al. conditions such as rh, temperature, and light intensity (li-cor model 185-b) were recorded and soil chemical contents were analyzed (page 1982). morphological trait assessments traits such as plant type, plant height (cm), flag leaf length (cm) and width (cm), culm diameter (cm), panicle length (cm), empty glume length (mm), spikelet length (mm), spikelet width (mm), spikelet weight (fresh weight of 100 seeds in gram), panicle awn, and number of tiller were assessed at the maturity stages. plant type (the degree of opening at the first node) and panicle awn were considered as qualitative characters visually rated on a scale from none (0), moderate (1) to severe (2) (park et al. 1995; yamasue 1997). in addition, growth characters such as emergence date, heading time and growth duration were also evaluated. specimen identification and statistical analysis specimen identification was carried out following the classification prepared by yabuno (1983), michael (1983), itoh (1991), and park et al. (1995) and compared with the specimens of the faculty of agriculture's herbarium of university putra malaysia. morphological descriptors were subjected to cluster analysis using euclidean distance coefficient of upgma analysis and associated dendrogram using ntsys-pc package version 2.1 (rohlf 2000). principal component analysis (pca) for step-wise observations among the groups of barnyard grass ecotypes was also analyzed. square root transformation was used to transform the raw data before analysis. the shan clustering program of the ntsys-pc was used to group the ecotype on the bases of those matrices. anova was used to determine significant difference in growth character of each location of barnyard grass ecotypes with five replications. means were separated using duncan's multiple range test (dmrt) at the 5% probability level using sas version 6.0 (sas 1996). results and discussion soil analysis and environmental conditions of the glasshouse a paddy field soil was taken from mardi research station, tanjong karang, selangor. the soil physical and chemical properties were as follows (sand 2%, clay 72%, dust 28%), c 3.13%, n 0.35%, ph 4.8, cec (mg/loog 29.74), c/n ratio 10 ca (bpj/loog = 8.37), and mg (bpj/loog =1.94). relative humidity of the glasshouse during experimental period ranged from 68% to 90%, and temperature ranged from 24°c to 34°c, while light intensity ranged from 660 to 930 um m^sec. these conditions were conducive for growth of barnyard grass. biotropia no. 22, 2004 variability in growth characteristics among barnyard grass ecotypes variability in growth characteristics among barnyard grass ecotypes is presented in table 2. the range of emergence was from 2.8 days to 7.2 days, that of heading time was from 42 to 56 days. while, the growth duration ranged from 89 days to 98.2 days. for emergence date, perlis and kedah ecotypes were the earliest (2.8 days to 3.0 days) and significantly different (p<0.05) from the rest of the ecotypes. the heading time (42 days and 44 days) was significantly earliest for perlis, kedah, penang, perak, kelantan and johor ecotypes. south sulawesi was the latest besides having the longest growth period of 98.2 days. while perlis, kedah, penang, perak, kelantan and johor growth were the shortest and significantly different from the rest of the ecotypes. results of the growth characteristics among barnyard grass ecotypes collected from malaysian or indonesian rice growing areas showed distinct variability. growth characteristics of the ecotypes such as heading time (42 days to 56 days) and growth duration (89 days to 98 days) were slightly similar to the growth duration of barnyard grass ( 44 dae to 45 dae and 90 dae to 95 dae) as reported by azmi et al. (1997) and itoh (1991). however, the values were slightly lower than the growth duration of barnyard grass in arkansas ( barret and wilson 1983). morphological variation of echinochloa crux-galli var. crux-galli l. beauv. ecotypes arifin tasrif el al. in general, growth characteristics variability among barnyard grass ecotypes might be affected by geographic origin. this observation was in line with the work of johnson et al. (1989) who reported that even though ecotype of some sweetvetch (hedysarum boreale nutt.) differed in their taxonomic distance values, this difference is not always closely related to specific characteristics of the collection sites. widespread species tend to have more genetic variability than close relation with narrow distribution (hamrick and godt 1990; karron 1987; maki et al. 1999). pylogenetic relationship within individual barnyard grass ecotypes within malaysian ecotypes dendrogram using upgma clustering methods from morphological traits is presented in figure 2. six groups were identified at the taxonomic distance of 0.016. the first group comprised 21 ecotypes mainly from perils, kedah, and johor. the 3rd and 4th groups comprised 14 ecotypes, respectively. groups 2, 5 and 6 consisted of 2, 3 and 1 ecotypes, respectively. two individualecotypes belonged to group 2. the only ecotype from selangor (s-02) was found in group 6. ecotypes from perlis, kedah, perak, and johor were found to be linked together and have closer relation widi each other. ecotypes from perlis and johor fell within group 1, while those of kelantan and negeri sembilan mostly belonged to group 3. penang, pahang, and selangor were dominant in group 4. principal component analysis of barnyard grass was applied to differentiate variability among the existing groups. the proportion of the first and second components were 88.76% and 7.01%, respectively (table 3). the first component was largely contributed by the size of characters such as plant height, plant type, and number of tillers. the second component was due to the shape and size characters of panicle awn, flag leaf width and spikelet length. scatter diagram between the first and second component showed that the individual plants present in different groups had characters different from the others (figure 4.a). most of them were separately distributed in the quadrants of the diagram. on the contrary, individual barnyard grass in group 6 were farther apart than the other groups, indicating that they vary largely. within indonesian ecotypes at the taxonomic distance of 0.08, three groups were classified from six locations representing three different islands. group 1 consisted of 19 ecotypes representing five locations ( two islands), except for ecotype from lampung. eight ecotypes were obtained in group 2, except for lampung ecotype. in contrast, only three south sulawesi ecotypes (ss-1, ss-2, ss-4) were found in group 3 (figure 3).   morphological variation ofechinochloa crus-galli var. crus-galli l. beauv. ecotypes arifin tasrif et al. principal component analysis among groups of barnyard grass is presented in table 3. the proportion of the first and second components were 95.22% and 61.22%, respectively. the first component was largely due to shape and size of characters such as plant height, panicle awn, and panicle length, respectively. the second component was mainly due to plant height and number of tillers. scatter diagram between the first and second components showed that plants in different groups had characters different from the others (figure 4.b). most of them were distributed in the first two components. however, ecotypes belonging to group 1 were farther distributed than other groups, indicating that group 1 had wide variation. the results of this study indicated that individual ecotype of malaysian and indonesian barnyard grass were found to be more variable (tables 2 and 3; figures 2 and 3). in general, ecotype of barnyard grass from malaysia and indonesia are separated by larger geographical distance and is expected to be different than those from the same locations. yamasue (1997) reported that variation in morphological characters were significantly different among barnyard grass collected from different habitats, even though they are from the same locality. on the contrary, weed population which are continuously associated with specific agricultural systems may evolve phonological patterns which optimize survival within the most favorable growing areas (barret 1983). the results demonstrated that genetic differentiations among ecotypes were apparently an important factor of the variation in weed management practices. the differentiation among local ecotypes was probably encouraged by self-pollinating reproduction of the barnyard grass ecotypes (honek biotropia no. 22, 2004 and martinkova 1996). on the contrary, phenotypic variations in barnyard grass ecotypes from indonesia and malaysia affected seedling emergence and growth characteristics. interestingly, south sulawesi ecotypes seemed to be specific in different habitats of different islands (figure 3). this finding is to be similar with the work of maki et al. (1999) who found that highly phenotypic and genetic differentiation of aster miyagii (asteraceae) existed among islands in japan, or high levels of genetic diversity among island populations of suzukia luchunsis (labiatae) in japan (maki et al. 2003). morphological variation of echinochola crus –galli .var.crus-galli l beauv . ecotypes – arifin tasrif et al. biotropia no. 22, 2004 morphological variations among barnyard grass ecotypes in malaysia and indonesia are not very high at present. if newer strains (biotypes) of the barnyard grass migrate from one to other regions and cross-pollinate, the genetic diversity may increase, and the control of barnyard grass in rice production may become more difficult. both genetic and environment play a role in the expression of barnyard grass phenotype. however, barnyard grass genotype maintained their general populations when grown in common site. interestingly, when barnyard grass population was grown at the uniform sites (glasshouse conditions) ecotypes from the most malaysian regions, maintained the highest plant size at short periods to reproductive maturity compared to barnyard grass from indonesian regions. the variability could be affected by local environmental heterogeneity, geographic distance and possibly agricultural practices. this finding is of importance in trying to explain the ecotype distribution among barnyard grass ecotypes from malaysian and indonesian rice fields. these results suggest that barnyard grass ecotypes have formed, and the ecotypes have adapted to specific geographic locations. the ecotype variations will affect successful management of barnyard grass using chemical or potential biocontrol agent. however, molecular traits analysis would be useful to characterize the level of genetic variability among barnyard grass ecotypes. acknowledgement acknowledgement is accorded to the integrated pest management for smallholder estate crop project (ipm-shecp) plant quarantine component and for the contribution of yayasan felda malaysia for a research studentship to arifin tasrif. we are also grateful to dr. osman osumanu, for his valuable suggestions and discussions during the preparation of this article. references anonimous .2001. echonichloa l. beauv. http:file/a:/echinochloa spp. htm azmi m. 1988. weed competitions in rice production. proc. of the national seminar and workshop on rice field management (eds. chang et al.) 141-152. azmi m. and b.b. baki. 1995. the succession of noxious weeds in tropical asian rice field with emphasis on malaysian rice ecosystem. proc. 15* apwsc.tsukuba. japan. july, 24-28, 51-67. azmi m. 1997. weed population in direct-seeded rice as affected by seeding rates. proc. 16* asian-pacific weed science society conference (rajan, eds). mpps. kuala lumpur, 251-256. azmi m. and b.b. baki. 2002. impact of continuous direct seeding rice culture on weed species diversity in the malaysian rice ecosystems. reg. symp. on env. and nat. res. 10-11 april 2002. kuala lumpur. malaysia. 11 p. barret s.c.h. 1982. genetic variation in weeds. in: biological control of weeds with plant pathogens (charudattan and walker eds). john wiley and son. 73 -98. 12 morphological variation of echinochloa crux-galli var. crus-galli l. beauv. ecotypes arifui tasrif et at. barret s.c.h. and b.f. wilson. 1983. colonizing ability in the echinochloa crusgalli complex (barnyard grass).e. seed biology. can. j. bot. 61: 556-562. barret s.c.h. 1983. crop mimicry in weeds. economic bot. 37(3): 225-282. brod g. 1968. untersuchgen zen biologic unal okologie der huknen-hirse (echinochloa crus-galli). weed res. 8: 115127. clayton w.d. and s.a. renvoize . 1986. genera grarninium. grasses of the world. p 280-281 in t.a. cope, ed. london: royal botanic garden, kew. degennaro p.p. and s.c. weller . 1984. growth and reproductive characteristics of field bindweed (convolvulus arvensis) biotype. weed sci. 32: 525528. hamrick j.l. and m.j. godt . 1990. allozyme diversity in plant species. in a.h.d. brown m.t. clegg a.l. kohler and weir b.s. 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(ed: rajan) mpps. kuala lumpur. 233-237. westra p.h. and d.l. wyse 1981. growth and development of quack-grass (agropyron repenx) biotypes. weed sci. 29: 4452. 14 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf iiventory on the alien invasif plants species of indonesia inventory of the invasive alien plant species in indonesia biotropia no. 25, 2005 : 60 – 73 sri sudarmiyati tjitrosoedirdjo dept. of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, and south east asian regional center for tropical biology (seameo biotrop) p.o. box 116, bogor, indonesia sudarmiyati@biotrop.org abstract an inventory of the alien plant species in indonesia based on the existing references and herbarium specimens concluded that 1936 alien plant species are found in indonesia which belong to 187 families. field studies should be done to get the complete figures of alien plant species in indonesia. based on the existing figures of the plant species, the invasive alien plant species can be identified, followed by studies on the assessment of losses, biology, management and their possible utilizations. alien plant species are imported to indonesia for cultivation, collection of the botanical garden, as experimental plants or other curiosities. aside from plants purposely imported, there are also introduced plant propagules contaminating imported agricultural products. these alien plant species can be beneficial or have a potential of being invasive. the alien cultivated species consisted of 67% of the total number. more than half of the cultivated plants are ornamental plants. some of the species are naturalized or escaped from cultivation and become wild and invasive. some other naturalized species, adapted well without any problems of invasion. there are 339 species or 17% of the species recorded as weeds. the highest record of weeds is found in the family of poaceae (57 species), followed by asteraceae (53 species) and cyperaceae (35 species). there are 6 families having more than 10 species of weeds: amaranthaceae, asteraceae, cyperaceae, euphorbiaceae, poaceae, and rubiaceae. three families have more than 100 species: asteraceae 162 species, poaceae 120 species, and papillionaceae 103 species. five species of aquatic and 20 species of terrestrial plants considered as important alien plant species in indonesia were identified and some of their distributions noted. keywords : alien plant species/invasive alien plants/weeds/environmental weeds/aquatic and terrestrial alien plants introduction indonesia, with its many large and small islands, has a great diversity of plants. however, natural disasters, logging and other activities destroy the habitat and reduce biodiversity. as people move around the world, they bring together plants with them (weber 2003). expanding global trade in agriculture, forestry and other industries that depend on raw materials has allowed the transport of species to various parts of the world including indonesia. not all of the alien species are harmful. most of them are crops or ornamental plants. some species are naturalized and adapted well to the local environment, while some become invasive. biological invasion by alien plant species constitutes one of the leading threats to natural ecosystems and biodiversity, and they also impose an enormous cost on 60 agriculture, forestry, fisheries, and other human enterprises, as well as human health. the effects of alien species on native species and ecosystems are numerous and usually irreversible. the impact is sometimes massive but often subtle. natural barriers such as oceans, mountains, rivers, and deserts that allowed the intricate coevolution of species and the development of unique ecosystems have been breached over the past five centuries, especially during the twentieth century, by rapidly accelerating human trade and travel. plane, ships, and other forms of modern transportations have allowed both intentional and unintentional movement of species between different parts of the globe, often resulting to unexpected and sometimes disastrous consequences. biotropia no. 25, 2005 the invasive plant species; some of them have been well recognized as weeds in agricultural production systems, while weeds in natural habitat have been recognized as environmental weeds. agricultural weed problems have received more attention than in other areas. weeds in agro-ecosystems differ in their ecology from invasive plants because the invaded ecosystems are different. agro-ecosystems are highly artificial and represent simple, species poor habitats with environmental homogeneity and predictable disturbance regimes. in contrast, natural habitats are mostly species-rich, environmentally heterogeneous and often unpredictable. plants invading agroecosystems represent mostly herbaceous species, often adapted to the crop system, whereas plants invading natural habitats comprise the full range of life forms (weber 2003). environmental weeds have received increasing attention, because their impact on native biodiversity has been increasingly recognized and the direct costs of controlling them have increased (groves 1999). invasive alien species are taxonomically diverse. many local species are extinct or at the risk of being out-competed by invasive alien plants species, and many native ecosystems have been irreversibly lost to invasion. alien plant species introductions might become invasive, and degrade the ecosystems as well as economic income. degraded environment due to alien plant invasions is not easy to rehabilitate. the extinction of local organisms is not easily renewable. the united nations convention on biodiversity or convention on biological diversity (cbd) which was declared in 1992 was ratified by the indonesian government in 1994. protecting our biodiversity will be very valuable to development in the future. seameo biotrop has been working on weeds for quite sometime. at present, not only weeds cause considerable agricultural losses, degrading catchments areas and fresh water ecosystem, invasive alien plant species which constitute one of the leading threats to natural ecosystem and biodiversity are studied in cooperation with the indonesian ministry of environment since 2003 (tjitrosoedirdjo & tjitrosemito 2005). 61 the records on the invasive alien plant species in indonesia have been dispersed in many publications. however, there are very few comprehensive information on invasive alien plant species. inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo there are some problems in studying the invasive alien plant species in indonesia i.e.: 1). people are not aware of the harmful effects of invasive alien species and the government is not prepared to handle the problems yet. 2). the limited information scattered among various institutes; fail to impress the government on the urgency of the matters although the government ratified cbd in 1994. 3). it takes a long time among the departments to lead coordination. it is now recognized that the focal point for invasive alien species is the ministry of environment which currently coordinates the activities. 4). the ministry of environment is currently studying the principles of handling invasive alien species. the objective of this work is to identify the invasive alien plant species and their distribution in indonesia materials and methods inventory and distribution a large amount of literature has been searched for inventory. the main sources of the list include the following: flora of java, flora malesiana, weeds of rice in indonesia, weed leaflets papers of the weed science society conference proceedings, various other publications in edited books and journals. other additional informations were searched from common floras and from the internet. the list included the number of families, species and the origin of each species. some of the important invasive alien plant species were analyzed for their distribution. the distributions were noted from the references and examination of the herbarium specimens conducted at the herbarium bogoriense (bo) and seameo biotrop herbarium (biot). important indonesian invasive plant species the decision on which species to consider as important is based on the availability, importance and distribution. the species included here is only a fraction of all invasive species in indonesia. only the species considered as seriously problematic and regarded to deserve attention in terms of monitoring and control are included. 62 results and discussions inventory based on the inventory of alien plant species as of january 2005, there are 1936 species belonging to 187 families extracted from the compilation of tjitrosoedirdjo & tjitrosemito 2005, as shown in table 1. the inventory was conducted from the existing references and information from the herbarium specimens. field studies were not conducted yet. if the field studies were conducted, the number of the species recorded will increase with additional records from the fields and new records will be available. new species and varieties of plants have been brought into indonesia since the colonization era. the demand to import new plants continues in a global environment of free trade and this demand is likely to increase. some of these alien species are beneficial and contribute to the quality of the indonesian life, a similar proportion have naturalized and become weeds in agricultural ecosystems or the natural environment. as indicated in table 2, the cultivated one and the ornamental plants are the highest containing 63% of the total numbers, followed by weed species 17.5%. more than half of the cultivated plants are ornamental plants. some of the alien species are naturalized or escaped from cultivation and become wild. there are 81 species whose benefits are unknown. table 1. introduced plant families in indonesia no. family alphabet name of the family no. of families no. of species 1. a acanthaceae, acoraceae, agavaceae, aizoaceae, alismataceae, alliaceae, aloaceae, amaranthaceae, amaryllidaceae, anacardiaceae, annonaceae, anthericaceae, apiaceae, apocynaceae, aponogetonaceae, araceae, araliaceae, araucariaceae, arecaceae, aristolochiaceae, aslepiadaceae, asparagaceae, asphodelaceae, asteraceae, aucubaceae, azollaceae 26 426 2. b balsaminaceae, basellaceae, begoniaceae, berberidaceae, betulaceae, bignoniaceae, bixaceae, bombacaceae, boraginaceae, brassicaceae, bromeliaceae, buddlejaceae, burseraceae, butomaceae 14 111 3. c cactaceae, caesalpiniaceae, campanulaceae, cannaceae, capparaceae, caprifoliaceae, caricaceae, caryophyllaceae, casuarinaceae, chenopodiaceae, chloranthaceae, clusiaceae, cochlospermaceae, colchiaceae, combretaceae, commelinaceae, convallariaceae, convolvulaceae, costaceae, crassulaceae, cucurbitaceae, cupressaceae, cyanastraceae, cycadaceae, cyclanthaceae, cyperaceae 26 237 biotropia no. 25, 2005 63 table 1. continued no. family alphabet name of the family no. of families no. of species 4. d dioscoreaceae, dipsaceae, dracaenaceae 3 8 5. e ebenaceae, elatinaceae, eriocaulaceae, ericaceae, erythroxylaceae, escalloniaceae, euphorbiaceae 7 56 6. f fagaceae, flacortiaceae 2 6 7. g geraniaceae, gesneriaceae, gleicheniaceae, goodeniaceae 4 15 8. h haemodoraceae, haloragaceae, hamamelidaceae, heliconiaceae, hemerocallidaceae, hyacinthaceae, hydrangeaceae, hydrocharitaceae, hydrophyllaceae, hypericaceae 10 35 9. i iridaceae 1 28 10. j juncaceae 1 1 11. l lamiaceae, lauraceae, lecythidaceae, lentibulariaceae, linaceae, loasaceae, loganiaceae, lythraceae 8 76 12. m magnoliaceae, malphigiaceae, malvaceae,marantaceae, marsileaceae, melastomataceae, meliaceae, menispermaceae, menyanthaceae, mimosaceae, molluginaceae, moraceae, moringaceae, musaceae, myoporaceae, myristicaceae, myrsinaceae, myrtaceae, 18 179 13. n nelumbonaceae, nyctaginaceae, nymphaeaceae 3 8 14. o oleaceae, onagraceae, orchidaceae, oxalidaceae 4 156 15. p pandanaceae, papaveraceae, papilionaceae, passifloraceae, pedaliaceae, philesiaceae, philydraceae, phormiaceae, phytolaccaceae, pinaceae, piperaceae, pittosporaceae, plantaginaceae, plumbaginaceae, poaceae, polemoniaceae, polygalaceae, polygonaceae, pontederiaceae, portulacaceae, primulaceae, proteaceae 22 315 16. r ranunculaceae, resedaceae, rhamnaceae, rhizophoraceae, rosaceae, rubiaceae, rutaceae 7 80 17. s salicaceae, salviniaceae, sapindaceae, sapotaceae, saururaceae, saxifragaceae, scropulariaceae, simarubaceae, simmondsiaceae, solanaceae, sterculiaceae 11 95 18. t taccaceae, tamaricaceae, taxodiaceae, theaceae, theophrastaceae, thymelaeaceae, tiliaceae, trapaceae, tropaeolaceae, turneraceae 10 18 19. u urticaceae 1 4 20. v velloziaceae, verbenaceae, violaceae, vitaceae 4 38 21. x xanthorrhoeaceae, xyridaceae 2 2 22. z zamiaceae, zingiberaceae, zygophyllaceae 3 12 total 187 1936 inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo 64 based on table 1, the introduced families which have 10 or more weedy species are listed in table 3. there are 17 families having more than 5 species of weeds, namely amaranthaceae, asteraceae, caecalpiniaceae, carryophyllaceae, cyperaceae, euphorbiaceae, lamiaceae, lytraceae, onagraceae, papillionaceae, polygonaceae, poaceae, rubiaceae, solanaceae and verbenaceae. three families have more than 100 species, asteraceae, poaceae and papillionaceae. the highest number was recorded from asteraceae 162 species, followed by poaceae 120 species and papillionaceae 103 species. biotropia no. 25, 2005 the highest number of cultivated plants is found in asteraceae. some 77% of the cultivated plants of asteraceae are ornamental plants. papillionaceae has 61 species of cultivated plants, followed by poaceae 42 species. the highest record of weeds is found in the family of poaceae 57 species, followed by asteraceae 53 species, cyperaceae 35 species, euphorbiaceae 16 species, rubiaceae 11 species, amaranthaceae 10 species, while the other families have only less than ten species (table 3). table 2. alien plant species in indonesia, their cultivation and weedyness no. cultivated/weedy ness number 1. cultivated 551 2. cultivated as ornamental 671 3. naturalized 252 4. escaped 60 5. weeds 339 6. curiosity 6 7. unknown 63 total 1936 origin of the alien species the alien plant species came from almost all parts of the world and continents, europe, africa, asia, australia, pacific and america. most of the species came from tropical america or other parts of america. the highest number came from america 40%, mostly from tropical america (figure 1), followed by asia 26%, and africa 12.8%. the lowest number of alien plant species came from australia and new zealand only 4%. 65 table 3. the families of introduced species with 10 or more weed species inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo no. family cultivated cultivated as ornamental plants naturalized escaped weeds not known total no. 1. amaranthaceae 0 3 3 0 10 0 16 2. asteraceae 19 63 20 4 53 3 162 3. caecalpiniaceae 15 22 5 0 5 6 53 4. carryophyllaceae 0 12 2 1 7 0 22 5. cyperaceae 0 2 0 0 35 2 39 6. euphorbiaceae 12 5 10 0 16 0 43 7. lamiaceae 12 10 12 0 8 0 42 8. lytraceae 5 3 2 0 7 1 18 9. onagraceae 7 0 3 0 5 0 15 10. papillionaceae 49 12 24 5 9 4 103 11. polygonaceae 6 1 2 1 5 0 15 12. poaceae 31 11 19 2 57 0 120 13. rubiaceae 15 19 2 0 11 0 48 14. solanaceae 14 5 17 2 5 1 44 15. verbenaceae 3 19 3 1 6 0 32 africa america tropic america (others parts) asia australia & new zealand europe not known 25 % 15 % 26 % 12.80 8.20 % 9 % 4 % fig 1. origin of the alien plant species in indonesia 66 important invasive alien plant species in indonesia biotropia no. 25, 2005 important invasive alien plant species in indonesia are classified into two different habitats: aquatic and terrestrial, as shown in tables 4 & 5. tabel 4. important aquatic alien invasive plants species no. species family origin 1. eichhornia crassipes (mart.) solms pontederiaceae south america 2. hydrilla verticillata (l.f.) royle hydrocharitaceae asia 3. mimosa pigra l. mimosaceae tropical america 4. pistia stratiotes l. araceae not known 5. salvinia molesta d.s. mitchell salviniaceae south america for a long time, we have been aware of the existence of infestation problems of aquatic alien plant species in open waters, such as natural lakes, man-made lakes, irrigation channels, fish ponds, and others (tjitrosoedirdjo & widjaja 1991). the first record of aquatic plants in java, bali and sumatra was made during the year 1928 to 1939 by the “deutschen limnologischen sunda expedition” which recorded over 80 species present in some open waters in indonesia (van steenis & ruttner 1933). most of the records came from the open waters in java with only few records from outside java. pancho and soerjani (1978), soerjani (1979) and tjitrosoedirdjo & widjaja (1991) reviewed that eichhornia crassipes, hydrilla verticillata, mimosa pigra, pistia stratiotes and salvinia molesta were the most important species of invasive aquatic plants in indonesia. there were some species specific to certain open waters such as polygonum barbatum which invaded ir. p.m. noor reservoir in south kalimantan (hisbi 1990); hanguana malayana invaded lake kerinci in sumatra and lake semayang in east kalimantan (staff university of indonesia 1970; sastroutomo & utomo 1985); phragmites karka invaded lake rawa danau, banten and lake curug, west java (sastroutomo & utomo 1985). the only native species is phragmites karka, while other species belong to the alien species. these alien species easily established themselves in their new environment, and spread so rapidly that native species were sometimes suppressed. this indicates that these plants possess a high power of adaptation when accompanied by a lack of natural predators. another characteristic of these plants is their rapid reproduction both vegetatively and generatively. 67 table 5. important invasive terrestrial alien plant species inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo no. species family origin 1. acasia nilotica (l.) willd. ex del. mimosaceae africa & asia 2. austroeupatorium inulaefolium (kunth) r.m. king & h. rob. asteraceae tropical america 3. chromolaena odorata (l.) king & h. rob. asteraceae central & south america 4. cryptostegia grandiflora r. br. asclepiadaceae india & madagascar 5. dicranopteris linearis (burm. f.) gleicheniaceae tropical america & subtropic 6. eupatorium sordidum less asteraceae mexico 7. jatropha gossypifolia l. euphorbiaceae central & south america 8. lantana camara l. verbenaceae tropical america 9. mikania micrantha kunth asteraceae central & south america 10. melastoma affine d. don melastomataceae asia 11. mimosa diplotrica c. wright ex sauvalle mimosaceae brazil 12. panicum maximum jacq. poaceae tropical africa 13. passiflora ligularis a. juss passifloraceae south america 14. pennisetum polystachion (l.) schult.) poaceae africa tropic 15. piper aduncum l. piperaceae south america 16. sida rhombifolia l. malvaceae asia 17. stachitarpeta indica (l.) vahl verbenaceae tropical america 18. stachytarpeta jamaicensis (l.) vahl verbenaceae tropical america 19. themeda arguens (l.) hack. poaceae not known 20. tribulus terrestris l. zygophyllaceae warm temperate most of the species listed in table 5 have been recorded as important weeds in agricultural crops. however, some additional species which have not been reported before are the important environmental weeds such as piper aduncum, jatropa gossiphifolia. two species, cryptostegia grandiflora, and tribulus terrestris are also included in table 5. rubber vine or c. grandiflora is included here, since it has become an important invasive species in australia after their introduction. new records in indonesia have to be monitored, although up to now, it is only reported in java. the other species t. terrestris also have to be monitored. t. terrestris is cultivated as medicinal plants in central java. the spread or escape from cultivation has to be prevented. passiflora ligularis is included due to its invasiveness in mount gede pangrango, where its climbing habit covers the crown of the trees and suppresses their growth. this species will be more problematic in the future than they are at present, if there is no effort to control it. 68 distribution of some important invasive alien plant species biotropia no. 25, 2005 the distribution of some important invasive alien plant species in indonesia are discussed in this paper. distribution of invasive alien plant species is necessary to be studied to prevent the spread to other areas and for the plant quarantine to decide which species is considered as optk a2 (organisme penganggu tumbuhan karantina a2)/ the invasive alien plant species which are found in indonesia but not widely distributed and being officially controlled and prevented from entering other parts of the islands. eichhornia crassipes it was introduced to beautify the ponds of bogor botanical garden in 1886. soon afterwards this plant had already spread all over the country (tjitrosoedirdjo & widjaja 1991). it is recorded in sumatra: south sumatra, lampung, west sumatra, jambi and north sumatra; kalimantan; south kalimantan; nusa tenggara: bali, lombok and flores; papua: jayapura and merauke. almost all open waters are infested by water hyacinth. the most well known and striking example is the water hyacinth problem at rawa pening lake, central java where the problem up to now is still unsolved. hydrilla verticillata it is recorded in three islands: java, sumatra and sulawesi. it is found in sumatra: lampung, south sumatra, west sumatra and jambi; sulawesi: south sulawesi; and no record yet from kalimantan and papua (figure 2). mimosa pigra it was first introduced in indonesia from mexico for botanical curiosity by the bogor botanical garden (thysman & binnendijk 1866). the earliest record of its presence was in 1844 from bogor, west java, (hasskal 1844) and it was spread and naturalized in java (tjitrosoedirdjo 1988). in sumatra, it was reported from solok, west sumatra and sibolangit in 1917. now it has spread widely from aceh to south sumatra (partomihardjo 1987). in kalimantan, it was reported since 1979 in samarinda at the lakes semayang, jempang and melintang as well as at the river sides of mahakam at east kalimantan (tjitrosemito 1997). in papua, it was firstly noticed in 1995 at the riverside of maro river between desa (village) poo and toray. apparently, the seeds contaminated materials and equipment transported by boat for constructing the people’s settlements at the places nearby maro river (barano 1999). m. pigra infested the riverside of maro river at the area of wasur national park up to wango river near the border of papua new guinea. in 1999 it was estimated that m. pigra covered approximately 15.6 ha, forming a discontinuous belt of 10 m wide (purba 1999). 69 as shown in figure 2 in indonesia, it has been recorded in sumatra: lampung, south, west and north sumatra, aceh; kalimantan: central, south and east kalimantan; papua: merauke: there is no record yet from sulawesi. inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo salvinia molesta it is recorded in java, sulawesi, sumatra, kalimantan, and papua. it is distributed in sumatra: lampung; kalimantan: south and east kalimantan; sulawesi: lake tempe; papua: danau sentani. acasia nilotica a native of africa and continental asia, it was introduced to java in 1850 and since long time out of cultivation and spread also outside java. java: west and east java; nusa tenggara: timor, and papua: wasur national park (figure 2). introduced in 1969 in baluran, east java, where a. nilotica was planted as fence to protect the teak forests near baluran national park. shortly after that, rapid expansion was begun, more than 5000 ha was occupied by a. nilotica. baluran is the only conserved savanna area left in java providing herbivore feed for banteng (bos javanicus). the invasion of a. nilotica reduces the herbage yield as well as the population of banteng. there is no record yet from sumatra, kalimantan and sulawesi. austroeupatorium inulaefolium a native of tropical america, it is commonly found in tea plantations in west java and naturalized at mount gede-pangrango. in jambi, bengkulu and west sumatra it is found in highlands. chromolaena odorata a native of central and south america, it is an aggressive invader. in central and south america, the genus chromolaena has about 165 species, but c. odorata is the only species which is now pan-tropically distributed. it is found for the first time in indonesia in 1934 in lubuk pakam, north sumatra, in tobacco plantations. after the war of independence, it had become quite common and very distinctive by its white violet flowers. it spreads very quickly all over the islands in indonesia from aceh, sumatra to papua. eupatorium sordidum a native of mexico, it was introduced in west java as ornamental plant. running wild it becomes a problem at gede-pangrango national park in west java at 1400-17 000 m altitude at the forest trails, forest-borders and waste places 70 biotropia no. 25, 2005 legend: mimosa pigra acacia nilotica hydrilla verticillata figure 2. distribution of acacia nilotica, hydrilla verticillata and mimosa pigra in indonesia mikania micrantha the genus mikania has about 400 species mainly of the warmer parts of the new world. the only indigenous species in asia is mikania cordata while mikania micrantha is the only new world species to have been introduced there. the latter species readily takes to disturbed areas and tends to be weedy (parker 1972). in 1949 it was imported from paraguay and planted in the bogor botanical garden. in 1956, this species was introduced as a non-legume ground cover in rubber plantations due to the scarcity of legume seeds. wirjahardja (1976) reported that by 1976, it occupied the greater part of rubber plantations and abandoned agricultural areas in west and east java and south sumatra. now it is widely spread in almost all the islands of indonesia. although m. cordata is a native species of southeast asia, m. micrantha is a more aggressive species and causes a lot of problems, especially the maintenance and harvest of the crop become difficult. it is also climbing the trees at the edge of the forest in open areas. in indonesia, it is now difficult to find m. cordata. their growth is suppressed by m. micrantha. the specimens can only be found at the herbarium bogoriense (bo) and biotrop herbarium (biot) (tjitrosoedirdjo 2002). 71 conclusions inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo based on the inventories of the existing references and herbarium specimens there are a total of 1936 alien plant species belonging to 187 families in indonesia. field studies should be done to get the complete figures of the alien plant species in indonesia. based on the existing figures of the alien plant species, the invasive alien plant species can be identified, followed by studies on assessment of losses, biology, management and their possible utilizations. most of the alien plant species are cultivated as ornamental plants. there are 67% of the total species number. the weed species consists 17.5% of the total species number. the alien plant species came from all continents of the world, the highest number of their origin was america mostly the tropical part, followed by asia, africa, europe and the smallest number from australia and new zealand. there are three families with more than 100 species, asteraceae 162 species, poaceae 120 species and papillionaceae 103 species. the highest number of the weedy species is found in the family of asteraceae followed by poaceae and cyperaceae. twenty-five important aquatic and terrestrial alien plant species in indonesia were identified and some of their distributions were noted. field studies should be conducted for completing the records. acknowledgements this work was supported by tropical biology development sub project (d.i.p. project funded by goi) through seameo biotrop 2004 and the ministry of environment, indonesia. thanks are due to ms. nenah suminah for her assistance in compiling the data and to mr. setiabudi for preparing the distribution map. references barano, s.s. 1999. persoalan dan penanganan eceng gondok, putri malu raksasa, kirinyu dan rusa di taman nasional wasur. proceeding lokakarya introduksi species asing dan permasalahannya, jayapura 29-30 july, 1999(in indonesian) . groves, r.h. 1999. environmental weeds-past, present and future. plant protection quarterly vol. 14 (3): 92-99 hasskarl, j.k.1844. catalogus plantarum in horto botanico bogoriense cultarum alter. ter lands drukkerij, batavia. hisbi, d. 1990. problems and control of polygonum barbatum in ir. p.m. noor reservoir, south kalimantan, indonesia. biotrop special publication no. 40: 63-68 mangunsoekarjo & kadnan. 1973. mikania eradication in immature rubber. proceedings 2nd indonesian weed science society conference. 72 partomihardjo, t. 1988. observation of some biological aspects of mimosa pigra to support its control in indonesia. proceeding ixth indonesian weed society conference, bogor, indonesia biotropia no. 25, 2005 purba, m. 1999. masalah penangan flora fauna eksotik di taman nasional wasur dan sekitarnya. proceeding lokakarya introduksi species asing dan permasalahannya, jayapura 29-30 july, 1999 (in indonesian). sastroutomo, s.s. and i.h. utomo. 1985. aquatic weeds problems in lake rawa danau, west java. biotrop internal report. soerjani, m. 1979. recent trend in aquatic weed management in indonesia. proceedings 7th apwss conference. supplement volume. steenis, c.g.g.j. van and f. ruttner. 1933. die pteridophyten und phanerogamen der deutschen limnologischen sunda expedition. archiv. fur hydrobiologie, suppl. band xi. tropische binnengewasser band iii. e. schweizerbartsche verlachhandlung, stuttgart. thysmann, j.e. & s. binnendijk. 1866. catalogus plantarum guae in horto botanico bogoriense colunter. ter lands drukkerij, batavia. tjitrosemito, s. 1997. infestation of mimosa pigra in mahakam river systems, east kalimantan, indonesia. weedwatcher 27:1-2. tjitrosoedirdjo, s.s. & f. widjaja. 1991. aquatic weeds management in indonesia. biotrop special publication no. 40: 25-36. tjitrosoedirdjo, s.s. 2002. notes on the asteraceae of sumatera. biotropia. no. 19: 65-84. tjitrosoedirdjo, s.s. & s. tjitrosemito. 2005. compilation of alien plant species in indonesia. research report cooperation between ministry of environment, republic of indonesia and seameo biotrop. doc. no.: biotrop/capsi/2005/1019. universitas indonesia staff. 1979. survei ekologi danau kerinci. laporan akhir tahun 1978-1979. lab. biologi wilayah, fmipa, universitas indonesia. doc. no. 05/lbw/79 (in indonesian). weber, e. 2003. invasive plant species of the world. a reference guide to environmental weeds. cabi publishing. cab international. wallingford. oxon ox 10 8de, uk. wirjahardja, s. 1976. autoecological study of mikania spp. biotrop internal report. 73 abstract introduction materials and methods inventory and distribution important indonesian invasive plant species results and discussions inventory origin of the alien species distribution of some important invasive alien plant species eichhornia crassipes hydrilla verticillata mimosa pigra salvinia molesta acasia nilotica austroeupatorium inulaefolium mikania micrantha acknowledgements references biotropia vol. 29 no. 3, 2022: 225 233 doi: 10.11598/btb.2022.29.3.1669 225 cadmium, nickel, and lead concentration of municipal dumpsite in western samar, philippines mariss bostrillo varona1, judy-ann ligo amistoso1 and pearl aphrodite bobon-carnice12* 1department of natural sciences, eastern visayas state university, tacloban city 6500, philippines 2office of research and development, eastern visayas state university, tacloban city 6500, philippines received 21 september 2021/accepted 8 march 2022 abstract heavy metal is one of the major problems due to its accumulation from the soil to the food chain, wherein dumpsites are the primary sources of heavy metal pollution. this study aimed to determine the presence of heavy metals in the soil of santa rita, western samar dumpsite and to quantify them to obtain knowledge on the possible high contamination that may affect the surrounding areas. this study focused on the presence and concentrations of heavy metals cd, ni, and pb. eighteen (18) soil samples were acquired within the three sampling sites: shoulder slope, main dumpsite, and foot slope. each sampling site has three sampling points with a depth of 0 30 cm and 30 60 cm. analysis showed that all heavy metals are present in the dumpsite, and the concentrations ranged from 0 0.1 mg/kg, 0.09 3.7 mg/kg, and 0.09 3.7 mg/kg for cadmium, lead, and nickel, respectively. in comparing heavy metals within the sampling sites and depths, only cadmium has a significant difference, while ni and pb have no significant difference. compared with who standards, all heavy metals tested still fall within the standard limit. therefore, the dumpsite is still at a safe level. however, residents should take measures to maintain the soil quality since heavy metal contamination in dumpsites is likely to exacerbate. keywords: cadmium, lead, municipal dumpsite, nickel, soil pollution introduction one of the global issues is improper waste management. open dumping and open burnings are the most implemented waste treatment and final disposal systems, mainly available in lowincome countries (ferronato & torretta 2019). the increased population and the rising demand for food and other essentials equate to increased waste generated daily in each household, wherein this waste eventually dumps in dumpsites and impacts the soil and the surface environment (mekonnen et al. 2020). uncontrolled disposal results in severe heavy metal contamination (vongdala et al. 2019). further, dumpsite operation is a source of heavy metal pollution that can affect the biosphere (sakawi et al. 2013). heavy metals naturally occur in the earth's crust with high atomic weight and density at least five times greater than water (tchounwou et al. 2012). heavy metal is one environmental contaminant affecting aquatic and terrestrial ecosystems (sakawi et al. 2013). heavy metals possess a severe risk because they tend to bioaccumulate, which means an increase in chemical concentration in a biological organism over time compared to the chemical concentration in the environment (helmenstine 2021). sources of heavy metals include mining and plating, fertilizers and pesticides, sludge dumping, and municipal waste (mudgal 2010). the liquid that exudes and percolates solid wastes eventually transfers to the soil. heavy metals, pesticides, and hydrocarbons are threatening substances that bind in liquid and constantly contaminate soil and water (adelekan & alawode 2011). dumpsite of santa rita is located at sitio canonay of barangay rosal, santa rita, western samar. the dumpsite is already 20 years old, and all kinds of waste are accepted from all *corresponding author, email: pearl.carnice@evsu.edu.ph biotropia vol. 29 no. 3, 2022 226 barangays of santa rita, western samar. the municipal government of santa rita owns the entire area of the dumpsite. the main dumpsite is 45 m from the nearby residences and 22 m away from the rice plants in sitio canonay. this study aimed to determine and quantify the concentrations of heavy metals present in the santa rita, western samar dumpsite soil and provide knowledge about the possible high contamination of heavy metals that may affect the surrounding areas. materials and methods study site the study was conducted in santa rita, western samar, where the dumpsite is located at sitio canonay of barangay rosal (fig. 1). the dumpsite was divided into three sampling sites: shoulder slope, main dumpsite, and foot slope, wherein three sampling points in each sampling site were randomly selected. the dumpsite comprises verdant hills covered with grassland and forest vegetation on the shoulder slope, a bulk of wastes that covers the main dumpsite with ipomoea aquatica (water spinach) plantation that surrounds some parts of the area, and a rice field that could be found at the foot slope of the dumpsite. sample collection, preparation, and heavy metal analysis a descriptive analysis of research was utilized in this study. each sampling point was dug up to at least 60 cm depth. then, two (2) composite samples in every sampling point were collected according to their depth (0 30 cm and 30 60 cm), wherein 18 soil samples were acquired within the three sampling sites. after sampling, all soil samples were air-dried, crushed using a mortar and pestle, sieved through a 2-mm sieve, and stored in a clean, resealable, properly labeled bag. figure 1 location map showing the sampling points cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 227 soil samples were digested using the dtpa acid extraction solution. the digested soil samples' cd, ni, and pb concentrations were analyzed using atomic absorption spectrophotometry (aas) equipped with pb hollow cathode lamp (varian spectraaa 220 fs with sips pump unit and autosampler sps-5). standard solutions of the heavy metals were utilized in the preparation of the calibration curve. the following formula was used to compute pb, cd, and ni concentration in the dumpsite soil. heavy metals in soil (ppm) = ml of extraction solution x conc. (ppm) grams of soil where: ml of extraction solution = volume of extraction solution grams of soil = weight of soil sample in grams conc. (ppm) = result of aas concentration in mg/l statistical analysis two-way analysis of variance (anova) was used in this study to determine the significant variation in the mean concentrations of identified heavy metals in soil concerning sampling depth and sampling site. natural logarithm was used to obtain the assumption of anova in which all data should be in a normal distribution and have homogenous variance. shapiro wilk's test was used for analyzing the normality, and levene's test was used to determine the homogeneity. in comparing the concentrations of identified heavy metals with standard, a two-tailed t-test was used in this study. all statistical computation was run in the program r project where in to run the statistical computing in r software, packages used were dplyr, car, rstatix, tidyverse, and ggpubr (r core team 2021). acceptance and rejection of the hypothesis were set at a 0.05 level of significance. results and discussion heavy metal contents of dumpsite soil cd, ni, and pb in different sampling sites at different depths were found in the study site (table 1). the environment and its components in santa rita have been seriously polluted by heavy metals, which have compromised the ability of the environment to foster life and take its intrinsic values. heavy metals commonly occur naturally on earth, but anthropogenic activities result in large quantities of different environmental components (masindi & muedi 2018). the presence of nickel could be attributed since nickel is a metal with widespread distribution in the environment, including minerals. it is an essential constituent with many industrial and commercial uses (iyaka 2011). nickel can exist in soils in several forms, such as inorganic crystalline minerals or precipitates, complexed or adsorbed on organic cation surfaces, or inorganic cation exchange surfaces, among others (cempel & nikel 2006). nickel is a ubiquitous pollutant and carcinogen toxic metal found in many hazardous waste sites, especially in dumpsite that receives different types of waste, such as municipal solid waste, clinical waste, and industrial waste. industrial waste materials, lime, fertilizer, and sewage sludge contribute to the significant nickel sources in the soil (das et al. 2018). table 1 concentrations of heavy metals in dumpsite soil at different sampling sites and depths sampling sites sampling points gps coordinates cd (mg/kg) ni (mg/kg) pb (mg/kg) 0 30 cm 30 60 cm 0 30 cm 30 60 cm 0 30 cm 30 60 cm shoulder slope point 1 n 11o28’9.52364” e 124 o57'7.20043" trace trace 3.717 3.054 0.246 0.327 point 2 n 11o28’10.09024” e 124o57’6.34467” trace trace 0.494 0.378 trace trace point 3 n 11o28’10.32682” e 124o57’5.89446” trace trace 2.715 3.679 0.795 0.566 mean 2.308667 2.370333 0.5205 0.4465 main dumpsite point 1 n 11o28’9.17942” e 124o57’6.63555” 0.0114 0.1094 0.446 1.078 1.51 58.642 point 2 n 11o28’8.81745” e 124o57’7.07731” 0.0014 trace 2.154 1.91 0.318 0.846 point 3 n 11o28’8.4058” e 124o57’6.42071” trace 0.0504 1.939 1.065 0.7 11.578 mean 0.0064 0.0799 1.513 1.351 0.842667 23.68867 foot slope point 1 n 11o28’9.05403” e 124o57’2.92525” 0.0024 0.001 0.167 0.175 2.291 1.893 point 2 n 11o28’8.94994” e 124o57’4.08035” trace 0.001 0.448 0.409 1.438 1.525 point 3 n 11o28’8.17159” e 124o57’12.86853” trace 0.0023 0.085 0.147 1.469 1.56 mean 0.0024 0.001433 0.233333 0.243667 1.732667 1.659333 biotropia vol. 29 no. 3, 2022 228 on the other hand, lead was found at all sampling sites and depths within the dumpsite of santa rita, western samar. lead could be found in the dumpsite because it has some unique physical and chemical properties from historical times and has become a common environmental pollutant. lead forms different complexes with soil elements. it only takes a small piece of the lead as these complexes within the soil are phytoavailable (pourrut et al. 2011). lead has remained in the ground for thousands of years, not biodegradable (epa 2019). it poses a severe environmental risk due to its continued usage in every part of the world due to the abundance of gasoline, industrial processes, lead-based painting, lead-containing pipes, and lead-acid batteries (wani et al. 2015). lead consumption has increased due to increased demand for lead-containing products, such as batteries, electronic products, cathode ray tubes, pvc stabilizers, and lead pigments. these leadcontaining products increase lead-containing waste (european commission dg env 2002). moreover, cadmium was also found within the dumpsite of santa rita. cadmium can be released into the environment through anthropogenic activities and can be used in many industrial uses, such as plastic, pigment, enamels, ceramics, and steel plating. in addition, industrial processes produce cadmium as a byproduct (hasan et al. 2019). results of our study showed that nickel is found at all sampling points and depths. lead was present in trace amounts at the shoulder slope point 2 and other sampling points and depths. cadmium was mainly found at the foot slope at 30 60 cm depth, sparsely found at the main dumpsite, and was present only in trace amounts at the shoulder slope. the mean concentrations of cadmium, nickel, and lead in soil ranged from 0 0.1 mg/kg, 0.09 3.7 mg/kg, and 0.09 3.7 mg/kg for cadmium, nickel, and lead, respectively. mean concentrations were 0.0084±0.0030 mg/kg, 1.3517±0.4382 mg/kg, and 1.0959±0.2475 mg/kg, respectively, for 0 30 cm depth and 0.0137±0.0122 mg/kg, 1.3217±0.4322 mg/kg, and 1.1195±0.2560 mg/kg respectively for 30 60 cm depth. the results agree with the result from orodu et al. (2017), who reported a range of 0.09 1.10 mg/kg, 0.19 1.84 mg/kg, and 0.47 14.33 mg/kg for cadmium, nickel, and lead, respectively with means of 0.22±0.08 mg/kg, 1.03±0.16 mg/kg, and 5.17±5.04 mg/kg. results of our study also agree with amostautua et al. (2014), which determines the concentration of heavy metals in the dumpsite and cadmium. they reported a range of < 0.000.1±0.01 mg/kg, while amadi et al. (2012) studied different heavy metals, cadmium, and lead, resulting from a mean concentration of 0.15 ppm and 16.0 ppm, respectively. essien et al. (2019) reported a range of 0.92 1.4 mg/kg, 1.8 3.7 mg/kg, and 0.79 0.98 mg/kg taken during the wet season while 0.124 1.4 mg/kg, 1.72 2.98 mg/kg and 0.94 4.12 mg/kg taken during the dry season for nickel, lead and cadmium, respectively. it can be seen that heavy metal concentrations followed the order ni > pb > cd, which is in agreement with the results from orodu et al. (2017), amadi et al. (2012), essien et al. (2019), and makuleke & ngole-jeme (2020). figure 2 shows the mean concentrations of the metals from each sampling site at different depths. the trends observed at a sampling depth of 0 30 cm were generally the same as those taken at 30 60 cm. differences in heavy metal concentration can be attributed to the amount of waste in the dumpsite carrying the different heavy metals. for example, nickel having the highest levels could be attributed to more nickelcontaining wastes being dumped, such as buttons, zips, coins, household appliance tools, and other consumer products (asemave & annwange 2013). extensive industrial use of nickel has led to widespread environmental pollution (das et al. 2018). on the other hand, cadmium generally has the lowest concentration. this could be due to the decrease in demand for cadmium-containing products in the industry. specifically, the use of cadmium for pigments, pvc stabilizers, and plating has been phased out. as an impurity in zinc and fertilizers, turnover of cadmium has also been significantly decreased due to refining and changes in raw materials (european commission dg env 2002), resulting in a decrease in cadmium-containing products that end up in waste. cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 229 figure 2 comparison of (a) cd, (b) ni, and (c) pb concentration means per sampling site and depth comparison of heavy metal contents according to sampling site and depth table 2 presents the statistical analysis results of comparing concentrations of cadmium, nickel, and lead at the different sampling sites and depths. significant differences are indicated by p ≤ 0.05. in case of significant differences, the results of post-hoc tukey's honest significant difference (hsd) are indicated in table 3. the mean values of cd, ni, and pb for all sampling sites were 0.008 mg/kg, 1.351 mg/kg, and 1.096 mg/kg, respectively, for 0 30 cm depth. while for 30 60 cm depth, the mean values of cd, ni, and pb were 0.033 mg/kg, 1.322 mg/kg and 1.119 mg/kg, respectively for all sampling sites (table 2). the computed p values showed no significant differences between the two sampling depths for all heavy metals tested. in addition, the computed p values of cd, ni, and pb in each sampling site for both sampling depths are 0.001, 0.003, and 0.012, respectively. it means that all sampling sites differed and suggested that dumpsite soil lacks the capacity to impede the downward migration of leachate from topsoil to subsoil (amadi et al. 2012). moreover, only cadmium had a depth-site interaction wherein two independent variables should be tested whether the effect of one independent variable is dependent or affects the other. biotropia vol. 29 no. 3, 2022 230 table 2 differences in cadmium, nickel and lead concentrations between different sampling sites and depths sampling depth heavy metals mean concentration (mg/kg) p value (α = 0.05) interpretation 0 30 cm 30 60 cm cd 0.0084 0.03282 0.9421 not significant ni 1.3517 1.32166 0.8945 not significant pb 1.095875 1.1195 0.9320 not significant sampling site heavy metals mean concentration (mg/kg) p value (α = 0.05) interpretation shoulder slope main dumpsite foot slope cd 0.04565 0.001675 0.0006 significant ni 2.3395 1.432 0.2385 0.0026 significant pb 0.4835 0.8435 1.13067 0.0115 significant depth sampling site interaction heavy metals p value (α = 0.05) interpretation cd 0.0207 significant ni 0.9767 not significant pb 0.9505 not significant table 3 presents results for multiple pairwise comparisons using tukey's honest significant difference test for the indicated heavy metals. this is employed to identify which groups are significantly different from each other. results was found that cadmium levels were approximately 3 times higher in the main dumpsite than in the foot slope. for nickel and lead, only the main effect of the sampling site was found to be significant. nickel concentrations at both the main dumpsite and shoulder slope are approximately 2 times higher than concentrations at the foot slope (table 3). this suggested a stronger upward lateral leaching (raji & adeoye 2017), leading to more nickel accumulation at the shoulder slope than at the foot slope. in the case of lead, levels at the foot slope were found to be 1.3 times higher compared to the shoulder slope, suggesting a slightly higher downward lateral leaching (raji & adeoye 2017). differences in leaching strength can be attributed to differences in soil properties (asemave & anhwange 2013; makuleke & ngole-jeme 2020). to better visualize the interaction between the depth effect and sampling site effect for cadmium, an interaction plot is presented in figure 3. an interaction effect is the simultaneous effect of two or more independent variables on one dependent variable. their combined effect is significantly greater (or substantially less) than the sum of the parts. the presence of interaction effects in survey research is essential because it tells researchers how two or more independent variables impact the dependent variable. table 3 tukey's hsd results for nickel, lead, and cadmium heavy metal sampling site difference p value (α = 0.05) interpretation ni main dumpsite foot slope 1.8365680 0.0092866 significant shoulder slope foot slope 2. 1108135 0.0035699 significant pb shoulder slope foot slope -1.3433094 0.0104619 significant cd main dumpsite foot slope 2.9373 0.0006842 significant depth sampling site interaction difference p value (α = 0.05) interpretation 60 cm main dumpsite 30 cm foot slope 3.4320323 0.0103995 significant 30 cm main dumpsite 60 cm foot slope 2.1559770 0.0191866 significant 60 cm main dumpsite 60 cm foot slope 4.0298647 0.0018821 significant 60 cm main dumpsite 30 cm main dumpsite 1.8738877 0.0421485 significant cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 231 cadmium concentration at the main dumpsite is 3 times higher than the foot slope (fig. 3). however, differences can be observed due to the interaction between depth and sampling site effects. specifically, cadmium concentration at the main dumpsite (0 30 cm) is 2 times higher compared to the foot slope (30 60 cm), 3 times higher at the main dumpsite (30 60 cm) than the foot slope (0 30 cm), and 4 times higher at the main dumpsite (30 60 cm) compared to foot slope (30 60 cm). comparison of heavy metal contents with standard permissible limits in soil table 4 presents the results for comparison of heavy metal concentrations with the maximum permissible limits set by the world health organization (who). the permissible limits of heavy metals in soil are 0.02 0.5 (mg/kg), 0.1 5 mg/kg, and 0.3 10 mg/kg for cadmium, nickel, and lead, respectively (who 2001). a soil sample is polluted with a particular heavy metal if the concentration exceeds the upper limit of the acceptable range (ogundele et al. 2015). therefore, to determine whether soil samples from santa rita, western samar dumpsite was polluted with the indicated heavy metals, the right-tailed t-test was employed to compare the sample's mean concentration with the maximum permissible. figure 3 depth sampling site interaction plot for cadmium table 4 t-test results for comparison of mean concentrations with who standard heavy metals sampling sites sampling depth (cm) mean concentration who standard p-value (α = 0.05) interpretation cd shoulder slope 0 30 n/a 0.05 n/a n/a 30 60 n/a 0.05 n/a n/a main dumpsite 0 30 n/a 0.05 n/a n/a 30 60 0.0799 0.05 0.2479 no significant difference foot slope 0 30 n/a 0.05 n/a n/a 30 60 0.0014 0.05 1 no significant difference ni shoulder slope 0 30 2.3087 5 0.9471 no significant difference 30 60 2.3703 5 0.9391 no significant difference main dumpsite 0 30 1.513 5 0.9885 no significant difference 30 60 1.351 5 0.9971 no significant difference foot slope 0 30 0.2333 5 0.9997 no significant difference 30 60 0.2437 5 0.9998 no significant difference pb shoulder slope 0 30 0.5205 10 0.9908 no significant difference 30 60 0.4465 10 0.9960 no significant difference main dumpsite 0 30 0.8427 10 0.9993 no significant difference 30 60 23.689 10 0.2606 no significant difference foot slope 0 30 1.7327 10 0.9994 no significant difference 30 60 1.6593 10 0.9999 no significant difference biotropia vol. 29 no. 3, 2022 232 the results obtained showed that all heavy metal concentrations were not significantly higher than the maximum permissible limits set by the who (table 4). as such, they still fall within the permissible limits of who. it means that the concentrations of all indicated heavy metals are still at safe levels. heavy metals in the dumpsite soil should not threaten anyone, particularly the surrounding susceptible environment. however, serious measures should still be taken to maintain soil quality since heavy metal contamination in dumpsites will likely exacerbate in the future (orodu et al. 2017). conclusion identified heavy metals are all found at different sampling sites and depths of santa rita western samar dumpsite soil. the mean concentrations (mg/kg) of ni is the predominant, followed by pb, and the least is cd. no significant differences were observed in the concentrations of pb and ni in different sampling sites and depths, but significantly different in the concentration of cd. in addition, no significant differences were observed between the concentrations of identified heavy metals in all sampling sites at different depths and the standard permissible limits. therefore, all concentrations of identified heavy metals were within the permissible limits in soil. acknowledgments the authors acknowledge prof fe t. piedad, prof adorne y. madera, and prof grechelle n. socias for their essential contributions during the final polishing of this manuscript. references adelekan ba, alawode ao. 2011. contribution of municipal refuse dumps to heavy metals concentrations in soil profile and groundwater in ibadan nigeria. j appl biosci 40: 2727-37. amadi n, olasehinde p, okosun e, okoye o, okunlala a, baba a, dan-hassan m. 2012 . a comparative study on the impact of avu and ihie dumpsites on soil quality in southeastern nigeria. am j chem 2(1): 17-23. amos-tautua bmw, onigbinde ao, ere d. 2014. assessment of some heavy metals and physicochemical properties in surface soils of municipal open waste dumpsite in yenagoa, nigeria. afr j enviro sci technol 8(1): 41-7. asemave k, amhwange, ba. 2013. evaluation of heavy metals in waste dumpsites. germany: lap lambert academic publishing. cempel m, nikel g. 2006. nickel: a review of its sources and environmental toxicology. pol j environ stud 15(3): 375-82. das kk, reddy rc, bagoji ib, das s, bagali s, mullur l, ..., biradar ms. 2018. primary concept of nickel toxicity-an overview. j basic clin physiol pharmacol 30(2): 141-52. environmental protection agency. 2019. learn about lead. [updated 2019 dec 15; cited 2020 jan 28]. available from: https://www.epa.gov/lead/learnabout-lead essien jp, inam ed, ikpe di, udofia ge, benson nu. 2019. ecotoxicological status and risk assessment of heavy metals in municipal solid wastes dumpsite impacted soil in nigeria. environ nanotechnol monit manag 11: 100215. european commission dg env. 2002. heavy metals in waste. department for environment, food & rural affairs. ferronato n, torretta v. 2019. waste mismanagement in developing countries, a review of global issues. int j environ res public health 16(6): 1060. hasan sa, fariduddin q, ali b, hayat s, ahmad a. 2019. cadmium: toxicity and tolerance in plants. j environ biol 30(20): 165-74. helmenstine a [internet]. 2021. heavy metal definition and list :thought.co; updated 2020 jan 26; cited 2020 feb 17]. available from: https:// www.thoughtco.com/definition-of-heavy-metal605190 iyaka ym. 2011. nickel in soils: a review of its distribution and impacts. j sci res essays 6(33): 6774-7. makuleke p, ngole-jeme vm. 2020. soil heavy metal distribution with depth around a closed landfill and their uptake by datura stramonium. appl environ soil sci 2020: 1-14. masindi v, muedi kl. 2018. environmental contamination by heavy metals. in: saleh hm, editor. heavy metals. intechopen 2020: p.1-14. mekonnen b, haddis a, zeine w. 2020. assessment of the effect of solid waste dump site surrounding soil and river water quality in tepi town, southwest ethiopia. j environ public health 2020: 9. cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 233 mudgal v, madaan n, mudgal a, singh rb, mishra s. 2010. effect of toxic metal on human health. open nutraceutical j3: 94-9. ogundele dt, adio aa, oludele oe. 2015. heavy metal concentrations in plants and soil along heavy traffic roads in north central nigeria. j environ anal toxicol 25: 6. orodu ve, leizou ke. 2017. determination of heavy metals ( pb , cd and ni ) in soils of municipal solid wastes dumpsite, yenagoa metropolis, bayelsa state. scholars acad j biosci 5(4): 320-4. pourrut b, shahid m, dumat c, winterton p, pinelli e. 2011. lead uptake, toxicity and detoxification in plants. rev environ contam toxicol 213: 113-36. raji wo, adeoye to. 2017. geophysical mapping of contaminant leachate around a reclaimed open dumpsite. j king saud univ sci 29(3): 348-59. r core team. 2021. r: a language and environment for statistical computing. vienna, austria. sakawi z, ariffin m, mastura sa. 2013. the analysis of heavy metal concentration per distance and depth around the vicinity of open landfill. res j appl sci eng technol 5(4): 8619-25. tchounwou pb, yedjou cg, patlolla an, sutton dj. 2012. heavy metal toxicity and the environment. molecular clinical and environment toxicology experientia supplementum 101: 13-6. vongdala n, tran h, xuan td, teschke r, khanh td. 2019. heavy metal accumulation in water, soil, and plants of municipal solid waste landfill in viantiane, laos. int j environ res public health 16(1): 21. wani al, ara a, usmani ja. 2015. lead toxicity: a review. interdiscip toxicol 8(2): 55-64. word health organization. 2001. standard for the contents of heavy metals in soils. [updated 2020 dec 14; cited 2020 jan 23]. available from: https://www.who.int/fact-sheets/detail/standardfor-the-contents-of-heavy-metals-in-soils http://www.who.int/fact-sheets/detail/standard-for-the-contents-of-heavy-metals-in http://www.who.int/fact-sheets/detail/standard-for-the-contents-of-heavy-metals-in microsoft word 69 biotropia vol. 13 no. 2,2006 : 69 74 embryo reconstruction by transplantation of the donor inner cell mass to the recipient bovine blastocyst arief boediono laboratory of embryology, department of anatomy, physiology and pharmacology, faculty of veterinary medicine, bogor agricultural university, darmaga 16680bogor, indonesia abstract in an attempt to produce the interspecies embryo transfer, this study was conducted to evaluate the efficacy of the production of reconstructed blastocyst by transferring the donor icm into the recipient trophoblast. icm cells were isolated from the donor blastocyst by immunosurgery method. zona-free blastocysts were incubated in the medium (tcm-199) containing 20% of the heat-inactivated rabbit anti-bovine-serum. the embryo reconstruction was produced by three different methods. recipient blastocyst was maintained on the holding pipette by gentle suction, with the icm in a 9 o'clock position to have the possibility of developing incorporate icms (method i), the icm was in a 3 o'clock position to break the original icm during injection (method 2); cutting the original recipient icm followed by insertion of the donor icm (method 3). reconstructed blastocysts were then cultured overnight and examined morphologically according to the re-expansion of the reconstructed blastocyst with or without developed donor icm. according to morphological observation in this study, 37.9% of the reconstructed blastocyst developed with the incorporation of two icm originally from recipient and donor (method i), 66.7% of the reconstructed blastocysts developed with a single icm (method 2), and 80.0% of the reconstructed blastocyst developed from the icm originally from donor icm (method 3). these results showed that the reconstructed blastocyst is better produced by cutting the original recipient icm followed by the insertion of the donor icm (method 3). key words: embryo reconstruction, immunosurgery, icm transfer, bovine. introduction embryo reconstruction for chimera production has been used in experiments oriented towards animal science such as the production of interspecies pregnancies in domestic animals (fehilly et al. 1984a; polzin et al. 1987). so far, chimeras have been obtained by the aggregation of the blastomeres (brem et al. 1984; boediono et al. 1993; boediono et al. 1999) or by inner cell mass transplantation (butler et al. 1987; polzin et al. 1987; picard et al. 1990). the aggregation of cell from embryos results in embryos with a more randomly distribution contribution of cells from each donor to the trophoblast and icm. fehilly et al. (1984b) in their production of interspecific sheep-goat chimeras produced one kid by the injection of a goat icm into a sheep blastocyst and one lamb from the reciprocal injection. rorie et al. (1994) produced two normal lambs after transferring nine reconstructed embryos into three recipient goats. the reconstructed embryos comprised trophoblast cells from the embryo recipient species (goat) and icm cells from the embryo donor species (sheep). the present study was conducted to evaluate alternative procedures corresponding author: abl@cbn.net.id 69 biotropia vol. 13 no. 2, 2006 for production of the reconstructed embryo by transplantation of the donor inner cell mass to the recipient bovine blastocyst. materials and method in vitro embryo production embryos were produced by standard in vitro maturation, fertilization and culture procedures (boediono et al. 2003). bovine ovaries were collected from a nearby abattoir and transported (32-35°c) to the laboratory in 0.9% physiological saline solution immediately following collection. follicular oocytes were allowed to mature for 22 hr at 38.5°c under 5% co2 in air. frozen-thawed sperm was washed twice with 2.5 mm caffeine in brackett-oliphant medium (b-o; brackett and oliphant 1975) without bovine serum albumin. sperm concentration was adjusted to 5 x 106 spermatozoa per ml in b-o supplemented with 0.3% bovine serum albumin (bsa, sigma) and 20 ̂ g/ml heparin (shimizu, japan). a100 ul aliquot of the sperm suspension was pre-incubated for 1 hr. in v//ra-matured oocytes were transferred into fertilization droplets for insemination (20-25 oocytes/droplet). after 18 hr of sperm exposure, oocytes were washed and transferred to a polystyrene dish (4-well multidish; nunclon, roskilde, denmark) containing tcm199 supplemented with 5% superovulated cow serum (boediono et al. 1994), 5 ng/ml insulin (wako, osaka, japan) and 50 ng/ml gentamicin sulfate for further development. adherent cumulus cells surrounding the embryos were removed by repeated pipetting 48 h post-fertilization. the cumulus cells adhering to the surface of the culture dish were not disrupted, and embryos were cultured on this somatic cell monolayer. the culture medium was replaced with fresh medium 96 h post-fertilization. icm isolation blastocysts produced in vitro were used in this experiment. an immunosurgical method for icm isolation was carried out by the similar method described by softer and knowles (1975) with modification. the zona pellucida was removed by a short incubation (<5 min) in tcm-199 containing 0.5% pronase. for immunosurgery, the embryos were incubated for 1 h in the medium (tcm-199) containing 20% of the heat-inactivated rabbit anti-bovine-serum. they were then washed in three successive baths of tcm-199 + 20% pcs and were transferred into tcm-199 containing 10% of reconstituted guinea pig complement for 10 min followed by two successive washes in tcm-199 + 20% pcs. embryos were aspirated into a pipette of a diameter slightly larger than the icm to remove any degenerating trophoblastic cell. embryo reconstruction the manipulator was arranged with a beveled injection pipette with an outer diameter of approximately 40-50 \\m on one side and an embryo holding pipette 70 embryo reconstruction a. boediono with an outer diameter of 150 urn on the other side (method 1 and 2). the donor icm was drawn into the injection pipette and held directly opposite the recipient blastocyst on the holding pipette. the embryo reconstruction was produced by three different methods. recipient blastocyst was maintained on the holding pipette by gentle suction, with the icm in a 9 o'clock position to have the possibility of developing an incorporate icms (method 1, figure 1a-b), the icm was in a 3 o'clock position to break the original icm during injection (method 2, figure 1c-d); cutting the original recipient icm followed by insertion of the donor icm (method 3, figure 1e-f). with a swift precise forward motion the tip of the injection pipette was introduced into the blastocoele and the icm was injected. reconstructed embryos were cultured overnight and then examined morphologically according to the re-expansion of blastocyst with the presence of original and donor icms (method 1 and 2) and the re-expansion of the blastocyst with or without donor icm (method 3). results and discussion embryo reconstruction was performed by injection of a total of 78 icm isolated from donor blastocysts into blastocoel recipient blastocysts. in an attempt to produce interspecies pregnancy, the development of the donor icm with the original trophoblast is most important to produce a baby originating from donor icm. according to morphological observation , 37.9% of the reconstructed blastocyst produced by method 1 developed from the incorporation of two icm originally from recipient and donor, while 34.5% developed with the corporation of two icm (table 1). using the same method, fehilly et al. (1984b) in their production of inter 71 biotropia vol. 13 no. 2,2006 species sheep-goat chimeras, produced one kid that developed from the donor icm, two chimeras and 10 lambs after transfer of 22 reconstructed blastocysts into 14 recipients. none of the reconstructed blastocyst produced by method 2 developed with two incorporated icm. a total of 66.7 percent of the reconstructed blastocysts developed with a single icm. the purpose of this method was to break the original recipient icm and insert the doonor icm. however, we are not sure that the broken original icm would be destroyed and the developed icm come only from donor icm. there was a possibility that the original icm could recover and develop 72 embryo reconstruction a. boediono incorporating both icms from donor and recipient. the transfer of these embryos may result in chimeric animals. to avoid the development of the original recipient icm into the reconstructed blastocyst, the removal of icm recipient blastocyst by cutting followed by insertion of the donor icm (method 3) is the most definite way to produce interspecies pregnancy. in this study, 80.0% of the reconstructed blastocyst developed with the icm originally from donor icm. the reconstructed blastocysts have been transferred to the three recipients resulting in one pregnancy. this method would be useful for production of the interspecies embryo transfer to rescue the endangered animals or for nuclear transfer produced embryo (sansinena et al. 2005). conclusions attempts to produce interspecies embryos for transfer showed that the reconstructed blastocyst is better produced by cutting the original recipient icm followed by the insertion of the donor icm (method 3 in this study) and transferring into recipient with the same species as recipient blastocyst. acknowledgments the author would like to thank dr. robert a. godke, professor of the department of animal science, louisiana state university, usa for his suggestions and helpful evaluation of the manuscript. references butler, j.e., anderson, g.b., bondurant, r.h., pashen, r.l, and m.c. penedo. 1987. production of ovine chimeras by inner cell mass transplantation. j. anim. sci., 65:317-324. boediono, a., oe, m, yamamoto, m, takagi, m., saha, s. and t. suzuki. 1993. production of chimeric calves by aggregation of in vitro bovine embryos without zonae pellucidae. theriogenology, 40:1221-1230. boediono, a., takagi, m., saha, s. and s. suzuki. 1994. the influence of day 0 and day 7 superovulated cow serum during development of bovine oocytes in vitro. reprod. fertil. dev., 6:261-264. boediono, a., suzuki, t., li, l.y. and r.a. godke. 1999. offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos. mol. reprod. dev., 53:159-170. boediono, a., suzuki, t. and r.a. godke. 2003. comparison of hybrid and purebred in v/fro-derived cattle embryos during in vitro culture. anim. reprod. sci.,78:l-l 1. brackett, b.c. and g. oliphant. 1975. capacitation of rabbit spermatozoa in vitro. biol. reprod., 12. 260-274. brem, g., tenhumberg, h., and karausslich. 1984. chimerism in cattle through microsurgical aggregation of morula. theriogenology, 22:609-613. fehilly, c.b., willadsen, s.m. and e.m. tucker. 1984a. experimantal chimaerism in sheep. j. reprod. fertil., 70:347-351. fehilly, c.b., willadsen, s.m. and e.m. tucker. 1984b. interspecific chimerism between sheep and goat. nature, 307:634-636. picard, l, chartrain, i., king, w.a. and k.j. betteridge. 1990. production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae. mol. reprod. dev., 27:295-304. polzin, v.j., anderson, d.l., anderson, g.b., bondurant, r.h., butler, j.e., pashen, r.l., penedo, m.c.t. and j.d. rowe. 1987. production of sheep-goat chimeras by inner cell mass transplantation. j. anim. sci.. 65:325-330. 73 biotropia vol. 13 no. 2,2006 rorie, r.w., pool, s.h., prichard, j.f., betteridge, k.j. and r.a. godke. 1994. a simplified procedure for making reconstructed blastocysts for interspecific and intergeneric transfer. vet. rec., 135:186-187. sansinena, m.j., hylan, d., hebert, k., denniston, r.s. and r.a. godke. 2005. banteng (bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer. theriogenology, 63:1081 1091. solter, d. and b.b. knowles. 1975. immunosurgery of mouse blastocyst. proc. natl. acad. sci. usa., 72:5099-5102. 74 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf biotropia vol. 30 no. 2, 2023: 242 252 doi: 10.11598/btb.2023.30.2.1938 242 indigenous bacillus species isolated from aedes aegypti larvae: isolation, larvicidal toxicity screening, phenotypic characterization, and molecular identification salamun1,2,3,4*, rizky danang susetyo1, hakimatul husniyah1, almando geraldi1,2,3, ni’matuzahroh1,2,3, fatimah1,2,3, farah aisyah nafidiastri5 and nabilatun nisa’6, muhammad fath alhaqqi sanis salamy7 1 laboratory of microbiology, department of biology, faculty of science and technology, universitas airlangga, surabaya, 60115, indonesia 2 research group for applied microbiology and bioresource technology, universitas airlangga, surabaya, 60115, indonesia 3 university of co-e-research center for bio-molecule engineering, universitas airlangga, surabaya, 60115, indonesia 4 laboratory of entomology, institute of tropical diseases, universitas airlangga surabaya, 60115, indonesia 5 laboratory of microbiology, department of biology, faculty of mathematic and natural sciences, universitas negeri surabaya, jl. ketintang surabaya, 60231, indonesia 6 laboratory of molecular genetics, department of biology, faculty of science and technology, universitas airlangga, surabaya, 60115, indonesia 7 department of medical physiology and biochemistry, faculty of medicine, airlangga university, kampus a jl. mayjen prof. moestopo 47, surabaya, 60131, indonesia received 7 march 2023 / revised 15 may 2023 /accepted 15 may 2023 abstract vector-borne diseases transmitted by mosquitoes are considered a significant public health problem worldwide. aedes aegypti is one of the mosquito species responsible for transmitting these diseases. one environmentally friendly method of vector control is the use of microbial agents such as bacillus species. this study aimed to explore investigate indigenous entomopathogenic bacteria of bacillus species isolated from a. aegypti larvae. larvae samples were collected from breeding sites of a. aegypti. all isolates underwent screening and affirmation confirmation tests to assess their larvicidal toxicity against a. aegypti larvae. phenotypic characterizations and molecular identifications were conducted to determine the species of the bacillus isolates based on similarity index and percent identity (%id). phylogenetic trees were used to compare the isolates with other bacillus species. the results revealed 120 isolates of bacillus species from a. aegypti larvae samples. among them, three isolates (ls3.3, ls9.1, and lsd4.2) exhibited the highest larvicidal toxicity in the confirmation test, resulting in larval mortality rates of 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. molecular identifications, showed that lsd4.2 had a 99.16% id with bacillus velezensis, ls3.3 had a 98.22% id with bacillus mojavensis, and ls9.1 had a 99.93% id with bacillus subtilis. these three bacteria from the bacillus genus have been reported to offer significant benefits to humans. keywords: aedes aegypti, bacillus mojavensis, bacillus subtilis, bacillus velezensis, dengue vector, larvicidal toxicity introduction dengue fever (df) is a vector-borne infection transmitted by mosquitoes, which is considered a significant public health problem worldwide (dahmana et al. 2020). aedes aegypti is the mosquito species responsible for transmitting this disease. various attempts have been made to address the issue of df, but the outcomes have fallen short of expectations. extensive research has been conducted on developing vaccines to prevent this disease; however, satisfactory results have yet to be achieved. one alternative to combatting this disease is controlling the population of the vector (melanie et al. 2018). several measures have been taken to suppress the population *corresponding author, email: salamun@fst.unair.ac.id indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 243 of a. aegypti, including the use of chemical insecticides. however, the use of chemical insecticides has negative implications for environmental quality and is toxic to non-target organisms present in breeding sites for a. aegypti larvae (dahmana et al. 2020). experts have suggested the development of bioinsecticides as biocontrol agents for disease vectors in response to the df problem (thomas 2018). bioinsecticides are known to possess advantages such as specificity and safety for non-target organisms and the environment. one of the biocontrol agents being developed is entomopathogenic bacteria from the genus bacillus. bacillus sp. has been proven to be effective and highly specific, particularly toxic to the a. aegypti mosquito. certain bacillus species are capable of producing protein crystals along with spores during sporulation (evdokimov et al. 2014). numerous studies have demonstrated that multiple bacterial strains within the bacillus genus have the potential to eliminate a. aegypti larvae, including b. thuringiensis and b. sphaericus (boyce et al. 2013). these species exhibit high toxicity towards mosquito larvae while being safe for other parasites, predators, and mammals, in addition to causing no environmental pollution (melanie et al. 2018). in general, bacillus sp. can form endospores when confronted with unsuitable growth conditions that compromise their survival structure (zeigler & perkins 2015). the isolation and characterization of indigenous strains of b. thuringiensis from saudi arabia have been carried out (el-kersh et al. 2016). sixtyeight isolates have demonstrated larvicidal potential against the malaria disease vector, anopheles gambiens (el-kersh et al. 2016). similarly, b. sphaericus was isolated and characterized on lombok island, showing potential as a bio-insecticide for controlling the malaria vector a. aconitus (suryadi et al. 2016). salamun et al. (2021) recently isolated a bacillus species, bacillus thuringiensis bk5.2, from baluran national park, east java, indonesia, which displayed high toxicity against a. aegypti larvae. b. thuringiensis strains isolated and characterized from lebanese soils have also proven to be effective (fayad et al. 2019). these strains have been developed as bioinsecticides targeting agricultural pest insects (kumar et al. 2021). numerous scientific studies have explored the role of biocontrol agents and their potential in disease vector control (thomas 2018). toxins produced by bacillus sp. exhibit specific activity against target insects (schünemann et al. 2014). microbial larvicides can be employed as environmentally friendly biological agents for disease vector control (benelli et al. 2016). building on previous studies utilizing natural soil samples collected in baluran national park, east java, indonesia (salamun et al. 2021), our research aims to identify the diversity of bacillus species isolated from a. aegypti larvae in df endemic areas. this study aims to isolate indigenous entomopathogenic bacillus sp. from samples of a. aegypti larvae in their breeding sites in df endemic areas, conduct screening and affirmation tests to determine the larvicidal toxicity of the isolates against a. aegypti larvae, perform phenotypic characterizations, and conduct molecular identification. the findings are expected to contribute to the development of diverse entomopathogenic bacillus species as potential agents for the biocontrol of disease vectors, plant diseases, and pests. materials and methods materials the materials and tools utilized in this study were employed for the isolation, larvicidal toxicity screening, phenotypic characterization, and molecular identification of bacillus sp. from the aforementioned isolation. samples of a. aegypti larvae were collected from water reservoirs serving as breeding sites for a. aegypti in gresik, surabaya, and sidoarjo, east java, indonesia. screening and affirmation of the larvicidal toxicity of bacillus sp. were performed using third-instar a. aegypti larvae. the a. aegypti larvae were obtained from the tropical disease institute, universitas airlangga, surabaya, indonesia. sampling of aedes aegypti larvae samples of a. aegypti larvae were collected from mosquito breeding sites, specifically water reservoirs. identification of a. aegypti larvae samples was conducted following the identification key of a. aegypti larvae (bar & biotropia vol. 30 no. 2, 2023 244 andrew 2013). previous studies have shown that bacillus can be isolated from various sources, including soil, aquatic environments, herbivorous droppings, forest soil, dead insects, and mosquito breeding sites (paul 2007; zeigler & perkins 2015; suryadi et al. 2016). five larvae per sample were extracted using a pipette and placed in sterile glass bottles. isolation of bacillus sp. bacillus sp. was isolated from the laboratory of microbiology, department of biology, faculty of science and technology, universitas airlangga, as conducted by suryadi et al. (2016). five a. aegypti larvae were sampled from a suspected location infected with entomopathogenic bacteria, where the larvae exhibited minimal or slow movement on the water's surface. all larvae were placed in a sterile test tube and macerated, followed by the addition of 9 ml of 0.85% nacl solution. the mixture was allowed to sit for 5 minutes. a 101102 dilution of the sample was prepared, heated at 70°c for 30 minutes, and then inoculated with 1 ml of nutrient agar (na) using the pour plate method onto a sterile petri dish. the solidified media was incubated at 30°c for 48 hours. the resulting colonies were subjected to spore stain. the bacillus colonies were isolated on na media using the streak method and stored at 4°c (suryadi et al. 2016). larvicidal toxicity screening of bacillus sp. a pure bacillus sp. isolate was inoculated into a sterile glass container containing 10 ml of nutrient yeast salt medium (nysm) and incubated on a rotary shaker incubator at room temperature (35°c) for 48 hours (suryadi et al. 2016). the absorbance value of the bacillus sp. suspension was measured using a spectrophotometer at a wavelength of 600 nm (od600nm). for the screening of larvicidal toxicity, ten third-instar larvae of a. aegypti reared at the entomology laboratory of the institute of tropical diseases, airlangga university, were inoculated with a 5 ml suspension of bacillus sp. in a bottle containing 45 ml of tap water. the control group consisted of 45 ml of well water, 5 ml of nysm, and 10 a. aegypti larvae (suryadi et al. 2016). the percentage of larvae that died after 24 and 48 hours of exposure was calculated. the screening for larvicidal toxicity was conducted with three replicates, using a mortality range of 60-100 larvae, and the absorbance was set to 0.8. phenotypic characterizations morphological characterizations were conducted to determine the macroscopic and microscopic characteristics, such as the colony shape and the spore location. three bacillus sp. isolates with the highest potential were cultured on petri dishes containing 8 ml of na media using the streak method. the plates were then incubated for 48 hours and stained using the spore staining method. physiological characterization included testing for indole production, motility, oxidase activity, starch hydrolysis, and salinity tolerance. additionally, the microbact 12a/12b kit was used for an additional test. for this test, 225µl of bacterial suspension was taken and added to each well of the kit. one drop of immersion oil was added to each well, and the results were observed after incubation at 37°c for 24 hours. molecular identification molecular identification of bacterial isolates was conducted through the 16s rrna gene (kumar et al. 2016; johnson et al. 2019). initially, an isolated culture in 20 ml of nb media was incubated at 120 rpm and room temperature (35°c) for 48 hours. dna extraction was performed using the ctab method. the concentration and purity of the dna were determined at 280 nm and 260 nm using the multiskan go. the 16s rrna gene was amplified using the eppendorf mastercycler tool and the pcr method. the process involved adding gotaq green master mix and primers 16s rrna, p0 (5'-gag agt ttg atc ctg gct cag-3') and p6 (5'-cta cgg cta cct tgt tac ga-3'). the steps included denaturation at 94°c for 2 minutes, denaturation at 92°c for 30 seconds, annealing at 55°c for 30 seconds, elongation at 72°c for one minute, and final elongation at 72°c for 5 minutes, repeated for 35 cycles of polymerase chain reaction (pcr). amplicons were sequenced, and similarity analysis was conducted by comparing the data in genbank using ncbi's blastn. the pcr visualization results were obtained by indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 245 electrophoresis of a 1% agarose gel stained with ethidium bromide and observed under uv light. bacterial relationship analysis was performed by constructing a phylogenetic tree using the mega 6.0 application (tamura et al. 2013). data analysis the results of the isolation and larvicidal toxicity screenings were analyzed using descriptive analysis. bergey's manual of systematic bacteriology (paul et al. 2009) was utilized to obtain data on morphological and physiological properties of the local bacillus sp. the similarity index percentage was calculated based on the positive and negative similarity of the characters of each isolate to determine the bacterial species of bacillus sp. (paul et al. 2009). based on phenotypic characteristics, the similarity percentage pointed towards bacillus thuringiensis and bacillus sphaericus, bacteria that have demonstrated larvicidal activity against a. aegypti larvae. the 16s rrna gene, which had been amplified by pcr and confirmed by electrophoresis, was further purified and sequenced to determine the sequence of the 16s rrna gene in bacterial isolates. the pcr results were then submitted to malaysia's first base dna sequencing service. the sequencing results were edited using bioedit sequence alignment editor software version 7.2.5, and the similarity of the isolated 16s rrna gene of bacillus sp. with the gene data of bacteria in genbank was determined using the basic local alignment search tools (blast). the nucleotide blast analysis was conducted by the national center for biotechnology information at the national library of medicine in washington, dc and can be accessed at https://blast.ncbi.nlm.nih.gov/. results and discussion sampling and isolation of bacillus sp. in the isolation of 30 samples (150 larvae) of a. aegypti larvae from surabaya, gresik, and sidoarjo, east java, indonesia, and the map of sampling locations shown in figure 1, there were 120 isolates of bacillus sp. (table 1). larvicidal toxicity screening results with varying od600nm values were obtained for isolates of bacillus sp., which exhibited potential diversity as entomopathogenic bacillus sp. (table 1 and figure 2a). figure 1 map of sampling locations: gresik (gr), surabaya (sb), and sidoarjo (sd), east java, indonesia https://blast.ncbi.nlm.nih.gov/ biotropia vol. 30 no. 2, 2023 246 table 1 potency of indigenous bacillus sp. isolates (od600nm varies) based on the results of larvicidal toxicity screening against aedes aegypti third instar larvae at 48-hour exposure sampling location (city) global positioning systems (gps) of sampling locations sample codes number of isolates collection screening results of larvicidal toxicity culture turbidity of bacillus sp. isolates (od600nm) up lp mp hp surabaya s07’03.293é112’42.460’ s07’03.293é112’42.452’ s07’03.293é112’42.447’ s07’03.293é112’42.434’ s07’03.293é112’42.438’ s07’03.293é112’42.455’ s07’03.293é112’42.446’ s07’03.293é112’42.445’ s07’03.293é112’42.452’ s07’03.293é112’42.443’ ls1 ls2 ls3 ls4 ls5 ls6 ls7 ls8 ls9 ls10 2 4 5 6 4 4 4 3 3 4 0 1 0 0 2 1 3 0 1 2 1 1 1 4 1 2 1 3 1 1 1 2 1 0 1 1 0 0 0 0 0 0 3 2 0 0 0 0 1 1 1.50 – 1.50 1.00 – 1.25 0.80 –1.50 0.80 – 1.30 1.00 – 1.20 1.10 –1.50 0.80 – 1.15 1.00 – 1.35 0.85 – 1.40 0.95 – 1.50 gresik s07’03.293é112’34.459’ s07’03.293é112’34.471’ s07’03.293é112’34.437’ s07’03.293é112’34.436’ s07’03.293é112’34.484’ s07’03.293é112’34.515’ s07’03.293é112’34.536’ s07’03.293é112’34.530’ s07’03.293é112’34.948’ s07’03.293é112’34.965’ lg1 lg2 lg3 lg4 lg5 lg6 lg7 lg8 lg9 lg10 2 4 4 4 4 6 5 2 5 6 0 3 0 0 4 2 0 1 1 0 2 1 2 2 0 4 3 0 2 4 0 0 2 2 0 0 1 0 2 1 0 0 0 0 0 0 1 1 0 1 1.10 –1.10 1.00 – 1.50 0.40 – 1.30 0.85 – 1.40 0.95 – 1.50 0.80 – 1.50 0.50 – 1.20 1.00 – 1.40 0.80 – 1.50 1.00 – 1.50 sidoarjo s07’03.293é112’45.472’ s07’03.293é112’45.578’ s07’03.293é112’45.491’ s07’03.293é112’45.264’ s07’03.293é112’45.624’ s07’03.293é112’45.623’ s07’03.293é112’45.432’ s07’03.293é112’45.536’ s07’03.293é112’45.542’ s07’03.293é112’45.541’ lsd1 lsd2 lsd3 lsd4 lsd5 lsd6 lsd7 lsd8 lsd9 lsd10 6 4 3 2 3 5 4 3 3 6 3 2 0 0 1 4 2 0 0 2 3 2 1 0 1 0 0 1 2 4 0 0 2 0 1 1 0 2 0 0 0 0 0 2 0 0 2 0 1 0 0.75 – 1.40 0.55 – 1.50 1.10 – 1.30 0.90 – 1.10 0.80 – 1.35 1.10 – 1.40 0.40 – 0.95 0.90 – 1.40 1.10 – 1.15 0.65 – 1.50 120 35 50 20 15 0.40 – 1.50 descriptions: up = un-potential, larval mortality 0%; lp = low-potential, larval mortality <30%; mp = mediumpotential, larval mortality 30-50%; hp = high-potential, larval mortality >50%. figure 2 results of the larvicidal toxicity screening (a) (od600nm varies) with one replication and the affirmative toxicity test (b) (od600nm = 0.80) with three replications, performed on 15 isolates of indigenous bacillus sp. from gresik (lg), surabaya (ls), and sidoarjo (lsd) against aedes aegypti third-instar larvae at 24and 48-hour exposure indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 247 larvicidal toxicity screening of bacillus sp. the results of the affirmative toxicity test (fig. 2b) were conducted at turbidity of 0.80 (od600nm) from cultures of bacillus sp. isolates. the correlation between turbidity and the concentration of bacillus sp. (cfu/ml) yielded a regression line of y=151.5+17.6, with a coefficient of determination (r2) of 0.9525, as depicted in figure 3. based on calculations, turbidity of 0.8 in bacillus sp. cultures is equivalent to a bacterial cell count of 13.8x107 cfu/ml. following the affirmative toxicity test (fig. 2b), the three isolates with the highest potential underwent phenotypic characterizations. the results of the phenotypic characterizations for these three isolates are presented in figure 4 and table 2. figure 3 standard curve for quantifying bacillus sp. cell count (cfu/ml) in lsd4.2 isolate culture using optical density (od600nm) variation phenotypic characterizations the ls3.3 and ls9.1 isolates exhibited colonies with irregular shapes and flat elevations, while the lsd4.2 isolate had circular colonies with raised elevations. the size of the colonies for all three isolates was moderate. the margins of ls3.3, ls9.1, and lsd4.2 isolates were lobate, serrate, and entire, respectively. microscopic characterization using spore staining (fig. 4) revealed that ls3.3 and lsd4.2 isolates had spherical spores located at the terminal end, while the ls9.1 isolate had ovalshaped spores located at the subterminal end. detailed phenotypic characterizations are provided in table 2. figure 4 spore location of endospores in local bacillus sp. isolates using spore staining. descriptions: a) lsd4.2 isolate; b) ls9.1 isolate; c) ls3.3 isolate . table 2 phenotypic characterizations based on the physiological tests of bacillus sp. isolates coded lsd4.2, ls9.1, and ls3.3 no. physiological tests characteristics of bacillus sp. lsd4.2 ls9.1 ls3.3 1. lysine + 2. ornithine 3. h2s 4. glucose 5. mannitol 6. xylose + + 7. onpg + + 8. indole 9. urease + 10. vp + + + 11. citrate 12. tda 13. gelatin + + + 14. malonate + 15. inositol 16. sorbitol 17. rhamnose 18. sucrose 19. lactose 20. arabinose + + 21. adonitol 22. raffinose 23. salicin 24. arginine 25. motility + + + 26. katalase + + + 27. oksidase + 28. salinity 5% + + 29. salinity 10% 30. hidrolysis of amylum + + + molecular identification the results of pcr amplification of the 16s rrna gene for three bacillus sp. isolates, confirmed by electrophoresis, are shown in figure 5. the third band of bacillus sp. appeared at approximately 1500 bp. biotropia vol. 30 no. 2, 2023 248 figure 5 confirmation of the 16s rrna gene in three bacillus sp. isolates using electrophoresis methods. (descriptions: s1 = lsd4.2; s2 = ls3.3; s3 = ls9.1; m = marker) table 3 shows the results of sequencing to identify the similarity of the 16s rrna gene for bacillus sp. using blast. isolate code lsd4.2 had a 99.16% identity with bacillus velezensis, ls3.3 had a 98.22% identity with bacillus mojavensis, and ls9.1 had a 99.93% identity with bacillus subtilis, respectively. the results of constructing the phylogenetic tree of bacillus sp. on genbank are shown in figure 6. table 3 similarity of bacillus sp. based on sequencing of the 16s rrna gene using the basic local alignment search tools (blast) program isolates code spesies name accession no. e value % id query cover (%) lsd4.2 bacillus velezensis strain cbmb205 nr_075005.2 0.0 99.16 99 bacillus velezensis strain fzb42 nr_116240.1 0.0 99.02 99 ls9.1 bacillus subtilis subs. inaquosorum strain bgsc 3a28 nr_104873.1 0.0 99.93 100 bacillus subtilis strain jcm 1465 nr_113265.1 0.0 99.86 100 ls3.3 bacillus mojavensis strain ifo15718 nr_024693.1 0.0 98.22 99 bacillus halotolerans strain lmg 22477 nr_115931.1 0.0 98.11 99 figure 6 phylogenetic tree of bacillus sp. isolates coded lsd4.2, ls9.1, ls3.3, and their relationship to other bacillus sp. in the genbank database https://www.ncbi.nlm.nih.gov/nucleotide/nr_075005.2?report=genbank&log$=nucltop&blast_rank=1&rid=ertby417016 https://www.ncbi.nlm.nih.gov/nucleotide/nr_116240.1?report=genbank&log$=nucltop&blast_rank=2&rid=ertby417016 https://www.ncbi.nlm.nih.gov/nucleotide/nr_024693.1?report=genbank&log$=nucltop&blast_rank=2&rid=erv37gap01r https://www.ncbi.nlm.nih.gov/nucleotide/nr_115931.1?report=genbank&log$=nucltop&blast_rank=3&rid=erv37gap01r indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 249 in this study, the initial objectives were to isolate b. thuringiensis or b. sphaericus and screen their toxicity to a. aegypti larvae. variations in the mortality rate of a. aegypti larvae due to exposure to bacillus sp. were observed. thirdinstar larvae of a. aegypti were used for both screening and confirming the larvicidal toxicity of bacillus sp. (table 1; fig. 2a and 2b). a total of 120 isolates could be isolated from 150 samples of a. aegypti larvae collected from surabaya, sidoarjo, and gresik cities in east java, indonesia. among them, 15 isolates showed high potency in the larvicidal toxicity screening. the affirmation test of larval toxicity (fig. 2b) revealed that three isolates exhibited the highest toxicity. the larvicidal toxicity screening using third-instar a. aegypti larvae was based on their sensitivity to entomopathogenic bacterial toxins (kim et al. 2017). the older the larval instar, the lower their sensitivity to the bacterial toxin. additionally, fourth-instar larvae exhibit less feeding habits compared to younger larvae, resulting in reduced consumption of bacterial toxins. furthermore, during the pupal phase, feeding activity ceases (aynalem 2022). in the affirmation test of bacillus sp. lsd4.2 (fig. 3), a concentration of 13.8 x 107 cfu/ml, caused 100% larval mortality after 48 hours of exposure, categorizing it as highly toxic. b. thuringiensis pwr4.32, isolated in malang, indonesia, exhibited a lethal concentration 50% (lc50) value of 22.79 x 107 cells/ml after 72 hours of exposure (gama et al. 2010). similarly, b. thuringiensis w.swh.s.k2, isolated in nganjuk, indonesia, had an lc50 value of 3.53 x 107 cells/ml after 48 hours of exposure (pratiwi et al. 2013). b. thuringiensis bk5.2, isolated from baluran national park in east java, indonesia, showed an lc50 value of 8.3 x 106 cells/ml after 48 hours of exposure (salamun et al. 2021). the results of this study indicate differences in larvicidal toxicity among different bacillus sp. isolates, suggesting that these isolates may belong to different species or strains. bacillus sp. larvicidal toxicity can be identified through two mechanisms of action. during sporulation, bacteria produce an insecticidal toxin stored in parasporal inclusions. during the vegetative stage, bacteria produce secondary metabolites, such as enzymes or other chemical compounds, that are also insecticidal. the entomopathogenic action of bacillus sp. involves the toxin produced during sporulation, which binds to intestinal cell receptors, causing pores to form in the intestinal cell membrane. this leads to the entry of ions to balance intracellular and extracellular fluids. consequently, intestinal cells experience rapid damage, resulting in the lysis of epithelial cells. infected larvae stop feeding for several hours, ultimately leading to their death (polenogova et al. 2022). the endotoxin in the parasporal inclusion of the entomopathogenic bacillus sp. also reduces the blood's acidity (ph), leading to larval death due to septicemia (poopathi et al. 2013). other actions of entomopathogenic bacillus sp. as bioinsecticides have also been reported. bacillus sp. produces secondary metabolites, including biosurfactants, during bacterial growth in suitable media. the biosurfactant produced by the b. subtilis strain is composed of a mixture of molecules, some of which are toxic to arthropods and vectors (sachdev & cameotra 2013). biosurfactant-producing bacteria have been found to be effective in controlling diseases in plants and insects (zhao et al. 2014). biosurfactants can affect the cuticle of insects due to their amphiphilic nature, which includes hydrophobic and hydrophilic molecules. this can damage cell membranes and epithelial cells and ultimately cause death (zhao et al. 2014). based on the phenotypic characteristics (table 2 and fig. 4) and identification using bergey's manual of systematic bacteriology, the isolates coded lsd4.2 and ls3.3 showed similarity indices of 82.6% and 63.3%, respectively, with b. sphaericus. isolate ls9.1 had a similarity index of 62.50% with b. thuringiensis. however, based on molecular identification using the 16s rrna gene, these three bacillus sp. isolates showed different results. they were identified as b. velezensis, b. mojavensis, and b. subtilis (table 3; fig. 6). b. velezensis fzb42t, previously classified as part of the b. subtilis group due to its 99% genetic similarity, was later included in a different phylogenomics category based on additional genetic characteristics. this strain of b. velezensis produces unique intracellular biomolecules that have the potential for development through genetic engineering in various industries, including health, pharmaceuticals, environment, and food, particularly in agriculture (adeniji et al. 2019). biotropia vol. 30 no. 2, 2023 250 studies have shown that b. velezensis nkg-2 is useful as a potential biocontrol agent and promoter of plant growth (myo et al. 2019). b. velezensis strain wlys23 has great potential as a biocontrol agent for disease control in freshwater aquaculture (zhang et al. 2021). b. velezensis 33rb is a potential alternative to chemical pesticides as a biological control agent for phytopathogens, offering environmentally friendly and sustainable properties (dawwam & sehim 2022). the search for new biocontrol agents focuses on bacillus subtilis and its related species, including bacillus mojavensis. the metabolites produced by the b. mojavensis ps17 isolate from wheat germ inhibit the growth of the plant pathogen fusarium spp., indicating its potential as a biocontrol agent for agriculture (diabankana et al. 2021). b. mojavensis shares similarities with b. subtilis but differs in fatty acid composition, dna sequences, and resistance to genetic transformation (bacon & hinton 2002). b. mojavensis produces surfactin, iturin, and fengycin, which belong to an antimicrobial and antifungal lipopeptide group (mounia et al. 2014; blacutt et al. 2016). according to jasim et al. (2016), the lipopeptide compounds surfactin and fengycin in b. mojavensis have antimicrobial activity against pathogenic bacteria, including both gram-negative and gram-positive strains. hmidet et al. (2017) reported that b. mojavensis produces surfactin and fengycin, with optimal production occurring in media containing glucose. b. mojavensis demonstrated hemolytic activity on blood agar, suggesting the production of biosurfactants (berekaa & ezzeldin 2018). b. mojavensis btcb15 is capable of producing 2.3 nm agnps and exhibits antibacterial activity against numerous drug-resistant pathogens (iqtedar et al. 2019). in their study, fanaei et al. (2021) discovered that b. mojavensis hf produces three types of lipopeptides: surfactin, fengycin, and kurstakin. they identified a wide variety and number of surfactin and fengycin isomers compared to previous reports and claimed to be the first to report the presence of kurstakin in bacillus mojavensis species. further research is needed to determine whether kurstakin is stored in parasporal inclusions or excreted as secondary metabolites. b. subtilis also produces biosurfactant as a mosquitosidal toxin (kumar et al. 2022). mosquitosidal toxin activity has also been reported from b. cereus (mani et al. 2017). biosurfactants, synthetic compounds produced by several strains of bacillus sp., have been used as biocontrol agents against insects (mani et al. 2017). for example, b. subtilis isolated from soil has been introduced as a biological control agent for insects due to its production of surfactin (kumar et al. 2022). b. subtilis (mw644765) mediated silver nanoparticles (agnp) have shown promising larvicidal activity against mosquito larvae, making them a potential biocontrol agent for reducing mosquito populations (wilson et al. 2022). b. subtilis is considered a universal cell factory for various industries such as agriculture, biomaterials, pharmaceuticals, and industry (su et al. 2020). molecular identification results have identified three high-potential bacillus species: b. subtilis (ls9.1), b. velezensis (lsd4.2), and b. mojavensis (ls3.3). commercial products derived from b. thuringiensis and b. sphaericus have been used for the control of a. aegypti larvae (boyce et al. 2013). the discovery of b. velezensis, b. mojavensis, and b. subtilis in this study is highly significant. these bacteria have been reported as multifunctional bacteria in various industries, including health, pharmaceuticals, environment, and food, and as biocontrol agents for disease vectors, plant pests, and disease control in freshwater aquaculture. conclusion the results of the isolation and larvicidal toxicity screenings of bacillus sp. against aedes aegypti larvae revealed a range of potential larvicidal toxicity levels, varying from low to high. screening 120 isolates of bacillus sp. for larvicidal toxicity identified 15 isolates with high potency. the confirmation test identified three isolates with the highest potential. the larval mortality rates due to exposure to isolates ls3.3, ls9.1, and lsd4.2 were 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. molecular identification using the 16s rrna gene revealed the diversity of the isolates, with isolate lsd4.2 sharing 99.16% identity with bacillus velezensis, ls3.3 sharing 98.22% identity with bacillus mojavensis, and ls9.1 sharing 99.93% identity with bacillus subtilis. these three bacteria, belonging to the bacillus genus, offer significant benefits for humans. indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 251 references adeniji aa, loots dt, babalola oo. 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biocontrol agent against fusarium oxysporum f. sp. spinaciae. j bas microbiol 54: 448-56. https://www.sciencedirect.com/journal/south-african-journal-of-botany/vol/151/part/pb https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=zhang%2c+df https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=xiong%2c+xl https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=wang%2c+yj https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=gao%2c+yx https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=ren%2c+y https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=wang%2c+q https://ami-journals.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=shi%2c+cb https://ami-journals.onlinelibrary.wiley.com/toc/13652672/2021/131/6 biotropia no. 5, 1991/1992: 22-25 antioxidant metabolism in water stressed peanut treated with diniconazole g.c. rivero institute of biology, university of the philippines, diliman, philippines and d.m. orcutt department of plant physiology, virginia polytechnique institute and state university, blacksburg, va 24061-0331, usa abstract the response of alpha-tocopherol, glutathione and ascorbate was studied in peanut (arachis hypogea l., cv. nc-17) subjected to water stress and treated with a triazole fungicide, diniconazole (dini). there was no significant difference in alpha-tocopherol levels between control and dini treated plants. however, a 14% increase was observed in alpha-tocopherol level in dini treated, water stressed plants compared to water stressed plants. total glutathione in dini treated stressed plants increased by 13 and 31% over control and water stressed plants, respectively. ascorbate levels decreased significantly in all treatments compared to the control. these results indicate that dini alleviates the oxidative damage caused by water stress by increasing total glutathione levels. however, dini does not seem to affect alpha-tocopherol and ascorbate levels in peanuts under water stress. introduction protection against harmful oxidation of biomembranes is provided by several antioxidative systems, including a large array of enzymes and a variety of small molecules such as glutathione, beta-carotene, and the vitamins c and e (finckh and kunert 1985). in plants, vitamin e (alpha-tocopherol) is located mainly in the chloroplast (bucke 1976) and a high concentration of the lipid-soluble vitamin is found in the chloroplast envelope (lichtenthaler et al. 1981). vitamin c (ascorbate), a reducing agent, is present in substantial amount in higher plants (jones and hughes 1983), and the water-soluble vitamin is generally regarded as a normal cellular constituent (finckh and kunert 1985). the tripeptide glutathione is widely distributed in plant cells (rennenberg 1982). it is implicated in the adaption of plants to environmental stress such as drought and extremes of temperature (alscher 1989). glutathione, ascorbate and alpha-tocopherol are of special interest because of their demonstrated association with stress resistance. the present study evaluated the response of alpha-tocopherol, glutathione and ascorbate in water stressed peanut plants treated with diniconazole. 22 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt materials and methods peanut (arachis hypogea l., cv. nc-17) seedlings were grown in controlled conditions, i.e. continuous illumination (392 uem-2sec-l) and controlled temperature (28 ± 2°c). treatments consisted of control, water stressed, dini treated and dini treated stressed plants arranged in a completely randomized block design with 10 replications. dini was applied foliary at a rate of 4 mg/plant (recommended field rate) every 2 weeks for 7 weeks commencing when the plants were 6 weeks old. drought stress treatment began 4 days following initial dini treatment and consisted of applying 125 ml of distilled water to plants subjected to drought every 3 days versus 250 ml to non-stressed plants. harvested plants (117 days old) were frozen in liquid nitrogen, freeze dried and stored at -23°c until analyzed for alpha-tocopherol, glutathione and ascorbate. alpha-tocopherol was extracted in 80% ethanol partitioned with hexane and the hexane layer analyzed using a hewlett packard model 1090 hplc, equipped with a fluorescence detector (294 nm excitation, 325 nm emission) following modified procedures of cort et al. (1983) and grumbach (1983). the hexane ran through a silica column (hypersil 5 um, 200 x 4.6 mm, hewlett packard). glutathione and ascorbate extraction was accomplished by homogenizing leaf tissue in 2% metaphosphoric acid. the homegenate was centrifuged at 17 000 g and the supernatant analyzed for glutathione (griffith 1980) and ascorbate (foyer and halliwell 1977) using a beckman du-65 spectrophotometer at wavelengths 412 nm and 523 nm, respectively. results and discussion the following table reflects the responses of alpha-tocopherol, glutathione and ascorbate in water stressed peanut plants treated with dini. alpha-tocopherol, total glutathione and ascorbate levels in water stressed peanut (arachis hypogea l., cv. nc-17) plants treated with diniconazole trt vitamin e glutathione vitamin c (% dw) (nmoles/gdw) (umoles/gdw) c 0.57a 835.01b 7.28a t 0.43b 880.40ab 3.91b cs 0.49ab 719.86c 4.65b ts 0.50ab 946. 14a 4.74b c = control; t = treated w/ dini; cs = water stressed; ts = treated w/ dini and water stressed. value with different letters are significantly different at α = 0.05 (lsd). 23 biotropia no. 5, 1991/1992 there was no significant difference in alpha-tocopherol levels between control and dini treated plants. however, a 14% increase was observed in alpha-tocopherol level in dini treated stressed plants compared to water stressed plants. earlier reports (senaratna et al. 1985, mackay et al. 1987) on antioxidant levels in stressed plants indicated an increase on the levels of total antioxidants. mackay et al. (1987) evaluated the antioxidant potential of microsomal membranes and reported an increase in antioxidant potential of triazole (s-3307) treated wheat plants exposed to ozone. however, their analysis was made on total antioxidant potential of the lipid fraction, as the ability of the lipid extract to inhibit the in vitro oxidation of exogenous linoleic acid. the oxidation reaction was similar to that seen for alpha-tocopherol and thus, the antioxidant capacity of their samples were expressed as alpha-tocopherol equivalents (mackay et al. 1987). in our study, alpha-tocopherol was measured directly by hplc. ascorbate levels decreased significantly in all treatments compared to the control. there is evidence that ascorbate represents a reservoir of antioxidant potential to regenerate directly, under conditions of stress, the lipid-soluble primary antioxidant alpha-tocopherol (packer et al. 1979; leung et al. 1981). this could explain the significant decrease in ascorbate levels in water stressed and dini treated water stressed plants compared to the control. total glutathione in dini treated stressed plants increased by 13 and 31% over control and water stressed plants, respectively. in pea, a chilling-resistant species, total glutathione decreased only slightly, but was still higher than that in cucumber, a chilling-sensitive species (wise and naylor 1987). the results presented here indicate that dini alleviates the oxidative damage caused by water stress by increasing the total glutathione levels. summary total glutathione levels increased in stressed peanut plants treated with diniconazole and could be a factor in drought resistance. diniconazole does not seem to affect alpha-tocopherol and ascorbate levels in peanuts under water stress. 24 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt acknowledgement this study was part of a research conducted during the senior author's us-aid post-doctoral fellowship at the department of plant pathology, physiology and weed science, virginia polytechnique institute and state university, blackburg, va 24061, usa from october, 1989 to august, 1990. special thanks are due to dr. r. madamachi and ms. n.r. hopkins from the same department for their invaluable assistance. references alscher, r.g. 1989. physiol. plant. 77: 457-464. bucke, c. 1976. phytochemistry 7: 693-700. cort, w.m., t.s. vicente, e.h. waysek and e.d. williams. 1983. j. agric. food chem. 31: 1330 1333. finckh, b.f. and k.j. kunert. 1985. j. agric. food chem. 33: 574-577. foyer, c.h. and d.o. hall. 1976. planta 133: 21-25. _____ and b. halliwell. 1977. phytochemistry 16: 1347-1350. griffith, o.w. 1980. anal. biochem. 106: 207-212. grumback, k.h. 1983. z. naturforsch. 38c: 996-1002. jones, e., and r.e. hughes. 1983. phytochemistry 22: 2493-2499. leung, h.w., m.j. vong and r.d. mavis. 1981. biochem. biophys. acta. 664: 266-272. lichtenthaler, h.k., u. presenzel, r. douce and j. joyard. 1981.. j. biochim. biophys. acta 641: 99-105. mackay, c.e., t. senaratna, b.d. mckersie and r.a. fletcher. 1987. plant cell physiol. 28 (7): 1271-1278. packer, j.e., t.f. slater and r.l. willson. 1979. nature (london) 278: 737-738. rennenberg, h. 1982. phytochemistry 21: 2771-2781. seneratna, t., b.d. mckersie and r.h. stenson. 1985. plant physiol. 78: 168-171. 25 22.pdf 23.pdf 24.pdf 25.pdf microsoft word 1 biotropia no. 24, 2005 : 1 19 aspergillus fla vus infection and aflatoxin contamination in peanuts at various stages of the delivery chains in cianjur regency, west java, indonesia okky setyawati dharmaputra seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia ina retnowati, santi ambarwati and erita maysra seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract a survey to obtain information on preand postharvest handling of peanuts at farmer, collector, wholesaler and retailer levels, including aspergillus flavus infection and aflatoxin bi contamination of peanuts collected in cianjur regency, west java, was conducted during the harvest period of the wet season of february 2004. the moisture contents and physical qualities of the peanuts were also determined. thirteen and 40 dry pod samples were collected randomly from 12 farmers and 23 collectors, respectively. seven dry kernel samples were also collected from collectors. five and 45 dry kernel samples were collected randomly from 2 wholesalers and 45 retailers in traditional markets, respectively. thus, a total of 110 dry peanut pod and kernel samples were collected. the results of interviews with farmers, collectors, wholesalers and retailers, and also the moisture contents and physical qualities of the peanuts arc described in this article. the percentages of samples infected by a. flavus were highest at the wholesaler as well as at retailer levels (100%, respectively), followed by those sampled at the collectors (85.0 and 85.7%, respectively), and farmers (84.6%). the mean percentage of infected kernels in infected samples of peanuts collected from retailers was the highest (87.6%), followed by those collected from wholesalers (72.4%), collectors in the form of kernels (23.3%) and pods (17.7%), and farmers (15.2%). the range of aflatoxin bi contents in peanut samples collected from farmers (dry pods), collectors (dry pods), wholesalers (dry pods and kernels) and retailers (dry kernels) were < 3.6 -114.2, < 3.6 -2999.5 and < 3,6 34.1, < 3.6 6065.9, and < 3.6 6073.0 ppb, respectively. the highest aflatoxin b, contents at the wholesaler and retailer levels were 6065.9 ppb (in one sample) and 6073.0 ppb (in one sample), respectively. the percentage of samples contaminated with more than 15 ppb of aflatoxin bi was the highest in peanuts collected from wholesalers (80.0% of samples), followed by retailers (75.6%), farmers (38.5%) and collectors (30.0 and 14.3%). in 1999 codex alimcntarius commission determined that the maximum total aflatoxin content in peanuts intended for further processing is 15 ppb, suggesting that an alarming proportion of peanuts throughout the indonesian food chain arc in excess of this maximum limit. key words : ,4,spergillius flavus/aflatoxin/peanuts/cianjur regency introduction peanuts are next to maize and soybean as the most important secondary crop in indonesia. since indonesia has a humid tropical climate, peanuts can easily be 1 biotropia no. 24, 2005 infected by moulds (including a. flavus) under drought stress before harvest, during the drying phase in the field, or under poor storage conditions. aflatoxin has been recognized as a human and domestic animal carcinogen, and is produced following infection of peanuts by certain strains of a. flavus. pitt and hocking (1996) reported that 45% of 215 peanut samples collected from farm storage, middlemen and retailers in bogor (west java), yogyakarta (central java), and their surroundings, contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22 % exceeded 1000 ppb. the total annual cost of aflatoxins in peanuts in 1991 in indonesia, philippines and thailand was estimated as about $ a 158 million. indonesia incurred 84% (= $ a 132 million) of this cost (lubulwa and davis 1994). according to dharmaputra et al. (2003a), in general, aflatoxin bi contents of peanuts collected from farmer's fields/pewefoas/collectors and processed samples in the pati regency of central java were low (less than 15 ppb). the highest aflatoxin b| contents were found in raw peanut kernels collected from retailers in traditional markets, ranging from 2-124 and < 4 342 ppb during the wet and dry seasons in 2002, respectively. the percentage of raw kernel samples contaminated with aflatoxin bi (exceeding 15 ppb) collected during the wet and dry seasons was 33 and 25%, respectively. another study was also carried out by dharmaputra et al. (2005) on aflatoxin bi contents of peanuts collected from farmer's fields, collectors and retailers in the wonogiri regency and specifically the city of surakarta (central java) during the wet and dry seasons in 2003. the results also showed that the highest aflatoxin b, contents were found in raw peanut kernels collected from retailers in traditional markets, with the range of < 3.6 1859.3 and < 3.6 5511.5 ppb during the wet and dry seasons, respectively. the percentage of raw kernel samples contaminated with aflatoxin bi (exceeding 15 ppb) collected during the wet and dry seasons was 33 and 76 %, respectively. the 23rd session of the joint fao/who food standards programme held in rome, italy (28 june-3 julyl999) reported that codex alimentarius commission has adopted a maximum level of total aflatoxins in peanuts intended for further processing at 15 ppb. on 9 september 2004 the national agency for drug and food control, republic of indonesia has determined that aflatoxin bi and total aflatoxin contents in processed peanut products should not be more than 20 and 35 ppb, respectively. to minimize or to reduce aflatoxin contamination in peanuts, appropriate post-harvest handling methods in each level of peanut delivery chain (farmer, collector, wholesaler, and retailer) should be carried out. the objective of this study was to obtain information on preand postharvest handling methods, aspergillus flavus infection and aflatoxin bi contamination of peanuts collected from different points of the delivery chains in cianjur regency of west java. the moisture contents and physical quality of peanut kernels were also determined. aspergillusflavus infection and aflatoxin contamination in peanuts — okky s. dharmaputra et al. materials and methods time and location of surveys surveys were conducted during the harvest of the wet season (february 2004) at cidaun, naringgul and sindangbarang districts located in cianjur regency, and the city of cianjur, west java. based on the information obtained from the indonesian government regional office for food crops of west java province, peanut production was high in cianjur regency and it ranks second after garut regency. peanut production was high at cidaun, naringgul and sindang barang districts according to the information obtained from the indonesian government regional office for food crops of cianjur regency. the surveys comprised: • interviews, using questionnaires, with farmers, collectors, wholesalers and retailers. the questionnaires consisted of questions relating to preand postharvest handling of peanuts. • random sampling of various kinds of peanut products collected from farmers, collectors, wholesalers and retailers who were interviewed. the moisture contents, physical quality of kernels, fungal (a. flavus) infection and aflatoxin b, contents from each sample were analyzed. sampling methods based on differences of storage duration, more than one peanut sample could be obtained from each farmer, collector and wholesaler. the kind of peanuts sampled included dry pods and kernels. samples of dry pods (about 2 kg each) were divided three times manually and homogeneously to obtain working samples (about 250 g each) for analyzing moisture contents, physical quality of kernels, percentage of kernels infected by a. flavus, aflatoxin bi content, and a reserve sample. the dry peanut pods were then shelled manually. samples of dry peanut kernels (about 1 kg each) were also divided three times using a box divider to obtain working samples (about 125 g each) for analyzing moisture contents, physical quality of kernels, percentage of kernels infected by a. flavus, aflatoxin bi content, and a reserve sample. moisture content, physical quality of kernels, a. flavus and aflatoxin bi analyses moisture contents of kernels (based on a wet basis) were analyzed using a sinar tm ap 6060 moisture analyzer. the moisture contents of some samples were confirmed using the oven method (bsi 1995). two replicates were used from each sample. biotropia no. 24, 2005  physical  quality  of  kernels  was  assessed  in  intact,  shriveled  and  damaged  kernels.  the  damaged  kernels included cracked, broken, discoloured, and damage caused by insects or fungi. the percentage of  each category of kernels was determined by counting them and dividing the total number of kernels used  for physical quality analysis.  the percentage of kernels infected by a. flavus was determined using a plating method (100 kernels per  sample) on aspergillus flavus and parasiticus agar (afpa) (p'metal. 1983).  aflatoxin bbt was analyzed because  it  is the most dangerous toxin. aflatoxin b, contents  in the kernels  were determined using the el1sa method (lee and kennedy 2002), with two replicates used for each sample.  results and discussion source, kind and number of samples  at the farmer level (12 farmers), 13 samples of dry peanut pods were collected, while at collector level (23  collectors),  40  samples  of  dry  pods  and  7  samples  of  dry  kernels  (1  kg/sample)  were  collected.  at  the  wholesaler and retailer levels (2 wholesalers and 45 retailers), 5 and 45 samples of dry peanut kernels were  collected, respectively. thus, the total number of samples was 110. details of the peanut delivery chain,  location and number of peanut samples are presented in table 1.      aspergillus flavus infection and aflatoxin contamination in peanuts — okky s. dharmaputra et at.  results   of  interviews   with   farmers,   collectors,   wholesalers   and   retailers concerning pre‐ and  postharvest handling of peanuts  interview with farmers  the results from farmer interviews (12 respondents) are presented in table 2. all farmers (100%)  planted a local variety of peanuts, with most of the seed sources from farmers (83% of respondents).  fanners harvested their peanuts at 90 to 100 days after planting. during planting, all of respondents  used fertilizer, such as urea, tsp and k.c1. weed control was conducted manually. farmers sun‐dried  peanut  pods  for  3  ‐  4  days.  most  of  the  farmers  used  woven  polypropylene  bags  (67%  of  respondents)  to  dry  peanuts.  based  on  their  experiences,  farmers  could  determine  when  their  peanuts  were  fully  dried  for  safe  storage.  sixty  seven  percent  of  respondents  stored  peanuts  in  woven polypropylene bags for 1 ‐ 7 days before selling to collectors. all respondents sold peanuts to  collectors  in  the  form  of  dry  pods.  interestingly,  all  respondents  were  not  aware  of  the  aflatoxin  problem in peanuts.        interview with collectors the result of interview with collectors (23 respondents) are presented in table 3.based on their experience the collectors knew whether their peanuts were fully dry. before selling to wholesalers, peanuts were stored in woven poly propylene bags (91% respondents), or by spreading them on paved floor (9% of aspergillus flaws infection and aflatoxin contamination in peanuts okky s. dharmaputra et ai. respondents) for 1-30 days. peanuts were sold to wholesalers in the form of dry pods (35% of respondents), or as dry pods and kernels to wholesalers and retailers (9% of respondents) in the city of cianjur. peanuts were also sold to wholesalers in the form of dry pods (43% of respondents), and to wholesalers and retailers in the form of kernels (4% of respondents) in the big cities of west, central and east java. all respondents shelled peanut pods using a diesel powered sheller. most collectors (87% of respondents) sorted peanuts manually before selling them to wholesalers. all respondents were not aware of aflatoxin problem in peanuts. interview with wholesalers the results of interview with wholesalers (2 respondents) are presented in table 4. at the wholesaler level peanuts were stored in woven polypropylene bags (50% of respondents), as well as in woven polypropylene and jute bags (50% of respondents). the stacks of bags containing peanuts were not placed on pallets. peanuts samples were stored for 1 month (50% of respondents) and 1-2 months (50% of respondents). fifty percents of the respondents sold only peanuts, while 50% of respondents sold peanuts and other commodities such as wheat flour and peanut oil. peanuts were sold to smaller wholesalers in the cities of bandung, bogor and jakarta. interview with retailers the results of interview with retailers (45 respondents) are presented in table 5. fifty three percent of respondents bought their peanuts from wholesalers, while 47% of respondent bought peanuts from farmers and collectors. peanuts were stored in woven polypropylene bags (71% of respondents) and jute bags (29% of respondents) for 1 7 days. containers used at the time of sampling were rectangular plastic basins (42% of respondents), winnowing trays (29% of respondents), wooden boxes (18% of respondents), round plastic basins (5% of respondents), woven polypropylene bags (4% of respondents) and jute bags (2% of respondents). aside from peanuts, retailers also sold other general commodities. peanut buyers included sellers of peanut sauce products for making gado-gado, pecel and sate, as well as house wives. retailers were also not aware of the aflatoxin problem in peanuts.   moisture contents, physical quality of kernels, the incidence of a. flavus and aflatoxin contamination  moisture contents according to diener and davis (1969) moisture content is an important factor affecting the growth of a. flavus and aflatoxin production. the range and mean moisture contents of kernels derived from various kinds of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6 and figure 1. the moisture contents of kernels collected from farmers was relatively similar to those collected from collectors and retailers, while the moisture contents of kernels collected from wholesalers was the lowest. this was due to the storage duration of peanuts at farmer and collector levels which were relatively shorter than those at the wholesaler level (tables 2, 3 and 4). the moisture contents of kernels were always in equilibrium with the relative humidity of storage room. the range and mean moisture contents of kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 5.9 9.9% and 8.4%; 7.8 -9.6% and 8.7%; 8.1 9.4% and 8.9%; 6.2 8.8% and 7.5%; 6.5 9.3% and 8.5%, respectively (table 6). in general, the mean of moisture content of peanut kernels in each delivery chain were considered safe for storage. sni (1995) determined that the safe moisture contents for storage of peanut pods and kernels were 9 and 8%, respectively. 10 aspergillus flavus infection aflatoxin contamination in peanuts – okky s. dharmaputra et al moisture content of pods and kernels are closely related with the drying process. sun-drying is the most critical postharvest handling procedure for peanuts, especially when the harvest coincides with the wet season. according to wongvirajtana et al. (1993) the duration of sun-drying can significantly affect fungal growth in grains, with the longer the period of sun-drying, the more chance for the fungi to infect the grains. physical quality of kernels range and mean of physical quality characteristics of kernels derived from various kinds of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6, and figure 2. the percentage of intact kernels of peanuts collected from farmers was the highest, followed by those collected from collectors, wholesalers and retailers. this was probably due to the peanuts which have not being shelled using mechanical or diesel powered sheller at the farmer level, while the duration of storage of peanuts was relatively short (< 7 days). the percentages of shriveled kernels in each of the delivery chain were relatively the same (more than 22%). sni (1995) determined the maximum percentage of shriveled kernels is 4%, consequently the percentage of shriveled kernels in each delivery chain can be categorized as very high. this was probably due to the early harvest. results of interviews with farmers showed that 59% of respondents harvest peanuts 90 days after planting (table 2). peanuts harvested before full maturity will tend to produce 12 shriveled kernels after drying, consequently the kernels could be more easily infected by fungi. the percentage of damaged kernels collected from retailers was the highest, followed by that collected from wholesalers, collectors and farmers. the damaged kernels could have been caused by insects, rodents and fungal attacks, and inappropriate equipment used for the shelling of pods. the highest percentage of damaged kernels at the retailer level was probably due to the longer duration of postharvest handling from farmer up to the retailer. the range and mean percentages of intact kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 59.6 83.9% and 74.8%; 42.2 83.0% and 70.0%; 56.2 77.3% and 69.0%; 37.8 -93.4% and 63.6%; 29.3 73.0% and 56.3%, respectively. the range and mean-percentages of shriveled kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 14.2 36.6% and 22.9%; 13.7 56.4% and 28.0%; 19.7 41.7% and 27.9%; 3.3 50.7% and 29.0%; 15.9 -39.0% and 29.0%, respectively. the range and mean percentages of damaged kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 0.6 4.9% and 2.3%; 0.6 5.6% and 2.2%; 2.1 4.5% and 3.1%; 3.2 15.6% and 7.4%; 4.1 40.3% and 14.7%, respectively. 13 biotropia no. 24, 2005 the incidence o/a. flavus the percentage of samples infected by a. flavus, range and mean percentages of infected kernels in infected samples of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6, figures 3 and 4. 14 aspergillus flaws infection and aflatoxin contamination in peanuts okky s. dharmaputra et al. the percentage of samples infected by a. flavus was highest in peanuts collected from wholesalers and retailers (100%, respectively), followed by those collected from collectors in the form of pods (85%) and kernels (85.7%), and farmers (84.6%). the mean percentage of infected kernels in infected samples of peanuts collected from retailers was the highest (87.6%), followed by those collected from wholesalers (72.4%), collectors in the form of kernels (23.3%) and pods (17.7%), and farmers (15.2%). the highest percentage of peanut samples and mean percentages of infected kernels in infected samples collected from retailers was related to the methods of postharvest handling from farmers up to retailers, as well as the duration of storage. peanut kernels were more easily infected by fungi compared to unshelled peanuts. the damaged kernels were more easily infected by fungi compared to intact kernels. dharmaputra and retnowati (1996) observed that the range in percentage of peanut kernels infected by a. flavus in samples collected from retailers in some locations in west java during the wet season was 83 100%. aspergillus flavus was found in 98% of 256 peanut kernel samples and in 61% of all examined kernels collected from retailers in some locations in west and central java (pitt e? al. 1998). according to dharmaputra et al. (2003) 24 raw kernel samples collected from retailers in traditional markets located in bogor, pati, yogyakarta and malang were 100% infected with a. flavus during both the wet and dry seasons, respectively. dharmaputra et al. (2005) also reported that 98 and 100% of 54 raw peanut kernel samples collected from retailers during the wet and dry seasons in wonogiri regency, west java, were infected by a. flavus, respectively. aflatoxin b: contamination the range of aflatoxin b| contents in peanut kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers is presented in table 6. the range of aflatoxin b, contents in peanut samples collected from retailers (< 3.6 6073.0 ppb) was the widest, but almost similar to those collected from wholesalers (< 3.6 6065.9 ppb). the percentage of samples contaminated with different levels of aflatoxin b| is shown in table 7. in australia the maximum allowable limit of aflatoxin in peanut and peanut products is 15 ppb (qdpi 2000). the percentage of samples contaminated with more than 15 ppb of aflatoxin bj was highest in peanuts collected from wholesalers (80% of samples), followed by retailers (75.6%), farmers (38.5%) and collectors in the form of pods and kernels (30.0 and 14.3%, respectively), although the highest percentage of samples infected by a. flavus was found in peanuts collected from retailers, followed by those collected from wholesalers, collectors and farmers. this may have been associated with differences in the existence of toxigenic strains of a. flavus. according to pitt and hocking (1997) aflatoxin production depends on the toxigenicity of strains of a. flavus. the presence of antagonistic fungi to toxigenic a. flavus could also inhibit aflatoxin production. dharmaputra et al. (2001) reported that a. niger was the most promising 15 biotropia no. 24, 2005  antagonistic  fungus  to  toxigenic  strains  of  a.  flavus,  because  it  inhibited  aflatoxin  production up to 80% under in vitro conditions. the percentages of peanut samples infected  by a. niger at farmer, collector, wholesaler and retailer levels in cianjur regency were 69.2,  80 and 85.7, 60 and 76.5%, respectively.  at the farmer  level, no sample was contaminated with aflatoxin bj at more than  1000 ppb. at  the  collector,  wholesaler and retailer  levels, 1  sample (2.5% of collected  samples), 3 samples (60% of collected samples) and 7 samples (15.6% of collected samples)  contained more than 1000 ppb of aflatoxin bi, respectively. at the collector level, aflatoxin  bi content in one sample was 3000 ppb. the highest aflatoxin bi content measured at the  wholesaler and retailer level was 6066 ppb (in one sample) and 6073 ppb (in one sample),  respectively. pitt and hocking (1996) concluded that more than 1000 ppb of aflatoxin  could cause acute toxic both in humans and animals.  although the percentage of samples contaminated with aflatoxin b i exceeding 15 ppb  at the farmer  level (38.5%) was higher than that at the collector  level (30%), aflatoxin bi  contents more than 500 ppb were found in 4 peanut samples collected from collectors,  while the highest aflatoxin b] content at farmer level was 114.2 ppb. this results showed  that aflatoxin bi contents at the collector level are generally higher than at the farmer  level, probably because the duration of storage at the collector level was longer, and  under poorer storage conditions compared to that at the farmer level (tables 2 and 3).  at the farmer level, 2 peanut samples were not infected by a. flavus, but they were  contaminated  with  aflatoxin  b,  (table  6).  it  was  therefore  assumed  that  these  peanuts  were infected by a. flavus before harvest, or during sun‐drying, and contaminated with  aflatoxin  b!  during  sun‐drying.  further  sun‐drying,  and  in  the  presence  of  antagonistic  fungi to a. flavus, could inhibit or kill a. flavus. therefore,  16    aaspergillus flavus infection and aflatoxin contamination in peanuts okky s. dharmaputra et al. no a. flavus was isolated using afpa media, but aflatoxin b, still existed. buchi and rae (1969) found that aflatoxin could only be degraded at 268 269°c. conclusions in cianjur regency farmers dried (sun-drying) peanut pods until safe moisture contents were obtained, and sold peanuts to collectors in the form of dry pods. in general, farmers stored peanuts in woven polypropylene bags for 1 7 days before selling to collectors. collectors sold the peanuts to wholesalers and retailers in the form of dry pods and kernels. collectors shelled the peanut pods using diesel powered shellers. at the wholesaler level, peanuts were stored in woven polypropylene and jute bags. in general, stacks of bags containing peanuts were not placed on pallets. retailers stored peanuts in woven polypropylene and jute bags for 1 7 days. most retailers used rectangular plastic basins as containers when the peanut sampling was being conducted, followed by winnowing trays, wooden boxes, round plastic basins, woven polypropylene bags and jute bags. the mean moisture contents of peanut kernels derived from various kinds of peanuts in each part of the delivery chain were considered to have safe moisture contents. the percentages of intact peanut kernels collected from farmers was the highest, followed by those collected from collectors, wholesalers and retailers. the percentages of shriveled kernels collected from different parts of the delivery chains were relatively similar. the percentage of damaged kernels collected from retailers was the highest, followed by that collected from wholesalers, collectors and farmers. the percentages of samples infected by a. flavus in peanuts collected from wholesalers and retailers were the highest (100%, respectively), followed by those collected from collectors and farmers. the mean percentages of infected kernels in infected samples collected from retailers were the highest, followed by those collected from wholesalers, collectors and farmers. the range of aflatoxin bi contents in peanut samples collected from retailers (< 3.6 6073.0 ppb) was the widest, but almost similar with that collected from wholesalers (< 3.6 6065.9 ppb). the percentage of samples contaminated with more than 15 ppb of aflatoxin b) collected from wholesalers (80.0% of samples) was the highest, followed by retailers (75.6%), farmers (38.5%) and collectors in the form of pods and kernels (30.0 and 14.3%, respectively). our results show that postharvest handling methods employed prior to peanuts being delivered to wholesalers and retailers will have a severe impact on the level of aflatoxin contamination in peanuts in different parts of the delivery chain. postharvest handling methods that minimize kernel infection by a. flavus and aflatoxin contamination, especially at wholesalers and retailers in traditional markets should be employed to minimize aflatoxin contamination for indonesian peanut consumers. 17 b1otropia no. 24, 2005 acknowledgements the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the indonesian government regional office for food crops of west java province, head and staff of the indonesian government regional office for food crops of cianjur regency , especially to mr. jayanudin for the information given relating to the survey; head and staff of the indonesian government subregional office for food crops at cidaun, naringgul and sindangbarang districts, cianjur regency, for their cooperation during the survey; to drs. ivan r. kennedy and nanju alice lee of the university of sydney, australia; to prof.dr. rizal syarief and dr. graeme wright who have reviewed this article; and to the technicians of the laboratory of plant pathology, seameo biotrop; mr. faizal arifin nur akbar and mr. pujo waluyo for their assistance. references bsi. 1995. oilseeds-determination of moisture and volatile matter content. british standard international. (supplement). buchi, g. and i.d. rae. 1969. the structure and chemistry of the aflatoxins. in goldblatt, l.a. (ed). aflatoxins: scientific background, control and implications. academic press, new york. p. 55 75. dhartnaputra, o.s and i. retnowati. 1996. fungi isolated from groundnuts in some locations of west java. biotropia no. 9: 15 25. dharmaputra, o.s., a.s.r.putri, i. retnowati and s. ambarwati. 2001. soil mycobiota of peanut fields in wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropia no. 17: 30 56. dharmaputra, o.s., i. retnowati, a.s.r. putri and s. ambarwati. 2003. aspergillus flavus and aflatoxin in peanuts at various stages of the delivery chain in pati regency, central java. paper presented at the 3rd apec/21st asean postharvest technology seminar. nusa dua, bali, 23 26 august 2003. dharmaputra, o.s., i. retnowati, a.s.r.putri and s. ambarwati. 2005. aspergillus flavus infection and aflatoxin contamination in peanuts at various stages of the delivery chain in wonogiri regency, central java, indonesia. paper presented at the international peanut conference. bangkok, 9-12 january 2005. diener, u.l. and n.d. davis. 1969. aflatoxin formation by aspergillus flavus. in goldblatt, l.a. (ed). aflatoxins: scientific background, control, and implications. academic press, new york. p. 13-54. lee, n.a. and i.r. kennedy. 2002. elisa workshop analysis of aflatoxin b, in peanuts. seameo biotrop, bogor, 12-13 february 2002. lubulwa, a.s.g. and j.s. davis. 1994. estimating the social costs of the impacts of fungi and aflatoxins in maize and peanuts. in highley, e., e.j. wright, h.j. banks and b.r. champ. (eds). stored product protection. vol. 2. proceedings of 6lh international working conference on stored-product protection. canberra, 17-23 april 1994. cab international, wallingford. p. 1017 1042. 18 aspergillus flavus infection and aflatoxin contamination in peanuts okky s. dharmaputra et al. pitt, j.i., a.d. hocking and d.r. glenn. 1983. an improved medium for the detection of aspergillus flavus and a. parasiticus. journal appl. bacteriology 54: 109-114. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia in highley, e. and g.i. johnson. (eds). mycotoxin contamination in grains. aciar technical reports 37, canberra, p. 5 10. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic and professional, london. pitt, j.i., a.d. hocking, b.f. miscamble, o.s. dharmaputra, k.r. kuswanto, e.s. rahayu and sardjono. 1998. the mycoflora of food commodities from indonesia. journal of food mycology i (1): 41 -60. qdpi. 2000. aflatoxin in peanuts; tips to reduce the risk. crop link. queensland department of primary industries and fisheries, farming systems institute, kingaroy. (see www.qld.gov.au/ fieldcrops/3027.html). standar nasional indonesia (sni). 01-39219-1995. 1995. kacang tanah. dewan standardisasi nasional, jakarta. wongvi raj tana, p., s. soponronnarit and a. nathakaranakule. 1993. feasibility study of in-store corn drying under tropical climates. in naewbanij, j.o., a.a. manilay and a.s. frio (eds). increasing handling, processing and marketing efficiency in the grain postharvest system. proceedings of the 16'h asean seminar on grain postharvest technology. phuket, 24 26 august 1993. asean grain postharvest programme, bangkok, p. 265 283. 19 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf microsoft word 22 biotropia vol. 13 no. i, 2006 : 22 36 integration of npp semi mechanistic modelling, remote sensing and cis in estimating co2 absorption of forest vegetation in lore lindu national park tania june0, andreas ibrom2), and gode gravenhorsr9 "biotrop-icsea, seameo biotrop, bogor. lndonesia;email: taniajune@biotrop.org: and laboratory ofagrometeorology, bogor agricultural university, bogor, indonesia. ''storma (stability of rainforest margin) scientists hltp:/avww. storma.de abstract net primary production, npp, is one of the most important variables characterizing the performance of an ecosystem. it is the difference between the total carbon uptake from the air through photosynthesis and the carbon loss due to respiration by living plants. however, field measurements of npp are time-consuming and expensive. current techniques are therefore not useful for obtaining npp estimates over large areas. by combining the remote sensing and gis technology and modelling, we can estimate npp of a large ecosystem with a little ease. this paper discusses the use of a process based physiological sunshade canopy models in estimating npp of lore lindu national park (llnp). the discussion includes on how to parameterize the models and how to scale up from leaf to the canopy. the version documented in this manuscript is called netpro model, which is a potential npp model where water effect is not included yet. the model integrates cis and the use of remote sensing, and written in visual basic 6.0 programming language and map objects 2.1. netpro has the capability of estimating npp of cs vegetation under present environmental condition and under future scenarios (increasing [co2], increasing temperature and increasing or decreasing leaf nitrogen level). based on site-measured parameterisation of vam* (photosynthetic capacity), /jj (respiration) and leaf nitrogen oni), the model was run under increasing co2 level and temperature and varied leaf nitrogen. the output of the semi-mechanistic modelling is radiation use efficiency (?). analysis of remote sensing data give normalized difference vegetation index (ndvi) and related leaf area index (lai) and traction of absorbed photosynthetically active radiation (/m>ak). climate data are obtained from 12 meteorological stations around die parks, which includes global radiations, minimum and maximum temperature. co2 absorbed by vegetation (gross primary production, gpp) is then calculated using the above variables and parameters with the following equation: estimating npp, while ecosystem respiration is set as a function of temperature for estimating nee. under present condition, the net absorption of co> by the vegetation of lore lindu national park (npp) is 1330.31 gcm"2year"' and at double co2 and temperature increased of 3.5 "c, it increased by 23 %, reaching 1638.80 gcm'2 year'1. key words : npp semi-mechanistic model, photosynthesis, carbon sequestration, net primary-production, tropical forest 22 biotropia vol. 13 no. 1,2006 introduction the global carbon balance has become an issue of great concern during the last decade due to its impact on climate. ipcc (2001) has implicated that the increase in co2 concentration from 285 ppm in year 1780 to 360 ppm in year 2000 has resulted in 0.6 °c global average temperature increase and it is projected that in the year 2100, the increase will be up to 5.8 °c. the concern over this problem has resulted in international agreements (e.g., rio 1992; unfccc 1994; and kyoto 1997) to reduce co2 concentration in the atmosphere. net primary production (npp) is an important quantitative characteristic of an ecosystem (churkina et al. 1999). it refers to the net production of organic carbon by plants in an ecosystem usually measured over a period of a year or more. it is the difference between carbon gain through photosynthesis and carbon loss through respirations. it constitutes the total annual growth increment (both above and below ground) plus the amounts grown and shed in senescence, reproduction or death of short-lived individuals in a stand plus the amounts consumed by herbivores. seasonal changes in npp will influence seasonal changes in net ecosystem exchange (nee; npp-soil respiration), and it is a principal cause of seasonal changes in atmospheric co2 (keeling et al. 1996). tropical forests are important sink of co2, representing 59 % of the global carbon pool in forests (dixon et al. 1994). although the area of tropical forests is only 22 % of the global forest area, they account for 32 43 % of the world's potential terrestrial npp (melillo et al. 1993; field et al. 1998). because conventional measurements (periodical destructive harvesting) of npp of all ecosystems at all times are impractical, models are needed to estimate npp. the model discussed in this manuscript is based on farquhar and caemmerer (1982), june (2002) and de pury and farquhar (1997). most part of the model equations are mechanistic and some are semi mechanistic. when using such models in estimating npp, prediction of environmental effects to npp become more reliable. this manuscript contained theoretical background in developing the models, at leaf and canopy level; parameterisation and integrating remote sensing (rs) technology for data input and geographical information system (gis) for display. parameterisation of model was conducted using photosynthesis system adclc4am. the model is run to answer the following question: (i). how much co2 is absorbed by vegetation in a protected forest like lore lindu national park ; (ii). how would changes in temperature, light, and co2 concentration in the atmosphere affect the absorption? model parameters and estimated npp of lore lindu national park are presented. integration of the model with remote sensing data and gis are done using map object 2.1 and visual basic 6.0 in an application software netpro. 23 integration of npp semi mechanistic-modelling t. june et al. modelling net primary production (npp) plant photosynthesis and net primary production plants fix carbon dioxide from the atmosphere during the process of photosynthesis. all the carbon fixed during photosynthesis is called gross primary production (or gross primary productivity in terms of rate, for example, tonnes of carbon per hectare per year, usually called gpp). some of the fixed carbon is used by the plants themselves for metabolic processes (largely respiration) and in this process, carbon dioxide is returned to the atmosphere. the carbon that is not used in respiration remains on the plant and adds to the biomass of the plant. this is net primary production (npp). npp represents the net new carbon stored as biomass in stems, leaves or roots of plants. it is the difference between the carbon assimilated during photosynthesis by plant leaves and carbon consumption through respiration by leaves, stems and roots. it is a quantitative measure of plant growth and carbon uptake. the knowledge of npp distribution provides information on the productivity of croplands, forest and grasslands and thus helps improve management strategies for sustainable development of natural resources. at the national scale, npp allows the estimation of the contribution of landmass to the global carbon budget which is important in global change studies. in agricultural system and forestry, npp is usually defined as the increase in the standing biomass plus losses through litterfall and through consumption by herbivore. there are three main ways of estimating net primary productivity first involves the estimation of biomass production and second through the measurement of gas exchange and modelling or third through co2 flux measurement using surface tower over forest canopy or agroforestry system or using mast installed with instrument directly measuring co2 fluxes over short type of vegetation like grassland or crop. npp modeling and data requirements. an attractive approach for estimating npp was firstly proposed by mont'ith (1972; 1977), in which he determined dc/dt (carbon accumulation over time;wnen time = 1 year dc/dt = npp) as a product of the efficiency of the canopy (e, mol co2 mol" 1 par or in unit gc mjt1), fraction of photosynthetically active radiation, par (/apak) absorbed by the canopy and the daily par reaching the top of the canopy (par) as: the original approach of monteith considered the value of e as a constant and based on net co2 absorption (determined through increased in biomass). on reality, 24 biotropia vol. 13 no. 1,2006  e changes through time due to the changing climatic and plant variables like leaf area  index, nitrogen level, and water status. in order to make the model to be responsive to  the changing environmental condition, or to be used for a climate change prediction  effect, e has to be mechanistically or semi mechanistically modelled using the approach  introduced in june (2002).  the outline of the semi‐mechanistic model to produce the e value introduced in this  manuscript is shown in figure 1.  01 i\rr scim mcciiaiiimii-inuueiuug — i. june tfi ui. i light intensity incident on leaf surface (nmolm"2 s"1) j rate of actual electron transport (̂ imol m"2 s"1) •/ma* maximum electron transport rate (̂ imol m'2 s"1) ke michaelis-menten constant for carboxylation by rubisco (|.ibar) ka michaelis-menten constant for oxygenation by rubisco (mbar) o ambient partial pressure of oxygen (mbar) r universal gas constant, 8.3144 j mol4 k"1 rt dark respiration of leaf which continues in the light (nmol m"2 s"1) t leaf temperature (°c) k™« maximum rate of rubisco activity in the leaf (umol m"2 s"') &„ nitrogen extinction coefficient k light extinction coefficient af0 leaf nitrogen concentration on top of canopy (mmolm" 2) ^c.kai total par absorbed by the canopy and leaf (umol m"2 s"') npp net primary production gpp gross primary production nee net ecosystem exchange rveg respiration by vegetation (0.45 gpp) reco respiration of ecosystem (as a function of temperature) the model used is a c^ photosynthesis model. it is chosen due to the fact that €3 plants dominate 95 % of earth vegetation. it is shown in the model that the responses of c3 leaf photosynthesis to light, temperature and co2 concentration can be described by the biochemical properties of just two steps in the process, the carboxylation reaction (shown by kcmax) and the regeneration of the acceptor for carboxylation (shown by j,mu). this mechanistic model, has been widely validated as an accurate predictor of photosynthetic carbon uptake by leaves with variation in environmental conditions. the scaling up to canopy to estimate npp is done using sun-shade model (de pury and farquhar 1997; june 2002). in the simulation, supply of cck (fj) into the leaf is modelled as 0.7 of ambient cck (ca). this is a condition where water is not a limiting factor and vapour pressure deficit is around 12.5 mbar. 7max is taken as 2.1 fcmax. to run the model the following groups of data are needed: (1) fixed parameters of leaf and canopy photosynthesis for €3 plants (table 1); (2) photosynthetic parameters based on measurements ( table 2) ; (3) hourly climate data of maximum and minimum air temperature and global radiation. these hourly data are generated from daily data. ra was determined by extrapolation of a linear regression at the lower end of the par response curve (at par = 0-100 ^mol m"2 s"1) (figure 2) and fcmm was estimated from the lower end of the q response curve at q around 100 jibar (table 2). 26 b1otropia vol. 13 no. 1,2006 to scale up the model result for the whole national park, input data (lai and /apar) derived from ndvi (normalized difference vegetation index) observed from satellite images (landsat tm) are used as follows: where nir and red is the amount of reflected light of visible near infrared, and red wavelengths, respectively; /apar is fraction of absorbed photosynthetically active radiation. both equations (3) and (4) are developed for tropical forest. model development with study case lore lindu national park netpro model is a prototype of potential net primary production model where water deficit effect is not included yet. the model integrates the use of remote sensing in obtaining leaf area index (lai) through a relationship with ndvi. the mathematical equations showing the linear regression between lai with ndvi and lai with /apar were obtained from tropical forest area. the lai is used as input to the photosynthesis model, while the fapm is used as input for eq. (1) to estimate 27 integration of npp semi mechanistic-modelling t. june et al. table 1. fixed photosyiithetic parameters for c3 plants and canopy parameters parameters value sources r* co: compensation partial pressure in the absence of dark respiration (|*bar) (at 25 42.75 bemacchi et al. (2001) e activation energy for carboxylation, oxygenation, respiration, rubisco activity and coi compensation point (j mol"') 79430,36380, 46390, 65330,37830 bemacchi et al. (2001) k, michaelis-menten constant for carboxylation by rubisco (pa) 40.49 bemacchi et al. (2001) ka michaelis-menten constant for oxygenation by rubisco (pa) 27840 bemacchi etal. (2001) r universal gas constant (j mol"' k"1) 8.3144 goudriaan (1977) p« canopy reflection coefficient for diffuse par 0.036 goudriaan (1977) k, diffuse and scattered diffuse par extinction coefficient 0.715 goudriaan (1977) ph reflection coefficient of a canopy with horizontal leaves 0.041 goudriaan (1977) *' beam and scattered beam par extinction coefficient (for random orientation of leaves) 0.69/sinp june (2002) n,, base level of nitrogen not associated with photosynthesis (rnmol n m"') 29 antene/a/. (1995) x. ratio of rubisco capacity to leaf nitrogen content 1.63 june (2002) pi the leaf reflection coefficient for par 0.10 de pury and farquhar (1997) t, the leaf transmissivity to par 0.05 de pury and farquhar (1997) 0, curvature factor of the light response curve 0.7 june (2002) note: 4-i/ip is sun elevation. 28 biotropia vol. 13 no. 1,2006 study site: lore lindu national park a. site information the netpro v. 1.0 is run for lore lindu national park, central sulawesi the whole national park is located in lat. 1° 107-1 °50s and long. 119°50?120°20?e (figure 5). vegetation that occurs in tnll includes species dominating the lower area (200-1000 m) such as mussaendopsis beccariana, ficus sp., myristica sp., pterospermum sp., canangium odoratum, arrenga pinata and species dominating the higher area (1000-2500 m, 90 % of tnll) such as castanopsis argentea and lithocarpus sp. other species includes podocarpus sp., elaeorpus sp., adinandra sp., litsea sp., callohylhim sp., eucalyptus deglupta and palmae (kartawinata 1985; mogea 2002). b. software used and data input for netpro sofware used for data preparation and to run netpro includes visual basic 6.0, ermapper 6.4, arcview 3.3 and mapobject 2.1. data input into netpro v 1.0 includes (1) shapefile of polygon with different characteristics of ndvi, minimum 29   biotrop1a vol. 13 no. 1, 2006 figure 5. lore lindu national park in central sulawesi, with red dots showing 12 meteorological stations around the park and red square shows the location the meteorological tower where direct measurement of co: fluxes are conducted. and maximum temperature and par classes of llnp; (2) daily averaged climate data (global radiation, maximum and minimum temperature) from year 2001-2005. ndvi is derived from landsat tm 7 dated 21 august 2001. the climate data, global radiation and daily temperature are zoned into several polygons ( figure 6). ndvi, leaf area index (lai), and /apar resulting from image analysis for year 2001 are shown in figures 7, 8 and 9. ndvi values are divided into 4 classes and based on the equation of ibrahim (2001), lai ranges from 0 to 10.04. lai values are used as input to photosynthesis model to obtain e value. /apar distribution (ranges from 0 to 65 %) is shown in figure 9, where these values are used as input to the model to obtain npp and nee. classes of average ndvi, temperature and global radiation are overlayed to form polygons with different characteristic of climate and ndvi. c. net primary production (npp) and net ecosystem exchange (nee) of llnp the value of simulated e changes with changing environmental conditions such as changes in global radiation, temperature, atmospheric co2 concentration, and nitrogen level of the leaf, and therefore result in varied npp and nee values (table 3). 31     integration of npp semi mechanistic-modelling t. june et al. conclusions this framework of model in estimating npp using remotely sensed ndvi combined with semi-mechanistic modeling is first introduced by the author at the scientific meeting on climate and weather prediction, center for climate and atmospheric science applications, national institute of aeronautics and space (lapan) in bandung, indonesia on 31th july 2002 where the proceedings was published in early 2003. the idea was then used by several undergraduate and post graduate students for thesis researches, using a constant value of radiation use efficiency (e) under the author's supervision for study sites in sumatera and sulawesi. this research idea is further extended to include a mechanistic estimation of the radiation use efficiency, and in 2004 it was approved by the integrated research award secretariat of the indonesian institute of sciences to be funded for the period of 2004-2006. however with limited availability of budget during that period, the funding was then cancelled after being approved by the reviewer panel. through seamed biotrop dipa2004, the development of the framework was realized and the prototype of the application software netpro is produced. model parameterization was conducted in lore lindu national park early 2005 supported by storm a sfb 552 (stability of rainforest margin) project. this work still needs improvement in display and modeling. it also needs inclusion of water effect, although application for lore lindu national park the assumption that water is not a limiting factor can be used. to be applicable to different site and type of vegetation, site specific parameterization (vcmax, e) and site variable measurement and estimation like leaf nitrogen, lai, /apar. climate data (diurnal global radiation, minimum and maximum temperature, diffuse radiation, vapour pressure deficit) are required. it is also important to develop relationship between ndvi and lai and /apar from different type of vegetation. direct measurement of co2 flux and e from eddy correlation and micrometeorology techniques for validation of model are needed. application of the model framework to other national parks in indonesia will be an ongoing effort. references anten, n.p.r., f. schieving, and m.j.a. werger. 1995. patterns of light and nitrogen distribution in relation to whole canopy carbon gain in c3 and c4 monoand dicotyledonous species. oecologia, 101:504-513. bernacchi, c.j., e.l. singsaas, c.pimentel, a. r. portis and s.p. long, 2001. improved temperature response functions for models of rubisco-limited photosynthesis. plant cell and environ., 24:253-259. churkina, g., s.w. running, and a.l. schloss, 1999. comparing global models of terrestrial net primary productivity: the importance of water availability. global change biol., 5 (suppl): 46-55. 34 biotrop1a vol. 13 no. 1, 2006 de pury, d. g. g. and g. d. farquhar 1997. simple scaling of photosynthesis from leaves to canopies without the errors of big-leaf models. plant cell and environ., 20:537-557. dixon, r. k., s. brown, r. a. houghton, a. m. solomon, m.c. trexler, and j. wisniewski, 1994. carbon pools and flux of global forest ecosystems. science, 263: 185-190. farquhar, g. d. and , s. von caemmerer 1982. modelling of photosynthetic responses to environmental conditions. physiological plant ecology. //. encyclopedia,of plant physiology, new series. o. l. lange, p.s. nobel, c.b. osmond and h. ziegler. berlin, springer-verlag. farquhar, g. d. and s. c. wong 1984. "an empirical model of stomatal conductance. aust j. plant physi., 11:191-210 farquhar, g. d., s. von caemmerer and j. a. berry 1980. a biochemical model of photosynthetic co2 assimilation in leaves of c3 species. planta, 149, 78-90. farquhar, g. d. and s. von caemmerer 1982. modelling of photosynthetic responses to environmental conditions. physiological plant ecology. ii. encyclopedia of plant physiology, new series. o. l. lange, p.s. nobel, c. b. osmond and h. ziegler. berlin, springer-verlag. field, c. b., m. .1. behrenfeld, j. t. randerson, and p. falkowski 1998. primary production of the biosphere: integrating terrestrial and oceanic components. science, 281: 237-240. hunt jr., e. r., j. t. fahnestock, r. d. kelly, j. m. welker, w. a. reiners, w. k. smith 2002. carbon sequestration from remotely-sensed ndvi and net ecosystem exchange. in. r. s. muthiah (ed.). from laboratory spectroscopy to remotely sensed spectra of terrestrial ecosystems, p 161-174. kluwer academic publishers, dordrecht, netherlands. goudriaan, 3. 1977. crop micrometeorology: a simulation study. pudoc, wageningen. goudriaan, j and h.h. van laar 1994. modeling potential crop growth processes. kluwer academic publishers. dordrecht/boston/london. ibrahim, 2001. environment and development in coastal region and in small island. assessing mangrove leaf area index and canopy closure. 1pcc. 2001. climate change 2001. the scientific basis. contribution of working group i to the third assessment report of the intergovernmental panel on climate change. cambridge university press. june, t. 2002. environmental effects on photosynthesis of cj plants: scaling up from electron transport to the canopy (study case: gtycine max l. merr). environmental biology, research school of biological sciences. australian national university. canberra. june, t. 2003 modelling net primary productivity (npp) using c3 photosynthesis model: theoretical approach. proceeding to the scientific meeting on climate and weather prediction. pusat pemanfaatan sains atmosfir dan iklim lapan) bandung, indonesia 31 july 2002 keeling, c. d., j.f.s chin,, and t.p. whorf 1996. increased activity of northern vegetation inferred from atmospheric co2 measurement. nature, 382,146-149. lind, m. and r. fensholt 1999. the spatio-temporal relationship between rainfall and vegetation development in burkina faso. dan. j. geogr., 1999, 2, 43-56. lloyd, j., j.grace, a.c. miranda, p. meir, s.c. wong, h.s. miranda, i. r wright, j.h.c gash, and j. mclntyre 1995. a simple calibrated model of amazon rainforest productivity based on leaf biochemical properties. plant cell and environ., 18:1129-1145. kartawinata, k. 1985. the tropical rainforest: phytogeography, ecology, and altitudinal zonation. in. remote sensing in vegetation studies. report of training course on remote sensing techniques applied to vegetation studies. bogor, 4 november-13 december, 1985. 35 integration of npp semi mechanistic-modelling t. june et al melillo, j.m., a.d mcguire, d.w. kicklighter, b. moore iii, c.j. vorosmarty and a.l. schloss 1993. global climate change and terrestrial net primary production. nature, 363:234-240. monteith, j. l. 1972. solar radiation and productivity in tropical ecosystems. j appl. ecol., 9:747-766. monteith, j. l. 1977. climate and the efficiency of crop production in britain. phill. trans. r. soc. londb., 281:277294. ochi, s. and r. shibasaki 1999. estimation of npp based agricultural production for asian countries using remote sensing data and cis. institute of industrial science. university of tokyo. japan. pinter p. j. 1992. solar angle independence in the relationship between absorbed par and remotely sensed data for alfalfa. remote sensing of environ., 46, 19-25. prince, s. d. and s. n. coward 1995. global primary production: a remote sensing approach. j. biogeogr., 22,815-835. ruimy, a., b. saugier, and g. dedier 1994. methodology for the estimation of terrestrial npp from remotely sensed data. j. geophy. res., vol. 99, (3): 5263-5283. von, caemmerer, s.j.r. evans, g.s. hudson and t.j. andrews (1994). the kinetics of rubisco inferred from measurements of photosynthesis in leaves of transgenic tobaco with reduced rubisco content. planta, 195: 33-47. 36 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf biotropia vol. 29 no. 2, 2022: 181 184 doi: 10.11598/btb.2022.29.2.1709 181 short communication ageing and the amount of dna extracted from bawean deer (axis kuhlii) feces adi nugroho*1, denni susanto1 and sena adi subrata2 1department of bioresources technology and veterinary, vocational college, universitas gadjah mada, yogyakarta 55281, indonesia 2department of forest resource conservation, faculty of forestry, universitas gadjah mada, yogyakarta 55281, indonesia received 4 december 2021/accepted 15 march 2022 abstract noninvasive genetic sampling is the most reliable approach to collect endangered species which are typically rare and elusive. however, the approach is hampered by poor quality and quantity of dna amount, and therefore, a pilot study is required. our current short communication article reports the first noninvasive genetic sampling approach using feces of critically endangered bawean deer (axis kuhlii) to test the effect of aging on the amount of dna extraction. fecal samples of the bawean deer were collected from the bawean deer captive unit in sangkapura village, bawean island, east java. the samples were grouped into two different categories, namely: 1) new samples: for feces that had just been expelled for less than a day and 2) old samples: for feces that were estimated to have been expelled for more than a day. there were 11 new samples and 16 old samples. the samples were extracted using the qiagen mini stool kit. the dna quantification was carried out by using a fluorometer. the results of the extraction between the two categories were analyzed by the kruskal wallis test. the results showed that the mean rank value of the new samples was better (15.27) than the old samples (13.13). the p value of the kruskal wallis test, however, was higher than the asymp significant value, indicating that there was no meaningful differences (p = 0.49) between the two samples categories. the work described in this short communication article is a preliminary result and an important step in the study of bawean deer population genetic. keywords: bawean deer, biodiversity, critically endangered, dna, noninvasive sampling introduction conservation strategies for endangered species require genetic information (whiteley et al. 2015) that can be difficult to obtain using a traditional method such as invasive sampling and destructive sampling. in small and elusive wildlife populations, noninvasive dna sampling was successfully employed for obtaining genetic data (kohn & wayne 1997). however, the effective use of noninvasive samples for genetic analyses of wildlife populations is hampered by typically poor quality and low quantity of the dna obtained (taberlet & waits 1999). in large animals, dna samples can be obtained from fluids, hair and feces gathered in the field. however, these types of samples usually provide dna of low quantity, quality, and integrity, causing the difficulties in analyzing some molecular markers (venegas et al. 2020). dna of the depositor is present in sloughed intestinal epithelial cells (waits & paetkau 2005). dna degradation in fecal dna is enhanced by ageing of the samples and by the action of variable environmental conditions (e.g., temperature, humidity, exposure to sun or rain) (nsubuga et al. 2004). the effectiveness of genetic analysis using fecal samples would be improved if some of the factors affecting dna yield could be identified and used as guidelines in sample collection. recent study has analyzed the effect of ageing and environmental condition on the amount of dna extraction from deer feces *corresponding author, email: adi.adinugroho@ugm.ac.id biotropia vol. 29 no. 2, 2022 182 (brinkman et al. 2010; ebert et al. 2021; gupta et al. 2018). however, these studies were conducted on temperate climate conditions. to our knowledge, such study has not been conducted in tropical environments which have different uv radiation from sunlight and humidity. our current short communication article reports the first noninvasive genetic approach using feces of critically endangered bawean deer (axis kuhlii) to test ageing on the amount of dna extraction. materials and methods sample collection the bawean deer feces were collected from the bawean deer captive unit at bawean island, east java. the fecal samples were categorized as new for feces that has probably been deposited for less than one day and old for feces that has been deposited for more than 1 day. the two categories of feces were differentiated by evaluating its appearance and by monitoring the depositor on the captivity. the feces were collected using a feces container added with silica granules and labeled. the fecal samples were collected on august 2021. the fecal dna were immediately extracted at the laboratory of bioresources and veterinary technology, ugm vocational college. dna extraction the dna extraction was carried out by using qiamp dna stool mini kit (qiagen) following the manufacturer’s protocol. the results of the extraction (double stranded dna only) were measured by a fluorometer (quantus fluorometer, promega). results and discussion the 27 collected bawean deer feces were categorized as the new and old feces. the new category is characterized by the moist and glittering appearance, while the old category is relatively dry and harsh in appearance (figs. 1 & 2). figure 1 bawean deer feces categorized as new with the moist and glittering appearance ageing and the amount of dna extracted from bawean deer feces – adi nugroho et al. 183 figure 2 bawean deer feces categorized as old with the dry and harsh appearance the dna extraction showed different quantities of dna at different sample conditions as shown in table 1. table 1 quantification of dna extraction from bawean deer feces new samples (ng/µl) old samples (ng/µl) 0.615 0.73 0.993 1.245 2.061 0.805 1.512 0.73 0.079 1.106 2.857 1.655 12.224 6.352 2.613 8.82 5.845 0.066 7.123 0.207 3.179 7.727 1.189 3.057 1.972 2.743 3.379 kruskall wallis test showed that the new feces samples resulted to better dna quantification compared to the old feces samples (mean rank = 15.27). however, there is no significant difference between the two categories (p = 0.477). table 2 kruskall wallis analysis sample n mean rank quantity new 11 15.27 old 16 13.13 total 27 table 3 kruskal wallis test (p > 0.05) quantity kruskal-wallis h 0.477 df 1 asymp. sig. 0.490 a. kruskal wallis test b. grouping variable: sample this finding is in line with the work of agetsuyama-hanigara et al. (2017) showing that it is recommended to use fecal samples not older than 3 days old for genetic analysis of the sika deer (cervus nippon yakushimae). this study shed the light on the importance of collecting the new fecal samples for studying bawean deer genetics in a natural population with a noninvasive sampling approach. however, the finding is only limited to dna quantity which can come from bacteria, fungi, or any other genetic sources in the feces. further research is required to explore the optimum condition for successful species-specific bawean deer dna amplification. biotropia vol. 29 no. 2, 2022 184 conclusion the data showed that the new fecal samples provided better results of dna extraction than the old fecal sample. nevertheless, the differences are not significant. acknowledgments the author would like to thank the research directorate of universitas gadjah mada for supporting this study through the young lecturer grant no. 2458/un1/ditlit/ditlit/pt/2021. references agetsuma-yanagihara y, inoue e, agetsuma n. 2017. effects of time and environmental conditions on the quality of dna extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest. mammal res 62(2):201-7. doi: 10.1007/s13364-016-0305-x brinkman tj, schwartz mk, person dk, pilgrim kl, hundertmark kj. 2010. effects of time and rainfall on pcr success using dna extracted from deer fecal pellets. conserv genet 11(4): 1547-52. doi: 10.1007/s10592-009-9928-7 ebert c, sandrini j, welter b, thiele b, hohmann u. 2021. estimating red deer (cervus elaphus) population size based on non-invasive genetic sampling. eur j wildl res 67(27):1-13. doi: 10.1007/s10344-021-01456-8 gupta sk, kumar a, angom s, singh b, ghazi mgu, tuboi c, hussain sa. 2018. genetic analysis of endangered hog deer (axis porcinus) reveals two distinct lineages from the indian subcontinent. sci rep 8(1):1-12. doi: 10.1038/s41598-018-34482-9 kohn mh, wayne rk. 1997. facts from feces revisited. trends ecol evol 12(6)l223-7. doi: 10.1016/s0169-5347(97)01050-1 nsubuga am, robbins mm, roeder ad, morin pa, boesch c, vigilant l. 2004. factors affecting the amount of genomic dna extracted from ape faeces and the identification of an improved sample storage method. mol ecol 13(7):2089-94. doi: 10.1111/j.1365-294x.2004.02207.x taberlet p, waits lp, luikart g. 1999. noninvasive genetic sampling: look before you leap. trends ecol evol 14(8):323-7. venegas c, varas v, vásquez jp, marín jc, de genómica l, de ciencias f, bio-bío u. 2020. non-invasive genetic sampling of deer: a method for dna extraction and genetic analysis from antlers. gayana 84(1):75-82. waits l, paetkau d. 2005. noninvasive: genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. j wildl manage 69(4):1419-33. whiteley ar, fitzpatrick sw, funk wc, tallmon da. 2015. genetic rescue to the rescue. trends ecol evol 30(1):42-9. doi: 10.1016/ j.tree.2014.10.009 biotropia no. 5, 1991/1992: 15-21 effect of light qualities and storage periods on the germination of pennisetum polystachion seeds*) verapongs kiatsoonthorn and soekisman tjitrosemito department of biology, faculty of science, srinakhrinwirot university (prasanmitr), bangkok 10110, thailand and seameo biotrop, jl. raya tajur km 6, p.o. box 17, bogor, indonesia. abstract seeds of the yellowish inflorescence strain of pennisetum polystachion, collected from the field in indonesia, were kept in the dark for 30 days, then germinated in 12-h light and 24-h light under various light qualities, namely, white, black, blue, red and far-red. there was no effect of photo-period to seed germination. percent of seed germination under white, red, far-red, blue and dark were 49, 43, 22, 11 and 2%, respectively. white and red light did not cause any difference to seed germination. seeds kept in 12-h light alternating with 12-h dark and 24-h dark for 15 and 30 days were tested for germination. results showed that light condition during seed storage did not effect seed germination. long storage period resulted in more seed germination. during seed germination test, effect of light played a great role on increasing seed germination. introduction pennisetum polystachion, or mission grass in english (wssa 1984), in thailand had two distinctive forms of immature inflorescence colour, namely purplish and yellowish. studies concerned with seed germination of the yellowish inflorescence strain had been reported (van rooden et al. 1970; noda et al. 1985; suppaphon et al. 1987; arunpu et al. 1991). fernandez (1980) without specifying the immature inflorescence colour, stated that seed of this species collected in indonesia was photosensitive for germination, especially in the first 70 days of storage after harvest; whereas, noda et al. (1985) reported that the response of this seed species to light was very low. arunpu et al. (1991) reported that this species was non-sensitive to light. in order to clarify these discrepencies, the following experiments were conducted. *)this article is part of a six-month research fellowship report submitted to seameo biotrop in december 1991. 15 biotropia no. 5, 1991/1992 materials and methods 1. light qualities and durations p. polystachion seeds harvested on 8 july 1991 from biotrop experimental field were removed from bristles, screened through a 1-mm-wire mesh to obtain uniform seed sizes and kept in the dark at room temperature (25 29°c) for 30 days until germination test. one hundred seeds were placed on a filter paper moistened with 4ml distilled water, in a 9-cm petridish covered with lid. treatments were designed in factorial of 2 factors: first factor was light period i.e. 12-h light and 24-h light, and another factor was the light qualities, namely white, red, far-red, blue and dark. for light quality treatments each petridish was wrapped with a blue, a red, a blue plus a red transparent polyethylene to obtain blue, red and far-red treatments, respectively (fernandez 1980). the dishes of dark treatment were wrapped with aluminium foil whereas the white lighted treatment was unwrapped. light transmission through those transparent polyethylene, measured by specto-photometer is shown in fig. 1. these dishes were placed in a 25-29°c room illuminated with 24-h light by cool fluorescent tubes philips tld 36 w. light fig. 1. percentage transmission of light through red, blue, and red plus blue transparent polyethylenes from tungstar light source 16 effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito intensity reaching the dishes through red, blue, and red plus blue transparent polyethylene was 0.066, 0.073, 0,004 uem -2 s -1 , respectively, whereas the controlled light was 0.775 uem -2 s -1 . for the 12-h lighted treatment, those dishes were covered with an opaque container for 12-h dark alternating with 12-h light. during the experimental period the dishes were randomly rotated in order to reduce the effect of different light intensities. germination was tested 6 days after treatments and was transformed to arcsine square root before statistical analysis. 2. storage period, light conditions during seed storage and germination test 2.1. effect of lighted conditions during storage and storage period lighted the experiment was replicated 4 times in factorial design with 2 factors: the first factor was the storage period of 15 and 30 days; the second factor was light conditions during storage consisting of the dark and light/dark conditions. p. polystachion seeds collected from the same location as in experiment 1, on 20 august 1991, were kept in a 24-h dark (dark) or 12-h light alternating with 12-h dark (light/dark). for the dark treatment, seeds were placed in polyethylene bag and wrapped with aluminium foil; while for the light condition, the polyethylene bag was not wrapped with aluminium foil. both treated seeds were separately kept in 9-cm petridishes, placed on a table near the window for 15 and 30 days until germination. germination test was done in a 30°c controlled incubator illuminated with 12-h light alternating with 12-h dark. light intensity during the day was 1.32*10 -4 uem -2 s -1 . the detailed methods were the same as mentioned in experiment 1. accumulated number of seed germination at 8th day were transformed to arcsine square root before statistical analysis. 2.2. effect of lighted conditions during storage and germination methods seeds stored in light/dark and dark conditions for 30 days, as in experiment 2.1, were used. the experiment was replicated 4 times in factorial design with 2 factors: the first factor was light condition, consisting of the dark, and the light/dark condition for 30 days; the second factor was germinating condition consisting of alternating 12-h lighted with 12-h dark with data collected every 2 days up to 8 days (light/2), dark condition with data collected every 2 days up to 8 days (dark/2), and dark condition with data collected 8 days later (dark/8). for seeds germinated in the dark/2 and dark conditions, petridishes were wrapped with aluminium foil. germinated seed counting was done in dark room under green light (40 watt incadescent bulb wrapped with a green transparent polyethylene). in both experiments, accumulated data of seed germinated at 8 days after treatments were transformed to arcsine and statistically analyzed. 17 biotropia no. 5, 1991/1992 results and discussion 1. effect of light qualities and durations results of this experiment showed that exposure of 24-h light and 12-h light did not cause any difference on seed germination. percent germination of seeds under white, red, far-red, blue light and dark conditions were 49, 43.1, 21.9, 11.1 and 1.6%, respectively, whereas red and white light treatments similarly effected seed germination (figure 2). percent (%) far + red l light qualities fig. 2. germination of p. polystachion seeds under various light qualities (30 days after seeds collection) light qualities and photo-period did not interact to effect seed germination (figure 3). the result of seed germination under various light qualities in this experiment was similar to fernandez's result (1980) except the effects of blue and far-red which were significantly different in this result but not different in fernandez's. one form of phytochrome in seed, phytochrome far-red (pfr), which is an active form for seed germination, can be transformed from phytochrome red (pr) when irradiated with red light; and pfr can be transformed back to pr when irradiated with far-red light (taylorson 1987). pfr can also degenerate to pr in the 18 0 blue dark red white effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito fig. 3. seed germination of p. polystachion effected by light qualities and photo-periods (nonsignificant between the interaction factors) dark. this explains why red and white lights showed higher germination than the dark condition. blue and red transparancies apparently permitted transmission of red light better than blue transparancy, therefore the former stimulated more germination than the later. although blue transparancy transmitted wavelength between 430-550 nm (fig. 1), this light has not been reported to affect germination. in fernandez's method, the light intensities were different from the other because some petridishes were covered with various transparancies, while the distance between the light source and petridishes were similar. 2. storage period, light conditions during seed storage and germination test 2.1. effect of lighted conditions during storage, and storage period p. polystachion seeds kept in room temperature for 15 days had lower germination percentage than those kept for 30 days (table 1). however, different light conditions during seed storage did not show any different effect on seed germination. light condition and period during seed storage did not interact to affect seed germination. 19 biotropia no. 5, 1991/1992 table 1. percent germination of seeds kept in two different lighted conditions for 15 and 30 days (8 days after treatments) 2.2. effect of lighted conditions during storage and germination methods the result of this experiment supports that of experiment 2.1., i.e. the light condition during seed storage did not affect seed germination. however, different germinating methods effected seed germination differently. more seeds germinated in light/2 than in dark/2 or dark/8. in dark condition, those seeds observed for germination every 2 days had higher germination than those observed only once at 8 days after germination (table 2). the green transparancy may be able to transmit some red light, which stimulates germination. however, both factors of light conditions during seed storage and of germinating methods did not have any interaction on the number of germinated seeds. the difference in seed germination under light and dark condition found in this experiment was similar to fernandez's result (1980) and noda et al. (1985); but was contrary to arunpu et al. (1991). it was reasoned that seeds used in the experiment of arunpu et al. were kept for too long (6 months at 5°c dark condition) so photo-sensitivity of seed was lost. table 2. percent germination of seeds effected by lighted conditions during storage and germinatimg methods (8 days after treatments) 20 effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito references arunpu, s., v. kiatsoonthorn and y. yingwiwatanapong. 1991. effect of some environmental factors on seed germination of p. setosum (swartz) l.c. rich. kasetsart journal (abstract in english). in press. fernandez, d.b. 1980. some aspects on the biology of pennisetum poylstachyon (l.) schult. philippine journal of weed science 7: 1-10. noda, k., l. chaiwiratnukul, s. kanjanajirawong and m. teerawatsakul. 1985. some biological characteristics of pennisetum spp. in thailand. proceeding of 10 tn asian-pacific weed science society conference: 75-80. suppaphon, j., p. roengapapong and chitpong. 1987. certain characteristics of flower and seed germination of pennisetum setosum. in: report of workshop on pennisetum setosum, held by prince of songkla university and ministry of agriculture and cooperatives in cooperation with agricultural science association of thailand under the royal patronage, pesticide business association of thailand, and weed science society of thailand: 19-32. (abstract in english). taylorson, r.b. 1987. environmental and chemical manipulation of weed seed dormancy. in: review of weed science. weed science society of america vol. 3: 135-154 van rooden, j., l.m.a. akkermans and r. van der veen. 1970. a study on photoblastism in seeds of some tropical weeds. acta. bot. neerl. 19(2): 257-264. wssa. 1984. composite list of weeds. weed science vol. 32 (supplement 2). 21 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf biotropia vol. 30 no. 2, 2023: 232 241 doi: 10.11598/btb.2023.30.2.1902 232 use of biological organic fertilizers and pesticides to improve potato cultivation in slope andisols tamad1*, loekas soesanto2 and akhmad rizqul karim3 1department of soil science, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia 2department of phytopathology, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia 3department of agribusiness, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia received 21 february 2023 / revised 13 march 2023 / accepted 13 march 2023 abstract in the 1990s, potato yield in the andisols of dieng, central java, indonesia, was approximately 30 t ha–1, but this value decreased rapidly to 12–15 t ha–1 in recent years. this rapid decline could be attributed to the use of unbalanced organic and chemical fertilizers, without the application of conservation techniques. therefore, this study aimed to sustainably improve the local potato cultivation pattern of farmers on andisols using biological organic fertilizers and pesticides (bofp). a randomized block design was used with two factors, namely: 1) 20 t bofp, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg lime ha–1, and 2) comparison with the pattern of farmers, consisting of 20 t of chicken manure, 1–t npk, and 250 kg za ha–1. the potato plant mounds were tilted 10% to the contour direction, and each treatment was carried out with 16 replications. granola seeds were used to plant potato during the rainy season from march-june 2022. the results showed that the plants cultivated using the local pattern of farmers were affected by wilt from fusarium spp, while the use of bofp decreased the incidence of the disease by 80%. furthermore, the bofp pattern significantly increased andisols organic-c from 1.78% to 3.83% and total soil p from 5.20% to 11.34%, compared to the pattern of farmers. it also increased potato yields from 12.31 t ha–1 to 22.93 t ha–1 and the r/c from 0.85 to 1.23, compared to the pattern of farmers. based on the results, the use of bofp pattern decreased wilt attacks by fusarium spp, improved the productivity of andisols, as well as increased potato production and profits of farmers. keywords: beneficial microbes, farmer income, manure, potato, soil productivity introduction farmers on the andisols slopes have been cultivating potato using unbalanced organic and chemical fertilizers, without applying conservation techniques. furthermore, potato plant mounds are often prepared toward the hill (figure 1), which causes a decrease in soil fertility due to nutrient imbalances and intensive erosion. this planting pattern has also led to a decrease in potato production of 10–15 t ha–1 compared to the volume recorded in the 1990s. the decline was due to a reduction in the productivity of andisols and an increasingly intensive potato plant disease. dieng, central java, indonesia, is situated on andisols land, which is known to have a high adsorption capacity for phosphorus (p), leading to low availability of p for plants. this high adsorption capacity is due to the presence of aluminol groups (al–oh and al–oh2 +), ferrihydrite, and al-humus complex (pizarra et al. 2008; delfim et al. 2018). the application of a large amount of p fertilizer to the andisols does not necessarily increase the availability of p to plants, as the efficiency of p fertilization is low, ranging from 10% to 20% of applied p (sacrc 2004; elásquez et al. 2016). however, the available p can be increased through the use of organic fertilizer enriched with phosphate microbial (pm), organic matter ameliorant (humic-fulvic), and signal quorum sensing (qs), as a regulator of the effectiveness of biological agents. *corresponding author, email: tamad_1965@yahoo.com; tamad@unsoed.ac.id mailto:tamad_1965@yahoo.com mailto:tamad@unsoed.ac.id potato cultivation in slope andisols – tamad et al. 233 figure 1 the potato cultivation system with mounds in the direction of the slopes in dieng, central java, indonesia pm, such as pseudomonas trivialis, p. putida, and p. fluorescens, has the potential to increase p availability (arcand and schneider 2006; tian et al. 2021; yadav et al. 2021) by dissolving inorganic–p and mineralizing organic–p. in andisols, these microbes have been found to increase the dissolution of inorganic–p by 535– 575%, mineralization of organic–p by 336– 387%, and reduced adsorbed–p to 15–30% (tamad et al. 2021). intensive disease infestation, including late blight, potato bacterial wilt, tuber rot, fusarium wilt, and dry spot caused by phytopthora infestans, ralstonia solanacearum, colleotrichum coccodes, fusarium sp., and alternaria solani, can contribute to low potato production. an alternative for controlling potato disease is the use of bio p60, a biological pesticide containing crude secondary metabolites of bacterial antagonist p. fluorescens p60 (soesanto et al. 2011). another critical factor that must be considered during cultivation in andisols is the need for soil amendments. humic materials include humin, humic acid c10h12o5n, and fulvic acid c12h12o9n. humic acid is brownish-black, resistant to degradation, and contains a negative charge that is dependent on ph. several studies showed that humic-fulvic had the potential to be used for soil amendment (stevenson 1994; baveye and wander 2019). however, the effectiveness of andisol amendments is greatly influenced by soil microbial activity. the biochemical process that regulates population density-dependent microbial behavior through crucial signaling molecules is known as quorum sensing (qs) (teplitski et al. 2011; ma et al. 2018; coquant et al. 2020; ward et al. 2001). gram-negative bacteria typically produce n-acyl–homoserine lactones (ahl) as a qs signal (ward et al. 2001; muras et al. 2020). a previous study also showed that plant root extracts could serve as a source of n–ahl (tamad et al. 2020). the enrichment of organic fertilizers with microbes and signal quorum sensing as biological organic fertilizers effectively increases soil fertility. tamad et al. (2020a) stated that using biological organic fertilizer (bof) of up to 20 t ha–1 increased potato yields by 2 t ha–1 compared to the cultivation pattern of farmers in kaligua, central java, indonesia. the use of bof in potato cultivation also increased r/c from 1.13 to 1.53, and profits rose by idr 29.995.573 ha–1 compared to the practice of farmers. therefore, this study aims to improve the local potato cultivation pattern of farmers on andisols using bofp. the results are expected to increase potato production, sustainably improve the quality of andisols, and increase the income of farmers. materials and methods this study was carried out in andisol dieng, central java, where the soil had slightly acidic ph h2o, medium organic–c, high k2o, high p2o5, and medium n of 5.63, 2.39%, 0.4%, 3.20%, and 0.34%, respectively. furthermore, the region had an area of 3000 hectares and an altitude of 1,000–2,500 m above sea level, which was very suitable for potato cultivation. this study utilized biological organic fertilizers and pesticides, which were applied using conservation techniques in partnership with local farmer groups. bofp formulation the bofp formula was produced using chicken manure inoculated with phosphate solubilizing microbial and bio p60 (107 cfu g–1) biotropia vol. 30 no. 2, 2023 234 at a concentration of 0.025% v w–1, followed by the addition of humic-fulvic acids and 1% v w–1 qs signal. the bofp components were then mixed with the granulator under moist conditions, as shown in figure 2. subsequently, the product obtained was incubated for four weeks with 15–20% moisture content. figure 2 the formulation of biological organic fertilizers pesticides (bofp) preparation of phosphate microbial (pm) culture pm consortium of pseudomonas trivialis, p. putida, and p. fluorescens was grown on molasse culture for one week (end of log phase) (tamad et al. 2020). the standard plate count (spc) method was then used to determine pm at a minimum population of 107 cfu ml–1. production of bio p60 culture bio p60 was an isolate of p. fluorescens p60 cultured in snail broth for one week (soesanto et al. 2013). the spc method was utilized to assess the microbial population at a minimum of 107 cfu ml–1. humic-fulvic acids extract humic-fulvic acids were obtained from harvested top potato plant waste. furthermore, extraction was carried out by soaking the waste in 1 m technical naoh for two weeks (modified tan 1998; dulaquais et al. 2018). n-ahl extract potato plant root waste was used as a source of quorum-sensing signals for microbes (tamad et al. 2020). n-ahl compound, a quorum sensing signal, was obtained by soaking the waste in 5% acetonitrile for two weeks (rani et al. 2011). bofp application this study used a randomized block design with two factors, namely: 1) the application of 20 t bofp, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg lime ha–1, and 2) comparison with the application pattern of the farmers, consisting of 20 t of chicken manure, 1–t npk, and 250 kg za ha–1, with 16 repetitions. the potato granola plant mounds were tilted at a 10% contour coverage rate per ha, as shown in figure 3. the seed potato was 800 kg ha–1, and the plant was spaced at 30 cm x 70 cm interval total obtain a total population of 47,000 ha–1. fertilization was performed every 20 days: a) at 20–40 days after planting (dap) when tuber growth begins; b) at 40–60 dap when tuber enlargement begins; c) at 60-90 dap (plant optimal enlargement); and d) at 90–110 dap after all the leaves dry up. furthermore, the variables observed included soil parameters, growth and yield components of potato (10% of the population were sample plants), and farming analysis (revenue and total cost). the variable of ph was measured with a ph meter, ec was assessed with an ec meter, c was analyzed with walkley and black, and p was evaluated with the hcl extraction method. figure 3 the design of potato mounds cutting a 10% sloping slope in dieng, central java, indonesia statistical analysis variance analysis was carried out on the data on soil, potato growth, and yield using fisher's test (p<0.05). subsequently, significant differences were determined by calculating the mean value using duncan's multiple range test. the statistical analysis was performed using the costat ver 6.451 application. the difference in the effect of the treatments on the economic value was carried out by assessing the production costs, income, and profits (r/c ratio). potato cultivation in slope andisols – tamad et al. 235 results and discussion soil, potato growth, and potato yield the growth of potato appeared normal up to 60 dap, as shown in figure 4. however, at 80 dap, the bofp pattern demonstrated better growth compared to farmers’ pattern, which was infested by diseases, as indicated by the falling and withering of leaves in figure 5. the plant often faced several challenges during the rainy season, including falling of leaves and intensive wilt disease. figure 4 the optimal growth of potato plant at 60 the day after planting (dap) in andisols dieng, central java, indonesia, with mound tilt of 10% contour direction figure 5 the differences in potato growth at 80 dap in andisols between farmers’ pattern and bofp pattern treatments this study showed significant improvement in soil, potato, and economic variables in bofp pattern compared to farmers’ pattern, as shown in table 1. it was observed that the plant was susceptible to fusarium infection, which caused wilt symptoms (thanaa et al. 2018). furthermore, fusarium is one of the most important genera of phytopathogenic fungi, causing potato wilt in the field (azil et al. 2021). the results showed that the application of bofp and conservation techniques to andisols improved the growth of potato plants and reduced fungi attack. p. fluorescens as bio p60 microbes produced secondary metabolites, which helped to inhibit fusarium sp wilt (soesanto et al. 2011; deveau et al. 2016). a previous study explored the antifungal activity of the extracellular metabolites of the most effective isolates of fusarium species infecting potato (trabelsi et al. 2016). table 1 the effect of bofp pattern and farmers’ pattern treatment in andisols on soil and potato variables average no variable n0a n1b 1 soil ph h2o 5.56 a 6.04 b 2 soil ph kcl 4.68 a 5.30 b 3 soil electric conductivity/ec (ms cm–1) 409.11 a 324.29 a 4 soil organic–c (%) 1.78 a 3.83 b 5 soil total–p (%) 2.27 a 4.95 b 6 plant height (cm) 54.88 a 63.19 b 7 leave number (pieces) 60.38 a 116.69 b 8 fall leave age (days) 85 a 100 b 9 fusarium spp attack (%) 80 20 10 tuber number 5.2 a 6.0 a 11 tuber diameter (mm) 51.24 a 56.74 b 12 tuber length (mm) 68.56 a 73.92 b 13 tuber volume (mmh3) 631.88 a 723.75 b 14 tuber weight (g plant–1) 301.69 a 561.81 b 15 tuber yield (t ha–1) 12.31 a 22.93 b 16 tuber p uptake (%) 0.23 a 0.23 a 17 tuber p (g plant–1) 5.46 a 8.81 b 18 r/c ratio 0.85 1.23 note: an0 = farmers’ pattern treatment is 20 t chicken manure ha–1, 1 t npk ha–1, and 250 kg za ha–1 bn1 = bofp pattern treatment is 20 t bofp ha–1, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg calcite ha–1 biotropia vol. 30 no. 2, 2023 236 enrichment of chicken manure with bio p60 led to a significant reduction in the intensity of the fusarium wilt attack on potato plants, thereby increasing their productive age. bio p60 is a secondary metabolite formula derived from the antagonist bacterium p. fluorescens strain p60, which has been tested to treat various plant diseases. furthermore, p. fluorescens produced secondary metabolites that contain several bioactive compounds. it also played an essential role in inhibiting microbial growth in the soil, including fusarium sp., the cause of wilt in potato (soesanto et al. 2011; deveau et al. 2016). p. fluorescens is a ubiquitous soil bacterium that promotes plant health, and some of its strains produce secondary metabolites, such as 2,4– diacetyl phloroglucinol (dapg), phenazines, and hydrogen cyanide, thereby inhibiting soilborne pathogens (neidig et al. 2011). the strain p. fluorescens p60 inhibited fusarium wilt of shallots (santoso et al. 2007) and tomatoes (soesanto et al. 2011), stem rot of peanuts, chili virus, tomato microbe wilt (soesanto et al. 2013), and stem base rot of dragon fruit (hamarawati et al. 2018). the results showed that bofp pattern (n1) increased the soil ph (h2o) from acid to slightly acid and decreased the electrical conductivity compared to the farmers’ practice (n0) (figure 6). furthermore, it significantly increased soil ph (kcl), organic–c, and total–p. the application of the bofp pattern was also shown to improve the quality of andisol by enhancing some chemical characteristics. the application of bofp improved the chemical characteristics of andisols. furthermore, the enrichment of chicken manure with pm and balanced chemical fertilization improved the soil ph, organic-c, and total–p. based on the results, the inoculation of phosphate-solubilizing microorganisms increased soil biomass and microbial activity (wang et al. 2022). pm had been reported to mediate the bioavailability of p by mineralizing organic–p and dissolving the inorganic variant. the soluble p in andisols was affected by inorganic–p solubility, mineralized organic–p, and adsorption–p (tian et al. 2021). phosphate solubilizing microorganisms as a component of the entire soil community can be manipulated to be more effective for plant nutrition (raymond et al. 2021). these microbes can increase phosphate availability through the dissolution of inorganic– p and mineralization of organic–p. the dissolution of inorganic–p was often carried out through the production of organic acids, such as sulfuric acid, nitric, and carbonate. the mineralization of organic–p occurred due to the production of extracellular enzymes, such as phosphatase, phytase, phosphonatase, and c–p lyase by pm. the phosphatase enzyme converts high molecular weight organic phosphate into low molecules by hydrolyzing the bonds with the release of ions (prabhu et al. 2018). figure 6 the effect of the farmers’ pattern (n0) and the bofp pattern (n1) treatments on the andisols variables potato cultivation in slope andisols – tamad et al. 237 pm significantly increased inorganic–p solubility and organic–p mineralization, while significantly reducing adsorption–p in andisols. the ability of pm ability to dissolve p was influenced by the type of species, p compounds, organic acids released, and microbe population. furthermore, its capacity to mineralize organic– p depended on the activity of phosphatase and phytase (tamad et al. 2021). the results showed that pm increased p availability, improved plant growth, and restored soil fertility. phosphate-solubilizing microorganisms have enormous potential as biofertilizers because they can increase the bioavailability of p for plants, offer sustainability, improve soil fertility, and increase crop yields (timofeeva et al. 2022). the pm isolates pseudomonas trivialis, p. putida, and p. fluorescence released citrate, lactate, malonic, oxalic, and acetic acid of 156.25 mg kg–1. the phosphatase and phytase yields obtained from phosphate microbe ranged from 12 to 47 mg po4 3– dm–3 h–1. the amount of p dissolved by pm was higher between 147.66 and 194.61 mg p kg–1 compared to the control (31.06 mg p kg–1). furthermore, pm inoculation produced mineralized organic–p of 63.69 mg p kg–1, compared to the control with 23.7 mg p kg–1 (tamad et al. 2021). it also increased the dissolution of p up to 300% compared to the control (tamad et al. 2020). based on these results, pm can be applied to plants to promote growth or increase p availability, while reducing the dependence on phosphate-based fertilizers and restoring soil fertility (yadav et al. 2021). bofp pattern significantly increased plant height, number of leaves, and age of potato plants, as well as decreased fusarium wilt attack compared to farmers’ pattern, as shown in figure 7. bofp pattern significantly improved tuber quality in terms of diameter, length, and volume, as shown in figure 8. it also significantly increased the number of potato plants and yield per ha compared to the farmers’ practice. the yield obtained in the rainy season from farmers and bofp patterns was 12 t ha–1 and 23 t ha–1, respectively. bofp pattern treatment significantly increased the total p of andisols (figure 6), which supported the uptake of p by potato grown on the soil. the results showed that p uptake in potato tubers was significantly higher in bofp pattern. however, the p content obtained from both patterns was the same, as shown in figure 9. bofp was found to significantly increase potato yield plant–1, as shown in figure 8. figure 7 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on potato variables in andisols biotropia vol. 30 no. 2, 2023 238 figure 8 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on the potato variables in andisols figure 9 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on the potato p variables and the r/c ratio in andisols the application of bofp in andisols improved p_absorption, the quality of tubers, and the yields of the plant. biological organic fertilizer (bof) is a chicken manure enriched with phosphate bacteria, humic–fulvic acid, and n–acyl homoserine lactone to effectively increase p solubility and increase yield. furthermore, potato yield in kaligua, central java, indonesia, using a bof dose of 20 t ha–1 increased tuber yield to 2 t ha–1 compared to chicken manure (tamad et al. 2020). the application of bopf technology to potato cultivation in andisol during the rainy growing season significant;y increased production and farmers' profits. profitability of potato cultivation farming based on the profitability analysis of potato farming, bofp pattern increased the r/c ratio compared to farmers’ pattern, as shown in table 2 and figure 9. based on the results, the total cost of cultivation with bofp pattern was greater, namely idr 5 million ha–1, which was 4% higher than farmers’ pattern. the total revenue for cultivation was 51% greater than farmers’ pattern. bofp potato cultivation provided a profit of idr 31.4 million ha–1, while farmers’ pattern led to a loss of idr 19.5 million ha–1, as shown in table 2. potato cultivation in slope andisols – tamad et al. 239 table 2 the difference in potato farming between farmers’ pattern and bofp pattern on revenues, total cost profits, and efficiency per hectare no. differences farmers’ pattern bofp pattern 1 revenue (idr) 109.030.000 165.096.000 2 total cost (idr) 128.536.667 133.700.667 3 profits (idr)a –19.506.667 31.395.333 4 efficiencyb 0.85 1.23 note: aprofits = revenue – total cost befficiency = revenue / total cost farming efficiency was calculated based on revenues and total expenses (anggraeni et al. 2021). potato cultivation with bofp pattern had better efficiency compared to the farmer’s pattern, as shown in table 2. the r/c value of 1.23 in bofp pattern showed that one unit of the cost used to cultivate potato provided 23% of the profit from the cost. meanwhile, farmers’ pattern showed that farming was inefficient, as indicated by r/c < 1. the results showed that the profits of farmers increased after the application of bofp. the increase in yield of potato cultivated with bofp pattern was shown to exceed the additional cost required. the profit obtained for the use of this pattern also exceeded that of farmers' pattern. during the rainy season, the use of farmers’ pattern led to losses due to rain factors and intensive fusarium wilt disease. the calculation of potato farming efficiency was aimed at comparing the results with the cost of using resources. potato cultivation with bofp pattern can provide better results than farmers’ pattern. the average profit from environmentally friendly farming within a year was also shown to be higher compared to conventional methods (saem et al. 2016). the yield quantity played a significant role in determining the significant gain from potato farming with bofp. the application of biofertilizers significantly correlated with increased outcomes (khoiriyah et al. 2018). conclusion the application of bofp pattern on potato cultivation in andisols decreased fusarium spp wilt attack, improved soil characteristics, enhanced vegetative component, and increased yields from 12.32 to 22.93 t ha–1. during the rainy season, farmers’ pattern caused a loss of idr 19.9 million, while bofp yielded a gain of idr 31.4 million. furthermore, bofp pattern increased the r/c ratio from 0.85 to 1.23, as well as andisols’ productivity, potato production, and farmers’ profits. acknowledgments the authors are grateful to the ministry of education, culture, research, and technology (kemdikbudristek) as well as the educational fund management institution (lembaga pengelola dana pendidikan/lpdp) of 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bogor agricultural university, bogor, indonesia phil dobie storage department, odnri, slough, uk brian m. gerard school of agriculture, faculty of science university of edinburgh, edinburgh, uk abstract ovipositional behaviour, development period, and density effect on adult survival of c. maculatus strains from indonesia, nigeria, and yemen, and c. chinensis strains from indonesia and kenya on cowpea and green gram were studied at 20°c and 70% relative humidity. variations on ovipositional behaviour were found among c. maculatus as well as among c. chinensis strains. variations on developmental period were found only among c. maculatus strains. the developmental period of callosobruchus spp. was shorter on green gram than that on cowpea. density effect was remarkably found only on adult survival of c. maculatus yemen strain. these results make useful contribution to the species biology, and have important implication if strains of these species are accidentally imported to countries, or when new legume crops are introduced. introduction beetles belonging to the family bruchidae are the most important insect pests of stored legumes. infestation by bruchids causes losses of weight, nutritional value and germination potential, and therefore the commercial value of the commodity may be reduced (southgate 1978; dick and credland 1986). the most economically important and widespread bruchids species are the cowpea seed beetle, callosobruchus maculatus (fabricius), and the adzuki bean beetle, c. chinensis (linnaeus) (southgate 1978; tdri 1984). the use of resistant varieties of cultivated legumes is one of the recommended control methods of bruchid infestations. varietal resistance against callosobruchus has been reported in cowpeas and chickpea (dobie 1981; raina 1971; singh 1978). 19 biotropia no. 4, 1990/1991 however, there were variations reported in the response of geographically different strains of c. maculatus to a resistant variety of cowpea (dick 1984; dick and credland 1986). further studies on variation on geographically different strains of c. maculatus revealed the occurrence of differences in their biology and behaviour (credland et al. 1986; credland and dick 1987; credland 1986). this study was made to seek more information on the occurrence of geographical variations among c. maculatus and c. chinensis strains, especially to compare strains from indonesia (asia) with those from other tropical countries. materials and method three strains of c. maculatus viz. strain from the iita cultures, nigeria (labeled as iita), yemen, and indonesia; and two strains of c. chinensis viz. strains from indonesia and kenya were used. they were obtained from the culture of odnri, slough, uk. two types of seeds were used as hosts i.e. californian black eyed cowpea (vigna unguiculata (l.)) and australian green gram (vigna radiata (l.)). daily egg production the surface area of cowpea seeds is larger than that of green gram. as oviposition of the species is assumed to be influenced by the surface area, the cowpea seed number used should therefore be different from that of green gram. after measuring the surface areas of both seeds, it was decided that the surface area of one cowpea seed was approximately equal to the surface area of three green gram seeds. one kernel of cowpea or three kernels of green gram were introduced into a 2.5 cm diameter and 5 cm high glass tube. a pair of adults (age < 1 day) of each strain was added into the tube, and the tubes were covered with foam bungs. twenty five replicates were made on both types of seed and all insect strains. the whole set of experiment was kept in the laboratory at 27°c and the r.h. at 70%. the following day, the number of eggs laid by each female was counted and recorded, and egg-laden seeds were replaced by fresh seeds. the observation and seed replacement were done daily until the 10th day. effect of seed availability on egg production different numbers of seeds i.e. 1 (low), 3 (medium), and 10 (high) kernels of cowpea; and 3 (low), 9 (medium), and 30 (high) kernels of green gram were introduced into the tubes. one pair of adults (age < 1 day) of each strain was placed 20 strain differences in two species of callosobruchus-th. roesli, p. dobie & b.m. gerard in each tube and the tube was covered with foam bungs. twenty replicates were made of all treatments and all insect strains, and kept in the experimental room. observation on the number of eggs laid by each female was done on the 7th day after treatment. developmental period eight to 10 adults of each insect strain were introduced into each petri dish containing cowpea or green gram seeds. the dishes were covered with the lids and kept in the experimental room for one night the adults were removed the following day. under a binocular microscope, seeds bearing a single egg were taken out and individually put into tubes. the tubes were covered with foam bungs and kept in the experimental room. twenty five replicates were made of all insect strains. observation on adult emergence were started on the 20th day after treatment. emergence of fresh adults was recorded daily until no more adults emerged. adult survival from seeds bearing different number of eggs to obtain cowpea seeds bearing 1, 2, 3, 4, and 5 eggs, the same procedure as in the developmental period experiment was applied. however, selection was done not only of seeds bearing 1 egg, but of those bearing 2, 3, 4, and 5 eggs as well. the replicate number of each treatment was designed to be inversely proportional to the egg density (giga 1982). thus, the number of eggs used were approximately the same in all treatments (table 1). table 1. the design of the experiment on adult survival from seed bearing different number of eggs no. of eggs/seed 1 2 3 4 5 no. of replicates 20 10 7 5 4 no. of eggs 20 20 21 20 20 emergence of adults was observed and recorded daily starting from the 20th day after treatment. result daily egg production some females were found to lay eggs on the seeds and on the tube walls and so both were recorded and analyzed separately. 21 biotropia no. 4, 1990/1991 the general pattern of daily egg production of all strains on seed was the same. high number of eggs were laid during the early period of the female's life. the maximum number was reached either on the second or third day, and then it gradually decreased (fig. 1). the sum of 10-day egg production per female of all strains was analyzed by two factors analysis of variance. oviposition on seed, on tube wall, or the total, over the period of 10 days was influenced by insect strain (table 2). c. maculatus iita strain was found to have the highest fecundity followed by c. chinensis indonesian, c. maculatus yemen, c. chinensis kenyan, and c. maculatus indonesian strain (table 2, column "total"). differences in the fecundity were found between each strain of c. maculatus, as well as between each strain of c. chinensis (table 2 and fig. 2). c. chinensis indonesian strain laid the highest number of eggs on seed, followed by c. maculatus iita strain. lower numbers were laid by c. maculatus yemen and c. chinensis kenyan strains with the lowest number laid by c. maculatus indonesian strain (table 2). large number of eggs on the tube wall were especially laid by c. maculatus iita and yemen strains. the rest of the strains laid very small number of eggs on the tube. the mean egg number was not significantly different from one another, but they differ significantly from those laid by c. maculatus iita and yemen strains. seed species did not affect oviposition of all strains over 10 days, either on seed, on tube wall or the total (table 3). table 2. means and standard errors of egg product ion of five strains of callosobruchus maculatus over 10 days on seed, on tube wall, and the total insect strains on seed on tube wall total cm* iita 63 .90 a ±4.57 17.02 a ±2.55 80.92 a ±4.98 cm yemen 45.90 b ±4.11 6.06 b ±1.35 51.96b ±4.67 cm indonesia 32.08 c ±2.08 0.50 c ± 0.30 32.55 c ±2.15 cc* indonesia 68.24 a ±1.86 0.47 c ±0.25 68.74 a ±1.91 cc kenya 38.33 bc ±3.20 0.02 c ± 0.02 38. 35 be ±3.20 a, b and c indicate level of significance at 5%; relevant only down the column. * cm for callosobruchus maculatus cc for callosobruchus chinensis. table 3. means and standard errors of insect oviposition over 10 days on seeds, on tube wall, and the total seed species on seed on tube wall total cowpea 48.91 ns ± 2.56 4.89 ns ± 0.96 53.80 ns ± 2.86 green gram 51.08 ns ± 2.38 4.98 ns ± 1.09 56.06 ns ± 2.77 ns : non significant. 22 strain differences in two speciess of callosobruchus – r. roesli, p. dobie & b.m. gerard figure 1. oviposition over 10 days of c. maculatus iita (1), yemen (2), indonesia (3) and c. chinensis on cowpea (cp) and green gram (gg) 23 biotropia no. 4, 1990/1991 figure 2. effect of seed availability on the fecundities of c. maculatus iita (1), yemen (2), indonesia (3) and c. chinensis on cowpea (cp) and green gram (gg). 24 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard effect of seed availability on egg production some females were also found to lay eggs on the tube wall. eggs laid on seeds and on tube walls were analyzed separately. tables 4, 5, and 6 show that the oviposition of callosobruchus over 7 days was influenced by seed availability and insect strain, but not by the seed species. the total number of eggs increased significantly with the increase of seed availability. c. maculatus iita produced the highest total number of eggs, followed by c. chinensis indonesia. lower number was produced by c. chinensis kenya, and the lowest number was produced by c. maculatus yemen and indonesian strains. the number of eggs laid on the seed increased significantly with the increase of seed availability (table 4). in contrast, the number of eggs laid on the tube wall decreased with the increase of seed availability (table 4), however the number of eggs laid on tubes with low seed number did not significantly differ from that laid on tube with medium number of seeds. table 4. mean and standard errors of callosobruchus spp. oviposition on seed, on tube wall, and the total at different seed availabilities seed number on seed on tube wall total low medium high 35.42 c±1.50 50.22 b±1.56 59.88 a ±1.46 8.55 b±1.14 .31 b±1.03 3. 63 a ±0.62 43.96 c± 1.78 57.51 b±1.81 63.51 a±1.54 a, b, and c indicate level of significance at 5%; relevant only down the column. table 5. mean and the standard errors of five strains of callosobruchus spp. ovipositions on seed, on tube wall and the total insect strains on seed on tube wall total cm* iita 58.98 a ±2.52 18.68 a ±1.85 77.64 a ±2.39 cm yemen 36.54 c ± 1.85 11.50 b ± 1.36 48.04 c ± 2.21 cm indonesia 34.10 c ± 1.48 1.21 c ±0.24 35. 29 d ± 1.48 cc* indonesia 63 .05 a ±1.92 0.84 c ± 0.32 63. 89 b ± 1.90 cc kenya 49.85 b ±1.31 0.24 c ± 0.07 50.09 c ± 1.33 a, b, c, and d indicate level of significance at 5%; relevant only down the column. * cm : c. maculatus cc : c. chinensis table 6. mean and standard errors of callosobruchus spp. oviposition on seed, on tube wall, and the total of two kinds of seed. seed kind on seed on tube wall total cowpea 49.29 ns ± 1.43 4 . 39 b ± 0.56 53 . .67 ns ± 1.51 green gram 47.72 ns ± 1.30 8 . .60 a ± 0.95 56 . .32 ns ± 1.45 a and b indicate the level of significance at 5%; relevant only down the column. 25 biotropia no. 4, 1990/1991 c. maculatus iita laid the highest number of eggs on tube wall followed by c. maculatus yemen. the rest of the three strains laid only a small number of eggs on tube wall and they did not differ significantly (table 5). the number of eggs laid on the tube containing green gram was higher than that on tube containing cowpea (table 6). the effect of seed availability on egg production of individual strains on cowpea and green gram is shown in figure 2. the effect of seed availability is remarkably seen in c. maculatus yemen and indonesian strains. development period the results indicated that there were differences in the development rate among the insect strains (table 7). the development period of c. maculatus iita strain was the longest, and was significantly longer than the others. development periods of c. maculatus yemen and indonesian strains did not differ significantly. the difference in the development period of the two strains of c. chinensis was not significant, however they were significantly shorter than the development periods of all c. maculatus strains (table 7). seed species significantly influenced c. maculatus and c. chinensis development periods. most of the insect strains developed faster on green gram than on cowpea, except the c. maculatus strain from yemen (table 8). table 7. mean and the standard errors of development periods of 5 callosobruchus strains table 8. mean and standard errors of development period of callosobruchus on cowpea and green gram a and b indicate the level of significance at 5%. 26 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard adult survival from seeds bearing different number of eggs the results indicated that egg density, from 1 to 5 eggs per seed, did not influence significantly the number of adult survival of most strains, except on c. maculatus yemen strain (fig. 3). remarkable decreases were noted on adult survival means of seeds bearing 4 and 5 eggs. figure 3. adult survival from seeds bearing different number of eggs of 5 different callosobruchus strains. discussion some females, especially those of c. maculatus females, were found to lay eggs on tube surface. however, there seemed to be less preference for oviposition on tube wall because the number of eggs laid on the tube wall was usually large only when there was a shortage of seeds for oviposition, such as during the peak days of the ovipositional period (fig. 1), or when there were few seeds for oviposition (fig. 2). females of callosobruchus spp. have been reported to control the successful development of their progenies by choosing an appropriate site for oviposition and distributing their eggs more or less equally over the available seeds. they avoid laying eggs on seeds with a rough surface and on seeds already bearing bruchid eggs if noninfested seeds are still available. if noninfested seeds are unavailable, the 27 biotropia no. 4, 1990/1991 females will lay eggs on seeds already bearing eggs, but will choose the bigger ones first. the females can also detect small differences in egg density, and prefer to oviposit on seeds bearing smaller number of eggs (yoshida 1961; avidov et al. 1965; nwanze et al. 1975; messina and renwick 1985a, b). the discriminating ability of female callosobruchus in uniformly distributing their eggs is highly developed, however, in some species the discrimination in choice of seeds suitable for larval growth is not developed. in other words, the ovipositional behaviour was not related to the suitability of seeds for the development of larvae (avidov et al. 1965; bhattacharya et al. 1977). therefore, the apparent preference for oviposition on the tube wall under these conditions could possibly be more accurately described as a strong repellence to ovipositing on seeds that are already bearing numerous eggs. it is difficult to say if the differences in the number of eggs laid on the tube wall by different strains of c. maculatus were due to differences in the ability of females of different strains to discriminate between suitable sites for larval development or simply because they have different fecundities. for example, the fecundity of c. maculatus iita female was the highest, and that strain female also laid the highest number of eggs on the tube wall. probably the female of that strain had relatively greater seed shortage problem than the other two strains. in contrast, c. chinensis appears to have more developed discriminating ability to choose suitable sites for larval development. it was found that although females of the c. chinensis indonesia had higher fecundity than those of the c. maculatus yemen and indonesian strains, fewer eggs were laid on the tube by c. chinensis indonesia. the number of eggs oviposited by females c. maculatus indonesian and yemen strain was suppressed when only a small number of seed was available. however, those oviposited by the other strains were not remarkably suppressed with the reduction of seed availability. credland (1986) stated that the conditions that determine the maximum fecundity differ within and between strains. the reduction in female fecundity as a response to low seed availability is perhaps due to deterrence effects, chemically or physically, of eggs already laid on the seed (messina and renwick 1985a). the development period of callosobruchus, in this experiment was slightly shorter on green gram than that on cowpea. this suggests that green gram is a slightly better host for callosobruchus. giga and smith (1978) reported that of the several pulses tested, including green gram and cowpea, green gram was the most favourable food species for oviposition, speed of development and survival of c. maculatus. when suitable host seeds are infested at numbers above the population's optimum density, there is a reduction in the number emerging of adults due to mortality which primarily took place during the larval stage (utida 1941; mitchel 28 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard 1975; giga 1982; dick 1984). the reduction in the number of adults produced due to high density was more pronounced in the c. maculatus yemen strain than those of other strains used in this experiment. variations in the effect of density on the number of adults produced have also been reported to occur among strains of c. maculatus. strains from brazil and nigeria (iita) can produce more than ten adults from a seed with numerous eggs, whereas a strain from yemen rarely produced more than three (dick 1984; dick and credland 1984; credland et al. 1986). the density effect on the number of adults observed in c. maculatus indonesian strain, c. chinensis indonesian and kenyan strains seems to be similar to that in c. maculatus iita. however, higher densities than the maximum density recorded in this experiment should have been used to be able to see the effect more clearly. the geographical variations on the biology and behaviour of c. maculatus and c. chinensis found in this experiment are possibly the result of either genetic evolution of a population which occupies a particular environment and is therefore subjected to that environment selection pressures; or genetic divergence among populations which is caused by chance fluctuations in its allele frequency; or the change in the gene pool (dick 1984; credland 1986). the occurrence of geographical variations among populations of an insect species should be noted when studying or referring to the species biology or behavioral characteristics. attention should also be given to the possibility that a less important species population might become a serious pest if a better plant host species or variety were introduced to the area. acknowledgement the authors would like to thank dr. c.p. haines for his assistance and advice, mr. d.j.b. calverley, head of storage department, odnri, slough, uk. for providing laboratory space and facilities, and the british council for the research funds. reference avidov, z., s.w. applebaum, and m.j. berlinger, 1965. physiological aspects of host specificity in the bruchidae ii: ovipositional preference and behavior of callosobruchus chinensis (l.). ent. exp. & appl. 8: 96-106. bhatacharya, a.k., p.k. pathak, and s.p. shah, 1977. oviposition and development of callosobruchus chinensis (linn) (coleoptera : bruchidae) on several host species. bull. grain technol. 15: 38-41. 29 biotropia no. 4, 1990/1991 credland, p.p. 1986. effect of host availability on reproductive performance in callosobruchus maculatus (f) (coleoptera : bruchidae). j. stored prod. res. 22: 49-54. credland, p.p. and k.m. dick. 1987. food consumption by larvae of three strains of callosobruchus maculatus coleoptera : bruchidae). j. stored prod. res. 23: 31-40. dick, k.m. 1984. bionomic variation among populations of southern cowpea weevil, callosobruchus maculatus and their responses to different varieties of the primary host. ph.d. thesis, bedford college. 305p. dick, k.m. and p.f. credland. 1984. egg production and development of three strains of callosobruchus maculatus (f.) coleoptera : bruchidae). j. stored prod. res. 20: 221-227. dick, k.m. and p.f. credland. 1986. variation in the response of callosobruchus maculatus (f.) to a resistant variety of cowpea. j. stored prod. res. 22: 43-48. dobie, p. 1981. the use of resistant varieties of cowpea (vigna unguiculata) to reduce losses due to post harvest attack by callosobruchus maculatus. in: the ecology of bruchids attacking legumes (pulses): 185-192. (ed. v. labeyrie). dr. w. junk publisher. london. giga, d.p. 1982. the comparative biology of four callosobruchus species with particular reference to competition in c. rhodesianus and c. maculatus. ph.d. thesis, university of reading. giga, d.p. and r.h. smith, 1987. egg production and development of callosobruchus rhodesianus pic and callosobruchus maculatus (f.) (coleoptera : bruchidae) on several commodities at two different temperatures. j. stored prod. res. 23: 9-15. messina, f.j. and j.a.a. renwick, 1985a. ability of oviposition seed beetles to discriminate seeds with differing egg loads. ecological entomology 10: 225-230. messina, f.j. and j.a.a. renwick, 1985b. mechanism of egg recognition by cowpea weevil callosobruchus maculatus. entomol. exp. appl. 37: 241-245. mitchel, r. 1975. the evolution of oviposition tactics in the bean weevil. callosobruchus maculatus (f.). ecology, 56: 696-702. raina, a.k. 1971. comparative resistance to three species of callosobruchus in a strain of chickpea (cicer arietinum l.). j. stored prod. res. 7: 213-216. singh, s.r. 1978. resistance to pests of cowpea in nigeria. in: pests of grain legumes: ecology and control: 267-279. (ed. singh et al.). academic press. london. southgate, b. j. 1978. the importance of bruchidae as pests of grain legumes, their distribution and control. in: pests of grain legumes: ecology and control: 219-229. (ed. singh et al.). academic press. london. tdri. 1984. insect and arachnid of tropical stored products : their biology and identification (atraining manual). tropical development and research institute. slough. utida, s. 1941. studies on experimental population of the azuki bean weevil callosobruchus chinensis (l.) i: the effect of population density on the progeny population. mem. coll. agr. kyoto imp. univ. 48: 1-31. yoshida, t. 1961. oviposition behaviour of two species of bean weevils and interspecific competition between them. mem. fac. lib. arts and educ., miyazaki univ. 11: 41-65. 30 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf microsoft word 99 biotropia vol. 13 no. 2, 2006 : 99 110 alimentary canal anatomy and histology of the worker termite neotermes bosei leksono ekopuranto hariprabowo, rika raffiudin and taruni sri prawasti department of biology, faculty of mathematics and sciences, bogor agricultural university, jalan ray a pajajaran, bogor, 16144 indonesia abstract as social insects, termites live in a colony that consist of reproductive (drone and queen), and non-reproductive (soldiers and workers) castes. workers obtain their food directly from wood, humus, and other substances that contain cellulose. the objective of this study was to examine the alimentary canal of the neotermes bosei workers. observations of gut transverse section were carried out through the length, perimeter, and area of each alimentary canal region. the results showed that total length of n. bosei alimentary canal was 13.71+1.28 mm. the canal was divided into fore-, mid, and hindgut which were 24, 28, and 48%, respectively of the gut total length. two types of alimentary canal epithelial cells were found, i.e. the squamous and transitional cells. areas covered with thick muscular tissues were crop, proventriculus, and rectum. proventriculus was characterized with six large dentitions. there was no gastric caeca in n. bosei midgut, which commonly occurred in chewing insect. secretory cells .were observed at proventriculus and ventriculus regions. cardiac valve was found at the anterior end of ventriculus. area with the largest outer perimeter was the rectum pouch. enteric valve had three internal folds. key words: drywood termites, alimentary canal, histology, kalotermitidae introduction termites were classified into the order of isoptera, which consist of six families i.e. mastotermitidae, kalotermitidae, hodotermitidae, rhinotermitidae, serritermitidae, and termitidae (krishna 1969). as social insects, termites live in a colony. each colony comprises reproductive (drone and queen) and non-reproductive (worker and soldier) castes. worker termites obtain their food directly from wood, humus and other materials that contain cellulose. soldier castes obtain the food from the workers through trophalaxis (stuart 1969). the ability of termites, particularly of the members of kalotermitidae to digest the cellulose is due to the mutualistic symbiosis with the flagellate protist (claveland 1925). hence, the food digestion process in termites is different with that of most other insects (tokuda et al. 2000; inoue etal. 2000). the alimentary canal is divisible into stomodeum (foregut), mesenteron (midgut) and proctodeum (hindgut). digestion process occurred in the midgut (dow 1986; inoue et al. 2000), whereas hindgut is the most important part in food absorptions (inoue et al. 2000). neotermes bosei (figure 1) is classified into the family of kalotermitidae (ahmad 1965). n. bosei is known as damp wood termites, because it lives in the dry and living wood (scheffrahn & su 1994). this species was selected for this current corresponding address : rika rafiudin.net.id 99 biotropia vol. 13 no. 2, 2006 termite histology study due to lack of information on kalotermitidae alimentary canal . the objective of this study is to examine the anatomy and histology of the termite n. bosei worker alimentary canal. materials and method neotermes bosei collection the n. bosei soldiers and workers imagoes were collected from the fishing shop in several traditional markets in bogor, west java (i.e. pasar anyar, pasar bogor, pasar ciawi, and pasar empang). the soldiers of n. bosei were needed for identification purpose up to species level. observation of n. bosei alimentary canal length of alimentary canal ten n. bosei alimentary canal were taken by dissectioning the lateral part of termite thorax and abdomen. termite alimentary canal was observed under stereo microscope (nikon fdx-35) and photographs were taken by using nikon smz1000 camera. the photographs were digitized for analysing canal total length (crop up to anus), the length of fore-, mid-, and hindgut. the measurements were carried out with imagej sofware (http://rsb.info. nih.gov/ii). diameter and the cell structure ofn. bosei alimentary canal six series of serial transversal section of the canals were made through paraffin embedding method (thickness of 6 um). the sections were stained by using double staining of haematoxylineosin (he). photographs were taken with compound microscope (olympus ch20) and olympus dp 12 camera. inner and outer peripheri 100 alimentary canal anatomy and histology l. e. hariprabowo et al. (ip and op), lumen area (la), and gut area (ga) were measured by imagej sofware (http://rsb.info. nih.gov/ij). embedding block with paraffin method (gray 1952) neotermes bosei alimentary canals were fixed with bouin fixative for two hours. then, they were washed and dehydrated in series of ethanol i.e. 70, 80, 90 %, and absolute ethanol, 15 min for each step. for slide clearing it was immersed in xylol for 1.5 hours. infiltration paraffin step was performed for three times, each step in 45 min and subsequently stained with he. results and discussion foregut the tv. bosei foregut was a slender canal. the mean of length from the crop up to proventriculus is 3.32 + 0.57 mm (figure 2). figure 2. the alimentary canal of n. bosei worker termite oesophagus oesophagus was the canal that connected termite's pharynx and its crop. this region consisted of several layers, i.e. intima, squamous cells (at the lumen wall), columnar cell (at vili), and thin muscle layer (figure 3). the ip, op, la, and ga values were listed in table 1. figure 3. oesophagus transversal section of n. bosei worker termite 101 biotropia vol. 13 no. 2,2006 crop neotermes bosei worker crop was divisible into three parts, i.e. the anterior, median, and posterior regions (figure 4). the anterior crop had six large vili which covered almost the entire lumen. this region had intima layer, columnar epithelial cells (at the vili) and squamous epithelial cells (at the lumen wall), circular and longitudinal muscle cells. mean of the ip, op, la and ga were listed in table 1. figure 4. crop transversal section of n. bosei worker termite (a) anterior, (b) median, (c) posterior region, (d) cell layers of the posterior crop note: oeso= oesofagus; crl= anterior crop; cr2= median crop; cr3= posterior crop; pvl= anterior proventriculus; pv2= posterior proventriculus; vl= anterior ventriculus; v2= posterior ventriculus; pl= short channel; p2= enteric valve; p3= rectum pouch; p4= colon; p5= rectum; = data not available 102 alimentary canal anatomy and histology l. e. hariprabowo et al. large number of vili covered the median region of the crops compared to that in the anterior region. squamous epithelial cells in the median crop were shorter than those in the anterior (figure 4b). the ip, op, la and ga values of median crop regions were higher than those in the anterior (table 1). the posterior crop region contained the thickest muscle, intima layer, squamous epithelial cells, circular and longitudinal muscle layers (figure 4c and 4d). proventriculus proventriculus was the last part of the foregut (figure 2) consisted of anterior and the posterior regions (figure 5a,b). four dentitions were observed at the lumen wall at the anterior proventriculus region (figure 5a), whereas six dentitions were found in the posterior region (figure 5b). figure 5. proventriculus transversal section of .m bosei worker termite (a) anterior region, (b) posterior region, (c) cell layers composed the anterior region, (d) layers composition of the posterior region; 1,2,3,4 = dentition type in the anterior proventriculus proventriculus ofn. bosei worker was covered by a thick intima, transitional epithelial cells, and a thick muscle layer (figure 5c,d). several secretory cells (figure 5d) were also found in this region. they were characterized by a cluster of larger cells compared to the other epithelial cells surrounding them. anterior and posterior proventriculus measurements were shown in table 1. as a chewing insect n. bosei worker has proventriculus canal (miller 1965). the proventriculus occurred in the chewing insect such as moth larva hofmannophila pseudospretella (lepidoptera: oecophoridae) (gerard 2002) and beetle dendroctonus (coleoptera: scolytidae) (diaz et al. 2003). the intima layer in the proventriculus acts as the food grinder (wigglesworth 1972). no proventriculus 103 biotropia vol. 13 no. 2,2006 occurs in the haustellate insect such as in the fly bactrocera dorsalis (diptera: tephritidae) (lee et al. 1998). midgut ventriculus the length ofn. bosei worker midgut (figure 2) was 3.86+0.61 mm, composed of only a region, the ventriculus. it was characterized by the occurrence of transitional epithelial cells and thin muscle cells. this was congruent with the result of noirot and noirot-timothee (1969) that ventriculus has one cell type, the transitional epithelial cell, as the absorption and secretion cells. however, nasutitermes takasagoensis worker ventriculus consisted of columnar cells (tokuda et al. 2001). a variety of cell occurs in the dendroctomts (coleoptera: scolytidae) ventriculus as well (diaz et al. 2003). the anterior ventriculus had a cardiac valve (figure 6a), which was an imagination structure at the foregut towards the midgut. this structure was not found in the posterior ventriculus (figure 6b). the cardiac valve covered the constricted region, hence it had low ip, op, la and ga values (table 1). antiperistaltic movement was prevented by the cardiac valve, a constriction structure in the anterior ventriculus as shown in n. bosei. it prevented the food at the midgut to flow back to the foregut (snodgrass 1935). this character was also reported in n. takasagoensis (termitidae) (tokuda et al. 2001). hence, in termites and most other insects, the digested food always moves f rom anterior towards the posterior region of the alimentary canal. there is no gastric caeca in the midgut of n. bosei worker; this structure is the same in n. takasagoensis worker (tokuda et al. 2001). in most chewing insects, gastric caeca lays at the anterior region of the midgut (romoser 1973) to enlarge the food absorption (wigglesworth 1972). the absence of gastric caeca in worker termites showed an adaptation of termite digestion system as well. termites do not 104 alimentary canal anatomy and histologyl. e. hariprabowo et al need an additional space in the midgut for food absorption because the cellulose will be degraded subsequently in the hindgut, particularly in the rectum pouch. hindgut the length ofn. bosei worker hindgut was 6.56+0.91 mm, composed of intima layer, columnar, transitional epithelial cells, and muscle cells. this alimentary canal region consisted of five parts, i.e. short channel that connected the midand the hindgut, enteric valve, rectum pouch, colon and the rectum. short channel this region could not be determined in the fresh alimentary canal, unless a histology preparation was made (figure 7a, b). this is due to the similar diameter and the same color with that of ventriculus region. short channel was covered with columnar epithelial cells and muscle layers (figure 7b). intima layer was not found in this region. the mean value of ip, op, la, and ga of these regions were shown in table 1. enteric valve enteric valve was the second region in the hindgut constructed of three longitudinal folds facing towards the lumen, performed a clep structure (figure 8a). it composed of intima layer, columnar epithelial cells, and muscle cells (figure 8b). mean of enteric valve op was 0.530+0,039 mm (table 1). rectum pouch the third part of the hindgut was the rectum pouch (figure 9a), consisted of intima layer, transitional epithelial cells, and thin layer of muscle cells (figure 9b,c). rectum pouch had the largest op value compared to others, that was 3.859+1.807 mm. 105 biotropia vol. 13 no. 2,2006 colon colon was the fourth part in the hindgut region (figure 10a,b), that composed of the same layers as shown in the rectum pouch. the op value of n. bosei worker colon was 1.508+0.370 mm. rectum rectum was the last part in the hindgut region (figure 11) with a thick muscle layer. other layers were the same as shown in the rectum pouch. there was no distinct region which separated the circular and longitudinal muscle layer, hence both were linked (figure lib). the op value of n. bosei worker rectum was 2.036+0.569 mm, it was the largest op diameter (see also figure 2). this big pouch in termite hindgut is an important region as mentioned by inoue et al. (2000), the hindgut harbours symbiotic flagellates which assist in cellulose digestion. cellulose is a polimer of 5000 -10 000 anhidrid glucosa connected with p(l,4) glycoside (feighl and hill 1983). most organism could not use p(l,4) glycoside as the energy source. those flagellates produce cellulase enzyme that degrade the cellulose into acetic acid, co2, and h2. hence, the enlargement of the termite hindgut is an adaptation mechanism of its behaviour and physiology. another difference of n. bosei worker to other termites is that it did not have a mixed segment. mixed segment has a variety of histologic structures, located between the midand the hindgut. it is reported to exist in the termitidae as an important character for identification (bignell 1994). in addition, the anatomy of n. bosei worker alimentary canal showed several differences compared to other insect alimentary canals. the hindgut of n. bosei worker was the longest region, which comprised 48% of the total length, whereas 24 and 28% for foregut and midgut, respectively. in most insects, the midgut is the longest part (wigglesworth 1972) as shown in fruit fly bactrocera dorsalis 106   biotropia vol. 13 no. 2,2006 (diptera: tephritidae) (lee et al. 1998; hung et al. 2000) and moth larva, hofmannophila pseudopretella (lepidoptera: oecophoridae) (gerard 2002). in dendroctonus (coleoptera: scolytidae) no difference in length among the fore-, mid-, and the hindgut were found (diaz et al. 2003). high variety value was observed in the outer and inner perimeter ratio (op/ip). in most measurements, ip was higher than op, because large vili were facing to the lumen. however, at the crop, ip was lower than op; this was due to the small vili size at the crop. ip was lower than op which also occurred at the anterior ventriculus as well. this is due to the existence of cardiac valve. muscle thickness in n. bosei worker alimentary canal was varied as well. thin muscle was found in oesophagus, ventriculus, and rectum pouch. this character showed that mechanic digestion was not a dominant process in those regions. thin muscle in oesofagus helps the peristaltic movement to bring the food towards the crop (snodgrass 1935; elzinga 1978). in ventriculus there was no thick muscle which means that the cells did not digest the food mechanically. on the other hand, food had been mechanically digested in the proventriculus. 108 alimentary canal anatomy and histology l. e. hariprabowo et al. the thick muscle layer occurred in the crop, proventriculus, and rectum. food were mixed with saliva and stored in the crop (wigglesworth 1972). thus, the thick muscle layers in the crop were needed to enhance the muscle contraction to bring the food forward. in the proventriculus, the thick muscle layer together with six dentitions in the proventriculus ground the food. moreover, the function of the thick layer in the rectum was to draw the feces out of the colon. in future studies, we need to collect more information about the anatomy and histology of other termite caste ofn. bosei alimentary canal, such as the soldier and reproductive castes. then, one can make a comparative study among those termite castes and determine the conserve canal structure. one can also examines the plasticity of the structure(s), if any, due to the different behaviour and physiology of each termite caste. for example, soldiers termites obtain their food from the workers through trophalaxis. based on the different types of food, do the crop of the soldier termites have the same histology structure as those of the workers? this basic histology study will be fruitful as well for a further researches such as to examine the influence of several pesticide at the digestive canal of worker termites. one can see whether the pesticide is able to change the cells structure and intima layer covered the worker termites gut. conclusions the total length of n. bosei alimentary canal was 13.71+1.28 mm. the canal was divided 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1972. the principles of insect physiology. london: chapman and hall. wilson e.o. 1965. chemical communication in the social insects. in: wilson eo. 1974. the insect societies. cambridge: the belknap univ pr. wilson e.o. 1974. the insect societies. cambridge: the belknap univ pr. 110 99.pdf 100.pdf 101.pdf 102.pdf 103.pdf 104.pdf 105.pdf 106.pdf 107.pdf 108.pdf 109.pdf 110.pdf microsoft word 57 biotropia no. 19, 2002 : 57 64 molecular identification of egg parasitoid, telenomus spp. (hymenoptera: scelionidae) from several locations in java using rapd -pcr netti yuliarti1, purnama hidayat 2, and damayanti buchori3 'agricultural polytechnic, andalas university, payakumbuh, west sumatera, indonesia mj department of plant pests and diseasestfaculty of agriculture, bogor agricultural university, bogor, indonesia abstract random amplified polymorphic dna (rapd) amplified by the polymerase chain reaction (pcr) was used to determine the differences of four telenomus species and five populations of t. rowani from several locations in java. amplification of genomic dna by using primer p2 (amersham pharmacia biotech) indicated that each telenomus species had a unique set of rapd bands. two bands which characterized the genus are estimated to be 300 and 430 bp. each species had three specific bright bands except t. dignoides which only had two specific bright bands. however, no bands are unique to any of the five populations of t. rowani and all of the bands are less than 500 base-pair. cluster analysis using upgma (unweighed pair group method with arithmatic mean) showed that the four telenomus species consist of two groups, t. rowani and t. remus in one cluster and t. dignus with t. dignoides belonging to another cluster. key words : pcr-rapd / clustering / telenomus spp. introduction telenomus (hymenoptera: scelionidae) are minute wasps, often black, about 1 mm in length, and feed exclusively on the eggs of other insects. the genus particularly attacks lepidoptera and hemiptera but they are also known to attack diptera and neuroptera (johnson 1984). the host range is relatively broad but the degree of host-specificity varies between species (polaszek & kimani 1990). telenomus often plays an important role in the natural control of insect pest populations. some biological control programs are presently being carried out on pyralid populations using egg parasitoids of the genus telenomus with good results. in the ivory coast, for instance, these parasitoids are used to control eldana saccharina wlk., maliarpha separatella rag., and scirpophaga melanoclysta mey., on sugar-cane, rice and maize; in india against chilo sacchariphagus (boyer) on sugarcane and in bolivia against diatraea rufescens box and d. saccharalis (f.) on sugarcane. the biology of these egg parasitoids is even more interesting and has a potential for preventing an outbreak of the pest population. some of them are phoretic upon the adults of their host (bin & johnson 1982). more than 500 species of telenomus are currently known. however, biological studies about the genus are few in number when compared to trichogramma (polaszek & kimani 1990; honda & trjapitzin 1995). there are a number of 57 biotropia no. 19,2002 unresolved taxonomic problems at species-level in this genus because of its small size and general morphological uniformity (nixon 1937 and polaszek 2001). the taxonomy of the genus has been based on morphological traits. however, these characters may display a great level .of plasticity and do not always reflect the phylogenetic relationships among species. molecular techniques provide efficient tools for the study of natural population genetics (hoy 1994). random amplified polymorphic dna polymerase chain reaction (rapdpcr) has been used to survey mosquito species and population (kambhampati et al. 1992) and population genetics of parasitoid wasp diaeretiella rapae (hymenoptera: braconidae) (vaughn & antolin 1998). previously, landry et al. (1993); masutti (1994) and meilin (1999) used rapd technique for tricho-gramma species identification. rapd-pcr is particularly valuable for genomic mapping in species for which little genetic information is available (hoy 1994) such as the genus telenomus. so far, no study has been done on the differentiation between and within telenomus species in indonesia. we employed molecular technique to see species differences and to see whether there are genetical variations within the same species from different locations. our objective was to differentiate four species of telenomus and five populations of t. rowani collected from several locations in java using the rapd technique. materials and methods sample collection and identification egg parasitoids of telenomus spp. were collected from various crops, e.g soybean, sugarcane and paddy in west java, central java and east java. parasitized lepidoptera and hemiptera eggs were collected and brought to the laboratory for subsequent parasitoid development. identification of parasitoid was based on nixon (1937), nishida and torii (1970) dna extraction dna was extracted using a technique described by goodwin et al. (1994) with the following modifications. one to three wasps of each telenomus species and five populations of t. rowani were crushed with a glass rod in 125 ul ctab (2 %). dna preparations were stored at 4 °c in 30 ul distilled water. pcr amplification pcr was performed using gene amp r pcr system 9700 (pe applied biosystem). ready to go ™ rapd bead (pharmacia biotech) was used for each pcr reaction. each of 25 ul reaction mixture contained a rapd bead, 5 ul of genomic dna, 5 ul of 25 pmol primer p2 (5' -d[gtttcgctcc]-3' ) and 15 ul 58 molecular identification of egg parasitoid, telenomus spp netti yuliarti et al. distilled water. pcr condition for the integron was amplified in 45 thermal cycles at 95 °c for 1 minute, 36 °c for 1 minute and 72 °c for 3 minutes. the pcr products were then held at 4 °c until recovered. dna visualization the dna fragment was separated using an agarose gel. agarose gel of 1.5 g was prepared in 100 ml of 1 x tbe (90 mm tris-borate buffer, 2 mm edta ph 8.0) and then heated until boiling point using microwave. electrophoresis eight ul of pcr product was mixed with 2.0 ul of gel-loading buffer (30% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol) and electrophoresed on 1.5% agarose gel (tbe) at 100 volt for about 2 hours. dna bands were stained with ethidium bromide and photographed with fast polaroid film using transluminator uv light. dna band size was determined by comparing with a 100 base-pair dna ladder run on gel. data analysis the data matrix was constructed with each line in the gel containing the identified species and rapd-pcr bands were scored 1 if it is present and 0 if absent. a cluster analysis was carried out using the unweighed pair group method with arithmetic means (upgma clustering) of similarity coefficient for all pairs of species and population. the phenogram was generated using a computer-based taxonomy program numerical taxonomy system, (ntsys-pc version 2.1) (rohlf 2000). results and discussion based on morphological characters, four telenomus species have been identified, t. rowani, t. remits, t. dignus and t. dignoides (yuliarti 2002). we have screened three primers (pharmacia biotech) as follows: pi (5'-d[ggtgcgggaa]-3'), p2 (5'-d[gtttcgctcc]-3') and p3 (5'-d[gtaga.cccgt]-3-). the results showed that primer p2 gave the best polymorphic bands. the results of genomic dna amplification using primer p2 (5' d[gtttcgctcc]-3') indicated that the four telenomus species can be genetically differentiated (fig. 1). each species of telenomus has a unique set of rapd bands. several clear and well-amplified bands were used for analysis. the result of the amplification showed that the size range of the bands are between 200 base-pair (bp) and 1500 bp. two bands approximately 300 and 430 bp seem to indicate the specificity of the genus telenomus. these two bands are monomorphic bands (fig. 1). in contrast, the polymorphic bands which indicate the specificity of each species are shown by different band size as seen in fig.l. 59 biotropia no. 19,2002 fig. 1 shows that each telenomus species has three specific bands except t. dignoides which has only two specific bands. specific bands of t. rowani are estimated to be 650, 850 and 1100 bp, whereas t. remus are 600, 850 and 1150 bp. unique bands of t. dignoides are estimated to be 700 and 1450 bp, whereas t. dignus are 550, 800 and 1050 bp. although only slight differences occur between each telenomus species, our results showed that rapd procedures can reveal species-specific banding pattern that can be used for a rapid and easy identification of telenomus species. this technique can potentially be very important for identification purposes, since polaszek and kimani (1990) stated that due to its smallness, telenomus spp. are most often difficult to identify correctly which then resulted in confusion and errors in identification. to overcome these difficulties, molecular technologies can be applied to allow the detection of diagnostic markers which are necessary to resolve ambiguities in taxonomical identification and systematics in the genus telenomus (polaszek 2001). 60 molecular identification of egg parasitoid, telenomus spp — netti yuliarti et al. rapd-pcr has some limitations i.e uncertain reproducibility, fragments of the same size are not necessarily of the same sequence and reveal dominant marker only. previously, masutti (1994) and meilin (1999) used rapd markers for trichogramma species identification. even though rapd-pcr has its own drawbacks, i.e bands of the same size are not necessarily of the same sequence, these bands are normally inherited as dominant traits. therefore, rapd is still useful for generating species-specific markers. hoy (1994) stated that rapd-pcr may be able to detect small differences in the genomes of individual insects or mites, different insect populations or species. in this study we could not find differentiation among the five t. rowani populations by using primer p2 (5 -d[gtttcgctcc]-3) (amersham pharmacia biotech), despite the fact that those populations originated from different locations in java (fig.2). we found no variations in dna banding patterns for all populations. in other words, all of the populations revealed the same bands with the same molecular weight and only three bright bands were estimated to be in the range of 300-480 bp. there are two interpretations that could be derived from these results. first, the null difference can be due to the limitations of rapd technique; second, the result reflected real condition of t. rowani populations. in this case rapd-pcr did not allow differentiation of t. rowani populations because there are no specific bands that can be detected using this particular primer. however, screening was conducted with only one primer, and it is likely that unique rapd markers could be found that discriminate among the populations if additional primers are tested. however, these studies can be used as a framework on which to base further studies which are required to fully elucidate species and population of telenomus. this result was contrary to previous studies on some populations of trichogramma evanescens (masutti 1994); t oidea armigera and t oidea cojuangcoi (meilin 1999) by using rapd-pcr in which differentiation among the populations were found. in another study, vaughn & antolin (1998) reported that diaretiella rapae (hymenoptera: braconidae) populations are genetically subdivided into a small spatial scale that corresponds to host-use patterns. cluster analysis cluster analysis based on upgma revealed that four telenomus species formed two groups (fig. 3). the dendrogram showed that t. remits and t. rowani belong to one cluster, whereas t. dignus and t. dignoides belong to another group. however, the two groups in this dendrogram were different in coefficient level (table 1). the dendrogram showed that t. rowani was closely related to t. remus, whereas t. dignus was closely related to t. dignoides. however, t. remus and t. rowani were rather far to t. dignus and t. dignoides. masutti (1994) stated that rapd banding patterns might be informative for the phylogenetic relatedness. in addition, nixon (1937) reported that t. dignoides was certainly very closely related to t. dignus. 61   references bin f, johnson nf. 1982. some new species of telenomus egg parasitoids of tropical pyralid pests. redia 65: 229-252 (+ 5 plates). goodwin dh, xue bg, kuske cr, sears mk. 1994. amplification of plasmid dna to detect plant pathogenic mycopiasma like organism. ann appl biol 36. 124-127. johnson nf. 1984. systematics of nearctic telenomus : classification and revisions of the podisi and phymatae group. bull of the ohio biological survey 6 (3): 1-113. honda jy, trjapitzin sv. 1995. a species description and biological comparison between a new species of telenomus haliday (hymenoptera: scelionidae) and trichogramma platneri nagarkatti (hymenoptera: trichogrammatidae): two egg parasitoids of sabulodes aegrotata (guenee) (lepidoptera: geometridae). pan pacific entomol 71 (4): 227-236. hoy ma. 1994. insect molecular genetics. an introduction to principle and applications. san diego. academic press. kambhampati s, black iv wc, rai ks. 1992. random amplified polymorphic dna of mosquito species and population (diptera: culicidae). techniques statistical analysis and applications. j med entomol 29 (6): 939-945. landry bs, dextraze l, boivin g. 1993. random amplified polymorphic dna markers for dna fingerprinting and genetic variability assessment of minute parasitic wasp species (hymenoptera: mymaridae and trichogrammatidae) used in biological control programs of phytophagous insects. genome 36: 580-587. masutti fv. 1994. molecular identification and phylogeny of parasitic wasp species (hymenoptera: trichogrammatidae) by mitochondrial dna rflp and rapd markers. insect mol biol 3 (4): 229-237. meilin a. 1999. keragaman karakter morfologi dan genetika populasi parasitoid telur, trichogramma spp. and trichogrammatoidea spp. (hymenoptera: trichogrammatidae) dari daerah geografis yang berbeda di pulau jawa. (tesis). bogor: program pascasarjana ipb. nishida t, torii. 1970. a handbook of field methods for research on rice stem-borers and their natural enemies. london. international biological programme. 63 biotropia no. 19,2002 nixon gej. 1937. some asiatic telenominae (hymenoptera: proctotrupoidea). ann & mag nat hist 10 (20): 444-475. polaszek a, kimani sw. 1990. telenomus species (hymenoptera: scelionidae) attacking eggs of pyralid pest (lepidoptera) in africa: a review and guide top identification. bull of entomol res 80: 57-71. polaszek a. 2001. an overview of the parasitoids ofspodoptera exigua. workshop on the management ofspodoptera spp. in vegetable crops. kuala lumpur. 14-16 march 2001. rohlf fj. 2000. ntsys pc, numerical taxonomy and multivariate analysis system, version 2.1. exeter software. new york. vaughn tyt, antolin mf. 1998. population genetics of an opportunistic parasitoid in an agricultural landscape, j heredity 80: 152-162. yuliarti n. 2002. karakter morfologi dan molekuler parasitoid telur, telenomus spp. (hymenoptera: scelionidae) dari beberapa daerah di jawa. (tesis). bogor. program pascasarjana ipb. 64 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf biotropia no biotropia no. 18, 2002 : 38 51 adherence and pathogenicity assay of vibrio harveyi in tiger shrimp (penaeus monodon) larvae for screening biocontrol agent yusminah hala1, antonius suwanto23, ridwan affandi4 and muhammad zairin jr.4 'department of biology, faculty of science and mathematics, makassar university, makassar 90221, indonesia 2 department of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia jseameo-biotrop, jl. raya tajur km 6, bogor, indonesia 4department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia abstract rifampicin-resistant marker was employed as a reporter to detect the adherence and colonization of v. harveyi in shrimp larvae. vibrio harveyi p1b and ya32.2 were isolated from dead shrimp larvae in besuki, northern coast of east java, while v. harveyi hb3, was isolated from pristine sea water in pacitan, southern coast of east java. vibrio metschnikovii used as biocontrol agent was isolated from healthy shrimp larvae in serang, west java. spontaneous mutation was conducted to generate v. harveyi p1b, ya32.2 and hb3 resistant to rifampicin. these mutants exhibited similar survival ability to their parental (wild type) strains. significant larval mortality was observed in shrimp larvae inoculated with ya32.2 than that of larvae inoculated with p1b. larvae inoculated with hb3 showed the lowest mortality. bacterial cell count of vibrio rf* in dead larvae were 103-104 cells/larvae. isolates of vibrio metschnikovii z and m as biocontrol candidates effectively reduced the growth and adherence ability of ya32.2 to shrimp larvae. larval mortality in rearing water inoculated simultaneously with ya32.2 and v. metschnikovii was lower than the one inoculated with ya32.2 alone. therefore, vibrio metschnikovii z or m could be developed as an effective probiotic or biocontrol agent for v. harveyi in shrimp hatcheries. key words : biological control/vibrio metschnikovii/shrimp \arvae/penaeus mwu«fon/pathogenicity assay/vibrio harveyi introduction shrimp farming is one of the most important activities in indonesia and other asian countries such as thailand, phillippines, and india (ruangpan 1998). up to 1997, indonesia was the second largest world producer of tiger shrimp (penaeus monodori) after thailand with a production of more than 99 000 metric tons of tiger shrimp (anonymous 1999). however, the exponential growth of shrimp culture in the last five years was not supported properly by a sufficient supply of shrimp larvae *corresponding author : e-mail address : asuwanto@indo.net.id: fax : 62-251-315107 38 38 biotropia no. 18, 2002 due to larval diseases and poor environmental quality of the hatcheries. among shrimp diseases, vibriosis is one of fatal bacterial diseases. this disease is caused by vibrio sp. that attacks the tiger shrimp at early larval or post-larval stage. mass mortality of shrimp larvae frequently associated with luminous vibrio (lavilla-pitogo et al. 1998; sunaryanto & mariam 1986) was identified as vibrio harveyi (karunasagar et al. 1994; lavilla-pitogo et al. 1990). although vibrio carchariae was reported as a pathogen in a brown shark (carcharinus plumbeus) that was found dead in captivity (grimes et al. 1984), it has not been reported as a pathogen of shrimp larvae. it was also found associated with a chronic skin ulcer on a shark (bertone et al. 1996). so far, it has not been determined whether luminous vibrio is a pathogen or saprophyte, even though it could frequently be isolated from dead larvae. therefore, the pathogenicity status of this group of bacteria is uncertain. pathogenicity assays based on koch's postulate (salyers & whitt 1994) is practically difficult to be conducted in tiger shrimp larvae due to its relatively small size and since so far, no germ-free larvae are available (hameed 1993). the source of luminous vibrio colonized shrimp larvae might originate from the midgut contents of the spawners (lavilla-pitogo et al 1992). bacterial pathogenicity is determined by several factors such as the ability to adhere to the host tissue to colonize and to secrete virulence factors (salmond et al. 1995). the aim of this study was to determine the adherence and colonization of vibrio isolates and their pathogenicity to shrimp larvae. bacterial isolates were molecularly tagged to distinguish it from vibrio naturally associated with the shrimp larvae. luminous vibrio isolated from hatcheries and coastal water in east java, indonesia, showed sensitivity to rifampicin (tjahyadi et al. 1994). therefore, we used rifampicin resistant (ri*) vibrio generated by spontaneus mutation as a marker for v. harveyi isolates used in this study. vibrio harveyi p1b and ya32.2 were isolated from dead larvae, while hb3 was isolated from pristine sea water (suwanto et al. 1998; teo et al. 2000). our study also indicates that adherence assay in this study could be developed to screen for potential biocontrol bacteria or probiotics against pathogenic vibrio. materials and method isolation and identification of vibrio carchariae the dead shrimp larvae were obtained in february 1997 from besuki in the northern coast of east java, indonesia. the larvae were rinsed twice with sterile sea water and placed aseptically on the thiosulphate citrate bile salt (tcbs) agar. the plate was incubated for 24 h at room temperature (28 ± 2 °c). isolated luminous colonies were restreaked'on vibrio harveyi agar (vha) (harris et al. 1996). 39 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. bacterial characterization was conducted employing cellular fatty acid analyses by microcheck, inc., microbial analyses laboratory in northfield, usa. bacteria were grown on trypticase soy broth agar (tsba) at 28 °c and then extracted for cellular fatty acid analysis (miller 1982). the fatty acid composition of each isolate was compared to a database of standard strain (v. carchariae atcc 35084) profile, using overlap coefficient which allows their identification according to their similarity index. two other v. harveyi isolates used in this study were isolated and characterized from dead larvae (p1b) and from pristine sea water (hb3) (suwanto et al. 1998). antibiotics sensitivity in order to tag vibrio with the antibiotics resistant markers, it is necessary to determine the natural antibiotic sensitivity of the isolates. all isolates used in this research were tested for antibiotic sensitivity against kanamicin (50 p.g/ml), ampicillin (50 |o.g/ml), tetracycline (10 |j.g/ml), gentamicin (20 ng/ml) and rifampicin (10 |j.g/ml) on luria bertani (lb) agar (10 g nad, 10 g tryptone, 0.5 g yeast extract, 15 g agar and 1 l distilled water). rifampicin-resistant vibrio harveyi spontaneous mutants of v. harveyi resistant to rifampicin (rf*) were selected on lb agar supplemented with rifampicin (50 (ig/ml) (eisenstad et al. 1994). the survival of rf* v. harveyi was evaluated and compared to the wild type strains in artificial larval-rearing water (3% nacl and 0.3% yeast extract in 1l distilled water= sye). growth pattern and survival assay vibrio harveyi rf" was inoculated into 100 ml sye media and incubated at 28°c. cell concentration was enumerated daily on tcbs and tcbs supplemented with 50 (j.g/ml rf (tcbs-rif). enumeration began just after v. harveyi was inoculated into sye media. the survival of v. harveyi on tcbs was compared with the survival on tcbs-rif to detect the stability of the rf* mutation. preparation of shrimp larvae two-day-old tiger shrimp postlarvae (pl2) and sea water for larval rearing were obtained from a hatchery in labuhan, west java. the larvae were maintained for acclimatization in a three-liter glass stock jar for at least two days in laboratory condition. sea water salinity was maintained at 2.5% and at a temperature of 2830°c. the larvae were fed on an artificial diet (lanzy pl; inve aquaculture, belgium). for adherence and pathogenicity assays, 50 larvae were kept in a threeliter glass jar containing 2.5 liters of autoclave-sterilized sea water. 40 biotropia no. 18, 2002 adherence assay tested bacterial strains used in the adherence assays were cultured in 50% sea water complex (swc) agar medium (ph 7.2) for 24 h at 28 °c. the culture was harvested by adding 5 ml sterile sea water onto the bacterial lawn, scraped, and collected into a tube, and then centrifuged at 5000 x g for two minutes. the bacterial cells were rinsed in sterile sea water and diluted to give a final concentration of 106 cfu/ml. the number of wild type vibrio and vibrio rf* in larval rearing water was enumerated daily on tcbs and tcbs-rif, respectively. the number of larval mortality was counted daily. the vibrio harveyi which colonized the dead larvae was enumerated daily on tcbs-rif. challenge test the isolates used for the challenge test in this study were vibrio metschnikovii isolates z and m, which were isolated from healthy zoea (z) and mysis (m), respectively (widanarni 1999). cell suspension of vibrio metschnikovii z and m at a concentration of 108 cfu/ml were inoculated into larval rearing jar 2 h before the shrimp larvae were introduced into the jar. after 6 h cocultivation of z or m isolate with shrimp larvae, pathogenic vibrio (ya32.2 rf*) were inoculated into the larval rearing jar. the treatments consisted of ya32.2 rf" which was challenged with v. metschnikovii z, m, or a combination of z and m. shrimp larvae cocultivated with ya32.2 rr* alone was employed as a control. the number of larval mortality as well as concentration of wild type vibrio and vibrio rf* in the dead larvae and in rearing water were enumerated daily after cocultivation. the total number of vibrio was enumerated on tcbs agar, while vibrio rfr was counted on tcbs-rif. each experiment was conducted in duplicates. the number of dead larvae and bacterial cell concentration were presented in average values. results isolation and identification of vibrio carchariae two vibrio isolates (ya32.2 and ya31.4) were isolated from dead larvae. colonies of these isolates were luminous, circular and appeared green with dark green in the center of the colony when grown on tcbs agar. they also grew on vha, a differential agar for vibrio harveyi (harris et al. 1996), and appeared as blue colonies due to decarboxylation of ornithin which shifted ph medium toward alkaline. when illuminated from below, yellow halos appeared, indicating cellobiose fermentation around the colonies. based on these specific characteristics, ya32.2 and ya31.4 were identified as v. harveyi. 41 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al cellular fatty acid analyses of ya32.2 and ya31.4 grown on trypticase soybroth agar indicated that similarity index to v. carchariae atcc 35084 were 0.955 and 0.944, respectively. these similarity indices were much higher than those of v. parahaemolyticus or v. alginolyticus (table 1). however bergey's manual classifies v. carchariae as a junior synonym (variant) to v. harveyi (baumann et al. 1994). four vibrio harveyi isolates used in this study, i.e.: p1b, hb3, ya32.2 and ya31.2, were resistant to ampicillin (50 tig/ml) and kanamicin (50 jig/ml) but sensitive to tetracycline (10'|ig/ml), gentamicin (20 (j-g/ml) and rifampicin (10 (j.g/ml) (tabel 2). vibrio harveyi mutant rf11 an amount of 50-100 u.1 of 108 cfu/ml v. harveyi was inoculated onto lb agar supplemented with 50 (j-g/ml rifampicin. isolated colony which showed similar morphology and luminescence to the wild type was further employed for survival assay. the growth pattern and survival of three mutants p1b rf*, ya32.2 rt* and hb3 rf* were similar to their parental wild type strains (data not shown). table 1. similarity index isolates ya32.2 and ya31.4 with vibrio sp based on cellular fatty acid profiles 42 biotropia no. 18, 2002 adherence assay about 106 cfu/ml of v. harveyi rf* was inoculated into larval rearing water for adherence assay on shrimp larvae. bacterial growth profile in larval rearing water showed similar patterns to that of the wild type (fig. 1). the three strains showed bacterial growth patterns that generally increased in cell concentration at first to second or third day and decreased the following days. the highest concentration of vibrio ri* in larval-rearing water was approximately 107 cfu/ml. larval mortality in response to the three vibrio rf* strains was different as presented in figure 2. isolate ya32.2 caused the highest larval mortality on the first day and the number of vibrio rf* found in the dead larvae was 2.3xl03 cfu/larvae. on the second day after inoculation, the number of dead larvae increased to 36 animals concomitant with the highest vibrio rf* concentration at 1.2xl04 cpu/ larvae. all of the larvae were found dead on the third day with vibrio rf" found in the larvae at concentration of 1.3xl04 cfu/larvae . 43 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. figure 1. comparison of vibrio rf* and their respective parental wild type growth pattern in the larval rearing water 44 biotropia no. 18, 2002 figure 2. correlation between the cumulative number of death larvae and the number of vibrio adhered in the shrimp larvae 45 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. larval mortality in the jars inoculated with p1b occured on the second day after inoculation. the number of dead larvae found on the second day was 19 animals. larval mortality reached its highest number on the third day when the concentration of vibrio rf* in the larvae was 8.7xloj cfu/larva. the lowest larval mortality was found in the larvae inoculated with hb3: larval mortality was found only after three days of coinoculation, and until the last day of observation, larval mortality occurred in only 13 animals carrying vibrio rf* at concentration of 1.75xl02 cfu/larva (fig. 3). challenge test shrimp larvae inoculated with ya32.2 supplemented with v. metschnikovii z or m, either alone or in combination, showed less mortality than the larvae treated only with ya32.2 alone as shown in figure 4. the number of dead larvae when treated with ya32.2 in the presence of v. metschnikovii m was 11 larvae. moreover, the number of dead larvae slightly increased if ya32.2 was inoculated in the presence of z and m, although the number of vibrio rf* which adhered to the larvae was relatively similar i.e. 1.2xl02 to 3.2xl02 cfu/larva. al l of the larvae treated with ya32.2 were found dead after three days of cocultivation when concentration of vibrio rf* in the larvae reached 104 cfu/larva (fig.4). figure 3. comparison between bacterial cell count of ya32.2 when challenged or when inoculated alone 46 biotropia no. 18, 2002 figure 4. correlation between the number of dead larvae and ya32.2 adherence in a challenge test experiment 47 adherence and pathogenecity assay of vibrio harveyi yusminah hala et ill. discussion fatty acid profile analyses indicated that isolates ya32.2 and ya31.4 were v. carchariae. the results indicated that vha media which was supposedly selective for v. harveyi (harris et al. 1996) actually could also support the growth of v. carchariae, although dna-dna hybridization analysis suggested that v. carchariae is a junior synonym for v. harveyi (baumann et al. 1994). in addition, ribotyping of v. harveyi and v. carchariae indicates very similar dna banding patterns (pedersen et al. 1998). physiology and genetic similarity might explain the ability v. carchariae to grow on vha media that was originally designed as a selective media for v. harveyi. one of the physiological characteristics of v. carchariae is its capability to hydrolyze urea (uh+). other reports indicated that brown shark (from where the original name of v. carchariae was derived) excreted urea for osmoregulation. it was suggested, therefore, that the adherence of v. carchariae to shark was due to urea-urease linkage (bertone et al. 1996). meanwhile, urea hydrolyzing activity was reported as an indication of potential pathogenicity of v. parahaemolyticus (kays-ner et al. 1994). in v. parahaemolitycus, uh+ is related to hemolysin production, which is a virulence factor of vibrio (osawa et al. 1996). the result of our study indicated that ya32.2, p1b and hb3 were uh+, and two of them (i.e. p1b and ya32.2) were isolated from the dead shrimp larvae. however, the shrimps were reported to excrete ammonia, instead of urea (baticados 1988). therefore, the adherence of v. harveyi to shrimp might not be mediated by urea-urease linkage. three isolates used in this research demonstrated similar sensitivity to the test antibiotics. they were resistant to ampicillin and kanamicin but sensitive to tetracycline, gentamicin and rifampicin. vibrio carchariae isolated from shark skin ulcer was sensitive to tetracycline and resistant to piperacillin and carbenicillin (bertone et al. 1996). vibrio harveyi which was the causal agent for mass mortality of shrimp larvae in india, was sensitive to tetracycline but resistant to erythromycin. on the other hand, v. harveyi generally showed similar sensitivity to gentamicin (austin et al. 1981) and rifampicin (tjahyadi et al. 1994). this study indicated that v. harveyi p1b and hb3 and v. carchariae ya32.2 were sensitive to tetracycline, gentamicin and rifampicin. this result showed that v. harveyi isolated from anywhere has similarity in sensitivity to tetracycline, gentamicin and rifampicin. bacterial cell count of v. harveyi rf* in larval-rearing water showed a different number from that of vibrio sp which occurred naturally in larval rearing water. the difference indicated the-number of vibrio sp originally colonized shrimp larvae. these vibrio sp yielded green colonies which could not be distinguished from green colony of v. harveyi rf* in rearing water. therefore, it is necessary to introduce molecular marker to distinguish the green colonies of vibrio rt* from the green colonies bf wi'd type vibrio. 48 biotropia no. 18, 2002 p1b, hb3 and ya32.2 were sensitive to rf. therefore, in this experiment, we used rf* as molecular marker to distinguish these three strains from the other v. harveyi isolates which occurred naturally in the shrimp larvae or larval rearing water. vibrio harveyi rf* mutants showed similar growth patterns to that of the wild type strain. this result indicated that the survival of v. harveyi rf* was similar to the wild type, and rf* character was stable even when it was kept in larval-rearing water. although the cell densities of each vibrio rf1* in larval-rearing water were similar at approximately 107 cfu/ml (fig. 1), larval mortality in response to the three rf* strains was different. the fastest and the highest larval mortality was due to ya32.2 inoculation which could be detected as early as one day after coin-oculation time. larval mortality due to inoculation with p1b was detected after two days of coinoculation although the number of the dead larvae which resulted from those treatments was relatively similar. probably, the two strains were potential pathogens because they were isolated from dead shrimp larvae. similarly observed mortality was found when shrimp larvae were inoculated with similar density of v. campbelli\ike bacterium (hameed 1995). our study showed that hb3 was not pathogenic. larval mortality in hb3 inoculation jar maybe due to the high density of vibrio. nonpathogenic or very weak pathogenicity of hb3 might be the reflection of its habitat where hb3 was originally obtained from pristine sea water in southern coast of java island. isolate ya32.2 was then selected for challenge test due to its potential pathogenicity. vibrio metschnikovii as a challenge strain is a vibrio species which usually adheres and colonizes shrimp larvae until the stages of post-larval stage. we assumed the strains of v. metschnikovii are not pathogen because they do not produce virulence factors (widanarni 1999). austin et al. (1995) used v. alginolyticus as a probiotic strain for a. salmonicida, v. anguillarum and v. ordalii. our results indicated that v. metschnikovii z or m could significantly reduce the growth and adherence of ya32.2 in shrimp larvae and significantly reduce larval mortality. this result demonstrated potential application of the adherence assay to screen for potential prdbiotics or biocontrol bacteria in shrimp hatcheries. acknowledgment this work was funded by grants from riset unggulan kemitraan (ruk), indonesia, and international foundation for science (ifs, a/2207-2), sweden, to antonius suwanto. 49 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. references anonymous. 1999. aquaculture production statistics 1988-1997. fao fisheries circular no 815, revision 11. fao, roma. austin b., d.a. morgan and d.j. alderman. 1981. comparison of antimicrobial agents for control of vibriosis in marine fish. aquaculture, 26:1-12. austin, b. l.f., p.a. stucken, w. robertson, 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(ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok, p:217-224. teo, j.w.p., a. suwanto, and c.l. poh. 2000. novel p-lactamase genes from two environmental isolates of vibrio harveyi antimicrob. agents chemother. 44: 1309-1314. tjahjadi, m.r., s.l. angka and a. suwanto. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial diseases in tiger shrimp larvae (penaeus numodon. fab.). as. pac. j. mol. biol. biotechnol. 2:347-352. widanarni and a. suwanto, 2000. genetic diversity of ampicilin-resistant vibrio isolated from various stages of tiger shrimp larvae development. biotropia. 15:36-47. 51 biotropia vol. 29 no. 2, 2022: 171 180 doi: 10.11598/btb.2022.29.2.1703 171 community structure of benthic foraminifera in eastern waters of segara anakan lagoon in cilacap tjahjo winanto*1, petrus hary tjahja s2, amron1, taufan harisan1 and hendrayana1 1marine science study program, faculty of fisheries and marine sciences, universitas jenderal soedirman, purwokerto 53122, indonesia 2aquaculture study program, faculty of fisheries and marine sciences, universitas jenderal soedirman, purwokerto 53122, indonesia received 30 november 2021/accepted 19 january 2022 abstract benthic foraminifera are types of organisms that are sensitive to environmental changes, so they are often used as a bioindicator for aquatic environmental conditions. the purpose of this study was to determine the community structure of benthic foraminifera community, sediment types and the relationship between the abundance of foraminifera with the sediment types in the eastern waters of segara anakan lagoon in cilacap. method used in this study was a survey method. samples were taken by using random sampling method. the study was conducted at 5 stations with 3 repetitions. laboratory observations carried out included the types and numbers of benthic foraminifera. community structure of foraminifera among stations were analyzed using one-way anova, while the relationship between community structures and water quality parameters was analyzed using pearson correlation. the results showed that in the the eastern waters of segara anakan lagoon there were 58 species of foraminifera which abundance ranged from 532 ind/m2 to 927 ind/m2. the diversity index of foraminifera was in the medium to high diversity categories. the uniformity index of foraminifera was in the high uniformity in a stable community. the dominance index of foraminifera was in the low category. the sediment types was fine sand, medium sand and coarse sand. the relationship between the abundance of foraminifera with the sediment types was strong with high r values (0.763-0.809). keywords: benthic, community structure, foraminifera, sediment, segara anakan lagoon introduction the segara anakan lagoon is located between the southern part of the java island and the nusa kambangan island (180º53' 109º30' w; 7º20' 7º35' s), with an area of about 240 km2, extending from west to east. this area is a protected water area with a closed tropical forest (setyowati 2005). industrial activities and residential areas that continue to increase, as well as the rapidly growing tourism areas causes adverse impacts on the surrounding ecosystem (setyowati 2005). one way to see the impact is to investigate the habitat of an organism in a community (ferawati et al. 2014). biological component analysis in an ecosystem is a measurement of biological responses to changes in environmental conditions, which can be studied through a community of organisms that are used as parameters of important biological components (fachrul 2007). one of the important organisms in marine ecosystems, especially in shallow seas, is benthic foraminifera. foraminifera organisms are shelled organisms that are abundant in various marine environments. in a volume of 1 cm3 of sediment, there can be hundreds of living foraminifera individuals, along with many dead shells (sadough et al. 2013; bawole et al. 2017). foraminifera is among the organisms in seabed sediments that can indicate the environment conditions of their living area. their way of life is by attaching themselves to sediments, rocks, marine plants and corals available at the bottom of the waters. as a result, benthic foraminifera are very sensitive to various environmental changes such as temperature, salinity, light and *corresponding author, email: tjahjo.winanto@unsoed.ac.id mailto:tjahjo.winanto@unsoed.ac.id biotropia vol. 29 no. 2, 2022 172 ph (gustiantini 2008). therefore, foraminifera are widely used by biologists, geologists and oceanographers in relation to various changes in marine environmental conditions (boltovkoy & wright 1976 in sidiq et al. 2016). foraminifera are single cell organisms having hard shells and most of their communities live in the sea. the numbers of foraminifera found in the waters worldwide, both planktonic and benthic, are around 12,000 species (puspasari et al. 2012). the wide distribution area of foraminifera in various types of aquatic environment has the potential to assist in understanding environmental conditions in a waters (rositasari 2006). the living condition of foraminifera are strongly influenced by microand macro-environmental conditions, which makes foraminifera potential to be used as an indicator for environmental changes (rositasari 2011). information on the types and community structure of foraminifera in the waters of the segara anakan lagoon is still very limited and not much studies have been done. on the other hand, knowledge about the community structure of foraminifera can be used to determine the ecological conditions of a waters (sen gupta 2003; sadough et al. 2013), because the distribution of foraminifera is strongly influenced by the surrounding environmental conditions, so that certain species can reflect certain ecological conditions (gustiantini 2008). this study aimed to determine: 1) the types and community structure of benthic foraminifera, sediment types and 2) the relationship between the foraminifera abundance and sediment types in the waters of the segara anakan lagoon. materials and methods the study was conducted in the eastern waters of the segara anakan lagoon in cilacap by using a survey method. data collection was done by random sampling method. foraminifera samples were observed in the laboratory of the faculty of fisheries and marine sciences of universitas jenderal soedirman. the laboratory observations identified the types and numbers of foraminifera collected from the sediment samples. the observational station consisted of 5 stations. samples were taken from each station with three repetitions. the measured water quality parameters were salinity, temperature, ph and brightness. sediment samples were taken using a sediment grab. the samples were stored in the sample sticks, transported to and analyzed in the laboratory. the collected sediment samples were washed, then ovendried. after the samples’ temperature reaching room temperature, the samples were sieved using a multilevel sieve. the sifted dry samples were weighed and sprinkled onto a surface, followed by identification of the foraminifera (armstrong & brasier 2005; barker 1960; loeblich & tappan 1994; yassini & jones 1994; sadough et al. 2013; gupta 2003). research parameters observed were the types and community structure of benthic foraminifera, such as abundance, diversity, uniformity and dominance. the abundance of foraminifera is the number of individuals of a species occupying a certain area. diversity index (h') is used to measure the degree of ecological instability in a system. the uniformity index is an index used to measure the even distribution of species abundance in a community. dominance index is used to determine the extent to which a group of biota dominates another group (bawole et al. 2014; insafitri, 2010; odum 1994). the data from the study were analyzed descriptively. differences in foraminifera community structure among stations were analyzed using one way anova, while the relationship between foraminifera abundance and sediment types was analyzed using pearson correlation. data processing was done by using spss software version 17.0. results and discussion foraminifera community structure the number of benthic foraminifera species found in the eastern waters of segara anakan lagoon was 58 species. at station 1 there were 22 foraminifera species with the most abundant community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 173 species at this station was elphidium craticulatum. there were 13 foraminifera species at station 2, while at station 3 there were 25 species of foraminifera. the most common species found at stations 2 and 3 was ammonia beccarii. at station 4 there were 26 species with the most species was operculina ammonoides. there were 31 species of foraminifera found at station 5 with the highest number of species being amphistegina hauerina (fig. 1). the highest number of foraminifera species was found at station 5 (31 species), while the lowest foraminifera species was found at station 2 (13 species). ammonia beccarii, ammonia falsobeccarii and elphidium craticulatum were foraminifera species found in all research stations. these species has high adaptability and high tolerance to various environmental conditions compared to other species. according to uthicke (2008), foraminifera of the genus elphidium are indicators of high turbidity levels and low brightness. this finding is in agreement with the work of toruan (2011) which stated that foraminifera from the genus elphidium and ammonia are included in the opportunist group which are tolerant to adverse environmental conditions compared to other foraminifera. the species amphistegina hauerina and operculina ammonoides were foraminifera species that were only found at stations 4 and 5. it is suspected that stations 4 and 5 are coral reef areas with the characteristics of clear waters with high brightness. foraminifera of the genus amphistegina are the most abundant foraminifera of the coral reef symbiotic group in waters with a fairly high level of brightness (toruan 2011). in general, foraminifera from the genus amphistegina are the most well-known species associated with coral reef areas (eichler et al. 2019). foraminifera of the genus amphistegina are important ecosystem builders because of their contribution to calcium carbonate production and thrive in a variety of shallow water habitats. the genus amphistegina is also a bioindicator to determine the quality of clean water in the coastal environment (weinmann et al. 2013). the abundance, diversity, uniformity and dominance indices were used to determine the foraminifera community structure in the eastern waters of segara anakan lagoon. foraminifera abundance the abundance of foraminifera found in the eastern waters of segara anakan lagoon ranged from 532 to 927 ind/m2 (fig. 2). figure 1 average numbers of benthic foraminifera in the eastern waters of segara anakan lagoon biotropia vol. 29 no. 2, 2022 174 figure 2 abundance of benthic foraminifera in the eastern waters of segara anakan lagoon the highest number of benthic foraminifera was found at station 5 with 927 ind/m2, while the lowest number was found at station 2 with 532 ind/m2. station 5 had the highest abundance of benthic foraminifera, presumably because this station is an open area that is directly affected by the water currents (higher water currents). station 2 had the lowest abundance of foraminifera, because this station tends to be more protected from water currents (lower water currents). high velocity of water currents can affect the distribution of benthic foraminifera in a waters. gustiantini (2008) stated that the distribution of benthic foraminifera is influenced by current patterns; in relatively speedy water currents conditions the foraminifera species will be very abundant and diverse. the presence of water current will evenly distribute the water temperature and salinity. in addition, water currents also play a role in carrying food for foraminifera (toruan 2011; uthicke 2008). diversity index (h') the biota diversity can be determined using the shannon-wiener index (h'). in this study, the diversity index of foraminifera in the eastern waters of segara anakan lagoon ranged from 2.29 to 3.11 or falls into the medium to high diversity categories. the diversity index at station 1 was 2.43. station 2 had diversity index of 2.29. station 3 had diversity value of 2.94, while the index for station 4 was 3.03 and the index for station 5 was 3.11 (fig. 3). the diversity index in this study showed that the stations have suitable water quality conditions as the habitat for foraminifera in terms of nutrients suppy availability, lighting, temperature, salinity and ph. this finding is supported by gustiantini (2008) which stated that the life of foraminifera organisms is strongly influenced by abiotic factors such as temperature, salinity, brightness, ph and sediment types. water quality measurements obtained in the eastern waters of segara anakan lagoon showed that the water temperature at all stations ranged from 26 oc to 29 ºc. this temperature range is suitable for the habitat of foraminifera organisms, which can be found at temperature range of 10 30 ºc (pranajaya 2015). according to rositasari (1997) temperature affects the metabolism of an organism and foraminifera can live and adapt to a certain temperature range. the critical point of temperature can be reached when the foraminifera reproduction process is no longer taking place (rositasari 1997). in this study, the water salinity ranged from 29 to 31 ppt. the salinity range corresponds to the habitat of benthic foraminifera which can be found in salinity range of 18 30 ppt (rositasari 1997). waters with normal salinity usually has a high diversity of foraminifera species (rositasari 1997). foraminifera organisms are also able to adapt to low salinity, such as bays, brackish waters and swamps, which are usually inhabited by foraminifera with agglutinin shell type. foraminifera are also able to live in waters having high salinity. the high salinity waters community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 175 figure 3 diversity of benthic foraminifera in the eastern waters of segara anakan lagoon (hypersaline waters) are favored by foraminifera species with porcelain shell types having high concentrations of calcium carbonate (rositasari 1997). the water brightness in this study was in the range of 1.1 2.1 m which is below the brightness quality standard of sea water of > 3 meters based on the kepmeneg lh (2004). the low brightness in the eastern waters of segara anakan lagoon is due to the fact that the area is close to the river mouth, which is a meeting place between river waters and sea waters. river waters carry wastes from the land and the process of mixing seawater and freshwater causes the waters to become muddy due to the mixing of particles from the mainland (saraswati et al. 2017). the ph values ranged from 6 to 7. water ph affects the growth of foraminifera organisms, because the degree of acidity of a waters can affect the biological activity of foraminifera organisms, one of which is the shell formation process (yanti 2016). waters with acidic water conditions can cause a reduced ability of foraminifera to secrete calcium carbonate in shell formation (brasier 1980). the analysis of variance (anova) results showed that there was a significant difference in diversity index of benthic foraminifera (p < 0.05) among stations. stations 1 and 2 had significant differences in diversity index to stations 3, 4 and 5. the differences are presumably due to the different characteristics of the study locations. stations 3, 4 and 5 have characteristics of open environment, while stations 1 and 2 have characteristics of a more protected environment. the open area is affected by the current velocity which tends to be greater than the one in the protected area. current velocity can affect the distribution of foraminifera in a waters. the distribution of foraminifera in the waters is influenced by currents, sediment types and the presence of coral reefs (gustiantini 2008). uniformity index (e) the uniformity index is an index used to measure the even distribution of species abundance in a community. this index provides information about the similarity of species living among stations. the uniformity index of foraminifera in the eastern waters of segara anakan lagoon enters the category of high uniformity, stable community, ranging from 0.82 to 0.94. the uniformity index at station 1 was 0.82 (the lowest). the uniformity index at station 2 was 0.92. station 3 had a uniformity index of 0.93. the uniformity index at station 4 was 0.94 (the highest). the uniformity index at station 5 was 0.93 (fig. 4). the uniformity index (e) from all stations was > 0.6 which means that the uniformity at each research station enters the high category with stable community. if the distribution of organisms in an ecosystem is evenly distributed, the ecosystem tends to be in a stable condition (odum 1994). the evenness of species presence biotropia vol. 29 no. 2, 2022 176 figure 4 uniformity index of benthic foraminifera in the eastern waters of segara anakan lagoon at each research station is supported by the morphology of foraminifera having the ability to exist in various ecosystems. benthic foraminifera as single-cell shelled organisms have the ability to occupy various marine areas ranging from the supertidal zone above the littoral to the deepest depths in the hadal abbysal zone (bawole et al. 2017). our study showed that there was a significant difference (p < 0.05) in the uniformity index of benthic foraminifera among stations, i.e., the uniformity index at station 1 was significantly different from that at stations 2, 3, 4 and 5. dominance index (c) the dominance index shows the dominant value of species abundance at each station. the greater the value, the dominant the species at the station. our study showed the dominance index ranging from 0.0535 to 0.1182 which enters the low category (fig. 5). the highest dominance index was found at station 1 (c = 0.1182), while the lowest index was found at station 5 (c = 0.0535). the dominance index values of all stations are in the range of 0.00 0.50, which enters the low category. the dominance index in a community structure is usually inversely proportional to diversity index. the low dominance value (< 0.5) indicates that the foraminifera organisms are highly diverse and more evenly distributed. the low dominance index shows that no species dominates (bawole et al. 2017). sediment types the sediment types in the eastern waters of segara anakan lagoon are fine sand to coarse sand. stations 1 and 2 had fine sand sediment type. station 3 had a medium sandy sedimen type, while stations 4 and 5 had coarse sand sediment type (fig. 6). community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 177 figure 5 dominance index of benthic foraminifera in the eastern waters of segara anakan lagoon figure 6 percentage of sediment types in the eastern waters of segara anakan lagoon the fine sand sediment was found at stations 1 and 2, presumably because these two stations are close to the river mouth, which is more protected than other stations. a relatively protected area has a lower water movement resulting to smaller size particles (aritonang et al. 2014). stations 4 and 5 have coarse sand sediment, presumably because these two stations are close to the beach, which are open areas and have relatively higher water currents. a strong water currents will cause the sediment to have coarser fractions, so that the sediment is not easily carried away by the water current (bayhaqi 2015). relationship between benthic foraminifera abundance and sediment types the pearson correlation analysis showed relationship between the foraminifera abundance with sediment types. the correlation values ranged from a low but definite relationship (r = 0.331) to a high and strong relationship (r = 0.763 0.809) (table 1). the foraminifera abundance was strongly related to the coarse sand sediment type (0.809) and fine sand sediment type (-0.763). on the contrary, a low but definite relationship occurred between the foraminifera abundance to the medium sand type (0.331). the negative biotropia vol. 29 no. 2, 2022 178 tabel 1 relationship between foraminifera abundance and sediment types coarse sand medium sand fine sand abundance pearson correlation 0.809** 0.331 -0.763** sig. (2-tailed) 0.000 0.228 0.001 n 15 15 15 note: ** = correlation is significant at p < 0.01. value in the correlation between foraminifera abundance and fine sand sediment type indicated inversely proportional relationship, which meant that the finer the sediment type, the less the foraminifera abundance is. these findings are supported by the work of natsir (2010) which states that foraminifera organisms generally occupy sediments containing sand; no foraminifera are found in mud and silt sediment types. the work of lacuna (2013) also supported our findings which stated that the sediment types affect the presence of foraminifera, because foraminifera generally are more abundantly found in the sandy sediment type and less abundant in a finer sediment type. conclusion the sediment types and water current affect the foraminifera abundance and distribution. water quality parameters among observation stations are relatively similar and have no significant effect on the abundance and distribution of foraminifera. acknowledgments the authors thank the lppm of universitas jenderal soedirman for funding the institutional research of universitas jenderal soedirman year 2021. the authors also deliver deep gratitude to the dean and colleagues at the faculty of fisheries and marine science of universitas jenderal soedirman for the tremendous support in our study. references anugrah py. 2012. foraminifera. teknik geologi. jakarta (id): universitas trisakti. aritonang ae, surbakti h, purwiyanto ais. 2014. laju pengendapan sedimen di pulau anakan muara sungai banyuasin, sumatera selatan. 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[ecology and environment]. gorontalo (id): universitas negeri gorontalo. weinmann ae, dennis r, stefan l, martin rl. 2013. traveling through time: the past, present and future biogeographic range of the invasive foraminifera amphistegina spp. in the mediterranean sea. mar micropaleontol 105 (2013):30-9. widhayanti a, ismanto a, yulianto b. 2015. sebaran tumpahan minyak dengan pendekatan model hidrodinamika dan spill analysis di perairan cilacap, jawa tengah. [the distribution of oil spill and hydrodynamics approach and spill analysis in the cilacap waters]. jurnal oseanografi, 4(4):641 -50. biotropia no biotropia no. 17, 2001 : 18-29 establishment of neochetina spp.: their pattern of local dispersal and age structure at the release site kasno, asmarina s.r. putri, sri widayanti and sunjaya seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract study on the distribution pattern and age structure ofneochetina spp. (coleoptera: curculionidae) at the release site was conducted to know a) the distribution pattern of the weevil, b) its establishment status, c) its survival rate in the field, and d) relationship between the attack of the weevil and the fungus, altemaria eichhorniae nag rag & ponappa (hyphomycetes), in causing damage to water hyacinth. this study was conducted at situ bagendit lake, garut, west java. a release and recapture method was employed to study the mode and rate of dispersal of the weevils under field conditions. regular sample collection at two-month interval was done to evaluate the pattern of distribution and to assess the age structure under field condition. another two months regular observation was done to assess damage severity due to adult weevils and the fungus, a. eichhorniae, on water hyacinth. results showed, that the weevils seem to disperse actively to all directions following the presence of water hyacinth. the data also showed that the dispersal rate of the weevils was about a few meters a week. under field conditions at situ bagendit lake, the weevils were about evenly distributed throughout water hyacinth mass. the density of the weevils fluctuated from time to time, but the trend slightly increased. the survival rate of the weevils at situ bagendit lake was estimated not more than 5%. it was suspected that various limiting factors such as various predators have caused the low population increase under field conditions. the population increase through time confirmed that the weevils have established at situ bagendit lake. field data showed that there were no interaction between the damage severity of the weed caused by both weevils and the fungus. introduction water hyacinth, eichhornia crassipes (mart.) solms laubach (pontederiaceae), is one of the most important aquatic weed and causes various serious problems in indonesian water bodies as well as in some other tropical and subtropical regions of southeast asia. various efforts to control water hyacinth have been attempted including the use of biotic agents. biotrop initiated researches on biological control of water hyacinth by conducting a seminar on aquatic weed management in 1974 followed by introducing water hyacinth weevil, neochetina eichhorniae warner (coleoptera : curculionidae) from florida in 1975. aside from n. eichhorniae, biotrop in cooperation with aciar and csiro has also imported in 1994 another weevil species, i.e., neochetina bruchi hustache (coleoptera : curculionidae) for the same purpose. both water hyacinth weevils have also been tested and released in the fields of many countries including indonesia where this weed has been introduced to those countries (julien and griffith 1998; barley 1990). 18 biotropia no. 17, 2001 research activities on biological control of water hyacinth have been conducted in many countries, generally using insects, fungi and fish as biological control agents (harley 1990). the significant role of neochetina spp. as biocontrol agent of water hyacinth has been reported from different countries such as queensland (wright 1979; room 1986), sudan (bashir 1984), bangalore (jayanth 1988) and louisiania (goyer and stark 1984). other studies showed that better control of water hyacinth may be achieved by the integrated use of water hyacinth weevils with other biocontrol agents such as fungi (saraswati 1980; charudattan 1986; galbraith 1987; gaunter and mohamed 1992). the release of n. eichhorniae has been conducted at some release sites in indonesia but after some time only partial control of water hyacinth has been achieved. for experimental purposes n. bruchi has been released at situ bagendit lake in august 1996 and october 1996 consisting of 100 and 125 pairs, respectively. further releases in the same site were done in september 1998, december 1998, january 1999, august 1999, december 1999 consisting of 265, 600, 262, 307, 700 adults ofn. bruchi and n. eichhorniae. another biological control agent is a fungus, alternaria eichhorniae nag raj & ponappa (hyphomycetes), found in the lake, but its role has never been recorded. the role of the fungi to control water hyacinth at experimental scale combined with n. bruchi resulted in a more serious damage. the aims of the study were : to study the distribution pattern of neochetina spp. at the release site and the composition amongst the developmental stages of the weevils; to evaluate the establishment of neochetina spp. at the release site; and to study the relationship between water hyacinth damage severity due to neochetina spp. and a. eichhorniae at the release site. materials and methods mode and rate of dispersal and distribution of the water hyacinth weevils under open field the release of the weevils was done at situ bagendit lake, garut regency. the water body of the lake was originally 124 ha, but when the research was carried out the estimated water body varied from 30 ha (in dry season) to 50 ha (in rainy season). there were few blocks of water hyacinth mats. the biggest water hyacinth mat was about 0.5 ha on which the experiment was carried out. the aim of this experiment was to evaluate the mode, dispersal rate and distribution pattern of the weevils at the release site. a total of 307 adults of neochetina spp. marked with spotted white color on their elytra consisting of 174 males and 133 females were released in the center of situ bagendit lake as the first release. three months later, the second release consisted of 350 marked adult males and 350 marked adult females. fortnight observation was conducted using transect method. from the release point, transects 19 establishment of neochetina spp. kasno et ai were drawn to the four cardinal directions, and hence the presence of adult weevils marked with spotted white color was observed. the parameters observed were distance from the release point and the number of adults at 1 m2 area. age structure of water hyacinth weevils in the field the structure of developmental stages or the age structure of neochetina spp. was determined at situ bagendit lake. the aim of this experiment was to assess the age structure of those weevils at the release site. ten units of water hyacinth samples measuring 50 x 50 cm each were collected for further examination on the number of eggs, larvae, pupae and adults. each sample was taken from the systematic points of diagonal imaginary lines crossing the compact floating water hyacinth mat. regular observations at monthly interval were conducted starting from june 1999 until the end of january 2000. the experiment was also conducted to prove the hypotheses that there were predaceous fish affecting the survival trend of the weevils under field conditions of the lake. floating screen cages, measuring 125 x 175 x 75 cm each, were used as unit plot of the experiment. each cage contained water hyacinth plants covering about 0.25 % of the water surface. each cage was provided with 2 pairs of adult weevils, 23 eggs and 25 larvae. there were 10 cages split into two groups. group 1 was equipped with screen net on the root areas under water surface in such a way that the net did not affect the root development, while group ii was not. the plots were arranged in a randomized layout into two rows. the space between the rows was about 1 m and space between cages in each row was about 0.5 m. observations were done on the number of adult weevils and pupae in each cage on the 60* and 120th day after setting. relationship between the attacks of water hyacinth weevils and leaf blight using the same collected samples given above, the damage severity of each sample unit was assessed by estimating the area of the leaves attacked by the weevils and the fungus. the collected data were then analyzed on its correlation. results and discussion mode and rate of dispersal and distribution of the water hyacinth weevils under open field condition when an exotic biotic control agent of a weed is released in an open field, they are free to express their true behavior. the release using adult weevils, both n. eichhorniae and m bruchi, instead of other developmental stages is due to the fact that adult weevils are the most suitable as well as practical in handling. 20 biotropia no. 17, 2001 if the phytophagous insects are collected from the field, and cultured under artificial condition in the laboratory, they are generally to some extent, suffering from stress. culturing insects under such condition for a longer period may reduce their vitality, the longer the worse. how many generations of insects suffering a significant reduction in its vitality vary from species to species (donnelly 1997). adult weevils of either n. eichhorniae or tv. bruchi to be released in an open field were originated from outdoor screen cage cultures. in the outdoor screen cage, the weevils were expected to start adapting to the new environment. such treatments were intended to provide opportunity to recover the loss of vitality, because good quality is more important than quantity of a weed biotic control agent (julien et al. 1999) . as soon as the weevils were released in the open field, they might express whatever they like such as feeding on the preferred available host plants, breeding and spreading naturally. it was expected that soon after the release the adult weevils would not move far away from the release point and settle far from each other. if they disperse far from each other it may reduce the opportunity to meet each other for a mating process. data on the dispersal of released weevils collected from the experimental release site and recapture showed that the adult weevils spread through all cardinal directions (figure 1). figure 1. distribution of adult n. eichhorniae and n. bntchi after 2 (*) and 4 (o) weeks from the release time at situ bagendit lake 21 establishment of neochetina spp. kasnoe/a/. data from the first release of 307 marked weevils showed that two weeks after release the weevils dispersed to all cardinal directions with various distances. data collected on the 4th week after release showed a similar pattern but with longer distance. the longest distance on most cardinal direction almost reached the border of the water hyacinth mat within four weeks after release. outside of the water hyacinth mat, one of the border areas was swampy but with no water hyacinth, the two other borders were grassland and agricultural land and the rest of the borders (west and north) was the open water surface of the lake. with the assumption that the rate of dispersal from the release point was about the same, if the area of water hyacinth mat was larger, it is estimated that the adult weevils would have dispersed about 15 m within 4 weeks. besides, the limited size of the water hyacinth mat prevented spotting of the marked adult weevils. therefore, the plan to conduct observation on the 6th week was not carried out. the release of 700 marked adult weevils showed similar results. however, some parts of the water hyacinth mat on the western and northern parts were moved away by the wind before the first and second observation, respectively. therefore, there was no data representing weevil's distribution on western and northern parts from the release point. within a fortnight they were observed to move several meters away from the release point farther from the original site. it seemed that the adult weevils moved by crawling from plant to plant amongst the connected plants instead of flying. probably they could fly to move to disconnected water hyacinth plants. the data indicated that within four weeks after release, the adult weevils spread unevenly. the presence of young leaves which started to open from folded stage probably attracted the weevils. the young leaf is softer than the older , because the nitrogen content of young leaf is much higher than the older leaf (center and wright 1991). the difference in nutrient content was suspected to be one of the reasons of feeding preference. young leaf stage was most preferred by the adults during the day. after about two and a half years from the release of the first batch in the situ bagendit lake, adult feeding scars on the leaves could be found on most of water hyacinth plants growing on almost all parts of the lake. generally, adult weevils prefer to feed on young laminas which have just started to open from closed stage. assuming that water hyacinth suffered from adult feeding scars, the weevils have spread almost all over the water hyacinth area. it seems that within a few weeks after the release most of the adult weevils were still confined in one area where the distance between their standing position is relatively close. with such position, it is believed that the possibility of mating is still high. therefore, releasing water hyacinth weevils in a new environment by liberating few hundred adults of mixed sex at one site is safe. age structure of the water hyacinth weevils in the field after releasing in an open field the weevils have to adapt to the local conditions as well as to encounter various limiting factors. the ability to adapt to the 22 biotropia no. 17, 2001 new environment may be shown by some indicators such as the presence of damage symptom as the result of feeding activity, the presence of various developmental stages, the sustainable generations, and the most important indicator is the increased population density through time. monthly interval sample collection was carried out to evaluate the age structure of the weevils at the rdease site. it was indicated that the weevils have spread to most water hyacinth plants in the lake. figure 2 shows the population trend of each developmental stage of the weevils under field conditions. a trend of increase through time was shown by adult and a slight increase was also shown later by the pupal stages. figure 2. population trend of each developmental stage of neochetina spp. from june 1998-february 1999 in situ bagendit lake the number of adults was mostly higher than the pupae because there was an accumulation of the survived adult of the overlapped generations as the average longevity of the adult was much longer than the average developmental period of both pupa and larva. it was questioned why the increase in the number of adults in september was not followed by the increase in the number of both eggs and larvae. the trend of the number of larvae from month to month was more or less stable which might be an indicator of the limit of carrying capacity of water hyacinth plant in the lake. the decreasing trend from the number of eggs to larvae and pupae is shown in figure 3. such curve may provide information on the age structure and the survival rate of the weevils at the release site. about 5 % of eggs could survive up to the 23 establishment of neochetina spp. kasno et al. pupal stage and less than 5 % may survive up to the adult stage. this estimated survival rate was much lower than the reproductive potential determined under laboratory condition. tjitrosoedirdjo et al. (1999) reported that the potency of weevil reproduction could reach about 50%. figure 3. structure of developmental stages ofneochetina spp. at situ bagendit lake. the difference between potential survival rate of the weevils in the laboratory and the fact in the field may be attributed to various limiting factors resulting in low survival rate. it was suspected that some predaceous fish could cause the low survival rate of the weevils in the situ bagendit lake. an experiment was then set up to proof the possible attack from predaceous fish. the results are presented in table 1. table 1. average number of pupal and adults stages of water hyacinth weevils and damage severity under simulation to test the role of predators in inhibiting the population increase 24 biotropia no. 17, 2001 table 1 indicates that the number of both pupae and adult weevils under screen cages equipped with screen net on its base after two and three and a half months were higher than the cages without screen net on the base. the cages which were equipped with screen net prevented from possible predation of the weevils. unpublished data collected from a simulation experiment showed that various fish may consume root-bearing pupae under laboratory conditions. there were various fish such as common carp (cyprinus carpio), tilapia, oreochromis mosambicus, cat fish, found in situ bagendit lake. spiders were reported to predate and prey on tv. bruchi larvae (tjitrosoedirdjo 1998). at situ bagendit lake, spiders were often found, so they might be one of the natural enemies of water hyacinth weevils. the slow population increase of the weevils at the release site may lead to a conclusion that neochetina spp. are not effective control agents. it was reported that effectiveness of an biotic control agent may be affected by various factors such as by climate (jayanth and visalakshy 1990; grodowitz et.al. 1991). although the population increase in situ bagendit lake was very low, the increasing trend during the observation period confirmed that neochetina weevils have established in the lake. therefore, additional releases of same biological control agents are not necessary. relationship between the attacks of water hyacinth weevils and fungus a plant pathogenic fungus may disperse from an initial point of infection to other host plants through a direct contact with the infected parts. aside of that, spores and mycelia may be transferred to other host plants by carrying agents. an adult weevil of neochetina may act as a carrying agent of the spores and mycelia of the fungus, a. eichhorniae. a monthly interval observation carried out to assess the damage severity of water hyacinth plants by each biological control agent in situ bagendit lake is presented in table 2. it was obvious that there was no interaction between the attack of adult weevils and the fungus. data obtained from laboratory experiment showed that a higher damage severity of water hyacinth due to both biotic control agents caused a more serious damage (kasno et al. 1998). probably the level of both weevil populations and the fungus colony were not high enough to cause such speculated effect. according to kasno et al. (1998) various fish such as tilapia and common carp at the feeding test showed that those fish predated on pupae which were in the roots of water hyacinth. aside from fish, frogs and eel fish might also attack the weevils. under the simulated experiment, the weevil's elytra were found in the crop of the frog. at situ bagendit lake, spiders were often found, so they might be the natural enemies of the weevils. according to tjitrosoedirdjo (1998) spiders were able to catch and prey on n. bruchi larvae. 25 establishment of neochetina spp. kasnoetal. correlation between the damage severity of water hyacinth due to the weevils and fungus the percentage of weevil attack and the number of feeding scars on the second youngest leaf of water hyacinth is shown in table 2 and appendix 1. the attack level of the pathogenic fungi a. eichhorniae and the weevil neochetina spp. at situ bagendit lake did not show significant correlation in causing damage to water hyacinth, because the attack was relatively low. table 2. correlation between damage severity of water hyacinth plants due to the attacks of weevils and fungus at situ bagendit lake by the year 1999 2000 note: figures followed by the same letter in the same column are not significantly different according to duncan multiple range test at 95% confidence level. conclusions in the spatial distribution, adult of water hyacinth weevils actively dispersed in all cardinal directions following the presence of water hyacinth. the distribution pattern of the adults of water hyacinth weevils was initially contagious or clumped, but later on they tended to spread uniformly. neochetina. bruchi and n. eichhorniae released at situ bagendit lake since 1996 have become widely established. the survival rate of water hyacinth weevils at situ bagendit lake being estimated by the age structure showed a much lower rate than its reproduction potential found in the laboratory. the damage severity of water hyacinth plants due to a. eichhorniae and neochetina spp. at situ bagendit lake was not significantly correlated. 26 biotropia no. 17, 2001 references bashir, m.o. 1984. the establishment and distribution of natural enemies of waterhyacinth released in sudan, short communication. tropical pest management 30(3):320-323. caunter, i.g. and s. mohamed. 1992. effect ofneochetina eichhorniae on waterhyacinth in malaysia and its interaction with myrothecium roridum. in: proc. of the 3rd international conference on plant protection in the tropics (eds. p.a.c. ooi. g.s. lim and p.s. teng) 6:261-264. mapps, kuala lumpur, malaysia. center, t.d., steward, k.k., and m.c. bruner. 1982. control of waterhyacinth (eichhorniae crassipes) with neochetina eichhorniae (coleoptera:curculionidae) and a growth retardant. weed science 30(5):453-457. center, t.d. and a.d. wright. 1991. age and phytochemical composition of water hyacinth (pontederiaceae) leaves determine their acceptability to neochetina eichhorniae (coleoptera: curculionidae). j. environ. entomol. 20(l):323-334. charudattan, r. 1986. integrated control of waterhyacinth (eichhorniae crassipes) with a pathogen, insects, and herbicides. weed science 34 (suppl.) 1):26-30. donnelly, g. 1997. mass rearing insects for biological control of weeds. in biological control of weeds: theory and practical application. m. julien and g. white (eds). aciar monograph no 49. canberra: 89-96 galbraith, j.c. 1987. the pathogenicity of an australian isolate of acremonium zonatum to waterhyacinth, and its relationship with the biological control agent, neochetina eichhorniae. australian journal of agricultural research 38:219-229. goyer, r.a. and j.d. stark. 1984. the impact of neochetina eichhorniae on waterhyacinth in southern louisiana. journal of aquatic plant management 22:57-61. grodowitz, m.j., r.m. steward and a.f. confrancesco. 1991. population dynamics of waterhyacinth and the biological control agent neochetina eichhorniae (coleoptera: curculionidae) at a southern texas location. environmental entomology 20(2):652-660. harley, k.l.s. 1990. the role of biological control in the management of water hyacinth, eichhornia crassipes. biocontrol news and information 11(1): 11-22 jayanth, k.p. 1988. successful biological control of waterhyacinth (eichhorniae crassipes) by neochetina eichhorniae (coleoptera: curculionidae) in bangalore, india. tropical pest management. 34(3):262266. jayanth, k.p. and p.n.g. visalakshy. 1990. studies on drought tolerance in water hyacinth weevils neochetina eichhorniae and n. bruchi (coleoptera: curculionidae). j. biological control 4(2): 116119. julien, m.h and w.w. griffith. 1998. biological control of weeds: a world catalogue of agents and their target weeds. 4th ed. cabi, uk. 223p. julien, m., w.w. griffith and a.d. wright. 1999. biological control of water hyacinth. aciar monograph no.60. canberra. 87p. kasno, okky s. dharmaputra, sunjaya, a.s.r. putri and h.s. handayani. 1998. establishment of water hyacinth weevil, neochetina bruchi. seamed biotrop. 26p. 27 establishment of neochetina spp. kasno et al. saraswati, r. 1980. the possible use of the combination neochetina eichhorniae warner — myrothecium roridum tode ex fr. to control waterhyacinth (kemungkinan penggunaan kombinasi kumbang moncong neochetina eichhorniae wamer dan jamur penyakit myrothecium roridum tode ex fr. untuk pengendalian eceng gondok eichhornia crassipes (mart.) solms. faculty of mathematic and natural science, padjajaran university, bandung. 44 p. tjitrosoedirdjo, s.s. 1998. biological control of water hyacinth, water fern and giant mimosa using insect natural enemies (pengendalian hayati eceng gondok, kiambang dan klampis air dengan menggunakan serangga musuh alami.) research report of competitive grant of higher education, ii/5. directorate general of higher education. department of education and culture. wright, a.d. 1979. preliminary report on damage to eichhornia crassipes by an introduced weevil at a central queensland liberation site. proceeding of the seventh conference of the asian pasific weed science society, p: 227-229. 28 biotropia no. 17, 2001 appendix 1. analysis of variance on the percentage of damage severity due to weevils and fungi, and the number of spots at the second youngest leaves of water hyacinth at situ bagendit, garut regency 29 microsoft word 29 bio fropia no. 22, 2004: 2 9 3 9 isolation and selection of alkaline proteolytic bacteria from leather processing waste and enzyme characterization budiasih wahyuntari', nisa r mubarikand marita anggarani2 agency for the assessment & application of technology, jakarta, indonesia ²bogor agricultural university, bogor, indonesia abstract the aims of this experiment were to isolate alkaline protease producing bacteria from leather processing waste, and to study the biochemical properties of the enzyme produced by the selected bacteria. nine bacterial isolates incubated at 37"c, revealed proteolytic activity on skim milk containing media. four isolates were grown at ph 9 and another four isolates at ph 10 and only one isolate at ph 11. however, in further subculture, there were only three isolates that showed proteolytic activity, namely, d2, d7, and d l l . among the three isolates, isolate d2 was the highest protease producer. the highest protease production (36.5u/l) was reached after a 36-hr fermentation at ph 9. the optimum activity of d2 protease was observed at ph 8 and 60"c. the enzyme was stable at ph range of 7-10, and at temperature of 52-62"c. in the presence of 5mm edta or pmsf, the crude enzyme activity decreased to 7.04% and 23.29% respectively, which indicated that the enzyme might be a metal dependent serine protease. zymogram analysis revealed the molecular weight of the enzyme was about 42.8kd. keywords: leather/ waste/protease/alkaline introduction alkaline protease, along with other alkaline enzymes such as amylase and cellulose, has been used on an industrial scale. microbial akaline protease is the enzyme that dominate commercial application, mainly for laundry detergents. alkaline protease with elastolytic and keratolytic activity is used in the tannery industry for dehairing and the bating of skin and hides (taylor et al. 1987). alkaline protease from bacillus spp. was reported to be used to decompose gelatin on x-ray film for silver recovery (horikoshi 1996). other uses of alkaline protease are for medical purposes, food and chemical industries, as well as in waste treatment plants. alkaline protease has been used in industrial scales and has been an attractive option for researchers for the last three decades since the first publication concerning the enzyme by horikoshi (1971). studies on exploration of alkaline protease producing microorganisms, which produce novel enzymes have been extensively carried out by researchers all over the world (banerjee et al. 1999; singh et al. 2001; johnvesly & naik, 2001; kanekar et al. 2002, joo et al 2002; gessesse et al. 2002; moreira et al. 2003). 29 biotropia no. 22, 2004 most of the alkaline protease producing microorganisms were isolated from natural, as well as manmade alkaline environments. such microorganisms were also isolated from non-alkaline habitats, such as neutral, and acidic soil (kumar et al. 1999). this experiment aims to isolate alkaline protease producing microorganisms from alkaline waste of leather processing and to study the biochemical properties of the enzyme of the selected isolate. materials and methods isolation, screening and identification of microbial strains the samples for screening were taken from the waste of different leather processing steps including soaking (ph 10), liming (ph 13), and deliming (ph 8.5-9.0) from pt. muhara dwi tunggal laju, and pt. gunung putri, in bogor. the microorganisms were enriched on agar plates containing (gram per liter) 2 gr yeast extract, 0.4 gr (nh4)2so4, 1 gr mgso4.7h2o, 0.5 gr cacl^.2 h2o, 0.8 gr kh2po4, 40 gr agar, and 8 gr skimmed milk. the ph medium was adjusted to 8-11 using naoh. the microorganisms were incubated at 24-40°c for 3 days. the colonies which revealed proteolytic activity were then isolated and screened further, using the same composition of medium. the isolates were screened based on proteolytic index which defined as a ratio of clear zone and colony diameter. the isolates that had the highest proteolytic index were screened further for protease production in luria broth containing 1% peptone, 0.5 gr yeast extract and 0.5% nacl at ph 9, 10 and 11. in the same medium composition, they were then incubated in a shaking incubator for 19 hours at 37°c. the fermented broth was then centrifuged at 4000 rpm for 20 minutes. the protease activity was finally assayed and the isolates were selected based on the protease production. protease assay protease activity was measured using a method suggested by walter (1984). the method was based on amino acid hydrolyzed by the enzyme and was calculated as tyrosine. one unit of enzyme activity was defined as the amount of enzyme which liberate one micromol tyrosine per minute at certain condition. preparation of protease the selected isolate was cultured in 5 l fermentor containing 2.5 l enzyme media production with the same composition as mentioned above with addition of 0.2% skim milk as an inducer. after a 36-hr incubation at 37°c, the fermented broth was filtered using microfilter, and then concentrated further using 10 kd membrane ultrafilter module (minitan, milipore). 30 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. effect of ph and temperature studies the effect of ph on protease activity produced by the selected isolates was determined at 37°c in 50 mm tris-hcl buffer at ph 7-8 and in glycine-naoh at ph 9-11. to determine the effect of temperature on protease activity, the assay was performed in 50 mm tris-hcl buffer (ph 8) at temperature range of 30-70°c. effect of protease inhibitors and metal ions the concentrated enzyme solution was incubated in 2 and 5mm edta or pmsf in 50 mm tris-hcl at ph 8 for 30 minutes at 5°c. the same concentration of 2 and 5 mm mgcl2, cacl2 , zncl2 , mncl2 , fecl3 and feso4 were also incubated with the enzyme at 5°c for 30 minutes. residual activity was measured under optimum ph and temperature. protease activity assayed in the absence of inhibitors and metal ions was considered as 100%. polyacrylamide gel electrophoresis and zymogram analysis zymogram method was employed to determine the molecular weight of the protease. the concentrated enzyme solution was loaded into page containing skimmed milk (0.2%) which copolymerized with 7.5% acrylamide. electrophoresis was run at 110 v, 90 ma for 1.5 hours. after completing electrophoresis, the gel was soaked in 50 mm tris-hcl containing 5 mm cacl2 then incubated at optimum ph and temperature of the enzyme. visualization of the enzyme staining activity was carried out by staining the gel in commassie brilliant blue solution. clear band on the skim milk containing electrophoresis gel indicated the protein band which had shown proteolytic activity. the standard molecular weight marker used was hmw protein standard marker (amersham). results and discussion isolation of protease-producing bacteria there were 9 isolates which showed proteolytic activity based on clear zone formation on skim milk containing media. four isolates grew well on media at ph 9m, another four isolates at ph 10, and only one isolate at ph 11. table 1 shows the physical properties, proteolytic index, and protease activity of the isolates. proteolytic activity of isolate t4 decreased in the following experiment, and therefore the isolate was excluded. isolate d2, d7 and d l l were selected based on their proteolytic index and ability to produce protease in submerged culture. biochemical characteristics of the selected isolates are displayed in table 2. based on their physical, and biochemical characteristics, it can be concluded that the isolates (d2, d7 and d l l ) most likely belong to bacillus spp. 31 selection of bacterial strain. further selection of the best proteolytic bacteria was based on the productivity of the isolate at die highest ph. all the isolates screened were grown at three different levels of ph (9; 10 and 11). however, all isolates did not grow well at ph 32 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari el al. 11. figures 1, 2 and 3 reveal growth and protease production rates of d2, d7, and d l l , respectively. the growth rate of isolate d2 was only slightly better at ph 9 as compared to ph 10. however, its protease production rate at ph 9 was higher than that at ph 10. the highest protease production of isolate d2 was at 36-hour fermentation with a protease concentration of 3.65 u/l. the growth rate of isolate d7 at ph 9 was slightly better than at ph 10. however, the protease production rate was higher at ph 10 after 36 hours incubation with the activity of 10.6 u/l. figure 3 shows that the growth rate and protease production of isolate dl 1 was better at ph 9 and with the highest protease production at 24 hour incubation time with enzyme activity of 5.8 u/l. the protease produced by all isolates in skim milk containing medium was much higher as compared to enzyme produced in luria broth used in early selection of isolates. the data indicated that the enzyme produced by all isolates observed were inducible ones. among the three isolates observed, isolate d2 produced the highest protease, which showed that the isolate was the most tolerable to alkaline condition. it was assumed that the enzyme belonged to alkaline protease family. therefore, t he enzyme of the isolate was selected for further study.   isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. effect of ph and temperature study on protease activity optimum activity of d2 isolate protease was observed at ph 8 and 60uc (figure 4). the enzyme relative activity was 70% at ph 7 and 9.5 and at 52 and 62°c. these data show that the enzyme is an alkalo and thermotolerant one. studies on isolation and characterization of alkaline protease have been ongoing for the last three decades (horikoshi 1996 and kumar and takagi, 1998). these are the most recent studies on the subject. singh et al. (2001) reported that optimum activity of bacillus sp. ssr1 was reached at the range of ph 8-11, 40°c, the optimum activity was shifted up to 45°c due to the presence of calcium ions. thermostable alkaline protease from bacillus sp. jb-99 reported by johnvesly and naik (2001) had an optimum activity at ph 11 and 70°c. this bacterium was isolated from sugarcane molasses at ph 5.8. but with a chemically defined medium, the bacterium was able to produce alkaline thermostable protease. gessesse et al. (2002) succeeded in isolating two bacterial strains from lake abjata (an alkaline soda lake in ethiopia), using chicken feather meal as the sole source of nitrogen and carbon. the bacteria were identified as nestemkonia sp. al-20 and b. pseudofirmus al-89. the optimum enzyme activities of al-20 and al-89 were observed at ph 10, 70°c and at ph 11, 50°c, respectively. the al-20 protease was calcium independent whereas the al-89 was calcium dependent. both enzymes were indicated as serine protease. bacillus, horikoshii protease had optimum activity at ph 9, 45"c, and the enzyme was stable at the ph range of 5.5-12 and temperature up to 50°c (joo et al, 2002). effect of protease inhibitor and divalent ions the presence of 5 mm phenyl methyl sulfonyl fluoride (pmsf) reduced the protease activity down to 23.3% which indicated the enzyme belongs to serine protease. five mm eot a almost totally inhibiting the enzyme with a remaining activity of 7% only, which showed that the enzyme activity depends on the presence of metal ion. the study on the effect of several metal salts revealed that calcium, zinc and manganese ion increased the activity of the enzyme up to 117.2%, 111.3%, and 114.4%, respectively. whereas, magnesium, ferry and ferrous ions at 5 mm decreased the enzyme activity down to 91.9%, 64.8%, and 71.3%, respectively. previous studies on alkaline proteases reported that for maximum activity, some serine alkaline proteases were dependent on divalent ion such as calcium, magnesium and manganese (kumar & takagi, 1998). however, gessesse et al (2002) reported that serine alkaline protease activity produced by nesternkonia sp was not dependent on the presence of calcium ion. the enzyme was not affected by pmsf, but, it was inhibited by another serine protease inhibitor 3,4-dichlorocoumarine at concentration of 100 (j.m,. 36 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. control edta pmsf mg2+ ca2+ zn 2+ mn 2+ fe 3+ fe 2+ inhibitor/cation d : 2 mm, • : 5 mm figure 6. et'fecl of protease inhibitor and cation on enzyme activity molecular weight of the enzyme zymogram analysis revealed that the molecular weight of the enzyme was approximately 42.8kd (figure 7). that molecular weight of most bacillus alkaline proteases reported ranges from 15 to 45 kd (kumar & takagi, 1998). a more recent report by singh et al (2001) also stated that the molecular weight of serine alkaline protease produced by bacillus sp ssr1 was 29kd which was also lower than the previous reports . figure 7. zymogram otisolate d2 protease 37 biotropia no. 22, 2004 conclusion in this exploration of protease-producing bacteria from leather processing waste, only few bacteria that produce alkaloprotease were discovered. among the three isolates screened. isolate d2 produced the highest protease in submerged culture medium. based on physical and biochemical properties of the isolate, d2 most likely belongs to bacillus sp. the optimum activity of the enzyme produced was observed at ph 8, 60°c. the enzyme retained 70% of its activity at the range ph of 7-10. at ph 8, the enzyme also retained more than 70% of its activity at the range temperature of 52-62°c. the enzyme was identified as serine protease and its activity was affected by the presence of calcium, zink and manganese ion. the molecular weight of the enzyme as determined using zymogram analysis was approximately 42.8 kd. further investigation is needed to identify the isolate using more reliable means such as 16rrna sequencing the enzyme can be used in a condition that is slightly alkaline at around ph 8 and at 50-60°c. however, to find suitable application of the enzyme, other considerations have to be taken into account such as type of substrate of the respected process. hence, it is necessary to investigate different kinds of protein substrates such as hemoglobin, collagen, gelatin, keratin etc. selecting the best medium to produce the enzyme is also necessary, as well as more thorough characterization of the enzyme such as the stability of the enzyme toward ph, temperature, and some additives that might be used in enzyme application. references banerjee uc, rk sani, w azmi and r soni. 1999. thermostable alkaline protease from bacillus brevi.t and its characterization as a laundry detergent additive, process biochem. 35:213-219. gessesse a, hk rajni, ba gashe arid b mattiasson. 2002. novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. enzyme & microbial tech. 6250:1-6. horikoshi, k, 1971. production of alkaline enzymes by alkalophilic microorganisms. part 1. alkaline protease produced by bacillus no. 221. agric. biol. chem. 36:1407-1414. horikoshi k, 1996. alkaliphiles from an industrial point of view, eems microbiol riev, 18:259-270. johnvesly b. and gr naik. 2001, studies on production of thermostable alkaline protease from thermophilic bacillus sp jb-99 in a chemically defined medium, process biochem. 37:139-144. joo hs, sg kumar, gc park, kt kirn, sr paik and cs chang. 2002. optimization of the production of an extracellular alkaline protease from bacillus horikoshii, process biochem, 38:155-159. kanekar pp, ss nilegaonkar, ss sarnaik and as kelkar. 2002. optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in india, bioresource tech. 85:87-93. kumar cg, and h takagi 1998. microbial alkaline proteases: from a bioindustrial viewpoint, biochem adv, 17:561-594. 38 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. moreira ka, ts porto, mfs teixeira, alf porto and jl lima filho. 2003. new alkaline protease from nocardiopsis sp partial purification and characterization, process biochem.oo: 1-6 . singh j, n batra, and rc sobti 2001, serine alkaline protease from newly isolated bacillus sp ssr1, process biochem, 36:781-785. taylor mm, dg bailey and sh feaiheller. 1987. a review of the use of enzymes in tannery, j. ame leather chem assoc, 82:153-165. walter he. 1984. proteinases in methods of enzymatic analysis, (ed. h.u. bergmeyer and m qrassl), verlag chemie. weinheim-deerfield, florida-basse!, vol. 5:271-276. 39 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf microsoft word 32 biotropia no. 21,2003 : 32 -44 control of aflatoxigenic aspergillus flavus in peanuts using nonaflatoxigenic a. flavus, a. niger and trichoderma harzianum okky setyawati dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia asmarina s.r. putri, ina retnowati and santiambarwati seameo biotrop, p.o. box 116, bogor, indonesia abstract the effects of nontoxigenic aspergillus flavus, a. niger and trichoderma harzianum inoculated into planting media on toxigenic a. flavus infection and its aflatoxin production in peanut kernels at harvest were investigated together with (1) the moisture content of planting media before peanut planting, at the time of inflorescence, and at harvest, (2) the population of aflatoxigenic and nonaflatoxigenic a. flavus, a. niger and t. harzianum in peanut planting media before peanut planting, at the time of inflorescence, and at harvest, (3) the moisture content of peanut kernels at harvest, and (4) toxigenic a. flavus invasion in peanut plant parts (roots, stems, petioles, leaves and flowers) at the time of inflorescence. the fungal isolates were inoculated into planting media at the same time with the planting of peanut seeds. peanut plants were grown under glasshouse conditions. treated planting media were inoculated with the combined use of (1) toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, and (3) toxigenic a. flavus and t. harzianum. planting media inoculated only with each fungal isolate and uninoculated planting media were used as controls. two watering treatments of peanut plants were carried out, i.e. watering until harvest and not watering for 15 days before harvest. the populations of the fungal isolates in the planting media and peanut kernels were determined using dilution method followed by pour plate method; the percentages of toxigenic a. flavus and test fungal colonizations in peanut plant parts were determined using plating method; the moisture content of planting media and peanut kernels were determined using oven method; the aflatoxin content of peanut kernels was determined using thin layer chromatography method. the results indicated that at the time of harvest the decrease in moisture contents of planting media not watered for 15 days before harvest was higher than those watered until harvest. the lowest population of toxigenic a. flavus was in planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus at the time of inflorescence and at the time of harvest. toxigenic a. flavus could invade the roots, stems and flowers of peanut plants. the lowest percentage of invasion was on the plant parts which planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus. the moisture content of peanut kernels originated from watered plants until harvest were higher than those not watered for 15 days before harvest. the population of toxigenic a. flavus in peanut kernels derived from the plants whose planting media were inoculated with the combined use of toxigenic a. flavus and each test fungi, was lower than those inoculated only with toxigenic a. flavus. it indicated that the test fungi inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin was only detected in peanut kernels originated from one plant whose planting medium was inoculated only with toxigenic a. flavus and the plant was watered until the time of harvest. toxigenic a. flavus infection and aflatoxin production were not influenced by planting media which were not watered for 15 days before harvest. keywords: biocontrol / aflatoxigenic / nonaflatoxigenic / aspergillus flavus i aspergillus niger i trichoderma harzianum i peanuts 32 biotropia no. 21, 2003 introduction aflatoxins are potential hepatotoxic and carcinogenic metabolites produced by the fungi aspergillus flavus, a. parasiticus, and a. nomius. contamination of afla-toxins occurs when peanut kernels become infected by the three fungal species under drought stress before harvest, during the drying phase in the field, or under unsuitable storage conditions. pitt and hocking (1996) reported that 45, 33 and 22% of 215 peanut samples collected from farm storage, middlemen and retailers in bogor and yogyakarta contained aflatoxins of more than 50, 300 and 1000 ppb, respectively. levels of 1000 ppb of aflatoxins will cause liver damage in man and animals. lower levels of aflatoxins consumed from peanut products can cause liver cancer and premature death in humans, as well as reducing productivity of livestock. based on the report of the 23rd session of the joint faoavho food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commissions adopted 15 ppb as the maximum level of total aflatoxins to be contained in peanuts intended for further processing. the entry of aflatoxin in the peanut plant could be from the roots, flowers, leaves damaged by insects, and from air or dust. the most important point of entry to the developing peanut, however, is directly from the soil surrounding it. so, the point of application of any biocontrol measure such as competitive exclusion should be in the soil of peanut growing fields. there are only few reports on the reduction of aflatoxins by competitive fungi. ehrlich et al. (1985) reported that by growing a. parasiticus in mixed culture with penicillium oxalicum, toxin productions of both species were reduced. to assess this effect under more practical conditions, wicklow et al. (1988) inoculated corn kernels with toxigenic a. flavus at preharvest, with or without a range of other fungi present. they found that much lower levels of preharvest aflatoxin were produced in the presence of some other fungal species. dorner et al. (1999) found that aflatoxin contamination of corn was reduced when the soil of corn plots were inoculated with nonaflatoxigenic strain of a. flavus. dharmaputra et al. (2001) reported that non-aflatoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130 were antagonistic to aflatoxigenic a. flavus bio 2128 in vitro. aspergillus niger was the most promising fungal antagonist, because it caused the highest percent inhibition of aflatoxin production of a. flavus bio 2128. the objectives of this study were to investigate the effects of nontoxigenic a. flavus, a. niger and t. harzianum inoculated into the peanut planting media on toxigenic a. flavus infection and its aflatoxin production in peanut kernels at harvest. investigations were also carried out on (1) the moisture contents of planting media before peanut planting, at the time of inflorescence, and at harvest, (2) the population of aflatoxigenic and nonaflatoxigenic a. flavus, a. niger and t. harzianum in peanut planting media before peanut planting, at the time of inflorescence and at harvest, (3) the moisture content of peanut kernels at the time of harvest, and (4) toxigenic a. flavus invasion in peanut plant parts (roots, stems, petioles, leaves and flowers) at the time of inflorescence. 33 control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. materials and methods planting media, peanut variety, isolates of toxigenic a. flavus and test fungi a mixture of soil (latosol type), sand and casting (2:1:1) was used as planting medium, while peanut variety komodo was obtained from the research institute for legumes and tuber crops (rilet) in malang. four fungal isolates were used: toxigenic a. flavus bio 2128, nontoxigenic a. flavus bio 2127, a. niger bio 2129, and t. harzianum bio 19130. the latter three fungal isolates were used as test fungi. preparation of a. flavus , a. niger and t. harzianum inocula mixtures of ground rice bran and tap water (4:3) were placed in transparent polyethylene bags (125 g/bag). they were then sterilized in an autoclave for 1 h, and incubated at room temperature for one night. for the time being, each fungal isolate was grown on potato dextrose agar in petri dishes (diam 9 cm) and incubated at room temperature for 6 days. five ml of spore suspension (106 spores/ml) of each fungal isolate was inoculated into the mixture of sterilized ground rice bran and tap water. they were then incubated at room temperature for 7 days to obtain the inocula of planting media. inoculation of toxigenic a. flavus and test fungi into planting media, and seed planting pasteurized planting media were placed in black polyethylene bags (15 kg/bag). the inocula of planting media were then mixed with planting media at the time of germinated-seed planting (1 seed^ag). treated planting media were inoculated with the combined use of (1) toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, (3) toxigenic a. flavus and t. harzianum. the comparison of toxigenic a. flavus and each test fungal isolate was 1:1 (one portion was equivalent with 30 g of inoculum). planting media inoculated only with each fungal isolate and uninoculated planting media were used as controls. six replicates (6 bags) were used for each treatment (including the controls). the bags with the peanut plants were placed under greenhouse conditions. two watering treatments of peanut plants were carried out, i.e. (1) watering until harvest and (2) not watering for 15 days before harvest. sterilized tap water was used for watering peanut plants. the individual plants were watered with the same volumes of sterilized tap water. sampling of planting media sampling of planting media were conducted : before peanut-seed planting, but after each treated planting medium as well as each control (except uninoculated planting media) have been mixed with fungal 34 biotropia no. 21,2003 inocula homogeneously. they were then placed in clean polyethylene bags (about 1 kg/bag/treatment or control). at the time of inflorescence and at the time of harvest. the peanut plants were pulled prior to sampling of planting media. each treated planting medium as well as each control was mixed homogeneously; they were then placed in clean polyethylene bags (about 1 kg/bag/treatment or control). each sample of planting media (about 1 kg) was divided several times manually to obtain working samples for moisture content and fungal analysis, and for reserved sample. determination of moisture contents of planting media; population of toxigenic a. flavus and test fungi in planting media before peanut seed planting, at the time of inflorescence and at the time of harvest moisture content of planting media derived from each bag was determined using oven method at 105°c for 12 hours (johnson et al. 1960). the population of each fungal isolate in planting media before peanut seed planting, at the time of inflorescence and at the time of harvest was determined using serial dilution method, followed by pour plate method (johnson and curl 1972) on pda containing chloramphenicol (100 mg/l media). sampling of peanut plants at the time of inflorescence at the time of inflorescence (25 30 days after planting), peanut plants grown on treated planting media as well as on the controls, were pulled. their roots, stems, petioles, leaves and flowers were then cut into pieces (10 pieces/plant) about 1 cm long, surface sterilized using 0.5% na-hypochloride for one minute, rinsed with sterile distilled water, dried on sterilized filter paper, and finally plated on potato dextrose agar (pda) containing chloramphenicol (100 mg/l media). the percentage of each fungal isolate colonization in various plant parts was determined using the following formula: number of plant part pieces colonized by each fungal isolate plant part pieces colonized = number of plant part pieces x 100% by each fungal isolate plated on agar media determination of moisture content of peanut kernels; population of toxigenic a. flavus and test fungi, and aflatoxin content in peanut kernels peanuts were harvested at 90 days after planting. they were then shelled manually and aseptically. peanut kernels derived from treated planting media and 35 control of aflatoxigenic aspergillus flaws in peanuts okky s. dharmaputra et ai the controls were divided using a box sample divider to obtain working samples for moisture content, fungal and aflatoxin analyses. the moisture content of peanut kernels was determined using the oven method (bsi 1995). the population of toxigenic a. flavus and test fungi was determined using serial dilution method, followed by pour plate method on pda containing chloramphenicol (100 mg/l media). aflatoxin content in peanut kernels was determined using thin layer chromatography method'(ao ac 1995). statistical analyses the experimental design used in this study was completely randomized factorial design with two factors, i.e. the kind of fungal inocula in planting media and watering of peanut plants. the kinds of fungal inocula consisted of (1) the combined use of toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, (3) toxigenic a. flavus and t. harzianum, (4) toxigenic a. flavus, (5) nontoxigenic a. flavus, (6) a. niger, (7) t. harzianum, and (8) uninoculated planting media.-peanut plants (1) were watered until harvest and (2) not watered for 15 days before harvest. results and discussion moisture content of planting media before peanut seed planting, at the time of inflorescence and harvest the fungal inocula did not give any significant difference on the moisture contents of planting media at the time of inflorescence. besides, the moisture contents of planting media before peanut planting were not significantly different from the time of inflorescence. it indicated that up to the time of inflorescence, the moisture contents of planting media were always stable, although the media were inoculated with various fungal inocula. the moisture content of planting media treated with various fungal inocula and the time of watering decreased from before peanut planting up to harvest. fungal inocula and their interaction with time of watering were not significantly different with the decrease of moisture contents, while time of watering showed significant differences. table 1 shows that the decrease in moisture content of planting media not watered for 15 days before harvest (22.9%) was higher than that watered until harvest (11.1%). 36 biotropia no. 21,2003 table 1. the decrease in moisture content of planting media before peanut planting until harvest time treatment decrease of moisture content (%) watered planting media 11.09 a not watered planting media 22.85 b numbers followed by the same letter do not differ significantly according to duncan multiple range test at 95% confidence level population of toxigenic a. flavus and test fungi in planting media before peanut planting, at the time of inflorescence and at the time of harvest in the planting media, populations of toxigenic a. flavus and test fungi in-creaseed at the time of inflorescence as well as at the time of harvest. at the time of inflorescence, fungal inocula gave significant differences on the increase of toxigenic a. flavus and test fungal populations. at the time of harvest, fungal inocula and time of watering gave significant differences on the increase of fungal populations. the increase of toxigenic a. flavus population in planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus was lower compared with planting media inoculated (1) only with toxigenic a. flavus, (2) with the combined use of toxigenic a. flavus and a. niger, and (3) with the combined use of toxigenic a. flavus and t. harzianum, either at the time of inflorescence or at the time of harvest (table 2). the increase of each test fungal population in either the planting media inoculated with each test fungi or in the planting media inoculated with the combined use of toxigenic a. flavus and each test fungi were higher than the increase of toxigenic a. flavus population in the planting media inoculated only with toxigenic a. flavus and in the planting media inoculated with the combined use of toxigenic a. flavus and test fungi, either at the time of inflorescence or at the time of harvest (table 2). it indicated' that the growth of toxigenic a. flavus was more inhibited compared with the growth of test fungi, either in the planting media inoculated with each fungal isolate or in the planting media inoculated with the combined use of toxigenic a. flavus and test fungi. at the time of harvest, the increase of fungal population in the watered planting media was higher than that not watered for 15 days before harvest (table 3). it was related to the decrease of moisture content which was higher in the not watered planting media compared with the watered planting media (table 1), consequently fungal growth was inhibited. 37 at the time of harvest in the uninoculated planting media, three fungal isolates were found: nontoxigenic a. flavus, cladosporium cladosporioides and t. harzianum. it was assumed that fungal contamination originated from the air or from peanut seeds. 38 biotrop1a no. 21,2003 toxigenic a. flavus and test fungal invasion in peanut plants at the time of inflorescence pitt et al. (1991) stated that the invasion of toxigenic a. flavus in peanut plants could originate from the soils or from peanut seeds grown under greenhouse conditions. toxigenic a. flavus invasion was observed in peanut plants where the planting media were inoculated with the fungus as well as its combination with the test fungi (table 4). its percentage of invasion occurring in plant parts adjacent to the soils (root and stem) was higher than the other parts (petioles and leaves). pitt et al. (1991) reported that a. flavus could invade all plant parts, with the highest invasion occurring in plant parts adjacent to the soils. the percentage of toxigenic a. flavus invasion in plants where the planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus, toxigenic a. flavus and a. niger was lower than that only inoculated with toxigenic a. flavus. nevertheless, the percentage of toxigenic a. flavus invasion was higher in plants where the planting media were inoculated with the combined use of the fungus and t. harzianum (table 4). in the planting media inoculated with the combined use of toxigenic a. flavus and nontoxigenic a. flavus, also the combined use of toxigenic a. flavus and a. niger, the invasion of toxigenic a. flavus were only in the roots, while in the planting media inoculated only with toxigenic a. flavus, this fungus could invade roots, stems and flowers. in the planting media inoculated with toxigenic a. flavus and t. harzianum, toxigenic a. flavus could invade all the plant parts. the percentage of toxigenic a. flavus invasions was related to the increase of toxigenic a. flavus population in the planting media (table 2). the lowest increase of toxigenic a. flavus population was in the planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus, while the highest was in the planting media inoculated with the combined use of toxigenic a. flavus and t. harzianum. in the planting media inoculated only with toxigenic a. flavus as well as those inoculated with the combined use of toxigenic a. flavus and t. harzianum, toxigenic a. flavus could also invade flowers (table 4). it is related to the position of peanut flowers, i.e. adjacent to the planting media. if the flowers come in contact with the planting media, they could be invaded by toxigenic a. flavus. toxigenic a. flavus derived from the planting media can systemically invade peanut plant parts adjacent to the soil up to the plant parts above the soil surface. the fungus can also invade young peanut plants. based on microscopic examination, little or no damage was observed in the cells of stems invaded by a. flavus (pitt et al. 1991). toxigenic a. flavus invasion did not cause any disease in peanut plants. 39 test fungi could also colonize roots, stems, petioles, leaves and flowers (table 4). some plant parts, either on planting media inoculated with toxigenic a. flavus, test fungi, or uninoculated planting media, were contaminated with other fungi such as exosporium sp., fusarium spp., gilmaniella spp., sphaeropsis sp. and tuber   biotrop1a no. 21,2003 cularia sp. (table 4). it was assumed that the fungal contamination originated from the air or peanut seeds. moisture content of peanut kernels at the time of harvest the fungal inocula and their interaction with time of watering did not give any significant difference on the moisture content of peanut kernels at the time of harvest, while only time of watering gave significant differences. the moisture content of peanut kernels derived from watered plants until harvest was higher than those of not watered plants for 15 days before harvest (table 5). the effects of nontoxigenic a. flavus, a. niger and t. hanianum on toxigenic a. flavus infection and aflatoxin production in peanut kernels from six replications derived from the planting media inoculated with toxigenic a. flavus and its combination with test fungi, in general only one replication of peanut kernels was infected by the fungus (table 6). it indicated that the capability of toxigenic a. flavus in infecting peanut kernels was relatively low. toxigenic a. flavus population in peanut kernels derived from the plants where the planting media were inoculated with the combination of toxigenic a. flavus and each test fungal isolate was lower than that of only inoculated with toxigenic a. flavus (table 6). it indicated that the test fungal isolates inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin was only detected in peanut kernels derived from the plants where the planting media were only inoculated with toxigenic a. flavus and the plants were always watered until harvest time. aflatoxin b] was produced with a concentration of 32 ppb (table 7). it indicated that toxigenic a. flavus (21 cfu/g dry weight) in peanut kernels (table 6) could produce aflatoxin. aflatoxin was not detected in peanut kernels derived from the plants where the planting media were inoculated with a combination of toxigenic a. flavus and test fungi, either in watered plants or unwatered plants for 15 days before harvest, because the test fungi could inhibit aflatoxin production of toxigenic a. flavus. inhibition of aflatoxin production caused by the test fungi could be affected by the control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. dose of biocontrol agent inocula. according to dorner et al. (1998), aflatoxin production in peanut kernels decreased with the increase of the dose of biocontrol agents inocula (nontoxigenic a. flavus and a. parasiticus) inoculated into the soils. aflatoxin content in peanut kernels with the dose of biocontrol agents inocula 0, 2, 10 and 50 g/m were 337.6, 73.7, 34.8 and 33.3 ppb, respectively. biotropia no. 21, 2003 conclusions at the time of harvest, decrease in the moisture content of peanuts in the planting media not watered for 15 days before harvest was higher than those watered until harvest. the lowest population of toxigenic a. flavus was in the planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus at the time of inflorescence and at the time of harvest. toxigenic a. flavus could invade the roots, stems and flowers of peanut plants. the lowest percentage of invasion was on the plant parts where the planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus. the moisture content of peanut kernels originated from plants watered until harvest was higher than those not watered for 15 days before harvest. the population of toxigenic a. flavus in peanut kernels derived from the plants where the planting media were inoculated with the combined use of toxigenic a. flavus and each test fungi, was lower than that only inoculated with toxigenic a. flavus. it indicated that the test fungi inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin b] was only detected in peanut kernels originated from one plant where the planting medium was only inoculated with toxigenic a. flavus and the plant watered until harvest. toxigenic a. flavus infection and aflatoxin bj production were not influenced by the planting media which were not watered for 15 days before harvest. 43 control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the research institute for legumes and tuber crops (rilet), malang, in providing peanut seeds var. komodo. we also thank ms. shanti saraswati, department of biology, faculty of mathematics and natural sciences, bogor agricultural university, and the technicians of the laboratory of pest and disease management, seameo biotrop for their valuable assistance in conducting the experiment. references aoac. 1995. natural toxins. in e. scott (ed.). official methods of analysis of natural poison. 6th ed. association of official analytical chemists, arlington. chap. 49. p. 1-5. bs1. 1995. oilseeds-determination of moisture and volatile matter content. british standard international. dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. soil mycobiota of peanut fields at wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropiano. 17: 3058 dorner, j.w., r.j. cole and p.d. blankenship. 1998. effect of inoculum rate of biological control agents on preharvest aflatoxin contamination of peanuts. biological control 12: 171-176 dorner, j.w., r.j. cole and d.t. wicklow. 1999. aflatoxin reduction in corn through field application of competitive fungi. journal of food protection 62(6): 650-656 ehrlich, k., a. ciegler, m.a. klich and l. lee. 1985. fungal competition and mycotoxin production on corn. experimentia 41: 691 693. johnson, l.f., e.a. curl, j.h. bond and h.a. fribourg. 1960. methods for studying soil microflora-plant disease relationships. burgess publishing company, minneapolis. johnson, l.f. and e.a. curl. 1972. methods for research on the ecology of soil-borne plant pathogens. burgess publishing company, minneapolis. pitt, j.i., s.k.. dyer and s. mccammon. 1991. systemic invasion of developing peanut plants by aspergillus flavus. letters in applied microbiology 13: 16-20. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia. in highley, e. and g.i. johnson (eds). mycotoxin contamination in grains. pp. 5-10. aciar technical reports 37, canberra. wicklow, d.t., b.w. horn, o.l. shotwell, c.w. hesseltine and r.w. caldwell. 1988. fungal interference with aspergillus flavus infection and aflatoxin contamination of maize grown in a controlled environment. phytopathology 78: 68 74. 44 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf the growth of cenococcum geophilum effect of mancozeb 80% concentrations on the growth of cenococcum geophilum fr. under in vitro condition biotropia no. 25, 2005 : 22 – 28 supriyanto seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of silviculture, faculty of forestry, bogor agricultural university, bogor, indonesia ujang susep irawan laboratory of silviculture, seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract fungicides, such as mancozeb 80% are used in nurseries to prevent the plant root against pathogenic fungi. these fungicides may have negative impacts on beneficial organisms such as ectomycorrhizal fungi. cenococcum geophilum is an important ectomycorrhizal fungus associated with some forest trees species. an in vitro experiment was conducted in laboratory condition. cenococcum geophilum was cultured on solid modified melin nokrans’ (mmn) medium containing mancozeb 80 % at different concentrations (0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μm). a completely randomized design was used with 8 replicates petri dishes. mancozeb 80 % decreased the growth of mycelia of c. geophilum. the radial growth of mycelia was not inhibited by mancozeb 80 % at 0 to 400 μm concentrations. fungi-static effect of mancozeb 80 % was found at 500 to 600 μm concentrations, meanwhile fungi-toxic effect of mancozeb 80 % was obtained at concentration more than 700 μm. a lethal level of mancozeb 80% to the growth of c. geophilum was not found. key word : fungicide/mancozeb 80 % /cenococcum geophilum/fungi-static, fungi-toxic introduction background seedlings production is one of the important processes for the success of forestry programs. good quality seedlings must be provided in sufficient number and in appropriate time prior to the planting activities. seedling quality is affected by genetic and environmental factors. the presence of mycorrhizal fungi on the root system of seedlings is one environmental factor that has been proven to increase plant growth. mycorrhizal research in indonesia has been promoted by the government, because of its potential to increase the quality of seedlings via growth acceleration (marx 1973), to control some root pathogenic microorganisms (marx 1973), to improve rooting due to hormone production (gay and debaud 1987), to increase nutrient and water uptake (boyle et al. 1987), to increase drought resistance (boyle 22 et al. 1987), and to increase survival and accelerate xylem formation of plants obtained by tissue culture techniques (supriyanto 1989, chang 1993). biotropia no. 25, 2005 the incidence of disease in tropical nurseries can lead to loss of nursery stock. the damping off diseases, caused by fusarium sp., rhizoctonia sp., or pythium sp., are of most concern. these fungi destroy the root system and curtail water and nutrition absorption. therefore, fungicides are commonly used in nurseries to protect the plant root against these pathogenic fungi. concentrations of mancozeb 80% normally used in forest nurseries, such as in pinus merkusii nurseries, are between 1.8 – 2.0 gram/liter. fungicides may inhibit the growth of the pathogenic fungi or kill them. however, non-target microorganisms, especially the beneficial ones such as mycorrhizal fungi, may also be killed. cenococcum geophilum is an important mycorrhizal fungus in the nursery. this fungus belongs to imperfect fungi, and is commonly found in the nursery as mycorrhizal fungi. cenococcum geophilum doesn’t produce a fruiting body. its isolation started from isolated mycorrhizal root tips. in nursery techniques for seedlings production, the nursery man used commonly pesticide and fertilizer intensively. therefore, it is possible that c. geophilum could survive under high concentration of pesticide and fertilizer, high water retention, and less oxygen. in such condition, c. geophilum became a promising mycorrhizal fungi to be used for seedlings production of forestry species. the use of fungicides such as mancozeb 80% (dithane m-45) could have a negative effect on the growth of c. geophilum. several studies have pointed out the negative effect of pesticides on the growth and development of mycorrhizal fungi (marx and rowan 1981). in pure culture, mancozeb 80 % has a strong toxic effect on the growth of mycorrhizal fungi including pisolithus tinctorius, laccaria laccata, rhizopogon luteolous, and corticium bicolor (thapar 1987). however, so far no information is available for c. geophilum. aside from the effect of fungicides on the growth of mycorrhizal fungi, the work on herbicides indicates opposite growth responses in scleroderma aurantius and pisolithus tinctorius (lake et al. 1981). hence, the objectives of this research were : to study the effect of different concentrations of mancozeb 80% on the growth of c. geophilum and to determine the fungi-toxic level of mancozeb 80% to c. geophilum under in vitro conditions. materials and methods source of culture come from the laboratory of silviculture seameobiotrop, that was isolated from pinus merkusii seedlings. cenococcum geophilum was propagated on solid modified melin norkrans’ medium (marx 1969). small pieces of dense mycelia (1 cm in diameter) were cultured on mmn media containing mancozeb 80 % at 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 23 1000 μm concentrations. mancozeb 80% was added in the culture media and autoclaved at 120o c, 1.5 bar, 30 minutes. the experiment was carried out in an incubator. the temperature in the incubator was 28o c to 30o c and in dark condition. radial growth of mycelia was observed every 5 days for two months. measurement of radial growth of c. geophilum mycelia was conducted at 8 different directions. the colour change of mycelia was also observed. morphological change of mycelia from different fungicide treatments was observed under a light microscope. the experiment was arranged in completely randomized design with 8 replicates petri dishes. analysis of variance was done followed by duncan’s multiple range test. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan results and discussions radial growth of mycelia of cenococcum geophilum the effects of different concentrations of mancozeb 80 % on the growth of mycelia of c. geophilum is shown in figure 1. at 0, 50, 100, 200, 300 and 400 μm concentrations, the growth of mycelia was always better than the other concentrations ( > 500 μm ). inhibition of mycelial growth was found clearly at 30 days after treatment. the mycelia of c. geophilum grew very well at 5 days after treatment, especially at 0, 50, and 100 μm concentrations. however, in general the increase of fungicide concentration significantly inhibited the growth of mycelia. finally, the effects of mancozeb 80 % on the growth of mycelia of c.geophilum can be classified into two groups as follows : fungi-static and fungi-toxic effects (figure 1). unit in μm 0 5 10 15 20 25 30 5 10 15 20 25 30 35 40 45 50 55 60 time (day) r ad ia l g ro w th (m m ) 0 50 100 200 300 400 500 600 700 800 900 1000 24 figure 1. effect of mancozeb 80 % on the growth of cenococcum geophilum over 60 days on mmn agar figure 2 shows the radial growth of two-month-old c. geophilum mycelia in different concentrations of mancozeb 80 %. biotropia no. 25, 2005 0 5 10 15 20 25 30 0 50 100 200 300 400 500 600 700 800 900 1000 r ad ia l g ro w th ( m m ) concentration of fungicide (μm) 26.3 a 27.3 a 24.8ab 22.8ab 22.4ab 21.8abc 19.9 c 16.3 c 4.1 d 3.8 d 2.2 d 1.1 d figure 2. radial growth of two-month old cenococcum geophilum mycelia in different concentrations of mancozeb 80 % the radial growth of c. geophilum mycelia in different concentrations of mancozeb 80 % was divided into three groups as follows: (1) the radial growth of mycelia at concentration of 0-400 μm (21.8 – 27.3 mm), (2) the radial growth of mycelia at concentration of 500-600 μm (16.3 -19.9 mm), (3) the radial growth of mycelia at concentration of 700 – 1000 μm (1.1 4.1 mm). inhibition effect of mancozeb 80% to radial growth of c. geophilum mycelia in the solid medium of mmn was not significant at 50 to 400 μm concentrations. the fungi-static effect (the concentration in which fungicide starts to inhibit the growth of mycelia) of mancozeb 80 % was started at 500 μm up to 600 μm concentrations. while, the fungi-toxic effect (the concentration in which fungicide starts to kill the fungi) of mancozeb 80 % was found at 700 μm concentration. figure 3 shows that c. geophilum mycelia could not grow in solid medium of mmn containing mancozeb 80% of 700 – 1000 μm. in this case, mancozeb 80 % disturbed cellular organ of c. geophilum. moreover, the normal process of fungal intracellular digestion did not run well. it will cause the dead of the fungi or disturb the fungal growth (finholt 1952). fungicides will cause the decrease of membrane permeability surrounding fungal hyphae. three systemic fungicides, benlate, aliette, and ridomil, showed 25 different effects on mycorrhizal infection. benlate reduces or eliminates mycorrhizal infection, aliette increases, while ridomil has been reported to have no consistent effects on the percentage of mycorrhizal infection (jabaji -hare and kendrick 1987). benlate, affected all aspects of fungal development, not only the fraction of the root length infected but also the total root length (carey et al. 1992). benlate severely reduced the number of intercellular hyphae and arbuscules per section and there was a small but non-significant effect with respect to vesicles (sukarno and smith 1993). this condition will give a negative effect on the intracellular digestion process. the decrease in membrane permeability caused inhibition of liquid material absorption through the fungal membrane. moreover, the fungicide could dis-activate extracellular enzymes (finholt 1952). enzyme availability is very important for fungal activity in decomposing its nutrient source to the simplest form to be absorbed. the inactivity of this kind of enzyme will inhibit the fungal nutrient absorption that may cause the disturbance on the fungal growth. finally, this condition will cause the death of the fungi. the effects of mancozeb 80 % on the performance and structure of mycelia of cenococcum geophilum is shown in figure 4. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan figure 3. radial growth of two-month old cenococcum geophilum mycelia in different concentrations of mancozeb 80 % in solid medium of mmn in solid medium of mmn containing mancozeb 80% most of the hyphae became fragmented dark black color thick and branching while in the control 26 treatment their hyphae were light black slurry healthy and no thickening of cell walls. biotropia no. 25, 2005 thickening of cell wall figure 4. two-month-old cenococcum geophilum mycelia in solid medium of mmn (40 x) without fungicide (a) and with fungicide addition (b) conclusions an in vitro experiment showed that mancozeb 80 % reduced the growth of cenococcum geophilum mycelia at more than 700 μm concentration as fungi-toxic level. mancozeb 80 % affected also the mycelial morphology (fragmented, dark black) of cenococcum geophilum. references boyle, c.d., w.j. robertson and p.o. solanius. 1987. use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree nurseries. can. j. for. res. 17 : 1480 –1486. carey pd, fitter ah, watkinson ar. 1992. a field study using the fungicide benlate to investigate the effect of mycorrhizal fungi on plant fitness. oecologia 90 : 550-555. chang, d. 1993. the study of va mycorrhizal effects on horticultural crops. in proc. of second acom, 11-15 march 1991, chiang mai, thailand. eds. i soerianegera and supriyanto. biotrop special publication no. 42. finholt, r.w., weeks, m. and hathaway, c. 1952. new theory on wood preservation. ind. eng. gay j.c. and j.c. debaud. 1987. genetic study on indole-3-acetic acid production by ectomycorrhizal hebeloma species : inter-and intra specific variability in homo and dikaryotic mycelia. appl. microbiol. biotechnol. 16 : 141-146. 27 jabaji-hare sh. kendrick wb. 1987. response of an endomycorrhizal fungus in allium porrun l to different concentrations of the systemic fngicides metalaxyl (rindomil) and fosetyl-al (alliete). soil biology and biocemistry. 19 : 95-99. lake d.b. ippoliti d.g., and brandow c.c. 1981. effect of herbicides on the growth of pisolithus tinctorius and scleroderma aurantrium in pure culture. fifth north american conference on mycorrhiza, quebec, canada. august 16-22, 1981. pp 66. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan marx, d.h. 1969. phytopathology 59, p 152-163. marx, d.h. 1973. mycorrhizae and feeder root diseases. in ectomycorrhizae their ecology and physiology. eds. g.c. marks and t.t. kozlowski. academic press, new eds. g.c. marks and t.t. kozlowski. academic press, new york. marx, d.h. and rowan s.j. 1981. fungicides influence growth and development of specific ectomycorrhizae on loblolly pine seedlings. for. sci. 27 : 167 – 176. sukarno, n. and smith, s.e. 1993. effect of fungicides on vesicular –arbuscular mycorrhizal symbiosis. the effect of vesicular-arbuscular fungi and plant growth. the new phytologist v. 123 (1) p 139147. supriyanto. 1989. micropropagation de pinus nigra et pinus sylvestris. application a leurs hybrides interspecifiques. phd dissertation, university of nancy i, nancy, france. thapar, h.s. 1987. effect of fungicides on ectomycorrhizal fungi in vitro. proceedings of the workshop on mycorrhiza round table, held in new dehli, 13-15 march 1987. p. 155-159. 28 supriyanto abstract background results and discussions conclusions references microsoft word 24 biotropia no. 20,2003: 24 35 influence of mevalonic acid and linalool on limonene accumulation in callus tissues of citrus grandis osbeck nik norulaini nik ab rahman', zarina zakaria2 .and mohd omar add kadirs 'school fur distance learning education, universiti sains malaysia, 11800 usm. penang, malaysia. 2 faculty of applied sciences, universiti teknologi mara, arau campus, 02600 arau, perils, malaysia. 3 school of industrial technology, universiti sains malaysia, 11800 usm, penang, malaysia. abstract effect on callus growth was studied for citrus grandis cultured with feeding of exogenous mevalonic acid (mva) at concentrations of 0.04, 0.08, 0.38, 0.77, 1.15 and 1.54 mm. similar effect with linalool ranging from 10 to 200 nl was studied under various incubation periods. the growth was proportional to the concentrations of precursors used meaning that higher precursors concentrations influenced more growth on c. grandis callus culture. mevalonic acid and linalool showed quite similar precursor feeding effects on limonene accumulation of c. grandis callus cultures. it was revealed that limonene production was triggered with the introduction of mva and linalool even at low concentration. limonene accumulation was detected as early as week four and continued to increase at about 0.0030 and 0.0032 mg/g with mva and linalool, respectively, after the seventh week incubation. in comparison to the unfed cultures, no limonene was detected from the callus up to eight weeks in incubation. keywords : c/rrusgrarafo/limonene/linalool/mevalonic acid abbreviations: 2,4-d: 2,4 — dichlorophenoxyacetic acid, aba: abscisic acid, mva : mevalonic acid introduction bioconversion using an exogenous supply of biosynthetic precursor is believed to improve the accumulation of desired metabolites compound where the productivity is limited because of the lack of particular precursors. naturally occurring as well as related synthetic compounds may be used as precursors. many attempts have been made to produce valuable compounds by adding precursors to several culture species. often the precursors undergo more than one bioconversion resulting in complex mixture of unknown products (kawaguchi et al. 1988). dorisse et al. (1988) had studied the bioconversion of papaverine in glycyrrhiza gabra cell suspension and found the bioconversion rate into papaverinol (31.5%), papa-veraldine (4%) and demethylpapaverine was rather slow. in other case, hydro quinone was glucosylated into arbutin after addition to cell suspension cultures of datura innoxia (suzuki et al. 1987) in production rate of 2.5 gl-1 after of incubation biotropia no. 20, 2003 feeding of 3 mm l-tyrosine or phenylalanine into lithospermum flavum cultures yielded a 2-3 fold increase in 5-methoxy podophyllotoxin accumulation (van uden et al. 1990). the byconversion rates were low; 0.34 and 0.5 mg g d , respectively. it is clear that there are significant differences in bioconversion rates. several reasons include that; the rate may be dependent on the solubility of precursors and/or the intracellularly present amounts of enzyme. furthermore, the precursors can be metabolized by other reactions and even a specific transport system may be involved. funk and brodelius (1990) have used precursor feeding and addition of metabolic inhibitors techniques as a tool to evaluate the possibility that involved to the undetactable vanillin from the cultivated cells of vanilla plan/folia. they found that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of v. plamfolia. extraction of citrus grandis or pomelo peel contained almost exclusively limonene (92.6%) and myrcena (1.9%) suggests that pomelo may be utilized as source material for the production of limonene (wong 1992). the precursors for the synthesis of monoterpenoids are obtained from acetate-mevalonate pathway, which supply to mevalonic acid. mevalonic acid is the primary precursor of all the terpenoids biosynthesized by plant, including limonene. orange juice vesicles (citrus sp., rutaceae) contain an enzyme system which is capable of phospho-rylating mevalonic acid and forming the final product, a monoterpene, linalool. intact fruits and leaves convert linalooi to limonene and other cyclic monoterpenoids. linalool is considered as an immediate precursor which is expected to readily converted to limonene under favourable conditions. there are numerous reports of experiments on mevalonic acid as a precursor. moreno et al. (1993) reported that feeding mevalonic acid did not increase alkaloid accumulation in cell suspension cultures of catharanthus roseus. mevalonic acid added to cultures at concentration of 3.3 mm was reported to saturate the 030 (steroid) biosynthetic pathway in nicotiana tabaccum suspension cultures (threfall & whitehead 1988). with the aim of shortening the period for limonene production and enhancing its accumulation, the effect of feeding primary and immediate precursors was investigated. materials and methods culture conditions young c. grandis fruits about five weeks after anthesis and 4-5 cm in diameter were obtained from established plantation. sterilization technique was established by immersing the whole fruit into 20% clorox® (5.2% sodium hypochloride) for 2 hours prior to peeling and cutting. the fruits were rinsed with sterile distilled water three times and cut transversely into small segments. callus was initiated on ms (murashige & skoog 1962) basal medium supplemented with 30 mg/1 sucrose. 3.0 me/1 2.4-d 3.0 mg/1 kinetin and 0.2 mg/1 aba and incubated in the dark 24 influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. hours/day at room temperature. callus were formed after 2-6 weeks with a success rate of 80-90%. callus were maintained on the media and subcultured to the same fresh media every 4 weeks interval. feeding of precursors mevalonic acid lactone ( mva) and linalool (analytical grades) were purchased from fluka ( japan). aqueous solution of mva was prepared by adding 50 ml distilled water to 1 g of mva powder. mva was filter sterilized and aliquots were added to different ms media to get a range of final mva concentrations of 0.04, 0.08, 0.38, 0.77, 1.15 and 1.54 mm in the media. various volume of linalool (~ 97% purity), 10, 50, 100, 150 and 200 μj were added to 1 liter ms media prior to autoclave. after 4 weeks, callus from maintenance media were cut into smaller pieces and transferred by spreading over the same ms medium with varying added precursors. callus growth determination callus fresh weight was taken as final weight started from the fourth week followed by fifth, sixth, seventh and eighth week of incubation period by direct measurement. to minimize the differences in growth, the initial weight of the callus prior transferring to the experimental ms media was ensured about equal according to each treatment. callus was cleaned from attached media and dried using tissue paper before weighing using analytical balance (sartorius, germany). several plates were sacrificed along the time course of the experiment, to measure the biomass accumulate. limonene determination and analysis extraction of limonene was carried out from four to eight weeks old callus. fresh callus weighing 2.0 3.0 g were extracted with approximately 70 ml methanol using soxhlet apparatus according to morris et al. (1985). identifications and quantification of limonene were made by comparing with standard purchased from fluka using gas chromatography (shimadzu gc 17a) equipped with fid detector. column used was bp-20 (0.25 (im, 30m x 0.25mm) from sge. the temperature was programmed at 60 c, 4 min, 140 c, 8 min. injector and detector temperature was 250 c respectively. the experiments were performed in triplicates and data were subjected to analysis of variance (anova). biotropia no. 20, 2003 results effect of various concentrations of mva feeding and incubation period on callus growth rate figure 1 shows the effect of various concentrations of mva feeding on callus fresh weight. comparisons of callus fresh weights, taken as an indicator of cell proliferation were determined within the eighth weeks period for all mva feeding tested. addition of exogenous mva enhanced the callus growth even at low level except for 0.04 mm compared to the control. however, as the concentration of mva feeding increased, the callus fresh weight increased concomitantly as pointed out in the figure. among the concentrations used, there was no significant difference (p>0.05) between 0.38 and 0.77 mm and between 1.15 and 1.54 mm (table 1). highest callus fresh weight was obtained with the feeding of 1.15 and 1.54 mm of mva where increase in fresh weight was about two times higher compared to control callus. influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. table 1 : effects of mevalonic acid concentrations on callus growth and limonene accumulation of citrus grandis after culture for 7 weeks. mva(mm) fresh weight (g) limonene (mg/g) 0.04 3.127a 0.0015c 0.08 3.900c 0.003 le 0.38 4.868d 0.0030e 0.77 4.807d 0.0020d 1.15 5.335e o.ooisc 1.54 5.574e 0.0013c control 3.218b 0 different alphabet for each data indicates a significant difference at a = 0.05 according to tukey hsd test. the figure also reveals that mva feeding exerts positive effect on callus growth throughout the eight-week culture duration. the most active growth occurred from fourth to sixth week in incubation. callus fresh weight was measured beginning the fourth week of incubation. first measurements of fresh weight were done at week four and at this point all mva feedings showed a higher callus fresh weight than control except for 0.04 mm mva feeding. however, subsequently each feeding concentration displays similar callus growth. higher concentrations of mva feeding illustrates greater callus growth from fourth to seventh week in culture. for all corcentrations, the fresh weights achieved constant level after the seventh week. effect of various linalool feeding concentrations and incubation period on callus growth rate all feeding concentrations of linalool enhanced callus growth rate, though to a different degree. figure 2 shows that only the addition of 10 to 100 jil/1 had resulted on distinctively different (p<0.005) in callus fresh weight proportional to linalool concentrations. feeding of higher than 100 (j.1/1 showed slightly reduction in callus growth but still higher than control callus. callus showed fresh weight increment at the beginning of incubation period at the fourth week and increased in fresh weight occurred after longer incubation period until the seventh week. after this period, fresh weight remained about level indicating callus had slowed down or reached constant growth. it was about twofold increment in callus fresh weight compared to control treatment when added with 50 (il/l and above of linalool. effect of incubation period and various mva feeding concentrations on limonene accumulation the combined effect of various concentrations of mva feeding and the incubation period is shown in figure 3. compared to cultures with no exogenous mva added, other cultures with mva feeding showed limonene as early as four weeks in culture. this accumulation continues up to the seventh week, whereby limonene accumulation displays a sudden decline. this phenomenon was exhibited in all mva concentrations used. however, cultures fed with 0.08 mm mva shows a more active limonene accumulation from fourth to seventh week and this is followed by 0.38 mm mva which after the fifth week, shows a drastic increase in limonene accumulation to match that accumulated in cultures fed with 0.08 mm mva. other concentrations of mva feeding profile displayed an increase from fourth to seventh week and a decline thereafter in much lower limonene accumulation. greatest effect of mva concentrations was observed on limonene accumulation using 0.08 and 0.38 mm, whereby there is no significant difference influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. between the two (table 1). outside these concentrations, the accumulation of limonene was significantly lower (p>0.05). for cultures with no exogenous mva added that is the control, no limonene was detectable. effect of various linalool feeding concentrations and incubation period on limonene accumulation results from figure 4 shows that limonene accumulation was detected from feeding of linalool at all concentrations studied. there was no particular pattern on limonene accumulation with various linalool feeding concentrations. limonene content seems to accumulate at a significant amount when 10 and 50 μl/1 of linalool was added. this amount slightly decreased with feeding of 100 μl/l of linalool and increased instantly with 150 μ.1/1 before dropped again at concentration of 200 μ.1/1 of linalool. the figure also demonstrated that limonene accumulation was highest at seventh week of incubation period. however, limonene accumulation with 200 μl/1 exogenous linalool addition gave maximum peak at week six. limonene value dropped after the seventh week of incubation regardless of linalool feeding concentrations. most rapid increase of limonene accumulation occurred between six to seven weeks of incubation and the feeding of 150 μl/1 of linalool giving the highest accumulation (table 2). influence of mevalonic acid and imalool on limonene accumulation nik norulaini nik ab rahman et al. discussion effect of precursor feeding on callus growth effect on callus growth was studied for c. grandis cultured with feeding of exogenous mva ( figure 1) and linalool ( figure 2) under various concentrations and incubation period. the growth was proportional to the concentrations of precursors used meaning that higher precursors concentrations influenced more growth on c. grandis callus culture except at very low concentration, which is 0.04 mm of mva. obvious callus growth was observed from week five to week six and began to reduce after week seven where the increase was about threefold by fresh weight. during this time, callus turned brown indicated that the accumulation of secondary metabolites in cells had caused toxicity and started to die. increase in callus growth showed that precursor supplied at the studied range did not inhibit callus growth in fact improve growth development. similar result by moreno et al. (1993) stated that feeding of the terpenoid precursors mevalonic acid, loganin, loganic acid or secologanin to suspension cultures of catharanthus roseus did not affect the culture growth, as determined by dry weight concentrations measured at 72 and 120 hrs after feeding. these results demonstrated no toxic effect for all precursors added to the cultures. decreased in callus fresh weight after the seventh week could coincide with the initiation of secondary metabolites synthesize as cell growth approached stationary phase. lindsey and yeoman (1983) concluded that there is indirect relationship between cells growth and secondary metabolites accumulation. it is expected that secondary metabolites accumulation was stimulated only when cells slow down their rate of growth i.e. at the end of the logarithmic phase and the beginning of the stationary phase. ketchum et al. (1999) showed that taxus suspension cultures accumulated maximum levels of taxoid at the end of the growth phase. this conclusion could be related to the finding of this study. interestingly, the rate of growth and highest fresh weight value attained for both precursors did not differ significantly (p>0.05). this concluded that both precursors have had same potential to serve as internal growth stimulator acting to shoot up various aspects of cell metabolism. it was suggested that rapid growth at the early stage of callus was due to synergistic effect of feeding precursors and added growth factors. this phenomenon was demonstrated when 2,4-d was reduced in immobilized cultures of catharanthus roseus cells that rised up the production of alkaloid (tom et al 1991). decrease in growth rate after week seven for fed cells was found a little greater than nonfed cells. reasonable explanation for this phenomenon is that the accumulation of limonene and linalool themselves might have caused cells toxicity which led to cell fatality (brown et al 1987; charlwood & brown 1987). biotropia no. 20,2003 effect of precursors feeding on limonene accumulation mevalonic acid lactone and linalool showed quite similar precursor feeding effects on c. grandis callus cultures. illustrations in figure 3 for mva feeding and figure 4 for linalool feeding revealed that limonene production was triggered with the introduction of mva and linalool at low concentration. limonene accumulation was detected as early as week four and continued to increase until the seventh week. the highest limonene accumulation was 0.0030 mg/g and 0.0032 mg/g obtained from cultures fed with 0.08 mm of mva for the former and 150 um of linalool for the later on the seventh week of incubation (tables 1 & 2). the results proved that precursor feeding had triggered limonene production in c. grandis cultures earlier than non fed cultures as suggested by moreno et al. (1993). they stated that exogenous supply of a biosynthetic precursor, secologanin, to the culture medium may improve metabolites, such as strictosidine and ajmalicine accumulation where the productivity is limited by lack of that particular precursor. it was showed when they added the terpenoid precursor during the growth phase, about 11-fold increase in strictosidine occurred 72 hrs after feeding and decreased to 5 fold after 120 hrs. similar pattern was observed from imbault et al. (1996) on mva feeding effects. they showed that the inhibition of endogenous mva production by pravastatin treatment blocked the alkaloid biosynthesis; supplying such cells with exogenous mva allowed accumulation of alkaloid, but did not result in incorporation of mva. however, moreno et al. (1993) did not find any significant increase in alkaloid level after mevalonic acid feeding, the basic precursor for all terpenoids, in catharanthus roseus suspension cultures. for both treatments, it is concluded that limonene production was not dependent on amount of precursors added. it showed that only a small amount of mva and linalool was needed as a stimulator for limonene production. other possibility that contributes to this finding was the facts that mva must be catalytically phosphorylated by atp and mevalonic kinase itself for the pathway to continue. therefore, the lack of those co-factor and enzyme had restricted limonene production eventhough with the presence of high amount of precursor (rohmer et al. 1996; lichtenthaler et al. 1997). the strong role of enzymes and its incorporation with feeding precursor in study of biosynthesis was briefly described by zenk (1991) in his review. besides, for accumulation of a secondary compound to occur in cultured cells, it is clearly necessary that the system retains the ability both to synthesize and to store the product. trend in limonene accumulation shows that cell maturity has direct relation to secondary metabolite accumulation with both precursor feeding. it was obvious that feeding of both precursors accumulated highest peak of same amount of limonene at the same incubation period. highest limonene accumulation at the seventh week suggested that cells had slowed down their division to prepare themselves for metabolite production. this result fulfilled the suggestion from lindsey and yeoman (1983) that rapidly growing cultures accumulate cells and not secondary products. a similar pattern of results was obtained from suspension culture of influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. podophyllum hexandrum on the production of podophyllotoxin (pras 1992). both situations demonstrated that the increase in metabolite compounds accumulation occurred only at the mature-death stage of the growth curve. consideration also has to take account for limonene itself an intermediate product and would be converted to other form of monoterpenoid. this situation is definitely uncontrollable because biosynthesis is a continuous process and when and how the bioconversion occurred need further and deeper study. potential compounds synthesize from limonene are a-terpeniol, perillic acid and carveon. further experiments on identifying the accumulation of those compounds in c. grandis cultures should be carried out to prove this statement. for future work on limonene improvement, it can be suggested that study on the regulation of enzymes involved in limonene biosynthesis could impose clearer picture of the biosynthetic pathway. the use of suspension cultures for fast limonene accumulation is among the future work that can be considered. conclusion the period of limonene production in c. grandis callus culture was shortened and accumulation was enhanced by precursor feeding with mva and linalool. callus growth rate improved significantly in the presence of the precursors. this result also reconfirmed the presence of mva and linalool in limonene pathway. acknowledgement the authors would like to thank universiti sains malaysia for the research grant and the ministry of science, technology & environment (moste) for national science fellowship scheme. references brown, j.t., hegarty, p.k., & b.v. charlwood. 1987. the toxicity of monoterpenoids to plant cell cultures. plant science 48,195-201. charlwood, b.v. & j.t. brown. 1987. transport and storage of secondary metabolites in tissue cultured plant cells. biochemistry society transactions. 61-63. dorisse, p., gleye, }., loiseau, p., puig, p., edy, a.m. & m. henry. 1988. papaverine biotransformation in plant cell suspension cultures. jour. natural products 120,211-234. funk, c. & p. brodelius. 1990. influence of growth regulators and an elicitor on phenylpropanoid metabolism in suspension cultures of vanilla planifolia. phytochemistry, 29(3), 845 848. imbault, n., thiersault, m., duperon, p., benabdelmouna, a. & p. doireau. 1996. pravastatin: a tool for investigating the availability of mevalonate metabolites for primary and secondary metabolism in catharanthus roseus cell suspensions. physiologica plantarium 98, 803-809. 34 biotropia no. 20, 2003 lichtenthaler, h.k., rohmer, m. & j. schwender. 1997. two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. physiologica plantarium 101, 643-652. lindsey, k. & m.m. yeoman. 1983. the relationship between growth rate, differentiation and alkaloid accumulation in cell cultures. jour. experimental botany, 34,1055-1065. kawaguchi, k., hirotani, m. & t. furuya. 1988. biotransformation of digitoxigenis by cell suspension cultures ofstrophanthus amboensis. phytochemistry. 27, 3475-3479. ketchum, r.e.b., gibson, d.m., croteau, r.b. & m.l. shuler. 1999. the kinetics of taxoid accumulation in cell suspension cultures of taxus following elicitation with methyl jasmonate. biotechnol. bioeng. 62(1), 97105. moreno, p.r.h., vander heijden, r. & r. verpoorte. 1993. effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of catharanthus roseus. plant cell reports 12, 702-705. morris, p., scragg, a. h., smart, n. j. & a. stafford. 1985. secondary product formation by cell suspension cultures. in: dixon, r. a. (ed.), plant cell culture. irl press, washington. murashige, t. & f. skoog. 1962. a revised medium for rapid growth and bio assays with tobacco tissue cultures, physiologica plantarium 15, 473. pras, n. 1992. bioconversion of naturally occurring precursors and related synthetic compounds using plant cell cultures. jour, of biotechnology 26, 29-62. rohmer, m., seeman, m., horbach, s., bringer-meyer, s. & h. sahm. 1996. glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. journal american chemical society 118, 2564-2566. suzuki, t., yoshioka, t., tabata, m. & y. fuyita. 1987. potential of datura innoxia cell suspension cultures for glucosylating hydroquinone. plant cell report 6, 275-278. threfall, d.r. & i.m. whitehead. 1988. co-ordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoid phytoalexin biosynthesis in cultures of nicotiana tabaccum. phytochemistry 27,2567 2580. tom, r., jardin, b., chavarie, c., rho, d. & j. archambault. 1991. effect of culture process on alkaloid production by catharanthus roseus cells. jour, of biotechnology 21, 21-42. van uden, w., pras, n. & th. m. malingre. 1990. on the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell cultures of podophyllum hexandrum royle. plant cell tissue organ culture 23,217-224. wong, k.c. 1992. flavour compounds from local aromatic plants, in: proceedings of asean workshop on the production of natural flavours for food, p. 66 67. zenk, m.h. 1991. chasing the enzymes of secondary metabolism: plant cell cultures as pot of gold. phytochemistry 30(12), 3861-3863. 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf biotropia no. 4, 1990/1991: 918 soil as a factor influencing the mangrove forest communities in talidendang besar, riau cecep kusmana forest ecology laboratory, department of forest management faculty of forestry, bogor agricultural university, bogor, indonesia abstract one transect of 10 m x 900 m was constructed perpendicular to the sea edge to know the pattern of mangrove forest communities from the sea edge through the inland area. then, one sample plot of 50 m x 50 m was established at each forest community to explore its vegetation structure and soil characteristics. the results show that in talidendang besar area, there are three mangrove forest communities stretching from the sea edge to the inland, namely bruguiera parviflora, b. sexangula and b. sexangula-nypa fruticans. the soil factors such as ph.ec (electrical conductivity), % k, % na, c-organic, n-total, nh4 (ammonia), and cec (cation exchange capacity) were regarded important in influencing the pattern of the mangrove forest communities. introduction darsidi (1987) reported that the mangrove forest in indonesia covers an area of approximately 4.25 million ha, of which about 276 000 ha are distributed in riau. most of the mangrove forests in riau are located at the east coast where the major tidal swamp land areas are under development for transmigration projects. numerous environmental factors operate in mangrove swamps, but the most important are soil type, salinity, drainage, and water currents (chapman 1975). steenis (1958) stated that the soil type is more important than the other factors in controlling the zonation of mangroves. in any area with fairly uniform climate, the chemical differences in the soil may produce marked changes in the vegetation (billing 1950). while the mangrove forest in indonesia is believed to be the largest in the world (christensen 1982), studies on the physico-chemical properties of mangrove soils are still few (soegiarto 1979). the present study was done to investigate the soil factors influencing the mangrove forest communities in talidendang besar, riau. it is hoped that the results of this research may contribute to the establishment of proper mangrove forest management in indonesia. 9 biotropia no. 4, 1990/1991 materials and methods this research was conducted in a mangrove forest concession area at tali-dendang besar belonging to the pt bina lestari which is located in the kateman district, indragiri hilir regency, riau province. geographically, this mangrove forest area is located at the east coast of sumatera with gentle topography and altitude of 03 m above sea level between long. 103° 28' to 103° 48' e and lat. 0° 21' to 1° n (figure 1). based on the systems of schmidt and ferguson (1951), the tem figure 1. location and the climatic diagram ofthe mangrove forest area of talidendang besar, riau. 10 soil as a factor influencing the mangrove forest communities c. kusmana bilahan research area has a b climate type with seven wet months, two dry months, and three humid months (badan meteorologi dan geofisika 1990). the soils of this area are organosol and glei humus (lembaga penelitian tanah 1964). to investigate the forest community from the sea edge to the inland, one transect of 10 m x 900 m was divided into 10 m x 20 m contiguous subplots and constructed perpendicular to the sea edge. within these subplots, the dbh (diameter at breast-height) of all trees (plants with dbh 10 cm up) was measured. then, one sample plot of 50 m x 50 m was established in each forest community type to explore its vegetation structure and soil characteristics. four soil samples up to a depth of 25 cm were collected randomly from the area within each adjacent sample plot of 50 m x 50 m. each soil sample was analysed for texture, ph, c-organic, ec (electrical conductivity), cec (cation exchange capacity), n-total, nh4 (ammonia), and exchangeable cations (k, na, mg, ca) at the soil laboratory of the faculty of agriculture, bogor agricultural university. the vegetation data were analysed using cox's method (1967) and the importance value index (curtis and mclntosh 1951) was used to determine the vegetational importance of a species within the forest community. results and discussion a. vegetation composition as shown in figure 2, the mangrove forest from the sea edge to the inland in talidendang besar could be divided into three different forest communities, namely, bruguiera parviflora community which occupied the area from the sea edge to about 180 m inland, b. sexangula community from about 180 m to 740 m inland, and b. sexangula-n. fruticans community from about 740 m to 900 m inland as transition area with swamp forest. table 1 shows that in the b. parviflora community, b. parviflora was considered the dominant species and b. sexangula the codominant one. in the b. sexangula community, b. sexangula was considered the dominant species and b. parviflora the codominant one, while in the b. sexangula-n. fruticans community, b. sexangula was dominant and tv. fruticans codominant. there is a marked tendency for the density and basal area of b. parviflora to decrease from the sea edge (b. parviflora community) to the inland (b. sexangula-n. fruticans community). the opposite occurred for the density and basal area of b. sexangula which tended to increase toward the inland area. in addition, the further from the sea edge, the more varied was the tree species richness. it is assumed that the less severe site conditions in the inland area give the chance to 11 figure 2. mangrove forest community from sea edge through inland in mangrove forest area of talidendang besar, riau. bp (b. parviflora), bs (b. sexangula), cd (c. decandra), hm (//. microcarpum), fb (f. benjamina), ra (r. apiculata), and nf (tv. fruticans). table 1. species density and species importance value index (ivi) of trees at three forest communities in a mangrove forest of talidendang besar, riau 12 soil as a factor influencing the mangrove forest communities-c. kusmana many species for a better growth. johnstone (1983) stated that the presence of terrestrial species in the back zone of mangal is more indicative of the salinity regime than representing an active process of colonization from the land as part of an integrated successional system. b. forest community occurrence as related to soil factors as shown in table 2, the soils which occupied each forest community have a high percentage of clay, an intermediate percentage of silt, and a low percentage of sand. this indicates that the mangrove forest area in talidendang besar receives much eroded soil containing fine soil particles through the stream flow from the upper river basin of talidendang besar. it is probably due to the extensive conversion of peat swamp forest to coconut plantation mainly by the bugis people who came from the southern part of sulawesi. the soils covered either by b. parviflora or b. sexangula communities were classified as clay, while the soils covered by b. sexangula-n. fruticans were classified as silty clay. the soils of this mangrove forest area were almost the same as those of the mangrove forest area in ujung karawang, cilacap (al rasyid 1971; soerianegara 1971), and bengkalis (dinas kehutanan propinsi dati i riau 1978). the ph of the soils which covered each of the forest community was generally neutral due to the decreasing percentage of exchangeable cation content in the soils from the sea edge of the inland area. similarly the salinity (electrical conductivity) of the soils tends to decrease toward the inland, but generally the salinity of the soils occupied by each forest community was considered low, and the adsorption site of the soils was dominated by cations in the order of ca > mg > na > k. this is suggested to be due to the fact that the mangrove forest in this area receives much fresh water through the stream flow of talidendang besar river, while the area toward inland was infrequently submerged by sea water. c-organic, nh4 (ammonia), n-total, and cec (cation exchange capacity) increased toward inland. it indicates that toward the inland area the organic matter and its decomposition process tends to increase. it is probably correlated with the maturity of the trees and the soil substrate condition in these forest communities. based on the average height and diameter of the trees in these forest communities (table 1), the inland area of the forest community appears to be occupied by more mature trees as suggested by the amount of litter fall on the forest floor. in addition, the dense aerial roots and the acrostichum aureum on the floor of the b. sexangula and b. sexangula-n. fruticans communities play an important role in trapping leaves and debris during tidal inundation, thereby contributing to the high organic matter content in these forest communities. the increasing content of n and nh4 of the soil covered by the forest community toward inland indicated a more rapid decom 13 14 soil as a factor influencing the mangrove forest communities c. kusmana position process of organic matter on sites further from the sea edge. it is probably due to the decreasing frequency of inundation of the sites toward inland so that the soils are rather stable and more or less well-drained. ponnamperuma (1972) stated that the accumulation of ammonia in anaerobic soils is due to the lack of oxygen to carry the oxidation from nitrite to nitrate, and so the mineralization of organic nitrogen in these soils stop at the ammonia stage. furthermore, broto (1984) reported that if anaerobic soil has a ph greater than 7.0 ammonia volatilization might take place through a denitrification process resulting in severe losses of nitrogen from the soils. there was a significant difference among the three forest communities for eight soil characteristics such as ph, c-organic, n-total, % k, % na, nh4, cec, and ec (table 2). these soil characteristics appeared to be important in influencing the occurrence pattern of mangrove forest communities in talidendang besar. based on a statistical test using least significant difference (table 3), the values of c-organic, cec, ec, % k, and % na were significantly different between forest communities, but the contents of n and nh4 were only significantly different between the b. parviflora and b. sexangula-n. fruticans communities. meanwhile, the ph of the soils was significantly different among the three forest communities, except between the b. parviflora and b. sexangula community. if the three mangrove forest communities were ranked according to their relative positions with regard to these important soil characteristics (table 4), the b. parviflora community tends table 3. least significant difference (lsd) test for soil characteristics showing significant difference among three forest communities no. soil characteristics community type 1 vs 2 1 vs 3 2 vs 3 1. ph ns ** * 2. c-organic ** ** ** 3. n-total ns * ns 4. nh4 ns * ns 5. % k ** ** ** 6. % na ** ** ** 7. cec ** ** ** 8. ec * ** * community type 1: bruguiera parviflora community community type 2: bruguiera sexangula community -community type 3: bruguiera sexangula-nypa fruticans community * significant difference at p < 0.05 ** significant difference at p < 0.01 ns non-significant. 15 biotropia no. 4, 1990/1991 table 4. relative position of three forest communities ranked according to mean values of eight important soil characteristics soil characteristics rank ph c-org. n-total *k %na cec nh4 ec 1 bp bs-nf bs-nf bp bp bs-nf bs-nf bp 2 bs bs bs bs bs bs bs bs 3 bs-nf bp bp bs-nf bs-nf bp bp bs-nf bp : bruguiera parviflora community bs : bruguiera sexangula community bs-nf : bruguiera sexangula-nypa fruticans community. to occupy soils which contain higher % na, % k, ec and ph, and lower c-organic, n-total, nh4, and cec compared to the others. on the contrary, the soils occupied by the b. sexangula-n. fruticans community contained lower % na, % k, ec and ph, and higher c-organic, ntotal, nh4, and cec. the b. sexangula community tends to occupy soils which contain intermediate values of these soil characteristics. it means that in talidendang besar, b. parviflora tends to grow on the rather saline and soft mud clayey soils in the area near the sea edge which is frequently submerged in sea water. b. sexangula can grow on various types of mud ranging from rather soft clayey soils in the area near the sea edge to the hard silty clayey soils in the inland area which is infrequently submerged in sea water. b. sexangula tends to grow optimally on rather hard silty clayey soil with ph 7.0 in the inland area, while the other species viz n. fruticans and ficus benjamina occur in the innermost zone of the mangrove with lower salinity. yamaha and sukardjo (1979) reported that in south sumatera, b. parviflora grows in soft mud, while b. sexangula grows mixed with b. gymnorrhiza and rhizophora apiculata in the innermost zone of mangroves on rather hard mud soil. conclusions the mangrove forest in talidendang besar could be divided into bruguiera parviflora community occupying an area from the sea edge to about 180 m inland, b. sexangula community from about 180 m to 740 m inland, and b. sexangula-n. fruticans community from about 740 m to 900 m inland. in the b. parviflora community, b. parviflora was considered the dominant species and b, sexangula the codominant one. in the b. sexangula community, b. sexangula was considered the dominant species and b. parviflora the codominant one, while in the b. sexangula 16 soil as a factor influencing the mangrove forest communities c. kusmana n. fruticans community, b. sexangula was considered the dominant species and tv. fruticans the codominant one. there were eight soil characteristics considered important in influencing the occurrence pattern of the mangrove forest community in talidendang besar, riau: ph, ec (electrical conductivity), cec (cation exchange capacity), c-organic, n-total, nh4, % k, and % na. references al rasyid, h. 1971. pemilihan jenis tanaman dalam rangka meningkatkan produksi hutan payau ujung karawang. laporan no. 134, lembaga penelitian hutan, bogor. badan meteorologi dan geofisika. 1990. keadaan curah hujan di propinsi riau. departemen perhubungan republik indonesia. badan meteorologi dan geofisika, jakarta. billings, w.d. 1950. vegetation and plant growth as affected by chemically altered rocks in the western great basin. ecology 31: 62-74. broto, k.g. 1984. waterlogged saline soils. in: snedaker, s.c. and j.g. snedaker (eds.). the mangrove ecosystem: research methods: 114-130. unesco, paris. chapman, v.j. 1975. mangrove vegetation. strauss and cramer gmbh, germany. 499 p. christensen, b. 1982. management and utilization of mangroves in asia and the pacific. fao environment paper 3. fao, rome. cox, g.w. 1967. laboratory manual of general ecology. brown company publishers, dubuque, iowa. 195 p. curtis, j.t. and r.p. mcintosh. 1951. an upland forest continuum in the prairie-forest border region of wisconsin. ecology 32 (3): 476-496. darsidi, a. 1984. pengelolaan hutan mangrove di indonesia. in: soemodihardjo, soerianegara, sutisna, kartawinata, supardi, naamin, and al rasyid (eds.). presiding seminar ii ekosistem mangrove, baturaden 3-5 agustus 1982: 19-28. mab-lipi, jakarta. dinas kehutanan propinsi dati i riau. 1979. hutan mangrove di propinsi riau. in: soemodihardjo, nontji, dan djamali (eds.). presiding seminar ekosistem hutan mangrove, jakarta 27 pebruari -1 maret 1978: 176-185. mab-lipi, jakarta. lembaga penelitian tanah. 1964. peta tanah eksplorasi sumatera bagian selatan, skala 1:1 000 000. departemen pertanian republik indonesia, lembaga penelitian tanah, bogor. johnstone, i.m. 1983. succession in zoned mangrove communities: where is the climax? in: teas, h.j. (ed.). biology and ecology of mangroves: 131-139. w. junk publishers, the hague. ponnamperuma, f.n. 1972. the chemistry of submerged soils. advances in agronomy 24: 29-96. schmidt, f.h. and j.h.a.ferguson. 1951. rainfall type based on wet and dry period ratios of indonesia with western new guinea. verhandelingen no. 42, kementerian perhubungan djawatan meteorologi dan geofisik, jakarta. soegiarto, a. 1979. the mangrove ecosystem in indonesia, its problems and management. paper prepared for the second international symposium on biology and management of mangrove, july 20-26, 1980, port moresby, papua new guinea. 17 biotropia no. 4, 1990/1991 soerianeoara, i. 1971. characteristics and classification of mangrove soils of java. rimba indonesia 16 (3,4): 141-150. steenis, c.g.g.j. van. 1958. ecology (the introductory part to the monograph of rhizophoraceae by ding hou). flora mal. 5: 431-441. yamada, i. and s. sukardjo. 1979. ecological study of mangrove and swamp forest in south sumatera, j. southeast asian studies 17 (3): 425-443. 18 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf biotropia no biotropia no. 17,2001 : 9 17 characterization of three benzoate degrading anoxygenic photosynthetic bacteria isolated from the environment dwi suryanto1, antonius suwanto2'3*, and anja meryandini3 ; dept. of biology, faculty of science and mathematics, north sumatra university, medan, indonesia 2 south east asian regional center for tropical biology (seameo-biotrop), bogor, indonesia 3 dept. of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract three anoxygenic photosynthetic bacteria, ds-1, ds-4 and cas-13, have been examinated for their morphological and physiological properties. all strains were rod-shape cells with a swollen terminal end, gram negative, motile, non-halophilic, non-alkalophilic and non-acidophilic, and capable of utilizing benzoate aerobically and photo-anaerobically. sequence analysis of part of 16s rrna genes showed that ds1 and cas-13 were closely related to rhodopseudomonas palustris strain 7 with a similarity of 97%, whereas ds-4 may not be closely related to the former two strains with a similarity of 78% based on the constructed phylogenic tree. spectral analysis indicated that the three bacteria had bacteriochlorophyl a and normal spirilloxanthin series. growth in medium enriched with vitamin and supplemented with benzoate as their sole c-sources was better than in medium without vitamin. benzoate degradation in medium with vitamin was accelerated. the ability to grow on benzoate without added vitamins indicated that the bacteria were able to synthesize their own vitamins. key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16s rrna gene. introduction some toxic compounds are slowly degraded in polluted aerobic zones and, therefore, may leach into anaerobic subsurface environments (kohring et al. 1989). anaerobic degradation of such compounds may play an important role in eliminating toxic substances. anaerobic bioremediation has been proposed as an inexpensive method for in situ removal of organic contaminants in the environment (kuo and genthner 1996). recent concern about the environmental fate of industrially produced organic compounds has prompted a resurgence of interest in the anaerobic degradation of aromatic compounds (harwood and gibson 1988). anoxygenic photosynthetic bacteria (apb) are nutritionally versatile in their ability to utilize diverse sources of carbon ranging from simple aliphatic organic acids to complex polysaccharides (hiraishi et al. 1995). the occurrence of these bacteria as common inhabitants in aquatic and some terrestrial habitats in nature ' corresponding author: e-mail address : asuwanto@indo.net.id 9 biotropia no. 17, 2001 (hiraishi et al. 1995) might be explained partly by the fact that these bacteria survive on various modes of energy-generating systems (hiraishi et al. 1995). the ability of phototrophic purple non-sulfur bacteria to grow aerobically and anaerobically in the presence of diverse aromatic compounds makes these good candidates for potential biodegradation of harmful compounds (hanvood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). commercialization of these bacteria for purification treatment plants was initiated about 15 years ago (kobayashi and kobayashi 1995). not many phototrophic purple non-sulfur bacteria species are known to degrade aromatic compounds. rhodopseudomonas palustris (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994), rhodomicrobium vannielii (wright and madigan 1991), rhodobacter capsulatus (blasco and castillo 1992; shoreit and shaheb 1994), rs. blastica, and rhodospirilium rubrum (shoreit and shaheb 1994) are able to degrade a variety of monocyclic aromatic compounds with or without other c-sources. benzoate and its derivatives are among the common aromatic compounds that can be completely mineralized (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). however, blasco and castillo (1992) noted that rhodobacter capsulatus e1f1 is able to degrade mononitrophenol and dinitrophenol with acetate as its carbon source. rs. palustris utilized several phenolic compounds, hydroxylated and methoxylated aromatic acids, aromatic aldehydes, and hydroaromatic acids (harwood and gibson 1988). other diverse aromatic compounds have also been reported to be utilized by this group (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). degradation of aromatic compound by phototrophic purple non-sulfur bacteria in general was emphasized in this investigation based on their ability to utilize diverse aromatic compounds. to date, this group of bacteria has not been subjected to intensive examination (harwood and gibson 1988). this study focused on the ability of three new isolates of apb to utilize benzoate. identification of the bacterial isolates based on their 16s-rrna genes as well as their morphological and physiological properties was also carried out. materials and methods bacterial cultures and cultivation three isolates of apb, designated as ds-1, ds-4, and cas-13 were studied. the two former strains were isolated from java, and the last was isolated from moluccas. all isolates were maintained on modified sistrom medium with benzoate as the sole carbon source. to determine the ability of the bacteria to utilize and degrade aromatic compounds, the isolates were grown in modified sistrom by omitting all carbon sources, including nitrilo-triacetic acid, supplemented with 5 mm benzoate as the c 10 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. source with or without vitamins in 100 ml completely filled screw-capped tubes. isolates also were grown in modified sistrom supplemented with vitamins and 5 mm succinate, or 5 mm acetate as their carbon sources to serve as a means of comparison. for cultures grown on benzoate, the inocula were obtained from 3-day old cultures of bacteria grown in modified sistrom with benzoate with an initial cell number of 5xl06 cells/ml. for cultures to test other c-sources, inocula were taken from 2-day old cultures grown in sistrom media with succinate as c-source. unless mentioned otherwise, all media were adjusted to ph 7.2. growth condition, measurement of growth, and quantitation of benzoate cultures were illuminated with a 40 w tungsten bulb at a distance of 30 cm. growth rates were determined by measuring turbidity at 660 nm every 24 hours. for cultures grown in 5 mm succinate and 5 mm acetate, growth rates were measured every 12 hours. for cultures grown in benzoate, residual benzoate levels were measured at 120 hours of incubation. benzoate concentration was measured at 276 nm using a hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) according to shoreit and shabeb (1994). cell density of the isolates grown in other c-sources was measured after 72 hours of inoculation. spectral analysis cells were harvested from 7-day old anaerob-phototrophic cultures grown on modified sistrom supplemented with vitamins and 5 mm succinate as sole carbon source, and suspended in icm buffer (10 mm phosphate buffer ph 7.0 and 1 mm naedta ph 7.0). sonication was carried out using soniprep 150 (msb, uk) at an amplitude of 2 u. for 2 minutes, three times with a time interval of 1 minute. cell extracts were centrifuged at 3000 rpm for 30 minutes at 4°c. protein concentration was determined according to the pierce bca* protein assay kit (rockford, iii, usa). spectral analysis was performed using hitachi u-2010 in protein concentration of ± 100 (ig/ml. amplification and sequencing of part of 16s rrna genes to sequence part of the 16s-rrna genes, the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna (200 ng) on readyto-go pcr beads (pharmacia-biotech, uppsala, sweden). phenol-chloroformisoamylalcohol (25:24:1) treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. total volume of the pcr reaction (25 ul) consisted of 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in a gene amp pcr system 2400 thermocycler (perkin-elmer cetus, norwalk, conn.). 11 biotropia no. 17, 2001 part of 16s-rrna gene was sequenced to infer the closest related organism from the ribosomal database project (rdp) maintained at the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purified by ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (pe applied biosystems, foster city, ca.). construction of phylogenetic trees for the construction of phylogenetic trees, cluster analysis of 16s-rrna gene was done by a computer program from the european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of the department of biochemistry, university of antwerp) was used to determine their phylogenetic relatedness based on their nucleotide sequences and mflp profiles obtained from pulsed-field gel electrophoresis. results and discussion the three new isolates described in this study were similar in their morphological properties. all were gram negative, non-halophilic, non-alkali or acidophilic, aerobic and anaerob-phototrophs that have motile rod-shaped cells with terminal swellings. under anaerobic photothrophic growth conditions, all new isolates produced brick-red and pink cultures in succinate and benzoate, respectively. spectral analysis of cell free extracts (figure 1) of photosynthetic pigments showed absorption maxima for ds-1 at 374, 493, 587, 803 and 852 nm, ds-4 at 371, 501, 585, 803 and 848 nm, and cas-13 at 373, 502, 585, 802 and 871 nm indicating the presence of bacteriochlorophyl a and normal spirilloxanthin series including lycopene and rhodopin (imhoff 1995). in this respect, the three isolates were nearly identical to each other and might be considered as one group. however, these isolates clearly differed from rhodobacter sphaeroides 2.4.1., which has different absorption maxima (376, 451, 453, 477, 507, 587, 746, 799, and 850 nm), with yellow-brown colonies grown anaerobically in succinate. the results from the analysis of partial sequencing of 16s-rrna (c.a. 500 bp) of ds-4, ds-1, and cas-13 demonstrated that ds-4 and cas-13 were closely related (figure 2). partial sequencing of the first 500 bp of 16s rrna gene could lead to a main line of descent (stackebrandt and rainey 1995). comparison with the rdp database of the university of illinois indicated that both ds-1 and cas-13 demonstrated 97% similarity with rs. palustris strain 7, and of 83% and 84% with rb. sphaeroides il106, respectively (table 1). similarity of those of ds-4 to rs. palustris strain 7 was 78%. these results suggested that ds-1 and cas13 are likely to be one species, rs. palustris. ds-4, however, is significantly different from ds-1 and cas-13 by 22%, and from rb. sphaeroides il106 by 30%. complete sequence analysis is needed to give more definitive information about the taxonomic position of these three isolates. 12 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 1. absorption spectrum of cell extracts ds-1, ds-4, cas-13, and rb. sphaeroides 2.4.1. figure 2. phylogenetic tree 16s rrna gene sequences. the number at the tree lines represented bootstrap values. 13 biotropia no. 17, 2001 table 1. percent similarity of the ds-1, ds-4, and cas-13 with other relative members of anoxygenic photosynthetic bacteria. unlike rb. sphaeroides 2.4.1, all of the three isolates were able to metabolize benzoate (figure 3). among the members of apb, rs. palustris is the most common species capable of utilizing benzoate (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994). the ability to grow in the presence of aromatic compounds such as benzoate without vitamins (figure 3) may suggest that the organisms were able to synthesize their own vitamins. however, supplemented vitamins could increase their potential in metabolizing benzoate. the rate of benzoate utilization for ds-1, ds-4, and cas-13 were 0.024 mm/hour, 0.02 mm/hour, and 0.03 mm/hour in media with vitamin supplements compared to 0.017 mm/hour, 0.012 mm/hour, and 0.018 mm/hour in media without vitamins. a relatively similar pattern of extended lag phase was observed in growth of the cultures. time was needed in preparation of producing a number of enzymes. carbon availability might affect cell growth. cell density was observed to be higher in media with benzoate (c7) (figure 3), followed by succinate (c4) and acetate (c2) (figure 4). relatively low cell density of all isolates was shown in 5 mm benzoate with no vitamins. vitamins were certainly necessary for their metabolic activity. 14 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 3. growht of ds-01, ds-4 and cas-13 in 5 mm benzoate with vitamins (above) and without vitamins (below). rb. sphaeroides 2.4.1. was used as negative control. 15 biotropia no. 17, 2001 figure4. growth of ds-1, ds-4, cas-4, cas-13, and r6. sphaeroides 2.4.1. in 5 mm succinate (above) and 5 mm acetate (below) with vitamins. 16 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. acnowledgement this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor indonesia. references blasco, r. and f. castillo. 1992. light-dependent degradation of nitrophenols by the phototrophic bacterium rhodobacter capsulatus 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t. madigan. 1991. photocatabolism of aromatic compounds by the phototrophic purple bacterium rhodomicrobium vannielli. appl. environ. microbiol. 57: 2069-2073. 17 microsoft word 11 biotropia no. 20, 2003: 11 23 potency of vibrio isolates for biocontrol of vibriosis in tiger shrimp (penaeus monodon) larvae widanarni1'2. a. suwantou',sukenda2,and b. w. lay4 'department of biology, faculty of science and mathematics, bogor agricultural university, bogor 16144, indonesia 2 department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia 3seameo-biotrop, jl. raya tajur km 6, bogor 16720. indonesia ^faculty of veterinary science, bogor agricultural university, bogor 16680, indonesia abstract this study was carried out to obtain vibrio isolates able to function as biocontrol of vibriosis in shrimp hatchery. thirty one vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i.e. labuan, pangandaran, and lampung, indonesia. pathogenic v. harveyi mr5339 was obtained from maros, south-sulawesi and was made as a rifampicin resistant mutant (rfr) to screen for those 31 vibrio isolates in in vitro assays and to allow us to monitor their presence in shrimp larvae and larval rearing water. almost all vibrio isolates could inhibit the growth of pathogenic v. harveyi mr5339 rfr. skt-b isolate from skeletonema was the most effective to inhibit the growth of v. harveyi mr5339 rf* and significantly reduced larval mortality in pathogen challenge assays. these prospective biocontrol bacteria, at concentration of 10" cfu/ml, did not show pathogenicity to shrimp larvae. skt-b was gram negative, short rod-shape, exhibited yellow colonies on tcbs and swarming on swc-agar media, motile, utilized glucose and sucrose but not lactose: produced extracellular protease and amylase, but did not produce chitmase. partial sequencing of 16s-rrna gene skt-b showed skt-b similarity to vibrio alginofyticus. keywords: shrimp larvae / biocontrol bacteria / vibriosis. introduction tiger shrimp (penaeus monodori) culture in indonesia has become more intensive and extensive because of high demand and economic value of this export commodity. however, the shrimp culture industry is associated with multiple problems such as diseases and poor environmental quality, which became the main constraint to reach the target of production. bacterial disease that attacked at hatchery stage is the most serious threat and often caused mass mortality in shrimp larvae which greatly influenced the sustainable supply of healthy fry. this disease is often caused by a luminous bacterium identified as vibrio harveyi (lavilla — pitogo et al. 1990; pedersen et al. 1998). to prevent such mass mortalities, shrimp hatcheries routinely use antibiotics. however, widespread antibiotics applications could result in antibiotic resistant pathogens (karunasagar et al. 1994; tjahjadi et al. 1994; tendencia and de la pena * corresponding author: asuwanto@indo.net. id biotropia no. 20,2003 2001). not only can resistant bacteria proliferate after an antibiotic has killed off the other bacteria, but also they can transfer their resistant genes to other bacteria which have never been exposed to the antibiotic (verschuere et al. 2000). furthermore, this approach is not predictable since v. harveyi strains that attack shrimp larvae are genetically diverse (suwanto et al. 1998). the development of vaccine was hampered due to the lack of immune response-memory or the degradation of the absorbed vaccines (alabi et al. 1999). an alternative method of controlling pathogenic bacterial strains in shrimp cultures could be supplementation of pure cultures of natural bacterial isolates (biocontrol) which might produce chemical substances inhibiting the growth of pathogens. the approach basically employs the activity of microorganism that could suppress or inhibit the growth of v. harveyi without causing bad impact on the equilibrium system in a particular microbial community. this has been an established practice in husbandry of terrestrial animals (fuller 1992; ohhira et al. 1996), and has only recently been applied to aquatic systems such as fish culture (gildberg et al. 1995) '-nd crustacean culture (riquelme et al. 1997; rengpipat et al. 1998). widanarni and suwanto (2000) reported the presence of several vibrio isolates associated with the shrimp from the egg stadia to post larva, as well as their rearing environment. based on their physiological and genetical characters, the isolates were distinguishable from v. harveyi that has been proved to be pathogenic on shrimp larva. therefore, further studies should be conducted to evaluate its potency as biocontrol in shrimp hatchery. similar finding was also reported by riquelme et al. (1997) in which vibrio isolates associated with scallop larvae were proved to be a potential probiotic in scallop culture in chile. the purpose of this research was to obtain new bacterial isolates as improved biocontrol agents which can be applied to solve the problem of bacterial disease in shrimp culture, and to study their ability to inhibit the growth of v. harveyi. materials and methods isolation of vibrio candidates for biocontrol vibrio isolates for biocontrol were isolated from tiger shrimp larvae and hatchery environments, i.e. labuan, pangandaran, and lampung, indonesia. samples were taken from eggs, larvae (nauplius, zoea, and mysis) and post-larvae of tiger shrimp, natural shrimp feed (i.e. artemia and skeletonemd), seawater, and rearing water of each stage of shrimp larvae. all samples were spread on thiosulphate citrate bile salt agar (tcbs, oxoid). the culture was incubated at room temperature (28-31)°c, for 24 hours. subsequently, different morphological types of colonies were randomly selected for further study. pathogenic v. harveyi mr5339 was obtained from balitdita (research institute for coastal aquaculture) maros, south-sulawesi. 12 potency of vibrio isolates for biocontrol of vibriosiswidanarni et al. sensitivity test of vibrio to rifampicin purified isolates of vibrio candidates for biocontrol and pathogenic v. harveyi mr5339 were grown in seawater complete agar (swc-agar) (5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water) supplemented with rifampicin (rf) 50 u.g/ml. after overnight incubation at room temperature (28-31)°c, the bacteria were scored for their antibiotic sensitivity by streaking on the appropriate antibiotic-containing media. rifampicin-resistant mutant of v. harveyi one milliliter of a 24 hour culture of v. harveyi mr5339 was centrifuged at 5000 rpm for 1 minute. following the removal of the supernatant, the bacterial pellet was resuspended in 100 (il of sterile seawater. the suspension was then spread onto swc-agar containing rf (50 u.g/ml). colonies of luminescent bacteria grown on swc-agar + rf were restreaked onto new media to obtain isolated v. harveyi mr5339 resistant to rifampicin. determination by in vitro test of biocontrol vibrio the inhibitory effects of each isolate candidate for biocontrol were tested against v. harveyi mr5339 rf*, known to be a pathogen of shrimp larvae. tested isolate candidates for biocontrol was added to 10 ml of swc-broth in test tubes at 106 cells/ml. onto the same test tubes were also added overnight liquid culture of v. harveyi mr5339 rf* at 102cell/ml. all test tubes were incubated overnight at room temperature. appropriate dilutions will be chosen for seeding the mixed culture on swc-agar supplemented with rf (50 |ag/ml). if the number of colony forming unit (cfu) of v. harveyi mr5339 rf* from the control tubes (i.e. tubes inoculated only with v. harveyi mr5339 rf1*) is larger than the number of v. harveyi of the mixed cultures (i.e. v. harveyi mr5339 rf* which is mixed with the candidate biocontrol isolate), this could identify potential candidates of biocontrol isolates that inhibit the growth of the v. harveyi mr5339 rf* pathogen. pathogenicity assay of biocontrol bacteria to shrimp larvae shrimp larvae were incubated with the isolates which showed positive growth inhibitory activity against v. harveyi mr5339 rf*. bacterial isolates as candidate of biocontrol agent were resuspended in sterile seawater. shrimp larvae were cultured in 2-liter shrimp rearing tank (20 individuals per tank). bacterial suspension was added into the tank at the final concentration 106 cfu/ml. after incubation at room temperature, survival rate of larvae at 5 days was recorded. biotropia no. 20, 2003 pathogen challenge test this test was carried out to study the efficacy of vibrio candidate biocontrol isolates towards pathogenic v. harveyi in shrimp larvae. the best three biocontrol isolates, based on in vitro test, were selected for pathogen-challenged assay. suspension of each isolate was placed in the shrimp rearing tank for 6 hours before shrimp larvae were introduced into the tank. after cocultivation of isolates with shrimp larvae for 6 hours, v. harveyi mr5339 rf* was introduced into the culture tank. this experiment was performed in three replications. tanks treated with only v, harveyi mr5339 rf* or without bacteria were employed as controls. total vibrio population (counted on tcbs media), v. harveyi mr5339 rf* (counted on tcbs media + rf) in the rearing media, larval mortality monitored daily for 5 days and the survival rate of shrimp larvae were determined as described previously (hala 2002). microbiological and physiological properties cell shape, motility, and gram staining were evaluated using an olympus bh2-rfc microscope. a number of physiological characteristics were analyzed using microbact kit test (medved science pry. ltd. australia) and production of extra-cellular protease, chitinase and amylase monitored on swc-agar supplemented with skim milk, colloidal chitin and starch were tested, respectively. the presence of extra-cellular hydrolytic enzymes could be used to provide further information on the pathogenicity or virulence of the isolates. amplification and sequencing of 16s-rrna gene since many strains of marine bacteria share similar physiological and morphological characteristics, identification of bacteria strictly based on such parameters is not reliable. therefore, it is essential to perform dna sequencing of the 16s-rrna gene in order to provide reliable additional information for bacterial identification. modified phenol-chloroform-isoamylalcohol treatment and ethanol precipitation were used to extract the genomic dna (sambrook et ul. 1989). the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna provided by ready-to-go pcr beads (pharmacia-biotech, uppsala, sweden). these primers were successful to work with a broad range of environmental samples (marches! et al. 1998). a part of 16s-rrna gene was sequenced to infer the closest related organism from ribosomal database project (rdp) maintained in the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purify with ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn). potency of vibrio isolates for biocontrol of vibriosis — widanami el al. results and discussion isolation of vibrio candidates for biocontrol thirty one vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i. e. labuan, pangandaran, and lampung (table 1). of these isolates, 25 isolates were found to be associated with eggs, larvae, post-larvae, and the rearing water of each stage; 1 isolate was obtained from seawater reservoir: 4 isolates were obtained from natural feed (i.e. 2 isolates from artemia and 2 isolates from skeletonema). all of the isolates were non-luminous yellow colonies on tcbs and swarming on swc-agar media. table 1. codes and sources of vibrio isolates no. code source location 1. sw seawater labuan 2. n-a nauplius labuan 3. nt nauplmstank water labuan 4. z-a zoea labuan 5. pl,,-a post-larvae 6 labuan 6. pl,,-b post-larvae 6 labuan 7. pu-c post-larvae 6 labuan 8. pl,,-d post-larvae 6 labuan 9. pln-a post-larvae 1 1 labuan 10. pl,,-b post-larvae 1 1 labuan 11. plnt-a post-larvae 1 1 tank water labuan 12. plnt-b post-larvae 1 1 tank water labuan 13. skt-a skeletonema labuan 14. skt-b skeletonema labuan 15. pl2 post-larvae 2 pangandaran 16, pl2t-a post-larvae 2 tank water pangandaran 17, pl2t-b post-larvae 2 tank water pangandaran 18. pl, post-larvae 3 pangandaran 19. pl,t post-larvae 3 tank water pangandaran 20. pl,, post-larvae 6 pangandaran 21. pl,,t-a post-larvae 6 tank water pangandaran 22. pl,,t-b post-larvae 6 tank water pangandaran 23. pl7 post-larvae 7 pangandaran 24. pl7t-a post-larvae 7 tank water pangandaran 25. pl7t-b post-larvae 7 tank water pangandaran 26. at-a artemia pangandaran 27. at-b anemia pangandaran 28. e egg lampung 29. n-b nauplius lampung 30. n-c nauplius lampung 31. z-b zoea lampung 32. mr5w) (v.harveyi) infected shrimp larvae maros-sulsel biotropia no. 20,2003 pathogenic v. harveyi mr5339 was obtained from balitdita (research institute for coastal aquaculture) maros, south-sulawesi and was isolated from infected shrimp larvae. this isolate formed green colonies on tcbs and became luminous on tcbs or swc-agar media. v. harveyi mr5339 was further employed for both in vitro and pathogen challenge assay to screen for potential biocontrol bacteria. sensitivity test of vibrio to rifampicin sensitivity testing of vibrio isolates including v. harveyi mr5339 showed that all of the isolates were sensitive to rifampicin. therefore, a rifampicin resistant mutant of v. harveyi mr5339 was employed in the assay for biocontrol in vitro and pathogen challenge to screen for potential biocontrol bacteria. tjahjadi et al. (1994) reported that almost all of the bacteria that were isolated from seawater and hatchery-rearing water at kalianget, east java, including v. harveyi, were resistant to various antibiotics except rifampicin. rifampicin, a bactericidal antibiotic, is active against gram-positive and some gram-negative bacteria. this drug interferes with transcription processes in bacteria and thus far was never used in shrimp hatchery. in vitro assay for biocontrol activity the inhibitory effects of each vibrio isolate candidate for biocontrol were tested against v. harveyi mr5339 rf (figure 1). three out of 31 isolates tested i.e. skt-b, pl2t-a and n-c were isolated from skeletonema, post-larvae 2 rearing water and nauplii showed the best result. after 24 hours of incubations, only very few colonies (102-103 cells/ml) of v. harveyi mr5339 rf* were grown on culture media inoculated with v. harveyi mr5339 rf* at concentration of 102 cells/ml and vibrio candidates for biocontrol at concentration of 106 cells/ml. the number of colonies in the control experiments (inoculated only with v. harveyi mr5339 rf*) could reach 5x108 cells/ml. the other tested isolates could not significantly inhibit the growth of mr5339 rf, therefore, only sktb, pl2t-a, and n-c were further studied in the pathogenicity challenge experiments. before performing challenge test with v. harveyi mr5339 rfr in shrimp larvae, some potential isolates candidate for biocontrol were analysed for their pathogenicity to shrimp larva. after 5 days of exposure with biocontrol isolates at 1q6 cfu/ml, it had confirmed that skt-b, pl2t-a and n-c isolates were not pathogenic to shrimp larvae. these facts were indicated by relatively similar values of survival rate to those of control group (table 2). pathogen challenge test three of the most potential vibrio isolates i.e. skt-b, pl2t-a and n-c for biocontrol based on in vitro test, were assayed for their activities in inhibiting colonization of v. harveyi mr5339 rf* in shrimp larvae. observations were carried out on larval survival rates and total population of vibrio and v. harveyi mr5339 rr* both in the rearing water and in the dead larvae. results showed that each of the three biocontrol candidates significantly (p<0.05) could increase survival rates of shrimp larvae reared in seawater inoculated with 103 cells/ml of mr5339 rf". larval survival rates in each of biocontrol treatment, i.e. skt-b, pl2t-a and n-c were 93%, 90% and 82%, respectively. on the other hand, treatment with mr5339 rf* inoculation without biocontrol isolate showed only 68% survival rate of shrimp larvae (figure 2). the isolates presumably increased survival rates of shrimp larvae as survival rates of control group (without addition of biocontrol or v. harveyi mr5339 rfr isolates) were lower than treatments with biocontrol isolates. however, observation on shrimp larvae fitness after inoculation is required to determine the physiological condition of larvae. rengpipat et al. (1998) reported the use of bacillus strain s l l as a probiotic administered in enriched artemia to larvae of the black tiger shrimp (p. monodon). at two weeks, p. monodon survival was significantly different between the control groups (85%) and the treated groups (89%). when challenged with a pathogenic v. harveyi strain d331, the shrimp treated with probiotics showed a higher survival (13%) when compared to the control group (4%). haryanti et el. (2000) also reported that inoculation with by-9 strain at a dose of 106 cfu/ml to shrimp larvae, resulted to a survival rate of 59.3%, while for the control only 14.7%. the increased survival rate might be due to the growth inhibition of v. harveyi mr5339 rf* on shrimp larvae by biocontrol bacteria. although no significant difference was observed in the number of v. harveyi mr5339 rf* either in culture media (figure 3) or dead larvae (figure 4), high mortality in shrimp larvae for treatment without biocontrol isolates indicated the effect of the inhibition. however, the way how the mechanisms occurred need to be studied further. according to verschuere et al. (2000), action mechanism of probiotic bacteria or biocontrol could be divided into several ways as follows: (1) production of inhibitory compounds, (2) competition for chemicals or available energy, (3) competition for adhesion sites, (4) enhancement of the immune response, (5) improvement of water quality, (6) interaction with phytoplankton. to get an insight about action mechanism of probiotic bacteria, especially on their competition for adhesion sites, biocontrol bacteria should be tagged with molecular marker so that the existence of bacteria in shrimp larvae could be figure 3. number of colonies of vibrio sp. and v. harveyt mr5339 rľ in larva rearing thanks   potency of vibrio isolates for biocontrol of vibriosiswidanarni et al. detected. one of the molecular markers extensively used for studying bacterial activity in the e nvironment is gf'p (green fluorescent protein) gene isolated from a jellyfish (aequorea victoria) (manning 1997). if expressed, the gene will produce green fluorescence gfp protein under uv light, so that the bacteria could be easily observed. moreover, as molecular gene marker, gfp could provide several advantages such as no requirement for exogenous substrate or energy source for their visualization. gfp assays were reported to be sensitive, stable, non toxic, and did not disturb cell function and growth (josenhans et al. 1998; ling et al. 2000). characterization and identification of biocontrol bacteria skt-b was gram negative, short rod-shape, produced yellow colonies on tcbs, and exhibited swarming activity on swc-agar. this isolate was motile, could utilize glucose and sucrose but not lactose; produced protease and amylase, but did not produce chitinase. partial sequencing (500 bp of the 5'-end) of 16s-rrna gene of skt-b showed that the isolate showed similarity to vibrio alginolyticus (83% of similarity). complete sequencing of the 16srrna gene, however, should give more definitive information about the taxonomic position of this isolate. austin et al. (1995) reported that v. alginolyticus was effective in reducing diseases caused by v. anguillarum and v. ordalii, however, other strains of this bacterium has been associated with vibriosis in shrimp. therefore, characterization and identification of biocontrol strain were crucial steps to be carried out to assess the pathogenicity of biocontrol bacteria to the shrimp larvae. moreover, characterization and identification of biocontrol strain were also important for mass production, quality control and patenting to protect commercial interest (gomezgil et al. 2000). according to riquelme et al. (1997), vibrio might be more promising to be developed as probiotics when compared to other probiotic species for hatchery applications, since vibrios are commonly associated with larvae in culture, and auto-inhibition could limit the growth of pathogenic vibrio. in conclusion, vibrio isolates from tiger shrimp larvae and hatchery environments have the ability in reducing shrimp larvae mortality in pathogen challenge assays. the isolates are potential to be developed as a biocontrol agent and therefore as an alternative to chemical treatment in preventing luminous bacterial diseases in shrimp hatcheries. acknowledgments this research was supported by dip-funded research biotrop 2001/2002 to as. biotropia no. 20, 2003 references alabi, a.o., d.a. jones and j.w. latchford. 1999. the efficacy of immersion as opposed to oral vaccination of penaeus indicus larvae against vibrio harveyi. aquaculture, 179:1-11. austin. ii l. f., p. a. stucken, w. robertson, i. effendi and d. r. w. griffith. 1995. a probiotic strain of vibrio alginofyticus effective in reducing diseases caused by aeromonas salmonicida, v. anguillarum and k ordain. j. fish diseases, 18:93-96. fuller, r. 1992. history and development of probiotics. in r. fuller (ed) probiotics the scientific basis, p.p: 1-8. chapman and hall. london. gildberg, a., a. johansen, and j. bogwald. 1995. growth and survival of atlantic salmon (salmo salar) fry given diets supplemented with, fish protein hydrolysate and lactic acid bacteria during a challenge trial with aeromonas salmonicida. aquaculture, 138:23-34. gomez^jil, b., a. roque and j.f. turnbull. 2000. the use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. aquaculture, 191:259-270. hala, y., a. suwanto, r. afemdi, and m. z. zairin. 2002. adherence and pathogcnicity assay of vibrio harveyi in tiger shrimp (penaeus monoclori) larvae for screening biocontrol agent biotropia, 18:8-51. josenhans, c., s. friedrich and s. suerbaum. 1998. green fluorescent protein as a novel marker and reporter system in helicobacler sp. fems microbiol. lett., 161:263-273. karunasagar, i., r. pai, g. r. malathi, and i. karunasagar. 1994. mass mortality of penaeus monodon larvae due to antibiotic-resistant vibrio harveyi infection. aquaculture, 128:203-209. lavilla-pitogo, c.r., l.l. baticados, e.r. cruz lacierda and l.d. de la pena. 1990. occurrence of luminous bacterial diseases of penaeus monodon larvae in the philippines. aquaculture, 91:1-13. ling, s.h.m., xh. wang, l. xie, t.m. lim, and k.y. leung. 2000. use of green fluorescent protein (gfp) to study the invasion pathways of edwardsiella tarda in in vivo and in vitro fish models. microbiol., 146:7-19.. manning, e. 1997. glow fish: an unusual glowing molecule from jelly fish is helping to illuminate cellular events. bioscience, 47:135-138. marchesi, j.r., t. sato, a.j. weightman, t.a. martin, j.c. fry, s.j. hiom, and w.g. wade. 1998. design and evaluation of useful bacterial-specific pcr primers that amply genes coding for bacterial 16s rrna. appl. environ. microbiol. 64:795-799. ohhira, i., t. tamura, n. fujii, k. inagaki, and h. tanaka. 1996. antimicrobial activity against methicillin-resistant sk/phyhcoccus aureus in the culture broth of enterococcus faecalis th10, an isolate from malaysian fermentation food. temph. japanese j. dairy and food sci., 45:93-96. pedersen, k.l., l. verdonck, b. austin, d.a. austin, a.r. blanch, p.a.d. grimont, j. jofre, s. koblavi, j.l. larsen, t. tiainen, m. vigneulle, and j. swings. 1998. taxonomic evidence that vibrio carchariae grimes et a!.. 1985 is a junior synonym of vibrio harveyi (johnson and shunk, 1936), baumann et al, 1981, int. j. sys. bacteriol., 48:749758. rengpipat, s., s. rukpratanporn, s. piyatiratitivorakul, and p. menasveta. 1998. probiotics in aquaculture: a case study of probiotics for larvae of the black tiger shrimp (penaeus monodon). in flegel, t.w.(ed.) advances in shrimp biotechnology. the national center for genetic engineering and biotechnology. thailand. potency of vibrio isolates for biocontrol of vibriosis — widanarni et al. riquelme. c., r. araya, n. vergara, a. rojas, m. guaita, and m. candia. 1997. potential probiotic strains in the culture of the chilean scallop argopecten purpwatus (lamarck, 1819). aquaculture, 154:17-26. sambrook. j., e.f. fritsch, and t. maniatis. 1989. molecular cloning. cold spring harbor laboratory press, cold spring harbor, new york. suwanto. a., m. yuhana, e. herawaty, and s.l. angka. 1998. genetic diversity of luminous vibrio isolated from shrimp larvae. in flegel t.w. 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(abstract). tjahjadi, m.r., s.l. angka and a. suwanto. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial diseases in tiger shrimp larvae (penaeus monodon fab.) aspac. j. mol. biol. biotechnol. 2:234-352. verschuere. l., g. rombaut, p. sorgeloos, and w. verstraete. 2000. probiotic bacteria as biological control agents in aquaculture. microbiol. mol. biol. rev., 64:655-671. widanarni and a. suwanto. 2000. genetic diversity of ampicillin resistant vibrio isolated from various stages of shrimp larvae development. biotropia, 15: 36^47. 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf biotropia vol. 29 no. 2, 2022: 142 149 doi: 10.11598/btb.2022.29.2.1680 142 distribution pattern and diversity of epiphytic orchids in the curug cibereum path, mount gede pangrango, indonesia bela prapitasari* and ardyan pramudya kurniawan laboratory of ecology-botany, universitas islam negeri sunan kalijaga, yogyakarta 55281, indonesia received 14 october 2021/accepted 18 december 2021 abstract the curug cibereum path as a tourist attraction in the mount gede pangrango area is dominated by a lush tree, making it a suitable place to find a diversity of epiphytic orchids. this study aimed to determine the distribution pattern and diversity of epiphytic orchids along the curug cibereum path which is influenced by environtmental parameters. the research method was carried out by plotting ten plots on the right and left sides of the path, with each plot measuring 500 x 5 m and the distance between plots was 100 m. the results showed that there were 31 species of epiphytic orchids from 12 genera. the distribution pattern of epiphytic orchids was mostly clustered except for oberonia lotsyana, which had a uniform pattern. the epiphytic orchid species with the highest inp were bulbophyllum gibbosum with an inp value of 35% and coelogyne speciosa with an inp value of 20%. species diversity (h ') was moderate (h '=2.54), the evenness index (e) was high (e=0.73), the dominance index (d) was low (d=0.13). the measurements of environmental parameters showed optimal conditions for the growth of epiphytic orchids, namely with a temperature of 24.5 oc, soil moisture of 76.5%, air humidity of 70%, ph 5.1, the light intensity of 6351 lux and wind speed of 0.03 m/s. keywords: curug cibereum, distribution, diversity, dominance, epiphytic orchid introduction orchids are unique plants because they have several benefits both economically and ecologically. the many uses of orchids for decoration, traditional medicinal ingredients and cosmetics make orchids have a high selling value in the world of trade and many fans because they have high aesthetics value. the number of orchids in the world is estimated at around 20,000-30,000 species consisting of 700 genera (shutleworth et al. 1970). orchids in indonesia are estimated at 5,000 species from 40 genera scattered in sumatra, kalimantan, papua, sulawesi and java. in java, there are about 731 species of orchids with the 231 species are endemic. in terms of orchid distribution, west java has 642 species, east java has 390 species, and central java has 295 species (chomber 1990). an epiphytic orchid is one type of natural orchid that has the characteristic of sticking to the host tree. ecologically, epiphytic orchids function as habitats for certain animals such as ants and termites. research activities on epiphytic orchids are currently considered very important because of the many damaged habitats caused by felling trees to clear forests used for repairs, animal husbandry and visitor attractions. according to the world conservation monitoring center (1995), orchids are plants with a high threat of extinction, around 39% (203 species) compared to native indonesian plants, which are also threatened. based on these conditions, many orchids may have become extinct, but no data has been collected (puspitaningtyas 2005). mount gede pangrango, west java, is a national park used as a conservation area, climbing or tourist attraction. one of the attractions in the area is curug cibereum which is located at resort selabintana, sukabumi. the *corresponding author, email: belaprapitasari@gmail.com distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 143 area consists of a natural forest where many orchids are found, both epiphytic and terrestrial. the types of orchids in the area have been recorded. namely, 46 species were found, consisting of 25 species of epiphytic orchids and 21 species of terrestrial orchids (prapitasari et al. 2020). the existence of tourist activities on the curug cibereum path, of course, can affect the reduction in the types of orchids in the area. based on the above explanation, this study was conducted to determine the distribution pattern and diversity of epiphytic orchids in the curug cibereum path which is influenced by environmental parameters. this research is also expected to provide data updates from previous studies. materials and methods study site the research was conducted in march 2021 in the selabintana resort area, sukabumi, which is on the route to curug cibereum mount gede pangrango, west java. geographically, selabintana resort is located at 106o57'41"e and 06o50'50"s with an area of 2,547.93 ha (fig. 1). this area has a topography of hills and mountains with slightly sloping areas, altitudes ranging from 1,130 to 3,019 masl. in general, selabintana resort is an area with a wet climate with an average rainfall of 3,000-4,200 mm/year. the rainy season lasts from october to may, and from december to march the rainfall can be more than 400 mm/month. the average temperature at resort selabintana is 18 oc with relatively high humidity throughout the year, which is around 80-90% (dendang 2009). based on these environmental parameters, it can be seen that the environment in the selabintana area is a suitable habitat for the growth of epiphytic orchids. it can be seen that the types of epiphytic orchids in the selabintana area are very plentiful, especially along the curug path. curug cibereum is the highest waterfall in the gunung gede pangrango national park area and is located at an altitude of 1,200 masl. the curug cibereum path is perfect for a leisurely walk or hiking. the waterfall can be reached with a distance of 3.1 km using gps or 1.5 h from the entrance to resort selabintana or pondok halimun. material and data collection the tools used in this research are tally sheet, camera, environmental parameter tools (lux meter, thermometer, hygrometer, anemometer, soil tester), global positioning system (gps), and orchid identification book, namely orchid of java book (chomber 1990), native orchids of indonesia (handoyo & ramadhani 2006) and orchids of indonesia (handoyo 2019). data collection is done by plotting along the right and left sides of the waterfall path. there were ten plots with each plot size of 500 m long and 5 m wide (fig. 2). the distance between plots was 100 m. the data were collected in the form of epiphytic orchids found (types of orchids, number of species of orchids, the number of individuals of each type of orchid, and data on environmental parameters). figure 1 research sites (the blue and white line is the curug cibereum path) biotropia vol. 29 no. 2, 2022 144 figure 2 the research sampling design of epiphytic orchids in the curug cibereum path data analysis data analysis was carried out qualitatively and quantitatively. qualitative analysis was done by describing the data that have been collected and processed from the research results. quantitative analysis was used to determine species composition and level of species diversity. determination of species composition was used to determine density (d), relative density (rd), frequency (f), relative frequency (rf) and important value index (ivi) (indriyanto 2006). the level of species diversity was determined by the shannonwiener index (h'), evenness index (e), morishita index (id) and dominance index (d) (ernst et al. 2002). 1. species composition a. density (d) = number of individuals of species total area b. relative density (rd) = density each species x 100% density all species c. frequency density (f) = number of individuals x 100% total number of plots d. relative frequency (rf) = number of frequency of each species x 100% frequency all species e. important value index= rd+rf 2. species diversity a. shannon-wiener diversity index (h') h'= -ʃ [pi ln (pi)]; pi: ni n description: h': shannon-wiener diversity index ni: number of individuals of each species n: number of all species shannon-wiener diversity index (h') determination criteria: h' >3 : the diversity is high h' 1-3: the diversity is moderate h' <3 : the diversity is low b. evenness index (e) e = h' ln s description: e : evenness index h' : shannon-wiener diversity index s : number of species evenness index (e) determination criteria: evenness index values ranged from 0-1. if the value is 0, it indicates the level of evenness of species is very uneven, whereas if the value is close to 1, almost all species have the same abundance (maguran 1988). c. morishita index (id) id = q (xi(xi – 1) n(n – 1) description: id : morishita index q : number of sample plots xi : number of individuals on the plots n : total number of species the criteria for determining the morishita index: id>1 : the pattern of distribution of individual types is clumped id=1 : the distribution pattern of individual types is random id<1 : the distribution pattern of individual species is uniform d. dominance index (c) c= ʃ[ni/n]2 description: c: the dominance index of a species ni: the number of individuals of a species n: number of individuals of all types 500 m 500 m curug cibereum path 3.1 km 5 m 1 3 5 7 9 2 4 6 8 10 5 m distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 145 dominance index determination criteria (c): when the value of c is close to 0, then no individual dominates so that there is excellent uniformity. the value of c is close to 1, indicates that several species dominate at specific locations so that the value of the uniformity index is getting smaller (odum 1993). results and discussion distribution pattern of epiphytic orchids the study results found as many as 31 species of epiphytic orchids consisting of 12 genera. comparing our results with the research of prapitasari et al. (2020) regarding the types of orchids found in the selabintana area (both on the curug cibereum path and the hiking trail), there was an increase in the number of epiphytic orchid species in the curug cibereum path from this study, which was 13 species. the epiphytic orchid species found were adenoncos virens, appendicula cornuta, bulbophyllum capitatum, bulbophyllum multiflora, bulbophyllum gibbosum, bulbophyllum sp. 1, bulbophyllum sp. 2, cerastostylis anceps, cerastostylis graminea, dendrochilum sp., oberonia lotsyana, oberonia similis, and phreatia sp. (table 1). table 1 comparison of the types of epiphytic orchids found in the curug cibereum path in 2020 and 2021 no types of epiphytic orchid 2020 2021 distribution index (id) 1 adenoncos virens √ clumped 2 agrostophyllum bicuspidatum √ √ clumped 3 agrostophyllum laxum √ √ clumped 4 appendicula angustifolia √ √ clumped 5 appendicula cornuta √ clumped 6 bulbophyllum capitatum √ clumped 7 bulbophyllum multiflora √ clumped 8 bulbophyllum gibbosum √ clumped 9 bulbophyllum sp. 1 √ clumped 10 bulbophyllum sp. 2 √ clumped 11 bulbophyllum sp. 3 √ √ clumped 12 cerastostylis anceps √ clumped 13 cerastostylis graminea √ clumped 14 cerastostylis sp. √ √ clumped 15 coelogyne speciosa √ √ clumped 16 dendrobium mutabile √ clumped 17 dendrobium rugosum √ √ clumped 18 dendrobium sp. 1 √ √ clumped 19 dendrobium sp. 2 √ √ clumped 20 dendrobium sp. 3 √ √ clumped 21 dendrobium sp. 4 √ √ clumped 22 dendrochilum sp. √ clumped 23 eria iridifolia √ √ clumped 24 eria monostachya √ √ clumped 25 eria multiflora √ √ clumped 26 eria sp. √ √ clumped 27 liparis eliptica √ √ clumped 28 liparis pallida √ √ clumped 29 oberonia similis √ √ unifrom 30 oberonia lotsyana √ clumped 31 phreatia sp. √ clumped 32 schoenorcis juncifolia √ √ clumped amount 20 31 biotropia vol. 29 no. 2, 2022 146 based on the analysis of the morishita index (id), most of the epiphytic orchids in the curug cibereum path showed a clumped distribution pattern (id>1), and there were only one species with uniform distribution, namely oberonia similis (id<1) (table 1). the distribution of clumped is influenced by several factors, such as the breeding process. epiphytic orchids reproduce by producing vast numbers of seeds and usually fall near their mother. besides that, they also reproduce by rhizomes that produce many vegetative tillers (barbour et al. 1987). environmental factors also affect the distribution pattern of epiphytic orchids due to non-uniform environmental conditions (wahyuni et al. 2017). the uniform pattern is caused by the intense competition for survival between epiphytic orchid species, resulting in the distribution of the same living space in an environment. the uniform distribution pattern is a non-random pattern indirectly caused by a limiting factor to the existence of a population. uniform dispersion comes from negative intuition between individual species, such as competition for nourishment or other specialties (onrizal et al. 2005). in addition, not all of the host trees in the curug cibereum patth area are suitable for epiphytic orchids to live. there are also epiphytic spikes that can inhibit the growth of epiphytic orchids because there are too many of them (paramitha et al. 2010). types and composition of epiphytic orchids the most dominant epiphytic orchid species found in the curug cibereum path were appendicula angustifolia (171 individual pieces/ 2.5 ha), bulbophyllum multiflora (142 individuals/ 2.5 ha), bulbophyllum gibbosum (670 individuals/ 2.5 ha), coelogyne speciosa (323 individual pieces/ 2.5 ha), eria iridifolia (119 individuals/2.5 ha), and eria multiflora (272 individuals/2.5 ha). while the few epiphytic orchids found were dendrobium sp. 1 (2 individuals/2.5 ha), dendrobium rugosum (2 individuals/2.5 ha), liparis elliptica (3 individuals/2.5 ha), oberonia similis (1 individual/2.5 ha), and oberonia lotsyana (2 individuals count/2.5 ha) (fig. 3). figure 3 the density of epiphytic orchids and important value index (ivi) in the curug cibereum path distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 147 the presence of several dominant species of epiphytic orchids will affect the value of the important value index (ivi). the ivi value is obtained from relative density (dr) and relative frequency (fr) results by adding the two parameters. the greater the level of dominance of the species in a community, the greater the important value index of that species (tahier et al. 2018). based on the graph above, it is known that the highest ivi value of epiphytic orchids is also found in the orchids which are the most abundant or dominant in the research location. epiphytic orchid species with high ivi were bulbophyllum gibbosum with an ivi value of 35% and coelogyne speciosa with ivi 20% (fig. 3). species with a high ivi dominate and have an influential role in an area, namely, play a role in ecosystem stability. meanwhile, epiphytic orchids with low ivi indicate that the species have a narrow distribution and a specific environment to grow (wulanesa et al. 2017). diversity of epiphytic orchids species diversity is a community-level characteristic based on its biology that can express community structure. at the same time, the diversity index is a value that can indicate the high and low population diversity of the species in the community. this value is obtained by comparing the number of species and individuals in a community or habitat (yuanda 2007). the species diversity index (h') of epiphytic orchids in the curug cibereum path is in the medium category with an h' value of 2.54 (fig. 4). the moderate diversity index indicates that the ecosystem is relatively balanced, with sufficient productivity and moderate ecological pressure (fitriana 2006). the medium category is also caused by the area having almost the same abundance of epiphytic orchid species. according to odum (1996), the greater the number of species found, the greater the diversity, whereas if the number of species found is small, the diversity will be low, and it is suspected that a few species only dominate the area. figure 4 diversity index (h'), evenness index (e), and dominance index (c) of epiphytic orchids in the curug cibereum path the evenness index, also known as the species abundance index, is used to determine the level of species abundance influenced by diversity. the evenness index value of epiphytic orchids in the curug cibereum path was e = 0.73, indicating the evenness of epiphytic orchids in the curug cibereum path was relatively high (because the value is close to 1). a reasonably high evenness index indicates that epiphytic orchids are evenly distributed with a stable number in a habitat (zulkhaidah et al. 2018). the high evenness index of epiphytic orchid species is also influenced by the reasonably stable value of the species diversity index (h'). species diversity (h') of epiphytic orchids in the curug cibereum path is moderate with abundant epiphytic orchid species so that the distribution of orchid species in the location is evenly distributed with the stable numbers of individual orchids. a species with a high level of stability has a more significant opportunity to maintain the sustainability of its species in an environment (odum 1993). the dominance index of a species illustrates that the species is very influential on the environment. the dominance index value of epiphytic orchids in the curug cibereum path was c = 0.13, which means that the epiphytic orchid dominance index in the curug cibereum path had a low-level dominating category. having a dominant species in the study location, it is suspected that the species is the most able species to adapt and survive in an environment. dominant species will affect the value of the uniformity index, which will decrease. the dominance index (c) value is related to the value of species diversity, in which biotropia vol. 29 no. 2, 2022 148 the higher the species diversity, the lower the dominance index or vice versa (rikardus et al. 2017). thus, moderate diversity index (h') of epiphytic orchids in the curug cibereum path means lower dominance index. environmental parameter factors the high level of diversity of epiphytic orchids in the curug cibereum path is strongly influenced by environmental parameters. environmental parameters are an essential factor in determining the existence of a plant. environmental parameters in the curug cibereum path showed normal conditions for the growth of epiphytic orchids (table 2). table 2 environmental parameters in the cibereum curug no environtmental parameters measurement results 1 soil ph 5.1 ± 0.96 2 soil moisture (%) 76.5 ± 15.11 3 air temperature (oc) 24.5 ±2.44 4 humidity (%) 70 ± 11.73 5 light intensity (lux) 6,321 ± 16,482.52 6 wind velocity (m/s) 0.03 ± 0.19 the optimal temperature for the growth of epiphytic orchids is 24.5 oc. the magnitude of the temperature is very suitable for the growth of orchids. the coldest temperature for orchid growth is 12.7 oc, and the average temperature for orchids is in the range of 15-28 oc (indarto 2011). furthermore, soil ph measurements were carried out under the epiphytic orchid host tree and not in the area around the epiphytic orchid growth because the location was difficult to reach. the result of soil ph measurement was 5.1, indicating that the ph is suitable for the growth of epiphytic orchid host trees. the normal ph for orchid growth is 5-6.5 (purbadi et al. 2005). soil ph condition at the study site tend to be acidic (ph = 5.1) due to high soil moisture, which was 76.5%. wet soil due to high humidity will cause a low ph value, and vice versa (yulia 2008). air humidity at the research site was 70%. the high humidity condition is very suitable for the growth of epiphytic orchids. the humidity required for optimal orchid growth is 50 80%. humidity that is too low causes the air around the orchid to dry out and cause a disturbance. conversely, if the humidity is too high, it will increase disease attacks, especially diseases caused by fungi and bacteria. air humidity on the curug cibereum path is high, also influenced by low wind speeds so that it tends to get wet. if the humidity is low, the wind speed is high, causing an area to experience dry condition. at the study site, there was almost no wind blowing. it was only encountered several times with very low speeds so that the results of the average wind speed at the study site were only 0.03 m/s. the wind speed is very suitable for orchid growth, where orchids like soft air circulation. (purwanto 2016). light intensity is one of the environmental parameter affecting the growth of epiphytic orchids. the living nature of epiphytic orchids attached to the host tree is one form of adaptation to get sunlight. therefore, epiphytic orchids require more light than terrestrial orchids (tirta & sutomo 2014). usually, epiphytic orchids require different light intensities depending on the type of epiphytic orchid itself. for example, the type of dendrobium requires a light intensity of 2,0003,000 lux. the light intensity for orchid growth is 1,000-2,000 footcandle or 10,000-20,000 lux (darmono 2007). the results of the light intensity measurement in the curug cibereum path were 6,321 lux. the amount of light intensity is still suitable for the growth of epiphytic orchids because it does not interfere with the growth of epiphytic orchids in the curug cibereum path. in addition, the light intensity at the study site is also influenced by the canopy density. several locations do not have a dense canopy causing relatively high light intensity. however, epiphytic orchids are more commonly found in low light intensity. conclusion there were 31 species of epiphytic orchids from 12 genera found in the curug cibereum path. curug cibereum has environment parameters suitable for epiphytic orchids. acknowledgments this study was funded by the research and community service institute of uin sunan kalijaga yogyakarta. the authors are gratefull to distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 149 the officials of the mount gede pangrango national park for the permission and facilities provided. special gratitudes are also presented to the field research team (ahmad aliwafa, dharfan ihlasul iman, and aulya nidaur rahmah) who assisted in data collection. references barbour sl, lam l, fredlund dg. 1987. transient seepage model for saturated-unsaturated soil systems: a geotechnical engineering approach. can geotech 24(198):565-80. boughey as. 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paliasa plant (kleinhovia hospita l.) in the bontobahari district. conservation media 22(1):11-8. world conservation monitoring centre. 1995. indonesian threatened plants. exploration 2(3):8-9. wulanesa wos, soegianto a, basuki n. 2017. exploration and characterization of epiphytic orchids in the coban trisula forest, bromo tengger semeru national park area. journal crop production 5(1):125-31. yulia dn. 2008. inventory and habitat characteristics of dendrobium capra j.j. in madiun and bojonegoro. biodiversitas 9(3):190-3. zulkaidhah, sri nm, ferdiansah. 2018. diversity of natural orchids species in lembantongoa village, palolo district, sigi regency. j forest sains 15(2): 58-66. biotropia vol. 30 no. 2, 2023: 206 219 doi: 10.11598/btb.2023.30.2.1850 206 preliminary study: feeding ecology and daily activity of three colored langur (presbytis chrysomelas ssp cruciger thomas, 1892) in danau sentarum national park nyoto santoso, sutopo*, lorenzo elton meo, natasya nurul fauziah and alfatheya margwita diva department of forest resources conservation and ecotourism, faculty of forestry and environment, ipb university, bogor, 16680, indonesia received 13 october 2022 / revised 8 may 2023 / accepted 8 may 2023 abstract three colored langur (presbytis chrysomelas ssp cruciger thomas, 1892) is a primate that has been recognized as a critically endangered species in accordance with the international union for conservation of nature, however still not protected and lacks substantial information about the bio-ecology of their natural habitat. the habitat plays a determining factor not only in space utilization but also the daily activities of the three colored langurs (p c cruciger). the aim of this preliminary study is to collect information regarding their habitat characteristic, feed species, daily activity, and canopy stratum utilization. this research was conducted between july and august of 2021 at bukit semujan, lupak mawang resort, danau sentarum national park. the method implemented was to collect the habitat characteristic by plot samples, and their daily activity data by scan sampling with consecutive recording. the study demonstrated that langur inhabited both primary as well as mixed forests (swamp, cultivation land, and secondary forest). there were 27 species as feeds of langurs and the most preferred types of feed are gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). the most preferred feed compositions consisted of leaves (50%), fruits (30%), and seeds (20%). the most frequently utilized stratum for activities was stratum c (70,49%) and b (27.87%). the highest daily activities were categorized into three parts of time, the morning was dominated by social (44,26%), the afternoon was dominated by rest (59,7 7%), and the evening was dominated by social (73,68%). the highest social activities shown by three colored langurs were agonistic (48,48%), followed by vocalization (39,39%), playing (10,61%), exploring (1,52%), and sexual (0%). keywords: daily activities, feeding ecology, presbytis chrysomelas, ranging pattern, three colored langur introduction three colored langurs locally known as lutung sentarum (presbytis chrysomelas ssp cruciger thomas 1892) and referenced as langur for this paper is one of the endemic primates of bornean island and danau sentarum national park, a place used as their natural habitat. nevertheless, it has been categorized as critically endangered species by the international union for conservation of nature (iucn), however it is yet to be registered as the protected species in accordance with the regulation of animal protection in indonesia. this can be attributed to the absence of adequate ecological information on the species, namely, habitat characteristics and daily activities. so, far there has been no research that specifically examines the ecology of the feed and space utilization for the langur habitat. the distribution of p c ssp cruciger was found at sungai pelaik sub-village but only with encounters notes, and do not provide comprehensive information on the ecology (rifki et al. 2019). research that has been done previously is still very finite on topics regarding habitat characteristics (musyafa and santoso 2020) and population estimates that still need to be repeated (aripin et al. 2019). sources *corresponding author, email: nyotosa@apps.ipb.ac.id mailto:nyotosa@apps.ipb.ac.id preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 207 indicate that this species are also inhabitants of the north borneo island additionally known in sabah, sarawak (malaysia) and brunei darussalam, however there is not adequate information available to understand the comprehensive ecology of this species in their natural habitat (nijman 2020). areas that are practiced as natural habitat of langur in danau sentarum national park can be found in the forest of bukit semujan. administratively, these habitats scope at lupak mawang resort and at government administration including the selimbau and jongkong district, kapuas hulu regency. the condition of feed resources in the habitat of the three colored langur also affects their daily activity pattern. habitat impacted the canopy utilization for distinct activities too, namely, moving, foraging, and social recreations (watanabe 1981; hadi et al. 2012). primates such as three colored langurs usually exhibit further characteristics of their activity with vocalization or agonism from intraspecies interactions (singh et al. 2011; houle et al. 2006). this study aims to identify ecological characteristics in the form of habitat and feed, as well as daily activity, and ranging patterns of three colored langurs. materials and methods study site and time this research was conducted at bukit semujan, lupak mawang resort, danau sentarum national park, west kalimantan, indonesia with geographic coordinates of 00°45'–01°02' n and 111°55'–112°26' e. primary data collected namely, group size, habitat characteristic, feeds, and daily activity were observed between july and august of 2021. bukit semujan has topography that ranges from flat (elevation of 60 above sea level) and continues to be wavy at an elevation of 60–80 and high cliff at an elevation of 80–300 above sea level. land cover condition in accordance with the on ground truthing and rupa bumi indonesia classification of kapuas hulu regency encompasses the primary forest at the top of the hills and mixed forest that includes secondary forest surrounding the sub-hills, swamp forest, and small area of the cultivation land. following figures exhibit the study area (figure 1a and 1b). figure 1a land cover condition at bukit semujan captured by an aerial photo taken by a drone biotropia vol. 30 no. 2, 2023 208 figure 2b map of the research location at bukit semujan, lupak mawang resort field data of habitat characteristic implemented the plot sample with purposive sampling for every single land coverage and ecotone areas (soerianegara and indrawan 1988). total sampling comprised of 12 plots, located in swamp forest, secondary, and mixed forest. plot sample size were prepared in blocks of 20 x 20 m for trees habitus, 10 x 10 m for pole habitus, 5 x 5 m for sapling habitus, and 2 x2 m for herbs and seedling habitus, and these samples were further divided into 4 plots at three habitats. these plots were distributed perpendicular to each hill gradient from swamp forest, mixed and primary forest. in order to analyze the habitat of three colored langurs, the established methods included vegetation analysis and the land coverage classification in accordance with the ministry of forestry and environment (2021) that consisted of three main habitat types namely, secondary forest, swamp forest, and mixed forest. the vegetation analysis was further categorized into relative density (2), relative frequency (4), relative dominance (6), and the importance value index (inp) (4, 5) on each growth level of vegetation. meanwhile, the availability of langur feed in the two ecosystems was determined based on the relative density value of each species. these values were utilized to establish the dominant species of a habitat (parmadi et al. 2016). density (k) =  ind.species_i ...................... (1) areas of plot sample relative density (kr) = density a species x 100% ................... (2) density all number species frequency (f) = number of plot sample that was found ...................... (3) total number of all plot sample relative frequency (fr) = frequency a species x 100% ................... (4) frequency all the species dominance (f) = basal areas species_i ...................... (5) areas of plot sample relative dominance (dr) = dominance a species x 100% ................... (6) dominance of all species preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 209 inp herbs, seedling, and sapling = kr + fr inp pole and trees = kr + fr + dr to illustrate the structure of habitat langurs the profile diagram by sexifs software (hardja and vincent 2008) was implemented. data collected to create the structure profile using this software consist x: the x position of the tree base (m), y: the y position of the tree base (m), species: the species label, if the label is match with the one in the species list, then it will be linked, otherwise new species definition will be created, dbh: the diameter at breast height of the tree (m), height: the height of the tree (m), cr depth: crown depth (m), cr curve: crown curve (m), cr radius: crown radius in vertical projection, can be more than one value separated by semicolon (m), rotation: a rotation of the vertical projection of the crown geometry (degree), cp: crown position index (0 1), cf: crown form index (0 1). this illustration will exhibit the vertical model of the ranging pattern by three colored langurs. the tree canopy stratum used by three colored langurs was further classified into several strata, namely stratum a (>30 m), stratum b (20 30 m), stratum c (4 20 m), stratum d (1 4 m), and stratum e (0 1 m) (soerianegara and indrawan 1988). method to identify the daily and social activities was use scan sampling with 1 minute interval time to record each activity of an individual in a group at a certain time cumulatively (hepworth & hamilton 2001). to study the behavior pattern of the three colored langurs, continuous recording was practiced. each behavior pattern was categorized into eating (take and eat foods to the mouth), moving (movements from one to another site using their quadrupedal), resting (off from all their activity and it can be indicated by the closed eyes or sleeping time), and social activity (napier and napier 1967). the social behavioral pattern was further classified into agonistic, vocalization, playing, exploring, and sexual. each behavior was calculated as a percentage of its frequency and duration. results and discussion group size of langur the field observation recorded three groups of langurs with total 16 encounters. each group consisted of 21 24 individuals and was widespread in several parts of bukit semujan. the first group recorded nine individuals, including an infant and a baby in the south hill.eanwhile, on the west side (second group) of the hill five individuals were observed. third group with seven individuals was recorded on the east of the hill, including one sole male with direct encounter who was observed more than two times always ranging alone at the surrounding camp research. this individual langur always observed around the group 2, so we notes the appearance at group 2. we have assumed that the alone male langur is a subadult individual and was rejected by the original group. it can be indicated and visible from body-size and genitals that are not too big and clearly visible. following is the group composition of langurs inhabiting the study site during the preliminary study (table 1). table 1 composition and time of appearance of three colored langurs in bukit semujan. group time of appearance 1. 12/7/2021 16/7/2021 18/7/2021 10 10 12 2. 10/7/2021 11/7/2021 12/7/2021 22/7/2021 24/7/2021 25/7/2021 26/7/2021 27/7/2021 5 2 5 1* 3 4 2 1* 3. 18/7/2021 7 notes: *alone male biotropia vol. 30 no. 2, 2023 210 habitat characteristic the habitat of the three colored langurs in bukit samujan, lupak mawang resort has been distinguished in two distinct ecosystems, namely, the primary forest, and the mixed forest that occurs between swamp and sub-hill forest. based on the results of vegetation identification in both ecosystems, there were 27 plant species that were found directly consumed as langur feeds. there are three species for each growth level that have the highest value index in both ecosystems, which signifies the cruciality of these species and its relevance in langur feeds for their future regeneration. the types of vegetation in the primary forest with the highest index value have been depicted in table 2 below. habitat characteristics of primary forest is that it has thicker and firmer tree canopy than mixed forest. these characteristics not only play a significant role as a feed source, but also functions as a sleeping tree and aids in easier movement during their locomotion (febriyanti 2008). arboreal primates will choose sleeping trees based on the proportion and thickness of branches (giovana 2015). three colored langurs use the lush and tall canopy to acquire shelter from the sun during the day. the mixed forest has a good continuity canopy but is divided into several segments. it restricts langurs’ movements to lower planes and constraints them to rotate to reach other tree crowns. table 2 highest index value of vegetation in primary forest growth level/habitus local name primary forest mixed forest scientific name inp (%) local name scientific name inp (%) seedling resak garcinia lateriflora 73,93 blaban bukit syzygium rostratum 54,87 keratih bukit cleistanthus sumatranus 43,50 keranji bukit garcinia rostrata 41,77 kretih shorea sp. 28,73 ubah bukit syzygium laxiflorum 12,87 sapling kemerawan d. rappa 22,71 ubah bukit syzygium laxiflorum 29,59 blitan fordia splendidissima 18,89 blaban pepah ptychopyxis bacciformis 21,85 resak garcinia lateriflora 18,89 kebesi memecylon myrsinoides 20,54 pole resak garcinia lateriflora 59,11 engkupak ptychopyxis bacciformis 45,97 masam unidentified 30,11 pau pimelodendron griffithianum 41,31 keranji tikus xerospermum norohianum 30,08 sikup bukit gardenia sp. 38,04 tree medang drepananthus havilandii 35,84 kelangsau dryobalanops lanceolata 59,67 mengkirai dryobalanops lanceolata 32,15 cempedak air artocarpus teysmannii 26,07 resak bara cleistanthus sumatranus 22,77 belaban whiteodendron moultonianum 18,41 figure 3 canopy projection of three colored langur’s habitat (a) mixed forest (b) primary forest preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 211 species and feed availability based on the results, there were 9 feed species that were recognized to be directly consumed by langurs in the study area during the observation, and the remaining 18 species were acquainted with the local community information. for the preliminary study, this is a big potential for diverse information and data about three colored langur’s feed species, especially during fruiting season. the following table contains the species list of langur’s feed and their respective fragments that were consumed during the preliminary study (table 4). the myrtaceae family tree was found to be the chief feed resource among the tree species. previous research found 19 species of feed trees in the same location. the dominant feed belonged to clusiaceae, moraceae, anacardiaceae, and euphorbiaceae (musyaffa 2020). the types of feed highly preferred were gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). based on these feed data, there is some unusual feed like h brasilliensis. h brasiliensis was present a long time before the study areas have become a national park, and previous status as a nature and wild reserve from 1981–19831. h brasiliensis was planted by the local community that has indigenous land surrounding the bukit semujan. nevertheless, consumption of rubber seed during the preliminary study was staggering, however, field observations confirmed that langurs not only eat the seeds but also the young leaves of the rubber tree. however, langurs can only consume the already planted ones now due to the new regulations set by the national park that restrict further planting of rubber trees in the protected land area. table 4 feed species and their consumed parts no. local name scientific name habitat habitat type parts eaten leaves fruit seed 1 tekam padi* polyalthia insignis annonaceae swamp √ 2 buah gita susu* willughbeia coriacea apocynaceae mixed forest √ √ 3 resak* garcinia lateriflora clusiaceae mixed forest √ 4 keranji bukit garcinia rostrata clusiaceae mixed forest √ 5 kenarin diospyros sp. ebenaceae swamp √ 6 karet* hevea brasiliensis euphorbiaceae cultivation land √ √ 7 temau cratoxylum grauncum hypericaceae swamp √ 8 putat barringtonia sp. lecythidaceae swamp √ √ 9 engkurung* grewia paniculata malvaceae swamp √ √ 10 empakan durio kutejensis malvaceae mixed forest √ 11 kebesi pternandra galeata melastomaceae mixed forest √ √ 12 ara* ficus spathulifolia moraceae secondary forest sub-hill forest √ 13 tenggelam timbul syzygium havilandii myrtaceae swamp √ 14 engkuni baccaurea parviflora phyllanthaceae swamp √ √ 15 sikup pantai gardenia sp. rubiaceae swamp √ 16 entangis ixora sp. rubiaceae swamp √ √ 17 sibau* nephelium unicatum sapindaceae mixed forest √ 18 kemerawan lempung* dipterocarpus rappa dipterocarpaceae mixed forest √ 19 merepat* unidentified unidentified mixed forest √ √ 20 tawun unidentified unidentified mixed forest √ 21 insubal bukit unidentified unidentified mixed forest √ 22 pregi bukit unidentified unidentified mixed forest √ 23 terap artocarpus odoratissimus moraceae mixed forest √ 24 merbemban xanthophyllum affine polygalaceae swamp √ 25 masung syzygium claviflora myrtaceae swamp √ √ 26 peregi unidentified unidentified mixed forest √ √ 27 jijab syzygium sp. myrtaceae mixed forest √ *based on primary observation data, and the local name refers to the melayu language by community within the study site 1 zonation books of danau sentarum national park, 2014 (not published) biotropia vol. 30 no. 2, 2023 212 figure 3 several documentations of feed species that are eaten by lutung sentarum such as a. grewia paniculata; b. bark from rubber seed (hevea brasiliensis), c. bark from buah gita (willughbeia coriacea). the feed part for consumption included leaves, fruits, and seeds. three colored langurs primarily consumed leaves (50%) in comparison to other parts, such as fruits (30%), and seeds (20%). the genus prebytis is one of the primates that eat fruits and leaves, however mainly prefers leaves parts (sumarni 2016). presbytis chrysomelas ssp cruciger is the same as their relative species in one genus, namely p. comata and p. hosei (ruhiyat 1983; mitchell 1994). the type of feed consumed by three colored langurs was dominant from tree species (89%) and lianas (11%). three colored langurs chiefly consumed leaf of rice tekam (polyalthia insignis). some leaf species have a complete source of nutrients including protein, carbohydrates, fat, tannins, and water (zulfahri and pohan 2016). the species that consumed the fruit were gita susu (willughbeia coriacea), sibau (nephelium unicatum), and karet (hevea brasiliensis). three colored langurs usually consume fruits that are small to adequately sized with range diameter 0,5 – 5 centimeter and lightly colored. primates did like fruits with hard skin, cracked, and yellow to brown color (leighton & leighton 1983). in addition to the leaves and fruits, three colored langurs also consume the seeds of several types of feed species. seeds of karet (hevea brasiliensis) are a rich source of forage for langurs. the water, protein, fat, and crude fiber content is beneficial for the optimum metabolism required for the growth of langur (syamsunarno & sunarno 2019). the potential of feed availability in both habitats can be determined through the relative density of each species. it was influenced by physical and biotic habitat factors and disturbances from destructive activities (violita et al. 2015). the relative density of a plant species will determine the dominance of that species in a community (putri & sudrajat 2017). the results of the relative density of forage plant species in the primary forest are shown in table 5. table 5 relative density of feed plant species in primary and mixed forest growth level type of ecosystem name species relative density (%) seedling primary forest resak garcinia lateriflora 36,4 kebesi memecylon myrsinoides 22,4 sibau nephelium unicantum 0,7 mixed forest keranji bukit garcinia rostrata 31,2 kenarin diospyros sp. 1,9 engkurung grewia paniculata 1,1 sapling primary forest kemerawan d. rappa 18,3 resak garcinia lateriflora 10,2 karet hevea brasiliensis 2 keranji itea macrophylla 2 resak batu cleistanthus sumatranus 2 mixed forest kebesi bukit pternandra galeata 11 kebesi m. myrsinoides 6,7 engkunik barringtonia macrostachya 6,1 kenarin diospyros sp. 6,1 keranji bukit garcinia rostrata 5,5 engkurung grewia paniculata 0,6 preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 213 pole primary forest resak garcinia lateriflora 30 keranji tikus xerospermum norohianum 10 keranji bukit garcinia rostrata 10 keranji itea macrophylla 5 mixed forest keranji bukit garcinia rostrata 10,3 engkurung grewia paniculata 9,6 kenarin diospyros sp. 9,4 sikup bukit gardenia sp. 8,2 engkunik dehaasia caesia 5 tree primary forest resak garcinia lateriflora 10,9 keranji itea macrophylla 2,7 terap tidak diketahui 1,3 keranji bukit garcinia rostrata 1,3 mixed forest engkurung grewia paniculata 4,7 sikup rimba garcinia rostrata 4,7 sikup bukit gardenia sp. 4,7 karet hevea brasiliensis 2,3 engkunik dehaasia caesia 2,3 the relative density of feed plants in the primary forest was 9 species of seedlings, 20 species of saplings, 11 species of poles, and 23 species of trees. the seedling and sapling density could be an indicator of feed availability in the future, while the pole and tree level indicate the current availability of feed (shankar 2001). the feed species with high regeneration capacity in a primary forest is resak (garcinia lateriflora). this species has a high relative density at every growth level. on the other hand, keranji (itea macrophylla) has an overall high relative density at all growth stages, except the seedling level. meanwhile, the relative density of mixed forest was 16 species of seedlings, 16 species of saplings, 13 species of poles, and 18 species of trees. the feed species with high regeneration capacity is engkurung (grewia paniculata). the other species with good regeneration and inadequate growth rates were keranji bukit (garcinia rostrata) and kenarin (diospyros sp.). then, the species that did not have good regeneration were sikup rimba (garcinia rostrata) and karet (hevea brasiliensis). the species like g panculata, g rostrata and diospyros sp with high regeneration need to increase, and species with low regeneration either. daily activity lutung sentarum begins activity at dawn (06.00) until early evening (18.00), adding the total time for their activity to 320 minutes. this diurnal primate will give signal communication to a movement for their group, called a morning call. this behavior exhibits in other species too such as p. thomasi, which emits a morning call from a sleeping tree (wich et al. 2002). during midday between 12.00–13.00, the three colored langurs will tend to find a place for rest after foraging in the morning. foraging in the morning would increase body temperature with air temperature (prayogo 2006). their activities were divided into foraging, moving, resting, and social. the percentage of each activity is divided into three parts of times, namely morning (06.00–10.00), afternoon (10.01–14.00), and afternoon (14.01– 18.00) in figure 3. biotropia vol. 30 no. 2, 2023 214 figure 4 daily activities of three colored langurs at different time three colored langurs allocate more time to do certain activities, especially social behavior and foraging in the morning. some activities usually coincide with another activity. in addition, the langurs will keep moving until they find suitable feed trees that the group needs (figure 4a). the movement gesture of langurs was rarely quadrupedal, walking using all four legs and arms. this movement could be down and up or to move to other trees by jumping. an adult male as the group leader always led the group movement. however, in some cases, it has been found that adult females with babies would lead the group movement. in all circumstances, juvenile or young individuals are restricted from leading the group (nursal 2001). lutung sentarum will rest more during the day after foraging (figure 4b). this can be attributed to several factors, ranging from the process in the body’s metabolism after eating to the influence of air temperature that affects body condition (alikodra 1990; prayogo 2006). lutung sentarum spent 1–2 hours resting and sleeping during the day. the rest location usually chosen had a sturdy branch or a dense canopy, such as mengkirai (dryobalanops lanceolata) species. the resting position of the lutung sentarum includes hunched sitting with one hand holding the trunk or branch of a nearby tree. it is also found in other primates, namely trachypithecus auratus. the positions found were bending over with the head tucked into the stomach between the two knees of the legs, the soles of the feet overlapping each other and the hands holding the branches (giovana 2015). preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 215 figure 5 three colored langurs’ behavior (a) moving (b) resting the activity of the lutung sentarum in the afternoon dominates by social behavior, namely a collection of behaviors carried out by two or more individuals and interconnected to survive. this activity could be carried out with other species. these activities are divided into agonistic and affiliative (sajuthi et al. 2016). agonistic is a form of negative response to something like fights and coalitions. as for affiliation, it is a form of positive response such as grooming, playing, vocalization, and sexual activity. based on the analysis, the social activity amount to 66 times with a total duration of 72 minutes. agonistic is the most active behavioral pattern (48.48%) of social activities. this behavior is a gesture of response to becoming steady and grinning position. the group leader (alpha male) would exhibit this position with vocalizations. this posture aims to display objects that make langur feel threatened and to repel. usually, this position can be sitting or bending the body toward other species to make the langur look big. chimpanzees (pan troglophytes) also exhibited it by straightening their body hair to make their posture look bigger and more dangerous (van hooff 1973). vocalization is an expression by making sounds for other interspecies and environments (irawan 2011). the vocals of three colored lutung sentarum were identified into two categories, its sounds with loud and long characteristics aimed at disturbance as a form of threat. then the voice with a shorter duration is addressed to group members as a form of disturbance warning. both types of vocalizations belong to loud calls, vocalizations that are loud and prominent in the primate vocal repertoire (delgado 2006). in addition, there are many functions of vocalization, namely as a marker of social status, efforts to maintain food sources, maintaining territory between groups, and maintaining cohesion between group members (wich et al. 2002, wich et al. 2003, wich &nunn 2022, chiarello 1995, riley 2005). the playing activities most carried out by young males, included chasing and wrestling each other. playing will train the motor nerves to avoid predators, protect themselves, and attract partners (spinka et al. 2001). then for grooming, it is usually done by adult female individuals to their babies. this activity shows proximity to each individual in groups. it is done by treating and searching for lice on the ears, neck, shoulders, and back with the hands, feet, teeth, and tongue (napier & napier 1985). grooming is also good for health maintenance (zamma 2002). activities that were not found in this research were sexual. the fundamental of living things was to obtain and maintain populations of their species according to the maturity level of an individual. lutung sentarum are polygamous and practices polygynous mating system, so mating does not refer to the mating season like other species, namely macaca fascicularis (setiawan 2002). stratum use on daily activity based on the encounters with three colored langurs, their distribution comprehends several areas of primary and mixed forest. this biotropia vol. 30 no. 2, 2023 216 movement has been influenced by factors of feed, shelter, and a safe place for langur groups. during afternoons, three colored langurs tend to move to the hills because of the tall trees that are more than 30 meters in height with lush canopy. using the stratum of three colored langurs in the primary and mixed forest had different purposes based on their activities. trees for three colored langurs as arboreal animals are important, especially for rest, selfprotection, foraging, mating, and socializing. the results showed that most of the canopy proportions used by three colored langurs were stratum c (70.49%) and stratum b (27.87%). it was similar to the previous study in which three colored langurs used stratum c (78%) dominantly (musyaffa 2020). the stratum utility is presented in figure 5. stratum c was a place for foraging activity that included seven encounters with the langur group there. the longest resting time is about 103 minutes. mengkirai (dryobalanops lanceolata) is one of the sleeping trees, and the langur group was found at an altitude of 30 meters on the mengkirai tree. in addition, there was a case in this study that lutung sentarum would descend to the stratum e (ground) to pick up fallen food. the moving activity of three colored langurs has been carried out in stratum b and c for supply needs that avoided disturbance. the canopy has a function to make it easier for the lutung sentarum to move through connected tree branches. other primates, such as the javan gibbon, use a dense canopy to support their cruising range (zanuansyah 2013). figure 5 stratum utility based on three colored langurs’ activities figure 6 stratum projection in ranging pattern (a) mixed forest (b) primary forest preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 217 in addition, the social activities mostly had a similar stratum to other activities. the langur appearance on stratum c and b amounted to 19 times and five times during the research respectively. vocalization was the most common activity; the sounds were louder and echoed further. other species, such as the trachypithecus auratus will choose trees with upper-middle crowns for vocalization so that the sound echoes further and clearer (oktaviani 2009). the use of ranging patterns by three colored langurs in primary and mixed forest habitats is exhibited in figure 6. in addition, this habitat is inhabited by other species, especially in the primate class. according to ecological theory, the concepts of niche and resource competition are central to the coexistence of sympatric species (gause 1934; tokeshi 1999). conservation implication for conservational efforts, feeding ecology is crucial including the type and composition of feed in its natural habitat not only for better understanding but also to conduct habitat enrichment, security and further research related to bio-ecology and conservation of langur in the natural habitat by the long-term national park managers and interested parties. as we are aware, primates do not receive the required research attention and are often neglected due to arduous and restricted collection of primary data in their habitat. this preliminary study is a progressive step forward toward future research and to draw the attention of researchers and observers to conduct numerous studies on primate species that are currently lacking the bio-ecology data. conclusion the habitat of the three colored langurs in bukit semujan, lupak mawang resort was discovered in both primary as well as mixed forests. the feed species for three colored langurs in both habitats amounted to 27 species, and the highly preferred are gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). its feed existed in study areas because of prior sowing by the local community before the land was distinguished as part of the national park. meanwhile, the highly preferred feed compositions were leaves (50%), fruits (30%), and seeds (20%). the highly preferred stratum for their activities was stratum c (70.49%) and b (27.87%). the identification of vegetation species for resting or sleeping was mengkirai (dryobalanops lanceolata). the highest daily activity of three colored langurs is divided into three parts of time, the morning dominated by social activity (44.26%), the day dominated by resting 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http://dx.doi.org/10.1163/15685390252902292 https://link.springer.com/journal/265 https://link.springer.com/journal/265 https://doi.org/10.1007/s00265-002-0541-8 https://doi.org/10.1007/s00265-002-0541-8 https://doi.org/10.1046/j.1439-0310.2003.00837.x https://doi.org/10.1007/bf02629575 https://doi.org/10.1007/bf02629575 microsoft word 11 biotropia vol. 13 no. 1, 2006 : 11 21 seedbank and seedling emergence characteristics of weeds in ricefield soils of the muda granary area in north-west peninsular malaysia mahfuza begum''*, abdul shukor juraimi'', syed omar bin syed rastan", rajan amartalingam^ and azmi bin man2) " faculty of agriculture, university putra malaysia, 43400 sci'dang. malaysia l, pulau pinang, malaysia abstract tlie experiment was conducted in the glasshouse of upm from march 2003 to june 2004 to determine the soil seedbank in the ricefields ot'muda rice granary area in peninsular malaysia. six soil cores of 5 cm in diameter and 10 cm depth were sampled from each of 24 fields. all samples from each individual field were bulked and placed in plastic trays of 38 x 25 x 10 cm. soil was moistened as required and emergence of weed seedlings were recorded over period of one year. after one year, remaining seeds were separated, removed and identified. the total seed bank was estimated at 1136.48 million/ha of which 62.35% (708.60 million seedlings ha"1) germinated within 12 months and 37.65% (427.88 million seeds ha"1) remained ungerminated. total of 20 taxa were recognized. based on importance value (i.v.) the five most dominant species in terms of emerged seedling were fimbristylis miliacea, leplochloa chinensis, litjwigia hyssopifolia, cyperus difformii and c. iria. of the remaining seeds the five dominant species with decreasing trend in ranking were f. miliacea, scirpus lateriflonis, monochoria vagina/is, l. hyssopifolia and l. chinensis. ranking of total seed reserves (seedlings+ remaining seeds) were similar to emerged seedling indicating that emerged seedlings reflect the actual weed flora in the muda area. among the dominant species f. miliacea accounted for 58.07% of emerged seedlings, 79.31% of remaining seeds and 66.07% of total seed bank. total seedling emergence of all species was higher in the first observation in april 2003 and cumulative seedling emergence showed no clear peaks. key words : seedbank, seedling emergence, weeds, ricefield soils, malaysia introduction the weed seedbank comprising viable seeds either on the surface or in the soil, is the principal source of annual weed infestations in field crops. the seedbank consists of new seeds recently shed by weeds and older seeds that have persisted in the soil for several years. changes in agricultural management practices alter the pattern of disturbance and produce changes in seedbank characteristics. changes in these seedbank characteristics often lead to changes in the size and species composition of the weed flora (roberts and neilson 1981; wrucke and arnold 1985; cardinal/. 1991; clementsetal. 1996). * corresponding author: mafupaz@hotmail.com 11 biotropia vol. 13 no. 1, 2006 in muda rice granary, the change in cultural practices and use of agrochemicals had led to a shift in species from broad-leaved sedge dominance weed flora to grasses (azmi et al. 1995). the building up of certain weed species with continuous use of particular herbicides may be due to inherent resistance to that herbicide or the continuous absorption of the herbicide at sub-lethal concentrations leading to gradual development of herbicide resistance. sometimes, elimination of the competitors of a particular weed favours the abundance and predominance of the weed in that particular environment (azmi and baki 1995; ho 1998). these weeds produce abundant viable seeds and generally the vast majority of seeds entering the seedbank come from such annual weeds growing on the land (roberts 1981; hume and archibold 1986), and would thus represent the potential weed flora (kott 1947; rahman et al. 1995). therefore, knowledge of the dynamics of the seedbank is necessary to determine whether weed seed population has changed and the rate of change. species composition of the flora may be sometimes more important than total number of seeds (roberts and ricketts 1979). however, weed seed populations in cultivated soils are generally composed of a few dominant species that are present in high numbers, a few others present at moderate levels, and a large variety of species present in the soil at low levels (vengris 1953; wilson and furrer 1996). knowledge of the size and species composition of this seedbank would be useful in predicting future weed infestations (carretero 1977). the fate of seeds in the soil is difficult to determine and comprehensive information on the seed pool dynamics of most species is sparse. high weed seed populations occur in tropical soils, but limited data is available on emergence patterns (zimdahl et al. 1988). most weeds show some periodicity of emergence (roberts and margaret 1980). practical knowledge of periodicity of germination is of significant importance, since it is a major factor in determining the association of weeds with cropping systems and to enable a degree of forecasting as to which weed species may occur in a seedbed. predicting potential weed emergence is fundamental in the development of integrated pest management strategies for weed control. predictions of emerged seedling densities allow estimations of weed competition, crop yield loss, need for herbicides, financial returns and weed seed production at the end of the growing season (forcella 1992). furthermore, the remaining seeds in the soil are also a major concern to understand the soil seedbank status (cardina and sparrow 1996). this study was conducted to determine the germinable seedbank, total seed reserve, species composition of the entire germinable seedbank, ranking of species and their emergence pattern in the soils of the muda rice granary area. materials and methods sample sites were located in northern parts of muda ricefields at kedah in the north-west of peninsular malaysia. soil samples were obtained from 24 fields in march 2003. six soil cores of 5 cm in diameter and 10 cm depth were sampled from 12 seedbank and seedling emergence characteristic of weeds m. begum etal. each field in a w shaped pattern. samples from each individual field were bulked and air-dried in the glasshouse. subsequently, the soil samples were passed through a 4-mm sieve to remove large debris and break up soil pods. samples from each of the 24 fields were placed in 38 x 25 x 10 cm plastic trays. each plastic tray was filled with 2.0 kg of soil. the samples were daily sprinkled with water as needed in order to keep them moist. weed seedlings that emerged were identified, counted, and removed at one-month intervals, throughout the one-year germination period. seedlings were identified using the seedling keys of chancellor (1966). weedy rice seedlings were excluded in this study because of the obvious difficulty of identifying between weedy rice and off-type rice. seedlings of questionable identity were transferred to pots and grown until maturity to facilitate identification. after the removal of each batch of seedlings, soils were air dried for 3 days, thoroughly mixed in order to expose the weed seeds to the upper layer of the soil, and rewetted to permit further germination. this process was repeated 12 times from march 2003 to february 2004. after one year any remaining seeds were separated by the method described by wilson et al. (1985). the soil in each tray was passed through a descending series of five sieves containing screens of the following sizes: 4 mm (5 mesh), 2 mm (10 mesh), 850 jim (20 mesh), 425 urn (40 mesh), and 250 urn (60 mesh). water was run through the sieves to enhance sample separation through the screens. the contents collected in each screen were removed, sun dried, and seeds were removed under a luminated magnifier. seeds from entire samples were sorted using a dissecting microscope and counted according to species. the seed counts were expressed in numbers per m2. the total of emerged seedling during one year period and the remaining seeds represent the total species composition and seed reserve of respective species. seed and seedling counts were converted to numbers per m2 within 10 cm soil depth. results and discussion seedbank density total seed reserve in the muda area was 113648 seeds m"2 equivalent to 1136.48 million ha"1, of which 70860 seeds m"2 germinated within 12 months and remaining seeds were 42 788 m"2 (table 1). the total number of buried seeds as well as germinable seeds reported here are higher compared with 29 551 viable seeds m"2 reported by pane (1997) and much lower than densities of 712228 to 930910 13 biotropia vol. 13 no. i, 2006 seeds m"2 recorded by ismail et al. (1995) in direct-seeded ricefields at kampung tandop in muda area. however, watanabe et al. (1997) noted that such abundance reported by the latter tar exceeds that usually observed in cropped land, including upland ricefields' which had two orders of magnitude lower. while in the philippines, vega and sierra (1970) reported more or less similar viable seeds as the present study, with 800 million seeds ha"' in ricefields within a plough depth of about 15 cm. species composition in seedbank a total of 20 taxa were recognized, of which 14 common weed species emerged from the soils collected in the muda area (table 1). based on dominance ranking, the highest population of seedlings that emerged were of f. miliacea followed by l. chinensis, c. difformis, c. iria, l. hyssopifolia, sphenoclea zeylanica, m. vaginalis, limnophila erecta, hedyotis diffusa, ceratopteris thalictroides, echinochloa crus-galli (complex), bacopa rotundifolia and eleocharis variegata and e. colona (table 1). pane (1997) reported 17 species in muda ricefields of which the higher population of seedlings emergence were l. chinensis, f. miliacea, c. difformis, s. zeylanica, l. octovulvis, m. vaginalis, c. iria, lindernia octovulvis, e. crus-galli, and sagittaria guyanensis. the dominant species were almost similar as in the present study. another seven species were rare and present in relatively low numbers. after the 12-month gennination period, the seeds remaining in soils comprised 13 weed species (table 1). of these weed species seven, namely f. miliacea, l. chinensis, c. difformis, c. iria, l. hyssopifolia, m. vaginalis and e. crus-galli (complex) had been recorded as seedlings during the 12month period, while seeds of 6 weed species, previously not found as seedlings, were from scirpus lateriflorus, s. juncoides, s. guyanensis, nymphoides indica, najas graminea and cleome viscosa. the non-emergence of these species may be due to the very low population of these species combined with low germination rate. ismail et al. (1995) had observed that although seeds of s. juncoides were the dominant species in volunteer seedling fields in the muda area, but its summed dominance ratio values of emerging seedlings was very small, which also indicate its low germination rate. seeds of s. lateriflorus, s. juncoides, s. guyanensis can persist in the dormant state in the soil and dormancy may be broken through straw burn and tillage (azmi 2005; pers. comm.). no remaining seeds were detected for seven weed species which had germinated during 12 months period, namely s. zeylanica, c. thalictroides, l. erecta, h. diffusa, e. variegata, b. rotundifolia and e. colona (table 1). ismail et al. (1995) also observed that although c. thalictroides, hedyotis sp., bacopa sp. emerged in direct seeded ricefields in muda, no seeds of these species was found in soil seed reserves. based on total seed reserve, the species composition in descending order was f. miliacea, l. chinensis, l. hyssopifolia, c. difformis, c. iria, m. vaginalis, s. zeylanica, s. lateriflorus, l. erecta, e. crus-galli (complex), c. thalictroides, h. diffusa, s. juncoides, s. guyanensis, b. rotundifolia, n. indica, e. variegata, n. graminea, e. colona and c. viscosa (table 1). however, 14 seeclbank and seedling emergence characteristic of weeds m. begum el al. table 1. germinable soil seedbank, remaining seed and total seed reserves in the muda rice granary area weed species seedbank germinable within 12 months (no.m"2) remaining seed reserves (no.m"2) total seed reserve (germinable seeds + remaining seeds) f. miliacea 41148 33936 75084 l. chiiienxis 7655 989 8644 c. difformis 4947 42 4989 c. iria 4530 428 4958 l. hyssopifolia 3633 1561 5194 s. zeylanica 2402 2402 m. vaginali.i 1481 1598 3079 l. erecta 1292 1292 h. diffusa 1129 1129 c. thalictroides 1045 1045 e. crus-galli 670 477 1147 b. mtundifolia 644 ' 644 e. variegata 227 227 e. colona 57 57 s. laterifloms 1613 1613 s.juncoides 943 943 s. guyanensis 742 742 n. indica 235 235 n. gramineu 201 201 c. viscosa 23 23 total 70860 42788 113648 due to the limited size of soil samples analyzed for seeds, only seeds of most predominant species were expected to be detected with consistency (forcella et al. 1992). occurrence of species frequency the frequency of occurrence of emerged seedlings and remaining seeds of individual component species in the seedbank are shown in table 2. f. miliacea, l. chinensis and l. hyssopifolia were most frequent, and found in all sampled fields (100% frequency), followed by c. difformis (95.83%), c. iria (95.83%), m. vaginalis (95.83%), s. zeylanica (91.67%), e. crus-galli (91.67%), l. erecta (83.33%), c. thalictroides (75%) and h. diffusa (58.33%) (table 2). these were found in large numbers up to 1046-41148 in"2, except e. crus-galli, which had a high frequency of occurrence but the number of seeds was 670 m"2 (table 2). watanabe et al. (1997) observed that echinochloa species were well controlled by herbicide and few adult plants, less than eight plants m"2 (mostly 0-3 plants m"2) were observed growing after rice heading. average number of spikelets/plant of e. crus-galli was 972 (watanabe et al. 1997) which was lower than other dominant species 15 biotropia vol. 13 no. 1,2006 (viz. l. chinensis, 1877 spikelets per plant). in spite of the frequency, total seed incorporation was lower than with other dominant species like l. chinensis, f, miliacea, l. hyssopifolia, c. iria, c. difformis etc. in the case of remaining seeds, the most frequent species were m. vaginalis (87.5%), f. miliacea (83.33%), l. hyssopifolia (83.33%), 5. giiyanensis (79.19%), s. lateriflorus (70.83%), e. crus-galli (complex) (58.33%) and l chinensis (54.17%). all species had large seed populations, except e. cms-galli (complex). bahtia et al. (1990) reported that seed reserves of e. crus-galli were mostly 97.7% exhausted during one season and only a small fraction (2.3%) carried over to the second season. no seed germination occurred during the third season and soil was free of viable seeds. similarly, azmi et al (1995) detected very few viable seeds of echinochloa crus-galli in the soil after six cropping seasons, suggesting seed longevity of barnyard grass in ricefields was shorter than three years. this study revealed that most frequent species appeared as a most dominant species. the percentage of occurrence of many species were high which have higher percentage viability than m vaginalis and f. miliacea (table 2). this indicates that these species are persistent in the seed bank, and hence become dominant in the above ground flora, even though there was a shift towards grassy weeds due to changes in the cultural practice from transplanting to direct seeding. dominant species in seedbank according to importance value, the five most dominant species in terms of emerged seedlings were f. miliacea (58.07%), l. chinensis (10.80%), c. difformis (6.98%), c. iria (6.39%) and l. hyssopifolia (5.13%) with another eight weed species sharing only 12.55% of the total emerged seedlings (figure la). in terms of seeds remaining in the soil after 12 months, the five most important species were f. miliacea (79.31%), s. laterifloms (3.77%), m. vaginalis (3.74 %), l. hyssopifolia (3.65%) and l. chinensis (2.31%), while other species shared only 7.22% (figure ib). the five most dominant weed species in terms of total seed reserves (seedling + remaining seeds) in the muda area were f. miliacea (66.07%), l. chinensis (7.61%), l. hyssopifolia (4.57%), c. difformis (4.39%) and c. iria (4.36%) while the rest of the species shared only 13% of the total seed reserve (figure ic). this was similar to the germinable seedlings of 12 months period, but with slight variation in ranking of species. pane (1997) reported that the predominant weed species in the muda area, with respect to emerged seedlings in the seedbank study, were l. chinensis, f. miliacea, c. difformis. s. zeylanica and l. octovulvis. the results were almost similar to the present study, but the relative percentage and ranking were slightly different. reserves of seeds in soil are typically dominated by two to four species (wilson and furrer 1996). at irri, zimdahl et al. (1988) reported that in lowland irrigated ricefields three weed species, namely c. difformis (52%), f. miliacea (26%) and m. vaginalis (18%) , covered 96% of total emerged seedlings. in this study emergence density, ungerminated seeds and total seed reserve of f. miliacea were much higher than other weeds. watanabe et al. (1996) also observed 16   note  :  fimmi‐  f.  miliacea;  lepch‐  l  chinensis;  cypdi‐  c.  difformis;  cypir‐  c.  iria  and  ludhy‐  l.  hyssopifolia;  monva‐ m. vaginalis; sirla‐ s1. lateriflorus.  17  biotropia vol. 13 no. 1, 2006 higher emergence of f. miliacea seedlings in the muda area. pane (1997) reported a f. miliacea germinable seedbank of 22.9%, which was lower than this study, but was the second most dominant species after l. chinensis (26.9%), which according to ho et al. (1995) occupied 80% of above-ground weed flora. the domination of the seedbank by a single annual species, in this case f. miliacea, is not unusual. a single species often comprises over half of the soil seedbank (thompson 1986; schott and hamberg 1997; navie et al. 2004). common lambsquarters comprised more than 50% of the seedbank (clements et al. 1996). these clearly explain the huge seedbank and persistence o'f f. miliacea in the area. emergence pattern in general, weed emergence is influenced by soil disturbance, temperature, rainfall, soil moisture and radiant energy. in the present study the highest percentage of emerged seedlings was recorded in the first observation in april (20.29%). seedlings emerged with each soil disturbance at emergence rates of between 4.45-20.29. seedlings continued to emerge irrespective of the time of year, but in reduced numbers. however, periodic seedling emergence showed no clear peaks during the 12-month period (figure 2). within the first two months 32.84% of seedlings had emerged and more than 50% seedlings emerged within 5 months. watanabe et al. (1996) observed that weed seedlings emerged mostly in the first thirty to forty days after rice seeding, although l. chinensis, f. miliacea, m. vaginalis, l. hyssopifolia, and broadleaved weeds often emerged over a longer duration. zimdahl et al. (1988) observed that one third of all weed species emerged within 3 weeks of tillage, and 57% emerged within 6 weeks in upland soils at irri, whereas 38 and 51% of total emergence occurred within 3 and 6 weeks after tillage, respectively. jensen (1969) also found seedling emergence accounted for \ 18 seedbank and seedling emergence characteristic of weeds m. begum et al. only about 25% of the seeds in the soil and that most of those that did so in the first month. he found the strong correlation between immediate seedling emergence in the glasshouse and field emergence suggests only the first flush of seedling emergence need to be considered. conclusions a total 20 taxa (including seedling + remaining seeds) were found of which 14 species were germinable, while six species did not germinate during the 12-month period in this study. total seed reserve was 1136.48 million ha"' of which 708.60 million ha"' was germinable within the 12month period. previous studies had only reported the germinable seedbank in sampled soils. no information was available on dormant seeds of different species, which would reflect the actual seed reserves of the muda area. f. miliacea, l. chinensis, l. hyssopifolia, c. difformis and c. iria appeared to be the most frequent species and made up the bulk of the seed population in the soil, with more than 80% occurring as a germinable seedbank. among these important species the domination of the seedbank by a f. miliacea comprised 58.07%, 79.31% and 66.07% of germinable seedbank, ungerminated seeds and total seed reserves, respectively. the result indicates that the very large size of this seedbank is probably due to both its prolific seed production and the ability of its seeds to persist more than a year. it would be valuable to predict the size of a weed infestation before it actually occurred, and this is especially true if reliance is to be placed on pre-emergence herbicides, which must be applied before the nature and severity of the potential infestation are visible. to determine whether site-specific weed management is practical, the first criterion is to decide whether weeds (density, species) vary enough from field to field. the second step is to obtain accurate and reliable information about weed species and density on specific field. the third step matches weed management solutions with problems. acknowledgement our appreciation goes to the third world organization for women in science (twows) trieste, italy and universiti putra malaysia under the intensification of research in priority areas (irpa) (no.: 01-02-04-0778-pr0068/05-05 ) who had provided the grant of postgraduate fellowship and research facilities for this study. references azmi, m. and b.b. bald. 1995. the succession of noxious weeds in tropical asian ricefields with emphasis on malaysian rice ecosystem. in: proceeding 15* asian pacific weed science society conference, tsukuba, japan, p. 51-67. azmi, m., m. mashhor, k. itoh, and h. watanabe. 1995. life cycle and seed longevity of echinochloa crus-galli complex in direct seeded rice in malaysia. in: proceeding of 15 asian pacific weed science conference, tsukuba, japan, p. 505-511. 19 biotropia vol. 13 no. i, 2006 bhatia r.k., k..s. sandhu and t. singh. 1990. geiinination and longevity of echinochloa crus-galli l. under natural conditions. j. res. punjab agric. univ., 27 (1):17-21. carretero, /.l. 1977. estimation del contenido de semillas de malas hierbas de un suelo agricola como prediction de su flora adventicia. ann. inst. biol. caranilles, 34: 267-278. cardina, j., e. regnier and k. harrison. 1991. long-term tillage effects on seedbanks in three ohio soils. weed sci., 39: 186194. cardina, j. and d.h. sparrow. 1996. a comparison of methods to predict weed seedling populations from the soil seedbank. weed sci., 44: 46-5 1 . chancellor, r.j. 1966. the identification of weed seedlings of farm and garden. blackwell scientific publication, oxford. clements, d.r., d.l. benoit, s.d. murphy and c.j. swanton. 1996. tillage effects on weed seed return and seedbank composition. weed sci., 44:3 14-322. forcella, f. 1992. prediction of weed seedling densities from buried seed reserves. weed res., 32: 29-38. forcella, f., r.g. wilson, k..a. renner, j. dekker, r.g. harvey, d.a. aim, d.d. buhler and j. cardina. 1992. weed seedbanks of the u.s. cornbelt: magnitude, variation, emergence and application. weed sci., 40: 636-644. jensen, h.a., 1969. content of buried seeds in arable soils in denmark and its relation to the weed population. dansk botanisk arkiv., 27: 7-57. ho, n.k. 1998. the rice agro-ecosystem of the muda irrigation scheme: an overview. in: nashriah et at. (eds.) rice agrosystem of the muda irrigation scheme, malaysia. malaysian institute of nuclear technology research (mint) and mada, bangi, kajang, malaysia, p. 1-24. ho, n.k., y.m. esa, and m. abu bakar. 1995. implementation of agricultural extension programme on integrated weed management in rice: malaysia approaches and experience. rice 1pm network workshop on weed management in rice production. 19-23 june 1993. ferringhi beach hotel, penang, malaysia, p. 14. hume, l. and o.w. archibold. 1986. the influence of a weedy habitat on the seed bank of an adjacent cultivated field. canadian j. of bot., 64: 1879-1883. ismail, s., z.n. faezah and n.k. ho. 1995. weed population and their buried seeds in ricefields of the muda area, kedah, malaysia. pertanika j. trop. agric. sci., 1 8( 1 ):2 1 -28. kott, s.a. 1947. the biological properties of weedy plants and the struggle against the wecdiness of soils. ogiz-sel khozgiz. moskva. navie, s.c., f.d. panetta, r.e. mcfadyen, and s.w. adkins, 2004. germinable soil seedbanks of central queensland rangelands invaded by the exotic weed parthenium hysterophoru.i l. weed biol. and manag.,4: 154-167. pane, h., 1997. studies on ecology and biology of red sprangletop [ leptocmoa chinensis) (l.) nees] and its management in direct seeded rice. ph. d. thesis. universiti sains malaysia, pp. 41-60. rahman, a., t.k. james, n. grbavac, and j. mellsop. 1995. evaluation of two methods for enumerating the soil weed seedbank. in proceedings 4s'1' n.z plant protection conference, www.hortnet.co.nz / roberts, h.a. and m.e. ricketts. 1979. quantitative relationships between the weed flora after cultivation and the seed population in the soil. weed res., 19:269-275. roberts, h.a. and e. margaret. 1980. emergence patterns of weed seedlings in relation to cultivation and rainfall. weed res., 20:377-386. 20 seedbank and seedling emergence characteristic of weeds m. begum et al. roberts, h.a. 1981. seed banks in soils. advances appl. biol., 6:1-55. roberts, h.a. and j.e. neilson. 1981. changes in the soil seedbank of four long-term crop/herbicide experiments, i. appl. ecol., 18: 661-668. schott, g.w. and s.p. hamberg. 1997. the seed rain and seedbank of an adjacent native tallgrass prairie and old field. can. j. bot., 75:1-7. thompson, k. 1986. small-scale heterogeneity in the seedbank of an acidic grassland. j. ecol., 74 : 733-738. vega, m.r. and j.n. sierra. 1970. population of weed seeds in a lowland rice field. phillipp. agric., 54:1-7. vengris, j. 1953. weed populations as related to certain cultivated crops in the connecticut river vally, ma. weeds, 2: 125134. watanabe, h., m. azmi, and md. z. israail. 1997. emergence of major weeds and their population change in wetseeded rice fields of the muda area, peninsular malaysia. in: proceedings of 16th asian pacific weed science society conference, malaysian plant protection society, rajan, a. (ed.), kuala lumpur, pp. 246-250. wilson, r.g., e.d. derr and l.a. nelson. 1985. potential for using weed seed content in the soil to predict future weed problems. weed sci., 33:171-175. wilson, r.g. and j. furrer. 1996. 1996. where do weeds come from? university of nebraska-lincoln cooperative extension, http : // www.iam-.unl.edu /pubs /weeds / g807.htm wrucke, m.a. and w.e. arnold. 1985. weed species distribution as influenced by tillage and herbicides. weed sci., 33; 853856. zimdahl, r.l., k.. moody and r.t. lubigan. 1988. patterns of weed emergence in tropical soil. weed sci., 36: 603-608. 21 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 3. imam (a species).cdr biotropia vol. 19 no. 1, 2012: 19 29 a species-specific pcr assay based on the internal transcribed spacer (its) regions for identification of , and on banana mycosphaerella eumusae m. fijiensis m. musicola iman hidayat recipient of biotrop research grant 2010/accepted 28 june 2012 a study on development of a rapid pcr-based detection method based on its region of , , and on banana was carried out. the main objecive of this study was to develop a fast and species-specific pcr-based detection method for the presence of species on banana. the methods include collection of specimens, morphological identification supported by molecular phylogenetic analysis, rflp analysis, species-specific primers development, and validation. two species of , namely, and , and one unidentified species were found in java island. three restriction enzymes used in the rflp analysis, viz, alui, haeiii, and taqi were capable to discriminate , , and . two species-specific primer pairs, viz, mfijf/mfijr and mmusf/mmusr have been successfully developed to detect the presence of and , respectively. banana, detection, fungi, leaf spot, phytopathology microbiology division, research center for biology, indonesian institute of sciences (lipi), cibinong 16911, west java, indonesia m. eumusae m. fijiensis m. musicola mycosphaerella mycosphaerella m. fijiensis m. musicola pseudocercospora m. eumusae m. fijiensis m. musicola m. fijiensis m. musicola mycosphaerella abstract introduction key words: indonesia is one of banana production zones in southeast asia. however, crop losses from global climate change and fungal pathogens pose a serious threat not only to indonesia, but also to global food security. therefore, these threats should not be underestimated. among the banana pathogens, three morphologically similar species, viz, (black leaf streak disease/black sigatoka), (yellow sigatoka disease), and (eumusae leaf spot) are well known as important plant pathogens (crous & mourichon 2002). in indonesia, these pathogens are considered as quarantine organisms (http://www.karantina.deptan.go.id/optk/ mycosphaerella fijiensis m. musicola m. eumusae * corresponding author : imanhidayat@yahoo.com 19 detail.php?id=731). therefore, it is important to prevent introduction (entry and establishment) and to limit dissemination of these pathogens in many indonesian banana-producing regions. correct and rapid identification is a fundamental step for limiting the dissemination of the plant pathogens (arzanlou 2007). failure to manage the pathogens would have far reaching effects on the industry. the 10-14 days incubation and classical isolation of the pathogens by culturing on appropriate media followed by morphological characters examination is a standard method currently used in indonesia for the imported crops inspection. however, an accurate detection and diagnosis of the and based on the conventional method are complicated due to the similarity in morphological characters (arzanlou 2007). consequently, this problem yield difficulties for indonesian quarantine in inspecting imported banana seeds or crops. many pcr-detection methods for fungi have shown to be accurate and sensitive in detection various plant pathogens (bonants 1997; mumford 2006). its sequence analysis has shown that , and are only distantly related in terms of phylogeny (crous 2002). however, the phylogeny method was still time consuming and lacked specificity to differentiate among the , and (arzanlou 2007). the lack of specificity was possibly due to the high variability among those three pathogens. therefore, it is necessary to develop a fast and specific pcr-based detection method with the aim of improving the specificity of the diagnostic procedure and increasing throughout readiness for outbreaks of the disease. fungal materials were collected from several locations in bogor and cibinong (west java), and one specimen was collected from wonosobo (central java). specimens with black leaf streak diseases symptoms of were collected during the course of field trips by using a 10×/20× magnifying lens. specimens were kept in resealable plastic bag. the bags were labelled by adding all necessary information such as location, collector/s, collection date, host name, etc. microscopic examination of materials was referred to hidayat . (2007). ascomata appearances of spp. and caespituli of anamorphic states ( spp.) on the host surface were observed by using stereo microscope (olympus szx7). detailed observations of morphological characters was carried out by means of an olympus cx31 light microscope using oil immersion (1000×). water and lactophenol were used as mounting media. measurements of all important characters and photographing/line drawings were conducted at a magnification of 1000×. single spore isolation was referred to choi . (1999). voucher specimens were deposited at the herbarium bogoriense, research center for biology, indonesian institute of sciences-lipi, cibinong, west java, indonesia. living cultures were deposited at the et al. m. fijiensis, m. musicola, m. eumusae et al. et al. et al. m. fijiensis, m. musicola m. eumusae et al. m. fijiensis, m. musicola m. eumusae et al. m. fijiensis et al mycosphaerella pseudocercospora et al materials and method fungal materials biotropia vol. 19 no. 1, 2012 20 lipimc microbial culture collection, microbiology division, research center for biology, indonesian institute of sciences-lipi, cibinong, west java, indonesia. fungal species found in this study were compared to isolate of the , , and obtained from cbs culture collection (table 1). dna from fungal cultures was extracted using cetyltrimethylammonium bromide (ctab) protocols (rogers & bendich 1994). the primers its1 (5'-gaagtaaaag tcgtaacaag-3') and its4 (5'cctccgcttattgatatgc-3') (white . 1990) were used to amplify the its area. the pcr of 10×concentrated reaction buffer containing 1.5mm mgcl taq dna polymerase (5 water . the pcr reaction was performed as follows: 1 cycle of 5 min at 94ºc followed by 40 cycles of 30s at 94ºc, 30s at 52ºc, and 30s at 72ºc. one cycle of 7 min at 72ºc was conducted. after amplification, 5 l of the reaction mixture was loaded onto a 1.0% agarose gel in 0.5×tbe buffer, separated by electrophoresis, stained with ethidium bromide, and viewed and photographed under uv light. a negative control (no dna target) was included in every experiment to test for contamination, as well as a positive control (dna from a reference strain of the pathogen). the amplicons was sequenced in both directions using the pcr primers and a dyenamic et terminator cycle sequencing kit (amersham, biosciences) according to the manufacturer's recommendations. the products were analyzed on an abi prism 3700 dna sequencer (perkin-elmer, foster city, ca). a consensus sequences were computed from the forward and reverse sequences with seqman from the lasergene package (dnastar, madison, wi). the sequences obtained from the respective primers (its5 and its4) were aligned in clustal x (thomson . 1997) and bioedit (hall 1999). phylogenetic analysis was performed in paup* (swofford 2002). ambiguously aligned sites were excluded from all analyses. unweighted parsimony (up) analysis were performed. gaps were treated as missing data. maximum parsimony analysis was performed for all data sets using the heuristic search option with 1000 random taxa additions and tree bisection and reconstruction as the branch-swapping algorithm. branches of zero length was collapsed and all multiple, equally parsimonious trees were saved. the robustness of the trees obtained was evaluated by 1,000 bootstrap replications. other measures calculated include tree length, consistency index, retention index, and rescaled consistency index (tl, ci, ri, and rc, respectively). the resulting phylogenetic tree was printed with treeview version 1.6.6 (page 1996). restriction digestion of pcr products was conducted directly without further purification with restriction endonucleases to obtain rflps; each sample was digested m. eumusae m. fijiensis m. musicola et al et al dna extraction and sequencing sequence alignment and phylogenetic analysis restriction fragment length polymorphism (rflp) analysis reaction mixture contained 5μl dna suspension; 2.5μl ; 2.5μl 600μmdntps; 0.25μl of each primer at 60μm; 0.2μl u/μl); 0.25μl internal control, and was filled up with milliq to a final volume of 25μl μ 2 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella 21 biotropia vol. 19 no. 1, 2012 with i, iii, i, or i in single enzyme digests. per each 20ml restriction digest, 10 ml of unpurified, amplified pcr reaction was mixed with the appropriate restriction reaction buffer and 10 u of the appropriate enzyme and then incubated for 6h at 37°c for the i, iii, or i digests or at 65°c for the i digests. restriction fragments were separated by electrophoresis in 2% (wt/vol) and 2.5% (wt/vol) sepharide gel matrix in 1× tae (40mm tris acetate, 1mm sodium edta) with etbr at 100 ng/ml in the gel and running buffer. dna bands were visualized by fluorescence under uv light and photographed. sequences obtained from its region were aligned with clustal x (thomson . 1997) dan bioedit (hall 1999). a series of species specific primers were designed using vector nti software (invitrogen, sigma-aldrich), based on sequence differences among the , , and . the robustness and specificity of various primer combinations were evaluated using dna from isolates of the , , and . dna extraction and pcr amplification of these isolates were performed as described above. alu hae taq rsa alu hae rsa taq et al m. fijiensis m. musicola m. eumusae m. fijiensis m. musicola m. eumusae development of specific pcr primers table 1. list of and obtained in this study.mycosphaerella pseudocercospora no. name origin culture collection number 1 mycosphaerella musicola (mycosphaerella sp.1) cibinong, west java, indonesia lipimc 0598 2 mycosphaerella fijiensis (mycosphaerella sp.2) cibalagung, west java, indonesia lipimc 0599 3 mycosphaerella musicola (mycosphaerella sp.3) wonosobo, central java, indonesia lipimc 0600 4 mycosphaerella eumusae unknown cbs 114825 5 mycosphaerella fijiensis cameroon cbs 120258 6 mycosphaerella musicola cuba cbs 116634 7 pseudocercospora sp. cibinong, west java, indonesia lipimc 0601 results and discussions fungal materials and phylogenetic analysis three isolates of and one isolate of were isolated from specimens collected. the cultures of species collected in this study were compared morphologically to the three species from banana obtained from cbs culture collection (netherlands). all isolates are listed in table 1. blast result from ncbi genbank database showed that sequences of sp.1 has 100% similarity to the (ay646445) (fig. 1), and sp.2 has 99% similarity to the (gq169763) (fig. 2). mycosphaerella pseudocercospora mycosphaerella mycosphaerella mycosphaerella m. musicola mycosphaerella m. fijiensis 22 the alignment data matrix of newly its sequences of three species and one speg. species from banana were aligned with sequences of , fresen., and fr. retrieved from ncbi genbank dna database. the alignment consists of 47 taxa including sequences of (fresen.) g.a. de vries and (de not.) crous & u. braun as outgroup. the data matrix yielded 510 total characters included in the analysis of which 320 characters were constant, 27 characters were variable and parsimony-uninformative and 137 characters were parsimony-informative. twentysix of the informative characters which were positioned within small insertion/deletions or ambiguous regions were excluded from the analysis. two maximum parsimonious trees were generated from the analysis. sum of minimum possible lengths is 232, and sum of maximum possible length was 1091. the best parsimonious tree selected by using kh test was generated in 360 steps (ci = 0. 644, ri = 0. 851, rc = 0.548, hi = 0. 356). the best phylogenetic trees obtained from unweighted maximum parsimony analysis is shown in figure 3 mycosphaerella pseudocercospora mycosphaerella cercospora pseudocercospora passalora cladosporium cladosporioides davidiella tassiana . figure 1. pairwise alignment showed 100% similarity between sp.1 and . (ay646445). mycosphaerella m musicola based on this analysis, three major clades were performed. these included clade with anamorph (clade i) with 61% bootstrap support. another clade was and anamorph (clade ii, 54% bootstrap support) which is also sister clade to clade with 61% bootstrap support. the last clade is and anamorph (clade iii) with 90% bootstrap support. the sp.1 from this study nested together with species in the clade with 100% bootstrap support. another species from this study, sp.3 nested together with species within clade with 92% bootstrap support. this information confirms the mycosphaerella pseudocercospora mycosphaerella cercospora mycosphaerella pseudocercospora mycosphaerella passalora mycosphaerella m. musicola mycosphaerella mycosphaerella m. fijiensis 23 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella biotropia vol. 19 no. 1, 2012 species name of sp.1 as , and sp.3 as . furthermore, this finding confirms that and exist in indonesian banana plantation (java). another isolate, sp.1 needs more detailed examination as this species does not form monophyletic group with any clades in the phylogenetic tree all species from banana, viz, , , and form a monophyletic clade with 52% bootstrap support. this finding has shown that species from banana is a distinct group of species among the species from various hosts. it has also indicated that the three species of from banana are host specific to the banana trees. further analysis such as pathogenicity test is necessary to carry out in order to justify the specificity of the three species from banana. mycosphaerella m. musicola mycosphaerella m. fijienssis m. fijiensis m. musicola pseudocercospora mycosphaerella m. eumusae m. fijiensis m musicola mycosphaerella mycosphaerella mycosphaerella mycosphaerella . figure 2. pairwise alignment showed two nucleotides differences between sp.2 and . (gq169763) (boxes). mycosphaerella m fijiensis restriction fragment length polymorphism ( ) analysisrflp polymorphism of fragment size of its regions was recognized as reported previously in other fungal group (gardes & bruns 1993; sreenivasadprasad 1996), and it was thought to be variable in the sequences of its region because of nucleotide deletions and insertions. in order to identify the species detected by pcr using primers its5 and its4, the rflps of the its region were generated using four restriction enzymes, namely, i, iii, i, or i. from the analysis, only i was not very useful because it did not cut the amplicon of all species et al. mycosphaerella alu hae taq rsa rsa mycosphaerella 24 from banana (fig. 4a). other restriction enzymes, namely, i, iii, and i, generated more fragment per digest, so that the its sequences of the , , and could be separated using each rflps profile. the iii had two recognition sites in the fragments of , and had three recognition sites in the fragments of and (fig. 4b). one isolate of also be separated from three other isolates of using the iii restriction enzyme because it only had two recognition sites in the fragments. a similar result was also found in the restriction fragments of generated by i and (fig. 4c-d). the majority of rflps profiles generated from iii were unique for each species. for , the given enzymes ( i and i) probably generated rflps profiles which separated isolates at the subspecies level (fig. 4c-d), but further analysis will be required to justify this result. alu hae taq mycosphaerella fijiensis m. musicola m. eumusae hae m. eumusae m. musicola m. fijiensis m. musicola m. musicola hae m. musicola alu taqi hae mycosphaerella m. musicola alu taq can figure 3. single parsimonious tree based on its nrdna sequence data representing placement of spp. and sp.1 found in this study within representatives of the family . the tree is obtained from heuristic search with 1000 random taxon addition of the sequences alignment. bootstrap values (>50%) from 1000 replicates of unweighted maximum parsimony (ump) analyses are shown above internodes. mycosphaerella pseudocercospora mycosphaerellaceae 25 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella biotropia vol. 19 no. 1, 2012 development of specific pcr primers understanding the banana sigatoka disease complex is a challenge for plant pathologists (arzanlou 2007). therefore, in this study we developed rapid and specific detection method with the feasibility of wide application. even though a pcr-based detection tool has been developed previously (johanson & jeger 1993), those primers could only differentiate from three speciesspecific primers were designed, namely, meuf/meur, mfijf/mfijr, and mmusf/mmusr, respectively (table 2). all primers were designed to operate at relatively high annealing temperatures (54°c-55°c), thereby preventing the coamplification of non-specific dna targets. primer sequences were compared against existing sequences in ncbi genbank data base (http://www.ncbi.nlm.nih.gov/) and ddbj dna data base of japan (http://www.ddbj.nig.ac.jp/), and a result of blast (basic local alignment search tool) showed 100% homology of the primers with sequences of strains belonging to the species of which primers were designed. single bands of correct size were obtained with species-specific primers from all strains belonging to the three species from banana. et al. m. fijiensis m. musicola. mycosphaerella table 2. primer pairs designed in this study. no. name species target notes 1 meuf (forward) (5’-catctttgcgtcggagttca-3’) mycosphaerella eumusae not species-specific (cross reactions with m. fijiensis )meur (reverse) (5’-ccgaagcgaattgaagaatcc-3’) 2 mfijf (forward) (5’-tctttgcgtcggagtttca-3’) mycosphaerella fijiensis species -specific mfijr (reverse) (5’-tccgaagcgaattgaaagatc-3’) 3 mmusf (forward) (5’-tccttaacactgcatctctacg-3’) mycosphaerella musicola species specific mmusr (reverse) (5’-tcagccgggagactttgg -3’) m. eumusae m. fijiensis m. musicola m. fijiensis m. musicola mycosphaerella m. eumusae m. fijiensis m. eumusae m. fijiensis m. fijiensis m. musicola validation assay on pure cultures of , , and showed that primer pairs of (mfijf/mfijr) (fig. 5a) and (mmusf/mmusr) (fig. 5b) are specific to the fungal pathogens as no cross-reactions with others species were observed in the amplification bands. unfortunately, primer pairs of (meuf/meur) failed to show specificity as cross-reactions were found with sequences of (fig. 5c). it is probably due to small nucleotide differences between dna sequences and from its region. the specificity of primer pairs of mfijf/mfijr to and mmusf/mmusr to is good indication for the development of molecular diagnosis technique and understanding of the sigatoka diseases complex of banana in indonesia. the molecular technique developed in this study may also significantly contribute to plant quarantine because of its reliability, specificity and simplicity. this assay could be done within 1-2 days laboratory works and analysis. 26 figure 4. restriction patterns of internal transcribed spacer (its) regions of ribosomal dna amplified from three species from banana. a. i b. iii c. i d. i (1-3: ; 4-7: ; 8-9: ). mycosphaerella rsa hae alu taq m. eumusae m. musicola m. fijiensis 27 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella figure 5. species-specific amplification of species from banana using specific primers. a. primer pairs mfijf/mfijr to the b. primer pairs mmusf/mmusr to the c. primer pairs meuf/meur to the and (2-4: ; 5-8: ; 9-10: ). mycosphaerella m. fijiensis m. musicola m. eumusae m. fijiensis m. eumusae m. musicola m. fijiensis biotropia vol. 19 no. 1, 2012 28 conclusions diagnosis of the banana sigatoka disease complex is a challenge for plant pathologists. rflp analysis using iii is capable in discriminating the and the rapid and specific pcr-based detection method using species-specific primers of mfijf/mfijr and mmusf/mmusr has been successfully developed to detect and , respectively, from pure cultures. further examination/validation directly on samples from infected banana leaves with diseases symptom are necessary to test the sensitiveness of this method this research was funded by seameo biotrop awarded to dr. iman hidayat. laboratory of plant molecular systematics, botanical division, research center for biology (lipi) is acknowledged for providing the authors with molecular facilities. the author also thanks sulistiani and aerma hastuty for laboratory assistance. hae restriction enzyme m. eumusae, m. fijiensis, m. musicola. m. fijiensis m. musicola mycosphaerella . acknowledgments references arzanlou m, abeln eca, kema ghj, waalwijk c, carlier j, de vries i, guzmán m, crous pw. 2007. molecular diagnostics for the sigatoka disease complex of banana. phytopathology 97: 1112-8. bonants p, hagenaar-deweerdt m, van gent-pelzer m, lacourt i, cooke d, duncan j. 1997. detection and identification of hickman by the polymerase chain reaction. eur j plant pathol 103: 345-55. choi yw, hyde kd, ho wh. 1999. single spore isolation of fungi. fungal divers 3: 29-38. crous pw, mourichon x. 2002. and its anamorph spp. nov.: causal agent of eumusae leaf spot disease of banana. sydowia 54: 35-43. crous pw, groenewald jz, aptroot a, braun u, mourichon x, carlier j. 2002. interacting morphological and molecular data sets on with specific reference to species occurring on . in: jacome l, lepoivre p, marin d, romero r, escalant jv, editors. proceedings of the 2 international workshop on leaf spot diseases. san jose, costa rica. p 43-57. gardes m, bruns td. 1993. its primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. mol ecol 2: 113-8. hall ta. 1999. bioedit: a user-friendly biological sequence alignment editor and analyses program for windows 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1-8. in : gelvin s, schilperoort ra, editor. plant molecular biology manual. 3 edition. kluwer academic publishers, boston. massachusetts, usa. sreenivasadprasad s, mills pr, meehan bm, brown ae. 1996. phylogeny and systematics of 18 species based on ribosomal dna spacer sequences. genome 39: 499-512. swofford dl. 2003. paup*: phylogenetic analyses using parsimony (* and other methods), version 4.0b 10. sinauer associates, sunderland, massachusetts. thomson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal_x windows interface: flexible strategies for multiple sequences alignment aided by quality analyses tools. nucleic acids res 25: 4876-82. hite tj, burns t, lee s, taylor j. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj, editors. pcr protocols: a guide to methods and application. academic press, san diego. p 315-22 rd colletotrichum w . biotropia vol. 29 no. 1, 2022: 81 89 doi: 10.11598/btb.2022.29.1.1662 81 neuston diversity and density as bioindicator for water quality imam safir alwan nurza*, jesslyn gitta vania, muhammad khatami reynaldi, zaki gunawan rasyid and ratna komala department of biology, faculty of mathematics and natural sciences, universitas negeri jakarta, jakarta 13220, indonesia received 9 september 2021 / accepted 12 december 2021 abstract lakes and waterfalls are freshwater ecosystems having important roles in ecology, tourism and economic aspects. among living organisms existing in lakes and waterfalls is neuston. neuston lives on the surface and below the surface of the waters. neuston can be used as a bioindicator for water quality due to the neuston’s high level of sensitivity toward pollutants. the purpose of this study was to determine the density and diversity of neuston as a bioindicator for water quality in the lake and waterfall. the methods used were survey and observation. the study was carried out by using purposive sampling at two locations, namely the lake and waterfall of situ gunung, sukabumi, with a sampling area of 1 x 1 m2. the samples obtained were put into bottles containing 70% alcohol to be identified in the laboratory. environmental parameters measured were air and water temperature, water ph, water depth, turbidity, water flow velocity, dissolved oxygen, substrate and weather conditions. the results obtained indicated that the lake and waterfall of situ gunung, sukabumi had highest neuston densities were shown by gerris lacustris and dineutus assimilis. the lowest neuston densities were shown by metrobates hesperius, gerris comatus, aquarius remiges and trepobates pictus. this study showed that the environmental parameters of the lake and waterfall of situ gunung, sukabumi can still support the survival of the existing neustons. keyword: density, diversity, neuston introduction freshwater ecosystems are divided into lentic/flooded freshwater ecosystems and lotic/flowing freshwater ecosystems. the examples of lentic freshwater ecosystems are lakes and swamps, while that of lotic freshwater ecosystems are waterfalls and rivers (diantari et al. 2017; ramadhan et al. 2016). the existence of the lake is considered important because it has an ecological, social, and economic roles for the surrounding environment (postel & carpenter 1997; chen et al. 2020; heino et al. 2021; robert et al. 2020). the lake also acts as a habitat for several living organisms, such as neuston (asnil et al. 2013; ramadhan et al. 2016). according to mulyono (2018), neuston floats on water (epineuston) or below the water surface (hyponeuston). hyponeuston lives at a depth of about 0 10 cm (rumnasih 2016). neuston has a level of sensitivity to several contaminants so neuston can be used as a direct indicator for water contamination (oktarina 2015) and as a bioindicator for water quality. the upper part of the waters is the most susceptible part to environmental exposure, which in turn will impact the neuston. pollution often causes the formation of artificial stratification in lakes, where a body of water (hypolimnetic zone) is isolated. however, the hypolimnetic zone caused the contaminants trapped within the bottom of the waters and sediment masses (sutrisno & hamdani 2013; williams 2001; winton et al. 2019; perron et al. 2014; donyinah 2019). waterfalls are a form of lotic freshwater ecosystem and are generally used as natural tourist objects (siswantini & mulyana 2017). the waterfall and lake in the situ gunung area, sukabumi have an important role as one of *corresponding author, email: imamnurza@gmail.com biotropia vol. 29 no. 1, 2022 82 tourist attractions in kadudampit district, sukabumi, west java province, indonesia (soetopo 2011). situ gunung as a tourist object cannot be separated from tourists activities which will affect the surrounding environment, including the neuston community. therefore, the purpose of this study was to determine environmental factors influencing the density and diversity of neuston as water quality bioindicator in the lake and waterfall in situ gunung, sukabumi. materials and methods the study was conducted in june 2021 at situ gunung, gunung gede pangrango, kadudampit district, sukabumi. the methods used were survey and observation. the tools used were rope, wooden stick, measuring tape, thermometer, turbidimeter, do meter, loop, field guide as an identification guide, stopwatch, plastic bottle, sample bottle, sample plastic, camera, net 1 x 1 m2, identification key book and stationery. the materials used were 70% alcohol, labeling paper and universal ph indicator paper (1 14). the study was conducted in two study locations, namely lake and waterfall in the shallowest part. each location was represented by three observation stations based on differences in environmental conditions. sampling was carried out three times at each station. neuston collection was carried out at locations and stations that were purposively determined as a pick-up point sized 1 x 1 m2. neuston samples were taken by using a 1 x 1 m2 net which was placed on the water surface. the net captured neustons which went through the filtration following the horizontal movement of the surface water in the lake and waterfall. subsequently, the neustons sample was put into a bottle containing 70% alcohol. samples were identified in the field and photographed for documentation. environmental parameters measured were air and water temperature, water ph, water depth, water turbidity, water flow velocity, dissolved oxygen, substrate and weather conditions. the air temperature was measured using a calibrated air thermometer. water temperature measurements were carried out using a do meter. the ph of the water was measured using universal ph indicator paper (1 14). the water depth of the lake and waterfall was measured using a long wooden stick. the mark of the water depth was then measured by using a measuring tape. water turbidity was measured by using a calibrated turbidity meter. water flow velocity was measured by using floating method from the edge of the lake and waterfall. a rope was tied to a bottle and a 1-meter-long wooden stick. the bottle was floating at the water surface. the time taken from the moment the bottle was tied up to the wooden stick until the rope was stretched straight, was recorded by a stopwatch. the water flow velocity is expressed in m⁄s unit. the formula for calculating the water flow velocity is as follows: flow velocity = rope length (m) time (s) dissolved oxygen was measured three times using a do meter. the substrate is the surface on which an organism lives. the substrate sample of the lake was taken using the ekman grab, while the substrate of the waterfall can be seen directly because the water depth is quite shallow. the substrate was determined by looking at its composition. the types of water substrates are sand, mud, rocks, lime, and others. weather was determined by looking at the weather situation at the lake and waterfall environment. there are three kinds of weather conditions, namely sunny, cloudy, and rainy. data analysis was carried out by determining the simpson index using the formula as follows: 1. density b = t x p a x s where: t = quadrant area (1 m2 = 10,000 cm2) p = area of the taking transect (m2) a = number of individual species s = number of taking transects 2. diversity ds = 1 – d  ds = 1 –  ni (ni – 1) n (n – 1) where: ds = simpson's diversity index neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 83 d = dominant index ni = number of individuals species n = total number of individuals results and discussion there were six species of neuston found in the lake and waterfall of situ gunung. four species were existed in the lake, namely gerris lacustris, metrobates hesperius, gerris comatus, and aquarius remigis (fig. 1), while two species were found in the waterfall, namely dineutus assimilis and trepobates pictus (fig. 2). neuston species found in the lake and waterfall are different, depending on the habitat. gerris lacustris and gerris comatus are found in lakes due to their lentic habitat preferences. according to ye et al. (2017), gerris species inhabit several lentic habitats such as ponds, lakes and backwaters of streams. the habitat preference of gerris lacustris is a high level of water depth, covered with vegetation, which is in agreement with a study conducted by olosutean and ilie (2013) who found that gerris lacustris is correlated with water depth and have adaptation benefits from the presence of vegetation cover. the surface of the lakeside of situ gunung is covered with aquatic plants and the depth is relatively higher (58 93 cm) compared to the water depth of the waterfall (25 64 cm). in addition, gerris comatus is a characteristic organism for lentic water. gerris comatus is often found together with gerris marginatus and gerris buenoi (damgaard et al. 2014; pintar & william 2020). however, the last two species mentioned are not found in the lake. figure 1 neuston species found in the lake of situ gunung notes: a. gerris lacustris; b. metrobates hesperius; c. gerris comatus; d. aquarius remigis figure 2 neuston species found in the waterfall of situ gunung notes: a. trepobates pictus; b) dineutus assimilis a b dc a b biotropia vol. 29 no. 1, 2022 84 aquarius remigis were found in the lake of situ gunung, which fact is different from the statement of ye et al. (2017) that aquarius species are mostly confined to lotic habitats, such as water springs and rivers. aquarius species can live in two different kinds of freshwater, both lentic and lotic. aquarius species are semi-aquatic insects and opportunistic predators that live on the water surface of lakes, rivers, and the border between rivers and lentic habitats (ditrich & papáček 2016; guterres et al. 2019). metrobates hesperius were found in the lake because there are aquatic plants on the surface of the lakeside of situ gunung. metrobates hesperius lays eggs on the leaves of the floating aquatic plants (taylor 2009; ikawa et al. 2012; finet et al. 2018). the lake as a habitat supports the survival and distribution of these species. dineutus assimilis and trepobates pictus were found on the surface of a waterfall. dineutus assimilis prefers both lotic and lentic waters, such as river surfaces, lakeshores and lakes (gustafson & miller 2015; maclean 2013; webster & demerchant 2012). habitat preferences of trepobates pictus were waters with slow water flow with eutrophic, muddy and rocky conditions in lotic waters (naranjo et al. 2010; taylor & mcpherson 2006; wooden 2019; chordas 2017). the diversity index value (ds) of neuston species obtained in the lake and waterfall for each station in situ gunung is presented in table 1. diversity index of 0.50 means low diversity, diversity index value of 0.50 to 0.75 means moderate diversity index, while diversity index of 0.75 to 1 means high diversity index (nento et al. 2018). table 1 neuston diversity and total individuals in the lake and waterfall of situ gunung location station 1 station 2 station 3 ds ti ds ti ds ti lake 0.00 4 0.11 18 0.70 5 waterfall 0.47 10 0.00 6 0.48 7 notes: ds = diversity, ti = total individuals table 1 shows that neuston diversity index value (ds) varies among stations. the highest neuston diversity index in the lake occurred at station 3 with a value of 0.70 (moderate) with a total of 5 inviduals and 3 neuston species, namely gerris lacustris, gerris comatus and aquarius remigis. neuston diversity index value in the lake at station 2 was 0.11 (low) with a total of 18 individuals and 2 neuston species, namely gerris lacustris and metrobates hesperius. the lowest neuston diversity index in the lake occurred at station 1 with a value of 0.00 (low). in station 1, only one neuston species was found, namely gerris lacustris with a total of 4 individuals. the highest neuston diversity index in the waterfall occurred at station 3 with a value of 0.48 (low), where 2 neuston species were found, namely dineutus assimilis and trepobates pictus with a total of 7 individuals. the lowest neuston diversity index in the waterfall occurred at station 2 with a value of 0.00 (low), where 1 neuston species was found, namely dineutus assimilis with a total of 6 individuals. the neuston diversity index in the waterfall at station 1 was 0.47 (low) with a total of 10 individuals and 2 neuston species, namely dineutus assimilis and trepobates pictus. the different values of diversity index with total individuals obtained was due to dominant species in the lake and waterfall of situ gunung, namely gerris lacustris and dineutus assimilis. table 2 neuston density in the lake and waterfall of situ gunung location neutston species neuston density (individu/m2) lake gerris lacustris 0.24 metrobates hesperius 0.01 gerris comatus 0.01 aquarius remigis 0.01 waterfall dineutus assimilis 0.18 trepobates pictus 0.05 the highest neuston density neuston in the lake was shown by gerris lacustris with a value of 0.24 individu/m2. the three other neuston species, namely metrobates hesperius, gerris comatus, and aquarius remigis showed the same density value of 0.01 individu/m2 (table 2). on the lake surface of situ gunung at stations 1 to 3, there are aquatic plants. gerris lacustris prefers to live on aquatic plants available on the lake surface of situ gunung. according to yee (2016), gerris lacustris spends most of its life on the surface of the water. in the lake of situ gunung there were two species of gerris, namely gerris lacustris and gerris comatus. gerris species can be found in almost all aquatic habitats from water springs to tropical seas (yurtseven et al. 2016). at the waterfall of situ gunung, the neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 85 highest neuston density was shown by dineutus assimilis with a value of 0.18 individu/m2 and trepobates pictus with a value of 0.05 individu/m2. the common aquatic insects found in indonesian freshwater are of the order odonata, coleoptera, trichoptera, hemiptera, ephemeroptera, plecoptera and lepidoptera (mahajoeno et al. 2001; candra et al. 2014). in the lake and waterfall of situ gunung, we found orders hemiptera and coleoptera. every species found in the lake of situ gunung belongs to gerridae family. according to dwitawati et al. (2015), gerridae family is classified as having a low tolerance for pollutants. meanwhile, gerris lacustris, gerris comatus, metrobates hesperius and aquarius remigis belong to the order hemiptera. hemiptera insects often move rather than being settled in an unwanted location (mercer et al. 2017; peterson et al. 2017; wooden 2019). the predominant presence of hemiptera insects indicates that the lake is relatively less polluted (majumder 2013). the pollution that contaminates the lake of situ gunung is thought to come from household wastes and human recreational activities. aquatic insects are a good bioindicator for detecting pollution. aquatic insects are also useful for fish food and biocontrol agents (dalal & gupta 2016; ito et al. 2017). therefore, long-term monitoring of aquatic insects is needed to evaluate water quality. table 3 shows that the ph values in the lake of situ gunung for all stations are relatively constant, namely 6 (acid). the standard ph for clean water quality ranges from 6.5 to 9.0. the ph value in the lake of situ gunung which is below the clean water standard is suspected to have been polluted by household wastes due to the closeness with the mainland, hence acidic ph value. the acidic ph value is not within the suitable environment for the neuston's life. neuston can develop well in the ph range of 6.8-8.5 (gundo 2010; pratami et al. 2018). ph affects dissolved oxygen levels. the lowest dissolved oxygen level for the lake of situ gunung location was found at station 2, which was 3.28 mg/l. the acidic ph causes an increase in toxic substances in the water and decreases dissolved oxygen levels (pratami et al. 2018). dissolved oxygen plays an important role in the respiration process of most aquatic organisms (hariyani et al. 2017; pratami et al. 2018). the average of dissolved oxygen level in the lake of situ gunung was 3.62 mg/l. dissolved oxygen levels also indicate the level of pollution waters. dissolved oxygen levels below 5 ppm mean low pollution levels (hariyani et al. 2017). the low dissolved oxygen level shown in the lake of situ gunung indicated that the pollution level was low enabling neuston organisms such as water insects to obtain the needed oxygen for the respiration process. the lake of situ gunung showed the highest water turbidity value at station 1, which was 11.97 ntu. the high value of water turbidity is presumably caused by the existing fishing activities by humans and aquatic plants on the lake surface at station 1. penetration of sunlight into the lake waters can be hindered by the high value of water turbidity (pratami et al. 2018). the lowest value of water turbidity was shown at station 2, which was 4.44 ntu. the different value of water turbidity at station 2 compared to the other two stations is presumably caused by the utilization of station 2, which function is docking place for boats. also, only a few of aquatic plants were existing in station 2. the measurement of environmental parameters of the waterfall of situ gunung showed air and water temperatures of 20.7 oc and 17.9 °c, respectively, indicating a cold-water habitat for insects (table 4). in the environment of the lake of situ gunung, the air and water temperatures of 29 oc and 28 °c, respectively, indicating a warm-habitat for insects (table 3). according to mujiono et al. (2019), the higher the place is, the colder the temperature. the average of water ph of the waterfall of situ gunung is 6 (acidic) with average of dissolved oxygen value of 7.77 mg/l. dissolved oxygen levels are high in the waterfall because the waterfall is located on a hill surrounded by natural forests and has low pollution levels. meanwhile, the lake water is murky indicating water pollution that affects dissolved oxygen levels. in addition, the murky water can cause the increase of oxygen uptake from the air into the water causing the increase of dissolved oxygen in the water (diantari et al. 2017; mujiono et al. 2019; irby et al. 2015). biotropia vol. 29 no. 1, 2022 86 table 3 environmental parameters obtained from the lake of situ gunung environmental parameter station mean 1 2 3 water ph 6 6 6 6 air temperature (oc) 29 29 29 29 water temperature (oc) 28.03 27.93 27.93 27.97 water depth (cm) 58 71 93 74 water turbidity (ntu) 11.97 4.44 11.85 9.42 water flow velocity (m/s) 0.009 0.022 0.0085 0.013 dissolved oxygen (mg/l) 4.24 3.28 3.33 3.62 substrate mud and rocky mud and rocky mud and rocky weather cloudy and rainy cloudy and rainy cloudy and rainy table 4 environmental parameters obtained from the waterfall of situ gunung environment station mean 1 2 3 water ph 6 6 6 6 air temperature (oc) 20.8 20.8 20.5 20.7 water temperature (oc) 17.9 17.9 17.9 17.9 water depth (cm) 25 53.33 64.33 47.56 water turbidity (ntu) 0.41 0.46 0.51 0.46 water flow velocity (m/s) 0.49 0.58 0.13 0.40 dissolved oxygen (mg/l) 7.54 7.71 8.05 7.77 substrate rocky rocky rocky weather sunny sunny sunny the water flow of the stations at waterfall of situ gunung showed an average of water flow velocity of 0.40 m/s which affects the presence of swimming-type aquatic insects. due to the high value of water flow velocity and the presence of rocky substrate, the water insects hide behind rocks (leba et al. 2013). according to diantari et al. (2017), the rocky substrate can affect the water flow velocity in a stream. the lake water's flow velocity of 0.01 m/s causes the lake to be predominated by neustons with floating ability. the average water turbidity of the waterfall of situ gunung was 0.46 ntu with an average water depth of 47.56 cm. meanwhile, the lake showed an average turbidity of 9.42 ntu with an average water depth of 74 cm (table 3). our study showed that sunlight easily penetrated the waterfall. however, the murky lake water hindered the sunlight penetration into the lake. small value of water turbidity can support filterfeeder organisms and affect the activities of the existing orders hemiptera and coleoptera (ebenebe et al. 2016; marpaung et al. 2014). conclusion six neuston species were identified in situ gunung, namely four species in the lake of situ gunung and two species in the waterfalls of situ gunung. the four neuston species in the lake namely gerris lacustris, metrobates hesperius, gerris comatus and aquarius remigis, while the two neuston species waterfalls namely dineutus assimilis and trepobates pictus. the highest neuston densities in the lake and waterfalls were represented by gerris lacustris and dineutus assimilis, respectively. neuston diversity in the lake and waterfalls of situ gunung was low. environmental parameters of the lake and waterfalls of situ gunung were still in the normal range, except low water ph indicating polluted waters environment. therefore, longterm monitoring at the waters of situ gunung is needed for evaluating water quality parameters that support the life of aquatic organisms. neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 87 acknowledgments the author would like to thank the assistant of the ecology laboratory of universitas negeri jakarta who assisted us in analyzing water quality parameters of our study. special gratitudes are also delivered to the officials of the taman nasional gunung gede 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[thesis]. baton rouge (us): louisiana state university. yee kl. 2016. occurrence of aquatic macroinvertebrates from paleik''in'', sintkaing township. yadanabon university research journal 7(1):1-12. ye z, zhen y, damgaard j, chen p, zhu l, zheng c, bu w. 2018. biogeography and diversification of holarctic water striders: cenozoic temperature variation, habitat shifting and multiple intercontinental dispersals. syst entomol 43(1): 19-30. yurtseven i, serengil y, pamukçu p. 2016. seasonal changes in stream water quality and its effects on macroinvertebrate assemblages in a forested watershed. appl ecol environ res 14(1):175-88. microsoft word 54 biotropia no. 24, 2005 : 54 61 effect of 3, 5, 3'-triiodothyronine (t3) hormone on nucleic acid and protein content of the muscle and the growth of giant gouramy, osphronemus gouramy lac. sri handayani1, m. zairin jr.2, ing mok.oginta2 and maria bintang3 ' department ofaquaculture, faculty of fisheries and marine science, mulawarman university, samarinda, indonesia 2 department ofaquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia 3 department of biochemistry, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract this experiment was conducted to study the effect of 3, 5, 3'-triiodothyroninc (tj) hormone on nucleic acid and protein content of the muscle and the growth of giant gouramy, osphronemus gouramy lac. five experimental diets, which contain isocaloric diets, but different in t3 hormone level were used in this experiment (0.0, 2.5, 5.0, 7.5, and 10.0 mg t3 hormone/kg diet). the experimental diets were tested to three different groups offish for sixty days feeding trial. fish body weight in groups i, ii, and iii were 0.39-0.42 g/fish; 19.11 -21.99 g/fish, and 37.52-40.79 g/fish, respectively. the results showed that the highest rna, dna concentration and rna/dna ratio of the muscle were produced by 10.0 mg t3 hormone/kg diet for group i and ii; and 2.5 mg t3 hormone/kg diet for group iii. similar results also were found for the protein content of the whole body, protein retention, and the daily growth rate of the fish. key words : osphronemus gouramy/ti hormone/nucleic acid/protcin/growth introduction giant gouramy is well known as a slow growing fish that has been traditionally cultured for a long time in west java, indonesia. intensification of the culture of this fish has been started recently. however, our studies indicated that the growth of the fish could be enhanced by fulfilling their nutrient requirements (mokoginta et al. 1994, 1995a & b, 1996). in addition to fulfilling their nutrient requirements, growth could be enhanced by hormone treatment as suggested by matty (1985). some studies on thyroid hormone treatment revealed that the hormone plays an important role on somatic growth of fish (donaldson et al. 1979; fagerlund et al. 1984; matty et al. 1982). it was assumed that thyroid hormone could activate protein synthesis in liver and muscle cells (jackim and la roche 1973; narayansingh and bales 1975; medda and ray 1979; matty et al. 1982) by increasing rna and dna concentration of liver and muscle cells. woo et al. (1991) showed that administration of 3, 5, 3'triiodothyronine (t3) hormone in diet 54 biotropia no. 24, 2005 of red sea bream increased the growth rate, feed efficiency, digestive enzymes activity in the intestine and activities of other key enzymes in various pathways of carbohydrate metabolism. t3 affects protein synthesis by biphasic mode i.e., anabolic at lower dose, but catabolic at higher dose (matty and lone 1985; medda and ray 1979). medda and ray (1979) showed that the administration of thyroxine (t4) hormone at the dose of 2 or 4 mg/g body decreased protein accumulation and rna content in the liver and brain, whereas dosage of 1 mg/g body weight increased protein accumulation and rna content in liver and brain. therefore, in this experiment, we study the effect of the administration of tj hormone in diet on nucleic acid and protein content of the muscle and the growth of giant gouramy. materials and methods fish experimental fish were obtained from a fish farmer in parung. the fish were divided into three groups based on body weight. group i was of 0.39 0.42 g body weight, group ii was of 19.11 21.99 g body weight and group iii was of 37.52 -40.73 g body weight. fish were reared in aquaria (50 x 30 x 30 cm, 20 1 volume) with ten fish in each aquarium (group i), while for fish of group ii and iii with one fish in each aquarium. fish were fed three times daily to satiation for 60 days. to maintain water quality, 70 80 % of total water volume was replaced and feces were siphoned out every day. aquaria were cleaned and water was totally replaced once a week. this experiment was conducted at water temperature of 27 30°c; ph 7.5 8.0; do 6.28 7.2 ppm; and ammonia 0.08 0.13 ppm. diets semi-purified diets were used in this study. the fish were fed with a diet of 43 % protein level (group i), while the other fish (groups ii and iii) were fed with 32 % protein level. all diets have protein-energy ratio of 8 kcal de/g protein. the different protein levels between groups were based on the protein requirement of giant gouramy (mokoginta et al. 1994,1995 a & b, 1996). the diet was supplemented with 0, 2.5, 5.0, 7.5, and 10 mg of t3/kg. before mixing, t3 was dissolved in 70 % alkaline alcohol (33 ml 90% ethanol 12 ml 0.1 n naoh) and the solution was sprayed on the diet. the control diet was sprayed with alkaline ethanol without t3. following supplementation of t3, the diets were air dried and then stored in a freezer (-20°c) until feeding. the composition of diets for fish in group i, ii and iii are shown in tables 1 and 2, respectively. 55 data collection and chemical analysis each group of fish was weighed and taken for proximate analysis at the beginning and the end of the experiment. rna and dna concentrations were determined at the end of the experiment. proximate analyses were determined using methods as described by takeuchi (1988). analysis of rna concentration was determined by the orcinol method, while for dna concentration by diphenilamin method (plummer 1979). calculation and statistical analysis daily growth rate (dgr) was calculated according to equation of huisman (1976) : wt = (wo + 0,01 a)1 where wo and wt are initial and final mean of body weight, respectively, t is the number of days, and a is daily growth rate (%/day). protein retention was calculated according to the equation of takeuchi (1988): protein retention (%) =( total protein content of body (final) total protein content of body (initial)) (g)/ total protein consumed (g) x 100. the study was designed using complete randomized design with five treatments and three replications. the effect of t3 hormone on protein retention, rna and dna concentration, and rna/dna ratio and growth rate were evaluated by using analysis of variance followed by polynomial orthogonal test (steel and torrie 1993). 56 biotropia no. 24, 2005 results and discussion rna and dna concentrations and rna/dna ratio in muscle are shown in table 3. the table shows that rna/dna ratio increased following t3 levels supplemented into the diet. increasing t3 level from 2.5 mg t3/kg diet to 10 mg t3/kg diet increased rna/dna ratio in groups i and ii. the highest level of t3 hormone (10 mg t3/kg diet) caused an increase in rna/dna ratio to 10.8 % and 4.4 % over control in groups i and ii, respectively. in group iii, increasing rna/dna ratio (2.3% over control) was obtained at the level of 2.5 mg t3/kg diet, but the effect of 3,5,3'-triidothyronine (t3) hormone sri handayani et al. several studies showed that rna concentration and rna/dna ratio were closely related to growth and nutritional status of fish. therefore, rna concentration and rna/dna ratio could be used as an indicator of fish growth and nutritional status. it is commonly accepted that rna concentration in liver or muscle of fish may reflect protein synthesis, where increase in rna concentration was followed by a rise in protein synthesis (yang and dick 1993). rna/dna ratio is index of metabolic activity and change in rna/dna ratio reflects change in protein synthesis (brafield 1985; widler and stanley 1983). correlation between administration of t3 and protein synthesis might be explained by the effects of t3 hormone on the increase of rna concentration through interactions between t3 hormone and receptor in nucleus which activated enzyme for rna synthesis (matty etal. 1982; djojosoebagio 1990). this study showed that administration of 10 mg t3/kg diet in groups i and ii increased rna and dna concentration, rna/dna ratio to 26.6 %, 14.4% and 10.8% over control, respectively, for group i; and 9.6%, 4.95%, and 4.4% over control, respectively, for group ii. while in group iii, the highest value of rna/dna concentrations and its ratio were reached at 2.5 mg t3/kg diet i.e. 5%, 2.5% and 2.3% over control, respectively (table 3). as mentioned above, increasing rna concentration in muscle was followed by a rise in protein synthesis, rna/dna ratio and growth rate of fish. alien et al. (1979) defined growth as increasing mass by two fundamental biological processes; protein accretion and proliferation cell. this study, clearly showed that administration of 10 mg t3/kg diet in groups i and ii increased rna concentration (26.6 % and 9.6% over control, respectively) and protein retention (30 % and 16.5 % over control, respectively). the ability to retain the body protein was followed by an increase in growth rate offish (9.3% and 14.4 % over control, respectively) and also reflected by an increase in rna/dna ratio (10.8 % and 4.4% over control, respectively) (tables 3 and 4). in group iii, higher rna concentration was attained at 2.5 mg t3/kg diet and followed by increasing body protein level (5 % over control). furthermore, protein retention and growth attained a higher value, an increase of 10 % and 17.5 % over control, respectively (tables 3 and 4). the effect of t3 hormone on protein synthesis is biphasic, i.e. anabolic at low doses and catabolic at higher dose ( medda and ray 1979). as rna and protein concentrations in a cell depend upon the synthetic and degradation rate, and as the thyroid hormones increase the turnover rate of this cellular constituents, it is possible that at higher doses the turnover rate becomes much higher and the degradation rate surpasses the synthetic rate (goldberg et al. 1980 in matty et al. 1982). in this study, biphasic effect of t3 was only shown in group iii, but not in other groups. in conclusion, all groups in this study showed that the effect of t3 hormone administration on rna and dna concentration, rna/dna ratio, body protein level, protein retention, and daily growth rate depend on fish size, in which the difference of potential growth rate was produced by fish size. 58 * percentage of increase/decrease compared to control (treatment a). 59 effect of 3,5,3'-triidothyronine (t3) hormone sri handayani et al. references alien, r.e., r.a. mcrkel and r.b. young. 1979. cellular aspect of muscle growth : myogenic cell proliferation. j. anim. sci. 49 : 115 127. brafield a. e. 1985. laboratory studies of energetics budgets. in tytler, p. and p. calow (eds), fish energetics, new perspectives. croom helm, london, p. 257-281. djojosocbagio s. 1990. fisiologi kclenjar endokrin i. pusat antar universitas ilmu hayat. institut pertanian bogor. 247 p. donaldson e.m., u. h. m. fagcrlund, d.a. higgs, j. r. mcbridge. 1979. hormonal enhancement of growth. in fish physiology. i w s woar, d. j. randall, and j.r. brett (eds.), vol. 8. academic press, new york. p. 453-597. fagerlund u. h. m., i. mccallum, d. a. higgs and j. r. mcbride. 1984. diet composition as a factor in the anabolic efficacy of 3, 5, 3'triiodo-l-thyronine administrated orally to steelhead trout (salmo gairdneri). aquaculturc, 36 : 49-59. huisman, e.a. 1976. food convcrtion cfficicnces at maintenance and production levels for carp, cyprinus carpio l and rainbow trout, salmo gairdneri. aquaculturc, 9 : 259 273. jackim and la roche. 1973. protein synthesis in fundulus leteroctilus muscle. comp. biochem. physiol. a., 44:851-866. matty a. j. 1985. fish endocrinology. croom helm, london. 267 p. matty a. j. and k. p. lone. 1985. the hormonal control of metabolism and feeding. in: fish energetics. tytler p. and p calow (eds.). new perspectives, croom helm, london, p. 185-209. matty a. j., m. a. chaudhry, and k. p. lone. 1982. the effect of thyroid hormone and temperature on protein and nucleic acid content of liver and muscle of sarotherodon mossambica. gen. comp. endocrinol., 47 : 497 507. medda, a. k. and a. k.. ray. 1979. thyroxine and muscle of lata fish, gen. comp. endocrinol., 9 : 473-480. mokoginta i., m. a. suprayudi, and m. setiawati. 1994. kcbutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan rcproduksi. lap. pen. hb. ii/2, dirbinlitabnas, dikti, dcpdikbud. ipb. mokoginta i., m.a. suprayudi and m. setiawati. 1995a. kebutuhan optimum protein dan energi makanan bcnih ikan gurame (oshpronemus gouramy lac.). jur. perikanan indonesia, 1 (3) : 82 94. mokoginta i., m. a. suprayudi, and m. setiawati. 1995b. kebutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan reproduksi. lap. pen. hb. 11/3, dirbinlitabnas, dikti, depdikbud. ipb. mokoginta i., m. a. suprayudi, and m. setiawati. 1996. kebutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan reproduksi. lap. pen. hb. ii/4, dirbinlitabnas, dikti, dcpdikbud. ipb. narayansingh t. and j. g. eales. 1975. effect of thyroid hormones on in vivo [1 14c] l icucine incorporation in plasma and tissue protein of brook trout (salvelinus fontinalis) and rainbow trout (salmo gairdneri). comp. biochem. physiol. b., 52: 399 405. 60 biotropia no. 24, 2005 plummer, d.t., 1979. an introduction to practical biochemistry. mcgraw-hill. new delhi. 362 p. steel r.; g. d., and j. h. torrie. 1993. prinsip dan proscdur statistika. pt gramcdia pustaka utama, jakarta. 748 p. takcuchi, t. 1988. laboratory work chemical evaluation of dietary nutrients. in: fish nutrition and mariculturc. watanabc (ed.). jica text book. the general aquaculture course, japan, p. 179-190. widlcr i. b. and j.g. stanley. 1983. rna dna ratio as an index to growth in salmonid fishes in the laboratory and in stream contaminated by carbaryl. j. fish biol., 22 : 165-172. woo, n. y. s., a. s. b. chung, and t. b. ng. 1991. influence of oral administration of 3, 5, 3'-triiodothyroninc on growth, digestion, food conversion and metabolism in the undcrycarling red sea bream (crysophrys major). j. fish biol., 39 : 459-468. yang x. and t. a. dick. 1993. effect of dietary fatty acids on growth, feed efficiency and liver rna and dna content of arctic charr, salve/inns alpinus (l). aquaculture, 116 : 57 70. 61 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf biotropia no. 4, 1990/1991: 49-56 a study on weed control in soybean s. tjitrosemito tropical agricultural pest biology programme, seameo biotrop, bogor, indonesia abstract two field experiments on weed control in soybeans were carried out at biotrop, bogor, indonesia from february to june, 1989. the critical period for weed control was found to be between 20 40 days after planting of soybean (c. v. wilis) grown at a planting distance of 40 x 10 cm. it did not coincide with the fastest growth in terms of trifoliate leaf number. further studies were suggested to understand the physiological growth of soybean related to weed control. pendimethalin at 6601320 g a.e./ha applied one day after sowing did not cause any phytotoxic effect to soybean and had good weed control performance. introduction with the establishment of the soybean yield gap analysis project (sygap) in indonesia (douphin et al. 1986), about 34.5 million ha were identified to be suitable for soybean planting (pasaribu & mclntosh 1986). it was assumed, therefore, that it was adequate area-wise but when yield/ha was considered, it was still very low (0.7-0.8 ton/ha; somaatmadja 1983). this low yield may be attributed to various factors like (1) poor stand, (2) poor growth, (3) weed problems, (4) excess water, (5) empty pods due to insect damage or drought (pasaribu & mclntosh 1986). fachurrozi et al. (1988) surveyed the production system of soybean at farmers level in east java (district of pasuruan). they reported that weeding was the most labour intensive operation. it took 51 % and 65% of the total work hours and 32% and 34% of the total cash production costs in two villages of oro-oro pule and sumberbanteng. however, although the cost of weeding was high, their studies indicated that yield could be increased by controlling weeds, pests and diseases. twice weeding resulted in marginal rates of return (mrr) of 118 147%, whereas mrr of controlling pests and diseases was 1012 — 1454%. these figures showed how beneficial the control of "pests" (insect, pathogens, weeds, vertebrate pests) was. recent work indicated that soybean crops planted at various planting distances from 30 x 10 cm to 40 x 20 cm could tolerate infestation of weeds up to 30% of sdr (tjitrosemito 1987). the growth of soybean was, however, not optimal as was also reported by pasaribu and mclntosh (1986). in this context, it is appropriate to emphasize that weed control (weed management) is an integral part of the whole production management. the success of weed control is to be judged also from its ability to increase yield with sufficient profit. 49 biotropia no. 4, 1990/1991 material and methods experiment 1 a field experiment was conducted at biotrop, bogor from november 1988-february 1989 to determine the critical period of weed control on soybean crops (c.v. wilis). fourteen treatments consisting of various manual control schemes were applied in plots measuring 4 x 5 m 2 replicated 3 x with randomized block design. the fourteen treatments were: 1. manual weed control from planting up to 10 days after planting (dap) 2. manual weed control from planting up to 20 dap 3. manual weed control from planting up to 30 dap 4. manual weed control from planting up to 40 dap 5. manual weed control from planting up to 50 dap 6. manual weed control from planting up to 60 dap 7. manual weed control from planting up to harvest 8. manual weed control from 10 dap to harvest 9. manual weed control from 20 dap to harvest 10. manual weed control from 30 dap to harvest 11. manual weed control from 40 dap to harvest 12. manual weed control from 50 dap to harvest 13. manual weed control from 60 dap to harvest 14. no weeding at all. the plots were manually cultivated before planting and fertilized at the rate of 45 kg n/ha, 50 kg p2o5/ha and 50 kg k2o/ha, applied by broadcasting along the planting rows. the planting distance was 40 x 10 cm. insect pest control was done by spraying azodrin 60 wsc at 2 ml/it solution; while dithane m-45 at 2 g/1 was used against pathogens. the soybean was harvested at 98 dap; and the yield was compared statistically using lsd at 5%. weeds were sampled before harvest using a quadrat of 50 x 50 cm 2x in each plot. the growth of soybean was recorded in terms of trifoliate leaf number. experiment 2 another field experiment was also conducted at biotrop from march-june, 1989. there were 12 treatments applied in plots measuring 2.5 x 10 m replicated 3x in a randomized block design. this experiment was a repetition of 50 a study on weed control in soybean-s. tjitrosemito the one of previous year, since some herbicidal treatments showed high crop mortality. the treatments were: 1. untreated plot 2. imazethapyr (i) : pre-emergence : 50 g a.e./ha 3. imazethapyr (i) : pre-emergence : 75 g a.e./ha 4. imazethapyr (i) : pre-emergence : 100 g a.e./ha 5. imazethapyr (i) : pre-emergence : 150 g a.e./ha 6. imazethapyr (i) : early post : 100 g a.e./ha 7. pendimethalin (p) : pre-emergence : 660 g a.e./ha 8. pendimethalin (p) : pre-emergence : 1320 g a.e./ha 9. p + 1 : pre-emergence : 660 + 50 g a.e./ha 10. p + 1 : pre-emergence : 1320 + 50 g a.e./ha 11. alachlor : pre-emergence : 1440 g a.i/ha 12. manual weeding : at 3 and 6 weeks : the spraying of herbicides was done using a cp 15 knapsack sprayer, calibrated to deliver 400 1 solution/ha, using yellow nozzle at high pressure. the pre-emergence spraying was done one day after planting. the samplings were carried out using a quadrat measuring 50 x 50 cm 2 . the plots were manually cultivated and fertilized with 60 kg p2o5/ha; 45 kg n/ha and 50 kg k2o5/ha at planting time, broadcast along the soybean row. to prevent damage to seedlings of o. phaseoli, furadan was utilized. the soybean was harvested at 98 days after planting. results and discussion the soybean yield from experiment i is shown in table 1. when expressed in the graph (figure 1) it indicates that the critical period of weed control is between 20 and 40 dap. lengthening the weed free period from 10 to 40 dap increased the yield i.e. from 0.62 ton/ha to 1.36 ton/ha, but further lengthening the period of control did not increase the yield any more. leaving the field unweeded for a period of 10 20 dap did not really matter, but leaving more than 20 days unweeded reduced the yield considerably i.e. from 1.54 ton/ha to 1.33 ton/ha. when weeding was done only 40 dap the yield was further reduced. the weed infestations were presented in table 2 (weed count) and table 3 (dry weight). the infestations of borreria alata, ageratum conyzoides, eleutheran 51 biotropia no. 4, 1990/1991 table 1. the mean yield of soybean at various control schemes treatment ton/ha 1. weeded up to 10 days after planting (dap) 0.62 2. weeded up to 20 dap 0.92 3. weeded up to 30 dap 1.25 4. weeded up to 40 dap 1.36 5. weeded up to 50 dap 1.36 6. weeded up to 60 dap 1.35 7. weeded up to harvest 1.50 8. weeded from 10 dap to harvest 1.50 9. weeded from 20 dap to harvest 1.54 10. weeded from 30 dap to harvest 1.33 11. weeded from 40 dap to harvest 1.26 12. weeded from 50 dap to harvest 1.05 13. weeded from 60 dap to harvest 0.85 14. no weeding at all 0.65 lsd (5%) 0.14 cv 10% figure 1. the effect of "weed free" and "weed present" periods on yield of soybean. the critical period appeared to be between 20-40 dap. 52 a study on weed control in soybean-s. tjitrosemito table 2. mean weed count/50 x 50 cm 2 at various weeding schemes (average of 3 blocks) b.a. : borreria alata (aubl.) dc a.c. : ageratum conyzoides l. e.r. : eleutheranthera ruderalis sch. m.sp. : mitracarpus sp. c.r. : cyperus rotundus l. d.c. : digitaria ciliaris (retz) koel. others include : celosea argantea leucas lavandosalia stachytarpheta jamaicensis euphorbia geniculata oxalis sp. phyllantus niruri l. cleome rutidospermum dc. emelia sonchifolia tridax procumbens eleusine indica thera ruderalis, digitaria ciliaris, and to some degree also cyperus rotundus were considerable when not weeded and they constituted the main weeds in the area. the growth performance of soybean is presented in figure 2. early in the growth stage, the growth was slow especially in the first 2-3 weeks in terms of leaf number. this was the period when the presence of weeds was not felt by the crops. from 3 to 6 weeks (20 40 dap) the growth was still slow compared to the period between 40 to 60 days when growth was from 9 to 16 leaves/plant. however, at 60 dap, the crop started to shed the leaves and the number of leaves went down. 53 biotropia no. 4, 1990/1991 table 3. mean dry weight of weed grains/50 x 50 cm 2 at various weeding schemes (average of 3 blocks) figure 2. the growth performance of soybean (c.v. wilis) in terms of number of trifoli ate leaves. 54 a study on weed control in soybean-s. tjitrosemito probably, availability of resources in the period of 20 40 dap was necessary to support the rapid growth in the following period. as pointed out by spitters and van den bergh (1982), once nutrients or moisture were taken up by the competing weeds, they were not available anymore to the crop. further works are certainly needed to really understand the physiological growth of soybean and its relation to weed control the soybean yields in the experiment are presented in table 4. contrary to the result of the previous year (tjitrosemito 1988) soybean treated with pendimethalin did not show any phytotoxicity with the pre-emergence spraying done one day after planting. at the same time, the control of weed was better, thus facilitating a higher yield. as was reported earlier (tjitrosemito 1988) borreria alata was the main weed. pendimenthalin at 1320 g a.e./ha was good in controlling digitaria ciliaris. table 4. the mean of soybean yield (ton/ha) under various weed control treatments references dauphin, f., j.w.t. bottema and a. rachim. 1986. soybean yield constraints, socio-economics or technologic. palawija news iii (2): 1-2. fachrurrozi, q. j. laumans and r. krisdiana. 1988. production economic aspects of soybean grown on legal: a case study in pasuruan district east java. penelitian palawija 3 (2): 105-115. s omaatmadja, s. 1983. development of soybean culture in indonesia in: international symposium on soybean in the tropics and substropics. tropical research center, ministry of agriculture, forestry and fishery japan. tropical agricultural research series no. 17: 23-35. 55 biotropia no. 4, 1990/1991 pasaribu and me. intosh. 1986. soybean production in indonesia. soybean in tropical and subtropical cropping systems. proc. symp. tsukuba, japan 26 sept.1 oct. 1983. avrdc: 1-11. spitters, c.j.t. and j.p. van den bergh. 1982. competition between crop and weeds. a system approach in: holzner and numata (eds) biology and ecology of weeds: 137-148. tjitrosemito, s. 1987. threshold level of weed control in soybean crop for small farmers. proc. xlth apwss conf. i: 247-258. ______, 1988. weed control on soybean crop using imazethapyr. paper presented to the second trop. weed conf. phuket, thailand. december 6-10, 1988. 56 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf biotropia vol. 30 no. 2, 2023: 147 157 doi: 10.11598/btb.2023.30.2.1765 147 production optimization, partial purification, and thrombolytic activity evaluation of protease of bacillus cereus hsfi-10 ainutajriani ainutajriani1, sri darmawati1, dewi seswita zilda2, muhammad ardi afriansyah3, ragil saptaningtyas3 and stalis norma ethica1* 1magister of clinical laboratory science, postgraduate program, universitas muhammadiyah semarang, central java, 50273, indonesia 2research center for deep sea, earth sciences and maritime research organization, national research and innovation agency (brin), jl. pasir putih raya pademangan, north jakarta city, jakarta, 14430, indonesia 3department of medical laboratory technology, faculty of nursing and health sciences, universitas muhammadiyah semarang, central java, 50273, indonesia received 17 june 2022 / revised 2 march 2023 /accepted 11 march 2023 abstract cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. bacterial isolate hsfi-10 (holothuria scabra fermented intestine-10) previously isolated from rusip fermented sea cucumber had demonstrated thrombolytic activity. this study aimed to produce crude protease of hsfi-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on a, b, ab, and o blood types. isolate hsfi-10 was first molecularly identified and found to be bacillus cereus with a homology level of 99.80% with bacillus cereus strain st06. the optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 u/ml, which increased to 438.84 u/ml after ammonium sulfate precipitation and dialysis. clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the abo system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. in conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the abo system, with only 1% activity variability between a, b, ab, and o blood types. keywords: bacillus cereus hsfi-10, blood system, clot lysis, partial purification, thrombosis introduction thrombosis is the leading cause of global death in cardiovascular disease (cvd) (scheres et al. 2018). cardiovascular is a group of diseases related to the heart and blood vessels. according to the world health organization (who), this disease causes 31% of death worldwide. who predicts that by 2030, cardiovascular disease will continue to increase to more than 23.6 million people. the number is two times higher than the death rate from cancer (who 2017). thrombosis causes blood to clot (forming a thrombus), so blood vessels in the heart and brain will be blocked (martina et al. 2019). thrombus occurs in the area of the injured vascular wall, thereby stimulating platelet adhesion in that direction. the attached platelets are activated and release adenosine diphosphate (adp) and thromboxane a2 (txa2), which causes other platelets to stick to the activated platelets. platelet aggregation is strengthened with the help of clotting factors in the form of fibrin threads to form a hemostatic plug (bartinelli et al. 2013; durachim and astuti, 2018). thrombosis therapy can use anticoagulant agents, anti-platelet drugs, fibrinolytic/thrombolytic enzymes, and surgery. however, these drugs have side effects such as headaches, urticaria (allergic reactions), increased clotting time, nausea, vomiting, bleeding complications, low fibrin specificity, and *corresponding author, email: norma@unimus.ac.id biotropia vol. 30 no. 2, 2023 148 relatively high price (saxena et al. 2015; krishnamurthy et al. 2018; sharma et al. 2019). research conducted by hidayati et al. (2021) successfully isolated protease-producing bacteria from the digestive organs of the sand sea cucumber coded hsfi-10 to -12 (holothuria scabra fermented intestine-10). the crude enzyme of bacterial isolate hsfi-10 showed higher thrombolytic ability than the positive control nattokinase (a commercial antithrombosis agent). however, the bacterial isolates were only known for their microscopic and macroscopic characteristics. in addition, the thrombolysis test does not explicitly mention blood groups a, b, ab, and o. in contrast, antigens a and b in the abo system blood group impact hemostatic balance and respond differently to thrombolytic therapy (separham et al. 2020; mehic et al. 2020). identification was done to determine bacterial species by amplifying the 16s rrna gene using polymerase chain reaction (pcr). this molecular diagnostic method is more sensitive, specific, efficient, and faster than manual identification (shahi et al. 2018). bacteria secrete proteases in the production medium. each bacterium has a different enzyme production time depending on the type of bacteria (sharma et al. 2015)—partial purification of the protease enzyme bacillus sp. hsfi-10 can be done by precipitating enzymes using ammonium sulfate. this salt has a fairly high ionic strength, does not cause damage to proteins, and has a high solubility in water (mukherjee, 2019). the remaining ammonium sulfate salt from the enzyme precipitate is removed by dialysis (razzaq et al. 2019). this study aims to determine the type of bacteria based on the 16s rrna gene sequence using the pcr method, to determine differences in protease enzyme activity based on the optimization of production time for 24, 48, and 72 hours and to determine the description of a, b, ab, and o blood smears after the addition thrombolytic protease enzymes of bacillus sp. hsfi-10. materials and methods bacterial origin bacterial strain hsfi-10 was previously isolated in an earlier study from sand sea cucumber h. scabra captured from its captivity at marine bio industry office (bbil), the indonesian institute of sciences (lipi), kodek gulf village, lombok, west nusa tenggara (figure 1). the isolate was then subcultured, and the purified colonies were further analyzed to identify and confirm its fundamental characteristics. figure 1 location of the captivity of sand sea cucumber h. scabra, which intestine was the source of hsfi bacteria including hsfi-10 strain: (red dot, coordinates 8°24'15.1"s 116°04'47.2"e) (google map 2022) thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 149 procedures bacterial subculture and crude protease production bacterial subculture was done by cultivating a loop-full of hsfi-10 bacterial single colony nutrient agar (na) medium and then incubated at 37℃ for 24 hours. a single colony of isolate hsfi-10 obtained was then tested to confirm its proteolytic activities by streaking it on skim milk agar (sma) media and re-incubated for 24 hours at 37°c. the clear proteolytic zone formed around the colony's growth was observed (fuad et al. 2020; hidayati et al. 2021). bacterial molecular identification by cloning 16s rdna bacterial colonies of hsfi-10 were inoculated into the bhib 1 ml medium. after incubation at 37°c for 2 × 24 hours. bhib media was centrifuged for 1 min at 12000 rpm. dna genome was extracted with a dna extraction kit from geneaid, namely presto™ mini gdna bacteria kit. the amplification process used go tag green master mix (promega). the universal primers used were 27f (5'-aga gtt tga tcc tgg ctc ag-3') and 1492-r (5'-ggt tac ctt gtt acg act t-3'). amplicon was visualized using the major science uv transilluminator (darmawati et al. 2015). cloning of the 16s rrna gene was done as previously reported by darmawati (2015) using plasmid vector pta2 transformed in e. coli zymo 5α competent cells. recombinants were screened using the blue-white method (green & sambrook 2021). plasmids were isolated according to the previously reported method, with the results of recombinant dna checked with electrophoresis on 0.8% agarose gel. sequencing was carried out using abi sequence programs with primers t7 and t3 5'taatacgactcactataggg-3' and 5'ccctttagtgagggttaatt-3' as previously reported (darmawati 2015). next, bioinformatics dna sequencing results are analyzed using bioinformatics devices, then processed manually and matched with data in www.ncbi.nih.gov through the blast program (ethica et al. 2018). optimization of bacterial crude protease production colony of bacillus sp. hsfi-10, which has proteolytic abilities inoculated in minimally synthetic medium (msm) (nacl 0.1%, k2hpo4 0.1%, ammonium sulfate 0.7%, mgso4.7h2o 0.01%, yeast extract 0.05%, skim milk 1%) and incubated at 37°c for 24,48 and 72 h (farooq et al. 2021). the extraction of thrombolytic protease enzymes is done by bacterial culture centrifugation at 10,000 rpm, where supernatant was regarded as a crude enzyme. it further measured the enzyme activity of the bergmeyer and grab method (1983) using a spectrophotometer at λ 600 nm. partial purification of bacterial protease partial purification was initiated by precipitation using 70% ammonium sulfate, as much as 400 g added slowly in 1 l of crude enzymes as previously described (natsir et al. 2015). cellophane bags were initially soaked in hot water at 60°c for 2 mins, then replaced with 0.2% sulfuric acid as previously reported. enzymes obtained from the deposition of ammonium sulfate were put in cellophane bags. both ends of the cellophane bag are tied, then the cellophane bag is soaked with a 500-700 ml phosphate buffer of 0.05 m ph 7 at ± 4°c for 2 h. the soaking buffer is replaced every 2 hours until all salts are separated (abbas et al. 2018). specific enzyme and clot lysis activity assay the protease-specific activity was measured using the modified bergmeyer & grab (1983) (natsir et al. 2015; si & jang 2018). in vitro, blood clot lysis tests were conducted on blood from 4 volunteers, each with blood types a, b, ab, and o, in 6 of the 1.5 ml microtubes previously weighed. each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type a), tube 4 (blood type b), tube 5 (blood type ab), and tube 6 (blood type o), and filled with 600 μl blood of each. as previously reported, gravimetry determined clot lysis percentage (fuad et al. 2020; hidayati et al. 2021; prasad et al. 2006). proteolytic activity test a single colony of bacillus sp. bacteria. hsfi10 obtained from the results of bacterial purification in na media tested its proteolytic abilities. bacteria that grow on na media were streaked on skim milk agar (sma) media and incubated for 24 h at 37°c then the clear zone biotropia vol. 30 no. 2, 2023 150 formed around the growth of the colony was measured (fuad et al. 2020; hidayati et al. 2021). optimization of crude enzyme protease production time from bacillus cereus culture colony of bacillus sp. hsfi-10 with proteolytic abilities was inoculated in minimal synthetic medium (msm) incubated at 37°c for 24, 48, and 72 h. extraction of thrombolytic protease enzymes was done by centrifugation at 10,000 rpm for 20 mins at 4°c. the obtained supernatant was regarded as a crude enzyme, and its activity was further measured by bergmeyer & grab method (1983) using a spectrophotometer at λ=600 nm (farooq et al. 2021). partial purification of crude protease 70% of ammonium sulfate (400 g) was inserted slowly in 1 l of crude enzymes in cold conditions until dissolved for 2 hours. the enzyme solution was kept in the refrigerator to precipitate the enzymes overnight. the enzyme solution was concentrated at 4°c at 10,000 rpm for 30 mins, and the formed pellet was kept. the pellet was then rushed with a 15 ml tris-cl buffer of 0.05 m ph 8 (natsir et al. 2015). cellophane bags were prepared by soaking in hot water and 0.2% sulfuric acid solution before use. enzymes obtained from the deposition of ammonium sulfate are put in cellophane bags. dialysis was conducted as previously described until all salts were separated (abbas et al. 2018). enzyme-specific activity assay the protease-specific activity was measured using the modified bergmeyer & grab (1983) folin reagent method (natsir et al. 2015; si and jang 2018). clot lysis (thrombolysis) activity in vitro assay in vitro blood clot lysis tests before and after enzyme purification were conducted on blood taken from 4 volunteers, each with blood types a, b, ab, and o. six 1.5-ml-microtube tubes that had been weighed were prepared. each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type a), tube 4 (blood type b), tube 5 (blood type ab), and tube 6 (blood type o). the percentage of % blood clots was determined following the previously reported method and was conducted in duplicates (prasad et al. 2006; fuad et al. 2020; hidayati et al. 2021). the effect of blood clot lysis was also observed microscopically by the eustrek (removal) technique according to the may grunwald-giemsa mixed method (geneser 1994). results were observed with magnification under a dino-lite digital microscope and documented with a camera (ethica et al. 2018). results and discussion bacterial subculture and morphology characteristics bacillus sp. hsfi-10 has a circular shape, edge (entire), size (3 mm), milk-white color, convex elevation, and smooth consistency (figure 2a)— bacillus sp. hsfi-10 has a short life in na media (figure 2b), a colony life of approximately 4 days of storage at cold temperatures, characterized by colony colors beginning to fade and cannot grow back on the new na medium. figure 2 characteristics of bacillus sp. hsfi-10 in agar nutrient (na) (a) colonies after 24-h incubation, (b) after stored for 4 days. c. on skim milk agar media with lugol-staining (clear zone showing proteolytic activity) thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 151 figure 3 similar characteristics of bacillus sp. hsfi-10 on blood agar plate media. (a) compared to (b) b. cereus indicates β-hemolysis (milojevic et al. 2019) gram staining showed the rod shape was gram-positive, lined, and had spores. b. cereus could produce a clear zone on the 7th day of cultivation on skim milk agar (sma) media with a diameter of 36 mm. sma contains casein as a protease enzyme substrate. bacterial growth in blood agar plate (bap) media indicates a βhemolysis pattern, characterized by an area of clear zone around the colony (figure 3) (bottone 2010; lu et al. 2018). as seen in figure 3, the macroscopic characteristics of bacteria on bap media were evaluated by the shape, color, size, edge, and elevation of the colony as well as discoloration in the media (e. g. hemolysis in the medium for blood) (pitt et al. 2012). the result aligned with the mogrovejo et al. (2020) study reporting that bacillus cereus can disintegrate red blood cells (forming a β-hemolysis pattern on bap). b. cereus hsfi-10 morphology and properties reported in this study following the results reported by hidayati et al. (2021). the proteolytic ability test aims to determine bacteria that have the potential to produce proteases, characterized by the formation of a clear zone around the bacterial colony (assaf et al. 2020). the clear zone produced by proteolytic bacteria occurs due to protease activity which breaks the peptide bonds of casein in skim milk medium by breaking the co-nh peptide bond with the entry of water into the molecule, thereby releasing amino acids (baehaki et al. 2011; ethica et al. 2018; artha et al. 2019). bacterial molecular identification dna extract of bacillus sp. hsfi-10 has a concentration of 232.6 ng/μl and a purity of 1.82. the purity of extracted dna was high if the absorbance ratio (λ260/λ280) was 1.83. if the absorbance ratio is less than 1.8, proteins still contaminate dna. if greater than 1.8, the dna is contaminated with rna (gupta 2019). the genomic 16s rrna gene cloning technique obtained the full-length 16s rrna gene sequence (green & sambrook 2021). the cloning process was done by ligating, transforming, and isolating recombinant dna from transformants (green & sambrook 2021). recombinant dna was then transformed in escherichia coli zymo 5α bacterial cells and grown on a solid medium. the success of cloning could be seen through white colonies (clones carrying recombinant dna). the white colony indicates the lacz region on the plasmid is inactive because it has been inserted by the inserted gene so that the galactose present in the media cannot be hydrolyzed by the -galactosidase enzyme which, if active, will form blue colonies (sambrook et al. 1989; darmawati 2015)— results of the 16s rrna gene cloning of bacillus sp. hsfi-10 is displayed in figure 4, with an amplicon size of 1517 bp. biotropia vol. 30 no. 2, 2023 152 figure 4 the complete sequence of the 16s rrna bacillus sp. hsfi-10 obtained from cloning and sequencing 16s rrna gene the consensus was made on the forward and reversed 16s rdna sequences using the bio edit program (hall 2004). the 16s rrna gene sequence data saved in fasta format were then analyzed and matched with the data available in the gene bank basic local alignment search tool (blast). the results of blast analysis of bacillus sp. hsfi-10 16s rrna gene fragments showed a homology level of 99.80% with bacillus cereus strain st06 (acc. no.: mh475925.1). optimization of crude production time for protease enzymes from bacterial culture crude enzyme production was optimized with various incubation times of 24, 48, and 72 hours. the results of this study indicate that bacillus cereus hsfi-10 can produce the most optimum crude enzyme at 48 hours with an enzyme activity of 222.52 u/ml. it has been reported that protease production was proportional to bacterial growth. in the early stages of the growth curve, bacteria produced few proteases and optimum production in the stationary phase (ahmadpour and yakhchali 2017; pagarra et al. 2020; suleiman et al. 2020). partial purification of protease and specific activity assay the protease activity of b. cereus hsfi-10 was calculated using the equation from hidayati et al. (2021): y = 0.0019 x + 0.0092. y = absorbance, while x = enzyme activity (u/ml). enzyme activity was expressed as the number of tyrosine amino acids released by the casein substrate per unit of time under test conditions (zainuddin et al. 2020). results of specific activity assay on crude and dialysate protease of b. cereus hsfi-10 are shown in table 1. table 1 absorbance and specific activity data of crude and dialysate protease of b. cereus hsfi-10 protease extract absorbance at λ=600 nm at incubation time (h)*: enzyme activity (u/ml) at incubation time (h)*: 24 48 72 24 48 72 crude 0,264 0,433 0,346 134,105 222,526 146,000 dialysate 0,843 438,842 *all tests were conducted in duplicate thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 153 based on data in table 3, it could be inferred that ammonium sulfate precipitation followed by dialysis could increase the enzyme-specific activity by almost doubling from 222.526 u/m to 438.842 u/ml. these results are to the research of mothe and sultanpuram (2016), reporting that enzymes purified by dialysis had high activity compared to crude enzymes, where the level of enzyme purity could reach 2.23 times higher. clot lysis activity assay on crude and partially purified protease crude and dialysate enzyme b. cereus hsfi-10 could lyse abo blood clot better than nattokinase as control (figure 5). a higher percentage of clot lysis characterized it. the % blood clot lysis of each sample is shown in table 2. nattokinase is a serine protease enzyme with fibrinolytic and antithrombotic activity. this ability can be used for cardiovascular treatment. several studies have stated that nattokinase can thin the blood and dissolve blood clots in experimental animals and humans (gallelli et al. 2021; chen et al. 2022). this study shows that crude enzymes and dialysate can lyse blood clots better than nattokinase (figure 5). hence, the protease enzyme from b. cereus hsfi-10 bacteria can be used as a substitute for nattokinase. as seen in table 2, part of the limitation of this preliminary study is that the thrombolysis ability test was conducted only in duplicates with the objective of screening. one of the reasons is that the gravimetry test, first described by prasad et al. 2006, is semi-quantitative. the qualitative part of the assessment lies in whether the lyse blood is still present before weighing the step of the method. thus, the more accurate thrombolysis test should be confirmed with the in vivo one involving precise crude and dialysate bacterial protease dosage followed by statistical analysis (dewi et al. 2022). the effect of clot lysis on o blood cells before and after adding crude and dialysate enzymes was also observed under a microscope with 400× magnification, with results displayed in figure 6. figure 5 crude protease enzyme thrombolysis test and dialysate on blood type o table 2 results of the thrombolysis ability test for crude protease enzymes and dialysates in abo blood type blood type initial clot weight (g)* final clot weight (g)* % clot lysis* nk crude dialysate nk crude dialysate nk crude dialysate a 0.358 0.358 0.299 0.141 0.111 0.055 60.614 68.994 81.605 b 0.333 0.334 0.282 0.133 0.101 0.051 60.060 69.760 81.914 ab 0.290 0.290 0.276 0.116 0.088 0.052 60.000 69.655 81.159 o 0.369 0.368 0.349 0.145 0.113 0.061 60.704 69.293 82.521 *all measurement was conducted in duplicates nk: nattokinase biotropia vol. 30 no. 2, 2023 154 figure 6 the observation results on protease clot lysis activity on o blood cells using a dino-lite digital microscope. (a) negative control, (b) positive control, (c) crude enzyme, (d) dialysate enzyme as seen in figure 6, platelets undergoing aggregation and erythrocytes experience changes in shape and overlap (figure 6a), indicating the occurrence of blood clots. while figures 6b, c, and d show that platelets did not undergo aggregation, erythrocytes did not change in shape and are evenly distributed. the platelet condition was due to favorable control treatment, crude, and dialysate, which can lyse blood clots. these results align with the research of fuad et al., 2020, where crude protease enzymes produced by staphylococcus hominis hsft-2, s. saprophyticus hsft-11, and bacillus aryabhattai hsft-5 caused erythrocytes not to create, spread evenly and platelets did not aggregate. our results showed that partial purification by ammonium sulfate precipitation and dialysis on produced bacterial crude protease is very beneficial. the partial purification could increase bacterial protease-specific activity from 222.52 u/ml to 438.84 u/ml. it could also increase clot lysis activity based on the gravimetry method on blood in the abo system from 68.99% 69.76% (crude protease) to 81.16% (crude 82.52% (dialysate protease). the next step will be enzyme purification techniques using various chromatography-based methods to maximize protease's specific and clot lysis activity from b. cereus hsfi-10 (westphal & van berkel 2021). in this study, bacterial proteases had a similar effect on the lysis of abo blood clots regardless of their different types of agglutination. despite the previously reported data that blood type did affect the formation of blood clots, it has been known that individuals with non-o blood type have been shown to have a higher risk of thrombus formation than individuals with blood type o. this was associated with levels of coagulation factors, especially von willebrand factor (vwf), vwf levels 30% higher in individuals with non-o blood type compared with blood group o (holle et al. 2020; mohamed et al. 2020; separham et al. 2020; pendu et al. 2021). conclusion the optimum production of crude enzyme of thrombolytic protease-producing bacterium, b. cereus hsfi-10, resulting in the highest specific activity, was at 48-h incubation. partial purification of bacterial protease increased both its specific and thrombolytic activities in human blood of the abo group system with only 1% activity variability between a, b, ab, and o blood types. acknowledgments the authors would like to express their sincere gratitude to the center for research and community service (lppm) universitas muhammadiyah semarang for financially supporting this research through an internal grant in 2021. thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 155 references abbas, n., siddique, h.n., masood, f., shehzadi, a., abbas, z. and ali, s., 2020. production of protease enzyme from bacillus subtilis using skimmed milk. science international (lahore), 32(2), pp.211-214. 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https://www.who.int/en/news-room/factsheets/ detail/cardiovascular diseases(cvds) biotropia no. 4, 1990/1991: 31-40 potential role of wild crucifers in the preservation of diadegma eucerophaga horstman (hymenoptera: ichneumonidae), a parasitoid of the diamondback moth, plutella xylostella linnaeus (lepidoptera: plutellidae) utomo kartosuwondo faculty of agriculture bogor agricultural university bogor, indonesia sunjaya tropical agricultural pest biology programme, seameo biotrop bogor, indonesia abstract laboratory and field experiments were conducted to evaluate the potential role of two species of wild crucifers in the preservation of diadegma eucerophaga horstman, a parasitoid of the diamondback moth, plutella xylostella linnaeus. in the laboratory, d. eucerophaga developed quite well on p. xylostella larvae fed on leaves of two species of wild cruciferous plants, nasturtium heterophyllum bl. and cardamine hirsuta l. these wild crucifers may serve as food and oviposition sites for p. xylostella. in the field, n. heterophyllum and c. hirsuta planted adjacent to insecticide-treated cabbage plots provided refuge for d. eucerophaga parasitoids. introduction the diamondback moth, plutella xylostella linnaeus, is a major pest of cole crops in indonesia (and other parts of the world). in indonesia, an introduced parasitoid, diadegma eucerophaga horstman, plays an important role as a natural control agent of diamondback moths. the parasitoid was established in the cabbage-growing regions in west java where the rate of parasitism was reported to be in the range of 5-86% (sudarwohadi 1984). in the absence of insecticide application, d. eucerophaga parasitoids can often give an effective natural control of the diamondback moth. since insecticides are regularly applied to cabbage fields, the role of d. eucerophaga in suppressing diamondback moth population may be severely affected by intensive insecticide applications. under such unfavourable condition, a sound ecological measure should be undertaken in order to preserve d. eucerophaga parasitoids. preservation of natural enemies can be achieved, among other means, through the provision of refuge for natural enemies by managing wild host plants growing 31 biotropia no. 4, 1990/1991 in the surrounding of the main-crop field. preservation of natural enemies through appropriate management of wild host plants is a feasible means which can be incorporated in any crop pest management (van emden 1976). wild alternative host plants growing in the vicinity of the main-crop field can enhance the survival of a limited number of pest individuals and its natural enemies in the absence of the main host plant (ullyett 1947 in van den bosch and telford 1973). it has been known that a number of wild cruciferous plant species can serve as alternative hosts for p. xylostella (harcourt 1957). these wild crucifers may serve as refugium for the d. eucerophaga parasitoids when the main crop is being treated with insecticides or during a fallow period, provided that the parasitoid can develop on diamondback moth larvae feeding on wild crucifers. preservation of natural enemies is an important component of pest management programme (luckmann and metcalf 1975). natural enemies can be affected by insecticide application in two ways: they die of insecticide intoxication or due to lack of food (hosts or preys) which are eliminated by insecticides. in order to assure the availability of food for natural enemies, it is necessary to adopt a control approach that will leave a pest residue, below its economic threshold. in the case of the diamondback moth, the pest residue may find refuge in the wild cruciferous plants growing in the surrounding of the main-crop field and in turn, the diamondback moth residue can support the development and survival of d. eucerophaga parasitoids. in java, it was reported that p. xylostella can live on the following cruciferous plants: capsellabursa-pastoris, cardamine hirsuta (everaarts 1981) and nasturtium heterophyllum (backer and van slooten 1924). the potential role of the last two wild crucifers, which can be found in bogor and pacet highland, west java, in the preservation of d. eucerophaga parasitoids was evaluated in this study. laboratory and field experiments were carried out to study: 1) the oviposition preference of the diamondback moth to n. heterophyllum, c. hirsuta and cabbage, 2) the development of the diamondback moth on n. heterophyllum and c. hirsuta, 3) the development and survival of d. eucerophaga parasitoids on diamondback moth larvae fed on leaves of n. heterophyllum and c. hirsuta, and 4) the occurrence of d. eucerophaga on n. heterophyllum and c. hirsuta planted adjacent to cabbage plots. materials and methods laboratory experiments were carried out in the tropical pest laboratory, seameo biotrop, bogor, from november 1988 to february 1989, while field 32 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya experiments were conducted in the horticultural experimental farm, segunung, facet, from january to april 1989. insects and food plants larvae, pupae and adults of the diamondback moth were collected from cabbage fields around facet (1100 m in altitude), cianjur, west java. insects were maintained in the laboratory on brassica juncea. d. eucerophaga parasitoids were collected from the same area as diamondback moth and reared in the laboratory on diamondback moth larvae. both adults, p. xylostella and d. eucerophaga were fed on 10% honey solution soaked in cotton wool. experimental host plants, cabbage (brassica oleracea var. capitata), nasturtium heterophyllum and cardamine hirsuta, were planted in an insecticide-free area in a green house at seameo biotrop, bogor. when used, the age of plants was about 40-60 days old. experiments the study consisted of three laboratory experiments (1-3) and two field experiments (4-5). the first three experiments served as a preliminary study for the last two. experiment 1 oviposition preference of the diamondback moth to n. heterophyllum, c. hirsute and brassica oleracea var. capitata n. heterophyllum, c. hirsuta and cabbage plants kept separately on small vials containing water were placed in a wooden-framed muslin cloth cage (30x30x30 cm). a pair of mated diamondback moth adults was then introduced into the cage. the number of eggs deposited by the female on the three plant species was recorded daily until the female died. food plants were replaced daily. each treatment (plant species) was replicated eight times and the experiment was arranged in a completely randomized design. experiment 2 development of the diamondback moth on n. heterophyllum, c. hirsuta and brassica oleracea var. capitata eggs deposited by diamondback moth females maintained in cages containing larval food plants were transferred separately into a plastic cage (15 cm in diameter and 20 cm high) containing the corresponding food plants. food plants were replaced daily. insect development was followed until adult emergence, and the duration of 33 biotropia no. 4, 1990/1991 the egg, larval and pupal development stages were recorded. the experimental design was the same as in experiment 1. experiment 3 survival of d. eucerophaga on diamondback moth larvae fed on leaves of n. hetero phyllum and c. hirsuta ten third-instar diamondback moth larvae were confined in a plastic cage (25 cm in diameter and 50 cm high) containing n. heterophyllum or c. hirsuta plants as food. a pair of mated adult of d. eucerophaga parasitoids was then released into the cage. the female parasitoid was allowed to oviposit on diamondback moth larvae for five hours and then the parasitoid pair were removed from the cage. the number of parasitoid adults emerged from parasitized diamondback moth larvae was recorded. the percentage of emergence was calculated based on the number of diamondback moth larvae used. the experiment was arranged in a completely randomized design with two treatments (two species of wild crucifers) and eight replications. experiment 4 occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and b. oleracea var. capitata in the field n. heterophyllum, c. hirsuta, and cabbage plants were planted separately on thirty field small plots (1x1 m) which were divided into ten blocks. thus, each block consisted of three small plots containing the three plant species (one species/plot). plants were not treated with insecticides. the experimental plots were examined regularly for evidence of diamondback moth infestation. infested plant leaves (containing diamondback moth larvae) were removed and transferred to plastic cages (25 cm in diameter and 50 cm high) in the laboratory. collected diamondback moth larvae were fed on the same food plants as their origin and food plants were replaced daily. diamondback moth larvae were examined for evidence of parasitization and the number of adult d. eucerophaga parasitoids which emerged was recorded. a randomized block design was used to arrange this experiment. experiment 5 occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and insecticide treated b. oleracea var. capitata in the field this experiment was similar to experiment 4, but in this experiment cabbage plots were treated with baythroid 50 ec at a rate of 0.4 kg a.i./ha for each application. at the time of sampling of infested plant leaves, cabbage plots had been treated with the insecticide four times. the experimental design and the parameter observed were the same as in experiment 4. 34 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya results and discussion oviposition preference of the diamondback moth to n. heterophyllum, c. hirsuta and brassica oleracea var. capitata the average number of eggs deposited by the female diamondback moth per day over a seven-day oviposition period on two wild crucifers and cabbage is presented in table 1. table 1. average number of eggs deposited by the female diamondback moth on three cruciferous plant species over a seven-day oviposition period day plants 1 2 3 4 5 6 7 n. heterophyllum 11.2 1) 2.50 2.75 0.75 0.25 0.12 0 c. hirsuta 16.7 9.12 3.62 6.87 2.25 1.75 1.87 b. oleracea 25.6 10.60 11.20 4.25 2.00 0.12 0 1) each value in the table is an average of eight replications. the number of eggs deposited on day-1 was the highest among the seven-day oviposition period. this result is in agreement with that obtained by vos (1953). the oviposition preference of diamondback moth females to three cruciferous plant species is shown in table 2. table 2. average number of eggs deposited by the diamondback female on three cruciferous plant species 1) means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to √ x+ 1 before analysis; each value in the table is an average of eight replications. in the laboratory, the three cruciferous plant species could serve as oviposition sites for diamondback moth females, with the order of preference: cabbage > c. hirsuta >n. heterophyllum. apparently, the three plant species contain secondary compound(s) that act as oviposition stimulant for diamondback moths, although 35 biotropia no. 4, 1990/1991 it may not be in the same amount. in crucifers, a compound that stimulates ovi-position in diamondback moths is known as mustard oil glucoside (gupta and thor-steinson 1960; harborne 1988). development of the diamondback moth on tv. heterophyllum, c. hirsuta and brassica oleracea var. capitata it was known from experiment 1 that diamondback moth females could lay eggs on the two wild crucifers tested, especially on c. hirsuta. results of experiment 2 indicated that the diamondback moth developed quite well on those wild crucifers. egg plus larval and pupal periods of the diamondback moth reared on three cruciferous plant species are indicated in table 3. table 3. average egg plus larval and pupal periods (days) of the diamondback moth reared on three cruciferous plant species food plants egg plus pupal period larval period 1) n. heterophyllum 11.0 a 4.75 b c. hirsuta 10.38 a 3.0 c b. oleracea 11.0 a 4.0 b 1) means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; each value in the table is an average of eight replications. the three host plants did not affect the combined duration of egg and larval stages of the diamondback moth significantly. the effect of host plants on the diamondback moth development was manifested in the duration of the pupal stage. the pupal period of the diamondback moth maintained on n. heterophyllum was comparable with that on cabbage, whereas on c. hirsuta the pupal period was the shortest. the obtained data suggest that the nutritional contents of the three cruciferous plant species can adequately support the growth and development of the diamondback moth. like in oviposition, a secondary compound mustard oil glucoside can also act as feeding stimulant for diamondback moth larvae (gupta and thorsteinson 1960; harborne 1988). survival of d. eucerophaga on diamondback moth larvae fed on leaves of tv. heterophyllum and c. hirsuta results of experiment 2 showed that diamondback moth larvae developed quite well on two wild crucifers, n. heterophyllum and c. hirsuta. from this experiment it can be inferred that diamondback moth larvae feeding on those wild 36 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya crucifers could support the development of its parasitoid, d. eucerophaga. the percentage of emergence of adult parasitoids resulting from a five-hour exposure often third-instar diamondback moth larvae to a mated parasitoid pair is presented in table 4. the data indicated that d. eucerophaga could survive on diamondback moth larvae feeding on wild cruciferous plants. the yield of food plants of diamondback moth larvae did not show a significant effect on the percentage of emergence of adult parasitoids. the percentage of emergence could be higher if the female parasitoid was allowed to oviposit on its host for more than five hours. table 4. average percentage of emergence of d. eucerophaga adults from diamondback larvae reared on two species of wild crucifers food plants of percentage of emergence diamondback larvae of parasitoid adults 1) n. heterophyllum 31.63 a c. hirsuta 40.0 a 1) percentage of emergence was calculated based on the number of diamondback moth larvae used. means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to arcsin x before analysis; each value in the table is an average of eight replications. occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and b. oleracea var. capitata in the field the average number of d. eucerophaga adults emerging from its host collected from three host plant habitats is shown in table 5. the number of d. eucerophaga adults which emerged from its host feeding on three cruciferous plants did not differ significantly from each other, although table 5. average numbers of d. eucerophaga adults emerged from its host collected from three cru ciferous plant habitats 1) means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to √x + 1 before analysis; each value in the table is an average of ten replications. 37 biotropia no. 4, 1990/1991 cabbage and c. hirsuta seemed to provide better habitats than n. heterophyllum. the data suggested that in the field, diamondback moths can lay eggs on the wild crucifers, n. heterophyllum and c. hirsuta. the larvae of diamondback moths can feed on the wild crucifers aside from cabbage. occurrence of d. eucerophaga on tv. heterophyllum, c. hirsuta and insecticide-treated b. oleracea var capitata in the field in experiment 5, cabbage plots were sprayed with insecticide, while wild crucifers plots were not. the results of experiment 5 are shown in table 6. the average number of d. eucerophaga in insecticide-treated cabbage plots was the lowest among the three plant habitats, although it did not differ significantly from that in n. heterophyllum habitat. the average number of d. eucerophaga in c. hirsuta habitat was the highest and significantly different from the other two. data from experiment 4 and 5 suggested that there might be a movement of diamondback moths and d. eucerophaga parasitoids from insecticide-treated cabbage plots to adjacent wild crucifer plots. thus, wild crucifers growing in the surrounding of the cabbage field may provide refuge for d. eucerophaga parasitoids when unfavourable conditions prevail. table 6. average numbers of d. eucerophaga adults emerged from its host collected from two wild cruciferous and insecticide-treated cabbage plant habitats 1) means in the same column followed by a common letter were not significantly different (p =0,05), using least significant difference test; data were transormed to √x+1 before analysis; each vaue in the table is an average of ten replications the experimental results discussed above provide information on the potential role of wild cruciferous plants in the preservation of d. eucerophaga, a parasitoid of the diamondback moth, p. xylostella. two species of wild crucifers, tv. heterophyllum and c. hirsuta, can provide oviposition sites for diamondback moths and can serve as larval food. d. eucerophaga parasitoid can survive on its host feeding on the aforementioned wild crucifers. 38 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya p. xylostella is an oligophagous insect feeding on a number of plant species of cruciferae (thorsteinson 1952) which contain a secondary compound known as mustard oil glucoside (thorsteinson 1952; gupta and thorsteinson 1960; harborne 1988). this compound serves both as oviposition and feeding stimulant for the diamond-back moth. p. xylostella larva is a specific host for d. eucerophaga parasitoids (vos 1953). therefore, in order to enhance the survival of the parasitoid under unfavourable conditions, such as during insecticide application or in the absence of cabbage plants in the field, it is necessary to ensure the availability of wild cruciferous plants refuge for the pest residue. this necessity was supported by data from the last experiment. when cabbage plants were treated with insecticide, the diamondback moth in wild crucifers could support the survival of d. eucerophaga parasitoids. the wild crucifer c. hirsuta seems to be more suitable than n. heterophyllum as refuge for the diamondback moth and its parasitoid. acknowledgement the research work was supported by seameo biotrop. the authors wish to thank prof. dr.ir. amris makmur for making possible to carry out the experiment and to prof.dr.ir. h. sitti soetarmi tjitrosomo for her guidance and criticism. references backer, c.a. and d.f. van slooten. 1924. javaansche thee onkruiden. batavia drukkerijen ruygrok & co. 287 p. everaarst, a.p. 1981. weeds of vegetables in the highlands of java. lembaga penelitian horti kultura, pasar minggu, jakarta. 121 p. gupta, p.d. and a.j. thorsteinson. 1960. food plant relationship of diamondback moth (plutella maculipennis curt.). ii. sensory regulation of oviposition of the adult female. ent. exp. appl. 3: 305-314. harborne, j.b. 1988. introduction to ecological biochemistry. academic press, london. 356 p. harcourt, d.g. 1957. biology of the diamondback moth, plutella maculipennis curt. (lepidoptera: plutellidae), in eastern ontario. ii. life history, behaviour, and host relation-ships. can. entomol. 89: 554-564. luckmann, w.h. and r.l. metcalf. 1975. the pest-management concept. in introduction to insect pest management (r.l. metcalf and w.h. luckmann, eds.), p . 3-35. john wiley & sons, new york. 587 p. 39 biotropia no. 4, 1990/1991 sudarwohadi, s. 1984. status pengendalian hayati hama plutella xylostella oleh parasitoid diadegma eucerophaga di jawa barat. seminar hama dan penyakit sayuran di cipanas, 29-30 mei 1984. 17 p. thorsteinson, a.j. 1952. the chemoctactic responses that determine host specificity in an oligophagous insect (plutella maculipennis curt., lepidoptera). can. j. zool. 31: 52-72. van den bosch, r. and a.d. telford. 1973. environmental modification and biological control. in biological control of insect pests and weeds (p. debach, ed.): 459-488. chapman and hall, ltd., london. 844 p. van emden, h.f. 1976. pest control and its ecology. edward arnold. 59 p. vos, h.c.a.a. 1953. introduction in indonesia of angitia cerophaga grav., a parasite of plutella maculipennis curt. cont. gen agric. res. stat., bogor. 32 p. 40 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf introduction in vitro conservation of coconut (cocos nucifera l.) embryos biotropia no. 25, 2005 : 11 – 21 in culture media sukendah1 and ma l. o. cedo2 1dept. of agronomy, faculty of agriculture, pembangunan nasional “veteran” university, surabaya, indonesia 2 assistant professor, dept. of horticulture, uplb, laguna 4031, los baños, philippines the possibility of delaying the germination of mature coconut embryos in vitro for medium-term conservation of coconut was investigated. embryos of coconut was stored in sugar free, full-strength and half-strength ms media for 3 months with minimal loss in viability. germinability of embryos decreased with prolonged storage. at the end of 9-month storage period, embryos kept in full-strength media were all non-germinable, while those stored in half-strength media still gave 15% germination. addition of mannitol up to 0.3m did show any significant effect on germination of embryos stored for 3 months. however, longer period of storage helps to a certain extent in the preservation of embryo viability. the presence of mannitol in the storage medium also induced morphological abnormalities in seedlings derived subsequently from the stored embryos. mannitol at 0.4m was totally deleterious to the embryo. significance of these findings on coconut genetic resources conservation are discussed. keywords: in vitro conservation/coconut embryo. introduction the safe conservation of genetic resources of coconut is faced with various problems. coconut is a species with a truly recalcitrant seed which has no dormancy (engelmann et al. 1995). because of this characteristic, there is no possibility of storing it ex situ even for short periods of time. genetic resource collections (germplasm) are conserved as special field genebanks (living plants) in national or regional centers. field genebanking, however, run the risk of being damaged by natural disasters, pests and diseases. it also requires more space. moreover, many sources of plant genetic materials (indonesia, the pacific, and philippines) are still almost unexplored, and the small proportion of coconut germplasm currently available in existing field collections and certain accessions are now in danger of dying. the in vitro technique addresses all these concerns. it offers the possibility of providing duplicate, healthy collections for storage and use with less space. since vegetative propagation of coconut is as yet not feasible, embryo culture technique is a promising alternative. coconut embryo culture protocols are now available in the literature (batugal and engelmann 1998). in all these protocols, embryo germination commences soon after the embryos are placed in the culture medium. subsequently, fully developed seedlings ready for acclimatization may be obtained within 5-7 months. for in vitro conservation, such quick growth response to the medium is not desired. thus, efforts 11 are now focussed on modifying the medium and other culture conditions in order to delay the germination while at the same time preserving the germination potential of the embryos during storage. biotropia no. 25, 2005 techniques which can be used to maintain collection in delaying the embryo germination are: the use of reduced temperature, minimized light, and modifications in media. ashmore (1997) was able to store sugarcane shoots at 18ºc on reduced mineral salts, and banana shoots at 16ºc in the presence of plant growth subtances. chatty and tissouni (1999) showed that the suitable temperature for major palm species is about 25ºc, whereas lack of germination was observed at the extreme temperature of 5ºc. the american palm germinated better at temperatures ranging from 25ºc to 35ºc, but did not germinate at 15ºc. meanwhile, ng and ng (1991) reported to slow down the growth rate, cultures are basically incubated under reduced light intensity or total darkness in conjunction with low incubation temperature. however, the modification of temperature and/or light to preserve cultures required a high cost because of electricity and equipment. ng and ng (1991) suggested that an alternative approach to retard growth was to use a reduced-growth medium. recent publications on slow-growth media indicate that a variety of techniques are still being utilized, such as the reducing concentration of mineral salt, increasing, decreasing or eliminating sucrose, adding mannitol or sorbitol and growth retardant to the culture medium (ng and ng 1991). one of the advantages of this method is that the modification of the culture medium requires no special equipment. preliminary studies on storage media conducted by assy-bah and engelmann (1993) have indicated that embryos of coconut can be stored for 6 months on a medium devoid of sucrose and containing 2 g/l activated charcoal with eventual germination of stored embryos at 100%. related study done by damasco (2000) on tagnanan tall showed that aba and mannitol at low concentrations (0.05 to 0.1m) might not be effective to inhibit growth in culture. however, high concentration of mannitol (0.2 and 0.3m) significantly reduced percent germination and subsequent growth of germinated embryos. in this report, results on the use of reduced-nutrient media and mannitol for coconut embryo storage are presented. materials and methods collection, sterilization, and excision of embryos mature (11-12 months) coconut palm variety ‘tagnanan tall’ were collected from the philippine coconut authority zamboanga research center. a cylinder of endosperm embedding the embryo was extracted from each nut using a 2-cmdiameter cork borer. the endosperm cylinders were washed in 95% ethanol (1-2 minutes) then sterilized in 100 % commercial bleach (5-6 % sodium hypochlorite) for 20 minutes. 12 under aseptic condition, the embryos were excised from the solid endosperm cylinders, then sterilized in two changes of 10% commercial bleach for 5 minutes at each time and finally rinsed three times with sterile distilled water. sterilized embryos were transferred to a sterile flask containing sterile distilled water for size selection. the same size embryos (± 1 cm) were then inoculated singly onto liquid storage medium according to the treatments. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo culture media and arrangement of treatments the composition of the storage medium used in the present experiment was that of assy-bah (1986), at full-strength or half-strength. the medium contained activated charcoal and had no sucrose. after the required period of storage (3, 6, and 9 months), embryos were germinated following the embryo culture protocol of the university of the philippines at los baňos or uplb (batugal and engelmann 1998). the experiment was conducted by culturing the embryos in test tubes containing 10 ml of full-strength or half-strength ms media without or with mannitol at 0.10 m, 0.2 m, 0.30 m, or 0.4 m. each treatment was replicated three times, each consisting of 35 embryos. the cultures were randomized and incubated in a dark room with temperature maintained at 25°c. observation of embryos viability of embryos was evaluated based on the following parameters: length of embryos, measured every 2 months since embryos have been incubated; percent germination, it was done after the embryos have been preserved for 3, 6, and 9 months of storage. the stored embryos from the experiment were transferred to the germination medium described in the uplb protocol; normal and abnormal seedlings, observed 3 months after embryos have been transferred to the germination medium. seedlings which showed well-developed roots & shoots were considered normal, and seedlings with no shoot, no root or stunted seedlings as abnormal seedling. data were collected based on: a) percent normal seedlings and abnormal seedlings, and b) morphological features, observed by taken their photograph. observations of shoot length, number of leaves, leaf width, leaf length, length of primary root, and number of adventitious roots for normal seedlings were done 5 months after the embryos have been transferred to the germination medium. results and discussions coconut embryos that preserved in full-strength or half-strength ms basal medium significantly increased in length even up to 6th month of storage. in overall, embryos maintained in half-strength media were significantly longer than embryos in the full-strength media (table 1). previous studies on reducing the mineral salt concentration showed that the cultures in general grow slowly or not at all (ashmore 13 1997). apparently the coconut embryos did not have the same response. no further study has reported on the response of coconut embryos treated in low-salt medium. interestingly, at the end of each storage period, it was noted that the final volumes of the spent half-strength media were lower than those of the full-strength media (data not presented). this supports the view that greater growth (in length) of embryos in the half-strength media could have resulted from greater nutrient and water uptake by embryos in those media. biotropia no. 25, 2005 by contrast, addition of mannitol to the storage medium tended to inhibit elongation of embryos during storage. increasing amounts of mannitol (0.1m to 0.4 m) increasingly reduced the final lengths of the embryos, particularly during the first two months of storage (table 1). beyond 6 months of storage, no significant difference on final length among embryos was noted. table 1. effects of level of nutrients and concentration of mannitol on elongation growth of coconut embryo during storage period of storage (month) treatments 2 4 6 final length of embryo (mm) level of nutrients: full-strength half-strength 9.76b* 10.37a 11.01ns 11.56 11.33ns 11.71 concentration of mannitol (m): 0.0 0.1 0.2 0.3 0.4 10.55 a 10.65 a 10.17 a 9.58 ab 9.38 b 11.85ns 11.83 11.28 11.37 10.12 12.27 ns 12.23 11.50 11.45 10.15 * means followed by the same letter in a column for each factor are not significantly different from each other using lsd at 5% level of significance when the stored coconut embryos are placed in a germination medium such as y3 (eeuwens medium), they would start germinating or may exhibit a slight increase in size but still remain ungerminated or the embryos may take a deteriorative path and eventually dies. table 2 shows that storing coconut embryos for 3 months in the modified ms medium, either at full-strength or half-strength, resulted in significant 10-30% reduction in percent germination. prolonging the storage period to 6 months reduced even further the germination to 10-30% in five of the treatments and to zero (no germination) in the other five treatments. with 9 months of storage all embryos kept in full-strength media become non-germinable, while those kept in half-strength media containing low amounts of mannitol showed 13-20% germination. mannitol when added to half-strength (but not to full strength) 14 table 2. percent germination of stored embryos observed 2 months after transfer to the modified y3 germination medium control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo period of storage (month) level of nutrients mannitol concentration (m) 3 6 9 full-strength 0.0 0.1 0.2 0.3 0.4 80.00 bc* 70.00 c 80.00 bc 90.00 ab 20.00 e 30.00 b 00.00 d 20.00 cb 00.00 d 00.00 d 00.00 c 00.00 c 00.00 c 00.06 c 00.00 c half-strength: 0.0 0.1 0.2 0.3 0.4 100.00 a 80.00 bc 100.00 a 90.00 ab 40.00 d 00.00 d 20.00 cb 30.00 b 10.00 cd 00.00 d 00.00 c 20.00 b 20.00 b 13.33 b 00.00 c unstored embryos 100.00 a 100.00 a 100.00 a * means followed by the same letter in a column are not significantly different from each other using dmrt at 5% level of significance. media up to 0.3m appeared to help preserve the viability of the stored embryos. it is possible that the hypertonic condition created by the addition of mannitol at this level could have helped maintain membrane integrity, thus prevent rapid cell death which otherwise occurs in untreated coconut embryos during prolonged storage. gomez et al. (1995) reported that mannitol significantly prevented ep-catalyzed hydrolysis of 40 and 50 kda polypeptides. ep (endopeptidase) is a monomeric enzyme (gomez et al. 1994) that has an important role in the digestion of cell wall protein. however, addition of high mannitol concentration (0.4m) significantly inhibited the germination of embryos. after the required storage period, embryos were transferred to the y3 germination medium. although high percentage of the stored embryos exhibited the early signs of germination and they were considered germinated (table 2), many of these embryos did not develop into normal seedlings. in fact, some 10-70% of them showed abnormal growth such as produced well-developed roots but without shoots or well-developed shoot but little elongation of roots or stunted seedling even up to the third month after transfer to the y3 medium (table 3 & fig. 1). of the remaining embryos, normal seedlings were obtained from those stored in media without mannitol or with mannitol at 0.1m (fig. 2). the presence of higher levels of mannitol (0.2 and 0.3m) in the storage medium seemed to have inhibited seedling development. seedlings tended to produce shorter shoots whether stored in full-strength or half-strength media. with 0.4m mannitol, development of shoot and root totally failed (table 4). several authors have reported similar inhibitory effect of mannitol on growth of several crops (ashmore 1997; babu et al. 1999; and damasco 2000). 15 table 3. percentage of normal and abnormal seedlings developed from embryos stored for 3 months in full and half-strength media with or without mannitol biotropia no. 25, 2005 abnormal seedlings mannitol concentration (m) total number of embryos cultured un germinated embryos normal seedling no shoot (a) no root (b) stunted (c) total abnormal seedlings (a +b+ c) fullstrength medium: 0.0 0.1 0.2 0.3 0.4 10 10 10 10 10 20 30 20 10 80 80 70 50 30 0.0 0.0 0.0 20 30 20 0.0 0.0 0.0 10 0.0 0.0 0.0 10 20 0.0 0.0 0.0 30 60 20 halfstrength medium: 0.0 0.1 0.2 0.3 0.4 10 10 10 10 10 0 20 0 10 60 80 70 60 20 0.0 0.0 0.0 20 50 30 10 0.0 0.0 0.0 0.0 10 10 20 20 10 20 10 40 70 40 cba figure 1. a. abnormal seedling with stunted growth; b. abnormal seedling with extensive root system but without shoot; c. abnormal seedling with two sword leaves and an enlarged base but without fully formed root 16 table 4. shoot and root development of seedling grown from embryos stored for 3 months in halfand full-strength ms medium with various concentration of mannitol control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo concentrations mannitol (m) shoot length (cm) total no. of leaves/plant average leaf width (cm) average leaf length (cm) length of primary root (cm) no. of adventitious root full-strength medium: 0.0 0.1 0.2 0.3 0.4 half-strength medium: 0.0 0.1 0.2 0.3 0.4 unstored embryos 11.23 ns 10.58 8.05 8.15 0.00# 11.48ns 10.02 10.90 6.80 0.00# 17.66* 2.50ns 2.17 2.00 3.00 0.00# 2.50ns 2.33 2.50 1.00 0.00# 3.00 1.47ns 1.43 1.13 1.20 0.00# 1.40ns 1.50 2.08 0.40 0.00# 3.03** 7.20ns 8.08 6.18 6.92 0.00# 8.33ns 6.93 6.68 2.40 0.00# 10.06 8.93ns 8.10 5.88 6.60 0.00# 6.08ns 5.13 6.25 2.40 0.00# 10.57 2.00ns 1.17 1.25 2.00 0.00# 1.00ns 1.17 1.50 1.00 0.00# 1.20 ns = not significantly different among the treatments in the same column * = significantly different from stored embryos in the same column ** = highly significantly different from stored embryos in the same column # = embryos remained germinated figure 2. normal five-month-old seedlings in full-strength medium with various concentrations of mannitol 17 inhibitory effect of osmotically active agents such as mannitol at high level has appeared during the storing period of embryos (table 1). these embryos, then were not able to germinate even though supported by enough nutrient. mannitol is a metabolically inactive sugar alcohol (dodd et al. 1991). it is one of compatible solutes that can decrease plant water potential by osmotic adjustment (the accumulation of compatible solutes that promotes acclimation to dry) (buchanan et al. 2005). the large amount of mannitol in the cell could result in the increase of the osmotic pressure of the medium drastically. a high osmotic potential in the external medium can induce the protoplast to lose water, causing the cells to become plasmolyzed. if kept in this medium for extended period, it can cause cells to become permanently damaged. study on leaf disks of maize under hyperosmotic condition by del longo (1993) revealed the cellular damage in both the sensitive and the resistant maize line. damage as indicated by, for example, the extension of lipid peroxidation, the destruction of chlorophyll and carotenoids, the decrease in levels of protein sulfhydryl groups and the leakage of electrolytes from cells was apparent in leaf disks as a consequence of the applied stresses (0.5 m mannitol). biotropia no. 25, 2005 it should be mentioned that the stored embryos were not washed off before they were transferred to the germination media. so that the mannitol in the storage media was carried over to the new media. this could have caused the abnormal growth of the seedling. the washing step was not done, however to avoid the removal of important metabolites from possibly already leaky cells as suggested by withers (1986; cited by benson 1990). microscopic observations of the paraffin sections of the embryos showed that high mannitol concentration caused plasmolysis of cells in the region of the apical meristem, which could have led to damage of shoot or root development (fig. 3). evidence from some researches showed that the damage occurred on the hypocotyls figure 3. plasmolyzed cells in 0.3 m mannitol medium 18 or in a critical number of cells of the axis would produce abnormal seedling or the germination would be retarded. thus, the symptoms of slow growth, abnormal growth, or no growth could result from fundamental changes in membranes and macromolecules. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo the results showed that delaying the germination of coconut embryos of the variety ‘tagnanan” was effective for up to 3-month-storage period. after 3 months, the deterioration of embryos occurred very fast. by the end of 9 months, most of the embryos showed significant loss in viability. however, some embryos still showed viability potential after 9 months of storage (indicated by percent germination of about 20-30%). clearly, there is still a need for further studies on the culture media of coconut embryos to increase the viability and germinability of embryos longer than 3 months period of storage. addition of mannitol at a low concentration, lower than 0.2 m, gave a high germination rate (70-100%) and relatively high percentage of normal seedlings (5060%), both in the full-strength and half-strength media free of sucrose. damasco (2000) reported that addition of mannitol at 0.05 to 0.1 m to the medium containing high level of sucrose (60 g/l) induced germination and growth of coconut embryos of the variety ‘tagnanan’ with the same germination rate as the control treatment. it appeared that mannitol was not effective in delaying germination. since the present study was able to delay the germination up to 3 months and maintain high viability, further studies using other substances which could delay germination should be done. sucrose analysis showed that coconut embryo contains very low amount of sucrose, only 42.49 mg per 100 g dry weight. apparently, addition of some amount of sugar in the storage medium may still be important. the study indicated that it is possible to prolong the inactive metabolic phase of coconut embryos before the active germination phase for at least 3 months by controlling the culture medium.technique of delaying the germination may be used as a complementary conservation strategy of coconut germplasm. this technique may have a role in one or more activities in the conservation-use cycle such as in in vitro collecting, in vitro exchange and distribution of coconut germplasm, and in vitro conservation. conclusions the overall results indicate that it is possible to delay the germination of coconut embryo in vitro for up to 3 months without loss in viability. ms medium at half-strength was found to be a better medium for embryo storage than the full-strength medium. addition of mannitol at low concentration (0.2m or lower) did not have any significant effect on storage. however, with 0.3 m or higher concentration of mannitol, the germination fell drastically to 30%. furthermore, high concentration of mannitol induced abnormality in the resulting seedlings (formation of shoot without root, root without shoot, and stunting). 19 acknowledgments biotropia no. 25, 2005 the author would like to express her sincere gratitude to searca and ipgri for the financial support to endure this study. she is also deeply grateful to the director and staff of department of horticulture, uplb, philippines for the assistance and facilities and also to mr. carlos b. carpio, deputy administrator, ardb, pca for the supply of the coconut embryos. references ashmore, s.e. 1997. status report on the development and application of in vitro techniques for the conservation use of plant genetic resources. ipgri. rome. 67p. assy-bah, b. 1986. culture in vitro d’embryos zygotiques de cocotiers. oleagineux 41:321-328. assy-bah, b. and f. engelmann. 1993. medium-term conservation of mature embryos of coconut (cocos nucifera l.). plant, cell, tissue and organ cult. 33:19-24. babu, k.n., s.p. geetha, d. minoo, p.n. ravindran and k.v. peter. 1999. in vitro conservation of cardamom (elettaria cardamomum maton) germplasm. plant genetic resources newsletter. 119:41-45. batugal, p.a and f. engelmann. 1998. coconut embryo in vitro culture. proceedings of the first workshop on embryo culture 27 –31 october 1997 banao, guinobatan, albay, philippines. benson, e.e. 1990. free radical damage in stored plant germplasm. international board for plant genetic resources. rome. buchanan, b.b., w. grussen, and r.l. jones. 2005. biochemistry and moleculer biology of plants. chapter 22: responses to abiotic stresses. aspb (american society of plant biologists). new york. chatty, y. and t. tissouni. 1999. efect of temperature on germination of ornamental palm tees in tunisia. in: ruano,m. c. (ed.). proc. of the 2nd int. symp. on ornamental palms and other monocots from the tropics. acta hortic. 486, ishs. damasco, o.p. 2000. utilization of embryo culture technology for gemplasm conservation: development of medium term conservation for coconut zygotic embryos. paper report of the 2nd inter. coconut embryo culture workshop, merida, 20-23 march, 2000. mexico. del longo, o.t., c.a. gonzalez, g.m. pastori, and v.s. trippi. 1993. antioxidant defences under hyperoxygenic and hyperosmotic conditions in leaves of two lines of maize with differential sensitivity to drought. plant cell physiol. 34 (7): 1023-1028. dodds, j.h., z. huaman and r. lizarraga. 1991. potato germplasm conservation. in: dodds, j.h. (ed.). in vitro methods for conservation of plant genetic resources, chapman and hall. london. p. 93-109 engelmann, f., b. assay-bah, s. bagniol, d. dumet, and n. michaux-ferriere. 1995. cryopreservation of date palm, oil palm, and coconut. in: bajaj, y.p.s. (ed.).biotecnology in agriculture and forestry. springer-verlag, berlin. p. 148-167. 20 gomez, l.d., l.m. casano, m.rouby, m.s. buckeridge, and v.s. trippi. 1994. proteolytic activity associated with cell wall. isolation and partial characterization of a protease from the extracellular fluid of bean hypocotyls. agriscientia. 11:3-11. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo gomez, l.d., l.m. casano, and v.s. trippi. 1995. effect of hydrogen peroxide on degradation of cell wall associated proteins in growing bean hypocotyls. plant cell physiol. 36 (7):1259-1264. ng, n.q. and s.y.c. ng. 1991. reduced-growth storage of germplasm. in: dodds, j.h. (ed.). in vitro methods for conservation of plant genetic resourcess. chapman and hall. london. p. 11-40. 21 introduction observation of embryos microsoft word 1 biotropia no. 19, 2002 : 1 25 fungal population, aflatoxin and free fatty acid contents of peanuts packed in different bag types sonia s.p. bulaong natural sciences research institute, university of the philippines, diliman, quezon city, philippines okky s. dharmaputra seameobiotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract shelled peanuts of gajah var. with initial moisture content of 7% were stored at 11 kg/bag in four bag types namely: jute bag, polypropylene bag, jute bag doubled with thin polyethylene (pe), and jute bag doubled with thick pe. storage was done for six months under warehouse conditions with monitoring of relative humidity and temperature. samples taken at the beginning of storage and every month thereafter were analyzed for moisture content, fungal population, aflatoxin and free fatty acid contents. statistical analyses showed that moisture content, fungal population, and free fatty acid contents were significantly higher in jute and polypropylene bags than in pe-dou,bled jute bags. no significant differences were obtained in aflatoxin contents among bag types but at the end of six months storage, toxin level in jute bag exceeded the 30 ppb limit. polypropylene had second highest toxin level at 23 ppb. the pe-doubled bags had 17 and 19 ppb total aflatoxins for thin and thick films, respectively. the results indicated that the immediate packaging of dried shelled peanuts at safe moisture level in plastic films with water vapor transmission rated of 1 g/m2/24 hr or lower is recommended. this packaging will delay critical increases in moisture content, fungal population, aflatoxin and free fatty acid contents of peanut kernels at ambient storage conditions. keywords: peanuts / bag types / fungal population / aflatoxin content / free fatty acid content. introduction peanuts (arachis hypogaea l.) is a valuable source of protein and fats for human and livestock. in 2000 the production of peanuts in indonesia was 718 000 tons (bps 2001). peanuts are subject to various types of deterioration during storage. one cause of deterioration is fungal growth which makes the material unacceptable to consumers. storage fungi can cause decrease of germination capability, loss in weight, discoloration of kernels, heating and mustiness, chemical and nutritional changes, and mycotoxin contamination (sauer et al. 1992). they can change fat quality of peanuts by hydrolytic enzymes producing free fatty acids and glycerol (pomeranz 1992). altogether, these changes lead to a lower quality or rejection of commodity biotropia no. 19,2002 as foodstuff. three fungal species, namely aspergillusflavus, a. parasiticus, and a. nomius produce aflatoxins as secondary metabolites (pitt and hocking 1997). the toxins are known to be carcinogenic, hepatotoxic and teratogenic in test animals. based on the report of the 23rd session of the joint fao/who food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commission adopted the maximum level of 15 ppb for total aflatoxins in peanuts intended for further processing. the problem of fungal growth and aflatoxin contamination of foodstuffs remains, especially in developing countries where handling and storage technologies are still being developed. pitt and hocking (1996) reported that aflatoxin exceeding 50 ppb contaminated 45, 22 and 25% of 215, 81 and 94 peanut samples collected from retailers in 1990/1991 in indonesia, philippines and thailand, respectively. in line with this study lubulwa and davis (1994) reported that the total annual cost of aflatoxin contamination in indonesia, philippines and thailand was about $a 158 million. indonesia incurred 84% (= $a 132 million) of this cost, thailand incurred 13% (= $a 21 million) and philippines 3% (= $a 5 million) of the cost. moisture content is the most important factor affecting fungal growth in stored products. peanuts are stable at 70% relative humidity between 7 9% moisture content, at which conditions fungal growth is arrested (icar 1987). when the product absorbs moisture from the environment or when the relative humidity exceeds the equilibrium rh of the kernels, fungal growth occurs. for storage, therefore, the packaging material used should have a water vapor transmission rate (wvtr) low enough to minimize moisture absorption from the environment. in commercial practice, 2 types of bags are commonly used to store peanuts, i.e. jute and polypropylene bags. jute bags easily absorb moisture but allow good airflow. polypropylene is non-absorptive but tends to trap heat inside (kennedy and devereau 1994). the ability of polyethylene (pe) bags, singly or in combination with polypropylene to inhibit fungal growth and aflatoxin production in wet corn for two weeks has been reported (siriacha et al. 1990). peanut kernels packed in pe bags then stored in jute bags were found to remain viable up to 7 months of storage (reddye/fl/. 1992). the objectives of this study were to determine the effects of different bag types on fungal population, aflatoxin and free fatty acid contents of stored peanuts together with change in moisture content. the effects of storage duration on the changes in the parameters mentioned above were also determined. materials and methods methods of harvesting, drying and shelling of peanut the peanut used in this study was the "gajah" variety, a cross between schwarz and spanish lines. the plants were cultivated in the citayam farm of the research institute for food crop biotechnology, bogor, indonesia. it was harvested fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra 100 days after sowing. the pods were stripped from plants and sundried to moisture content of about 8%. pods were shelled manually to minimize damage to kernels. before storage, shelled peanuts were fumigated with phosphine for 5 days at 2 grams/ton to control insect pests that may exist. prior to bagging, damaged or moldy kernels were hand picked from the batch. storage of peanut shelled peanuts with initial moisture content of about 7% were packed at 11 kg/bag in 0.42 m x 0.47 m bags. the bag types used were (1) jute bag (jb), (2) polypropylene bag (pb), (3) jute bag doubled with thin polyethylene (pe) bag (jb+1), and (4) jute bag doubled with thick pe bag (jb+2). the physical properties of the pe bags are given in table 1. each bag type (treatment) was replicated 3 times (4 bags per replication or stack). each stack was arranged randomly and placed upright on wooden pallets. the bags were stored under warehouse conditions for 6 months. ambient temperature and relative humidity were recorded using a wilh. lambercht thermo-hydgrograph type 252. sampling method samples were taken from each bag at the beginning of storage and subsequently every month thereafter until 6 months storage. initial samples were taken from 3 points (top, middle and bottom) in each bag using a sampling spear. every sampling, the samples were taken from the same points. about 250 grams samples from each bag in a stack were mixed homogeneously to get a 1000 gram primary sample. the primary sample was divided twice using a sample divider to obtain working samples for moisture content, fungal population, aflatoxin and free fatty acid contents. a 500 gram portion was set aside as reserved sample. samples for fungal population were refrigerated until analysis. samples for aflatoxin and free fatty acids tests were frozen until analysis. moisture content was carried out on the day of sampling. samples were analysed in duplicates. biotropia no. 19,2002 moisture content analysis moisture content of kernels was determined using oven method (bsi 1980), 2 replicates per sample. the kernels were ground and dried in an oven at 130°c for 2 hours. the moisture content was calculated using the following formula: determination of fungal population the dilution method followed with pour-plating using dichloran 18% glycerol agar (dg18) (pitt and hocking 1997) was adopted. a 25 gram sample was ground for 2 minutes and mixed with 225 ml sterile distilled water to make an initial dilution of 10"1. this initial suspension was shaken manually for 2 minutes, then settled for 30 seconds before aliquots were taken from the surface. dilutions from 10"2 and higher were shaken for 1 minute before sampling. platings were done from 10"' to 10"4 at the beginning and after 1 month of storage. later samplings were plated up to 10"5 dilution. four replicate plates were used per dilution. plates were incubated at ambient temperature for 6 days. populations of individual species were determined using the following formula: fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra aflatoxin analysis the thin layer chromatography (tlc) method of blaney et al. (1984) was used for aflatoxin analysis. a 10 gram portion of ground peanut was mixed with 50 ml acetonitrile, 5 ml 4% kcl and 1 ml 5% hcl. the mixture was stirred for 45 minutes over a magnetic stirrer. the mixture was filtered and defatted twice with 25 ml n-hexane in a separator/ funnel. the acetonitrile phase was extracted with two (25 ml) portions of dichloromethane. water was removed by filtering through 2 gm anhydrous sodium sulphate into a round-bottom flask. dichloromethane extract was evaporated to dryness on a rotary evaporator. the residue was dissolved in 0.5 ml chloroform and spotted on tlc plate at 20 uj per spot. a series of standard solutions of aflatoxin bj, b2, gi and g2 were spotted on the same plate for comparison. the plates were developed in a chamber containing chloroform: acetone (9:1). plates were air-dried and viewed under uv 366 nm. aflatoxin concentration was calculated using the following formula (bainton et al. 1980): free fatty acid analysis the titration method of joslyn (1970) for peanut oil was adopted. peanut oil was extracted from a 30 gram sample using a soxhlet apparatus. a 10 gram portion of oil was dissolved in hot 100 ml neutralized ethanol. the solution was titrated using 0.1 n naoh solution with phenolpthalein indicator. free fatty acid was calculated as oleic acid and reported as fatty acid value (fav) or mg kom/g sample. statistical analysis the data were analyzed using a completely randomized factorial design with two factors. the first and second factors were bag type and duration of storage, respectively. biotropia no. 19,2002 results and discussion the effect of bag type and duration of storage on moisture content based on statistical analysis, type of bags, duration of storage and their interaction gave very significant differences on moisture content of peanut kernels (table 2). at 95% confidence level, the moisture contents (m.c.) in both polypropylene bag (pb) and jute bag (jb) were significantly higher than m.c. in jute bag doubled with thin pe (jb+1) and jute bag doubled with thick pe (jb+2) (table 3). the m.c. of jb and pb were not significantly different but significant difference exists between m.c. of jb+1 and jb+2. the m.c. of peanut kernels packed in jb, pb, (jb+1) and (jb+2) were 8.23, 8.26, 7.55 and 7.49%, respectively. the lack of significant difference between m.c. in jb and pb suggests that moisture transfers through both types of packaging were at similar rates. as expected, jb+2 had lower m.c. than jb+1 due to its lower wvtr. the difference between m.c. in plastic-doubled bags was only 0.06%. this slight yet significant difference might be due to the condition of the bags during storage. since sampling was done by spear method, 3 points on one side of each bag were punctured at the start of storage. these openings were covered with a plastic tape which shrank with time and did not completely sealed the holes. at these points, the kernels were exposed to the ambient. under this condition, jb+1 and jb+2 bags increased to 1.04% and 0.87% m.c., respectively during 6 months of storage (table 3). the critical m.c. for peanuts at which a. flavus grows and produces aflatoxin is at 9 -10% corresponding to water activity (aw) of 0.85 (who 1979). based on these results, jb+2 is the better packaging material since it allowed less moisture increase from the initial 7% m.c. of the kernels. bulaong (1998) reported that at 25°c the sorption isotherm of peanut kernels var. gajah approximates the isotherm of other fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra peanut varieties. at this temperature and erh of 85%, the kernels of peanuts var. gajah have an equilibrium moisture content (emc) of about 12.5%. the m.c. in the different bags changed significantly with duration of storage (table 3). from the start of storage until month 2 the m.c. were relatively constant. significant increases were observed from month 3 until month 6. the slight insignificant decrease in m.c. during months 1 and 2 could be due to the low rh -high temperature in the warehouse during the dry season (appendix 1). the vapor pressure (v.p.) of the kernels might have been higher than the v.p. of the surrounding air inside the warehouse. upon the onset of the wet season in month 3, significant increases in m.c. were observed. the respective means of m.c. for months 0, 1, 2, 3, 4, 5 and 6 were 7.20, 7.17, 7.12, 7.60, 8.42, 8.74 and 8.97%, respectively. biotropia no. 19,2002 significant differences in the m.c. for bag type-duration of storage interactions were observed mostly at month 3. for jb, the first significant increase was observed at month 4 at 9.26%. in jb, the succeeding 2 months still had significant increases at 9.58% and 9.97%. in pb, the first significant increase in m.c. was at month 3 at 7.77%. this was followed with significant increases in months 4, 5 and 6 with 9.15, 9.51 and 9.70% m.c., respectively. for jb+1, there was a significant decrease at month 2 from the initial 7.19% m.c. in the other bag types, the decreases in month 2 were insignificant. in pb and jb which are both porous, the kernels might be equilibrating with the .ambient at such a fast rate that moisture content appeared relatively stable. in the case of jb+1, the film puts a barrier to moisture migration such that after the kernels have lost moisture to low rh environment over a period of time, the regain of moisture proceeds of a slower rate than in the porous bags. the m.c. in jb+1 for months 3 and 4 were not different at 7.71%. significantly different increases occurred in months 5 and 6 with 8.0 and 8.23% respectively. in the case of jb+2, it appeared that this was the only system where m.c. did not decrease at month 1. moisture migration into jb+2 appeared to be slow but consistent. means from month 0 to month 2 were not significantly different. a significant increase occurred at month 3. the increase in month 4 was not significantly different from month 3. moisture increases in months 5 and 6 were significantly different from month 4. 8 fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra at month 6, the final m.c. in the bags were significantly different with jb > pb > jb+1 > jb+2, they were 9.97, 9.70, 8.23 and 7.98%, respectively (table 3). the results suggested that moisture migration through jb was faster than at pb. this may be due to the difference in pore size and permeability of the 2 bags. pb is made of synthetic material less absorbent than jute fiber. as expected, jb+1 allowed more moisture migration than jb+2 because of its higher wvtr. of all the bag types, jb+2 showed consistent but slow increase in m.c. during storage. moisture increases in jb and pb were drastic at the times rh was high. moisture content in jb+1 during these months of high rh also significantly increased at 8 and 8.23% for months 5 and 6, respectively. at the same period, jb+2 did not have significant moisture increase. with the final moisture content of 9.97%, jute bag was already within the critical moisture limit of 9 10% for peanuts. jb+2 with final 7.98% was still in save moisture level for storage up to six months. the changes in m.c. during storage in the four treatments are illustrated in figure 1. the effect of bag type and duration of storage on fungal population bag types, duration of storage and their interaction produced very significant differences on fungal population of stored peanuts (table 2). at 95% confidence level, the fungal populations during 6 months of storage were in the order of jb > pb > jb+2 > jb+1 (table 4). biotropia no. 19.2002 there was a significant decrease in fungal count at month 1. this coincided with a slight insignificant decrease in moisture content at the same month (table 3). at months 2 and 3, the fungal population increased significantly from month 1. the means between months 2 and 3 were not significantly different although at month 3, the moisture content significantly increased. at month 4 was another significant decrease in the population. this occurred despite the significant increase in the moisture content at 8.42% (table 3). at months 5 and 6 significant increases in fungal counts coincided with ncreases in moisture content. fungal counts were 32 535 and 94 856 cfu/g (db), respectively. the fungi have been described to be sensitive detectors of moisture changes in stored grains. their sensitivity was described to be comparable to sophisticated equipment (sauer et al. 1992). the decrease in fungal count at month 1 may be attributed to the significant decrease of 0.03% moisture content (table 3). with a further (insignificant) decrease of 0.03% moisture content for month 2, there was however a significant increase in the fungal population (table 4). this was caused by the growth of penicillium funiculosum which was the dominant species in all bag types at the beginning of storage (table 5). penicillium funiculosum was common in peanuts both before and after harvest (joffe 1969). the occurrence of p. funiculosum in peanuts before harvest indicated that this fungus is also a field fungus. dharmaputra and putri (1997) found that p. funiculosum was a dominant species in peanuts with different percentages of splitted kernels (0, 25, 50, 75 and 100%) stored for 2 months under laboratory conditions. its population was between 0.7x10 1.2xl03 cfu/g (wb). pitt et al. (1998) reported that p. funiculosum were found in 4% of 256 retail samples examined and 9% of all kernels examined (50 kernels/sample) in bogor and yogyakarta, indonesia.     fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra it was assumed that the dominance of p. funiculosum was due to a little change in moisture content. according to sauer et al. (1992) some fungal species were observed to become dominant with as little as 0.2% change in moisture content. the significant decrease in the fungal population at month 4 may be attributed to a physiological change in the fungi. although there was a significant increase in moisture content at this time, the population decreased. it has been suggested that vegetative growth and secondary metabolism are two competitive processes in the life cycle of an organism (garraway and evans 1984). the onset of secondary metabolism, such as aflatoxin synthesis, requires additional physiological resources like moisture, enzymes and nutrients. hence, it competes with vegetative growth. this may account for the decrease in fungal count despite moisture increase. secondary metabolism is triggered by accumulation of primary metabolic intermediates. these intermediates could have started in the kernels from the start of fungal colonization during preor post-harvest of peanuts until month 3 of storage. secondary metabolism is viewed by some as a way for microorganisms to remove excess primary metabolites intermediates so that the primary processes, like growth, may continue in times of environmental stress (garraway and evans 1984). aflatoxins were first detected at month 4 when fungal count was lowest and the moisture content had significantly increased to 8.42%. aspergillus flavus constitutes a minor percentage of the total population, its range was from 0.001 -1.45% of population during month 4 to month 6 (table 5). it was possible that secondary metabolism occurred as well in the other fungal species. significant differences were observed in the bag type-duration of storage interactions. for jb, pb and jb+1, fungal counts were significantly different from month 0 to month 6. only at jb+2 during months 2 and 3 the fungal counts were not significantly different (table 4), despite the significantly higher moisture content at month 3 in jb+2 (table 3). it could be explained in the light of competition for moisture during secondary metabolism as discussed in earlier part. the changes in fungal population in the treatments during storage are illustrated in figure 2. for all bag types, the significant increases in fungal count was attributed to 2 fungal species, i.e. penicillium funiculosum and aspergillus penicillioides. in jb, the dominant fungus was p. funiculosum from month 0 to month 4. at month 5 and 6, the dominant species was a. penicillioides (table 5). for pb, the dominant species from month 0 to month 4 was p. funiculosum, while a. penicillioides became dominant in pb from month 4 to month 6. eurotium amstelodami was the second dominant species from month 5 to 6 in jb and pb. in jb+1 p. funiculosum was dominant from month 0 until month 6. eurotium chevalieri was the second dominant species at 1.70 and 17.6% of population for months 5 and 6, respectively. in jb+2, p. funiculosum was dominant from month 0 to month 6. eurotium chevalieri contributed 2.08 and 0.75% to total population at months 5 and 6 respectively. the changes in the fungal population in each bag type are presented in figures 3 and 4. 13   fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra in the complex system where several fungal species exist together, the dominance of a species may not be solely due to its ability to tolerate existing moisture conditions. several types of interactions exist among fungi. aside from competition for space, moisture and nutrients, there could be inhibitory interactions as well due to release of metabolites of derailing of biochemical path ways (choudhary 1992). the sudden succession between p. funiculosum and a. penicillioides in jb and pb during the latter months of storage may be due to a combination of these interactions. a possible reason for the continued dominance of p. funiculosum in pe-doubled bags may be its ability to compete with other species when both moisture and o2 are less than normal. aspergillus penicillioides was observed in conditions of higher moisture content and o2 in the porous bags. the effect of bag type and duration of storage on aflatoxin content of peanuts two kinds of aflatoxins were detected, i.e. aflatoxin bi and b2. based on the analyses of variance there were no significant differences in total aflatoxin content among bag types and the interaction between bag types and duration of storage (table 2). the means for jb, pb, jb+1 and jb +2 were 13.1, 8.9, 8.7 and 9.2 ppb, respectively (table 6, figure 5). 15 biotropia no.19,2002 aflatoxins were first detected at month 4 at concentration of 2.73 ppb. there was a significant increase to 4.69 ppb at month 5. at month 6, the increase to 22.58 ppb was highly significant (table 6, figure 5). at month 4, jb and pb had higher aflatoxin levels than jb+1 and jb +2. the levels were 4.6, 3.6, 2.7 and 0 ppb, respectively (table 6). at month 5, jb+2 and jb+1 had higher levels than jb and pb. the levels were 8.7, 6.7, 3.3 and 0 ppb, respectively. at month 6, jb had higher levels than pb, jb+2 and jb+1. the values were 31.5, 23.1, 18.9 and 16.8 ppb, respectively. aflatoxins were not detected in the samples until month 3 of storage. it was assumed that aflatoxigenic a. flavus population in the soil of peanut planting field was low. the moisture content of the samples remained relatively stable from month 0 to month 2 at values 7.12 7.19% (table 3). this moisture content corresponds to water activity of 0.65, insufficient for aflatoxin production. other works have reported a similar absence of aflatoxins in peanuts at low moisture content conditions. at 6.9% m.c., aflatoxin in shelled spanish peanuts was not detected after 8 months stored at cold storage (wilson and jay 1976). significant increases in moisture content started from month 3. at month 4, aflatoxins were detected in 3 bag types in small amounts. the moisture contents at month 4 for jb and pb were 9.26 and 0.15%, respectively, within the 9 10% range, of lower limit for aflatoxin production in peanuts (who 1979). jb+1 at month 4 had only 7.7% moisture content yet it was positive for aflatoxin production. jb+2 also had a moisture content less than 8% during storage, and it was positive for aflatoxins (tables 3 and 6). it appears that aflatoxin synthesis could occur at moisture levels lower than normal range for a. flavus growth. 17 biotropia no. 19,2002 aspergillus flavus count did not correlate with aflatoxin production (table 7). one reason is that only a minor portion of the total fungal population is responsible for the toxin production. aflatoxin production depends among others on the strain of a. flavus (kozakiewicz 1996; pitt and hocking 1997). an interesting correlation may exist between free fatty acid contents and aflatoxins. glycerol was reported to be an inducer of bbh b2 + g2 production by a. parasiticus (abdollahi and buchanan 1981). it induced higher levels of aflatoxin than glucose, galactose, fructose and xylose at controlled conditions. sufficient levels of glycerol might have been produced in stored peanuts during hydrolysis of triglycerides into free fatty acids and glycerol. it follows that in samples with high levels of free fatty acids, glycerol would also be high and so would aflatoxin level. it also follows that after some aflatoxin synthesis, glycerol level (and free fatty acid) would be lower. such a correlation may explain the decrease in free fatty acid contents during months 5 and 6 when aflatoxin synthesis increased (tables 6 and 8). the higher increase in free fatty acid contents in jb and pb from month 0 to month 3 compared with jb+1 and jb+2 could also result to the observed differences in aflatoxin levels of the bag types (table 6). the role of seed lipases in free fatty acid production is discussed in the next section. the major role   biotropia no. 19,2002 of lipases in the kernels in lipid hydrolysis may offer explanation why aflatoxin occurred in jb+1 and jb+2 at moisture levels lower than the minimum level reported for a. flavus growth. the means of free fatty acids and final aflatoxins levels in each bag are illustrated in figure 6. fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra the effect of bag type and duration of storage on free fatty acid contents bag types, duration of storage and their interactions gave very significant differences on free fatty acid (ffa) contents of stored peanuts (table 2). at 95% confidence level, the ffa contents in the 4 bags were significantly different. jb and pb had insignificantly different means at 0.51 and 0.52 fav, respectively. jb+1 and jb+2 had significantly different means at 0.47 and 0.43 fav, respectively (table 8). significant differences in ffa contents were observed during storage. the lowest level was at month 0 at 0.32 fav. a significant increase to 0.45 fav was observed at month 1. this was relatively unchanged until month 2 (table 8). significant increases were again observed in months 3 and 4. these were followed with significant decreases in months 5 and 6. the values for months 0, 1,2, 3, 4, 5 and 6 were 0.32, 0.45, 0.42, 0.54, 0.57, 0.56 and 0.50 fav, respectively. interactions between bag type and duration of storage resulted to significantly different ffa contents. at month 0, the 3 bag types contained kernels with approximately same ffa contents at 0.33 0.34 fav. only pb had a significantly lower level at 0.28 fav (table 8). at month 1, pb had the highest increase in ffa with 0.20 fav increase from month 0. jb had the second highest increase at month 1 with 0.18 fav increase from month 0. jb+1 had 0.12 unit increase while jb+2 had only 0.04 fav increase. the highest increase in ffa contents in jb, pb and jb+1 occurred at month 1. this was followed with a significant decrease at month 2 for the same bag types. jb+2 showed a significant increase from month 1 to month 2. from month 3 to month 4, significant increases were observed in jb and jb+1. in jb+2, the means for months 3 and 4 were not significantly different. in pb, there was a decrease in ffa at month 4. significant decreases in ffa contents were observed in jb from month 4 to months 5 and 6. in jb+1, the decrease was observed from month 5 to month 6. in jb+2, the decrease was from month 4 to month 5 (table 8). free fatty acids are released from the hydrolysis of triglycerides. the rapid formation of ffa during month 1 of storage for jb, pb and jb+1 was due to the activity of lipases in the kernels (acker 1969; pomeranz 1992). these upases are known to be active even at low water activity or moisture content levels. at month 1, moisture content was generally lower than at month 0 and the fungal population was significantly lower than month 0 (tables 3 and 4). this is another proof that the lipase activity must be largely due to seed enzymes rather than fungal enzymes at the low moisture level. at month 2, there were significant decreases in ffa contents in jb, pb and jb+1. only jb+2 has a significant increase to 0.43 mg koh/g sample (table 8). jb+2 was the only bag system where ffa contents were more consistently increasing with duration of storage. the fluctuations in month 2 in the 3 other bag types could be attributed only to the higher permeability to o2 and moisture. during months 3 and 4, increases in ffa in jb, pb and jb+1 were significant, but not in jb+2. at month 5, significant decreases were observed in jb 21 biotropia no. 19,2002 and jb+2. pb at month 5 regained significantly higher ffa contents compared with month 4 when it lost ffa. jb+1 at month 5 still had an increasing ffa content. at month 6, a significant drop was observed in both jb and jb+1. pb had a significant increase at month 6 to 0.80 fav. jb+2 had an insignificant increase at month 6 to 0.40 fav. the possible correlation between free fatty acids and aflatoxin synthesis was discussed in the previous section. essentially, analysis in this study indicates that high free fatty acid contents correspond to high glycerol levels since both are byproducts of lipid hydrolysis. since glycerol induces aflatoxin synthesis, a bag system with high free fatty acid may also produce high levels of aflatoxin. this appears to be true in the case of jb and pb which had the highest initial increases in ffa content at month 1. table 6 shows that at month 6, peanuts packed in jb had the highest aflatoxin content at 31.5 ppb. pb had the second highest content at 23.1 ppb. jb+1 and jb+2 with less increase in ffa at month 1 had 16.8 and 18.9 ppb of aflatoxin, respectively. it appears from the analysis of data that a packaging relatively impermeable to o2 and moisture would allow slower lipid hydrolysis than porous materials. in this respect, it may be concluded that immediate packing of peanuts in protective films may be a way to slow down aflatoxin production in peanuts. conclusions the use of polyethylene lining in jute bags effected lower moisture gains than either jute or polypropylene bag alone in peanut storage. in the order of decreasing moisture gains pb > jb > jb+1 > jb+2. the dominant fungal species found in peanuts during storage were aspergillus penicillioides and penicilliumfuniculosum. bag type and duration of storage caused significant differences in fungal populations both in quantity and quality. in the order of decreasing fungal populations, jb > pb > jb+2 > jb+1. fungal populations generally increased with duration of storage, except at the beginning of secondary metabolism (aflatoxin synthesis). level of free fatty acids changed significantly according to bag type and duration of storage. in the order of decreasing free fatty acid, pb > jb > jb+1 > jb+2. aflatoxin level was not significantly affected by bag type. in the order of decreasing total aflatoxin, jb > jb+2 > pb > jb+1. aflatoxin level changed significantly with duration of storage. in the order of decreasing aflatoxin level, month 6 > month 5 > month 4. aflatoxin level was formed at water activity and moisture content lower than previously reported. aflatoxin level was apparently related to free fatty acid level. 22 fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra recommendation shelled peanuts with moisture content of 7% should be immediately packed in polyethylene bags with water vapor transmission rate of 1 gm/m2/24 hr or lower in order to preserve its quality. such packaging will delay moisture increase, minimize fungal growth, aflatoxin production and free fatty acid formation. acknowledgements the authors gratefully acknowledge the financial support of the canadian international development agency (cida) through seameo biotrop. we also thank the other scientists and technicians of the laboratory of pest and disease management, and laboratory of chemistry, seameo biotrop who have in one way or another contributed to this research; and dr. gloria l. enriquez, former deputy director for programme and marketing of seameo biotrop for her suggestions and encouragement. references abdollahi, a. and r.l. buchanan. 1981. regulation of aflatoxin biosynthesis. induction of allatoxin production by various carbohydrates. j food sci. 46: 633-635. acker, l.w. 1969. water activity and enzyme activity. food tech. 23: 27-40. bainton, s.j., r.d. coker, b.d. jones, e.m. morley, n.j. nagler and r.l. turner. 1980. mycoioxin training manual. tropical products institute, london. blaney, d.j., c.j. moore and a.l. tyler. 1984. mycotoxins and fungal damage in maize harvested during 1982 in far north queensland. austr. j. agric. res. 35: 463-471. bps. 2001. statistik indonesia 2000. badan pusat statistik, jakarta. in indonesian. bsi. 1980. methods of test for cereals and pulses. part 3. determination of moisture content of cereal.-, and cereal products (routine methods). british standards institution. isbn 0580 11 4333. bulaong, s.s.p. 1998. sorption isotherm of peanut kernels var. gajah. seameo biotroi'. unpublished. choudhary, a.k. 1992. influence of microbial co-inhibitors on aflatoxin synthesis of aspergillus flavim on maize kernels. letters appl. microb. 14: 143-147. dharmaputra, o.s. and a.s.r. putri. 1997. the relation between splitted kernels and population of storage fungi in peanuts. seameo biotrop. unpublished. fardiaz, s. 1989. food microbiology. inter university center for food and nutrition. bogor agricultural university, indonesia, p. 268. garravvay, m.o. and r.c. evans. 1984. fungal nutrition and physiology. john wiley and sons, new york. 23 biotropiano. 19,2002 icar. 1987. aflatoxin in groundnut-technologies for better crops. publications and information division, indian council of agricultural research, new delhi, india. joffe, a.z. 1969. the mycoflora of fresh and stored groundnut kernels in israel. oleagineux 27: 489-491. joslyn, m.a. 1970. acidimetry. in: joslyn, m.a. (ed.). pp. 401-446. methods in food analysis; physical, chemical and instrumental methods of analysis. academic press, new york. kennedy, l. and a. devereau. 1994. observations on large-scale outdoor maize storage in jute and woven polypropylene sacks in zimbabwe. in: highley, e., e.j. wright, h.j. banks and b.r. champ (eds.). pp. 290295. proceedings of the 6th international working conference on stored-product protection. canberra, australia, 1723 april 1994. kozakiewicz, z. 1996. occurrence and significance of storage fungi and associated mycotoxins in rice and cereal grains. in: highley, e. and g.i. johnson (eds.). pp. 18-26. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17th asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. lubulwa, g. and j. davis. 1994. estimating the social costs of the impacts of fungi and aflatoxins in maize and peanuts. in: highley, e., e.j. wright, h.j. banks and b.r. champ (eds.). pp. 1017-1042. proceedings of the 6th international working conference on stored-product protection. canberra, australia, 17-23 april 1994. lubulwa, g. and j. davis. 1996. completed-project economic assessment of two aciar projects on fungi and aflatoxins: a discussion of methodology issues and some estimates of potential benefits. in: highley, e. and g.i. johnson (eds.). pp. 66-98. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17lh asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxin assessment of food commodities in south east asia. in: highley, e. and g.i. johnson (eds.). pp. 5-10. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17"' asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic & professional, london. pitt, j.i., a.d. hocking, b.f. miscamble, o.s. dharmaputra, k.r. kuswanto, e.s. rahayu and sardjono. 1998. the mycoflora of food commodities from indonesia. journal of food mycology 1(1): 41-60. pomeranz, y. 1992. biochemical, functional and nutritional changes during storage. in: sauer, d.b. (ed.). storage of cereal grains and their products. amer. assoc. cereal chemists, inc. st. paul, minnesota, pp. 55-141. reddy, p.s., m.s. basu, m.a. khaleque, m.s. haque, a. ali, h. malek, h. than, t. soe, b. regunathan, b. mishra, t. murthy and s. nigam. 1992. status of groundnut research and production in south asia. in: nigam, s.n. (ed.). groundnuta global perspective. proceedings of an int'l. workshop 25-29 november 1991. icrisat center, india. icrisat. sauer, d.b., r.a. meronuck and c.m. christensen. 1992. microflora. in: sauer, d.b. (ed.). storage of cereal grains and their products. amer. assoc. cereal chemists, inc. st. paul, minnesota, pp. 313-340. siriacha, p., k. kawashima, m. saito, p. tan-boon-ek and d. buangsuwon. 1990. prevention of thai maize from the infection by aspergillus flavus and aflatoxin contamination in various packages. proc. jpn. assoc. mycotoxicology 32: 41-46. wilson, d. and e. jay. 1976. effect of controlled atmosphere storage of aflatoxin production in high moisture peanuts (groundnuts). j. stored prod. res. 12: 97-100. who. 1979. mycotoxins. world health organization, geneva. 24 appendix 1. average temperature and relative humidity in warehouse during storage of peanuts month temperature (°c) relative humidity (%) july august september october november december january 25.6 26.9 27.4 27.2 26.7 27.3 68.5 66.7 70.4 75.5 78.7 76.9 25 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf biotropia no. 5, 1991/1992: 26-40 effects of carbofuran on the reproductive capacity of a freshwater snail, radix quadrasi, under laboratory conditions imelda f. pagulayan, gloria l. enriquez and virginia s. caring college of science, university of the philippines diliman, 1001 quezon city, philippines abstract the effects of 4 sublethal concentrations of carbofuran (250, 500, 1000 and 2000 ppm) on the reproductive capacity of r. quadrasi was determined. results showed that incubation period is delayed and inhibited by 1000 and 2000 ppm carbofuran but not by lower concentrations. the hatching period is longer in treated snails and not all eggs hatch in the 1000 and 2000 ppm treatment. the percentage of hatching is inversely proportional to the carbofuran concentration. oviposition was delayed in all the treated stages and at all dosages. the higher the carbofuran concentration, the later the onset of oviposition. the reproductive period is shortened. fecundity was decreased in snails treated at emb and sm. however, only the 2000 ppm carbofuran concentration showed an adverse effect on the snails exposure at psm. introduction pesticides are continuously finding widespread use and increasing amounts are being introduced into the aquatic environment. researchers worry that this increasing use of pesticides and the farming industry's dependence on it may eventually lead to health problems for both consumers of farm products and farm workers (allmon 1985). moreover, this will pose a potential hazard not only to human beings but also to aquatic organisms of economic importance (mane et al. 1979). invertebrates constitute a very large portion of the fauna in both the aquatic and terrestrial system. from an ecological viewpoint, these animals play an important role in the energy flow in these ecosystems. the feeding habit of invertebrate animals varies from detritus feeding to herbivorous and carnivorous food consumption. disruptions caused by pesticide stress in one or more organisms may directly interfere with other inter-dependent components of the food chain (gunther et al. 1968). carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-n-methylcarbamate) developed by the niagara chemical division of fmc corporation is commercially known as furadan. it is a systemic insecticide, nematicide with broad spectrum activity (metcalf et al. 1968; yu et al. 1974; cremlyn 1980; handa 1980) although hydro 26 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt lyzed rapidly in water. it is an effective contact toxicant and is non-corrosive and compatible with other pesticides (rangaswany et al. 1976). carbamate insecticide is a popular choice since it is less toxic, less persistent and has lower residual effects in animals than other classes of pesticides. nevertheless, it is accumulated to a certain extent. this work aims to determine the levels of carbofuran concentrations that are toxic or deleterious to the embryonic (emb), presexually mature (psm), and sexually mature (sm) r. quadrasi; to determine the effects of sublethal concentrations of carbofuran on the fecundity and reproductive period of the snail; and to determine the particular age group that is most sensitive to carbofuran. materials and methods organism radix quadrasi, (fig. 1) the snail used in this study, were obtained from stock cultures started in 1981 and have been maintained and reared continuously in the snail room of the natural sciences research institute, university of the philippines, diliman, quezon city. 27 figure 1. radix quadrasi biotropia no. 5, 1991/1992 laboratory maintenance and culture of snails, maintenance of eggs and young snails followed that of de lara and enriquez (1981) and pagulayan and darvin (1986). preparation of working solutions carbofuran in the form of 3g was prepared by dissolving the granules first in acetone before adding distilled water. the solution was aerated overnight before use. concentration parts in per million (ppm) were used. all glasswares and plastic containers were acid washed and rinsed with de-ionized water. petri dishes were oven-dried before use. experimental set-up thirty egg masses were used as the source of snails that were observed for fecundity and reproductive period. ten of these egg masses were allowed to hatch under normal conditions (control). the other experimental groups consisting of 10 egg masses each were treated with 250 ppm and 500 ppm carbofuran for a period of one week in separate petri dishes. two-week treatment durations and 1000 and 2000 ppm concentrations were not used because preliminary work showed that survival of experimental snails would be less than 50%. a. treatment at the larval stage two weeks after the eggs hatched into young snails, 48 snails were taken at random from the untreated egg masses and were placed in groups of four in 12 separate basins. the same was done for egg masses treated with 250 and 500 ppm. the average size and age of the snails at first and last oviposition were recorded. the total number of egg masses laid per group were recorded weekly in the entire length of the reproductive period. the length of the reproductive or egg laying period was recorded. b. treatment at week four (psm) forty five newly hatched snails from egg masses under normal laboratory conditions were separated at the age of two weeks. they were placed in 9 separate basins in groups of five. the above procedure was followed. however, the snails were exposed to the four carbofuran concentrations applied at the end of the 4th week to the end of the 6th week after hatching. 28 effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. c. treatment at week seven (sm) the same procedure was followed except that the snails were treated at the end of the 7th week to the end of the 9th week after hatching. in the two-week duration of treatment, the medium was changed after the first week. possible toxic material in the medium like the feces were removed regularly. results effects of fecundity fecundity is the total number of the eggs produced by the individual during its reproductive period. reproductive period, on the other hand, is defined as the number of days from the first to the last day of egg-laying. a. treatment at embryonic stage (emb) table 1 shows the size range and average at the onset and end of oviposition, the reproductive capacity and the reproductive period of r. quadrasi that were hatched from untreated embryos and embryos exposed to 250 and 500 ppm concentrations of carbofuran. the average size length: 10.62, 10.56 and 10.45 mm for the control, 250 ppm and 500 ppm treatment respectively, are not significantly different from each other. however, the age range and average in days of the treated snails differ greatly from the untreated. the earliest oviposition took place in the control at day 29, although the average age was 39 days. the earliest oviposition for both treatments was 43 days with an average age of 49.5 days. the treated snails oviposited at a much older age but the size of the snails were almost the same as that of the untreated ones. the average size length at last oviposition differ between the control and the treated snails; for the control it took place at an older age range with an average of 224 days. oviposition stopped at an average age of 196.5 days for the 250 ppm treatments; and 193 days as average age for the 500 ppm treatment. there is no significant difference in the size and average for the two treatments. the number of egg masses, number of eggs (table 1 and fig. 2) and average number of eggs per egg mass are highest in the control followed by the 250 ppm and lowest in the 500 ppm treatment. the reproductive period was shorter in the treated snails. 29 biotropia no. 5, 1991/1992 30 t a b le 1 . s iz e a n d a g e a t th e o n se t a n d e n d o f o v ip o si ti o n , re p ro d u c ti v e p e ri o d o f r . q u a d ra si t h a t w e re h a tc h e d f ro m e m b ry o s e x p o se d t o tw o c o n c e n tr a ti o n s o f c a rb o fu ra n efects of carbofuran on the reproductive capacity – imelda f. pagulayan et al. 31 f ig u re 2 . t o ta l n u m b e r o f e g g m a ss e s a n d e g g s p ro d u c e d i n r . q u a d ra si t re a te d a t e m b b y 2 c a rb o fu ra n c o n c e n tr a ti o n biotropia no. 5, 1991/1992 b. treatment at week 4, pre-sexual maturity (psm) table 2 shows the effect on the size and age of the snails treated at the end of week 4 to the end of week 6 at the onset and end of oviposition. the total number of egg masses, eggs (fig. 3) and the average eggs per egg mass observed during the entire reproductive period is also shown in the table. with the exception of the 2000 ppm treatment wherein the average snail size at initial oviposition is smaller, the size average of the rest of the treatments and control are not significantly different from each other. the average age for initial oviposition in the control is 40.44 days, while the treated snails at 4 carbofuran concentrations are 53.36, 55.0, 56.5 and 55.84 days. this is an indication that treated snails oviposited at an older age. the average size in the last oviposition of the treated snails is smaller than in the control except in the 2000 ppm treatment. the average age at last oviposition is higher in the control and significantly lower in the treated. the 500, 1000 and 2000 ppm treatments were not significantly different from each other but significantly different from the 250 ppm group. the number of egg masses and eggs in the snails exposed to 2000 ppm is significantly low. there is no significant difference though in the total number of eggs and egg masses among the other experimental set-ups including the control (fig. 3). the reproductive period is longest in the control, and decreasing with higher concentrations. those exposed to 500, 1000 and 2000 ppm are lower but not significantly different from each other. c. treatment at week 7, sexual maturity (sm) table 3 shows the effect of the 4 carbofuran concentration on the size of the snails treated at week 7 at the onset and end of oviposition. the total number of egg masses laid and the total number of eggs for the entire reproductive period is also shown (fig. 4). the average number of eggs per egg mass at the end of the observation period is likewise indicated. it is shown in table 3 that untreated snails start to oviposit at an average length of 9.6 mm. the average size of the snails at the onset of oviposition in all four treatment groups as well as in the control does not significantly differ from each other. the average age, however, was older in the treated snails, indicating that while sizes are more or less the same, oviposition occurred at an older age. there are snails in the untreated that oviposited in as early as 35 days of age, while the youngest snail that oviposited in the experimental group was 45 days old, or a difference of 10 days. some snails did not oviposit: 1 snail in the untreated, 10 in the 250 ppm treatment, 8 in the 500 ppm treatment, 6 in the 1000 ppm treatment and 32 . effects of carbofuran on the reproductive capacity – imelda f. pagulayan et al. 33 t a b le 2 . s iz e a n d a g e a t th e o n se t a n d e n d o f o v ip o si ti o n , e g g -l a y in g c a p a c it y a n d r e p ro d u c ti v e p e ri o d o f r . q u a d ra si e x p o se d to fo u r c o n c e n tr a ti o n s o f c a rb o fu ra n a t w e e k 4 ( p re -s e x u a l m a rt u ri ty ) fo r a d u ra ti o n o f tw o w e e k s t a b le 3 . s iz e a n d a g e a t th e o n se t a n d e n d o f o v ip o si ti o n , e g g l a y in g a n d r e p ro d u c ti v e p e ri o d o f r . q u a d ra si e x p o se d t o f o u r c o n c e n tr a ti o n s o f c a rb o fu ra n a t w e e k 7 ( s e x u a l m a tu ri ty ) fo r a d u ra ti o n o f 2 w e e k s) biotropia no. 5, 1991/1992 34 f ig u re 3 . t o ta l n u m b e r o f e g g m a ss e s a n d e g g s p ro d u ce d b y r . q u a d ra si t re a te d a t th e p s m b y 4 c a rb o fu ra n c o n ce n tr a ti o n s effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. 35 f ig u re 4 . t o ta l n u m b e r o f e g g m a ss e s a n d e g g s p ro d u c e d i n r . q u a d ra si t re a te d a t s n b y 4 c a rb o fu ra n c o n c e n tr a ti o n s biotropia no. 5, 1991/1992 7 in the 2000 ppm treatment. average age at first oviposition in the untreated is 42 days, while in the treated the range is from 58.4 to 60.73 days (table 3). the average size of the snails at last oviposition was smaller in the treated but not significantly different from control and from each other. the average age at last oviposition, however, is highest in the control (169.42 days) and lowest in the 2000 ppm treatment (120.36 days) in a decreasing order with increasing concentration. the total number of egg masses and eggs was highest in the untreated snails, 1284 and 76559, respectively. the number of egg masses and eggs produced in the treated was lower and significantly different from the control but not significantly different from each other. the average number of eggs per mass is highest in the control and lower in the treated. the numbers of snails that reached the stage of oviposition in the treated set-ups were reduced. the reproductive capacity of the treated snails increases weeks after treatment. the observation is more evident in the treated than in the untreated snails. however, the above increase is still not enough to equal the number of egg masses and eggs produced by the control group. the reproductive period in the treated snails is significantly lower than in the control. d. comparative fecundity results in the three designated stages (emb, psm, sm) table 4 is the summary of the effects of all the treatments on the size and age at the onset and end of oviposition, on the reproductive capacity and length of the reproductive period in the three stages of treatment. the size range in days at the start of oviposition was 29 50 days for the emb control; 35 52 days for the psm control; and 35 50 days in the sm control. the total range using 138 snails was from 29-52 days. the smallest in size average at the same time oldest in average for the initial oviposition in all the treatments was on the sm treated snails, followed by the psm and the largest but the youngest to oviposit was in the emb. while table 4 shows that the psm group is the earliest to stop ovipositing, it cannot be concluded that it is the most sensitive to the treatment since the control group follows the same trend. the reproductive capacity is highest in the emb treated snails. while sm treated snails had a longer reproductive period than the psm, their reproductive capacity is very much less. 36 effects of carbofuran on the reproduction capacity – imelda f. pagulayan et al 37 t a b le 4 . c o m p a ra ti v e e ff e c t o f c a rb o fu ra n a p p li e d o n t h e t h re e d e si g n a te d s ta g e s in t h e l if e o f r . q u a d ra si o n i ts f e c u n d it y biotropia no. 5, 1991/1992 discussion the results of previous work (de lara and enriquez 1981) gave a range of 35 -42 days under controlled condition as the start of oviposition. in the current work, however, the total range in these 138 snails was 29-52 days. the current results did not exactly sustain the earlier findings, perhaps because of the effect of cold temperature when these experiments were done, since the expected oviposition periods coincided with the cold months of december and january. boray (1964) observed in his work that no copulation occurred in l. tomentosa in temperatures below 16°c in the laboratory cultures or in the field. the variations apparently reported in the reproductive biology of lymnaeids in general may be due to the different culture methods used. however, remigio and pagulayan (1984) contradict this finding. start of oviposition was delayed in all the treated snails while size range and average do not differ much in both the untreated and treated snails in the initial oviposition. the treated snails are older which means that it took a greater number days to reach the ovipositing size. it seems that it is more the size rather than age that determines the onset of oviposition. this is true in all the 3 age treatments. since growth is delayed by the treatment, delay in the oviposition could be attributed to it. it can be stated that the higher the concentration, the later is the start of oviposition. this could also be due to a delay in the rate of gonadal maturation. the higher the concentration, the earlier the cessation of oviposition. the number of egg masses and eggs produced at initial oviposition is highest at the control and lowest at the 2000 ppm treatment. the treatment not only delayed but also inhibited the reproductive capacity of most of the snails. inhibition increased with increasing carbofuran concentration. however, the actual number of egg massesat first oviposition could not be correctly determined. the fact that the number of egg masses at its first appearance is less than the number of snails clearly indicates that not all oviposited. there is no way of determining which of the snails in the basin did or did not oviposit since they are in groups of 4's and 5's. it is possible that the first oviposition in some may have been included in later weeks and counted as later ovipositions. while it is true that there is no accurate method of counting the number of egg masses for first oviposition, there is no other way, unless observations will be done on individual snails cultured in separate basins; such condition, however, is not conducive to fecundity. the results showed that the carbofuran maybe more effective in a particular age group, and the results could have altered the individual's competitive ability, i.e. food getting, mating. this could be the reason for the decreasing fecundity with increasing concentration in the emb; there was a drastic effect only 38 effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. of the 2000 ppm on the egg laying capacity of the psm treated snails, and a drastic effect of all the treatments on the egg laying capacity of the sm treated snails. it may also be possible that the psm treated snails could have recovered after the effect of the 250, 500 and 1000 ppm carbofuran concentrations had diminished. recovery brought them back on equal footing with the untreated ones, thus the egg production was higher, counteracting the low egg production at the start. the initial effect, therefore, seems to be delaying in this particular stage instead of totally inhibiting with the exception of the 2000 ppm which partially inhibited the egg laying capacity. godan (1983) stated that the periods of greatest sensitivity occur when the gastropods are very young and again when they are adults, at the peak of their reproductive activity. resistance is greatest among juveniles before the onset of sexual maturity. this reinforces the results of this work, as shown by the effect on fecundity of the sm treated snails. sexual maturity is the most critical stage to carbofuran treatment when it comes to reproductive capacity. the reproductive period is shorter in the treated snails and is again inversely proportional to the carbofuran concentration. the snails had to attain a bigger size and older age before oviposition took place. the snails used as control in the emb, psm and sm treatments had a wide range of variation for the first oviposition. the said control set-ups were not done at the same time. that could explain the wide variation in 29 50 for the emb control; 35 52 for the psm control; 35 50 for the sm control. the final average, though, for all the control is still 40.48 days which still supports the upper range of the results of earlier works, 35-42 days (de lara and enriquez 1981). the pesticide decreased the reproductive capacity of the snails and shortened the reproductive period. unlike its effect on growth, the sm treated snails were the ones adversely affected. there were snails that did not oviposit at all and a great number did not reach the size/age of oviposition. the carbofuran, depending on the concentration and/or the stage of application must have direct and subtle impacts on behaviour that influences the snail's competitive success i.e. small changes in courtship or mating rituals induced by the carbofuran may lead to mating failure, thus affecting the reproductive capacity and period. references allmon, f. 1985. pesticides and unhealthy dependence. science 6(8): 14. cremlyn, r. 1980. pesticides: preparation and mode of action. john wiley and sons, new york. de lara, a.v. and g.l. enriquez. 1981. life history of laboratory reared radix quadrasi (gastropoda: pulmonata). kalikasan 10(2-3): 242-254. 39 biotropia no. 5, 1991/1992 godan, d. 1983. pest slugs and snails, biology and control. springer-verlag, berlin. gunther, f.a., w.e. westtake and p.s. jaolan. 1968. reported. handa, s.k. 1980. spectrophotometric determination of carbofuran residues in water. assoc. chem. 64(2): 200-201. mane, u.h., m.s. kachole, and s.s. pawar. 1979. effect of pesticides and narcotants on bivalve mollusks. malac. 18: 347-360. metcalf, r.l., t.r. fukoto, c. collins, k. berck, s. abd el-aziz, r. munoz and c.c. cessrl. 1968. metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7-n-methyl carbamate (furadan) in plants, insects, and mammals. j. agr. food. chem. 16(2): 300-311. pagulayan, i.f. and d. darvin. 1986. the systematic relationship of radix quadrasi and mixas cumingiana (pulmonata: lymnaeidae). part iii genetics and part iv ecology. final report on a research project submitted to the natural sciences research institute, diliman, quezon city. rangaswany, j.r., y.d. vijayashanakar, and s.r. prakish. 1975. a simple spectrophotometric method for the determination of carbofuran residues. j. aoac 59(6): 1276-1278. yu, c., g.m. booth, d.j. hansen, and l.r. larden. 1974. fate of carbofuran in a model ecosystem. j. food chem. 22(3): 431-434. 40 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf microsoft word 37 biotropiavol. 13 no. 1, 2006 : 37 -48 identification of oceanographic parameters for determining pelagic tuna fishing ground in the north papua waters using multi-sensor satellite data vlncentius slregar1'and harold joppie davido waas2) "seameo b1otrop, bogor, indonesia 2> faculty of fisheries, university ofpattimura, ambon, indonesia abstract the north papua waters as one of the important fishing grounds in the world contribute approximately 75% of world production of pelagic tunas. these fishing grounds are still determined by hunting method. this method is time consuming and costly. however, in many areas determination of fishing ground using satellited data lias been applied by detecting the important oceanographic parameter of the presence of fish schooling such as, sea surface temperature and chlorophyl. mostly these parameters are used integratedly. the aim of this study is to assess the important oceanographic parameters detected from multi-sensor satellites (noaa/avhrr, seawifs and topex poisedon) for determining fishing ground of pelagic tunas in the north papua waters at east season. the parameters include sea surface temperature (stt), chlorophyl-a and currents. the availability of data from optic sensor (seawifs: chl-a and avhrr: thermal) is limited by the presence of cloud cover. in that case, topex poseidon satellite data can be used to provide the currents data. the integration of data from multi-sensors increases the availability of the oceanographic parameters for prediction of the potential fishing zones in the study area. key words : identification, oceanographic parameter, sea surface-temperature, chlorophyl-a, multi sensors, pelagic tuna, north papua-waters introduction the north papua waters in west pacific is one of the important fishing grounds for tuna in the world, beside the east pasific waters, japan and west coast of africa. it has contributed 75 % of world production of skipjack (katsuwonus pelamis) and yellow fin (thunnus albacares) (lewis and williams 2001). the fishing activities of local fishermen and the fishing fleet of a national company in this area is characterized by hunting method, eventhough actually using echo-sounder to detect fish schools of tunas. they use natural features such as birds, floating woods, shools of dolphines and whales to locate the tuna schools. this feature having been in use since longtime, has convinced experts to consider as an indicator of the existence of pelagic tunas. however, this method is practically inefficient because it is time consuming and costly. according to previous researches, it is known that the presence of tunas in the waters is possible due to the availability of food in the area, which is indirectly trigged by the occurrence of thermal front, current (divergent and convergent) and eddys (dwivedi 2001 ; lehodey 2003). in line with the development of remote 37 integration of npp semi mechanistic-modelling t. june et al. sensing technology, these oceanography parameters, in real time, can be detected by sensors of satellite remote sensing. this study is intended to determine oceanography parameters as a key factor in the detection of potential fishing grounds of the skipjack and yellowfm tuna in north papua waters in west pasific using multi-sensors of satellite data. materials and methods the study was conducted in the north papua waters with coordinate 3° 40' s -6° 40' u and 130° e 142° e, from august to october 2003 (figure 1). satellite data used to identify oceanographic parameters for determining skipjack and yellowfm tuna fishing ground are collected from multi-sensors satellite (noaa/avrr, seawifs and topex poisedon). local area coverage (lac) of noaa/avhrr data received from lap an (indonesian agency of aerospace), biak ground station are used to map sea surface temperature (sst) distribution using the equation of me millan dan crosby (1984), as follows: sst data derived from noaa/avhrr were validated using triton mooring bouy data at the position of (02° n ; 137° e) , (05° n ; 137° e). these mooring data were received from the website 38   images of topex poisedon and seawifs received in the form of globale area coverage (gac) from website htlp://www-ccar.colorado.edu/ and http://seawifs.gsfc.nasa.gpy/. are utilized for current analysis (divergent, convergent and eddys), and chlorofill-a distribution. the analysis is performed according to pickard and emery (1990). meanwhile, fishing catch data are collected from the fishing fleet of purse seiner 1025 gt(amount :15) of pt.biak mina java. this purse seiner has dimension of 1800 m in length and 300 m in width. the fishing catch data is in the form of cpue (catch per unit of effort ton"1 per ship hour"1). 39  biotropia vol. 13 no. 1, 2006 results and discussions sst validation validation of sst derived from noaa/avhrr data using triton buoy data (in situ) showed that the sst from these two different systems has similar pattern with certain variations of value of the sst. this variation could happen depending on the oceanographic condition that controls the temperature structure at depth measurement (robinson 1985). t-test (95 %) showed that there is a strong correlation ( r=0.72) between the sst of these different systems (figure 2). therefore, we used this algorithm to map the distribution of sst for further analysis and discussion. fishing ground condition the oceanographic parameter of skipjack and yellowfm tunas fishing grounds detected from multi-satellite sensors showed that in the east season, the distribution of the fishing grounds is concentrated at warm water centroids with temperature range varying between 27.02 and 30.86°c, except during the first week of august where the temperature is lower than before, varying between 26.64 and 27.45 °c. low sst during this month is probably due to the appearance of equatorial upwelling which attains its peak in june-july (vinogradov 1981), with the lowest sst detected varying from 26.62 to 26.87 °c (waas 2004). from this range of sst, it is presumed that the appearance of two tuna species in july and august is related with the period of their spawning season, where the ideal temperature is limited to 26 °c (lehodey 2003), thus this water is a good spawning ground for skipjack and yellowfm tuna (loukos et al. 2003). chlorophyll-a (chl-a) is an indicator of the productivity of the waters which has indirect relationship with the tuna food found at high concentration in fishing grounds every month during the period of study. the concentration of chl-a detected varies between 0.21 and 0.35 ingm"3. the high concentration of chl-a 40 indenlification of oceanographic parameters v. siregar and h.j.d. waas (>0.2 mg m"3) indicates that the presence of this phytoplankton in the waters is suitable to support or to maintain the sustainability of the development of fisheries, such as skipjack and yellowfin tunas (gower 1972). lehodey (2003) and wcnno et al. (2001) found that there is a high concentration of chl-a near the coastal zone of north papua. this high concentration of chl-a is contributed not only by the upwelling (equatorial upwelling) process which occurred in that area, but also by the river flow (e.g.mamberamo river and matabori river) which bring nutrient to the north papua waters, and by halmahera eddies (lipi 1992). the surface current detected by topex poseidon satellite in the fishing grounds of skipjack and yellowfin tunas is classified as south equatorial current (sec) system, concentrated at 0° 2.5° n. the appearance of those tunas in this zone has strong relationship with the occurrence of divergent and convergent currents detected. the high concentration of phyto planktons during the equatorial upwelling at 0° 0.5° n, is predicted indirectly as a factor of the appearance of tunas at this system of currents. this condition confirms the hypothesis of uda (1973) in laevastu and hayes (1981) that the good fishing ground for tuna is found in the system of sec. oceanographic parameter derived from satellite data due to cloud cover, the number of images available from optic sensor used is limited. the number of images available are thermal: 10, chl-a distribution: 4 and currents : 12 images. those images were used for the analysis, but in this paper the number of images presented is limited to 2 for each type of data. the important parameter analysed from the available images are described as follows : thermal front analysis of thermal front using 10 images available showed that fronts could occur and develop in the north papua waters. the thermal front is detected from august until october 2003 in different forms and patterns. at the end of august the front is in the form of line streched from north of monokwari towards the northeast as long as 840 nautical miles. the position of fishing activities spread out near the thermal front in the warmest water mass. during september, the thermal front appears clearly. this front has a length varying from 550 920 nautical miles, move away off the waters of monokwari towards the northeast, forming a smaller width of meandering. intensive fishing was found concentrated at the meandering zone, which has a role as barrier, so that the phyto planktons could not move away from the front zone (dwivedi 2000). on the 2nd and 11th september (figure 3a,b), fishing grounds were clearly found near the thermal front. this phenomenon confirms narain's study (1993) that the high intensity of fishing occurs at the thermal front. the thermal front still exists during the month, move and change its form following the currents pattern. high concentrations of phyto planktons occur at the front due to two different water masses (warm and cold) and divergent currents (shixing et al. 1993). these currents can be seen from image of topex poseidon at the same date of acquisition. along the thermal front, a high 41   ^identification of oceanographic parameters v. siregar and h..i.d. waas concentration of chl-a was recorded, and at the same time, convergent current and eddys were taken place. in october, the thermal front has different pattern compared to the front of the previous month. this front forms cold water pockets with a width of ± 9.02 71.35 n.mile square and surrounded by warm water mass from the pacific ocean. the position of the fishing ground was generally concentrated outside of the thermal front zone. in the front zone, strong divergent current and eddys were taken place so that the cold water mass was spreading out following the direction of the current chlorophyll-a distribution during august and september, the images of the area were not realy clear due to the cloud cover. however, from those images and by plotting the area of fishing activities, it showed that skipjack and yellowfin tunas were found in the area of higher chl-a concentration and along the front (figure 4). in august the chl-a front is not clear. meanwhile, in october the images of chl-a is not available. in most of the cases, in order to locate accurately the potential fishing grounds, the images of chl-a distribution and sea surface temperature are used integratedly. this method could be proposed as an important technique for identifying the potential fishing ground. the method also provides indirect information on the horizontal mixing of water masses, which contributes to high productivity or high concentration of phyto plankton (chl-a) in the waters (dwivedi 2000). in this study, these images from different sensor optics (noaa/avhrr and seawifs) are also used together. currents currents (convergent, divergent and eddies) are specific oceanographic parameters in the north papua waters. these parameters play an important role in the development of thermal front, the distribution of water mass and the concentration of chlorophyll-a in the area. together, the convergent and divergent these currents are favorable for the development of marine organisms. the area with divergent currents has high productivity, and convergent current forms a mechanism of phyto plankton aggregation at the front of currents. meanwhile, eddies as the result of moving cycle (form a ring) of the water masses increase the intensity of vertical mixing of the waters followed by upwelling which occurs at the outer side of the ring, triggering the increase of water mass productivity (dwivedi 2000). according to mean and lazier (1990), currents in the study area are classified into a). sec (south equatorial current) with direction to the west b). necc (north equatorial counter current) is a narrow current with the direction to the east, and c). euc (equatorial upwelling current) is subsurface water current with the direction to the east. the dynamic of these currents is due to the coriolis force, which effects divergent and convergent currents along the borders of the currents in different directions. these currents occurred intensively in the papua waters. the illustration 43   ^identification of oceanographic parameters v. siregar and h.j.d. waas of this mechanism is shown in figure 5: (a.l) currents in the northern hemisphere have oposite direction, west-east; (a.2) shows the effect of corriolis force, the currents are divergent and the subsurface water upwell replaces the surface water as a result. this mechanism occurs in the system current of nec (northern equatorial current) and necc; (b.l) showing the convergent current as the result of necc and sec encounter; (c.l) in the equator, sec with direction towards the west will create divergent currents, inversly, with directions towards the east will c reate convergent currents (d. 1 dan d.2). the analysis of fishing ground based on available data set of topex poisedon (augustoctober) showed that the pelagic tunas are mostly catched in all types of current such as divergent, convergent and eddies . in august, most of the fishing grounds were found at the outer ring of eddys and divergent current (fig 6 a). at the end of the month, most of the fishing grounds were only found at the convergent current system. in september and october, the fishing grounds are found mostly at the boundary on divergent current system. several fishing grounds were also found at the outer ring of eddys (figure 6). from analysis of available satellite data in figure 6, it can be concluded that currents (divergent, convergent and eddies) are also an parameter important for identification of fishing ground of the pelagic tuna in the papua waters. these oceanographic parameters were derived from satellite altimeter data (microwave-radar). compared to optic data, the detection of these parameters with radar were not influenced by cloud cover. therefore, utilization of microve data which provides information on currents is proposed to be used together with optic data (visible and thermal), in order to increase the accuracy of fishing ground determination. in the case of unavailability of optic data, the use of radar data can be used as an alternative. 45   indentification of oceanographic parameters v. siregar and h.j.d. waas conclusions analysis of oceanographic parameters detected from several satellite data and fishing catch data of the north papua waters showed that the important oceanographic parameters which can be used to identify fishing ground of small pelagic tunas (skipjack and yellow fin tunas) are thermal and chlorophyl-a front, and currents (divergent, convergent and eddies). the combination of these parameters increase the accuracy of fishing ground identification. thermal (sst), chlorophyll-a and currents data are derived from optic and microwave (radar) sensors respectively. availability of data from sensor optic is limited due to cloud cover, which in this case is very frequent in papua and indonesia in general. in the case of cloud cover, where the optic data is not available, radar data can be used to provide currents information an alternative to determine fishing ground. the use of multi-sensor satellite data such as noaa-17/avhrr, seawifs and topex poseidon can be used integratedly as a promising approach in determination of potential fishing ground of the pelagic tunas in the north papua waters. references dwivedi, r.m.2000. ocean colour as a tool for potential fishing zone identification and forecast. porsec 2002. pre-conference training. national institute of oceanography. dona paula, goa -403 004, india, p. 17-22. gower, j.f.r.1972. opportunities and problems in satellite measurements of the sea. unesco tech. pap. 46, 70 p. laevastu, t., and m. l. hayes. 1981. fisheries oceanography and ecology. fishing news books ltd., 199 p. lehodey, p., 2003. sepodym application to albacore (thunnus alalunga). oceanic fisheries programme. noumea, new caledonia, p. 1-16. lewis, a.d., p.o. williams. 2001. overview of the western and central pasific ocean tuna fisheries, 2000. oceanic fisheries programme (ofp) 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and j.h. torrie, 1989. principles and procedures of statistics. second edition. me gi hill int.book co., 633 p. vinogradov, m.e. 1981. ecosystems of equatorial upwelling, in: analysis of marine ecosystei a.r.longhurst (ed). london: academic press, p 69-93 waas, h.j.d. 2004. analisis daerah potensial penangkapan cakalang (katsuwonus pelamis) ( madidihang (thunnnx albacares) di perairan utara papua, pasifik barat. tesis. seko pascasarjana ips, 102 p. wenno, l.f., hadikusuma and nurhayati, 2001. hubungan antara beberapa parameter fis oseanografi terhadap distribusi kandungan klorofil-a di perairan mamberamo irian jaya, agus 2000. dalam perairan indonesia, oseanografi, biologi dan lingkungan. lp3o-li jakarta, p. 9-19. 48 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf biotropia vol. 30 no. 2, 2023: 158 170 doi: 10.11598/btb.2023.30.2.1778 158 identification of the moluccan megapode (eulipoa wallacei) natural habitat in haruku island, indonesia and its vegetation composition handy erwin pier leimena1*, achmad sjarmidi2 and tati suryati syamsudin2 1biology department, faculty of mathematics and natural sciences, pattimura university, m. putuhena street, pattimura university campus, poka, ambon, 97233, indonesia 2school of life science and technology, institut teknologi bandung, ganesa street no. 10, bandung, west java, 40132, indonesia received 7 july 2022 / revised 9 april 2023 /accepted 11 april 2023 abstract the characteristics of island vegetation greatly influence the activities of endemic birds in island areas, such as the moluccan endemic megapodes (eulipoa wallacei) on haruku island. therefore, it is essential to identify the specific location of the habitat utilized by individual birds for their daily activities on haruku island and to analyze the composition of the vegetation and the variety of plant species. identification of bird habitat locations using radio-tracking on four newly hatched chicks and four adult birds. a total of 330 individual tracking points were recorded during the study period. the vegetation sample used a total of 420 plots for seedlings, saplings, poles, and trees which were then analyzed for importance value index (ivi), diversity, evenness, and similarity. we found that the tanjung maleo forest was their nesting habitat, while the marunimei and lalean forests were their daily habitats. a total of 91 plant species and 60 plant families were discovered with the vegetation diversity value of the three habitats was moderate (h’mean = 3.07) and tended to be dominated by air plant (kalanchoe pinnata), sword fern (nephrolepsis exalta), cogongrass (imperata cylindrica), coco-grass (cyperus rotundus), indian camphorweed (pluchea indica), and lanzone (lansium domesticum) (emean = 0.88), and have a relatively low level of species similarity between habitats (simean = 38.30%). we found that the daily habitat of the moluccan megapode on haruku island was around their nesting sites and has a complex structure because it was composed four vegetation strata. therefore, for conservation purposes, habitat management must prioritize preserving forest habitats around the bird nesting sites. keywords: diversity, evenness, moluccan scrubfowl, momoa, maleo, similarity, vegetation composition introduction the maluku islands, indonesia, located in the wallacea region, support endemic bird species, such as the moluccan megapode, through various habitat types (yuni & yuda 2020). tropical forests provide complex habitats rich in composition and structure of vegetation for shelter, foraging, roosting or sheltering, and breeding (baldeck et al. 2012; bu et al. 2014; velasques-trujillo et al. 2021). therefore, the composition and diversity of habitat vegetation support the diversity of endemic, rare, threatened, and endangered wild birds (sodhi et al. 2011; rocha et al. 2015; devenish‐nelson et al. 2019; feng et al. 2020). the structure of a bird’s habitat, which includes vertical structure and floristic composition, is an essential component of its habitat. vegetation structure and floristic composition have been used to predict the presence and diversity of bird species for a long time. some bird taxa are influenced by vegetation structural factors (e.g., the complexity of stand configuration or architecture), while natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 159 others are influenced by vegetation composition factors (e.g., vegetation type) (tews et al. 2004; tassicker et al. 2006; gao et al. 2014; rutten et al. 2015; melo et al. 2020; moudry et al. 2021). island bird species in the tropics are threatened by several environmental challenges, including habitat change and destruction (duncan & blackburn 2007; taylor & kumar 2016; radley et al. 2020; dri et al. 2021). habitat destruction causes changes in the structure and composition of vegetation in bird habitats, thus affecting the survival and reproduction of bird populations (ruiz-gutierrez et al. 2008; matthews et al. 2015; tulloch et al. 2016; tchoumbou et al. 2020; atikah et al. 2021). one of the native and endemic bird species in the maluku islands is the moluccan megapode (eulipoa wallacei), which is only found in northern maluku and central maluku. like other megapodes, the moluccan megapode is super-precocial and classified as a terrestrial bird (colar et al. 1994; argeloo & dekker 1996; dekker et al. 2000; heij & rompas 2011). moluccan megapodes inhabit coastal areas through tropical rain forests of more than 750 m asl (dekker et al. 1995; heij et al. 1997; heij 2001; heij & rompas 2011; birdlife international 2022). the occurrence of the birds in coastal areas is related to their nesting behavior, which involves digging holes in the sand substrate to lay their eggs (dekker et al. 1995; jones et al. 1995; dekker et al. 2000; heij et al. 1997; heij & rompas 2011). the population of moluccan megapodes has been decreasing due to habitat destruction and egg harvesting (colar et al. 1994; argeloo & dekker 1996; dekker et al. 2000; heij & rompas 2011), so it has been designated as a vulnerable species by the iucn since 1994 (birdlife international 2016). studies in 1997 by heij et al. (1997) confirmed that habitat destruction caused the moluccan megapode to become rare on several large islands, such as seram island and bacan island, and smaller islands, such as ambon island and ternate island. likewise, several nesting sites have been abandoned and are no longer used by birds due to the destruction of their nesting habitat. the effect of habitat destruction on the decline of the moluccan megapode indicates that habitat quality and composition play an essential role in the bird populations. adult moluccan megapodes use vegetation around their nesting site for perching before and after nesting activities. meanwhile, newly hatched chicks require vegetation cover as shelter from predators (heij et al. 1997; heij & rompas 2011). in central maluku, one of the largest nesting sites of the moluccan megapode is tanjung maleo in haruku island (heij et al. 1997; heij & rompas 2011). until recently, information about the habitat used by moluccan megapodes on haruku island has been scarce and limited to habitats around bird nesting sites (heij et al. 1997; heij & rompas 2011; sjafani et al. 2015), while daily habitat use studies have never been reported. better knowledge of the specific habitats used by moluccan megapodes allows conservation managers to carry out appropriate engineering to protect bird habitats from expanding settlements near nesting habitats or opening new agricultural fields in their daily habitats. habitat protection engineering can be carried out by delimiting habitat boundaries, maintaining the composition of vegetation strata, or providing corridors for the distribution of chicks and pathways for adult birds to and from their nesting habitat in tanjung maleo. this study investigated habitat use by moluccan megapodes on haruku island. we also analyze the composition of the vegetation and the diversity of plant species in these habitats to understand the influence of habitat on birds’ daily activity patterns. materials and method study area the study was conducted on haruku island in central maluku district (3.567os, 128.483oe) (figure 1). haruku island has an area of 274 km2 and a topography of mountains and hills, with the highest point reaching 554 meters above sea level. the climate on haruku island is locally influenced by tropical marine climate and monsoon climate (aldrian & dwi susanto 2003; wirjohamidjojo & swarinoto 2010) with relatively high rainfall of 224.58 mm per year in 2021 (bps propinsi maluku 2022). biotropia vol. 30 no. 2, 2023 160 figure 1 study sites used to identify natural habitats and analyze the composition and diversity of the vegetation of the moluccan megapode (eulipoa wallacei) habitat in haruku island, indonesia [dotted line boxes indicate the location of daily bird habitats]. identification of bird habitat bird habitat identification was conducted by using radio-tracking. during 40 days of observation from december 2020 until january 2021, 330 points of bird presence were recorded. individual females and chicks were tracked from their nesting sites to collect data on the relationship between nesting habitats and daily foraging habitats. this study did not use adult males because only adult females carried out nesting activities at nest sites in tanjung maleo, while adult males remained in their daily habitat (heij et al. 1997; heij & rompas 2011). adult male birds were challenging to catch in their daily habitat because of their camouflage amidst the surrounding understory vegetation. eight birds were utilized, including four newly hatched chicks and four adult females. chicks and adult females were caught using a hand capture technique (de beer et al. 2001; bloom et al. 2007; whitworth et al. 2007; busse & meissner 2015) during nesting and when the chicks emerged from their nesting holes. for tracking purposes, the sirtrack v8009 transmitter was utilized for chicks, and the sirtrack p04347 transmitter for the adult birds. using loctite cyanoacrylate (bowman et al. 2002; diemer et al. 2014), the transmitter was attached to the base of the wing. the movement and position of individual birds were tracked using biotrack sikka vsr 04, receiver, yagi antennae, and gps garmin 64s. the birds were tracked every day on foot (ground tracking). the position of the universal transverse mercator coordinate for each bird was plotted on a map of the study site to determine the area used by birds using arcgis 10.3 and arcmap 10.3. natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 161 vegetation sampling intensive floristic inventory and plot-based vegetation survey (krebs 1998; colwell 2009) was conducted at forest sites on haruku island that were used by individual moluccan megapodes, specifically in tanjung maleo, marunimei, and lalean. vegetation sampling was carried out at the same time as bird habitat identification. twelve transect lines measuring 500 and 1000 meters were utilized, and the distance between transects was 250 meters. the distance between plots was 100 meters. the plot sizes were 1 m x 1 m (seedlings), 5 m x 5 m (saplings), 10 m x 10 m (poles), and 20 m x 20 m (trees) (barbour et al. 1987; woodward et al. 2009; rahman et al. 2016; kusmana 2017; peng et al. 2018). the observed variables for each stage were as follows: (i) seedling stage: germinated seeds to < 1.5 m in height, (ii) sapling stage: height >1.5 m to a diameter at breast height (dbh) < 10 cm, (iii) pole stage: 10 cm < dbh < 20 cm (barbour et al. 1987; woodward et al. 2009; kusmana 2017). there were 84 plots in tanjung maleo, 168 in marunimei, and plots in lalean. vegetation parameters included species name, number of species, number of individual species, and diameter at breast height (dbh). plant species were identified using a collection of identification guides (whitmore 1978; soerianegara & lemmens 1994; lemmens et al. 1995; llamas 2003; gunawan et al. 2019). data analysis all coordinate positions of bird presence points were plotted onto the haruku island land cover map to obtain the moluccan megapode habitat land cover type. the land cover map of the bird habitat area was determined using a 2021 haruku island land cover map combined with 2021 google earth imagery data, a 1:50,000 scale map of haruku island in 2021, and a 2020 landsat 5 tm image (composite color band 4 band 3 band 2) with a spatial resolution of 30 meters. land cover types on bird habitats were created using arcmap 10.3. the land cover classification was based on the indonesian national land cover standards agency (national standardization agency of indonesia 2010). vegetation structure and floristic composition were determined based on the analysis of the importance value index (ivi) (bendre & kumar 2010), species diversity (shannon – wiener index) (shannon & weaver 1963; odum 1983), species evenness (pielou index) (odum 1983), and species similarity (sorensen index) (sorensen 1948). the importance value index (ivi) is the sum of relative frequency, density, and dominance values. the species diversity was calculated by using the shannon-wiener index: h’ = -∑ pi ln pi where: pi = proportion of individuals of the i-th species to individuals of all species found. the species evenness index was calculated by using the pielou index: e = h’/ln (s) where: h’ = shannon-wiener diversity indeks; s = total number of species in the sample; e = 1 if all species are represented equally in the sample, and e is close to zero if one species predominates strongly. the species similarity index (si) between sites was calculated using the sorensen index: si = (2c/(a+b)) x 100%, where: c = standard number of species at the two sites; a = number of species at site a; b = the total number of species at site b. the f test was conducted using one-way anova to compare the composition of vegetation growth strata between bird habitat forests. result and discussion habitat of moluccan megapode the radio-tracking record of individual chicks and adult females of moluccan megapodes on haruku island indicated that bird activity occurred in forest areas near their nesting sites. birds were active in their nesting biotropia vol. 30 no. 2, 2023 162 site at tanjung maleo, marunimei, and lalean forests. marunimei forest is ± 1.60 km northeast of tanjung maleo, and lalean forest is ± 2.50 km southeast of tanjung maleo. the moluccan megapodes habitat was classified as moor-farm-field and dryland forest based on the land cover type. the type of land cover around the bird’s nesting site in tanjung maleo was classified as a development area because it was adjacent to a residential area. however, around the bird nesting site in tanjung maleo, there were still various types of vegetation specially guarded by the community as part of the bird’s nesting habitat. meanwhile, the marunimei forest, as the bird’s daily habitat, was classified as a moor-farm field, while the lalean forest was classified as a dry land forest (figure 2). these results confirm previous research indicating that moluccan megapodes could survive and thrive in various habitat types ranging from secondary forests to open beaches, lowland forests, and tropical rain forests (dekker et al. 1995; heij et al. 1997; heij 2001; heij & rompas 2011). in contrast to their diverse daily habitats, bird nesting habitats were always found in coastal areas, specifically open beach areas surrounded by forest areas (dekker et al. 1995; jones et al. 1995; dekker et al. 2000; heij et al. 1997; heij & rompas 2011). megapode species in island areas are known to be more active in tropical forests and use coastal areas for breeding. therefore, both habitat types are essential for survival (goth & vogel 1995; jones et al. 1995; dekker et al. 2000; pangauadam & brodie 2019; paguntalan et al. 2021). the daily activities of the moluccan megapode within a radius of ± 2.50 km from their egglaying locations in tanjung maleo indicated that most of their daily activities are not far from their egg-laying locations. several studies of other bird species also show that most of the birds' daily activities occur in areas not far from their nesting sites (ryan & jamieson 1998; yaremych et al. 2004; bosch et al. 2010; rechetelo et al. 2016). adult female moluccan megapode used the forest in tanjung maleo for perching before starting nesting activities or as a perch after nesting, before flying away from the nest site (heij et al. 1997; heij & rompas 2011). for chicks, the forest around their nesting site in tanjung maleo was used as a shelter from the moment they hatch (heij et al. 1997; heij & rompas 2011) because of their super-precocial nature (colar et al. 1994; argeloo & dekker 1996; starck & ricklefs 1998; dekker et al. 2000). consequently, vegetation around the nesting site was crucial to the chick’s survival. it has been reported that moluccan megapode chicks immediately moved to nearby vegetation after hatching (heij et al. 1997; heij & rompas 2011). the use of vegetation around the hatchery as a shelter for chicks has also been reported among the australian brush-turkey chicks (alectura lathami) (goth & jones 2001; goth & vogel 2002; goth & vogel 2003; goth & evans 2005). natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 163 figure 2 the point of presence of individual moluccan megapodes (eulipoa wallacei) in haruku island based on radiotracking results. vegetation structure and floristic composition of the three forest locations used by the moluccan megapodes, the marunimei forest has the highest number of plant species (48 species). in contrast, the tanjung maleo forest has the most diminutive plant species (14) (table 1). the number of plant species in the tanjung maleo forest is currently fewer than the study results in 1997. in 1997, approximately 20 species were found in the tanjung maleo forest (heij et al. 1997; heij & rompas 2011). these results indicated that in the twenty-four years since 1997, the number of plant species in the tanjung maleo forest has decreased. because tanjung maleo's forest was close to the villages, it was believed that the expansion of the residential area was responsible for the decline in species. the number of plant species in the maluku megapod habitat on haruku island from this study was higher than the results of a survey conducted by ahmad (2014) around bird nesting sites on halmahera island, which only found as many as 13 to 17 plant species. this study also found that the highest number of plant families was found in the marunimei forest area (30 families), and the lowest was in the tanjung maleo forest area (14 families) (table 1, figure 3). no dominant plant species families were found in the tanjung maleo forest; each family only consisted of one species. in contrast, in marunimei and lalean forests, members of the fabaceae family dominated the vegetation. the current number of plant families in the tanjung maleo forest is the same as the 1997 study by heij et al. (1997) and heij & rompas (2011), but in 1997 the fabaceae family had more than one species. table 1 the number of species, individual species, and plant families in forest habitats utilized by moluccan megapodes (eulipoa wallacei) in haruku island forest areas species (n) individual species (n) family (n) tg. maleo 14 49 14 marunimei 48 1401 29 lalean 28 345 16 total 91 1795 60 biotropia vol. 30 no. 2, 2023 164 figure 3 comparison of the number of plant species per family in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island (a) tanjung maleo, (b) marunimei, and (c) lalean table 2 comparison of vegetation structural composition in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island forest areas vegetation growth stratum tree pole sapling seedling number of individuals (n) percentage (%) number of individuals (n) percentage (%) number of individuals (n) percentage (%) number of individuals (n) percentage (%) tg maleo 34 69.39 4 8.16 2 4.08 9 18.37 marunimei 605 43.18 226 16.13 165 11.78 405 28.91 lalean 109 31.59 69 20.00 14 4.06 153 44.35 average 249 100 60 189 the strata of vegetation growth in the three moluccan megapodes habitat forest areas included trees, poles, saplings, and seedlings (table 2). the tree strata dominated the forest area in tanjung maleo (63.39%) and marunimei (43.18%), while the seedling strata dominated the lalean forest area (44.35%). the f test showed that the vegetation's composition significantly differs between habitat forests (fcount = 8.82 > ftable = 4.26). this study showed that the forest regions surrounding bird nesting sites were classified as having complex vegetation because they contain four growth strata: trees, poles, saplings, and seedlings. the moluccan megapode habitat on halmahera island also consists of four growth strata (ahmad 2014). studies of several megapode species show that their habitat tends to be dominated by tree strata compared to other growth strata (goth & vogel 1995; khairuddin & yamin 2019; pangau-adam & brodie 2019; paguntalan et al. 2021; dhafir et al. 2022 ). however, several species of megapodes, such as the australian brush turkey (alectura lathami) (goth & vogel 2003), the wattle brush turkey (aepypodius arfakianus) (pangau-adam & 0 1 2 3 4 5 acathaceae amaranthaceae annonaceae burseraceae crassulacea lamiaceae meliaceae myristicaceae anacardiaceae apocynaceae lauraceae malvaceae myrtaceae arecaceae asteraceae fabaceae number of species per family 0 1 2 3 4 5 6 acathaceae amaranthaceae apocynaceae burseraceae crassulacea cyperaceae elaeocarpaceae flacoutiaceae meliaceae myristicaceae nephrolepidaceae oxalidaceae phyllanthaceae plantaginaceae poaceae rubiaceae rutaceae santalaceae selaginellaceae annonaceae arecaceae lauraceae moraceae asteraceae lamiaceae malvaceae myrtaceae anacardiaceae fabaceae number of species per family 0 1 2 anacardiaceae annonaceae apocynaceae burseraceae calophyllaceae clusiaceae convolvulaceae crassulacea lauraceae lecythidaceae malvaceae moraceae nephrolepidaceae sapindaceae number of species per family (b) (c) natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 165 brodie 2019), and the orange-footed scrubfowl (megapodius reindwartii) (khairuddin & yamin 2019) are also found in a sapling or seedling strata. complex vegetation contributes to the survival of wild species populations by providing a suitable microclimate, shelter, and a place to rest and reproduce (baldeck et al. 2012; bu et al. 2014; bergner et al. 2015). in tanjung maleo, the percentage of tree strata is currently higher than in 1997. in this study, the tree strata in the tanjung maleo forest reached 70 percent, whereas the 1997 study found only 67 percent (heij et al. 1997; heij & rompas 2011). likewise, tree strata dominated the marunimei forest. on the other hand, the seedling layer dominated the lalean forest. tree strata in the tanjung maleo forest function as perching for birds when they come to the nest or leave their nesting sites, while the strata of seedlings and saplings serve as protection for the chicks after hatching (heij et al. 1997; heij & rompas 2011). observations in the study area show that the moluccan megapodes also use trees in their habitat as perching to move between habitats or escape from predators. individual birds used the tree layers in their habitat as perches, resting spots, and shelters. moluccan megapodes' activity primarily occurs on the forest floor (mackinnon & wind 1980; del hoyo et al. 1994; strange 2012) and causes variations in vegetation strata to be essential for the survival of individual birds (bucklin et al. 2015; walther & pirsig 2017; velasques-trujillo et al. 2021). species composition and diversity based on the importance value index (ivi), the dominant species in each forest area were different (table 3). the dominant species in the tanjung maleo forest were air plants (kalanchoe pinnata) (ivi = 67.96) and sword ferns (nephrolepsis exalta) (ivi = 61.47). marunimei forest was dominated by cogongrass (imperata cylindrica) (ivi = 36.80) and coco-grass (cyperus rotundus) (ivi = 32.59), and the lalean forest was dominated by indian camphorweed (pluchea indica) (ivi = 24.23) and lanzones (lansium domesticum) (ivi = 18.30). the dominant plant species in the tanjung maleo forest differed from the dominant species around the moluccan megapodes nesting site on halmahera island in north maluku. the vegetation around the nesting site at galela on halmahera island was dominated by more varied species, namely bay table 3 the vegetation composition of the nine most abundant plant species in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island no forest areas tanjung maleo marunimei lalean species name % inp species name % inp nama ilmiah % inp 1 kalanchoe pinnata 21.93 imperata cylindrica 12.27 pluchea indica 8.08 2 nephrolepsis exaltata 20.34 cyperus rotundus 10.86 lansium domesticum 6.10 3 planchonia valida 9.91 nephrolepsis exaltata 8.49 amaranthus spinosis 6.07 4 cananga odorata 7.58 selaginella doederleinii 4.04 tectona grandis 5.61 5 alstonia scholaris 6.08 ficus benjamina 3.84 leucaena leucocephala 4.82 6 ipomoea pes caprae 5.72 lannea grandis 3.51 durio zibethinus 4.58 7 garcinia bancana 5.34 durio zibethinus 3.23 calameae sp 4.56 8 eusideroxylon zwageri 4.06 cocos nucifera 3.21 myristica fragrans 4.55 9 mangifera indica 3.69 artocaphus heterophyllus 3.07 cocos nucifera 3.94 10 others (6 species) 15.36 others (39 species) 47.47 others (19 species) 51.70 total percentage 100.00 100.00 100.00 hops (ipomoea pescrapae), clover (marsilea creanata), mangrove (rhizophora sp), and tropical almond (terminalia catapa) (sjafani et al. 2015). these results indicated that plant species around bird nesting sites did not significantly affect bird arrivals for nesting. dominant plant species in both forest areas adjacent to nesting sites (marunimei and lalean forests) act as shelters and support the daily activities of moluccan megapodes on haruku island. the highest diversity of plant species in the three forest areas around the moluccan megapodes site was in the marunimei forest (h’ = 3.87), while the lowest was in the tanjung maleo forest (h’ = 2.27) (table 4a). in terms of diversity, the plant species diversity in biotropia vol. 30 no. 2, 2023 166 the three forest regions was categorized as moderate. in addition to its diversity, species abundance distribution in the three forest areas, as measured by an evenness index, was close to one. the lalean forest had the most notable species distribution equality (e = 0.92) (table 4a). the evenness index indicates that the distribution of plant species abundance in the three locations tends to be relatively even and does not indicate the dominance of one or more species. nevertheless, according to the importance value index, each type of bird habitat contains several dominant plant species. tanjung maleo forest has a greater variety and more even distribution of plant species than halmahera island’s forest (sjafani et al. 2015). the diversity and evenness of plant species in the moluccan megapode habitat were higher than the orangefooted scrubfowl habitat on moyo island, west nusa tenggara (h’ = 2.42 dan e = 0.86) (khairuddin & yamin 2019). table 4 comparison of ecological index values between the three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island a. species diversity and evenness index between forest areas forest areas shannon-wiener index (h’) pielou index (e) tg. maleo 2.27 0.86 marunimei 3.87 0.86 lalean 3.06 0.92 b. similarity and dissimilarity between forest areas forest areas similarity (%) dissimilarity (%) tg. maleo – marunimei 25.81 74.19 tg. maleo – lalean 28.57 71.43 marunimei – lalean 60.53 39.47 average 38.30 61.70 this study found that from the three forest areas, the similarity of plant species between tanjung maleo forest, a bird nesting site, with the other two forests was low (is = 25.81% and is = 28.57%). on the other hand, the similarity of plant species between marunimei and lalean forests was high (is = 60.53%) (table 4b). therefore, the dissimilarity of plant species between the bird’s nesting site in tanjung maleo and the other two forest areas indicates the difference in vegetation between the bird’s nesting and daily habitats. the difference in species diversity between the forests surrounding bird nesting sites on haruku island and halmahera island indicates that the habitat types of moluccan megapodes were diverse and not influenced by specific plant species. the low average similarity between the three forests (simean = 38.30 percent) reflects the diversity of vegetation in the forest surrounding the haruku island bird nesting site. however, the similarity index between the three bird habitats revealed vegetation type differences between nesting habitats and the habitat of the moluccan megapodes on haruku island. conclusion this study enhances our understanding of the habitat conditions of moluccan megapodes on indonesia’s haruku island. it was determined that the moluccan megapodes on haruku island were active in the forest area approximately 2.50 kilometers from their nesting sites. based on the distance from the nesting site, the forest area in tanjung maleo serves as a bird nesting habitat. in contrast, the other two forests (marunimei and lalean) are a bird’s daily habitat. haruku island’s bird habitat consists of four layers of vegetation that support the activities of chicks and adult birds during nesting, foraging, and perching. variations in plant species diversity among the three habitats indicate moluccan megapodes’ ability to occupy diverse habitat types. regarding plant species diversity, nesting habitats and daily habitats of natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 167 birds differ by more than 70% of plant species, indicating differences in vegetation between nesting habitats and daily habitats of birds. an important finding from this study was that conservation efforts for the moluccan megapodes on haruku island should prioritize nesting and daily habitats in the forest area around the bird's nest sites. acknowledgments special thanks to d. usemahu and kailolo village for their assistance and cooperation during fieldwork, r. huda, and international animal rescue indonesia foundation for providing a tracking receiver and a yagi antenna and school of life science and technology, institut teknologi bandung for supporting facilities during the study. this research was funded by the directorate general of higher education, ministry of education, culture, research, and technology of the republic of indonesia as a scholarship and doctoral research grant to the first author. references ahmad z. 2014. selection strategy for laying eggs by mamoa bird (eulipoa wallacei gray, 1980) in galela. 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(a) microsoft word 75 biotropia vol. 13 no. 2,2006 : 75 84 preovulatory changes and ovulation in cattle undergoing spontaneous or cloprostenol-induced luteolysis bambang purwantara1'3, rene h0ier2, mette schmidt2, and torben greve2 1 department of veterinary clinic, reproduction and pathology, faculty of veterinary medicine, bogor agricultural university, darmaga 16680 bogor, indonesia ²department of clinical studies, section reproduction royal veterinary and agricultural university, bulowsvej 13, 1870 frederiksberg c. denmark ³seameo biotrop, jl. raya tajur km 6, p.o. box 116bogor, indonesia abstract the follicular population, diameter of the ovulatory and subordinate follicles, corpus luteum (cl) size, concentration of progesterone and estradiol-17p were studied following spontaneous or cloprostenol-induced luteolysis. a total of 14 heifers received cloprostenol treatment on day 9-11 of the cycle to synchronize their estrus. subsequently, they were divided into two groups, one group which was allowed to undergo spontaneous luteolysis and the other group in which estrus was induced from days 9 to 12. in the induced-group, transrectal ultrasonography were performed daily started two days prior to injection until the onset of estrus. in the spontaneous-group, ultrasonography was done daily from day 15 until the onset of estrus. in both groups scanning were performed every 4 h from the onset of estrus until ovulation was ascertained. small (sf, 2-4 mm), medium (mf.5-9 mm ) and large (lf,>9mm) size follicles were recorded. the diameter of largest and subordinate follicles were measured and blood were drawn from jugular vein at approximately around scanning and the plasma were used for measurement of progesterone (p4) and estradiol-17p (e2) concentration. there was no different in term of number of sf, mf and lf (p>0.05) between the two groups. similarly, no effect of side (left vs. right ovary) and cl position (ipsivs contralateral to the ovary) was found (p>0.05). however, it was demonstrated that mean number of ovulatory follicles was higher (po.01) in the spontaneously ovulating group while the regressing-cl size was larger in the cloprostenol induced animals (p<0.05). occurrence of time of ovulation in relation to initial signs of estrus was observed in both groups of animals. this variation could be attributed to the existence of a large preovulatory follicle which enhanced the time ovulation. conversely, when subordinate follicles showed grow up and replace the large follicle the interval from heat to ovulation was prolonged. progesterone experienced a more steep decrease than the spontaneous group of animals and a positive correlation was observed between the diameter of cl and the p4 concentration. for e2 there was a positive correlation between the e2 and follicular size. it is concluded that the variation in follicular among animals contributed to the variability in timing of ovulation, particularly prostaglandin-induced animals. the diameter of ovulatory follicle in spontaneous group was larger as compared to inducedgroup. key words : follicular development, ovulation, cloprostenol,cattle reproduction introduction preovulatory follicular development and its regulation in cattle has so far remained unclear (assey et al. 1993; lindsay et al. 1996; ireland et al. 2000; evan 2001; burn et at. 2005). detailed studies of the pattern of follicular development during the estrous cycle has been based on slaughter house material (rajakoski 1960; dufour et al. 1972) and ultrasonography (pierson and ginther 1989a; pierson corresponding author: b.purwantara@biotrop.org biotropia vol. 13 no.2,2006 and ginther 1989b; fortune et al. 1988; rivera et al. 2001; ginther et al. 2001; webb et al. 2003) however, the processes by which the ovulatory follicles is selected is still not clear (staigmiller and england 1982; pierson and ginther 1988 and quirk et al. 1986; mihm et al. 2000; evan 2003; kobayashi et al. 2006), although india ink marking of preovulatory follicles combined with cauterization of certain larger follicles (matton et al. 1981) has improved the understanding of the underlying mechanisms. the development of clinical ultrasonogaphy (quirk et al. 1986; pierson and ginther 1989a; pierson and ginther 1989b; savio et al. 1988; sirois and fortune 1988; purwantara et al. 1993; purwantara et al. 1994; ginther et al. 2001; frike et al. 2002) which allows visualization of ovarian follicles over time, has given even a better insight. it is, however, still an open question when the follicle destined to ovulate can be recognized and how this affect the subordinate follicular population. the timing of ovulation in cattle has in most studies been determined in spontaneous non superovulated cows/heifers either by rectal palpation (schams et al. 1977) or by ultrasonography (larsson 1987), and in superovulated cattle (savio et al. 1990; fortune et al. 1991; purwantara et al. 1994). however, no detailed studies are available concerning a possible relationship between the occurrence of ovulation and preovulatory hormonal parameters (progesterone and estradiol 17p in neither spontaneous nor prostaglandin induced luteolysis and ovulation. the aim of this study was, therefore during the preovulatory periods in spontaneous vs. induced luteolysis (1) to monitor the development of ovulatory and non-ovulatory follicles at various times prior to ovulation (2) to characterize the regression of the corpus luteum, and (3) to determine progesterone (p4) and estradiol (e2) profiles and relate those to the time of ovulation. materials and methods animals fourteen heifers of mixed beef cattle breed between 2-3 years of age and weighing between 300-500 kg were used in the experiment. the animals had normal estrous cycles at least once prior to the study. subsequently, the animals were divided equally into two groups, the spontaneous-group and the induced-group. spontaneous-group: following induced estrus using a single injection of cloprostenol (estrumat vet., cooper animal health ltd., denmark) and ovulation, the animals were left alone for approximately 19-20 days at which time close observation for a spontaneous heat was performed three times daily. induced-group: on day 9-12 of estrous cycle (day estrus = day 0) the heifers received a single injection of cloprostenol (estrumat vet., cooper animal health ltd., denmark) to induce estrus and ovulation. 76 preovulatory changes and ovulation in cattle b. purwantara et al. ultrasonography a real-time b-mode diagnostic ultrasound equipments (concept ultrasound scanner, dynamic imaging ltd., uk) equipped with a linear array 7.5 mhz transrectal transducer was used. detailed procedures of the ultrasonographic examination of the ovaries were performed as described earlier by purwantara et al. (1994) and the schedule was as follows: in the spontaneous-group, scanning was performed daily from day 15 of the estrous cycle until onset of heat and then every 4 h. in the induced-group daily ultrasonographic scanning were done two days prior to cloprostenol injection and until the onset of heat. from the onset of heat and until ovulation was confirmed the animals were scanned every 4 h. the number of small (sf, 2-4 mm), medium (mf, 5-9 mm), and large (lf, >10 mm) size ovarian follicles were counted and the diameter of the largest, subordinates and the diameter of corpus luteum were measured by using built-in integral caliper. corpus luteum integrity were scored as 3 (highly intact), 2 (moderate) and 1 (less intact, difficult to discriminate) depending on the gray shade and the demarcation line between the corpus luteum and the stroma. each ovarian scan was recorded by means of video recorder and diagram of each ovary were drawn. individual preovulatory and its subordinate follicles were closely examined. ovulation was defined as the disappearance of the largest non-echogenic area (dominant follicles) between two consecutive examinations. blood sampling and hormone measurement daily blood sampling were drawn from jugular or coccygeal vein using heparinized vacuum tube. blood plasma was separated by centrifugation at 3000 rpm for 15 min and stored at -20°c until analysis was performed. progesterone (p4), estradiol-17p (e2) and lh concentration were analyzed in all samples by ria according to h0ier (1989), respectively. the interand intra-assay coefficient of variance (cv) of p4 measurement was 3-10% and 715% respectively, and <10% for e2 depending on the position of displacement of curve. the sensitivity (least detectable concentration) was 0.25 ng/ml and 4 pg/ml for p4 and e2 measurement, respectively. statistical analysis data were analyzed using general linear model for repeated measurement analysis of variance of statistical analyses system (sas 1986). the effect of group (spontaneous vs. induced) and day on mean number of sf, mf, lf and total follicle (tf) were determined. the similar effect were examined on the diameter of ovulatory follicle and its subordinate, corpus luteum diameter, progesterone (p4) and estradiol-17p (e2) concentration. regression analysis and pearson correlation coefficient were determined to relate cl diameter and p4. correlation analysis were also performed to examine relationships between the occurrence of ovulation and the rate of cl regression and the changes on diameter of ovulatory follicle. 77 biotropia vol. 13 no.2, 2006 results and discussion occurrence of estrus was subject to a great deal of individual variation but it was in general observed on between day 18 and 23 of the estrous cycle in heifers undergoing spontaneous luteolysis and on the third day after cloprostenol injection. in the spontaneous-group, the interovulatory interval ranged from 19 d 2 h to 23 d 10 h. in the induced-group, ovulations occurred from pg-injection, ranging from 64 (62-66) h to 126 (124-128) h. in 4 animals where the lh surge was clearly identified, the interval between lh surge to ovulation ranged from 24 to 26 h. a pronounced individual variation occurred in terms of the time which lapsed between prostaglandin injection (induced-group) or cl regression (spontaneous-group) and subsequent ovulation. it is anticipated at least in the induced-group that the stage of follicular development plays an important role on these differences. savio et al. (1990) and ginther et al. (2001) observed that the majority of ovulation originated from the follicles which was dominant on day 7 at the time when prostaglandin was injected. moreover, fortune el al. (1991) and evan (2003) confirmed that the majority of the dominant follicle undergo ovulation when luteolysis was induced during the growing or early plateau phase. in contrast, when prostaglandin was injected during the late plateau phase or atresia phase, the largest follicles failed to ovulate and its subordinate became the ovulatory follicles. in two heifers ovulating within 62-66 h (<3 days) after cloprostenol injection, the ovulatory follicle was generally not difficult to determine as a growing larger follicle where the subordinate had diameters less than 5 mm during the last 3 days prior to ovulation. no other large and medium size follicle were detected in these animals, the follicle which became the largest follicle during the 3 days prior to ovulation. this follicle underwent ovulation while the other became atretic. the other two of these late-ovulating groups developed two large follicles >10 mm which both increased in size until ovulation. ovulation occurred from the follicles which remained the largest during the last 5 days prior to ovulation. in the animals where ovulation occurred within 3 and 4 days after cloprostenol injection shift in dominance was observed only in one animal, while in the remaining, the dominant ovulatory follicles suppressed their subordinates which underwent atresia. evidence from this study showed that a shorter interval between injection of cloprostenol and ovulation occurred when a single large follicle with no medium size subordinate follicles were present during the period of luteolysis. in contrast, the interval was longer when the dominant follicle had to be or was replaced by one of the subordinate or when more than two large follicles developed during the phase of luteolysis. this is in agreement to larsson (1987) who found an increase in the length of proestrus period when 2 follicles greater than 10 mm in diameter were present in that period. some developmental changes during the transitional phase from early to late plateau phase of growth, characterized by a loss in the ability of the dominant follicle to ovulate may contribute to this variation (sirois and fortune 1990; ginther et al. 2001; evan 2003; burn et al. 2005). ovulation was not dependant upon the side. ovulations occurred on the right ovary in 55 % of the induced-group (n=ll) and in 56 % of the spontaneous-group 78 preovulatory changes and ovulation in cattle b. purwantara et al (n=14). the trend was not statistically significant (p>0.05). ovulations occurred ipsi-lateral or contra-lateral to the corpus luteum in 55% or 45%, respectively of the spontaneous ovulations and 64% or 36%, respectively of the induced ovulations. one heifer developed two large size growing follicles on the same ovary and they both ovulated. these ovulations occurred within a 4 h interval and no extended preovulatory period was observed. no significant difference were demonstrated on the mean number of sf, mf and lf between the groups (p>0.05). it was noticeable as seen on fig. 1 (a), that the ovulatory follicle in the spontaneous-group attained a larger mean diameter than the ovulatory follicles in the induced-group. mean diameter of ovulatory follicle was different between group (p<0.01) and day (p<0.01). in the spontaneous-group as seen on fig. 1 (b), the mean diameter of subordinate follicles remained fairly stable until 2 days prior to ovulation at which time they decreased, a pattern which was contrary to the induced group the interovulatory interval was subject to a wider variation in the group undergoing spontaneous ovulation. perhaps, the number of waves has influenced this variation. it was reported by ginther et al. (1989a) that the length of the interovulatory interval was in average of 2.4 days longer in 3-wave than in 2-wave cycles. other studies on the other hand (savio et al. 1988; sirois and fortune 1988) were unable to find differences in the interovulatory interval between the 2-wave and 3-wave pattern animals. since this study was limited to the preovulatory period, these contradictory findings could not be confirmed. in this particular period, however, it was reported that the length of interval from luteal regression to ovulation (ginther et al. 1989a; ginther et al. 1989b) and growth of follicle during luteolysis (savio et al. 1988; sirois and fortune 1988) did not differ between 2-wave and 3-wave pattern animals. the number of small, medium and large size follicle was not different between the two groups and days. this indicated a similar pattern of folliculogenesis in spontaneous and induced ovulations. in addition, during luteolysis no major changes 79 biotropia vol. 13 n0 .2, 2006 in follicular population was observed except the disappearance of ovulatory follicles at the time of ovulation. no ovulation occurred when the diameter of follicle was less than 10 mm. the larger mean diameter of ovulatory follicles which was observed in spontaneous-group is not in agreement to quirk et al. (1986) who found no significant difference of ovulatory follicle development in spontaneous vs. cloprostenol-induced ovulation. this emphasizes that the size alone is not a good indicator of readiness to undergo ovulation. an interesting pattern was observed concerning the mean diameter of subordinate follicles in the spontaneous and induced-group. in the spontaneous group, the mean diameter remained stable until proestrus and then decreased dramatically, while in the induced -group the subordinate follicles tended to increase in size until estrus and then decreased slightly. it is speculated, therefore, that the ovulatory follicles in the spontaneous-group had exhibited a clear dominancy several days before estrus, while those in the induced-group was not selected until the latest stage of the cycle. in this study ovulations occurred with an equal frequency on left and right ovary. this is different from previous studies (rajakoski 1960; pierson and ginther 1989b; purwantara et al. 1992) which reported that ovulations occurred more frequently on the right than the left ovary. in addition, we found no ipsior contralateral effect of cl on ovulation in the spontaneous-group and only a slightly greater number of ovulation from ipsilateral cls in the induced-group. this is conflicting to rajakoski (1960) who found a significant greater number of ovulation occurred from cl bearing ovary. as depicted in fig. 2 (a), the mean diameter of corpus luteum was affected significantly by the group (p< 0.01) and the induced-group maintained a higher diameter. a similar trend was indicated on the effect of day (p<0.01). 80 the cl was scored as 3 at the day of cioprostenol injection, then it changed to 2 sometime between the second and third days and reached score 3 at the day or 1 day before -ovulation. in the spontaneous group score 3 existed 5-4 days before ovulation, then attained score 2 during the following 2-3 days and reached score 1 at 1-2 days prior to and until the day of ovulation. the mean concentration of p4 decreased following cioprostenol injection as seen on fig. 2 (b) and more dramatically in the induced-group. however, in some heifers, p4 concentration did not decline to reach a level under 1 ng, which is defined as the level compatible with luteal regression. on the other hand, the concentration of e2 increased and reached a maximum level 48 h prior to ovulation, and then decreased (fig. 3). a positive correlation was found to exist between p4 concentration and cl diameter during the regression period (r=0.51, po.01). similarly was plasma p4 and cl integrity (r=0.49, p<0.01), and cl diameter and cl integrity (r=0.86, po.01). the mean diameter of the regressing cl was larger in the induced-group compared to spontaneous-group and although subject to great variation, the concentration of p4 was also greater in inducedgroup until 3 days prior to ovulation. it is interesting to note that the dramatic decrease of p4 in the induced-group between day 5 and day 3 prior to ovulation was not accompanied with a similar trend in the decrease of cl diameter, but rather related to the cl integrity. we speculate that the decrease of p4 concentration during luteolysis in the induced-group occurred ahead of decrease in cl diameter, and was different from the spontaneous luteolysis. concentration of e2 reached the peak 2 days prior to ovulation or about 1 day before the lh surge which it is in agreement to other studies (harrison et al. 1985; dutchen et al. 1994; rhodes et al. 1995; singh et al. 1997; valdez et al. 2005). conclusions it was concluded that the timing of ovulation following luteolysis varied between animals both in spontaneous and inducedgroup. this variation was predominant in the induced-group and it was shorter when the single large 81 biotropia vol. 13 no.2,2006 preovulatory follicle existed, and tended to occur later when a subordinate follicle had to replace a large follicle and became the preovulatory follicle. no effect of side (left or right ovary) and ipsior contralateral position of cl to the ovulatory follicle was found. population of small, medium and large size follicles was not different between induced and spontaneous-group. however, mean diameter of ovulatory follicle was larger in spontaneous compared to induced-group. moreover, the diameter of subordinate follicles and cl regression varied between the two treatment groups. acknowledgments this study was supported by animal biotechnology research center, royal veterinary and agricultural university copenhagen denmark and danida. the authors also would like to thank mr. ben s01oy for providing assistance for the hormonal analysis. references assey, r.j., b. purwantara, t. greve, p. hyttel and m. schmidt. 1993. corpus luteum size and plasma progesterone levels in cattle after induction of luteolysis. theriogenology, 39 (6): 1321-1330. burns, d.s., f. jimenez-krassel, j.l.h. ireland, p.o. knight and j.j. ireland. 2005. numbers of antral 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functional correlates. j. reprod. fertil., 109:35^4. sirois, j. and j.e fortune. 1988. ovarian foilicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. biol. reprod., 22. 308-317. staigmiller, r.b. and b.c. england. 1982. folliculogcnesis in the bovine. theriogenology, 17: 43-52. 83 biotropia vol. 13 no.2, 2006 valdez k.e., s.p. cuneo, p.j. garden and a.m. turzillo. 2005. the role of thecal androgen production in the regulation of estradiol biosynthesis by dominant bovine follicles during the first folicular wave. j anim. sci., 83: 597-603 webb, r., b. nicholas, j.g. gong, b.k. campbell, c.g. gutierrez, h.a. garverick and d.g. armstrong. 2003. mechanism regulating follicular development and selection of dominant follicle. reprod. suppl., 61:71-90. 84 75.pdf 76.pdf 77.pdf 78.pdf 79.pdf 80.pdf 81.pdf 82.pdf 83.pdf 84.pdf 1 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al.biotropia vol. 15 no. 1, 2008 : 1 11 * corresponding author: mafupaz@hotmail.com growth and development of fimbrist ylis miliacea (l.)vahl mahfuza begum1*, abdul shukor juraimi 2, rajan amartalingam 2, syed omar bin syed rastan3 and azmi bin man4 1department of agronomy, bangladesh agricultural university, bangladesh 2department of crop science, faculty of agriculture, upm, 43400 serdang, malaysaia 3department of land management, faculty of agriculture, upm, 43400 serdang, malaysia 4mardi, pulau pinang, malaysia abstract this experiment was conducted in the glasshouse of universiti putra malaysia, to determine the growth and development of fimbristylis miliacea (l.) vahl. twenty f. miliacea seeds were surface sown in ten plastic buckets of 18 cm diameter filled with 3 kg soil. after germination only one plant/bucket was retained. time of first seedling emergence, time and number of leaves appearing until first tiller formation, time of tiller formation, first inflorescence, the first 10 inflorescences appearance and their maturity were recorded for each plant. plant height and the number of inflorescence per plant was recorded weekly for up to 4 months after sowing. the first ten inflorescences for each plant were tagged after emergence, subsequently mature inflorescences were collected and the numbers of spikelets/inflorescence, seeds/inflorescence, seeds/ plant and 1000 seed weight were determined. statistical analysis was performed as complete randomized design on weekly observed plant height and inflorescence number using the sas statistical software and means were tested using tukey’s studentized range test at the 5% level of probability. fimbristylis miliacea seedlings emerged at 3 days after planting of seeds. approximate times required for the sequential production of 10 leaves, tillers, first 10 inflorescence and their maturity were 28 days after emergence (dae), 35 dae, 49 dae, 63 dae, respectively. plant height increased rapidly from 3-8 wae and maximum plant height (64.05 cm) was attained at 10 wae. this species had three important growth stages: a slow growth stage during the first 4 weeks after emergence (wae); a rapid growth stage from 4-9 wae; and finally, a maximum growth stage from 9-17 wae. within this first 4 weeks after emergence would be the most appropriate time for controlling this species with early post-emergence herbicides. each f. miliacea plant produced on average of 2.3 tillers/plant and a total of 134 inflorescences, with 84 inflorescences/plant ripening within this period. each inflorescence comprised 48 spikelets with 511 seeds and matured after 3 weeks of emergence. total seeds/plant and 1000 seed weight were 42275 and 0.035 g, respectively. time required for seed ripening was 76 days after emergence. key words: life cycle, growth, development, fimbristylis miliacea 2 biotropia vol. 15 no. 1, 2008 introduction species differ substantially in life form and timing of stages of development and reproduction. each of these processes occurs at a measurable, requisite rate. successful individuals grow rapidly or largely, develop through the various stages of their life cycle, and eventually are replaced in the environment by their progeny (radosevich et al. 1997). the components of the life cycle such as timing of germination, seedling emergence, adult survival, age of flowering, and seed numbers are pre-requisites to be determined during the life span of the plant (sabrig 1980; silvertown and doust 1993). the plant developmental phases are fundamental to the understanding of plant function, that is, the manner of interaction within the environment. fimbristylis miliacea is a serious weed in ricefields in south-east asia (moody 1989; smith 1983). in spite of the importance of this weed little is known about the species growth and development. in several annual and perennial weed species, studies on growth and developmental characteristics have increased the understanding of the weed’s morphological characteristics that aid in success, and or exploited in a control program. smith and fick (1938) studied the life history of purple nutsedge (c. rotundus) and related several growth characteristics to possible control methods. they found that effective control can be obtained if control measures are taken early in the life cycle of the plant. mcwhorter (1961) conducted similar experiments with johnsongrass [(sorghum halepense) (l.) pers.]. they concluded that eradicating johnsongrass plants must be accomplished before new rhizomes are formed (6 to 8 weeks) to be effective. chandler et al. (1977) stated that in moonflower weed the total number of seed per pods and seeds produced per plant were 2730 and 9350, respectively, with peak production occurring 5 through 7 weeks after initiation of anthesis. controlling this weed within this period is to eliminate seed production. thus, the time of flowering and seed production are thus important to know. growth and development study have been observed in several weed species such as echinochloa crus-galli (azmi 1994; itoh 1991; arifin 2004), leptochloa chinensis (pane 1997), ischaemum rugosum (bakar and nabi 2003), cyperus esculentus and c. rotundus (williams 1982). even though, biological study of f. miliacea has been reported to some extent by burkill (1935) and holm et al. (1977). but, in recent past growth and development studies on f. miliacea have not been reported. an understanding of the phenology of a weed is important for developing strategies for its control. differences in the behavior of species at each stage reveal to differences in susceptibility to control by a particular approach. information on occurrence of emergence, leaf formation, tillering, flowering, fruiting and other developmental processes would facilitate the timing of effective control measures. therefore, the study on the growth and development of f. miliacea was undertaken with the objectives of (i) to determine f. miliacea growth characteristics from seed germination to seedling establishment and subsequent seed production and (ii) to quantify the time of occurrence of all growth-developmental stages of fimbristylis miliacea. 3 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. materials and methods the experiment was conducted in the glasshouse at universiti putra malaysia, from november 2003 to march 2004. seeds of f. miliacea were collected from a ricefield at the malaysian agricultural research development institute (mardi) research station, in bertam, pulau pinang on august 2003. the soil used was collected from a tanjung karang ricefield, which was a silty clay (49.71% clay, 49.56% silt and 0.73% sand) of bakau series (typic hydraquents) with ph 4.98. twenty seeds were surface sown in 18 cm diameter plastic buckets filled with 3 kg soil. ten buckets were used for this experiment. after germination, only one plant/bucket was retained. all other seedlings were removed as soon as they emerged. one third of the urea, whole triple super phosphate (tsp) and muriate of potash (mop) were applied as basal fertilizer treatments at rates equivalent to 370, 167 and 250 kg/ha, respectively. another two thirds of the urea was applied at 35 and 55 days after sowing. based on area basis each bucket received a total of 0.93 g urea, 0.42 g tsp and 0.63 g mop. the buckets were irrigated with tap water and maintained at saturation condition. malathion was applied when required to control insect pests. time of first seedling emergence, time and number of leaves appearing until first tiller formation, time of tiller formation, and first inflorescence appearance were recorded for each plant. plant height was measured weekly from the soil surface to the tip of the leaf. time of appearance of the first 10 inflorescences and their maturity was also recorded to determine the average total time to maturity of fimbristylis miliacea. the number of inflorescence per plant was recorded weekly for up to 4 months after sowing to correlate with the life span of cultivated rice. the first ten inflorescences for each plant were tagged when they emerged. these inflorescences were collected when mature and the numbers of spikelets/inflorescence were determined. these ten inflorescences were then dried for 1 week, threshed by hand and the seeds separated by winnowing. the seeds were weighed and number of seeds per inflorescence were estimated using 1000 seed weights. the number of seeds/ inflorescence was derived from the average number of seeds/10 inflorescence per plant which was as follows (charotte et al. 1990): weight of seeds from 10 inflorescence (g) no. of seeds per inflorescence = --------------------------------------------------- × 1000 seeds weight of 1000 seeds (g) × 10 thus total seeds per plant were estimated from the number of seeds per inflorescence multiplied by the number of matured inflorescence per plant. statistical analysis was performed on weekly observed plant height and inflorescence number using the sas statistical software and means were tested using tukey’s studentized range test at the 5% level of probability. 4 biotropia vol. 15 no. 1, 2008 aaab b bc d e f fg g ghh gh fgh efg def de cd bc ab a 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 week after emergence (wae) p la nt h ei gh t ( cm ) 0 20 40 60 80 100 120 140 160 in flo re sc en ce n um be r plant height (cm) inflorescence number results and discussion growth characteristics of fimbristylis miliacea growth rate as measured by increases in plant height over time was significant. initially, the plants were stunted and increase in plant height was slow for the first 2 weeks (figure 1). figure 1. weekly measured plant height and inflorescence number of f. miliacea (means within week after emergence (wae) with same alphabets are not significantly different at p ≥ 0.05 (tukeys test)) however, at the 3rd week after emergence, a significant rapid increase in plant height (10.5 cm) was observed and significantly higher weekly increasing rate was observed for up to 8 weeks. during this time the plant reached 96.57% of its maximum height. at 10 weeks after emergence the plant reached maximum height (64.05 cm) (figure 1). beyond this period, height did not increase due to onset of leaf tip senescence. a similar pattern was obtained in l. chinensis, where for the first 20 days, plants grew slowly, while during 20 – 50 days after sowing (das) the plant height increased rapidly to 100 cm and with increases of 2-3 cm/day. at 70 das the plants reached maximum height (pane 1997). pennisetum polystachyon which grows to a height of 201 cm, also showed slow initial growth for up to 8 weeks followed by a rapid increase in height at 14 weeks, resulting from the elongation of the internodes at the onset of reproduction (fernandez 1982). in contrast the fast growing weed species, purple moonflower reached 85% of its height within by 4 weeks after emergence (chandler et al. 1977) and scirpus maritimus increased it height at the rate of 2.7 cm/day during the first 40 days of growth (visperas and vergara 1978). differences in plant height are an important 5 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. criteria determining species competitiveness as well as to timing of control measures. growth studies on giant burred (sparganium eurycarpum englem.), a perennial weed in annual wild rice, demonstrated that during early season growth a height difference existed between the weed and the crop which allowed for selected herbicide application ( john and ervin 1990). in some crops, efficient and safe use of herbicides, requires a height differential between the crop and the weed in order that the weed can be sprayed with minimal contact with crop foliage. tiller formation in f. miliacea was 1-4 per plant and average tiller production was 2.3 per plant (table 1). total production of inflorescences/plant was 134 within the 4-month period (table 1). positive correlation was found between tiller numbers and number of inflorescence produced (figure 2). when tiller production increased from 1 to 4, the inflorescence production showed a linear increase from 67 to 210. weekly increases in inflorescence production were significant (figure 1). the lowest inflorescences number was observed at 7-week after emergence (wae). inflorescence number then gradually increased from 10 to 16 wae. there were no significant differences between 16 and 17 wae. during the 4 months period a total of 84 inflorescences/plant matured, and percentage of ripened inflorescence was 63.22% (table 1) table 1. growth characteristics of f. miliacea plants growth parameter mean ± se plant height (cm) 64.05 ± 6.07 no. of tillers/plant 2.3 ± 0.483 no. of produced inflorescence/plant 133.8 ± 42.39 no. of ripened inflorescence/plant 84 ± 25.33 % ripened inflorescence/plant 63.22 ± 4.57 no. of spikelets/inflorescence 48.96 ± 6.82 seed no./inflorescence 511 ± 76 seed weight/plant (g) 1.374 ± 0.330 seed no./plant 42275 ± 6696 1000 seed weight (g) 0.035 ± .00215 each inflorescence was laddered with 49 spikelets and 511 seeds (table 1). the total seed and 1000 seeds weight were 1.37 g and 0.035 g, respectively. total number of seed/plant was 42 276. although in the phillipines holm et al. (1977) observed that seed production of each f. miliacea plant was 10 000. actually, seed output is a highly plastic character and is responsive to local growing conditions particularly nutrient availability, day length, and plant density. echinochloa crus-galli seed production per plant ranged from 10 776 – 47 850 (itoh 1991). whereas, azmi and itoh (1991) reported that the 6 biotropia vol. 15 no. 1, 2008 e. crus-galli complex produced less seeds 12 129 seeds/plant in the ricefield due to the impact of competition with the direct-seeded crop. most annual weeds produce a few thousand seeds per individual when growing with minimal competition, though some produce 10 000 to 25 000 seeds per plant (salisbury 1942), while others like e. crusgalli under some situations may produce over 100000 seeds per plant (young 1986; norris 1992). a few annuals (e.g. varonica hederifolia) produce fewer than 100 seeds per individual plant (salisbury 1942; boutin and harper 1991). the number of seeds produced by a plant is the product of three variables: (i) the size of the plant (ii) the proportion of photosynthate allocated to seeds, and (iii) the mean seed weight. the seed production capacity of weeds varies greatly both within and between species. williams (1982) reported that the growth of c. esculentus and c. rotundus as characterized by their total dry weight, inflorescence dry weight, root and rhizome dry weight and number of shoots per pot was similar, but they differed in the manner in which the dry weight was partitioned to reproductive structures. each species partitioned less than 2% of its dry weight into floral formation. however, c. esculentus partitioned only 38% of its dry weight to tubers, whereas c. rotundus partitioned 50% of its dry weight to fewer and larger tubers. in the case of i. rogusum, seeds/spike, seed weight/plant (g) and seed number/plant and 1000 seed weight (g) was 57, 24.5, 6000 and 4, respectively (bakar and nabi 2003), which indicates much bigger seed size and lower seed number/plant than fimbristylis miliacea. growth stages of fimbristylis miliacea emergence of f. miliacea started at 3 days after planting of seeds (table 2). approximate times required for the sequential production of leaves, tillers, first 10 inflorescence and their maturity are shown in table 2 and figure 2. at 7 days after emergence (dae) the plants were at the 3-4 leaf stage. table 2. approximate time required for seedling emergence, leaf, tiller and inflorescence formation and inflorescence ripening growth parameter mean time (days after seedling emergence) ± se 1st seedling emergence 3 ± 0 2nd leaf formation 2.82 ± 0.40 3rd leaf formation 5.27 ± 0.65 4th leaf formation 7.65 ± 0.84 5th leaf formation 10.32 ± 0.95 6th leaf formation 12.18 ± 0.75 7th leaf formation 14.5 ± 0.58 8th leaf formation 18.33 ± 0.82 7 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. growth parameter mean time (days after seedling emergence) ± se 9th leaf formation 20.67 ± 0.52 10th leaf formation 25.0 ± 1.05 11th leaf formation 29.0 ± 0.82 12th leaf formation 33.43 ± 1.72 1st tiller formation 35.2 ± 3.68 2nd tiller formation 40.41 ± 3.36 3rd tiller formation 41.57 ± 3.36 4th tiller formation 42 ± 0 1st inflorescence 49 ± 2.78 5th inflorescence 57 ± 2.70 10th inflorescence 63 ± 1.94 inflorescence ripening (after formation) 17.90 ± 1.62 inflorescence ripening (after emergence) 76 ± 6.71 at 14 dae the plant had 6-7 leaves and within 28 dae the plants had reached the 10-11 leaf stage. at 4 weeks (28 days) the plant started to produce tillers. observation at 35 dae showed that the plant produced the first tiller from the 5-7 leaf node while at the 11-12 leaf stage. the first 4-6 leaves senesced before first tiller formation. obviously, leaf formation with time varies from species to species. pane (1997) observed that l. chinensis produced 4-6 leaves at 20 das and produced 2-3 tillers with 1-3 inflorescences at 30 das. whereas, a very fast growing species like e. crus-galli produced the first 2 to 3 leaves within 4 to 7 days after emergence, and seedlings started producing tillers at 14-18 days after emergence (itoh 1991; azmi et al. 1995). tiller formation stopped at the onset of the reproductive stage. all tiller formation occurred 42 days (7 weeks) after seedling emergence, followed by the emergence of 1st inflorescence within one week, whereas in i. rugosum, production of new tillers ceased 3-4 weeks after floral initiation, but resumed at 16 weeks when most of the spikes had ripened. new tillers were usually unbranched, reduced in height with less seeds/spike (bakar and nabi 2003). azmi et al. (1995) reported that e. crus-galli headed at 44-55 dae. in this study, f. miliacea produced 10 inflorescences within 63 dae (9 weeks). the inflorescence matured after 18 dae (3 weeks) of emergence (table 2 and figure 2). total time required for emergence to seed maturation was 76 days. in i. rugosum approximately 4 weeks was required for spikes to ripen (bakar and nabi 2003). in l. chinensis, panicles ripened at 50-60 days after seeding (pane,1997), while 42 – 56 days table 2. continued 8 biotropia vol. 15 no. 1, 2008 y = 43.901x + 32.827 r2 = 0.9652 0 50 100 150 200 250 0 1 2 3 4 5 tiller number in flo re sc en ce n um be r were required to heading and 89 98 days were required for maturity in e. crus-galli (itoh 1991; arifin 2004). in this study, 80% of f. miliacea leaves were dead at harvest (120 days). the time required to reach reproductive maturity in weeds varies considerably and often similar to the companion crop or sometimes considerably shorter. in the tropics, weed life cycles may be extremely short. echinochloa colona, setaria verticillata and d. aegyptium approach flowering in 30-45 days whilst rottboellia cochinchinensis produced mature seeds within 50 days of establishment (fisher et al. 1985). similar short duration life cycles may also be observed in weeds of temperate latitudes (e.g. capsella bursa-pastoris l.), but weeds of major importance tend to have an extended growing season approaching to at least 6 months. from comparative studies, m. vaginalis, c. difformis, f. miliacea and e. glabrescens have been reported to begin flowering even when plants have achieved less than 10% of their maximum biomass, with seed production continuing for more than four months (kim and moody 1989). the growth stages of f. miliacea could be classified into 3 stages (a) slow initial growth stage, from emergence to 4 weeks after emergence (wae), (b) active growth stage, from 4 to 9 wae, (c) maximum growth stage, from 9 to 17 wae (figure 2). figure 2. growth and development of fimbristylis miliacea (l.) vahl in the first 4 weeks after emergence, the plants grew slowly and attained 10-11 leaf stage. this would be the most appropriate time for controlling this species with early post emergence herbicides. during the active growing period plant height was maximum, tiller formation ceased and the plant produced up to 10 inflorescences. in annual plants the foregoing phases of the life cycle follow one another in uninterrupted sequence (larcher 1975). beyond 10 wae f. miliacea reached maximum flowering stage, and leaves, and some parts of flowering stalks and inflorescences gradually became senescent. the phase of seed production in annual plants is often associated with the rapid death of leaves and the appearance of symptoms usually associated with mineral deficiency as if the leaves have been “sucked dry” of some essential nutrient which has been trans-located to meet the greater needs of the seeds. the evolutionary development of annual plants have major activities that require resource allocation, i.e. starting from 9 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. seed, and continuing with the processes of dispersal, dormancy, germination, seedling establishment, vegetative phase, resource capture, growth, flowering, seed production and lastly seed maturity (radosevich and holt, 1984). evidently, f. miliacea also has the same growth pattern. holm et al. (1977) repeatedly emphasizes the ability of weeds to compete against crop species and most of the weeds allocate a large proportion of resources to seed production. thus importance of early weed control for optimum crop production has been recognized for many years by agronomists. perhaps the basis for this view is the morphological and physiological adaptations through common evolutionary origin among weed species and crops that cause early onset of competition. species growth characteristics provide an indication of competitive ability, and understanding weed seedling growth rates is also important when considering weed management strategy that includes the use of herbicides. improper application timing of herbicide can result in poor weed control and the need for remedial treatment. conclusions fimbristylis miliacea seed emergence occurred 3 days after sowing. plant height increased rapidly from 3-8 wae and maximum plant height of 64.05 cm was attained at 10 wae. first tiller formation was at 11-12-leaf stage after 4 wae and the range of tiller production was 1-4 (average 2.3) per plant. inflorescence production was proportional to number of tillers produced. total numbers of 134 inflorescences/ plant were produced, of which the ripened inflorescences/plant and seeds/plant was 84 and 42 276, respectively. one thousand seed weight was 0.035 g. this species has three important growing stages: a slow growth stage during the first 4 wae; a rapid growth stage that generally took place from 49 wae; and finally, a maximum growth stage from 9-17 wae. the inflorescences generally developed 7-8 wae and subsequently matured 11 -12 wae. the time requirement for inflorescence maturation was 3 weeks after inflorescence emergence. agricultural weeds generally share certain properties, including small seed size, high relative growth rate, low early absolute growth rate, tolerance to stress and high reproductive capacity. these differences form the basis for a variety of weed management tactics for successful control. thus, growth and development study of a specific weed species is an organizing principle for the integration of weed management practices. acknowledgments this study was a part of ph d research work, funded as postgraduate fellowship sponsored by third world organization for women in science (twows), trieste, italy. all other research facilities were provided by the irpa project no. 01-02-040778-pr0068/05-05, universiti putra malaysia. 10 biotropia vol. 15 no. 1, 2008 references arifin, t. 2004. morphological and molecular variability amongs ecotypes of barnyardgrass (echinochloa crus-galli var. crus-galli (l.) beauv) : implication for biocontrol. ph. d. thesis. universiti putra malaysia. p. 4.14.28. azmi, m. 1994. biology and control of echinochloa crus-galli (l.) beauv. in direct seeded rice. ph. d. thesis, school of biological sciences, universiti sains malaysia. p. 333. azmi, m. and k. itoh. 1991. echinochloa crus-galli complex. in : life cycles of rice field weeds and their management in malaysia. tropical agriculture research centre, ed. itoh, k., tsukuba, japan, p. 17-27. azmi, m., m. mashhor., k. itoh and h. watanabe. 1995. life cycle and seed longevity of echinochloa crus-galli complex in direct-seeded rice in malaysia. in: proceeding of 15th asian pacific weed science conference, p. 505-511. tsukuba, japan. bakar, b.h. and l.n.a. nabi. 2003. seed germination, seedling establishment and growth patterns of wrinklegrass (ischaemum rugosum salisb.). weed biol. and manag., 3:8-14. boutin, c. and j. l. harper. 1991. a comperative study of the population dynamics of five species of veronica in natural habitates. j. ecol., 79: 199-221. chandler, j.m., r.l. munson, and c.e. vaughan. 1977. purple moonflower: emergence, growth, reproduction. weed. sci., 25(2): 163-167. charrote, v.e., l.l. edith, l.m. timothy and l.m. janis. 1990. growth and development of wild-proso millet (panicum miliaceum) biotypes. weed tech., 4: 415-419. fisher, h.h., f. lopez, l.margate, p. elliot and l. burrill. 1985. problems in control of rottboellia exaltata in maize in bukidnon province, mindanao, philippines. weed res., 25:93-102. holm, l.g., d.l. pluknett, j.v. pancho and j.p. herberger. 1977. the world’s worst weeds: distribution and biology. the university press of hawaii. honolulu. p. 273-279. itoh, k. 1991. life cycles of rice field weeds and their management in malaysia. tropical agriculture research centre, tsukuba, japan, p. 92. john, w.l. and a.o. ervin. 1990. growth and development of giant burreed (sparganium eurycarpum). weed tech., 4:849-854. kim, s.c. and k. 1989. adaptation moody, strategy and seed production of rice and weed species. korean j. weed sci., 12:183-200. larcher, w. 1975. physiological plant ecology. springer-verlag. new york. p.252. mcwhorter, c.g. 1961. morphology and development of johnsongrass plants from seeds and rhizomes. weeds, 9:558-562. moody, k. 1989. weeds reported to occur in rice in south and southeast asia. in: weeds reported in south and southeast asia. international rice research institute. los banos, laguna, p.o box 933, 1099 manila, philippines, pp. 1-80. norris, r.f. 1992. relationship between inflorescence size and seed production in barnyard grass (echinochloa crus-galli). weed sci., 40: 74-78. pane, h. 1997. studies on ecology and biology of red sprangletop [leptochloa chinensis) (l.) nees] and its management in direct-seeded rice. ph. d. thesis. universiti sains malaysia. pp. 1-235. radosevich, s.r. and j.s. holt. 1984. weed ecology: implications for vegetation management. john wiley & sons. new york. p. 265. 11 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. sabrig, o.t. 1980. demography and evolution in plant populations. botanical monogr. vol. 15. oxford. blackwell sci. pub. p.222. salisbury, e.j. 1942. the reproductive capacity of plants. london: bell. silvertown, j.w. and j.l. doust. 1993. introduction to plant population biology. oxford. uk. blackwell sci. pub. p. 210. smith, e.v. and g.l. fick 1938. nut grass eradication studies: i. relation of the life history of nut grass, cyperus rotundus l., to possible methods of control. j. am. soc. agron. 10:107-113. smith, jr.r.j. 1983. weeds of major economic importance in rice and yield losses due to weed competition. in : proceedings of conference on weed control in rice. international rice research institute. los banos, laguna philippines. pp. 19-36. visperus, r.m. and b.s. vergara. 1978. autecology of scirpus maritimus l. growth characteristics and competition with rice. philip. weed sci. bull., 3: 1-13. williams, r.d. 1982. growth and reproduction of cyperus esculentus l. and cyperus rotundus l. weed res., 22: 149 -154. young, f.l. 1986. russian thistle (salsola iberica) growth and development in wheat (triticum aestivum). weed sci., 34: 901905. biotropia no biotropia no. 18, 2002 : 21 37 morphological characterization of malaysian wild banana musa acuminata muhammad asif javed*, mak chai and rofina yasmin othman division of genetics & molecular biology, institute of biological sciences, university of malaya, 50603 kuala lumpur, malaysia abstract fourteen populations of musa acuminata ranging from populations in the lowlands of northern (ssp. siamea) to central malaysian region (ssp. malaccensis) and highland banana (ssp. truncata) were characterized based on chromosome number and 46 morphological characters. a large amount of variation was observed within the populations. however, only highland bananas appeared morphologically distinct. lowland populations both from northern and central malaysia were found to be overlapping and no distinguishing pattern was observed. the morphological characters found variable within these populations were related to developmental changes and mutations. the results obtained in this study were not revolutionary. however, the survey of a large number of characters treated with multivariate techniques further sharpened the existing groupings of the musa acuminata subspecies. key words: musaceae/ malaysia / ssp. malaccensis i ssp. truncata i ssp. siamea introduction banana and plantains are one of the most important tropical fruit crops. they are staple for rural and urban consumers in the humid tropics and an important source of rural income. bananas are grown in 122 countries of the world with a cultivated area of 3.8 million ha and total production of 56.4 million metric tons both for export and local consumption (fao 1999). banana monoculture cultivation provided a powerful driving force in disease epidemics. it was observed in the case of gros michel attacked by fusarium oxysporum f. sp. cubense (foc) race 1 resulted in the collapse of the banana industry. gros michel was then replaced by cavendish bananas resistant to foc race 1. however, recent outbreak of a new foc race 4 attacking cavendish in subtropics and tropics again threatened the banana industry. there is no chemical control available and the only way is to plant cultivars resistant/tolerant to the pathogen (buddenhagen 1990). malaysia is one of the centers of diversity for both wild and cultivated bananas. musa acuminata is the most variable species and progenitor of cultivated bananas (simmonds 1962). m. acuminata was reported to be resistant to fusarium wilt race 1 (vakili 1965). a survey of local wild species has been reviewed by simmonds (1955) and kiew (1987) based on morphological characters. only a few morphological charac 21 biotropia no. 18, 2002 ters were used and characters found to be useful for subspecific classification have been further questioned (hari 1968; shepherd 1988 and 1999). therefore, the subspecific grouping of local musa acuminata has been complicated by taxonomic revisions. recent renewed emphasis on the utilization of these resources for disease resistant studies for molecular banana breeding elucidated the need to characterize different wild musa acuminata forms in malaysia. for breeding and ease of field identification, taxonomic differentiation based on morphological traits seems to be convenient and useful for evaluating germ-plasm. hence the present study aims to describe intra-and interspecies variation in different musa species, using both qualitative and quantitative morphological traits. multivariate techniques are used to describe relationship and to observe the most variable characteristics. materials and method fourteen populations of wild seeded musa acuminata species and two of musa violascens and musa balbisiana (fig. 1) were surveyed for this study (table 1). suckers collected were all planted on the university malaya farm under the same environmental conditions to reduce variation due to environmental factors. suckers were planted in holes to a depth of 30-40 cm with 2 x 2 m spacing within and between rows. weeding was done manually and fertilizer was applied occasionally. 22 marphological characterization of malaysian wild banana – muhammad asif javed et al. *geographical grouping northern peninsular malaysia = kelantan and kedah central peninsular malaysia = pahang, perak, selangor higlands = genting and cameron highlands (pahang) a mean number of five specimens were examined for each sample. the data on different morphological characters were taken during first crop cycle. all accessions planted grow well except bc3 (highland banana) and musa violascens (bc1) which were dead; therefore, morphological data on these accessions were taken in-situ. fruit bunches were also collected for the development of open pollinated seed populations. initial studies on seed germination in the greenhouse were not successful; therefore, in vitro embryo culture protocol was standardized (asif et al. 200la). ploidy was determined using flow cytometery (fcm) as previously described (asif et al. 2001). all samples used are diploid where musa acumlnata and m. balbisiana have (2n = 22) and m. violascens (2n = 20) chromosome number as suggested by simmonds (1962). the vegetative characters were scored on plants with emerged inflorescence. during this time, maximum development of vegetative parts has taken place (purseglove 1972). forty-six characters were used including those suggested by simmonds (1955) and ipgri descriptors (1996). thirty-five qualitative and 11 quantitative characters were included. simple two-state qualitative characters ordered multi-state characters were coded as series of discrete states (table 2). characters related to color were examined using a standard color chart (ipgri 1996). mean values for continuous quantitative characters for the minimum five randomly selected healthy plants per accession were calculated. phonetic analyses were carried out using spss-pc for windows version 9.0 (norusis 1985). cluster analysis and principal component analysis were carried 23 biotropiano. 18,2002 out on a data set including both metric and binary data. a distance matrix of squared euclidean distance was calculated. this distance was used as a measure of phenetic difference. clustering was carried out by single linkage method. table 2. description of morphological characteristics based on banana descriptor (ipgri 1996) and simmonds (1955). 24 marphological characterization of malaysian wild banana – muhammad asif javed et al. 25 biotropia no. 18, 2002 principal component analysis was conducted as it has the advantage of abstracting factors, one of which may represent some underlying variables like overall size, which may affect many characters. there were several significant factors but only the first four were extracted. factor scores were calculated for each otu and the first two plotted in a scatter diagram. results a single linkage cluster method grouped m. acuminata samples into four where bc2 (musa violascens) and gala (musa balbisiana) were clearly separated (fig. 2). the highland banana was clearly differentiated from lowland banana forms based on deep purple brown color of the pseudostem, petioles, and male bracts, a non-waxy petiole and lamina with both sides of lamina bases rounded and creamy colored internal bract face. the lowland populations were found to be overlapping irrespective of their geographical origin and no distinct separation was observed. flava and segun were collected from central region of peninsular malaysia while sintok and j-4 found in the northern region were grouped together and showed large morphological similarities. bd1, bd2, ri and iptj populations were from 26 morphological characterization of malaysian wild banana muhammad asif javed et al. southwest of the peninsula, while bc1, kra and ppc were from the central region (fig. 1). these accessions were morphologically similar possibly reflecting the overlapping of the geographical ranges in the central lowland regions. geographically, rangis and perak were of very diverse origin but showed high morphological similarity, differing only in bract imbrication. rangis is characterized by young bracts greatly overlapping compared to the convolute male bud observed in perak. the last group comprised gala and bc2. these were well separated from m. acuminata with little or no similarity. the position of all musa accessions on the first and second principal components as derived from the original pairwise correlations among the accessions are shown in figure 3. the first factor accounted for 36.73% variation while the second factor showed only 11.14% variation. a significant percentage decrease was observed with components three (10.36%) and four (8.79%). fig 2. phenogram of 16 musa accessions based on morphological characters and chromosome number. musa acuminata accessions were separated into four groups based on single linkage and squared euclidean distance. the highland banana bc3 was well separated at the bottom from lowland accessions. the two species bc2 and gala were also clearly separated from the musa acuminata. 27 biotropia no. 18, 2002 fig 3. principal component analysis. pca showing relative positions on the first and second principal components of 16 accessions of mum species based on 46 morphological characters and chromosome number. musa acuminata samples showed large morphological variation and grouping of the different accessions was in agreement with that of cluster analysis. component one clearly separated the samples ppc and bc3, the highland banana, from rest of the accessions of m. acuminata, whereas second component separated kra. the major groupings observed were bd1, iptj, bc1, bd2 and ri, a second group composed of segun, j-4, sintok, and flava, and finally rangis and perak (fig. 3). table 4 shows that of the 46 characters used in the analysis 13 had loadings of more than 0.5 on the first component. four of these characters i.e. chromosome number, bract apex shape, male bract lifting and fruit apex were found to be useful 28 morphological characterization of malaysian wild banana muhammad asif javed et al. in separating the three species samples m. acuminata, m. balbisiana (gala) and m. violascens (bc2), respectively. five characters could differentiate both interspecific and intraspecific accessions were bunch posture, bunch appearance, bract base shape, bract imbrication and transverse section of fruit. m. acuminata accessions could be differentiated based on these four characters e.g. rachis type, bract behaviour before falling, male flower behaviour before falling and remains of floral relicts . table 3. fruit bunch (five bunches/sample) characteristics of different wild musa acuminata bananas eight characters which accounted for much of the variation were contributed by the second component. the characters like leaf and petiole waxyness, insertion point of leaf blade clearly separated the highland form bc3 from the lowland form of m. acuminata accessions. six characters contributed largely on component three which separated the ri accession based on male bud degeneration at maturity and yellow color of compound tepal while kra and ppc were separated based on the rachis position and free tepal development. the fourth component showed four characters with loadings of more than 0.5. pseudostem color had the highest loading compared to the other three characters and clearly separated the highland banana bc3 from the other accessions. the grouping of accessions in pca corresponded well to that of cluster analysis. 29 biotropia no. 18, 2002 table 4. characters found most variable based on first four principal components. thirty characters with loadings of more than 0.5 (in bold) on components one, two three and four separating different accessions and groups of accessions of musa samples in pca. the majority of samples from the central region, bc3, bc1, bd2, ri and kra tended to be tall. perak, rangis, ppc, sintok, j4, and segun were semi-dwarf with plant heights of less than 2 m. deep purple brown color of pseudostem and petioles were found useful to differentiate highland banana form (bc3). characteristically, 30 morphological characterization of malaysian wild banana muhammad asif javed et al. highland banana showed non-waxy leaves and petioles compared to waxy types in all m. acuminata samples. generally, the leaf sheath in wild bananas is narrow on both sides forming the petiole, which is rounded beneath and channeled above, retaining the crescent shaped section of the sheath. petiole canal of perak, ppc and ri was observed to have wide and erect petiole margins compared to curve or straight erect margins in other samples. sample bc3 had both sides of the lamina pointed, whereas j-4 showed one side rounded and the other side pointed while other samples had round lamina bases. samples bci, bd1, bd2, ri, iptj and bc3 (a highland banana) were characterized with very hairy fruit peduncles. the perak, sintok, j-4 and segun samples have hairless fruit peduncles with the exception of rangis, ppc and flava having slightly hairy peduncles. ppc had a very compact fruit bunch compared to compact or lax fruit bunches in other samples. rachis position was also found to be a useful character to differentiate both kra and ppc, which had rachis falling vertically, compared to the normal horizontal position. fruits were biseriate; rachis and male bud were persistent in all accessions until the fruit maturity except the sample ri where male bud was degenerated before fruit maturity. fruit shape was straight in kra or slightly curved or curved in all other samples. observations made on fruit transverse section showed that sample ppc had a rounded transverse section, while iptj showed pronounced ridges. all samples studied showed a normal male bud (male bud and rachis persisted till fruit maturity) with the exception of ri where the male bud degenerated before fruit maturity. perak, bc3, bci, ppc and flava are characterized with medium size of bract. a strong imbrication, with young bracts overlapping at the apex of the male bud was observed in rangis, whereas flava and segun showed slight bract imbrication. other samples of m. acuminata showed convolute bract behavior. the highland banana bc3 could be differentiated from other forms with a distinct deep purple brown external and creamy color of internal bract face. similarly, flava, a yellow bracted mutant of m. acuminata, added more complexity to bract color characterization. bract curling was also found to be useful for differentiating m. acuminata accessions from m. balbisiana. all samples had basal flowers which were functionally female and contain stamens reduced to staminoids, whereas distal flowers had functionally male flowers and were female infertile. the male flowers fell off before bract dispersal in rangis, bd1 and iptj. compound tepal basic color was observed to be creamy except the sample ri (yellow). free tepal development was observed for samples kra and ppc, whereas all other samples had little or no development. number of male flowers per row ranged from 5 to 11. sample iptj had the minimum number of male flowers per row. the length of the male flowers ranged from 3 cm in the case of iptj and 7 cm for sintok. the maximum filament length was observed in the sample bci (4.4 cm) and the minimum of 1.1 cm in bc3. 31 biotropia no. 18, 2002 as agronomic characters are greatly affected by the environment, they are not useful for classification purposes but are of great importance for breeding new cultivars. the agronomic characters recorded were bunch weight, number of hands per bunch, number of fingers per hand, finger length and finger diameter. fruit bunch weight ranged from 1.96±0.02 to 9.86±0.10 kg. large fruit bunches were observed in samples bc1, bd2, bc3, kra and ppc, respectively (table 3). samples with relative large fruit hands were observed in bc3, bc1, and bd2. for fingers per hand, the maximum fruits were produced by ri (22.8+0.5) compared to a minimum of 12.8±0.5 in bd2. large finger length was observed in the samples ri (14.94±0.2 cm), bc3 (14.90+0.04 cm), and bc1 (14.0±0.32 cm). finger diameter ranged from 1.44+0.03 to 3.5±0.03 cm where large finger diameters were observed in ppc followed by bd2 and bc3, respectively. m. acuminata fruit bunches were found to contain a large number of seeds ranging 30-120 per fruit. seeds range between 5-6 mm in diameter and irregularly subglobose in shape. seeds were grayish-brown in color. germination studies in sand beds resulted into 1-2% germination. however, in vitro embryo culture resulted into more than 90% seed germination (asif et al. 2001a). iptj produced albino seedlings when cultured in vitro. discussions morphological characters used for wild banana characterization are taxonomic and not agronomic. harlan (1975) argued that it will be breeders who will make use of the classifications of crop germplasm and they will be mainly concerned with the genetic compatibility, while morphological characters will be secondary. however, engels (1986) stated that the importance of a germplasm collection to a breeding program is strongly dependent on the availability of accurate descriptions of the accessions and on the taxonomic identification of the germplasm. in this study, 10 quantitative characters were used and only four were found useful, according to pca (table 4). in general, most of the north malaysian accessions were semi-dwarf. however, this character is not useful for classification because of variation due to environmental factors. moreover, for breeding purposes, it appears that plants with semidwarf pseudostem height are preferred and capable of withstanding both stress conditions and fruit bunch weight at maturity without propping. similarly, there is less loss from wind damage, harvesting is easier and broadly, they imply high yields and easy care at a little extra expense for planting material (simmonds 1986). the number of male flowers per row was found to be varying within the same male bud similarly, flowers and style length could be modified by environmental factors. hence, these characters are not useful for characterization (ortiz & sevilla 32 morphological characterization of malaysian wild banana muhammad asif javed et at. 1997). quantitative characters might not be appropriate for such task in musa, because all were significantly affected by genotype environmental interactions. pseudostem and petiole sheath color was observed to be useful (simmonds 1955) in the identification of the highland form bc3 from the other two groups. the sheath color is the result of environment or due to genetic factors, could not be further differentiated, as the highland form did not survive under lowland conditions. lamina bases are also found to be interesting characters. skutch (1930) reported that the meristem of one lamina half frequently extends further down the petiole than the other. there is no rule as to which side is produced furthest basally. in cases of smaller size of the lamina, the inequality is much less conspicuous. the basal inequality of the lamina halves is a lasting testimony that they are of independent origins. very late appearance of the lamina halves, compared with the other portion of the leaf, also points to a phylogenetically later origin. a large amount of variability was observed in the fruit shape of different musa acuminata accessions. however, it was observed to be variable even within the same fruit bunch and thus, did not seem to be a reliable character. persistent style and staminodes were observed only in the perak. however, this character could be related to slow maturity. karamura (1999) stated that clones growing at high altitudes usually have non-persistent styles and stamens, which may take a long time to drop. rachis position could be used to differentiate kra and ppc from other accessions. the absence of the rachis and male bud observed in ri was considered a mutation (de langhe 1961). bract imbrication was regarded as an interesting character both at interspecific and intraspecific levels. bract imbrication was used by simmonds (1955) mainly to differentiate 'kedah form' (ssp. siamea) from the selangor and cameron forms. in the present study three samples, rangis, flava and segun, were observed with imbrication. rangis showed greater imbrication compared to the other two samples. in this study, three samples, sintok, j-4 and rangis, were collected from kedah and kelantan, respectively. whereas the samples flava and segun with slight imbricated bracts were collected from pahang states, the center of diversity of 'selangor form' (simmonds 1955). bract imbrication was found to vary within the accessions of 'kedah form' even within the ^ame geographical range. hari (1968) also recommended that bract imbrication should be rejected as taxonomic character in the classification of musa acuminata accessions subject to morphological changes during bud development. the data confirmed that bract imbrication could not be used as a reliable character for classification. ppc was differentiated by having a very compact fruit bunch hanging at an angle. it was observed that accessions having a pendulous rachis had compact fruit bunches. similar observations were made by de langhe (1961) where he found a pendulous bunch which commonly has fruits requiring towards the peduncle or rachis. however, karamura (1999) observed clones that showed no geotropic reaction, had pendulous bunches. a sub-horizontal bunch posture (angle of female 33 biotropia no. 18, 2002 axis) is under the control of additive gene action of dominant alleles with threshold effect (ortiz 1993). this character is of economic importance as pendulous bunch is the most symmetrical and is therefore better adapted to transportation. great variability also accounted for bract color, both at interspecific and intraspecific levels. however, the situation was more complex at intraspecific level where different samples of m. acuminata had different bract colors. highland banana sample bc3 (ssp. truncatd) was classified based on the bract color. bract color characterization based on anthocyanin analysis using high performance liquid" chromatography (hplc) showed very distinct anthocyanin pattern in highland banana compared to similar anthocyanin composition observed in lowland populations (asif et al. 200 ib). flava, a yellow bracted mutant, showed the absence of anthocyanin (simmonds 1955). during the current study, several of the observed abnormalities included the transformation of the free tepals and the different number of male flowers in alternate rows. different number of male flower rows were also varied in the same male bud of the sample bd2. in the case of sintok, two male flowers were fused together and had a total of 11 anthers instead of 10 (5 each). similarly, sample ri showed fusing of the fruits and degeneration of male bud and rachis. nair and karunakaran (1962) and jacob (1952) have reported similar abnormalities in cultivated bananas. cluster analysis grouped all m. acuminata accessions into four groups where north malaysian accessions were closely related with selangor group and highland banana sample was well separated. pca produced similar grouping of the accestsions and also showed that they were phonetically related. it thus gave a more accurate representation of how similar accessions were phenetically related to each other, although the overall picture was very similar to that of cluster analysis. the first group contained accessions flava, segun, sintok and j-4. they were collected from different geographical regions but showed large morphological similarities. most of the characters were observed to overlap with other lowland m. acuminata accessions (groups 2 & 3). this was similarly observed in the case of rangis and perak, which were collected from kelantan and perak. rangis, a typical kedah form (ssp. siamea), showed similarities with perak, a sample collected from perak (from geographical range of ssp. malaccensis). these characters were variable developmentally (hari 1968; karamura 1999). the second group comprised a large number of accessions mainly collected from the ssp. malaccensis range. they shared many morphological characters and were, as expected, closely clustered together. free tepal development and rachis position could be an interesting character for the identification of ppc and kra. the highland banana accession (bc3) growing at an altitude of 2000 m was clearly separated from the lowland accessions and showed great ecological and morphological variability from the lowland accessions. this accession was also found to be 34 morphological characterization of malaysian wild banana muhammad asif javed etal. different in growth habit, anthocyanin composition (asif et al. 200 ib) and genomic dna content (asif et al. 2001c) compared to lowland forms. it was suggested that the highland banana form ssp. truncata (simmonds 1955), could be separately classified based on morphology since the large morphological variability observed among lowland accessions made it difficult to group them into other distinctive groups. most of these accessions had overlapping morphological characteristics irrespective of their geographical relationship. the variation observed among different forms of musa acuminata could be related to their partial geographical isolation (highlands and lowlands) and changes in gene frequencies due to the small wild banana population sizes resulting from genetic drift and hybridization of different subspecific forms. mutations could also be an important factor contributing to the total variability. different subspecific forms of musa acuminata had been reported to cross frequently within the same geographical range. it had been repeatedly demonstrated that hybridization between species and subspecies belonging to the same section or between species in different sections occur readily in musa. some of these hybrids are fertile. two well-known examples include the m. balbisiana and m. textilis interspecific hybrids referred to as "canton" cultivar; and the other is m. textilis and m. balbisiana hybrid known as "pacol" (tezenas du montcel 1987). the observations based on cytological and field experiments (e.g. dodds & simmonds 1948) showed that several forms are interfertile, thus the whole assemblage formed a 'panmictic unit' (so far as geography permits). simmonds (1955) further suggested that field evidence of hybridity was most striking where populations of the ssp. malaccensis and ssp. truncata occur together. furthermore, the occurrence of mutations further increased the range of morphological variability among the populations (gonzales de leon & faure 1992). the fourteen m. acuminata populations studied consisted of a small number of individuals. small populations may not be stable because of dispersive processes which lead to the division of the populations into groups or sub-populations as observed in the case of wild bananas, where different populations observed in the same area appeared variable. secondly, it reduces the genetic variability and increases homozygosity. since in sub-populations mating between similar individuals takes place (i.e. inbreeding), genetic variability decreases within the groups, some genes are fixed and some eliminated, thus increasing homozygosity (wright 1951). this was observed in most of the wild banana populations where little variation was observed within the populations compared to among the populations and the variation observed was likely due to mutations appearing as a result of inbreeding. the development of different local populations of open pollinated plants is an example of the dispersive processes at work in natural populations. brieger (1950) found a number of local populations of maize in south america where populations were quite distinguishable among themselves but each population was highly uniform. 35 biotropia no. 18, 2002 m. acuminata produced a large amount of seeds as a result of open pollination. majority of the seeds observed had normal embryos, whereas few abnormal seeds showed the absence of embryo/endosperm or both. more than 90% embryos were germinated, thus could enable to study a large number of hybrid populations for banana breeding. bananas are cross pollinated species and it will be interesting to know the effect of inbreeding on seed bearing and germination. morphological characters studied although found useful at interspecific level was less so at intraspecific level. there were only a few morphological characters found to be useful to differentiate highland banana population from lowland, whereas there was no distinct pattern observed within lowland banana populations. therefore, it seems important that more sensitive markers be used to characterize these populations. genomic dna markers may offer the useful systems to increase our knowledge about wild 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(persley, g. j. and e. a. de langhe ed.). proceedings of an international workshop held at cairns, australia, 13-17 october, 1986. skutch, a. f. 1930. on the development and morphology of the leaf of the banana. (musa sapientum l.). amer. j. bot, 17:252-71. tezenas du montcel, h. 1987. plantain bananas. cta. paris and macmillan. london. vakili, n. g. 1965. fusarial wilt resistance in seedlings and mature plants of musa species. phytopathology, 55: 135-140. wright, s. 1951. the genetic structure of populations. ann. eugenetics, 15: 323-354. 37 3. annisa (microclimate).cdr biotropia vol. 19 no. 2, 2012: 80 91 microclimate preference and habitat of begonia in bedugul, bali annisa satyanti and hartutiningsih m. siregar received 17 januari 2012/accepted 21 may 2012 a study on ecology of was conducted in two forest sites, a nature reserve and reboisation forest, in bedugul, bali. the objective of the study was to describe the species found in these forest sites, to gather information on microclimatic variables and to find the influences of these variables on the abundance of species. three species were identified, , and . there were two forms of , white and red. the multivariate analysis (pca) showed that microclimatic variables measured were relatively similar among plots and hence, no particular microclimatic variable influenced species abundance. however, the multivariate analysis implied that has a different microclimate preference between forms (white and red). f. white was highly correlated to axis 1 (99%) and f. red was highly correlated to axis 2 (92%) of the pca graph. the abundance of all species in the two forest sites was similar (t-test, p=0.061). sp., microclimate, natural habitat, bedugul, multivariate analysis 1,2 1 1 2 centre for plant conservation, bogor botanical garden, indonesian institute of sciences visiting scholar (humboldt fellow) at institut für botanik, universität regenburg germany begonia begonia begonia b. multangula b. baliensis b. longifolia b. longifolia begonia b. longifolia b. longifolia b. longifolia begonia abstract introduction key words: begoniaceae begonia begonia begonia et al begonia ex situ begonia is easily distinguished from other forest herbs due to its distinctive habit. species can be found in several forms, either erect or creeping herbs, with succulent stems and asymetric leaves. species are common to secondary and primary forest, along rivers or creeks, mostly in humid places, and around waterfalls (kiew 2005). many species have potential as ornamental plants, either as species or hybrids (tebbit 2005; purwantoro . 2010). the main risks of plant species loss are possibly forest conversion and land use change as well as illegal logging. fluctuating microclimate conditions caused by forest clearance and possibly by climate change may alter the germination and hence, composition of forest understorey. the genus should be a priority in conservation, potentially to allow reintroduction activity. species have * corresponding author : a.satyanti@gmail.com 80 considerable economic value as ornamental plants and some have potential as medicinal plants and as a vegetable (tebbit 2005; chiew 2005, girmansyah 2008). in banten, west java, , known as 'krokot', is popular among locals as vegetable (djarwaningsih 2010). in bali, is used for curing cough (hartutiningsih 2005). in west java, the sundanese called and as hariang or asam-asam for substituting sour taste from in local dishes (purwantoro 2010; wiriadinata 2002) and it is also used for traditional medicine (priyadi 2010). a number of species are close to extinct due to habitat destruction (chiew 2005). research in is challenging as species are really under pressure in their natural habitat; research has to keep up with the pace of species loss due to habitat loss. logging can destroy populations, and limestone quarries have contributed to the extinction of those endemic to limestone hills. the vulnerability of extinction for is compounded by the fact that many are extremely local endemics (kiew 2005). in peninsular malaysia, of nearly 60 species, 26 are known from single localities and some of their populations are small (kiew 2005). therefore, conservation finds its relevance here. kebun raya eka karya in bali, an conservation location in indonesia, harbours more than 60 indigenous species (thomas 2009) and is regarded as the richest collection among botanical gardens in the world (hartutiningsih 2005). conservation efforts begin with exploration activity, which involves the inventory and collection of species, and habitat study. in addition to collection and taxonomic studies, there is a need for ecological study, in particular which environmental factors determine the existence of particular species in its natural habitat. there is very scant microclimate information available for tropical habitats, especially in the case of . therefore, this study aims at describing: (i) which species are found in two forest sites in bedugul, bali, (ii) measure environmental variables in each habitat, and (iii) look for correlation between the recorded environmental parameters and the frequency of species. observation on populations and environmental data were conducted in two locations, namely bukit tapak (08°16'00” s and 115°08'00”e) and (reboisation forest, 08°16'00” s and 115°09'00”e). bukit tapak is mainly secondary forest with somewhat heavier canopy cover which is represented by lower solar radiation lux values in general (table 1, fig. 1). most of the in bukit tapak already produce either flower or fruits whereas in reboisation forest, the observation was done along small open roads which is intensively managed by mowing, leading to shorter plants with less reproductive organs (fig. 2). b. multangula et al. b. multangula b. multangula b. robusta tamarindus indica et al. et al. begonia begonia begonia begonia ex-situ ex-situ begonia et al. begonia ex-situ begonia begonia begonia begonia hutan kontrak begonia materials and methods study site 81 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. bukit tapak, batukaru nature reserve, bali batukaru nature reserve is located in baturiti subdistrict, tabanan and sukasade subdistrict, buleleng. the topography ranges variably from flat to steep, approximately 8% to 45% slope. the area of the reserve is 8557.98 ha, which consists of 3 sections which are resort pemangkuan hutan (rph) pupuan, rph penebel, and rph candikuning (ksda-bali 2009). the access to the nature reserve is via wangaye gede village, subdistrict penebel. the dominant vegetation consists of cemara pandak ( ), cemara geseng ( ), kepelan ( sp.), juwet manting ( ), and seming ( sp.). on forest floors, several species of can be found. dacrycarpus imbricatus casuarina junghuhniana magnolia crypteronia paniculata pometia begonia figure 1. ; hutan tapak siteb. multangula figure 2. (left, middle) and (right); reboisation forest siteb. longifolia b. baliensis biotropia vol. 19 no. 2, 2012 82 reboisation forest area at “eka karya” botanical garden, bali species and data collection data analysis the reboisation area (hereafter also written as ) consists of flat, and hilly topography at an altitude of 1250-1400 m asl (purwantoro 2010). the forest comprises 22-52 years old rasamala ( ), gintungan ( ) and cempaka ( ). other trees and herbs naturally exist including lateng ( ), , sp. and spp. the submontane cool climate of bali botanical garden facilitates good growth of species. the climate is type b with precipitation rate of 2000-3000 mm/year; a 7-9 month rainy season, and 1-3 month dry season, intercepted solar radiation of 4660%, relative humidity of 78-96% and mean wind speed 7.27 km/hour (hartutiningsih 2005). the exploration is using a random purposive method, meaning that one track is taken for each site and standing from the line and observing the existing population, we decided where to establish the plot. we sampled 40 plots, 20 plots in each site. the size of the plot was 0.5 m × 0.5 m. within the plot several measurements were taken for number of individuals as well as the following microclimatic factors: temperature (°c), solar radiation (lux), air humidity (%), soil ph, soil moisture (%) and altitude (m). temperature, solar radiation, and air humidity were measured using lutron lm-8100. to measure temperature, solar radiation, and air humidity the device was located directly above individuals, at about 1.3 m above ground. soil ph and soil moisture were measured using soil tester takemura dm-5. the soil tester was placed about 10 cm (max 30 cm) from the main clumps. gps garmin 60 was used to indicate altitude and geographical coordinates. all measurements were carried out between 10 am and 3 pm. in addition, we collected plant samples within the plot, adjacent to for further identification in the lab. plant size, including diameter and height, number of seedlings, number of individuals in reproductive phase, were also measured (data not shown in this paper). differences between plots of each environmental factor, viz. temperature (°c), solar radiation (lux), air humidity (%), soil ph, soil moisture (%) and altitude (m) were observed using univariate analysis, viz. t-test performed in spss for windows (version 19.0). in addition, t-test was also performed to find whether there was difference between species abundance of the two forests, bukit tapak and . for this analysis, the species are considered as four variables: , , f. white and f. red. subsequently, separate analysis for each forest site was also conducted. within each forest site, species were treated as four ( , , f. white and f. red) or three variables ( , , and ) which will be further explained in the discussion. for the analysis in each forest, analysis of variance, onehutan kontrak altingia excelsa bischofia javanica michelia champaca laportea microstigma eugenia uniflora garcinia begonia begonia et al. begonia begonia begonia begonia begonia begonia univariate analysis begonia hutan kontrak begonia b. multangula b. baliensis b. longifolia b. longifolia begonia b. multangula b. baliensis b. longifolia b. longifolia b. multangula b. baliensis b. longifolia 83 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. way anova was planned to be employed. however, due to the nature of the data which were not normally distributed and the variance was not homogeneous, while the data remained the same upon transformation efforts, non-parametric data analysis was then carried out. kruskal wallis test was hence performed to investigate whether there was difference in species (considered as either four or three variables) within each forest. multivariate analysis was used to see the simultaneous correlation between species and the measured environmental factors across forty sites and how these variables may explain the abundance of species. pcord5 for windows was employed for detrended correspondence analysis (dca). the dca showed that the length of gradient resulted as 1.880, and based on this we re-conduct the analysis using principal component analysis (pca). to observe how species abundance is explained by environmental factors we established a graph by determining the main matrix as species abundance in each plot and the second matrix as environmental factors of each plot. as a rule of thumb, when given r2-cutoff ≥ 0.300 the environmental variables shown in the graph are considered significant enough to explain the abundance of species. however, it was not possible to get the environmental variables vectors with r2-cutoff ≥ 0.300 from our data consequently, we chose r2-cutoff 0.004 to show the vectors in the species abundance and microclimate graph. blume (fig. 1) herbs, height 100 150 cm. green stem, hairy, segment swollen, segment 10 12 cm. leaves hairy, clear venation, length 10 15 cm, width 8 10 cm, margins clearly serrate. flower white, fruit capsule, without wing, smooth. flowering whole year, distinguishable from large leaf, growth in group and abundant in certain patches. distribution: mountain forest in java, sumatra, and bali, altitude up to 2400 m asl. for nomenclatural information and a full description, refer to hughes (2008) and hughes and girmansyah (2011). blume (fig 2, left and middle) erect herb, cane like growth, 50 cm height, smooth stem. leaves surface smooth, tip either sharp or blunt, size 12 20 × 6 12 cm. flower emerges from axillary leaf, short; male flower white, tepals 4; female tepals 5 6. fruits without wing, surface with dots. commonly found on humid places, slopes, grow solitary. in indonesia, frequently found in the islands of java, sumatra, bali and sulawesi. for nomenclatural information and a full description, see kiew (2005), hughes (2008), hughes and girmansyah (2011). begonia begonia begonia begonia . begonia . b. longifolia multivariate analysis results and discussions species descriptions b. multangula b. longifolia 84 biotropia vol. 19 no. 2, 2012 begonia baliensis begonia longifolia b. baliensis b. multangula girm. b. baliensis b. multangula b. longifolia hutan kontrak per se begonia begonia baliensis smilax elaeocarpus diplazium esculentum clautylon b. longifolia homalanthus giganteus ardisia cyrtandra procris ruhlandii adiantum alyxia adiantum smilax glochidion digitaria diplazium flacourtia nephrolepis biserrata homalanthus giganteus polygonum chinense raphidopora elatostema stigosum eupatorium triplinerve diplazium pilea syzygium b. multangula meliosma peruginea digitaria (fig. 2, right) erect and cane-like, seldom branched, 15 100 cm tall, stem brownish green to reddish brown; nodes brownish green to reddish brown, swollen. leaves distant, lamina oblique, broadly ovate, asymmetric, basal lobe rounded, 3.5 7.5 cm long. inflorescences axillary, few flowered. fruits berry, 4 5 mm long, green when ripe, fleshy, globose, elongated into a fleshy beak, glabrous, 3-lobed, with one larger wing, not splitting. seeds barrel-shaped, 0.25 0.3 mm long. distribution: bali. for nomenclatural information and a full description, see girmansyah (2008). the number of individuals found and recorded in each forest is shown in figure 3, where it depicts the total number of individuals recorded across plots. , and (red and white forms), were all found in bukit tapak and . as we did not conduct association study, we will not present association for in the respective forest. instead, we will elaborate list of other species found in the plot as follows. was found with sp. (smilacaceae), sp. (elaeocarpaceae), (pterydophyta), sp. (euphorbiaceae). was recorded adjacent to (euphorbiaceae), sp. (myrsinaceae), sp. (gesneriaceae), (urticaceae), sp., sp. (apocynaceae), sp. (adiantaceae), sp. (smilacaceae), sp. (euphorbiaceae), sp. (poaceae), sp. (denst.), sp. (flacourtiaceae), (nephr.), (euph.), (polygon.), sp. (araceae), (urticaceae), (asteraceae), sp. (denst.), sp. (urticaceae), sp. (myrtaceae). was found with (sab.), and sp. (poaceae). , , in their natural habitat and their association figure 3. species average abundance across 40 plots in (20 plots) and (20 plots); error bars show 95% ci. begonia hutan tapak hutan kontrak 85 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. microclimate conditions the average value of microclimatic factors, viz. air humidity, temperature, solar radiation, soil ph and moisture between two habitats of in bedugul differed (table 1), except for soil moisture or humidity. the overview of the environmental variables or microclimate can be found in figure 4. the habitat conditions in both forests were somewhat different. was frequently disturbed by mowing or herbs cutting and received more abundant light and is relatively warmer (table 1, figure 4). in order to understand the habitat preferences of each species, with respect to microclimate, we used multivariate analysis. for the analysis, species abundance was used as main matrix whereas environmental variables recorded across forty plots were used as second matrix. in general, we found that the three species of occupy similar microhabitats, as shown by the short vectors of the environmental variables (figure 5). in other words, in each plot there was no characterizing environmental variable that distinguished species preference. other factors that were not measured, on the other hand, may determine habitat preference. such factors may refer to for example, nutrient availability, soil texture, mineral structure, root competition, litter layer, topsoil depth, allelopathy or competition with bryophytes, etc. in fact, species are able to occupy wide range of habitat type and microclimate, as in the case recorded for in thailand (phutthai 2009). figure 6 depicts the distribution and abundance of species across plots. main matrix and second matrix were both species abundance across plots. when r2-cutoff is set to higher threshold i.e. 0.600, only f. white and f. red vectors were able to show up indicating that plots significant characters the frequency of found in . when r2-cutoff reduced to 0.300, vectors produced were for f. red, f. white, and . thus, the presence of these species were able to be grouped based on plots, except for begonia hutan kontrak begonia begonia begonia begonia begonia begonia begonia et al. begonia begonia b. longifolia b. longifolia b. longifolia b. longifolia b. longifolia b. baliensis begonia table 1. microclimatic features of two natural habitats of . ttest was applied to define statistical difference of each factor between two sites begonia abiotic factor forest site mean se of mean p-value temperature (°c) bukit tapak 23.6 0.169 0.000 reboisation forest 27.1 0.475 ** solar radiation (lux) bukit tapak 472.95 107.0367 0.000 reboisation forest 2246.197 502.2649 ** air humidity (%) bukit tapak 71.86 0.4245 0.043 reboisation forest 70.175 0.6871 * soil ph bukit tapak 6.225 0.571 0.043 reboisation forest 6.02 0.793 * soil moisture (%) bukit tapak 78.057 1.652 0.801 reboisation forest 78.759 2.203 (ns) altitude (m asl) bukit tapak 1428.95 2.259 0.000 reboisation forest 1351.1 7.021 ** p-value indicates * as significant; ** as higly significant; (ns) as not significant at 95% confidence interval 86 biotropia vol. 19 no. 2, 2012 . however, from figure 5, environmental variables to characterize each plot were not found. f. white had a high correlation with axis 1, and on the other hand, f. red had a high correlation with axis 2. interestingly, within , white and red form has a tendency to be present in different plots. further investigation on how these two forms differ in occupying site shall be carried out. based on figure 6, and were relatively frequent to occupy similar plots, even though not significant (higher r2-cutoff= 0.106). , , and f. red were somewhat found to occupy similar sites, but completely apart from f. white. b. multangula b. longifolia b. longifolia b. longifolia b. baliensis b. multangula b. baliensis b. multangula b. longifolia b. longifolia figure 4. the microclimatic features of the 40 study plots (20 plots in bukit tapak and 20 plots in or reboisation forest)hutan kontrak f re q u e n c y altitude (m asl) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 15 10 5 0 15 10 5 0 1325 1350 1375 1400 1425 f re q u e n c y b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s air humidity (%) 66,0 68,0 70,0 72,0 74,0 76,0 78,0 80,0 6 4 2 0 6 4 2 0 f re q u e n c y soil ph b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 8 6 4 2 0 5,0 5,5 6,0 6,5 7,0 7,5 8 6 4 2 0 f re q u e n c y temperature ( c) o b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 12 10 8 6 4 2 0 20 22 24 26 28 30 12 10 8 6 4 2 0 f re q u e n c y soil humidity (%) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 50 60 70 80 90 100 12 10 8 6 4 2 0 12 10 8 6 4 2 0 f re q u e n c y solar radiation (lux) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s ,0 2000,0 4000,0 6000,0 8000,0 20 15 10 5 0 20 15 10 5 0 87 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. figure 5. principal component analysis of species abundance and measured environmental factors in bukit tapak (bt) dan hutan reboisasi (hk) (r2cutoff=0.04; length of gradient axis 1=1.996, axis 2=1.763). these environmental variables were not significant (when r2-cutoff ≥ 0.300, no vectors was produced) related to plots and hence, could not explain species abundance. begonia begonia figure 6. principal component analysis of (form red and white), and (r2cutoff=0.004). correlation of , , f. red and f. white to axis 1were 0.285, 0.235, 0.243, and -0.989, respectively; whereas the correlation value of species to axis 2 were -0.263, -0.620, 0.923, and 0.04, respectively. b. longifolia b. multangula b. baliensis b. multangula b. baliensis b. longifolia b. longifolia begonia 88 biotropia vol. 19 no. 2, 2012 b. multangula begonia b.longifolia b. baliensis begonia begonia b. multangula b. baliensis b. longifolia b. longifolia b. longifolia hutan kontrak begonia et al b.multangula b. longifolia b. multangula b. robusta b. longifolia b. muricata b. bracteata b.multangula b. multangula begonia et al. b. multangula b. longifolia b. longifolia b. longifolia b. longifolia b. multangula b. baliensis b. multangula begonia b. robusta is actually known to have a wide distribution range and is present in java, sumatra and lesser sunda islands (hughes 2008). girmansyah (2008) in his study on of bali and lombok described the distribution of which extends from the himalayas (india) to south china, vietnam and through thailand, peninsular malaysia, and indonesia (sumatra, java, bali, and lombok). is endemic to bali and is known to occupy humid forest along trails at 1300 – 1800 m either in small colonies or large populations (girmansyah 2008). through univariate analysis, the difference between species abundance between the two forest types was similar (t-test, p=0.0061). subsequently, for bukit tapak the four species ( , , f. red, f. white) abundance did not differ among species (chi-square=1.913, p=0.591) neither when the two forms of was pooled together (chi-square=2.018, p=0.365). the results were similar to , species did not differ among each other, either way when considered as four (chi-square=0.556, p=0.906) or three (chisquare=0.476, p=0.788) different species. a plant diversity study in resort cidahu gunung halimun salak, west java (larashati . 2010) pointed out that is abundant in areas which intercept higher solar radiation, whereas within the same research site tended to prefer areas with heavier shade such as the forest floor. and were found to be abundant along paths, and around an open, frequently disturbed helicopter pad. furthermore, , , and were found mostly in forest under heavy shade. however, on the forest floor, was also found. it seems that has a wide range of preference and therefore it confirms our pca result (figure 6) that the correlation to axis 1 and axis 2 were the lowest amongst other species observed, which were 0.235 and -0.263, respectively. another study in gunung halimun national park by wiriadinata (2002) recorded that was very abundant. the distribution of this species is within ground cover of mountainous forests in java up to 2400 m altitude. in gunung halimun, it was always found to grow in clumps or groups at a wide range of altitudes, 900-1800 m. the flowering and fruiting period is all year round, and it prefers medium soil moisture and humidity. surprisingly, they also found that is on the contrary rare (only found in two spots in cikaniki and ciptarasa), grows solitary, and prefers very moist substrates or semi-waterlogged. was found only at 9001000 m altitude in this study. these studies did not mention any differentiation (colour) of in the forest in java. (both forms) and are cane-like, whereas is shrub like. this trait can be further analyzed using correlation with specific abiotic factors, such as light interception or shade tolerance. however, a study by shiodera and kohyama (2005) showed that in gunung halimun salak, at 1000-2000 m altitude, population of observed were found generally under medium light intensity. unfortunately, no further information refers to the extent of how light intensity determines the abundance of sp. on the other hand, its close relative, i.e. , present in similar forest, showed a tolerance to a wider lowmediumand high light intensity. further ecological studies on habitat preference should address more features of the environment such as nutrient availability, 89 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. competition, and disturbance level as many species are known for their narrow endemism, but a small number of species have a wide distributional range as well. as it has been hypothesized in several previous studies that light might determine the abundance of and , further research is needed to address this aspect in addition to that presented here. the abundance of all species between two forest types and within each forest were not significantly different. microclimate factors of two observed habitats of in bedugul were relatively equal between two forests and do not characterize habitat. however, there was a strong habitat separation between f. white and f. red. even though not statistically evident, (f. red), , and tend to occupy similar plots. unfortunately, environmental variables measured in the study did not appear significant to characterize each plot, and hence we could not able to identify which abiotic factors were able to determine the occurrence of species. several previous studies emphasized light intensity as explaining abiotic factor that distinguish the abundance in and in java, and hence, further elaborative study should address this matter. the project work was funded by the indonesian higher education department or dikti (2010-2011) research grant for “potensi lamiaceae, begoniaceae, dan lamiaceae sebagai bahan baku obat dan aspek konservasinya”. made ardaka and i wayan mastra (bali botanical gardens) are acknowledged for their technical assistance in the field, while didit okta pribadi (bogor botanical gardens) and maria hanauer (university of regensburg)for the advise on multivariate analysis. we would like to thank to an anonymous reviewer and dr. mark hughes (royal botanic garden edinburgh) for the constructive comments on the manuscript. begonia b. multangula b. longifolia begonia b. longifolia b. longifolia b. multangula b. baliensis begonia b. multangula b. longifolia conclusions acknowledgments references chiew h. 2005. begonias under threat. accessed from bgci (botanic gardens conservation international). webpage ( ) on 15 january 2011. djarwaningsih t, sulistiarini d, sunarti s, aerida ih, dewi, mahyuni r. 2010. karakterisasi tipe vegetasi dan keanekaragaman jenis flora/ jamur di cagar alam gunung tukung gede serang banten. laporan akhir program insentif peneliti dan perekayasa, lipi tahun 2010. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. girmansyah d. 2008. a taxonomic study of bali and lombok (begoniaceae). reinwardtia 12(5): 419-34. hartutiningsih. 2005. kebun raya bali, candikuning: upt balai konservasi tumbuhan kebun raya bali, lipi. isbn: lipi press. 797-26-2410-4. http://www.bgci.org/worldwide/news/0087/ begonia begonia 90 biotropia vol. 19 no. 2, 2012 hartutiningsih, ardaka im, siregar m. 2007. masa berbunga 22 jenis alam di kebun raya eka karya bali. biodiversitas 8(3): 192-96. hughes m. 2008. an annotated checklist of southeast asian . royal botanical garden edinburgh, uk. hughes md. girmansyah. 2011. a revision of sect. (hassk.) warb. from sumatra. gardens' bulletin singapore 62(2): 27-39. ksda-bali. 2009. cagar alam batukahu. accessed online from on 13 january 2012. kiew r. 2005. begonias of peninsular malaysia. natural history publications (borneo), sdn. bhd. kota kinabalu, sabah, malaysia. larashati, mirmanto ie, mansur h, wiriadinata h. 2010. penelitian ekologi jenis tumbuhan sebagai dasar pengelolaan dan pengembangan taman nasional gunung halimun-salak. laporan akhir program insentif peneliti dan perekayasa, lipi tahun 2010. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. purwantoro rs, hartutiningsih, siregar m, sudarmono, fijridiyanto ia, satyanti a. 2010. potensi lamiaceae, begoniaceae, dan lamiaceae sebagai bahan baku obat dan aspek konservasinya. laporan akhir tahun program insentif peneliti dan perekayasa lipi. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. priyadi h, takao g, rahmawati i, supriyanto b, nursal wi, rahman i. 2010. five hundred plant species in gunung halimun salak national park, west java: a checklist including sundanese names, distribution and use. cifor, bogor, indonesia. phuttai t, sands m, sridith k. 2009. field surveys of natural populations of l. in thailand. thai forest bulletin (bot.) special issue: 186-98. shirodea s, kohyama t. 2005. tradeoff and diversity of leaf/ shoot traits among plant life forms in non seasonal environment. in environmental conservation and land use management of wetland ecosystem in southeast asia annual report for april 2004-march 2005; core university programme between hokkaido university japan and research center for biology lipi, indonesia. sponsored by japan society for promotion of science (march 2005). tebbitt mc. 2005. . cultivation, identification, and natural history. published in association with brooklyn botanic garden. timber press, usa. thomas dc, ardi wh, hartutiningsih, hughes m. 2009. two new species of (begoniaceae) from south sulawesi, indonesia. edinburgh j bot 66 (2): 229-38. wiriadinata h, girmansyah d, hoover s, hunter j. 2002. kekayaan taman nasional gunung halimun. berita biologi 6(1) edisi khusus “biodiversitas taman nasional gunung halimun” (ii): 91-97. begonia begonia begonia sphenanthera begonia begonias begonia begonia http://www.ksda-bali.go.id/?page_id=11 91 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. 6. ristianti (notes) rev.cdr biotropia vol. 18 no. 2, 2011: 123 128 notes on the distribution of invasive freshwater snail (lamarck, 1822) and ( d'orbigny, 1835) in indonesia pomacea canaliculata p. insularum ristiyanti m. marwoto & nur r. isnaningsih received 24 november 2011/accepted 10 december 2011 the freshwater snails and have been reported as important invasive species causing damage to crops and predominantly wetland rice in asia. these snails are known as “golden apple snail” (gas), an introduced species from argentina. or known as “keong mas, keong murbei” was introduced in indonesia around 1983, and after more than 20 years, it now can be found very abundant at various habitats such as marshes, ponds, irrigations, lakes and rice fields in almost all places in indonesia. based on the collections of these snails deposited in the mzb (museum zoologicum bogoriense, research center for biology) and secondary data (references), the distribution of these two snails was studied. is widely distributed, while is only found at lake semayang and lake balikpapan in kalimantan. the distribution map is presented and will be useful as a basic information to manage these invasive snails. distribution, snail, invasive, indonesia research center for biology, gedung widyasatwaloka, jalan raya jakarta bogor km 46, cibinong, bogor 16911 pomacea canaliculata p. insularum pomacea canaliculata pomacea canaliculata p. insularum pomacea canaliculata, p. insularum, abstract introduction key words: the freshwater snails and have been reported as important invasive species causing damage to crops and predominantly wetland rice in asia. reports on the serious damage to rice in malaysia, philippines, japan, vietnam and indonesia have been published (hyunh 2006; cuong 2006; adalla & magsino 2006; wada 2006; suharto . 2006; yahaya . 2006). these snails are known as “golden apple snail” (gas), an introduced species from argentina. or named as “keong mas, keong murbei” was introduced in indonesia around 1983, and after more than 20 years, the snail has spread and became pomacea canaliculata p. insularum et al et al pomacea canaliculata * corresponding author : rist001@lipi.go.id 123 very abundant in various habitats such as marshes, ponds, irrigations, lakes and rice fields in almost all places in indonesia. in 2004, the first author visited lake semayang, lake loa kang and lake balikpapan in east kalimantan and collected shells of and unexpectedly shells that have similar characters with (d'orbigny 1835) were found which have not been recorded before. this finding added the number of invasive snails in indonesia to two species. hayes (2008) did not list from indonesia but he reported that this snail is present in singapore and malaysia. rawling (2007) already stated that this snail is distributed widely in southeast asia and assumed that it was introduced to indonesia from malaysia. the occurrence of in indonesia also have been reported by suharto 2006; isnaningsih & marwoto (2011), but based on our examination, the shells are similar to as mentioned by cowie 2006 who also noted that has not been detected in asia. detailed study on the occurrence of in indonesia is still needed. damage to ricefields in indonesia caused by have been reported in medias such as newsletters, radios, and televisions (see appendix). presently, even the data on their distribution in indonesia is largely a speculation. the public awareness about the danger of the gas (golden apple snails) in general is very limited. identifying the freshwater snails found in rice fields or irrigations is also difficult for the farmers and the local people. usually they only recognised the occurrence of based on the color of the egg capsules which are pink or bright reddish. preliminary study on the distribution of and is needed to evaluate the distribution of these two species. the aim of this study is to present a basic data on the distribution of and in indonesia. the data would be necessary for future studies and also to manage the invasive snail becoming pest in rice and threatens some native snails. the author used about 500 specimens deposited in the mzb (museum zoologicum bogoriense, research center for biology) and secondary data from references dated from 2005 up to 2011. all information of the localities where the snails have been collected from 1990 to 2011 were recorded and marked on the distribution map. yellow dots represent the localities based on references and red data for the localities based on mzb's specimens of , while the occurrence of was indicated by star. of mzb collection came from 90 localities in indonesia, distributed from the northern sumatra (aceh, bukit kese, bengkulu sibaganding,manggung pariaman, danau kerinci, jambi, danau ranau, lampung, sigarung-garung, tanggamus, lampung, krakatau), java (bogor, tasikmalaya, pomacea, p. insularum et al. p. insularum p. paludosa et al. p. canaliculata et al. p. paludosa p. paludosa p. canaliculata p. canaliculata p. canaliculata p. insularum p. canaliculata p. insularum p. canaliculata p. insularum pomacea canaliculata materials and methods results and discussion 124 biotropia vol. 18 no. 2, 2011 figure 1: map of the distribution of and in indonesiap. canaliculata p. insularum the distribution map (based on mzb collections & secondary datas) shows that is more widely distributed compared to that of found only in lake loa kang and semayang in balikpapan, kalimantan. according to reports from newspapers and magazines the invasive snail has damaged about 10 ha of ricefields in some places such as in java, sumatra, sulawesi, kalimantan, sumbawa, lombok or totally about more than 100 ha mainly in aceh north sumatra and some areas in java. it seems that possesses less physiological adaptability compared to and need a specific habitat such as big lakes with muddy substrates and water hyacinth or water plantations like in lake loa kang, semayang and balikpapan. cazzaniga (2006:39) explained about the pattern of distribution and habitat of in argentina basically tropical and subtropical and fail to thrive in salty, very alkaline, poorly vegetated environments, with high risk of desiccation. furthermore, he concluded that the fast expansion of this snail in southeast asia might be caused by some biological reasons but the main factor is human actions, since the snail has potential economic value. can be usually found in irrigated rice fields areas, or marshes and ponds. the populations will increase in rainy season and the juveniles or young snails will spread widely. the expansions of in indonesia are usually caused by human activities, especially in jawa, sumatra and sulawesi where people have cultured the snail to be consumed (as edible snail) without realising the risk of the invasive snail (isnaningsih & marwoto 2011). on the contrary, the local people in balikpapan, east kalimantan, generally prefer to consume freshwater fishes rather than snails. p. canaliculata p. insularum p. canaliculata p. insularum p. canaliculata p. canaliculata pomacea canaliculata p. canaliculata cianjur, sukabumi, krawang, depok, bekasi, rawapening, tuban, jogya), bali (lake tamblingan, lake bratan), sulawesi (maros, manrepo, buton, palu, bone, pangkep), kalimantan (tau lumbis, lake semayang, lake balikpapan, malinau), and papua (wamena, biak). while was only recorded from three locations i.e lake semayang, lake loa kang and lake balikpapan in east kalimantan. the distribution of and is presented on figure 1. p. insularum p. canaliculata p. insularum notes on the distribution of invasive freshwater snail ..... ristiyanti m. marwoto .et al 125 conchological et al. p. canaliculata p. insularum p. canaliculata p. insularum et al. p. canaliculata et al. pomacea canaliculata . the shell morphology description is based on general description to compare the two species, for detailed morphology and anatomy study see cowie 2006. the invasive snail and have similar shells. the shell differ mainly in having relatively higher spire and less broadly in compared to that of (rawling 2007), and the shell size of is relatively smaller (cowie 2006). the shell characters of both species are described as follows :. (lamarck, 1822) shell (fig.2) globose, somewhat thin or transparent with smooth surface. dextral coiling. yellowish or dark brown, around the suture shell's color become pale. sometimes there are dark spiral bands that become brighter at the body whorl. spire high and pointed. whorls 5.25 5.50, and rapidly increasing in size, the body whorl more globose. umbilicus perforate. suture curved to form deep channel. apertural shape elongate cylindrical and the columellar lip not thickened. measurements: height of shell 12.58 69.66 mm; width of shell 4.94 64.90 mm; height of body whorl 11.20-61.20 mm; length of aperture 8.58 49.7 mm; width of aperture 6.50 34.31 mm fig. 2. ariation of from different localities.pomacea canaliculatashell v 126 biotropia vol. 18 no. 2, 2011 pomacea insularum (d'orbiny, 1839) m . shell (figs.3) globose, somewhat thick compared to . dextral coiling. brown and become darker near the umbilicus.spire low and commonly erroded. whorls 5, and rapidly increasing in size, the body whorl more globose. umbilicus perforate. suture with shallow channel. apertural shape elongate cylindrical and the columellar lip not thickened. easurements: height of shell 66.16-85.00 mm; width of shell 63.32-79.6 mm; height of body whorl 64.60 -77.55 mm; length of aperture 46.05-59.25 mm; width of aperture 33.24-44.45 mm p. canaliculata figure 3. shell variation of from lake loa kang. adalla cb, magsino e. 2006. understanding the golden apple snail ( ): biology and early initiatives to control the pest in the philippines. : global advances in ecology and management of golden apple snails. rc joshi & ls sebastian (eds), 199-213. philrice. philippines. cazzaniga nj. 2006. harmless and useless in its natural realm (argentina). global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 3760. philrice. philippines. cuong dn. 2006. the golden apple snail in vietnam. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 243-254. cowie rh, hayes ka, thiengo sc. 2006. what are apple snails? confused taxonomy and some preliminary resolution. : global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 3-23. philrice. philippines. hayes ka, joshi rc, thiengo sc, cowie h. 2008. out of south america: multiple origins of non-native apple snails in asia diversity and distributions, 14 (4), 701-712. pomacea insularum pomacea canaliculata in pomacea canaliculata : in: in in . acknowledgements we would like to express our thanks to the anonymous reviewers who gave the comments and corrections on the first draft of this manuscript. this work was supported by dipa of research center for biology lipi (2009). references 127 notes on the distribution of invasive freshwater snail ..... ristiyanti m. marwoto .et al huynh nk. 2006. golden apple snails in vietnam. : global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 255-266. philrice. philippines. isnaningsih nr, marwoto rm. 2011. keong hama pomacea di indonesia: karakter morfologi dan sebarannya (molluska, gastropoda, ampullariidae). berita biologi, 10(4): 441-447. marwoto rm. 2006. adaptasi moluska air tawar di danau loa kang dan balikpapan kalimantan timur. fauna indonesia, 6 (2): 59-64. rawling ta, hayes ka, cowie rh, collins tm. 2007. the identity, distribution, and impacts of non-native apple snails in the continental united states. bmc evolutionary biology, 7: 1-14. suharto h, marwoto rm, heryanto, mulyadi, siwi ss. 2006. the golden apple snail spp. in indonesia. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 231-242. philrice, philippines. wada t. 2006. impact and control of introduced apple snail, (lamarck), in japan. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 181195. philrice. philippines. yahaya h, nordin m, muhamad hisham mn, sivapragasam a. 2006. golden apple snails in malaysia. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 215230. philrice. philippines. in pomacea in pomacea canaliculata in: in: 128 biotropia vol. 18 no. 2, 2011 microsoft word 111 biotropia vol. 13 no. 2, 2006 : 111 121 antagonistic bacteria against schizophyllum commune fr. in peninsular malaysia antarjo dikin', kamaruzaman sijam', jugah kadir' and idris abu semanz 'department of plant protection, faculty of agriculture, universiti putra malaysia, 43400 upm serdang, selangor d.e, malaysia 2plant pathology and weed science group, biological research division malaysian palm oil board, 43000 kajang, selangor, d.e. malaysia abstract schizophyllum commune fr., is one of the important fungi, causes brown germ and seed rot of oil palm. biodiversity of antagonistic bacteria from oil palm plantations in peninsular malaysia is expected to support in development of biopesticide. isolation with liquid assay and screening antagonistic bacteria using dual culture assay were carried out in the bioexploration. a total of 265 bacterial isolates from plant parts of oil palm screened 52 antagonistic bacterial isolates against 5. commune. bacterial isolates were identified by using biolog* identification system i.e. bacillus macroccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, sternotrophomonas maltophilia. key words : schizophyllum commune, biodiversity, antagonistic bacteria introduction schizophyllum commune fr. causes brown germ and seed rot. heavy infection decreased seed germination of oil palm about 60 percent (dikin et al. 2003). proper seed treatments are required for the control of s. commune in the oil palm seeds. some synthetic fungicides were applied to reduce the loss of germination due to this pathogen, but the negative impact from toxic chemicals to the environment was difficult to avoid. the utilization of bacteria as biological control agents successfully controlled plant pathogen (sharga and lyon 1998; bapat and shah 2000). many studies in exploration of beneficial organisms have been carried out such as pseudomonas fluorescent for the control of fusarium wilt of tomato (dekkers et al. 1998). streptomyces halstedii (k122) and s. coelicolor (k139) to inhibit the fungi belonging to oomycetes, zygomycetes, deuteromycetes, ascomycetes and basidiomycetes (frandberg and schnurer 1998). bacillus subtilis suppressed phytopathogenic microorganism (phae et al. 1990). the isolation of antagonistic bacteria was early stage for development of biopesticide such as pseudomonas fluorescent from rhizospheres (dekkers et al. 1998), bacillus licheniformis from leaves of citrus orchard at letaba estates, corresponding address : antario_dikin@yahoo.com 111 biotropia vol. 13 no. 2,2006 tzaneen (jager and kosten 1998), streptomyces halstedii (k122) and s. coelicolor (k139) from cereal grains (frandberg and schnurer 1998), bacillus subtilis from the composts (phae et al. 1990), and burkholderia cepacia from infected oil palm seeds (dikin et al. 2003). the liquid assay technique was a simple method for isolation of bacteria. fluorescent pseudomonas from pythiumdiseased tulip roots was isolated by extraction of infected root in sterilized water (weststeijn 1990). malaysia is well known as a mega-biodiversity country with complex microbial association. the exploration of beneficial bacteria from oil palm plantations is expected to utilize the antagonistic bacteria from the same ecology of the oil palm pathogen itself. the purposes of the study were to isolate and to screen the antagonistic bacteria from different plant parts of oil palm for the control of schizophyllum commune. materials and methods cultural schizophyllum commune fr. and plant part of oil palm the culture of s. commune was isolated from heavy infection of oil palm seeds. the fungus was confirmed based on their morphological characteristics and the pathogenic fungus of oil palm (alexopoulus et al. 1996). the fungal isolate was sub-cultured onto pda medium for further study. randomized samples of plant parts such as fruits, seeds, rhizosphere, and plant debris were collected from oil palm fields in selangor (upm, bangi, kajang and seri kembangan), guthrie, layang-layang, johor in peninsular region, malaysia. plant parts were used for isolation of potential bacteria. isolation of antagonistic bacteria plant parts of oil palm such as fruits, seeds, rhizospheres and plant debris were rinsed with tap water to remove the adhered soil on surface. mesocarp of fruits, endosperm of seeds, and rhizosphere were sliced into 0.5-1.0 cm2 and then 50 g sliced plant parts were placed into 250 ml erlemeyer flask added with 100 ml distilled water. plant debris with 5. commune was collected under oil palm tree. ten g of plant debris were cut off in size 1 cm and then transferred into a 250 ml erlemeyer flask added with 100 ml sterilized water. sample materials in flasks were placed on electric rotator at 100 rpm overnight at 26 ± 2°c. fold serials (lo^-lo"4) dilutions of suspension were made, 0.5 ml suspension from each diluted suspension was streaked on king's b (kb) and nutrient agar (na) agar media plates. plates were incubated at 26-28°c for 48 hours. bacterial colonies on plate were purified by streaking single bacterial colony onto na medium plates. each pure culture of bacteria was screened for the antagonistic bacteria based on dual culture (dikin et al. 2002;montealegree/a/. 2003). 112 antagonistic bacteria a. dikin et al. screening the antagonistic bacteria screening of bacterial antagonist was carried out using dual culture assay. one 6-mm diameter of s. commune agar plug was placed at the centre of pda medium in a petri dish with 9 cm diameter. bacterial isolate was streaked on pda medium with a distance of 2.5 cm between s. commune agar plug and bacterial isolate. plates were incubated for 7 days at 26 ± 2°c. the percentage of radial inhibition growth was measured with the formula: pirg (%) = (1 (fungal growth near to bacterial isolate /fungal growth other side at the same plate as control)) x 100%. each treatment was replicated 3 times. receded data were analyzed using sas® software. treatment effect was tested by anova and the means compared using least significant different test at 5% probability level (okamoto et al. 1998; anonymous 1999; montealegreefa/. 2003). identification of antagonistic bacteria potential antagonistic bacterial isolates were identified by biolog® identification system which followed the biolog's procedures. bacterial suspension was inoculated into gn or gp micro plates depending on gram reaction cluster, 145 ul per well using the 8-channei repeating pipette. microplate was covered with its lid and incubated at 28-30°c for 24 hours to allow the utilization of carbon sources. reading result was directly done after inserting the incubated microplate into the biolog's reader apparatus and its installed micro soft ware of biolog® identification system for identifying bacteria up to the species level (anonymous 2001). results and discussion isolation of antagonistic bacteria the number of bacterial isolates was extracted from samples of seeds, fruits, rhizospheres and plant debris of oil palm which grew on kb and na media. a total of 265 bacterial isolates from plant parts of oil palm were found from different locations, peninsular region, malaysia. separation of bacterial isolates was based on the morphological colony performance such as colony colour, elevation, the margin of colony and colony surface (hayward 1983). isolation of potential bacteria from plant parts of oil palm using liquid assay was effective and simple technique. the liquid assay and the agar plate media are commonly used for isolation of pathogenic bacteria from infected plant parts. bacterial isolates from different plant parts of oil palm on na and kb media grew well on the cultural plates. dual culture assay screened 52 out of 265 bacterial isolates against s. commune. the number of antagonistic bacteria from each location isolated from different plant parts is presented in table 1. 113   antagonistic bacteria a. dikin et al. based on table 1, 96 bacterial isolates as the highest number were obtained from the rhizosphere followed by plant debris, 89 isolates. the average number of bacterial isolates from rhizosphere was 9.6 followed by 6.8 from plant debris, 6.1 from fruits, and 5.2 from seeds. the bacterial isolates obtained from plant debris were more diverse with the number of bacterial isolates higher than other plant parts such as rhizophere, fruit, and seed. eighteen out of 89 antagonistic bacterial isolates were obtained from plant debris, followed by 15 out of 96 isolates from rhizosphere, 10 out of 43 isolates from fruit, and 9 out of 37 isolates from seed. the probability for isolation of antagonistic bacteria from each plant part was 20.2 percent, 15.6 percent, 23.2 percent, and 24.3 percent, respectively. more dominant bacteria in the rhizosphere and plant debris than seeds and fruits were due to the different available nutrition and the requirement for bacterial growth. dominance of bacteria in the rhizospheres and plant debris was due to complex interaction between microorganisms and plant parts. plant debris such as decayed empty bunch, fronds, and rachis were good media for the fungal growth. blotching symptom with water soak and brown colour in the fruiting bodies of s. commune was the indication of interaction between fungus and bacteria. dual culture assay of s. commune against antagonistic bacteria is presented in figure 1. figure 1. a. burkhoderia multivorans (bacterial code-50) inhibits the growth of s. commune on dual culture of pda medium at 7-day after incubation at 26 ± 2°c b. burkholderia cepacia inhibits the growth of s. commune on dual culture of pda medium at 7-day after incubation at 26 ± 2°c among 52 isolates from different plant parts and different sampling locations inhibited the mycelial growth of s. commune with various percentages of radial inhibition. each bacterial isolate with radial growth inhibition of s. commune is presented in table 2. the range of radial growth inhibition of antagonistic bacteria was 3.3 percent up to 95.2 percent from the bacterial code 29 and 10, respectively. in table 2, there are 7 bacterial isolates with highest mean percentage of radial growth inhibition with the bacterial code 10, 8, 9, 14, 50, 7, and 2 with the percentage of inhibition of 95.2, 115 biotropia vol. 13 no. 2,2006 90.6, 83.2, 83.1, 83, 81.8, and 81.5, respectively. a number of antagonistic bacteria were isolated from plant parts with varied mean percentages of radial growth inhibition against s. commune. the bacterial isolates had high radial growth inhibition which were obtained from plant debris, rhizospheres, and fruit. many authors have reported that certain antagonistic bacteria suppressed the growth of pathogenic fungus. in vitro study showed that burkholderia cepacia from tomato's rhizospheres suppressed the growth of fusarium oxysporum f.sp. lycopersicae stronger than s. commune. in contrast, b. cepacia from rhizhosperes of oil palm suppressed s. commune stronger than f. oxysporum f.sp. lycopersicae (kamaruzaman and dikin 2005). in this case , targeted potential antagonistic bacteria against s. commune should be isolated from the area of oil palm plantation. identification of antagonistic bacteria identification of antagonistic bacteria against s. commune based on biolog® identification system is presented in table 3. 116 antagonistic bacteria a. dikin et al. table 3. antagonistic bacteria based on biolog* identification system plant part bacterial code bacteria rhizosphere 1 burkholderia cepacia 6 corynebacterium agropyri 7 microbacterium testaceum 17 pseudomonas citronellolis 20 bacillus macroccanus 23 b. cepacia 24 burkholderia spinosa 47 b. cepacia 49 staphylococcus sciuri 50 burkholderia multivorans 51 burkholderia pyrrocinia 52 b. cepacia fruit 3 b. cepacia 5 serratia marcescens 9 c. agropyri 25 pseudomonas aeruginosa 34 p. aeruginosa 38 p. aeruginosa 40 p. aeruginosa seed 4 serratia sp. 12 bacillus thermoglucosidasius 18 b. pyrrocinia 22 b. cepacia 26 p. aeruginosa 37 sternotrophomonas maltophilia plant debris 2 s. mallophilia 8 burkholderia gladioli 10 b. gladioli 11 b. cepacia 14 c. agropyri 16 serratia ficaria 19 enterobacter aerogenes 28 b. gladioli 30 p. aeruginosa 33 corynebacterium masitidis 42 m. testaceum 43 rhodococcus rhodochrous table 3 presents the identified bacteria and non-identified bacteria by using biolog identification system. in the rhizosphere 12 identified species and 3 non-identified species were found. the identified species from rhizosphere were b. cepacia, c. agropyri, m. testaceum, p. citronellolis, b. macroccanus, b. spinosa, s. sciuri, b. multivorans, and b. pyrrocinia. among 9 species, the dominant identified species in the rhizosphere was b. cepacia. 117 biotropia vol. 13 no. 2,2006 seven identified species and 3 non-identified species of antagonistic bacteria were found in fruits. the identified species from fruits were b. cepacia, s. marcescens, c. agropyri, and p. aeruginosa. among the 4 species, the dominant identified species in the fruit was p. aeruginosa. p. aeruginosa was isolated from the rhizospheres and plant debris. this bacteria was recognized as the supplier of mineral which was required for metabolism process of plant from the access of bacterial metabolites (hofte et al. 1993; abdullah et al. 2003). there were complex microorganisms around rhizospheres to compete with each other for survival which showed the synergism and antagonism interaction. infected plant debris with s. commune around the rhizosphere was to bait the potential antagonistic bacteria. six identified species and 3 non-identified species of antagonistic bacteria in seeds were found. the identified species from fruits were serratia sp., b. thermoglucosidasius, b. pyrrocinia, b. cepacia, p. aeruginosa, and s. maltophilia. in the plant debris 12 identified species and 6 non-identified were found. the identified species were s. maltophilia, b. gladioli, b. cepacia, c. agropyri, s. ficaria, e. aeogenes, p. aeruginosa, c. masitidis, m. testaceum, and r. rhodochrous. the dominant species from plant debris was b. gladioli. b. cepacia and b. gladioli were dominantly found in the rhizospheres and plant debris, respectively. the presence of b. cepacia in the rhizospheres of oil palm was the same evident with the presence of b. cepacia in the rhizospheres of banana to protect plant infection caused by fusarium oxysporum f. sp cubense. b. cepacia colonizes the surface of hyphae and fungal macrospores (pan et al. 1997). b. gladioli was found in the rhizospheres and potential antagonistic bacteria against s. commune, however the implication for biological control was less recognized. the identified species of antagonistic bacteria from different plant parts were bacillus macmccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, and sternotrophomonas maltophilia. out of these species were new recorded species of antagonistic bacteria i.e. b. thermoglucosidasius, b. multivorans, b. spinosa, c. agropyri, c. misitidis, enterobacter aerogenes, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, and staphylococcus sciuri. however, b. cepacia, b pyrrocinia p. aeruginosa, s. marcescens and s. maltophilia were reported as biocontrol agents (burkhead et al. 1994; kobayashi et al. 1995; suparman et al. 2002; szczech and shoda 2004). avirulent isolate of b. gladioli strain 1064a is used for suppressing the incidence of bacterial seedling blight of rice caused by b. plantarii (miyagawa 2000). p. aeruginosa is grouped as fluorescent pseudomonads based on the production of a fluorescens pigment on kb medium (sand et al. 1980). the bacterium produces siderophores as plant growth promoter (hofte et al. 1993) and broad spectrum antagonistic bacteria against pathogenic fungi (haas et al. 1991). 118 antagonistic bacteria a. dikin et at. several species of fluorescent pseudomonads were known to be antagonistic bacteria and used as biological control agents. p. aeruginosa 7nssk2 was able to suppress pythium splendens, the causal pre and post-emergence damping-off and root rot of many crops such as tomato (tambong et al. 1998). p. aeruginosa and s. marcescens were isolated from plant part of oil palm, these isolates were confirmed as biocontrol agent for suppressing sclerotium rolfsii and rhizoctonia solani (ordentliche/a/. 1987). conclusions a number of bacterial isolates from plant part such as seeds, fruits, rhizospheres, and plant debris under oil palm trees were potential antagonistic bacteria against s. commune. a total of 52 out of 265 bacterial isolates were identified as the antagonistic bacteria against s. commune. the identified antagonistic bacteria using biolog® identification system were as follows : agrobacterium agropyri, bacillus macroccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, and sternotrophomonas maltophilia. some of bacterial isolates were recognized as biocontrol agents of plant pathogenic fungi and the rest of isolates have yet to be studied for their status. all of these species are required for further studies on the production of their secondary metabolites which might be potential substances to inhibit the growth of s. commune. acknowlegdment the study is partially supported by irpa project, malaysian government (vote no. 54400) as part of the ph.d. thesis of the first author. references abdullah, h., h.m. saud and c.e. fong. 2003. evaluation of the combined effect of pseudomonas aeruginosa and penicillmm sp. on the development of bacterial wilt caused by ralstonia solanacearum on tomato. abstract. third federal of asia pacific microbiology societies conference. kuala lumpur, october 15-18,2003 alexopoulos, c.j., c.m. mims and m. blackwell. 1996. introductory mycology. phyllum: basidiomycota. order: aphyllophorales. polypores, chantharelles, 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dikin, a., kamaruzaman sijam, zainal abidin mior ahmad, mohamad zakaria hussin and idris b abu seman. 2002. interaction between schizophyllum commune and antatonistic bacteria isolated from rotted fruits of oil palm. malaysian microbiology symposium. kota bahru. september, 8 11,2002. dikin, a., kamaruzaman sijam, zainal abidin mior ahmad and idris b abu seman. 2003. association of schizophyllum commune fr. with oil palm seeds. the 3"1 national seed symposium, putrajaya, malaysia. april 8-9,2003. frandberg, e. and j. schnurer. 1998. antifungal activity of chitinolytic bacteria isolated from airtight stored cereal grain. canadian j. microbiol., 44:121-127. haas, k., c. keel, j. laville, m. maurhofer, t. orbenhan-sli, u. schnider, c. voisard, b. wuthrich, g. defago. 1991. secondary metabolites of pseudomonas fluorescens strain chad involved in the suppression of root diseases. in advances in molecular genetics of plant-microbe interactions. vol 1. proceedings of the 5th international 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(3): 385-388. kobayashi, d.y., m. guglielmoni and b.b. clarke. 1995. isolation of the chitinolytic bacteria xanthomonas maltophilia and serratia marcescens as biological control agents for summer patch disease of turfgrass. soil biol. and chemy., 27: 1479-148. miyagawa, h. 2000. biocontrol of bacterial seedling blight of rice caused by burkholderia gladioli using its avirulent isolate. japan journal of phytopathology 66: 232-238. montealegre, j.r., r. reyes, l.m. perez, r. herrera, p. silva and x. besoain. 2003. selection of bioantagonistic bacteria to be used in biological control of rhizoctonia solani in tomato. electron. j. biotech., 6(2):115-127. okamoto, h., m. sato, z. sato.y. koiso, s. iwasaki and m. isaka. 1998. identification of antibiotic red pigments of serratia marcescens f-l-1, a biocontrol agent of damping-off of cucumber, and antimicrobial activity against other plant pathogens. annual phytopathology society. japan, 64:294-298. ordentlich, a., y. elad and i. chet. 1987. rhizosphere colonization by serratia marcescens for the control of sclerotium rqflsii. soil biol. and biochemy., 19: 747-751. pan, m.j., s. redeman, k.kunert and j.w. hastings. 1997. ultrastructural studies on the colonization of banana tissue and fusarium oxysporum f.sp. cubense race 4 by the endophytic bacterium burkholderia cepacia. j. phytopathol., 145: 479-489. phae, c.g., m. sasaki, m. shoda and h. kubota. 1990. characteristics of bacillus subtilis isolated from composts suppressing phytopathogenic microorganisms. soil sc. pi. nutr., 36: 555-586. sand, d.c., m.n. schroth and d.c. hildebrand. 1980. pseudomonas. in laboratory guide for identification of plant pathogenic bacteria, ed. n.w.schaad. aps st. paul. minnesota, p. 72. sharga, b.m. and g.d. lyon. 1998. bacillus subtilis bs107 as an antagonist of potato blackleg and soft rot bacteria. can. j. microbiol., 44: 777-783. szczech, m. and m. shoda. 2004. biocontrol of rhizoctonia dampingoff of tomato by bacillus subtilis combined 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2005 : 20 29 phytotoxicity and field efficacy of exserohilum longirostra jc/min the control of barnyardgrass ecotypes (echinochloa crus-galli var. crus-galli(l.) beauv) abdul shukor juraimi, arifin tasrif, jugah kadir*, suhaimi napis' and soetikno slamet sastroutomo2 *faculty of agriculture, universiti putra malaysia, 43400 serdang, malaysia ; 'faculty of biotechnology and science ofbiomolecular, 43400 serdang, malaysia cab international sea regional centre, 43400 serdang, malaysia; abstract five selected ecotypes of bamyardgrass (echinochloa crus-galli var. crus-gatti) from several rice growing areas in malaysia and indonesia were tested for their susceptibility to the potential bioherbicide (exserohilum longirostratum). bamyardgrass seedlings at the 2-3-lcaf stage were treated with 2.5xl07 conidia/ml from e. longirostratum at different application frequencies (single, double and triple). in addition, aqueous extract assays were evaluated for the presence of a phytotoxic compound responsible for the virulence of the bioherbicide. results of the study showed that disease severity significantly increased 20 days after treatment and resulted in mortality of the seedlings. ecotypes from perak and lampung were most susceptible to the bioherbicide upon triple applications. percentage dry weight reductions were 86.34 and 83.14%, respectively. other ecotypes (melaka, banten and south sulawesi) were observed to have a relatively similar response. moreover, aqueous extracts of e. longirostratum increased mortality up to 92.50% of bamyardgrass seedlings. these findings suggest that regular (double and triple) applications of e. longirostratum at a concentration of 2.5xl07 conidia/ml significantly increased mortality among bamyardgrass ecotypes. mortality of the seedlings was attributed to the presence of a secondary phytotoxic metabolite. key words : field efficacy / phytotoxicity / exserohilum longirostratum i echinochloa crus-galli var. crusgalli i ecotypes. introduction bamyardgrass (echinochloa crus-galli var. crus-galli (l.) beauv) is the most important weed occurring in rice (holm et al. 1977). although various management strategies are available for bamyardgrass control in rice (e.g., cultural measures, hand-weeding, mechanical control and herbicides), each has its limitations (matsunaka 1983). following some recent successes in using plant pathogens as biological agents to control weeds (charudattan 2000), the possibility of using the bioherbicide approach to control bamyardgrass in rice-based cropping systems was investigated in the philippines and japan (zhang and watson 1997; tsukamotoe? al. 1999). as *) correspondent to : jugah kadir, department of plant protection, faculty of agriculture, universiti putra malaysia, 43400 serdang, selangor, malaysia email : kadirj2000@yahoo.com 20 biotropia no. 24, 2005 such exserohilum monoceras has since been selected to be the potential biocontrol agent for echinochloa species (zhang et al. 1997; tzukamoto et al. 1999). in addition, high virulence of e. monoceras on barnyardgrass species was due to the production of a phytotoxic compound by this fungus (zhang and watson 2000). furthermore, another species, exserohilum longirostratum was shown to be a potential biocontrol agent of itchgrass (ahmad et al. 2002). the possibility of using exserohilum longirostratum as a mycoherbicide to control barnyardgrass was investigated under glasshouse conditions. this fungus has also been recorded as a potential biocontrol agent for grassy weeds (chandramohan and charudattan 2001) and is mainly host specific to grassy weeds, while broadleaf weeds and crops are highly resistant (kadir et al. 2003). previously, we reported that e. longirostratum suppressed the growth of barnyardgrass and significantly reduced above ground biomass when applied at i x l o 7 conidia/ml under glasshouse conditions (tasrif et al. 2003). based on the glasshouse studies, we decided to examine the efficacy of this pathogen in field conditions and assess the possibility that a phytotoxin may be responsible for the virulence. thus, the objective of this study was to quantify the efficacy of exserohilum longirostratum in suppressing ecotypes of barnyardgrass at different frequencies of application and its phytotoxic effect on barnyardgrass seedlings. materials and methods seed sources and inoculum production the five selected ecotypes of barnyardgrass for field experiments were collected from malaysian and indonesian rice fields. the distance between sampling locations were within 5 to 10 km (table 1). table 1. selected ecotypes of barnyardgrass for field efficacy studies ecotype location region pk-04 perak malaysia m-01 melaka malaysia l-01 lampung indonesia b-04 banten indonesia ss-01 south sulawesi indonesia small pieces of mycelium from the stock culture were aseptically transferred to potato dextrose agar (pda) in petri dishes. each pda culture was sealed with parafilm and incubated at room temperature (approximately + 30°c) for 7 days. agar plugs with mycelium (6 mm diameter) from the margins of these young 21 phytotoxicity and field efficacy of exserohilum longirostratum abdul shukor juraimi et al. colonies were used as source of inoculum. v-8 juice (v-8 juice of 200 ml, agar 18 g and distilled water 1 liter) medium were used for inoculum production following tray-mass method. the inoculum of selected potential fungal pathogen was produced on v-8 juice agar added with 3.7 mg/1 of streptomycin sulfate and 2.5 mg/1 of choloramphenicol. the antibiotic was added to prevent bacterial contamination. approximately 50 ml of mycelia suspension were sprayed on the food trays (size 20 x 30 cm) containing about 300 ml of v-8 juice agar. then the inoculated trays were incubated at room temperature (25°c to 30°c) for 2 to 3 days under 12 hour light/12 hours dark cycle. conidia were collected from a 2-day-old culture by scraping the agar surface with sterile water and rubber spatula, and filtering conidial suspension through a single sterile cheesecloth (kadir and charudattan 2000). field trials field trials were conducted in field 2 of the faculty of agriculture, universiti putra malaysia. plastic buckets (20cm diameter and 30 cm height) were filled each with 15 kg soil (jawa series) from tanjong karang paddy fields. each bucket was planted with pre-germinated seeds of barnyardgrass ecotypes. basal fertilizer (45 kg n/ha, 40 kg p/ha and 30 kg k/ha) was applied before planting (pane and masshor 1997). one week after emergence, the 2-3-leaf stage seedlings were selected and thinned to 5 seedlings/bucket. an e. longirostratum concentration of 2.5xl07 conidia/ml as counted using haemocytometer was used with 0.05% tween 20 as wetting agent diluted in water. each treatment was sprayed with 100 ml conidial solution using hand sprayer. tween 20 has been used in field trials as an additive to spore suspension to improve plant kill (klein and auld 1995). disease assessment was determined at 5-day intervals for 20 days after spraying. disease assessment was done visually based on a disease severity scale as developed by kadir and charudattan (2000). the disease ratings were: 0: 0% infection, 1: 1 to 10% leaf area are damaged, 2: 11 to 20% leaf area are damaged, 3: 21 to 30% leaf area are damaged, 4: 31 to 40% leaf area are damaged, 5: 41 to 50% leaf area are damaged, 6: 51 to 60% leaf area are damaged, 7: 61 to 70% leaf area are damaged, 8: 71 to 80% leaf area are damaged, 9: 81 to 90% leaf area are damaged and 10: 91 to 100% leaf area are damaged (plant died). environmental conditions such as relative humidity, rainfall and temperature during the study were noted. this was a factorial experiment in a randomized complete block design. barnyardgrass ecotypes (pk-04, m-01, l-01, b-04 and ss01) were selected as factor one, while the frequency of applications (single, double and triple applications) was selected as the second factor. each treatment combination was repeated four times. all control plants were sprayed with water containing 0.05% tween 20. a completely randomized design was carried out to determine the phytotoxic effect. 22 biotropia no. 24, 2005 phytotoxic effect of aqueous extracts agar plugs (6 mm diameter) of the fungus taken from the edge of 7-day-old pda cultures (inoculum) were placed into 500-ml erlenmeyer flasks containing 100 ml of modified fries liquid medium (30.0 g of sucrose, 5.0 g of kh2po4, 0.5 g of ammonium tartrate, 1.0 g of mgso4, 0.1 g of nacl, 0.1 g of cacla, 0.5 g of casein hydrolysate, and 1.0 g of yeast extract in 1000 ml of distilled water) (zhang et al. 1996). flasks were then placed on rotary shakers at 100 "rpm" and flask containing only modified fries medium served as control. after a 2-week incubation, cultures collected from each flask were separately centrifuged at 6000 "rpm" for 10 min. the supernatant was filtered through cheesecloth, followed by two layers of whatman no.2 filter paper to obtain a cell-free culture filtrate. the 3 concentrations of the e. longirostratum aqueous extract used were full-strength (v/v) (250 ml/1) half-strength (125 ml/1) and quarter-strength (62.5 ml/1). dilutions were made with sterilized distilled water. the roots of the barnyardgrass seedlings at the 2-leaf growth stage were immersed in plastic containers containing 25 ml of the aqueous extract. observations were carried out 48 hours later. disease severity was assessed based on disease incidence (percentage of diseased plant) (kadir and charudattan 2000). necrosis and wilting indicated the presence of the phytotoxic compound in the aqueous extract. statistical analysis anova was used to test for the determination of significant differences among treatment combinations, while mean separation was resolved using duncan's new multiple range test at p < 0.05 (sas 2000) for both experiments. disease severity was determined according to area under disease progress curve (audpc) (campbell and madden 1990). the audpc is the disease intensity between two periods. the higher the audpc value, the more severe the disease is. dry weight above ground biomass reduction (percent control) was taken 20 days after spraying. disease assessments were carried out based on disease index scale (0: immune, 1-2: resistant, 3-4: tolerant, 5-7: susceptible and 8-10: highly susceptible) (chang and hwang 2003). results field efficacy studies means relative humidity (%), temperature (°c) and rainfall at the experimental sites were 94%, 24 to 33°c and 8 mm, respectively. location of the trials had no significant effect on growth of barnyardgrass ecotypes. the disease severity that was observed for all ecotypes tested as expressed by audpc ranged from 590 to 1118 units 20 days after spraying (table 2). the results 23   audpc-area under disease progress curve (campbell and madden 1990); column means followed by the same small letter and row means followed by the same capital letter arc not significantly different at the 5% level by duncan's new multiple range test (dnmrt). indicate that the audpc for ecotype of pk-04 (1118) and l-01(1083) were more severely affected by treatment with e. longirostratum conidia than ecotypes m-01(775), b-04 (883), and ss-01(773). it is interesting to note that the effects of application frequencies for pk-04 and l-01 ecotypes were similar (table 2). effects of application frequency on seedling response as shown by the audpc also were observed. ecotypes pk-04 and l-01 gave similar responses for all spray frequencies and were significantly different (p < 0.05) compared to other ecotypes. ecotypes m-01 and ss01 gave similar responses at triple application frequencies. ecotype of b-04 and ss-01 had similar response at double application frequencies. the effect of application frequency on dry weight reduction of above ground biomass for all treatments is presented in table 3. the range of dry weight reduction was 37.58 to 86.34%. ecotypes of pk-04 and l-01 yielded the highest dry weight reduction (86.34 and 83.14%) at triple applications and was significantly different compared to other ecotypes. the least dry weight reduction was recorded for the ss-01 ecotype at all three levels of applications. the single and double application frequencies that reduced ecotypes biomass by more than 50% were observed for the pk-04 and l-01 ecotypes. however, double and triple applications reduced dry weight biomass below 50% for ss-01 ecotypes. in general, ecotypes pk-04 and l-01 were found to be most susceptible to. e. longirostratum injury. at triple applications, pk-04 disease severity increased up to 15 days after spraying but leveled off afterwards. at a single application, the disease severity increased up to 10 days following applications then leveled off afterwards (figure 1). however, at triple applications the level of disease severity increased beyond values obtained for those receiving double and single applications. 24     phytotoxicity and field efficacy of exserohilum longirostratum — abdul shukor juraimi et al. phytotoxic effect of aqueous extracts three aqueous extract concentrations at full, half, and quarter-strength prepared from the fungus produced necrotic, chlorosis and wilting in e. crus-galli seedlings within 48 hours after immersion. at full strength, the solution reduced living leaf area by a disease incidence of 92.5% as compared to controls and was significantly different compared to the other two treatments. half strength and quarter strength aqueous extracts caused a disease incidence of 85.0% and 57.5%, respectively (table 4). the results indicated that higher aqueous extract concentration increased seedling mortality. discussions field efficacy the virulence of the fungus e. longirostratum (upm isolate) was evaluated on 5 barnyardgrass ecotypes from different geographic locations under field conditions. among the ecotypes studied, pk-04 and l-01 were most susceptible to e. longirostratum toxicity compared to ecotypes m-01, b04 and ss-01. in this study, varied responses among barnyardgrass ecotypes in disease severity and dry weight reduction were obtained following single, double, and triple applications of e. longirostratum conidia. the conidial isolate of e. longirostratum was pathogenic to all ecotypes of barnyardgrass. the response ranged from highly susceptible (pk-04 and l-01 ecotypes) and moderately susceptible (m-01, b-04, and ss-01 ecotypes) was recorded in our field experiments. similar variability in pathogenic response among different ecotypes has also been reported for other plants. leisner and howel (1992) reported variation in symptoms from highly susceptible to resistant among 18 ecotypes of arabidopsis thaliana infected by cauliflower mosaic virus (cmv). in another study, chang and hwang (2003) reported significant differences in level of disease response among 6 adlay cultivars to fungal bipolaris coicis from different locations in south korea. 26 biotropia no. 24, 2005 e. longirostratum reduced growth and dry weight of barnyardgrass ecotypes pk-04, l-01 and b-04. however, efficacy of this fungus to control barnyardgrass depends on conidial concentration, frequency of application, and the genetic variability of barnyardgrass ecotypes. several publications on other fungal bioherbicides suggest an inoculum concentration of 106 conidia/ml to be the effective concentration in their studies (wymore and watson 1989; kadir et al. 2000). rosskopt et a/.(1999) also observed that the optimal level of control over amaranthus species by phomopsis amaranticola occurred at 6xl07 conidia/ml. accordingly, we agreed on an inoculum density of 2.5xl07 conidia/ml for e. longirostratum for our studies. regular application of certain bioherbicides has been reported to provide long-term control of the target weed. for example, kadir and charudattan (2000) reported that dactylaria higginsi provided excellent long-term control of purple nutsedge (cyperus rotundus l.) when it was applied regularly. in this study, regular application of e. longirostratum significantly controlled barnyardgrass. however, the severity of the disease caused by this fungus for barnyardgrass ecotypes in the field did not always correlate well with the aggressiveness observed in glasshouse studies. this may suggest that other factors such as increased inoculum concentration, improved formulation and barnyardgrass density influence disease severity. phytotoxic effect of aqueous extracts e. longirostratum causes leaf blight in echinochloa crus-galli ecotypes and is presently being evaluated as a potential mycoherbicide for the control of barnyardgrass. inoculation with aqueous extracts from e. longirostratum resulted in a blight-like reaction characterized by chlorosis appearing 12 hours after immersion. this was followed by rapid necrosis of the affected leaf tissue, often with an absence or weak expression of typical lesions (tasrif et al. 2003). the symptoms induced by e. longirostratum appear to be due to the presence of a phytotoxic compound. zhang and watson (2000) characterized the phytotoxic compound of e. monoceras as toxin-1, shown to be a potent and host-specific principle responsible to disease expression in echinochloa species. also, the toxin tryptophol appear to be a major metabolite in culture filtrates of drecshlera nodulosa, a pathogen of goosegrass (eleusine indica (l.) gaerth.) reported by sugawara and strobel (1987). microscopic observation following treatments with the full and half-strength aqueous extracts caused plasma-membrane breakdown in leaf tissue of the highly susceptible ecotype pk04. these results would support the presence of a phytotoxin in the aqueous extracts, that may have affected plasma membrane integrity of susceptible ecotypes in the infection process of the fungus e. longirostratum. ismail et al. (1993) have reported similar results from full-strength of aqueous extracts of legume cover crops that decreased germination and seedling growth of asystasia intrusa and paspalum conjugatum weeds attributed to an allelopathic principle. 27 phytotoxicity and field efficacy of exserohilum longirostratum abdul shukor juraimi et al. in this study, our results demonstrate that e. longirostratum at a concentration of 2.5xl07 conidia/ml effectively increased the mortality of barnyardgrass ecotypes at the 2-3-leaf stage. conclusions the fungal pathogen e. longirostratum seems to be a potential candidate as mycoherbicide on barnyardgrass. phytotoxic compound of this fungus might also enhance virulence on barnyardgrass. however, for satisfactory control on barnyardgrass, further studies need to be addressed on the increase in conidial concentration, use of different surfactant, formulation, and time of application. acknowledgement our appreciation goes to the integrated pest management for small-estate crop project (ipm-schep), ministry of agriculture in indonesia and felda foundation of malaysia who had funded this study. we also thank ms. christina stephensons for her valuable contribution in correcting the manuscript. references ahmad, a., j. kadir, m. sariah, a.s. juraimi, 2002. evaluation for drechslera sp. for potential bioherbicidc control of itchgrass (rottboellia cochinchinesis; poaccac). screening and host range test. proceedings third international conference on biopesticidcs, ed. e.m. mulla, the department of entomology, university of california, riverside, usa. p. 181 -184. campbell, c.l. and l.v. madden, 1990. introduction to plant disease epidemiology. john wilcy & son. 532 p. chandramohan, s. and r. charudattan, 2001. control of seven grasses with a mixture of three fungal pathogens with restricted host range. biological control, 22: 246-255. chang, s.w. and b.k. hwang, 2003. evaluation of virulence to adlays of korean isolates of bipolaris colds using a disease rating scale. plant disease, 726-731. charudattan, r. 2000. current status of biological control of weeds: in emerging technologies for integrated pest management. concepts, research and implementation, cds c.g. kennedy and t.b. sutton, st. paul minnesotta, aps press, p.269-288. holm, l.g., d.l. plucknett, j.v. pancho and j.p. herberger, 1977. the world's worst weeds. distribution and biology. hawaii, honolulu. ismail, b. s., a. tasrif, sastroutomo, s.s. and a. latif, 1993. allelopathic potential of legume cover crops on selected weed species. plant protection quarterly, 86: 81-87. kadir, j. b. and r. charudattan, 2000. dactylaria higginsi, a fungal bioherbicide agent for purple nutsedge (cyperus rotundas), biological control, 17: 113-124. 28 biotropia no. 24, 2005 kadir, j., r. charudattan and r.d. berger, 2000. effects of some epidemiological factors on levels of disease caused by dactylaria higginsi on cyperus rotundus. weed science, 48:61-68. kadir, j., a. ahmad, m. sariah and a.s. juraimi, 2003. potential of drechslera longirostrata as bioherbicide for itchgrass (rottboellia cochinchinensis). proceedings of the nineteenth asian pacific weed science society conference. weed science society of the philippines, manila, philippines, p 450-455. klein, t. a. and b.a. auld, 1995. evolution of twccn 20 and glycerol as additives to mycoherbicides suspensions applied to bath rust burr. plant protection quarterly, 1: 14-16. lcisner, s.m. s.h. howell, 1992. symptoms variation in different arabidopsis thaliana ecotypes produced by cauliflower mosaic virus. phytopathology, 82: 1042-1045. matsunaka, s. 1983. evolution of rice weeds control practices and research: world perspective in weed control in rice. international rice research institute. manila, philippines, p 5-17. pane, h. and m. masshor, 1997. fenoxaprop-p-ethyl is an effective herbicide in controlling red sprangletop (leptochloa chinensis l. ness). proceedings 16lb asian pacific weed science society conference, -malaysian plant protection society, ed. rajan, a. kuala lumpur, malaysia.p.272-277. rosskofpf, e.n., r. charudattan and j.b. kadir, 1999. use of plant pathogens in weed control in handbook of biological control, eds. t.s. bellows, t.w. fisher, l.e. caltagirone, d.l. dahlsten, g. gordt and c.b. huffaker, san diego, san fransisco, new york, boston, sydney, tokyo. academic press, p.891-919. 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disease 80: 1053-1058. zhang, w.m. and a.k watson, 1997. host range of exserohilum monoceras, a potential bioherbicide for the control of echinochloa species. canadian journal botany, 75: 685-692. zhang, w.m. and a.k . watson, 2000. isolation and partial characterization of phytotoxin produced by exserohilum monoceras, a potential bioherbicide for control of echinochloa species. proceedings of the tenth international symposium on biological control of weeds, ed. n.r. spencer, montana, usa. p.125-130. 29 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf biotropia vol. 30 no. 2, 2023: 183 194 doi: 10.11598/btb.2023.30.2.1784 183 ethnobotanical study of medicinal plant usage during covid-19 pandemic: a community-based survey in indonesia ni made dwi mara widyani nayaka1*, putu era sandhi kusuma yuda1, dwi arymbhi sanjaya2, desak ketut ernawati3, erna cahyaningsih1, ni luh kade arman anita dewi1 and maria malida vernandes sasadara1 1department of natural medicine, faculty of pharmacy, universitas mahasaraswati denpasar, denpasar, 80233, indonesia 2departement of pharmacology and clinical pharmacy, faculty of pharmacy, universitas mahasaraswati denpasar, denpasar, 80233, indonesia 3departement of pharmacology and therapy, faculty of medicine, udayana university, denpasar, 80232, indonesia received 21 july 2022 / revised 1 june 2023 /accepted 4 june 2023 abstract before the availability of a vaccine, indonesian population relied on traditional medicines to prevent covid19. any species used by indigenous people could lead to further investigations in modern pharmacology, to preserve ancient knowledge, and to plan for plants’ conservation. the study aimed to discover and record species, methods of preparation, route of administration, and motivation in using medicinal plants by the indonesian population during the covid-19 pandemic. participants of survey were selected from the people who live in java and bali for responding to an online structured questionnaire. relative frequency of citation (rfc) was employed in the quantitative analysis of the collected data. the pharmacological relevance of the five plants with the highest rfc was further reviewed. the results showed that respondents used 59 plants from 28 families. five species with the highest rfc were curcuma longa (0.707), zingiber officinale (0.674), cymbopogon citratus (0.269), kaempferia galanga (0.174), and curcuma zanthorrhiza (0.165). most plants were prepared by boiling (77.97%) and administered orally as a single ingredient or mixed with other herbals. respondents believed that the plants were beneficial as immune-booster (71.26%), maintain good health (24.85%) and stamina (12.28%), and prevent viral infection, including covid-19 (5.39%). the most commonly used plants might be scientifically based to boost immunity. however, their usage against covid-19 and the medicinal value of herbal mixtures should be further investigated. keywords: covid-19, ethnobotany, indonesia, medicinal plants introduction corona virus disease 2019 (covid-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (sarscov-2). it was found in china in december 2019 and spread to other countries, including indonesia. the first two confirmed covid-19 cases in indonesia were reported on march 2, 2020, and the numbers keep rising since then (djalante et al. 2020). based on history, previous sars-cov coronavirus also caused an outbreak in china in 2003. the genetic sequence analysis showed that sars-cov-2 was similar around 79% to sarscov. thus, most of the studies on its prevention and medication were adopted from the previous outbreak (ghaffari et al. 2020). the indonesian ministry of health released health protocols to prevent and control covid-19 (hk.01.07/menkes/382/2020), which included the suggestion to wear masks, washing hands frequently, social distancing, and immunity enhancement through clean and healthy living behavior. during the sars outbreak, natural medicine showed beneficial effects in preventing and treating patients, particularly in high-risk subjects (boozari & *corresponding author, email: nimade.nayaka@unmas.ac.id; nimade.nayaka@gmail.com biotropia vol. 30 no. 2, 2023 184 hosseinzadeh 2020; y. li et al. 2020). the usage of medicinal plants as a prophylaxis measure against covid-19 was also recommended by ayurveda and traditional chinese medicine (boozari & hosseinzadeh 2020; khanal et al. 2020; vellingiri et al. 2020). further, many studies have proven the antiviral, antiinflammatory, and immunomodulatory properties of medicinal plants that are potentially helpful to combat viral diseases (lin et al. 2014; khanna et al. 2020). indonesia has abundant natural resources in plant species and the local people have used them as herbal remedies. some of the ethnobotanical studies recorded the importance of indonesian biodiversity as traditional medicines in different health conditions (nahdi & kurniawan 2019; taek et al. 2019; jadid et al. 2020). those studies are significantly important to converse precious indigenous knowledge and publish them as academic literature. in the present study, we conducted an online survey to identify the use of medicinal plants by the indonesian population during the covid-19 pandemic. the comprehensive data from respondents on the species of medicinal plants, method of preparation and administration, as well as motivation to use, were documented. while antivirus and vaccines are vital, the research on natural medicine regarding covid-19 may be used as a reference to develop new drug candidates and as homebased remedies in the future that are inexpensive, commonly, and easily implemented in society. the study aimed to discover and to record species, methods of preparation, route of administration, and motivation in using medicinal plants by the indonesian population during the covid-19 pandemic. materials and methods study area a survey was conducted in java and bali island (figure 1), which consist of 7 provinces, namely special capital region of jakarta (6°12′s, 106°49′e), banten (6°30′s 106°15′e), west java (6°45′s 107°30′e), central java (7°30′s 110°00′e), special region of yogyakarta (7°47′s 110°22′e), east java (7°16′s 112°45′e), and bali (8°20′06″s 115°05′17″e). all the regions have diverse ethnicities such as bantenese in banten, balinese in bali, and javanese in other regions. respected to the ethnicity, each of the regions has its traditional language. however, natives speak indonesian in their daily lives. figure 1 study area of the ethnobotanical survey in 7 provinces in indonesia medicinal plants usage during covid-19 in indonesia – nayaka et al. 185 data collection a cross-sectional study was undertaken using a self-administered and structured questionnaire. the questionnaire consisted of three parts that aimed to collect the respondents demographic characteristics, medicinal plants data, and respondents’ motivation using the plants during the pandemic. the data collection was carried out by online survey from june to august 2020. the questionnaire was in indonesian and examined by two experts in pharmacy and bahasa indonesia fields then piloted among 30 participants to ensure its validity before being used to collect data. a guide to estimate the minimum sample size of respondents required for this study based on the formula (pourhoseingholi et al. 2013). n = z2 p(1 – p) d2 where: z = the statistic corresponding to level of …….confidence. p = expected prevalence of covid-19. d = precision. in this study, the respondents included indigenous people living in java and bali islands and consuming medicinal plants during the covid-19 pandemic in indonesia. the respondents’ motivation to use the plants was also recorded. any dubious data that could not be confirmed was excluded. the questionnaire and methodology for this study were approved by the faculty of medicine, udayana university (ethics approval number: 1195/un14.2.2.vii.14/lt/2020). plant identification the scientific names of medicinal plants reported by respondents were determined using cross-references between their local names and database in indonesian herbal pharmacopeia, indonesian herbal formulary (permenkes no.6/2016), and indonesian traditional medicine formulary (hk.01.07/menkes/ 187/2017). herbal specimens could not be collected due to the strict travel restrictions regulated by the indonesian government during the covid-19 pandemic in the study area. the scientific names of the reported plants were checked with the plant list website (accessed on january 8, 2021, http://www.theplantlist.org). data analysis the collected data were evaluated by using microsoft office excel (2016) spreadsheets. further, quantitative data analysis to show the local importance of each plant species was demonstrated by using the relative frequency of citation (rfc) (aziz et al. 2017). with the formula below: rfc = fc/n (0 < rfc < 1) where: fc = number of informants mentioning a ………particular species. n = total number of respondents results and discussion indonesia is an inhabitant of about 80% of the world’s medicinal plants which local people use to prevent and cure many ailments (elfahmi et al. 2014). in the current study, an online survey was conducted to collect data regarding medicinal plant usage by indonesian during the covid-19 pandemic. the respondents were limited to those who were native and living in 8 provinces in the two most densely populated islands in indonesia (java and bali islands) (table 1). moreover, based on the indonesian government’s official website (www.covid19.go.id), most of the confirmed covid-19 cases were located in both islands and the prevalence reached 82.2%. based on the prevalence of covid-19 and the statistic corresponding to the level of confidence is 1,96 with a precision of 5%, the minimum number of respondents for this study was 224 respondents. however, in this study, 344 respondents participated and this number exceeded the minimum sample. as shown in table 1, 82.04% (274) of respondents were female. this result is in line with several studies (villena-tejada et al. 2021; brahmi et al. 2022; odebunmi et al. 2022). which indicated the domination of females in using medicinal plants. this predominance is probably related to several factors such as women being more familiar with medicinal plants because they biotropia vol. 30 no. 2, 2023 186 are also being used as cooking ingredients. correspondingly, in most populations, women are believed to bear the responsibility for the family health needs causing them to be more informed about using medicinal plants than their male counterparts (torres-avilez et al. 2016). in the current study, most of the respondents came from the province of bali. a hindu-most populated area in indonesia (statistics indonesia 2010). the balinese are well known for their local wisdom called usada, a traditional medicine inspired by the hindu holy book ayurveda (muderawan et al. 2020). age is another sociodemographic factor contributing to the use of medicinal plants. several studies found that older age was the main user of traditional medicine (rahayu et al. 2020). on the contrary, 67.37% (225) of respondents in this study were 20 to 40 years old. indicating the younger age group was also interested in using medicinal plants during the pandemic of covid-19. similar survey studies conducted in algeria and morocco during the pandemic also showed similiar results (belmouhoub et al. 2021; brahmi et al. 2022; chebaibi et al. 2022). table 1 demographic characteristics of respondents characteristic number of respondents (n = 334) percentage (%) gender male 60 17.96 female 274 82.04 province of origin special capital region of jakarta 13 3.89 banten 2 0.60 west java 35 10.48 central java 8 2.40 east java 24 7.19 special region of yogyakarta 6 1.80 bali 246 73.65 age (years) < 20 35 10.48 20 – 40 225 67.37 > 40 74 22.16 religion islam 66 19.76 hindu 247 73.95 protestant 12 3.59 catholic 8 2.40 buddha 1 0.30 table 2 medicinal plants used by respondents during the covid-19 pandemic in indonesia family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd acanthaceae andrographis paniculata (burm.f.) nees sambiloto green chiretta leaves boil 0.030 a, b, d, e 1, 5, 7 amaryllidaceae allium cepa l. bawang merah shallot bulb boil, burning, eaten directly 0.006 b 6,7 allium sativum l. bawang putih garlic bulb eaten directly 0.063 a-e 3-7 anacardiaceae spondias pinnata (l.f) kurz. cemcem common hog-plum leaves cold infusion 0.006 b 7 annonaceae annona muricata l. sirsak soursop leaves boil 0.015 a-d 3,7 apiaceae apium graveolens l. seledri celery leaves boil 0.003 b 3 centella asiatica (l.) urb. pegagan asiatic pennywort leaves boil, eaten directly 0.021 a,b,d,e 7 coriandrum sativum l. ketumbar coriander fruit boil 0.012 a,b,d,e 3, 5, 7 foeniculum vulgare adas fennel fruit boil 0.003 b 4 medicinal plants usage during covid-19 in indonesia – nayaka et al. 187 family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd mill. arecaceae cocos nucifera l. kelapa coconut fruit (water & oil) eaten directly 0.009 b 7 asteraceae blumea balsamifera (l.) dc. sembung buffalo-ear leaves boil 0.006 b,d 7 gynura procumbens (lour.) merr. sambung nyawa longevity spinach leaves boil 0.003 b 6 pluchea indica (l.) less. beluntas indian camphorweed leaves boil 0.009 b 7 sonchus arvensis l. tempuyung perennial sow-thistle leaves boil 0.003 b 7 basellaceae anredera cordifolia (ten.) steenis binahong gulf madeiravine leaves boil 0.003 b 1 caricaceae carica papaya l. pepaya papaya leaves boil 0.003 e 3 fabaceae caesalpinia sappan l. secang brazilwood wood boil 0.027 a-e 3-7 clitoria ternatea l. bunga telang asian pigeonwings flower boil 0.009 a,d 5, 7 erythrina variegata l. dadap tiger’s claw leaves boil 0.003 a,d 7 tamarindus indica l. asam jawa tamarind fruit boil 0.060 a-e 1, 3-7 lamiaceae mentha piperita l. pipermin peppermint leaves hot infusion 0.009 a,b,d,e 2, 3, 7 peronema canescens jack sungkai false elder leaves boil 0.003 b 3 lauraceae cinnamomum burmanni (nees & t.nees) blume kayu manis batavia cinnamon bark, leaves boil, burning, cold and hot infusion 0.114 a-e 1-7 malvaceae hibiscus sabdariffa l. rosela roselle flower boil 0.003 b 7 meliaceae azadirachta indica a.juss mimba neem leaves eaten directly 0.003 b 7 moraceae artocarpus altilis (parkinson ex f.a.zorn) fosberg sukun breadfruit leaves boil 0.003 b 7 moringaceae moringa oleifera lam. kelor drumstick tree leaves boil, hot infusion 0.045 a-e 3-7 myrtaceae melaleuca cajuputi powell kayu putih cajuput oil n/a 0.003 c 1,7 syzygium aromaticum (l.) merr. & l.m.perry cengkeh clove flower boil, hot infusion 0.048 a-e 2-7 syzygium polyanthum (wight) walp. salam indonesian bay leaf leaves boil 0.021 a-e 3, 5-7 oleaceae olea europaea l. zaitun olive fruit (oil) eaten directly 0.003 b 3 pandanaceae pandanus amaryllifolius roxb. pandan pandan leaves boil 0.003 a,d 2 phyllanthaceae sauropus androgynous (l.) merr. katuk sweet leaf leaves cold and hot infusion 0.021 a,b,d,e 7 phyllantus niruri l. meniran gale of the wind leaves boil, hot infusion 0.009 a,b 5, 7 piperaceae piper betle l. sirih betel leaves boil, eaten directly 0.072 a-e 1, 3 piper crocatum ruiz & pav. sirih merah celebes pepper leaves boil 0.072 b 7 piper nigrum l. lada black pepper seed boil 0.003 b 3 piper retrofractum vahl cabai jawa javanese long pepper fruit boil 0.003 e 7 poaceae cymbopogon citratus (dc.) stapf. serai dapur lemongrass stem, leaves boil, hot infusion 0.269 a-e 1-7 oryza sativa l. beras rice starch cold infusion 0.024 a,b,d,e 3-7 ranunculaceae nigella sativa l. jintan hitam black seed seed boil 0.012 a,b,e 3, 5, 7 rutaceae citrus aurantiifolia (christm.) swingle jeruk nipis egyptian lime fruit juiced, boil, hot and cold infusion 0.177 a-e 1-7 citrus hystrix dc. jeruk purut kaffir lime leaves, fruit boil 0.006 b,e 6 citrus limon (l.) osbeck lemon lemon fruit cold and hot infusion 0.108 a-e 1, 3-5, 7 citrus reticulata jeruk mandarin fruit eaten directly 0.021 a,b,e 4, 7 biotropia vol. 30 no. 2, 2023 188 family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd blanco rubiaceae morinda citrifolia l. mengkudu noni fruit juiced 0.015 b-d 7 schisandraceae illicium verum hook.f. bunga lawang star anise flower boil 0.009 a,b,e 4, 5, 7 theaceae camellia sinensis (l) kuntze teh tea plant leaves boil, hot infusion 0.012 b-d 1, 3, 7 zingiberaceae alpinia galanga (l) willd. lengkuas greater galangal rhizome boil, hot infusion 0.054 a-e 1, 4, 5, 7 boesenbergia pandurata (roxb.) schltr. temu kunci chinese keys rhizome boil 0.006 b,e 7 curcuma longa l. kunyit turmeric rhizome boil, burning, hot infusion 0.707 a-e 1-7 curcuma zanthorrhiza roxb. temulawak javanese turmeric rhizome boil, hot infusion 0.165 a-e 1-7 curcuma zedoaria (christm.) roscoe temu putih zedoary rhizome boil 0.006 a,d,e 5, 7 amomum compactum sol. ex maton kapulaga javanese cardamom seed, flower boil, hot infusion 0.018 b,d,e 3, 5, 7 kaempferia galanga l. kencur cutcherry rhizome boil, burning, hot infusion 0.174 a-e 1-7 kaempferia rotunda l. kunyit putih peacock ginger rhizome juice, hot infusion 0.021 a,b,d,e 5, 7 zingiber officinale roscoe jahe ginger rhizome boil, burning, hot infusion 0.674 a-e 1-7 zingiber officinale var.rubrum theilade jahe merah red ginger rhizome boil, hot infusion 0.039 a,b,d,e 3, 5-7 zingiber zerumbet (l.) roscoe ex sm. gamongan /lempuyang gajah bitter ginger rhizome boil 0.003 e 7 notes: aall plants were prepared with water and administered orally, except cajuput by inhalation. brfc = relative frequency of citation, ca = to keep healthy, b = to boost the immune system, c = to avoid virus infection, including covid-19, d = to build stamina, e = other, d1 = special capital region of jakarta, 2 = banten, 3 = west java, 4 = central java, 5 = east java, 6 = special region of yogyakarta, 7 = bali, n/a = not available, table 3 plants mixtures used by respondents during the covid-19 pandemic in indonesia plants mixture no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a. sativum + c. asiatica + c. nucifera + p. indica + t. indica + + + + c. burmanni + + s. aromaticum + p. amaryllifolius + + p. betle + c. citratus + + + + + + + + o. sativa + c. aurantiifolia + + + + + + + + c. lemon + + + + + c. sinensis + + a. galangal + c. longa + + + + + + + + + + + + + + + + + + + + c. zanthorrhiza + + + + + + + k. galangal + + + + k. rotunda z. officinale + + + + + + + + + + + + + + + + + + total plants mixed 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 notes: + = plants available in mixtures. * all mixtures were prepared by boiling and administered orally medicinal plants usage during covid-19 in indonesia – nayaka et al. 189 table 3 plants mixture used by respondents during the covid-19 pandemic in indonesia (continued) plants mixture no.* 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 c. asiatica + + c. sativum + f. vulgare + c. sappan + + + + + c. ternatea + t. indica + + + m. piperita + p. canescens c. burmanni + + + + + + + + + + a. altilis + s. aromaticum + + + + + + + s. polyanthum + o. europea p. amaryllifolius s. androgynous + p. niruri + p. betle + + + p. crocatum + p. retrofractum + c. citratus + + + + + + + + + + + + + + + + + + + + c. aurantiifolia + + + + + + + c. lemon + + i. verum + a. galanga + + + + b. pandurate + c. longa + + + + + + + + + + + + + + + + + + + + + + + + + + c. zanthorrhiza + + + + + + + + + + + c. zedoaria a. compactum + + + k. galangal + + + + + + + + + + + + + + + k. rotunda + + + + z. officinale + + + + + + + + + + + + + + + + + + + + + + + + + + + z. officinal var. rubrum + z. zerumbet + total plants mixed 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 8 9 10 notes: + = plants available in mixtures. * all mixtures were prepared by boiling and administered orally table 4 method of preparation for medicinal plants used during the covid-19 pandemic in indonesia category frequency %* boil 46 77.97 eaten directly 6 10.17 cold infusion 18 30.51 hot infusion 8 13.56 burning 5 6.78 notes: *some of the plants were prepared by more than one method. thus, the total percentage may not add up to 100%. table 5 the motivation for medicinal plants used by respondents during the covid-19 pandemic in indonesia category frequency %* to boost the immune system 238 71.26 to keep healthy 83 24.85 to build stamina 41 12.28 other motives 41 12.28 to prevent virus infection, including covid-19 18 5.39 notes: *some respondents reported more than one reason for herbal use. thus, the total percentage may not add up to 100%. biotropia vol. 30 no. 2, 2023 190 the present study revealed that respondents used 59 species of medicinal plants from 28 families (table 2) both singly or in herbal mixtures (table 3). additionally, most of the plants were prepared by boiling and then consumed orally (table 4). indonesian traditional medicine in the form of polyherbal drinks has existed for generations so called loloh (in bali) and jamu (in java). some of the reported plants in the present study that are also used in loloh formulation namely s. pinnata, b. balsamifera, e. variegata., c. burmanni, c. asiatica, a. indica, p. amaryllifolius, p. niruri, s. androgynous, p. betle, c. aurantiifolia, c. zanthorrhiza, k. rotunda, z. officinale, and z. zerumbet (sujarwo et al. 2015). similarly, other reported plants were commonly available in jamu formula such as c. verum, c. aurantifolia, z. officinale var. rubrum, t. indica, a. galanga, c. longa, c. zanthorrhiza, p. niruri. k. galanga, o. sativa, and p. amaryllifolius (elfahmi et al. 2014; hartanti et al. 2020). these plants were empirically used for various medicinal purposes (elfahmi et al. 2014; sujarwo et al. 2015). further, the current study revealed that respondents consumed jamu kunyit asam and jamu beras kencur during the pandemic (table 3, mixture no. 5 and no. 9, respectively). the composition of herbal mixtures could be varied according to individual preferences and local recipes. for example, mixture no.20, 29, 45, 52, and 59 in table 3 showed the variation of jamu kunyit asam. however, there was limited data related to the efficacy of the modified version of jamu formula. further research should be conducted to support the use of those herbal mixtures in term of efficacy test. most respondents believed that the plants’ consumption was beneficial during the pandemic because they could enhance immunity, maintain health and stamina, and prevent viral infection (table 5). a study showed that the interest in and use of immunerelated herbals worldwide increased during the covid-19 pandemic (hamulka et al. 2020). other ethnopharmacological studies confirmed the usage of medicinal plants such as a. cepa, a. sativum, c. asiatica, c. papaya, t. indica, c. burmanni, and c. longa to boost immunity by traditional healers and society in various health conditions (siew et al. 2014; anywar et al. 2020; oladele et al. 2020; lin et al. 2021). meanwhile, review studies confirmed the benefit of natural immune enhancer intakes such as a. panniculata, a. sativum, m. piperita, m. cajuputi essential oil, c. sinensis, n. sativa, and z. officinale to prevent covid-19 and improve overall patient health (boozari & hosseinzadeh 2020; sen et al. 2020; silveira et al. 2020). some of the medicinal plants reported in the current study have been also recommended by the indonesian ministry of health (hk.02.02/iv.2243/2020) to maintain wellbeing and prevent illness during the pandemic. in the official announcement, six herbal mixtures consisting of z. officinale var. rubrum, c. aurantiifolia, c. verum, c. longa, a. galanga, c. asiatica, c. zanthorrhiza, k. galanga, p. amaryllifolius, m. oleifera, and a. sativum. the mixtures were recommended to boost the immune response and also have similar preparation and administration methods as reported in this study. likewise, c. longa, z. officinalle, c. verum, and p. nigrum also have been recommended by the indian ministry of ayush (ayurveda, yoga, and naturopathy, unani, siddha, and homeopathy) to boost immunity as a prophylaxis measure against covid-19 (khanal et al. 2020). the beneficial effect of medicinal plants as immune-enhancer against covid-19 should be confirmed scientifically. in viral diseases, the infection could be fought by the host's immune response. when viruses infect the host cells, innate immunity blocks virus replication, promotes virus clearance, stimulates tissue repair, and activates a prolonged adaptive immunity (g. li et al. 2020). moreover, viral infection and inflammation of lung tissues are observed in covid-19. thus, the antiviral and inflammatory activities of medicinal plants are essential properties to combat covid-19 (khanal et al. 2020). on the other hand, it should be noticed that the immune system is complicated and highly regulated by numerous molecular and cellular events. therefore, immunity enhancement may be either valuable or destructive to the organism, depending on the overall degree of modulation and the pathophysiological condition (gertsch et al. 2011). based on the calculation of rfc, five plants had the highest scores: c. longa, z. officinale, c. medicinal plants usage during covid-19 in indonesia – nayaka et al. 191 citratus, k. galanga, and c. zanthorrhiza. therefore, further literature review in the current study was highlighted for those plants. turmeric (c. longa) contains curcumin, a polyphenol with various pharmacological actions. the compound showed immunomodulation activity through several mechanisms, especially by regulating inflammatory factors (tasneem et al. 2019; behl et al. 2021). likewise, the polysaccharide extract from turmeric could enhance the immune system (yue et al. 2010). a computational study regarding anti-sars-cov-2 showed that curcumin exhibited a high potency to block the virus's main protease (c19mpro), which plays an important role in the viral replication process. curcumin had lower binding energy to c19mpro than other compounds from p. nigrum, z. officinale, n. sativa, s. aromaticum, a. sativum and a. cepa (ibrahim et al. 2020). ginger (z. officinale) contains some compounds with anti-inflammatory and immunomodulatory activities such as 6-gingerol, 6-shogaol, zingerone, and 6-paradol (choi et al. 2018). an alcohol extract was reported to induce phagocytosis by macrophages in mice while crude extract increased humoral and cellmediated immune responses (gautam et al. 2020). meanwhile, another molecular docking evaluation showed the ability of zingiberene, 6gingerol, zingerone, gingerenone-a, 6-shogaol, and 6-dehydrogingerdione to block c19mpro. but their potencies were considerably low due to higher binding energies than n3 inhibitor as control (garg et al. 2020). an in vivo and in vitro study revealed the immunomodulatory effect of water extract and essential oil from lemongrass (c. citratus). the water extract with linalool oxide and epoxylinalool as major compounds could prevent the production of il-1β but induce il-6 production by macrophages. meanwhile, its essential oils which contained neral and geranial could inhibit cytokine production in vitro (sforcin et al. 2009). moreover, geraniol, another compound in its essential oil, inhibited the s1 subunit in spike proteins of sars-cov-2 through a docking simulation (wani et al. 2020). the rhizome part of cutcherry (k. galanga) is rich in bioactive compounds such as ethyl-pmethoxycinnamate and diarylheptanoids with anti-inflammatory and immunomodulation activity (jagadish et al. 2016; yao et al. 2018). its polysaccharides isolate enhanced the immunoregulation capability of cd4+ t cells (yang et al. 2018). furthermore, a computational study exhibited the activity of its bioactive compounds (kaempferol, kaempferol glycosides, and acylated kaempferol glucoside derivatives) to block the 3a channel protein of sars-cov. inhibition of this channel would inactivate virus production and allow the host to build up its immunity system (schwarz et al. 2014). another docking investigation indicated that kaempferol, due to its hydroxyl, ketone, and ether groups, was a stronger c19mpro inhibitor than other tested natural compounds (khaerunnisa et al. 2020). the crude polysaccharide extract of javanese turmeric (c. zanthorrhiza) could enhance the immune system by activating of nf-kappab (kim et al. 2007). xanthorrhizol and c. xanthorrhiza extract significantly inhibited the production of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6 and 1𝛽, and c-reactive protein (kim et al. 2014). moreover, curcumin, demethoxycurcumin, and bisdemethoxycurcumin in c. xanthorrhiza (similar compounds also contained in c. longa) showed their potential as c19mpro inhibitors (khaerunnisa et al. 2020; sumaryada & pramudita 2020). however, another study revealed that their inhibition actions were lower than nelfinavir, a protease inhibitor used as a drug standard (khaerunnisa et al. 2020). meanwhile, another docking investigation on the similarity of active sites exposed that bisdemethoxycurcumin had a greater ability to inhibit the binding pocket of c19mpro than n3 inhibitor, as the control ligand (sumaryada & pramudita 2020). other plants with lower rfc values (table 2) such as a. galanga and a. paniculata, citrus sp., c. sinensis, s. androgynous, f. vulgare, o. europea, and a. graveolens also confirmed to have immunomodulatory properties and potential against covid-19 (elfahmi et al. 2014; khaerunnisa et al. 2020; utomo et al. 2020). regarding covid-19, most of the antiviral studies of medicinal plants and their compounds were based on computational methods and resulted in a preview of their potential against covid-19. though some of the reported medicinal plants showed low molecular potency in blocking target sites, it is necessary to biotropia vol. 30 no. 2, 2023 192 highlight that the immunomodulatory actions support their beneficial role during the covid19 pandemic. further pre-clinical and clinical investigations are needed to warrant their efficacy as health-promoting agents against covid-19. these ethnobotany research results may be necessary to anticipate another transboundary animal or plant diseases pandemic by studying their bioactive compound for pharmacopeia studies in more detail. conclusion during the covid-19 pandemic, there were 59 medicinal plants belonging to 28 families used by indonesian. the plants were prepared mainly by boiling and administered orally. based on the rfc value, the most important plants were c. longa, z. officinale, c. citratus, k. galanga, and c. zanthorrhiza. also, respondents believed that those plants could boost immunity, maintain health and stamina, and prevent covid-19. in general, the medicinal plants reported in the current studies were confirmed by scientific literature to be beneficial as immune-booster during the covid-19 pandemic. meanwhile, their ability to block sars-cov-2 infection was mainly studied only through molecular docking evaluations. more research should be conducted to ensure their potency against sars-cov-2 and their efficacy when used as a single ingredient or in mixtures with other herbs. also, actions should be taken to preserve the community’s traditional knowledge of using medicinal plants. acknowledgments the authors are grateful to the authorities of the faculty of pharmacy, universitas mahasaraswati denpasar and indonesia government, through the directorate general of politics and public administration, 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and department of biology , faculty of mathematics and natural sciences, bogor agricultural university, jl. raya pajajaran, bogor, indonesia abstract aflatoxin is a human carcinogen that could contaminate foodand feedstuffs, and hence is a major food quality problem throughout the world. afiatoxin is produced by certain strains of aspergillusjlavus and //. parasiticus. a number of studies have been carried out in indonesia on atlatoxin contamination in indonesian foodand feedstuffs and their products from 1990 up to present. they were maize, maize product, peanuts, soybean and soybean meal, black and white pepper, feed ingredients; chicken and duck feeds. samples were collected from farmers, traders (middlemen), retailers (markets), supermarkets, exporters; poultry and duck community-based farms; and feed mill industries. high levels of aflatoxins were often found in maize, peanuts, chicken feed derived from markets, and duck feed. low levels of aflatoxins were found in soybean meal and chicken feedstuff. aflatoxins were not detected in soybean, black and white pepper. other studies have also been carried out on the effect of carbondioxide (co2), phosphine, black pepper extract and antagonistic fungi on aflatoxin production of a. flavus in vitro\ and the effect of airtight storage, phosphine, ammonium hydroxide, fermentation process, bag types, and phosphine in combination with different bag types on atlatoxin contents of maize, peanuts and soybean meal. some of these methods reduced aflatoxin contents significantly. keywords: aspergillus flavus i aflatoxin / food-and feed stuffs / product introduction aflatoxin is a human carcinogen that contaminates food-and feedstuffs, produced by the common fungi aspergillus flavus and a. parasiticus. the aflatoxin problem is a worldwide phenomenon, but it is particularly severe in developing countries, where food safety and security systems are not well developed to protect consumers against unsafe food products. in more developed countries, consumers are more aware of food safety issues such as aflatoxin, and are increasingly demanding those foods meet strict regulatory standards. aflatoxin limits for some commodities by importing countries are presented in table 1. among the foodstuff, peanuts is easily contaminated with aflatoxin. based on the report of the 23rd session of the joint fap/who food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commission adopted the maximum level of total aflatoxins in peanuts intended for further processing at 15 ppb. 26 biotropia no. 19,2002 this paper reviews the researches that have been undertaken in indonesia from 1990 to date on aflatoxin contamination in foodand feedstuffs and their products, as well as control ofa.flavus and aflatoxin. aflatoxin in food-and feedstuffs and'their products aflatoxin in maize and maize products in indonesia, maize is the second most important crop after rice. purwoko et al. (1991) studied aflatoxin content of 34 maize samples collected from poultry farms and poultry feedmills located around jakarta and bogor. aflatoxins were found in 91% of maize samples, and the predominant form was aflatoxin b|. the total concentration of aflatoxins in maize samples ranged from 22 to 6171 ppb, of which 91% contained aflatoxin bbh ranging from 22 to 4074 ppb, while aflatoxin b2 (71%) ranged from 11 to 3021 ppb. aflatoxin g, was present in two samples, at concentration of 101 and 528 ppb, and only one sample contained aflatoxin g2 at concentration of 144 ppb. maryam (1994) studied aflatoxin contamination of 32 maize samples derived from diagnostic samples sent to the research institute for veterinary science, 27 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra bogor. it was reported that the percentage of maize samples contaminated with aflatoxin b,, b2, g, and g2 were 78.1, 78.1, 20.0 and 20.0%, respectively. their ranges were between 1.1 63.7, 0.5 12.6, 1.2 51.7, and 0.6 4.2 ppb, respectively (table 2). a study by dharmaputra et al. (1995) on aflatoxin content of 35 maize samples collected from farmers and traders in lampung province in november 1992 reported that all of the samples (100%) contained aflatoxin b i, and 31% contained aflatoxin b2 (table 3). eleven samples obtained from farmers contained 25.4 367 ppb of aflatoxin bbh and 2 samples contained 12.5 and 12.7 ppb of aflatoxin b2, respectively; 4 samples obtained from village traders contained 119.7 276.7 ppb of aflatoxin b! and 11.9 41.6 ppb of aflatoxin b2, 4 samples obtained from middle traders contained only aflatoxin b! (25.6 97.6 ppb); 16 samples of big traders contained 23.4 278.7 ppb of aflatoxin b, and 5 samples contained 24.2 61.2 ppb of aflatoxin b2b . 28 biotropia no. 19,2002 another study on aflatoxin content of 108 maize samples collected from farmers, and 32 samples from village traders during the dry and wet seasons in central lampung (lampung province) and kediri (east java province) regencies was conducted in 1993 1994 (dharmaputra et al. 1996a). at farmers level, the total aflatoxin b| contents was between 5.3 291.4 ppb, while at village trader level, between 9.7 115.2 ppb (table 4). 29 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra a study by dharmaputra and putri (1996b) on total aflatoxin b, content in 11 samples of maize and maize products collected from some supermarkets in bogor, west java, reported that 1 sample of popcorn contained 15 ppb of the toxin; 1 sample of maizena flour contained 20 ppb of the toxin; 2 samples of maize for popcorn contained 40 and 40 ppb of the toxin, respectively; 1 sample of "maize chip" contained 19 ppb of the toxin. non detected aflatoxin was observed in 2 samples of popcorn, 1 sample of "marning", 2 samples of maize oil, and 1 sample of tortilla chips (table 5). biotropia no. 19,2002 aflatoxin in peanuts and peanut products peanuts is next to maize and soybeans as secondary crop of indonesia. dharmaputra et al. (1991) reported the aflatoxin contents in 35 peanut samples collected from 15 retailers located in three markets in bogor, west java, in september 1988. the aflatoxin contents were between 0 1154 ppb, while 80% of the peanut samples contained more than 30 ppb of aflatoxin. another study carried out by haryadi and setiastuty (1994) on aflatoxin contamination in 30 samples of raw and processed peanuts collected from various traders (big, medium and small) and processors during rainy and dry seasons in and around bogor (west java) and denpasar (bali), reported that aflatoxin b, was detected in 8 out of 15 samples collected during the rainy season, and in 5 samples collected during the dry season. aflatoxin b2 was detected in some samples contaminated with aflatoxin bi (tables 6 and 7). the possibility of aflatoxin contamination was higher among small traders, although big traders cannot guarantee that their products were free from aflatoxin contamination. the study also revealed that in processed peanuts, aflatoxins could still be present. 31 review on aflatoxin in indonesia food-and feedstuff's and their products — okky s. dharmaputra dharmaputra et al. (2002) reported that only one out of 15 samples of roasted pod and flour, and 14 samples of coated kernels of peanut collected from supermarkets in bogor, malang, pati and yogyakarta contained total aflatoxin of 15.54 and 20.72 ppb, respectively. table 8 shows the levels of aflatoxin found in maize and peanut samples from retailers located around bogor and yogyakarta. forty five percent of 215 peanut samples contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22% exceeded 1000 ppb (pitt and hocking, 1996). aflatoxin levels exceeding 300 ppb must be considered unsatisfactory, while those exceeding 1000 ppb must be considered totally unsatisfactory, capable of inducing acute toxic effects in both human and animals. an amount of 1% of maize and 17% of peanut samples, contained more than 1000 ppb aflatoxin. biotropia no. 19,2002 aflatoxin in soybean and soybean meal in indonesia, soybean ranks next to maize in importance as secondary food crops. the protein content of soybean is relatively high (42 50%), thus soybean meal is an important component of feedstuff. purwoko et at. (1991) studied aflatoxin contents of 10 soybean samples collected from some poultry farms and poultry feed mills located around jakarta and bogor in july 1990. aflatoxins were not detected in the samples. dharmaputra et al. (1997) reported that only 5 soybean meal samples were contaminated by aflatoxin from a study on aflatoxin content of 13 soybean meal samples collected from 10 feed mills factories, three bulog warehouses and one soybean crushing plant factory. the contents of aflatoxin were 7.9, 11.3, 14.5, 28.5 and 34 ppb, respectively. according to venkitasubramanian (1977), phytic acid in soybeans can bind zinc ions, while zinc ions are the important elements of aflatoxin synthesis. aflatoxin in pepper among spices, black and white pepper are important export commodities in indonesia. aflatoxins were not detected in 40 samples of black and white pepper (20 samples/commodity) collected from some retailers and supermarkets in bogor, and some exporters in bangka island and lampung province (dharmaputra et al. 1999). aflatoxin in chicken feed, duck feed, mixed duck feed, and rice bran dharmaputra and putri (1996b) reported that the total aflatoxin b[ content of 39 chicken feed samples collected from 3 markets in bogor during dry and wet seasons was between 0 — 200.73 ppb. twenty six out of 39 samples (67%) contained aflatoxins more than 30 ppb. a study by zahari and tarmudji (1995) on the aflatoxin content of 19 duck feed, 8 mixed duck feed and 8 rice bran samples collected from a duck farm in south kalimantan reported that in duck feed 100, 100, 52.6 and 15.8% of the samples were contaminated with aflatoxins b:, b2, g, and g2, respectively. their contents were between 4.0 160.0 ppb, 4.8 60.0 ppb, 0 60.0 ppb, and 0 8.0 ppb, respectively. in mixed duck feed, only one sample was contaminated with aflatoxin b,, and its content was 8.0 ppb. in rice bran, 75% of the samples were contaminated with aflatoxin b|. their contents were between 0 20.0 ppb. two out of 8 samples (25%) contained 7.2 and 72.0 of aflatoxin bb2. only one sample of rice bran was contaminated with aflatoxin g, (3.2 ppb). no aflatoxin g2 was detected in all samples. 33 review on ailatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra control of aspergillus fla vus and aflatoxin the effects of carbondioxide on mycelial growth and aflatoxin production of a. flavus in pure culture dharmaputra et al. (1992) studied the effects of co2 concentrations of 20, 40, 60 and 80% on mycelial growth and aflatoxin production of three isolates of a. flavus. as a control, these fungal isolates were maintained in air. the co2 concentrations used significantly affected both mycelial growth and aflatoxin production of a. flavus isolates. co2 at 20% started to inhibit the two parameters (figures 1 and 2). the growth of a. flavus and aflatoxin production decreased with the increase of co2 concentrations. figure 1. mycelial growth of a. flavus bio-16, bio-17 and bio-18 after treatment with different concentrations of co2 for 7 x 24 h on potato dextrose agar (dharmaputra et al. 1992). 34 biotropiano. 19,2002 the effect of carbondioxide on aflatoxin production in maize an investigation was carried out by dharmaputra et al. (1990) on the effect of co2 on aflatoxin production in stored maize. stacks of stored maize were sealed with pvc sheeting and treated with co2 for storage periods varying from 10 to 120 days. the concentration of co2 used was 2.4 kg/t. the control groups consisted of both stacks of maize sealed in plastic sheets, but not treated with co2, and stacks not sealed in plastic sheets. the aflatoxin b, content of maize, whether in plastic sheets and treated with co2 or only in plastic sheets, was lower than that of the unsheeted and untreated stacks (figure 3). the control showed that the aflatoxin content increased with the increasing duration of storage. 35 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra the effect of phosphine on mycelial growth and aflatoxin production of a. flavus in pure culture dharmaputra et al. (1991) studied the effect of phosphine on mycelial growth and aflatoxin b, production of two a. flavus isolates. the concentrations of phosphine used were 0.5, 1.5, 2.5 and 3.5 mg/l. as a control, these fungal isolates were maintained in air. the phosphine concentrations significantly affected both the mycelial growth and aflatoxin production of the isolates of a. flavus. mycelial growth and aflatoxin production decreased with increasing phosphine concentrations (figures 4 and 5). inhibition of mycelial growth commenced at 0.5 mg/l (figure 4). although the two isolates were still able to produce aflatoxin after treatment with 3.5 mg/l of phosphine, the amounts were low (figure 5).   review on aflatoxin in indonesia food-and feedstuffs and their products — okky s. dharmaputra the effectivity of phosphine to maintain the quality of maize packed in two different bag types another study was conducted by putri et al. (1999) on the effectivity of phosphine to maintain the quality of maize packed in two different bag types. maize with initial moisture content of ± 14% was stored in two bag types, namely jute and polypropylene bags, and then laid on the wooden pallet for 130 days. phosphine fumigation was carried out for 5 days at the beginning of storage and after 95 days of storage. maize samples (treated with phosphine and untreated one) were taken just before and immediately after fumigation, and thereafter every 30 days for a period of 130 days. the results indicated that the highest total aflatoxin b, content was found in unfumigated jute bag, followed by unfumigated polypropylene bag, fumigated jute bag, and fumigated polypropylene bag (tab'le 9). the effect of phosphine on aflatoxin production of soybean meal an investigation was carried out by dharmaputra et al. (1993) on the effect of phosphine on aflatoxin production of stored soybean meal. soybean meal was stored in polypropylene bags for 190 days. four stacks were treated with phosphine (2.1 g/t) for 5 days, once at the beginning of storage and again after 95 days of storage. four untreated stacks served as control. 38 biotropia no. 19,2002 there was a significant difference in aflatoxin b, content between the treated and the untreated samples (table 10). aflatoxin b, content increased during prolonged storage in both treated an untreated soybean meal (figure 6). the results indicated that phosphine somehow inhibits aflatoxin bi production. review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra the effect of airtight storage on aflatoxin production in maize an investigation was carried out by dharmaputra et al. (2000) on the effect of airtight storage on aflatoxin production in maize. the maize was placed in polyethylene bag under airtight condition (initial o2 content was 1.4 ± 0.1%), and stored for six months under laboratory conditions. the initial moisture contents of maize were 14 ± 0.3, 17 ± 0.2 and 20 ± 0.2%. as control, maize with the same initial moisture contents was placed in polyethylene bag under normal condition (air) with o2 content of 21%. the total aflatoxin b, content on maize with the three initial moisture contents packed under normal condition was higher than under airtight condition (figure 7). the total aflatoxin b! content increased with the increase of storage duration. the effects of black pepper extract on mycelial growth and aflatoxin production of a. flavus in pure culture retnowati et al. (1998) studied the effects of black pepper extract on mycelial growth and aflatoxin production of a. flavus. the concentrations of black pepper extracts used were 0.25, 0.50, 0.75 and 1.00% v/v media. aspergillus flavus was grown on smky liquid medium containing different concentrations of black pepper. as a control, smky liquid medium did not contain black pepper extract. black pepper extract at 0.25% concentration started to inhibit mycelial growth and aflatoxin production of a. flavus. mycelial growth and aflatoxin contents decreased with the increase of the extract concentrations (figures 8 and 9). no aflatoxin was detected on smky liquid medium containing 1% black pepper extract. 40 biotropia no. 19,2002 the effect of ammonium hydroxide on aflatoxin production of maize the effect of ammonium hydroxide on aflatoxin production of maize stored under laboratory conditions was investigated by dharmaputra and ambarwati 41 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra (1999). maize var. arjuna was treated with different concentrations of ammonium hydroxide, i.e. 0.5, 1.0, and 1.5% (v/v). the durations of the treatments were 12, 24 and 36 hours. the result showed that ammonium hydroxide decreased the aflatoxin content. the lowest aflatoxin content was detected when the maize was treated with 1.5% ammonium hydroxide for 36 hours (table 11). 42 biotropia no. 19,2002 the effect of fermentation process on the aflatoxin production of peanuts the effect of traditional fermentation process on aflatoxin production of peanuts was studied by edi et al. (1990) and fardiaz et al. (1993). peanuts were contaminated with aflatoxin-producing a. flaws, and incubated at 30°c for 6 days. the oil was extracted by hydraulic pressure, and the peanut presscake processed to make a fermented product called "oncom". to make "bla'ck oncom", the presscake was fermented with rhizopus oligosporus, while to make "red oncom" the presscake was fermentated with nenrospora sitophila. soaking of peanut presscake in water for 24 hours reduced the aflatoxin content to 48.6%, and it was reduced further to 42.9% after steaming at 95°c for 90 minutes. fermentation of the peanut presscake by r. oligosporus reduced the total aflatoxin content to 13.4%, while fermentation by n. sitophila reduced the total aflatoxin content to 41.1% of the original aflatoxin content in peanut presscake (figure 10). aflatoxin b, appeared to be the most sensitive to soaking and steaming processes. the decrease in aflatoxin content during soaking and steaming might be due to leaching, since aflatoxin is relatively resistant to heat. the effect of bag types on aflatoxin production of peanuts bulaong and dharmaputra (2002) studied the effect of four bag types namely jute bag, poly-propylene bag, jute bag lined with thin polyethylene (pe), and jute bag lined with thick pe, on aflatoxin production of peanuts with initial moisture content of 8%. storage was done for 6 months under warehouse conditions. 43 review on aflatoxin in indonesia food-and feedstuffs and their products okky s. dharmaputra no significant differences were obtained in aflatoxin levels among bag types, but at the end of six months storage, aflatoxin contents of peanuts packed in jute bag, polypropylene bag, jute bag lined with thin pe, and jute bag lined with thick pe were 31.5, 23.1, 16.8 and 18.9 ppb, respectively. based on the results, the immediate packaging of dried shelled peanuts at safe moisture level in jute bag lined with thin pe is recommended. the water vapor transmission rate of the thin pe is 1 g/m2/24 h. the effect of antagonistic fungi on aflatoxin production ofa.flavus in vitro investigation was carried out by dharmaputra et al. (2001) on the effect of fungi isolated from soil of peanut fields at wonogiri regency, central java, on aflatoxin production of a. flavus. figure 11 shows that a. niger was the most promising fungal antagonist, because it caused the highest percent inhibition (80%) of aflatoxin production of a. flavus isolate 102, followed by non-toxigenic a. flavus isolate 612 (61%), a. tamarii (60%) and non-toxigenic a. flavus isolate 36, (59%). conclusions only very limited analyses and surveys on the extent of aflatoxin contamination in indonesian foodand feedstuffs and their products have been carried out. high levels of aflatoxins were often found in maize and peanuts. consequently, 44 biotropia no. 19, 2002 researches have been concentrated on the effects of postharvest handling and fumigation on fungal infection and aflatoxin production in those two commodities. in soybean meal, aflatoxin contents were relatively low. no research has been carried out on aflatoxin contamination in relation with human health, cultural practices, peanut trading and industry. it is essential that a rigorous assessment of the critical points of entry of aflatoxin into the food and feed chains should be determined to more clearly assess the importance of pre-versus post-harvest management strategies. references bulaong, s.s.p. and o.s. dharmaputra. 2002. fungal population, aflatoxin and free fatty acid contents of peanuts packed in different bag types. biotropia no. 19 : 1 25. dharmaputra, o.s., h.s.s. tjitrosomo, f. aryani and m. sidik. 1990. the effect of carbondioxide on storage fungi of maize. proceedings of the 5"' international working conference on stored-product protection, bordeaux, france, 9-14 september 1990,291-301 dharmaputra, o.s., h.s.s. tjitrosomo, h. susilo and sulaswati. 1991. aspergillusflavus and aflatoxin of peanuts collected from three markets in bogor, west java, indonesia. proceedings of the 12"' asean seminar on grain postharvest technology, surabaya, indonesia, 29-31 august 1989, 110 -123. dnarmaputra, o.s., h.s.s. tjitrosomo, t.s. wardani and h. halid. 1992. the effect of carbondioxide on some biological aspects of aspergillus flavus. proceedings of the 13"' asean seminar on grain postharvest technology, bandar seri begawan, brunei darussalam, 4-7 september 1990, 280-293 dharmaputra, o.s., h.s.s. tjitrosomo, m. sidik and r.c. umaly. 1991. the effects of phosphine on some biological aspects of aspergillus flavus. aciar proceedings no. 36. proceedings of an international conference on fungi and mycotoxins in stored products, bangkok, thailand, 23-26 april 1991,244-248 dharmaputra, o.s., i. retnowati, m. s i d i k and h. halid. 1993. the effects of phosphine and length of storage on fungi, aflatoxin and protein content of soybean meal. proceedings of the 15'1' asean seminar on grain postharvest technology, singapore, 8-11 september 1992, 125-136 dharmaputra, o.s., i. retnowati, sunjaya and s. ambarwati. 1995. aspergillus flavus population and aflatoxin content in maize collected from farmers and traders in lampung province, south sumatra. proceedings of the 12"' national congress and scientific seminar, indonesian society for phytopathology, yogyakarta, indonesia, 6-8 september 1993. vol. 1, 560-566. (in indonesian). dharmaputra, o.s., i. retnowati, h.k. purwadaria and m. sidik. 1996a. surveys on postharvest handling, aspergillus flavus infection and aflatoxin contamination of maize collected from farmers and traders. paper presented at the 17"' asean technical seminar on grain postharvest technology, lumut, perak, malaysia, 25-27 july 1995. aciar technical reports 37, 38-53. dharmaputra, o.s., and a.s.r. putri. 1996b. aspergillus flavus population and aflatoxin content in maize, maize products and chicken feed. paper presented at the national microbiology seminar and annual scientific meeting, malang, indonesia, 12-13 november 1996. (in indonesian). dharmaputra, o.s., h. susilo and m. sidik. 1997. population of storage fungi and aflatoxin content of soybean meal. proceedings of the 14"' national congress and scientific seminar, indonesian society for phytopathology, palembang, indonesia, vol. 2,241-249. (in indonesian). 45 review on aflatoxin in indonesia food-and feedstuffs and their products okky s. dliarmaputrn dharmaputra, o.s., a.s.r. putri and w. setiawati. 1999. fungal infection and possibility of aflatoxin contamination in black and white pepper. hayati 6(3): 70-73. (in indonesian with abstract in english). dharmaputra, o.s. and s. ambarwati. 1999. detoxification effects of ammonium hydroxide on aflatoxin and total nitrogen content of maize. hayati 6(2): 25-28 dharmaputra, o.s., 1. retnowati, m. amad and s. ambarwati. 2000. airtight storage of maize: its effect on fungal infection and aflatoxin production. proceedings of the 19"' asean and 1st apec seminar on postharvest technology, ho chi minh city, vietnam, 9-12 november 1999, 474-482 dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. antagonistic effect of soil mycobiota on aflatoxin-forming aspergillus flavus in vitro. paper presented at the 20"' asean and 2"d apec seminar on postharvest technology. chiang mai, thailand, 11-14 september 200 1 . dharmaputra, o.s., a.s.r. putri and lilieanny. 2002. population of storage fungi and aflatoxin content in peanut products. internal report. seameo biotrop. (in preparation). edi, s. fardiaz and d. fardiaz. 1990. production of aflatoxin by aspergillus flavus and its destruction during fermentation of peanuts by neurospora sitophila. buletin penelitian ilmu dan teknologi pangan 1(1): 18-31. (in indonesian). fardiaz, s., s. wulan and b. setiyadi. 1993. effect of fermentation by rhizopus oligospoms on aflatoxin production by aspergillus flavus in peanut presscake. proceedings of the 5'1' asean food conference, akarta, indonesia, february 1992, 858-864 haryadi, y. and e. setiastuty. 1994. characterisation of aflatoxins bi, bb2, d and g2 in groundnuts and groundnut products. proceeding of the 6"' international working conference on stored-product protection, canberra, australia, vol. 2, 996-998. maryam, r. 1994. kontaminasi asam siklopiazonat dan aflatoksin pada jagung. makalah dibawakan pada kongres nasional perhimpunan mikologi kedokteran manusia dan hewan indonesia i dan tcmu ilmiah. bogor, 21-24 juli 1994, 289-293 pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and hiycotoxins associated with food commodities in southeast asia. mycotoxin contamination in grains. aciar technical reports 37,5-10 purwoko, h.m., b. hald and j. wolstrup. 1991. aflatoxin content and number of fungi in poultry feedstuff's from indonesia. letters in applied microbiology 12: 212-215 putri, a.s.r., o.s. dharmaputra, sunjaya and m. sidik. 1999. the effectiveness of phosphine to maintain the quality of maize packed in two different bag types. proceedings of the 7'1' international working conference on stored product protection, beijing, p.r. china, 14-19 october 1998, 274-279 retnowati, i., o.s. dharmaputra, h. susilo and h. affandi. 1998. inhibitory effects of black pepper and turmeric extracts onf the growth and aflatoxin production of aspergillus flavus . proceeding of the national microbiology seminar and annual scientific meeting, bandar lampung, indonesia, 14-15 december 1998, 210-220. (in indonesian with abstract in english) ventikitasubramanian, t.a. 1977. biosynthesis of aflatoxin and its control. in rodricks, j.v., c.w. hesseltine and m.a. mehlman (eds.). mycotoxin in human and animal health. pathotox publishers inc., part forest south, illinois, 83-98 zahari, p. and tarmudji. 1995, aflatoksikosis pada ternak itik alabio di kalimantan selatan. presiding seminar nasional teknologi veteriner untuk meningkatkan kesehatan hewan dan pengamanan bahan pangan asal ternak. cisarua, bogor, indonesia, 22-24 maret 1994, 408-41 1 46 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf microsoft word 15 biotropia no. 22, 2004: 15-28 soil quality changes following forest clearance in bengkulu, sumatra i.p. handayani department of soil science, agriculture college, university of bengkulu bengkulu, sumatra 38371 a, indonesia abstract intense destruction and degradation of tropical forests is recognized as one of the environmental threats and tragedies. these have increased the need to assess the effects of subsequent land-use following forest extraction on soil quality. therefore, the objective of this study is to evaluate the impacts of land-use type on soil quality properties in bengkulu province, sumatra. soil samples were collected from adjacent sites including natural secondary forest, bare land, cultivated land and grassland. the results show that land-use following forest clearance lowered saturated hydraulic conductivity (85%), porosity (10.50%), soil water content at field capacity (34%),c organic (27%), n total (26%), inorganic n (37%), soil microbial biomass c (32%), mineralizable c (22%), and particulate organic matter (50%), but slightly increased water soluble organic c. specific respiration activity rates increased about 14% in cultivated soils compared to natural forest soils, indicating greater c turnover per labile c pool in the form of soil microbial biomass, thus decreased biologically active soil organic matter. forest conversion tends to reduce the c,ffg/crer for all deforested sites. all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the c^g/crd of cultivated fields was about 24% less than that of remnant forest (1.07). grassland apparently maintains only slightly higher soil c levels than the bare land. on average, degradation index of soil following forest clearance was 35% with the highest deterioration occurred in the bare land (38%). fallowing the fields by naturally growth of imperata cylindrica for about 15 yr in abandoned land after 3-5 years of cultivation did not improve the soil quality. moreover, forest clearance has an impact on soil quality as resulted in the loss of a physically protected organic matter and reduction in some labile c pools, thus declined biological activity at disturbed ecosystems. keywords: degradation index / forest / imperata cylindrica grassland / soil quality/ soil organic matter introduction the integration among the primary natural resources vegetation, soil and water, are important factors for maintaining terrestrial ecosystem functions and productivity. human poverty and a continuous decline in the amount of agricultural area per family have led to exploitation of natural resources and deforestation on secondary forest in developing countries, such as indonesia. consequently, more forest land are converted to cropland at an alarming rate (riswan and hartanti 1995). these trends have caused a need to determine the effects of forest conversion and deforestation on soil quality. subsequent land-use change following deforestation or cultivation of deforested land may rapidly decline soil quality, as ecologically sensitive indicators of the tropical ecosystem are not able to buffer the impacts of land management practices. as a result, severe degradation in soil quality may lead to a permanent loss of land productivity (handayani 2001;mcdonald et al. 2002). evaluation of soil 15 biotropia no. 22, 2004 properties upon land conversion tor varying agricultural purposes is most crucial to assess early changes in soil quality. therefore, the specific objectives of this research were:(i) to determine and compare the changes in the physical, chemical and biological soil properties of soils in response to different land-use; and (ii) to calculate the degradation index of each tropical disturbed ecosystem. materials and methods study site the study site is located around the rajo lelo forest garden (kawasan taman hutan raya rajo lelo), 15 km north of bengkulu city, sumatra. the area was originally covered with typical tropical forest speces, such as arthocarpus champede, parkia spesiosa, durio zibethinus, pithecellobium latum, cinnamomum porectum, spondias pinnata kurt, rhodomnia cinerea, areca sp. and aporosa aurita. the forest have been cleared through cutting the wood to meet the increasing demands for timber and agricultural land. for more than 25 years, people living in the surrounding forest garden have often encroached upon and cultivated agricultural crops in the deforested land without using any fertilizer and soil conservation practices. these agricultural activities have caused regeneration of existing residual vegetation on clear-land and degraded deforested areas. the climate of the area is tropical monsoon with rainy season occuring from september to march and dry season from april to august. the mean annual temperature is 26uc, while the mean rainfall is 200 mm/month. the soils are classified as very fine, mixed, isohyperthermic, typic palehumult, according to us soil taxonomy. these soils are poorly to moderately drained and occur on undulating to flat uplands. the elevation is below 50 m above sea level. soil sampling surface soil samples at the depth interval of 0 to 15 cm were collected from three sites under each of four adjacent land-use/land cover types: (1) secondary forest ('natural forest'), (2) secondary forest cleared, and soil subsequently maintained weed-free ('bare land '), (3) secondary forest cleared, burned and soil planted with annual vegetable crops for about 6 years ('cultivated land') and (4) secondary forest cleared, burned and soil cultivated for about 5 years then abandoned, thus naturally grown by imperata cylindrica for about 15 years ('grassland') (figure 1.). for each site, 16 soil cores (1.9 cm diameter each) were randomly sampled and mixed to obtain a composite sample that was sealed in a plastic bag. field-moist soil samples were gently sieved through a 2 mm mesh to remove stones, roots, and large organic residues and sealed in plastic bags to store at 4°c. soil biological analyses were carried out within 10 days of sampling after an overnight acclimatization period at room temperature. 16   biotropia no. 22, 2004 soil physical, chemical and biological analyses saturated hydraulic conductivity (ksat) was determined by the constant-head method according to klute and dierksen (1986). soil bulk density (bd) was assessed by the core method and total porosity was calculated assuming a particle density of 2.65 g cm"3. gravimetric water field capacity was measured by pressure plate apparatus method (scott et al. 1994). soil particle size analysis was conducted by the hydrometer method. soil ph was determined in 1:2.5 soil-water slurry, using a combination of glass electrode. total c (tc) and nitrogen (tn) contents were determined on finely ground air-dried soils by wet combustion according to the method of kandeler (1995). soil microbial c (smbc) was determined by the fumigation-incubation method (jenkinson and powlson 1976) with the following modifications. moist soil (30 g dry-weight equivalent) was placed in 50-ml beakers, fumigated, brought to a water potential of approximately -30 j kg"1 with de-ionized water (0.3 kg kg"'), and incubated in 1-l air-tight canning jars in the presence of 10 ml of 0.5 m koh at 26°c for 10 days. the quantity of co2-c absorbed in the alkali was determined by titration (anderson 1982). soil microbial biomass c was determined from the following equation: smbc = [mg c02-c kg¯¹ soil (10 dy)¯¹]funigatjkc where kc = 0.41 (voroney and paul 1984). mineralizable c was estimated from the quantity of coi-c and net nkt-n + no3-n, respectively, released from an unfumigated sample during 10-day incubation at 26°c and a soil water potential of -30 j kg-1 (campbell et al. 1991). specific respiratory activity of soil microbial biomass c was estimated by dividing the net potential microbial activity (i.e., mineralizable c) by the size of the smbc (campbell et al. 1991). particulate organic matter (pom-c) was determined by a modified version of the cambardella and elliot (1992) method. pom-c was isolated by dispersing 30 g of soil in sodium hexametaphosphate and passing the dispersed sample through a 53-(am sieve, which retains the pom fraction + sand and allows the passage of mineral associated soil organic matter. the sand and pom fraction was dried at 50dc, finely ground and subsampled for total organic c. water soluble organic c (wsoc) was determined by the method of mazzarino et al. (1993). soil suspensions (1:2 soil:distilled water) were shaken for 30 minutes at 150 rev/min then centrifuged for 5 minutes at 2500 rev/min and filtered through whatman no 42 filter paper that had been rinsed with distilled water. carbon in the extract was determined following nelson and sommers (1982). current method for inventory of soil organic matter is based on an estimate of the soil c stored under natural vegetation and the relative changes due to aspects of human land use. in this case, calculation of a ratio of the measured soil organic c (corg) and a reference corg value for forest (top) soils of the same texture and ph was 18 soil quality changes following forest clearance i.p. handayani needed (van noordwijk et at. 1997). the ratio of corg/cret could be used as a 'sustainability indicator'. if the value of cqrg/cref ratio is 1, this means the soil is similar to that of forest, and/or is a "fertile soil"; values towards 0 mean "infertile soil". the current equation for cref for upland soils in sumatra (excluding peat and wetlands soils as well as recent volcanic andisols) is : cref =(zs/12.5)¯ 0'58 exp (1.333+0.00994*clay%+0.00699*silt%-0.156*ph-kcl) where zs=soil sampling depth, cm statistical analysis one-way analyses of variance (anovas) procedures were performed to compare the effects of different land-use/ land management on physical, chemical and biological properties of soil. the lsd procedure was used to separate the means of the soil properties at the 0.05 probability level as significant. results and discussion soils under cultivation had higher bulk densities than other land-use type (table 1.), with an associated decline in porosity and saturated hydraulic conductivity. the bare and cultivated soils were slightly lower in silt and clay than adjacent soils under natural forest, most likely as a result of preferential removal of silt by accelerated erosion during the rainy season (handayani 2001). the lowest saturated hydraulic conductivity was found in bare soils and the highest occurred in natural forests. beginning tillage practices resulted in higher saturated conductivity in cultivated soils compared to bare soils and grassland. lower plants residue/ organic input probably accounts for the higher bulk density and decreased porosity under cultivation as compared to the natural forest, bare land and grassland soils (table 1.). enhanced soil water content at field capacity and saturated hydraulic conductivity in natural forest is consistent with greater input of labile c contributed by the high quality litter-fall and root exudates as indicated by higher wsoc and smbc (table 3.). during the processes of c turnover, extracellular polysaccharides were produced and with association of root extension have created better soil aggregation (elliot 1983; cambardella and elliot 1992), which caused higher soil water content at field capacity and saturated hydraulic conductivity. on the other hand, organic matter in cultivated soils is less physically protected than that of bare and grassland soils because tillage periodically breaks up macroaggregates and exposes previously protected organic matter in soil macroaggregates (gupta and germida 1988), causing more compacted soils (poorer soil aggregation). total nitrogen content of soils under cultivation were lower compared to levels in natural forest soils. however, cultivated soils have higher total n than that in bare land and grassland soils (table 2.), as might be expected in a system dominated by 19 biotropia no. 22, 2004 nitrogen fixing crops such as peanut and bean. the organic c levels were significantly higher in natural forest than those of bare land, 'cultivated land and grassland soils. the c/n ratios were wider under undisturbed ecosystems such as natural forest and grassland, indicating higher c accumulation or slower decomposition resulted in c stabilization inside soil aggregates (handayani et al. 1995; feller and beare 1997). bare land and cultivated soils had lower inorganic n content than that of natural forest and grassland. these proved that less available-n were released through mineralization under disturbed sites; because these ecosystems had lower n stock from labile n pools, such as in soil microbial biomass n and paniculate organic matter n (pom-n) (unpublished results 1999). the lower levels of total c and n in bare land and cultivated soils may have resulted from a combination of lower c inputs because of less biomass c returned and greater c losses because of aggregate disruption, increased by tillage, plant residue burning, and enhanced soil erosion during rainy season (scott et al. 1994; handayani 2000). the trends toward lower total c and n in the disturbed land is probably caused by the breakdown of aggregates (gupta and germida 1988; blair et al. 1995), and greater organic matter oxidation following deforestation (handayani et al. 2001) and continuous tillage (handayani and coyne 1999). 20 biotropia no. 22, 2004 from biomass burning on the bare land and cultivated soils could have returned base-forming cations to increase ph of surface soil. the ratios of corg/cref were almost similar in bare land, cultivated land and grassland (table 2) with value of 0.92, 0.81 and 0.92, except for natural forest is 1.07. the average corg/cret ratio of 1.07 under forest, suggesting that the soil c status of this forest has increased. forest conversion tends to reduce the corg/crct for all deforested sites. as suggested by van noordwijk et al. (1997), if the value of corg/cref ratio is 1, this means the soil is similar to that of forest, and/or is a "fertile soil"; values towards 0 mean "infertile soil". therefore, all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the c,,rg/cref of cultivated fields was about 24% less than that of remnant forest. grassland apparently maintains only slightly higher soil c levels than the bare land. as may be expected that grassland have higher total n and available n compared to bare land (table 2.). the ratio of corg/cref in the forest for this study was higher compared to the study in sumberjaya, west lampung sumatra. hairiah et al. (2002) reported the value of corg/cref under forest condition was 0.73, suggesting that the soil c content in the forest has declined from the undisturbed condition. in addition this ration in coffee farming systems was about 50% less that of remnant forest. the values of all of the measured biological properties were significantly higher in natural forest than in cultivated soils, except for mineralizable c (table 3.). mineralixable c rates did not vary significantly among sites, but tended to be somewhat higher in the soils under natural forest. high rates of mineralizable c can occur either as a result of large pool of labile c substrates or rapid oxidation of a smaller pool. thus high mineralizable c may indicate a high level of ecosystem productivity and soil bioactivity (mazzarino et al. 1993). a more clearly interpretable parameter is the rate of mineralizable c per unit smbc (specific respiratory activity of soil microbial biomass c = srac), high levels of which have been associated with ecosystem stresses (killham 1985; handayani and coyne 1999). the highest srac was found in cultivated soils (24.03%) and the lowest was in natural forest soils (19.74%) (table 4.). enhanced microbial activities in soils under natural forest are related to greater levels of substrates c, which resulted in the increase of the labile fraction of c organic. as a result, soil microbial communities under cultivated soils are less biologically active and more stressed than in natural forest. in addition, relatively higher rates of srac for the cultivated soils suggest that intense competition for the available c may favor those microorganisms which use more c energy for cell integrity and maintenance than for growth under perturbed or disturbed ecosystems. consequently, cultivated soils favor bacteria-based food webs which have low c assimilation efficiencies and faster turnover rates than the more efficient fungal-based food webs dominant in untilled or natural ecosystems (hendrix et al. 1986). a lower srac implies a more stable and mature system (turco et al. 1994), thus this calculation may be a good indicator of status of a soil and therefore, its soil quality (insam and hasselwandter 1989). the highest decline of smbc was in cultivated soils (42%) and the lowest occurred in bare land soils (22%). the effect was more pronounced on pom-c 22 soil quality changes following forest clearance i.p. handayani (50% reduction). undisturbed ecosystems (natural forest and grassland soils) have the lowest wsoc compared to cultivated and bare land soils. data in table 3 describe the variety amount of labile c pools under different land-use types, but the availability of labile c pools are shown in table 4. microbial biomass c is often limited in size by the availability of c-labile substrates and is sensitive to variations in land-use and soil management practices, so a lower proportion of smbc in cultivated and grassland soils is an indication of degradation of available pool c in soils under cultivated and grassland soils. clearing and burning tend to decrease root production in bare land and cultivated soils. lower root biomass and removal of clipping resulted in lower substrates availability, and thus lower smbc. wsoc appears to be the intermediate organic substrate for soil microorganisms (mcgill et al. 1986). turnover of smbc requires repleshnisment of wsoc supplies. repleshnisment mechanisms include desorption from soil colloids, dissolution from litter, exudation sloughing and exfoliation from plant roots, or hydrolisis of insoluble soil organic polymers (mcgill et al. 1986). changes in soil environment, soil management and cropping practices would be expected to affect the above mechanisms, thereby altering wsoc supply and both amount and activity of smbc.the lower proportion of wsoc in c org under undisturbed ecosystems indicates more soil organic matter was in protected condition, so they are not readily soluble or not readily available to microorganisms. pom-c has been closely associated with the active soil organic matter pool and has been successfully used as an assay of nutrient availability (dalai and mayer 1987). loss of pom-c is an important aspect of soil organic matter degradation. pom-c declined in newly bare soils and cultivated fields and lowest under grassland soils. our study suggests that pom-c characteristics may also serve as early indicators of soil organic matter aggradation. pom-c accumulated as soil organic matter was restored in natural forest and it is important for c and nutrient reservoir in soils (cambardella and elliot 1992). values of ratio pom-c/c org indi 23 biotropia no. 22, 2004 cate similar trend with values of pom-c which show the highest under natural forest soils and lowest in grassland soils. this implies that availability of pom-c continuously declined when the forest is converted to other land-use type (table 4.). this study gives an implication that bare land, cultivated and grassland soils have less physically protected soil organic matter compared with natural forest soils, resulting in soil deterioration over time. the calculation of degradation level determined by scaling technique (scott et al. 1994), reflects the percent changes in soil properties from their standard values under natural forest (table 1.). scaling of soil properties was used to relate the characteristics of one land-use to the same characteristics of another land-use by dividing the mean values of each soil property from the natural forest. therefore, the values from the natural forest would have a scale of 1.0 and other land-use would have a scale value less than 1.0, indicating the decline of soil quality. all scale values were averaged at each land-use, then subtracted by 1.0 and converted to percentage as indicated by degradation level. in this study, soil properties scale was made on saturated hydraulic conductivity, gravimetric soil water content at field capacity, porosity, c organic, n total, inorganic n, mineralizable c, soil microbial biomass c, and particulate organic matter (table 5). the calculated soil degradation index reflects the percent changes in soil properties from their values under forest (table 5). soils under bare land had the highest degradation index up to 38% compared to grassland and cultivated soils (33%) with an average of 35%. the data also indicated that fallowing the land by natural growth grassland of imperata cylindrica for about 15 years have not improved the soil quality compared to cultivated soils. in some cases, grassland ecosystem often created more deterioration in several soil properties (table 5.). these soil degradation indices clearly show that degradation of soil quality occurs when the forest systems are converted for agriculture without the use of appropriate soil conservation practices. 24 biotropia no. 22, 2004 conclusions clearing, burning and cultivation of tropical secondary forest lands resulted in degradation of soil quality as indicated by the changes in physical, chemical and biological properties. land-use following forest clearance decreased saturated hydraulic conductivity by 85%, porosity by 10.50%, soil water content at field capacity by 34%, c organic by 27%, n total by 26%, inorganic n by 37%, soil microbial biomass c by 32%, mineralizable c by 22%, and particulate organic matter by 50%, but slightly increased water soluble organic c. specific respiration activity rates increased about 14% in cultivated soils compared to natural forest soils. forest conversion tends to reduce the corg/cret for all deforested sites. all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the corg/clef of cultivated fields was about 24% less than that of remnant forest. grassland apparently maintains only slightly higher soil c levels than the bare land. bare land and cultivated soils had higher bulk density, lower saturated hydraulic conductivity, porosity and soil water content at field capacity. based on calculation for the ratio of corg/cref, all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. degradation index of soil following secondary forest clearance was about 35% in average, with the greatest deterioration occurring in bare land. fallowing the abandoned land by natural grass has not improved the soil quality. therefore, improvement of soil properties under imperata grassland with well-adapted and fast growing vegetative species to compete with these grass is needed to gradually improve soil quality as well as regenerate degraded grassland. acknowledgements this work is a part of the research supported by the government of indonesia through the project under the department of research and technology (menris-tek) and the department of national higher education (dikti). the technical assistance of meizar and yulian hardi is gratefully acknowledged. we also thank the local government and local farmers for providing their fields for this study. special thanks are due to dr. meine van noordwijk for his help in reviewing the manuscript. references anderson, j.p.e. 1982. soil respiration, p. 837-871. in a.l.page et al. 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(ed.) in situ and ex situ conservation of commercial tropical trees. gmu and itto. yogyakarta. indonesia. handayani, i.p., and m.s.coyne. 1999. dynamics of biologically active c pools in relation to soil organic matter content. proceedings international seminar towards sustainable agriculture in humid tropics facing 21sl century. bandar lampung, indonesia. handayani, i.p. 2000. changes in soil organic matter pool during shifting cultivation in bengkulu, sumatra. jurnal penelitian unib vi(17):22-27. handayani, i.p., m.s.coyne, and r.l. blevins. 1995. soil biologically active carbon under long-term no till and conventional tillage. agron. abst. asa and sssa annual meeting, st. louis, usa. hendrix, p.e., r.w.parmele, d.a.jr. crossley, d.c.coleman, e.p.odum. and p.m.groffman. 1986. detritus food webs in conventional and no-tillage agroecosystems. biosci. 36:374-433. insam, h., and k.hasselwandter. 1989. metabolic quotient of the soil microfloral in relation to plant succession. oecologia 79:174-178. jenkinson, d.s., and d.s. powlson. 1976. the effects of biocidal treatments on metabolism in soil. v. a method for measuring soil biomass. soil biol. biochem. 8:209-213. kandeler, e. 1995. methods in soil chemistry, p. 397-398. in. e.schinner et al. 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(ed.). mineral nutrients in tropical forest and savanna ecosystems. blackwell, oxford, p. 325-336. nelson, d.w., and l.e.sommers. 1982. total carbon, organic carbon, and organic matter, p. 539-594. in a.l.page et al. (ed.) methods of soil analysis. part 2. 2nd ed. agron. monogr.9.asa and assa, madison, wi. riswan, s., and l.hartanti. 1995. human impacts on trical forest dynamics. vegetatio 121: 41-52. scott, h.d., i.p.handayani, a.mouromostakos, and d.m.miller. 1994. temporal variability of selected properties of loessial soil as affected by cropping. soil sci.soc.am.j. 58:1531-1538. shiddieq, d. 1993. effect of tropical rainforest conversion to agricultural farms on ultisols in subanjeriji. phd. thesis, university of los banos, philippines. turco, r.f., a.c.kennedy, and m.d.jawson. 1994. microbial indicators of soil quality, p. 73-90. in j.w.'doran et al. (ed.) defining soil quality for a sustainable environment. sssa special publication no. 35., madison, wi. van noordwijk, m., c.cerri, p.l.boomer, k.nugroho and m.bernoux. 1997. soil carbon dynamics in the humid tropical forest zone. geoderma 79:187-225. voroney, r.p., and e.a.paul. 1984. determination of kc and kn in situ for calibration of the chloroform fumigationincubation method. soil biol. biochem. 16:9-14. 28 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf result and discussion production and utilization of cellulase from biotropia no. 25, 2005 : 50 – 59 trichoderma viride r. hidayat1, s. wulandari2, k.g. wiryawan3 and suryahadi3 1department of animal nutrition and feed science, padjadjaran university, bandung, indonesia. 2faculty of animal science, jember agricultural polytechnique, jember, indonesia. 3department of animal nutrition and feed science, bogor agricultural university, bogor, indonesia. abstract an appropriate preservation technology for forage such as silage needs to be developed in order to overcome the shortage of tropical forage during the dry season. a good quality of silage is obtained by decreasing the fibre contents (neutral detergent fibre, acid detergent fibre and lignin). the research was conducted in two stages: 1). production and activity test of crude enzymes from trichoderma viride and 2). comparative test using crude enzymes from trichoderma viride strain qm 9414 (cetv) and commercial cellulase (cellulase “onozuka r-10”, heidelberg) on rice straw silage. the treatments were arranged as follows : p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. all treatments were enriched with 5% molasses. the result of the research indicated that: 1). the activity of crude enzymes from trichoderma viride strain qm 9414 (cetv) was 1.52 iuml-1. 2). the addition of both enzymes did not decrease dry matter of rice straw silage; however, organic matter significantly decreased (p<0.05) by addition of commercial cellulase. 3). the addition of both enzymes decreased fibre contents (ndf), furthermore increased (p<0.05) in sacco digestibility of dry matter and organic matter of rice straw silage. keywords : cellulase/trichoderma viride/rice straw/silage/fibre introduction the main problem of improving ruminant production in indonesia is the shortage of high quality forage during dry season causing the animal production become inefficient, because of an increase in feed cost. on the other hand, the forage is abundant during the wet season. application of forage preservation technology in the farm may solve the problem, as the forage becomes more available all the year. there are two forage preservation techniques namely dry preservation (hay) and wet preservation (silage). for some matters, ensiling has many advantages, as forage quality may be maintained for a few months. however, the silage production process still has weakness, as the dry matter decreases and the fibre content of the forage is not influenced by the process. rice straw is one of the agricultural by-products which is a very potential source of energy for ruminants. availability of rice straw increases as the rice straw silage production increases. rice straw is usually abundant at the same time as the 50 period of high forage production occurring in the wet season. farmers choose forage as feed rather than rice straw. farmers store rice straw to be used during dry season when forage production is low. rice straw as feed has been used generally in the tropic and sub-tropical areas (jackson 1977), especially in the hot season. in indonesia, rice straw has been used for feed at least 31-39%, but most of it was burnt or used as fertilizer (36-62%) and the rest is used for industries (komar 1984). biotropia no. 25, 2005 rice straw has low nutritive values. rice straw contains 80% organic material which can be potentially digested; but in reality, it can be digested by ruminants only 45-50% (lubis 1963; jackson 1977). according to doyle et al. (1986), rice straw nutrient content is generally low consisting of crude protein 2.2-9.5%, hemicelluloses 21-29%, cellulose 35-49%, with coefficient digestibility value of organic materials 31-59%, and lignin 4-8%. dry storage condition (hay) makes the quality of rice straw becomes worse, therefore, a better method is needed to store the rice straw. the present techniques in silage production are focused mainly on feed stuff preservation rather than improvement of its quality. it needs an effort to improve the process, and ensiling may be improved by both preservation and increase of the quality of forage. addition of enzymes as an active substance may disperse cell wall (ndf and adf compounds) (stokes and chen 1994), increase digestibility and release the soluble carbohydrate portion of the forage. enzymatic pre-hydrolysis of forage component may promote bacterial growth, enhance lactic acid production and decrease ph of silage (bolsen and sapienza 1993). the present experiment is focused on application of commercial cellulase enzyme and crude enzyme produced by trichoderma viride to improve nutritive value of rice straw. materials and methods materials the materials used in this experiment were: rice straw (cultivar ir 64), a commercial cellulase (onozuka r-10 from trichoderma viride with activity 1.0 umg-1 produced by serva electrophoresis gmbh d-69115 heidelberg carl-benzstr.7) and trichoderma viride strain qm 9414. growth media for trichoderma viride growth media composition that were used as substrates for trichoderma viride to produce raw enzymes were : media i, one litre media contains : 5 g nh4no3, 0.5g kcl, 0.01g feso4. 7h2o, 0.5g mgso4.7h2o, 0.001g cuso4.2h2o, 1g yeast extract and 6g pepton ,the ph of media was 5.45. media ii, one litre media contains : 5g nh4no3, 0.5g kcl, 0.01g feso4 .7h2o, 0.5g mgso4.7h2o, 0.001g cuso4.2h2o, 1g yeast extract and 15g rice straw. (montesqrit 1998). 51 experimental design production and utilization of cellulose from trichoderma viride – r. hidayat et al. the research was conducted in two stages as follows: stage i: production and activity test of crude enzymes from trichoderma viride. one loop of trichoderma viride was cultured in a reaction tube containing 3-5 ml media i for four days and then it was transferred to an erlenmeyer glass containing 250 ml media ii and incubated for 14 days with shaking. the crude enzymes were obtained by centrifugation at 4000 rpm, at 50 c for 30 minutes and the supernatant was used as an additive in the production of rice straw silage. stage ii: a comparative test using crude enzymes from trichoderma viride strain qm 9414 (cetv) and a commercial cellulase (cellulase “onozuka r-10”, heidel-berg) on rice straw silage. the experiment used completely randomized design with 5 replicates. a total of 25 silos with 1 kg per silo of fresh material were prepared. the treatments were based on enzyme activity and arranged as follows : p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. all treatments were enriched with 5% molasses to provide ready available carbohydrate (rac) as a stimulant fermentation. the ph before ensiling was normal. silage preparation the rice straw silage was made as many as 1 kg for each sample. the ensiling procedure is as follows: the rice straw was chopped into pieces of ± 5 cm length. the fresh and chopped rice straw was mixed with either commercial enzymes or crude enzymes from trichoderma viride (spraying). the mixture was pressed and compacted by hand, and placed into a silo (glass jar). then the silo was closed tightly so that the condition became free from o2, air and waterproof. chemical analysis silage samples were collected for chemical analysis on the 30th day of ensiling. the physical characteristics; colour, presence of fungi and smell of the silage were studied. hence, the following parameters were measured : dry matter (dm), organic matter (om), crude protein (cp) according to proximate analysis, ph (ph-beckman model φ 40). ndf, adf and lignin content were analysed according to van soest and wine (1967). lactic acid was determined with an hplc technique (shimazu, japan 3081-09202-20atd-e). dm and om digestibility were determined by nylon bag technique (in sacco) in a fistulated buffalo. the size of nylon bag is 140 x 90 mm. one gram sample of silage was placed into nylon bag and incubated in the rumen for 24 hours. one week before and during the experiment, the buffalo was fed on 40 kg elephant grass and 5 kg rice bran per day. 52 statistical analysis biotropia no. 25, 2005 the data were subjected to analysis of variance (anova). when the ftest was significant (p<0.05), the contrast orthogonal test for paired comparisons was used. results and discussions production and activity test of crude enzymes from trichoderma viride on medium i, fungal growth was visible at the second day of cultivation. on medium ii, fungal growth was detected at the second day which was shown by the mycelial growth in the media after transferred into erlenmeyer glass. on the fourth day, the growth of mycelium was maximum and it was harvested. from 25 glasses of erlenmeyer, 21 of them were harvested and others were thrown away because they were contaminated by unexpected micro-organisms or had imperfect growth. the cellulase activity of crude enzymes from trichoderma viride (cetv) was 1.52 iuml-1. this result was higher than previous results (montesqrit 1998) by the same procedure, which showed that the cmc-ase activity from t. viride fermented for 14 days was 0.673 iuml-1. this was caused by a different strain of t. viride used, while the present experiment used t. viride strain qm 9414. comparative test using crude enzymes from trichoderma viride (cetv) and commercial cellulase on rice straw silage. the influence of treatments on the physical quality of rice straw silage generally, rice straw silage had the same physical characters, either in colour, smell, texture or the presence of fungi. rice straw silage had the same colour that was yellow brownish and moulds were seen on the top of the silage. the colour of silage was not different from the fresh rice straw. the silage had good smell, fresh and crumb texture for all treatments. it showed that the fermentation had taken place by producing lactic acid. the utilization of dry rice straw as silage material also had positive impact on silage quality i.e. the silage liquid was little and there was not much soluble nutrient wasted into silage liquid. other reports indicated that making silage in wet condition produced much liquid (mcdonald et al.1991), so dryer condition was needed to make easy handling and produced higher and drier silage. 53 the influence of treatments on the chemical quality of rice straw production and utilization of cellulose from trichoderma viride – r. hidayat et al. 1. nutrients content of rice straw silage table 1 presents the nutrients content of rice straw silage. the dry matter content of rice straw silage treated with enzymes (p1, p2, p3 and p4) was not significantly different from dry matter of untreated rice straw silage. this result was different from rice straw directly treated by t. viride in which it caused degradation of rice straw dry matter 8.4% (soetjiharto 1997). however, dry matter of rice straw silage treated by commercial enzymes was significantly (p<0.05) reduced compared to that of silage treated by cetv. table 1. nutrient content of rice straw silage treatments nutrients p0 p1 p2 p3 p4 dry matter (%) 24.3±0.4 23.9±0.6 24.6±0.1 24.6±0.2 24.9±0.6 organic matter (%) 79.1±0.2 79.2±0.2 79.2±0.3 79.6±0.1 79.5±0.4 crude protein (%) 5.9±0.5 5.8±0.3 5.4±0.2 5.3±0.3 5.6±0.3 p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. organic matter content of rice straw silage treated with enzymes (p1, p2, p3 and p4) was 79.4 %, and higher (p<0.05) compared with control (p0=79.1 %). this result was different from direct microbiological treatment as stated by djunaidi (1988) that organic matter content of rice straw silage put on the ground was descending. organic matter content of rice straw silage treated with commercial enzymes (79.2 %) was not significantly different from organic matter of rice straw silage treated with cetv (p2, p3, and p4 = 79.4%). it was different from stokes and chen (1994) observation that the increase of commercial cellulase addition to corn silage decreased the silage organic matter content. crude protein of rice straw silage treated with enzymes (p1, p2, p3 and p4) was 5.5 % and significantly lower (p<0.05) than crude protein of untreated rice straw silage (p0=5.9%). this was caused by cetv which still contained protease, so that some crude protein of rice straw was degraded to become ammonia. a different result was reported by stokes and chen (1994) in which crude protein content of corn silage treated with commercial cellulase was increased. this was caused by microorganism growth as source of protein. 2. fibre content of rice straw silage the effects of addition of commercial cellulose and cetv to fibre content of rice straw silage are presented in table 2. the neutral detergent fibre (ndf) 54 content of rice straw silage treated with enzymes (p1, p2 ,p3 and p4) was 72.3 %, and significantly lower (p<0.05) compared to untreated silage (p0=79.0%). reduction of ndf content of rice straw silage treated with cetv followed the equation of y= 77.75-1.37x. corn silage treated with cellulase had lower ndf content (46.7%) compared to untreated corn silage (53.1 %) (stokes and chen 1994). biotropia no. 25, 2005 there is no significant difference (p<0.05) between acid detergent fibre (adf) content of rice straw silage treated with enzymes (p1, p2 ,p3 and p4=56.2 %) and control (p0=59.5%), but the adf content of rice straw silage treated with enzyme tended to be lower (p<0.05) than adf content of untreated rice straw. enzyme treatment decreased adf content of rice straw silage about 5.6% from control. table 2. fibre content of rice straw silage treatments p0 p1 p2 p3 p4 significance ndf (%) 79.0±3.4 76.4±1.2 74.2±2.7 71.2±3.1 67.4±2.9 p < 0,05 adf (%) 59.5±4.4 59.2±2.9 57.6±7.6 53.6±2.7 54.2±2.1 ns lignin (%) 7.7±.0.8 8.4±.1.7 6.6±.1.7 6.2±.1.5 5.7±.0.5 ns ndf= neutral detergent fibre, adf=acid detergent fibre, p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. the acid detergent fibre (adf) content of rice straw silage treated with cetv (p2, p3, and p4) was 55.1 %, and it was not significantly different from adf content of rice straw silage treated with commercial enzyme (p1=59.2%). this result was different from that reported by stokes and chen (1994), that adf content of corn silage treated by commercial enzyme decreased about 11 – 13 %. it appears that adf content of rice straw silage treated with cetv was lower compared with commercial enzyme treatment. this means that cetv had higher potential than commercial cellulase in degrading adf of rice straw silage. rice straw is difficult to be degraded because rice crop is harvested at mature stage with high cell wall content and perfect lignification level. the lignin content of rice straw silage treated with enzymes (p1,p2,p3 and p4= 6.6%) was not different from control (7.7%). however, the content of rice straw silage lignin treated by commercial enzyme (p1=8.4%) was significantly higher compared with that treated by cetv (6.2 %). the decrease in lignin content by cetv treatment might be caused by: crude enzymes cetv which possess many enzymatic activities including lignase, and production of organic acid during ensilage. 55 lignin can be depolymerised by microbial enzymes produced by trichoderma sp. (paterson 1986). on the other hand, commercial enzyme is purely cellulase enzyme that has no lignase activities, so increasing the level of commercial enzyme did not affect the lignin content of rice straw silage. production and utilization of cellulose from trichoderma viride – r. hidayat et al. 3. ph and lactic acid production of rice straw silage there were no differences between ph of rice straw silage treated with enzyme (p1, p2, p3 and p4 = 3.72) than that of control (p0 = 3.72). this result is in line with stokes and chen (1994) who reported that enzyme application has no effect to corn silage ph. nevertheless, rice straw silage ph treated with commercial enzyme (p1 = 3.68) had significantly lower (p<0.05) ph than rice straw silage treated by cetv (3.73). the effect on the addition of commercial cellulase and cetv to ph and lactic acid production of rice straw silage is presented in table 3. table 3. ph and lactic acid production of rice straw silage treatments p0 p1 p2 p3 p4 significance lactic acid (%) 0.23±0.05 0.35±0.04 0.27±0.01 0.26±0.03 0.25±0.04 p < 0.05 ph 3.72±0.03 3.68±0.02 3.71±0.01 3.75±0.01 3.74±0.02 ns p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. ph of rice straw silage treated with commercial enzyme had the lowest ph. this was related to silage lactic acid production, where silage treated with commercial enzyme had the highest concentration of lactic acid (0.35 %). rice straw silage ph treated with cetv tended to increase as the enzyme levels were increased. this might be due to a carry-over of some buffers from the media of trichoderma viride during centrifugation for enzyme preparation. in addition, protease activity of cetv could degrade protein into ammonia. the increase in ph of rice straw silage treated with cetv follows the linear equation of y=3.696+ 0.0072x. in general, rice straw silage ph was very good, i.e. under 4. this means that rice straw silage can be stored in silos for a long time. good silage had ph between 3.5 and 4.0 (aak 1983). lactic acid production of rice straw silage treated with enzyme (p1, p2, p3 and p4) was 0.29 % higher than the control (p0=0.25 %). addition of enzyme could improve lactic acid production of rice straw silage. low moisture content probably influenced the relative growth of homofermentative and heterofermentative lactic acid bacteria during ensiling, which in turn lowered the ph of silage, minimizing 56 deterioration (mcdonald et al. 1991). a different result was reported by man and wiktorsson (2002) in which lactic acid production of cassava and gliricidia tops silage was 0.95-0.99 %, respectively. biotropia no. 25, 2005 lactic acid concentration of rice straw silage treated with commercial enzyme (p1=0.35 %) was significantly higher than lactic acid concentration of rice straw silage treated with cetv (p2,p3 and p4=0.26 %). the lactic acid concentration was parallel to rice straw silage ph. the higher production of lactic acid, the lower became the silage ph. ph of rice straw silage treated with commercial enzyme was the lowest. while the ph of rice straw silage treated with cetv increased parallelly to cetv dosages. digestibility of dry matter and organic matter of rice straw silage the digestibility of dry matter (ddm) of rice straw silage treated with enzyme (p1, p2, p3 and p4=37.5%) was significantly higher than the digestibility of dry matter of untreated rice straw silage (p0=28.0%). a similar result was obtained for digestibility of organic matter (dom) (table 4.). the increased degradation might have to do with the increased damage of feed surface caused by exogenous enzyme treatment of feed before feeding (nsereko et al. 2000). hong et al. (2003) stated that nutrient degradation rate and effective degradability of dm increased by addition of enzyme mixture consisting of mainly cellulase and xylanase. the enzyme applied to forages immediately before in vitro incubation also improved digestion of dm and ndf, suggesting that fibrolitic enzymes applied for feeding (direct fed) may enhance digestion of forage by cattle (feng et al. 1996). beauchemin et al. (2001) reported that applying fibrolytic enzymes prior to feeding enhances ruminal fibre digestion by altering the structure of the feed, thereby making it more susceptible to degradation. exogenous enzymes treatment, prior to ingestion, increase bacterial colonization and thereby improves dm disappearance of forage (yang et al. 1999) because exogenous enzymes increased microbial attachment of ruminal microbes to feed and increased activity of enzymes associated with feed particles (wang et al. 2001). the digestibility of dry matter of rice straw silage treated by cetv (p2, p3 and p4=37.1%) was not significantly different from the digestibility of dry matter of rice straw silage treated with commercial enzyme (p1=38.8%). the improvement in the digestibility of silage treated with enzymes was caused by decreasing ndf, adf and lignin content in the silage. decreasing fibre content (adf and ndf) and lignin of rice straw silage made the silage easier to be degraded by rumen microbes. the increase of ddm of rice straw silage treated with cetv followed the linear equation of y=21.31+3.14x. meanwhile, dom followed the equation of y=17.43+3.46x. it showed that cetv dosage had not reached the maximum level as the digestibility was still increasing. 57 table 4. digestibility of dry matter and organic matter of rice straw silage production and utilization of cellulose from trichoderma viride – r. hidayat et al. treatments digestibility of dry matter (%) digestibility of organic matter (%) p0 28.6±3.3 25.6±3.6 p1 38.8±2.5 36.8±2.6 p2 28.3±2.0 25.0±2.5 p3 38.9±1.3 37.0±1.7 p4 44.0±3.5 42.3±4.0 p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. digestibility of dry matter (ddm) and organic matter (dom) of rice straw silage treated with enzyme (p1, p2, p3) was significantly higher than digestibility of dry and organic matter of untreated rice straw silage (p0). the present study clearly indicates that the most promising method to improve the nutritive value of agricultural by-products in terms of increasing the digestibility is to treat rice straw with cellulase enzyme addition during ensiling. conclusions fungi of trichoderma viride strain qm9414 produced crude enzymes with high cellulolytic activity i.e. 1.52 iu/ml. cetv was able to improve the biological value of rice straw silage and it was better than commercial cellulase. addition of commercial cellulase (onozuka r10 from trichoderma viride ) and crude enzymes from trichoderma viride (cetv) have positive influence on physical quality of rice straw silage and did not cause negative effect on dry matter and organic matter content of rice straw silage. acknowledgement the researcher would like to thank seameo searca for financing this research. references aak. 1983. the forage. yayasan kanisius. yogyakarta. beauchemin, k.a., d.p. morgavi, t.a. mcallister, w.z. yang and l.m. rode. 2001. the use of enzymes in ruminant diets. in: recent advances in animal nutrition (ed. p.c. garnsworthy and j. wiseman). nottingham university press. nottingham. p. 298-322. bolsen, k. k., sapienza. 1993. silage technology. planting, making and feeding to the livestock. pioneer seeds. usa. 58 djunaidi, i.h. 1988. improvement of rice straw through degradation of fungi. bogor agricultural university, bogor (desertation). biotropia no. 25, 2005 doyle, p.t., c. devendra, g.r. pearce. 1986. rice straw as a feed for ruminant. idp, australian universities and college, canbera. feng, p., c.w. hunt, g.t. pritchard and w.e. julien. 1996. effect of enzyme preparations on in situ and in vivo degradation and in vivo digestive characteristics of mature cool-season grass forage in beef steers. j. anim. sci. 74:1349-1357. hong, s.h., b.k. lee, n.j. choi, s.s. lee, s.g. yun and j.k. ha. 2003. effect of enzyme application method and levels and pre-treatment 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nutr. 85:325-332. yang, w.z., k.a. beauchemin and l.m. rode. 1999. effect of enzyme feed additives on extent of digestion and milk production of lactating dairy cow. j. dairy sci. 82:391-403. 59 r. hidayat1, s. wulandari2, k.g. wiryawan3 and suryahadi3 abstract introduction the main problem of improving ruminant production in indonesia is the shortage of high quality forage during dry season causing the animal production become inefficient, because of an increase in feed cost. on the other hand, the forage is abundant during the wet season. silage preparation chemical analysis results and discussions production and activity test of crude enzymes from trichoderma viride the influence of treatments on the physical quality of rice straw silage 1. nutrients content of rice straw silage 2. fibre content of rice straw silage hong, s.h., b.k. lee, n.j. choi, s.s. lee, s.g. yun and j.k. ha. 2003. effect of enzyme application method and levels and pre-treatment times on rumen fermentation, nutrient degradation and digestion in goats and steers. asian-aust.j.anim.sci. vol 16, no. 3:389-393. jackson, m.g. 1977. the alkali treatment of straw, anim. feed sci. and tech. 2:105-130. komar, a. 1984. technology of rice straw preservation as feed. yayasan dian grahita. indonesia, bandung. lubis, d.a. 1963. feed science. yayasan pembangunan jakarta. man, n.v. and h. wiktorsson. 2002. effect of molasses on nutritional quality of cassava and gliricidia tops silage. asian-aust. j. anim. sci. 5(9):1294-1299. mcdonald, p., a.r. henderson., s.j.e. heron. 1991. the biochemistry of silage. chalcombe publication, 13 highwoods drive, mariow bottom, marlow, bucks sl73pu. montesqrit, 1998. cellulase extraction from the mold and its application to improve fibrous waste product for ruminant. m.sc. thesis. post graduate programe. bogor agricultural university. bogor. nsereko, v.l., d.p. morgavi, k.a. beauchemin and l.m. rode. 2000. inhibition of ruminant feed enzyme polysaccharidase activities by extracts from silage. can. j. anim. sci. 80:523-526. 2. okky ds (aspergillus).cdr biotropia vol. 20 no. 2, 2013: 81 88 aspergillus flavus population and aflatoxin b content in processed peanut products in municipality of bogor, west java, indonesia 1 okky setyawati dharmaputra *, santi ambarwati , ina retnowati and amanda windyarani received 10 february 2013/accepted 22 october 2013 the objective of this study was to document the population of and aflatoxin b content of five processed peanut products collected from different retailers in (subdistrict of central bogor), municipality of bogor. a total of 129 samples of processed peanut products were collected. the products consisted of roasted nut-in shell peanuts (33 samples), flour-coated peanut kernels (33), sauce (18), sauce (33) and sauce (12). sample size varied from 2000 g for roasted nut-in shell peanuts and flour-coated peanut kernels, to 1500 g for sauce, sauce as well as sauce samples. the samples were mixed homogeneously. it was then divided into two parts manually, and then each part was also divided into two parts to obtain working samples to determine population, afb1 content and a reserve sample. kernels of roasted nut-in shell peanuts and flour-coated peanut kernels were obtained by shelling their skin pods and removing the seed coat and the batter coat of tapioca flour manually, respectively. in peanut processed products was isolated using a serial dilution method, followed by pour plate method on aspergillus flavus and parasiticus agar (afpa). afb content was determined using thin layer chromatography method. two replicates were used for each sample.the results showed that the population of in roasted nutin shell peanuts, flour-coated peanut kernels, sauce, sauce and sauce were 0.3, 0.1, 0.3, 13.2 and 0.4 cfu/g (wet basis), respectively. the highest afb content of 43.2 ppb was found in roasted peanut nut-in shell, followed by flour-coated peanut kernels (34.3 ppb), sauce (23.2 ppb), sauce (17.1 ppb) and sauce (4.4 ppb). aflatoxin b , processed peanut products, municipality of bogor 1,2 1 1 2 1 2 seameo biotrop, jalan raya tajur km. 6, bogor 16134, indonesia department of biology, facultas of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor16680, indonesia a. flavus kecamatan bogor tengah siomay pecel/gado-gado satai siomay pecel/gado-gado satai a. flavus aspergillus flavus a. flavus siomay pecel/gado-gado satai satai pecel/gado-gado siomay aspergillus flavus, abstract 1 1 1 1key words: * corresponding author : okky@biotrop.org; okky_sd@yahoo.com doi: 10.11598/btb.2013.20.2.5 81 introduction in indonesia peanuts ( ) is the third most important secondary crop after maize and soybean (bps 2013). they are consumed as human dietary supplements and processed into various snack foods such as, roasted nut-in shell peanuts, flour-coated kernels, siomay, and sauces. according to bps (2010) in indonesia in 2009 the production of peanuts was 763 507 tonnes. in tropical and humid countries such as indonesia, fungal infection can occur before as well as after harvest. according to sauer . (1992) fungal infection after harvest could decrease the physical quality and nutritional content, discolouration of seeds (grains) and mycotoxin production, among others aflatoxin. aflatoxin can cause liver cancer in human and domestic animals, produced by certain strains of . there are four kinds of aflatoxins which are generally found in foodstuff and their processed products, i.e. aflatoxins b , b , g and g . the most dangerous of the toxins is afb1. lilieanny . (2005) reported on the percentage of infection and total aflatoxin content in roasted nut-in shell peanuts (47 samples), flour-coated peanut kernels (22), sauce (12), and (4) obtained from several factories, supermarkets, and traditional markets in bogor, malang, pati and yogyakarta. the percentages of samples infected by in roasted nut-in shell peanuts, flourcoated peanut kernels, sauce and were 38.3, 27.3, 50.0 and 100%, respectively. total aflatoxin content in the products was 1.8, 5.2, 41.6 and 20.8 ppb, respectively. in indonesia the maximum tolerable limit of afb1 content in peanuts and their processed products is 15 ppb (sni 2009). the objective of this study was to investigate the population of and afb content of five processed peanut products collected from retailers in (subdistrict of central bogor), municipality of bogor. based on rankings given by 342 respondents in 11 subdistricts of central bogor who consumed 11 processed peanut products, roasted nut-in shell peanuts was ranked as first choice, followed by flour-coated peanut kernels; , and sauces (dharmaputra . 2010). the five processed peanut products were obtained from 11 shops in (subdistrict) of central bogor from 23 july until 21 august 2009. subdistrict of central bogor was selected for conducting survey and sampling, because it is the most populated subdistrict in the municipality of bogor. roasted nut-in shell peanuts, flour-coated peanut kernels and sauce were obtained from (small shops). was obtained from traveling salesman, was obtained from both arachis hypogaea gado-gado satai et al aspergillus flavus et al a. flavus pecel enting-enting gepuk a. flavus pecel enting-enting gepuk a. flavus kecamatan bogor tengah kelurahan siomay pecel/gado-gado satai et al kelurahan pecel/gado-gado warung siomay satai 1 2 1 2 1 materials and method processed peanut products location of sampling 82 biotropia vol. 20 no. 2, 2013 warung pecel gado-gado satai siomay pecel gado-gado satai siomay siomay pecel/gado-gado satai siomay pecel/ gado-gado satai pec l gado-gado siomay satai a. flavus and traveling salesman. and are salad made from cooked vegetables. is small pieces of meat roasted on skewer. is steamed ravioli filled with meat and open on the top. , , and are served with peanut sauces. a total of 129 processed peanut products were collected. they consisted of roasted nut-in shell peanuts (33 samples), flour-coated peanut kernels (33), sauce (18), sauce (33), and sauce (12). roasted nut-in shell peanuts and flour-coated peanut kernel samples were a labeled products, while pasta sauces samplings were collected randomly without knowing their processing and their sanitations during processing. at the time of purchasing, the sauces of , and were packed separately from the main materials. locations of sampling, the kinds and numbers of processed peanut products are presented in table 1. each sample consisted of five portions of processed peanut products in the form of pasta ( e / , and sauces) and 2 kg (100 @ 20 g packets) of roasted nut-in shell peanuts and flour-coated peanut kernels. one portion of gadogado, siomay and satai sauces contained 75, 50 and 60 g of peanut sauces, respectively. each sample was mixed manually and homogeneously, and it was then divided into two parts manually. each part was also divided into two parts to obtain working samples for the determination of population and afb1 content, and a reserve sample. peanut kernels of roasted nut-in-shell peanuts and flour-coated peanut kernels were obtained by shelling their skin pods and peeling the batter coat of tapioca flour manually, respectively. sampling and to obtain working samples 83 table 1. location of sampling, kinds and number of processed peanut product samples location of sampling number of samples total roasted nut-in shell peanuts flourcoated peanut kernels siomay sauce pecel/ gado-gado sauce satai sauce kelurahan tegallega 3 3 2 3 1 12 kelurahan babakan 3 3 2 3 11 kelurahan sempur 3 3 3 3 2 14 kelurahan panaragan 3 3 1 3 3 13 kelurahan gudang 3 3 1 3 3 13 kelurahan kebon kelapa 3 3 2 3 1 12 kelurahan ciwaringin 3 3 1 3 10 kelurahan cibogor 3 3 3 9 kelurahan babakan pasar 3 3 1 3 10 kelurahan pabaton 3 3 2 3 2 13 kelurahan paledang 3 3 3 3 12 total 33 33 18 33 12 129 note : = no sample aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 determination of population determination of afb1 content aspergillus flavus aspergillus flavus et al a. flavus siomay pecel/gado-gado satai aspergillus flavus a. flavus a. flavus was isolated using serial dilution method followed by plating method on aspergillus flavus and parasiticus agar (afpa) (pitt . 1983, 1992). the kernels derived from roasted nut-in-shell peanuts and flour-coated peanuts were ground. a 25 g of ground sample was placed in an erlenmeyer flask (volume 500 ml), and then sterile distilled water was added to make up the volume to 250 ml. this process resulted in dilution of 1 : 10. the erlenmeyer flask with the suspension was shaked vigorously using a shaker kottermann 4020 for two minutes as much as 250 times to obtain a homogeneous suspension. a 10 ml of the suspension was taken using a volumetric pipet, then it was placed in a separate 250 ml erlenmeyer flask containing 90 ml distilled water, to make a 1 : 100 dilution. the same step was repeated to obtain a serial dilution of 1 : 1 000.two replicates were used for each sample. a 1 ml of each dilution of each sample was transferred in a petri dish (9 cm in diameter) using a volumetric pipet, then 15 ml afpa media (45 c) was poured into the dish. three petri dishes were used for each dilution . the petri dishes were shaked manually to obtain a homogeneous dispersion of the suspension in the media, they were then incubated at room temperature (28 c) for 4 days. population of per gram kernels of roasted nut in-shell peanuts or that of flour-coated peanut kernels; , or sauces (based on wet basis, w.b.) from each replicate was determined using the following formula: x x y where: afp = population per gram each processed peanut product per replicate x = volume of each processed peanut product suspension placed in each petri dish y = dilution which gives colony separately z = mean of colony number of from three petri dishes afb1 content was determined using thin layer chromatography (aoac 2005). this method is applicable to determine 5-25 ng/g afb1 in processed peanut products. dilution is needed for higher concentration of afb1. limit of detection for this method is 0.5 ng/g, while that of recovery and precision are 87-101% and 12.6%, respectively (as rsd intra laboratory study). afb1 stock standard solution 1000 ppm was made from 1 mg crystalline afb1 using 1 ml methanol. lower concentrations of afb1 (100 and 20 ppm) was prepared from 1000 ppm stock solution and were verified using a spectrophotometer (aoac official method 970.44). extraction: as much as a mixture of 1100 g of kernels of roasted nut-in shell peanuts or flour-coated peanut kernels of each sample, 1500 ml distilled water and 22 g nacl were ground using a blender for three minutes. a 200 g of this mixture was then packed in a polyethylene bag and stored in a freezer as retain samples. about 130 g of each kernel sample which has been in the form of pasta and each sample of processed peanut o o 84 biotropia vol. 20 no. 2, 2013 afp = cfu/g (w.b.) 1 85 products in the form of sauces was placed in a 250 ml erlenmeyer flask. fifty ml nacl 2.2%, 150 ml methanol p.a. and 100 ml nhexane were added and the mixture was stirred using a magnetic stirrer for 30 minutes. it was then left for 30 minutes to obtain a good separation. as much as 25 ml methanol phase was taken using a volumetric pipette, it was then filtered and placed in a separated funnel (volume 250 ml). this part was extracted using 25 ml chloroform p.a. after separation, chloroform fraction in bottom layer was placed in a 100 ml vial. the liquid as the result from extraction was evaporated until it was almost dried. the obtained residue was dissolved again using chloroform p.a., it was then transferred into a vial and re-evaporated. before processing for tlc, the residue left after evaporation was dissolved again using 500 ul chlorofom solution p.a. identification was carried out using a chromatography tank containing eluent, i.e. chloroform p.a. : acetone p.a. (9 : 1). a 5 and 10 ul samples of aliquots were spotted on chromatography plate using a 10 ul microsyringe. on the same plate afb1 standard solutions 1-10 ul were also spotted. the known concentrations of aflatoxin b standards used were also spotted between 1-4 ul. chromatography plate was placed in chromatography tank containing eluent, it was then eluted from the bottom up to the top until the eluent attained the limit of the top. the result of elution was then dried using a hair dryer and was observed under long wave (365 nm) ultra violet length. qualitative test was conducted by comparing the retention factor (rf) of sample and standard spots, while quantitative test was carried out by comparing the fluorescence intensity of sample spot and standard spot. if afb1 was not detected, the fluorescence intensity of sample spot was compared with that of standard spot. if the fluorescence intensity of sample spot was too intense to match the standards, the sample extracts should be diluted and re-chromatographied.afb1content was determined using the following formula: aflatoxin b content (ppb) = w x z where: s = volume of afb1 standard (µl) which gives fluorescence equivalent with z ul of sample y = concentration of standard afb1 (µg/ml) z = volume of sample extract (µl) required to give fluorescence equivalent with s µl afb1 standard w = weight (g) of extracted sample v = volume of the solvent (µl) required to diluted final extract fp = dilution factor 150/25. . the percentage of roasted nut-in-shell peanuts and flour-coated peanut kernels; , and sauce samples infected by were 15.2, 6.1, 5.6, 1 1 s x y x v x fp results and discussion aspergillus flavus population and afb1 content of processed peanut products siomay pecel/gado-gado satai a. flavus aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 86 biotropia vol. 20 no. 2, 2013 57.6 and 8.3%, respectively, while those contaminated by afb were 42.4, 30.3,11.1, 27.3 and 16.7%, respectively. the percentage of processed peanut product samples infected by and those of non-detected afb contents are presented in table 2. all product types were infected by , but the highest population of was found in sauce, followed by and sauces, roasted nut-in shell peanuts and flour-coated peanut kernels. the range and the mean of population, afb1 content and the percentage of processed peanut product samples contaminated by afb1 exceeded 15 ppb are presented in table 3. afb1 contents in processed peanut products were varied. the mean of highest afb1 content was found in roasted nut-in shell peanuts (43.2 ppb) (table 3). the percentage of roasted nut-in shell peanuts samples containing afb1> 15 ppb was also 42.4%. the percentage of processed peanut product samples contaminated by afb1exceeded 15 ppb were as follows: roasted nut-in shell peanuts 42.4%, flourcoated peanut kernels 30.3%, sauce 11.1%, sauce 21.2%, and sauce 16.7%. 1 1a. flavus a.flavus a. flavus pecel/gado-gado satai siomay a. flavus siomay pecel/gado-gado satai table 2. percentage of processed peanut product samples infected by , contaminated by aflatoxin b , and non-detected aflatoxin b content a. flavus 1 1 * = afb1 content < detection limit for afb1 (0.5 ppb) using thin layer chromatography method table 3. range and mean of population, aflatoxin b content, and percentage of processed peanut product samples containing aflatoxin b exceeded 15 ppb a. flavus 1 1 processed peanut products number of sample of samples infected by a. flavus number (%) of sample contaminated by afb1 number (%) of non-detected afb1 content samples (0.0 ppb)* roasted nut-in shell peanuts 33 5 (15.2) 14 (42.4) 19 (57.6) flour-coated peanut kernels 33 2 (6.1) 10 (30.3) 23 (69.7) siomay sauce 18 1 (5.6) 2 (11.1) 16 (88.9) pecel/gado-gado sauce 33 19 (57.6) 9 (27.3) 24 (72.7) satai sauce 12 1 (8.3) 2 (16.7) 10 (83.3) processed peanut products range (mean) of a. flavus population (cfu/g (w.b)) range (mean) of afb1 content (ppb) samples containing afb1 exceeded 15 ppb (%) roasted peanuts with skin pods 0.0 – 3.3 (0.3) 0.0 – 316.8 (43.2) 42.4 flour-coated peanuts 0.0 – 1.7 (0.1) 0.0 – 160.0 (34.3) 30.3 siomay sauce 0.0 – 5.0 (0.3) 0.0 – 39.9 (4.4) 11.1 pecel/gadogado sauce 0.0 – 255.0 (13.2) 0.0 – 197.8 (17.1) 21.2 satai sauce 0.0 – 5.0 (0.4) 0.0 – 198.6 (23.2) 16.7 87 fungi can be killed by heating, while aflatoxins could not be easily degraded by heating, because they have high melting point. the melting point of afb1 is 267 c (buchi & rae 1969). therefore, afb1 was able to be detected on the processed peanut products used in this study, although they were heated during processing (table 2). although the percentage of samples infected by was low, the processed peanut products could be contaminated by afb1 due to the toxin produced during post-harvest handling (drying and shelling). afb1 can only be produced by toxigenic strains of . afb1 contents were lower than 0.5 ppb in some samples. the mean of highest population was found in sauce, followed by and sauces, roasted nut-in shell peanuts and flour-coated peanut kernels (table 3). dharmaputra (2010) reported that population in raw peanut kernels collected from two traditional markets in bogor was as high as 4865.8 cfu/g wet basis. in this study population in the processed peanut products were lower than those in raw peanut kernels, because the kernels were heated during processing. population in sauce was much higher compared to that in the other processed peanut products (table 3). at the time of purchasing, sauce was not ready to be consumed, but it was prepared by the seller when people come to buy . it was assumed, that was not only derived from the peanut kernels, but it also from the ingredients and tools that could have been contaminated, for example, a mortar to grind the peanut kernels or a glass jar to store the kernels could be the source of contamination.the level of population in the products could also be due to the sanitation during processing, which was not tested in this study. the mean of population in roasted nut-in shell peanuts and flour-coated peanut kernels, and sauces were very low, i.e. < 1 cfu/g (w.b.), because raw peanut kernels were already heated. he roasted nut-in shell peanuts had high aflatoxin levels as a consequence aflatoxin production could have occurred in the post-harvest storage was still found in processed peanut products, although they were heated during processing. certain fungal species have dormant structures. fungi can also contaminate processed peanut products during production, packaging and transportation. the mean of highest afb1 content was found in roasted nut-in shell peanuts, i.e. 43.2 ppb (table 3). afb1content was affected among others by the quality of the peanuts and the method of processing. bankole and eseigbe (2004) reported that 43.4% of 106 fried peanuts without oil samples collected in nigeria were infected by . the range of afb1 content of 64.2% of all samples positive contaminated by the toxin was 5 106 ppb. the peanuts were in the form of kernels and they were processed traditionally by roasting using hot sands on fire place made from clay. o a. flavus a. flavus a. flavus pecel/gado-gado satai siomay et al. a. flavus a.flavus aspergillus flavus pecel/gado-gado pecel/gado-gado pecel/gado-gado a. flavus a. flavus a. flavus siomay satai t aspergillus flavus a. flavus aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 88 biotropia vol. 20 no. 2, 2013 conclusions acknowledgements references the population in roasted nut-in shell peanuts, flour-coated peanut kernels / and sauces were relatively low (0.1-13.2 cfu/g wet basis). the highest afb1 content was found in roasted nut-in shell peanuts (43.2 ppb), followed by flour-coated peanut kernels (34.3 ppb), sauce (23.2 ppb), sauce (17.1 ppb) and sauce (4.4ppb). to minimize aflatoxin contamination in processed peanut products, it is important to conduct a good handling practice from farmer up to table. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the office of ( ) for the permission in conducting processed peanut products sampling, to the office of and 11 offices of belongs to for the information and cooperation during the sampling, and to mrs. elly sunarsih and mrs. ratnaningsih for their assistance in conducting afb analyses. of a. flavus , siomay, pecel gado-gado satai satai pecel/gadogado siomay kesatuan bangsa dan perlindungan masyarakat kesbanglinmas kecamatan bogor tengah kelurahan kecamatan bogor tengah 1 [aoac] association of official analytical chemist. 2005. natural toxins. di dalam: horwitz w, editor. . ed ke-18. gaithersburg: aoac. p 11. [bps] badan pusat statistik. 2013. . jakarta: bps. bankole sa, eseigbe da. 2004. aflatoxins in nigerian dry-roasted groundnuts. 34(6):268-271. büchi g, rae id. 1969. the structure and chemistry of the aflatoxins. in: goldblatt la, editor. . new york: academic pr. p 55-75. dharmaputra os, retnowati i, ambarwati s, windyarani a. 2010a. population and aflatoxin b content of raw peanut kernels collected from traditional markets in bogor, west java, indonesia. paper presented at international mycotoxin conference, penang, malaysia, 1 4 december 2010. dharmaputra os, ambarwati s, retnowatii. 2010b. dietary exposure assessment for aflatoxin b from processed peanut products in municipality of bogor, west java, indonesia. 18(1):1-8. lilieanny, dharmaputra os, retnowati i, putri asr. 2005. . (population of storage mold and aflatoxin content of processed peanut products). 10(1):17-20. pitt ji, hocking ad, glenn dr. 1983. an improved medium for the detection of and . 54: 109-114. pitt ji, hocking ad, samson ra, king ad. 1992. recommended methods for mycological examination of foods. in: samson ra, hocking ad, pitt ji, king ad, editor. . amsterdam: elsevier. p 365-368 [sni] standarnasionalindonesia. 7385:2009. 2009. . jakarta: badan standardisasi nasional. sauer db, meronuck ra, christensen cm. 1992. microflora. in: sauer db, editor. . ed ke-4. minnesota: american association of cereal chemist. p 313-340. official methods of analysis of aoac international produktivitas padi dan palawija di indonesia nutr food sci aflatoxins; scientific background, control, and implications aspergillus flavus biotropia populasi kapang pascapanen dan kandungan aflatoksin pada produk olahan kacang tanah j mikrobiol indones aspergillusflavus a. parasiticus j appl bacteriol modern methods in food mycology batas maksimum kandungan mikotoksin dalam pangan storage of cereal grains and their product 1 1 microsoft word 11 biotropia no. 21,2003 : 11 18 karyomorphology of the philippine rock goby, glossogobius giuris (gobiidae) from lake taal and some rivers of cavite, luzon island jimmy t. masagca1 and jose a. ordonez2 'center for multidisciplinary research and linkages, graduate school of education, arts and sciences de la salle university-dasmarinas, philippines 2 biological sciences department, college of science de la salle university-dasmarinas, philippines abstract the karyomorphology of glossogobius giuris (gobiidae) obtained from lake taal and some rivers of cavite in luzon island, philippines was described. metaphase chromosome analysis (colchicine-sodium citrate-carnoy's fixationgiemsa staining procedures) of the hematopoitetic cells in the anterior kidneys revealed that the diploid chromosome number was 2n=46 (46a). fundamental number (fn) is also 46, since all chromosomal morphology were acrocentrics without any distinguishable heteromorphic pair of chromosomes in the metaphase spreads from both dry and wet preparations. this study confirms previous reports on the chromosomal sets of g. giuris from india. keywords : philippines / lake taal / genetics / karyomorphology / glossogobius giuris introduction gobies such as glossogobious giuris and g. celebius have been the subjects of rapidly increasing number of pollution and genetic investigations because of their readily adaptable characteristics to laboratory studies. the present karyomorpho-logical study of this popular food fish (locally named as "biya" in the philippines) was conducted to fill in the wide gaps on fish cytogenetics in the country. several species of gobies and other gobioid fishes (e.g. eleotrids) have been described in terms of their karyomorphological characters such as boleophthalmus pectinirostris, gobius abei and periophthalmus cantonensis (kirpichnikov 1981; nogusa 1960). this study sought to provide relevant information on the basic cytogenetics of glossogobius giuris (gobiidae), specifically, 1) to ascertain the chromosome number and fundamental number of g. giurus; 2) to construct the tentative karyo-type or ideograms of representative samples of the test fish under consideration; and 3) to describe the karyomorphological characters of the test fish. glossogobius giuris was the test fish selected for our chromosomal investigation since no study so far has been reported on its karyomorphology in the philippines. selecting g. giuris from the teleosts of lake taal and rivers of cavite can be justified since cytogenetical investigations of these fishes would explain possible changes in the genetic constitution brought about by incipient speciation in our ongoing chromosome evolution studies. moreover, empirical data gathered in this study will add to the growing body of literature on fish chromosomes that are 11 biotropia no. 21,2003 needed in future studies related to cytotaxonomy, aquaculture breeding and genetoxic testing with the use of chromosomes as biomarkers. methodology fish samples of g. giuris were obtained in the waters off the volcano island or "pulo" in lake taal and some rivers of cavite (dasmarinas-indang area and maragondon riverine area). the methods used in the study basically followed the rapid flame drying techniques (with colchicinesodium citrate-carnoy's fixation-giemsa staining procedures) used in the previous works of masagca (2000) and masagca & sumantadinata (1994). sample preparation fish specimens were pre-treated by intra-muscular injection with colchicine (0.05% in 0.8% nacl) at 1 ml/loog body weight and allowed to swim in well-aerated glass aquaria (40 liters) for 5-6 hours (h). after treatment, the specimens were sacrificed by decapitation or hypothermia (in cracked ice) and kidneys were dissected out, carefully cleared of blood vessels and placed in a petri dish with 0.5-0.6% sodium citrate for hypotonization. anterior kidney tissues were minced into smaller pieces for 16 to 20 minutes in the dish with the hypotonic solution. cell suspensions are transferred to a 10-ml polypropylene tubes and centrifuged for 4-5 minutes at 2500-3500 rpm. the supernatant was removed using a pasteur pipette without disturbing the cell pellet or cell button. about 4 ml of the cold freshly prepared carnoy's fixative (3 absolute methanol: 1 glacial acetic acid) was poured into the tube. after 15 minutes of periodic agitation, the cells were centrifuged again, the supernatant was removed and replaced with fresh fixative. the cell pellet was disturbed gently with a fine point needle of a disposable syringe or dissecting needle, the tube was labeled and stored in a refrigerator for 24 to 30 h before slide plating. slide plating, staining and chromosome analysis pre-cleaned microscope glass slides previously soaked in a 50% ethanol and chilled in the refrigerator overnight were used. after final centrifugation, cells were re-suspended in a small volume of the fixative (about 0.5 to 0.95 ml, depending on the size of the cell button). three to four drops of the suspension were plated on the chilled slide with a pipette and air-dried. wet and dried slides were stained by dipping them into staining jars containing 4% giemsa stock solution (at ph 6.8) for 30-40 minutes. stained glass slides were rinsed with de-ionized water and dried for 12 karyomorphology of the philippine rock goby jimmy t. masagca & jose a. ordonez 30 minutes in an improvised slide dryer. the slides are then placed in a xylene (or xylol) for 10 minutes. slides were air dried for 15 minutes. some slides were mounted using entellan b. stained slides were examined under lpo (10 x) and hpo (loox) to locate well-spread metaphase chromosomes. suitable or well-spread chromosomes were screened to count the diploid chromosomes. results and discussion table 1 shows the frequency distribution of chromosome counts of g. giurus fish samples obtained from lake taal and selected rivers of cavite. using three (3) samples of g. giurus obtained from the dasmarinas-indang riverine areas, a total of 71 well-suited metaphase cells were screened to determine the chromosomal number (cn) as shown by the modal chromosome count. out of this number, 52 cells or 73.24% showed a characteristic count of 2n=46; 12 cells or 16.9% with 2n=44; 5 cells with the characteristic count of 45; and 1 cell each for counts 43 and 47. of the six (6) fish samples of g. giurus obtained from maragondon area, there were 126 metaphase plates with 104 or 82.54% showing a cn of 2n=46, 8 or 6.35% with 2n=44, 10 or 7.94% with 2n=45; and 4 cells with 47. from the 12 samples of g. giurus from lake taal, a total of 314 well-spread metaphase cells were obtained for chromosomal analysis. out of this number, 246 cells (78.1%) have the characteristic count of 2n=46; 39 cells or 12.4% with 2n=45; 12 or 3.8% with 2n=47; 11 or 3.5% with 2n=44 and 6 cells or 1.9% with a chromosome count of 43. in sum, of the 511 metaphase cells (71 metaphase cells from fish samples obtained in dasmarinas-indang areas, 126 metaphase cells from maragondon areas and 314 metaphase cells from lake taal) screened, there were 402 cells or 78.7% have the diploid number of 46; 54 cells (10.5%) have 45; 31 cells (6.1%) have 44; 17 cells (3.3%) have 47 and 7 cells (1.4%) have 43 chromosomes. modal chromosome number based on the data presented, the chromosomal number of the philippine rock goby, g. giurus obtained from 3 areas showed that the diploid modal chromosome number is 2n=46. nf is also 46 (fn=46), since all chromosomes are mono-armed. the predominant chromosome number from the 3 locations was consistently observed at 2n=46 (figure 1). this finding confirms the work of manna (1989) from indian samples and earlier reports of kaur & srivastava (1965, as cited by denton 1972). although characteristic counts of 43, 44, 45 and 47 were noted in wet preparations of slides, the majority of the metaphase cells showed the chromosome count of 46. the chromosomal count of 45 was known in 39 cells or 12.42% of the total number of metaphase spreads. in counting chromosomes, there are instances when 13 biotropia no. 21,2003 overlapping cannot be avoided. there are also possible technical reasons like a missing chromosome during slide plating. denton (1972) reported that within family and genus there seems to be a tendency of reduction in chromosome number to parallel speciations. furthermore, gold (1979) surmised that chromosome numbers and variations in chromosome number do distinguish certain taxonomic groupings, as in the case of salmoniformes (e.g. salmo truttd). generalizations have already been made on chromosome numbers among the members of orders cyprinidontiformes, cypriniformes, siluri-formes and perciformes (rishi 1989). however, it seems that gobies and eleotrids tended to show the common chromosome number of 44 to 46. karyomorphology of the p h i l i p p i n e rock goby jimmy t. masagca & jose a. ordonez figure 1. chormosome counts of g. giuris from different locations in cavite and lake taal. the karyotype of g. giuris consists of 46 acrocentric chromosomes. since an acrocentric chromosome is counted as only one arm, the total number of arms will also be 46. the centromere of acrocentric type of chromosome is terminal on which produces a chromosome with one long arm. the karyotypes of the gobies boleoph-thalmus pectinirostris, gobius abei and periophthalmus cantonensis were also found to have 2n=46 (all acrocentrics) as reported in the studies of kirpichnikov (1981) and nogusa (1960). karyomorphological characters of g. giuris as shown in tables 1 and 2, the diploid chromosome number, 2n=46 obtained for g. giuris was consistent in 3 locations (dasmarinas-indang areas, maragondon areas and pulo (i and ii) in lake taal. in the gobiidae family, most of the genera have chromosome number of 2n=44 to 2n=48, such as in bathygobius fuscus (2n=48) and chaetogobius annularis (2n=44). the chromosomes of gobies and eleotrids show variability from 2n=43 to 62 (masagca 2000), with most of the chromosome numbers are 2n=44, 46 and 48. in another study, the karyotype of g. microdon has a diploid number of 2n=56 and nf of 66 with a chromosome formula of 4m+6sm+46st, a. 15 biotropia no. 21, 2003 variability in chromosomal counts would lead to certain generalization of the possibility of changes in chromosomal number due to fusions, translocations and other mechanisms. however, this is not conclusive in the present study since there is a need for further chromosomal banding studies and constancy of variation in the counts. in some studies, like the paedomorphic goby, aphia minuta (gobiidae) wherein the diploid complement ranged from 44 to 41 due to robertsonian fusions (nf=44). data on spermatogenesis suggest that structural heterozygotes are fertile and that these chromosomal changes are not involved in speciation process (caputo et al. 1999). table 2. summary of chromosome counts for g. giuris from 3 locations. location diploid (2n) chromosome number total no. of metaphase cells 43 44 45 46 47 dasmarinnas indang area 1 12 5 52 1 71 maragondon areas 0 8 10 104 4 126 pulo i & 11, lake taal 6 11 39 246 12 314 total 7 31 54 402 17 511 percent (%) (1.4) (6.1) (10.5) (78.7) (3.3) (100) in this study, characteristic counts of 41, 42, 43, 44 and counts higher than 46 in the test animals (g. giurus) were observed. variations in chromosome number maybe attributed to several factors: (1) handling techniques; (2) chemically induced; and (3) inherent genetic characteristic of the test fishes. handling techniques would explain the variability in chromosome counts. the karyotypes of two other teleosts, g. giurus previously described in japan, india and elsewhere could permit the researchers to have further comparison using the conventionally stained chromosomes and in the future the banded chromosomes from fully elongated chromosomes. diploid chromosome number of the philippine common goby, g. giuris is 2n=46 and nf of 46. all of the chromosomes are acrocentric (mono-armed). the chromosome number of 46 is common to the order perciformes. diploid chromosome number of oxyeleotris aporos is 2n=46, which is also similar to the chromosomes of selene setapinnis (family carangidae) as described by netto & pauls (2000). in fishes, 48 rod-like chromosomes have been considered to be the modal number as shown in the works of nogusa (1960), roberts (1967) and ohno & atkin 16 karyomorphology of the philippine rock goby jimmy t. masagca & jose a. ordonez (1968). recently manna (1989) advocated that 48 chromosomes mixed morphology and only rods were the modal ones from which the evolution of different karyotypes can be envisaged. conclusion this study concluded that the philippine rock goby, g. giuris has a diploid chromosome formula of 2n=46 (a) and having the fundamental number of 46. karyomorphological characters of the goby under study reveal that majority of the chromosomal spreads consist of all acrocentric chromosomes, which are common among the gobiids and eleotrids. no heteromorphic pair of chromosomes was observed in the ideograms prepared. characteristic chromosome counts of g. giuris range from 41 to 48. acknowledgment the authors express their thanks to the dlsu-d administrators (dr. h.d. torres, dr. mf. ramos, dr. v.l. de leon, dr. n.m. medina, ms. c. cervillon and dr. j. samonte for the ufro grant. references arai, r. and y. sawada. 1974. chromosomes of japanese gobioid fishes (i). bulletin of natural science tokyo 17:97-102. arai, r. and a. fujiki. 1979. chromosomes of japanese gobioid fishes (iv). bulletin of the natural science museum. series a 5 (2): 153-159. caputo, v., m. l. caniglia and n. machella. 1999. the chromosomal complement of aphia minuta, a paedomorphic goby. the fisheries society of the british isles, p. 455-458. denton, t.e. 1972. fish chromosome methodology. charles c. thomas publisher, springfield, illinois. 166 p. kaur, d. and m.d.l. shrivastava. 1965. the structure and behavior of chromosomes in freshwater teleosts. cited by denton, t.e.1973. fish chromosome methodology. berlin: charles c. thomas publishing. springer verlag. kirpichnikov, v.s. 1981. genetic bases for fish selection. springer verlag: berlin. kligerman, a.d. and s.e. bloom. 1977. rapid chromosome preparations from solid tissues of fishes. journal of the fisheries research board canada, 23:266-269. levan, a., k. fredga and a. sanberg. 1964. nomenclature for centromeric position on chromosmes. hereditas, 52:201. manna, g.k.i 989.fish cytogenetics related to taxonomy, evolution and monitoring genotoxic agent. pages 2146, in: das and jhingran, eds. fish genetics in india. today and tomorrow printer & publishers: new delhi, india. 17 biotropia no. 21,2003 masagca, j.t. 2000.karyology of the south-east asian marble sleeper, oxyeleotris marmorata bleeker 1852. asian international journal of life sciences journal, 9(2):181-188. masagca, j.t. and k. sumantadinata. 1994. chromosomal characters of the indonesian sand goby, oxyeleotris marmorata blkr. 1874 (eleotridae), biotropia-southeast asian journal of tropical biology (7): 41-46. masagca, j.t. 1993. karyotypic differentiation among natural populations of sand goby. o. marmorata blkr. (eleotridae) in the indo-malayan region. proceedings of the symposium on fish genetics and its application to fishery management held on dec. 8-11, 1992, seameo-biotrop bogor, indonesia. medina, f.i.s. iii. 1984. a simple method for studying the mitotic metaphase of tilapia nilotica. proc. symp. research trends phils. nrcp bull. no. 98. netto, m.r.c.b. and e. pauls. 2000. family carangidae: cytogenetical and evolutionary aspects (international workshop on marine genetics, 1998 (brazil). nogusa, s. 1960. a comparative study of the chromosomes in fishes with particular considerations on the taxonomy and evolution. memoir of hyogo university of agriculture 3(1):1.cited by denton, t.e.i973.fish chromosome methodology. charles c. thomas publishing. springer verlag, berlin. 166 p. ohno and atkin. 1968. evolution from fish to mammals by gene duplication. hereditas, 59:169-187. pagulayan, r.c., n.c. lopez and f.s. magbanua. 1997. littoral fishes of lake taal. sylvatrop tech. j. of philipp. ecosystems and mat res. 7(l&2):84-93. reddy, p.v.g.k. and g. john. 1987. a method to increase mitotic metaphase spreads in permanent chromosome preparations for karyotype studies of fishes. pages 199-205. in: proceedings of the world symposium on selection, hybridization and genetic engineering in aquaculture held in bordeaux. 27-30 may 1986. vol. ii. rishi, k..k. 1989. current status of fish cytogenetics. pages 1-20, in: das and jhingran (eds.), fish genetics in india. today and tommorow's printers and publishers, new delhi, india. rivlin, k., j.w. rachlin and g. dale. 1985. a simple method for the preparation of fish chromosomes applicable to field work, teaching and banding. journal of fish biology, 26:267-272. roberts, e.l. 1967. chromosome cytology of the osteichthyes. progressive fish culturist, 29:75-83. sumner a.t., h.j. evans and r.a. buckland. 1971. a new technique for distinguishing between human chromosomes. nature new biol. 232:31-32. 18 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf microsoft word 13 biotropia no. 23, 2004 : 13 39 fisheries associated with mangrove ecosystem in indonesia: a view from a mangrove ecologist sukristijono sukardjo the centre for oceanological research and development, indonesian institute of sciences, jl.pasir putih 1 ancol timur, p.o.box 4801 jktf jakarta 11048, indonesia. e-mail: s_sukardjo@telkom. net and s_sukardjo@yahoo. com abstract blessed with mangrove area of some 9.6 million ha in extent, indonesia represents an important country with fishery resources being a source of food and nutrients. the fishery resources utilized by man, such as fishes, crustaceans and mollusks that are found in the mangrove ecosystem/swamp area arc enormous. there is a range of species caught in the mangrove and surrounding areas with over 70 species. however, commercially valued species are limited to a few such as rabbit fish, snapper, grouper, marline catfish, fringe-scale sardine, and anchovy. leaf detritus from mangroves contribute a major energy input into fisheries. but information about the study on the relationship between fishery species and mangroves, ecologically and biologically, arc scanty. the mangrove is a physiographic unit, the principal components of which arc organisms. therefore, the problems are predominantly of a biological nature (e.g., mangroves fishery relationship). positive correlation between the mangrove area and penaeid shrimp catch found in indonesia, the philippines, australia and mexico. finally, the most important part of the variance of the msy (maximum sustainable yield) of penaieds (53% of the variance) could be explained by a combination of area of mangrove habitats and latitude. keywords : indonesia/mangrove/ecosystem/fisheries/ecology/coastal areas/fishes/molluscans/ crustaceans. introduction indonesia (6° n 10° s and 95° e 142° e) is an archipelagic state in the tropical area with 18,110 islands and almost more than 108,000 km of coastlines (sukardjo 1997; anonim 2003). these areas form an important and valuable natural resource with potential economic value, and a potentially important production area for food. people have long relied on coastal waters as a source of food. the coastal zone of the indonesian waters includes a number of bays and gulfs into which large and small rivers empty, creating estuarine conditions in the inshore areas. the extent of the estuarine zone in the sea varies according to the size of the river and the volume of its freshwater discharge, the steepness of the gradient, and the tidal range. also, these coastal zones contain diverse productive ecosystems that include mangroves, sand beaches, several riverbasins and freshwater bodies. each of these ecosystems has components that are exploited in a wide variety and used with different intensities because of socio-economic and socio-cultural demands. therefore, indonesia, is considered as one of the largest maritime country in the world. 13 biotropia no. 23, 2004 with almost more than 75% of indonesian territories being seas, marine fishery is one of the important renewable resources for the nation. for instance, 1,450,000 tons of pelagic fish had been caught for whole indonesia (eps 1995). ironically, depletion of the marine resources is starting to become evident in indonesia. the fishery resources utilized by man, such as fishes, crustaceans and mollusks (oyster, mussel, cockle), that are found in the mangrove swamp area are enormous. indigenous people in indonesia have exploited the biota of mangrove waters for centuries, and fish and shrimp are still one of the major products harvested from this mangrove swamp (schuster 1952). thus, aquatic resources play a vital role in the social, economic and ecological well-being of coastal communities in indonesia. furthermore, the value of the mangrove ecosystems is not just of academic interest, it also relates to their cultural and commercial value. the full economic value should be determined in order to make intelligent decisions about their future use. thus the understanding of both the roles and the functions of mangrove ecosystem in terms of fishery in indonesia is also of considerable importance. the following paper is, therefore, an effort to review and methodize existing information on the mangrove-fishery association subject in indonesia. the indonesian mangroves: a fishery feature situated at the two continents, asia and australia, and two oceans, pacific and indian, the indonesia archipelago, has an ecologically and economically strategic position with distinct culture that took shape on these 18,110 islands. the climate of indonesia is tropical and generally governed by the monsoonal situation, resulting in fairly regular climate variations. during the winter, the northern part of the earth has air pressure around asia higher than that of australia. north of the equator, the wind blows from north and east the ne monsoon, at and near the equator, it becomes more north and north nw monsoon. south of the equator the winds turns from nw via w to sw the west monsoon. consequently, indonesia is blessed with high annual rainfall (>1500 mm/year) and comfortable tropical temperatures. moreover, indonesia's waters are homogeneous to an unusual degree. the country lies entirely within the tropics, straddling the equator from roughly 5°n to 10°s latitude. oceanographic conditions are uniform in this equatorial zone, characterized by lack of seasonal changes and consistently warm waters. the indonesian waters are ideal monsoon region. these are very favorable for mangroves development. consequently, large areas of the mangrove ecosystem in indonesia are to be found and estimated at about the range of 4.25 million ha (1982) to 9.6 million ha (2002). therefore, mangrove forests are a major feature of the coastline in many islands of indonesia. however, this figure and its distributional extent is still in debates for further clarifications. this type of forest vegetation is common in the malesia (comprising the political states of indonesia, malaysia, singapore, brunei darussalam, the philippines and png) and has stimulated the interest of various systematics and ecologists as can be learned from comprehensive 14 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo studies by watson (1928) in malay peninsula, and by brown and fisher (1918) in the philippines, and by becking et al. (1922) in indonesia. a substantial body of information exists on the biology, ecology and other aspects of mangroves or mangrove ecosystem or mangrove forests or mangals (macnae 1968). the bibliography lists of mangrove forests in indonesia can be found in e.g., rollets (1997). however, recent records of mangrove literatures can be learned through a series of the proceedings of the national seminar on mangrove ecosystem (i-vi) and others. but our knowledge of the mangrove fish fauna in contrast, is limited to a handful of papers that deal with inventory. a similar state of knowledge exists on the association between mangroves and fisheries (see: macnae 1974 for general information); and the economic value of the fisheries function of mangroves in the bintuni bay, irian jaya, has been discussed by ruitenbeek (1991). commercial fisheries in indonesia or indonesia's artisanal commercial fisheries are largely dependent upon coastal and estuarine fishes. in indonesia, mangrove forests reach their greater structural and floristic diversity in all biggest islands e.g., sumatra, kalimantan, and irian jaya, and tropical forests generally have higher rates of litter production than temperate forests (bray and gorham 1964). mangrove forests in indonesia are floristically rich with at least 94 species represented (sukardjo 1994), and produce large quantities of litterfall e.g., in east kalimantan estimated to be about 21.10 to 29.35 dry weight t/ha/year (sukardjo 1995). they produce a large amount of fixed carbon which is used by man for timber, firewood and charcoal. it is hypothesized that this carbon may also support detritus-based food webs both in the swamp and in surrounding coastal waters. in a global context, mangrove swamps have been recognized as extremely productive ecosystems, which not only have a high rate of primary productivity, but also export organic matter and support a variety of aquatic organisms (odum and heald 1972); and most of the aquatic organisms are valuable economically for man. it is generally supposed that mangroves play an important role in supporting a wide range of marine life in near-shore waters and in sustaining coastal fisheries. these highly productive mangrove forests provide critical habitat and food for many organisms, including important fisheries species. however, human population concentrates in the coastal zones, imposing enormous pressure on these mangrove forests. in indonesia moreover, mangrove swamp communities or mangals (after macnae 1968) occupy warm coastal areas where land is becoming increasingly valuable for farming, mariculture, and recreation. thus men's use of mangrove areas in indonesia for the establishment of ponds for the culture offish and prawns, and other fishery activities, and for timber etc., is practically common. in indonesia, mangrove ecosystems or mangroves are found in tidal wetlands under varying conditions of salinity, water-logging, anaerobic substrate, often hyper-saline (>33 %) and acidic. many of the largest mangrove areas of indonesia occupy a significant portion of the deltas of rivers, large and small. here, mangrove ecosystems are delicate and complex, intensely dynamic and fragile; and their principal components are the physical and chemical environments, the biotic elements (flora and fauna), and human interferences. each component of the 15 biotropia no. 23,2004 environment namely, climate, salinity, freshwater supply, siltation, erosion, substrate and nutrients, act on flora and fauna, and, in-turn, these influence the environment. for instance, reduced freshwater flow through a mangrove forest probably not only reduces the nutrient input from surrounding terrestrial areas, but also results in higher soil salinities. thus, it becomes a stress for mangroves itself and their associated meiofauna. mangrove ecosystems are open systems and the cycles of material transport in mangrove forests are driven by physical and biological factors that control the rate of input and output of inorganic and organic components. changes in the quality of water entering and leaving the mangroves are particularly important in determining natural cycles. in addition, they support a characteristic fauna which includes crabs, shrimps and molluscs. the waterways support a varied fauna of fish, shrimp and plankters. the general acceptance is prawn fisheries are important commercial fisheries in many developing countries and developed countries. an ecological importance of the mangrove ecosystem fishery aspects 1. ecological features of mangroves as fish habitat mangrove forests are a common type of pan-tropical coastal vegetation. in indonesia, characteristics of mangrove sites varied provincially and locally, but it does have the same serve as fish habitats and refuges for a large number of species of conservation concern. for instance, in the estuary of the rokan river in sumatra, hardenberg (1950) has reported the occurrence of some 175 species, including occasional migrants. the number of individuals is, however, high. in indonesia, there are large marine mangal areas in an environment characterized by tidal ranges of 2-5 m, fluctuating salinities of 28-33%o, high atmospheric humidity and constant subequatorial temperatures; and mangrove forests of embankments, lagoons, and harbors are less dominated by hydrological factors. for instance, the coast of java experiences strong semi-diurnal tides, with a range of 5 m and above during extreme spring tide. the coast may be classified as a high mesotidal environment. the waters are well mixed vertically, and salinities range from 20 to 32 %o. major dilution occurs from the discharge of freshwater by 11 rivers. also, wind blowing over the sea surface can cause the disturbance of hydrodynamic sea stability. this condition, furthermore, will result in a rising of sea water towards the surface from the below regions and oceanographically known as upwelling; and it is very beneficial for fishery. in sumatra and java, the color of estuarine water may be tea-brown or blackish, due to outflow from enormous peat forests (hardenberg 1950). the ph of this water is reported to be as low as 5 or less. there is a marked increase in the number of planktonic organisms as a result of increase in nutrient salts. copepods diminish to be replaced by an increased amount of diatoms and noctiluca. finally, mangrove ecosystems by their nature include an aquatic element; and 16 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo marine species are replaced by coastal forms. for instance, the position of burrowing animals within the substrate is most closely linked to the state of the tide. this may be merely the temporary intrusion of seawater along open coasts at high tide, or it may be the waters of tidal creeks or estuaries surrounded by mangroves. thus, the unique association of animals and plants, for instance, that dwell upon the roots of mangroves in indonesian seas are interesting and has long been attracting the attention of naturalists and ecologists. the structural-functional diversity of the true-mangrove species complex is probably best expressed in the remarkably high diversity of animals for which the ecosystem provides a variety of physical habitats and food sources. for example, the rhizophora mangroves that predominate the fringe habitat have a well-developed prop-root system that is flooded semi-diurnally by tides and may provide habitat to fishes. also, some structurally heterogeneous habitats in tidal waters have been shown to provide fish with protection from predators and enhanced feeding opportunities (rozas and odum 1988) as can be learned that a large proportion of fish found in mangroves are considered juveniles and species of a small size at maturity (bell et al. 1984). thus, the mangroves-fisheries connection may, therefore, lie in the nursery function through provision of food and shelter from predation (hatcher et al. 1989); and protection from predation is offered by physical structure (e.g., mangrove roots, substratum, and turbidity), in combination with for example, shrimp behavior such as hiding and burying (dall et al. 1990). moreover, the estuaries of the indonesian coastal zone support a varied fish fauna, but it is rather poor in species. the fish fauna consists of species that spend all or a major part of their life in the estuaries, and marine or freshwater species which migrate seasonally into or through the estuaries (table 1). consequently, the fish or ichthyoids fauna occurring in and/or within mangroves and/or mangrove environment, have been placed in various ecological groups: as permanent residents, temporary residents or rare species. table 1. types of fish species recorded from apar mangrove bay areas, east kalimantan, and from banyuasin mangrove estuary areas, south sumatra . type estuarine species marine species riverine species total *) adults 73 69 25 145 juveniles**) 45 31 15 68 total 73 75 26 151 notes : * as some species can be classified under several headings, totals given arc not the sums of columns. ** as the specific identification of juvenile fish is frequently difficult the numbers of species given mere approximations. 17 biotropia no. 23, 2004 the mangrove forest and aquatic ecosystems are independent, with biological production processes different from those of purely terrestrial forest ecosystems. due to their characteristic location, the mangroves play an important role in the ecology of the coastal zone area and in support of the marine species that utilizes the mangrove environment during part or all of their life cycles. for instance, some prawn species may breed and complete their life cycle in shallow coastal mangrove waters. many species require the more saline deep offshore waters to spawn. the larvae then migrate back to the food rich and protective coastal mangrove waters to mature. adults of the commercially important penaeid prawns usually spawn at sea and after a short larval life (2 to 3 weeks), the post-larval stage settles in the near inshore areas and estuaries (dall et al. 1990). roles of the mangrove forests in interactions with neighboring coastal and marine ecosystems in indonesia are summarized in table 2. there are features in common, including physical, chemical characters and effects of seasonality, of the mangrove areas as habitats for fish and other aquatic animals. for certain species of fishes, areas of higher salinities may provide suitable habitat for developing juvenile fishes. also, the fish of muddy estuaries show affinities to deep-sea fauna in some of their features. some genera of clupeids and polynemids have prolonged fin-rays, and others have small-diminutive eyes. hardenberg (1950) has recorded even a species of blind sole from the rokan estuary. the organic nitrogen compounds of the falling leaves of mangroves are either incorporated into the sediment or converted into peat for detritus feeding shellfish, or broken up to become food for bacteria, fungi and finfish. the importance of bacteria both as a food source in marine and estuarine sediments and as mineralizing agents has been widely commented upon. thus exported detritus also provides an important habitat for juvenile prawns in near-shore sub-tidal mudflats (daniel and robertson 1990). johannes (1978) pointed out that many fish in tropical marine environments move inshore to spawn. in this context, he mentioned the gerridae, sparidae, engraulidae, pomadasydae and sciaenidae, all of which are demersal coastal forms and spawn in estuaries. for example, in the palau (8° n, 135° e) spawning migrations, only herklotsichthys sp. migrated from its usual seagrass and/or lagoon habitat to spawn in mangrove creeks (johannes 1978). one common mangrove-deniser, lutjanus argentimaculatus, migrated to the lagoon and reef slope to spawn. lethrinus sp and siganus lineatus were the only other fish which occasionally inhabit mangroves that migrated to spawn on reef slopes. estuaries play a role in energy transfer between a river and the sea, which is especially important for many commercial coastal fishes whose fish larvae and juveniles are dependent on the estuary as a nursery and feeding grounds. as discussed by hambrey (1996a, b), the nursery function of mangrove ecosystem is likely to be highly variable, with some areas being of far greater value, and others of far lower value than the global estimates would suggest. for example, mangrove swamps of reef flats are generally important as nurseries for mullet (mugilidae) and baitfish (clupeidae and others), and, together with inland mangroves, are a source of crab (for instance scylla serrata and sesarma spp.). the dominant mullet in all 18 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo mangrove creeks is liza subviridis. large mullet, valamugil sp and l. argentea appear to be more abundant in the open main channels of indonesian mangrove system. ambassis gymnocephalus is the dominant zoo-planktivore in mangrove creeks. thus, mangroves are often considered to be important to coastal fisheries both in terms of their role as breeding and nursery grounds for various fish and prawn species, and as a source of food. table 2. roles of the mangrove forest in indonesia: an experience with the mangrove forests in south china sea, east kalimantan, south sumatra and segara anakan cilacap. physical interactions nutrient interactions biotic interactions 1.1. filtering terrestrially derived sediment, and 1 .2. reduction in sediment load reaching the sea. 1. export of dissolved and paniculate organic matters (dom, pom). 1 . export of mero-planktonic larvae to marine food chains. 2.1. buffering salinity changes. 2.2. reduction in volume of freshwater reaching the sea. 2. sinks for paniculate organic matters (pom). 2.1. provision of feeding habitat (pfh) -marine invertebrates and fin fish (diurnal and seasonal migration). 2.2. pfh seabirds, shorebirds, marine mammals. 3.1. provision of juvenile (nursery) habitat (pjh) crustaceans (shrimp, lobster) and mollusks. 3.2. pjh fin fish (reef fish, sea-grass bed fish, pelagic). 3.3. pjh — seabirds and shorebirds nesting (also roosting). 3.4. pjh marine mammals (dolphins). 2. aquatic ecology the ecological importance of mangrove forests (e.g., thayer et al. 1987; robertson and duke 1987; 1990) in estuarine and coastal ecosystems throughout the world is well established. estuaries in indonesia contain diverse habitats, including mangrove forests, sea-grass beds, mudflats and open water channels, all of which may differ in depth, structural heterogeneity, substratum type and tidal exposure. it is widely accepted that mangrove communities are important components of estuarine ecosystems (odum 1961; odum and de la cruz 1967). elsewhere in indonesia, mangroves are associated with estuaries; and most estuaries in indonesia are mangrove-lined for much of their lengths. thus, mangrove forests contribute a great amount of structural heterogeneity to these estuaries. mangroves in the estuaries are able to absorb inorganic compounds from fresh water runoff for photosynthesis and thus play an important role as primary producers. mangroves are 19 biotropia no. 23, 2004 a source of primary productivity for food webs that serve as a base for the production of marine and estuarine organisms such as lobsters, finfish and shellfish. mangrove estuaries and creeks usually have significant salinity fluctuations. the great changes in salinity occurring during rainy seasons may cause periodic mass mortality of marine organisms. on the other hand, changes in salinity will also result in soil salinity of root zones in the mangrove forests. the mangrove forests and/or association in indonesia may be considered of great importance too, for example, the south china sea, including the estuaries, adjacent coastal waters and the offshore waters, as its high productivity probably makes it the major source of organic matter. these communities act as nurseries for many organisms and produce large quantities of organic matter (e.g., litter-fall) which forms the base of estuarine food chains. moreover, their detritus and nutrients are exported out of the ecosystem through tidal flushing and these form a food base for marine micro-organisms which in turn support the valuable estuarine and near-shore fisheries. the vast mangrove system in indonesia may be considered as very important in terms of high productivity and as a major source of nutrients to the ecosystem of the estuaries, the adjacent waters and the offshore waters. in apar bay east kalimantan, sukardjo (1995) found that the total flux of organic matters (woody detritus and other mangrove litter components) estimated to be about 21.11 to 29.34 dry weight t/ha/year. this is exported to adjacent waters in the form of fine suspended materials. in florida, ray (1974) found that 90% of water-borne debris within the mangrove zone is derived from mangrove vegetation. about half of this is exported to adjacent waters in the form of fine suspended material so that about 35-60% of all suspended matter on the off-shore olithic banks is of mangrove origin. the waters in the deltas, as well as those inshore, carry a large amount of suspended, as well as large plant materials, such as leaves, branches and trunks. teredinid mollusks play an important role in many mangrove swamps by rapidly breaking down wood and releasing nutrients into the food chain. since teredinids use wood as a substratum for burrowing and, in many species, as food (turner 1966, rayner 1977), the numbers of teredinids may be directly related to the amount of wood available. on the other hand, the major food of sesarmid crabs in mangrove forests is leaf litter (e.g., malley 1978). these crabs carry leaves down their burrows as well as consuming them on the sediment surface, and macnae (1968) suggested that feeding by sesarmid crabs may account for the scarcity of leaves on the floors of mangrove forests. it is interesting than that in inland mangroves most of the detritus must be broken down in situ. the high productivity of associated estuaries and banks is due to such suspended matter. in indonesia, my field observations have shown that fish in the mangrove environment utilize virtually all sources of food available, as follows : 1. insects and fruits from mangroves and from terrestrial sources (e.g., toxotes and some catfish). 20 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo 2. detrital mud (e.g., scatophagus, mullets, some catfish). 3. small invertebrates (e.g., gudgeons, gobies, ambassids, anchovies, etc.). 4. prawns and crabs (e.g., polydactylus, johnius, pristia, lates calcarifer, kurtus, scutengraulis, some catfish). 5. mollusc (e.g., cinetodus,acanthopagrus, tokifugu). 6. fish (e.g., carcharhinus, lates, johnius, polydactylus, some catfish). some fish are primarily estuarine, with the major portions of their population residing in the mangrove areas. others appear to enter the estuaries from either the sea or rivers mainly to forage, while others again pass through the area on breeding migrations or breed within the area or in adjacent coastal waters. those forms which breed within the mangrove belt or in the adjacent sea frequently utilize the area as nursery grounds for juveniles during various stages of development. marine and riverine forms penetrate the mangrove zones to varying degrees either as small peripheral populations or occasional individual stragglers. table 2 summarizes the data available in the reports. it gives some idea of the inter-linking of the mangrove community with the adjacent marine and riverine communities and the presence of juveniles indicates which species utilize the mangrove area for breeding or nursery grounds. thus, the productivity of the mangrove ecosystem is not measured simply by the productivity of plant material by mangroves, but must include the production of fisheries that are dependent on the mangrove detritus. 3. ecological productivity of mangroves as habitat and fisheries in indonesia, mangroves represent the dominant soft bottom plant communities of the marineterrestrial transition in the coastal zone; and considered to be areas of high primary productivity which support economically important detrital-based marine food webs. as a consequence of the range of spatial and temporal variation in physical and chemical factors found in such environments, the biological communities display remarkable adaptations which permit them to survive under such harsh environmental condition. for instance, the extreme ranges of ph, do, temperature and salinity are caused by freshwater influx or conversely, by the shallowness of mangrove waters and high incident radiations are common features. the plant species are members of terrestrial families which have adaptations to survive under conditions of high salinity, low oxygen and nutrient availability in the soil, wind and wave action, and substrate instability. the animals are, like the plants, representatives of largely terrestrial groups. in contrast the aquatic animal community is dominated by members of essentially marine families which are adapted to viable salinity, turbid conditions and to feeding directly or indirectly on materials from the dominant primary producers. in a global context, mangroves are one of the more productive ecosystems in the world in terms of both primary productivity and the productivity of marine animals. the primary productivity is manifested in terms of the accumulation of 21 biotropia no. 23, 2004 biomass (wood, root etc.) and the production of leaves. in indonesia, sukardjo < yamada (1992) reported that the biomass of r. mucronata amounted to be 93.73 dry weight/ha and mangroves produce a large quantity of organic debris in the fo of leaf litter, viz 7.56-10.82 dry ton/ha/year (sukardjo 1995). generally, the tc biomass, height, litterfall, decomposition, and fresh water turnover increase fn dwarf to riverine mangrove forests (pool et al. 1975, 1977; brown and lugo 198 the significance of this detritus in the food web has been recently focused upon some workers (leh and sasekumar 1980; thong and sasekumar 1980). mullet a some juvenile fishes, and penaeid shrimps utilize this material in mangrove estuar almost exclusively. leaves which are continually being shed by mangroves under decomposition and result in pom (particulate organic matters) and do (dissolved organic matters) that enter into the near-shore estuarine or mari environment. the pom, which consists of small particles, is consumed by larval a; juvenile marine organisms that utilize the mangrove habitat as a nursery and feedii ground. the dom, which consists of a wide range of soluble organic compounds, consumed by filter feeders (e.g., clams, oysters, mussels, etc.) that are common the near-shore environment, and who retain the phyto-nano-and micro-plankton th is vastly composed of primary producers who need dom to synthesize organ matter. the production of bacterial biomass from dom is an important step carbon and energy flow through marine environments and potentially serves as tl mechanism by which dilute dissolved organics become available to metazoc trophic levels (azam et al. 1983). in addition, any organic material that transported seaward is flocculated by the increasing salinity. the flocculated organ matter (fom) becomes a substrate for benthic feeders and scavengers. therefor mangroves are known to be very productive in terms of primary organic mated! and recycling of mineral nutrient (lugo and snedaker 1974). also, the richness c mangroves in organic materials provides a wide variety of food at different levels c the food web for organisms that will , in turn, serve as fish food (beumer 1978 mugil cephalus and cyprinodon variegatus are mentioned as such. also, mud crat scylla serrata occupies a niche between the primary producers and tertiar producers and is thus, an important organism in the energy-flow of the mangrov ecosystem. sukardjo (1995) reported that the canopy closure by mangrove trees range 87.70-99.00%. macnae and kalk (1962) considered that shading by mangrove tree protects animals, particularly juvenile and larval forms, in shallow water (e.g. mangrove creeks) from direct sun. in addition, the complex and entangled mangrovi roots and stems offer refuge and protection from predators. thus, the importance o mangroves as habitat for fish and prawns continues to be a topic of debate amonj marine ecologists (macnae 1974; robertson and duke 1987). the detritus is eithei refractory and sinks to the bottom, presumably never enters the food chain, or is dispersed over a large area without any local impact (e.g., magnification of benthic secondary production). however, it should note that marine ecologists generall} agree that inshore areas constitute a crucial part of the life support systems oi offshore populations by providing nursery and feeding areas (jansson et al. 1988). 22 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo therefore, biologists have recently paid much attention to the mangrove areas as nursery areas for shrimp and fish, such as certain species of penaeids that are dependent on mangrove forests during their juvenile stages. these species include penaeus monodon, p. indicus, p. merguiensis and most species of metapenaeus, p. semisulcatus and p. latisulcatus spend larval and juvenile stages. the life cycle of these prawns and environmental factors influencing it, are summarized in figure 1. it has been shown that the mangroves and the environment they create is vital to the continuation of many commercial penaeid prawn fisheries. also, many commercially important fishes such as chanos sp., mtigil sp., hilsa sp., and pomadasys sp. use mangrove water ways as nursery grounds. moreover, mangrove communities function as a solar-powered, tidal subsidized and pulse stabilized ecosystem (odum 1974). there is a large energy surplus in mangroves in the form of detritus. the detritus forms the basic component in the marine primary consumers diet, and thus, a basic support for many commercial fisheries. therefore, the role of mangroves as a habitat for fish and prawn is interesting, even today. p. merguiensis is an important component of the commercial catches of penaeid prawns throughout indonesia. the population dynamics of juvenile p. merguiensis in estuarine systems in indonesia have been studied intensively by toro and sukardjo (1990), and mangrove-lined mud banks have been shown to be the main nursery area for this species. however, until recently, little research had been carried out on the behavior of juvenile p. merguiensis or the degree to which they utilize the mangroves. from the inventory of various sources, hundreds species of fish and prawn belonging to residents and non-residents will be found in the mangrove environment in indonesia (table 2). but not all non-resident fish found in mangroves inhabit them as fry or juveniles. in indonesia, six habitat groups can be identified in the mangrove environment. not all of the species will be found in any one of the habitat group. different resident species occupy different habitat in the mangrove ecosystem (table 3). for instance, in indonesia generally, the mudflat fish community consisted of mainly ambassids, ariids, clupeids, cynoglossids, engraulids, mugilids and sciaenids. five residents species found only on the mudflats were the catshark hemiscyllium indicum, the grey mullet liza argentea, the silver pennah croaker pennalia argentata, the spotted croaker protonibea diacanthus and the anchovy stolephorus macroleptus. also, the mangrove inlets and creeks were dominated by schooling fish species belonging to the families ambassidae, eleotridae, engraulidae, clupeidae, leiognathidae and mugilidae. among those, the dominant species in terms of weight were arius sagor, ambassis gymnocephalus, liza subviridis, toxotesjaculator, sphyraena barracuda and lates carcarifer. by using gillnet, 70% of the total catches consisted of sardinella melanura, thryssa kammalesis, t. hamiltonii and stolephorus indicus. also, several demersal fish species reportedly inhabit the creeks. thus, only 29 species are recorded to be commonly found in all of the habitat groups (table 4). 23 figure 1. summary of the factors afecting penaeid prawn in the mangrove ecosystem       biotropia no. 23, 2004 table 4. (continued) species family mangrove creeks & inlets mudflat near inshore waters far inshore waters 14. saurida tumbil synodontiidae + + + + 15. sccutor insidiator leiognathidac + + + + 16. sctipinna taty clupcidae + + + + 17. sillago sihama sillaginidac + + + + 18. stigmatogobius sadanundio gobiidac + + + + 19. stolcphorus tri engraulidae + + + + 20. tcnulosa sinensis clupeidac + + + + 21. thcrapon jarbua theraponidae + + + + 22. thryssa hamiltonii engraulidae + + + + 23. t. kammalcnsis engraulidae + + + + 24. trichiurus savala trichiuridac + + + + 25. trypauchcn vagina gobiidae + + + + 26. mctapenacus affinis penaeidac + + + + 27. m. brevicornis pcnacidae + + + + 28. parapcnacopsis sculptilis penaeidac + + + + 29. pcnacus merguiensis penaeidac + + + + discussions and concluding remarks: an indonesian perspective a positive correlation between near-shore catches of shrimp or fish and mangrove area has been documented for indonesia (martosubroto and naamin 1977), malaysia (gedney et al. 1982), australia (staples et al. 1985), the philippines (camacho and bagarinoa 1987) and elsewhere (cf. table 5). also, there exists an obvious link between the fishery in the immature mangrove system and the one in the adjacent marine system. fish populations in both estuaries and mangrove ecosystems can be abundant with a wide diversity of species. it is well known to fishermen that many species of fishes occur in mangrove estuaries and creeks, and it has been observed many times that these systems act as nursery areas for larval and juvenile fishes. a comprehensive listing of fish species that actually spend their juvenile years in mangrove environments has yet to be made. nor is it known to what degree the relationship is obligate or facultative for particular species. much of the evidence is circumstantial. moreover, the structural heterogeneity of habitats in tidal waters has been shown to provide fish with protection from predators and 28 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo table 5. quantified relationships between mangroves and coastal resources. no. study site formula references remarks 1. indonesia y = 0.1 128 x + 5.473 (r2 = 0.79, n = na) martosubroto and naamin 1977 y = shrimp production (x 1000 tons) x = mangrove area (x 10,000 ha) 2, philippines y = 0,8648x + 0.0991 (1^ = 0.66^= 17) paw and chua 1989 y = log 10 of penacid shrimp catch (tons) x = log 1 0 of mangrove area 3. 38 regions of the world log 10msy = 0.4875 logam-0.0212l + 2.41 pauly and ingles 1986 msy = maximum sustainablc yield of pcnacids am = area of mangroves l = degrees of latitude 4. australia y= 1.074x + 218.3 (r2 = 0.58, n = 6) staples et al. 1985 y = banana prawn catch (tons) x = mangrove shoreline (km) 5. northeastgulf of mexico and in lousiana y= 1.96x-4.39 (r2 = 0.92, n = 7) turner 1 977 y = percentage of brown shrimps x = % of saline vegetation in an hydrological unit 6. 27 locations in asia, america and africa no equation given (r2 = 0.69, n = 5; 8) in: turner 1977 y = annual shrimp yield x = hectare of vegetated estuary 7. gulf of mexico ln y = 0.496lnx + 6.070 (r2 = 0.48,n= 10) yancz-arancibia et al. 1985 y = fish capture (tons) x = coastal marshes in km2 enhanced feeding opportunities (rozas and odum 1988). indeed, the supposed connection between mangroves and juvenile nekton is often advanced as one of the key arguments for the conservation of mangrove forests in indonesia. unfortunately, the use of fringing mangrove habitats by commercial and recreational fishery in indonesia has not been well documented. also, no reliable catch statistics are available for both commercial and recreational fish production from mangroves. moreover, in indonesia, very little and/or no listing is made of various teleost families, such as the apogonidae, bothidae, chandidae, chanidae, dorosomidae, elopidae, engraulidae, gerridae, latidae, leiognathidae, megalopidae, platy-cephalidae, pseudomugilidae, soleidae, syngnathidae and toxotidae, all of which 29 biotropia no. 23, 2004 contain species occurring in mangrove systems. while the ichthyo fauna occurring within mangrove environment may be broadly categorized into those fishes that exist there permanently, those that intermittently enter them as adults, and those that seasonally occur there as eggs, larvae, or juveniles. thus, one of the principal reasons for rehabilitating mangrove ecosystem in indonesia is to increase biodiversity and conserve the ecosystem. mangroves are often considered as an ecosystem per se, due to their strong specificities (twilley et al. 1996) but they belong to intermittently brackish tidal zone and can be seen as a part of estuarine systems. young fish utilize estuaries, near shores marine areas and mangrove ecosystem in order to benefit from the availability of food and perhaps also to gain protection from predators. in indonesia, estuaries can be considered ecosystems because they are composed of numerous subsystems or habitats. the term habitat refers to the place occupied by an entire community of organisms. thus estuaries are known to be important as feeding, nursery, or habitat areas, in the life cycles of many fish species, while the role of estuaries, particularly mangrove, in producing commercially important fishes is being recognized, their value for fishes of little or indirect commercial or angling importance has not been accepted. it seems that estuaries, all over the world are mainly nursery for many marine animals. as a matter of fact, the problem of exact relationship existing between ichthyology and mangroves is far from being solved. even in temperate estuaries there are groups of important fishes which spend a part of their life either in the estuary itself or in the ocean during their life cycles. in recent years, the importance of the mangroves is well established (e.g., critical feeding, rearing and nursery habitat for economically important marine species, providing a buffer against storms, filtering pollutants from upstream sources, and preventing coastal erosion). also, quantitative relationships between fish yield and area of mangrove have been well established. for instance, according to turner (1977) who has analyzed the data for 27 locations in america, asia and africa, the abundance of penaeid shrimp is directly related to the absolute area and type of estuarine vegetation. however, in indonesia, the degree of importance to commercial ecological functions of the mangroves are often under pressure from economic uses of this coastal area. for instance, conversion of mangrove forest which destroys the natural nursery grounds of fish and shellfish aggravated by the conversion of inland freshwater swamp forests which diminished the area of freshwater habitat and affects the supply of water and nutrient to the spawning and nursery areas. studies in mangrove communities (e.g., austin 1971; odum and heald 1972; lasserre and taffort 1977; janez-aranbicia et al. 1980; bell et al. 1984) have generally concluded that the fish fauna has a low species diversity and high proportion of temporary residents occurring mainly as juvenile. there is however, little evidence of fish spawning or breeding inside mangrove areas. recorded evidence refers only to gobiid fishes (penridge 1971) including mudskippers (boleophthalmus, periophthalmus, periophthalmodon, scartelaos). the abundance 30 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo and species composition of fish and crustaceans in mangroves and their adjacent near-shore habitats (sea-grass, sand-flats, mudflats etc.) at several sites in particular islands of indonesia are necessary to answer the questions regarding mangroves-fishery relationship. for instance, the correlation between offshore prawn catches and area extent of mangroves (macnae 1974; turner 1977; martosubroto and naamin 1977) has been suggested to be indication of the dependence of juvenile prawns on mangroves rather than of a food chain link (hatcher et al. 1989). though prawns offshore did not carry the mangrove carbon signal, it was shown that several species of juveniles prawns collected from a mangrove inlet consumed mangrove -derived carbon (rodelli et al. 1984). another example is, the environmental conditions in the estuaries are not conducive to the survival of the first-stage zoea of scylla serrata and berried females, therefore, migrate to sea to spawn. thus, program aimed to test the belief that mangroves as major nursery grounds for juvenile's fish and crustaceans are needed. in indonesia, since mangrove forests sustain marine life in estuaries and ponds and act as shelter belts against tropical cyclones, the fresh water needs of mangrove forests should be evaluated in water management practices along the coasts. moreover, the estuarine is one of the common features in the coastal mangrove belts, and represent an important zone for many marine organisms to feeding, breeding, spawning, and other behavior. in addition, it is essential to determine, for a given local fishery, the real dependency of fish resources on estuarine environment by answering the following questions in regard to the relation with fishery, as follows: 1. is the estuarine zone essential for a given species? 2. are there alternative areas for its development? 3. which parameters are critical in its life-history? 4. what are its trophic relationships with other species? in indonesia, major fishing grounds are located in the coastal areas in east sumatra, south and east kalimantan, and south irian jaya where there are extensive virgin mangrove forests (table 6) (chong et al. 1990). fortunately, ecological information for fisheries fauna associated with mangroves in indonesia is scant. fisheries resources in the mangrove environment have usually been examined in terms of their economic potential, and thus only the commercial species such as p. monodon has received only detailed bioecological study (e.g., toro and sukardjo 1990, 1995). the prawn species of creeks were represented by juvenile penaeus penicillatus, p. merguiensis, p. indicus, p. monodon, metapenaeus brevicornis and m. ensis (affinist). also, sediment type and organic carbon have been known to influence the distribution of prawns (e.g., william 1958; branford 1981; toro and sukardjo 1997), as do the effects of salinity (e.g., gunter et al. 1964; mair 1980 ; dall 1981) and the presence of coastal vegetation (e.g. young 1978; de freitas 1986). i he fluctuations and success of prawn fisheries appears more dependent on the juvenile stage, and the conditions in the mangroves which influence it (e.g., salinity, water discharge, sediment type etc.), than any other part of its life history. biotropia no. 23, 2004 thus, the small number of prawn species as observed in mangrove habitats in indonesia could be due to the following three factors: 1. niche occupation being limited to the epibenthic surface of the substrate. 2. the substrate being comparatively homogenous (in this case, a largely silt-clay one), and 3. a lack of euryhaline species. traditionally mangrove ecosystems have provided some of the richest fishing grounds for much of the islands in the country. also, fishery activity within the mangroves is mainly at the subsistence level (cf. table 6). for instance, crabs belonging to different genera and species are abundant along the estuarine shores and mangrove swamps, but those of economic importance affording minor fisheries are the swimming crabs belonging to the genera scylla and neptumis. scylla serrata, neptunus pelagicus and n. sanguinolentus are the most common forms. therefore, methods and sampling time, and type of nets will catch different species composition by weight and diversity. fishing gear utilized includes fixed traps (sero and kelong), hook and line, cast nets, gillnets, beach seine, trammel net, stationary lift net and stationary tidal seine (logo), e.g. in muna (table 7). the gill nets for instance, were not effective in catching the demersal fish and prawns. main demersal fish caught are ariidae, carangidae (excluding megalaspis spp. and decapterus spp.), leiognathidae, lutjanidae, polynemidae, sciaenidae, serranidae. main pelagic fish caught are rastrellinger spp., megalaspis cordyla, decapterus spp., and sardines (clupeidae). multiple tidal nets are usually set up in the main river streams for catching shrimp.trammel nets are used in river mouths for catching coastal shrimp such as p. merguiensis, p. monodon and m. brevicornis. traps (sero) are placed at river mouths or at the edge of the mangrove forests. crab nets are used at the river edge or in small canals and streams for catching mangrove crabs. for instance crab nets are very valuable and efficient economically for fishermen in muna (tables 7, 8). thus, the people who live within the mangrove ecosystems and exploit them have adapted their life-style and landuse patterns in a variety of ways along the indonesian coasts, e.g., in south sumatra for general illustration (table 9). the results of investigations of fisheries fauna associated with mangroves in indonesia enable some generalizations to be drawn regarding the structure and composition of mangrove associated fish communities. such communities appear to be characteristically composed of 13 to 14 families with only 10 families recorded being dominant (tables 2-4). some fish families, whose taxonomy is still under review, such as mugilidae (thomson, in lal et al. 1984), leiognathidae and lutjanidae (alien, in lal et al. 1984) and sphyrocnidae (rose, in lal et al. 1984). fao (1994) reported that 64 fish species, 74 crustacean species and 71 mollusk species were harvested from the mangrove area in malay peninsula. the main facts that emerged from the discussions on the fisheries associated with mangroves were as follows : 32 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo 1. the life cycles, reproduction cycle, food habits and growth rates of mangrove species are poorly known and have only recently been the object of attention in relation to the mangrove ecosystem. the species being investigated are those of economic importance, such as sesarmids crabs, penaeid prawn, fin fish and certain molluscs. 2. research on food webs indicates complex pathways, which qualitative measurements of conversion efficiency are available. 3. migrations, behavior, eco-physiology and habitat requirements of species are partly known but generally poorly understood. 4. studies on the relationships with adjacent ecosystems such as sea-grass beds and coral reefs are being studied, but only a few are described in any detail. 5. the question was addressed as to what extent forest structure has an effect on fauna populations and trophic relationships, and it was re-emphasized that more studies are urgently needed on this area. 6. the often quoted factors which influence fisheries potential and production in mangrove systems are only described, seldom experimented upon and not quantified. 7. the level of assimilation efficiency has been measured for a few species of animals but measurements are still crude. at this stage we are still looking at quantitative rather than qualitative changes. 33 biotropia no. 23, 2004 table 6. fishery regulations (applied to all of indonesia): gear and vessel restriction. zone (distance from shoreline) vessel size and power maximum gear not allowed in zone 1 : 0-3 miles 5 gross tons, 10 hp purse seines, nets > 120 m length 2: 3-7 miles 25 gross tons, 50 hp nets > 300 m length 3: 7-12 miles 1 00 gross tons, 200 hp nets > 600 m length 4:> 12 miles no limit no limit table 7. fishery production in the mangrove forest in muna project sites in 1996. fishing gear (100% used in the mangrove area) number of fishing gear (unit) number of fishermen (h.hold) production (t/year) economic value (rp/year) 1. togo (multiple tidal net) 60 40 87.70 500,976,000 2. jala (cast net) 8 8 0.52 2,254,000 3. rokkang (crab net) 1,300 260 135.20 540,800,000 table 8. fishery product from mangrove forest by species in muna fish collector in 1996. species production (ton/year) prize (rp/kg) economic value (rp/year) 1. shrimps 7.30 5,500 40,150,000 2. scylla scrrata 60.00 4,500 270,000,000 3. portunus pclagicus 57.36 1,000 57,360,000 4. sea cucumber (teripang) 8.50 25,000 212,500,000 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo table 9. coastal zone resource use compatibility matrix: compatibility of resource use options in south sumatra (numbers correspond to resource use). c = complementary, h = harmful, e = exclusive sector item 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 fisheries 1 . coastal fishery * 2. inland fishery * 3. aquaculturc h e * forestry 4. large scale forestry h c * 5. husbandry of nipa h e * 6. husbandry of nibung c h e c * 7. husbandry of jelutung c e c c * 8. crocodile hunting c c e e c c c * 9. monitor lizard trapping h * agriculture 10. transmigration scheme h e h h h * 1 1. irrigation scheme e # transport 12. port construction h h * tourism 13. small scale tourism c c c * conservation 14. habitat & endangered species c c e e c c h h h h c * 15. migratory shore birds c c e c c h h h c c * 1 6. breeding water birds c c e c c h h c c c * acknowledgments the author is very grateful to anonymous reviewers who read critically the manuscript. biotropia no. 23, 2004 references anonim. 2003. kebijakan dan stratcgi pengelolaan pesisir dan laut (ocean policy). makalah disajikan pada lokakarya 10 tahun pengelolaan lingkungan pesisir dan laut di indonesia: 1993-2003. jakarta 20-21 oktober2003. austin, m.h. 1971. a survey of the ichthyofauna of the mangroves of western puerto rico during december 1967august 1968. caribbean j. ofsci. 11:427-455. azam, f., t. fenchel, j.g. field, j.s. gray, l.a. meyer-reil and f. thingstad. 1983. the ecological role of microbes in the sea. mar. ecol. 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(cd.), estuarinc perspectives, 465-485. ny and london: acad. press. yanez-arancibia, a., g. soberon-chavcz and p. sanchcz -gill. 1985. ecology of control mechanisms of natural fish production in the coastal zone. in: yancz-arancibia, a (cd.), fish community ecology in estuaries and coastal lagoon towards an ecosystem integration, 571-595. unam press, mexico. young, p.c. 1986. morcton bay, queensland: a nursery area for juvenile penaeid prawns. aust. j. mar. freshwater res. 29:55-75. 39 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 1. jimmy (occurance).cdr biotropia vol. 18 no. 2, 2011: 61 73 occurrence of arboreal-climbing grapsids and other brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca received 06 august 2010/accepted 02 may 2011 despite the obvious importance to ecosystem functioning, the most prominent groups belonging to the grapsidae are generally regarded as less studied in the philippines. in this study, the occurrence of arboreal-climbing grapsids and other brachyurans associated with the mangals of quezon and catanduanes was considered including some aspects on climbing, burrowing and feeding behaviour of selected grapsids represented by and . the non-grapsoid taxa are represented by varunidae ( ), portunidae ( , , , ); and eriphiidae ( ). (white 1847) [ ] is an exclusive mangrove tree climber (emtc), while (a. milne edwards 1873) is described here as occasional mangrove tree climber (omtc). (de haan 1835) is a non-mangrove arborealclimbing species (ntc) only seen on crevices of the mangrove areas. creates burrows most often than . likewise, the study provides information on the presence of the portunid orange mud crab ( ); the green mud crab ( ); the varunid ( ); and extremely abundant xanthiid crab, in the mangroves of catanduanes but not in pagbilao, quezon. grapsid crabs, brachyurans, mangroves, philippines catanduanes state colleges, virac 4800, catanduanes, the philippines; pacific island institute for pedagogy, technology, arts & sciences, inc. (pacifictech) 82 a constantino street, virac 4800, catanduanes, philippines; de la salle university-d, cavite, philippines) hemigrapsus, pseudograpsus metopograpsus ptychognathus charybdis portunus scylla thalamita epixanthus metopograpsus latifrons grapsus pseudograpsus elongatus hemigrapsus (hemigrapsus) penicillatus [grapsus (eriocheir)] p. elongatus m. latifrons scylla olivacea s. paramamosain ptychognathus altimana epixanthus dentatus abstract introduction key words: the brachyurans are interesting to study in terms of their association with mangrove flora, behavior, feeding and ecology (khan . 2005). this group makes up as much as 80% of the macro−faunal biomass in mangroves and densities can even reach to 80 to 90 sq. m. reports attest that the mangrove forest constitutes the habitat et al * corresponding author : pacifictechjtm@yahoo.com 61 with the richest diversity of land dwelling crabs (hartnoll 1988, fratini . 2005). it is also indicated that the most important functional role of mangrove crabs which received greater attention is their ability to process as much as 70% of the leaf litter (leh & sasekumar 1985, slim . 1997, dahdouh-guebas . 1999, ashton 2002). it was reported by jones (1984) and lee (1998) that brachyurans are important in the mangrove ecosystem structure and function. members of family grapsidae are possibly one of the most important components of the fauna of mangrove forests globally, in part because of their influence in nutrient cycling by feeding on litterfall (salgado-kent & mcguinness 2010). unfortunately, it appears that there is still a dearth of detailed published information on the structural components as to the occurrence of these grapsoidal families like grapsidae and other brachyurans in the mangroves of the philippines. there are few reports in this country that deal with the ways in which these crabs use the mangrove resources compared to other southeast asian countries wherein numerous crab literature are available (sivasothi 2000, sivasothi . 1993, tan & ng 1994, ng & liu 1999, leh & sasekumar 1985, soemodihardjo & soerianegara 1989, rahayu & davie 2002, poovachiranon 1986, lee & leung 1993, lee 1998, kwok & tang 2005). except for some previous reports (mcnae 1968, banaag 1972, zamora 1989a, zamora 1989b, dolar 1991, dólar 1991), there are scanty reports on occurrence, ecology and physiology of the mangrove-dwelling arboreal-climbing grapsid crabs in the philippines. this paper presents the occurrence of arboreal-climbing grapsid crabs and other brachyurans associated with the mangrove areas in quezon and catanduanes island, luzon (philippines). some insights on the dependence to mangrove trees as habitats, climbing skills and burrowing behavior of these grapsids are also noted. crab specimens were handpicked and scooped using nets and locally made traps during daytime and at night time in two mangrove areas of southern luzon, philippines: (1) palsabangon mangrove area, pagbilao, quezon; and (2) and the palnab-pajo mangrove area in catanduanes island. the collection sites include areas along rivers, creeks, inlets and the buffer zones or marginal strips of the coastline from june 2005 to february 2006. in addition nearby rivers, backshores and inside mangrove forests were also surveyed in may 2007 for these arboreal-climbing grapsid crabs. one female and several male specimens were obtained in each study area. measurements of the crab specimens were represented by maximum carapace width ( ); carapace length ( ); body height ( ); and chelar palm height (cph). ratios of cal/mcw, boh/mcw and cph/mcw were computed. all measurements are made with vernier calipers and ratios are in two decimal places following ng and liu (1999) as used by masagca (2009). observations on the feeding ecology of the crabs under study were carried out each of the mangrove study areas modifying the methods of gillikin (2000). in the mangrove areas covered, presence or absence of crab species were determined by visual inspection in 10 m diameter plots along a transect et al et al et al et al et al. agojo inlet mangrove reserve project mcw cal boh materials and methods 62 biotropia vol. 18 no. 2, 2011 perpendicular to the coastline, covering the full width of the forest. the study investigated at least 10 plots along a 100 to 200 m long transect in the study areas. table 1 presents arboreal-climbing mangrove grapsid crabs described in the present study, while table 2 shows the other brachyurans (or non-grapsids) occurring in the areas under investigation. based on the field surveys conducted in banks of the streams or rivers, backshores and inside mangrove forests, the families of brachyurans included in this report are the (1) grapsidae, (2) portunidae, (3) varunidae and (4) eriphiidae. as shown in the said tables these brachyurans include 3 genera ( , and ) for family grapsidae; a single genus ( ) for varunidae; 4 genera ( , , and ) for portunidae and a single genus ( ) for eriphiidae. results and discussion occurrence and some taxonomic diagnosis descriptions on the morphometry metopograpsus pseudograpsus hemigrapsus ptychognathus scylla thalamita portunus charybdis epixanthus table 1. summary of the different taxa of grapsoid sesarmid crabs identified in selected mangrove areas in quezon and catanduanes. brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca 63 family genus species occurrence/location grapsidae hemigrapsus metopograpsus pseudograpsus hemigrapsus penicillatus metopograpsus latifrons pseudograpsus elongates quezon catanduanes quezon table 2. summary of taxa of non-grapsoid sesarmid crabs obtained from different locations. family genus species occurrence/location varunidae ptychognathus ptychognathus altimana catanduanes, quezon portunidae charybdis portunus scylla thalamita charybdis affinis portunus pelagicus scylla serrata scylla olivacea thalamita crenata quezon, catanduanes quezon quezon, catanduanes quezon quezon, catanduanes eriphiidae epixanthus epixanthus dentatus catanduanes based on field surveys, the different arboreal-climbing grapsid crabs associated with the mangrove areas under study include the three genera: (1) 1851 (2) h. milne edwards, 1853 ) and (3) h. milne edwards, 1837 these grapsid crabs were known to occur both in the lowland portions of streams, estuaries, and backshores of the. the grapsid (white 1847) [ ] in quezon and catanduanes island was observed to be associated with the sesarmid crabs, (de haan 1835) and h. milne edwards, 1853 [ ]. this means that these mangrove crabs occupy the same spots in the mangrove habitats that include feeding as shown in their climbing and burrowing behavior. hemigrapsus dana, (hemigrapsus penicillatus), metopograpsus (metopograpsus latifrons pseudograpsus (pseudograpsus elongatus). m. latifrons grapsus perisesarma bidens neosarmatium smithii sesarma biotropia vol. 18 no. 2, 2011 64 the same observation that greater number of grapsid crabs occur in the banks of the stream and at the backshore of the mangrove rather than inside or within the forests of the 2 mangrove areas confirming the previous made by tam and wong (2000), showing a significant difference in occurrence or diversity of grapsoid sesarmids and other brachyurans. the succeeding paragraphs present some taxonomic descriptions and morphometry of the arboreal climbing grapsids and other brachyurans. = h. milne edwards, 1853 (type species forskal, 1775, subsequent designation by davie, 2002; gender masculine) white, 1847 [nomen nudum] h. milne edwards, 1853 de haan in herklots, 1861 (nomen nudum) de man, 1879 a. milne-edwards, 1867 metopograpsus metopograpsus latifrons grapsus h. milne-edwards, 1853 (grapsidae) (white, 1847) [ ] metopograpsus cancer messor = grapsus latifrons = metopograpsus maculatus = grapsus (grapsus) dilatatus = grapsus (grapsus) dilatatus = metopograpsus pictus figure 1. from a maqueda channel mangrove area (palnab-pajo mangrove) in catanduanes island metopograpsus latifrons this arboreal-climbing grapsid has squarish carapace, slightly converging backwards; with 3 maxilliped not meeting in the middle line; and one tooth behind the antero-lateral one. carapace of appears to be converging backwards. this grapsid attacks the collector during several field works in quezon and catanduanes. this crab is conspicuously found in sluice gates of fish ponds in the mangrove area, prop roots of and tree trunks. as an arboreal climbing grapsid crab, this opportunistic animal was observed to assume an inverted or downward position (facing the water) when found on trunks of mangrove trees. some samples were also collected in crevices of trees during low tides. this grapsoid crab feeds on leaves, algae mollusks (vannini . 1997) and crustaceans (jones 1984). it is also stressed that in another species of the genus is less dependent on the leaves of mangrove plants (dahdouh-guebas 1999). in singapore, 3 species ( and have been the subject of several reports. h. milne edwards, 1837 (type species latreille, 1817, subsequent designation by holthuis, 1977; gender masculine) rd m. latifrons rhizophora et al metopograpsus, m. oceanicus et al. m. gracilipes, m. frontalis m. latifrons) = pseudograpsus grapsus penicilliger genus pseudograpsus h. milne edwards, 1837 65 = pachystomum nauck, pachystomum philippinense = pseudograpsus erythraeus pseudograpsus elongates mcw boh 1880 (type species nauck, 1880, by monotypy; gender neuter) kossmann, 1877 table 3 shows the mean values of morphometric data of grapsid crab from the mangroves under study. males tend to be larger in terms of body size as to maximum carapace width ( ) and body height ( ). pseudograpsus elongatus heterograpsus(a. milne-edwards, 1873) [ ] table 3. mean values ( ) of the morphometry of from catanduanesin mm p. elongatus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 32.95 37.23 1.13 18.62 0.56 14.82 0.45 female 31.19 37.82 1.21 10.67 0.34 11.62 0.37 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). table 4. mean values ( ) of some of the morphometrics of female crab samples of , showed a mean mcw = 31.19 mm, while males = 32.95 mm. in terms boh, females mean showed mean boh of 10.67 mm and males gave a mean boh of 18.62 mm. body form for females (boh/mcw = 10.67mm/31.19 mm = 0.34± 0.01, n=2), while for males (boh/mcw = 18.62 mm/32.95 mm = 0.56± 0.01, n=8) the body form is relatively vaulted. chelipeds equal and sexually dimorphic. male chelae larger (cph/mcw = 14.82mm/ 32.95mm = 0.45) and more strong than females (cph/mcw = 11.65mm/31.19mm = 0.37). identifying characters of are two distinct teeth behind the anterolateral one, carapace converging backwards. carapace squarish, slightly converging backwards; 3 maxilliped not meeting in the middle line; legs and carapace not hairy, carapace slightly convex; two distinct teeth behind the antero-lateral one. = dana, 1851 (type species dana, 1851, subsequent designation by rathbun, 1918; gender masculine) = a. milne-edwards, 1869 (type species h. milne edwards, 1837, subsequent designation by rathbun, 1918; gender masculine) = yokoya, 1928 p. elongatus p. elongatus hemigrapsus hemigrapsus crassimanus lobograpsus cyclograpsus crenulatus brachynotus brevidigitatus rd genus hemigrapsus hemigrapsus penicillatus grapsus eriocheir dana, 1851 (de haan, 1835) [ ( )] in mm h. penicillatus sex of crabs mcw boh boh/mcw cph cph/mcw male 28.72 17.02 0.59 9.39 0.33 female 27.29 12.07 0.34 9.02 0.33 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). table 4 shows the identity of the grapsid, de haan, 1858 from catanduanes which was confirmed by ms. marivene manuel from the pnm in manila. h. penicillatus brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca biotropia vol. 18 no. 2, 2011 66 varunidae h. milne edwards, 1853 genus stimpson, 1858 rathbun, 1914 [ ] ptychognathus ptychognathus altimanus varuna = stimpson, 1858 (type species stimpson, 1858, by monotypy; gender masculine) [opinion 85, direction 37] = nauck, 1880, (type species coelochirus crinipes nauck, 1880, by monotypy; gender masculine) table 5 shows the summary of the mean values of selected morphometrics of males are bigger than females. carapace pitted but glabrous, a little broader than long ( =39.54/36.08); lateral margin with two sharp teeth; legs fringed on the last 3 joints. male chelipeds are larger (cph/mcw=0.38) than the females (cph/mcw=0.22). ptychognathus ptychognathus glaber coelochirus p. altimanus. mcw/cal table 5. mean values ( ) of the some morphometrics ofin mm p. altimanus sex of crabs mcw cal cal/mc w boh boh/mcw cph cph/mcw male 39.54 36.06 0.91 15.56 0.56 15.02 0.38 female 33.09 32.56 0.98 15.36 0.34 7.37 0.22 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). samples were collected near the canals connected to a small stream inundated during the high tides. this varunid crab is abundant in the backshore portions of the mangroves. found in the back mangroves of quezon and catanduanes (near the rice paddies) and at the edges near the areas where freshwater streams are flowing. ng . (2008) notes that is being revised by n.k. ng p.k.l. ng. several groups of species are now recognizable and new genera will be established for them. = forskal, 1775 = herbst, 1794 = marion de proce, 1822 = var. shen, 1932 as shown in table 6, males of are bigger than females. buccal frame rectangular; carapace much wider than long (mcw/cal 116.57/48.44= 2.41), bow fronted, and much serrate, drawn out into lateral spikes. chelae strong but slender. buccal frame rectangular, last pair of walking legs paddle-like; 9 antero-lateral spines. samples of this portunid crab were obtained at the outer margins of the mangrove forest areas in quezon and catanduanes. the use of baited lift nets (local name= “bintol”) allowed for the collection of this crabs. et al ptychognathus cancer pelagicus cancer cedonulli portunus denticulatus portunus pelagicus sinensis p. pelagicus portunidae rafinesque, 1815 subfamily portuninae rafinesque, 1815 genus weber, 1795 linnaeus, 1758) [cancer] portunus portunus (pelagicus) pelagicus 67 table 6. mean values ( ) of the some morphometrics ofin mm p. pelagicus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 116.57 48.44 0.42 25.73 0.22 17.78 0.152 female 112.65 48.65 0.43 20.38 0.18 17.35 0.154 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). genus forskal, 1775) [ ] scylla de haan, 1833 scylla serrata ( cancer = scylla cancer serratus achelous crassimanus scylla tranquebarica oceánica lupa lobifrons de haan, 1833 (type species forskal, 1775, subsequent designation by rathbun, 1922; gender feminine) = macleay, 1838 = var. dana, 1852 = h. milne edwards, 1834 a b figure 2. (a, carapace) pagbilao quezon and maqueda channel catanduanes.scylla serrata as presented in table 7, male samples of obtained from the study areas are smaller than the female samples. samples obtained were heavy with moderately convex carapace ( 63.54/40.46 = 1.57), with 4 teeth; antero-lateral margin with 7 teeth, periopods/pleopods smooth, no hairs. table 7. mean values ( ) of the some morphometrics of s. serrata mcw/cal in mm s. serrata sex of crabs mcw boh boh/mcw cph cph/mcw male 63.54 21.56 0.34 8.07 0.13 female 67.85 23.45 0.35 9.23 0.14 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). scylla olivacea cancer(herbst, 1796) [ ] summary data on selected morphometrics of are presented in table 8. ratios obtained for boh/mcw and cph/mcw show almost the same values, which may indicate that sexual dimorphism is not that intense. s. olivacea brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca biotropia vol. 18 no. 2, 2011 68 table 8 mean values of the some morphometrics of. (in mm) s. olivacea sex of crabs mcw boh boh/mcw cph cph/mcw male 62.02 20.34 0.33 7.09 0.11 female 63.52 21.57 0.34 7.26 0.12 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). genus charybdis harybdis (charybdis) affinis de haan, 1833 c dana, 1852 =? gordon, 1931charybdis barneyi figure 3. general view of from quezon. table 9. mean values ( ) of the some morphometrics of charybdis affinis in mm c. affinis carapace of more or less hexagonal (mcw/cal 40.09/29.63= 1.353), antero-lateral margins diverging backwards, fronto-lateral much less than maximum width, bow-shaped front cut into 6 teeth. table 9 shows that the ratio of cph/mcw for both male and female samples are almost the same. ward (1941, cited by ng 2008) described from davao as: carapace is broader than long, bare and glossy, granulated under lens. c. affinis et al. c. philippinensis sex of crabs mcw boh boh/mcw cph cph/mcw male 43.34 16.8 0.39 17.78 0.41 female 40.09 15.64 0.39 16.52 0.41 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). genus latreille, 1829 ruppell, 1830 [ , sic] thalamita thalamita crenata talamita = thalamita cancer adnete = thalamonyx goniosoma danae t. crenata mcw cal latreille, 1829 (type species herbst, 1803, by monotypy; gender feminine) a. milne edwards, 1873 (type species a. milne edwards, 1869, subsequent designation by rathbun, 1922; gender masculine) as shown in table 10, carapace of (figure 4) is much more or less hexagonal ( = 50.63 mm, = 32.02 mm), but antero-lateral margins sub-parallel; fronto-orbital not much less than maximum carapace width; chelipeds strong ( =11.35 mm, male; 12.06 mm, female); transverse ridges usually distinct. carapace is rounded with five antero-lateral teeth. cph figure 4. from pagbilao, quezon. table 10 mean values of the some morphometrics of thalamita crenata . (in mm) t. crenata sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 50.63 47.01 0.93 17.93 0.35 11.34 0.22 female 31.04 32.02 1.03 17.43 0.56 12.06 0.39 legend: mcw=maximum carapace width cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm) superfamily eriphioidea macleay, 1838 family eriphiidae macleay, 1838 genus heller, 1861 (white, 1848) [ ] epixanthus epixanthus dentatus panopeus = heller, 1861 (type species heller, 1861, by monotypy; gender masculine) = de man, 1879 = haswell, 1881 the mangrove crab, (figure 5) displays two visible spines on the upper internal face of claw carpus; 5 big teeth on the antero-lateral carapace margins, carapace widely mottled. celipeds are unequal (right larger than left). table 11 shows that chelipeds of male samples (0.40) are larger compared to the female samples (0.26) epixanthus epixanthus kotschii epixanthus dilatatus panopeus acutidens e. dentatus a b c figure 5. (carapace) from palnab-pajo mangrove area in virac, catanduanes (a), frontal view (b) and forcept-like claws (c). epixanthus dentatus 69 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca table 11. mean values ( ) of the some morphometrics ofin mm e. dentatus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 45.02 32.41 0.72 19.32 0.43 18.21 0.40 female 50.01 29.45 0.59 17.05 0.34 13.01 0.26 legend: mcw=maximum carapace width; cal= carapace length; boh= body height; cph= chelar palm height (all values are in mm). samples of were obtained under drift woods, buried on the mud. the right claw of this crab is stout and consists of a special tooth which it uses to open gastropods. plate 17b (fig. 5) shows the forcept-like claw of the crab. this crab is omnivorous (dahdouh-guebas . 1999), but preys mostly on crabs (cannicci . 2008). on the arboreal-climbing behavior, several individuals of the mangrove crabs, (white, 1847) were observed as exclusive tree-climber (emtc) in mangrove canopies. this grapsid, invariably stays longer on the branches of mangrove trees with mostly upside down position. during the study, climbing height range of 50 grapsid crabs (in each study area) observed from 0.065 m to 2.35 m above the water lining. majority of these grapsid crabs climb at the main trunks of and sometimes on the branch of when the tide is rising and when insects (e.g. spiders) are also found, since they are omnivorous feeders (jones 1984). although some are seen on the lateral branches, these are only happening when these grapsids were antagonized. it was observed that when are being caught by hand picking at the bottom of the trunk of the mangrove tree submerged in water, some of these crabs rushed to the upper portion of the trunk evading from the capturist. in catanduanes, a greater number of occur in the mangrove areas studied supporting the high biomass and density report in segera anakan, indonesia (geist 2011). this climbing behavior was not observed in the grapsid crabs of quezon. another observation refers to the tendency of the grapsid, to climb in the fronds of when chased or antagonized while they are in the water. individuals of this grapsid, (and also the sesarmid crab, ) tend to escape or evade the researchers by climbing fast to the trees. the other grapsid, is known to be an occasional mangrove tree-climber (omtc), while is non-arboreal species (nas) that was seen only in crevices of the mangrove areas. on burrowing behavior, the grapsid also creates burrows and so with , but the former is more active compared to the latter. burrowing activities have a pronounced effect on sediment properties, contributing immensely in rendering changes in the properties of mangrove sediments. as noted by nagelkerken (2008) changes in biochemical processes can be observed by enhancing the porosity and water flow through the sediment, assisting in flushing toxic substances. crab burrows provide an efficient mechanism for exchanging water between the anoxic substrate and the overlying tidal water. jones (1984) described the feeding habits of mangrove crabs and divided into seven groups: herbivore, carnivore, omnivore, deposit feeder, omnivore/deposit e. dentatus et al et al m. latifrons rhizophora sonneratia m. latifrons m. latifrons m. latifrons et al. m. latifrons nypa fruticans m. latifrons selatium elongatum p. elongatus, h. penicillatus p. elongatus m. latifrons et al. arboreal-climbing, burrowing and feeding behavior of the grapsid crabs 70 biotropia vol. 18 no. 2, 2011 feeder, specialized filterer, and filterer/omnivore. the grapsids are herbivores and omnivore/deposit feeders, eating mangrove litter and water plants. nordhaus (2011) described extensively the food preferences, diet and food consumption of grapsoid crabs in indonesia. this will become an important reference for studying further the food and feeding habits of grapsids in the philippines. a total of 3 genera belonging to the family grapsidae ( and ) are reported here possessing tree-climbing abilities. (white 1847) [ ] is an exclusive mangrove tree climber (emtc), while (a. milne edwards 1873) is described here as occasional mangrove tree climber (omtc). (de haan, 1835) on-mangrove arboreal-climbing species (ntc) only seen on crevices of the mangrove areas. the non-grapsoidal brachyurans are represented by 3 families [(varunidae ( ), portunidae ( , , , ); and eriphiidae ( )]. to what extent this tree-climbing abilities of the said grapsid crabs in quezon and catanduanes relate to the feeding behaviour of the grapsid crabs reported in the present study awaits further studies. likewise, food preference, diet and consumption of these grapsid crabs from the philippines indicate future needs. the author acknowledges with thanks to the de la salle university-dasmariñas, university faculty research office (ufro) for funding a research on the mangrove crabs. profound thanks to asst. prof. rico masagca of dlsu-manila and president & ceo of pacifictech, mrs. rose m. gianan, tersy m. flores, mrs. margie m. sabino, mrs. elsie m. almonte, yule, mark, kate and marby jean for their unending support in this work on mangrove brachyurans. the head of the catanduanes state colleges, dr. minerva morales (suc president iii) is also greatly appreciated. m. latifrons, p. elongates and n. penicillatus et al. hemigrapsus, pseudograpsus metopograpsus metopograpsus latifrons grapsus pseudograpsus elongatus hemigrapsus (hemigrapsus) penicillatus [grapsus (eriocheir)] is a n ptychognathus charybdis portunus scylla thalamita epixanthus conclusions acknowledgements references a . b . c shton ec. 2002. mangrove sesarmid crabfeeding experiments in peninsular malaysia. journal of experimental marine biology and ecology, 273: 97119 anaag jf. 1972. vegetational composition and association in a mangrove forest ecosystem in puerto galera, oriental mindoro. natural and applied science bulletin, 29(1-2): 1-40 annicci s, burrows d, fratini s, smith tj, offenberg j, dahdouh-guebas f. 2008. faunal impact on vegetation structure and ecosystem function in mangrove forests: a review. aquatic botany, 89 (2): 186-200. 71 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca chen gc, ye y. 2010. changes in properties of mangrove sediment due to foraging on leaves by crabs (grapsidae: sesarminae). dahdouh-guebas f, giuggioli m, oluoch a, vannini a, cannicci s. 1999. feeding habits of non-ocypodid crabs from two mangrove forests in kenya. 64: 291297. dolar mll. 1991. a survey on the fish and crustacean fauna of the seagrass beds in north bais bay, negros oriental, philippines, p. 367-377. : proceedings of the regional symposium on living resources in coastal areas. quezon city: university of the philippines marine science institute. dolar mld, alcala ac, nuique j. 1991. a survey on the fish and crustaceans of the mangroves of the north bais bay, philippines, p. 513-519. : alcala, a.c. 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(eds.), proceedings of a symposium on mangrove management: its ecological and economic considerations held in bogor, indonesia, august 9-11, 1988. biotrop special publication no. 37. tam fy, wong ys. 2000. hong kong mangroves. city university of hong kong press. tan cgs, ng pkl. 1994. an annotated checklist of mangrove brachyuran crabs from malaysia and singapore. 285: 75-84. vannini m, oluoch a, ruwa rk. 1997. the tree-climbing crabs of kenyan mangroves. : (b. kjerfve, b.l. de lacerda and e.s. diop, eds.), p. 325338. unesco technical papers in marine sciences. new york: unesco. zamora pm. 1989a. management policies and political awareness, p. 189-191. : cd field and m. vanucci (eds.) proceedings of the symposium on new perspectives in research and management of mangrove ecosystems, 11-14, nov. 1986, sri lanka. chiromanthes bidens c. maipoensis in proceedings of the second international marine biological workshop: the marine flora and fauna of hong kong and southern china, hong kong perisesarma crustaceana, in mangrove fisheries and connection terebralia palustris journal of experimental marine biology and ecology, in et al hydrobiologia, in mangrove ecosystems studies in latin america and africa in zamora pm. 1989b. mangroves of the philippines. biotrop special publication (bogor, indonesia), 37: 43-65. 73 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca 1. rudolf v kuhn.cdr biotropia vol. 20 no. 1, 2013: 1 9 diversity of plasmodial myxomycetes from anda island, pangasinan, philippines rudolf v. kuhn , anton oliver m. javier , coleen p. rodillas , christian m. parra , liwayway hiyas m. corpuz , anthony t. buaya , and thomas edison e. dela cruz * received 20 december 2011/accepted 10 january 2013 the unique life cycle and fascinating fruiting bodies of myxomycetes make them ideal model organisms for the study of cellular differentiation and ecological patterns. our research study then focuses on the diversity and abundance of myxomycetes found in anda island, pangasinan in northern philippines. a total of 180 moist chambers were prepared from ground leaf litter and twigs collected from a 15 m quadrat within the study site. twenty four species of myxomycetes belonging to 11 genera were collected and identified from the moist chambers: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) of all moist chambers, 55% yielded myxomycetes. ground leaf litter (29%) yielded more myxomycetes than twigs (26%). assessment of species diversity (h =1.15), richness (h =5.33) and evenness (e=0.56) showed rich assemblage of myxomycetes. among the collected species, one for each of the genera , , and were recorded to be abundant. interestingly, three species of myxomycetes are new records for the philippines: and . this is the first report of myxomycetes in anda island, pangasinan, philippines. myxomycetes, species abundance, diversity, biogeography, philippines 1 1 1 1 1 2 1,2 2 department of biological sciences, college of science and fungal biodiversity and systematics group, research center for the natural and applied sciences university of santo tomas españa 1015 manila, philippines arcyria collaria , comatricha , craterium diachea diderma , didymium , elaeomyxa perichaena , physarum stemonitis . arcyria craterium diderma, physarum, craterium microcarpum, physarum decipiens elaeomyxa miyazakiensis 1 2 abstract introduction s g key words: the philippines is a tropical archipelago of 7107 islands. the geographic isolation of many of these islands resulted in various unique flora and fauna. it has been estimated that more than 6 000 species of animals and plants are endemic to the country (myers . 2000). therefore, it is not surprising that new species of plants and animals are still being discovered recently in the country, e.g. forest mice ( et al apomys * corresponding author : tedelacruz@mnl.ust.edu.ph 1 aurorae a. banahao a. brownorum a. magnus a minganensis a. sierrae a. zambalensis) et al. (varanus bitatawa) et al. (nepenthes attenboroughii) et al. (drosera ultramafica) et al. craterium retisporum et al. et al et al. hemitrichia serpula, perichaena depressa, physarella oblonga, stemonitis fusca arcyria cribraria, lycogala stemonitis et al. et al. arcyria globosa comatricha robusta, craterium atrolucens, lamproderma cacographicum, oligonema schweinitzii perichaena microspora , , , , , , and (heaney 2011), monitor lizard (welton 2010), giant pitcher plant (robinson 2009), sundew plant (fleischmann 2011) including those belonging to the myxomycetes, e.g. (moreno 2009). however, studies on island myxomycetes are relatively few in spite of their role in wood decay and nutrient cycling in temperate forests and in bio-accumulation of heavy metals (zhulidov . 2002; takahashi 2004). rojas and stephenson (2008) surveyed different habitat elevations and substrates types in cocos island, costa rica and reported 41 species belonging to 19 genera. beltran (2010) also evaluated the species diversity of myxomycetes between arid and semi-arid zones in the canary islands in spain and reported 63 species belonging to 21 genera. the study also highlighted the similarities between slime molds collected from the arid and semi-arid zones in the canary islands to the desserts of central america. in the philippines, and species of , and were reported from the islands of palawan and palaui, cagayan (reynolds 1981; quimio 2002). dela cruz (2011) also reported 30 species of myxomycetes from hundred islands national park, pangasinan. macabago (2012) identified 45 species and 13 genera from lubang island, occidental mindoro. interestingly, this geographically isolated island yielded six new records for the philippines: , and . in this research study, we reported 24 species including three new records of myxomycetes from specimens collected in anda island, pangasinan, philippines. this is the first report of myxomycetes in anda island, pangasinan in northern philippines. anda island (16°17'00.16”n, 119°58'00.08”e; land area= 83.80 sq.km) is located near the town of bolinao in the province of pangasinan, northern philippines. climate data indicate an average monthly rainfall range of 6.1 mm in february to 608.6 mm in august. the island has an average minimum and maximum temperature of 23.3 °c and 32.4 °c, respectively, and average monthly humidity between 71 85%. it has a type i climate season, i.e. wet from june to november and dry from december to may. the study site is characterized by a tropical secondary forest, with patches of rice fields and established residential houses. a small patch of this secondary forest along the local highway and near a rice field was used as the collection site. the island is also inhabited by man and may be useful in looking at the effects of human civilization on myxomycete assemblages. materials and methods study site 2 biotropia vol. 20 no. 1, 2013 collection of substrates and preparation of moist chamber set-ups and herbaria characterization and identification of plasmodial myxomycetes moist chamber productivity and ecological analysis ground leaf litter (30) and twigs (30) were collected in brown paper bags within a 15 m quadrat. prior to the preparation of moist chambers, the collected substrates were allowed to air-dry for few days. a total of 180 moist chambers were then set-up following the protocol of stephenson and stempen (1994). the moist chambers were placed inside wooden cabinets, not directly exposed to sunlight, and incubated at room temperature for up to 8 weeks. all moist chambers were observed regularly (at least twice a week) for the presence of plasmodia and/or fruiting bodies. the number of moist chambers with plasmodia and/or fruiting bodies was recorded and used in ecological analysis. fruiting bodies with their respective substrate were removed from the moist chamber set-up, allowed to air dry and glued on previously prepared herbarium boxes. herbarium boxes were then labeled with the collection number, substrate, collection sites and dates, the species name and the names of the collector. all herbaria were deposited at the pure and applied microbiology laboratory, research center for the natural and applied sciences, university of santo tomas. to identify the collected myxomycetes, the specimens for each species were described based on their fruiting body descriptions and spore morphology under a dissecting and a compound light microscope. identification was done following comparison of these morphological characters with published literature (stephenson & stempen 1994; keller & braun 1999), web-based identification keys (http://slimemold.uark.edu/) and an electronic, computer-based identification key, (mitchell 2008). to check for the current valid names of the identified myxomycetes, an online nomenclatural information system (http://nomen. eumycetozoa.com) was used. the productivity of the moist chambers (mc) was computed by determining the percent yield for the collection site and each of the substrate types. plasmodial/or fruiting body growth on a moist chamber was counted here as positive for myxomycetes and was recorded as one positive collection. then, the total positive collection was divided by the total number of moist chamber prepared. thus, the percentage yield (py) was computed as follows: percent yield (py) = (1) total number of mc prepared then, the relative abundance (ra) was also determined for each of the collected species of myxomycetes. initially, the number of moist chambers positive for a species of myxomycetes was counted and divided by the total number of collections. 2 synkey number of mc positive for myxomycetes 3 diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. the relative abundance was computed as follows: relative abundance (ra) = (2) total number of myxomycete collection abundance indices (ai) were then assigned to all of the species represented among the collections. initially, the percentage of a particular species among the total number of collections, i.e. the relative abundance, was determined. then, a “breaking point” was assigned based on these percentage values (s. l. stephenson, pers. comm., 04 january 2009). each species was then categorized as: (1) abundant (a) if their relative abundance (ra) is ≥10% of the total collections, (2) common (c) if ra is ≥5% but <10% of the total collections, (3) occasionally occurring (o) if ra is ≥3 but <5 of the total collections, and (4) rare (r) if the myxomycetes had an ra of <3% of the total collections. finally, the myxomycete diversity of the study site was also calculated using different diversity indices as described by dagamac . (2012). these indices were computed based on ra and as follows: shannon index diversity (3) where p = is the proportional abundance of the th species gleason index of species richness (4) where n = the total number of species n = the total number of individuals in th species pielou's index of species evenness / (5) where h = shannon index of diversity h = the maximum value of hs of 180 moist chambers set-up from ground leaf litter and decaying woody twigs collected from anda island, 55% yielded myxomycetes (fig. 1a). fruiting bodies were recorded more in moist chambers than plasmodia. between the two collected substrates, ground leaf litter (29%) yielded more myxomycetes than twigs (26%) (fig. 1b). schnittler (2002) also found a higher occurrence of myxomycetes on the ground leaf litter, though macabago (2010) recorded a higher yield in aerial than ground leaf litter. it was previously reported that myxomycetes associated with woody twigs are not necessarily same as those associated with ground litter in terms of their occurrence and abundance (stephenson 2008). stephenson (1989) implicated that the myxomycetes assemblage in ground leaf litter was mediated by factors that is favorable for plasmodial or amoeba development. these factors included acidic ph of the decaying leaf litter, soil nutrients, moist and the available food microorganisms, bacteria, yeast and algae. as shown in this study, the high myxomycetes yield recorded in ground leaf litter as compared to twigs indicated that ground leaf litter is a favorable substrate for myxomycetes. number of collections for a particular species of myxomycetes et al et al. et al. et al. e.g. (h ) = -∑ (p ln p ) (h ) = n -1/ln n e = h h percent yield s i i i g p i s max i p i s max i i results and discussion 4 biotropia vol. 20 no. 1, 2013 5 b a figure 1. percent yield of myxomycetes in moist chambers (a) and between the two collected substrates (b) d b ca e f figure 2. representative species of myxomycetes recorded in anda island, pangasinan: (a) (b) , (c) and (f) craterium retisporum, diachea leucopodia perichaena pedata, (d) elaeomyxa miyazakiensis, (e) perichaena depressa physarum bivalve diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. 6 species occurrence, abundance and diversity arcyria collaria , comatricha , craterium diachea diderma , didymium , elaeomyxa perichaena , physarum stemonitis . physarum phy. bivalve, phy. bogoriense, phy. cinereum, phy. compressum, phy. decipiens phy. echinosporum arcyria cinerea, collaria arcyrionema diachea leucopodia, diderma hemisphaericum, didymium nigripes, elaeomyxa miyazakiensis, per. depressa, comatricha stemonitis fusca craterium cra. retisporum cra. microcarpum, craterium microcarpum, physarum decipiens elaeomyxa miyazakiensis a. cinerea, cr. microcarpum, diderma hemisphaericum, phy. echinosporum arcyria physarum stemonitis et al. et al et al et al. collaria arcyrionema diachea leucopodia, physarum decipiens stemonitis fusca comatricha craterium retisporum, diderma ., didymium nigripes, elaeomyxa miyazakiensis, physarum bivalve, phy. bogoriense, phy. cinereum, phys. compressum, physarum physarum physarum physarum diderma comatricha et al. arcyria, physarum, diderma stemonitis et al. craterium craterium leucocephalum craterium et al , a total of twenty four species of myxomycetes were collected from anda island, pangasinan (table 1). the identified species belong to the following genera: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) of the collected species, six were identified as and . , sp. and were also reported in anda island. two species of , i.e. and were also noted among collected substrates. interestingly, three species of myxomycetes are new records for the philippines: and . these species have been previously reported in other countries but not in the philippines until now. of all collected myxomycetes, and were the most abundant (table 1). species of , and were also reported to be widely abundant in temperate and tropical forests, and in island habitats occurring in ground leaf litter or parts of living trees such as twigs and old bark (schnittler 2002; stephenson . 2004; everhart & keller 2008; stephenson . 2008; tran 2008; adamonyte & kastanje 2011). , and were recorded as common in this study, while most of the species were recorded as rarely occurring (table 1). sp.1, sp sp.1, sp.2, sp.8, sp.9, and two unidentified myxomycetes were all recorded as rare. similarly, species of and has been found to be abundant in temperate than in tropical forests (stephenson 1989; novozhilov 2000). the genera and are also abundantly diverse and distributed in island habitats (beltran 2004; rojas & stephenson 2008). rojas and stephenson in 2008 described the genus as rare in cocos island, costa rica; however, adamonyte and kastanje in 2011 abundantly recorded in the island of saaremaa, estonia. differences in climatic conditions in these two islands play a crucial part in the abundance and distribution of species. this may also be a factor for the differences in myxomycete distribution in anda island. though anda island is inhabited by man, the influence of anthropogenic activities to myxomycete distribution was not correlated, and therefore, is recommended for future studies. in this study, the computed diversity indices of myxomycetes in anda island, pangasinan showed high value for species diversity (h =1.15), species richness (h =5.33) and species evenness (e=0.56). similarly, high species diversity was also recorded in lubang island, occidental mindoro (macabago . 2012). however it was observed that geographic isolation does not greatly influence species composition, and endemism seems not to occur in for myxomycetes since species recorded in an island habitat also occur in mainland (eliasson 1991; rojas & s g biotropia vol. 20 no. 1, 2013 7 stephenson 2008). but the wide range of diverse habitats in island ecosystems provides diverse microhabitats for myxomycetes (rojas & stephenson 2008). thus, the diverse assemblage of myxomycetes recorded in this study further supports other studies conducted in island ecosystems. a total of 24 species of myxomycetes belonging to 11 genera were collected and identified in anda island, pangasinan philippines: ( var. ) , , and conclusions arcyria cinerea a. cinerea digitata , collaria arcyrionema, craterium microcarpum, cra. retisporum diachea leucopodia, diderma hemisphaericum didymium nigripes, elaeomyxa miyazakiensis, perichaena depressa, physarum bivalve, phy. bogoriense, phy. cinereum, phy. compressum, phy. decipiens, phy. echinosporum, table 1. species abundance of myxomycetes collected from anda island, pangasinan taxa % abundance arcyria cinerea (a. cinerea var. digitata) a arcyria sp. 1 o collaria arcyrionema c comatricha sp. 1 r craterium microcarpum a craterium retisporum r diachea leucopodia c diderma hemisphaericum a diderma sp. r didymium nigripes r elaeomyxa miyazakiensis r perichaena depressa o physarum bivalve r physarum bogoriense r physarum cinereum r physarum compressum r physarum decipiens c physarum echinosporum a physarum sp. 1 r physarum sp. 2 r physarum sp. 3 o physarum sp. 8 r physarum sp. 9 r stemonitis fusca c unidentified myxo 6 r unidentified myxo 7 r note : abundant indices, where abundant (a) = ≥10% of the total collections, common (c) = ≥5% but <10% of the total collections, occasional (o) = ≥3 but <5 of the total collections, and rare (r) = <3% of the total collections a diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. 8 biotropia vol. 20 no. 1, 2013 stemonitis fusca. arcyria comatricha diderma physarum craterium microcarpum, physarum decipiens, elaeomyxa miyazakiensis, physarum arcyria craterium diderma one species each of , , and five species of were identified up to the genus level only. three species, i.e. and are new records for the philippines. higher productivity was also recorded in the study as well as in ground leaf litter than twigs. eleven species of and one species for each genus , , and were recorded abundantly. the authors would like to thank the research center for the natural and applied sciences, university of santo tomas for the research grant, prof. dr. steven l. stephenson, university of arkansas, usa for his assistance in the identification of our species, and nikki heherson a. dagamac and edward e. dela cruz for their assistance in collecting our substrates. acknowledgments references adamonyte g, kastanje v. 2011. myxomycetes of the island of saaremaa, estonia. folia cryptog estonica fasc 48:1-4. beltran e, lado c, barrera j, gonzález e. 2004. myxomycetes diversity in the laurel forest of garajonay national park (canary islands, spain). syst geogr pl 74: 159-73. beltran e, mosquera j, lado c. 2010. myxomycete diversity from arid and semiarid zones of the canary islands (spain). mycotaxon 113:439-42. dela cruz tee, kuhn rv, javier aom, rodillas cp, parra cm, corpuz lhm, mchugh rd. 2011. occurrence and distribution of plasmodial myxomycetes in hundred islands national park, pangasinan, philippines. acta manilana 59:65-74. eliasson uh. 1991. the myxomycete biota of the hawaiian islands. mycol res 95: 257-67. everhart se, keller hw. 2008. life history strategies of corticolous myxomycetes: the life cycle, plasmodial types, fruiting bodies, and taxonomic orders. fungal div 29: 1-16. fleischmann a, robinson as, mcpherson s, heinrich v, gironella e, madulid da. 2011. 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biodiversity hotspots for conservation priorities. nature 403: 853-858. diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. biotropia vol. 29 no. 2, 2022: 161 170 doi: 10.11598/btb.2022.29.2.1690 161 monitoring forest area change using quickbird sri endayani1, andrew stefano2*, fathiah3, purbawati4 and ida rosanti4 1faculty of agriculture, forestry study program, universitas 17 agustus 1945, samarinda 75123, indonesia 2department of engineering and informatics, politeknik pertanian negeri samarinda, samarinda 75242, indonsia 3department of forest management, politeknik pertanian negeri samarinda, samarinda 75242, indonsia 4faculty of engineering, universitas nahdlatul ulama kalimantan timur, samarinda 75251, indonesia received 16 november 2021/accepted 14 january 2022 abstract a study was conducted to compare the urban forest management in three urban forests in samarinda city. the application of gis (geographic information system) is one of the alternatives to conduct a variety of processes such as: providing geographical information system, identifying the areas of urban forests in samarinda, helping to plan the process of map digitalization and performing overlay process. the main method used for the data analysis process on the map was the overlay process data analysis technique. the research findings showed that: 1) the appointment of urban forests as the initial step of urban forest development needed more implementation from the government; 2) the urban forest determination needed more socialization to the owner of the urban forest and the public in 1992 and 2019; 3) the urban forests needed more management. there were some similarities and differences in the management of urban forests in the three study locations. the similarities among the three locations were that the three locations had already met the minimum standards of one urban forest location even though there was still one location outside of these three locations which did not meet the minimum standard. the differences were in managing the urban forests. these differences indicated that the urban forest policy was not fully implemented in samarinda city. keywords: comparison, geographic information system, management, policy implementation, urban forests introduction the increasingly rapid growth of population and development nowadays causes a lot of changes in the appearance of samarinda city (angel et al. 2019; caddeo et al. 2019; cao et al. 2019). the rapid development has turned most of the green spaces in samarinda city into shopping centers, stores, housing areas, mining areas and other places for anthrophogenic activities (tunas & maadji 2018). these changes caused extreme environmental degradations and destructions (carrer et al. 2018; daoed et al. 1997; deng et al. 2020). the city’s development emphasizes on the aspect of economic growth (diodato & bellocchi 2020; fernándezguisuraga et al. 2019; guo et al. 2020), leading to several environmental problems (hafeez & khan 2012; hong et al. 2017; de jager et al. 2019), including water crisis, floods and pollution resulting from the traffic and the decreasing number of green spaces in samarinda city (daoed et al. 1997). certainly, the current conditions of samarinda city are no longer compatible with the city’s slogan a “tepian” city, which is the abbreviation of ‘teduh’ (shady), ‘rapi’ (tidy), ‘aman’ (safe), and ‘nyaman’ (comfortable). environmental degradation, of course, should be ceased to continue (lagacherie et al. 2020; lister & leites 2018; liu et al. 2017). one of the solutions to overcome the environmental degradation is by applying environmentally sound developments through urban forest development (liu et al. 2019; morales & perry 2017; nguyen et al. 2019). the decree of samarinda mayor number 178/hk-ks/2017 showed that the size of the existing urban forests is 690.237 ha of the size of samarinda city which is 718 km2. clearly, the size of the urban forests is still far from sufficient to meet the minimum 10% of the urban area based on *corresponding author, email: andrew.stefano@politanisamarinda.ac.id biotropia vol. 29 no. 2, 2022 162 the regulation stated in article 8 of the government regulation number 63 year 2002 on urban forests (windusari et al. 2017). in order to meet the 10% minimum percentage, the samarinda city should have 7,180 ha of urban forests. one of urban forest locations in samarinda city owned by pt. gani mulya is only 0.097 ha and this is not in line with the article 8 of the government regulation number 63 year 2002 on urban forests which stated that the size of urban forest in one compact stretch is at least 0.25 ha. unfortunately, this regulation also mentioned about the adjustment of the urban forest size with the local condition of each region (abdullahi et al. 2017), which seems to give a room for the local government to ignore the existence of urban forests. most city planner is still unaware of the significance of urban forests (nuddin et al. 2019; podlaski 2019; reza et al. 2020). if the regulation stated in the government regulation number 63 year 2002 on urban forests is properly followed, then all of the existing environmental problems faced by the government of samarinda city may be minimized (hong et al. 2017). urban forest as an open green space or ruang terbuka hijau (rth) should actually get attention from the government in order to make samarinda city an environmentally-sound city (tunas & maadji 2018; viccaro et al. 2019; wiggins et al. 2019). the population of samarinda deserves to have a comfortable, healthy and aesthetic environment (windusari et al. 2017; zhao et al. 2020). the city needs to be protected from a variety of negative environmental impacts (tunas & maadji 2018; viccaro et al. 2019; wiggins et al. 2019). among techniques to achieve a better quality of environment is by increasing the quality and quantity of city greeneries suitable with the city’s urban forests (hafeez & khan 2012). urban forest as an element of rth is expected to overcome environmental problems in the urban areas by absorbing pollutions caused by the anthrophogenic activities (silveira et al. 2019; sinha et al. 2019; soma & kubota 2018). the development of urban forest in big cities in indonesia indicates the policy makers’ awareness on environmental issues (sameen et al. 2019; schwede et al. 2018; shang et al. 2020). the issues of urban forest need a special attention from the government considering the rapid development in samarinda city which causes adverse impact on the environment and the decreasing number of green spaces (nuddin et al. 2019; podlaski 2019; reza et al. 2020). following the changing of times and the rapid development of technology, there are many methods to retrieve information on location in the form of map, one of which is using gis (geographic information system) (liu et al. 2019; morales & perry 2017; nguyen et al. 2019). by using gis we can capture a map of a location which provides detailed information (lagacherie et al. 2020; lister & leites 2018; liu et al. 2017). with the existence of geographic information system, it is expected that people will know more about the urban forests available surrounding the samarinda city. this study aimed to obtain comprehensive understanding on urban forests as the basis of policy making in developing urban forests in samarinda city (hafeez & khan 2012; hong 2017; de jager et al. 2019). the interview conducted in this study focused on the urban forest areas of samarinda city, guard posts, and standardization of forest preservation (balachandran 2017). this research applied literature study by collecting and studying issues related to the geographic information system of forestry data (relevant institutions) (sulistyo et al. 2017). basic theoretical framework public policy is a specific goal or a series of specific principles or actions taken by the government in a certain period of time with regard to one subject or as a response toward a crisis condition (wahab 2008). in addition, rose (1990) defined public policy as a series of less or more related activities and their consequences to the people concerned rather than a separate decision. furthermore, van meter and van horn (1975) defined policy implementation as an action taken by individuals or officials or groups of governmental or private organizations directed to achieve goals which have been stated in the policy decision. from this definition it can be identified that policy implementation covers three aspects, namely: 1) goals and targets of policy; 2) activities or policy goals and objectives; 3) outcome of the activities (agustino 2006). public policy has goals, monitoring forest area change using quickbird – sri endayani et al. 163 objectives and is behavior-oriented. public policy refers to what the government really acts on, not merely a statement or desired target of action. public policy is a directed target, meaning that the action is followed by an actor or a number of actors working together to solve problems. implementation study is an analysis on the policy implementation process. in its practice, policy implementation is a considerably complex process; even it usually has political contents because of intervention of various interests. when an issue which addresses common interests is considered necessary to be regulated, then the formulation of the issue becomes a public policy which needs to be implemented, prepared and approved by all the authorized officials. when the public policy has been stated as a public policy, then it turns into a law which needs to be carefully observed. policy in urban forest management urban development is usually reflected by the physical development of a city which is considerably determined by the existing facilities and infrastructure. the past and current urban development tends to minimize the open green spaces and to eliminate the face of nature. the condition of urban environment develops economically, but degrades ecologically. indeed, the ecological balance of urban environment has the same significance as the development of economic value in urban areas. this condition creates unharmonious relationship between urban community and its environment. being aware of the inharmony and considering the negative environmental impacts, there should be an effort to improve the environment through urban forest management. urban forest is one of the open green spaces. its existence functions as hydrological system, creating micro climate, maintaining oxygen (o2) and carbon dioxide (co2) balance, reducing pollutant and absorbing noise. in addition, urban forests also function to add the aesthetic values and the beauty of the city, thus giving positive impacts on the quality of environment and the life of the community (sibarani 2003). there are some municipal government policies which regulated the appointments of some urban forest locations. samarinda city has an urban forest policy which is stated in the samarinda mayor’s decree number 178/hk-ks/2019. the detailed regulation which addresses urban forests in the form of local regulation has not been made until now in samarinda city. however, the regulation governing the management of urban forests can be seen in the higher-order regulation, namely the government regulation number 63 year 2002 on urban forests. in addition, there is also the minister of forestry regulation number p.71/menhutii/2009 on the guidelines for urban forest management. article 4 of the government regulation number 63 year 2002 on urban forests stated that urban forest management covers: 1) appointment; 2) construction; 3) determination; and 4) management. however, this research only covered urban forest management which focused on the appointment of urban forests which included the sizes and the locations of the urban forests in samarinda. the highlight of this study were: 1) the determination of urban forests and 2) urban forest management which includes maintenance, protection and security, utilization, monitoring and evaluation. the policy implementation policy of urban forests were analyzed in 3 locations based on their gradient distances from downtown, namely in the area of city hall, lempake village and samarinda botanical garden of mulawarman university. materials and methods data retrieval this study used primary data collected from field observations and systematic recording using gps garmin 60. secondary data were collected from various kinds of relevant literatures and social phenomena, namely administrative maps of samarinda city, vegetation, plantations, and contours. data processing administrative maps, vegetation, plantations and contours were superimposed over the samarinda urban forest map (bpkh region iv) (first process). field checking was conducted to biotropia vol. 29 no. 2, 2022 164 obtain the most up-to-date information on the urban forest (second process). subsequently, the results of the two processes were combined. the image correction was carried out using quick bird software to reduce geometric, radiometric and atmospheric problems (fig. 1). the image analyses were subsequently carried out, by first making criteria and scoring assessments in tabular form as part of the data preparation process. visual interpretation of upto-date urban forest maps was used to get raster data in the form of land-use conversion. figure 2 presents the flowchart of samarinda urban forest map preparation. the samarinda's urban forest vegetation was determined based on the results of image analyses. results and discussion the determination of one particular area as an urban forest can be in the form of designation within the urban area and it can be a piece of land owned by the government or owned privately with a land ownership right. the designated urban forest location is a part of the open green space of the city. the importance of urban forest functions is regulated in the government regulation number 63 year 2002 on urban forests in articles 7, 8 and 9. the designation is based on the programs of the government of samarinda city through bapedalda (local board of environmental impact management). in 2001 2019 the government of samarinda city through bapedalda had planned, prepared and implemented urban forests which was funded by dakdr budget (special fund allocation fund for forestation). in 2003 the city government and samarinda local house of representative enacted the local regulation number 28 year 2003 on protected area in samarinda city. even though there is no obvious regulation concerning urban forests, the government of samarinda city keeps continuing the development of urban forests based on the local regulation number 28 year 2003. through bapedalda, the samarinda city planned, prepared, and implemented urban forests using dak-dr budget (special fund allocation – fund for forestation). figure 1 quickbird satellite image of samarinda city monitoring forest area change using quickbird – sri endayani et al. 165 figure 2 flowchart of samarinda urban forest map preparation after planning, preparing and implementing urban forest in samarinda, bapedalda provided a recommendation to the government of samarinda city to issue a mayor decree concerning the location of urban forests in samarinda city. then, the decree of samarinda mayor number 178/hk-ks/2005 on the determination of urban forest locations in samarinda city was issued. there were 25 locations which were appointed as urban forests in the urban area of samarinda. the decree of samarinda mayor number 178/hk-ks/2005 showed that the area of urban forests was 690.237 ha with the percentage of 0.96% from the total urban area. samarinda city has the area of 718.00 km2, so that to meet the minimum 10% of the urban area size, samarinda should have urban forests with the size of 7,180 ha. this means that samarinda still requires 6,489.763 ha or 9.04% to fulfill the required minimum 10% of the total urban area. in addition, the decree also mentioned one of the urban forests, namely pt gani mulya with the extent of 0.097 ha. the size of urban forest owned by pt gani mulya does not meet the standard based on the criteria stated in article 8 section (2) that the size of urban area in one compact stretch should be at least 0.25 ha. furthermore, the distribution of urban forest in each subdistrict in samarinda is not equal because there are still 2 subdistricts from the total of 10 subdistricts which do not have an urban forest in their district areas, such as palaran and sungai pinang subdistricts. each subdistrict also still has a significant shortage from the required minimum total area. the criteria of urban forest in each subdistrict is presented in table 1. table 1 criteria of urban forest area for each subdistrict no subdistrict urban forest area (ha) area (km2) minimal forest area (ha) shortage (ha) percentage by the zone (%) 1. palaran 221.29 2,212.9 -2,212.9 0 2. samarinda ilir 6 17.18 171.8 -165.8 0.35 3. samarinda kota 11.56 11.12 111.2 -99.64 1.04 4. sambutan 187 100.95 1,009.5 -822.5 1.85 5. samarinda seberang 1.5 12.49 124.9 -123.4 0.12 6. loa janan ilir 8.697 26.13 261.3 -252.603 0.33 7. sungai kunjang 69.75 43.04 430.4 -360.65 1.62 8. samarinda ulu 8.98 22.12 221.2 -212.22 0.41 9. samarinda utara 306.75 229.52 2,295.2 -1,988.45 1.37 10 sungai pinang 34.16 341.6 -341.6 0 samarinda city 690.237 718.00 7,180 -6,489.763 0.96 source: processing data from the mayor’s decree number 178/hk-ks/2017on the determination of some urban forest locations in samarinda city. map of samarinda city samarinda city forest area overlay samarinda city + urban forest area distribution of forest types map of samarinda city forest area overlay biotropia vol. 29 no. 2, 2022 166 city hall urban forest the city hall urban forest was appointed as an urban forest in 1992 through the mayor’s decree number no. 224 year 1992, followed by the mayor’s decree number 178/hk-ks/2019. the city hall environment was appointed as an urban forest after meeting the requirements, and in this case, the city hall urban forest has an area of 7.64 ha. the area is more than enough to fulfill the minimum requirement of one location to be selected as an urban forest, namely 0.25 ha. the city hall urban forest is located on the state-owned land whose land status and ownership right belongs to the government of samarinda city. land ownership is proven with a land certificate number: p-24, number: 305/1981 which was issued by agrarian office of samarinda city on 29 june 1981. lempake urban forest the appointment of lempake urban forest was based on the land status owned by the municipal government since the status of the village was changed into kelurahan. this location was appointed by bapedalda in 2004 by involving the local community. lempake urban forest has an area of 3.5 ha and this size has met the requirement of a minimum size of 0.25 ha for one location of urban forest. the determination of urban forests policy in the determination of urban forest has been issued twice by the government of samarinda city. the first one was the mayor’s decree number 224 year 1992 and the second one was the mayor’s decree number 178/hkks/2019. the determination of urban forest in 1992 was issued by the cleaning and landscaping agency and the determination of urban forest in 2017 was issued by the bapedalda which is now recognized as blh (environmental agency). the urban forests which were stated in the mayor’s decree number 178/hk-ks/2019 in samarinda city were determined after the bapedalda accomplished the procedure of planning, preparation, and implementation of urban forest. the documented procedures was then proposed to the government of samarinda city in order to issue a letter of determination for the locations of urban forests in the region of samarinda city. from 1992 to 2005 there was an increase in urban forests both in their sizes and their total number. in 1992 samarinda had only 12 locations of urban forests with an extent of 218.177 ha, while in 2019 the number of urban forests increased up to 25 locations with an area of 690.237 ha. this indicates that samarinda has an additional 13 urban forests and an additional area of 472.06 ha. unfortunately, the determination policy for urban forests has not been reviewed at least once in two years following the rule stated in point three of the mayor’s decree number 178/hk-ks/2019. city hall urban forest the area of city hall was first designated as an urban forest on 17 december 1992 through the mayor’s decree number 224 year 1992 with an area of 6.9 ha. this determination was then renewed by the mayor’s decree number 178/hk-ks/2019. in the decree number 178/hk-ks/2019 the area of city hall had an additional area which was allocated for urban forest, from an area of 6.9 ha to 7.64 ha. the determination of the city hall area as an urban forest was well-socialized to the public because of its strategic location in the middle of samarinda city. lempake urban forest the determination of kas lempake as an urban forest was not known by the public or the village government. even though the location status is on the state-owned land, the determination of the urban forest should have been informed to the public, especially to the community where the urban forests are located. the fact that the public do not know the existence of the urban forest is caused by some factors. first, the appointment of kas lempake as an urban forest only involved some people following the rapid growth of population. second, the determination of urban forest had lasted for a long time without any socialization from the former government. third, there have been a lot of replacements of lurah and personnel in the lempake village since the urban forest determination. last, there is no activity initiated by the government in relation to to the management of kas lempake. monitoring forest area change using quickbird – sri endayani et al. 167 urban forest management urban forests are not managed uniformly because the managements are only conducted in several locations which are only owned by the government such as the area of city hall, segiri softball field, garden tombs of heroes, and samarinda city library, which are easily accessed by the government because these locations are in the downtown area. one of the efforts made by the government in urban forest management is by re-registering the existing urban forests. the result of re-registration can be seen table 2. the city still needs 9.12%. the local office of agriculture, plantations and forestry of the samarinda city (distanbunhut) also makes a plan for locations which are going to be appointed into urban forests. after doing reregistration on the size of urban forest stated in the mayor’s decree number 178/hk-ks/2019, the area of urban forest was decreased by 57,167 ha from the original 633.07 ha. therefore, the percentage of urban forest nowadays is only 0.88% from the previous percentage of 0.96%, namely 71,800 ha. the minimum requirement for an urban forest is 10% of the total urban area. this means that samarinda still needs urban forests. the planned locations include the center for dipterocarp, land of municipal government in makroman, polytechnic of agriculture campus (poliagro samarinda), and kaltim cultural park. the government regulation number 63 year 2002 and the regulation of the minister of forestry number p.71/menhut-ii/2009 which is supported by the mayor’s decree number 178/hk-ks/2019 is sufficient to be the basis for urban forest management. table 2 re-registration of urban forests in samarinda city no. city forest location large sk 2019 (ha) re-registration data (ha) 1. smu 10 melati 5 5 2. krus 300 300 3. tanah pemkot 5 5 4. hutan kota belakang rumah jabatan walikota 1.75 1.8 5. asih manuntung 0.25 0.25 6. pesantren hidayatullah 1 0.38 7. tanah pemkot di makroman 167 167 8. tanah pertanian terpadu 20 20 9. kas desa lempake 3.5 3.5 10. fakultas pertanian unmul 6.5 3.84 11. pesantren nabil husein 9.75 9.75 12. pesantren syachona cholil 0.25 0.25 13. rumah potong hewan 2 5 14. hotel mesra 2.3 0.7 15. jalan pembangunan voorfo 0.48 2.6 16. lingkungan balai kota 7.64 3.26 17. lingkungan lapangan softball gor segiri 0.5 0.25 18. perpustakaan kota samarinda 0.6 0.5 19. ujung timur jembatan mahakam 1.5 2 20. pt. hartaty 60 21. pt. gani mulya 0.097 2.75 22. pt. sumber mas 85 85 23. pt. sumalindo 3.6 24. taman makam pahlawan 0.52 1.04 25. pt. kiani (teluk cinta di selili) 6 13.2 total 690.237 633.07 source: agriculture, farming and forestry service office of samarinda city (2019). biotropia vol. 29 no. 2, 2022 168 city hall urban forest the urban forests which are located on the state-owned lands are managed by three government institutions at the same time, i.e., the agriculture, farming and forestry service, the cleaning and landscaping agency, and the city planning service. the forms of management include providing seedlings and plants. the construction of shopping center next to the urban forest location is believed not to use the land of the urban forest because it is still within the border of the land owned by the owner of the building. the city hall urban forest is one of the urban forest locations having decreasing in area size because of the building construction and parking lots which take the green space in this location. lempake urban forest the government policy on the management of urban forests which are located in the land owned by kas lempake has not been implemented. this is not in line with the regulation of minister of forestry number p.71/menhut-ii/2009 in article l32 through article l43, which technically governs the urban forest management. kas lempake village is under the responsibility of the government, but until now this urban forest location has never been managed properly. the fact that this urban forest is not managed by the community is caused by the status of the location that is owned by the government. the municipal government has never formed a coordination with the local village to involve the community in the management of urban forests. conclusion there are some similarities and differences in managing urban forests in samarinda city. their similarities can be found in the three research locations in which they have met the required minimum standard of one location, i.e., 0.25 ha. in addition, the urban forests also provide the same benefits to the surrounding environment. the city hall urban forest was established as an urban forest in 1992 and it was extended in 2017, while lempake urban forest and the urban forest of universitas mulawarman botanical garden were just established in 2017 through the mayor’s decree number 178/hk-ks/2019. those urban forests have differences in the status of land ownership. the city 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therefore, this study aims to characterize the molecular profile of native a. flavus isolated from several indonesian agricultural products, with a major focus on its toxigenicity and toxin production. a total of 18 a. flavus collections were isolated from nutmeg, ground peanut, cacao, coffee bean, corn, white pepper, and soil peanut plantation. species identification was carried out using molecular and morphological approaches. the toxigenicity of isolates was characterized based on the amplification of aflatoxin gene clusters, while toxin production was assessed through growth simulation on a 10% coconut broth media followed by hplc quantification. the result showed that all isolates were confirmed as a. flavus based on the morphological and sequence analysis of the its region. a total of 11 isolates (61%) were confirmed as toxigenic and produced 1-2 types of aflatoxin, in varying concentrations of high, moderate, or low levels of afb1. high levels of afb1 produced by seven isolates namely bio3313, bio33212, bio3361, bio33404, bio3338, bio3352, and bio3344, had concentration levels ranging from 76.78 to 2241.06 µg/kg, while three isolates (bio3314, bio3312, and bio3381) produced afb1 below 1 µg/kg. twenty-nine pairs of aflatoxin gene-specific sequences were successfully amplified as a single band, while some produced non-specific patterns in several low toxigenic and non-toxigenic isolates. based on the results, it was concluded that completed gene clusters and variations of gene deletion were observed in both toxigenic and non-toxigenic isolates. however, no specific target gene could effectively distinguish the two groups. two non-toxigenic isolates namely bio3393 and bio33403 exhibited a large deletion and could be potential candidates for biocontrol agents. keywords: aflatoxin, aspergillus flavus, its, pcr, toxigenic introduction aflatoxin contamination in agricultural products is a food safety concern in indonesia and worldwide. this carcinogenic mycotoxin is produced mainly by toxigenic aspergillus flavus and a. paraciticus during various stages of agricultural production including pre-harvest, harvest, post-harvest, transportation, and storage. the prevalence of aflatoxin is particularly high in tropical and subtropical regions due to optimal humidity and temperature conditions for toxin production (bhat et al. 2010). in indonesia, aflatoxin contamination has been reported in agricultural products such as nutmeg, corn, peanuts, pepper, and cocoa (dharmaputra 2002). this contamination poses a significant challenge for export-targeted commodities, leading to economic losses for farmers and exporters. for instance, upon arrival in the importing country, nutmeg was found to have a high level of aflatoxin due to the growth of toxigenic a. flavus (anidah et al. 2020). the growth was concluded to have *corresponding author, email: anidah@biotrop.seameo.id biotropia vol. 30 no. 2, 2023 196 occurred during transportation based on the low levels of the toxin detected before shipping. the activities of regulatory proteins and enzymes encoded by more than 25 genes cluster play an essential role in aflatoxin production (yu et al. 2002). non-toxigenic a. flavus was reportedly associated with the deletions of a part or the entire aflatoxin genes cluster (chang et al. 2007). however, the molecular mechanisms underlying the loss of the aflatoxin production ability in a. flavus are currently not well understood (rao et al. 2020; schmidt-heydt et al. 2008). pcr detection for afld (nor1), aflp (omta), aflm (ver1), and aflr genes have been applied as a marker to differentiate toxigenic and non-toxigenic isolates in different studies. these include isolated native to indonesia (anidah et al. 2020), animal feed in iran (davari et al. 2014), aspergillus species in korea (kim et al. 2011), maize in italy (degola et al. 2006), and an herbal product in italy (criseo et al. 2001). the results showed various deletion patterns for nontoxigenic isolates which could be distinguished from toxigenic ones having 4 complete genes or no deletion. however, it was observed that the toxigenic isolate exhibited variations in deletion patterns when amplified using a complete set of aflatoxin biosynthetic genes, rendering the use of 4 genes as a marker irrelevant. the characterization of deletion patterns within all the aflatoxin biosynthetic gene clusters has been elucidated in non-toxigenic isolates to find and assess the biocontrol agent candidate (rao et al. 2020; wei et al. 2014; donner et al. 2010; yin et al. 2009; chang et al. 2005). however, the genetic variability assessment in toxigenic isolates has not been widely carried out, despite its significance in distinguishing between toxigenic and non-toxigenic isolates. a molecular approach that characterizes the presence or absence of aflatoxin biosynthetic genes can indicate the functional status of the biosynthesis pathway gene (tran-dinh et al. 2014). this approach could serve as an effective marker for the identification of toxigenic isolates in food commodities. therefore, this study aims to characterize the molecular profiles of toxigenic and no-toxigenic a. flavus isolates from agricultural commodities in indonesia by using an aflatoxin biosynthetic genes cluster. materials and methods a. flavus isolates the a. flavus isolates used in this study (table 1) were from the phytopathology laboratory of seameo biotrop. these isolates were sourced from various regions in indonesia and included samples from nutmeg, ground peanut, cacao, coffee bean, corn, white pepper, and soil peanut plantation. all collected samples were grown in the potato dextrose agar/pda (himedia, india) with parafilm oil as a preservative, and were reinoculation in pda at 25oc for seven days before use. table 1 a. flavus isolates from agricultural products isolates commodities (origin) bio3376 nutmeg (manado – north sulawesi) bio33211 bio33212 bio33403 bio33404 bio3313 ground peanut (bogor – west java) bio3381 bio3334 ground peanut (wonogiri – central java) bio3338 bio3342 bio3344 bio3312 cacao (south sulawesi) bio3314 coffee bean (jember – east java) bio3393 coffee bean (toraja – south sulawesi) bio3382 corn (bogor – west java) bio3383 white pepper (bogor – west java) bio3361 soil (wonogiri – central java) bio3352 morphological characterization the morphological characterization of the isolates was carried out according to samson et al. (2004) and pitt & hocking (2009). czapek yeast autolysate (cya) agar was used for macromorphological observation. each isolate was inoculated at two points, and incubated at temperatures of 25 and 37oc in the dark for seven days. the colony color was observed, and the diameter was measured. meanwhile, the micromorphological observation was conducted by preparing microscopic mounts in lactic acid with cotton blue from cya colonies. the excess conidia and air bubbles were removed by adding a drop of 70% ethanol, then conidia morphology and presence, size of sclerotia, and head seriation were analyzed. molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 197 dna extraction the mycelia from a 3-day-old culture in 50 ml potato dextrose broth/pdb (himedia, india) were harvested and rinsed using sterile distilled water and then dried using filter paper. the sample was crushed using a mortar and pestle with continuous addition of liquid nitrogen until fine mycelia powders were obtained. dna was extracted using dneasy plant kit (qiagen, germany) according to the manufacturer’s instructions and the quality was assessed through electrophoresis in 1% agarose gel (invitrogen, usa) with sybrsafe dye (invitrogen, usa) in tris-acetate-edta (tae) buffer. the absorbance was measured at 260 nm wavelength for quantification, and dna was stored at -20 oc until further use. species identification molecular species identification was carried out using its 1 (tccgtaggtgaacctg cgg) and its 4 (tcctccgcttattgata tgc), as forward and reverse primers against all isolates and sequencing of the its region (white et al. 1990). the amplification reaction was carried out by mixing 100 ng of dna template, 0.2 m of each primer, and 1x gotaq hot start pcr master mix (promega, usa), in a total volume of 40 µl. the amplification program was performed in the genamp pcr system 9700 (abi, usa) thermal cycler with specific conditions including pre-denaturation for 5 minutes at 95oc, followed by 35 cycles of denaturation at 94oc, annealing at 5oc, and extension at 72oc for 30 seconds each. an additional final extension at 72oc for 7 minutes was also included. the amplified product was visualized by 1.2% agarose gel electrophoresis, followed by sequence analysis in 1st base (singapore). the sequences were analyzed for similarity using blast (http://blast.ncbi.nlm.nih.gov/blat.cgi) and submitted to the ncbi database. the neighborjoining method with 10000 bootstrap replicates in mega x software was used for tree construction (kumar et al. 2018). differentiation of toxigenic and nontoxigenic isolates toxigenic and non-toxigenic isolates were determined by growth simulation on an aflatoxin-inducing media (davis et al. 1987). total aflatoxin was extracted from a ten-day inoculated culture in 10% (v/v) coconut broth media/cbm, according to the aoac method 991.31 (aoac, 2000). for extraction, a 25 ml filtrate of the culture was mixed with 5 g nacl and 125 ml of methanol: water (70:30; v:v). the mixture was filtered through a glass microfiber filter after 2 times dilution with purified water. aflatoxins b1, b2, g1, and g2 from the extract were selectively isolated using the aflatest affinity column (vicam, usa). furthermore, the content of the extract was determined using hplc with post-column derivatization (vicam 2007). all the extraction chemicals and solvents used were from merck, germany, while the aflatoxin standard was from sigma, usa. pcr amplification using aflatoxin biosynthesis genes pcr amplification using aflatoxin biosynthesis genes was carried out according to chang et al. (2005). the isolated dna was amplified by pcr to detect the presence or absence of aflatoxin biosynthesis gene fragments using 29 pairs of primers as shown in table 2. table 2 list of aflatoxin biosynthesis primer sequences primer pair sequence aflu (norbcypa) f: gtgcccagcatcttggtcca r: aggacttgatgattcctcgtc aflt f: atgacatgctaatcgacgag r: aggcgcatgctacggatc aflc (pksa) f: actttgagggcgttctgtgc r: ctttcggtggtcggtgattc afld (nor1) f: agcacgatcaagagaggctc r: gatctcaactcccctggtag afla (fasa/hexa) f: tcctatccagtccacctcgta r: cacatctttgtcttgcccgc aflb (fasb/hexb) f: acaatcgaatgacaacactgc r: ccaccgaatccactacctaca aflr f: atggtcgtccttatcgttctc r: ccatgacaaagacggatcc afls f: cttcaacaacgacccaaggtt r: agatgagatacactgccgca aflh (adha) f: cctcgtgggagagccaaatc r: ggagcaagaaggttacagcg aflj (esta) f: cgatgggactgacggtgatt r; accacgccgctgactttat afle (nora) f: gtgttcgtgtgtcgccctta r: gtcggtgcttctcatcctga aflm (ver1) f: catcggtgctgccatcgc r: cctcgtctacctgctcatcg afln (vera) f: ccgcaacaccacaagtagca r: aaacgctctccaggcacctt aflg (avna) f: gcgatagaactgacaaaggca r: gaatgagtctccaaaggcgag afll(verb) f: ttcagtgacaaaggtcttcgc r: ggcagcgtt attgagcatct biotropia vol. 30 no. 2, 2023 198 afli (avfa) f: attcaaatcctcgttcggtcg r: tagcccgttggttgtgttcc aflo (omtb) f: acagacgatgtgggcaaacg r: acgcagtccttgttagaggtg aflp (omta) f: caggatatcattgtggacgg r: ctcctctaccagtggcttcg aflq (orda) f: aaggcagcggaatacaagcg r: acaagggcgtcaataaagggt aflk (vbs) f: aacgagcagcgtaagggtct r: tcagccagagcatacacagtg aflv (cypx) f: ggagcctaccattcgcaaca r: ggctttgacgaacagattccg aflw (moxy) f: tgctactggaacgaagaccg r: cgacgacaaccaaacgcaa aflx (ordb) f: gctgctactggaatgaagacc r: atgcgacgacaaccaaacg afly (hypa) f: cgcaagacggcagagatact r: gctccttcagttccacacca nada f: tgacgaggcctgcgagctgt r: aagcctcttcagaacggtca hexa f: tgtcctcacctctggcgtat r: agaccaaccactcttatgggc glca f: agacacagtcatcgcctgtt r: ggtgcgaataggtgcaggta sugr f: tcagctgaagcgctcgagag r: gtattgccgcactatgtatg c4 f: atcgtgcagacaggaacac r: ggtgccttggcctatgcgct a total of 20 µl of the reaction mixture was prepared for each isolate by mixing 50 ng dna, 0.2 µm each primer pair, 1x gotaq hot start pcr master mix (promega, usa), and molecular grade water. pcr amplification was performed in a thermal cycler geneamp pcr system 9700 (abi, usa) with specific conditions of pre-denaturation for 5 minutes at 94oc, followed by 30 cycles of denaturation at 94oc, annealing at 55oc, and extension at 72oc for 1 minute respectively. an additional final extension was carried out at 72oc for 6 minutes. the pcr products were visualized by electrophoresis on a 1.5% agarose gel in tae buffer, with sybrsafe dye (invitrogen, usa). the presence or absence of amplicon was observed, and the results were scored for the presence (1) or absence (0) of aflatoxin genes. to construct the phylogenetic tree, cluster analysis was performed using the unweighted pair-group method with arithmetic mean (upgma) functionality in ntsyspc 2.1 software (department of ecology and evolution, state university of new york, ny, usa). results and discussion morphological and molecular identification of a. flavus all isolates were identified as a. flavus based on macro and micromorphology observations, following the guidelines of pitt & hocking (2009). the colony diameter range was 3.8 5.6 cm and 4.2 6.4 cm when grown on cya agar at 25 and 37oc, respectively. the colony colors were greyish-green, yellow-green, and oliveyellow, while the reverse side was uncolored to reddish-brown. conidia were observed as smooth to finely rough with biseriate conidial heads, globose to subglobose, and 3 5 µm in diameter. moreover, the stipes were hyaline, smooth, variable in length, mostly 350-600 µm, and the diameter just below vesicles was 3 8 µm. the vesicles were globose to sub-globose, and 10-30 µm in diameter. isolates grew well at 25 and 37oc and no sclerotia were observed as shown in figure 1. molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 199 figure 1 colonies of the 18 a. flavus on cya (7 days at 25 oc) notes: a = bio3313; b = bio33212; c = bio3361; d = bio33404; e = bio3338; f = bio3352; g = bio3344; h = bio3334; i = bio3314; j = bio3312; k = bio3381; l = bio3382; m = bio3383; n = bio3376; o = bio33211; p = bio3342; q = bio33403; r = bio3393. j k l m n p q o r b a c d e f g h i biotropia vol. 30 no. 2, 2023 200 species identification at the molecular level was conducted by amplifying the its region as the official locus for fungal dna barcoding. amplification using its 1 and its 4 yielded a single amplicon of 600 bp in all isolates. the amplified products were sequenced and analyzed for similarity using blast (http:// blast.ncbi.nln.nih.gov/blast.cgi), and all the generated sequences were submitted to the ncbi database (table 3). afterward, a phylogenetic tree was constructed using mega 6, which included sequences from genbank for a. flavus (nr111041.1), a. paraciticus (nr151784.1), a. niger (nr111348.1), and a. fumigatus (nr121481.1). all isolates were found to have 100% homology to a. flavus (figure 2). the neighbor-joining method with 10000 bootstrap replicates in mega x software was used for tree construction. bootstrapping was translated as the accuracy value of the phylogenetic tree against randomization of 10000 repetitions (tamura et al. 2007). the morphological and molecular analysis, both confirmed all isolates identified as a. flavus (figures 1 and 2). toxigenic and non-toxigenic isolate differentiation toxigenic a. flavus was induced to produce aflatoxins in the cbm containing 10% coconut milk due to the presence of fat and fatty acids (lin & dianese 1976). during incubation, a. flavus mycelia grew on the media’s surface, releasing the toxin in the solution. the aflatoxin content produced was extracted from the cbm filtrate, followed by identification and quantification by hplc compared to respective standards as afb1, afb2, afg1, and afg2. a total of 11 isolates from a. flavus (61%) produced aflatoxin with varying concentrations, while seven (39%) did not, as shown in table 3. the toxigenic isolates yielded two forms of aflatoxins with a combination of afb1-afb2 (three isolates), and afb1-afg1 (three isolates), while the remaining five only produced afb. however, none of the toxigenic isolates produced afg2 as shown in table 3. figure 2 phylogenetic tree of a. flavus isolates using neighbor-joining method by mega x software (10000 x bootstrap) http://blast.ncbi.nln.nih.gov/blast.cgi http://blast.ncbi.nln.nih.gov/blast.cgi molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 201 table 3 aflatoxin contents from 18 isolates of a. flavus a. flavus isolate accession number aflatoxin (µg/kg) b1 g1 b2 g2 bio3313 on619457 2241.06 nd 66.8 nd bio33212 on619471 702.72 nd nd nd bio3361 on619464 607.67 1081.15 nd nd bio33404 on619473 255.27 nd nd nd bio3338 on619460 217.34 1486.52 nd nd bio3352 on619463 126.96 200.06 nd nd bio3344 on619462 76.78 nd 2.60 nd bio3334 on619459 7.68 nd 0.14 nd bio3314 on619458 0.62 nd nd nd bio3312 on619456 0.31 nd nd nd bio3381 on619466 0.10 nd nd nd bio3382 on619467 nd nd nd nd bio3383 on619468 nd nd nd nd bio3376 on619465 nd nd nd nd bio33211 on619470 nd nd nd nd bio3342 on619461 nd nd nd nd bio33403 on619472 nd nd nd nd bio3393 on619469 nd nd nd nd notes: nd = not detected; below the loq (b1 = 0.0202; b2 = 0.0171; g1 = 0.0220; g2 = 0.0183) in µg/kg all toxigenic isolates exhibited the capacity to produce afb1 in various concentrations, ranging from low, medium, to high levels. a total of seven toxigenic isolates had a high concentration of afb1 ranging from 76.78 to 2241.06 µg/kg, while one isolate had a moderate concentration of 7.68 µg/kg. furthermore, three other toxigenic isolates produced afb1 below 1 µg/kg as shown in table 3. the presence of toxigenic isolates in food commodities poses a significant threat due to their ability to produce aflatoxins. the maximum value in food commodities permitted by regulations in indonesia is 15 µg/kg afb1 (bpom 2012). based on the results, the potential of a. flavus to produce aflatoxin varied significantly among both strains. in nature, the percentage of the non-toxigenic strain varies from 0 to more than 80% worldwide (rao et al. 2020; yin et al. 2009; chang et al. 2007). molecular profile of toxigenic and nontoxigenic a. flavus isolates although the species identification by morphological and molecular approaches confirmed the same result, they could not distinguish between the toxigenic and nontoxigenic isolates. the toxigenicity of a. flavus was determined by the presence of genes encoding aflatoxin biosynthesis which was detected by pcr (yu et al. 2002). the pcrbased molecular technique offers the advantage of rapid diagnosis with a high level of sensitivity and specificity, compared to the conventional method (mamo et al. 2017). based on the results, twenty-nine pairs of primers were successfully amplified in 18 a. flavus isolates. the majority of the isolates produced single amplicons, while some yielded non-specific amplicons as multiband patterns in several low toxigenic and non-toxigenic isolates (figure 3). the presence of multiband amplicons indicates the formation of nonspecific product and suggested that the absence of aflatoxin production may be due to base-pair substitution mutations, resulting in the formation of non-functional gene products (levin 2012; criseo et al. 2001). the amplification results of 3 toxigenic isolates with high to low levels of afb1 showed no deletion of the amplicon. bio3313 which exhibited high afb1 production, as well as bio3334 and bio3381 with moderate and low afb1 production, respectively, had the complete aflatoxin gene cluster. similarly, the complete amplicons were also found in bio3382 as a non-toxigenic isolate. this result was consistent with previous studies which showed a complete set of genes in the nontoxigenic a. flavus (wei et al. 2014; kim et al. 2011; criseo et al. 2001). although amplification using dna-based pcr confirmed the presence of the target gene in the genomic dna, it was unable to explain the level of gene expression. this suggested the need for a real-time pcr as a biotropia vol. 30 no. 2, 2023 202 more proficient method but the gene expression result strongly depends on the reference stain, culture condition, and media used to induce the mrna expression (rao et al. 2020). figure 3 profile of all amplicons of aflatoxin biosynthesis genes in toxigenic and non-toxigenic isolates notes: * = non-toxigenic isolates; a = bio3313; b = bio3338; c = bio3352; d = bio3361; e = bio3376; f = bio33211; g = bio33212; h = bio33403; i = bio33404; j = bio3312; k = bio3314; l = bio3334; m = bio3342; n = bio3344; o = bio3381; p = bio3382; q = bio3383; r = bio3393. afld/nor1 aflu/norb a aflt aflc/pksa afla/fasa aflb/fasb aflr afls aflh/adha aflj/esta afle/nora aflm/ver1 afln/vera aflg/avna afll/verb afli/avfa aflo/omtb aflp/omta aflq/orda aflk/vbs aflv/cypx aflw/moxy aflx/ordb afly/hypa nada hexa glca sugr c4 a b c d e* f* g h* i j k l m* n o p* q* r * molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 203 variations in the deletion of aflatoxin biosynthetic genes were observed in both toxigenic and non-toxigenic isolates. the deletion involved one to a maximum of four genes in toxigenic isolates, except for bio3312 which exhibited a low level of afb1 production below 1µg/kg and experienced nine genes deletion, with some appearing as non-specific amplicons. meanwhile, non-toxigenic isolates showed one to 26 gene deletions. two nontoxigenic isolates, namely bio 33403 (90% deletion) and bio3393 (79% deletion) exhibited significant gene deletions, providing scientific data to address the safety issues in the application of biological control. according to a previous study, non-toxigenic isolates with extensive gene deletions are potential candidates for biocontrol agents (donner et al. 2010). moreover, extensive aflatoxin gene deletion is preferred to prevent adverse genetic recombination (chang et al. 2005). molecular characterization of non-toxigenic a. flavus using partial or completed aflatoxin biosynthetic genes has been carried out. pcr detection using afld (nor1), aflp (omta), aflm (ver1), and aflr as partial target genes were used in several studies to discriminate non-toxigenic isolates (anidah et al. 2020; davari et al. 2014; degola et al. 2006; criseo et al. 2001). in this study, afld, aflp, and aflr genes were found to be deleted in toxigenic isolates, while the aflr gene appeared in several non-toxigenic isolates. similar results were observed by rao et al. (2020), suggesting that these partial gene detections may not serve as reliable markers. genetic variations of non-toxigenic a. flavus isolates based on completed aflatoxin biosynthetic genes cluster have also been reported. chang et al. (2005) amplified 25 genes from 38 non-toxigenic a. flavus isolates in the united states and found eight deletion patterns. yin et al. (2009) analyzed the presence of 11 genes and found five deletion patterns from 11 non-toxigenic isolates in china. furthermore, donner et al. (2010) examined 21 non-toxigenic isolates in nigeria by amplifying 21 genes and found nine deletion patterns. wei et al. (2014) also found 25 deletion patterns by amplifying 29 genes from 76 non-toxigenic isolates in china. all the aforementioned studies focused on the variation of deletion patterns in non-toxigenic isolates with a major emphasis on their use as biocontrol agents in the field. the results indicated that the genetic variability from the aflatoxin biosynthetic gene cluster is diverse in non-toxigenic isolates. a comparison of the deletion patterns might contribute to a better understanding of specific markers for differentiating isolates based on toxigenicity. a cluster analysis was performed to identify the similarities among the toxigenic and nontoxigenic isolates based on the presence of aflatoxin genes. the genetic similarity coefficients (gsc) ranged from approximately 0.28 to 1.00. the phylogenetic tree assembled using the upgma grouped the 18 isolates into two main clusters. one group had the largest number of isolates (16 isolates) including toxigenic as well as non-toxigenic isolates. another group had 2 non-toxigenic isolates with large deletion, namely bio3393, and bio33403. this indicates that no specific genes could distinguish between toxigenic and non-toxigenic isolates (figure 4). biotropia vol. 30 no. 2, 2023 204 figure 4 dendrogram illustrating genetic relationships among 18 isolates of a. flavus, generated by the upgma cluster analysis (ntsys) calculated from 29 primers of aflatoxin biosynthesis genes notes: ** = highly toxigenic isolates; * = low toxigenic isolates. a. flavus isolates were identified at the species level through morphological and molecular analysis, (figures 1 and 2), while the toxin analysis was used to distinguish between toxigenic and non-toxigenic isolates (table 3). molecular characterization results based on aflatoxin biosynthetic genes provided an overview of the diversity profile at the molecular level (figures 3 and 4). the results also contributed to a better understanding of toxigenicity mechanisms. the genetic variations observed in each toxigenic and non-toxigenic isolate are important to explore the specific markers candidate for detection purposes and the development of biological agents. conclusion all isolates involved in this study were confirmed as a. flavus based on morphological and molecular identifications, while hplc assays confirmed their ability to produce aflatoxins. twenty-nine pairs of primers were successfully amplified, and the majority produced a single amplicon in toxigenic isolates. however, certain low toxigenic and nontoxigenic isolates produced non-specific amplicon patterns. deletion variations in several target genes were found in both isolates but no specific target gene could distinguish the toxigenicity based on the genetic similarity data. this study provides an overview of the molecular profiles of toxigenic and nontoxigenic a. flavus isolates. two non-toxigenic isolates with significant gene deletion were characterized and identified as potential candidates for biocontrol agents. the information about the molecular characterization of a. flavus based on toxigenicity could support the development of effective biological control strategies to prevent aflatoxin contamination. acknowledgments the authors would like to acknowledge seameo biotrop for providing financial support through daftar isian pelaksanaan anggaran (dipa) 2021. thanks to prof. dr. okky setyawati dharmaputra from fitophatology laboratory of seameo biotrop for their a. flavus collection isolates. references anidah, rahayu wp, nurjanah s. 2020. screening of toxigenic aspergillus flavus strains and aflatoxin content from agricultural commodities in indonesia. in: sitanggang az, rahayu wp, antara ns, santoso u, giyatmi, ardiansyah, haryono a, eds. proceedings of the 16th asean food conference-16th afc, isbn 978-989-758-467-1, p. 221-6. doi: 10.5220/ 0009981202210226 molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 205 aoac. 2000. section 49.2.18 (aoac method 991.31) for corn, raw peanut, and peanut butter. in official method of analysis. 17th edition. gaithersburg (us): aoac international. bhat r, rai rv, karim aa. 2010. mycotoxins in food and feed: present status and future concerns. compr rev food sci food saf 9: 57-81. [bpom] badan pengawas obat dan makanan. 2018. indonesian pom agency regulation no. 8 of 2018. the maximum limit of chemical contamination in processed food. jakarta (id): badan pengawas obat dan makanan. chang pk, horn bw, dorner jw. 2005. sequence breakpoint in the aflatoxin biosynthesis gene cluster and flanking region in nonaflatoxigenic aspergillus flavus isolates. fungal genet biol 42(11): 914-23. criseo g, bagnara a, bisignano g. 2001. differentiation of aflatoxin-producing and non-producing strains of aspergillus flavus group. appl microbiol 33(4): 291-5. davari e, mohsenzadeh m, mohammadi gh, rezaeiandoloei r. 2014. characterization of aflatoxigenic aspergillus flavus and a. paraciticus isolates from animal feedstuffs in northeastern iran. ijvr 16(2): 150-5. davis nd, iyer sk, diener ul. 1987. improved method of screening for aflatoxin with a coconut agar medium. appl environ microbiol 53(7): 1593-5. degola f, berni e, dall’asta c, spotti e, marchelli r, ferrero i, restivo fm. 2007. a multiplex rt-pcr approach to detect aflatoxigenic strains of aspergillus flavus. j appl microbiol 103(2):409-17. doi: 10.1111/j.1365-2672.2006.03256.x dharmaputra os. 2002. review on aflatoxin in indonesian food and feedstuffs and their products. biotropia 19: 26-46. donner m, atehnkeng j, sikora, ra, bandyopadhyay r, cotty pj. 2010. molecular characterization of a toxigenic strains for biological control of aflatoxins in nigeria. food addit contam 27(5): 576-90. doi: 10.1080/19440040903551954 kim dm, chung sh, chun hs. 2011. multiplex pcr assay for the detection of aflatoxigenic and nonaflatoxigenic fungi in meju, a korean fermented soybean food starter. food microbiol 28: 1402-8. kumar s, stecher g, li m, knyaz c, tamura k. 2018. mega x: molecular evolutionary genetics analysis across computing platforms. mol biol evol 35(6):1547-9. doi:10.1093/ molbev/msy096 levin re. 2012. pcr detection of aflatoxin producing fungi and its limitations. int j food microbiol 156(1): 1-6. lin mt, dianese jc. 1976. a coconut-agar medium for rapid detection of aflatoxin production by aspergillus spp. phytopathology 66: 1466-9. mamo ft, selvaraj jn, wang y, liu y. 2017. recent developments in the screening of atoxigenic aspergillus flavus towards aflatoxin biocontrol. appl environ microbiol 5(1): 20-30. pitt ji, hocking ad. 2009. fungi and food spoilage. 3rd edition. new york (us): springer. 519 p. qiagen. 2019. dneasy plant handbook. germany. rao kr, vipin av, venkateswaran g. 2020. molecular profile of non-aflatoxigenic phenotype in native strains of aspergillus flavus. arch microbiol 202(5):1143-55. doi:10.1007/s00203-020-01822-1 samson ra, hoekstra es, frisvad jc. 2004. introduction to food and airborne fungi. 7th edition. utrecht (nl): centraalbureau voor schimmelcultures (cbs). schmidt-heydt m, magan n, geisen r. 2008. stress induction of mycotoxin biosynthesis genes by abiotic factors. fems microbiol lett 84(2): 142-9. doi:10.1111/j.1574-6968.2008.01182.x tamura k, dudley j, nei m, kumar s. 2007. mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol biol evol 24(8): 1596-9. tran-dinh n, pitt ji, markwell pj. 2014 selection of nontoxigenic strains of aspergillus favus for biocontrol of afatoxins in maize in thailand. biocontrol sci technol 24: 652-66. wei d, zhou l, selvaraj jn, zhang c, xing f, zhao y, wang y, liu y. 2014. molecular characterization of a toxigenic aspergillus flavus isolates collected in china. j microbiol 52:559-65. doi:10.1007/ s12275-014-3629-8. white tj, bruns td, lee sb, taylor jw. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj, eds. pcr protocols. a guide to methods and applications. san diego (us): academic press. p. 315-20. yin y, lou t, yan l, michailides tj, ma z. 2009. molecular characterization of toxigenic and a toxigenic aspergillus flavus isolates, collected from peanut fields in china. j appl microbiol 107: 1857-65. yu j, bhatnagar d, ehrlich kc. 2002. aflatoxin biosynthesis. iberoam micol 19: 191-200. biotropia no. 5, 1991/1992: 1-9 production structure of main commercial tree species in a mangrove forest in east sumatera, indonesia cecep kusmana*) faculty of forestry, bogor agricultural university, bogor, indonesia and hiroyuki watanabe faculty of agriculture, kyoto university, kyoto 606, japan abstract production structure of main commercial tree species was studied in a mangrove forest in east sumatera, indonesia. this research was carried out in january 1991 using the estimation of standing biomass with stratified clipping method in order to know the production structure of the main commercial tree species in this mangrove forest, i.e. rhizophora apiculata, bruguieraparviflora and b. sexangula. the results obtained show that r. apiculata tended to have a sparser foliage of thicker leaves along the stem than b. parviflora or b. sexangula; therefore, r. apiculata is regarded as a shade-intolerant tree species. in contrast, either b. parviflora or b. sexangula tended to have a larger proportion of leaves and branches along the stem; consequently, those species are recognized as shade-tolerant tree species. introduction a substantial proportion of about 81 000 km coastal line in indonesia is covered by mangrove formations of various extents, from several meters to several kilometers (soegiarto 1979). in terms of tree size and extent, mangroves are more developed in the five big islands, i.e. java, sumatera, kalimantan, sulawesi, and irian jaya (darsidi 1984). in sumatera, the mangrove covers an area of 667 000 ha of which 276 000 ha are distributed in riau. recently, about 87 000 ha of mangrove forest in riau have been designated as forest concession areas belonging to the bina lestari company (42 000 ha), thai rayvithi company (40 000 ha) and silva saki company (5000 ha). for many years, the mangrove trees in riau were cut for fuelwood and used for charcoal either by forest concessionaires or local people inhabiting the region surrounding the mangroves. however, recently, most of the commercial tree species, especially bruguiera spp. and rhizophora apiculata are being exploited for the production of chipwood. *)present address: faculty of agriculture, kyoto university, kyoto 606, japan 1 biotropia no. 5, 1991/1992 it is highly necessary to establish a proper management system based upon biological knowledge to secure a sustained yield of mangrove resources. therefore, the production structure of mangrove tree species must be studied to understand the biological characteristics of mangroves. this research was undertaken in an attempt to analyze the production structure of the main commercial tree species (bruguiera parviflora, b. sexangula and r. apiculata) in the mangrove forest of talidendang besar, riau, east sumatera. study area and methods this research was carried out in january 1991 in a mangrove forest concession area belonging to the bina lestari company at talidendang besar (long. 103°28' to 103°48' e, lat. 0°21' to 1°n), which lies on the east coast of sumatera, riau province, indonesia (fig. 1). no meteorological data were available for talidendang besar. the rainfall data of mandah, the nearest area to talidendang besar, provide a good approximation of the climatic conditions (fig. 1). the average annual rainfall is about 1335 mm. according to the schmidt and ferguson system (1951), the climate of this area belongs to the type b rainfall with seven wet, two dry and three humid months. the mangrove forest in this area is characterized by the bruguiera species. the seaward fringe is strongly dominated by b. parviflora and landward zones are numerically dominated by b. sexangula. in the transition zone with a fresh-water swamp forest, the stand of b. sexangula is invaded by nypa fruticans, in which a few large individual trees of r. apiculata form an emergent layer above an even canopy of b. sexangula. in this zone, a few trees of a marginal species, ficus benjamina, occur in open areas created by fallen or dead trees and sometimes intermingled with n. fruticans along the river bank. sample trees of b. parviflora, b. sexangula and r. apiculata which have similar diameter sizes were felled. stem diameter at breast-height (dbh) and tree height of sample trees were 20.1 cm and 19.4 m for b. parviflora, and 19.2 cm and 17.6m for b. sexangula, respectively. however, the diameter at 20 cm above the highest prop-roots and the height of a felled sample tree of r. apiculata were 19.5 cm and 22.8 m, respectively. sample trees were felled at ground level and cut into horizons of 1 m length along the stem. fresh weight of stems, branches, flowers and fruits, and leaves of each horizon were separately weighed. a small sample of each organ from each horizon was taken and dried in an oven at 80°c for 48 hours to obtain a constant dry weight. the leaf area of a small sample of leaves from each horizon was measured with a leaf-area meter (type aam-7, hayashi denko). 2 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe figure 1. location and climatic diagram of the research area 3 biotropia no. 5, 1991/1992 results and discussion results tables 1, 2 and 3 show the estimated aboveground biomass of anatomical organs of each tree, the leaf area and specific leaf area of a sample tree of r. apiculata, b. sexangula and b. parviflora, respectively. the aboveground biomass of sample trees of r. apiculata, b. sexangula and b. parviflora were estimated at 381 kg d.wt, 387.70 kg d.wt and 296.36 kg d.wt, respectively. compared to the sample trees of r. apiculata and b. sexangula, a sample tree of b. parviflora has a smaller aboveground biomass, although its diameter (dbh) was similar to the sample trees of the two former species. the leaves as a photosynthetic part contributed very little to biomass matters, i.e. 2.48%, 3.61% and 4.48% for r. apiculata, b. sexangula and b. parviflora, respectively. the total leaf area as well as the mean specific leaf area of r. apiculata were smaller than those of the other species. it was estimated that the mean specific leaf areas were 61.19 cmvg d.wt for r. apiculata, 77.57 cmvg d.wt for b. sexangula, and 80.80 cmvg d.wt for b. parviflora. it indicates that the leaves of r. apiculata were thickest, while the leaves of b. sexangula were thicker than those of b. parviflora. the production structure of the main commercial trfee species is shown in fig. 2. the branches and leaves of r. apiculata were shallower and sparser than those of b. sexangula and b. parviflora. the large amount of leaves and branches of r. apiculata in the stem horizon of 13 to 14 m was due to the existence of large main branches with a diameter of 13.5 cm. in addition, b. sexangula has a larger proportion of branches than the other species. discussion as indicated by the mean specific leaf area (table 1) and vertical production structure (fig. 2), r. apiculata is recognized to have a sparser foliage of thicker leaves than b. sexangula and b. parviflora. it is suggested that one characteristic of/?, apiculata is to be a shade-intolerant tree species as reported by macnae (1968). in the mangrove forest of halmahera, maluku province in indonesia, komiyama et al. (1988) reported that r. apiculata has a sparser foliage of thicker leaves than b. gymnorrhiza. similar results were also reported by ninomiya et al. (1989) for r. stylosa and b. gymnorrhiza in the mangrove forest of funaura bay, iriomote island in okinawa, and by nakasuga (1979) for r. mucronata and b. conjugata in the ryukyu islands, southern japan. 4 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe table 1. estimated aboveground biomass, leaf area (la) and specific leaf area (sla) of the sample tree of rhizophora apiculata horizon aboveground biomass (kg d.wt) la sla (m) leaf flowers and branch stem prop-roots total (m 2 ) (cm 2 /g d.wt) fruits 01 9.57 16.74 26.31 12 22.65 13.77 36.42 23 22.33 22.33 34 19.46 19.46 45 18.82 18.82 56 18.82 18.82 67 18.50 18.50 7g 0.07 0.01 6.68 16.59 23.35 0.36 66.09 89 16.27 16.27 9-10 0.08 0.01 0.33 15.63 16.05 0.52 62.23 10-11 14.36 14.36 11-12 14.67 14.67 12-13 12.44 12.44 13-14 5.34 0.71 63.67 10.53 80.25 21.15 50.49 14-15 0.03 0.01 0.17 5.42 5.63 0.09 67.87 15-16 5.30 5.30 16-17 0.14 0.01 0.45 4.72 5.32 0.62 50.38 17-18 0.45 0.04 1.67 4.15 6.31 2.08 50.43 18-19 0.50 0.07 2.17 3.13 5.87 2.12 68.11 19-20 1.37 0.16 4.07 2.04 7.64 6.44 63.37 20-21 0.71 0.10 1.70 1.40 3.91 3.63 62.56 21-22 0.59 0.11 1.11 0.64 2.45 2.46 63.87 22-22.8 0.17 0.03 0.18 0.14 0.52 0.68 67.68 total 9.45 1.26 82.20 257.58 30.51 381.00 40.15 673.08 average 61.19 the prop-roots constituted about 8% of the aboveground biomass of a sample tree of r. apiculata. it is supposed that in this area the stand of r. apiculata grows in estuarine conditions. lugo and snedaker (1974) also reported the low proportion of prop-roots (2%, 15%) in estuarine forest in the mangroves of florida. in addition, christensen (1978) stated that the small proportion of prop-roots of rhizophora stand growing in the estuarine conditions might be due to a better nutrient supply or reduced salinity regime. the existence of the prop-roots on the stem may be regarded as a characteristic to distinguish the production structure of r. apiculata from that of b. sexangula and b. parviflora. on the other hand, b. parviflora and b. sexangula tended to have a greater proportion of leaves and branches along the stem than r. apiculata. 5 biotropia no. 5, 1991/1992 table 2. estimated aboveground biomass, leaf area (la), and specific leaf area (sla) of the sample tree of bruguiera sexangula horizon aboveground biomass (kg d.wt) la sla (m) (m 2 ) (cm 2 /g d.wt) leaf flowers and fruits branch stem total 0 1 40.52 40.52 12 23.73 23.73 23 23.79 23.79 34 21.90 21.90 45 20.44 20.44 56 0.15 0.01 0.77 18.62 19.55 1.53 97.96 67 17.16 17.16 78 0.21 0.03 2.71 18.25 21.20 2.34 79.87 89 0.46 0.05 3.84 17.16 21.51 4.36 75.73 910 0.27 0.04 2.07 15.70 18.08 2.55 77.36 10-11 2.86 0.37 29.68 16.79 49.70 21.22 80.49 11-12 4.09 0.61 39.65 12.05 56.40 21.48 74.75 12-13 0.14 0.02 0.89 6.75 7.80 1.51 75.29 13-14 1.69 0.27 11.21 5.48 18.65 10.86 80.41 14-15 1.62 0.24 6.93 3.87 12.66 9.59 74.99 15-16 1.56 0.34 6.84 1.61 10.35 8.83 71.12 16-17 0.11 0.12 2.04 0.91 3.88 5.54 72.84 17 17.6 0.14 0.02 0.15 0.07 0.38 0.70 70.05 total 14.00 2.12 106.78 264.80 387.70 90.51 930.86 avera ge 77.57 conclusion while the leaves of b. parviflora were thinner than those of b. sexangula, rhizophora apiculata has a sparser foliage of thicker leaves than the two former tree species. in addition, either b. parviflora or b. sexangula has a larger proportion of leaves and branches along the stem than r. apiculata. it is suggested that one characteristic of r. apiculata is a shade-intolerant tree species whereas b. parviflora and b. sexangula are shade-tolerant tree species. 6 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe table 3. estimated aboveground biomass, leaf area (la), and specific leaf area (sla) of the sample tree of bruguiera parviflora horizon aboveground biomass (kg d.wt) la sla (m) (m 2 ) (cm 2 /g d.wt) leaf flowers and fruits branch stem total 0 1 28.81 28.81 12 21.11 21.11 23 18.43 18.43 34 17.76 17.76 45 17.42 17.42 56 16.08 16.08 67 15.08 15.08 78 14.41 14.41 89 0.50 0.07 4.43 13.40 18.40 5.41 109.16 9-10 4.30 0.39 23.78 12.40 40.87 31.89 73.13 10-11 0.64 0.11 2.71 9.72 13.18 5.04 81.96 11-12 8.71 8.71 12-13 0.09 0.02 0.21 7.71 8.03 0.73 99.01 13-14 1.66 0.32 9.68 8.04 19.70 14.36 85.18 14-15 1.99 0.35 8.64 4.69 15.67 12.91 75.75 15-16 1.11 0.22 4.43 3.02 8.78 6.97 71.12 16-17 2.06 0.45 6.23 1.34 10.08 13.50 85.03 17-18 0.83 0.18 1.80 0.67 3.48 5.56 73.67 18-19 0.08 0.01 0.11 0.10 0.30 0.47 68.36 19 19.4 0.03 0.01 0.01 0.01 0.06 0.15 66.47 total 13.29 2.13 62.03 218.91 296.36 96.99 888.84 average 80.80 acknowledgements we wish to thank mr. subkhan, student of the faculty of forestry, bogor agricultural university for helping in the collection of data in the field; dr. dudung darusman, dean of the faculty of forestry, bogor agricultural university for his kind assistance in providing admission letters; prof .dr. ishemat soerianegara and ir. syafii manan, msc., faculty of forestry, bogor agricultural university for their encouragement throughout this research. 7 biotropia no. 5, 1991/1992 8 f ig u re 2 . p ro d u c ti o n s tr u c tu re o f a s a m p le t re e o f r . a p ic u la ta ( le ft ), b . p a rv if lo ra ( m id d le ), a n d b . se x a n g u la ( ri g h t) production structure of main commercial tree species cecep kusmana & hiroyuki watanabe references christensen, b. 1978. biomass and primary production of rhizophora apiculata bl. in a mangrove in southern thailand. aquatic botany 4: 43-52. darsidi, a. 1984. mangrove forest management in indonesia. in: soemodihardjo, s., i. soerianegara, m. sutisna, k. kartawinata, supardi, n. naamin and h. al rasyid (eds.), proceedings symposium ii on mangrove ecosystem: p. 27-37. mab, lipi, jakarta. komiyama, a., h. moriya, s. prawiroatmodjo, t. toma and k. ogino. 1988. forest as an ecosystem, its structure and function: 2. primary productivity of mangrove forest. in: k. ogino and m. chihara (eds.), biological system of mangroves: a report of east indonesian mangrove expedition 1986: 97-106. ehime university, ehime, japan. luoo, a.e. and s.c. snedaker. 1974. the ecology of mangroves. ann. rev. ecol. syst. 5: 39-65. macnae, w. 1968. a general account of the fauna and flora of mangrove swamps and forests in the indo west pacific region. adv. mar. biol. 6: 73-270. nakasuga, t. 1979. analysis of the mangrove stand. sci. bull. coll. agric. univ. ryukyus 26: 413 519 (in japanese with english summary). ninomiya, i., a. komiyama, h. moriya, y. tadaki, and k. ogino. 1989. production structure of rhizophora stylosa and bruguiera gymnorrhiza in funaura, iriomote. galaxea 8: 65-68. schmidt, f.h. and j.h.a. ferguson. 1951. rainfall type based on wet and dry period ratios of indonesia with western new guinea. verhandelingen no. 42, kementrian perhubungan, djawat an meteorologi dan geofisika, jakarta. 77 p. soegiarto, a. 1979. the indonesian marine environment: its problems and management. ber. ilmu peng. teknol. 23 (1): 9-21. 9 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 3. suryo wiyono.cdr biotropia vol. 20 no. 1, 2013: 19 28 powder formulation of antagonistic yeasts, and as biofungicides cryptococcus albidus cryptococcus terreus suryo wiyono* and widodo recipient of biotrop research grant 2009/accepted 14 may 2012 this study aimed 1) to investigate the compatibility of yeast antagonists i.e. and 2) to obtain suitable carrier in powder formulation for those two yeasts, 3) to get appropriate formulation additives for those yeasts, and 4) to obtain optimum powder formulation consisting of yeasts, carrier materials and additives. compatibility of and were tested through bio-assay against on detached banana fruit and on detached tomato leaves. compatibility was indicated by no reduction of antagonistic activity. benomyl-resistant mutant of and cycloheximide-resistant mutant of were mixed with sterilized tapioca, talc and kaolin to get initial yeasts density of 8.5 log cfu/g and water content of 15%, then packed in plastic bag and stored under room temperature. survival of formulated yeasts was assessed monthly by planting on pda medium containing 150 ppm cycloheximide for , and 150 ppm benomyl for . yeasts population was expressed in log cfu/g materials. to determine the effect of carrier materials on antagonistic activity, io-assay of formulated yeasts against pathogens was conducted after 3 months of storage. tested additives cacl , pure chitin and crab shell powder were added into suspension of and to get concentration of 1.25%, 0.5 % and 0.1% (w/v). sterilized distilled water and yeasts without additives was used as control. then, the treatments were examined for the antagonistic activity through bio-asaay on detached banana fruits and tomato leaves. appropriate additive(s) was determined by its ability to increase antagonistic activity of yeasts. storability and antagonistic activity of and in the mixture of best carrier and additive were examined. was compatible to . talc was the best carrier material supporting highest survival of , without contamination of other microorganisms. talc was the best carrier material for formulation by maintaining its survival for six months of storage. additives able to increase antagonistic activity of were cacl 0.1 %, pure chitin 0.1 % and crab shell powder 0.5%. all of tested additives materials did not affect antagonistic activity of supplementation of chitin and crab shell, both at the rate of 1.25% into talc-base powder formulation increased survival of and . formulation, yeast antagonists carrier agent, talc, tapioca, kaolin, cacl , chitin, crab shell powder 1 department of plant protection, faculty of agriculture, bogor agricultural university darmaga, bogor-indonesia c. albidus c. terreus, c. albidus c. terreus lasiodiplodia theobromae alternaria solani c. albidus c. terreus c. terreus c. albidus b i.e. c. albidus c. terreus c. albidus c. terreus cryptococcus terreus c. albidus c. terreus c. albidus c. terreus c. albidus. c. terreus c. albidus , cryptococcus terreus, cryptococcus albidus, abstract 2 2 2 key words: * corresponding author : suryow@hotmail.com 19 introduction a group of novel promising biocontrol agents of plant disease are antagonistic yeasts. the advantage of yeast as biocontrol agent is dry and heat tolerance, therefore, adaptable on leaves and other aerial plant parts. it is fast growing, easy to be mass cultured and socially more acceptable (spadaro 2003). research in foreign countries showed the effectiveness of yeast in controlling plant disease. a yeast antagonist was reported effective to control apple rot disease caused by (spadaro 2003). moreover, near harvest application of effectively control post harvest disease of grapefruit (karabulut . 2003). on tomato plants was reported to be effectively controlled by the use of epiphytic yeasts strains 101 and us 7 and (saligkarias . 2002). and collected by the authors have been screened for their antagonistic activity , bio-assay , field test and partially characterized. and were effective yeast antagonists against stem end rot of mango caused by and petal blight of orchid caused by , respectively (wiyono 2008; sugiprihatini 2009). recently, the second yeast is also effective tested against leaf blight of tomato caused by and chrysanthemum white rust caused by . in addition, fan and tian (2001) reported that is also effective in controlling apple post harvest diseases i.e. grey mold and blue mold. after obtaining superior isolates of antagonists, technology for mass production and formulation is required to develop an antagonist as biocontrol agent on commercial scale. since those two yeasts are relatively easy to be mass cultured in a cheap medium (potato dextrose broth pdb), formulation technology is a critical step. suitable formulation technology is the main limiting factors in developing microbial pesticides in indonesia, even high numbers of effective antagonists had been isolated (santoso . 2005). appropriate formulation technology will facilitate storage, transportation, application technique and also bio-performance of antagonists. solid formulation is chosen to be developed for easier transportation and handling. formulation of beneficial microbes contains microbes, carrier and additives. combination of microbes will broaden the target of bio-fungicide. optimization of whole components ensures the quality of formulation and further commercial application. some minerals such as kaolin and talc can be used as carrier in formulation of and (kinay & yildiz 2008). additives play also an important role in biocontrol performance. calcium chloride and chitin were used as additive enhancing antagonistic activity of yeasts antagonists and . compatibility of the two yeast antagonists, appropriate as carrier materials and additives for and has not been investigated yet. the objectives of the research were: 1) to assess the compatibility of two yeast antagonists and , 2)to obtain cheap material as carrier agent which ensure long time survival and bio-performance of and 3) to get formulation additives supporting storability and bioperformance of yeast antagonist metschnikowia pulcherrima penicillium metschnikowia fructicola et al botrytis cinerea candida guilliermondii candida oleophila et al cryptococcus albidus cryptococcus terreus in vitro in vivo cryptococcus albidus cryptococcus terreus lasiodiplodia theobromae curvularia pallescens alternaria solani puccinia horiana crypococcus albidus et al metschnikowia pulcherrima pichia guilliermondii candida guilliermondii, pichia membranefaciens, cyptococcus laurentii c.albidus c. terreus c. albidus c. terreus c. albidus c. terreus, 20 biotropia vol. 20 no. 1, 2013 c. albidus c. terreus cryptococcus albidus c. terreus c. albidus c. terreus lasiodiplodia theobromae alternaria solani lasiodiplodia theobromae alternaria. solani c. albidus c. terreus l. theobromae a. solani c albidus, c. terreus c albidus c. terreus c. albidus c. terreus and ., and 4) to obtain optimum solid formulation in combination with yeasts, carrier material and additives antagonistic yeasts used were and obtained from the author's collection. for formulation purpose, benomyl-resistant mutant of which is similar to antagonistic activity was generated. in addition, resistant to cycloheximide was used in formulation experiment. and collections of the author were used for bio assay of yeasts. both of the two yeast antagonists were cultured in potato dextrose broth (pdb difco) and harvested at early stationary phase of growth, centrifuged at 5000 g (jouan centrifuge br4i), washed with sterilized distilled water, mixed with saline solution adjusted to appropriate density. was cultured on pda (difco) ph 5.5 for seven days and the conidia harvested by soaking water on the surface and filtered with cheese cloth. was cultured on s-medium and incubated for seven days under nuv exposure to induce sporulation (abadi 1987), prior to harvesting of conidia io-assay of yeast antagonists was conducted based on previous technique developed by the authors using detached banana fruits and detached leaves of tomato. the detached organs were placed on moistened plastic pans (30 cm x 25 cm x 5 cm), one plastic pan contained five banana fruits or tomato leaves. detached banana fruits were dipped in cell suspension of at 7 log cfu /ml, while the detached leaves were dipped in cell suspension of 7 log cfu/ml, both added with wetting agent tween 20, 0.005 %. then the treated banana fruits and tomato leaves were air dried. sterilized distilled water was used as control. conidia suspension at 50 µl of and , both at the density of 10 conidia /ml was placed on the surface of fruit and leaves, respectively. inoculated fruits and leaves were stored under dark condition for 24 hours and then incubated under room temperature (27 c) and photoperiods of 12: 12 (d:l). disease severity was assessed by estimating of necrosis part at five days after inoculation and expressed in percent. effective treatment was indicated by low disease severity he treatment consists of , a mixture of and , and untreated (water). each treatment was replicated five times. yeast concentration used for compatibility test was 5 x 10 cfu/ml for and 5 x 10 cfu/ml , therefore obtaining final concentration of 7 log cfu/ml, with ratio of 1:1, and added with wetting agent tween 20, 0.005 %. bio-assay was conducted on detached fruit of banana and detached leaves of tomato (see bio-assay of antagonistic activity). . . b . t materials and methods yeast and pathogen preparation bio-assay of antagonistic activity of yeasts compatibility of and 4 o 6 6 c. albidus c. terreus 21 powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. if the two yeasts are compatible, it will be further tested in a formulation experiment aterials tested for carrier were kaolin powder, talc powder and tapioca. each material was regarded as treatment and replicated five times. the materials were sterilized by standard autoclaving. suspension of two yeast antagonists and its mixture ( preparation mentioned above) was mixed by spraying (hand sprayer yoto 1-l, made in indonesia) yeast suspension on the tested materials in running blender, then air dried to get final yeast density of 8.5 log cfu/g with 15% of water content of formulation. materials containing yeast were then packed in plastic bags and stored under room temperature for 6 months. each material containing yeast was assessed for yeast survival and antagonistic activity every 30 days for six months. survival of yeast in each carrier materials was determined by plating on pda (difco) ph 5.5 containing 150 ppm benomyl for , and 150 ppm cycloheximide for colony isolated from each carrier material of three month storage was then tested for its antagonistic activity using the technique as described in bioassay of antagonistic activity aterials screened for additives are pure chitin (sigma), natural material containing chitin i.e. crab shell powder, and calcium chloride. methods for testing the formulation additives are based on previously developed technique (wiyono . 2008). each material was tested with water-based suspension/solution at the rate of 1.25 %; 0.5%; 0.1% (w/v). suspension of and at density of 7 log cfu/ml mixed with the tested additives and final concentration of additives was adjusted to the tested concentration. yeast suspension without addition of tested materials was used as control. yeasts with various additives treatment was furthermore tested for their antagonistic activity using the technique as described in bioassay of antagonistic activity est results obtained from previous experiment with yeasts compatibility, carrier materials and additives were then continued to test for suitable powder formulation . combination of yeasts, selected carrier materials, selected additives in powder formulation was conducted. the survival and antagonistic activity were also assessed every 30 days for four months he research showed that was compatible to . effectiveness . m . m . b . t screening for carrier materials screening for additives combination of suitable yeast composition, carrier materials and additives compatibility of dan c. albidus c. terreus. et al c. albidus c. terreus c. terrreus c. albidus results and discussion c. terreus c. albidus 22 biotropia vol. 20 no. 1, 2013 23 of the two yeast antagonists did not decrease in mixed application (tables 1 and 2). the compatibility of the two yeasts make broader spectrum of the mixture. one important advantage of antagonists combination is the possibility to obtain a broader spectrum (burges & jones 1998). survival of dan in various carrier materialsc. terreus c. albidus c . e . t arrier materials are important component of formulation for maintaining microbes survival and antagonistic activity. among materials tested for , tapioca provided the highest survival. overall, could survive in all tested carriers for four months. during five months of storage could not be detected anymore in all of the tested materials (table3). talc powder provided highest survival for (table 4). up to six months of storage, talc powder still resulted in relatively high survival (4.60 log cfu/g) ven though tapioca is the best material for storage, it has high contamination level of other fungi. tapioca is an organic flour, hence it can act as nutrition for some other fungi and bacteria. this was not the case with mineral powder such as kaolin and talc. kinay and yildiz (2007) stated that talc in granular formulation is able to provide storability of antagonistic yeasts and for more than 6 months. storability of talc was better than kaolin. storability of talc in this experiment was lower than that reported by kinay and yildiz (2007), because this experiment used powder formulation instead of granular form he use of talc as carrier material in powder formulation is also able to maintain bio-performance of the two yeasts in storage (tables 5 and 6). antagonistic activity of c. terreus c. terreus c. terreus c. albidus c. albidus c. terreus metschnikowia pulcherrima pichia guilliermondii table 1. compatibility of and in controlling fruit rot of banana caused by cryptococcus albidus c. terreus l. theobromae treatment disease severity (%) untreated 34.53 b cryptococcus albidus 26.00ab cryptococcus terreus c. terreus+ c. albidus 7.53 a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 2. compatibility of and in controlling alternaria leaf blight of tomato caused by cryptococcus albidus c. terreus alternaria solani treatment disease severity (%) untreated 10.58 b cryptococcus terreus 5.55 a c. terreus+ c. albidus 5.48 a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. 24 biotropia vol. 20 no. 1, 2013 table 3. survival of on various carrier materials in powder formulationc. terreus 5 carrier materials yeast density (log cfu/g) in i-th month 0 1 2 3 4 tapioca 8.70 a 7.06 b 7.23 b 7.36 b 4.18 b talc 8.65 a 6.00 a 6.54 a 5.69 a 4.50 b kaolin 8.65 a 6.67 a 6.24 a 6.26 a 3.82 a note: = undetected numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 4. survival of on various materials in powder formulationc. albidus carrier materials yeast density (log cfu/g) in i-th month 0 1 2 3 4 5 6 tapioca 8.24 a 5.95 a 6.98 a 4.21 a talc 8.72 a 5.70 a 6.65 a 5.98 b 6.17 4.20 4.65 kaolin 8.60 a 5.25 a note: -= undetected numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 5. antagonistic activity of formulated with different carrier materials against alternaria leaf blight of tomato (after 3 months storage) c. terreus carrier materials alternaria leaf blight severity (%) control (water) 7.64 b unformulated fresh yeast 2.13 a tapioca 0 a talc 2.12 a kaolin 1.13a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 6. antagonistic activity of formulated with different carrier materials against fruit rot of banana (after 3 month storage) c. albidus carrier materials fruit rot severity (%) control (water) 16.23 b unformulated fresh yeast 5.24 a tapioca 9.14 a talc 5.22 a kaolin note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test 25 c. albidus c. terreus pseudomonas fluorescens et al c. terreus c. albidus, and in all tested materials (except kaolin) did not decrease after 3 months of storage indicated by no significant difference to unformulated fresh yeast ormulation additives such as additional nutrients are often important in formulation of beneficial microbes. some formulation additives are able to increase antagonistic activity of antagonistic microbes, for example zinc and manganese can improve antagonistic activity of b5 (wiyono . 2008). among tested additives, ca cl 0.1%, pure chitin 0.1% and crab shell powder 0.5% significantly increase antagonistic activity of (table 7). even though there was no significant difference among the three treatments, crab shell powder provided highest increase of antagonistic activity dditives treatment did not increase significantly antagonistic activity of however cacl 0.5%, crab shell powder 1.25% and pure chitin 1.25% tend to increase . f . a additives-mediated enhancement of antagonistic activity of and . c. terreus c. albidus 2 2 table 7. effect of additives on the antagonistic activity of c. terreus treatment conc. (% w/v) alternaria leaf blight severity (%) water 25.45 e yeast without additives 9.52 cd ca cl2 1.25 7.12 abcd ca cl2 0.5 5.35 abc ca cl2 0.1 4.52 ab chitin 1.25 10.00 d chitin 0.5 8.35 bcd chitin 0.1 4.25 ab crab shell powder 1.25 4.80 ab crab shell powder 0.5 3.14 a crab shell powder 0.1 3.94 ab note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 8. effect of additives on the antagonistic activity of c. albidus treatment conc. (% w/v) fruit rot severity (%) water 55.00 c yeast without additives 35.00 ab ca cl2 1.25 41.25 ab ca cl2 0.5 21.25 a ca cl2 0.1 43.75 ab chitin 1.25 15.75 a chitin 0.5 53.75 b chitin 0.1 43.75 ab crab shell powder 1.25 21.25 a crab shell powder 0.5 41.25 ab crab shell powder 0.1 43.75 ab note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. 26 biotropia vol. 20 no. 1, 2013 antagonistic activity (table 8). furthermore, crab shell powder 1.25% was used in powder formulation containing mixture of and , because its effect is not significant to chitin and its price is far cheaper than pure chitin the research result was in line with previous researches in other yeasts. antagonistic activity of yeast (various species or isolates) can be enhanced by addition of calcium chloride (tian . 2002; abadias . 2003), pure chitin (vivekananthan . 2004; yu . 2007). calcium chloride could increase the biocontrol efficacy of tested yeast due to its ability to enhance plant resistance against plant diseases (biggs . 1997; droby . 2003). the same mechanism is similar for chitin (yu . 2007). this research resulted in a new finding that crab shell powder, a chitin-containing material, provides the same level of enhancement compared to pure chitin. the mechanism how crabshell increase antagonistic activity of is not exactly known, probably it involves induction of plant resistance. harti (2010) reported induced resistance of banana against fusarial wilt diseases after treated with crab shell powder. aside from chitin, crab shell contains protein, calcium, phosphate, and other elements such as iron, manganese and zinc (multazam 2002), therefore effect of composing elements of crab shell powder on antagonistic activity could not be ignored. this is an advantage since using crab shell powder is cheaper than pure chitin. the use of crab shell powder does not need chemicals for processing, so it is costeffeective. ne strategy to improve storability and bio-performance of formulated microbes is by providing additives (burges & jones 1998; fravel 2005; wiyono 2008). some additives of antagonistic yeasts are metal ion (calcium), sugar (trehalose), biopolymer (chitin) and calcium (abadias . 2003). addition of crab shell 1.25% into talc-based c. terreus c. albidus et al et al et al et al et al et al et al c. terreus et al. et al . o effect of crab shell powder as additives in talc-based powder formulation of and c. terreus c. albidus table 9. survival of in talc formulation and supplemented with additivesc. terreus yeast density (log cfu/g) in i-th month 0 1 2 3 talc 8.7 a 6.15 a 6.54 a 5.02 a talc + chitin 8.19 a 7.89 b 6.75 a 5.02 a talc +crab shell powder 8.73 a 7.00 ab 6.07 a 6.07 b numbers followed by same symbol are not significantly different at significant level of 0.05 with dmrt test table 10. survival of in talc formulation and supplemented with additivesc. albidus yeast density (log cfu/g) in i-th month 0 1 2 3 talc 8.87 a 6.06 a 6.50 a 5.26 a talc + chitin 9.31 a 7.56 b 6.48 a 5.14 a talc + crab shell powder 9.56 a 7.30 b 6.22 a 6.06 b numbers followed by same symbol are not significantly different at p<0.05 with drmt test 27 formulation increased survival of and (tables 9 and 10). further studies on the exploration and optimization of various additives are needed to prolong survival rate of formulated yeasts is compatible to therefore it can be used in a mixture formulation. best carrier materials in powder formulation of was talc, able to maintain yeast survival for four months of storage without contamination of other microorganisms. talc powder provided best survival of , with survival more than 5 months of storage. crab shell at concentration of 1.25% can be used as additive in powder formulation containing a mixture of and based on its enhancement of antagonistic activity on and survival of both antagonistic yeasts he authors would like to acknowledge seameo biotrop for providing financial support through dipa 2009. moreover, the authors express their gratitude to people involved in this research, ms ratih munawaroh and mr. dadang surahman at the laboratory of plant mycology dept of plant protection, faculty of agriculture, bogor agricultural university c. terreus c. albidus . terreus c. albidus, c. terreus c. albidus c. terreus c. albidus, c, terreus . . t . conclusions acknowledgments references c abadi al. 1983. antagonism between leaf surface microorganism and causal agent of early blight of tomato ( ). master thesis. graduate school bogor agricultural university, bogor (in bahasa indonesia). abadias m, usall j, teixidó n, viñas i. 2003. liquid formulation of the postharvest biocontrol agent cpa-1 in isotonic solutions. phytopathology 93:436-42. biggs ar, el-kholi mm, el-neshawy s, nickerson r. 1997. effects of calcium salts on growth, polygalacturonase activity, and infection of peach fruit by plant dis 81:399-403. burges hd, jones ka. 1998. trends in formulation of microorganisms and future research requirements. in: burges, hd (ed.), formulation of microbial biopesticides, beneficial microorganisms, nematodes and seed treatment. kluwer academic publication, dordrecht, pp. 311-332. droby s, wisniewski m, el ghaouth a, wilson c. 2003. influence of food additives on the control of postharvest rots apple and peach and efficacy of the yeast-based biocontrol product aspire. post harvest biol technol 27: 127-35. fan q, tian sp. 2001. postharvest biological control of grey mold and blue mold on apple by (saito) skinner. postharvest biol technol 21: 341-50. fravel dr. 2005. commercialization and implementation of biocontrol. ann rev phytopathol 43: 337-59. harti h. 2010. control of fusarial wilt ( f.sp. (e f smith)) of banana by using crabshell powder and chitosan. master thesis. bogor agricultural university, indonesia. alternaria solani, lycopersicon esculentum candida sake monilinia fructicola. cryptococcus albidus fusarium oxysporum cubense powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. karabulut oa, smilanick jl, mlikota gabler f, mansour m, droby s. 2003. nearharvest applications of , ethanol, and sodium bicarbonate to control postharvest diseases of grape in central california. plant dis 87:1384-9. kinay p, yildiz m. 2008. the shelf life and effectiveness of granular formulations of and yeast isolates that control postharvest decay of citrus fruit. 433-40. multazam. 2002. prospect for utilization of shell of crab ( sp.) as supplement of fish feed. department of fishery product technology. bogor agricultural university. bogor (in bahasa indonesia). saligkarias id, gravanis ft, epton has. 2002. biological control of on tomato plants by the use of epiphytic yeasts strains 101 and us 7 and strain i-182: ii. a study on mode of action. 25: 345-6. santoso t, prijono d, wiyono s, buchori d. 2005. development, uses and application of biopesticides in indonesia: current research and future challenge. paper presented on international conference on biopesticides. chiang-mai thailand, 13-18 february 2005. spadaro d. 2003. biological control of postharvest diseases of pome fruit using yeast antagonists. phd thesis. torino university. torino. sugiprihatini d. 2009. the use of yeasts antagonist and chitosan to control stem end rot disease of mango caused by pat. in storage (in bahasa indonesia). master thesis. graduate school. bogor agricultural university. tian sp, fan q, xu y, jiang al. 2002. effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen . l 51:352-8. wiyono s, schulz df, wolf ga. 2008. improvement of the formulation and antagonistic activity of b5 through selective additives in the pelleting process. biol control 46 : 348-57. wiyono. s. 2008. biological control of petal blight of dendrobium caused by research report (in bahasa indonesia) departemen of plant protection faculty of agriculture ipb. bogor. vivekananthan r, ravia m, saravanakumara d, kumarb n, prakasama v, samiyappan r. 2004. microbially induced defense related proteins against postharvest anthracnose infection in mango. crop prot 23: 1061-7. yu tl, yin wy, wang y, zheng x. 2007. effect of chitin on the antagonistic activity of against in pear fruit. 122: 44-8. metschnikowia fructicola metschnikowia pulcherrima pichia guilliermondii portunus botrytis cinerea candida guilliermondii candida oleophila botryodiplodia theobromae rhizopus stolonifer pseudomonas fluorescens curvularia pallescens. cryptococcus laurentii penicillium expansum biol control 45: biol control plant patho int j food microbiol biotropia vol. 20 no. 1, 2013 28 4. agus nuryanto.cdr edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. user2 callout rapd edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. user2 strikeout user2 callout gen24 user2 note hilangkan spasinya edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 microsoft word 46 biotropia no. 24, 2005 : 46 53 effect of antanan (centella asiatica) and vitamin c on the bursa of fabricius, liver malonaldehide and performance of heat-stressed broilers engkus kusnadi1, reviany wldjajakusuma2, toha sutardi3, peni.s.hardjosworo4 and arifien habibie5 'department of animal production.faculty of animal husbandry, andalas university(unand), padang, indonesia 2department of physiology and pharmacology,faculty of veterinary medicine, bogor agriculture university(ipb), bogor, indonesia 3department of animal nutrition & feed science, faculty of animal husbandry, bogor agriculture universirty(ipb), bogor, indonesia 4department of animal production, faculty of animal husbandry, bogor agriculture university (ipb), bogor, indonesia 1 deputy assistant for agroindustry-jakarta, coordinating ministry for economic affairs, jakarta, indonesia abstract high environmental temperatures may cause heat stress in poultry. this may increase water consumption, decrease feed consumption and in rum, decrease productivity level. in addition, high temperature contributes to oxidative stress, a condition where oxidant activity (free radicals) exceeds antioxidant activity. in our research, antanan (centelta asiatica) and vitamin c were utilized as anti heat-stress agents for heat-stressed broilers. we used 120 male broilers 2 6 weeks old, kept at 31.98 ± 1.94 °c during the day and 27.36 ± 1.31 °c at night. the data collected were analyzed with a completely randomized factorial design of 2 x 3 (2 levels of vitamin c, 3 levels of antanan at 4 replications) and continued with the contrast-orthogonal test when significantly different. the results indicate that the treatments of 5 and 10% of antanan with or without 500 ppm of vitamin c and vitamin c alone significantly (p<0.05) decreased the heterophil/lymphocyte (h/l) ratio and liver malonaldchydc (mda). these treatments, however, significantly (p<0.05) increased the bursa of fabricius weight, feed consumption and body weight gain. it could be concluded that basal ration administered with 5% antanan and 500 ppm vitamin c could effectively prevent broilers from heat stress. the results support the conclusion that a basal ration supplemented with 5% antanan and 500 ppm of vitamin c or their combinations, effectivelly reduces heat stress in broilers. key words : heat stress/ centella asiatica i vitamin c introduction high environmental temperatures may result in the accumulation of body heat load so that the body suffers from heat stress. as one of the homeothermic species, poultry could maintain their body temperature relatively constant by increasing respiration rate and water consumption and/or decreasing feed consumption. as a result, their growth rate and productivity will decrease. 46 biotropia no. 24, 2005 may and lott (2001) showed that body weight gain of 3 to 7-week-old male broilers raised at a temperature of 30°c was 1869 g significantly lower than for those raised at 22°c with body weight gains of 2422 g and feed conversion decreased from 3.28 to 2.54. the lower performances of broilers raised at high temperatures may have occurred as a result of lowered secretion of thyroid hormones (geraert et al. 1996), decreased blood hemoglobin and hematocrit levels (yahave/a/. 1997), or increased excretion of some minerals (belay et al. 1992) and some amino acids (tabiri et al. 2001). in addition, heat stress may also cause oxidative stress in the body and develop abundant free radicals, promoting the occurrence of peroxidation of membrane lipids and hence attacking dna and protein membranes (rahman 2003). takahashi and akiba (1999) indicated that feeding oxidized lipids to broilers significantly decreased feed consumption, body weight gain, plasma vitamin c, and plasma a-tocopherol. in fact, the results were followed by an increase in plasma malonal-dehyde (mda) and blood heterophyl/lymphocytes (h/l) ratios as biological indices of stress in avian species. antanan/pegagan (centella asiatica (l.) urban), one of the medicinal plants containing active materials such as asiatic acid, asiaticoside, and madecasic, is readily available and evidently eliminates stress in rats (kumar and gupta 2003). shukla et al. (1999) reported that placing asiaticoside on rats wound increases curability and accelerates enzymatic and nonenzymatic antioxidant activities of new-growing tissues. in addition, vitamin c reportedly eliminated cold stress (sahin and sahin 2002) and heat stress in poultry (puthpongsiriporn et al. 2001) and showed synergism with some active materials contained in antanan (bonte et al. 1994). with this in mind, we have examined the effect of antanan (centella asiatica) and vitamin c on the bursa of fabricius, liver malonaldehide and performance in heat-stressed broilers. materials and methods this research used 2 to 6-week-old male broilers placed in several pens located in an open poultry house. each pen was flitted with a 40-watt lamp and a zinc-plate backing functioning as a heat reflector. temperature and relative humidity measurements obtained at noon and afternoon were 31.98 ± 1.28°c and 78.82 ± 5.43%, respectively. temperature and relative humidity measurements at night and early morning were 27.36 ± 0.88°c and 86.23 ± 3.93%, respectively. the levels of 500 ppm vitamin c and 10% antanan (all plant parts) of ration used were established during preliminary trials. vitamin c was dissolved in drinking water and served in the morning, two hours after the broilers received their last waterfeeding. 47 effect of antanan (centella asiatica) and vitamin c ‐ engkus kusnadi et al.  one‐hundred‐and‐twenty‐two‐week‐old male broilers were randomly allocated into 24 pens,  5  broilers  each.  antanan  (5%  and  10%)  was  mixed  with  other  ingredients  to  make  three  different  rations  as  follows:  1)  the  control  ration  contained  the  calory  as  metabolizable  energy  (me)  3245.02  kcal/kg  and  20.84%  crude  protein  ,  2)  a5  =  5%  antanan  contained  me  3222.95 kcal/kg and crude protein 20.91%, and 3) a10 = 10% antanan contained me 3202.87  kcal/kg  and  crude  protein  20.99%.  the  ration  formulation  and  nutrient  composition  of  treatments are presented in table 1.                  the broilers were subjected to six treatments, 20 broilers each, as follows:  1) k (control)/ration neither contained antanan nor vitamin c.  2) a5/ration was supplemented with 5% antanan  3) alo/ration was supplemented with 10% antanan  4) c, drinkwater contained 500 ppm vitamin c  5) a5c, combination of a5 and c, and  6) a10c, combination of a10 and c  48  biotropia no. 24, 2005 variable measurements: 1) relative bursa of fabricius weight taken from 4-week-old broilers, by weighing the organ and divided by body weight (puvadolpirod and thaxton 2000). 2) heterophyl/lymphocyte ratio was taken from 4-week-old broilers, by hemo-cytometer method. blood was diluted 1:101 in a red blood cell pipette with nat and herrick diluent. the total leucocyte count includes heterophils, lymphocytes, monocytes, basophils, and eosinophils is divided by the number of lymphocytes. 3) liver malonaldehyde (mda) taken from 6-week old broilers, by measuring the thiobarbituric acid (tea) value using tarladgis method (apriyantono et al. 1989). destilate from liver sample with ph: 1.5, is added to the tba reagents, covered, mixed, and incubated in boiling water bath for 35 minutes. after cooling, the absorbance of filtrate (d) was determined at 528 nm wave length. the tbars values = 7.8 d were expressed as mg/kg of malonaldehyde per kg of tissue. 4) feed consumption, body weight gain, and feed conversion are measured for 4 weeks (from 2 to 6-week-old broilers). feed consumption was determined by weighing the given ration minus the leftover. body weight gain was measured by weighing the final body weight at 6 weeks old minus the body weight at 2 weeks old. feed conversion was measured by dividing feed consumption with body weight gain. statistical analysis the collected data were analyzed using a completely randomized factorial design (crd) 2 x 3 (2 levels of vitamin c i.e. 0 and 500 ppm and 3 levels of antanan i.e. 0, 5 and 10% of rations at 4 replications), and where applicable, continued with orthogonal contrast test according to steel and torrie (1980). results and discussion the results of the effect of antanan and vitamin c administration on bursa of fabricius weight, h/l ratio and liver mda contents are presented in table 2. the data for feed consumption (fc), body weight gain (bwg), and feed conversion are presented in table 3. from table 2, the average relative of 4 week bursa of fabricius weight for controls (k) is significantly lower than those treated with a5, a10, c, a5c, or a10c. the bursa of fabricius weight is similar for all treatments from a5 to a10c. 49 effect of antanan (centella asiatica) and vitamin c ‐ engkus kusnadi et al.  these results suggest that antanan, vitamin c, or their combinations increase burs of fabricius  weight of broilers that suffered heat stress.  the ability of antanan to stimulate lymphoid gland weight was reported i stressed rats  by sharma et al. (1996). antanan contains phenol compounds th< potentially can prevent  peroxidation of lipid membranes including t‐ and e lymphocyte membranes. t‐lymphocytes  produce cellular immunities while e lymphocytes produce humoral immunities produced by the  bursa of fabricius in bir species. phenol compounds of tea extracts were potentially capable of  stimulatin the production of  lymphoid cells  in rats (murtini et al. 2003). on the other hanc  vitamin c acts, as a water‐soluble antioxidant capable of protecting lymphocyte from suffering  heat  stress  (puthpongsiriporn  et  al.  2001).  as  a  result,  the  number  o  circulating  lymphocytes  increased so that the h/l ratio decreased.  table 2 shows a relationship between the increase of the bursa of fabriciu weight and  decrease in h/l ratio because the bursa of fabricius is a lymphoid orgai producing lymphocytes.  thus, the smaller the bursa of fabricius size, the fewe lymphocytes will be produced, and in  turn, the higher the h/l ratio will be. increase of the relative lymphoid organ weight occurred as a  result of antanan feeding whicl was demonstrated by sharma et al. (1996), while increase of the  relative bursa o fabricius weight as a result of supplementing vitamin c to broilers was reported  b] anim et al. (2000).                                  heat stress generating oxidative stress may increase mda content as a result of lipid  peroxidation, especially for unsaturated fatty acids of membrane cells. feeding  50    biotropia no. 24, 2005 antioxidants, i.e. antanan and vitamin c, significantly decreases liver mda content. besides of being able to relieve free radicals by releasing an electron and a proton (a hydrogen ion), phenol compounds are also able to provide a chelating effect in such a way that phenols bind to transition ions. unbound metals might increase free radicals (pietta 2000). in addition, phenol compounds are characterized by flavonoids that are able to decrease fluidity of cell membranes so that it may decrease diffusion of free radicals and mda contents. this is also true for vitamin c, a water-soluble antioxidant with 2 hydroxyl groups at €2 and €3 that are readily oxidized (sediaoetama 1987).                  in addition, it is unmistakable that feeding antanan and vitamin c increases feed consumption and body weight gain of broilers over four weeks (2-6 weeks of age) but not for feed conversion (table 3). this agrees with the reports of anim et al. 2000) for broilers and sharma and sharma (2002) for rats suffering from stress. antanan contains antioxidants such as phenol compounds that are capable of eliminating oxidative stress processes (blokhina 2000), as is apparent from the decrease of h/l ratios, liver mda levels and increase of bursa of fabricius weights reported here.  conclusions feeding antanan or vitamin c or the combination of antanan and vitamin c increases bursa of fabricius weight, feed consumption, and body weight gain but decreases h/l ratio and liver mda levels. the combination of 5% antanan and 51   effect of antanan (centella asiatica) and vitamin c — engkus kusnadi et al. vitamin c tends to increase feed consumption and body weight gain and , therefore this treatment tends to be very effective in alleviating heat stress in broilers. acknowledgements the authors wish to thank the department of higher education, nations department of education, republic of indonesia and seameo searca regional center for graduate study and research in agriculture, college, lagun 4031 philippines for their financial support. references anim aj, tl. lin, py. hester, d. thiagarajan, ba. watkins and cc. wu. 2000. ascorbic aci supplementation improved antibody response to infectious bursal disease vaccination in chickens poultry sci. 79: 680-688. apryantono.a, d.fardiaz, nl.puspitasari, scdarnawati and s.budiyanto. 1989. analisis pangar departcmen pcndidikan dan kcbudayaan dircktorat jcnderal pcndidikan tinggi pusat anta universitas pangan dan gizi institut pcrtanian bogor. belay t, wicmusz cj and rg. teeter. 1992. mineral balance and urinary and fecal mineral excrctioi profile of broilers housed in thcrmoncutral and heat-distressed environments. poultry sci. 71: 104 1047. blokhina o. 2000. anoxia and oxidativc stress: lipid pcroxidation, mitochondria! functions in plant antioxidant status and mitochondria! functions in plants http://ethesis.helsinki.fi/iulkaisut/mat/bioti/vk/blokhina/anoxiaan.html. [20 dcscmbcr 2003]. bonte f, dumas m, chaudagnc c and a. mcybcck. 1994. influence of asiatic acid, madccassic acid, am asiaticoside on human collagen i synthesis. planta med. 60: 133 135. geraert pa, padilha jcf and s. guillaumin 1996. metabolic and endocrine changes by chronic hca exposure in broiler chickens: biological and endocrinological variables. br. j. nutr.75:205-216. gross wb and hs. siegel. 1983. evaluation of the heterophil/lymphocyte ratio as a measure of stres in chickens. avian dieseasc.27: 972 979. kumar vmh and yk. gupta. 2003. effect of centella asiatica on cognition and oxidative stress in ai intracerebroventricular streptozotocin model of alzheimers disease in rat. clin exp pharmaco physiol 30: 336342. may jd and bd. lott. 2001. relating weight gain and feed:gain of male and female broilers to rearini temperature. poultry sci 80: 581-58444. murtini s, murwani r, bunawan a, handharyani e and f. satrija. 2003. effects of inoculation route am dose level of tea mistletoe (scurrula oortiana) stem extract on the development of lymphoi< follicle of bursa fabricius in chick embryonated eggs. international symposium on biomedicincs 18lhand 19th september 2003. ipb. 52 biotropia no. 24, 2005 pietta pg. 2000. flavonoids as antioxidants. reviews. j nat prod 63: 1035-1042. puthpongsiriporn u, scheidelcr se, sell jl and mm. beck. 2001. effects of vitamin e and c supplementation on performance, in vitro lymphocyte proliferation, and antioxidant status of laying hens during heat stress. poultry sci 80: 1190-1200. puvadolpirod s and jp. thaxton. 2000. model of physiological stress in chickens 1. response parameters. poultry sci 79: 363-369. rahman.i. 2003. oxidative stress, chromatin remodelling and gene transciption in inflammation and chronic lung desease. j.biochcm. mol. biol. 36: 95-109. sahin k and n. sahin. 2002. efcct of chromium picolinatc and ascorbic acid dietary supplementation on nitrogen and mineral excretion of laying hens reared in low 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304-310. yahav s, straschnow a, plavnik i and s. hurwitz 1997. blood system response of chickens to changes in environmental temperature. poultry sci 76: 627 633. 53 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf microsoft word 56 biotropia vol. 13 no. 1,2006 : 56 67 current microbiological aspects in high mountain lake research munti yuhana0* and kurt hanselmann2)* "faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia. 21 institute of plant biology, university of zurich, 8008 zurich, switzerland abstract remote and normally unpolluted high mountain lakes provide habitats with no or very limited anthropogenic influences and, therefore, their hydrodynamics are mostly regulated by the natural conditions. researches in high mountain lakes deal with measuring and modeling the response of the habitats to environmental changes especially correlated to acid deposition, pollutants influx and climatic variability. the microbial world has also become a focus in many studies of these extreme ecosystems. despite the pressure of harsh and extreme conditions, microbial communities living in these habitats developed flexible strategies and show quick adaptation to climate oscillation. new aspects in microbiological studies in recent high mountain lake research are presented in this paper. key words : high mountain lake, extreme environment, microbial communities, microbial diversity, psychrotolerant microbe, molecular microbe introduction in recent years, interests in the presence and survival of microorganisms which inhabit extreme environments have been increasing. attempts to discover novel microorganisms possessing special metabolic characters have encouraged microbiologists to intensive researches on microorganisms which are able to survive and sustain in extreme habitats. extremophiles dwelling in extreme environments have attracted the interest of microbial ecologists because of the uniqueness of peculiar physiological properties. life strategies of microbes evolved in low temperatures and nutrient limited environments have also attracted the attention of those who are studying conditions for life on other planets, such as mars (monastersky 1997). naturally cold ecosystems, such as antarctic and arctic regions, cryoconite holes, ice-covered lakes, the deep sea, high altitude mountain environments and glaciers (morgan-kiss et al. 2006) may represent analogs on earth to the cold extra terrestrial habitats. high mountain ecosystems are also of ecological interest because of their richness in natural resources, their biodiversity, the clean water, healthy climate, original soil, and diverse geology which are relevant to issues of environmental protection. on the other hand, the utilization of the mountain areas e.g. for the tourist industry is an issue of socio-economic importance. * corresponding authors: myuhana@botinst.unizh.ch or hanselma@botinst.unizh.ch 56 biotropia vol. 13 no. 1, 2006 microbiologists are mainly interested in the following questions: • what microbial diversity is present in high mountain lake habitats? are there unknown microorganisms which are characteristic for the extreme conditions? • are psychrotolerant organisms phylogenetically closely related to each other? • do microbes present in oligotrophic cryo-habitats possess special life strategies? • do the communities possess physiological and ecological flexibilities with which they can respond to the fluctuating physico-chemical conditions? • what are the ecological parameters determining the community structures and how rapidly do they respond to environmental changes? in tropical regions, the extreme cold environment can be found for instance in areas at high elevations of ice-covered mountains, i.e. mount jayawijaya in papua island, indonesia, mount kilimanjaro in tanzania, and the tropical andes in south america. despite of the cold temperature and oligotrophic characteristics, these ecosystems are different to those found in temperate and polar regions. they exist only at altitudes of 4500 m a.s.l. and higher, whereas in temperate and polar regions they normally begin at lower altitudes (e.g. > 1 400 m a.s.l) depending on the season. strong uv radiation, because of the continuous sun ray exposure throughout the year and thinner ozone layer at higher altitudes, might influence the physico-chemical characteristics of tropical mountain habitats. in addition, atmospheric deposition of pollutants (acidity and toxic air pollutants) that strongly influences the ecology of temperate high mountain lakes (mosello et al. 2002), may occur less in tropical mountain habitats, because of the higher elevation. so far, very limited information is available on (sub) tropical high mountain lake habitats. therefore, this article presenting various studies on temperate high mountain areas may encourage for new research and exploration of our tropical high mountain lake habitats. high mountain lake habitats as indicators of environmental changes global warming indicators for decades mountain areas have been used as sites to evaluate possible effects of global warming and its consequences. they are sensitive indicators for the early detection of the impacts of rapid climatic changes on the hydrological cycle, water chemistry and ice melting. even a small temperature change can strongly influence their hydrological cycle (schindler et al. 1990). the climatic warming observed in the last 25-30 years seems to be most pronounced in the mountain regions all over the world (beniston et al. 1997; rogora et al. 2003). the temperatures measured in these areas have increased by 1.5 2.0°c on the average since 1980, whereas an increase of approximately 0.5°c was observed on a global scale (beniston et al. 1997). the climatic changes and environmental fluctuations particularly affect open waters of high mountain lakes (psenner and schmidt 1992; sommaruga-wograth et al. 1997; koinig et al. 1998). in addition, other factors such as the atmospheric dust 57 current microbiological aspectsm. yuhana et al. deposition (psenner 1999) and long periods of anthropogenic impacts (kamenik et al. 2000) also shape the ecology of these areas. the duration and extent of the snow cover in a lake catchment area are dominant factors governing the release of weathering products from rocks and soils into the water (wright and schindler 1995). a recent study shows that the effect of climate warming has influenced the weathering rate in the mountains (rogora et al. 2003). through the chemical and physical degradation of rocks and soil minerals, solute contents increased. warmer temperatures are also enhancing biological processes by increasing the primary productivity. biochemical characteristics and dynamics of high mountain lake habitats trophic condition of high mountain lake habitats high altitude lakes are normally situated above the timberline and have catchment areas with little or no vegetation. consequently, they are often oligotrophic as defined by the concentration of nitrogen and phosphorus. examples include the lakes which became subject of the european project molar (mountain lake research) such as in the high tatra mountains (slovakia, 2 655 m a.s.l.) (kopacek et al. 1995), and the sumava mountains (czech, 1456 m. a.s.l.); schwarzsee (austria, 2799 m a.s.l.), lake paione superiore (italy, 2269 m a.s.l.) (mosello et al. 2002), yellow belly lake (u.s.a, 2157 m a.s.l.) (pilati and wurtsbaugh 2003), gossenkollesee (austria, 2417 m a.s.l.) (kamenik et al. 2000), jori lakes iii and vii (switzerland, 2557 m a.s.l.) (hinder et al. 1999a). lake jori xiii does not meet the oligotrophy criteria especially in summer season. low n:p ratios were measured at the end of the ice cover period, whereas the highest ratios were measured during the ice free period (iqbal-nava 2003). the physico-chemical conditions strongly depend on the duration of the ice and snow cover. the nutrient conditions in lake jori xiii initiate high biomass and remarkable microbial activities and population dynamics. atmospheric deposition influences mountain lakes' hydrochemistry atmospheric depositions have been demonstrated to be one of the key factors to influence longterm changes in the chemical characteristics of high mountain lakes (kamenik et al. 2000). airborne desert dust depositions contain major basic cations, carbonates, sulphates and other anions of nutrient value for residents of remote lakes (psenner 1999). the high calcium contents of the saharan aerosols, which are also found in aerosols from other deserts, might significantly contribute to the biogeochemical cycles involving acid neutralization (de angelis and gaudichet 1991). airborne dusts in snow and rain have also contributed to the high buffering capacity of some mountain lakes and to the elevated seasonal nutrient levels (de angelis and gaudichet 1991; psenner 1999). 58 biotropia vol. 13 no. 1, 2006 environmental changes demonstrated by microbial community succession physicochemical conditions in high mountain ecosystems are exposed to strong environmental fluctuations, seasonally as well as diurnally. these habitats (figure 1) are good models for studies of microbial dynamics of cold environments with a simple and short food web. during the long ice-cover period, they are exposed to cold temperatures, nutrient limitation, darkness below the thick ice cover, and anoxic conditions at larger water depths. whereas in the short summer season, they are exposed to high sun radiation, oxic conditions and wind-driven water masses upwelling. despite low nutrient input and commonly limited concentrations, some lakes also showed self-trophication capability, which is particularly observable during the summer season (iqbal-nava 2003). these environmental pressures provide heterogeneous ecological niches in the habitats. therefore, the microbial community adapts itself by continuously selecting for those members which are best adapted to the particular conditions in the ecosystem. a recent study observes dynamic changes in planktonic bacterial community composition, seasonally as well as spatially (yuhana 2005). these changes are a consequence of strongly fluctuating environmental conditions in which the communities show a quick response to fulfill the requirements for various ;**. photo by munii yuhami ecological niches. their dynamics were not only demonstrated by the seasonal changes in the genera diversities, but also their abundances. the quick adaptation was also observed in the planktonic microalgal populations. the autotrophic and heterotrophic flagellate as well as ciliate communities showed dynamics and high activities as indicated by high [3h]thymidine and [3h]leucine or [14c]leucine uptake rates during the short ice-free period (felip et al. 1995; hinder et al. 1999a). their population was supported with the availability of organic matters as nutrients which originate from allochthonous and autochthonous sources. two different external 59 current microbiological aspects m. yuhana et al sources can contribute to nutrient enrichment inside a lake: atmospheric input or input from the catchment. the input can consist of dissolved organic matter, organisms, particulate matter, vegetation debris including pollen grains, and insects (fclipetal. 1995). the role and the physiological adaptation of microorganisms in high altitude habitats the role of microorganisms in high altitude habitats in aquatic ecosystems, the bacterioplankton community is an integral part of the food web, which is essential for protists and mctazoans (cho and azam 1988). temporal and spatial alterations of the microbial community in pelagic food webs of high mountain lake habitats have been studied (felip et al. 1995). recent studies focus on more specific subjects such as the effect of the uv radiation on the bacteriovory (sommaruga et al. 1996), microbial diversity and activity (alfreider et al. 1996) and palaeolimnology (koinig et al. 1998). compared to eutrophic ecosystems, the pelagic food webs in these oligotrophic ecosystems appeared to be less complex and the microbial loops might play an essential role in recycling nutrients for the higher trophic levels (hinder et al. 1999b). the significance of the pelagic microbial assemblage increases with the level of oligotrophy of the lake water (hahn et al. 1999). this is true for pelagic food webs in circumcontrol as well as in acidified high mountain lakes (wille et al. 1999). microorganisms in high mountain habitats play a key role in biogeochemical cycles such as subglacial rock weathering and nutrient cycling. microorganisms living in phosphate-limited mountain lakes are able to mobilize particulate iron phosphates from sedimentary deposits and biofilm-associated microbes can develop on iron-phosphate-oxyhydroxide coated surfaces (amberg-brunner 2002). microbially mediated pyrite oxidation occurs at low temperature and glacier bed microbial communities contribute to sulfate release to the environment (sharp et al. 1999). from these biochemical processes, furthermore the microbial population derives energy from the oxidation of reduced mineral or organic carbon within the sediments. physiological adaptation to low temperature organisms that live and actively grow at near freezing temperatures and limited-nutrient conditions face a number of growth constraints. under the cold conditions the enzyme reaction rates are generally lower, uptake and transport systems function more slowly, membranes become less fluid and nucleic acid structures become more stable (feller et al. 1996; graumann and marahiel 1996). however, microorganisms have evolved various strategies to adapt to these hindrances. they range from molecular level to cell and ecosystem levels (gerday et al. 1997). the evolution of cold shock and antifreeze proteins, the modulation of the kinetic key enzymes, and the development of more fluid biological membranes 60 biotropia vol. 13 no. 1,2006 through the accumulation of polyunsaturated fatty acyl chains are among the means of adaptation to low temperature (morgan-kiss et al. 2006). adaptation to high-level uv exposure attenuation coefficients (kd) of the photosynthetically active radiation (par) and uv were used for indirect determination of the chlorophyll concentrations. kd values, which are based on the upper and lower uv radiation intensities, are used to illustrate the strength of uv intensity at different wave lengths i.e. 305, 320, 340, and 380 nm. results from iqbal-nava's study (2003) showed that the diffuse attenuation coefficients (kd) measured in swiss alps lake jori xiii (ca. 2640 m a.s.l.) were ranging from 0.68 to 1.54. whereas kj values of the austrian lake gossenkolle at 2417 m a.s.l were ranging from 0.14 to 0.32 (sommaruga and psenner 1997), and the mean kd values of 13 oligotrophic lakes in the bariloche region in argentina (ca. 2000 m a.s.l.) were between 0.3 and 0.8 (morris etal. 1995). microorganisms living at high altitudes are challenged by intense uv radiation. uv b radiation (280-320 nm) is potentially the most damaging for living cells (morris et al. 1995). the uv b level in the winter cover of high mountain lakes can reach up to 50% higher than at sea level (psenner and sattler 1998). the strong uv exposure can lead to growth inhibition of benthic diatoms (bothwell et al. 1994); causes damage in heterotrophic flagellates (sommaruga et al. 1996), and inhibits the rate of nutrient uptake by bacterioplankton in the water column (sommaruga et al. 1997). some adaptive strategies against strong uv radiation, that were found in permanently low temperature high mountain habitats are, for instance, the capability to screen uv and the photosynthetically active radiation (par) by chlamydomonas sp. in addition, chloromonas sp. posseses uv-screening pigments called mycosporine-like amino acids (maa), whereas some cyanobacteria like nostoc, phormidium, and anabaena spp. segregate a mucopolysaccharide matrix (morgan-kiss et al. 2006). molecular approaches applied to study the microbial succession in high mountain aquatic ecosystems pcr-based methods provide information on the nucleic acid composition of microorganisms isolated from different habitats which mostly are still not (yet) cultivable. when carl woese introduced the use of 16s rrna sequences for molecular phylogeny (woese 1987), only 12 microbial phyla could be phylogenetically compared. currently, 26 phyla of approximately 52 identifiable major phyla within the bacterial domain have cultivated representatives (rappe and giovannoni 2003). the number of environmentally retrieved bacterial 16s rrna genes has been increasing rapidly and now is exceeding 30 000 (rappe and giovannoni 2003; wagner 2004). carl woese's approach has also uncovered a new domain of life, the archaea. formerly, archaea were thought to exclusively consist 61 current microbiological aspects m. yuhana et al. of thermophiles, halophiles, and strictly anaerobic methanogenic microorganism inhabiting extreme environments. today, archaea are recognized as ubiquitou microorganisms, also present in high mountain lake habitats (yuhana 2005). in recent years, advanced techniques have been developed to study thi composition of microbial communities (figure 2). pcr-based community fingerprinting techniques such as denaturing gradient gel electrophoresis (dgge or temporal temperature gradient gel electrophoresis (ttge) allow us to monitoi the microbial structure based on community banding patterns. these techniques an based on the separation of the pcr products of genes isolated from mixec populations possessing different nucleotide sequences (muyzer et al. 1993). nucleic acid is extracted from the natural samples and dna amplification is performed by pcr with primers targeting for instance the small subunit ribosomal rna genes. the pcr products are subsequently analyzed by loading them onto a polyacrylamide gel containing a linearly increasing gradient of denaturants (in dgge) or a linearly increasing temperature gradient (in ttge). by using dgge or ttge, the diversity and microbial community composition can be described without enriching them or performing the cloning techniques (muyzer and smalla 1998; muyzer 1999). the number of bands on a gel may not accurately reflect the number of different species in their habitat, but the dna of the most abundant representatives of the communities normally gets amplified and should be represented in the band pattern (muyzer et al. 1993). these techniques can also be applied for the screening of clone libraries (e.g. bosshard et al 2000). both techniques provide an alternative way for microbial community fingerprinting without application of cloning strategies which is more time consuming. dgge and ttge combine a direct visualization of community diversity and the opportunity for subsequent identification of microbial population members by sequence analysis or hybridization experiments using taxon specific probes (muyzer 1999). this community fingerprinting technique has been successfully applied in high mountain lake ecosystems. the population dynamics and the community succession can be followed seasonally as well as spatially which has been done for example in the alpine shallow lake jori xiii (10.4 m, maximum depth). while this lake was temperature-stratified, it showed distinct ttge banding patterns, whereas the patterns were identical at all depths during summer upwelling events (yuhana 2005). a different study was carried out in the meromictic lake cadagno (bosshard et al. 2000). this lake shows a permanent stratification, chemically as well as physically, represented by an oxic mixolimnion, a chemocline water column and an anoxic, monimolimnion of high salinity. the spatial community structure, as revealed by ttge banding patterns, of the chemocline and the monimolimnion were highly similar whereas the mixolimnion showed a distinctly different pattern. for the temporally community shift, the authors reported that community structure of these three zones varied with time. in the mixolimnion and the chemocline, the community composition changed greatly during the sampling period, whereas in the monimolimnion, the microbial community shift was less distinctive. the distribution of microbial populations was mainly correlated to the spatial or temporal fluctuation 62 biotropia vol. 13 no. 1,2006 in their micro-environmental conditions, such as organic matter concentration (crump et al 2003) or nutrient availability (yuhana 2005). it has also been demonstrated that the spatial distribution of the phototrophic community present in a dense layer changed diurnally by active vertical movement in response to the light conditions and the chemical gradient (egli et al. 2004). a direct and rapid detection technique to taxonomically identify the members of microbial communities is fluorescent in situ hybridization (fish). the use of specific rrna-targeted oligonucleotide probes allows one to visualize the morphotype and size of hybridized cells; as well as to determine quantitatively the species, sub groups, or domains among the dapi-stained cells (amann et al. 1990; amann et al. 1995). the in situ assessment for the abundances and the microbial composition has been widely applied to various habitats. different studies were carried out to investigate the community compositions of habitats from high mountain lakes (alfreider et al. 1996; pernthaler et al. 1998). alfreider et al. (1996) 63 current microbiological aspects m. yuhana et al. studied the microbial composition in the different snow, slush, and pelagic layer c lake gossenkolle in the tyrolean alps (2417 m a.s.l.). they reported that th application of probes specific for the alpha, beta, and gamma subclasses c proteobacteria and the cytophagaflavobacterium group showed a very distinc bacterial community composition within different habitats (snow, slush, and lak water). the community, in most cases, was dominated by members of the bet subclass (6.5 to 11.6% of the bacteria detectable with the probe hub). the pcr-based (culture-independent) analyses, however also have thei limitations. without having appropriate culture representatives, it is quite difficult ti predict phenotypic properties of the detected but uncultured microorganisms. it i still a challenge for microbial ecologists to assign functions and activities t< populations within those communities in complex ecosystems (wagner 2004; paei and steppe 2003). the microbial communities, which consist of mixed groups o microbial species having different functions and metabolic activities, are responsibli for maintaining the ecosystem fitness (paerl and steppe 2003), i.e. microbiall; mediated chemical transformations and habitat alterations (boetius et al. 2000). ii spite of extreme conditions, highly active and diverse microbial communitiei occupying the ice and snow cover of high mountain lakes have been demonstratec (felipefaz. 1995). conclusions although mountain environments have been exploited for a number 01 purposes, the giant microbial gene pool present in these extreme habitats has beer neglected so far. their aquatic areas are environments often characterized by simple food chains, low in species diversity and species richness. appreciation of microbiai diversity, their genomic richness and metabolic capabilities is very much supported by advances in molecular techniques. these techniques allow us to phenotypically and genotypically characterize microbes, as well as to assign their ecological roles, without the need for cultivation processes. hence, the remote mountain habitats which typically contain low nutrient concentrations can no longer be underestimated as genetic pools. this untouched reservoir of genomes might be exploited for biotechnological purposes, one day, e.g. for cold active enzymes. acknowledgments we would like to thank thomi horath for his valuable suggestions on the article. the fruitful comments from the reviewers were also extremely appreciated. references alfreider, a., j. pernthaler, r. amann, b. saltier, p.o. glockner, a. wille and r. psenner. 1996. community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake using in situ hybridization. appl. environ. microbiol., 62: 2138-2144. 64 btotropia vol. 13 no. 1, 2006 amann, r.l, l. krumholz and d.a. stahl. 1990. fluorescent-oligonucleotide probing of whole cells for determinative, 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65.pdf 66.pdf 67.pdf microsoft word 122 biotropia vol. 13 no. 2,2006 : 122 -131 assessment of coastal land use changes in banten bay, indonesia using different change detection methods puvadol doydee¹ and vlncentius slregar² 'department of fishery management, faculty of fisheries, kasetsart university, bangkok, 10900, thailand 2seameo biotrop, jl. raya tajur km 6, p.o box 116, bogor indonesia e-mail:vincents@biotrop.org abstract many types of the coastal land use in banten bay have been assessed in order to know the change as evidently detected by landsat imagery in 1994 and 2001. image processing such as, supervised classification and various change detection techniques are performed to the satellite images. red green method showed the best result for detecting the coastal land use change. this method is suitable for detecting the increasing areas of the paddy fields and settlement. image differencing method is capable to detect the increasing areas in agriculture, decreasing in fishponds and natural areas. image ratioing method can be considered suitable for detecting the increasing area of fishponds, decreasing of paddy fields and agriculture areas. each coastal land use type has increased, except for the natural area/brushwood. most of agriculture and paddy fields areas have been converted to fish ponds. key words: coastal land use/land cover, change detection method, remote sensing introduction as a transition area between land and sea, coast is characterized essentially by geologic nature of the land. sometimes, coastal zone is defined depending on the project purposes, for example loicz project (holligan and de boois 1993). this zone in many cases is relatively unstable and fragile (carter 1989), but became an important place for the human activities, such as culture, economic, fisheries, industry, tourism and social (rais and de boois 1997). more than 60 % of the human population lives in coastal area and it will increase in the future, hi many cases, coastal area is the best place or the only available space for city development, agriculture, fisheries, industry and tourisms. this condition will cause the increasing demand for space for such activities. however, in coastal zone a land use change is obviously a phenomenon (loicz 1994; williams et al 1997; noaa 1998) this change is an essential matter that should be monitored for planning or avoiding any further changes that can damage or harm the environment. remote sensing technique is one of the effective tools in monitoring phenomena that change continuously overtime and cover large areas (aronoff 1991). one of the major applications of remote sensing technique is change detection method. many readymade algorithms are available to perform that method. despite of their differences in identification, an accurate spatial registration of the various dates of imagery is a requirement for all methods used. corresponding author: vincents@biotrop.org 122 asessment of coastal land use changes p. doydee and v. siregar this study is intended to assess the capability of four methods of change detection in detecting the change in coastal land use, and to compare the result of those methods with each other. location of this study is the coastal zone of banten bay. banten bay is an area most frequently used for various researches, since in this region many activities are taken place, such as agriculture, fishing and mariculture, harbor, and industry. there is no doubt that these activities will influence the coastal habitats and resources. materials and methods the study area is located in banten bay at the northern coast of banten province (figure 1). banten bay has an area of about 120 km2 with water depth of less than 13 m. geographically, this bay lies on 05°55' s-06°05' s and 106°05'e -106°15'e and the distance from jakarta is about 175 km. this bay is also characterized as a lowland area (saptarini 2000). data used comprised satellite data of landsat-5 tm (with the path/row 123/064 and acquisition date of april 6, 1994), and landsat-7 etivt (with path/row 123/064 and acquisition date of august 7, 2001, nasa, 2002), topographic and geomorphological maps. different software have been used such as image processing er mapper (er mapper 1997), banten bay information system 3.0 and geographic information system. supervised classification method used has the objective to identify and classify the features in an image quantitatively (richards 1995; lillesand and kiefer 1994; congalton and green 1999; yusuf 2001). this involves the analysis of multi-spectral image and the application of statistically based decision rules for determining the coastal land use type of each pixel on an image (middlekoop and lif 1991). many different techniques have been developed for change detection. these methods involve change extraction and change classification. (coppin et al. 2002; jensen 1986). the use of change detection techniques in this study comprised four methods, including red green, image differencing, image ratioing and principal component analysis methods. image analysis was performed to assess the coastal land use change between two different dates by overlaying the images of landsat-5 tm in 1994 and landsat-7 etm+ in 2001, and applying the change detection techniques as stated above. prior to the application of change detection techniques , the images utilized have to be normalized using atmospheric correction technique of histogram adjustment in order to eliminate error due to different atmospheric conditions of the images of different periods (wallace and campbell 1996). image of 1994 was rectified to image of 2001 which has been already corrected (geodetic datum wgs 84 and map projection sutm 48). 123 coastal land use in 1994 and 2001 the type of coastal land use in 1994 and 2001 are grouped into 5 classes such as, paddy fields, fishponds, settlement, agriculture and natural areas (brushwood). the maps of coastal land use were prepared from the result of supervised classification using the band combination of 542/rgb (figure 2.) change detection techniques red green method this method involves displaying simultaneously one dataset in green and one dataset in red, namely image in 1994 was displayed as red and image in 2001 was displayed as green. the red color means that these areas only exist in image 1994 and the green color in image 2001. for example in lontar district (figure 3) as zoomed-in, paddy fields in 1994 have been converted to fishponds in 2001. the red color is represented as paddy fields before and those that have been converted into fishponds in 2001. this could be confirmed by using geomorphological map and thematic coastal land use map resulted from supervised classification. 124 asessment of coastal land use changes p. doydee and v. siregar image differencing method the digital number value of image in 2001 was subtracted from digital number value of image in 1994. the basic concept of this method is that, in the raster data, each band has its digital value (dn). therefore, if it is subtracted from the same dn value of each pixel in both images, the output is equal to 0. the output might be minus or plus in the case that those pixels do not have the same dn values, this means that there is a change of value between the two images. this change might be due to the change of characteristic of the object. the output of this method was displayed as pseudo color area (figure 4-left). prior to data overlay, the raster data should be converted from raster cells into vector polygon and then overlaid that vector (change portion) with the original image so that the raster and vector polygon could be distinguished (original image), the result is presented in figure 4 (right). figure 4. the result of image differencing method. 125 biotropia vol. 13 no. 2, 2006 image ratioing method actually, this is a simple method as image differencing method where in this method the formula is changed from subtraction to division. the data from two registered images are divided pixel by pixel. therefore, the areas in different dates that have the same value of dn is equal to 1, it means that those areas do not change. bands of tm1, tm2 and tm3 are highly correlated. the lower the correlation between two bands the greater the information content. ratio of tm2 and tm5 provide some subtle wetland information (jensen 1986). the result of this method is presented in figure 5. figure 5. the result of image ratioing method principal component analysis method the pc#2 has maximum variance of the original data set, therefore it is used for analysis. pc#2 data consist of two bands inputs, where input 1 and input 2 were images of 1994 and 2001, respectively. the result showed that the bright blue color appears covering the areas (figure 6-left). the output should be converted from raster cells into vector polygon in order to facilitate the interpretation (figure 6right). figure 6. the result of principal component analysis method 126 asessment of coastal land use changes p. doydee and v. siregar change analysis the entire study area was 46 785.69 ha consisting of 23 437.26 ha terrestrial zone and 23348.43 ha water. the classification of coastal land use (type) between two dates was processed by post-supervised classification method (cho 2000) and the result is presented in table 1 . table 1 shows that the area of each land use type has increased, except for the natural area/brushwood which shows the contrary. the main factor caused by this change is the increasing numbers of the population and human activities in this area. from the field check, it was observed that some parts of agriculture and paddy fields are converted into fishponds. it is known that in 1990's fishponds were very promising, and for that reason most of villagers and farmers converted their agriculture area and paddy fields into fishponds in the coastal zone. according to table 2, there are two types of changes, that is the increasing and the decreasing area. obviously, the total areas of coastal land use converted was 7706.79 ha .these changes not only happen from one type of land use to other types, but also in the area of the same land use type. for example of decreasing area, it can be noted as follows: paddy fields as in 1994 have been converted into different land use types, such as fishponds (812.46 ha), settlements (70.16 ha), and agriculture areas (712.66 ha), respectively in 2001. fishponds as in 1994 have been converted only into settlement (8.15 ha) in 2001, while for settlement there is no change in terms of land use type and the area between the two periods. agriculture as in 1994, has been converted into paddy fields (1015.06 ha), fishponds (761.39 ha) and settlement (317.18 ha), respectively in 2001. natural areas (brushwood) in 1994 have been converted into paddy fields (998.51 ha), fishponds (727.27 ha), settlement (320.19 ha) and agriculture (1963.76 ha) in 2001. meanwhile, the increasing areas of the land use types from 1994 to 2001 are as follows: paddy fields are increased as 127 biotropia vol. 13 no. 2,2006 much as 2013.57 ha from agricultural (1015.06 ha) and natural areas (998.51 ha); the area of fishponds are increased to 2301.12 ha from paddy fields (812.46 ha), agricultural (761.39 ha) and natural areas (727.27 ha); settlement areas are increased 715.68 ha which comprised paddy fields (70.16 ha), fish ponds (8.15 ha), agriculture (317.18 ha) and natural areas (320.19 ha) and; agriculture areas are increased to 2,676.42 ha that is from paddy fields 712.66 ha and natural areas about 1903.76 ha. the total of land use type (7706.79 ha) then is considered as a reference for selecting the best method of change detection to be applied. using the four converted detection methods, the converted areas of each method were as follows: the red green method (7094.97 ha), image differencing method (6185.25 ha), image ratioing method (8490.96) ha and principal component analysis (pc#2) (5920.47 ha), respectively. from the result presented in table 3, it could be concluded that red green method is better for detecting the coastal land use change compared to other methods. saptarini (2000) has also concluded that red green method is simple and easy method to detect the change of areas in coastal zone of banten bay. this method shows that the total number of converted areas closely agrees with the number of converted areas of the reference. as stated above, the total of each land use type has increased from 1994 to 2001, except for natural areas/brushwood which show the contrary. 128 asessment of coastal land use changes p. doydee and v. siregar the increasing and decreasing areas of each type of land use were derived from four change detection methods. for increasing area in general, red green method is the best compared to the other methods. but, if the increasing area of each type of land use is observed, we found that only pc#2 has no land use types which has a wide close to the reference (table 4). it means that each change detection method is suitable to detect certain land use type. table 4. the increasing area of each land use type using the four change detection methods compared to the reference. type red green image differencing image ratioing pc#2 references paddy fields 1,969.86 1,680.39 2,088.15 1,802.88 2,013.57 fishponds 2,161.82 1,806.74 2,394.97 1,988.55 2,301.12 settlement 804.84 439.41 827.13 493.20 715.68 agriculture 2,158.44 2,258.72 3,180.70 1,635.85 2,676.42 total 7,094.97 6,185.25 8,490.96 5,920.47 7,706.79 therefore, it could be concluded that red green method is suitable for detecting the increase of paddy fields and settlements. differencing method is better in detecting the increasing agriculture areas. meanwhile, the ratioing method could be considered as the best method to detect the increasing fishpond areas (table 5). for decreasing areas, all the change detection methods used showed that the area of each type of land use is far less under the area of reference (table 6). meanwhile, if the decreasing area of each type of land use provided by the change detection methods used is compared with the reference, it could be 129 biotropia vol. 13 no. 2,2006 concluded that image differencing method is better for detecting the decreasing area of fishponds and natural areas compared with other methods and image ratioing method could be considered as the better method for monitoring the decrease of paddy fields and agriculture areas (table 7). this study showed that no specific change detection method could be used to detect all land use types. each method could detect better a specific land use type compared to the other methods. conclusions supervised classification of remotely sensed data (landsat data in!994 and 2001) of the coastal zone of banten bay provides 5 land use types such as paddy fields, fishponds, agricultural area, settlement and brushwood (natural area). these land use types served as the reference for land use change during those two periods. to detect the change of land use types in those areas, several change detection methods are performed to the landsat satellite data. change analysis showed that among various change detection methods used, the red green method has provided the best result for detecting the coastal land use change. this method is suitable for detecting the increasing areas of the paddy fields and settlements. meanwhile, image differencing method is capable to detect increasing areas in agriculture, decreasing in fishponds and natural areas. image ratioing method could be considered suitable for detecting the increasing area of fishponds, decreasing of paddy fields and agriculture. each coastal land use type in the study areas had increased, except for the natural areas, and most of agriculture and paddy fields areas have been converted to fishponds. certain change detection methods used has shown a good performance for detecting the change of certain coastal land use types in banten bay, however, several tests in different areas are needed to be conducted in order to know the level of accuracy of each method used. acknowledgments the authors would like to acknowledge the funding support of the seameo regional center for graduate study and research in agriculture (seameo searca), and technical support and assistance of mr. nyoman sukmantalya in providing all the data needed. 130 asessment of coastal land use changes p. doydee and v. siregar references aronoff, s. 1991. geographic information systems. a management perspective, wdl publications, ottawa, 294 p. carter, r.w.g. 1988. coastal environment. an introduction to the physical, ecological and cultural systems of coastlines, academic press, toronto, 617 p. cho, s.h.2000. digital change detection by postclassification 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131.pdf 6. okky (postharvest).cdr biotropia vol. 19 no. 2, 2012: 115 129 postharvest quality improvement of sorghum ( (l.) moench) grainssorghum bicolor okky setyawati dharmaputra , santi ambarwati , and ina retnowati received 29 june 2012/accepted 26 october 2012 the objectives of this study were (a) to investigate the effect of postharvest handling (threshing and storing) methods on the quality of sorghum ( (l) moench) grains variety numbu, in terms of the percentages of damaged grains and seed germination, population growth of , and ; fumonisin b and carbohydrate contents, and the percentage of weight loss during storage. the change of moisture contents of sorghum grains was also recorded. threshing was conducted using wooden stick and a paddy thresher. sorghum grains were packed in hermetic plastic bags. the conditions inside of the bags were airtight and normal. each bag with different conditions inside was infested with 10 pairs of (1-14 days old). sorghum was stored for one, two and three months under warehouse conditions. the results showed, that the moisture contents of sorghum were lower than its standard safe moisture content (<14%) during storage. at the beginning of storage, the percentage of damaged grains caused by threshing using wooden stick was higher than that of using a paddy thresher. the increase of percentage of damaged grains was caused among others by increase population under normal oxygen concentration inside of the bag (about 21%), consequently the percentage of weight loss was also increased. the percentage of seed germination of sorghum threshed using wooden stick was lower than that of threshed using a paddy thresher. the percentage of seed germination decreased with the increase of storage duration. population of and . decreased with the increase of storage duration. fumonisin b content of sorghum threshed using wooden stick was higher than that of using a paddy thresher during one, two and three months of storage. fumonisin b contents were considered low. in general, carbohydrate content of sorghum threshed using either wooden stick or paddy thresher from the beginning up to three months of storage were not significantly different. threshing using a paddy thresher was better in comparison to threshing using wooden stick. postharvest quality, sorghum, 1,2* 1 1 1 2 seameo biotrop, jl. raya tajur km. 6, bogor, 16134, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, dramaga campus, bogor 16680, indonesia sorghum bicolor sitophilus zeamais fusarium proliferatum f. verticillioides s. zeamais s. zeamais f. proliferatum f verticillioides sorghum bicolor abstract 1 1 1 key words: * corresponding author : okky@biotrop.org 115 introduction as foodstuff, sorghum ( (l) moench) is the fifth most important cereal after rice, wheat, maize, and barley in the world. sorghum can adapt to broad agroecology, gives high production, and is more resistant to drought, pests and diseases, compared to other food crop. it has also high nutritional content which is consequently good to be used either as alternative food or feedstuff. according to suarni (2001) sorghum has carbohydrate content of 80.42% as compared to 86.45% in milled rice and 79.95% in maize. in terms of protein, sorghum has slightly higher content (10.11%) than milled rice (9.28%), but slightly lower than maize (11.02%). sorghum's lipid content value (3.65%) is between milled rice (1.88%) and maize (5.42%). the use of sorghum as foodstuff in the form of flour is more beneficial, because it is easier to be processed into food products such as cake, cookies, bread and noodle. the capability of sorghum flour substitution upon wheat flour varied, i.e. for cookies, cake, bread and noodle was 50-75%, 30-50%, 20-25% and 15-20%, respectively (suarni 2004). sorghum grains could substitute maize for feed stuff, because the nutritional content of sorghum is not so different from that of maize, (sirappa 2003). since long time ago sorghum plant has been recognized by farmers in indonesia, especially in java, west nusa tenggara and east nusa tenggara. nevertheless, the production of sorghum is still low. although in indonesia the production of sorghum is still low compared to other countries such as india, china and united states, it is equally important to pay attention to the postharvest handling of this commodity to maintain its good quality during storage. cultivation and development of sorghum are important to anticipate food crisis caused by global warming. in indonesia, the low production of sorghum grain is due to (a) the nonavailability of appropriate technology for sorghum handling and processing after harvest, (b) the limited knowledge on how to process sorghum grain into various food products, (c) the lower price of sorghum grains compared to milled rice, maize and peanuts. the postharvest handling (drying, threshing and storing) can affect the quality of sorghum. dharmaputra . (2010) reported that based on the surveys conducted in demak and wonogiri regencies in 2010, the postharvest handling of sorghum practiced by farmers and collectors was not carried out appropriately, therefore sorghum grains were easily attacked by insects. the dominant insect species found were and . the most common fungi found in sorghum were , and . during storage, sorghum could be infested by insects, microorganisms, mites and rats. insects are considered the most significant cause of postharvest losses. among microorganisms, fungi are the most important cause of deterioration of stored grains. the role of insects in fungal infection cannot be disregarded. aside from injuring grains, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce heat and moisture (especially during longterm storage) which stimulate fungal growth. fungal infection in grains can cause discoloration, decrease sorghum bicolor et al sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum f. verticillioides 116 biotropia vol. 19 no. 2, 2012 in germination, physical quality and nutritional contents, and also mycotoxin contamination (sauer 1992). fumonisins are important mycotoxins, produced mainly by and in several agricultural products worldwide, especially in maize and sorghum (marasas . 1988; abdel-hafez . 1990; da silva . 2000). the former name of is . according to desjardins (2006) there are four kinds of fumonisins occurring at significant levels in naturally contaminated grain, i.e. fumonisin b , b , b and b . the most toxic among these four kinds of fumonisins is fumonisin b which can cause leukoencephalomalacia in equines (marasas . 1988 ) and rabbits, pulmonary edema in swine (bucci . 1996; harrison . 1990) and it has been reported as a probable cause of esophageal cancer in humans (marasas 2001). a high incidence of esophageal cancer has been observed in certain geographic areas and ethnic groups in africa, asia and latin america. in indonesia, no research has been done on fumonisin level in sorghum. the method of postharvest handling is one of the factors that can affect the quality of sorghum. however, the postharvest practices in indonesia are still inadequate, it is important to conduct research on the effects of different postharvest treatments on the quality of sorghum grain. this study is a continuation of the research conducted last year. the objectives of the research were: (1) to investigate the effects of postharvest handling (threshing and storing) on the quality of sorghum grains, in terms of the percentages of damaged grains and seed germination, population growth of , . and . , fumonisin b and carbohydrate contents, and the percentage of weight loss; (2) to record the change of moisture contents of sorghum grains during storage, and (3) to recommend proper postharvest handling methods to ensure the quality of sorghum grains during storage. the research results should provide recommendations to farmers, collectors, food and feed industries on the most adequate postharvest handling practices (threshing and storing) for sorghum to maintain good quality of grain during storage the variety of sorghum used in this study was numbu, cultivated by pt tri fondasi indonesia in balaraja subdistrict, tangerang regency, west java. the numbu grains were harvested 95 days after sowing. sorghum was harvested by cutting the panicle from the standing stalk using a sickle. sorghum in the form of panicles was sun-dried on tarpaulin. drying was conducted up to moisture content of about 13%. threshing was done using wooden stick and a paddy thresher. after threshing, grains were separated from dirt and chaff by winnowing. sun-drying, threshing and winnowing were conducted in the location et al. f. proliferatum f. verticillioides et al et al et al f. verticillioides f. moniliforme et al et al et al s. zeamais f proliferatum f verticillioides 1 2 3 4 1 1 materials and methods variety of sorghum methods of harvesting, drying, threshing and winnowing . 117 postharvest quality improvement of sorghum okky s. dharmaputra– et al. where sorghum was cultivated. sorghum grains were then transported to seameo biotrop, bogor, by a car equipped with air conditioning. prior to packaging, sorghum grains were: (a) fumigated with phosphine for five days at 2 grams/ton of sorghum to control insect pest that may exist, (b) checked for the percentage of damaged grains. damaged grains included cracked and broken grains caused by threshing. the grains were packed using hermetic bags under airtight conditions, i.e. oxygen concentration inside was about 18%, and under normal conditions (oxygen concentration inside was about 21%). each bag type of packaging material contained 2 kg of sorghum grains. characteristics of plastic packaging material used to pack sorghum grains are presented in table 1. in three replicates, inside of each bag type with different oxygen concentrations were treated as follows: (a) infestation with ten pairs of adult (1-14 days old) , (b) different method of threshing, and (c) different storage duration. the bags designated as “control” were not infested with . z s, but were also subjected to different methods of threshing as well as storage durations. sorghum grains were stored for one, two and three months under warehouse conditions. bags containing sorghum were placed on wooden shelves randomly. the temperature and relative humidity of the storage were recorded using a thermohygrograph. grain samples were collected from each bag before storage and subsequently every month thereafter until three months of storage. in samples infested with . , the insects were separated from the sorghum grains using a sieve, they were then preserved in vials containing 70% ethanol. after that, each sample was divided three times using a box sample divider to obtain working samples for the determination of moisture content, percentages of damaged grains and seed germinations, population of and ; fumonisin b and carbohydrate contents, and a reserve sample. the following quality parameters were determined in the experiments: moisture content, percentages of damaged grains , seed germinations, populations of , and , fumonisin b and carbohydrate contents, percentage of weight loss. methods of packaging and storing sampling methods determination of quality parameters sitophilus zeamais s eamai s zeamais f. proliferatum f. verticillioides s. zeamais f. proliferatum f. verticillioides 1 1 118 biotropia vol. 19 no. 2, 2012 statistical analyses moisture content of sorghum (based on wet basis) was determined as soon as samples were collected using delmhorst model g-7 moisture meter. this instrument has been caliberated and cross-checked using oven method. two replicates were used for each sample. damaged grains were collected after one, two and three months of storage including cracked, broken and damaged grains (damage caused by . z or fungi). the number of grains used for the determination of damaged grain percentage was 300 per sample. percentage of seed germination was determined based on blotter method (mathur and kongsdal 2001). population growth of was determined based on the number of adult insects per kg of each sample. and were isolated and enumerated using a serial dilution method followed by pour plate method on dichlorane chloramphenicol peptone agar (dcpa) (pitt & hocking 2009). fumonisin b and carbohydrate contents were determined using liquid chromatography-mass spectrophotometry (lc-ms) (zöllner & mayer-helm 2006) and sni (1992) methods, respectively. the percentage of weight loss was determined at the end of storage. grain samples contained in each bag were weighed before and at the end of storage. grain samples were taken periodically from each bag and weighed. the data were analyzed using completely randomized factorial design with four factors. the first, second, third and fourth factors were the method of threshing, oxygen concentrations inside of the bags, presence or absence of , and storage duration, respectively. s eamais s. zeamais fusarium verticillioides f. proliferatum s. zeamais 1 119 table 1. laboratory analyses of plastic packaging material* parameter test condition of sample unit test method test result uncertainty at 95% confidence level, k=2 wvtr (water vapour transmission rate) good g/m2 day astmf 124920062 2.8946 ± 0.3619 temperature = 37.8 oc rh = 100% thickness of sample = 82.5 μm *analysed by laboratory of test and calibration, the agency for chemical and packaging, jakarta, indonesia postharvest quality improvement of sorghum okky s. dharmaputra– et al. / results and discussions moisture content percentage of damaged grains moisture content of grains is one of the important factors that affected the deterioration of grains during storage. high moisture content will give an opportunity for fungal growth. sni (1992) defines 14% as the maximum moisture content of sorghum grains during storage. method of threshing, duration of storage and their interaction gave very significant differences in moisture content of sorghum grain. moisture content of sorghum threshed using wooden stick at the beginning of storage, subsequently after 1, 2 and 3 months of storage was significantly different from samples threshed using a paddy thresher during the same periods. the lowest moisture content (13.30%) was found in sorghum grain threshed using wooden stick before storage and after two months of storage (table 2). the highest moisture content (13.72%) was found in sorghum grain threshed using wooden stick after three months of storage. it was significantly different from that threshed using wooden stick before storage or after two months of storage. according to christensen . (1992) moisture content is always in equilibrium with the relative humidity of storage room. bala (1997) reported, that moisture content is also affected by the temperature of storage room. in this study, the range and mean of the temperature of storage room during storage were 26.8 29.3 c (28.1 c), while the range and mean of relative humidity of storage room were 47.5 70.8% (64.5%) (table 3). lacey and magan (1991) reported that among the microorganisms which colonize grains, fungi are the most tolerant to low relative humidity, consequently fungi has an important role in the deterioration of grain. method of threshing, duration of storage and their interaction gave very significant differences (at 99% significance level) in the percentage of damaged grain, while the interaction among method of threshing, infested with insects, oxygen concentration and duration of storage affected less significantly (95% significance level) the percentage of damaged grain. at the beginning of storage, the percentage of damaged grain of sorghum threshed using a wooden stick was higher and significantly different from that threshed with a paddy thresher. the percentage of damaged grain in each treatment combination increased and was significantly different during three months of storage. the sorghum grains threshed either using wooden stick or a paddy thresher, not infested with insects, packed under low or normal oxygen concentrations did not gave any significant difference on the percentage of damaged grains after three months of storage (table 4). the lowest percentage of damaged grain was found in sorghum threshed using a paddy thresher, not introduced with insect and stored under low oxygen concentration (ms0h) at the beginning of storage (5.00%), while the highest was found in sorghum et al o o 120 biotropia vol. 19 no. 2, 2012 threshed using wooden stick, infested with insect and stored under normal oxygen concentration (ks1n) after three months of storage ( 21.47%) (table 4). according to dobie (1991) is an important insect pest in stored grain. in indonesia could be a dominant insect species in milled rice and maize. the adult can live for several months up to one year. their eggs, larvae and et al. s. zeamais s. zeamais 121 table 2. moisture content of sorghum grains threshed using wooden stick and a paddy thresher during storage duration of storage (month) moisture content (%) wooden stick paddy thresher mean ± std mean ± std 0 13.30 ± 0.08 a 13.38 ± 0.05 b 1 13.55 ± 0.05 c 13.67 ± 0.15 de 2 13.30 ± 0.09 a 13.52 ± 0.12 c 3 13.72 ± 0.07 e 13.64 ± 0.09 d note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 3. range and mean values of temperature and relative humidity during storage duration of storage (month) range and mean of temperature (oc) range and mean of relative humidity (%) 0 1 27.4-29.3 (28.3) 54.0 -70.8 (59.8) 1 2 26.8-28.9 (28.3) 47.5 -68.6 (57.7) 2 3 27.2-28.2 (27.8) 48.7 -58.6 (57.9) table 4. percentage of damaged grains of sorghum caused by various treatments treatment duration of storage (months) 0 1 2 3 ks0h 9.90 ± 0.00 fegd 10.63± 2.63 fcegd 12.08 ± 2.23 cebd 20.20 ± 0.08 a ks0n 8.44 ± 1.66 fhgi 11.62 ± 0.16 fcebd 14.60 ± 0.00 b 18.48 ± 2.53 a ks1h 7.66 ± 1.26 hjgi 12.53 ± 2.80 cbd 13.33 ± 1.54 cb 19.73 ± 1.62 a ks1n 9.38 ± 0.35 fhegd 10.03 ± 3.03 fegd 11.99 ± 2.95 cebd 21.47 ± 0.83 a ms0h 5.00 ± 0.22 j 8.98 ± 0.22 fhegi 9.76 ± 0.19 fegd 10.45 ± 0.00 fcegd ms0n 5.04 ± 0.36 j 5.92 ± 0.12 ji 7.79 ± 0.82 hjgi 9.45 ± 0.45 fhegd ms1h 6.20 ± 0.45 hji 9.01 ± 0.00 fhegi 8.93 ± 2.06 fhegi 10.05 ± 0.05 fegd ms1n 5.07 ± 0.47 j 7.74 ± 4.46 hjgi 8.91 ± 1.63 fhegi 9.87 ± 0.56 fegd note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level k = wooden stick ; s0 = not infested with ; n = normal oxygen concentration (±21%) m = a paddy thresher ; s1 = infested with ; h = low oxygen concentration (± 18%) s. zeamais s. zeamais postharvest quality improvement of sorghum okky s. dharmaputra– et al. pupae are found inside of grain. dharmaputra . (2010) reported, that the dominant insect species of sorghum stored at farmer and collector levels in wonogiri and demak regencies were and . the existence of damaged grain before storage was due to the different threshers. during storage, the increase of damaged grain in sorghum infested with insects could be caused by insect and fungal attacks, while that not infested with insects may be due to fungal infection. effects of threshing and storage duration was highly different in the percentage of seed germination. percentage of seed germination in sorghum threshed using wooden stick (91.04%) was lower and significantly different from that of sorghum threshed using a paddy thresher (93.33%) (table 5). it was stronger correlated with the percentage of damaged grain in sorghum threshed using wooden stick than in grain threshed with a paddy thresher. percentage of seed germination during storage decreased with the increase storage duration (table 6). percentage of seed germination at the beginning of storage (93.88%) was significantly different from that after one (91.96%), two (91.88%) and three months of storage (91.04%). percentage of seed germination after one, two and three months of storage was not significantly different. according to neergard (1979) fungal infection in the embryo of seed is a factor causing the decrease of seed germination during storage. percentage of seed germination after three months of storage was more than 90%, thus the percentage is still higher than the minimum standard germination of seed, i.e. 70% (directorate general of food crops 1984). the capability of seed to germinate is affected by the atmosphere composition of storage room, but its effect is lower compared to the moisture content and the temperature of the storage ( priestley 1986). et al s. zeamais t. castaneum percentage of seed germination 122 biotropia vol. 19 no. 2, 2012 table 5. percentage of seed germination in sorghum threshed with wooden stick vs a paddy thresher thresher seed germination (%) a wooden stick 91.04 a paddy thresher 93.33 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 6. percentage of seed germination in sorghum grains during storage duration of storage (month) seed germination (%) mean ± std 0 93.88 ± 4.07 a 1 91.96 ± 2.20 b 2 91.88 ± 2.47 b 3 91.04 ± 3.22 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level population of andf. proliferatum f. verticillioides population of f. proliferatum s. zeamais f. proliferatum f. proliferatum f. proliferatum f. proliferatum f. prolliferatum population of f. verticillioides f. verticillioides f verticillioides f. verticillioides f. verticillioides fusarium aspergillus, eurotium penicillium f. proliferatum the duration of storage gave very significant difference (99% significance level) to the population of , while the interaction between the infestation by insects and the duration of storage, and the interaction among the four factors (method of threshing, presence of , oxygen concentration and duration of storage) gave significant difference (95% significance level) to the population of . population of in sorghum subjected to different postharvest treatments differed in the samples examined immediately after harvest but no clear trend could be detected. in contrast, no significant difference could be detected between treatments after 1, 2 and 3 months in storage (table 7). the population of in sorghum subjected to various treatments and storage duration fluctuated considerably resulted to very high standard deviation. the fluctuation was probably due to the existence of other fungal species which were antagonistic or synergistic to the growth of . the population of in grain subjected to various treatments decreased with the increase of storage duration. f. verticillioides the duration of storage gave significant difference in population. population of decreased significantly with the increase of storage duration (table 8). the population of . at the beginning of storage (1370 cfu/g) was significantly higher than that after three months of storage (433 cfu/g). the decrease of population was probably due to the existence of other fungal species which were antagonistic to the growth of . according to neergaard (1979) there are two groups of fungi infecting grains, i.e. field fungi and postharvest fungi. belongs to field fungi, while and belong to postharvest fungi. during storage, the activity of field fungi often stops, because they need high relative humidity (≥ 90%). 123 table 7. population of (cfu/g) in sorghum grains related to various treatments fusarium proliferatum treatment duration of storage (month) 0 1 2 3 ks0h 7333.33 ± 10540.44 ba 433.33 ± 133.50 c 289.00 ± 329.02 c 311.00 ± 371.55 c ks0n 1855.67 ± 650.07 c 933.33 ± 851.25 c 133.33 ± 88.44 c 77.67 ± 134.52 c ks1h 755.67 ± 680.58 c 578.00 ± 943.70 c 1444.67 ± 1924.89 c 333.33 ± 296.52 c ks1n 2677.67 ± 2117.54 bc 377.67 ± 77.36 c 533.33 ± 695.72 c 300.00 ± 185.71 c ms0h 1700.00 ± 1010.79 c 411.00 ± 416.61 c 500.00 ± 433.35 c 266.33 ± 57.74 c ms0n 9155.67 ± 8592.37 a 167.00 ± 0.00 c 113.00 ± 72.11 c 255.33 ± 385.09 c ms1h 1516.50 ± 118.09 c 1167.00 ± 707.11 c 2033.50 ± 2781.05 c 233.50 ± 47.38 c ms1n 1333.25 ± 585.33 c 358.50 ± 421.22 c 933.25 ± 968.68 c 1025.00 ± 1540.18 c note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level postharvest quality improvement of sorghum okky s. dharmaputra– et al. fumonisin b content carbohydrate content 1 1 1 1 1 1 1 1 1 method of threshing and duration of storage, and their interaction gave very significant differences in fumonisin b content. fumonisin b content in sorghum threshed using wooden stick was higher and significantly different from that of sorghum threshed with a paddy thresher after one, two and three months of storage (table 9). fumonisin b content in sorghum threshed using wooden stick fluctuated and showed significant differences at various points in time during storage. the content in sorghum threshed with a paddy thresher also fluctuated, but showed no significant differences between sampling times. the fluctuation of fumonisin b content in sorghum was probably due to the existence of certain strains of which can produce fumonisin b . the lowest fumonisin b (1.58 ppb) was found in sorghum threshed using wooden stick at the beginning of storage, while the highest in sorghum also threshed using wooden stick after one month of storage (27.55 ppb). fao (2004) reported the maximum allowable fumonisin b content in some countries. in france the maximum fumonisin b content allowed in cereals and their processed products is 1000 ppb, in cuba for maize and milled rice 1000 ppb, in switzerland for maize 1000 ppb. the existence of f and other toxigenic species of , and fumonisin production in the field and their products are determined by environmental factors in the field, during transportation and storage (gamanya & sibanda 2001). the duration of storage and the interaction between method of threshing were very significant (99% significance level). the carbohydrate content in sorghum before storage (67.33%) was not significantly different from that after two (68.64%) and three months (67.02%) of storage, but it was significantly different from that after one month of storage (70.81%) (table 10). the carbohydrate content in sorghum threshed using wooden stick and a paddy thresher after one and two months of storage was significantly different, while the content at the beginning of storage and after three months of storage was not significantly different. the lowest carbohydrate content was found in sorghum threshed using a paddy thresher at the beginning of storage (65.98%), while the highest content was found in sorghum threshed using a paddy thresher after one month of storage (74.11%). f. verticillioides . moniliforme fusarium biotropia vol. 19 no. 2, 2012 table 8. population of during storagefusarium verticillioides duration of storage (month) population of f. verticillioides (cfu/g) mean ± std 0 1370.88 ± 1320.68 a 1 1293.04 ± 2202.69 ba 2 1157.38 ± 1491.77 ba 3 433.00 ± 590.17 b numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level 124 suarni (2001) reported that carbohydrate content in sorghum was 80.42%. in this study the range of carbohydrate contents in samples that had been threshed using wooden stick was 66.69 71.28% during storage, while the content in sorghum threshed using a paddy thresher was 65.98 74.11% during storage (table 11). the difference of carbohydrate contents in this study and that of suarni (2001) was probably due to the difference of sorghum variety and the method of cultivation. garraway and evans (1984) reported, that carbon source needed for fungal growth were (among others) the carbohydrates (e. g. monosachharides). the capability of fungi to use sugars could be different among fungal species, even sometimes among different strains of the same species. the method of threshing and duration of storage differ very significant by (99% significance level) in the population of adult , while the interaction between the two factors gave a significant difference (95% significance level). population of in sorghum threshed using wooden stick after one and three months of storage was higher and significantly different from that of samples threshed with a paddy thresher (table 12). in general population either in sorghum threshed using wooden stick or a paddy thresher increased during storage. according to dobie (1991) the actual length of the life cycle of depends also upon the type and quality of grains being infested, i.e. in different varieties of maize, mean development periods of at 27°c and relative humidity 70% have shown to vary from 31 to 37 days. the duration of storage gave very significant difference (99% significance level) in weight loss of sorghum. the percentage of weight loss increased and differed significantly with the increase of storage duration (table 13). the weight losses of sorghum at the beginning of storage, subsequently after one, two and three months of storage were 1.62, 1.92, 2.21 and 2.30%, respectively. the increase of weight loss was due among others by the increase of the percentage of damaged grain (table 4) and population of (table 12) during storage. sitophilus zeamais population percentage of weight loss s. zeamais s. zeamais s. zeamais et al. s. zeamais s. zeamais s. zeamais duration of storage (month) wooden stick paddy thresher mean ± std mean ± std 0 1.58 ± 0.81 a 1.94 ± 1.47 a 1 27.55 ± 12.87 d 3.22 ± 2.71 a 2 20.43 ± 7.56 c 4.65 ± 4.66 a 3 12.57 ± 12.25 b 2.43 ± 3.32 a numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 9. fumonisin b content (ppb) in sorghum grains threshed using wooden stick and a paddy thresher during storage 1 125 postharvest quality improvement of sorghum okky s. dharmaputra– et al. biotropia vol. 19 no. 2, 2012 table 10. carbohydrate content in sorghum grains during storage duration of storage (month) carbohydrate content (%) mean ± std 0 67.33 ± 3.33 b 1 70.81 ± 5.57 a 2 68.64 ± 4.05 b 3 67.02 ± 1.45 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 11. carbohydrate content (%) in sorghum grains threshed using a wooden stick and a paddy thresher during storage duration of storage (month) w ooden stick paddy thresher mean ± std mean ± std 0 68.68 ± 4.18 ab 65.98 ± 1.32 a 1 67.51 ± 3.57 a 74.11 ± 5.34 c 2 71.28 ± 2.28 b 66.00 ± 3.71 a 3 66.69 ± 1.48 a 67.36 ± 1.41 a note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 12. population of adult (number/kg) in sorghum grains threshed using wooden stick and a paddy thresher during storage tests sitophilus zeamais duration of storage (months) wooden stick paddy thresher mean ± std mean ± std 0 10.00 ± 0.00 a 10.00 ± 0.00 a 1 20.38 ± 3.37 c 13.62 ± 1.81 b 2 15.98 ± 3.73 b 15.29 ± 1.97 b 3 21.11 ± 2.40 c 15.02 ± 1.16 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 13. percentage of weight loss of sorghum grains during storage duration of storage (months) weight loss (%) mean ± std 0 1.62 ± 0.44 a 1 1.92 ± 0.54 b 2 2.21 ± 0.51 bc 3 2.30 ± 0.49 c note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level 126 conclusions acknowledgments 1. the moisture content of sorghum grain subjected to various postharvest treatments was lower than that of safe moisture content of sorghum for storage (< 14%). 2. at the beginning of storage the percentage of damaged grains of sorghum threshed using wooden stick was higher than that of threshed using a paddy thresher. the percentage of damaged grain in each treatment increased with the increase of storage duration. after three months of storage the highest percentage of damaged grain was found in sorghum threshed using wooden stick, infested with , and stored under normal oxygen concentration (± 21%). 3. the seed germination percentage of sorghum threshed using wooden stick was lower than that of samples threshed with a paddy thresher. during storage the percentage of seed germination decreased with the increase of storage duration. 4. populations of and decreased with the increase of storage duration. fumonisin b content in sorghum threshed with wooden stick was higher than that of samples threshed with a paddy thresher during one, two and three months of storage. fumonisin b contents were considered low. in general carbohydrate content in sorghum threshed using either wooden stick or a paddy thresher from the beginning of storage up to three months of storage was not significantly different. after three months of storage, population either in sorghum threshed using wooden stick or a paddy thresher was higher and significantly different from that of at the beginning of storage. the percentage of weight loss of sorghum increased with the increase of storage duration. after three months of storage the percentage of weight loss was higher and and significantly different compared to that at the beginning of storage or any other sampling time before three months. the two different oxygen concentrations inside of the bags did not produce any significant difference in the quality of sorghum. threshing using a paddy thresher was better in comparison to threshing using a wooden stick. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to pt tri fondasi indonesia for providing sorghum grains, to the agency for chemical and packaging, jakarta for analyzing plastic packaging material, to pt angler biochemlab, surabaya for analyzing fumonisin b content, to indonesian center for agricultural post harvest research and development for analyzing carbohydrate content; to mr. edi suryadi, ms. nijma nurfadila, ms. lia amelia, ms. dwi ayu kencana ungu, for their assistance. we would like also to express our appreciation to the reviewers of this manuscript. sitophilus zeamais fusarium proliferatum f. verticillioides s. zeamais 1 1 1 127 postharvest quality improvement of sorghum okky s. dharmaputra– et al. references abdel-hafez sii, moubasher h, shoreit m, ismail m. 1990. fungal flora associated with combine harvester wheat and sorghum dusts from egypt. j basic microbiol 30: 467-9. bala bk. 1997. drying and storage of cereal grains. new hampshire: 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american association of cereal chemist. p 313-40. sirappa mp. 2003. prospek pengembangan sorgum di indonesia sebagai komoditas alternatif untuk pangan, pakan, dan industri. j litbang pertanian 22 (4): 133-140. 1 1 1 1 1 1 1 in fusarium moniliforme f. verticillioides in in th nd th 128 biotropia vol. 19 no. 2, 2012 [sni] standar nasional indonesia. 1992. sorgum. sni 013157 1992. jakarta: badan standardisasi nasional. suarni. 2001. tepung komposit sorgum, jagung dan beras untuk pembuatan kue basah (cake). risalah penelitian jagung dan serealia lain. vol.6, p 55-60. maros: balai penelitian tanaman jagung dan serealia. suarni. 2004. pemanfaatan tepung sorgum untuk produk olahan. j litbang pertanian 23 (4): 145-51. zöllner p, mayer-helm b. 2006. trace mycotoxin analysis in complex biological and food matrices by liquid chromatography atmospheric pressure ionization mass spectrometry. j of chromatography a 1136: 123-69. 129 postharvest quality improvement of sorghum okky s. dharmaputra– et al. 2. diah ratnadewi.cdr biotropia vol. 20 no. 1, 2013: 10 18 enhanced production level of quinine in cell suspension culture of cinchona ledgeriana moens by paclobutrazol 1 2 3 diah ratnadewi *, dedi satriawan , and sumaryono 1 department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia 2 department of biology, faculty of mathematics and natural sciences, bengkulu university, kandang limun campus, bengkulu, indonesia 3 indonesian biotechnology research institute for estate crops, jalan taman kencana no. 1, bogor 16151, indonesia received 10 januari 2013/accepted 22 may 2013 abstract quinine is one of the major alkaloids in cinchona spp., and it is used both as a medication and as a drink additive. the plant produces most of its alkaloids in the bark after 6-8 years of age. repeated harvests can be performed until the plant dies, but only after every 3-5 years. we tested an improved method for culturing cell suspensions of cinchona ledgeriana investigate the possibility of increasing production of quinine. the clone qrc 315 was treated with either a growth retardant (abscisic acid or paclobutrazol) or precursor feeding of ltryptophan. to generate stress, we applied mannitol at 5.3 g/l combined with sucrose at a lower concentration (20 g/l), and we used sucrose at 30 g/l as the control. paclobutrazol (7 mg/l) significantly suppressed cell growth and produced the highest level of quinine (11%) after 7 weeks of culture. l-tryptophan also reduced cell growth, but without any positive effect in the production of quinoline. the highest amount of quinine per culture flask, however, resulted from cells treated with 3 mg/l abscisic acid. key words: cinchona ledgeriana, suspension culture, quinine, paclobutrazol introduction more than 30 alkaloids have been isolated from the bark of cinchona spp. but the most economically important cinchona alkaloids are the quinolines: quinine, quinidine, cinchonine, and cinchonidine (mccalley 2002). quinine is the major alkaloid and has been used for hundreds of years as an anti-fever agent and for prevention and treatment of malaria. malaria still exists in many tropical areas of the world. the world health organization (2011) reported 36 000-655 000 deaths . . * corresponding author : diahbiologi.ipb@gmail.com 10 doi: 10.11598/btb.2013.20.1.10 between 2009 and 2010 in 106 malaria-endemic countries. apart from its use to treat malaria, quinine also has analgesic and anti-inflammatory properties; its sulfate salt is used in eye lotions as an antibacterial and anesthetic agent (taylor 2005, kurian & sankar 2007). south americans use quinine as a natural remedy for cancer, amoebic infections, heart problems, diarrhea, dysentery, varicose veins, and nocturnal leg cramps (taylor 2005). in addition, quinine salts are added to tonic drinks and beverages to achieve a bitter taste. taylor (2005) reported a commercial market of approximately 300-500 metric tons of quinine alkaloids, which are extracted annually from 5000-10000 metric tons of harvested bark. at the time of that report, half of the harvest was directed to the food industry. the bark of wild cinchona yields 7% quinine, whereas cultivated crops yield up to 15%, none of which can be obtained until after the plant is 6-12 years old (barrett 1928). quinine production from its cultivation is, therefore restricted by these growth limitations. cell suspension culture of cinchona spp. could solve the above problem. several groups of researchers have attempted enhancement of cinchona alkaloid production in cell suspension cultures by using stressing agents, precursors, elicitors, auxins, and enzymes (harkes et al. 1986, wijnsma et al. 1986, payne et al. 1987, robins et al. 1987, toruan-mathius et al. 2006). previous research on c. ledgeriana clone qrc 313 increased quinine content to 0.12%, which was 11-fold more than that in control cells (ratnadewi & sumaryono 2010). although promising, this result is still far from satisfactory. in this study, we sought to improve quinine production methods by testing a selected potential clone of c. ledgeriana in treated cell-suspension cultures. materials and method preparation of cell-suspension cultures lamina of young leaves (the second or the third leaf from the shoot tip) from the c. ledgeriana clone qrc 315 were surface-sterilized with 0.2% dithane m-45 for 10 minutes, followed by treatment with 20% commercial naocl for 15 minutes. these lamina were used as explants for callus production on semi-solid woody plant (wp) media (lloyd & mccown 1981), which contained 15 µm picloram, 2 µm benzyladenin (ba), 1 µm phloroglucinol, and was solidified with 3.5 g/l gelrite (mb cell). sucrose, at a standard level (30 g/l), was added to the media. after 8 weeks in the initiation and proliferation media, fast-growing calli were transferred to baffle flasks that contained wp liquid media, and homogenized for 1 week before being used for cell suspension culture. liquid media composition was the same as the semi-solid, except that ba was reduced to 0.5 µm. this procedure was taken from that described by sumaryono and riyadi (2005), with slight modification. one spatula (0.2-0.3 g) of cells, which had been filtered through 1000 and then 50µm filter mesh, was transferred into erlenmeyer flasks containing 20 ml liquid wp media, of which the composition was the same as for cell homogenization. this was designated as the basic media for this research. we varied the basic media by addition of certain growth regulators: either (1) abscisic acid (aba) or paclobutrazol (pbz); 11 enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. and (2) a precursor, l-tryptophan (trp), in combination with sucrose (at either a standard or lower concentration) with or without mannitol. we applied the following treatments to the basic media: (1) 1 mg/l aba + 30 g/l sucrose(aba1), 3 mg/l aba + 30 g/l sucrose (aba3), 0.2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol (trp0.2-m); (2) 2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol (trp2-m); 5 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol (pbz5-m); and 7 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol (pbz7-m). the molarity of 30 g/l sucrose was equivalent to that of 20 g/l sucrose + 5.3 g/l mannitol. the cell suspension cultures were maintained for 7 weeks on a horizontal shaker, at 100 rpm, 26 ± 1°c, 2 under light intensity of 20 µmol photon/m /second for 12 hours per day. cell growth was measured once per week until the cells were harvested. we used cell volume after sedimentation (cvs) to represent cell growth, according to the methods described by blom et al. (1992).cell volume (a ml) was then calibrated into fresh weight (g) using the following formula: {(a 0.4565) 0.3252}. quinoline extraction and analysis at weeks 6 and 7, five flasks of suspension culture were simultaneously harvested from each treatment for quinoline alkaloid content because cinhona ledgeriana cells achieve their maximum growth at week 6 (sumaryono & riyadi 2005). reversed-phase high-performance liquid chromatography (hplc; varian-usa, type 940) was used to measure the four major quinolines (quinine, quinidine, cinchonine, and cinchonidine). quinoline extraction and analysis was performed as described previously by klink (1979), with some modifications. briefly, as much as 0.5 g oven-dried cell powder was taken for the extraction and purification, boiled in 95 ml aquadest for 25 minutes, and then allowed to cool at room temperature. the remaining liquid was then adjusted to 100 g by adding aquadest. a 5-ml aliquot was taken from decanted solution and filtered using a 0.45-µm millipore filter then injected into an hplc column (pursuit xrs3 µ c-18, column length 150 cm x 4.6 mm). the column temperature was adjusted o to 30 c. aquadest, acetonitril, and glacial acetic acid (ratio 81:18:1, respectively) served as an eluent using a 0.6 ml per minute flow rate with six attenuation. standard quinoline alkaloids (sigma, germany) served as positive controls. the chromatogram was detected by uv-vis at 250 nm. data from each treatment were collected from duplo analysis. results and discussion cell growth one week after being transferred into treated liquid media, the cells grew well. treatment with aba with the normal rate of sucrose (30 g/l) demonstrated the highest growth, whereas trp0.2-m, trp 2-m, pbz5-m, and pbz7-m resulted in lower performance, even compared to the control. pbz7-m greatly restricted cell growth, whereas pbz5-m slightly reduced cell growth. in general, the highest fresh weight was reached at the sixth week in the treated media, and it decreased afterwards (fig. 1-a, 12 biotropia vol. 20 no. 1, 2013 -b, -c). this was consistent with the results of sumaryono and riyadi (2005). in previous work (ratnadewi & sumaryono 2010), the incorporation of aba and pbz into the media at the fifth week of cell culture had no negative effect on cell growth. in this research, aba treatment from the beginning of the culture even increased growth. aba can both promote and inhibit plant growth (finkelstein & rock 2002). it has a complex role in various cellular processes, such as embryo maturation and germination, vegetative growth, and tolerant response to drought stress (xiong & zhu 2003 hirayama & shinozaki 2007, hubbard et al. 2010). figure 1. cell growth in suspension culture of c. ledgeriana treated with aba (a), tryptophan (b) and pbz (c). data are expressed as mean ± sd (n = 10). c, control media/basic media; aba1, basic media treated with1 mg/l aba; aba3, basic media with 3 mg/l aba; trp0.2-m, basic media with 0.2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol; trp2-m, basic media with 2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol; pbz5-m, basic media with 5 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol; pbz7-m, basic media with 7 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol. enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. 13 in contrast, we observed that growth inhibition of pbz, applied from the starting point of culture, reduced cell growth. we hypothesized that the inhibiting action of pbz required more time to cause an effect. by increasing the concentration of pbz to 7 mg/l, combined with 5.3 g/l mannitol and 20 g/l sucrose, the cell growth rate diminished significantly (fig. 1-c). pbz is known to be an antagonist of gibberellins. it inhibits gibberellin biosynthesis at the point when kaurene is oxidized into kaurenoic acid (graebe 1987, chaney 2004), because gibberellin and aba biosynthesis begin at the same initial pathway (i.e., from geranyl pyrophosphate),both growth regulators may be affected by pbz along their synthesis pathways. also, blockage of the terpenoid pathway causes accumulation of aba (chaney 2004) and could therefore, lead to unbalanced cellular metabolism. this, in turn, would reduce the rate of cell division (graebe 1987). chaney (2004) suggested that these cells will still divide and differentiate at the same rate but simply do not elongate. this may explain why cell growth was lower in the cultures treated with pbz. one of the precursors in the biosynthesis of quinoline alkaloids is tryptophan (facchini 2001). we used it in combination with mannitol as a partial substitute of sucrose in the culture media with the expectation that it would increase the production of quinoline in cells. tryptophan is also involved in the first step of the auxin biosynthesis pathway. in this study, cell growth rate in tryptophan-treated media, however, was less than that of untreated cells. this may be because tryptophanderived auxin either was not at an optimal level or simply auxin synthesis from tryptophan did not occur. sucrose is the main source of energy and carbon in in vitro cultures. substitution of some sucrose in media with mannitol reduces the supply of carbon and energy to cells because mannitol is an alcohol sugar that cannot replace sucrose (although it determines the osmotic potential value of media). mannitol has been used as an osmotic stressing agent in many experiments (hassanein 2004, 2010; hussein & aqlan 2011). le clere et al. (2010) confirmed that sugar concentration in in vitro-cultured maize kernels regulates the expression of the gene, zmyuc, which is involved in the biosynthesis of auxin through hydroxylation of tryptamine. reduced levels of sucrose in the culture media, along with mannitol substitution, combined with tryptophan, might explain the lower growth rate of the corresponding treated cultures, which may have used less tryptophan for the synthesis of auxin. quinine in the suspension cultures in normal culture media, cinchona cells produced quinoline alkaloids as demonstrated by robins et al. (1986) and ratnadewi and sumaryono (2010). quinine appeared to be the main alkaloid produced by the cells, occurring at levels of 4-7% in the control media (c). we found all four alkaloids at the sixth week of culture in all treatments (table 1). cinchonidine was the least abundant, followed by cinchonine, quinidine, and quinine, which was the most abundant. this percentage of quinine was further augmented 1 week later, at the seventh week of culture, which was in contrast to our results for quinidine, cinchonine, and cinchonidine. the four types of alkaloid are readily 14 biotropia vol. 20 no. 1, 2013 transformed from one to another type due to their similar molecular structure (tadeusz 2007). all the treatments applied to the suspension cultures were best at producing quinine rather than the other types of quinoline. cell suspension treated with pbz7-m presented a remarkably high percentage of quinine content (11%). this value was 1.5-fold higher than that of the untreated cultures at the seventh week. in cells treated with aba, trp-m or pbz5-m, the quinine level was either at an average level or less than that contained in the untreated cells (c). precursor feeding of tryptophan did not improve quinoline alkaloid production in cinchona cell cultures. this is consistent with the results obtained by robins et al. (1987) in c. pubescens and by payne et al. (1987) in a transformed line of c.ledgeriana. harkes et al. (1986) and robins et al. (1987) found an accumulation of β-carboline alkaloids and indole-3-aldehyde in tryptophan-fed growth media, with and without cinchona cells. according to cao et al. (2007), β-carboline alkaloids (a large group of indole alkaloids) are easily transformed from tryptophan, tryptamine, pyruvate, or acetate precursors by pictet-spengler reactions, even in foods and beverages. this indicates that the transformation of tryptophan to quinoline alkaloids needs more particular conditions in living cells. moreover, payne et al. (1987) suggested that growing cultures in darkness is more favorable than under light when attempting to enhance alkaloid accumulation. ratnadewi and sumaryono (2010) found that providing 0.2 mg/l tryptophan to the c ledgeriana clone qrc 313 increased total alkaloids (predominantly cinchonidine) 35fold more than those in untreated cultures after 6 weeks. cinchona plants naturally have variable types of alkaloids. according to taylor (2005), quinine content averages 1-3% in c. pubescens and 3-13% in c ledgeriana, whereas c. succirubra can reach 4-14% but only 6-8 years after planting. when we examined the dry weight cells collected from each culture flask and the quinine content percentage, we found that cells treated with aba which had the highest rate of cell growth produced the highest amount of quinine at 6 and 7 weeks of culture at the end, compared to any other treatment (table 2-a and -b) and aba3 had the greatest abundance of quinine (almost 3 times more than that produced by pbz7-m per flask). it remains challenging to improve quinine production in cells treated with pbz7-m. table 1. quinoline content in cells of c. ledgeriana at 6 and 7 weeks of culture (% dry weight) quinine quinidine cinchonine cinchonidine treatment 6 weeks 7 weeks 6 weeks 7 weeks 6 weeks 7 weeks 6 weeks 7 weeks c 4.60 7.12 2.61 1.40 0.69 0.42 0.40 0.18 aba1 3.97 5.7 2.40 2.32 0.57 0.56 0.27 0.20 aba3 5.08 6.83 3.11 2.19 0.46 0.37 0.51 0.15 trp0.2-m 3.72 5.12 2.45 1.48 0.80 0.49 0.29 0.22 trp2-m 4.36 6.04 1.97 1.26 0.62 0.50 0.32 0.34 pbz5-m 5.01 5.75 2.80 2.07 0.73 0.71 0.19 0.28 pbz7-m 4.15 10.90 3.66 2.39 2.90 0.65 0.28 0.30 note : these data are means of duplo analysis, collected each from 5 flasks of cell culture enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. 15 conclusions the growth retardant paclobutrazol at 7 mg/l combined with 5.3 mg/l mannitol and a lower concentration of sucrose (20 mg/l) significantly increased the accumulation of quinine in the cells of c. ledgeriana clone qrc 315. quinine approached the level extracted from productive c. ledgeriana bark. precursor feeding by l-tryptophan to the cell culture media increased neither quinine nor the other major quinoline alkaloids. the way in which it transforms to quinoline alkaloids remains incompletely understood. the high growth rate of cells treated with 3 mg/l aba and sucrose at normal concentration (30 g/l) resulted in the most abundance of quinine per culture flask. acknowledgments the authors thank the iaard of the indonesian ministry of agriculture for sponsoring this research through the incentive program. table 2. production of quinine per flask of cell culture of c.ledgeriana treated with various substances after 6 (a) and 7 weeks (b) treatment means of cell dry weight/flask (g)* quinine (%)** quinine (mg/flask) c 0.122 ± 0.042 4.6 0.561 aba1 0.201 ± 0.024 3.97 0.722 aba3 0.182 ± 0.046 5.08 1.021 trp0,2-m 0.091 ± 0.018 3.72 0.338 trp2-m 0.106 ± 0.032 4.36 0.462 pbz5-m 0.116 ± 0.078 5.01 0.581 pbz7-m 0.064 ± 0.041 4.15 0.265 treatment mean of cell dry weight/flask (g) * quinine (%) ** quinine (mg/flask) c 0.115 ± 0 .045 7.12 0.819 aba1 0.175 ± 0 .032 5.70 0.997 aba3 0.206 ± 0 .043 6.83 1.407 trp0,2 -m 0.091 ± 0 .023 5.12 0.466 trp2 -m 0.111 ± 0 .030 6.04 0.67 0 pbz5 -m 0.097 ± 0 .064 5.75 0.558 pbz7 -m 0.050 ± 0 .037 10 .90 0.545 a b 16 biotropia vol. 20 no. 1, 2013 references barrett. 1928. the tropical crops. new york: the mcmillan. blom tjm, kreis w, van iren f, libbenga kr. 1992. a non-invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures. plant cell rep 11:146-9. cao r, peng w, wang z, xu a. 2007. β-carboline alkaloids: biochemical and pharmacological functions. curr med chem14: 479-500. chaney wr. 2004. paclobutrazol: more than just a growth retardant. pro-hort conf. peoria il: febr 4th. department of forestry and nat res, purdue university.www.ina online.org/prohort/treegrowth. pdf. facchini pj. 2001. alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation and metabolic engineering applications. ann rev plant physiol plant mol biol 52:29-66. finkelstein rr, rock cd. 2002. abscisic biosynthesis and response. in: the arabidopsis book. new york. amer soc plant biologists1-52. graebe je. 1987. gibberellin biosynthesis and control. annu rev in plant physiol 38: 419-65. harkes paa, de jong pj, wijnsma r, verpoorte r, van der leer. 1986. the influence of production media on cinchona cell cultures; spontaneous formation of β-carbolines from l-tryptophan. plant sci 47:71-6. hassanein am. 2004. effect of relatively high concentration of mannitol and sodium chloride on regeneration and gene expression of stress tolerant (alhagi graecorum) and stress sensitive (lycopersicon esculentum l) plant species. bulg j plant physiol 30: 19-36. hassanein am. 2010. establishment of efficient in vitro method for drought tolerance evaluation in pelargonium. j hort sci & ornament plant 2: 8-15. hirayama t, shinozaki k. 2007. perception and transduction of abscisic acid signals: keys function of the versatile plant hormone aba. trends in plant sci 12: 343-51. hubbard ke, nishimura n, hitomi k, getzoff ed, schroeder ji. 2010. early abscisic acid signal transduction mechanisms: newly discovered components and newly emerging questions. gene develop 24:1695708. hussein ea, aqlan em. 2011. effect of mannitol and sodium chloride on some total secondary metabolites of fenugreek calli cultured in vitro. plant tiss cult biotech 21:35-43. klink f. 1979. determination of caffeine and theobromine in chocolate product. in: varian instrument at work. walnut creek 94-7. kurian a, sankar ma. 2007. medicinal plants. in: horticultural science series-2. edited by peter kv. new delhiindia: new india publ agency. le clere s, schmelz ea, chourey ps. 2010. sugar levels regulate tryptophan-dependent auxin biosynthesis in developing maize kernels. plant physiol 153: 306-18. lloyd g, mccown b. 1981. commercially feasible micropropagation of mountain laurel kalmia latifolia by use of shoot tip culture. comb proc intl plant prop soc 30: 421-27. mccalleydv. 2002. analysis of the cinchona alkaloids by high-performance liquid chromatography and other separation techniques. j chromatogr a 967:1-19. payne j, rhodes mj, robins rj. 1987. quinoline alkaloid production by transformed cultures of cinchona ledgeriana. planta med 53: 367-72. ratnadewi d, sumaryono. 2010. quinoline alkaloids in suspension cultures of cinchona ledgeriana treated with various substances. hayati j biosci 17: 179-82. robins rj, hanley ab, richards sr, fenwick gr, rhodes mjc. 1987. uncharacteristic alkaloid synthesis by suspension cultures of cinchona pubescens fed with l-tryptophan. plant cell, tiss organ cult 9: 49-59. sumaryono, riyadi i. 2005. growth of callus and cell suspension cultures of cinchona (cinchona ledgeriana moens). menara perkebunan 73:1-9. enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. 17 tadeusz a. 2007. alkaloid-secrets of life.in alkaloid chemistry, biological significance, applications and ecological role. elsevier. taylor l. 2012. the healing power of rainforest herbs. [tropical plant database: file for quinine (cinchona officinalis). rainforest database.com/plants/quinine.htm. 2005]. cited 13 august 2012. toruan-mathius n, haris n, santoso j, ade-heri. 2006. effect of elicitation on growth and alkaloid quinoline production in hairy root of cinchona plant (cinchona succirubra pavon ex klotzsch). menara perkebunan 74:10-22. wijnsma r, verpoorte r, harkes paa, van vliet tb, ten hoopen hjg, svendsen ab. 1986. the influence of initial sucrose and nitrate concentration on the growth of cinchona ledgeriana cell suspension cultures and the production of alkaloids and anthraquinones. plant cell tiss org cult 7: 21-9. world health organization: world malaria report. [http://www.who.int/malaria/world_malaria_report2011/en/2011]. cited 26 august 2012. xiong l, zhu j-k. 2003. regulation of abscisic acid biosynthesis. plant physiol 133: 29-36. 18 biotropia vol. 20 no. 1, 2013 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 12 biotropia vol. 15 no. 1, 2008biotropia vol. 15 no. 1, 2008 : 12 24 somatic embryogenesis from meristem explants of ginger otih rostiana* and sitti fatimah syahid *division of plant breeding, indonesian medicinal and aromatic crops research institute jalan tentara pelajar no. 3 bogor 16111, indonesia abstract the use of planting materials from in vitro culture, especially derived from somatic embryos has some advantages such as genetically stable and pathogen-free. meristem culture of ginger through somatic embryogenesis could be a potential method for producing pathogen-free planting materials. somatic embryogenesis on ginger was performed to obtain vigorous plantlets having the same rhizome size as the mother plant. callus was induced from meristem tissue of inner bud of indonesian ginger rhizome var. cimanggu-1 and consecutively subcultured into certain media at each steps of experiments. the vigorous embryogenic calli were observed on ms medium containing 100 mgl-1 glutamine and 2% sucrose with addition of 1.0 mgl-1 2,4-d + 3.0 mgl-1 ba. the highest number of somatic embryos (about 82.0.g-1 friable calli) was achieved on that medium, 4 weeks after culturing. furthermore, the optimum growth of embryogenic calli containing somatic embryo was obtained on ms medium enriched with 6% sucrose. the highest number of mature somatic embryos (57.2 embryos) was achieved on ms medium, 18 days after incubation. the regeneration potency of somatic embryos obtained from ginger meristem was 51.20%.g-1 friable callus. the valuable result of this study was the achievement of normal rhizome size of regenerated plantlets, instead of micro rhizome. key words: zingiber officinale rosc., meristem culture, somatic embryogenesis. introduction ginger (zingiber officinale rosc.) is one of the most important export commodities of indonesia. this crop is also one of the important components of indonesian herbal medicine, locally called jamu, phytopharmaca and is contributing to the indonesian foreign exchange and work opportunity for labors. the lack of market stock and serious damages due to pest and disease, have caused high fluctuations to indonesian ginger exports and prices within the last decades. to overcome the limiting factors, the use of healthy planting material is necessary to be developed. plant tissue culture has been adopted for the purpose of in vitro mass propagation on various crop species. in case of the big-white ginger cultivar of indonesian variety cimanggu-1, the use of shoot bud-derived plantlets, either by direct organogenesis or through callusing, has been developed to obtain disease-free planting materials (mariska * corresponding author : otihrostiana@yahoo.com 13 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid and syahid 1994). in further field experiments, the healthy plantlets and vigorous plants were not able to produce normal rhizomes (the same-big rhizome as their mother plants), eventhough they were repeatedly planted in three consecutive generations (syahid and hobir 1996). it is suggested that genetic alteration or epigenetic change during the in vitro culture and regeneration have been performed. in order to eliminate the genetic change during the in vitro culture, other regeneration pathway should be considered. somatic embryogenesis has been accomplished in some species for producing millions of seeds. the form of somatic seeds through somatic embryogenesis, is more efficient than organogenesis. besides, somatic embryogenesis is more preferable in plant genetic improvement through in vitro culture and genetic transformation as well, because single cell derived-plant is eassier to be controlled as somatic embryo derived-plant. in general, somatic embryogenesis and meristem derived plants are true-to-type and genetically identical to the mother plants (evans and sharp 1986; jimenez 2001), however, certain differences might appear according to the plant species characteristics. the true-to-type of somatic embryo derived plantlet has been found in in vitro culture of picea abies (heinze and schmidt 1995). on the other hand, among recalcitrant species such as serealia crops and conifers, good results have been performed by the induction of somatic embryogenesis. achievements in inducing somatic embryogenesis in monocots are mostly derived from generative explants. meanwhile, in dicotyl vegetative explant, such as leaves, are commonly applied for inducing somatic embryo derived-plantlet. leaf explant of orchard grass completely induces somatic embryo through the formation of embryogenic calli with addition of a strong auxin like dicamba (bhojwani and razdan 1996). on the other hand, kackar et al. (1993) performed the somatic embryo culture of indian ginger var. eruttupetta by using an aseptic leaf-explant with the addition of 2,4-d and dicamba. the same explant source was also performed in inducing somatic embryogenesis of fingerroot (boesenbergia rotunda l.) and galangal (kaempferia galanga l.) (tan et al. 2005; rahman et al. 2004). this study was aimed at obtaining the normal-size of rhizome (the same size as their mother plant) through induction of somatic embryos from meristem explant of indonesian var. cimanggu-1. most meristematic cells of meristem tissue derivedsomatic embryo are genetically stable and not easy to be mutated (bach and pawlowska 2003). therefore, to eliminate somaclonal variation and other genetic alteration during the in vitro culture, meristem tissue will be applied for the induction of somatic embryogenesis on ginger. 14 biotropia vol. 15 no. 1, 2008 materials and methods explant preparation the inner shoot bud (meristem) of indonesian var. cimanggu-1, was excised and simultaneously sterilised by using sterilizing agents such as 70% etoh, 5% sodium hypochlorite and 0.2% mercury chloride, for about 5-10 minutes, followed by rinsing with sterile-aquadest. callus induction sterilized meristems were placed on ms basal medium (murashige and skoog 1962) consisting of 8% agar, 2% sucrose, 100 mg l-1 l-glutamine, 1.0 to 3.0 mg l-1 2,4-dichlorophenoxy acetic acid (2,4-d) and 0 to 5.0 mg l-1 n6-benzyl adenin (ba). a single factor experiments were arranged in completely randomized design, replicated three times. subculturing. embryogenic calli were formed 8 weeks after culturing on callus induction medium, and then transferred on to hormone-free ms or n6 basal media with the addition of 3% mannitol for calli proliferation. a single factor experiments were arranged in completely randomized design, replicated four times for each treatments. pro-embryos were then subcultured into the best basal medium, either ms or n6 basal media, according to the best result of the previous stage of experiment, with the addition of 6% sucrose (6s) for obtaining mature embryo. single-factor experiments were arranged in completely randomized design, replicated four times. regeneration mature embryos (torpedo-like structure-embryos) were subcultured into regeneration medium for further development. ms basal medium with addition of 3% sucrose and 200 mgl-1 l-proline were applied in combination with pgrs such as ba at the concentration of 0, 0.1, 0.5 and 1.0 mgl-1, ga3 at the concentration of 0, 0.1, 0.3, 0.5 and 1.0 mgl-1. single-factor experiments were arranged in completely randomized design, replicate three times. histology the callus at each stage of development was subjected to histological analysis according to sass (1951) by using paraffin-embedded callus dissection following formaldehyde-glacial acetic acid-alcohol (faa) fixation series. statistical analysis all collected data were analyzed by using anova (p0.05) followed by duncan’s multiple range test (p. 0.05) (windows computers, 2000). observation results on embryo proliferation were analyzed by using t-test according to furlong et al. (2000). 15 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid results and discussion callus induction callus initiation was on tract when the basal-edge of the meristems enlarged, followed by the change of shoot-dome colour from white to yellowish, at two weeks after culturing. eight weeks after culturing, friable callus (embryogenic callus) formation of about 93.33%/explant was formed on ms basal medium enriched with 1 mg l-1 2,4-d in combination with 3 mg l-1 ba. though, that combination was statistically the same as the treatment without ba (table 1). table 1. percentage of embryogenic calli from ginger meristem cultured on ms medium enriched with various concentration of 2,4-d and ba, 8 weeks after culturing treatment percentage of embryogenic calli (%) 2,4-d (mg.l-1) ba (mg.l-1) 1 0 86.67 a*) 1.0 8.33 e 3.0 93.33 a 5.0 70.00 b 2 0 46.67 c 1.0 25.00 d 3.0 63.33 b 5.0 61.67 b 3 0 28.33 d 1.0 36.67 cd 3.0 63.33 b 5.0 61.67 b note: numbers followed by the same letters are not significantly different according to dmrt (5%). the application of 2,4-d on ginger meristem culture showed that the higher the concentration applied, the more compact calli were observed. furthermore, when 2,4-d was applied at 2-3 mg.l-1 browning and necrotic calli were observed. an addition of 1.0 mg.l-1 ba into medium containing 2,4-d neither vigorous embryogenic calli were obtained, low quantity and compact calli were observed (table 1). embryogenesis from the subcultured callus for obtaining globular somatic embryos structure the formed embryogenic calli were cultured on either ms or n6 basal media with the addition of 3% mannitol. transparent globular embryos were developed on both media in one week after subculturing. the shape of ginger embryos at globular phase was generally round or oval (fig. 1a). 16 biotropia vol. 15 no. 1, 2008 a c e f g b d mm mm cm cm cm mm mm figure 1. the development of ginger meristem into newly regenerated plant with normal rhizome size through somatic embryogenesis. a. the structure of globular somatic embryo of ginger, 4 weeks after subculturing into proliferation medium (30 times enlargement). b. globular embryo, 2 weeks after proliferation (40 times enlargement). protoderm layer started to differentiate (arrowed). c. the structure of torpedo somatic embryo of ginger, 18 days after subculturing into maturation medium (10 times enlargement). d. torpedo embryo, 18 days after subculturing into maturation medium (40 times enlargement). arrowed: differentiated procambium. e. embryo somatic-derived seedlings on ms medium supplemented with 1 mgl-1 ba (left) and embryo somatic-forming adventitious roots on hormone free-ms medium (right), 30 days after subcultured (1 : 1.4 scaled). f. embryo somatic-derived normal plantlet, 8 weeks after subcultured on to hormone-free ms medium (1: 1.3 scaled). g. normal rhizome size of ginger-regenerated plants obtained from meristem culture through somatic embryogenesis. 17 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid numbers of globular phase of somatic embryos increased due to the age of cultures. the highest number of somatic embryo was obtained at 4 weeks after subculturing, 82.00 ± 12.25 embryo.g-1 embryogenic callus on ms medium and 70.00 ± 19.26 embryo.g-1 embryogenic callus on n6 medium. statistical analysis showed that ms basal medium was more capable in inducing higher numbers of somatic embryo than that of n6 basal medium (p0.05). the number of somatic embryo decreased by the increase of age of cultures (table 2). histological analysis conformed that the somatic embryo was induced from cortex tissue and that the globular phase developed at 2 weeks after culturing on proliferation medium (fig. 1b). table 2. number of somatic embryos derived from ginger meristem culture on ms and n6 basal media enriched with 3% mannitol period (weeks) average number of somatic embryos/g of embryogenic callus ms n6 1 32.75 ± 11.97 39.75 ± 5.31 2 54.50 ± 20.16 51.50 ± 9.66 3 70.50 ± 31.97 48.75 ± 15.16 4 82.00 ± 12.251) 70.00 ± 19.261) 5 53.75 ± 16.32 36.25 ± 9.28 6 51.50 ± 10.92 22.75 ± 15.28 7 43.50 ± 19.55 11.00 ± 7.65 note: based on t-test at 5% level. mature embryo formation was performed on ms basal medium, enriched with 6% sucrose. cylindrical form of mature embryo started to develop into 2 different domes, i.e. cotyledons and apical shoot-forming dome as well as root-forming dome. two days after subculturing into ms medium enriched with 6% sucrose, about 10.60 ± 4.76 torpedo-shape somatic embryos were initiated from 1 g of embryogenic calli (table 3). 18 biotropia vol. 15 no. 1, 2008 table 3. number of mature somatic embryos on ms basal medium supplemented with 6% sucrose age(day) average number of mature embryo/g of embriogenic calli1) 2 10.60 ± 4.76 6 33.40 ± 6.74 10 55.80 ± 12.73 14 38.60 ± 6.53 18 57.20 ± 15.99 22 45.80 ± 10.50 26 28.00 ± 8.65 30 16.20 ± 8.91 the highest number of torpedo-shape somatic embryo (57.20 ± 15.99 embryos) was obtained at 18 days after subculturing. mature somatic embryo was capable to form two different domes, i.e. shoot and root (fig. 1c-d). however, in some cases apical meristem structure was more dominant than shoot meristem. histological section showed that during the formation of torpedo-shape somatic embryo, root-apical, procambium and scutellum differentiations were performed (fig. 1c-d). regeneration germination of somatic embryo was characterized by the formation of the triangle-shape at meristem apical dome with yellow greenish in color, at 6 days after subcultured. at the second week of culturing, germinating seeds started to appear and developed to become normal seedlings/plantlets, especially when ba was added into ms basal medium. three weeks after culturing, various shapes of somatic embryos either a single normal or fused embryos, had been differentiated into plantlets. meanwhile, the abnormal embryos underwent necrosis and then aborted. embryo germination was indicated by the presence of shoot and root buds. four weeks after culturing, the morphogenesis of somatic embryos were clearly visible, nevertheless the low ability of somatic embryos to germinate was still a limiting factor in meristem culture of ginger. ms basal medium supplemented with 1 mgl-1 ba was found to be the best culture medium for obtaining normal plantlets at the age of 30 days, to which the highest number of seedlings was obtained (22.1 seedlings). on the other hand, instead of growing plantlet, vigorous adventitious roots were obtained on hormone-free ms medium (fig. 1e). furthermore, it was found that both ba and ga3 significantly affected the somatic embryo regeneration (p 0.05). however, the higher the concentration of ga3, the lower the number of seedlings was obtained (table 4). 19 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid table 4. number of seedlings derived from somatic embryo of ginger meristem cultured on ms medium supplemented with ba and ga3 treatment average number of seedlings ba (mgl-1) ga3 (mgl -1) 0 0 7.3 cd* 0.1 16.4 ab 0.3 16.4 ab 0.5 9.3 bcd 1.0 9.3 bcd 0.1 0 7.3 cd 0.1 10.0 bcd 0.3 3.7 de 0.5 16.1 ab 1.0 11.5 bc 0.5 0 0.5 f 0.1 0.5 f 0.3 0.5 f 0.5 12.8 abc 1.0 17.4 ab 1 0 22.1 a 0.1 12.6 abc 0.3 12.5 abc 0.5 0.5 f 1.0 1.2 ef notes : numbers followed by the same letter are not significantly different according to dmrt (5%). for statistical analysis data were transformed into √(y + 0.5). based on this study, it was found that regeneration potency of meristem culture of ginger through somatic embryogenesis was 51.20%.g-1 of embryogenic calli when cultured on ms medium supplemented with 1 mg l-1 ba. the addition of ga3 into ms basal medium produced only 34.13% of regeneration potency, and 1.13 to 31.84% of regeneration potency when ba was combined with ga3. therefore, to gain an optimal germination of ginger somatic embryo, higher concentration of ba (> 1 mg.l-1) without addition of ga3, was necessary. germinating somatic embryos from ms medium supplemented with either 1 mg l-1 ba, 0.5 mg l-1 ba, 0.1 mg l-1 ba + 0.3 mg l-1 ga3 or 0.1 mg l -1 ga3 were then transferred onto hormone-free ms medium with the addition of 3% sucrose. among them, the most vigorous plantlets were obtained from ms + 0.1 mg l-1 ba + 0.3 mg l-1 ga3 culture medium-derived seedlings (fig. 1f ), at 8 weeks after subculturing. however, the number of embryo somatic-generated plantlets remained low, of which only about 2-6 plantlets were observed. 20 biotropia vol. 15 no. 1, 2008 abundant embryogenic calli (93.33%/explant) from meristem culture of ginger were achieved on ms basal medium supplemented with 1 mg l-12,4-d in combination with 3 mg l-1 ba, and 100 mg l-1 l-glutamine, at 8 weeks after incubation. this result showed that 2,4-d was necessary for initiation of embryogenic callus of ginger, with the addition of ba at proper concentration. the same result was also found in embryogenic callus initiation of kaempferia galanga l. (vincent et al. 1992). callus proliferations were obtained on hormone-free ms medium consisting of 3% mannitol. a globular-shape pro-embryo started to develop at the first week after subculturing into proliferation medium. quantities of globular embryo increased due to the age of culturing up to seven weeks, then gradually decreased. ammonium-rich basal media such as ms, which consists of high concentration of nh4no3 and myo-inositol, supported well the differentiation of ginger somatic embryo, though an adverse effect was observed on other grammineae species (talwar and rashid 1989; adkins et al. 2002). salt nutrients in ms medium with addition of 3 to 12% sucrose are usually sufficient to support embryo differentiation (raghavan 2003; anbazhagan and ganapathi 1999). an addition of 3% mannitol into ms basal medium, assumed that it acted as an osmotic regulator which in turn affected the rate of cell differentiation and stimulated embryogenic cells morphogenesis in meristem culture of ginger. torres et al. (2001) reported that the development of synchronized embryo and the increase of its quantity were observed when mannitol was added into ms medium. the same results were also found on embryogenic culture of papaya, celery, alfalfa and maize (ziv 1999; bronsema et al. 1997). somatic embryo maturation in meristem culture of ginger was indicated by the change of embryo color and formation of the brown spots. these brown spots suggest the presence of an active substance i.e. amylum, protein and lipid, secreted by somatic embryo from the osmotic cell pressure. the increase of secretion of active substances suggests its correlation with the desiccation process when somatic embryo enters the germination phase. bach and pawlowska (2003) suggested that the presence of amylum, protein and lipid around the vascular cells was an indicator in somatic embryo development. similarly, oropeza et al. (2001) reported that the development of embryogenic calli was associated with the number and type of intracellular protein and callus cells. hence, somatic embryo maturation from proliferated calli of ginger meristem is interrelated with the type and number of active substance among the embryogenic cells. the growth and development of ginger mature somatic embryo into complete plantlets (seedlings) sometimes go along with the abnormal germinated seeds, such unexpected growth of hairy roots, hereafter we named as an early germinating seeds. according to bronsema et al. (1997), somatic embryo maturation is associated with the presence of scutellum, a coleoptile and root-bud like structures. somatic embryo maturation was also affected by the composition of applied culture medium. the presence of ammonium ion and nitrate at a high concentration is a prerequisite in ginger somatic embryo development. high concentration of ammonium ion supported the process of 21 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid somatic embryo differentiation into normal plantlets (george 1993). these results were in agreement with the findings of krikorian (1995), adkins et al. (2002) and ramage and williams (2002). however, the needs of ammonium ion for the growth of somatic embryo and its morphogenesis depend on the explant sources and the initial plant growth regulators applied. according to percy et al. (2000), high osmotic pressure in culture medium would increase the somatic embryo formation. sucrose, usually applied as a carbon source, is also a potent osmotic agent (van creij et al. 1999). another potential osmotic agent is abscisic acid (aba), that at a proper concentration it would enable to control the medium osmolarity (raghavan 2003; mohan and krishnamurthy 2002). to overcome the early seeds germination (the formation of unexpected hairy roots) in somatic embryogenesis of ginger, an experiment on addition of aba, aside from sucrose, into culture medium of somatic embryo maturation is now in progress. somatic embryo germination was indicated by the simultaneous formation of shoots and roots. goh et al. (1999) stated that embryo germinated when the plumullae started to emerge. in this research, adventitious roots formation was observed at first stage of regeneration and increased by the increase of age of culture up to the fourth week. addition of plant growth regulators into regeneration medium would accelerate the germination process and increase the quantity and quality of seedlings. in this research, a high percentage of somatic embryo germination was observed on the medium containing 1 mg.l-1 ba, at 30 days after culturing. regeneration potency of ginger meristem somatic embryo reached 51.20%.g-1 of embryogenic calli cultured on ms basal medium enriched with 1 mgl-1 ba, of which the number of seedlings was 8.98 times higher than that of the control medium. meanwhile, ga3 alone or in combination with ba, remained ineffective in supporting somatic embryo germination of ginger. however, to obtain the optimum growth of plantlet, the presence of ba is a prerequisite in meristem culture derived somatic embryo of ginger. similar result was also found in the development of ginger leaf derived embryogenic callus (kackar et al. 1993), though simultaneous development of somatic embryo derived either root or shoot meristems remained unclear. the development of somatic embryo into normal plantlet commonly occurs through four stages i.e. callus induction, callus proliferation, embryo maturation and germination. in case of somatic embryo derived from ginger meristem culture, its development into normal plantlet needs a transfer process of germinated seeds from the regeneration medium into new medium. such new medium is called growth medium for plantlet development. in this experiment the medium used for the growth of plantlet was hormone-free ms medium with the addition of 3% sucrose. although the development of protocol in ginger somatic embryogenesis required more researches, the results of this experiment, somehow, have a definite advantage in in vitro studies of ginger, especially in generating healthy planting material with normal rhizome size, a bigger size than the usual micro rhizomes resulted from shoot 22 biotropia vol. 15 no. 1, 2008 tip-derived in vitro plantlets (fig. 1 g). therefore, the encouraging results of this study gave great possibility for development of ginger resistant variety either through in vitro selection, somatic hybridization or genetic transformation. conclusions the addition of 1 mg.l-1 2,4-d and 3 mg.1-1 ba into ms basal medium enriched with 100 mg.l-1 of glutamine and 2% sucrose, was effective in inducing embryogenic calli from meristem explants of indonesian ginger. in further embryo development, ms medium was found to be more capable to increase mature somatic embryo than n6 medium. the regeneration potency of somatic embryos obtained from indonesian ginger meristem was 51.20%/g friable callus. though, further researches are needed to be conducted, especially to eliminate early germinating seeds. the protocol for generating somatic embryo derived plantlet from meristem culture of ginger was developed. the most valuable result of this study was the achievement of normal rhizome size of regenerated plantlet, instead of micro rhizome. acknowledgement the authors would like to thank dr. ika mariska for her assistance and encouragement during the research work and the manuscript preparation and also to dr. d. sitepu for his critical reading of the manuscript, and ms. r.r. sitinjak for her contribution on the research work. references adkins, s.w., a.l. adkins, c.m. ramage and r.r. williams. 2002. in vitro ecology: modification of headspace and medium conditions can optimize tissue and plant development. in: taji a, r. williams (eds) the importance of plant tissue culture and biotechnology in plant sciences. university of new england unit, australia, pp. 55-77. anbazhagan v.r. and a. ganapathi. 1999. somatic embryogenesis in cell suspension cultures of pigeon pea (cajanus cajan). plant cell, tissue organ cult. 56: 179-184. bach, a and b. pawlowska. 2003. somatic embryogenesis in gentiana pneumonanthe l. acta bio. crac. series bot. 45 (2): 79-86. bhojwani, s.s. and m. razdan. 1996. plant tissue culture: theory and practice. elsevier amsterdam, oxford, new york, tokyo. 23 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid bronsema, f.b.f., w.j.e. van oostveen and a.a.m. van lammeren. 1997. comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines a188 and a632. plant cell, tissue organ cult. 50: 57-65. evans, d.e and w.r. sharp. 1986. somaclonal and gametoclonal variation. in: evans et al. (eds) hand book of plant cell culture, vol. 4, technique and application. macmillan pub. co., new york, pp. 97-132. furlong, n.e., e.a, lovelace and k.l. lovelace. 2000. research methods and statistics an integrated approach. harcourt college, tokyo. george, e.f. 1993. plant propagation by tissue culture. 2nd ed. exegetics ltd, england. goh, d.k.s., n. michaux-ferriere, o. monteuuis and m.c. bon. 1999. evidence of somatic embryogenesis from root tip explants of the rattan calamus manan. in vitro cell. dev. biol. plant. 35: 424-427. heinze, b and j. schmidt. 1995. monitoring genetic fidelity vs somaclonal variation in norway spruce (picea abies) somatic embryogenesis by rapd analysis. euphytica 85: 341345. jimenez, v.m. 2001. regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. r. bras. fisiol. veg. 13 (2): 196-223. kackar, a., s.r. baht, k.p.s. chandel and s.k. malik. 1993. plant regeneration via somatic embryogenesis in ginger. plant cell. tissue organ cult. 32: 289-292. krikorian, a.d. 1995. hormones in tissue culture and micropropagation. in: p.j. davies (ed.) plant hormones: physiology, biochemistry and molecular biology. kluwer academic, dordrecht, boston, london, pp. 774-793. mariska, i and s.f. syahid. 1994. propagation of ginger through meristem culture. j. indust. crops. res. 7 (1): 1-6. murashige, t and f. skoog. 1962. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiol. plant. 15: 473-497. mohan, m.l and k.v. krishnamurthy. 2002. somatic embryogenesis and plant regeneration in pigeon pea. biol. plant. 45 (1): 19-25. oropeza, m., a.k. marcano and e. de garcia. 2001. proteins related with embryogenic potential in callus and cell suspensions of sugarcane (saccharum sp.). in vitro cell dev. biol. plant. 37: 211-216. percy, r.e., k. klimaszewska and d.r. cyr. 2000. evaluation of somatic embryogenesis for clonal propagation of western white pine. can. j. for. res. 30: 1867-1876. raghavan, v. 2003. one hundred years of zygotic embryo culture investigations. in vitro cell. dev. biol. plant. 89: 437-442. rahman, n.n., m.n. amin, t. ahamed, m.r. ali and a. habib. 2004. efficient plant regeneration through somatic embryogenesis from leaf base-derived callus of kaempferia galanga l. asian j. plant sci. 3 (6): 675-678. ramage, c.m and r.r. williams. 2002. mineral nutrition and plant morphogenesis. in vitro cell. dev. biol. plant. 38: 116-124. sass, j.e. 1951. botanical microtechnique. iowa state university press, ames. 24 biotropia vol. 15 no. 1, 2008 syahid, s.f and hobir. 1996. the growth and rhizome yield of ginger derived from in vitro culture. j. indust. crops. res. 2 (2): 95-100. talwar, m and a. rashid. 1989. somatic embryo formation from unmerged inflorescences and immature embryos of a graminaceous crop echinochloa. ann. bot. 64: 195-199. tan, s.k., r. pippen, r. yusof, h. ibrahim, n. rahman and n. khalid. 2005. simple one-medium formulation regeneration of fingerroot [boesenbergia rotunda (l.) mansf. kulturfl.] via somatic embryogenesis. in vitro cell dev. biol. plant 41: 757-761. torres, a.c., n. mfëe-ze and d.j. cantliffe. 2001. abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato. in vitro cell. dev. biol. plant. 37: 262-267. van creij, m.g.m., d.m.f. kerckhoffs, s.m. de bruijn, d. vreugdenhil and j.m. van tuyl. 1999. the effect of medium composition on ovary-slice culture and ovule culture in intraspecific tulipa gesneriana l. crosses. available at <http://www.liliumbreeding.ne/crey-med.htm>[12/06/04]. vincent, k.a., m. hariharan and k.m. mathew. 1992. embryogenesis and plantlet formation in tissue culture of kaempferia galanga l. a medicinal plant. phytomorphology. 42 (3 & 4): 253-256. ziv, m. 1999, developmental and structural patterns of in vitro plants. in: soh w, bhojwani ss (ed.) morphogenesis in plant tissue culture. kluwer academic, dordrecht, boston, london, pp. 235-253. the effect of the light gradient inside a soybean leaf on the curvature factor of the light response curve and the estimation the light gradients inside soybean leaves and their effect on the curvature factor of the light response curves of photosynthesis biotropia no. 25, 2005 : 29 – 49 tania june biotrop icsea, seameo biotrop, btic building, jl. raya tajur km. 6 bogor, indonesia; email: taniajune@biotrop.org; and laboratory of agrometeorology, bogor agricultural university, bogor, indonesia abstract light gradients within leaves are not included in the model of farquhar, although a steep light gradient does exist within leaves. for a bifacial leaf, the model shows good agreement with measured data, but for an isobilateral leaf the model may underestimate photosynthesis measured by conventional gas exchange. isobilateral leaves easily developed when plants were grown in growth chambers where some light were reflected from the growth chamber metal base onto the lower surface of the leaves during growth, resulting in adjustment of the photosynthetic capacity inside the leaves. this could also happen in the field when canopy is very sparse and lower surface of leaves was exposed to reflected light from soil surface. complications occurred when fitting the light response curves of the electron transport rate, due to the interaction between the quantum yield of electron transport (a2) and the curvature factor (θ). it is suspected that there may be an interaction with the light gradient within the leaf. this manuscript discusses the effect of a light gradient inside a soybean leaf on the estimation of θ. it is shown in the manuscript how the light curves of the isobilateral leaves (at different degree) responded when measured using conventional gas exchange and how it affected the estimation of θ and the electron transport capacity, jmax. an experiment was conducted to prove the hypothesis that this “out of ordinary” estimate of θ (and hence jmax) was due to the unmatched distribution of photosynthetic capacity with distribution of absorbed light. keywords : light gradient / photosynthetic capacity (jmax) / curvature factor (θ) / gas exchange introduction anatomically, there are two types of leaves, bifacial and isobilateral. bifacial leaves have different adaxial and abaxial surfaces (i.e. top and bottom), while in isobilateral leaves these differences do not exist (kirschbaum 1986). these differences are due to the way the leaves receive irradiance. bifacial leaves, which are usually planophile (perpendicular to the stem), receive most light from the adaxial surface while isobilateral leaves receive light from both sides. the distribution of rubisco and electron transport components within these two types of leaves are different. besides these biochemical differences, lloyd et al. (1992) and syvertsen et al. (1995) found that the mesophyll of leaves exposed to different light intensities also changes, and so it might be that these two types of leaves differ anatomically as well. the optimal distribution of the photosynthetic machinery (rubisco activity and electron transport) would follow the gradient of irradiance in a theoretical bifacial 29 mailto:taniajune@biotrop.org leaf, receiving irradiance from one direction, as shown mathematically by farquhar (1989), while in isobilateral leaves a bimodal distribution would be expected as light comes from both sides of the leaves (kirschbaum 1986). any functional isobilaterality will affect the interpretation of the gas exchange data as gas exchange measurement normally was done with irradiance given to the upper side of the leaf (adaxial surface) only. biotropia no. 25, 2005 light gradients in the leaves were not included in the model of farquhar et al. (l980), although a steep light gradient does exist within leaves (terashima & saeki l983; vogelmann & björn l984). for a bifacial leaf, even without the consideration of the light gradient within the leaves, the model shows good agreement with measured data (harley et al. l985; june 2002), presumably for the reason noted above. however, for isobilateral leaves the model may not give a good representation of the relationship between capacities (e.g., rubisco and electron transport) and actual photosynthesis rate as function of irradiance, in a conventional gas exchange system. materials and methods plant materials indeterminate soybean (glycine max [l.] merr.) were grown in 12 liter plastic pots containing sand and vermiculate mixture (1:1, v/v). plants were exposed to a controlled environment in a growth chamber where relative humidity was kept constant at 60/70 % day/night and three temperatures: 20/15, 25/20 and 32/27 day/night oc with [co2] of 350 μmol mol -1. models of leaf photosynthesis leaf photosynthesis can be described by the equations developed by farquhar et al. (l980) and farquhar & von caemmerer (1982). the rate of photosynthesis is controlled by rubisco (rubp carboxylase-oxygenase), the rate of regeneration of rubp, and the relative partial pressures of co2 (ci) and o2 at the site of co2 fixation. under a given set of environmental conditions, the net co2 assimilation rate, a, is taken as being either the rubisco-limited rate, av, or the predicted rubpregeneration limited rate of photosynthesis, aj, whichever is the lower at a particular ci. (this holds for ci > γ*, the co2 compensation partial pressure in the absence of dark respiration.) a has units of μmol m-2 s-1. a j c c rj i i d= − + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ − 4 2 γ γ * * (1) 30 the light gradients inside soybean leaves and their effect on the curvature factor – tania june a v c k o k c rv c i c o i d= − +⎛⎝ ⎜ ⎞ ⎠ ⎟ + ⎛ ⎝ ⎜ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ ⎟ −max *γ 1 (2) a = min (aj, av) (3) where ci = partial pressure of co2 in the leaf (μbar); γ* = co2 compensation partial pressure in the absence of dark respiration (μbar); rd = dark respiration by the leaf which continues in the light (μmol m-2 s-1); o = ambient partial pressure of oxygen (mbar); kc and ko are michaelis-menten constants for carboxylation and oxygenation by rubisco (μbar and mbar, respectively); vcmax is the maximum rate of rubisco activity in the leaf (μmol m-2 s-1); and j is the actual electron transport rate (μmol m-2 s-1). the temperature dependence of kc and ko follows the arrhenius unction: f ( ) ⎥⎦ ⎤ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ + −= 273 2.298 1 2.298 exp25, tr e kk ccc (4) ( ) ⎥⎦ ⎤ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ + −= 273 2.298 1 2.298 exp25, tr e kk ooo (5) where r is the universal gas constant, 8.3144 j mol-1 k-1, and t is temperature in oc. ec and eo are the apparent activation energies and the 25 subscript refers to the value at 25oc. the temperature effect on the co2 compensation point of photosynthesis in the absence of mitochondrial respiration follows the equation of von caemmerer et al. (l994): ( ) ( )225036.02588.19.36* −+−+=γ tt (6) the parameters kc and ko indicate the intrinsic kinetic properties of rubisco. they are relatively constant, varying only with temperature for all c3 species (berry & björkman l980; jordan & ogren l984), and hence in this analysis the values presented by badger & collatz (l977) and von caemmerer et al. (l994) were used. the values for kc, ko and γ* (pa) at 25 oc are 40.4, 24800, 3.69 and the activation energies (j mol-1) for kc and ko are 59400, 36000, respectively, assuming wall conductance gw = (von caemmerer et al. l994; badger & collatz l977). the rate of electron transport, j, follows the equation by farquhar & wong (l984): ∞ 31 biotropia no. 25, 2005 ( ) j ia j ia j ia j = + − + −2 2 2 24 2 max max maxθ θ (7) where jmax is the maximum light-saturated rate of electron transport of the leaf (μmol m-2 s-1), θ is the curvature factor of the light response curve that varies from 0 (rectangular hyperbola) to 1 (two straight lines quasi blackman), a2 is the quantum yield (in terms of incident par) of electron transport at low light and i is the light intensity (μmol m-2 s-1) incident on the leaf. results and discussions example of light response curves from the measurement of the light response curves, where the incident light ranged from 0 to 1650 μmol m-2 s-1, rd was determined by extrapolation of a linear regression at the lower end of the response curve (at i = 0 150 μmol m-2 s-1). using this interpolated rd along with γ* corrected for each temperature using equation (6), j was calculated from eq. (1) and then jmax, θ and a2 were estimated by fitting the jirradiance curve with equation (7). figure 1 shows example of the light response curves of the electron transport rate for plants grown at [co2] of 350 μmol mol -1 and air temperatures of 32/27oc and 20/15oc (day/night) and measured at the gas exchange at three different temperatures. the electron transport rate was calculated using eq. (1). 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 2 / 2 7 o c ( 3 5 0 ) j, μ m ol m -2 s -1 i o , μ m o l m 2 s 1 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 0 / 1 5 o c ( 3 5 0 ) j, μ m ol m -2 s -1 300 250 200 150 100 50 0 0 500 1000 1500 2000 0 500 1000 1500 2000 lo, μmol m-2 s-1 32/27oc(350) 20/15oc(350) figure 1. light response curves of the electron transport rate measured at three temperatures (δ: 15oc; : 25oc and ∇: 35oc). plants were grown under different conditions as indicated in the graph and measured at [co2] of 700 μmol mol-1. 32 the light gradients inside soybean leaves and their effect on the curvature factor – tania june the negative curvature factor (θ) and its effect on the estimation of jmax when these soybean plants were grown in the growth chambers, about 11 % of the incident light was reflected from the metal base of the chamber onto the lower surface of the leaf, resulting in a changed distribution of light and hence, presumably, of photosynthetic capacity inside the leaf. in other words it became partially isobilateral. when the light response curve of this leaf was measured by gas exchange, with light reaching only the upper surface of the leaf, the upper and lower surfaces of the leaf will have saturated at different “times”, i.e. at different irradiances on the adaxial surface. this means that chloroplasts near the abaxial surface continue to increase in irradiance at levels where the adaxial chloroplasts are light saturated. this continuous response at high irradiance will result in a very low fitted curvature factor, θ, as shown in the following parts. the value of rate of bending, θ, tended to be 1 (leverenz 1987; oya & laisk 1976) as the distribution of light given during the gas exchange measurement was made more nearly proportional to the distribution of the photosynthetic capacity (developed during growth). the soybean data showed that when gas exchange measurement with the light source aimed at the top part of the leaf was done, the value of θ was -1.86 at 15oc, 0.6 at 35oc and then decreased to -0.39 at 40oc (june 2002). i hypothesise that the very low value of θ was due to the proportion of light given to each surface of the leaf during measurement not being similar to what the leaf received during growth. if some levels of light reached the lower leaf surface during gas exchange measurements, then this low value of θ should increase as was suggested by oya & laisk (1976), leverenz (1987) and farquhar (l989). fitted parameters (jmax, θ, a2 ) the fitted parameters as functions of the growth and measurement temperatures are listed in table 1 (a2 and θ fitted freely) and table 2 (a2 constrained) using eq. (7) with j from eq. (1) and gw = ∞, and varying the fitting procedure as described below. the fitted parameters a2, jmax and θ in table 1 resulted from the fitting procedure where all the parameters vary freely. the estimation of θ (curvature factor) was very low, reaching -4.85 for plants grown at 32/27oc when measured at 15oc. a negative value of θ has never been observed before, as the normal values range from 0 to 1 (farquhar & wong 1984). the estimation of jmax becomes unrealistically high if θ is very low. in this case, it reached 407 μmol m-2s-1 at 15oc, which is 85 % higher than the jmax value at 25 oc and 17 % higher than jmax at 35 oc; this is implausible as jmax is expected to increase with temperature from 15 to 35oc, as with other growth conditions in table 1. 33 table 1. list of model parameters with + s.e, for each measurement at 3-leaf temperatures and a co2 concentration of 700 μmol mol-1. fitting of the light response curve was done using eq. (7) by letting all the parameters fit freely and assuming gw = ∞. biotropia no. 25, 2005 parameters of light response curve, measured at 700 μmol mol-1 [co2] growth condition day t/[co2] leaf temperature (oc) rd a2 jmax θ 20/350 15 0.65 + 0.25 0.25 + 0.01 268 + 101 -1.70 + 1.30 25 1.15 + 0.05 0.24 + 0.01 291 + 28 0.35 + 0.20 35 2.60 + 0.20 0.28 + 0.01 614 + 258 -0.24 + 0.76 20/700 15 0.35 + 0.25 0.27 + 0.07 185 + 22 -0.40 + 0.51 25 0.55 + 0.21 0.25 + 0.01 283 + 50 0.78 + 0.10 35 2.15 + 0.15 0.27 + 0.02 303 + 17 0.90 + 0.02 25/350 15 0.12 + 0.12 0.15 + 0.04 200 + 115 -1.00 + 1.87 25 1.01 + 0.44 0.22 + 0.01 212 + 43 0.70 + 0.05 35 1.74 + 0.06 0.27 + 0.02 284 + 42 0.84 + 0.01 32/350 15 0.00 + 0.00 0.34 + 0.06 162 + 4 -1.20 + 0.20 25 0.80 + 0.20 0.28 + 0.02 277 + 21 0.56 + 0.04 35 2.25 + 0.15 0.31 + 0.02 449 + 0.2 0.31 + 0.05 32/700 15 0.00 + 0.00 0.21 + 0.00 407 + 181 -4.85 + 3.55 25 1.00 + 0.04 0.25 + 0.04 220 + 21 0.00 + 0.49 35 2.15 + 0.07 0.24 + 0.01 347 + 31 0.76 + 0.10 leverenz (l988) discussed the strong correlation between the estimate of a2 and that of θ, which in some cases results in an unrealistically low estimate of a2 and an unusually high estimate of θ. based on table 1, there is no definite trend in a2 with increasing temperature, although there is a tendency for the value to be lower at 15 oc than at 35oc as shown by 60 % of the data. the parameter a2 (quantum yield of electron transport) has a theoretical maximum of 0.5 in red light (due to two photosystems) and 0.375 in white light. in practice, these values are usually lower (for example, by 15%, kirschbaum & farquhar l987). several studies have shown that the quantum yield of co2 assimilation decreases as temperature increases between 15 and 35oc (when measured at ambient [co2]). for example, ehleringer & björkman (1977), with encelia california; ku & edwards (1978) with triticum aestivum; ehleringer & pearcy (1983) with avena sativa; osborne & garrett (1983) with lolium perenne; leverenz & oquist (1987) 34 with pinus sylvestris. these yields are complicated by the increasing proportion of light energy used for photorespiration at higher temperatures. there are few temperature studies that have eliminated the effects of rubp oxygenation by either working at low [o2], high [co2], or by correcting the measurements using light given both from the top and bottom surfaces of the leaves. the light gradients inside soybean leaves and their effect on the curvature factor – tania june however, harley et al. (l985) working with soybean found that a2 increased from 0.16 at 15oc, 0.21 at 20oc, 0.27 at 25oc, 0.26 at 30oc, 0.25 at 35oc and down to 0.22 at 40oc. there may be some difficulties in comparing these values with ours as they used a different equation from eq. 3.7 that had no independent cuvature term. wang et al. (1996), working with scots pine, found that a2 increased from 0.18 at 6oc to 0.30 at 21oc and decreased to 0.23 by 32oc. there are no consistent differences in a2 observed between the growth treatments in this experiment. osborne & garrett (1983) and leverenz & oquist (1987) showed evidence that low temperature stress and frost hardening may change membrane properties and result in lower a2. according to wilkins et al. (l994), the difference in the values of a2 between growth treatments could reflect changes in the structure of the needle tissue, the chlorophyll content and the structure of the thylakoid membrane. with all parameters fitted freely in table 1, there was only a slight tendency for a2 to increase with short-term temperature variation from 0.24 ± 0.06 at 15 oc, to 0.25 ± 0.02 at 20oc and 0.27 ± 0.02 at 35oc. the mean value is 0.26 ± 0.04. this value is slightly higher than the mean value (0.23) across all temperatures found by harley et al. (1985) for soybean, and slightly less than that, 0.28, found by kirschbaum & farquhar (1987) in eucalyptus pauciflora, at 25oc, when allowance is made for an absorptance of 0.9. it is identical to the value found by ehleringer & björkman (1977) for quantum yield in white light as a mean of seven species. in 2 % [o2] they obtained a quantum yield of co2 assimilation 0.0733. multiplying by 4ē/co2 and by an assumed absorptance of 0.9 yields a2 = 0.26. other data on quantum yield in white light and low [o2] of co2 assimilation of sets of c3 species summarised by evans (1987) are 0.296 and 0.324 [with 0.328 and 0.388 in two individual studies]. to reduce the effect of “noise” in a2 on the estimates of θ, a second set of fitting procedures was used holding a2 constant at 0.26. the resulting fitted parameters θ and jmax are shown in table 2. with a2 held constant, the effect of temperature on θ becomes more consistent. θ increases with temperature in all cases and so does the estimate of jmax, except for the data coming from plants grown at 32/27oc, 700 μmol mol-1 [co2], where jmax is still very high at 15 oc associated with a probably due to the very low fitted value of θ. in most cases the value of θ was still very low, and negative in almost half of the cases. a total of 67 % of the data set shows a θ value lower than the commonly accepted value of 0.7 (farquhar & wong 1984, evans & terashima l988, evans & farquhar 1991). 35 table 2. list of model parameters with + standard error, for each measurement at 3-leaf temperatures and a co2 concentration of 700 μmol mol-1. fitting of the light response curve was done using a2 = 0.26 and assuming gw = ∞. biotropia no. 25, 2005 parameters of the light response curve, measured at 700 μmol mol-1 [co2] growth condition, day t/[co2] leaf temperature (oc) jmax θ 20/350 15 277 + 98 -2.01 + 1.14 25 331 + 11 0.002 + 0.003 35 444 + 115 0.39 + 0.26 20/700 15 190 + 18 -0.56 + 0.77 25 311 + 48 0.70 + 0.10 35 300 + 35 0.92 + 0.03 25/350 15 197 + 67 -5.75 + 3.39 25 264 + 58 0.16 + 0.02 35 274 + 18 0.84 + 0.10 32/350 15 134 + 18 -0.05 + 0.54 25 267 + 35 0.65 + 0.08 35 326 + 14 0.81 + 0.08 32/700 15 490 + 154 -9.10 + 4.20 25 236 + 25 -0.16 + 0.30 35 585 + 14 -0.04 + 0.07 however, apart from my data there is supporting evidence that the best fit of θ can be lower than 0.7, as shown by wang et al. (1996) who found that θ ranges from 0.32 to 0.66 for scots pine grown and measured at different conditions. however, in contrast to the results obtained in this worl, they found that θ decreased with increasing temperature across the whole range examined. smaller values of θ are observed when the light response curves are measured using light given solely from the abaxial rather than the normal adaxial surface (oya & laisk 1976; terashima & saeki 1985; terashima 1986). leverenz (1988) found that θ values increased as the leaf acclimated to the light environment inside an integrating sphere (measurement was done at ambient co2 concentration). the values of jmax were obtained from fitting the light response measurements at temperatures of 15, 25 and 35oc and for plants with different growth conditions using eqs. (1), (2), (7) and (8). 36 the light gradients inside soybean leaves and their effect on the curvature factor – tania june what is the meaning of a negative value of θ? what does the fitted curve look like with a negative θ? to answer these questions, potential electron transport rate, j, was calculated for values of θ ranging from 1 to -9, with i2 ranging from 0 to 4000 μmol m -2 s-1 for each θ, as shown in figure 2. it can be seen that for curves with a negative value of θ, the electron transport rate, j, does not saturate (i.e. level off) even at i2 = 4000 μmol m-2 s-1. figure 2. the effect of changing the curvature factor (θ) on the response curve of electron transport at (a) low and (b) high jmax. curves were generated using eq. (7). the value of i2 where j starts to saturate will determine the fitted value of θ, as shown in figure 3. as saturation starts earlier (at lower i2), θ will become higher. for example, when saturation started at i2 = 200 μmol m -2 s-1, the estimated θ was 0.999, while when saturation started at i2 = 1000 μmol m -2 s-1, θ was 0.77. it can be argued that the light response curves which were measured here at i up to 1650 μmol m-2 s-1 in soybean leaves did not reach saturation, in particular at 15oc, and hence the estimation of θ was too low. as an example, when the j value from the θ = 0 curve at i2 = 4000 μmol m -2 s-1 in fig. 2 (b) was increased by only 4.6 % and then refitted using eq. (7) to find θ again, the value of θ dropped from 0 to -0.36 (see top two curves in fig. 3). one interpretation of the above results is that as light becomes more available (higher i), more light will be distributed to the bottom part of the leaf where photosynthetic capacity is probably increasing. this may be due to the leaf receiving 0 1000 2000 3000 4000 0 20 40 60 80 100 -9 -4 -0.9 0 0.7 1 e le ct ro n tr an sp or t r at e, μ m ol m -2 s -1 0 1000 2000 3000 4000 1 0 100 200 300 400 (b) -9 -4 j max = 400 -0.9 0 0.7 (a) j max = 100 absorbed light, mol m-2 s-1μ 37 some light from the lower metal base of the growth chamber and adjusting its photosynthetic capacity accordingly. this will increase j and, therefore, decrease the value of θ. when the value of θ becomes too small (negative), significant overestimation of jmax using eq .(7) will occur. biotropia no. 25, 2005 0 1000 2000 3000 4000 5000 0 100 200 300 400 θ -0.36 0.999 0.77 0.41 0.00 e le ct ro n tr an sp or t r at e, µ m ol m m -2 s -1 absorbed light, μmol m-2 s-1 figure 3. the effect of the starting point of saturation of j on the estimated value of θ. data wer heoretical simulation in the following part i will explain the mechanisms of the negative θ by means f si e generated initially with θ = 0 as in figure 2 (b). arrows indicate point of saturation. t o mulation. in the simulation, the leaf is divided into 10 layers. division of layers is based on equal amounts of chlorophyll in each layer. the contribution of each layer to the leaf co2 assimilation rate is determined by the amount of light absorbed and the photosynthetic capacity. the photosynthetic capacity is given by jmax. according to kirschbaum (l986), who applied a modified kubelka-munk theory, the pattern of space irradiance within the leaf can be approximated by an exponential curve: ( )iccii o 21i exp −= (8) where i is the space irradiance at any layer , o is irradiance incident on the top of i ii the leaf, c1 (>1) and c2 are parameters specific for each leaf (dimensionless) and i is the layer number where i is 0 < i < d, in units of 1/d x total absorptance (d is total number of layers in leaf). note that the space irradiance in the top layer, i1, is 38 generally greater than io because of internal reflections. the space irradiance in the top layer of the leaf (i1) and in the bottom layer of the leaf (id), can be calculated (kirschbaum l986): the light gradients inside soybean leaves and their effect on the curvature factor – tania june ( )( ) ( )u u oo o r r rrr i i − + −+−= 1 1 11 (9) ( ) ( )l l o d r r t i i − + = 1 1 (10) here ro is reflection at the air-to-leaf interface, ru and rl are internal reflectivities at layer, i, will be aio exp(-ic2) / σ =1 w the upper and lower leaf-to-air interfaces, and r and t are total reflectance and transmissivity. values for ru, ro and rl (0.37, 0.037 and 0.37 respectively) are given by jenkins & white (l957), for a refractive index of 1.48 from calculations based on the theory of geometrical optics. using equations (9) and (10) for incident irradiance of 1200 μmol m-2 s-1, r = 0.0741 and t = 0.0143 gives i1 = 1252 μmol m -2 s-1and i10 = 37.32 μmol m-2 s-1 (june, 2002). these values are then inserted into eq. (8) taking 10 layers (d = 10) to give c1 = 1.2806 and c2 = 0.372. equation (11) says that the light absorbed by any d i 2 2 exp (-ic ) where a is absorptance of the leaf given by 1-r-t. light effectively absorbed by photosystem ii at each layer, i (i), would then be given by multiplying the above by 0.5(1-f), and the electron transport rate for each layer would become ( ) j i j i j i j i i i i = + − + −2i 2i 2 2i4 2 max( ) max ( ) max( ) θ θ . (11) in the case of light incident only on one side of the leaf (upper surface), if ( ) ( ) j j i i c i c i k i i ai ai max( ) max( ) exp exp∑ ∑ ∑ = = − =2 2 1 (12) where all summations are for i =1 to 10, then 1 (13) and ther j ∝ jmax(i) ∝ iai (farquhar 1989). in this case, the shape (θ) of the total − j k j= ∑i imax( ) max( ) efore i j would be the same as the shape for an individual layer, ji. this is true in the ideal case for bifacial leaves as shown in figure 4. 39 0 500 1000 1500 2000 2500 0 20 40 60 80 5 4 3 2 1 j i 0 500 1000 1500 2000 2500 0 4 8 12 10 9 8 7 6 0 500 1000 1500 2000 2500 0 50 100 150 200 250 layer total layer layer jmax a2 theta 1 79.59 0.109 0.7 2 54.87 0.075 0.7 3 37.82 0.052 0.7 4 26.07 0.036 0.7 5 17.97 0.025 0.7 6 12.39 0.017 0.7 7 8.54 0.012 0.7 8 5.89 0.008 0.7 9 4.01 0.006 0.7 10 2.80 0.004 0.7 total 249.99 0.342 0.7 j i i o biotropia no. 25, 2005 figure 4. simulated ji light response curves of a bifacial leaf. data were generated with jmax = 250 μmol m-2 s-1, a2 = 0.3 and θ = 0.7. it shows that the shape (θ) of the curve for each layer is the same as the shape of the total. the fitting result, represented by the solid line (eq. (11)), is shown in the inset table. for a completely isobilateral leaf with, over a period of time such as a day, light reaching the leaf from both sides equally, the time-average of light in each layer would be as shown in figure 5. 40 0 2 4 6 8 10 0 2 4 6 8 10 layer 0 2 4 6 8 10 0 200 400 600 800 1000 1200 =+ i i the light gradients inside soybean leaves and their effect on the curvature factor – tania june figure 5. theoretical light gradient inside an isobilateral leaf (right plot), which is the average of the light gradient from the upper surface (left plot) and the light gradient from the lower surface (middle plot). the average light in these leaves determines the distribution of jmax(i), where ( ) ( ) ( )( ) ( )( ) j j c i c i c i c i i i max( ) max( ) . exp exp exp exp∑ ∑ ∑ = − − + − − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟0 5 11 11 2 2 2 2 ( ) ( )( ) ( ) = − + − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟∑ 0 5 112 2 2 . exp exp exp c i c i c i . (14) in the case of soybean leaves, where they are not completely isobilateral (50 % light absorbed from the upper surface and 50 % from the lower surface), eq. (14) should be modified as follows: ( ) ( )( ) ( ) j j a c i b c i c i i i max( ) max( ) exp exp exp∑ ∑ = − + − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟ 2 2 2 11 (15) where a is the proportion of light reaching the upper surface and b (=1-a) is the proportion of light reaching the lower surface of the leaves. the light absorbed effectively by the photosystem ii at each layer would again be 0.5 (1-f) times the total light absorbed in the layer and ji would still follow eq. (11). 41 for a leaf which has already adjusted its photosynthetic capacities (jmax) to its light growing condition (as given by the values of a and b in e biotropia no. 25, 2005 q. (15)), the light respo by the values of a and b but whic able 3. fitted parameters of simulated data generated with jmax= 50 and 250 μmol m-2s-1, a2 = 0.3 and θ = 0.7, then these parameters were fitted freely using eq. (11). χ2 shows the goodness of the max 2 nse curve of each layer will have a different shape compared to the total when light comes from only one side. the curvature factor, θ, decreases as the proportion of jmax distributed to the lower part of the leaf, b, increases. this happens because the [b exp(-c2(11-i))] term in eq. (11) gets bigger and results in increasing that part of the curve at high i0, and hence θ becomes smaller. table 3 shows the simulation results for a theoretical leaf which has a distribution of its photosynthetic capacity as shown h is given light reaching only the upper surface. as the proportion of light on the top surface of the leaf during growth (a) deviates more from the measurement condition (a = 1), the curvature factor becomes smaller. this is consistent with the hypothesis that a low value of θ indicates a mis-match of light distribution during measurement with that experienced during growth. t fitting. jmax a b j a θ χ2 1 0 5 0. 0. 0.884 0.0 34 70 50 0.9 0.1 50.0 0.35 0.64 0.001 250 0.8 0.2 49.9 0.37 0.47 0.022 0.7 0.3 49.6 0.42 0.15 0.081 0.6 0.4 49.3 0.50 -0.50 0.150 0.5 0.5 49.2 0.68 -2.09 0.187 1 0 250.0 0.34 0.70 0.788 0.9 0.1 248.4 0.35 0.66 0.027 0.8 0.2 241.6 0.35 0.59 0.176 0.7 0.3 231.8 0.36 0.51 0.432 0.6 0.4 220.8 0.37 0.39 0.778 0.5 0.5 209.3 0.38 0.20 1.184 the light resp rv the ph nthet stem has b pot sed to e in the form of a quasi-blackman response, where the curvature factor should appr onse cu e of otosy ic sy een hy hesi b oach 1.0 (leverenz 1987; leverenz l988; oya & laisk l976; terashima & saeki l985). therefore, i further simulated the j vs light response curve using a curvature factor equal to 1.0, and the result is shown table 4. 42 the light gradients inside soybean leaves and their effect on the curvature factor – tania june table 4. change in the parameters of the light response curve with changing distribution of photosynthetic capacity (as shown by the a value) at jmax total = 50 μmol m-2 s-1 and 250 μmol m-2 s-1. data were generated with a2 = 0.3 and θ = 1.0 and fitted using eq. (11). χ2 shows the goodness of the fitting jmax a b jmax a2 θ χ2 50 1 0 50.0 0.34 1 0.015 0.9 0.1 50.1 0.36 0.98 0.034 0.8 0.2 50.2 0.39 0.90 0.392 0.7 0.3 50.4 0.46 0.70 1.446 0.6 0.4 51.1 0.65 -0.003 1.958 0.5 0.5 52.7 1.74 -4.89 1.421 250 1 0 250.0 0.34 1 0.9 0.1 245.9 0.35 0.99 5.857 0.8 0.2 236.3 0.36 0.98 17.009 0.7 0.3 225.4 0.38 0.94 20.620 0.6 0.4 214.8 0.40 0.88 24.577 0.5 0.5 204.0 0.42 0.75 26.370 table 4 shows that at low jmax the biggest change in the light parameters as the proportion of light coming from the upper and the lower surfaces changes are in θ and a2, not in jmax. jmax increases by 5.4 % (at low jmax) when light coming from the lower surface increases from 0 to 50 %. when jmax is high, both θ and jmax change as the light from the lower surface increases. jmax decreases by 18 % (at high jmax) when light from the lower surface increases by 50 %. a most interesting feature is that the reduction in the apparent value of θ is greatest at low jmax, and this may explain why θ was so low at 15oc in data shown in table 1. methodology to validate the hypothesis in order to test the theory discussed above, another set of soybean plants were grown in a growth chamber at 25oc and [co2] of 350 μmolmol -1. the leaves grew with either a black or a reflective surface underneath them during their growing period. the black surface only allowed up to 1.6 % of the incident light (which is reflected from the metal base of the growth chamber) to reach the lower surface of the leaf while the reflective one allowed up to 48 %. some plants were left untreated as control plants. the control plants received 10.9 % of the incident light on the lower surface of its leaves. these three treatments enabled different light gradients inside the leaf to be compared. leaves from each treatment were then measured by gas exchange, with light reaching only the upper surface and with light reaching both the upper and lower surfaces of the leaf. 43 light response curves of the co2 assimilation rate with total light intensity from around 50 μmol m-2s-1 to around 1500 μmol m-2s-1 were measured with the following conditions: biotropia no. 25, 2005 1. at 25oc, the light response curve for one leaf from each of the three conditions (black, control and reflective surface) was measured with 100 % of the light incident on the upper surface. three replications were done on a single leaf for each growth condition. the data obtained from these measurements were then fitted using eq. (11) to obtain estimations of jmax and a2 for each growth treatment. 2. the same leaves measured as in point 1 were then measured again by giving an optimum percentage of light to the lower surface at 25oc (two replications were done for each growth condition). this optimum percentage was obtained by increasing the proportion of light given to the lower surface of the leaf stepwise until a maximum co2 assimilation rate and a further decrease with increasing proportion of light were observed. the percentage of light given where this maximum assimilation rate occurred was then used to measure the light response curve. the light intensity used to obtain this optimum percentage was i2 = jmax, where the maximum bending of the j vs light response curve occurred. jmax was estimated using eq. (11) with the data set from the earlier measurements in point 1 above. as i2 = i0 a2 , the light intensity used was i0 = jmax/a2. 3. light response curves at 15 and 35oc were then measured using the optimum proportion of light given to each side of the leaves as obtained in point 2 (at 25oc). the results show that the distribution of incident light needed for maximum photosynthesis follows closely the condition the plant experienced in the growth chamber. the leaf grown with the black surface underneath, which received the least light at the lower leaf surface in the growth chamber, needed only 7.6 + 3.0 % of the total light on the lower surface during the gas exchange measurement to reach its maximum value of co2 assimilation rate. the leaf grown with the reflective surface underneath, which had the highest proportion of light received at the lower leaf surface in the growth chamber, needed 40.0 + 10.0 % of the light intensity reaching the lower leaf surface during the measurement to reach its maximum value and the control leaves needs 23.0 + 2.1 %. directing an optimal percentage of the incident light to the lower surface of the leaf during a gas exchange measurement not only increases photosynthesis compared to when light is given to the upper leaf surface only, but it also changes the curvature factor (θ) of the light response curve (table 5, figure 6). when light was given only to the upper surface of the leaf, θ of the reflective leaf was lower than θ of the black leaf with the control leaf value in the middle. when measurements were conducted with light given optimally to both sides of the leaf, θ increased for all leaves and the value did not differ significantly between growth treatments. the magnitude of this change in θ, from measurement with upper light 44 only to measurement with upper and lower light, becomes larger as the differences in light distribution between growth and measurement conditions become smaller. hence the change in θ with incident light distribution is greater for the reflective leaf than for the black leaf. the light gradients inside soybean leaves and their effect on the curvature factor – tania june table 5. the effect of giving an optimum percentage of light to the lower surface of the leaf during gas exchange measurements on the j vs light response curve parameters (using ci) (± s.e). measurements were conducted at 25 oc and 700 μmol mol-1 [co2], with 2 replicates for each measurement. parameters θ a2 black leaf upper light only lower and upper light 0.86 ± 0.06 0.93 ± 0.01 0.3 ± 0.08 0.3 ± 0.04 control leaf upper light only lower and upper light 0.73 ± 0.04 0.90 ± 0.06 0.3 ± 0.05 0.3 ± 0.03 reflective leaf upper light only lower and upper light 0.56 ± 0.07 0.93 ± 0.04 0.3 ± 0.04 0.3 ± 0.03 figure 6 shows one example of the j vs light response curves from gas exchange measurements with light reaching the upper side only and with light given to both surfaces of the leaf, for each leaf treatment (note the change in the curvature factor). 45 figure 6. light response curve of the electron transport rate (j) of (a) black leaf, (b) control leaf and (c) reflective leaf measured with light reaching the upper surface only (triangle, fitted by segmented line) and light reaching both upper and lower surfaces (circle, fitted by solid line). measurements were done at 25oc and co2 concentration of 700 μmol mol-1. parameters of the response curves are (a) θ = 0.84, 0.94; a2 = 0.3, 0.3; jmax = 215, 207, (b) θ = 0.73, 0.95; a2 = 0.3, 0.3; jmax = 276, 223, (c) θ = 0.49, 0.96; a2 = 0.3, 0.25; jmax = 259, 210, segmented and solid line, respectively. the optimum percentage of light directed to the lower surface was 10.0 %, 20.4 % and 50.0 % for black, control and reflective leaf respectively. 0 400 800 1200 1600 0 50 100 150 200 250 c b 0 50 100 150 200 250 a j, µ m ol m -2 s -1 io, µmol m-2 s-1 io , µ m ol m -2 s -1 biotropia no. 25, 2005 conclusions as found and discussed earlier, θ is partly an artefact of the distribution of light intensity in relation to the distribution of photosynthetic capacity, where θ will reach 1 if the two distributions match. during gas exchange measurements, if the proportion of light incident on the upper and lower surfaces of the leaf is similar to the light condition experienced by the leaf during its growing period, then the curvature factor (θ) of the light response curve moves closer to 1, confirming the work by leverenz (1988), although the 46 value of 0.7 has been commonly used in fitting the light response curve as first suggested by farquhar & wong (1984) and evans & terashima (1987) and used by farquhar & evans (1991). the light gradients inside soybean leaves and their effect on the curvature factor – tania june as the proportion of light given to both sides of the leaf deviates further from the leaf’s growing condition, the apparent whole leaf curvature factor becomes lower. in the case of a bifacial leaf measured with light only given to the upper surface, the upper and lower parts of the leaf would reach saturation at different light intensities causing the light response curve to bend more slowly and resulting in a lower value of the curvature factor. this finding has to be taken into consideration when modelling the effect of light on canopy photosynthesis. for example, when using the sun-shade model (de pury & farquhar l997), the curvature factors for the sun and the shade parts of the canopy would change during the time course of the day. estimation of jmax depends on the curvature factor value, and hence it is important to have a degree of certainty in this value. this experiment and associated modelling reveal the effect of a differential distribution of photosynthetic capacity throughout a leaf from the distribution of irradiance. the same concepts apply at the canopy level, which the sun-shade model addresses to an extent. references badger, m.r. and g.j. collazt. 1977. studies on the kinetic mechanism of ribulose-1,5 bisphosphate carboxylase reactions, with particular reference to the effect of temperature on kinetic parameters. carnegie institute of washington yearbook, 76: 355-361. berry, j. and o. bjorkman. 1980. photosynthetic response and adaption to temperature in higher plants. annual review of plant physiology, 31: 491-543. de pury, d.g.g and g.d, farquhar 1997. simple scaling of photosybthesis from leaves to canopies without the errors of big-leaf models. plant cell and environment, 20: 537-557. ehleringer, j., and o. bjorkman. 1977. quantum yields for co2 uptake in c3 and c4 plants:dependence on temperature, co2 and o2 concentration. plant physiology, 59: 86-90. ehleringer, j., and . w. pearcy. 1983. variation in quantum yield for co2 uptake among c3 and c4 plants. plant physiology, 73: 555-559. evans, j.r. 1987. the dependence of quantum yield on wavelength and growth irradiance. australian journal of plant physiology, 14: 69-79. evans, j.r. and g.d. farquhar. 1991. modelind canopy photosynthesis from the biochemistry of the c3 chloroplast. in “modeling crop photosynthesis-from biochemistry to canopy”. 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k. laitinen. 1996. acclimation of photosynthetic parameter in scots pine after three years exposure to elevated temperature and co2. agricultural and forest meteorology, 82: 195-217. wilkins, d., van oosten, j. j. and r.t. besford. 1994. effects of elevated co2 on growth and chloroplast protein in prunus avium. tree physiology 14: 769-779. 49 plant materials models of leaf photosynthesis example of light response curves the negative curvature factor (() and its effect on the estimation of jmax fitted parameters (jmax, (, a2 ) growth parameters biotropia vol. 30 no. 2, 2023: 220 231 doi: 10.11598/btb.2023.30.2.1896 220 diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia muhammad mansur1,2*, andi salamah3*, edi mirmanto2 and francis q. brearley4 1postgraduate biology study program, faculty of mathematics and natural sciences, universitas indonesia, depok, west java, 16404, indonesia. 2research center for ecology and ethnobiology, indonesian national research and innovation agency (brin), jalan raya jakarta-bogor, km 46, cibinong science center, cibinong, west java, 16911, indonesia. 3cellular and molecular mechanisms in biological system (cembios) research group, department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok, west java, 16404, indonesia. 4department of natural science, manchester metropolitan university, chester street, ,,,,jmanchester, m1 5gd, uk received 14 february 2023 / revised 11 april 2023 /accepted 12 april 2023 abstract nepenthes is the largest carnivorous plant genus present in indonesia. there are 39 species of nepenthes pitcher plants recorded in sumatra from lowland to montane forests, and 34 of them are endemic; this represents the greatest species diversity of nepenthes after borneo. field studies were conducted in 2021 and 2022 to increase our knowledge of the diversity, habitats and distributions of nepenthes in west sumatra province. twenty-three species of nepenthes were recorded from the province, consisting of 15 highland species, 4 mid-elevation species and 4 lowland species. ecophysiological studies conducted at bukit malalak showed clumped distributions of n. bongso, n. dubia, n. eustachya and n. rhombicaulis. foliar and pitcher fluid nutrient concentrations were found to be similar to those cited in other recent studies although growth rates were slightly more rapid than at gunung talang. bukit malalak is a new locality for two threatened species, namely n. dubia (cr) and n. rhombicaulis (vu), enlarging their extents of occurrence. in total, nine species from west sumatra are threatened and conservation actions are urgently needed for these and other nepenthes species remaining on the island. keywords: associations, carnivorous plants, diversity, ecology, foliar nutrients, indonesian nepenthes, montane forest, red list, west sumatra introduction nepenthes is the only genus in the nepenthaceae family (phillipps & lamb 1996; clarke 2001). nepenthes are climbing lianas that produce characteristic fluid-filled pitchers extending from tendrils at the end of leaf-like phyllodes. the pitchers have a range of adaptations to attract, catch, retain and digest mostly insect prey (moran & clarke 2010). the indonesian archipelago, with a land area of 1,919,440 km2, is in the centre of the distribution of nepenthes (which extends from madagascar to new caledonia). in 2021, 80 species, or approximately 44 % of all nepenthes (181), were recorded from indonesia (mansur et al. 2021), with diversity concentrated in borneo and sumatra. over the past few decades, many new species have been described, particularly in indonesia, malaysia and the philippines. in the last three years alone, three new species have been described in sumatra: n. putaiguneung al farishy, metusala & jebb in kerinci seblat national park (metusala et al. 2020), n. longiptera victoriano in aceh (victoriano 2021) and n. harauensis hernawati, r. satria & chi.c.lee in west sumatra (hernawati et al. 2022b). there are now 39 nepenthes species recorded in sumatra (hernawati et al. 2022a). mansur et al. (2022a) recently documented the 22 species found in the province of north sumatra, but information from other provinces remains poor. this is particularly true of the provinces through which the barisan mountains run, as these are likely to harbour the highest diversity of *corresponding author, email: mansurhalik@yahoo.com/ salamah@sci.ui.ac.id mailto:mansurhalik@yahoo.com mailto:salamah@sci.ui.ac.id diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 221 nepenthes due to their topographic variation (nerz 2005). furthermore, there are few ecophysiological studies of nepenthes species endemic to sumatra, in contrast to those in borneo (but see pavlovič et al. 2009, 2010; moran et al. 2012; mansur et al. 2022b). west sumatra has a diverse topography, ranging from lowlands (< 500 m asl) to mountains (> 2000 m asl), with a broad range of associated microclimates and geologies. these conditions allow this province to support a high diversity of nepenthes species. the estimated forest cover in west sumatra is 1,897,911 ha, of which 41.7% is protected forest, 15.8% is production forest and 42.5% is within nature reserves and nature conservation areas (dinas kehutanan sumatra barat 2018). the aim of this study was therefore to determine the species diversity, distribution, population, habitat and ecology of nepenthes in west sumatra province. materials and methods field studies were conducted in october 2021 and october 2022 (totalling four weeks) in agam regency (bukit malalak and gunung singgalang), limapuluh kota regency (air putih nature reserve, batu karang-harau, kelok sembilan and palupuah), solok regency (gunung talang) and around the city of padang (figure 1). exploration was conducted to inventory the species of nepenthes in each study location. further study of literature, along with examination of herbarium specimens at herbarium bogoriense-cibinong, took place in may and june 2022 to add information about the species of nepenthes in west sumatra; pasaman regency was only studied through a review of literature and herbarium specimens. we further focused on at bukit malalak (00°09 s; 100°23' e) where three plots of 0.09 ha each (10 m × 90 m) were established to determine the abundance of nepenthes and the tree species growing in their habitat. each species of nepenthes in the nine subplots (10 × 10 m each) and their positions (x and y) were recorded. trees (ø ≥ 5 cm) in each subplot were identified and their trunk diameter, total height and positions (x and y) were measured. all data collected was processed and analysed according to the mueller-dombois & ellenberg method (1974) to obtain values for basal area (ba), relative frequency (rf), relative density (rd), relative dominance (rdo), and importance value index (ivi). a few individuals of each nepenthes species were numbered with small aluminium tags, and the top leaf of each individual was punched with a small hole using a paper hole-punch. after 12 months, the plants still living were re-censused and the stem length and number of leaves of each individual were re-recorded. figure 1 map of six study sites in four regencies/cities in west sumatra province (study site 1: around padang city (00°60' s; 100°23' e); study site 2: gunung talang (00°59' s; 100°41' e); study site 3: gunung singgalang (00°24' s; 100°20' e); study site 4: bukit malalak (00°09' s; 100°23' e); study site 5: kelok sembilan (s: 00°04' s ; 100°42' e), air putih nature reserve and palupuah (00°03' s; 100°41' e); study site 6: batu karang-harau (00°03' s; 100°44' e). biotropia vol. 30 no. 2, 2023 222 interspecific associations between plant species were calculated using a 2 × 2 contingency table (ludwig & reynolds 1988) on species that had an ivi > 10 % (zulkarnaen et al. 2017). the calculated χ2 value was then compared with the χ2 table at the 5 % test level. if the value of χ2 was greater than that in the χ2 table at the 5 % test level, then there was an association between the two species, whereas if the value of χ2 was less than that in the χ2 table, then there was no association (mueller dombois & ellenberg 1974). the strength of the associations were then calculated using the ochiai (1957) index with values ranging from 0 to 1, wherein the stronger the association between the two plant species, the closer the index value is to 1 (ludwig & reynolds 1988). equations are presented in more detail in mansur et al. (2022a). finally, to determine the spatial distribution of each nepenthes species (i.e. regular, random or clumped), morisita’s (1959) index of dispersion was calculated using a quadrat size of 10 m × 10 m. laboratory procedures analysis of nutrient concentrations was carried out on samples of n. bongso korth., n. dubia danser and n. rhombicaulis sh.kurata from bukit malalak. leaf samples were dried in an oven at 60 °c for three days and then ground with a pestle and mortar. two ml of mixed acid (sulphuric acid, nitric acid and perchloric acid) was added to 0.2 g of leaf material and heated on a hotplate at 170 °c until the solution was clear; samples were then diluted to a final volume of 10 ml before analysis. phosphorus concentration was determined using a colorimetric method, in which 1 ml of sample was added to 3 ml of distilled water and then reacted with 1 ml of p dye; the yellow colour of the sample was measured using a shimadzu biospec-mini 1240 uv-vis spectrophotometer at 450 nm. nitrogen concentration was also determined by a colorimetric method, where 2 ml of the sample reacted with 4 ml of sodium phenoxide and 4 ml of 5% naocl; the blue colour of the sample was measured using a spectrophotometer as above but at a wavelength of 636 nm. other elements were determined with atomic absorption spectrophotometry using a shimadzu aa-6800. pitcher fluid was analysed as above after being filtered to remove any debris. results and discussion diversity in total, 23 species of nepenthes were recorded from west sumatra province, comprising 18 species documented during the field surveys, two species represented by herbarium specimens (n. jamban chi c.lee, hernawati & akhriadi: bo.1979938 and n. lingulata chi c.lee, hernawati & akhriadi: bo.1979937), two species documented from the region in literature records, i.e., n. izumiae troy davis, c.clarke & tamin (clarke et al. 2003) and n. jacquelineae c.clarke, troy davis & tamin (clarke 2001), and one species also indicated for the region via a credible secondhand source, namely n. naga akhriadi, hernawati, primaldhi & m.hambali (putra-rsn nepenthes nursery, pers. comm.; table 1). the majority of species documented are considered highland species (15), with four species each of mid-elevation and lowland species. seven species are restricted to west sumatra, 11 are found across other provinces of sumatra (in addition to west sumatra) and five are also found on other indonesian islands. nine species have been assessed as having a threatened status (2 × vu, 2 × en, 5 × cr) against the international union for conservation of nature (iucn) 3.1 criteria (iucn 2001) (clarke et al. 2000a, b, c; clarke 2014; hernawati & clarke 2014; hernawati et al. 2014; cross et al. 2020). limapuluh kota regency had the greatest number of species (9), followed by pasaman regency (8 species), solok and agam regencies (7 species each) and then padang city, with just 2 species. we also found four natural hybrids: n. talangensis nerz & wistuba × n. bongso and n. talangensis × n. inermis danser at gunung talang, and n. eustachya miq. × n. albomarginata w.lobb ex lindl., and n. gracilis korth. × n. eustachya in air putih nature reserve. diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 223 table 1 habitat, distribution, elevation and conservation status of the 23 nepenthes species recorded from west sumatra province. species in bold were recorded from the field survey nepenthes habitat regency elevation distribution iucn red list category ag lm pd ps sl adnata tamin & m.hotta ex schlauer forest ● m/h ws en [d] albomarginata w.lobb ex lindl. forest/shrubland ● ● m/h indo lc ampullaria jack shrubland ● l/m indo lc bongso korth. forest ● ● h s lc dubia danser mossy forest ● h ws cr [b1+2e] eustachya miq. forest/shrubland ● ● ● m s lc gracilis korth. shrubland ● ● ● l indo lc harauensis hernawati, r.satria & chi.c.lee forest ● h ws inermis danser mossy forest ● h s lc izumiae troy davis, c.clarke & tamin forest/shrubland ● h ws lc jacquelineae c.clarke, troy davis & tamin mossy forest ● h ws cr [b2ab(v) c2a(i)] jamban chi c.lee, hernawati & akhriadi: mossy forest ● h s cr [b2ab(v) c2a(i)] lingulata chi c.lee, hernawati & akhriadi: forest ● m s cr [b2ab(v)] longifolia nerz & wistuba forest/riparian ● m s lc mirabilis (lour.) druce shrubland ● l indo lc naga akhriadi, hernawati, primaldhi & m.hambali forest ● h s vu [d2] pectinata danser forest ● ● ● h s lc reinwardtiana miq. shrubland ● ● l/m indo lc rhombicaulis sh.kurata forest ● h s vu [d2] singalana becc. forest ● h s lc spathulata danser forest/mountain peak ● ● h s lc talangensis nerz & wistuba forest/mossy forest ● h ws en [c2b] tenuis nerz & wistuba forest ● h ws cr [a2] notes: ag = agam, lm = limapuluh kota, pd = padang city, ps = pasaman, sl= solok; l = lowland, m = midelevation, h = highland; ws = west sumatra only, s = sumatra only, indo = other islands in indonesia (in addition to sumatra); lc = least concern, vu = vulnerable, en = endangered, cr = critically endangered, = not yet assessed against iucn red list criteria. in our survey, conducted across five regencies in west sumatra, we recorded 23 species of nepenthes, 18 of which are endemic to the island of sumatra. this number is comparable to our survey in north sumatra (22, including two taxa yet to be formally described; mansur et al. 2022a), with 12 species common to both surveys. nepenthes eustachya, n. gracilis and n. pectinata danser are three species whose distribution is quite broad, as they are found in three regencies/cities; indeed, n. gracilis is widespread not only in western indonesia, but also in peninsular malaysia and southern indochina. other species, however, are more limited, notably n. talangensis, which is found only on gunung talang (solok regency; nerz & wistuba 1997), n. jacquelineae in pasaman regency (clarke 2001) and n. harauensis in limapuluh kota regency (hernawati et al. 2022b), all three of which are the type localities for those species, while n. jamban, n. lingulata and n. naga are found not only in pasaman regency (west sumatra), but also in mandailing natal regency (north sumatra), on the border between west sumatra and north sumatra. population nepenthes population measurements were only carried out at bukit malalak. in the study plots, n. bongso generally grew as an epiphyte under a shady canopy with a small population (plot 2), while in open areas (mountain peak), it grew terrestrially with a larger population (plot 1). in a total plot area of 0.27 ha, seven individuals of n. biotropia vol. 30 no. 2, 2023 224 dubia were found: four in plot 1 and three outside of the plot in mossy forest (mountain peak), growing terrestrially under a slightly open canopy (table 2). nepenthes eustachya was abundant in plot 3 and grew terrestrially in shady areas. thirteen individuals of n. rhombicaulis were found in plot 1 and ten were found in plot 2, where they grew terrestrially in shady areas. morisita’s index was greater than 1 for all species in all plots, indicating a clumped distribution; the exception was of n. rhombicaulis in plot 2, which had a value of 0.80, indicating a more random distribution. these clumped distributions are in keeping with earlier studies (adam 2002; damit et al. 2017), which is likely due to similar habitat requirements for light or sufficiently moist soil, for example. more advanced statistical techniques, such as those based on ripley’s k, are likely to be more informative regarding spatial patterns if sufficient individuals are measured (brearley et al. 2023). table 2 number of individuals of four nepenthes species in three plots each of 0.09 ha at bukit malalak, agam regency (sumatra). nepenthes elevation (m) plot 1 (1586 m asl) 2 (1329 m asl) 3 (1186 m asl) total bongso 1300–1600 64 6 0 70 dubia 1550–1600 4 0 0 4 eustachya 1150–1300 0 0 50 50 rhombicaulis 1300–1600 13 10 0 23 figure 2 nepenthes distributions in three 0.09 ha plots on bukit malalak, agam regency (sumatra). open circles show locations of each tree species with larger circles indicating trees of larger stem diameter. coloured circles indicate locations of nepenthes individuals of different species diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 225 habitat at the bukit malalak study site, nepenthes plants grew in primary forest with very steep sloping. the forest’s condition was intact, with few signs of human disturbance. in the total plot area of 2700 m2, there were 416 individual trees (ø ≥ 5 cm) recorded, comprising 34 species, 29 genera and 20 families. myrtaceae was the most species-rich family, with eight species, five of which were syzygium. the five dominant tree species in the nepenthes habitat were ternstroemia gymnanthera (wight & arn.) bedd. (ivi = 42.1) followed by syzygium zeylanicum (l.) dc. (28.5), myrsine avenis (blume) a.dc. (24.0), rhodoleia championii hook. (22.1) and leptospermum javanicum blume (19.1; table 3). table 3 list of tree species and their abundance in 0.27 ha of forest plots containing nepenthes species at bukit malalak, agam regency (sumatra) species family ba (m2) rd (%) rf (%) rdo (%) ivi (%) ternstroemia gymnanthera (wight & arn.) bedd. pentaphylacaceae 1.330 22.1 6.04 14.0 42.1 syzygium zeylanicum (l.) dc. myrtaceae 1.100 11.5 5.37 11.6 28.5 myrsine avenis (blume) a.dc. primulaceae 0.720 10.3 6.04 7.60 24.0 rhodoleia championii hook. hamamelidaceae 0.860 7.69 5.37 9.02 22.1 leptospermum javanicum blume myrtaceae 1.170 3.37 3.36 12.3 19.1 syzygium sumatranum (miq.) widodo myrtaceae 0.760 4.33 6.04 8.01 18.4 castanopsis costata (blume) a.dc. fagaceae 0.500 4.33 6.04 5.26 15.6 syzygium bankense (hassk.) merr. & l.m.perry myrtaceae 0.440 4.09 4.70 4.65 13.4 gaultheria heterophylla var. latifolia (blume) kron & p.w.fritsch ericaceae 0.360 3.85 5.37 3.80 13.0 pittosporum ferrugineum w.t.aiton pittosporaceae 0.350 2.16 5.37 3.71 11.2 carallia eugenioidea king rhizophoraceae 0.210 3.61 4.70 2.17 10.5 eriosolena composita (l.f.) tiegh. thymelaeaceae 0.310 3.13 3.36 3.29 9.77 neolitsea cinnamomea (ridl.) kosterm. lauraceae 0.200 1.92 3.36 2.06 7.34 tristaniopsis merguensis (griff.) peter g.wilson & j.t.waterh. myrtaceae 0.200 1.92 2.68 2.11 6.72 heptapleurum sp. araliaceae 0.060 1.44 3.36 0.58 5.38 dacrydium elatum (roxb.) wall. ex hook. podocarpaceae 0.160 0.96 2.68 1.68 5.32 adinandra dumosa jack pentaphylacaceae 0.150 1.68 2.01 1.54 5.24 pterophylla fraxinea d.don cunoniaceae 0.170 0.96 2.01 1.76 4.74 syzygium acuminatissimum (blume) dc. myrtaceae 0.100 1.68 1.34 1.07 4.1 calophyllum teysmannii miq. calophyllaceae 0.070 0.96 2.01 0.75 3.73 polyosma ilicifolia blume escalloniaceae 0.050 1.20 2.01 0.49 3.71 archidendron bubalinum (jack) i.c.nielsen fabaceae 0.020 0.96 2.01 0.18 3.15 symplocos adenophylla wall. ex g.don symplocaceae 0.020 0.72 2.01 0.25 2.99 schima wallichii (dc.) korth. theaceae 0.020 0.72 2.01 0.21 2.95 elaeocarpus mastersii king elaeocarpaceae 0.020 0.72 2.01 0.20 2.93 litsea sp. lauraceae 0.050 0.48 1.34 0.51 2.34 wendlandia densiflora (blume) dc. rubiaceae 0.020 0.72 1.34 0.26 2.32 lithocarpus conocarpus (oudem.) rehder fagaceae 0.020 0.72 1.34 0.23 2.29 myrica javanica blume myricaceae 0.040 0.48 1.34 0.39 2.21 vaccinium lucidum (blume) miq. ericaceae 0.010 0.24 0.67 0.09 1.00 rhodamnia cinerea jack myrtaceae 0.010 0.24 0.67 0.07 0.98 syzygium antisepticum (blume) merr. & l.m.perry myrtaceae 0.010 0.24 0.67 0.06 0.98 garcinia lateriflora blume clusiaceae 0.004 0.24 0.67 0.04 0.95 timonius flavescens (jacq.) baker rubiaceae 0.003 0.24 0.67 0.03 0.94 grand total 9.517 note: ba = basal area, rd = relative density, rf = relative frequency, rdo = relative dominance, ivi = importance value index biotropia vol. 30 no. 2, 2023 226 interspecific associations the ochiai index showed that n. bongso was positively associated with n. dubia and strongly positively associated with ternstroemia gymnanthera in plot 1 (table 4). in plot 2, n. rhombicaulis was positively associated with n. bongso, but they were weakly associated in plot 1. nepenthes rhombicaulis was also strongly positively associated with five species with a very high ochiai index in plot 2 and, in plot 3, n. eustachya had positive associations with seven species with a very high ochiai index (table 4). adam (2002) also found positive associations between n. × kinabaluensis sh.kurata and n. villosa hook.f. and leptospermum recurvum hook.f. (myrtaceae) in montane forest in northern borneo, so it would be valuable to determine the traits of the trees that nepenthes tend to associate with. furthermore, understanding which habitats and which particular tree species or communities certain nepenthes species associate with may provide further insight into where these nepenthes species may be found in other localities. table 4 chi-square (χ2) and ochiai index (oi) values showing inter-specific associations between nepenthes bongso (plot 1), n. rhombicaulis (plot 2), and n. eustachya (plot 3) and other nepenthes species and the ten tree species with the greater ivis at bukit malalak, agam regency (sumatra) species plot 1: association with n. bongso plot 2: association with n. rhombicaulis plot 3: association with n. eustachya chi-squared (χ 2) ochiai index (oi) chi-squared (χ 2) ochiai index (oi) chi-squared (χ 2) ochiai index (oi) nepenthes dubia + 0.61 (high) absent absent absent absent nepenthes bongso na na + 0.74 (high) absent absent nepenthes rhombicaulis 0.25 (low) na na absent absent ternstroemia gymnanthera + 0.88 (very high) + 0.88 (very high) + 0.67 (high) syzygium zeylanicum absent absent 0.44 (low) + 0.94 (very high) myrsine avenis + 0.50 (high) + 0.88 (very high) + 0.88 (very high) rhodoleia championii 0.25 (low) + 0.77 (very high) + 0.82 (very high) leptospermum javanicum + 0.53 (high) absent absent absent absent syzygium sumatranum absent absent absent absent + 1.00 (very high) castanopsis costata absent absent + 0.80 (very high) + 0.56 (high) syzygium bankense absent absent (low) + 0.75 (very high) gaultheria heterophylla + 0.89 (very high) + 0.66 (high) + 0.75 (very high) pittosporum ferrugineum absent absent 0.00 (very low) + 0.82 (very high) diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 227 growth rates leaf production rates and stem growth rates were quite variable within species. there were no significant differences in the rates among the four species measured over one year (only one individual of n. bongso could be measured, because the others died; table 5). the mean stem growth rate was significantly greater on bukit malalak (42.1 ± s.e. 4.8 cm yr-1) than on gunung talang (29.9 ± 3.6 cm yr-1) (t = 2.06, p = 0.05), although the leaf production rate was not (9.8 ± 1.2 leaves yr-1 versus 7.3 ± 0.6 leaves yr-1, respectively; mansur et al. in review); this could be due to the lower elevation of the plants studied on bukit malalak or simply the fact that different species were measured at each site (with the exception of n. bongso). it would be interesting to further examine the possible tradeoffs between leaf and stem production rate and plant survival (mansur & brearley 2008), as has been done in tropical tree seedling studies (brearly et al. 2016). nutrient concentrations macronutrient concentrations in the leaves were greater than those in the nepenthes pitcher fluid, and there were no significant differences in nitrogen or potassium concentrations between species, but phosphorus was greatest in the leaves of n. dubia and least in n. rhombicaulis. the concentrations of foliar magnesium were not significantly different between the three species; however, calcium was greatest in n. bongso and sodium was greatest in n. dubia and least in n. bongso. pitcher fluid concentrations were variable, and there were few significant differences among species, although n dubia had notably lower p, k and mg concentrations (table 6). our ecological studies on bukit malalak showed that the habitat of nepenthes was an infertile latosol (unpubl. data), indicated by the presence of a large number of syzygium species and tristaniopsis merguensis (griff.) peter g.wilson & j.t.waterh. this compares with more fertile volcanic andosols as found on other mountains in the region, such as gunung singgalang and gunung talang. however, these differences were not reflected in the foliar n, p or k concentrations when compared with material from gunung talang (mansur et al. in review), although n was greater and k was lower than the species from north sumatra (mansur et al. 2022b). in contrast, less mg was noted in the leaves from bukit malalak than those from gunung talang (but more than those from north sumatra) and less ca but more na was found in the nepenthes leaves from gunung talang and north sumatra. if the plants obtain sufficient nutrients from their insect prey, this may be one reason nutrient concentrations in the sampled leaves do not differ between locations despite contrasting soil fertilities. indeed, we earlier showed that foliar n concentrations of nepenthes did not vary along elevation gradients, although there was an impact on foliar p concentrations (mansur et al. 2022b). determinants of foliar nutrient concentrations will differ by species and locality. how they influence the performance of different nepenthes species would be valuable further research. table 5 growth rates of four nepenthes species in their natural habitat at bukit malalak, agam regency (sumatra) over an twelve-month period (oct 2021 to oct 2022). also shown are the environmental conditions and survival rates. values are mean ± standard error nepenthes plot conditions no. of individuals survival (%) no. of new leaves produced length of new stem produced (cm) bongso 2 slightly shaded 1 33 5.0 24.6 dubia 1 fairly open 3 100 9.7 ± 0.3 48.9 ± 7.8 eustachya 3 shaded 3 100 13.3 ± 2.9 52.2 ± 8.0 rhombicaulis 2 slightly shaded 4 100 8.5 ± 1.6 33.7 ± 7.6 biotropia vol. 30 no. 2, 2023 228 table 6. nutrient concentrations in leaves and pitcher fluid of three nepenthes in bukit malalak, agam regency (sumatra). values are mean ± standard error with letters indicating significant differences (at p < 0.05) with a tukey’s test element leaves (%) pitcher fluid (mg l-1) bongso dubia rhombicaulis bongso dubia rhombicaulis n 1.09 ± 0.02 a 1.01 ± 0.01 a 1.36 ± 0.22 a 0.97 ± 0.22 a 3.52 ± 1.44 a 1.09 ± 0.04 a p 0.10 ± <0.01 ab 0.14 ± 0.02 b 0.08 ± 0.01 a 5.04 ± 0.03 a 0.18 ± 0.05 a 4.51 ± 2.35 a k 1.32 ± 0.02 a 1.08 ± 0.18 a 1.10 ± 0.05 a 721 ± 363 a 6.72 ± 1.23 a 283 ± 163 a ca 0.16 ± 0.02 b 0.05 ± 0.01 a 0.09 ± 0.02 a 491 ± 27 b 294 ± 4.6 ab 172 ± 86 a mg 0.14 ± 0.01 a 0.12 ± 0.01 a 0.13 ± <0.01 a 92 ± 11 c 4.9 ± 0.2 a 50 ± 5.6 b na 0.42 ± 0.04 a 0.92 ± 0.14 b 0.70 ± 0.02 ab 0.85 ± 0.03 a 1.48 ± 0.07 a 516 ± 296 a conservation status of the 23 species reported from west sumatra, nine have threatened conservation status, namely n. dubia (cr), n. jacquelineae (cr), n. jamban (cr), n. tenuis (cr), n. adnata tamin & m.hotta ex schlauer (en), n. talangensis (en), n. tenuis nerz & wistuba (en), n. naga (vu) and n. rhombicaulis (vu). at nearly 40 % of the known species in the province, this is a worryingly high proportion. our research extends the ranges of n. jamban and n. lingulata from their type locality in north sumatra (lee et al. 2006) to west sumatra based on herbarium collections deposited at herbarium bogoriense (bo) by wewin tjiasmanto and that of n. naga through personal communication (putra). previously, n. dubia was only known to grow on gunung talakmau, pasaman and west pasaman regencies, at an elevation of 1800 to 2700 m asl (clarke 2001), and mandailing natal regency, north sumatra (sahal, pers. comm.). our research also extends the known range of n. dubia to include bukit malalak, thereby increasing the size of the small, originally documented population and its extent of occurrence (eoo). this is positive, as species with larger ranges and population sizes are less prone to extinction; this species is currently listed as critically endangered, satisfying the red list criteria b1+2e, i.e. it has a small geographic range and small population size (clarke et al. 2000a; cross et al. 2020). the eoo of n. rhombicaulis is also extended, having been formally documented only from gunung pangalubao near lake toba in north sumatra, about 300 km distant. this species is currently assessed as vulnerable under the red list criterion d2, i.e. it has a restricted area of occupancy (clarke et al. 2000b). cross et al. (2020) listed n. tenuis as cr under criteria a2, i.e. a greater than 80% reduction in population over the last 10 years. they further considered that it might be extinct; however, we found this species growing in the batu karang-harau area, albeit with a very small population of about 20 individuals. despite extending the range and population of these species, they are still threatened, as we discuss below. the other six species are listed as threatened because of their restricted ranges and an observed decline in their population in the cases of n. jacquelineae, n. jamban and n. talangensis (clarke et al. 2000c; cross et al. 2020). nepenthes harauensis has only recently been described (hernawati et al. 2022b) and has not yet been formally assessed for the iucn red list; nevertheless, the restricted range of this species coupled with the rapid rate of environmental change in sumatran ecosystems strongly suggests that it is likely to be threatened in its natural environment and would likely fall under the endangered category if formally assessed (although we would need a better estimate of of its populations’ size and trajectory). other species that might be found in west sumatra province include n. putaiguneung, which was recently described from an unknown location in the extensive kerinci seblat national park (metusala et al. 2020), as well as n. aristolochioides jebb & cheek, which is known diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 229 from three locations in the park where it is under threat from collectors (mcpherson 2009), underlining the problems of plant conservation even in formally protected areas (linkie et al. 2010). with the exception of the two latter species, most of the other species described from sumatra (hernawati et al. 2022a) and not recorded from the north (mansur et al. 2022a) or west sumatra provinces (in this study) are found in the very north of the island in aceh or in the more central jambi province. although rates of land-use change are very high in indonesia (margono et al. 2012; austin et al. 2019), many rare and often endemic species remain in the sumatran mountains due to the protected status of some localities, combined with their poor accessibility. many of these species are not in ex-situ cultivation and a stochastic environmental event or agricultural invasion could rapidly cause the extinction of the entire global population of some of these locally endemic species. conservation needs and responsibilities are therefore extremely high. further exploration and formal records would add to the quality of existing assessments. beyond assessing their conservation status, the key threats to the majority of nepenthes in indonesia are land-use change and collection for the horticultural trade (clarke et al. 2018; cross et al. 2020), so practical conservation methods need to address these challenges that will only be successful with the support of local communities and governments. these fascinating restricted range and threatened nepenthes species in sumatra need further study, especially regarding their distribution and population status. a more detailed exploration of the mountainous regions of sumatra may well find further populations, as we have done here. figure 3 nepenthes species recorded from west sumatra: (a) n. adnata (lower pitcher), (b) n. albomarginata (lower pitcher), (c) n. ampullaria (rosette pitcher), (d) n. bongso (lower pitcher), (e) n. dubia (upper pitcher), (f) n. eustachya (lower pitcher), (g) n. gracilis (upper pitcher), (h) n. harauensis (lower pitcher), (i) n. inermis (upper pitcher), (j) n. izumiae (upper pitcher), (k) n. jacquelineae (upper pitcher), (l) n. jamban (upper pitcher), (m) n. lingulata (upper pitcher), (n) n. longifolia (upper pitcher), (o) n. mirabilis (upper pitcher), (p) n. naga (upper pitcher), (q) n. pectinata (rosette pitcher), (r) n. reinwardtiana (upper pitcher), (s) n. rhombicaulis (upper pitcher), (t) n. singalana (upper pitcher), (u) n. spathulata (upper pitcher), (v) n. talangensis (upper pitcher), (w) n. tenuis (upper pitcher). photos a & p by putra, photos b–i, n, o, q–s, v & w by m. mansur, photos j–m by sahal, photo t by sunardi, photo u by yusran e. ritonga. authorities are noted in table 1 biotropia vol. 30 no. 2, 2023 230 conclusion our research documented 23 species of nepenthes in west sumatra province through direct observation (18 species) and the remainder through herbarium specimens, literature review and second-hand information. of that total, 18 species are endemic to sumatra, nine of which are threatened with extinction (2 × vu, 2 × en and 5 × cr). we examined nepenthes at one of our study sites (bukit malalak) in more detail and compared ecological patterns and processes with those occurring elsewhere. nepenthes species require further observation, especially regarding their distribution and population, and additional exploration on poorly 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https://doi.org/10.2331/suisan.22.526 https://doi.org/10.1093/aob/mcp121 https://doi.org/10.1093/aob/mcp121 https://doi.org/10.1007/s11099-010-0028-1 https://doi.org/10.14203/reinwardtia.v20i1.3932 https://doi.org/10.14203/reinwardtia.v20i1.3932 https://doi.org/10.20956/jal.v8i16.2985 microhabitat influence on growth distribution pattern of ramin ( ) in siak, riau gonystylus bancanus province** didit okta pribadi* and yayan wahyu c. kusuma center for plant conservation, botanic gardens, indonesian institute of sciences, bogor, indonesia r 4eceived 12 april 2012/ accepted 31 december 201 abstract plant growth distribution patterns are influenced by habitat characteristics, ability of adaptation and association with other plant or animals. the influence of those factors, especially habitat characteristic, needs to be species defined to support plant conservation management. this study was aimed to: 1) measure plant growth dependence on their microhabitat; 2) define microhabitat variables that significantly influence the growth; and 3) develop suitable conservation measures at species level. ramin ( ) is one of major timber species that has been facing gonystylus bancanus high exploitation in indonesia. this species is usually found on specific “peat swamps” ecosystem. data were collected through primary surveys in riau province and analyzed by clustering the adult based on total height and basal area variables and describing the distribution pattern of cluster. then, discriminant function analysis the (dfa) was used to overlay the cluster with the distribution of microhabitat characteristic consist altitude, slope, ing soil humidity, soil ph, peat depth and canopy cover (measured in percentage). the results showed that distribution of microhabitat matched with 67.4% of height distribution and 78.3% of width distribution of tree basal area. altitude and canopy cover percentage had significant correlation with total height distribution (α=0.05). meanwhile, altitude, canopy cover and slope had significant correlation with basal area (α=0.1). however, peat depth variable showed an interesting pattern since shallower peat depth was followed by wider basal area. high correlation between plant growth and its microhabitat suggested that to conserve , conservation offered better strategy g. bancanus in-situ than conservationex-situ . keywords: discriminant function analysis, microhabitat, peat swamp forest, plant growth gonystylus bancanus, distribution, ramin introduction ramin ( ) wood is one of the gonystylus bancanus most valuable timbers in indonesia. it has various local names such as gaharu buaya, medang keladi or pulai miang. it is known as an expensive wood which is similar to merbau ( spp., especially intsia intsia bijuga intsia palembanica shorea and ), meranti ( spp.), bangkirai ( spp. and spp.), shorea hopea eboni ( spp.) and cendana (diospyros santalum album l.). people usually use it as material for construction, making doors and windows and producing various kinds of furniture. ramin wood is produced from genus , a gonystylus membe r of t hymelaeac eae f amily and gonystyloideae subfamily (sidiyasa 2005). approximately, 30 species of are gonystylus distributed in brunei darussalam, fiji, indonesia (kalimantan and sumatera), malaysia (peninsular malaysia, sabah and sarawak), singapore, nicobar island, solomon island and the philippines (soerianegara . 1994). there are et al ten species which are found in indonesia namely g. affinis g. bancanus g. radlk., (miq.) kurz., brunnescens g. confusus g. airy shaw, airy shaw, forbesii g. keithii g. macrophyllus gilg., airy shaw, (miq.) airy shaw, hook f., g. maingayi g. velutinus airy shaw, and airy shaw (shaw g. xylocarpus 1954; purba 2002).et al. biotropia vol. 22 no. 1, 2015: 1 10 doi: 10.11598/btb.2015.22.1.284 1 * corresponding author : diditpribadi@yahoo.com **this paper was presented at association for tropical biology and conservation (atbc) conference 2010 on tropical biodiversity: surviving the food, energy and climate crisis (19-23 july 2010). sanur-denpasar, bali, indonesia mailto:diditpribadi@yahoo.com biotropia vol. 22 no. 1, 2015 2 gonystylus bancanus is the most valuable ramin wood traded in international market. it can be found in peninsular malaysia, sabah, sarawak, sumatera, bangka, kalimantan and brunei darussalam (lim 2004; sidiyasa 2005). it et al. grows in peat swamp forest having wet climate and waterlogged soil. it mostly becomes dominant tree at peat depth of 350-600 mm (istomo 2006; rostiwati . 2007).et al in the early 1980s, indonesia had become the biggest exporter of ramin followed by sarawak an d pe n i ns ul ar ma lay si a ( s oe r i an e g a r a et al. 1994). in the 1970s the annual harvested volume of ramin in indonesia was about 1.5 million m , and it decreased drastically to 131,000 3 m in the year of 2000 (lim . 2004) which 3 et al indicated that exploitation of ramin wood had exceeded their regeneration capacity (bismark et al. 2005). therefore, ramin protection and conservation efforts have been started by including spp., especially , in gonystylus g. bancanus appendix ii cites in 2004 (samedi 2005). it means that the trade of spp., especially gonystylus g. bancanus, has been restricted by certain quota every year. trade restriction is among policies which are expected to reduce ramin wood's exploitation. however, since illegal logging activities and the loss of forestland are still continuing in indonesia, it should be coupled with direct conservation measures in the field. bogor botanical garden is one of the indonesian conservation institutions having responsibility to conserve such kind of plants. therefore, this research becomes important to analyze the growth pattern of g. bancanus in its habitat, thus conservation management could be defined properly. plant growth distribution patterns are influenced by habitat characteristics, ability of adaptation and association with other plant species or animals. recently, studies on the natural distribution of plants and animals species are growing not only to assess the impact of anthropogenic effect or climate change on their habitat, but also to define appropriate conservation management of the species (guissan & thuiller 2005; sinclair . 2010). et al ever y species has different strength of relationship with its habitat, thus some species have endemism distribution and the others have cosmopolitan distribution. the endemicity level of a species will determine the effectiveness of conservation management effort either in ex-situ or conservation.in-situ based on the above reasons, the aims of this research were: 1) to measure growth dependence of on their microhabitat; 2) to define g. bancanus microhabitat variables that significantly influence the growth of ; and 3) to formulate g. bancanus suitable conservation measures for . g. bancanus few parts of the research had been orally presented during atbc (association for tropical biology and conservation) 2010 conference in bali. materials and methods site description the research was conducted in danau pulau besar/danau bawah wildlife sanctuary that encompasses an area of 25,000 ha. this area is located in dayun village, siak district, siak municipality, riau province. geographically, this area lies between 00 35'-00 45' n and 102 10'-0 0 0 102 19' e (ipb & uksda riau 2000). it is 0 managed by balai besar konservasi sumber daya alam, riau province. generally, this area consists of peat swamp area which has flat topography, low altitude from 2 to 6 m above sea level and slope ranging from 00 to 3 (ipb & uksda riau 2000). it is situated in 0 an alluvium area which was produced by siak river sedimentation, thus it contains high organic and inorganic matters. based on climate type of schmidt-fergusson, this area is classified into type a which has rainfall from 2,200 to 2,600 mm per year. temperature of the area is 26.2 c per year on 0 average, with maximum temperature of 32.4 c 0 and minimum temperature of 21.7 c. air 0 humidity in this area can reach 84% on average with maximum humidity of 97% and minimum humidity of 60% (ipb & uksda riau 2000). research time and location the research was conducted from 5 to 17 december 2007. the location of the study site was danau pulau besar/danau bawah wildlife sanctuary, siak municipality, riau province. microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. survey methods field survey was conducted to collect primary data which consisted of spatial distribution of g. bancanus, total height and diameter breast height o f a d u l t t r e e , i n c l u d i n g m i c r o h a b i t a t characteristics in each location of g. bancanus comprising altitude, slope, soil humidity, soil ph, peat depth and canopy cover (in percentage). to investigate the microhabitat characteristics, unbound plots of ca. 100 m were applied with at 2 least one individual of centered within g. bancanus the plot. the sample was taken based on purposive random sampling method because the research was only focused on as the g. bancanus target species. data analysis data were analyzed by clustering the adult based on total height and basal area variables. the criteria used to define the cluster were as follows:  cluster 1: more than average + standard deviation  cluster 2: average ± standard deviation  cluster 3: less than average – standard deviation furthermore, discriminant function analysis (dfa) was used to overlay the cluster with the distribution of microhabitat characteristics consisted of altitude, slope, soil humidity, soil ph, peat depth and canopy cover. dfa is one of multivariate analyses used to evaluate the accuracy of cluster formation based on several quantitative variables. in this study, cluster of growth formation based on height and width of basal area was evaluated by microhabitat characteristics. the correlation between cluster of growth (based on height and width of basal area) and microhabitat characteristics were used to estimate influence value of microhabitat characteristics on growth distribution of . in addition, dfa was g. bancanus also used to discover which microhabitat characteristics best explained differences on distribution of the species (mcgarigal . 2000). et al when correlation between the plant and its habitat is relatively high, it means that the adaptation of the plant in different habitat is relatively low. results and discussion based on height variable, was g. bancanus divided into three clusters: 1) cluster 1 consisted of 9 individuals with 32-35 m of total height; 2) cluster 2 consisted of 29 individuals with 22-30 m of total height; 3) cluster 3 consisted of 8 individuals with 15-20 m of total height. based on basal area, was divided into three g. bancanus clusters: 1) cluster 1 consisted of 7 individuals with 2,428-2,307 cm ; 2) cluster 2 consisted of 31 2 individuals with 594-1,698 cm ; 3) cluster 3 2 consisted of 8 individuals with 154-330 cm .2 each cluster of height and basal area had microhabitat characteristics that were shown in table 1 and 2, respectively. there were 6 measured microhabitat characteristics i.e. altitude, slope, soil humidity, soil ph, peat depth and canopy cover. each cluster had respective mean and standard deviation for each microhabitat characteristic. based on the above results, dfa was conducted to evaluate whether clustering of height matched with distribution of microhabitat characteristics. t he result showed that distribution of microhabitat characteristics only 67.4% matched with cluster of height. however, f ur the r dfa an aly sis on mic rohabit at characteristics expressed that the individuals of g. bancanus could be grouped only in 2 clusters with detailed explanation as follows (table 3): a. nine individuals member of cluster 1 of height should be included in cluster 2 of microhabitat characteristics; b. from 29 individuals member of cluster 2 of height, 26 individuals should be included in cluster 2 of microhabitat characteristics, while the other 3 individuals should be included in cluster 3 of microhabitat characteristics; c. from 8 individuals member of cluster 3 of height, 3 individuals should be included in cluster 2 of microhabitat characteristics, while the other 5 individuals should be included in cluster 3 of microhabitat characteristics. since the correlation between distribution of height with microhabitat characteristics was only 3 biotropia vol. 22 no. 1, 2015 4 table 1. microhabitat characteristics of each cluster for total height group statistics total height mean standard deviation valid n (l istwise) unweighted weighted 1 alt 23.8889 1.90029 9 9.000 slope 12.2222 6.96020 9 9.000 srh 94.4444 11.30388 9 9.000 sph 5.3556 .46934 9 9.000 pd 119.8889 26.47666 9 9.000 cc .0000 .00000 9 9.000 2 alt 23.1724 2.81665 29 29.000 slope 12.7241 5.94406 29 29.000 srh 95.7931 10.23083 29 29.000 sph 5.4069 .59036 29 29.000 pd 112.3966 29.87510 29 29.000 cc 18.2759 24.79472 29 29.000 3 alt 18.7500 4.97853 8 8.000 slope 9.6875 4.78791 8 8.000 srh 94.3750 10.50085 8 8.000 sph 5.1125 .41897 8 8.000 pd 121.0625 34.62290 8 8.000 cc 55.6250 29.20830 8 8.000 total alt 22.5435 3.55094 46 46.000 slope 12.0978 5.95410 46 46.000 srh 95.2826 10.26897 46 46.000 sph 5.3457 .54353 46 46.000 pd 115.3696 29.69824 46 46.000 cc 21.1957 28.65862 46 46.000 notes: alt = altitude; slope = slope; srh = soil humidity; sph = soil ph; pd = peat depth; cc = canopy cover 67.4%, it was predicted that there were other factors affecting plant total height distribution such as genetic factors, age differences, or even other physiological factors (ryan . 2006). et al although the influence of age differences was minimized by selecting only adult plants which had similar appearance, however, the age of the plants was not measured quantitatively (e.g. tree rings approach) study. in this another result of dfa showed that there were only two variables having significant influence on the tree height distribution consisting altitude and canopy cover (table 4). based on information from table 1, it was described that increasing altitude was followed by increasing tree total height, while decreasing canopy cover was followed by increasing total height. figure 1 shows that cluster 2 (higher tree height) was located in higher altitude and lower canopy cover, whereas cluster 3 (lower tree height) was located in lower altitude and higher canopy cover. this explained light sufficiency is important to support the height growth of . higher g. bancanus altitude and less canopy cover gave more open space to the plant for getting more light intensity. no, 5 table 2. microhabitat characteristics of each cluster for basal area group statistics basal area mean standard deviation valid n (l istwise) unweighted weighted 1 alt 23.8571 2.85357 7 7.000 slope 17.8571 4.94734 7 7.000 srh 95.7143 11.33893 7 7.000 sph 5.2000 .46547 7 7.000 pd 106.0571 38.28607 7 7.000 cc 4.2857 11.33893 7 7.000 2 alt 23.1613 2.55730 31 31.000 slope 11.2903 5.74424 31 31.000 srh 96.0645 9.93960 31 31.000 sph 5.4194 .58276 31 31.000 pd 113.8097 27.25907 31 31.000 cc 15.4839 24.60877 31 31.000 3 alt 19.0000 5.31843 8 8.000 slope 10.1875 5.02805 8 8.000 srh 91.8750 11.31923 8 8.000 sph 5.1875 .42908 8 8.000 pd 129.5625 30.02075 8 8.000 cc 58.1250 24.19231 8 8.000 total alt 22.5435 3.55094 46 46.000 slope 12.0978 5.95410 46 46.000 srh 95.2826 10.26897 46 46.000 sph 5.3457 .54353 46 46.000 pd 115.3696 29.69824 46 46.000 cc 21.1957 28.65862 46 46.000 note: alt = altitude; slope = slope; srh = soil humidity; sph = soil ph; pd = peat depth; cc = canopy cover table 3. evaluation result for cluster of total height membership based on microhabitat characteristics by using dfa classification results a total height predicted group membership 1 2 3 total original count 1 0 9 0 9 2 0 26 3 29 3 0 3 5 8 % 1 .0 100.0 .0 100.0 .0 89.7 10.3 100.0 3 .0 37.5 62.5 100.0 notes: 1. = 67.4% of original grouped cases were correctly classifieda 2. cluster of height are represented by row, while cluster of microhabitat characteristics are represented by column no. microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. 2 biotropia vol. 22 no. 1, 2015 6 table 4. significant influences from microhabitat characteristics on tree total height distribution tests of equality of group means wilks' lambda f df1 df2 significance level alt .748 7.236 2 43 .002 slope .964 .811 2 43 .451 srh .996 .093 2 43 .911 sph .959 .918 2 43 .407 pd .982 .386 2 43 .682 cc .627 12.772 2 43 .000 figure 1. plot of cluster of tree height based on altitude and canopy cover variables simultaneously, dfa was also conducted to evaluate whether clustering of basal area matched with distribution of microhabitat characteristics. the result showed that distribution of microhabitat characteristics only matched 78.3% with the cluster of basal area. detailed explanation of the result was as follows (table 5): a. from 7 individuals member of cluster 1 of basal area, 2 individuals should be included in cluster 1 of microhabitat characteristics, while the other 5 individuals should be entered in cluster 2 of microhabitat characteristics; b. from 31 individuals member of cluster 2 of basal area, 1 individual should be included in cluster 1 of microhabitat characteristics, 28 individuals should be included in cluster 2 of microhabitat characteristics, while the other 2 individuals should be included in cluster 3 of microhabitat characteristics; c. from 8 individuals member of cluster 3 of basal area, 2 individuals should be included in cluster 2 of microhabitat characteristics, while the other 6 individuals should be included in cluster 3 of microhabitat characteristics. note: significance level of respective variable is shown by value in the sixth column which is less than 0.05 7 distribution of microhabitat characteristics had higher correlation with variable of basal area than with variable of height. it described that 78.3% of basal area distribution were influenced by microhabitat characteristics and the rest was influenced by other factors such as genetic factors or age differences. as mentioned before, we did not measure the influence of age differences. table 5. evaluation result for cluster basal area width membership based on microhabitat characteristics by using dfa classification resultsa basal area predicted group membership 1 2 3 total original count 1 2 5 0 7 2 1 28 2 31 3 0 2 6 8 % 1 28.6 71.4 .0 100.0 2 3.2 90.3 6.5 100.0 3 .0 25.0 75.0 100.0 notes: 1. = 78.3% of original grouped cases were correctly classifieda 2. cluster of basal area are represented by row, while cluster of microhabitat characteristics are represented by column table 6. significant influences of microhabitat characteristics on basal area distribution tests of equality of group means wilks' lambda f df1 df2 significance level alt .781 6.035 2 43 .005 slope .823 4.609 2 43 .015 srh .976 .525 2 43 .595 sph .961 .870 2 43 .426 pd .942 1.319 2 43 .278 cc .623 12.995 2 43 .000 note: significance level of respective variable is shown by value in the sixth column which is less than 0.05 figure 2. plot of cluster of basal area based on altitude, slope and canopy cover microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. biotropia vol. 22 no. 1, 2015 8 another result of dfa showed that there were three variables having significant influence on basal area distribution consisting of altitude, slope and canopy cover (table 6). based on information from table 2, the increasing altitude, slope and decreasing canopy cover were followed by increasing basal area. this could also be seen from figure 2 where cluster 1 (the widest basal area) was located in higher altitude, higher slope and lower canopy cover, whereas cluster 3 (the narrowest basal area) was located in lower altitude, lower slope and higher canopy cover. peat depth variable showed an interesting pattern since shallower peat depth was followed by wider basal area, although not significant. the above result indicated that the distribution of microhabitat characteristics had 67.4% correlation, with distribution of g. bancanus vertical growth. microhabitat characteristics which could support vertical growth of g. bancanus were higher altitude and decreasing canopy cover. this meant that needs g. bancanus sufficient light intensity to support its vertical growth. therefore, sufficient light intensity should be provided if would be g. bancanus cultivated in conservation to produce ex-situ longer log. a research by jans . (2012) showed et al that the seedling growth of was the g. bancanus highest in partial sunlight and reduced with decreasing light intensity. afterwards, big-sized tree will flourish in full sunlight (partomihardjo et al. 2008). the result also indicated that distribution of micr ohabitat characte ristics had higher correlation with distribution of basal area than with vertical growth. the correlation of microhabitat characteristics with basal area was 78.3%. microhabitat characteristics supporting basal area growth of were higher g. bancanus altitude, increasing slope, decreasing canopy cover, while level and shallower peat depth could also support basal area growth. peat swamp ecosystem is usually developed in a basin area. therefore, the result actually indicated that would have maxim basal area if g. bancanus um they grew at the edge of the basin area which had higher altitude, higher slope, lower canopy cover and shallower peat depth. the result followed istomo (1998) and bismark . (2005) where et al deeper peat depth gave higher density, thus shallower peat depth gave lower density and higher basal area. it showed that the range of their habitat actually was relatively narrow. the distribution of adult at the edge of the g. bancanus basin area, recorded by gps, in danau pulau besar/danau bawah wildlife sanctuary could be seen in figure 3. from the correlation between microhabitat characteristics and distribution of vertical growth and basal area, it could be estimated that planting of without any treatments in the g. bancanus outside area of their habitat would not be optimal. the survival rate would be low, and if they were successfully grown, the growth rate would be very slow s . ismail in term of height and tree basal area et al. g. bancanus (2007) found that planting of in non-peat swamp area after 11 years resulted to low survival rate (52%), low average of total height (8.16 m), and low average diameter breast height (9.1 cm). therefore, even though g. bancanus can be planted in non-peat swamp areas, limiting factor on utilization will be the relatively small diameter and log length. new or existing technology that can utilize smaller size logs may solve the problem (ismail . 2007). apparently, et al planting of is not easy and its growth g. bancanus is relatively very slow. according to the above discussion, although there is a possibility to conserve in an g. bancanus ex-situ in-situ conservation area, but the role of conservation becomes more important to conserve in an optimal condition. g. bancanus therefore, massive exploitation and land use change in peat swamp ecosystem become two important challenges in conserving . g. bancanus however, a technolog breakthrough to y cultivate outside their habitat is g. bancanus still needed to strengthen the conservation of this species. as suggested by krigas . (2010) et al this kind of study can provide valuable information to facilitate the transfer from wild habitats to man-made habitats like botanical garden. this transfer is important to produce material for reintroduction and reinforcement of this species when its existence in nature becomes increasingly rare (guerrant 2004). et al. bogor botanical garden has been planting seedlings of since year 2007, but g. bancanus their growth are still insignificant despite their survival. figure 3. distribution of within the study area in riau province in 2008 g. bancanus conclusions high correlation between the distribution of tree growth and microhabitat characteristics suggested that conservation offers better in-situ strategy than conservation to conserve ex-situ g. bancanus , especially when an appropriate planting method is not yet developed. acknowledgements we thanked our colleagues (yupi isnaini, enda suhenda and m. madhari) for their assistance in various aspects of this study. we also thanked the people in dpb-dw (sutrisno, jumaat, beni ishak silalahi) for their hospitality, and bksda riau for the permission support. the study was funded by dipa center for plant conservation bogor botanical gardens-lipi. references bismark m, kalima t, wibowo a, and sawitri r. 2005. growing stock, distribution and conservation of ramin in indonesia. technical report no. 01. itto pro.87/03 rev. 2 (f). identification of gonystylus spp. (ramin) potency, distribution, conservation and plantation barrier. ministry of forestry, center for forest and nature conservation research and development in cooperation with international tropical timber organization. guerrant eo, havens k, maunder m. 2004. ex-situ plant conservation: supporting species survival in the wild. washington dc(us):island press. guisan a, thuiller w. 2005. predicting species distribution: offering more than simple habitat model. ecol lett 8: 993-1009. institut pertanian bogor, unit konservasi sumberdaya alam riau. 2000. laporan akhir rencana pengelolaan suaka margasatwa danau pulau besar/danau bawah kabupaten siak propinsi riau. bogor (id):unit konservasi sumberdaya alam riau dan fakultas kehutanan institut pertanian bogor. ismail p, shamsudin i, abdul rahman k, hashim ws, ismail h. 2007. planting of in gonystylus bancanus non peat swamp area. jtfs 19 (1): 50-6. istomo. 1998. penyebaran pertumbuhan pohon ramin ( (miq.) kurz.) di hutan rawa gonystylus bancanus gambut: studi kasus di hph pt. inhutani iii kalimantan tengah. laboratorium ekologi hutan. buletin manajemen hutan. p 33-9. istomo. 2006. kandungan fosfor dan kalsium pada tanah dan biomassa hutan rawa gambut (studi kasus di wilayah hph pt. diamond raya timber, bagan siapi-api, provinsi riau). jmht 12 (3): 40-57. jans wwp, dibor l, verwer c, kruijt b, tan s, van der meer pj. 2012. effects of light and soil flooding on the growth and photosynthesis of ramin (gonystylus bancanus) seedlings in malaysia. jtfs 24(1):54-63. krigas n, mouflis g, grigoriadou k, and maloupa e. 2010. conservation of important plants from the ionian islands at the balkan botanic garden of kroussia, n greece: using gis to link the collection data in-situ with plant propagation and cultivation. ex-situ biodivers conserv 19:3583-603. 9 microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. lim t w, soehartono t, keong ch. 2004. framing the picture: an assessment of ramin trade in indonesia, malaysia and singapore. malaysia(my): traffic southeast asia. mcgarigal k, cushman sa, stafford s. 2000. multivariate statistics for wildlife and ecology research. new york(us): springer verlag. partomihardjo t, prajadinata, s, hidayat a. 2008. current status of ramin seeds source in sumatra. technical report itto project po 426/06 rev. 1 (f). indonesia: ministry of forestry. purba c, suparno, hariyono, zulfahmi, seventri ad. 2002. laporan studi perdagangan domestik dan internasional kayu ramin sebagai bagian dari pengumpulan informasi dasar dalam kerangka pelestarian jenis ramin di indonesia. bogor(id): forest watch indonesia/telapak. rostiwati t, murniati, hendromono. 2007. growth of ramin ( (miq.) kurz.) plantation gonystylus bancanus on various peat swamp forests in indonesia. ijfr 4 (2): 73-81. ryan mg, phillips n, bond bj. 2006. the hydraulic limitation hypothesis revisited. plant cell environ (29): 367–81. samedi. 2005. kontrol perdagangan ramin ( spp.) gonystylus internasional. dalam: semiloka nasional konservasi dan pembangunan hutan ramin di indonesia. prosiding. 28 september 2005; bogor (id): puslitbang hutan dan konservasi alam-itto. pp 60-72. shaw a. 1954. gonystilaceae. in: van steenis, cggj editor. flora malesiana i, vol. (4). jakarta(id): noordhoffkolff n. v. p 350-65. sidiyasa k. 2005. potensi botani, ekonomi dan ekologi ramin ( spp.). dalam: semiloka nasional gonystylus konservasi dan pembangunan hutan di ramin indonesia melalui regulasi perdagangan dan pemacuan alih teknologi konservasi, penanaman dan tehnik silvikultur. prosiding: 28 september 200 5; b og or (id): p usat peneliti an dan pengembangan hutan dan konservasi alam bekerja sama dengan itto dan ppd. p 9-34. sinclair sj, white md, newell gr. 2010. how seful re u a s d m mpecies istribution odels for anaging b f ciodiversity under uture limates? ecol soc 15(1): 8 [online] url: http://www.ecologyandsociety. org/vol15/iss1/art8/ soerianegara i, sambas en, martawijaya, sudo s, groen le. gonystylus teijsm. & binnend. in: 1994. prosea: plant resource of south-east asia 5 (1) timber trees: major commercial timber. edited by soerianegara i and rhmj lemmens. wageningen (nl):prosea, pudoc. p 221-30. 10 biotropia vol. 22 no. 1, 2015 http://www.ecologyandsociety. microsoft word 85 biotropia vol. 13 no. 2,2006 : 85 98 bacterial community shifts of a high mountain lake in response to variable simulated conditions: availability of nutrients, light and oxygen munti yuhana1, thomas horath2 and kurt hanselmann2 'department ofaguamlture, faculty of fisheries and marine science, bogor agricultural university, kampus ipb darmaga 16680 bogor, indonesia. 1 department of microbiology, institute of plant biology, university of zurich, zollikerstrasse 107, 8008 ziirich, switzerland. abstract we studied bacterial population composition shifts by exposing natural water samples to variable simulated environmental conditions. the samples were taken from lake jori xiii (2640 m a.s.l), an oligo-to mesotrophic cold freshwater lake, located in the eastern swiss alps. the jori lakes are characterized as remote, unpolluted high mountain lakes with a long period of ice cover and typically low nutrient concentrations. culture independent techniques (pcr-based analyses) were used for detection and molecular characterization of a large number of bacteria most of which are still uncultivable. bacterial community shifts over three ecological conditions (nutrients, light and oxygen availability) were detected by using temporal temperature gradient gel electrophoresis (ttge) of a pcr-amplified part of the 16s rrna gene. the bacterial populations responded differently to the variable conditions, as revealed by ttge pattern shifts during the experiment. key words: temporal temperature gradient gel electrophoresis (ttge), arb, small subunit ribosomal rna gene (ssu rrna gene), alpine freshwater lake jori xiii, pcr introduction lake jori xiii is located in canton graubiinden, switzerland. this lake has a maximum depth of 10.9 m and its surface area is approximately 1 5400 m2. lake xiii presently is one among 22 medium and small lakes in the jori catchment. some of them are several thousand years old, and others were formed when the glacier retreated during the last 150 years. the habitats are still evolving. they are situated in the eastern part of swiss alps, at 46°46'n latitude and 9°58'e longitude, at altitudes between 2489 m to 2750 m above sea level. the iron-rich area is almost free of vegetation. several lakes (for example lakes i, ii and iii) are still influenced by streamlets from the melting glacier, whereas many others, which are mostly situated on higher places, are no longer under glacier influence, including lake jori xiii (figure 1). remote, unpolluted high mountain lakes are ecosystems in which their hydrochemical conditions are characterized by extremely variable fluctuations and often sudden changes. these intrannual and interannual environmental conditions corresponding author: myuhana@botinst.unizh.ch or hanselma@botinst.unizh.ch 85 biotropia vol. 13 no. 2,2006 photo by kurt hanselmann figure 1. view of lakes jori i, ii, iii, xii and xiii during summer, ice-free period. can be related to the atmospheric or climate variability. the alpine lakes can be considered as relatively simple aquatic ecosystems with short food chains, restricted environmental variables and low species diversity. organisms living in these habitats are challenged by harsh temperatures (below freezing to +15°c maximally), by seasonal and diurnal radiation differences between darkness and high levels of uv, by nutrient deprivation and bursts during snow melt phases, and by anoxia and darkness in the hypolimnion below a long lasting ice cover. during the short summer period, the epilimnion experiences high diurnal temperature fluctuations, strong light penetration and high uv radiation (bothwell et ol. 1994). maximal water temperature during this period can reach 15°c but it could suddenly drop to 0°c in case of snow fall. bacteria are known to thrive under extremely broad-range conditions including such that would kill other organisms. they are further able to perform various metabolic processes, and their capabilities are more diverse compared to higher organisms. therefore, we focused on the adaptation of the bacterial community in this extreme habitat. we were interested in how the bacterial population composition shifts under fluctuating environmental conditions. normally, in the environments which get exposed to diurnal changes and annual variation in physicochemical and trophic conditions, the communities are forced to adapt quickly. the bacterioplankton community structure changes were studied in vitro by simulating three environmental determinants i.e. light, oxygen, and nutrients. temporal temperature gradient gel electrophoresis (ttge) has been used to follow the bacterial community composition changes. ttge is a method which does not require a cloning step for profiling microbial populations, therefore, it is a fast monitoring method. the small subunit (ssu) ribosomal rna gene is widely used as a comparative phylogenetic marker (ward et al. 1990; muyzer et al. 1993). the ribosomal rna gene has several features that make it an ideal phylogenetic marker. first, as the backbone inside the ribosome it is ubiquitous in the living world. 86 bacterial community shifts m. yuhana et al. further it has the same function in all organisms, it is functionally homologous. also it is more sensitive to mutations than most of the other genes since its gene product is used directly without any further translation into a protein where some mutations could keep silent. the gene sequence itself has regions of broad homology but also of heterogeneity compared with different organisms (it is moderately well-conserved) which makes it easy to align it in almost all levels of phytogeny. concerning the nucleic acid length of the rrna gene, the sequence of the ssu rdna has the advantage that it is long enough for a reasonable alignment but quite shorter than the sequence of the large subunit rdna (lsu rrna gene) which makes it cheaper and faster to work with. the community shifts were monitored by comparing the changes of dna fingerprints of pcr-generated 16s rdna fragments in a ttge gel. the electrophoretic separation of the community dna molecules in ttge is based on the temperature gradient. the separated bands can then be excised, pcr-amplified, and sequenced to identify the members of the community. this technique has been applied already for assessment and monitoring of bacterioplankton community dynamics in diverse aquatic environments (bosshard et al. 2000; pearce 2000; casamayor et al. 2002). the three environmental determinants chosen nutrient supply, light, and redox condition strongly affect biological processes. combinations of these factors were used for in vitro simulations. the results illustrate the population flexibility and further support the validity of the selective adaptation hypothesis for ecosystems under harsh and highly variable environmental conditions (elena and sanjuan 2003). materials and methods sample collection sampling was carried out in late autumn in october 2002 in lake jb'ri xiii. samples were taken from the lake water column, at 5 different depths of 1, 3, 5, 7, and 9 meters, respectively. water samples were collected with a 500 ml niskin sampler. the sampler was pre-rinsed with the lake water from the desired depths. the actual water samples were filled in sterilized 500 ml glass bottles and were kept at low temperature in a cooling box for transportation. sub samples for the experiments under the different conditions were taken in the laboratory (table 1). volumes of 100 ml for each experiment were placed aseptically in sterile 200 ml glass bottles. all samples were incubated at 4°c under a combination of the following conditions: with or without additional nutrients, in the light and in the dark, with oxygen (stirring) and anoxically (figure 2). the final concentration after adding nutrients was 10 fold diluted luria bertani (lb) medium consisting of: tryptone 0.5 g i"1, yeast extract 1.0 g i"1, and nacl 0.5 g i"1. the ph was adjusted to 7.2. a sample set was incubated in the dark (the bottles were wrapped in aluminum foil). samples that needed light were incubated under two 15 watt tube lamps (osram) i n a 3 0 x 3 0 x 7 0 cm3 refrigerator, with continuous illumination. aerobic incubation was done on stirrer plates. each bottle of this 87 biotropia vol. 13 no. 2,2006 figure 2. incubation of samples treated under a series of different simulated conditions at 4°c. treatment was stirred continuously using a magnetic bar and the bottles were covered by sterile cotton plugs. for anoxic treatment, the bottles were purged with o2-free n2 gas, completely filled and tightly sealed. resazurin was used as redox indicator in the anaerobic cultures. nucleic acid extraction, pcr, and ttge analysis for community profiling preparation for dna extraction of initial samples (without treatment) was processed directly in the field laboratory. water samples of 75 ml were filtered 88 bacterial community shifts m. yuhana et al. aseptically through 0.22 fim pore size filters (millipore gvwp, 25 mm in diameter) using a sterile syringe and a "swinnex®" disc filter holder (millipore, sxoo 025 00). the filters were placed in sterile 1.5 ml tubes and kept at -20°c until further processing, i.e. dna extraction, pcr amplification and ttge analyses. after 2 weeks of incubation, the cultures were harvested for total genomic dna extraction, pcr amplification, and further ttge analysis. cells were collected by filtration using 0.22 urn pore size filters (millipore, gvwp, 25 mm in diameter). the sample quantity varied, depending on the turbidity of the culture. normally, 40 to 50 ml from the treatments with nutrients and 75 ml from cultures without added nutrients were filtered. samples were treated applying protocol b of the qiagen dneasy® tissue kit for gram positive bacteria with an additional bead beating lysis step. the microorganisms on the filters were rinsed with lysis buffer and further processed according to the qiagen protocol. the cells were lysed with lysozyme and disrupted physically by bead beating. bead beating was done by adding 100 mg of glass beads (0.1 mm in diameter) to each tube and shaking at 80% of maximum speed in a retsch mm2000 bead beater (f. kurt retsch gmbh & co. kg, 42781 haan, germany) for 1 min. the extracted dna was diluted in 100 ^1 sterilized h2o. tubes containing extracted genomic dna were stored at -20°c until further processing. to amplify the 16s rrna gene fragments from the total community dna, we used the general bacterial primers 27f (= s-d-bact-0008-b-s-20; 5'-aga gtt tga tcm tgg ctc ag) and 1524r (= slightly modified 1525r, s-d-bact-1524-a-a-18; 5'-aag gag gtg atc car ccg) (lane 1991). pcr reactions were prepared in a volume of 25 ul, containing (final concentration): dh2o, taq buffer (ix) (sigma), 0.1 mg ml"1 dnase-free bovine serum albumin (amersham, pharmacia biotech inc.), 0.2 mm dntps, 200 nm of each primer, 40 u ml'1 taq polymerase (sigma), and approximately 50-100 ng template dna. pcr was performed with a techne thermocycler (techne ltd, duxford cambridge, u.k.). pcr was run under the following conditions: initial denaturation at 94°c for 130 sec. the next steps were 10 cycles of 94°c for 15 sec, 63°c for 30 sec and lowering the temperature by 0.5°c in every cycle (touch down), then 72°c for 80 sec with increasing the duration by 1 sec every cycle. these steps were followed by 20 cycles of 94°c for 15 sec, 56°c for 30 sec, 72°c for 90 sec with increasing the period by 1 sec every cycle followed by a final extension step at 72°c for 10 min. pcr products were analyzed by electrophoresis in a 1% agarose gel and 0.5x tab running buffer [20 mm trizma base, 10 mm glacial acetic acid, and 1 mm na2edta]. first pcr amplifications yielded approximately 1'500 bp length of the 16s rrna gene (data not shown). the primary pcr products were used as templates for the nested pcr. this method was used to increase the sensitivity and signal strength of the pcr amplification. universal primers 3 57f (slightly modified) (= s-d-bact-341-b-s-17; 5'-cct acg gga ggc agc ag) and gc-907r (= gc-univ-907-a-a-20; 5'-cgc ccg ccg cgc gcg gcg ggc ggg gcg ggg gca cgg ggg g ccg tca att cmt ttr agt tt) (lane 1991; muyzer et al. 1998) were used to amplify 89 biotropia vol. 13 no. 2, 2006 the 16s rrna gene fragments. pcr was performed with initial denaturation at 94' for 5 min, followed by 75°c for 15 sec. the next steps were 20 cycles of 94°c f 20 sec, annealing at 65°c lowered by 0.5°c every cyclefor 30 sec (touch dowr and elongation temperature 72°c for 1 min. these steps were followed by 15 cycl< of 94°c for 20 sec, 52°c for 30 sec, 72°c for 70 sec, and a final extension step ; 72°c for 10 min. nested pcr amplification yielded 16s rrna gene fragments c approximately 560 bp length (figure 3). figures. products after nested pcr with 357f and gc-907r of the 16s rrna gene fragments. only a part of the pcr products from several samples in this study is shown. m: marker (a dna digested with ecorl/himllu). (1-5: samples from depth 1 m; 1: initial, 2: hdo, 3: hlo, 4: ndo, 5: nlo; 6-10: samples from depth 3 m, 6. initial, 7: hda, 8: hla, 9: nda, 10: nla, 11-15: samples from depth 5 m; 11: initial. 12: hdo, 13: hlo. 14: ndo, 15: nlo; 16-20: samples from depth 7 m, 16: initial, 17: hda, 18: hla, 19: nda, 20: nla). initial: initial population, before treatments were applied. h: 10-fold diluted lb medium added, n: no nutrients added, d: incubation in the dark, l: incubation in the light, a: anoxic condition, o: oxic condition. 90 bacterial community shifts — m. yuhana et al. ttge was carried out in a dcode™ mutation detection system (bio-rad iboratories). 10 ul of the pcr samples and 10 ul of 2x loading buffer (70% [v/v] ycerol, 0.05% [w/v] bromophenol blue) were loaded onto 6% polyacrylamide gels :rylamide: n,n'-methylene bis-acrylamide 37.5:1 [w/w]; 7 m urea, ix tab). the is were run at temperatures starting at 54°c and raising to 64°c, temperature ramp te was +1.1°c h"1, the voltage was 90 v (4.0 v/cm), and running time was about 9 the gels were stained in 1 ng ml"1 ethidium bromide solution for 15 min, stained in water for 45 min, visualized, and photographed under uv insillumination (x = 312 run). •quencing of the 16s rrna genes from pcr-ttge bands and phylogenetic ee construction the representative bands of the dominant bacteria which appeared distinctly in fge gels were excised and dna was recovered by putting the gel fragments gether with 20 ul of h2o into a 1.5 ml tube, freezing twice, and taking the .pernatant. pcr amplification was performed by using 341f and the non-gc-907r imers. pcr products were bidirectionally sequenced using abi prism® big dye™ !.0 (applied biosystems). the pcr products were purified by centrifugation in icrocon filter devices (microcon ym 100, millipore, bedford, mass., usa), and ere used for a 10 ul-single pcr reaction: 5 to 20 ng dna template, 3 (jl big dye applied biosystems) and 3 |al of 1.5 um primer were used. after the sequencing 3r, the products were purified with sephadex g-50 (amersham, pharmacia iotech ab) and loaded onto the sequencing machine (abi prism 377 dna squencer). the blast search tool available from ncbi (http://www.ncbi.nlm.nih.gov/ ast) (altschul et al, 1998) was used to list the closest neighbors of the sequences, he new 16s rrna gene sequences were imported to the ssu rrna database :trieved from the technical university of munich (release january 2004) using the rb phylogeny program (http://www.arb-home.de/) (ludwig et al. 2004) and ligned automatically employing the fast aligner vi. 03 of the are software nvironment. subsequently the alignment was corrected manually according to the jcondary structure. the sequences were then added to the general tree of the arb su rrna database by calculating distances with a maximum-parsimony algorithm nd using the appropriate sai (sequence associated information) filters. results and discussion during the short summer period and autumn, the lake was homothermic i.e. the ike water masses were completely mixed. therefore, the physical and chemical larameters of the lake water column did not differ much among various depths table 2). the initial ttge profile from depth of 1 m is the same as those from lepths of 3 m, 5 m, 7 m, and 9 m. the community patterns of lake jqri xiii differ 91 biotropia vol. 13 no. 2,2006 generally, it can be seen that incubations under high nutrient conditions showed fewer bands than the initial community and also fewer when compared to those without nutrients addition. low numbers of ttge bands indicate a limited number of species, i.e. only those microorganisms from the community which possess the highest competitive ability, are growing well. environments with low nutrients lead to a more diverse species composition, and community shifts between the initial and the final state are less pronounced. cultures kept in the light did not yield distinctly different patterns from those ones kept in the dark regardless whether they were grown with or without nutrient addition, except for the aerobic condition with no additional nutrients. certain bacterial communities develop different life strategies as an adaptation to various environmental conditions. their population would be present predominantly even under markedly different environmental pressures which results in similar ttge banding patterns. however, it cannot be ensured that those co-migrating bands have the same sequence identity. therefore, it is helpful to identify the communities appearing in the ttge bands. in this experiment, the representative ttge bands of dominant communities were recovered and sequenced. the bands are phylogenetically affiliated to alpha-, and beta-proteobacteria, actinobacteria, chloroplasts, and candidates of division op 10 (figure 5). the communities are phylogenetically diverse and respond quickly to various environmental fluctuations. some sequences represented by bands a, b, c, 6 and 8, showed very low similarity values (86 to 94 % sequence similarities) to known 16s rdna sequences of cultured bacteria present in the databank (table 3). cultivation efforts for those microorganisms could become valuable concerning their physiological properties. the results suggest that community competition and adaptation processes are common in ecosystems exposed to strong physicochemical fluctuations. physiological abilities of the microorganisms present in their gene pool allow the community to respond rapidly to environmental changes and thus ensure the functioning of microbially-mediated processes in the habitat. microorganisms possessing a high physiological flexibility, so-called niche generalists, will dominate the population in their communities under various conditions (elena and sanjuan 2003), while niche specialists will get selected under more specific conditions. environmental changes express themselves as physical and chemical instabilities, which exert selection pressures for different microorganisms. these changes promote the dynamic microbial community transitions in fluctuating environments (rainey and travisano 1998). 94 bacterial community shifts m. yuhana el al. added) rather than to nlo. under low nutrient conditions, the majority of ttge bands still appeared. some became less intense after the two-week incubation period. a few bands are missing at the end of the experiment (e. g. bands a and b). initial: initial population, before treatments were applied. h: 10-fold diluted lb medium added, n: no nutrients added, d: incubation in the dark, l: incubation in the light, a: anoxic condition, o: oxic condition. s: start of gels, e: end of gels. the bacterial diversity decreases when the community is exposed to high nutrient concentrations and anoxic conditions. this can be detected from experiments designated hda and hla at depths of 3 m and 7 m, respectively. in these treatments, light apparently is an essential determinant. in the light, bands 8 (3m depth) and 10 (7m depth) became prominent, in the dark, band 7 (3m depth). based on ncbi-blast of 16s rrna gene sequences, the closest known relative of band 7 (community exposed to hda treatment) was an anaerobic, sulfate reducing bacterium, desulfotomaculum sp. dem-kme99-2, aj276565 (with 88% sequence similarity). in the hla culture, band 8 dominated the community, which is phylogenetically related to the alphaproteobacterium sphingomonadaceae bacterium n, dq497241 (99% sequence similarity). the sequence of band 10 was closest related to the betaproteobacterium li2-55 from lake loosdrecht water column, netherlands, aj964892 (97% sequence similarity). 93 biotropia vol. 13 no. 2, 2006 generally, it can be seen that incubations under high nutrient conditions showed fewer bands than the initial community and also fewer when compared to those without nutrients addition. low numbers of ttge bands indicate a limited number of species, i.e. only those microorganisms from the community which possess the highest competitive ability, are growing well. environments with low nutrients lead to a more diverse species composition, and community shifts between the initial and the final state are less pronounced. cultures kept in the light did not yield distinctly different patterns from those ones kept in the dark regardless whether they were grown with or without nutrient addition, except for the aerobic condition with no additional nutrients. certain bacterial communities develop different life strategies as an adaptation to various environmental conditions. their population would be present predominantly even under markedly different environmental pressures which results in similar ttge banding patterns. however, it cannot be ensured that those co-migrating bands have the same sequence identity. therefore, it is helpful to identify the communities appearing in the ttge bands. in this experiment, the representative ttge bands of dominant communities were recovered and sequenced. the bands are phylogenetically affiliated to alpha-, and beta-proteobacteria, actinobacteria, chloroplasts, and candidates of division op 10 (figure 5). the communities are phylogenetically diverse and respond quickly to various environmental fluctuations. some sequences represented by bands a, b, c, 6 and 8, showed very low similarity values (86 to 94 % sequence similarities) to known 16s rdna sequences of cultured bacteria present in the databank (table 3). cultivation efforts for those microorganisms could become valuable concerning their physiological properties. the results suggest that community competition and adaptation processes are common in ecosystems exposed to strong physicochemical fluctuations. physiological abilities of the microorganisms present in their gene pool allow the community to respond rapidly to environmental changes and thus ensure the functioning of microbially-mediated processes in the habitat. microorganisms possessing a high physiological flexibility, so-called niche generalists, will dominate the population in their communities under various conditions (elena and sanjuan 2003), while niche specialists will get selected under more specific conditions. environmental changes express themselves as physical and chemical instabilities, which exert selection pressures for different microorganisms. these changes promote the dynamic microbial community transitions in fluctuating environments (rainey and travisano 1998). 94 bacterial community shifts m. yuhana et figure 5. phylogenetic affiliation of representative dominant ttge bands of water samples from lake jori xi11 incubated at different environmental conditions. ne\v sequences are written in bold (band_xx, accession no., length ibpj). closest known relatives are written in italic, followed by their accession nos. and sequence lengths (bp). a yeast 18s rrna gene of saccharomyces cerevisiae is used as outgroup. the scale bar indicates 10% sequence divergence 95 biotropia vol. 13 no. 2,2006 conclusions it is possible to follow the bacterial community shifts by applying the pcr-based molecular technique of temporal temperature gradient gel electrophoresis (ttge). the physicochemical determinants applied in the experiments triggered the changes in the bacterial community composition originating from a high mountai bacterial community shifts m. yuhana et al. lake jo'ri xiii. 16s rrna gene sequencing and phylogenetic analysis revealed that the predominant populations are affiliated to the alphaand betasubgroups of the proteobacteria, to actinobacteria (high gc gram + bacteria), chloroplasts and candidates of division op 10. acknowledgments we are grateful to hanne grob, ignachio fernandez and gabriela iqbal-nava for excellent technical assistance. we thank the reviewers for helpful comments on the article. references altschul, s.f, t.l. madden, a.a. schaffer, j. zhang, z. zhang, w. miller and dj. lipman. 1997. gapped blast and psi-blast: a new generation of protein 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evolution: biogeochemical nutrient scavenging is a prerequisite. diploma thesis, university of basel, switzerland. lane, d.j. 1991. 16s/23s rrna sequencing, p. 115-175. in e. stackebrandt and m. goodfellow (ed.), nucleic acid techniques in bacterial systematics. john wiley & sons ltd., chichester, united kingdom. ludwig, w., o. strunk, r. westram, l. richter, h. meier, y. kumar, a. buchner, t. lai, s. steppi, g. jobb, w. forster, i. brettske, s. gerber, a.w. ginhart, o. gross, s. grumann, s. hermann, r. jost, a. konig, th. liss, r. lubmann, m. may, b. nonhoff, b. reichel, r. strehlow, a. stamatakis, n. stuckmann, a.vilbig, m. lenke, th. ludwig, a. bode, and k.-h. schleifer. 2004. arb: a software environment for sequence data. nucleic acids res, 32:1363-1371. muyzer, g, b.c. de waal and a.g. uiterlinden. 1993. profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna. appl. environ. 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bacterioplankton community dynamics, applied to atlantic freshwater lakes. polar biol. 23:352-356. rainey, p.b. and m. travisano. 1998. adaptive radiation in a heterogeneous environment. nature., 394: 69-72. ward, d.m., r. weller and m.m. bateson. 1990. 16s rrna sequences reveal numerous uncultured microorganisms in natural community. nature, 345: 63-65. 98 85.pdf 86.pdf 87.pdf 88.pdf 89.pdf 90.pdf 91.pdf 92.pdf 93.pdf 94.pdf 95.pdf 96.pdf 97.pdf 98.pdf 5. shahabuddin (effectiveness... biotropia vol. 21 no. 1, 20114: 48 58 effectiveness of dung beetles as bioindicators of environmental changes in land-use gradient in sulawesi, indonesia shahabuddin , uswah hasanah , and elijonnahdi received 13 june 2013/accepted 09 november 2013 bioindicators have been widely accepted as useful tools for monitoring and detecting changes in the environment or habitat condition. by using bioindicators, it is possible to assess the impact of human activities on the biota, instead of examining the entire biota. in this paper we analyzed diversity of dung beetles (coleoptera: scarabaeidae) across land use gradient in central sulawesi and tested the suitability of dung beetles as bioindicators for environmental changes. ninety baited pitfall traps were placed and several habitat parameters were measured at five land-use types ranging from natural forest to cacao agroforestry systems to open areas in 2009 and 2012. the effectiveness of dung beetles as bioindicators of environmental changes was evaluated by the method, a method combining the specificity and fidelity of certain species with particular types of habitat or environmental conditions. surprisingly, the results showed that the diversity of dung beetles in two types of cacao plantations were similar to the forest sites and were significantly higher than the open cultivated area. of the 16 dung beetles species analyzed only four species could be suggested as indicator (characteristic) species while the majority of collected species were categorized as detector species. two of them ( and ) were associated with natural forest and cacao agroforestry system, thus were suggested as the indicator of shaded and cooler habitats whereas and can be suggested as indicator of unshaded and warmer habitats (bare land area). bioindicators, diversity, scarabaeidae, habitat preferences, . 1* 1 2 1 2 department of agrotechnology faculty of agriculture university of tadulako, palu 94118, central sulawesi, indonesia department of biology faculty of mathematics and science university of tadulako, palu 94118, central sulawesi, indonesia indval copris saundersi onthophagus forsteni o. limbatus o. trituber indval abstract keywords: * corresponding author : shahabuddin_slh@yahoo.com doi: 10.11598/btb.2014.21.1.5 48 introduction the conversion of natural habitat to other land uses and its consequences on environmental changes has been acknowledged as the main driver of biodiversity loss at the southeast asia and global scales (sodhi . 2004) and by 2100 the impact of land use changes on biodiversity is likely to be more significant than that of climate change, nitrogen deposition, species introductions and changing atmospheric concentrations (sala 2000; young 2009). therefore, detecting environmental changes due to land-use change is needed in order to avoid the continuous loss of biodiversity. bioindicators have been proven to be useful tools for monitoring and detecting changes in the environment. species have different ecological requirements and their reactions to environmental variation are different from one another. therefore, some species are better indicators than others (dufrêne & legendre 1997). some species are generalists occurring in a wide range of habitats (ubiquitous), while others are more specialized, requiring certain habitat characteristics ( ). a bioindicator can be defined as a species or a species group that reflects the abiotic or biotic state of the environment ( ), represents the impact of environmental change on a habitat, community or ecosystems ( ), or indicates the diversity of other species ( ) (mcgeoch 1998). one of the reasons for using bioindicators is their cost-effectiveness. by using bioindicators it is possible to assess the impact of human activities on the biota, instead of examining the entire biota. especially useful are species that provide early warning of change (spellerberg 1993) environmental changes can cause different kinds of effects in the indicator, including physiological changes or changes in species number or abundance. the response of the species can be seen within the organism (e.g. heavy-metal concentrations), at the species level (species number and abundance) or at the community level (relations between species, e.g. pestpredator). increase or decrease or abundance of species number might be directly caused by change in abiotic and/or biotic factors or indirectly by change of species assemblage of other species (davis . 2001; rainio & niemela 2003; hambler . 2011; gerlach . 2013). a good bioindicator must fulfill several criteria. it has to be well-known taxonomically and ecologically, be distributed over a broad geographic area, have specialization to certain habitat requirements, provide early warning of change, be easy and cost-effective to survey, be relatively independent of sample size. its response should reflect the response of other species, one should be able to differentiate between natural cycles or trends and those induced by anthropogenic stress, and it should be of potential economic importance (e.g. noss 1990; pearson & cassola 1992). however, it is difficult to find species or species groups which would have all of these criteria (noss 1990; pearson & cassola 1992). requirements needed depend on the goal of the survey and the sensitivity to the anthropogenic disturbance is the most important criteria for monitoring environmental changes (kremen . 1993). et al et al stenotopic environmental indicator ecological indicator biodiversity indicator . et al et al et al et al . 49 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. in this study dung beetles (coleoptera: scarabaeidae) was selected as a bioindicator group because it has been widely known as one of the best bioindicator groups (e.g. halffter & favila 1993; mcgeoch 2002). dung beetles have been proven to be very suitable to assess effects of disturbances on tropical ecosystems (nichols . 2007; shahabuddin 2010) and human habitat modification (shahabuddin 2010; harvey 2006) due to their abundance, highly varied with respect to species traits, and rapid responses to environmental change (slade . 2011; shahabuddin . 2005 & 2010; spector 2006; slade . 2011). another advantage is their relatively complete species inventories and their data on the abundance of individual species can be achieved rapidly with standardized methods (larsen & forsyth, 2005). recently, dung beetles have been identified as one of the most cost-effective group for biodiversity survey in tropical forests (kessler . 2011) and contributors in improving the level of soil carbon stocks (kessler . 2012). one method used to quantify the 'bioindicator value' of a range of taxa is the indicator value ( ) method developed by dufrene and legendre (1997). this method combines measurements of the degree of specificity of a species to an ecological state, for example a habitat type, and its fidelity within that state (dufrene & legendre 1997). species with a high specificity and high fidelity within a habitat will have a high indicator value. high fidelity (frequency of occurrence) of a species across sample sites is generally associated with large abundance of individuals. both characteristics facilitate sampling and monitoring, which are important requirements for a useful bioindicator (kremen . 1994; mcgeoch . 2002). the indicator value method is important to conservation biology because it is conceptually straightforward and allows researchers to identify bioindicators for any combination of habitat types or areas of interest, e.g. existing conservation areas, or groups of sites based on the outcome of a classification procedure (mcgeoch & chown 1998). accordingly, method has become the most robust and popular method used to measure indicator species analysis (mc.geoch . 2002; aydin & kazak 2010; negro . 2011). in central sulawesi, forest habitats especially in the interior of lore lindu national park (llnp) are still relatively undisturbed while the margins of the park are characterized by a mosaic land-use type such as near-primary forests, secondary forests, forest gardens and plantations of cacao, maize and paddy rice fields (gerold . 2004). this study aimed to evaluate the effectiveness of dung beetles as bioindicator of environmental changes across land-use gradient in the margins of llnp. this study was carried out at the northern margin of the llnp in central sulawesi, indonesia. the park, a local biodiversity hot spot covers an area of 229,000 ha and is located southeast of palu, the capital of central sulawesi province. all study sites were selected at the surrounding of the palolo valley in the vicinity of the villages of bobo et al. et al et al. et al. et al et al et al et al et al indval et al et al indval et al et al et al materials and methods study area 50 biotropia vol. 21 no. 1, 2014 (01 07'0.46" s 119 59'702" e) and were situated at an altitude between 790 and 985 m asl. dung beetle communities were studied in five land-use types: natural forest (nf); secondary forest (sf); cacao agroforestry systems (cacao cultivated under natural shade tree at the forest margin (ac); cacao plantation under monospecific shade tree dominated by (cp); and open area (oa), cultivated either by or . three replications sites for each land-use type were selected with distance at least 50 m from each other dung beetles were sampled in 2500 m plots at 15 sites all using baited pitfall traps as described in shahabuddin (2010). six traps were set up at the centre of each plot and placed with an interval of 10 m. the traps were baited with ca. 30 g of fresh cattle ( ) dung and exposed six times from april to july in 2009 and march to june in 2012. cattle dung has been widely used as bait for dung beetles, aside from human faeces (e.g. erroizi 2004; andresen 2005; mendoza 2005). our previous study also showed that at the same weight of bait (ca. 30 g) cattle dung attracts the dung beetles with similar species composition found in the dung of anoa ( ), an endemic herbivore of sulawesi (shahabuddin . 2010). the trapped specimens were removed after two days and preserved in scheerpelz solution (krell 2007). later on, the samples were identified in the laboratory using available identification keys (e.g. balthasar 1963) and by comparing to the reference collection of the center for biodiversity research tadulako university. species which could not be identified, were sorted to morphospecies. several habitat parameters (i.e. vegetation structure and microclimate) affecting the dung beetles diversity (see davis . 2001; shahabuddin 2010) were measured to characterize the land-use types including air temperature, relative humidity, canopy cover, and herb layer coverage. the relative humidity at the start and end of the exposure period were measured using a digital thermo-hygrometer (corona model: gl 99) 1 m above ground while the canopy cover was visually estimated at four locations per site for a corridor of ca. 10 m inside the plot. the herb coverage was estimated at four plots of 2x2 m randomly placed at ca. 5 m inside the plot. based on the environmental variables measured all land-use types were then grouped using a two-dimensional scaling (clarke 1993; statsoft 2001). the three most widely used measures of species diversity were species richness, shannon-wiener index and simpson index (si = ∑ pi ) (lande 1996). species richness of dung beetles was estimated using the second-order jackknife extrapolation method (colwell, 2004), one of the best species richness predictor with respect to accuracy (e.g. brose . 2003). as units for estimating the total species richness of land-use type, samples from all traps and replicates were pooled for individual sample o o 2 r 2 2 gliricidia sepium zea mays morus alba et al. bos taurus et al. et al. bubalus deppresicornis et al et al et al specimens collection environmental variables measured data analysis . 51 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. times ( = 6) due to the close proximity between each trap and site. effects of habitat type on diversity were tested using one-way anova. abundance data were transformed by log (n+1) before analysis (zar 1999). statsoft 6.0 software (2001) was used to perform all statistical analyses. all diversities measured were computed with estimates version 7.00 program (colwell 2004) by randomizing the ranking of samples 50 times. only species sampled in both sample periods (2009 and 2012) were analyzed. the effectiveness of each dung beetles species as bioindicators were identified for each habitat type using the indicator value ( ) method (dufrene & legendre 1997).this method combines measures of specificity and fidelity and provides an for each species, as a percentage (dufrene & legendre 1997). specificity measure: = / where is the mean number of species across sites of group , and is the sum of the mean numbers of individuals of species over all groups. fidelity measure: = / where is the number of sites in cluster (habitat) j where species is present, and is the total number of sites in that cluster. the percentage indicator value for species in cluster (habitat) is then: = × × 100. the indicator values are the highest (100) when all individuals of a species are found in a single habitat (high specificity) and when the species occurs in all samples of that habitat (high fidelity). species with between 50% to less than 70% is categorized as the detector or generalist species while those species with significant of greater than 70% were regarded as characteristic indicator species for the particular habitat type (mc.geoch . 2002). a total of 1996 dung beetles specimens were collected during the study period. they belongs to four genera (dominated by ) and 28 species (for complete species list see shahabuddin 2013). however, only 16 species were recorded in both sample periods (2009 and 2012). the diversity of dung beetles changed from natural forest, to agroforestry cacao and to open area. interestingly, the diversity of dung beetles in both forest types and the two types of cacao plantations tend to be similar but significantly higher than that in open cultivated area (table 1). hence, this study showed that secondary forest and agroforestry system may support a high portion of tropical dung beetles species than in the bare land and thereby in line with the findings by nichols . (2007) and our previous study (shahabuddin . 2010). the fact that agroforestry system has high potency for conserving high biodiversity supported by previous study (e.g. mcnely & scroth 2006; schulze . 2010). however, the results may also be related with the spatial distribution of our study sites. the agroforestry cacao sites were closer to the natural and secondary forest than to the open area and has a high opportunity to be colonized by dung beetles coming from the forest sites. therefore, the high diversity of dung beetles at the cacao agroforestry system is also related to their close proximity to the forest sites. it has n indval indicator value a nindividuals nindividuals nindividuals i j nindividuals i bij nsites nsites nsites i nsites i j indval a b indval indval et al onthophagus et al et al et al ij ij i ij i ij j ij j ij ijij results and discussions diversity of dung beetles 52 biotropia vol. 21 no. 1, 2014 been reported that neighboring forest or isolation from forest may determine insect communities in tropical land-use systems (tscharntke . 2005; klein . 2006). agroforestry systems can be part of the habitat for many forest species using it for foraging, but they may also harbour largely independent populations. nonetheless, this study suggests that the preservation of environmental heterogeneity should be encouraged for conserving dung beetles in the llnp, central sulawesi. this is important because high diversity of dung beetles in tropical land-use will enhance its ecosystem function and this ecological services will be diminished by increasing human dominated land-use (shahabuddin 2011; slade . 2011; kudavidanage 2012). in more natural and heterogeneous habitats, such as natural forest and agroforestry system, dung removal, biological control and seed dispersal activities of dung beetles were higher than in homogenous or disturbed habitats (slade . 2007; nichols . 2008; shahabuddin 2011; slade . 2011). because the dung beetles diversity at the forest sites (natural forest and secondary forest) and the cacao plantation sites (agroforestry cacao and cacao plantation) were similar to each other but significantly higher than in the open area (table 1), the indicator species analysis land-use type is only classified into three groups, : forest, agroforestry cacao and open area. the high similarity of environmental parameter measured among these three groups of land-use types was supported by anova showing that temperature and herb coverage decreased from the natural and secondary forest to the open area while canopy and humidity showed a reverse pattern. however, a highly significant difference of environmental parameter measured was only recorded in the open area (fig. 1). this land-use type grouping was also supported by the ordination technique using multidimensional scaling (fig. 2). based on analysis of 16 dung beetles species collected in two sampling years (2009 and 2012), this study recorded four species having less than 70% and therefore, can be used as indicator (characteristic) species that are: at forest sites, at cacao plantation, as well as and at open area (table 2). and are suggested to be used as indicators of cooler and shaded habitats such as forest sites and cacao agroforestry et al et al et al et al. et al et al et al i.e indval indval copris saundersi onthophagus forsteni o. trituber o. limbatus copris saundersi onthophagus forsteni indicator species table 1. diversity of dung beetles at five habitat type. nf = natural forest; sf = secondary forest; ac = cacao agroforestry system ; cp = cacao plantation; oa = open area land-use type estimated species richness (jack-2) (f4,10=6.27, p <0.05) number of species recorded f4,10 =3·60, p < 0·005 h' simpsom nf 14.3a 9ab 1.6 4.2 sf 14.4a 10.7ab 1.7 4.2 ac 15a 11a 1.7 4.3 cp 13.6a 10ab 1.7 4.5 oa 8.7b 6.3b 1.3 3.1 53 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. system, while and most likely indicate warmer and unshaded habitats (e.g. open cultivated area). these characteristic species ( of > 70%) are unlikely to move from their requisite to other habitat types, even under changing conditions within this habitat. accordingly, populations of these species need only to be monitored within the specific habitat. beside indicator species, this study has recorded several generalist or moderate species with between 50% to less than 70%. these species were therefore, not characteristic species, as they do not have high of more than 70% for any particular habitat. species meeting these criteria are unlikely to respond very rapidly to changing habitat conditions. they can invade either close canopy or moist habitat e.g. natural forest, cacao agroforestry or cacao plantation but also open or warmer environment such us open area. furthermore, these species are less likely to become more vulnerable than indicator species, because a variety of habitats or ecological states, rather than only a single one, provide suitable resources for them and accordingly this group of species will be useful for longer-term monitoring. o. trituber o. limbatus indval indval indval figure 1. the effects of land use change on a) temperature (anova: f(4, 10)=25.15, p<0.01), b) relative humidity (anova:f(4,10)=62.88, p<0.01), c) canopy cover (anova:f(4,10)=139.81, p<0.01), and d) herb layer coverage (anova: f(4, 14)=52.15,p<0.01). all variables were averaged per land-use type. nf= natural forest; sf= secondary forest; ac= agroforestry cacao; cp= cacao plantation; oa= open area. significant differences between habitat types were indicated by different letters over the standard error (based on tukey's hsd post-hoc test) 54 biotropia vol. 21 no. 1, 2014 nf1 nf2 nf3 sf1sf2sf3 ac1 ac2 ac3 cp1cp2cp3 oa1oa2oa3 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 dimension 2 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 d im e n s io n 1 stress=0.007 open area forest sites agroforestry cacao figure 2. multidimensional scaling (mds) placing the five land-use type studied in three groups based on several habitat parameters measured. nf= natural forest; sf= secondary forest;, ac= agroforestry cacao; oa= open area table 2. indicator values ( percentage) for dung beetle species recorded in three group of habitats. species with > 70 % is categorized as a characteristic species (c) while species with 50 ≤ 70 % is a detector species (d) of certain habitat type indval indval indval species* forest sites agroforestry cacao open area species category 2009 2012 2009 2012 2009 2012 copris macacus 67.4 13.3 22.6 20.0 0.0 0.0 copris punctulatus 12.1 22.2 30.3 44.4 24.2 0.0 copris saundersi 95.8 91.7 0.0 0.0 0.0 0.0 c onthophagus cf.wallacei 22.4 41.5 10.5 41.5 67.0 1.5 onthophagus forsteni 0.0 3.7 33.3 74.1 0.0 0.0 c onthophagus fulvus 0.0 16.7 53.6 61.8 7.1 21.5 d onthophagus rectecornutus 0.0 0.0 0.0 33.3 66.7 0.0 onthophagus ribbei 60.5 32.4 8.4 37.1 0.4 1.0 d onthophagus rudis 5.3 39.5 29.3 51.2 13.3 9.3 d onthophagus scrutator 1.3 37.5 67.8 57.1 5.8 0.0 d onthophagus sp.1 63.6 62.8 19.4 30.2 6.9 4.7 d onthophagus sp.2 33.3 0.0 0.0 33.3 0.0 0.0 onthophagus sp.3 0.0 46.9 66.7 38.6 0.0 14.5 d onthophagus trituber 0.0 4.2 7.1 4.2 92.9 75.0 c phaechrous emarginatus 58.3 35.7 0.0 41.9 0.0 22.4 onthophagus limbatus 0.7 0.3 0.2 19.6 97.3 77.3 c * only species recorded in both sample years (2009 and 2012) were included in the analysesindval 55 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. conclusions acknowledgements references the fact that diversity of dung beetles at secondary forest and agroforestry system has no significant differences with natural forest has important implications on landscape management aiming at maintaining a high biodiversity. besides natural forest sites, certain agroecosystems like cacao agroforestry also have potency to maintain a high local diversity. dung beetles 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s178-s186. zar jh. 1999. biostatistical analysis. 4th edition. prentice-hall inc. new jersey.usa et al biodiversitas biological conservation . 58 biotropia vol. 21 no. 1, 2014 biotropia vol. 29 no. 3, 2022: 263 271 doi: 10.11598/btb.2022.29.3.1768 263 yeast probiotics with potential to assimilate cholesterol invitro yan ramona1,2* and ni luh putu ariwathi1 1department of biology, faculty of mathematics and natural sciences, universitas udayana, denpasar 80361, indonesia 2integrated laboratory for biosciences and biotechnology, universitas udayana, denpasar 80361, indonesia received 21 june 2022/accepted 3 october 2022 abstract in the last two decades the use of yeasts as new probiotics has increased significantly. therefore, our current research was focused on the investigation of yeasts for novel probiotic development in bali. the main objectives of this research were to isolate and characterize yeasts isolated from ragi tape (dried mix cultures of microorganisms normally used in the fermentation of rice or cassava in indonesia) and tape ketan (fermented sticky rice) for possible use as yeast-based novel probiotics, with capability to assimilate cholesterol in vitro. in this study, the potential yeast isolates were evaluated for survival at low ph conditions (ph 2, 3, or 4) and in high levels of sodium deoxicholic (nadc), at concentrations of 0.2, 0.4, or 0.6 mm. in addition, the yeast isolates were also evaluated for their ability to assimilate cholesterol in vitro and to elucidate biotransformation of cholic acid into deoxycholic acid. this study led to 10 isolates that were resistant to ph levels of 2, 3, or 4 and to nadc at concentration of higher than 0.4 mm. most of those isolates were also found to assimilate cholesterol in vitro at the rate of between 18% and 76% in 24 hours incubation. in the biotransformation test, none of those isolates transformed cholic acid into deoxycholic acid, indicating that they are safe and have potential to be developed into novel probiotics, either for human or cattle. keywords: bali, cattle, cholesterol assimilation, probiotics, yeast introduction intestinal probiotic has been defined as beneficial microbes residing along the intestinal tract of human or animals that provide beneficial effect to their host’s intestinal health (stasiak-różańska et al. 2021). these exclude commensal microbes residing in the intestine that do not provide health benefit to their host (lorenzo & lucio 2012). such commensals can be claimed as probiotics only if their health benefit can be demonstrated to their hosts. probiotics appear for the first time in the intestinal tract of human or animals either during delivery of babies through reproductive tract of their mothers or at the first time of lactation (quin et al. 2018). depending on the types of foods in the infant period of their growth and development, alteration of microbial composition in their intestine occurs (zhang et al. 2018). the role of prebiotics and fibers in their food has also been extensively reviewed by holscher (2017) to understand the effect of prebiotics and fibers to the composition of human intestinal tract. according to zommiti et al. (2020) probiotics applied as a single or mixed culture at the rate of 109 cells per day can improve the health of animal or human digestive system by maintaining the natural balance of the microbiota in the host’s digestive tract. besides, they can suppress or even exclude pathogenic microbes entering intestinal tract through mouth (markowiak & sli˙zewska 2017). the mechanisms of pathogenic growth suppression by probiotics in-vitro or in-vivo have been extensively reviewed by maldano-galdeano et al. (2019). probiotics are normally developed from nonpathogenic microorganisms that provide positive effects on the physiology of the intestine, if they are regularly consumed at sufficient density (maldano-galdeano et al. 2019). many of them play important roles in the elimination of toxic compounds in the intestinal track (petrova et al. 2022) resulting in an improved degree of host’s digestive tract health. *corresponding author, email: yan_ramona@unud.ac.id biotropia vol. 29 no. 3, 2022 264 many probiotic species also provide functional effects, such as preventing diarrhea (mcfarland 2010), reducing cholesterol content in the blood (nocianitri et al. 2017), or inducing the immune system of their hosts (maldanogaldeano et al. 2019). besides these benefits, the role of probiotics as possible anti-cancer agents has been reviewed by bhuvan and saroj (2018) and sankarapandian et al. (2022). until recently, commercial probiotics have been dominated by lactic acid bacteria (lab) isolated from healthy infants, aged under 3 months old (ramona et al. 2015). these labs have been recognized to be nontoxic, although some of them have been reported to produce antimicrobial compounds, such as bacteriocin (gonzales-perez et al. 2018). this ability enables the labs to control the balance of normal microbiota in the intestinal tract of human or animals, so that they can prevent pathogenic microbes from infecting the host’s intestinal tract. besides lab, yeasts have recently been investigated for novel probiotic development, although research on yeast, such as saccharomyces spp., is not as intensive as the study on lab. before being developed as potential and novel probiotics, some important specific characteristics of the yeasts, such as pathogenic properties, tolerance to upper part of intestinal tract conditions (e.g. low ph and high concentration of deoxicholic acid), and ability to convert cholic acid into deoxicholic acids need to be elucidated. in addition, the probiotic candidates should be nontoxic and provide beneficial effects, such as ability to reduce blood cholesterol (nocianitri et al. 2017) content and improve immune system in their hosts (especially human) so that they can be consumed in a long period of time to derive benefits (castellano et al. 2017). based on the above rational, yeasts (saccharomyces spp.) were isolated from ragi (dried mix cultures of microorganisms normally used in the fermentation of rice or cassava in indonesia) and tape ketan (fermented sticky rice), with the main objective to investigate some important characteristics (including their ability to assimilate cholesterol) of these isolates before being developed as novel and potential probiotics, in our present study. materials and method isolation of yeasts (saccharomyces spp.) from ragi and tape ketan the yeasts, saccharomyces spp., were isolated from ragi and tape ketan obtained from traditional markets around denpasar city, bali, indonesia. the samples which had been processed through dilution and spread plate methods as specified in ramona et al. (2015) were applied in the isolation and purification of yeasts with probiotic potential. sample size of 1 g of ground ragi or tape ketan was diluted in 9 ml of saline solution to obtain dilution rate of 10-1. this was further diluted to 10-5 in the saline solution. subsequently, 100 µl of suspension was taken from test tubes with dilution rates of 10-4 and 10-5 and was spread on plates containing potato dextrose agar (pda), followed by incubation at 37 oc for 24 hours. colonies with yeast morphological characteristics were purified by conducting streak culture for single colonies and stored at -20 oc in potato dextrose broth (pdb) supplemented with 30% v/v glycerol, before being characterized for the survival test at low ph condition and survival at high concentration of deoxicholic acid as well as being tested for transformation of cholic acid into deoxicholic acid, for assimilating cholesterol in order to see the yeasts potential as novel probiotics. growth of yeast isolates at ph 7 following exposure to low ph conditions (acidic conditions) one full loop of each yeast isolate was inoculated into 5 ml of potato dextrose broth (pdb) and incubated at 37 oc for 24 hours to obtain cell density of approximately 108 cells/ml (according to mcfarland scale). a volume of 100 µl of this suspension were then transferred into 900 µl of the same medium with various ph levels, i.e., ph 2, 3, and 4, and then incubated for 3 hours at 37 oc in a water bath, centrifuged at 7,000 rpm until a pelletform was obtained. the pellet was then washed twice with 300 µl saline solution. the washed pellet was then re-suspended in 300 µl pdb at ph 7, from which 50 µl was transferred into 6.5 ml pdb at ph 7, incubated at 37 oc for 24 hours, and finally, the optical density of the yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 265 pellet was measured at wavelength of 660 nm (od660). the survival of the yeast in this pdb medium at ph 7 after being exposed to various low ph levels in the same medium was indicated by an increase in turbidity (od reading) of the yeast suspensions. triplicate experiments per isolate were carried out to obtain representative data. survival at high concentration of sodium deoxicholic (nadc) a volume of 50 µl suspension of yeast isolates were inoculated to pdb that each containing three different concentrations of nadc, i.e., 0.2 mm, 0.4 mm, and 0.6 mm. pdb without nadc served as control. all test tubes were incubated at 37 oc for 24 hours and then measured for turbidity using a spectrophotometer at the wavelength of 660 nm (od660). triplicates per treatment were prepared to obtain representative data. an increase in od reading of the yeast isolates in pdb containing various concentration of nadc indicated the yeast’s survival in such medium following exposure with high level of nadc. in-vitro evaluation of yeast isolate’s ability to assimilate cholesterol the ability of yeast isolates to assimilate cholesterol in-vitro was conducted by following the method specified by buck and gilliand (1994). each isolate was initially grown in potato dextrose broth (pdb) at 37 oc for 24 hours. the yeast suspensions of 100 µl was next inoculated to 9.9 ml pdb supplemented with 2% sodium thioglycollate, 0.3% oxygall, and 0.10% cholesterol so that the total volume of each system was 10 ml, and then incubated at 37 oc for 24 hours, centrifuged for 20 minutes at 3,500 xg to obtain cell-free supernatant. following these steps samples were analyzed for cholesterol residue in each culture using o-pthalaldehyde method as the indicator reagent. un-inoculated medium served as control. the difference of cholesterol residue in the control and in the inoculated media was assumed as the amount of cholesterol assimilated by the yeast isolates. tests for biotransformation of cholic acid into deoxicholic acid this test adopted the method specified in uni (2012). a volume of 50 µl of yeast stock culture in pdb-glycerol medium was transferred into 5 ml pdb and incubated at 37 oc for 24 hours to produce cell suspension with approximate density of 108 cells/ml. subsequently, a suspension of 50 µl was grown at 37 oc for 24 hours in the same medium supplemented with cholic acid (ca). after that, 1 ml of the suspension was centrifuged at 5,000 rpm. a volume of 0.1 ml of the supernatant was subsequently transferred to a new eppendorf tube to which 500 µl of ethyl acetate and 20 µl hcl was added and then centrifuged at 5,000 rpm for 5 minutes. subsequently, the supernatant was evaporated at room temperature for 48 hours, and finally added with 15 µl methanol. this sample was then used for thin layer chromatography (tlc) to determine the extent of transformation. the eluent for the tlc consisted of a mixture of 10 ml cyclohexane, 15 ml ethyl acetate, and 4 ml acetic acid. before being used, this eluent was left for 30 minutes in a chamber in order to obtain saturated vapor of the mixture in the chamber. the tlc was run on a piece of aluminium silica gel, in a chamber previously prepared. samples and controls (cholic acid and deoxicholic acid) amounted at 1 µl were spotted on the aluminium silica gel and dried using a hair dryer. on the completion of the tlc run, the silica gel was removed from the chamber, air-dried at room temperature, sprayed with molibddophosporic acid, heated in an oven until black spots appeared on the silica gel, and photographed for documentation. results and discussion ten yeast isolates with potential as probiotics were successfully isolated in this study (table 1). all yeast isolates were found to ferment glucose and produce ethanol as well as gas from this metabolism. the colony morphology of our isolates was either rough or smooth, with pseudohyphal morphology. based on their cell morphology and biochemical evaluation, all isolates were preliminary identified as yeasts belonged to genus saccharomyces. these characteristics are in line with those reported by reis et al. (2014). yeasts belonging to this genus have been reported by many previous studies to have potential to be used as food supplements biotropia vol. 29 no. 3, 2022 266 for human or animal probiotic. pais et al. (2020) reported that s. boulardii plays important roles to maintain and control the balance of human or animal intestinal normal microbiota when included as food supplements. besides, yeast belongs to genus saccharomyces was also reported to prevent clostridium difficile from infecting human digestive tract (mills et al. 2018). in the recent years, the beneficial properties of such yeast strain have been extensively reviewed by tomičić et al. (2016). this strain was also engineered genetically by liu et al. (2016) so that the strain is able to produce several products of interest, including lysozyme which is important to improve and maintain intestinal tract health. this opens the possibility for our yeast isolates to be developed as potential and novel probiotics for cattle or human in the near future, although further evaluation is needed to elucidate their probiotic potencies. before being developed as novel and potential probiotics, isolates must show a high level of viability following exposure to extreme conditions of the upper part of the intestinal tract, such as low ph and high concentration of nadc). in the actual application, such probiotic candidates must flow along this part of the intestinal tract before reaching the lower part of the intestinal canal where they normally provide beneficial effects to their hosts (fuochi et al. 2015). the shown ability to transform cholic acid into deoxicholic acid must be excluded for further screening (jia et al. 2018). the yeast isolates in our study showed the capability to survive exposure to low ph conditions in-vitro (table 1). all isolates were found to grow in such medium conditions, although their growth was slightly inhibited due to 3-hour exposure to low ph conditions. the 3-hour incubation period was chosen in this study, because the probiotic candidates will experience three hour contact in the upper part of the intestinal tract when applied in the actual situation and this was in line with that reported by oozer et al. (2006). surprisingly, all isolates were found to survive in the exposure to ph 2 for 3 hours, although low turbidity was recorded after being incubated in new medium having ph 7 for 24 hours. the results of this study indicated that all yeast isolates may have the potential to be developed as probiotics, because under certain condition (such as empty stomach), the ph of the stomach may decrease to 2 or even 1.5 (beasley et al. 2015). knowledge about the mechanism by which microbial cells survive in a low ph medium is still limited. however, some studies reported that in order to survive under an extremely low ph medium, the cells must maintain their internal ph to be higher than that of their surroundings. (guan et al. 2020) by metabolizing glucose through which they derive energy so that they can activate proton pump to exclude h+ ions out from their cytoplasm. this process requires a significant amount of energy in the form of adenosine triphosphate or atp (anandakrishnan & zuckerman 2017). failure to maintain internal ph condition to be higher than that of cell’s environment will lead to cell’s death. table 1 growth of yeast isolates at ph 7 after being exposed to low ph conditions isolate codes optical density at 660 nm (od660)* control (ph 6.5) ph 2 ph 3 ph 4 n1 ++ (0.623 ± 0.141) ++(0.515 ± 0.064) ++(0.586 ± 0.057) ++(0.509 ± 0.092) n2 ++(0.653 ± 0.142) +(0.328 ± 0.057) ++(0.598 ± 0.201) ++(0.588 ± 0.061) n3 ++(0.559 ± 0.160) +(0.326 ± 0.050) ++(0.644 ± 0,177) ++(0535 ± 0.047) n4 ++(0.529 ± 0.075) +(0.339 ± 0.040) ++(0.600 ± 0.033) ++(0.585 ± 0.128) n5 +++(1.148 ± 0.291) +(0.478 ± 0.136) ++(0.530 ± 0.068) ++(0.549 ± 0.185) g1 ++(0.970 ± 0.183) ++(0.553 ± 0.060) ++(0.691 ± 0.106) ++(0.620 ± 0.203) g2 +++(1.110 ± 0.022) +(0.445 ± 0.038) ++(0.664 ± 0.340) ++(0.747 ± 0.068) g3 ++(0.865 ± 0.122) ++(0.610 ± 0.140) ++(0.750 ± 0.215) ++(0.582 ± 0.197) g4 +++(1.033 ± 0.051) ++(0.541 ± 0.154) ++(0.756 ± 0.138) ++(0.893 ± 0.029) g5 +++(1.061 ± 0.049) ++(0.514 ± 0.122) ++(0.654 ± 0.098) ++(0.628 ± 0.086) notes: * = each value ± standard deviation in table 1 is an average of triplicate measurements of optical densities (absorbance/a) of the cell suspensions which indirectly measure the growth of the cells in the low ph medium. the growth indication follows the following criteria: = od of < 0.1 (sensitive to low ph conditions); + = od of 0.1 0.5 (slightly resistant to low ph conditions); ++ = od of 0.5 1.0 (resistant to acid conditions); +++ = od of > 1.0 (highly resistant to acidic conditions). http://en.wikipedia.org/wiki/clostridium_difficile yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 267 in addition to resistance to acidic conditions, the yeasts isolates were also found to survive in medium containing high concentration of nadc (table 2), which is also a good indication for their possible use as potential probiotic candidates. on their way to colon, the probiotic must experience an extreme condition in the small intestine where high concentration of bile is secreted by the liver (darilmaz 2013). bile is toxic for microbes because of its function as biodetergent in the intestinal tract of human and other animals (urdaneta & casadesús 2017). the effects of this bile for bacterial cells can be fatal (cell death) as it can cause disintegration of the cell membrane system (urdaneta & casadesús 2017). other mechanism by which bile can disturb cell function is related to dca uptake by the cells. once exposed to neutral ph within the cell cytoplasm, the dca will be in a undissociated form and it is difficult for the cells to pump it out using proton motive force mechanism (chatterjee et al. 2004). accumulation of this compound in the cell can cause significant damage on dna and this will result in cell death (urdaneta & casadesús 2017). all yeast isolates showed resistance to various levels of nadc as indicated by high od reading (ranging from 0.5 to 1.0) (table 2). although it was not investigated in the present study, tolerance property to nadc exhibited by our yeast isolates might be related to glycosidase activity (kwun et al. 2017), the composition of membrane protein and fatty acid (gueimonde et al. 2005), and exopolysaccharide production (nguyen et al. 2020). the ability of cells to prevent damage on their cell membrane and dna during exposure to high level of nadc also plays important roles to survive in such condition (merritt & donaldson 2009). according to kusada et al. (2021) enzyme activity (bile salt hydrolase activity) that converts the physicochemical properties of the bile salt reduces toxicity of this compound to the cells. the potential of the promising isolates to assimilate cholesterol in-vitro is shown in figure 1. all isolates showed their ability to assimilate cholesterol in the medium with various degrees of degradation rates (between 18 76% reduction/24 hours incubation). when compared to control treatment (where the reading was constant or no reduction in cholesterol in the medium), these values were statistically significant (p < 0.05). only two isolates (n1 and n5) assimilated cholesterol at the rate of less than 50% (fig. 1). the others assimilated cholesterol at the rate of between 52% and 76%, relative to control, indicating that these isolates showed potential for further probiotic development. the data presented in figure 1 is in line with that mentioned by previous studies which found that some yeast species modified fat and its derivatives in the process of fermentation. aloglu et al. (2015) reported that yeast, including saccharomyces cerevisiae, had the ability to metabolize sterol. other yeast species, such as candida lipolytica was also reported by ali et al. (2010) to metabolize fats. in addition to yeasts, bacterial probiotics (lactic acid bacteria) have also been reported by tokatli et al. (2015) as having the capability to assimilate cholesterol. this yeast characteristic is important to reduce cholesterol absorption in the small intestine, which leads to reduced level of blood cholesterol. table 2 growth of yeast isolates in a medium containing various levels of nadc isolate codes optical density at 660 nm (od660)* control 0.2 mm nadc 0.4 mm nadc 0.6 mm nadc n1 ++(0.89 ± 0.24) ++(0.97 ± 0.25) +++(1.20 ± 0.26) +(0.58 ± 0.03) n2 ++(0.81 ± 0.21) ++(0.84 ± 0.34) ++(0.67 ± 0.10) +(0.34 ± 0.16) n3 ++(0.87 ± 0.10) ++(0.74 ± 0.05) ++(0.85 ± 0.05) +(0.29 ± 0.19) n4 ++(0.88 ± 0.90) ++(0.86 ± 0.01) ++(0.82 ± 0.06) ++(0.69 ± 0.29) n5 ++(0.99 ± 0.09) ++(0.84 ± 0. 04) +++(1.10 ± 0.14) +(0.21 ± 0.02) g1 ++(0.55 ± 0.09) ++(0.75 ± 0.11) ++(0.79 ± 0.06) +(0.49 ± 0.17) g2 ++(0.59 ± 0.06) ++(0.81 ± 0.01) +++(1.03 ± 0.09) ++(0.54 ± 0.37) g3 ++(0.73 ± 0.19) ++(0.98 ± 0.21) +++(1.09 ± 0.23) +(0.29 ± 0.21) g4 ++(0.66 ± 0.10) ++(0.87 ± 0.35) ++(0.95 ± 0.08) +(0.44 ± 0.08) g5 +++(1.04 ± 0.06) ++(0.92 ± 0.04) +++(1.02 ± 0.14) +(0.43 ± 0.38) notes: * = each value ± standard deviation in table 2 is an average of triplicate measurements of optical densities (absorbance/a) of the cell suspensions which indirectly measure the growth of the cells in various levels of nadc. the growth indication follows the following criteria: = od of < 0.1 (sensitive to nadc); + = od of 0.1 0.5 (slightly resistant to nadc); ++ = od of 0.5 1.0 (resistant to nadc); +++ = od of > 1.0 (highly resistant to nadc). biotropia vol. 29 no. 3, 2022 268 figure 1 percentage of cholesterol reduction in the medium after being inoculated with isolates of yeast and incubated for 24 h in pdb medium supplemented with cholesterol. note: values in the figure ± standard deviation bars are average value of triplicates. microbial isolates intended to be developed as potential probiotics should not transform cholic acid into deoxicholic acid in the intestinal track of their hosts. according to ajouz et al. (2014) and farhana et al. (2016), the accumulation of deoxicholic acid in the digestive track has been suspected to induce colon cancer. although yeast has the potential and has positive properties to be a probiotic candidate, the yeast will be rejected if it has the ability to transform cholic acid into deoxicholic acid. in our present study, the 10 yeast isolates (table 1 & 2) were evaluated for biotransformation of cholic acid into deoxicholic acid. the chromatogram of this evaluation indicated that none of the yeast isolates in our study transformed cholic acid into deoxicholic acid (fig. 2). figure 2 a chromatogram of test for biotransformation of cholic acid into deoxicholic acid for the 10 yeast isolates in our study. yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 269 the chromatogram confirmed that all yeast isolates obtained in our present study are safe and have the possibility to be developed as potential probiotics without harming human or cattle as their hosts. in the intestinal track, biotransformation of cholic acid into deoxicholic acid is commonly performed by clostridium scindens (guzior & quinn 2021). high concentration of deoxycholic acid in the intestinal tract was reported by wu et al. (2018) as having significant effect on the enlargement of colorectal tumor. conclusion all 10 yeasts isolates used this study were shown to be resistant to low ph conditions and high concentration of bile salt (nadc). the isolates were also found to reduce cholesterol content in-vitro at the rate of between 18 76% following 24 hours incubation. besides these positive attributes, none of the isolates transformed cholic acid into deoxicholic acid, indicating that they all have probiotic potential and are safe to be developed as novel probiotics in bali, either for human or cattle. acknowledgments the authors would like to acknowledge lppm universitas udayana and the integrated laboratory for biosciences and biotechnology, universitas udayana for the financial support and provision of consumables and equipment, respectively for this research. the authors’ thanks are extended to dr i nengah sujaya for his critical thinking on the subject of this manuscript prior to being submitted. 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handayani , alimuddin , utut widyastuti , emma suryati and andi parenrengi received 19 november 2013/accepted 6 september 2014 ice-ice disease is the biggest problem in the cultivation of seaweed . the disease is caused by bacterial infection and induced by drastic changes of water quality. lysozyme has the ability to break down bacterial cell wall. the purpose of this research was to construct of a binary vector pmsh1-lys carrying chicken lysozyme (lys) gene and to introduce pmsh1-lys on . the binary vector expression was transformed into lba4404 by triparental mating. thallus was inoculated with carrying pmsh1-lys and then the transformed thallus was selected by adding 20 mg/l hygromycin to the culture medium. polymerase chain reaction (pcr) analysis showed that the construction of the binary plasmid pmsh1-lys was established. percentage of pmsh1-lys transformation on was 23.56%, while the efficiency of regeneration was 11.32%. pcr analysis showed that three of the regenerated thallus contained lysozyme gene. thus, transgenic was successfully produced. these findings can be useful for studying the mechanisms of seaweed defense against bacterial infection. , genetic transformation, , lysozyme 1,2* 1 3 4 4 1 department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university, bogor 16680, indonesia research center for oceanography,indonesian institute of science, jakarta 14430, indonesia research center for bioresources and biotechnology, bogor agricultural university and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor 16680, indonesia research institute for coastal aquaculture, maros,south sulawesi, indonesia kappaphycus alvarezii k. alvarezii agrobacterium tumefaciens a. tumefaciens k. alvarezii k. alvarezii agrobacterium tumefaciens kappaphycus alvarezii 2 3 4 abstract introduction keywords: kappaphycus alvarezii (doty) is economically important red tropical seaweed highly demanded for its cell wall polysaccharides, being the most important source of kappa* corresponding author : umuamel@gmail.com doi: 10.11598/btb.2014.21.2.2 82 carrageenan in the world (bixler 1996). carrageenan is used in industries including food, textile, cosmetic, pharmaceutical and photography (yu . 2002). kappacarrageenan is used as stabilizers, thickeners and emulsifiers (bixler 1996). seaweed production can be increased by extensification and improvement of cultivation method. however, ice-ice disease is the biggest problem in seaweed cultivation. ice-ice infection can spread in broad area of cultivation for a week, and cause thallus damage about 60-80% within 1-2 months (sulistijo, personal communication, 2002). carrageenan content in the ice-ice infected seaweed decreases (amiluddin 2007), and this leads to loss for farmers (yulianto & mira 2009). bacteria are suspected as causative agents of ice-ice disease, including , and . shows the highest pathogenicity (nasution 2005). largo (2002) also found sp. on ice-ice infected thalli. furthermore, aris (2011) reported five bacteria on ice-ice infected thalli, namely and . genetic engineering techniques can be used to make seaweed resistant to bacterial infections. as the first step, takahashi . (2010) had transformed glucuronidase gene into thallus by particle bombardment. huddy . (2012) had also successfully introduced the lacz gene into the thallus by particle bombardment. in plant transgenic production, a foreign gene is generally transferred using . the method has advantages such as having relatively low cost, small copy number of gene and reproducibility (hiei . 1997). contains binary expression vector. the first vector is part of the virulent but without helper t-dna, while the second vector is smaller and contains a t-dna gene to be inserted (loeidin 1994). lysozyme has been used to generate transgenic fishes that are resistant to bacterial infections, such as zebrafish (yazawa . 2006), shrimp ( ) (burge . 2007) and salmon ( l.) (fletcher . 2011). lysozyme is small ubiquitous antibacterial enzyme that hydrolyzes gram-positive bacteria (li . 2008). in addition, lysozyme is also able to kill gram-negative bacteria infecting bivalves and shrimp (burge . 2007). yazawa (2006) established a transgenic zebrafish strain expressing chicken lysozyme gene. in the challenge experiment, 65% of the f2 transgenic zebrafish survived when infected by and 60% fish survived when infected by , whereas all non-transgenic fish were died. the lysozyme lytic activity of f2 in transgenic salmon was 40% greater than that in non-transgenic siblings (fletcher . 2011). chicken lysozyme possessed lytic activities against and (yazawa . 2006). thus, chicken lysozyme is an important component of immune defense against diverse bacterial infections that can be exploited to increase seaweed resistance against pathogens. the aims of this study were to construct a binary plasmid carrying chicken lysozyme gene and to transfer it into thallus by using . et al pseudomonas nigricaciens p. fluorescens, vibrio granii, bacillus cereus v. agarliquefaciens vibrio agarliquefaciens vibrio k. alvarezii flavobacterium meningosepticum, v. alginoliticus, pseudomonas cepacia, p. diminuta plesiomonas shigelloides et al porphyra yezoensis et al gracilaria gracilis agrobacterium tumefaciens et al a. tumefaciens a. tumefaciens, et al litopenaeus vannamei et al salmo salar et al et al et al et al. flavobacterium columnare edwardsiella tarda et al micrococcus lysodeikticus, flavobacterium columnare, aeromonas hydrophilla vibrio anguillarum et al k. alvarezii a. tumefaciens β-1, 4-linked glycoside bonds of peptidoglycan, a major cell wall component of 83 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. materials and methods kappaphycus alvarezii and sterilization construction of lysozyme-expressing plasmid kappaphycus alvarezii kappaphycus alvarezii et al. not spe not spe escherichia coli et al e.coli not spe thalli with green color were obtained from lampung, sumatera island, indonesia. thalli were maintained in fiber tanks with a flow-through water system, aerated and under a 12/12 hours (light/dark) photoperiod at room temperature. thalli were cut about 3 cm in length and then sterilized by 1% iodine solution and detergent. the thalli were maintained in prevasoli (pes) medium liquid until they were ready to be transformed. binary vector pmsh1 (nara institute of science and technology, japan; fig. 1) was used for the construction of lysozyme (lys) expression vector. lys gene was amplified from pjfker-lys (yazawa 2005; fig. 2) using specific pcr primers lysf: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc-3'. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator) and 1 minute of extension at 72 c. the amplified products were electrophoresed on a 2% agarose gel with 1x tae buffer. the dna of pmsh1 and pcr product of lys gen were cut by restriction enzymes i and i. lys gene was ligated with i and i-digested pmsh1. this recombinant binary vector was designated as pmsh1-lys. plasmid pmsh1-lys was then transformed into . 2002). transformant lys specific primer, and restricted using i and i enzymes. o o o o o dh5α by heat shock (suharsono dh5α was identified by pcr method using 84 biotropia vol. 21 no. 2, 2014 figure 1. map of the t-dna plasmid pmsh1 (naist, japan). npt ii: neomycin phosphotransferase ii, a selection marker gene. hpt: hyg romycin phosphotransferase, a selection marker gene. mcs: multi cloning site, regions of target genes that is controlled by the cauliflower mosaic virus 35s promoter (camv 35s) and terminator (t) nopaline synthase (nos), containing i, i, i, i, i, i, i, i xba xho sac sma kpn spe not bamh transformation of pmsh1-lys into agrobacterium tumefaciens plasmid pmsh1-lys was transformed into by tri-parental mating (liberty . 2008). tri-parental mating was carried out using , hyg ) as the donor strain, dh1 carrying prk2013 (kan ) as a. tumefaciens et al e. coli e. coli dh5α carrying pmsh1-lys (kan r r r 85 figure 2. map of pjfker-lys (yazawa . 2005). chicken lysozyme gene is controlled by the promoter of keratin (keratin) japanese flounder ( ). sv40: simian virus 40 terminator. npt ii: neomycin phosphotransferase, gfp: green flourescense protein et al paralichthys olivaceus binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. the helper strain, and lba4404 (strep ) as the recipient. was grown at room temperature on lb medium with 50 μg/l streptomycin. c on lb medium with 50 mg/l kanamycin and 50 mg/l hygromycin. dh1 bearing helper plasmid prk2013 was grown at 37 c on lb medium with 50 μg/l kanamycin. about 20 μl bacteria consisted of dh1 and were cultured together (conjugation=”konjugasi”) for 36 hours at room temperature. conjugated product was identified by antibiotic selection (kanamycin 50 mg/l, hygromycin 50 mg/l and streptomycin 50 mg/l), and pcr method using specific primers: for lys gene was lys-f: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc-3'; for 35s camv promoter was 35scamv-f: 35s-f: 5'-atg gct gga gta tta gct ggg-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc -3; for nos terminator was lys-f: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and nos-r: 5'-ctc ata aat aac gtc atg cat tac a-3'. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator) and 1 minute extension at 72 c. the amplified pcr products were electrophoresed on 2% agarose gel with 1x tae buffer. a single colony of containing pmsh1-lys was incubated in 5 ml of lb medium and grown for 36 hours on a 200 rpm shaker at room temperature. the bacterial culture was refreshed in 10 ml of lb medium and grown for 18 hours on a 200 rpm shaker at room temperature to an od600 of 0.5-1.0. the bacterial culture was centrifuged at 5,000 rpm and the pellet was resuspended in 25 ml of liquid suspension medium containing pes and 100 μm acetosyringone. the suspension was used for thalli transfection. a. tumefaciens a. tumefaciens e. coli e. coli e. coli e. coli a. tumefaciens a. tumefaciens a. tumefaciens r o o o o o o o dh5α containing pmsh1-lys was grown at 37 dh5α, preparation of suspension for co-cultivationagrobacterium 86 biotropia vol. 21 no. 2, 2014 production of transgenic detection of lysozyme gene in transgenic construction of lysozyme expressing plasmid kappaphycus alvarezii k. alvarezii et al a. tumefaciens k. alvarezii e. coli not spe e. coli e. coli e. coli a. tumefaciens transgenic were produced as previously reported (cheney . 2001). seaweed thalli (1-2 cm in length) were wounded by sterile syringe and infected by carrying pmsh1-lys for 30 minutes in suspension medium containing pes and 100 μm acetosyringone. the infected thalli were transferred to co-cultivation medium (pes medium containing 100 μm acetosyringone) for 3 days and then transferred to recovery medium (pes medium without acetosyringone) for 7 days. finally, transgenic thalli were selected in pes medium containing hygromycin 20 mg/l for 14 days. genomic dna was extracted from putative bud of transgenic using ctab reagent (doyle & doyle 1987). dna sample (1 μl) was used in a 10 μl pcr mixture. the pcr primers for lys detection were lys-f and lys-r; 35s camv-f and lys-r; and lys-f and nost-r. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator) and 1 minute extension at 72 c. the amplified pcr products were electrophoresed on a 2% agarose gel with 1x tae buffer. construction of binary plasmid was done by ligating chicken lys gene (460 bp) and pmsh1 (12,986 bp). result of the ligation was 13,449 bp (fig.3a lane 1). verification of containing pmsh-lys was performed by digesting pmsh1-lys with i and i restriction enzymes. the restriction products were two fragments in size of 12,986 bp and 460 bp (fig.3a lane 2). the 12,986 bp fragment was the size of pmsh1, and 460 bp was lys gene fragment. , respectively (fig. 3b). based on the results, it was concluded that the binary vector pmsh1-lys was established. pcr verification and enzyme restriction performed above also confirmed that transformation of pmsh1-lys into d been successful. plasmid pmsh1-lys contained genes encoding protein resistant to antibiotics. thus, bacterial colony survived and grew in selective medium containing kanamycin and hygromycin was a pmsh1-lys transformed ould . k. alvarezii o o o o o results and discussion dh5α dh5α carrying pmsh1-lys was also identified using pcr with primers lys-f and lys-r; 35s-f and lys-r, and lys-f and nos-r. the results of pcr analysis using those primers were 460 bp, 670 bp and 580 bp dh5α ha dh5α. the pmsh1-lys c then be used for transformation into 87 figure 3. a. pattern restriction of pmsh1-lys using i and i enzymes. m: 1 kb dna ladder marker (fermentas). lane 1: plasmid of pmsh1-lys, lane 2: i and i digested pmsh1-lys and lane 3: pcr product of lysozyme gene (lys). b. identification of lysozyme gene using pcr with primers lys-f and lys-r (lanes 1, 2 and 3), 35s-f and lys-r (lanes 4 and 5) and lysf and nos-r (lanes 6 and 7). m: 100 bp dna ladder marker (fermentas), lanes s. lane 2: positive control, pjfker-lys. lanes 3, 5 and 7 are the negative c not spe not spe escherichia coli dh5α containing 1, 4 and 6 are dh5α containing pmsh1-ly ontrol (non-transformant dh5α) figure 4. triparental mating. a. results of tri-parental mating grown on la medium without antibiotics. b. lba 4404 transformants on selective medium containing 50 mg/ hygromycin, 50 mg/ kanamycin and 50 mg/ streptomycin. c. lba 4404 non-transformants did not grow on selective medium agrobacterium tumefaciens a. tumefaciens l l l binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. transformation of pmsh1-lys into agrobacterium tumefaciens transformation of pmsh1-lys into was performed by tri-parental mating (fig.4). plasmid pmsh1-lys in (as a recipient) through the conjugation process by prk2013 in dh1 as a helper (fig. 4a labelled as “konjugasi”=conjugation). wa was resistant to streptomycin, but susceptible to kanamycin and hygromycin. thus, bacteria grew on the selective medium was containing pmsh1-lys derived from tri-parental mating (fig.4b). non-transformant was not grown on the selective medium (fig. 4c) a. tumefaciens e. coli a. tumefaciens e. coli e. coli a. tumefaciens a. tumefaciens a. tumefaciens dh5α (as a donor) was transferred into dh5α containing pmsh1-lys s resistant to kanamycin and hygromycin, but susceptible to streptomycin. . figure 5. identification of containing pmsh1-lys by pcr method. lanes 1, 2 and 3: pcr product using primers lys-f and lys-r. lanes 4, 5 and 6: pcr product using primers 35s-f and lys-r. lanes 7, 8 and 9: pcr product using primers lys-f and nos-r. m: 100 bp dna ladder marker (fermentas). lanes 1, 4 and 7 are ontaining pmsh1-lys). lanes 3, 6 and 9 are a negative control (nontransformant lba4404) agrobacterium tumefaciens a. tumefaciens a. tumefaciens lba4404 from tri-parental mating. lanes 2, 5 and 8 are a positive control (dh5α c identification of transformants was performed by pcr method. as shown in figure 5, pcr amplification products using lys-f/lys-r primer was 460 bp (lanes 1 and 2), using primers 35s-f/lys-r was 670 bp (lanes 4 and 5), and using lysf/nos-r primer was 580 bp (lanes 7 and 8). this result showed that contained pmsh1-lys, and it could then be used for transformation into . a. tumefaciens a. tumefaciens k. alvarezii 88 biotropia vol. 21 no. 2, 2014 transformation of pmsh1-lys into kappaphycus alvarezii k. alvarezii a. tumefaciens a. tumefaciens gracilaria changii k. alvarezii agrobacterium et al thalli were adapted on the pes culture medium (liquid and solid), and then were transformed with pmsh1-lys by (fig.6). the result showed that after 30 minutes infection with at od600 of 0.5 to 0.8, the transformed thalli could grow in pes medium containing 100 acetosyringone (fig.6). the transformed thalli could grow on selective medium containing 20 mg/l hygromycin (fig. 6a-c). non-transformant thalli was gradually dead on selective medium containing 20 mg/l hygromycin (fig. 6d-f). wild type thalli could grow on pes medium (fig. 6g-i). in total, 53 out of 225 transformed thalli (23.56%) grew on hygromycin selective medium (table 1). this number was lower compared to percentage of gene lacz transformation on using particle bombardment methods (80-94%). the result found in this study was thought to be affected by the difference of transformation method. in the next study, genetic transformation protocols of need to be improved for increasing the transformation percentage. there were 3 (11.32%) thalli succeeded to germinate. efficiency of putative sprouting ratio was determined by the number of putative sprouting thalli grew on hygromycin selective medium. the result showed that the efficiency of putative germination transformed thalli was lower than those of non-transformed ones (22%). it might be caused by infection and antibiotic selective medium. this finding was in accordance with suma . (2008) that the addition of antibiotics in selective medium μm figure 6. development of transformed thalli at 4 (a), 8 (b) and 12 (c) weeks culture in selective medium containing 20 mg/l hygromycin. non-transformant thalli at 4 (d), 8 (e) and 12 (f) weeks culture in selective medium containing 20 mg/l hygromycin. non-transformant thalli in the medium without hygromycin at 4 (g), 8 (h) and 12 (i) weeks culture. in d-f, green color indicates survived thalli, while the white color indicates dead thalli agrobacterium tumefaciens kappaphycus alvarezii table 1. transformation percentage and putative bud of containing lysozyme gene kappaphycus alvarezii treatment number of thalli number of hygromycin resistant thalli percentage of transformation a) number of putative bud number of positive by pcr efficiency of putative bud b) transformation 225 53 23.56% 6 3 11.32% control -1) 50 0 0 0 0 0 control +2) 50 0 11 0 22% notes: the percentage of the number of hygromycin-resistant thalli versus the total number of thalli transformed the percentage of the number of thalli that sprouts putative versus number of hygromycinresistant thalli non-transgenic thalli in hygromycin selective medium non-transgenic thalli in non-hygromycin medium a) b) 1) 2) 89 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. ba c d e f g h i can cause thallus growth and development decrease. optimum infection duration and different treatment methods on selective medium in future research is expected to result in higher germination efficiency. transformation percentage in this study was 23.56% (table 1). figure 7. lysozyme gene detection in transformed thalli by pcr method. lanes 1, 2 and 3: pcr products using lys-f and lys-r primers; lanes 4, 5 and 6 using 35s-f and lys-r primers; lanes 7, 8 and 9 using primers lys-f and nos-r primers. m: 100 bp dna ladder marker (fermentas). lanes 1, 4 and 7 are transgenic thalli; lanes 2, 5 and 8 are positive control (plasmid of pmsh1-lys); lanes 3, 6 and 9 are negative control (wild type of seaweed). agrobacterium tumefaciens kappaphycus alvarezii presence of lys gene in was confirmed by pcr method using three primers set. as shown in figure 7, transgenic thalli (lanes 1, 4 and 7) possessed pcr amplicon in the same size with the positive control of pmsh1-lys plasmid (lanes 2, 5 and 8), while the non-transgenic thalli showed no amplicon. this indicated that the putative bud derived from transformation was transgenic carrying lys gene. k. alvarerzii a. tumefaciens 90 biotropia vol. 21 no. 2, 2014 this study is the first transgenic production using infection method and lys gene as the transgene. analysis of lys gene activity in transgenic againts ice-ice bacterial agent is in progress. this transgenic can also be useful for studying the mechanisms of seaweed defense against bacterial infection. in addition, transgenic seaweed resistance to ice-ice disease can be useful to ensure seaweed production in the season when ice-ice disease frequently infects. transformation method developed in this study is beneficial to produce other transgenic seaweed expressing protein that regulates important traits in aquaculture. examples of interesting genes that can be used to generate transgenic seaweed are cooper/zinc superoxide dismutase (cuzn-sod) and omega-3 highly unsaturated fatty acids (n-3 hufa) metabolic enzymes. those enzymes had been used in plant transgenic production. cuzn-sod had successfully been transformed in to increase its resistant to environmental stress (hannum 2012). transgenic expressing n-3 hufa metabolic enzymes had also been generated (robert . 2005). construction of the binary plasmid pmsh1-lys was established with size of 13,449 bp. mediated transformation of lysozyme gene could produce transgenic . the efficiency of putative bud was 11.32 %, while percentages of positive transformants were approximately 23.56%. k. alvarezii a. tumefaciens k. alvarezii k. alvarezii nicotiana tabacum arabidopsis et al agrobacterium tumefaciens kappaphycus alvarezii conclusions 91 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. acknowledgements references this research was supported by the collaboration between research institute for coastal aquaculture, maros and research center for bioresources and biotechnology, bogor agricultural university. our thanks to department of aquaculture, faculty of fisheries and marine science, bogor agricultural university for technical support. amiluddin nm. 2007. a study of growth and carrageenan content of seaweed that are affected by ice-ice disease in pari island, thousand islands. . bogor (id): institut pertanian bogor. aris m. 2011. identification, pathogenicity of bacteria and the use of gene 16s rrna for ice-ice detection onseaweed aquaculture ( ). . bogor (id): institut pertanian bogor. bixler h.1996. recent developments in manufacturing and marketing 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nutritionally important dha in seed oil. functional plant biology 32: 473-9. kappaphycusalvarezii phd thesis kappaphycus alvarezii phd thesis litopenaeus vannamei agrobacterium porphyra yezoensis salmo salar .). melastoma malabathricum phd thesis agrobacterium tumefaciens gracilaria gracilis mytilus galloprovincialis . pathogenic of bacteria isolates on seaweed in pari island arabidopsis 92 biotropia vol. 21 no. 2, 2014 suharsono u, fujisawa y, kawasaki t, iwasaki y, satoh h, shimamoto k. 2002. the hetero stance of rice. proc. natl. acad. sci 99: 13307-12. suma b, keshavachandran k, nybe ev. 2008. mediated transformation and regeneration of ginger ( rosc.). j. trop. agri 46 (1-2): 38-44. takahashi m, uji t, saga n, mikami k. 2010. isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga . electronic j biotechnol 13(2): 1-4. yazawa r, hirono i, aoki t. 2005.characterization of promoter activities of four different japanese flounder promoters in transgenic zebrafish. mar. biotechnol 7: 625-33. yazawa r, hirono i, aoki t. 2006. transgenic zebrafish expressing chicken lysozyme show resistance against bacterial diseases. transgenic res 15: 385-91. yu g, guan h, ioanoviciu as, sikkander sa, thanawiroon c, tobacman jk, toida t linhardt rj. 2002. structural studies on k-carrageenan derived oligosaccharides. carbohydrate res 337: 433-40. yulianto k, mira s. 2009. a study of (doty) culture using the vertical line method and the symtomps of ”ice-ice” disease in pari island waters. oldi 35(3): 323-32. trimeric g protein α subunit acts upstream of the small gtpaserac in disease resi agrobacterium tumefaciens zingiber officinale porphyra yezoensis kappaphycus alvarezii microsoft word 1 biotropia no. 20, 2003: 1-10 synergistic activity of enzymes produced by eupenicillium javanicum and aspergillus niger nrrl 337 on palm oil factory wastes tresnawati purwadaria', noni nirwana 2, pius p. ketaren', dyah iswantini pradono2, and yantyati widyastuti3 ' research institute for animal production, p. o. box 221, bogor 16002, indonesia, 2 department of chemistry, faculty of science and mathematics, bogor agricultural university, jl raya pajajaran bogor, indonesia, 3 research centre for biology-lipi. jl. juanda 18, bogor 16002, indonesia. abstract the use of palm kernel cake (pkc) and palm oil mill effluent (pome), substances from palm oil factory wastes, for monogastric is limited by their high cellulose and mannan contents. hydrolytic enzymes have been supplemented to increase the nutrient digestibility. the maximal digestibility was obtained in the synergistic action of all enzyme components including b-d-endoglucanase (cmcase), b-d-glucosidase, b-d-mannanase, p-d-mannosidase, and oc-dgalactosidase. two kinds of enzymes produced by eupenicillium javanicum and aspergillus niger nrrl 337 on the submerged culture containing 3% coconut meal were selected to hydrolyze pkc or dry pome. enzyme from e. javanicum contained higher cmcase, b-d-mannanase, and a-d-galactosidase activities, while that from a. niger nrrl 337 contained more p-d-glucosidase and p-d-mannosidase activities. saccharification (hydrolytic) activities of enzyme mixtures on pkc and pome were determined at ph 5.0, the optimal ph for p-d-mannanase from e. javanicum, and at 5.4 the optimal ph for a-d-galactosidase from e. javanicum and p-d-glucosidase from a. niger nrrl 337. the enzyme proportions of e. javanicum and a. niger nrrl 337 were 100 : 0, 80 : 20, 60 : 40, 40 : 60, and 0 : 100%. the highest saccharification activity on both substrates was observed on the mixture of 80% a. niger nrrl 337. the ph levels did not significantly affect saccharification activity. fiber components in pkc were more digestable than in pome. further analysis on the reducing sugar components using thin layer chromatography showed that more monomers were produced in the 60 or 80% of a. niger nrrl 337. the glycosidases of a. niger nrrl 337 played more important role in the saccharification activity. keywords: synergistic activity/ palm kernel cake/palm oil mill effluent/ eupenicillium javanicum/ aspergillus niger nrrl 337 introduction feeding costs account for 65-70% of the total cost of animal production. therefore, it is important that most part of the feed can be utilized. feeding of commercial chickens in indonesia usually follows the formula applied in their original country (united states). normally, the feed contains corn, soybean meal, and fishmeal. these feedstuffs are not abundant in indonesia and have to be imported. on the other hand, rice bran, coconut meal, and palm oil factory wastes: palm kernel cake (pkc) and palm oil mill effluent (pome) are available. pkc is a residue of the oil extraction of palm fruit and constitutes up to 45% of palm oil bunches, while pome is the sludge of crude palm oil process and amounted to 2% biotropia no. 20, 2003 of palm oil bunches. the fiber content of pkc exceeding 20% could occur if shells and fruit fiber are not removed in the extraction process and over its half part is neutral detergent fiber (ndf) containing galactomannan and mannan (swick and tan 1995). the crude fiber (cellulose and lignin) of steamed pkc was 21.7% (supriyati et al. 1998). the cellulose, hemicellulose (galactomannan and mannan), and lignin of steamed pome were 17.3, 22.0, and 15.8% (purwadaria et al. 1998). supplementation of mannanase (galactomannanase) enzyme complex to improve the nutritional value is potential for improving feed quality. the components of the enzymes are pd-endoglucanase (cmcase), p-d-glucosidase, p-d-mannanase, p-d-mannosidase, and a-dgalactosidase. eupenicillium javanicum isolated from palm seed (purwadaria et al. 1994) produced less p-d-glucosidase and more p-d-mannanase, p-d-mannosidase, and a-dgalactosidase than aspergillus niger nrrl 337 known as mannanolytic fungus (araujo and ward 1990; haryati et al. 1997). the different activities of the enzyme component open up the possibilities of using the enzyme mixtures to improve the saccharification (hydrolysis) activity in pkc and pome. the synergistic effect using enzyme mixture of trichoderma viride and a. ustus on alkali treated bagasse had been reported (manonmani and sreekantiah 1987). the saccharification on the substrate was 63% when using enzyme mixture of 1:1, while when individual enzymes were used 13.5 and 22.9%, respectively. the information on optimal mixture of such enzyme composition will be useful in enzyme application for animal feeding. the objective of the present study is to determine the synergistic activity of enzymes produced by e. javanicum and a. niger nrrl 337 to hydrolyze pkc and pome. materials and methods palm kernel cake (pkc) and palm oil mill effluent (pome) pkc was obtained from palm oil factory by solvent extraction, while pome was obtained from the centrifugation of palm oil mill effluent. after centrifugation pome was dried under the sun. both dry materials were ground to a fine powder (0.5 mm) using wiley mill. enzyme production enzymes were produced by e. javanicum (riap collection) or a. niger nrrl 337 in the medium containing yeast extract 3g/l, coconut meal 30g/l and minerals in g/1 (nh4)2so4 1.4, kh2po4 2.0, mgso4 0.3, urea 0.3, and cacl2 0.3 and in ppm feso4 5, mnso4 16, znso4 14, and cocl2 20. molds were cultivated in 50 ml medium in a 250 ml flask at 29°c using reciprocal shaker (150 rpm), after inoculation with 2 ml of spore suspension (5 x 1013 and 30 x 1013/ml for e. javanicum and a. niger, respectively) from five-day pda culture slant. the synergistic activity of enzymes on oil palm factory wastes — tresnawati purwadaria et al incubation time for e. javanicum was five days, while that for a. niger was six days (haryati et al. 1997). sodium azide was added at 0.2% final concentration. the culture was then centrifuged (12000 rpm, 20 min, 4°c) and supernatant was collected for enzyme assays and saccharification. enzyme activities the activity of carboxymethylcellulase (cmcase) and β-d-mannanase were assayed by determining the reducing sugars produced from cmc and gum locust bean (mannan) as glucose or mannose, respectively (haggett et al. 1979; araujo and ward 1990). one unit was defined as enzyme which liberates one p.mol glucose or mannose per minute. the glycosidase activities (β-dmannosidase, β-d-glucosidase, and α-d-galactosidase) were assayed using nitrophenyl glycosides as substrates and one unit was defined as enzyme which liberates one umol nitrophenol per minute (ide et al. 1983). specific activity of all enzymes was calculated in unit/mg extracellular protein. determination of protein and fiber component concentration protein concentration was determined by bradford method (1976) and bovine serum albumin was used as a standard. the concentrations of fiber components (cellulose, hemicellulose, lignin and silica) were calculated as neutral and acid dietary fiber according van soest and robertson (1968). determination of optimum ph and temperature the activities of both enzymes were determined at 50°c at different ph (4.6, 5.0, 5.4, 5.8, and 6.2) to obtain the optimum ph, while for determination of the optimum temperature, the enzyme assays were carried out at maximum ph and different temperatures (35, 40, 45, 50, 55, and 60°c). saccharification activity towards pkc and dry pome saccharification activities were determined following determination of avicelase (haggett et al. 1979) using pkc and dry pome as substrates. the incubation time of the reaction was two hours and reducing sugars produced was determined with dns method (miller 1959). the activity value was expressed in unol glucose/ml liberated in one minute. this reaction was also used to determine the synergistic activity of the enzyme mixtures. several proportions of enzymes from e. javanicum and a. niger nrrl 337 (0:100, 20:80, 40:60, 80:20, and 100:0%) were used. aside from the concentration of reducing sugar produced in the reaction, the sugar components of the product were also determined using thin layer chromatography (lestari et al. 2001). the samples and standards (glucose, cellobiose, mannose, mannobiose, and biotropia no. 20, 2003 galactose) were spotted on plates of silica gel 60 (20 x 20 cm). the reducing sugar concentrations of spots from samples were 3-8 чg, while the concentration of each standard was 8 чg. elution was carried out using the mixture of ethanol, n-propanol and water (30:150:20). the elution was stopped when the solvent reached 2 cm from the top. the gel was then dried, and the elution was repeated to get better resolution. spots were detected by spraying the plates by a mixture of aniline (1 ml), diphenylamine (1 g), 80% h3po4 (7.5 ml) in 50 ml acetone and heated at 100°c for 1 hour. results and discussion the activities of enzyme components (cmcase, (β-d-mannanase, (β-d-manno-sidase, β-dglucosidase, and α-d-galactosidase) involved in the fiber hydrolysis were determined (table 1). the enzymes produced from e. javanicwn contained higher activities of cmcase, β-dmannanase, and a-d-galactosidase, while those produced by a. niger contained higher β-dmannosidase and p-d-glucosidase activities. it is already known that aspergillus spp. produce more glycosidases (ghose et al. 1985; manonmani and sreekantiah 1987; haryati et al. 1997). the different major enzyme components of both enzymes suggested the possibility of combination between enzymes. table 1. specific activities of cmcase, p-d-mannanase, p-d-mannosidase, p-d-glucosidase, and a-d-galactosidase of enzymes pr oduced by e. javanicum and a. niger nrrl 337 grown on coconut meal. specific activities (u/mg) molds [protein] cmcase (ug/ml) p-d-man nanase p-dgluco sidase a-dgalacto sidase p-d manno sidase e. javanicum 435 18.6 1172.2 1.5 16.8 1.1 a. niger nrrl 337 498 9.9 14.1 3.1 2.3 3.3 the optimal synergistic activity was obtained when the optimal ph and temperature conditions were applied. the optimal ph and temperature of e. javanicum β-d-mannanase and αd-galactosidase were at 5.4 and 50°c, and 5.0 and 55°c respectively, while those of a. niger βd-glucosidase were at 5.0 and 55°c (figures 1 and 2). the temperature difference gave less effect than the ph difference. a temperature increase from 50 to 55°c reduced β-d-mannanase activity at 22.7%, while the temperature reduction from 55 to 50°c reduced activities of α-d synergistic activity of enzymes on oil palm factory wastes tresnawati purwadaria et al. galactosidase and (β-d-glucosidase at 11.3 and 10.9%, respectively. an increase of ph from 5.0 to 5.4 increased 39.5% of the β-d-mannanase activity, and reduced 24.9 and 13.5% α-dgalactosidase and β-d-glucosidase, respectively. further analyses at these conditions were used to determine saccharification activity towards pkc and pome (table 2). biotropia no. 20,2003 enzymes produced by e. javanicum showed higher saccharification activity towards pkc or pome than those from a. niger due to high activities of e. javanicum cmcase, β-dmannanase and α-d-galactosidase in hydrolyzing cellulose and hemicellulose compounds. the hydrolysis activity upon pkc was also higher than pome which contains higher fiber and minerals. the cellulose and lignin contents of pkc were 14.2 and 20.5%, respectively, while those of pome were 20.8 and 25.6%. the hemicellulose (mannan and galactomannan) contents of pkc and pome were almost similar, being 5.3 and 5.6%, respectively. the microenvironment of ph influenced more significantly saccharification activity than the temperature, therefore the synergism activities towards pkc and pome were carried out at ph 5.0 and 5.4 which were the optimum ph for glycosidases and β-d-mannanase, respectively. both ph conditions were applied at 50°c. all mixtures of enzymes at ph 5.0 or 5.4 showed the synergistic saccharification either towards pkc or pome (figures 3 and 4). the highest synergistic action of the mixture was obtained when higher volumes of a. niger (60 or 80%) were used. for example, in the mixture of 80% a. niger the hydrolysis upon pome at ph 5.0 produced the reducing sugar at 162 uj/ml (figure 4a). without synergistic activity the reducing sugars produced from the 80% of a. niger (86.4 uj/ml) and 20% of e. javanicum (17.8 uj/ml) would be 104.2 uj/ml. the synergistic activity increased the reducing sugars up to 55%. the better activity in the higher a. niger rations indicated that its β-d-glucosidase and β-d-mannosidase play more important role in the hydrolysis system. it was already known that the digestion of disaccharides (cellobiose and mannobiose) by glycosidases reduced the feed back inhibition effects on endoglucanase and endomannanase. it was reported that the highest synergistic action in the culture mixture of t. reesei d-16 and a. went// pt 2804 to produce cellulase and hemicellulase was also observed at composition of 1 (t. reesei): 4 (a. wentii) (ghoseetal. 1985). the optimum ph of e. javanicum β-d-mannanase and α-d-galactosidase were at 5.4 and 5.0, respectively, while that of a. niger β-d-glucosidase was at 5.0 (figure 1). which of the enzyme component had more important role in the synergistic activity was not clearly indicated by varying ph conditions. all ph conditions and substrates produced similar pattern and showed the highest synergistic activity at 60 or 80% a. niger application. moreover, considering the reducing synergistic activity of enzymes on oil palm factory wastes tresnawati purwadaria et a 0:100 20:80 40:60 60:40 80.20 100:0 a. niger : e. javanicum (%) 0:100 20:80 40:60 60:40 80:20 1 a. niger : e. javanicum (%) figure3. synergistic saccharification towards pkc by enzyme mixtures of a. niger and e. javanicum at ph 5.0 (a) and 5.4 (b). the dotted lines represent the theoretical reducing sugar values expected for a non-synergistic saccharification. 0:100 20:80 40:60 60:40 80:20 100:0 a. niger : e. javanicum (%) 0:100 20:80 40:60 60:40 80:20 100:0 a. niger : e. javanicum (%) fiaure4. syncrmstic saccharification towards pome by enzyme mixtures of a. niger and e. javanicum at ph~5.0 (a) and 5.4 (b). the dotted lines represent the theoretical reducing sugar values expected for a non-synergistic saccharification. sugar produced by pkc and pome at ph 5.4 by 100% a. niger (143 jg/ml and 130 uj/ml, respectively) was higher than that of 100% e. javanicum (78 uj/ml and 69 fg/ml, respectively), then the proportion of a. niger should be higher than 80% on pkc and 60% on pome (figures 3 and 4). although the optimum ph of a. niger β-d-glucosidase was at 5.0, only saccharification upon pome showed a better synergy at ph 5.0, while that upon pkc was better at ph 5.4. the obtained result might be due to the high mineral concentration in pome that influenced the activity of each enzyme component differently. the ash and silica contents of pkc were 8.5 and 1.7%, respectively, while those of pome were 17.6 and 9.5%. the high mineral or cation contents in pome might have influenced the buffering capacity of the substrate and affected the ph in the reaction. biotropia no. 20,2003 the role of every enzyme component in the synergistic action ws difficult to be detected since all enzyme components took part in the reaction. detailed successive reaction in the synergistic action had been clearly observed in the pure enzyme components (purwadaria 1995). however, results from tlc plates indicated that different concentrations of sugar components were produced on pkc and pome by the hydrolysis action of the mixtures of e. javanictan and a, niger (figure 5). k5 85 ke s« figure 5. thin layer chromatogram of reducing sugars produced from synergistic sachanfication of e. javanicum and a. niger at ph 5.0. saccharification activities were determined towards pkc (a) and pome (b). glucose (gi), cellobiose (gi), mannose (mi), mannobiose (m2), and galactose (ga) were used as reference. the concentration of each standard was 8 \%. ki, kj, k3, kj, k5, and k,, were controls without incubation, while si, s2, s3, s4, s5, and s6 were samples from the composition of a. niger : e. javanicum at 0:100, 20:80,40:60, 60:40, 80:20, and 100:0%. the chromatogram of pkc digestion was clearer than that of pome due to its higher mineral content which had disturbed the separation (figure 5). the reducing sugars of controls produced from the mixture of enzymes and substrate without incubation were compared with samples from the mixture of enzymes and substrate with incubation. smaller amounts of reducing sugars were detected from the controls (substrates) compared to the samples. oligosaccharides (trimers and more) resulted from endoglucanase or endomannanase activities especially by higher composition of e. javanicum in the incubated samples (figure 5a-s], 82, and 83) were much more than those of controls. the oligosaccharides produced were further digested by glycosidases of e. javanicum and the addition of glycosidases from a. niger (figure 5a and b). addition of 60 or 80% a. niger to the enzyme mixtures produced more monomers and dimers including mannose, glucose, galactose, and cellobiose. the possibility of higher hydrolysis activity resulted from synergistic action will contribute a beneficial effect in the enzyme application on monogastric. the synergistic activity of enzymes on oil palm factory wastes ~ trtsnawati purwadaria tl at, incorporation of carbohydratases known as inducer enzymes is inhibited by the feed soluble sugar content prepared for energy source. the addition of glycosidases produced by a. niger (β-dglucosidase and β-d-mannosidase) on top of α-d-galactosidase from e.javanicum might have increased the digestion of the dimmers such as cellobiose and mannobiose that reduced the inhibition effect of the dimmers (short olygosaccharides) on endoglucanase and endomannanase. therefore, the application of enzyme cocktails in animal di»ts is considered to be more appropriate than single enzyme. references araujo, a. & , o. p. ward. 1990. extracellular mannanases and ;.alactanases from selected fungi. j. ind. microbiol. 6:171178. bradford, mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254. ghose, t.k., t panda, and v. s, bissau. 1985. effect if culture phasing and mannanase on production of cellulose and hemicellulase by mixed culture ot trichoderma reesei d 1-6 and aspergillus wendtii pt 2804. biotechnol. bioeng. 25: 1353-1361. haggett, k.d., p.p. gray and n.w dunn. 1979. crystalline cellulose degradation by a strain of cellulomonas and its mutants derivatives. eur. j. appl. microb. biotechnol. 8: 183-190. haryati, t, t. purwadaria, j. darma, and b. tangendjaja, 1997. production of extracellular glycosidase by eupenicillium javanicum and aspergillus niger nrrl 337 on the coconut meal substrate. proc. second conf. on agricultural biotechnology, jakarta, indonesia, 13-15 juni 1995.aard, indonesia, p. 517-522 ide, j.a., j. m. daly, and p.a.d. rickard. 1983. production of glycosidase activity by cellulomonas during growth on various carbohydrate substrate. eur. j. appl. microb. biotechnol. 18: 100-102. lestari, p., a. a. danvis, k. syamsu, n. richana, and d.s. damardjati. 2001. analisis gula reduksi hasil hidrolisis enzimatik ubi kayu oleh a-amylase termostabil dari bacillus stearothermophilus til 12 (reducing sugar analyses on the enzymatic hydrolytic product of cassava by thermostabil a-amylase from bacillus stearothermophilus til 12). j. mikrobiol. indon. 6: 23-26. manonmani, h.k. and k.r. sreekantiah. 1987. saccharification of sugar cane bagasse with enzymes from aspergillus ustus and trichoderma vinde. enzyme microb. technol. 9: 484-488. miller, g.l. 1959. use of dinitrosalicylic acid reagent for determination of reducing sugar. anal. chem. 31:426-428. purwadaria, t. 1995. synergism in the hydrolysis of cellulose by endoglucanase i and ii (endo 1 and ii) and cellobiohydrolase (cbh i) purified from cellulomonas cs1-17. annalcs boaoricnses. 3: 12-24. purwadaria, t., a.p. sinurat, t. haryati, i. sutikno, supriyati and j. darma. 1998. korelasi antara aktivitas enzim mananase dan selulase terhadap kadar serat lumpur sawit hasil fermentasi dengan aspergillus niger (the correlation between mannanase and cellulose activities towards fibre content of palm oil sludge fermented with aspergillus niger). jitv 3: 230-236. purwadaria. t., t. haryati, and j. darma. 1994. isolasi dan seleksi kapang mesofilik penghasil mananase (isolation and selection of mesophylic molds producing mannanases). ilmu & peternakan 7(2): 26-29. biotropia no. 20,2003 supriyati, t. pasaribu, h. hamid, and a.p. sinurat. 1998. fermentasi bungkil inti sawit secara substrat padat dengan menggunakan aspergillus niger (solid substrate fermentation of palm kernel cake using aspergillus niger). jitv 3: 165-170. swick, r.a. and p.h. tan. 1995. considerations in using common asian protein meals. tech. bull. asa mita(p) no. 083/12/94. vol. po25 van soest, p.j. and j.b. robertson. 1968. system of analysis for evaluating fibrous feeds. in w.j. pigden ed. standardization of analytical methodology for feed. cent. canada, idrc. 134e. 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf biomass and carbon stock estimation inventory of indonesian bananas ( spp.) and musa its potential for nrole land rehabilitatio setyawan agung danarto* lia hapsariand purwodadi botanic garden – indonesian institute of sciences, jalan surabaya-malang km. 65 purwodadi, pasuruan, east java 6716, indonesia received 24 february 2014/accepted 30 november 2015 abstract bananas ( spp.) are widely cultivated in indonesia. they are extensively grown in backyards, home gardens, musa intercropped with short term crops and also in agroforestry sy . stem the potential of bananas to sequester carbon has been reported but there is limited knowledge on the performance of various cultivars. an inventory of biomass and c-stock estimation on banana accessions has been conducted in germplasm plots purwodadi botanic garden, musa pasuruan. estimation on biomass and c-stock have been conducted for 42 individual banana accessions, comprised 5 wild banana species and 37 cultivars using non-destructive method i.e. allometric equation for banana. the objectives ere conduct inventory on the biomass and c-stock estimation of indonesian bananas in of this study w to germplasm collection of purwodadi botanic garden, to make the projections of time average above ground for cstock of banana farming system in indonesia and to discuss the potential role of bananas in land rehabilitation. the results showed that estimation value of biomass and c-stock varied from one to another. wild banana accession musa balbisiana musa acuminataspecies had higher biomass and c-stock value than wild species. banana cultivars containing one or two “b” genome (abb and aab) were more vigorous and contributed higher biomass and c-stock than aaa and aa cultivars. among cultivars, the highest c-stock was contributed by pisang kepok bung (average of 6.92 kg c/plant) whereas the lowest c-stock was contributed by pisang rayap (average of 0.67 kg c/plant). in average, various indonesian bananas studied contributed around 2.26 kg c/ or 0.98 tonnes c/ha. plant the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014, contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 with a decrease to 98.97 tonnes c in 2014. these numbers are still limited only to the recorded areas. anana plants in combination with b woody tree crops, are potential as important component of agroforestry r and mixed farming systems , home ga dens to rehabilitate and reforest landscape, to decrease carbon emission in atmosphere in the form of biomass and cstocks and to meet the economic needs for local surrounding community. keywords: banan stock estimation, purwodadi a, biomass, carbon (c-stock), , , musa acuminata musa balbisiana botanic garden r, ehabilitation introduction climate change is environmental issue an that has been discussed limate change is always . c caused by energy absorbed from sun as short wave then reflected in atmosphere as infrared long wave radiation. greenhouse gas absorb es effect s infrared radiation which is retained in atmosphere as heat energy causing the in of the earth's crease temperature, , efforts mitigate therefore to greenhouse gas needed saharjo & es are ( wardhana 2011 . world bank stated that ) in 2003, the co about 2earth's concentration was 27 billion metric ton, which a 9% increase from was 1 the year ipcc noted that 1990. (2014) the earth's temperature 1880 to 2012 showed from an increas from 0 65 c to 1 06 c. tudy by o oe . . a s ollivier (2014) reported countries et al. that contributing large amount of co in 2a atmosphere re china (29%), (15%), we usa european union (11%) india 4 4% , brazil , ( . ) ( . ) ( . the forests 6 2% and indonesia 2 3%). peat and fire in indonesia responsible for s were estimated the released of 2 co emission to the atmosphere a b o u t c t o c0 . 8 1 g t 2 . 5 7 g t in 1997 (page 2002). particular in borneo et al.* corresponding author : setyawan.10535@gmail.com biotropia vol. 22 no. 2, 2015: 102 108 doi: 10.11598/btb.2015.22.2.376 102 mailto:setyawan.10535@gmail.com island, the annual average carbon emission from forest fires was estimated about 0.02 to 0.06 gt c per year (kuntoro . 2015)et al . process amount 2 of decreasing co in the atmosphere through the process of plant photosynthesis is called c sequestration. in arbon the photosynthetic process, co in the 2 atmosphere a by plant, transformed to is bsorbed carbohydrate compound dispersed to alls and parts of plant. carbon sequestration describes long-term storage of co or other forms of 2 carbon to either mitigate or defer global warming and avoid dangerous climate change. it has been proposed as a way to slow the atmospheric greenhouse gases, which are released by burning fossil fuels (hairiah & rahayu 2007). therefore, it is important to conduct the study to examine ability of plant to a carbon, different species bsorb especially in the climate change mitigation efforts. agroforestry system is cultivation a practice combining trees and annual crop or other farm activities adopted by smallholders to meet their needs for food, medicine, timber, fuel, fodder and market commodities provides valuable . it also environmental services such as soil fertility replenishment, water catchment protection, carbon sequestration, conservation biodiversity and landscape restoration (garrity 2004) bananas . ( spp.) are extensively grown in backyards, musa home gardens, agroforestry system and are intercropped with short term crops. anana b s are the favorite s grown most plant in agroforestry system intercropped with other imp rtant tree o crops commodities (coffee, cacao, rubber), fruit trees timber bout 9 5% and . a . of bananas species occur in home gardens system contributing the to ecosystem services individual . only a handful of smallholder agroforestry systems store small amount of carbon per area basi , while the c systems store as much arbon as secondary c several forests (roshetko 2002). et al. the potential of bananas to sequester carbon has been reported by daphine (2014) on east african highland bananas, but there is limited knowledge on the performance of various cultivars in indonesia. being part of the primary center of origin and diversity so that has large number of bananas (musaceae) both wild seeded species and edible seedless or cultivar with many local names and synonimies (espino 1992; et al. valmayor . 2000)et al . there is no less than 200 local cultivars cultivated and developed across indonesia archipelago (nasution & yamada 2001). objectives erethe of this study w to conduct inventory on the biomass and c-stock estimation of indonesian bananas in germplasm collection of purwodadi botanic garden, to make the projections of time average above ground for c-stock of banana farming system in indonesia and to discuss the potential role of bananas in land rehabilitation. materials and methods study site the was conducted at banana collection study plots of purwodadi botanic garden – indonesian institute of sciences from . april to may 2012 purwodadi botanic garden collect has a ion of musa germplasm both wild and cultivated species varieties o ethr ugh exploration, plant exchang , grants community or personal contribution and from several regions all over indonesia, mostly from eastern indonesia. current collections in 201 accessions comprise 7 wild 4 is about 134 d species 127 cultivated varieties.and materials the materials were 4 selected banana studied 2 accessions of purwodadi botanic garden co lections comprise 5 wild species and 3 l d 7 cultivars. the diameter at breast height (dbh) of the i o wasndividual pseud stem measured at mature age (already flowering) using tape meter with of replicationsminimum two per accession. as reported by daphine (2014) that c-stock of banana plants was significantly influenced by growth stages in which maturity stage was the optimal stage to be measured (daphine 2014). biomass and c-stock estimation biomass was estimated using non-destructive method i e. allometric equation for banana . ( ) and then -stock was kurniawan . 2010 c et al estima e by its mass tot d crossing bio average value of in plants which is 0.46 (hairiah c-stock et al. 2010). the development of allometric equations has been investigated specific , based on condition species and or communit (ketterings plant ies et al. 2001; wibowo 2010):et al. biotropia vol. 22 no. 2, 2015 103 y = 0.0303 x d2.1345 z= y x 0.46 w :here y = plant biomass ( g)k d = diameter at breast high (cm) z = c-stock (kg c/plant). results and discussion biomass in was affected by interaction plants of genetic and environmental factor. in this study, the purwodadi botanic garden provides homogenous environmental condition, i.e. soil type p and . , water su plies culture practices therefore, the biomass results were mostly affected by its genetic factor. results showed the that the estimation value of biomass and c-stock tended to vary from one to banana accession another. pseudostem dbh was confirmed as the best predictor for biomass estimation in banana plants and it is recommended to be used in most carbon related studies. the more vigorous banana plants estimation value of contribute higher biomass and c-stock . (fig. 1) c stock related to photosynthesislevel is plant process its. based on photosynthesis pathway, banana c3 plant. in c3 plants are classified as plants 2, co and water from the environment are enzymatically combined with a five-carbon acceptor molecule to contribute two molecules of a three-carbon intermediate. c3 plants respond favorably to higher concentrations of carbon dioxide than c4 and cam. c4 plants include corn, sugar cane and many other tropical grasses, whereas cam plants include orchids, bromeliad and succulent plants (taiz & zeiger 2002). biomass and c-stock inventory results in wild banana species wild bananas are pioneer plants and can grow in various conditions. it commonly grows wild in the forests, road sides and river banks musa . wild balbisiana species is also being cultivated by farmers to get the leaves for various wrapping purposes he immature fruits also edible for . t are any traditional side dishes ild is . w musa acuminata rarely cultivated. there are also some other species of bananas (not studied here) such as musa velutina musa ornata musa borneensis , , etc. that are commonly cultivated for ornamentals due to its beautiful performances nasution yamada ( & 2001; hapsari . 2015a).et al among the wild studied, species musa balbisiana much species contributes higher level of biomass and c-stock estimation than musa acuminata musa balbisianaspecies (table 1). is considered to be more vigorous robust and , as well as , while musa drought and disease resistant acuminata species is slender but has attractive more morphology .et al(daniells 2001; nasution & yamada 2001). species (pisang musa balbisiana figure relation of pseudostem dbh to biomass and c-stock 1 positive cor values in banana plants 104 biomass nd carbon stock estimation inventory f ndonesian bananas ( spp.)a o i danarto and hapsarimusa – klutuk wulung) has pseudostem diameterlarge ( ) contribut21.47 cm ing around 21.09 kg/plant biomass and around 9.7 kg/plant c-stock. m . acuminata utilifes. r var with slender pseudostem (4 99 cm) contributed 0 94 . around . kg/plant biomass around . kg c/plant c-stock and 0 43 (table 1). biomass and c-stock inventory results in various banana cultivars edible banana cultivars biomass have lower and c-stock s than bananas. the genetic value wild composition of is the combination musa balbisiana of wild (donor a genome) and musa acuminata musa balbisiana (donor b genome). genomic composition can be identified using morphology (jumari & pudjoarinto 2000) and genetic (hapsari et al. 2015b). banana cultivars containing one or two “b” genome (abb and aab cultivars) are more vigorous and contribute higher biomass and c-stock than the aaa and aa cultivars. pisang the kepok bung (abb) is most vigorous cultivar with pseudostem diameter of 18.32 cm ing around 15.04 kg/plant contribut biomass and around 6.92 kg c/plant c stock. pisang slender cultivarrayap (aa) is the most with pseudostem diameter of 6.14 cm contribut ing around 1.46 kg/plant biomass and around 0.67 kg c/plant c stock. pisang triolin having aab genome biomass and contributed c-stock values in between pisang kepok bung (abb) and pisang rayap (aa) (f . . the ig 2) average value of c-stock contributed by banana plants per genome group from the highest to the lowest as follows: bb wild (7.22 kg values are c/plant), abb cultivars (2.74 kg c/plant), aab cultivars (2.11 kg c/plant), aaa cultivars (1.73 kg c/plant), aa cultivars (1.55 kg c/plant) and aa wild (0.72 kg c/plant). in traditional home gardens and in agroforestry, farmers plant various local cultivars. however, the commercial scale farmers plant bananas cultivars based on consumers' preference and agroclimate condition in an area. pisang kepok (abb) is the most favorite cultivar to be cooked, while pisang raja (aab), pisang ambon (aaa) and pisang mas (aa) are often processed for dessert. wild species and cultivars studied contributed an average of 2.26 kg c/plant cstock. t a in banana fime verage c-stock arming s ystem in indonesia banana s farming system recognize 3 different planting distance based on its canopy size s s, i.e. 6 x 6 m for wide canopy, 5 x 5 m for medium canopy and 4 x 4 m for small canopy (cahyono 1996). banana plants may contribute an average of 0.98 c tonnes/ha c-stock. this number is quite high if compared to c-stock contributed by understory of agroforestry system which only contributed 0.2-0.3 c tonnes/ha. however, agroforestr y of coffea plants contributed higher c-stock than banana plants, i.e. 2.0-12.0 c tonnes/ha (forda 2010). the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014 (ministry of agriculture 2015), contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 with a decrease to 98.97 tonnes c in 2014. these numbers are still limited only to the recorded areas. potential role of banana plants for land rehabilitation tropical forests in southeast asia are constantly changing as a result of and logging land such asconversion logging activities, complete deforestation conversion from forest , to grassland or annual crops, tree plantations and other woody perennial crops (lasco 2002; monde 2009). those vast area of degraded land are in need of rehabilitation. agroforestry system may become approach prevent deforestation by an to biotropia vol. 22 no. 2, 2015 table estimation 1 pseudostem dbh, biomass and carbon stock of wild banana species species local name pseudostem dbh (cm) biomass (kg/plant) c-stock (kg c/plant) musa balbisiana klutuk wulung 21.47 21.09 9.70 musa balbisiana klutuk ijo 15.35 10.30 4.74 musa acuminata var. alasensis pisang hutan 6.78 1.80 0.83 musa acuminata var. rutilifes pisang cici hutan 4.99 0.94 0.43 musa acuminata var. tomentosa unti darek 7.07 1.97 0.91 105 figure pseudostem dbh, biomass and c-stock of various indonesian banana cultivars 2 estimation 106 providing on-farm source trees s. agroforestry system provides better carbon storage than the usual annual crops farming system because agroforestry system intercropped trees with annual crops, continuously giving much higher biomass and litters in varied quality (utami 2003).et al. home garden as smaller level of agroforestry is species rich and tree-based system producing wood and non-wood products and therefore, producing high biomass. due to high biomass produced, this system potentially offers carbon storage. in terms of aboveground biomass, home garden contains more carbon per hectare than grasslands, cassava fields and imperata young rubber agroforestry (roshetko . et al 2002). biomass nd carbon stock estimation inventory f ndonesian bananas ( spp.)a o i danarto and hapsarimusa – biotropia vol. 22 no. 2, 2015 107 table 2 ime average c-stock in indonesia banana farming systems t in 2000-2014 planting distance number of plants/ha c-stock (c kg/ha) time average c-stock contribution year 2000 (c tonnes) year 2010 (c tonnes) year 2014 (c tonnes) wide canopy 6 x 6 m 278 628.67 46.23 64.01 63.24 medium canopy 5 x 5 m 400 905.29 66.57 92.18 91.07 narrow canopy 4 x 4 m 625 1,414.52 104.02 144.03 142.30 average 434 982.83 72.28 100.07 98.87 banana as a component of mixed agroforestry system has moderate c-stock contribution. this study showed that one hectare of banana plants store more carbon (0.98 tonnes) than cassava (0.5 tonnes) and grassland (0.7 tonnes) imperata (hairiah 1997). banana plants store much less carbon than perennial woody plants or trees, however, banana has high economic value and provide shades to tree crops commodities (coffee, cacao, rubber), fruit and timber (roshetko . 2002). also, et al banana plants produce fruit all year round which continuously contribute food to smallholder farmers in the area (hapsari 2011). the role of agroforestry in absorbing co as 2 well as in storing and maintaining carbon stocks is lower than that of natural forests, but this system can increase carbon stocks on degraded lands (widianto . 2003)et al . conclusions banana accessions contributed varied estimated per value of biomass and c-stock accessions depend on their characteristic ing s performance. pseudostem dbh was confirmed as the best predictor for biomass estimation in banana plants and it is recommended to be used in most carbon related studies. the more vigorous banana plants estimation value contribute higher of biomass and c-stock the c-stock value . ranged from / / . 0.67 kg c plant to 6.92 kg c plant in average, various indonesian bananas studied contributed around 2.26 kg c/ or 0.98 plant tonnes c/ha. the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014, contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 and decreasing to 98.97 tonnes c in 2014. agroforestry is species rich and tree-based system producing wood and non-wood products and therefore, producing high biomass. due to high biomass produced, this system potentially offers carbon storage. anana plants in combination b with woody tree crops, are potential as important component of agroforestry r and , home ga dens mixed farming systems to rehabilitate and reforest landscape, to decrease carbon emission in atmosphere in the form of biomass and c-stocks and to meet the economic needs for local surrounding community. acknowledgements the authors would like to acknowledge ahmad masrum and lamiran for their technical guidance during the study observation in banana collection purwodadi botanic garden. field of references cahyono b. 1996. . pisang (budidaya dan nalisis saha ani)a u t yogyakarta (id): kanisius. p 80. daniells j, jenny c, karamura d tomekpe k. 2001. , musalogue: a atalogue of musa ermplasm. diversity in c g the enus musag . montpellier (fr): international network for the improvement of banana and plantain (inibap). the international plant genetic resources institute (ipgri). daphine k. 2014. arbon equestration otential f ast c s p o e a h b c i ufrican ighland anana ultivars n ganda. dissertation. 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(icraf) sea regional office. 39 .p http://aplikasi.pertanian.go.id/bdsp/hasil_ko microsoft word 65 biotropia no. 19, 2002 : 65 84 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo dept. of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor and south east asian regional center for tropical biology (seameo biotrop) p.o. box 116, bogor, indonesia. abstract an account of the tribe composition, endemic taxa, comparison with adjacent areas and weedy asteraceae of sumatera is given. based on the records of january 2000, there are 133 species of 74 genera in 11 tribes. the tribe heliantheae is the largest, with 28% of the total number of the genera, followed by astereae with 15%, inuleae 12%, senecioneae 10%, anthemideae, eupatorieae and lactuceae 8%, the other tribes are represented by 4% or less. the most diverse genus is blumea with 14 species. other genera are only represented by 10 species or less, usually 4, or 3, or 2, and mostly by 1 species only. thirty nine or about 53% are exotic genera and the native ones are less than half of the total number of the genera. in terms of indigenous and endemic species, sumatera is richer than java. there are 1 genus, 7 species and 2 varieties of asteraceae endemic to sumatera. a number of 43 important weed species were introduced from tropical america, africa, asia and europe. among these chromolaena odorata and mikania micrantha are reported as the most noxious ones. list of the genera and species recorded in sumatera is provided in this paper. key words : asteraceae/sumatera/compositions/endemic species/distribution/weedy asteraceae introduction asteraceae is one of the largest families of flowering plants which has not been revised for the flora malesiana (ross 1993). this paper is a report on the results of the study on the asteraceae of sumatera based on the records up to 2002. fundamental work of this family was done by cassini (1817,1818, 1826-1834), and bentham (1873) and bentham & hooker (1873). bentham's tribal classification has stood the test of time, and some modifications were introduced by hoffmann (1890-1894), dalla tore and harms (1907) and melchior (1964). although the 13 tribes recognized by bentham & hoffman have been largely accepted up to the present, they are obviously in need of modification considering recent discoveries in biochemistry, palynology analysis, micromorphology, anatomy, cytology, micromolecular chemistry, and semantide analysis especially cpdna. cladistic method has been widely applied, subsequent more precise generic, and tribal concepts have been developed. it has become clear that not only quite a number of genera have been misplaced, but others require a transfer to other tribes (jeffrey 1995). 65 biotropia no. 19,2002 miquel (1856) made the first revision of the asteraceae of the malay archipelago followed by van hasselt & boerlage (1884), and boerlage (1891) gave an enumeration of the species. the latest partial revision of the asteraceae of the malay archipelago was made by koster, who elaborated the tribes eupatorieae and vernonieae (koster 1935, 1941, 1948, 1952, 1953, 1958). she treated 5 species of adenostemma, 2 of ageratum, 1 of centratherum, 2 of elephantopus, 8 of eupa-torium , 2 of ethulia, 1 of mikania. in total, 57 species were described of which 11 species are introduced. she also revised the compositae of java (koster 1965) and new guinea (koster 1966; 1970; 1976; 1979; 1980). van royen (1983) treated compositae component of the alpine flora of new guinea for the species above 3000 m. after these publications, there has been no other comprehensive one on the asteraceae of malesia. the first initial survey of the flora of sumatera began with the publication of jack in 1820. it was followed by several authors e.g. miquel (1856-1861) who published them separately in a number of publications. but after miquel there has been no attempt yet to bring together the vascular flora of sumatera. the only exception is the revision of the euphorbiaceae of sumatera by shaw in 1981. whitmore and tantra (1986) listed only those species with either a bole of at least 35 cm diameter, and/ or 20 m in height, they cited only eight out often species of vernonia reported for sumatera by koster (1935). vernonia cinerea, v. cymosa, v. forbesii, v. patula and v. vagans which were cited by whitmore and tantra as a tree are actually herbs, climbers or shrubs. vernonia is the only genus where four out of the ten species are trees up to 30 m in height: v. arborea, v. durifolia, v. patentissim and v. subdentata, while the other six species of vernonia are herbs or shrubs. other asteraceae genera consist of only herbs and shrubs. miquel (1861) treated 25 species of 20 genera in 7 tribes of asteraceae in sumatera. he described 5 species of blumea, 2 species of artemisia, and 1 species of several genera i.e. adenostemma, asteromoea, conyza, eclipta, elephantopus, emilia, erigeron., gnaphalium, gynura, lagenophora, microglossa, myriactis, sonchus, sphaeranthus, wedelia, vernonia, xanthium and youngia. boerlage (1891) in his enumeration of the asteraceae of sumatera listed 41 species of 28 genera in 8 tribes. he listed 5 species of blumea, 3 species of senecio and vernonia, 2 species of adenostemma, anaphalis, conyza, wedelia, and 1 species of several genera i.e. asteromoea, bidens, cosmos, dichrocephala, eclipta, emilia, enydra, erigeron, microglossa, myriactis, sphaeranthus, spilanthes and xanthium. koster (1935) in her treatment of the tribes eupatorieae and vernonieae in sumatera described 9 species of vernonia, 3 species of adenostemma, 2 species of ageratum and 1 species of elephantopus, mikania and eupatorium . nasution (1984) described 5 species of weedy asteraceae at the plantations of aceh and north sumatera, while soerjani et al (1987) described 25 species of asteraceae in the ricefields of sumatera. in total, there were 69 species of asteraceae in sumatera known by several authors: miquel 1861; boerlage 1891; koster 1935 and soerjani et al. 1987. the records up to december 1998 showed that 66 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo 67 genera and 122 species in 10 tribes of asteraceae in sumatera were recognized (tjitrosoedirdjo 2001). up to january 2000, the author has described 133 species of 74 genera in 11 tribes in sumatera (table 1 and appendix 1). four new taxa were recognized by tjitrosoedirdjo (2002) namely prenanthes steenisii, prenanthes sumatrana, senecio dewildeorum and one variety emilia sonchifolia var. lanceolata. in sumatera, asteraceae has been recorded in a wide variety of habitats including, montane, highlands, lowlands, fmperata-fields, open spaces of the plantations, and agricultural fields. representatives may be found in nearly every type of habitat, but few taxa are present in tropical rain forests, swampy, mangrove and aquatic areas. the species are predominantly perennial herbs and shrubs. materials and methods herbarium specimens of the asteraceae of sumatera and the surrounding islands were studied from several herbaria. the herbarium bogoriense (bo) and biotrop herbarium (biot) served as the major sources. most of the specimens at bo were collected during the colonial times, while those at biot was gathered after 1970's and some specimens were collected by the author. selected specimens were kindly obtained on loan from herbarium of andalas university ('anda') representing taxa from west sumatera and also some from frim (forest research institute malaysia's/kep) were also studied. some specimens of blumea, lactuca, and senecio were provided on loan from the national herbarium of netherlands (the former rijksherbarium:l). additional specimens were collected during the course of this study from aceh, north sumatera, riau, jambi, west sumatera, south sumatera and lampung. data and information on the morphology, habitat, and distribution were collected from the specimens, literature and field observations. results and discussions the tribe composition based on the studies up to january 2000, there are 133 species of 74 genera in 11 tribes of asteraceae in sumatera that have been recorded (table 1 & appendix 1). there is an increase compared to the records as listed in 1998, where 122 species of 67 genera are in 10 tribes (tjitrosoedirdjo 2001). the 11 tribes are anthemideae, astereae, cardueae, eupatorieae, heliantheae, inuleae, lactuceae, mutisieae, senecioneae tageteae, and vernonieae. the tribe cardueae is a new record. thirty-nine genera or about 53% are exotic (table 1), and the native genera are less than half of the total number of the genera. more than 61% of these genera 67 biotropia no. 19,2002 contain only one species, 9% have three species and 5% have 4 species. others such as vernonia and blumea have ten and fourteen species, respectively. three tribes are found in java but not in sumatera: arctoctideae, calenduleae, and helenieae. notes on distribution of selected genera of asteraceae in sumatera anaphalis is a genus with a disjunct range distribution: it has been recorded from europe, asia and north america. in sumatera it is represented by two native montane species. blumea has an african-asiatic-australian range. most of the species are found in malesia with 14 in sumatera which makes it the largest genus in sumatera. the other genera have 1 to 4, rarely up to 10 species. blumeopsis is a monotypic endemic genus of eastern asia. in malesia, it is only found in sumatera (aceh and north sumatera). carpesium is a genus of the northern temperate or subtropical europe through asia to japan. one possibly introduced species has been found in the mountains of aceh. lactuca is a cosmopolitan genus, with three species in sumatera. launaea is a new generic record for sumatera. launea sarmentosa was found during the course of this study at a sandy beach near uelele, aceh. kilian (1997) reported that the species has a scattered distribution from east africa to south china (guangdong). it is found in the andaman nicobars and in thailand. it is also found in northeast australia. in malesia, it is very rare and until now only known notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo autochthonously in malesia with the sumateran collection closing the gap. its occurrence in southwest java and northwest australia concurs with the ancient sites of european sailing ships that carried sand as ballast which had been obtained at beaches in east africa or sri lanka. these initially landed in pelabuhan ratu (wijnkoopsbaai) where ballast was removed, or floundered on the shores of northwest australia thus introducing the launaea. senecio is the largest genus of asteraceae, and a very heterogeneous one. its species are found in all parts of the world. in sumatera, there are two endemic montane species. sonchus is another cosmopolitan and weedy genus. there are three species in sumatera. sonchus oleraceous is commonly found at the mountain regions in java. in sumatera, it is only found in takengon, aceh. taraxacum is a cosmopolitan genus of weedy species and an extremely complex genus. there are few regularly amphimictic species, some that are occasionally amphimictic, but a vast number appear to be exclusively apomictic, often with 69 biotropia no. 19,2002 defective or quite abortive pollen. the apomictic forms are polyploids which have presumably arisen from primary interspesific hybridization. the only species found in sumatera is tentatively named taraxacum javanicum here, but may very well represent an undescribed (micro) species. vernonia is a wide ranging genus with species in tropical america, africa, madagascar. jones (1979; 1981), followed by jeffrey (1988), has suggested that actually two main subgenera, if not genera or even subtribes may be involved, an old world one, vernonia subgen. orbivestus, and satellite taxa, and one from the new world, vernonia s.s. and satellites. if correct, this means that malesian species attributed to sect. lepidaploa actually belongs to a possibjy undescribed section. in sumatera, there are ten species of which two are endemic. endemic taxa and their distribution in sumatera the asteraceae is generally a temperate to subtropical family, yet it forms an important part of the malesian flora. being temperate, it is therefore not surprising that the mountain areas in sumatera are richer in taxa than the lowlands. of the 133 species recorded in sumatera, nearly 50% have been introduced, while there are only 7 endemic species and 2 endemic varieties (table 1). these montane endemics are temperate genera and it supports the opinion of van steenis (1933) that in the tropics such as malesia, they are restricted to the mountains and represent only small offshoots from their generic centers. when compared for instance with india which has 242 endemic taxa (out of c. 900) the number in sumatera is very low. in india too many endemics (116) are confined to the montane regions of the himalayas (rao & dart 1996). aceh and west sumatera showed the highest degree of endemism, apparently due to the considerable areas of high mountains. there are one variety and five species endemic to aceh i.e. adenostemma lavenia var. sessilifolium, senecio dewildeorum, s. sumatranus, prenanthes steenisii, p. stenolimba, and p. sumatrana which are concentrated at the mount leuser nature reserve and its surrounding areas (fig. 1). three endemic species of prenanthes: p. steenisii, p. stenolimba, and p. sumatrana, have been found in aceh above 2000 m altitude (tjitrosoedirdjo 2002). in west sumatera four endemic taxa have been found on the mountains of kerinci, merapi, sago, talamau and talang namely emilia sonchifolia var. lanceolata, senecio sumatranus, vernonia durifolia and vernonia forbesii. emilia, sonchifolia var. lanceolata a newly described variety is endemic to mount kerinci (tjitrosoedirdjo 2002). jambi, south sumatera, and lampung have only one endemic species each, while there are no records of endemics in bengkulu and riau. the two endemic species of senecio, s. sumatranus and s. dewildeorum have been found at altitudes above 2500 m. senecio dewildeorum collected by van steenis from mount goh lembuh, aceh, and by de wilde and de wilde-duyfjes from mount leuser appears to be endemic to the mountain area of aceh (tjitrosoedirdjo 2002). 70   biotropia no. 19,2002 vernonia, has two endemic species: v. durifolia and v. forbesii are new records for west sumatera. vernonia durifolia is restricted to west sumatera at the mountain regions of mount sago, mount merapi, mount malintang and mount talamau which are closely located to each other at the altitude of 2000-2600 m. a comparison on the asteraceae of sumatera with adjacent areas in terms of indigenous and endemic species, sumatera is richer than java. out of 133 species, seven species and two varieties are endemic, while java has only one endemic species (phyllocephalum frutescens) out of nearly the same number (132) of species (koster in backer & bakhuizen f. 1965). although, sumatera is larger than java, it has less number in genera: 74 compared to 107. one of the reasons is that probably sumatera is less explored than java. in addition, during the colonial times, there was more international trade in java and many attempts were made to acclimatize exotic species for horticulture (e.g. cabbage, potatoes), plantation crops (oil palm, rubber, tea, tobacco) and plants with medicinal values. the presence of the kebun raya (botanical garden) in bogor certainly played an important part in this process. cargo, packing material, contaminated grain, introduction of exotic species etc. also inadvertently included the introduction of incipient weeds. among these were of course asteraceae. there are 41 genera of asteraceae which are known in java but have not yet been recorded for sumatera. most of these were introduced in table 2. the malay peninsula has less species than sumatera. turner (1995) listed 61, species, an increase of nearly 40% as compared to ridley's (1923) list of 44 species. of these 3 are endemic: erigeron oreophilus, gynura malaccensis, and vernonia rupicola. notwithstanding the suggestive epithet of the erigeron, there are no high mountains in the malay peninsula. few are over 2100 m. it appears that previously there was a connection between the himalaya and the mountain chain in sumatera. van steenis (1933b) listed ainsliae latifolia, prenanthes scandens, and rhynchospermum verticillatum which are recorded both in the himalayas and sumatera without significant difference in altitude range, about 1300-2900 m. in general, some himalayan/malesian taxa are found only in aceh and north sumatera, while others extend to central and south sumatera, or even further to java, south sulawesi and nusa tenggara. the leuser complex is especially rich in the himalayan elements, while there is also a floristic connection over the kinabalu to luzon, and over sulawesi and seram with new guinea. it is curious to note that these high-montane species (non-asteraceae) were able to reach the leuser complex, but did not venture further down south. the fact that the complex is nonvolcanic while the bukit barisan and the mountains in java are igneous, may offer a clue to this •unsolved question. other species are apparently not hampered by the occasional devastation that occurs on volcanic summits and were able to travel south. there are three genera, known in asia and sumatera which have not been recorded in java: ainsliaea, blumeopsis, and prenanthes. other species known from 72 notes on the asteraceae ot'suniaterasri sudarmiyati tjitrosoedirdjo asia and java but not yet recorded from sumatera and borneo are gnaphalium indicum l., inula cappa and senecio araneosus. the latter species turned out to be identical with cisampelopsis volubilis. inula cappa is apparently currently called as synotis cappa (yeffrey & chen 1984) or duhaldea cappa [pi. syst. evol. 176 (1991) 104]. however, koyama (1988) did not mention java, bali, lombok. he reported that the distribution of /. cappa did not reach as far as north thailand. montane genera known from sumatera and java but which have not been recorded from borneo are : anaphalis, gnaphalium, and prenanthes (van steenis 1933). weedy asteraceae of sumatera a large number of asteraceae have been introduced and became naturalized in sumatera. most of them were introduced from tropical america and the others came from elsewhere. many of the species become an adventive weeds (table 3). the diversity of climate, soil types, altitude and other factors have favoured the establisment and spread of asteraceae weeds from many regions of the world. most of the asteraceae in sumatera were introduced during the colonial times, among these were, for instance, the most noxious and dominant chromolaena odorata and mikania micrantha. both species came from tropical america and now have spread to almost all agricultural lands and plantations. 73   biotropia no. 19,2002 among the sumateran asteraceae, the records in 1996 showed that there are 32 species considered as weeds (tjitrosoedirdjo 1996). while in this report based on the observation in 2000, there are 43 species of weedy asteraceae or thirty two percent of the total number of asteraceae in sumatera (table 3). four of these species have not been reported before for sumatera: eupatorium capilifolium, son-chus oleraceus, shagneticola trilobata. and taraxacum javanicum . eupatorium capilifolium found in payakumbuh, west sumatera has a potential to become noxious weeds. sonchus oleraceous is a native of europe, north africa and continental asia. it is now a common weed in java beween 200-2700 m altitude, especially at 800 m altitude. in sumatera it is not yet so common. so far it is only known from two collections made in takengon (aceh) and in bukit gundaling, berastagi (north sumatera) at 1200-1400 m altitude. sphagneticola trilobata, a native of central and south america, is usually called wedelia trilobata. it seems that the plant was introduced and naturalized in sumatera long time ago where it was used for ground ccrver and as an ornamental plant. it has escaped from these applications and now is a weed at low to high altitudes from aceh to lampung. although it is now common, its occurrence was not reported before or neglected by the botanist collectors. taraxacum javanicum was first found in 1996 in north sumatera at bukit gundaling, berastagi, along a road side at 1400 m altitude (tjitrosoedirdjo 310, biot). in west java, it is found in montane areas between 1200-2500 m altitude, where it was first collected in 1888. in view of the tendency in taraxacum to form local microspecies rapidly this may be an undescribed one, so that a specialist will have to look into this. for this time the name t. javanicum is employed here. according to van soest in grierson (1980), this taraxacum species does not belong to the groups of indigenous to europe as was originally assumed. taraxacum javanicum has now been found in several parts of india and sri lanka. in java, however, it occurs in tea plantations and it may be introduced through this industry, the same way as it has been introduced in sri lanka (grierson 1980) among the three new weedy asteraceae found in indonesia reported by dekker (1981): acanthospermum hispidum, calyptocarpus vialis and elephantopus mollis, only e. mollis was found firstly in north sumatera in 1980 in s. bejingkar, kisaran, north sumatera (megia 230, biot). it is also known from sukarami in west sumatera where it was found in 1987 (tjitrosoedirdjo 50, biot). this species should not be confused with elepantopus scaber. it has alternate leaves and white flowers, whereas e. scaber l. has rosulate leaves and purple flowers. ageratum houstonianum mill, can easily be mistaken for the closely related a. conyzoides especially because the two are often found in the same habitat. it is also native to tropical america. in sumatera, it was first fo'und in 1928 by lorzing (13843, bo) collected in berastagi, north sumatera at 1200 m alt. it has now spread to sukarami, solok, west sumatera at an altitude of 900 m. (tjitrosoedirdjo 45, biot) and lampung at batu keramat, between gisting and kota agung at 500 m altitude (tjitrosoedirdjo 415, biot). a. conyzoides is commonly found everywhere in sumatera. 75 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo in central and south america, the genus chromolaena has about 165 species, but c. odorata is the only species which is now pantropically distributed. in sumatera, the first collection was made in deli, lubuk pakam in 1932 (van der meer-mohr 4004, bo) in tobacco plantations. after the war for independence, it had become quite common and being very distinctive by its white violet flowers, and known as "semak merdeka" (the independence shrubs) or "putihan" (whitish flowers). it has now become an important weed throughout sumatera. clibadium surinamense is another native of south america, and was first reported for sumatera by jochems who collected the first specimen in 1932 in tobacco plantations near medan. conyza sumatrensis, which is previously known as erigeron sumatrensis, is a common weed in open places of fields and plantations, and distributed widely throughout sumatera. in indonesia crassocephalum crepidioides has been confused with erechtites valerianifolia (tjitrosoedirdjo 1987). jochem (1931) noticed it for the first time in 1926 near medan. it had probably come from africa through sri lanka and from there to sumatera (van steenis 1938). within a few years, it then rapidly spread over the whole island. in java it was introduced from sumatera by tea planters, and it spread widely. it has become even more common than erechtites hieracifolia and e. valerianifolia which were introduced much earlier. crassocephalum crepidioides was identified by backer & van slooten (1924) in their "geillustreerd handboek der javaansche thee-onkruiden (no. 233)" as e. 'valerianifolia', so that many pamphlets, books, publications refer to this species as e. valerianifolia instead of c. crepidioides. in the orient c. crepidioides is sometimes confused with e. hieracifolia, as most of the labeled specimens are actually c. crepidioides (belcher 1955). the genus mikania has about 400 species mainly in the warmer parts of the new world. the only indigenous species in asia is m. cordata ,while m. micrantha is the only new world species to have been introduced there. the latter species readily takes to disturb areas and tends to be weedy (parker 1972). it was imported from paraguay in 1949 and planted in the bogor botanical garden. in 1956 this species was introduced as a non-legume ground cover in rubber plantations. wirjahardja (1976) reported that around 1976, it occupied the greater part of rubber plantations and abandoned agricultural areas in west and east java and south sumatera. recently, in west java and sumatera, m. cordata could not be found easily since it is suppressed by m. micrantha. porophyllum ruderale, a native of mexico and south america, was first reported in bogor in 1945, a new record for malesia (tjitrosoedirdjo 1991). in 1978 the first herbarium specimens from sumatera were collected from the transmigration areas of lampung (dekker & wirjahardja 2626, biot) and south sumatera (dekker & wirjahardja 2591, biot). currently, it is commonly found in sumatera, east and central java. its occurrence has also been reported for malaysia and singapore (tan & ibrahim 1992; turner 1995). 76 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo some of the weedy species of asteraceae which is currently not known in sumatera i.e. praxelis clematidea (eupatorium catarium ) and parthenium hyster-oporus have a potential to become noxious weeds. praxelis clematidea a native of south america, has been introduced in south china and queensland, australia (veldkamp 1999). partenium hysterophorus a native of the carribean islands has spread to australia, south africa, china, the pasific islands (sastroutomo & mahyudin 1990). the importation of grass seeds, cover crop seeds, wheat, grains and cattle from australia might result to the introduction of these weeds in sumatera. we have to be aware that those species have a potential in spreading rapidly. conclusions there are 133 species of 74 genera in 11 tribes of asteraceae in sumatera, based on the records in 2000. the most diverse genus is blumea with 14 species. other genera are only represented by 10 species or less, usually 4, or 3, or 2, and mostly by 1 species only. there are 1 genus, 7 species and 2 varieties endemic to sumatera in terms of indigenous and endemic species sumatera is richer than java. the high mountain region in sumatera is shown to be richest in the general asteraceae flora as well as endemic species.. thirty nine or about 53% are exotic genera and the native genera are less than half of the total number. there are 43 important weed species, which were introduced in sumatera from tropical america, africa, asia and europe. the exotic genera and species are introduced and naturalized in sumatera. the diversity in climate, soil types, altitude and other factors have favoured the establishment and spread of asteraceae weeds from many regions of the world. acknowledgements part of the dissertation of the author is supported by tmpd (team pengem-bangan program doctor) and seameo biotrop bogor, indonesia, through funding of the dip project. the author would like to express her sincere appreciation to all her supervisors , especially dr. j.f. veldkamp (l) for his suggestions and comments. thanks are also due to the directors of bo, 'anda', kep for giving the facilities to work at their herbarium and l for the loan specimens. references backer, c.a. 1918. indische duinplanten. trop. nat. 7: 59 backer, c.a. & d.f. van slooten.1924. geillustreerd handboek der javaansche thee-onkruiden. algemeen proefstation voor thee. batavia, drukkerijen roygrok & co. 77 biotropia no. 19,2002 belcher, r.o. 1955. the typification of crassocephalum moench and gynura cass. kew bull. 3: 455-465 bentham, g. 1873. notes on classification, history and geographical distribution of compositae. j. linn. soc. bot. 13: 335-577 bentham, g. & j.d. hooker. 1873. genera plantarum. 2: 1-224. lovell reeve & co., london. boerlage, j.g. 1891. flora van nederlandsch indie (compositae). e.j. brill, leiden. cassini, h. 1816. troissieme memoire sur le synantherees, analyse de la corolle. j. phys. chim. hist. nat. arts. 82: 116-146 cassini, h. 1817. quatrieme memoire sur la familie des synantherees, contenant 1'analyse de 1'ovaire et de ses assessoires. j. phys. chim. hist. nat. arts 85: 5-21 cassini, h. 1818. composees. in g. cuvier, diet. sci. nat. 10: 131-159 cassini, h. 1826-1934.opuscules phytologiques. 3 volumes. paris dalla tore, c.g. de & h. harms. 1907. genera siphonogamarum. lipsieae, berlin. dekker, r. j. 1981. notes on new or remarkable indonesian weed species. proc. konp. higi medan. jack, w. 1820. the malayan miscellanies. bencoolen jeffrey, c. & y. l. chen 1984. taxonomic studies on the tribe senecioneae (compositae) of eastern asia. kew bull. 39, 2:319 jeffrey, c. 1986. the senecioneae in east tropical africa. kew bull. 43: 195-277 jefrey, c. 1988. the vernonieae of east tropical africa. kew bull. 43:195-277. jeffrey, c. 1995. compositae systematic 1975-1993. developments and desiderata. in advances in compositae systematics d.j. n. hind, c. jeffrey & g.v. pope (ed.). the royal botanic garden kew. vol i. jochems, s.c.j. 1931. de invoer van twee nieuwe composieten in deli (gynura sp. en clibadium surinamense). trop. nat. 20: 5-10 jones, s.b. 1979. synopsis and pollen morphology of vernonia (compositae vernonieae) in the new world. rhodora 425-447. jones, s.b. 1981. synoptic classification and pollen morphology of vernonia (compositae: vernonieae) in the old world. rhodora 83: 59-75 grierson, a.j.c. 1980 in dassanayake, m.d. revised handbook of the flora of ceylon 1 (111). amerind, new delhi. hasselt, a.l. van 1884. bijdragen tot de kennis der flora van midden sumatra. flora in veth. midden sumatra 4(2): 1:49 hoffmann, o. 1890-1894.liguliflorae-cichorieae in engler & prantl. die naturlichen pflanzenfamilien kilian, n. 1997. revision oflaunaea cass. (compositae, lactuceae, sonchinae). englera 17: 288-296 koster, j.th. 1935. the compositae of the malay archipelago. blumea 1(3): 352-538 koster, j.th. 1941. notes on malay compositae 1. blumea 4(3): 482-492 78 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo koster, j. th. 1948. notes on malay compositae ii. blumea 6(1): 264-365 koster, j. th. 1952. notes on malay compositae iii. blumea 7(1): 288-291 koster, j. th. 1953. some annotation to the compositae collected by o/beccari in borneo & sumatra. koster, j. th. 1958. notes on malay compositae iv. blumea supplement iv: 170-177 koster, j. th. 1965.compositeae. in backer and bakhuizen van den brink f. fl. of java vol. ii: 362-437 koster, j. th. 1966. the compositae of new guinea i. nova guinea bot. 24: 479-614 koster, j. th. 1970. the compositae of new guinea ii. blumea 18(10: 137-145 koster, j. th. 1975. the compositae of new guinea iii. blumea 22(2): 207-217 koster, j. th. 1976. the compositae of new guinea iv. blumea 23: 163-175 koster, j. th. 1979. the compositae of new guinea v. blumea 25(1): 249-282 koster, j. th. 1980. the compositae of new guinea vi. blumea 26(l):233-243 koyama, h. 1988. taxonomic studies in the compositae of thailand. acta phytotax. geobot. 39: 161 melchior, h.a. 1964. engler's syllabus der pflanzen familien ii. band. berlin gebruder borntragen miquel, f.a.w. 1856. flora van nederlandsch indie 2: 1-116. tweede deel. fried. fleischer, leipzig. amsterdam, utrecht. miquel, f.a.w. 1861. flora van nederlandsch indie, eerste bijvoegsel, sumatra:210-213, 535-537. c.g. van der post, amsterdam. nasution, u. 1984. gulma dan pengendaliannya di perkebunan karet sumatera utara dan aceh (weeds and their control in rubber plantations in north sumatera and aceh) pp4m-tanjung morawa (in indonesian) parker, c. 1972. themikania problems. pans 18(3): 312-315 rao, r.r. & b. datt. 1996. diversity, phytogeography of indian compositae. in proceedings compositae conference. vol. i: 445-461. ridley, h.n. 1923. the flora of the malay peninsula. l. reeve & co., ltd. london. vol. ii.: 177-197 ross, m.c. 1993. annual report foundation flora malesiana 1992. fl. malesianabuli.il(2): 131-142 royen, p. van. 1983. the alpine flora of new guinea 4: 3139-3451. j. cramer, vaduz. satroutomo, s.s. & a.a. mahyudin. 1990. partenium hysterophorus l. pest data sheet no.: 43. asean pest plant quarantine center and training institute malaysia. shaw, h.k. airy. 1981. the euphorbiaceae of sumatera. kew bull. 36(2): 239-373 steenis, c.g.g.j. van 1933. on the origin of the malaysian mountain flora. part 1. fact and statement of the problems. bull. jard. bot. buit. 13(3): 125-417 steenis, c.g.g.j. van 1938. gymtra crepidioides bth. a recently introduced weed in s.e. asia. current science vii: 285 soerjani, m, a.j.g. kostermans & g. tjitrosoepomo. 1987. weeds of rice in indonesia. balai pustaka, jalarta tan, h.t.w., ibrahim, ali bin & chua, k.s. 1992. addition to the flora of singapore i gard. bull of singapore 44: 129 79 biotropia no. 19,2002 tjitrosoedirdjo, sri s. 1987. field characterization of the confused species crassocephalum crepidioides and erechtites valerianifolia in indonesia. proceed. apwss. conf. taipei. tjitrosoedirdjo, sri s. 1991. the present status and potential of porophyllum ruderale var ruderale in indonesia. proc. apwss conf. jakarta. tjitrosoedirdjo, sri s. 1996. gulma anggota suku asteraceae di sumatera (the weedy asteraceae species of sumatera). proc. higi, bandar lampung tjitrosoedirdjo, sri s. 2001. progress on the studies of asteraceae in sumatera. taxonomy: the cornerstone of biodiversity. proceedings of the fourth international flora malesiana symposium 1998. forest research institute malaysia, p: 181-186 tjitrosoedirdjo, sri s. 2002. four new taxa of asteraceae in sumatera. reinvvardtia 12 (1): 125-128 turner, i.m. 1995. a catalogue of the vascular plants of malaya. card. bull, of singapore 47(1): 168-172 veldkamp, j.f. 1999. eupatorium catarium a new name for eupatorium clematidea griseb. non. sch. brp. (compositae), a south american species naturalized and spreading in s.e. asia and queensland, australia. card. bull. singapore 51(1): 119-124 whitmore, t.c. & i.g.m. tantra. 1986. tree flora of indonesia. checklist for sumatra. forest research and development research center, bogor, indonesia wirjahardja, s. 1976. autecological study ofmikania spp. biotrop internal report. 7p. 80 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo appendix 1 list of the genera and species of asteraceae in sumatra. accepted genera and species in bold, synonyms in italic. achillea l. achillea millefolium l. acmella rich, ex pers. acmella paniculata (wall, ex dc.) r.k. jansen spilanthespaniculata wall, ex dc. spilanthes acmella auct. non murr. acmella uliginosa (sw.) r. cass. jaegera uliginosa (sw.) baker spilanthes iabadicensis a.h. moore spilanthes uliginosa sw. adenostemma forster & g. forster adenostemma lavenia (l.) kuntze adenostemma fastigiatum (blume) dc. lavenia vastigiata blume verbesina lavenia l. adenostemma macrophyllum (blume) dc. adenostemma parviflorum (blume) dc. adenostemma ovatum miq. lavenia parviflora blume ageratum l. ageratum houstonianum mill. ageratum conyzoides l. ainsliaea dc. ainsliaea latifolia (d. don) sch. bip. ainsliaeapteropoda dc. liatris latifolia d. don anaphalis dc. anaphalis javanica (reinw. ex blume) sch. bip. antennaria javanica (reinw. ex blume) dc antennaria saxatilis dc. gnaphalium javanicum reinw. ex blume gnaphalium saxatile blume anaphalis longifolia (blume) dc. gnaphalium longifolium blume artemisia l. artemisia scoparia waldst. & kit. artemisia vulgaris l. aster l. aster novae-angliae l. symphyotrichum novae-angliae (l.) nesom virgulus novae-angliae (l.) reveal & keener aster novi-belgii l. symphyotrichum novi-belgii (l.) nesom asteromoea blume asteromoea indica (l.) blume aster indicus l. boltonia indica (l.) benth. austroeupatorium r m. king & h. rob. austroeupatorium inulifolium (kunlh) r.m. king & h. rob. eupatorium inulifolium kunth eupatorium javanicum auct. non blume eupatorium pallescens dc. bidens l. bidens biternata (lour.) merr. & sherrf ex sherff bidens chinensis (l.) willd. coreopsis biternata lour. bidens pilosa c blumea dc. blumea balsamifera (l.) dc. blumea grandis (wall.) dc. conyza balsamifera l. blumea bicolor merr. blumea clarkei hook. f. blumea lessingii merr. blumea densitlora dc. blumea hieraciifolia (d. don) dc. blumea chinensis walpers blumea subsericans elmer erigeron hieraciifolius d. don blumea intermedia j. koster blumea junghuhniana (miq.) boerl. blumea dacycoma (miq.) boerl. conyza dacycoma miq. conyza junghuhniana miq. blumea korthalsiana (miq.) boerl. conyza korthalsiana miq. blumea lacera (burm. f.) dc. blumea lactucifolia dc. blumea runcinata dc. conyza lacera burm. f. blumea laciniata (roxb.) dc. blumea crepidifolia dc. blumea javanica (blume) zoll. blumea runcinata dc. blumea sonchifolia dc. conyza javanics blume conyza laciniata roxb. blumea lanceolaria (roxb.) druce blumea laxiflora elmer blumea longifolia dc. blumea myriocephala dc. blumea spectabilis dc. conyza lanceolaria roxb. blumea riparia (blume) dc. 81 biotropia no. 19,2002 blumea semivestita dc. conyza riparia blume blumea sylvatica (blume) dc. blumea sessilifolia (blume) dc. conyza sessilifolia blume conyza sylvatica blume blumea tenella dc blumea humifusa (miq.) boerl. conyza humifusa miq. blumeopsis gagnep. blumeopsis flava (dc.) gagnep. blumea flava dc. blumeopsiafalcata auct. non merr. laggera flava (dc.) benth. boltonia l'her boltonia asteroides (l.) l' her matricaria asteroides l. carpesium l. carpesium cernuum l. centaurea l. centipeda lour. centipeda minima (l.) a. br. & aschers. artemesia minima l. centipeda minuta (g.forst.) benth. ex cb. clark centipeda orbicularis lour. cotula minima (l.) willd. cotula minuta g. forst. grangea minima (l.) poir chromolaena dc. chromolaena odorata (l.) r.m. king & h. rob. eupatohum odoratum l. chrysanthemum l. chrysanthemum coronarium l. bupthalmum oleraceum lour. chrysanthemum roxburghii (desf.) dc. glebionis coronaria (l.) tzvel. pyrethrum roxburghii desf. chrysanthemum morifolium ramat. matricaria morifolia ramat. pyrethrum sinense (sabine) dc. cissampelopsis (dc.) miq. cissampelopsis volubilis (blume) miq. cacalia volubilis blume senecio araneosus dc. senecio blumei dc. senecio walkeri auct. non arn. clibadium allamand ex l. clibadium surinamense l. conyza less. conyza japonica (thunb.) less, ex dc. escenbachiajaponica (thunb.) j. koster conyza leucantha (d. don) ludlow & raven blumeopsis falcate (d. don) merr. conyza vicidula wall, ex dc. escgenbachia vicidula (dc.) j. koster erigeron falcatum d. don erigeron leucanthum d. don conyza sumatrensis (retz.) walker erigeron sumatrensis retz. erigeron linifolius auct. non willd. coreopsis l. coreopsis grandiflora hogg ex sweet coreopsis tinctoria cav. cosmos cav. cosmos bipinnatus cav. cosmos caudatus kunth cosmos sulfureus cav. bidens. sulfurea (cav.) sell. bip. cotula l. cotula anthemoides l. crassocephalum moench. crassocephalum crepidioides (benth.) s. moore crassocephalum diversifolium hiern gynura crepidioides benth. dahlia cav. dahlia pinnata auct. non cav. dicrocephala l. her. ex dc. dicrocephala integrifolia (l.f.) kuntze cotula bicolor roth dicrocephala bicolor (roth) schlechtend. dicrocephala latifolia (pers,) dc. grangea latifolia (pers) poir. eclipta l. eclipta prostrata (l.) l. eclipta alba hassk. eclipta erecta l. verbesina prostrate l. elephantopus l. elephantopus mollis kunth elephantopus martii graham ex sch. bip. elephantopus lomentosus auct. non l. elephantopus sea her l. eleutheranthera poit. eleutheranthera ruderalis (sw.) sch. bip. eleutheranthera. prostrata (sw.) sch. bip. kegelia ruderalis sch. bip. melampodium ruderale sw. ogiera ruderalis (sw.) griseb. emilia cass. e m i l i a coccinea (sims) g. don cacalia cocinea sims cacalia sagitata willd. emilia javanica auct. non c.b. rob. emilia sagittata dc. emilia prenanthoidea dc. emilia sonchifolia (l.) dc. cacalia sonchifolia l. 82 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo crassocephalum sonchifolium (l.) less. emilia purpurea cass. emilia rigidula dc. enydra lour. enydra fluctuans lour. meyerafluctuans (lour.) spreng. erechtites raf. erechtites hieraciifolia (l.) raf. ex dc. senecio hieraciifolius l. erechtites valerianifolia (wolf) dc. crassocephalum valerianifolium less. erechtites petio/ata auct. non benth. gymtra rosea ridl. eupatoriutn l. eupatorium capillifolium (lam.) small artemesia capillifolia lam. chrysosoma capillacea michx. eupatorium foenicoloides walt. gaillardia foug. gaillardia pulchella foug. galinsoga ruiz & pavon galinsoga parvillora cav. gerbera cass. gerbera jamesonii bolus ex adlam glossocardia cass. glossocardia leschaenaultii (cass.) veldk. chrysanthellum indicum auct. non dc. chrysanthellum leschenaultii (r. cass.) backer ex kostcr chrysanthellum procumbens auct. non l.c.m. rich. gnaphalium l. gnaphalium japonicum thunb. gnaphalium pensylvanicum willd. gamochaetapensylvanicum (willd.) cabrera gnaphalium perigrinum fern. granges adans. grangea niaderaspatana (l.) poir. artemesia maderaspatana l. gynura cass. gynura aurantiaca (blume) dc. gynura densiflora miq. gynura lyrata sch. bip. ex miq. gynura mollis sch. bip ex zoll. gynura sumatrana miq. gynura procumbens (lour.) merr. cacalia procumbens lour. cacalia sarmentosa blume gynura sarmentosa (blume) dc. helianthus l. helianthus angustifolius l. helianthus annuus l. helianthus tuberosus l. hclichrysum mill. helichrysum orientale (l.) gaertn. gnaphalium orientale l. lactuca l. lactuca indica l. pterocypsela indica (l.) c. shih lactuca laevigata (blume) dc. aracium laevigatum (blume) miq. ixeridium laevigatum (blume) pak & kawano prenanthes laevigata blume lactuca saliva l. launaea cass. launaea sarmentosa (willd.) sch. bip. ex kuntze ammoseris sarmentosus (willd.) dc. lactuca sarmentosa (willd.) dc. launaea pinnatifida cass. prenanthes sarmentosa willd. laggera sch. bip. ex koch laggera alata (d. don) sch. bip. ex oliver blumea alata (d. don) dc. conyza alata (d. don) roxb. erigeron alatum d. don lagenophora cass. lagenophora lanata a. cunn. lagenophora gracilis steetz lagenophora stipitata auct. druce lagenophora sundana miq. leucanthemum mill. leucanthemum vulgare lam. chrysanthemum leucanthemum l. microglossa dc. microglossa pyrifolia (lam.) kuntze conyzaprolifera blume conyza pyrifolia lam. mycroglossa volubilis dc. mikania willd. mikania cordata (burm.f.) b.l. rob. eupatorium cordatum burm. f. eupatorium volubile vahl. mikania scandens auct. non willd. mikania volubilis (vahl.) willd. mikania micrantha kunth myriactis less. myriactis javanica (reinw. ex blume) dc. bellisjavanica reinw. ex blume notonia dc. notonia grandiflora wall, ex dc. senecio grandiflorus (dc.) jacobsen senecio indicus backer ex heyne pluchea cass. pluchea indica (l.) less. baccaris indica l. porophyllum guett. porophyllum ruderale (jacq.) cass. cacalia ruderalis (jacq.) sw. 83 biotropia no. 19,2002 kleinia ruderalis jacq. porophyllum ellipticnm cass. prenanthes l. prenanthes scandens hook. f. ex benth. prenanthes steenisii tj. prenanthes stenolimba steenis prenanthes sumatrana tj. rhyncospermum blume rhyncospermum verticillatum reinw. ex blume senecio l. senecio dewildeorum tj. senecio sumatranus martelli senecio korintjianus boerl. sigesbeckia l sigesbeckia orientalis l. solidago l. solidago canadensis l. sonchus l. sonchus arvensis l. sonchus asper (l.) hill. sonchus malaianus miq. sonchus oleraceus l sphaeranthus l. sphaeranthus africanus l. sphaeranthus indicus l. sphagneticola o. hoffm. sphagneticola calendulacea (l.) pruski complaya chinensis (osbeck) strother telechitonia chinensis (osbeck) h. rob. & cuatrecasas verbesina calendulacea l, wedelia calendulacea (l.) less. wedelia chinensis (osbeck) merr. sphagneticola trilobata (l.) pruski complaya trilobata strother seruneum trilobatum (l.) kuntze silphium trilobatum l. thelechitonia trilobata (l.) h. rob. & cuatrecasas wedelia trilobata (l.) a. hitchc. struchiuni p. br. struchium sparganophorum (l.) kuntze ethulia sparganophora l. ethulia struchium sw. sparganophorus vailantii crantz sparganophorus struchium poir. synedrella gaertn. synedrella nodiilora (l.) gaertn. verbesina nodiflora l. tagetes l. tagetes erecta l. tagetes major gaertn. taraxacum wigger taraxacum javanicum soest. taraxacum offlcinale wigger tridax l tridax procumbens l. tithonia desf. ex juss. tithonia diversifolia (hemsl.) a. gray mirasolia diversifolia hemsl. urbanisol tagetiflora (desf.) kuntze tithonia rotundifolia (mill.) s.f. blake tagetes rotundifoliam\\\. desf. tithonia tagetiflora desf. vernonia schreb. vernonia arborea buch.-ham. gynanthemum arboreum (buch.-ham) h. rob. strobocalyx arborea (buch.-ham) sch. bip. vernonia cinerea (l.) less. conyza cinerea l. cyabthillium cinereum (l.) h. rob. senecioedes cinerea (l.) post & kuntze vernonia coronata j. koster vernonia cymosa blume vernonia durifolia j. koster vernonia forbesii s. moore vernonia patcntissima j koster vernonia patula (dryand.) merr. conyza patula dryand. cyanthillium villosum blume vernonia chinense (less.) less. vernonia subdentata j. koster vernonia vagans dc. gymnanthemum vegans dc. wedelia jacq. wedelia biflora (l.) dc. seruneum biftorum (l.) kuntze seruneum strigulosum (k. schum.) kuntze wolastonia biflora (l.) dc. wolastonia slrigulosa (k. schum.) kuntze wedelia montana (blume) boerl. seruneum montanum (blume) kuntze wolastonia montana blume verbesina montana blume wedelia urticifolia (blume) dc. seruneum urticifolium (l.) kuntze verbesina urticifolia blume wolastonia urticifolia (blume) hassk. xanthium l. xanthium indicum koenig. ex roxb. xanthium inequilaterum dc. youngia cass. youngia japonica (l.) dc. zinnia l. zinnia elegans jacq. 84 65.pdf 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf 75.pdf 76.pdf 77.pdf 78.pdf 79.pdf 80.pdf 81.pdf 82.pdf 83.pdf 84.pdf 4. sri sulandari (mitochondri... biotropia vol. 19 no. 2, 2012: 92 102 mitochondrial dna variation of the sumatran elephant populations in sumatera, indonesia sri sulandari and moch. syamsul arifin zein recipient of biotrop research grant 2010/accepted 11 september 2012 genetic analysis of mitochondrial dna diversity in sumatran elephant ( ) was conducted. a 630 bp segment of mitochondrial dna was amplified from 105 different sumatran elephant samples from 5 locations in sumatera (bentayan, sugihan, bukit salero lahat, seblat, way kambas) using a set of primers: mdl3 (5'-cccacaattaatgggccc-ggagcg-3') and mdl5 (5'-ttacatgaattggcagcca-accag3'). the objectives of this study were to generate mitochondrial dna d-loop sequences for all available sumatran elephant samples and to define haplotypes and nucleotide sequence diversity of the different sumatran elephant populations. the nucleotide sequence of a total of 105 pcr products were successfully determined with an average length of 616 bp. however, mitochondrial dna fragments for this analysis used the first 601 bases. six different haplotypes (bp, bt, bs, br, bx and by) were identified in sumateran elephant populations. the majority of the sampled individuals carried haplotype bt. bx and by are most likely novel derived haplotypes. all haplotypes, except for the haplotype bp belong to the sumatera clade. the haplotype bx was derived from the haplotype bt, and the haplotype by was derived from the haplotype bs by one transversion, respectively. all the other substitutions identified in this network were transitions. the haplotype bp is widely distributed from sri lanka, sumatera, peninsular malay and china. although reported to be distributed in sumatera and peninsular malay the haplotype bu was not detected among the samples analysed in this study. genetic distances within populations in bentayan, bukit salero lahat, seblat, sugihan and way kambas ranged from 0.0000 0003, and the genetic distance between the populations that is 0.0000 0.0022. the distance between haplotypes of different sumatran elephant populations was shown to be low. the diversity of haplotypes and nucleotides in sumatera island were low, the highest diversity was found in elephants sampled in the region of bukit salero lahat and the lowest was found in elephants from bentayan and sugihan. overall, the results of analysis of fu and li's f*test statistic indicates that the population of sumatran elephants in sumatra is -0.78871, which suggests that there is no inbreeding. however, the results are not significant (p> 0:10) and additional studies are required to confirm this finding. sumatran elephant, , mitochondrial dna, haplotype * research center for biology (rcb), the indonesian institute of sciences (lipi) cibinong science center, jalan raya jakarta bogor km.46, cibinong 16911, indonesia elephas maximus sumatranus elephas maximus sumatranus abstract key words: * corresponding author : ssulanda@yahoo.co.id 92 introduction the asian elephant is an endangered animal and listed on appendix i of cites (convention on international trade in endangered species) of wild fauna and flora. sumatran elephants ( ) is one of four different asian elephant subspecies in indonesia. today, the sumatran elephant is found in seven main provinces, namely nanggroe aceh darussalam, north sumatra, riau, jambi, bengkulu, south sumatra, and lampung (soehartono . 2007). the main serious threats to sumatra's elephant populations are among others forest/habitat loss, illegal hunting, poaching for ivory, and habitat degradation due to conversion of critical forest habitat to agriculture/plantation areas. also many elephant populations are trapped in small pockets which are not enough to support their life, and the condition has sparked conflict between humans and elephants. elephants raid people's crop, destroy their houses, and sometimes kill or injure people. in response, elephants are killed and the local communities persuade the government to catch and remove elephants from the wild. therefore, information on status and distribution of sumatran elephant populations, including information on genetic diversity of the remaining populations are urgently needed to determine conservation policy. molecular technology development at the moment has increased the efficiency and accuracy in the genetic characterization study among breeds of animals. mitochondrial dna (mtdna) sequences have been extensively and successfully used to determine genetic diversity. mitochondrial dna is passed to offspring by matrilineal inheritance of mitochondria via the oocyte. different regions of the mtdna evolve at different rates. the area displacement-loop (d-loop) has variation which is quite high, more polimorphic compared to other area mtdna (ishida . 1994; quinn & wilson 1993). as statement revealed by brown . (1982), quinn dan wilson (1993) and akhisinomiya (1994), d-loop area is often used for phylogenetic analysis, both inside the species and among species. therefore, much genetic variation can be expected between individuals of the same species. molecular techniques have been used to evaluate the diversity of asian elephants across or within populations. maternal inherited mitochondrial dna (mtdna) (fernando & lande 2000; fernando . 2000, 2003; fleischer . 2001; vandebona . 2002; vidya & sukumar 2005; vidya . 2005a, b, 2007; fickel . 2007) revealed several haplotypes, which belong to two haplogroups or clades. in thailand, eight haplotypes from two clades were found in 82 captive elephants (lertwatcharasarakul 2003), while mtdna control region sequences from 78 captive elephants represented 20 haplotypes (fickel . 2007). fernando . (2000, 2003) identified 27 different haplotypes within the asian elephant population by sampling over three hundred elephants from sri lanka, bhutan/north india and laos/vietnam, malaysia, thailand, bangladesh and cambodia. these haplotypes are/clustered into two well-differentiated assemblages/clades, α and β. these assemblages corresponded to the clades that were found by vidya . (2005) using mtdna sequencing. fleischer . (2001) also found these two distinct assemblages by sequencing mtdna. elephas maximus sumatranus et al et al et al et al. et al et al et al et al et al et al. et al et al et al et al 93 mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. however, studies that analyze the sumatran elephant based on mitochondrial dna are still limited, such as the studies of fernando . (2003) and fleischer . (2001). there was also a study among 27 elephants (17 samples from alas nepal and 10 samples from way kambas) reported by okayama . (2001) on mitochondrial dna analysis of sumatran elephant, stated that sumatran haplotypes were more similar to sri lanka haplotypes than mainland asian haplotypes. haplotype frequencies were different between populations of alas nepal and way kambas. unfortunately, there were no results on genetic differentiation of sumatran population, because sampling location and size are too small to estimate genetic differentiation in sumatra. furthermore, genetic analysis of the sumatran elephant populations comprehensively sampled from aceh to lampung has not yet been reported. therefore, a research on mitochondrial dna analysis of genetic diversity within maternal lines of different populations of sumatran elephant ( ) was conducted in this study, in order (1) to generate mtdna d-loop sequences for all the sumatran elephant samples under this study and (2) to provide information on haplotypes and degree of nucleotide sequence diversity of sumatran elephant populations. to conduct the study, the highly variable d-loop region of mtdna on 105 samples of sumatran elephant from 5 locations in sumatra was analyzed in this study. all results obtained in this study should be useful for conservation strategies, particularly for sumatran elephants. it is believed that diversity is only secure when diverse conservation strategies are employed. a total of one hundred and five (105) dna material blood of sumatran elephants ( ) were collected in 5 different locations of elephant conservation center ( table 1). the samples were preserved in 96 % absolute ethanol and stored at the bank of material dna for indonesian fauna, genetic laboratory, division of zoology, research center for biology-lipi. the samples were collected in 2000 by t. okayama in collaboration between biodiversity conservation project (bcp) japan international cooperation agency (jica) and research center for biology (rcb) lipi. a list of the samples is shown in table 1. et al et al et al elephas maximus sumatranus elephas maximus sumatranus et al. materials and methods samples and study areas table 1. list of samples of sumatran elephants ( ) collected from 5 locations. elephas maximus sumatranus no. elephant conservation center province total samples 1 sugihan (su) south sumatra province 18 2 bentayan (be) south sumatra province 20 3 seblat (se) bengkulu province 21 4 bukit serelo lahat (bsl) south sumatra province 24 5 way kambas (wk) lampung province 22 total samples 105 biotropia vol. 19 no. 2, 2012 94 dna extraction, amplification and sequencing data analysis dna was extracted from whole blood using qiagen ”dneasy®blood & tissue kit. amplification of mitochondrial dna was conducted by the method of polymerase chain reaction (pcr), using thermal cycler 2700 (applied biosystems). a 630 bp segment of mtdna was amplified using a set of primers, mdl3 (5'-cccacaattaat-gggcccggagcg-3') and mdl5 (5'-ttacatgaattggcagccaaccag-3') (fernando ., 2000), and subsequent nucleotide sequencing of the amplified fragment. these primers amplify a 630-bp fragment of mtdna, including the d-loop. fernando (2000 & 2003) stated that the first 109 bp of the fragment code is for the c terminal end of cytochrome b, the next 135 bp code for threonine and proline trna's, the rest of the fragment corresponds to the non-coding control region that is the d-loop. pcr amplification was performed using a mixture of 30 l reactions containing of 50 mg sample dna, pcr buffer 1 x 200 um dntps, 2 mm mgcl and 1 unit taq dna polymerase (fermentas, native with bsa). the amplified fragment was purified, and subsequently the nucleotide sequence was determined. the nucleotide sequences obtained, were aligned, edited and analyzed including definition of genetic diversity, genetic distance (estimate of the number of nucleotide substitutions per nucleotide site between two sequences), and phylogenetic analysis by the neighbour joining method. the haplotypes determined in this study was compared to the haplotypes identified by both fernando (2000 & 2003). and vidya (2005). the accession numbers for the fernando . haplotypes as deposited in genbank ( ) are: ay245538 and ay245802 to ay245827, and for vidya haplotypes as deposited in genbank are ay365432 and ay365433. trees were rooted using wooly mammoth (genebank nc007596, krause . 2006 and dq316067, rogaev . 2006), african forest elephant (genebank jf827275, ishida ., 2011) and african savanna elephant genebank af527654, eggert . 2002 , and genebank dq316069, rogaev . 2006) as an outgroup. a fragment of the d-loop mtdna corresponding to the first 601 bp was used for analysis in this study. nucleotide sequence data was obtained after editing of the fragment d-loop sequence. sequence data was analysed with various kinds of computer software. chromas which available on http://www.technelysium.com.au/chromas.html was used for and editing the sequence result. the clustalx 1.83 was used for multiple alignment of sequences (thompson 1997; and obtained from ftp://ftpigbmc:u-strasbg.fr/pub/clustalx). macclade 4.0 was used to make the polymorphic sites (maddison and maddison, 2000 and available on http://ag.arizona.edu/ macclade/macclade.html). molecular evolutionary genetic analysis (mega) version 3.0 was used for phylogenetic and analysis of the molecular evolution (kumar . 2004, available on http://www.megasoftware.net/), while network analysis was used for illustrating haplotype diversity i.e. network 4.1.0.8 (bandelt . 1999). et al et al. et al. et al. et al et al. et al et al el al et al et al viewing et.al. et.al et.al � 2 http://www.ncbi.nlm.nih.gov mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 95 genetic diversity indices of sumatran elephants including haplotype diversity, haplotype frequencies, nucleotide diversity, and fu and li's f*test (1993), were carried out using dnasp version 4.0 (rozas ., 2003; available at http://www.ub.es/dnasp). a total of 105 pcr products from the samples of sumatran elephants were successfully sequenced, with an average length of about 616 base pairs. the mtdna fragments used for this analysis corresponds to the first 601 bases. table 2 and figure 1 show the six (6) haplotypes found in sumatran elephants (bp, bt, bs, br, bx and by) and identified at 23 the sites of the polymorphic (variable sites) (table 3). all haplotypes except for the haplotype bp (bsl-20 and bsl-23) are belonging to the sumatera clade. the haplotype bp which belongs to the continental clade (fig. 2) is widely distributed from sri lanka, sumatera, peninsular malay (fernando . 2000, 2003, vidya . 2009), and china (yonezawa . unpublished). although fernando . (2000 & 2003), and vidya . (2009) reported that the haplotype bu is distributed in sumatera and peninsular malay, but bu haplotype could not be detected in the samples analysed in this study. et al et al et al et al et al et al results and discussions table 2. haplotype distribution of 105 sequence of mitochondrial dna no haplotype individual with same haplotypes number haplotypes % 1 bt be8,be19,be17,be15,be5,be7,be2,be4, be10,be1,be13,be6,be16,be14,be20, be18, be3,be9,be11,be12 20 91,40% bsl17,bsl8,bsl12,bsl1,bsl7,bsl16, bsl13,bsl2,bsl4, bsl11,bsl15,bsl19,bsl14, bsl10,bsl6, bsl9, bsl3, bsl5, bsl18 19 su2, su5,su11, su6,su15,su3,su10,su17,su8,su12,su13,s u7,su9,su1,su4,su14, su16, su18 18 wk19,wk16,wk1,wk20,wk21,wk18,wk 7,wk14,wk15, wk9,wk12, wk17, wk5, wk11, wk3, wk8, wk13 19 se9,se20,se12,se4,se14,se18,se2,se13, se5,se3,se17,se15,se21,se8, se7,se6,se10,se16,se1,se11 20 2 bs wk10,wk22 2 1,90% 3 br bsl21,bsl22,bsl24 3 2,90% 4 bp bsl20,bsl23 2 1,90% 5 bx se19 1 0,95% 6 by wk4 1 0,95% total 105 100% notes: wk = way kambas, su = sugihan, se = seblat, bsl= bukit selero lahat,be = bentayan biotropia vol. 19 no. 2, 2012 96 the genetic diversity (haplotype diversity, nucleotide diversity and haplotype frequencies) were analyzed for each region (be, bsl, se,su, and k), and whole sumatera island (table 3). these analyses were carried out by using dnasp version 4.10 (rozas . 2003). based on the whole data sequence, the nucleotide diversity in sumatra is 0.00092, in way kambas (wk) 0.00056, in seblat (se) 0.00016, the bukit salero lahat (bsl) 0.00309, and zero (0) in bentayan (be ) and sugihan (su). haplotype diversity on the island of sumatra is 0.164, way kambas (wk) is 0.255, seblat (se) is 0.095, the bukit salero lahat (bsl) 0.366, and zero (0) in bentayan (be) and sugihan (su). the highest haplotype and nucleotide sequence diversity was found in elephants from the bukit salero lahat (bsl) region, and the lowest is zero (identity) which was found in elephants from the bentayan (be) and sugihan (su) regions. this means, there is a lack of mtdna diversity in elephants sampled from this region. there is only one type of haplotype, and consequently the haplotype frequency is 100%. the conditions in the region of sugihan and bentayan might indicate inbreeding. unfortunately, the analysis of fu and li's f * test cannot be performed in the regions of sugihan and bentayan, because genetic diversity = 0. overall, the results of analysis of fu and li's f * test statistic indicates that the population of sumatran elephants is -.78871, which means there is no inbreeding, but not significant at p> 0:10. the result of genetic distance calculations, showed that genetic distances within regions/ populations in bentayan, bukit salero lahat, seblat, sugihan and way kambas ranged from 0.0000 0003, is the genetic distance between the populations that is 0.0000 0.0022. thus, both genetic distance within populations and among the different populations of sumatran elephants are very low. the distance is also counted among the eight haplotypes, consisting of 6 haplotypes (bp, bt, bs, br, bx, by) which are found in the sumatran elephant, bd (borneo haplotype) and bu haplotypes reported by fernando . (2000 & 2003), and vidya (2009 in sumatra. the highest haplotype distance shows 0.17 and the lowest shows 0.0002. a limitation with this type of analysis concerns the maternal inheritance of mitochondria as well as the population structure of elephants with dominant females. asian elephants live in matriarchal societies consisting of family groups of several related females and their (juvenile) offspring. males leave these groups when they reach puberty (fernando & lande 2000; vidya & sukumar 2005). hence, although less commonly used, ychromosome characters are required in its context as male lineage (paternal lineages) just like mitochondrial dna in female lineage (maternal lineages). therefore, additional study based on y chromosome may be needed to obtain a more comprehensive results in this study. figure 2 shows the median joining network (bandelt . 1999) by using the network version 4.5.1.6 (http://www.fluxus-engineering.com). most of the sampled individuals are the haplotype bt. the haplotype bx and by are novel haplotypes. from figure 1, the haplotype bx was derived from the haplotype bt, and the haplotype by was derived from the haplotype bs by one transversion, respectively. the other substitutions in this network were transitions. et al et al et al. ) et al mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 97 f ig u re 1 . n ei g h b o u r jo in in g t re e (n j tr ee ) o f s u m at ra n el ep h an ts an d o th er a si an el ep h an ts fr o m m al ay ,c o n ti n en ta la si a an d b o rn eo . s e 1 9 w k 2 1 w k 2 0 w k 1 9 w k 1 8 w k 1 7 w k 1 6 w k 1 5 w k 1 4 w k 1 3 w k 1 2 w k 1 1 w k 9 w k 8 w k 7 w k 6 w k 5 w k 3 w k 2 w k 1 s u -1 8 s u -1 7 s u -1 6 s u -1 5 s u -1 4 s u -1 3 s u -1 1 s u -1 0 s u -9 s u -8 s u -7 s u -6 s u -5 s u -4 s u -3 s u -2 s u -1 s e -2 1 s e -2 0 s e -1 8 s e -1 7 s e -1 6 s e -1 5 s e -1 4 s e -1 3 s e -1 2 s e -1 1 s e -1 0 s e -9 s e -8 s e -7 s e -6 s e -5 s e -4 s e -3 s e -2 s e -1 b s l -1 9 b s l -1 8 b s l -1 7 b s l -1 6 b s l -1 5 b s l -1 4 b s l -1 3 b s l -1 2 s u -1 2 s u m a te ra biotropia vol. 19 no. 2, 2012 98 b s l -1 1 b s l -1 0 b s l -9 b s l -8 b s l -7 b s l -6 b s l -5 b s l -4 b s l -3 b s l -2 b s l -1 b e -2 0 b e -1 9 b e -1 8 b e -1 7 b e -1 6 b e -1 5 b e -1 4 b e -1 3 b e -1 2 b e -1 1 b e -1 0 b e -9 b e -8 b e -7 b e -6 b e -5 b e -4 b e -3 b e -2 b e -1 h a p lo ty p e b t h a p lo ty p e b u h a p lo ty p e b s w k -1 0 w k -4 w k -2 2 h a p lo ty p e b r b s l -2 1 b s l -2 2 b s l -2 4 h a p lo ty p e b q h a p lo ty p e b v h a p lo ty p e b p b s l -2 0 b s l -2 3 h a p lo ty p e b k h a p lo ty p e b j h a p lo ty p e b i h a p lo ty p e b h h a p lo ty p e b m h a p lo ty p e b f h a p lo ty p e b a h a p lo ty p e b b h a p lo ty p e b l h a p lo ty p e b e h a p lo ty p e b n h a p lo ty p e b o h a p lo ty p e b d h a p lo ty p e a f h a p lo ty p e a g h a p lo ty p e a e h a p lo ty p e a h h a p lo ty p e a c h a p lo ty p e a d h a p lo ty p e a b h a p lo ty p e a a w o o ly m a m m o th fo re s t e le p h a n t s a v a n n a e le p h a n t 0 .0 0 5 9 8 9 7 9 9 6 3 5 6 6 8 7 4 6 5 5 2 6 6 8 59 6 9 6 9 7 7 4 6 0 m a la y c o n ti n e n ta l c o n ti n e n ta l b o rn e o mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 99 table 3. genetic diversity indices of sumatran elephants region* hd (haplotype diversity) (nucleotide diversity) number haplotipe haplotype ** way kambas/wk (22) 0.255±0.116 0.00056±0.00028 3 bs(0.09), bt(0.864), by (0.045) sugihan/su (18) 0 0 1 bt(1.00) seblat/se (21) 0.095±0.084 0.00016±0.00014 2 bt(0.952), bx (0.048) bukit salero lahat/bsl (24) 0.366±0.115 0.00309±0.00098 3 bp(0.792), br(0.125), bt(0.08 bentayan/be (20) 0 0 1 bt(1.00) sumatera (105) 0.164±0.049 0.00092±0.00032 6 bp(0.019), br (0.01), bs(0.019), bt(0.914), bx(0.01), by (0.01) note: hd: haplotype diversity (expressed as average ± 1se) nd: nucleotide diversity (expressed as average ± 1se) * the number of individuals sampled are shown within parentheses ** the frequencies of the haplotypes are shown within parentheses. the bold characters indicate the novel haplotypes. figure 2. median joining network bt bs br bp bx by continental typesumatera type wk se bsl su be transition transversion note: sampling locations: wk = way kambas, su = sugihan , se = seblat, bsl = bukit selero lahat, be = bentayan haplotypes: br, bx, bt, by biotropia vol. 19 no. 2, 2012 100 conclusions acknowledgments references based on the analysis of mitochondrial dna d-loop sequence from 105 samples of sumatran elephants, it was concluded that sumatran elephants within populations and among populations in sumatra exhibit only limited or very low genetic diversity. this study provides a first definition of mtdna diversity and is an important contribution. bp, br, bs, and bt haplotypes, and 2 new haplotypes (bx and by) are found in the sumatran elephant. the distance between haplotypes of sumatran elephant's population is also low. moreover, additional samples may be needed to obtain significant conclusions. a more comprehensive conclusion may be obtained if also y chromosome markers and autosomal markers are analyzed. many people contributed generously to this study. we are grateful to prof. m. hasegawa and t. yonesawa, ph.d for encouragement and their assistance in data analysis and a the processing of this manuscript. this study was supported by 2010 grant through seameo biotrop to sri sulandari,. we express our appreciation to t. okayama, ph.d for collecting material samples of sumatran elephants. ms. anik dhamayanthi, m.si. and ms. inda natalia, m.si. are also acknowledged for laboratory assistance. dipa akishinonomiya f, miyake t, sumi s, takada m, ohno s, kondo n. 1994. one subspecies of the red jungle fowl ( ) suffices as the matriarchic ancestor of all domestic breeds. in: proceedings national academy of sciences usa. 91: 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(2005a). population differentiation within and among asian elephant ( ) populations in southern india. journal heredity 94 (1), 71-80. vidya tnc, fernando p, melnick dj, sukumar r. 2005b. population genetic structure and conservation of asian elephants ( across india. anim conserv 8: 377-88. vidya tnc, varma s, dang nx, thanh tv, sukumar r. 2007. minimum population size, genetic diversity, and social structure of the asian elephant in cat tien national park and its adjoining areas, vietnam, based on molecular genetic analyses. conserv genet 8:1471-78. vidya tnc, sukumar r, melniek dj. 2009. range-wide mtdna phylogeography yields insights into the origins of asian elephants. proc r soc b 276: 893-902. doi:10.1098/rspb.2008.1494, published online 18 november 2008. evolution elephas maximus elephas maximus elephas maximus elephas maximus) biotropia vol. 19 no. 2, 2012 102 3. iman rusmana page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 3. etty riani.cdr page 1 page 2 page 3 page 4 page 5 microsoft word 47 biotropia no. 19, 2002 : 47 56 plojdy analysis and dna content of mutant banana "pisang berangan" using flow cytometry abdelgadir rayis shadia, rofina yasmin othman and mak chai division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, kuala lumpur, malaysia abstract mutagens cause random changes in the nuclear dna or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations and hence, create variability. in this study, flow cytometry (fcm) was used to determine ploidy levels and dna content in gamma-irradiated variants of mutated pisang berangan (cv. intan, aaa) a local banana genotype. induced variants such as short plant stature (stunted growth), late flowering plants (late maturity) and abnormalities in bunch characters were selected to study possible changes at the dna level. the study showed that dna content of mutated plants differed from non-irradiated control and that irradiation had the most effect at high doses (40 and 60 gy). the increase of dna content in 20 gy and 30 gy treated plants was not more than that of the control plants. the values of genomic dna content of gamma-irradiation variants decreased as the dose of irradiation increased from 20 to 60 gy, indicating that the high dose of gamma-irradiation had a significant effect on the genome of the plants. the analysis further showed that phenotypic variation due to mutagenesis was reflected in the dna content of the plants. the results also showed that ploidy levels were not affected by gamma-irradiation even at high doses. keywords: musa spp./mutation breeding/ flow cytometry/ ploidy level/ dna content introduction banana is the most important fruit crop in the world (kaemmer et al. \ 997). it forms the primary food source for millions of people in many parts of the world and ranks next to rice and wheat (inibap 1992). the cultivated bananas are mostly triploid (2n=3x=33) and exhibit a marked degree of sterility. sterility is a main problem of banana breeding, which is very complex depending on meiotic irregularities (novak 1992). therefore, genetic improvement has been difficult due to lack of useful genetic variability and low level of female fertility. hence, the potential of biotechnological techniques to complement conventional musa breeding has been widely investigated (wiame et al. 2000; vuylsteke 1998; ortiz & vuylsteke 1996). these techniques involved in vitro multiplication, mutation breeding and genetic manipulation. popular clones planted in malaysia on a commercial scale are mainly cavendish bananas and pisang berangan cv. intan (aaa) (mak et al. 1995). of the total cultivated areas almost half are cultivated with pisang berangan and the cavendish types for both local consumption and export (siti hawa 1998). pisang berangan has good fruit quality, colour, texture, size and shelf-life, in addition to its ease of ripening without cold treatment. the plants are however, relatively tall with mode 47 biotropia no. 19,2002 rate yield and are very susceptible to a number of diseases particularly fusarium wilt caused by fusarium oxysporum f. sp. cubense. pisang berangan is a sterile tri-ploid banana; therefore, complementary techniques such as tissue culture and mutagenesis are of great potential. induced mutation in bananas is an attractive means to generate genetic variability. mutation induction coupled with in vitro techniques induces morphological changes and also increases variability in quantitative traits. physical mutagens, such as gamma ray, are tools for enhancing and generating genetic variations by inducing mutations at the gene, chromosome and genome level, in nuclear and cytoplasmic organelle dna (larkin 1998). somaclonal variation provides additional source of variation. off-types have been noted for plant stature (mainly dwarf), leaf foliage, pseudostem and bunch characters (small bunches, short fingers, long peduncle and elongated male buds). the dna nuclear content of various banana varieties were not explored or determined until the use of flow cytometry method for plant was introduced in the early 1980s. flow cytometric analysis of nuclear dna content provides an alternative for individual and largescale ploidy screening (dolezel 1991). the technique first developed for analysis of human cells has been adopted as a convenient tool for estimation of nuclear dna content and ploidy level constitutions in plants (dolezel 1991; dolezel et al. 1994; dolezel et al. 1997). dolezel et al. (1994) modified the technique for analysis of nuclear genome of bananas and plantains. the method that was used to screen ploidy levels of plants regenerated from in vitro culture was also used to estimate genome size and its variation in musa. this paper presents the results of a study to evaluate the variability in pisang berangan cv. intan (aaa) generated by gamma irradiation in terms of changes in nuclear dna content and chromosome level by flow cytometric analysis. materials and methods vigorous and disease-free suckers of pisang berangan (aaa) were selected from fieldgrown, true-to-type, fruit-bearing mother plants. shoot tip meristem (approximately 5x5x5 mm) was removed with a sterilized scalpel, then transferred directly to modified ms medium. subculturing was carried out after 4-6 weeks or earlier depending on blackening of explants. shoots initials developed after 3 weeks and were proliferated on induction medium with the bap concentration of 4.5 mg/l. multiplication of propagules was carried out by subdividing the newly formed shoots or bud clusters and reculturing them on fresh medium at 4-6 week intervals up to sub-culture 6. ms medium was then supplemented with 2mg/l 1aa and 5mg/l kinetin for rooting. root generation took 6-8 weeks until plantlets reached the optimal size of 4 -5 cm. the shoot-tip meristem pieces (about 1cm x 2mm) were aseptically excised from micropropagated plantlets of pisang berangan. each shoot-tip was cut longitudinally into two pieces and then transferred to sterile moist petri dishes. each 48 ploidy analysis and dna content of banana mutant — abdelgadir rayis shadia et al. petri dish contained 10 meristem pieces and was sealed with parafilm. gamma induction was then carried out at the malaysian institute of nuclear technology research (mint). the meristem pieces were irradiated in a gamma cell with a cobalt-60 (60co) source (gc 400a, 10 kci). the doses included 0 (control), 20, 30, 40 & 60 gy at a dose rate of 0.54 gy/sec. the irradiated meristem pieces were washed thoroughly with sterile distilled water and cultured on modified ms solid medium (ho & tan 1990). sub-culturing was carried out at 4 6 week intervals until generation m|v4 i.e. three sub-cultures. the explants after mivj were allowed to root in rooting medium containing ms salts. after three to four weeks in rooting medium, the plantlets were deflasked and transplanted in the nursery for acclimatization or hardening (8-9 weeks). the plantlets were maintained under 70% -80% shade (using shading net) and high humidity. these materials were generated for field evaluation at the farm. the plants were grown in rows spaced at 2.5m x 2.5m in a triangular pattern with completely randomized design. abnormal and mutant plants identified from different gamma-dose treatments (20, 30, 40, and 60 gy) were selected from in vitro plantlets, glass house-grown and field-grown plants. plant materials and sample preparation for determination of ploidy level and identification of nuclear dna content by fcm, nuclei were isolated from young leaves following -the method developed by gailbraith et al. (1983). approximately 40 50 mg of young leaves from each sample (mutated & glycine max var. palmetto) were chopped up together with a sharp scalpel blade in a glass petri dish containing 1 ml lboi lysis (dolezel 1991), supplemented with 50 ug/ml each of propidium-iodide (pi) and rnase. glycine max was used as internal reference standard for identifying the nuclear dna content by fcm. an intact interphase nucleus was released from the cut surface directly into 2-ml lysis buffer supplemented with 0.1 mg/ml of 4,6-diamidino-2-phenylindole (dapi). the homogenate-stained nuclei were filtered through 50 jam nylon mesh into the analysis tube and its fluorescence was analyzed by fcm. instrument setting and alignment the fluorescence intensity of dapi-stained nuclei was measured using a partec ca11 flow cytometer (partec gmbh, munster, germany), that uses high-pressure mercury arc lamp (hbooow/2l), as an excitation light source. these were filtered through a combination of optical filters: kgi, bg38 and ugi for excitation: tk560 as dichronic mirror, and a long-pass emission filter of rg590. autoclaved deionized water was used as a sheath fluid. sample analysis about 2 ml of the homogenate nuclei was analyzed in the cell counter analyzer cytometer (model partec cca-ii). the fluorescence was measured with a 100 w high-pressure mercury arc lamp as a light source filtered as mentioned above. 49 biotropia no. 19,2002 the dna genome size of the sample was then estimated using the ratio of gl peaks. the nuclear dna content was quantified by the following formula 2c nuclear dna content fpg] = 2.5/fluorescence ratio* * fluorescence ratio = peak mean of musa____ peak mean of glycine max symbol c is used for dna content of haploid set of chromosomes. glycine max has 2c dna content = 2.5 pg (tiersch et al. 1989). results and discussion mutagenic treatment may cause histogenic disturbances. at the molecular level, mutagens cause random changes in the nuclear dna or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations that create variability. the changes may be a result of alterations of the dna sequence of gene such as gain, loss or substitution of one base pair by another. therefore, monitoring of mutagen-induced dna damage is potentially very useful in mutation breeding studies. in this study, mutation induction was used to generate variability in pisang berangan (aaa). at high doses of irradiation, too many mutational events per cell may be induced, with increased risk that a favourable mutation is accompanied by one or more unfavourable genetic changes. however, in vegetatively propagated crops (bananas) it is impossible to separate favourable from unfavourable mutations by cross-breeding mutants among each other or by back-crossing with the original material. therefore, the evaluation of mutants is very difficult unless alternative techniques such as analysis of dna content or molecular fingerprinting are used. to a certain extent, the evaluation of mutants can also be phenotypically possible depending upon certain characteristics when compared with control plants. flow cytometry (fcm) was used in this study to determine ploidy levels and dna content in gamma-irradiated (pisang berangan) variants. the variants chosen for dna analysis include those with short plant stature (stunted growth), late flowering (or late in maturity) and abnormal bunch characteristic (table 1). the selection of short stature plants was based on two main categories, severely stunted and weak (plant height were < 75 cm) and short plants measuring 150-170 cm. while the selection of lateness in flowering was compared with control plants. the late flowering types ranged from 380 to 465 days. some plants showed more than one variant character such as short plants that were also late in maturity or even failed to bunching. the number of variants with bunch and finger abnormalities recorded was few and included those with short compressed bunch with few hands or combs (< 4), bunch with long peduncle and abundant persistent flowers at the distal end of the bunch stalk, bunch with male flowers only or bunch with gaps (missing combs), fused fingers and long and thin fingers. ploidy analysis and dna content of banana mutant abdelgadir rayis shadia et at. flow cytometric analysis for ploidy and dna content the most reliable conventional method of ploidy level analysis is the counting of chromosomes of metaphase plates by using root tips. however, the preparation and microscopic analysis is time consuming, and a difficult task as banana chromosomes are extremely small, indistinguishable and frequently few cell divisions are visible in a single root tip. this has led to the development of flow cytometric techniques for rapid routine ploidy determination in musa (dolezel et al. 1994). in addition, flow cytometry is recommended for the accurate estimation of nuclear dna content (novak 1992; dolezel et al. 1989). flow cytometry involves the analysis of intact nuclei, hence dividing cells are not required, and the analysis is not limited to meristematic tissues. moreover, a small amount of fresh leaf tissue (5 50 mg) is sufficient, that means this method could be applied to plants at the early stage of development. analysis of the relative fluorescent intensity of propidium-iodide stained nuclei yielded a histogram showing two dominant peaks corresponding to g\ and g2 nuclei of pisang berangan and glycine max cv. palmetto, respectively. the majority of cells in full-grown plants does not participate in cell division and reside in a so-called go (g is gap) stage of the cell cycle. at this stage, the nuclear dna content reflect the ploidy state of the plant. cells, which are involved in divisions, start from a comparable so-called gi state and subsequently pass through s (= dna-synthesis), g2 (= an interphase nuclear stage with a doubled dna content preceding the actual nuclear division). accordingly nuclear dna content in absolute units (genome size in picogram-pg dna) was adopted for all samples used. such estimation requires comparison with a reference standard having a known dna content. in this study, glycine max was used as an internal standard because its genome size is relatively constant (dolezel et al. 1994, vinderlov et al. 1983) and to avoid bias due to staining and instrumental changes when estimating nuclear dna 44 biotropia no. 19,2002 ploidy by flow cytometric analysis. the precision of the ratio between the standard and the sample (pisang berangan) reflects the accuracy of dna content measurements. estimation of ploidy level was done by external standardization procedure comparing the position of the g| peak on a histogram to that of a reference plant with known ploidy. the results showed no significant differences in ploidy level between all samples used (3n), indicating that irradiation at the. doses used did not have an effect on ploidy. differences were, however, found between gamma-irradiated berangan variants with respect to dna content. dolezel (1995) reported similar findings in his analysis of a large populations of nuclei (5-2000) of mutated african plantains in which the occurrence of even small subpopulations differing in ploidy levels (mixoploidy) can be detected. the applications envisaged include control of ploidy level stability and screening for novel ploidy levels. figure (1) shows flow cytometric analysis of genomic dna content of pisang berangan nuclei and from the reference (glycine max cv. palmetto) stained with propidium iodide (pi). the nuclei isolated from musa and glycine, were analyzed simultaneously. most of the nuclei were in g, phase of the cell cycle. the first peak (channel 75) represented g, nuclei of musa, while the second peak represented g, nuclei of glycine. g, peak of pisang berangan was positioned on channel 75, and the other peak of glycine max was on channel 100. a third peak also appeared at channel 200 reflecting the g2 phase of the reference internal standard nuclei. ploidy analysis and dna content of banana mutant abdelgadir rayis shadia i et al. a measurement for the resolution of the histogramscoefficient of variation (cv)shows the variation of the analysis from the control values. overall means of g] peaks of pisang berangan ranged from 1.3% to 2.34%, whereas in glycine plants, it ranged from 1.02% to 1.67%. the estimated values of peak ratio of these samples could be used to discriminate between gamma-irradiated variants and non-irradiated plants. the peak appeared in the histogram, g[ for pisang berangan and g| for glycine max, indicated that soybean has more dna content than berangan. higher cv in berangan indicated that variation due to mutagenesis occurred within these samples. the discrepancies between observed and expected values could indicate the occurrence of changes in dna content due to effects of gammairradiation. all selected variants (short stature, late flowering and bunch abnormalities) showed dna content different from control plants. to investigate the significance of differences between observed frequencies arranged in these three characters the use of contingency x2-test for independence was performed. the test showed that there is a differential effect of gamma irradiation on these characters, but the two sets of variables are independently acting. dna content of different types of variants induced by gamma-irradiation (mutants) all selected mutants (short stature, late flowering and bunch characteristics) showed variation in dna content. an effect of irradiation appears to be reflected by an increase in variation of nuclear dna content (increase of coefficient of variation of g| and g2 peaks). the mean of the distribution of g0/g, nuclei represents a 2c (presynthetic level) dna amount in relative units. the ratio of g0/ g, peak means (musal glycine) was calculated and dna content of musa was estimated according to the formula: dna content (pg) = 2.5/peak ratio (dolezel et al. 1994). the ratio of gl peak means for non-irradiated control plants was gi of glycine/ g, of musa i.e. 100/75 = 1.333. the 2c dna content of pisang berangan (aaa) was calculated using the ratio of gl peak means: 2.5/1.333=1.875 pg dna or 1.875 ± 0.02 pg. the estimated value of peak ratio of these plants did not differ from the mean value obtained by fcm analysis (1.85 pg). the estimated value of dna content (pg & mbp) for different variants chosen from each group is shown in table 2. short plant variants (gamma-irradiated at different doses) displayed a wide range of dna content (1.0% 8.0%) as compared with non-irradiated plants (control). the variants showing lateness in flowering showed moderate changes in dna content as compared to short stature variants (0.8% 2.7%). bunch abnormalities variants showed slight to moderate changes in dna content as compared to control plants with dna content differences ranging from 0.1% to 1.1%. the mann-whitney test (non-parametric and distribution-free statistics) showed that there were no significant differences between the three characters in 53 biotropia no. 19,2002 dna content at (0.005 >p >0.001). on the other hand, phenotypic variation due to mutagenesis is reflected in the dna content of plants. table 3 demonstrates that the degree of variation in dna content values increased as the dose of gamma-irradiation was increased from 20 gy through to 60 gy. the dna content at 20 & 30 gy treatments differed from that of nonirradiated plants by 2.5% and 2.8%, respectively. whereas, 40 gy and 60 gy-variants showed more changes (4.7% and 4.5% for 40 gy & 60 gy treatments, respectively) (table 3). 54 ploidy analysis and dna content of banana mutant — abdelgadir rayis shadia et al. the relationship between short stature variants and high doses of irradiation was apparent. the majority of short stature or stunted growth variants were selected from 40 & 60 gy treatments. (table 1). other variants such as late maturity and bunch-abnormalities also showed decrease in genomic dna content ranging from 2% to 4%, suggesting that the variation in dna content has phenotypic effects via its influence on cell size and mitotic cycle time (van harten 1998). the results indicate that gamma ray has a direct effect at the dna level of pisang berangan and that this variation can be effectively measured using the flow cytometric technique. the high range of phenotypic changes observed at 40 gy and 60 gy treatments might be due to broken segments lost or translocated or may originate from deletions in the nucleotide sequences in structural genes, changes in the promoter sequences, deletions of the introns, regulating gene and repressers, which may either cause frame shift mutations or lead to the production of modified gene expression. it is estimated that at least 90% of the radiationinduced mutations refers to deletions (van harten 1998). in addition, duplication may occur resulting in an increase in the amount of dna content control (insertion). this phenomenon might be due to jumping genes or double replication in dna (copied twice) induced by irradiation. this technique has immediate application for selection of potentially useful mutants for pisang berangan breeding programme. it may be expected that the number of practical applications will increase and plant breeders will even more extensively use flow cytometry. acknowledgements i would like to express my gratitude to the malaysian institute for nuclear technology research (mint) and mr. azhar mohamad for the facilities and assistance throughout the duration of experiment. also to prof. t. k. mukheraji (institute of biological science) for his advice in statistical analysis. references dolezel, j. p. binarova and s. luretti. 1989. analysis of nuclear dna content in plant cells by flow cytometry. bio. plant. 31: 133-120. dolezel, j., m.dolezelova and f. novak. 1994. nuclear dna amount in diploid bananas (musa accuminata and m.balbisiana). biol. plant. 36: 351-357. dolezel, j. m. a. lysak, i. van den houwe, m. dolezelova, n. roux. 1997. use of flow cytometry for rapid determination in musa species, infomusa. 6: 6-9. dolezel, j., 1991. flow cytometric analysis of nuclear dna content in higher plants phytochem. analysis 2: 143154. 55 biotropia no. 19,2002 dolezel, j. 1995. application of karyology and cytometry in mutation breeding of african plantain in vitro. fao/iaea research co-ordination meeting on: cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes. fao/iaea, 1995, p. 13-22. gailbraith, d. w., k. r. harkins, j. m. maddox, n. m. ayres, d. p. sharma and e. firoozabady. 1983. rapid flow cytometric analysis of the cell cycle in intact plant tissues. science, 220: 1049-1051. ho, y. w. and y. p. tan. 1990. performance of tissue cultured bananas. perak planters annual report 1990: 6775. inibap, 1992. banana and plantainfood for thought. in: annual report inibap, montpellier, france. kaemmer, d., d. fisher, r. l. jarret, f. c. baurens, a. grapin, d. dambier, j. l. noyer, c. lanaud, g. kahl and p. j. l. lagoda 1997. molecular breeding in the genus musa: a strong case for stms marker technology. euphytica 96: 46-63. larkin, p. j.. 1998. induced mutation for crop improvement. in: somaclonal variation and induced mutations in crop improvement, (eds) by jain, s. m.; d. s. brar and b. s. ahloowalia (1998). p 3 13. mak c., y. w. ho, y. p. tan and r. ibrahim. 1995. novariaa new banana mutant induced by gamma irradiation. infomusa 4: 1. novak, f.j., 1992. musa (banana and plantains) in: biotechnology of perennial fruit crops, p.449-488 (eds f.a. hammerschlag and r.e litz) wallingford: c.a.b. international. ortiz, r. and d. vuylsteke. 1996. recent advance in musa genetics, breeding and biotechnology. plant breeding abst. 1996,66: 1355-1363. siti hawa, j. 1998. commercial exploitation of the banana diversity in malaysia. proceedings of the first national banana seminar (genting highland) 23-25 nov. 1998. tiersch, t. r., r. w. chandler, s. s. watchel and s. elias. 1989. reference standards for flow cytometry and application in comparative studies of nuclear dna content. cytometry, 10: 706-710. van harten, a. m., 1998. mutation breeding. theory and practical applications, cambridge univ., p. 163-203. vinderlov l. l., i. j. christensen and n. i. nissen. 1983. standardization of high-resolution flow cytometric dna analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. cytometry 3: 328-331. vuylsteke, d. r., 1998. field performance of banana micropropagules and somaclones. in: somaclonal variation and induced mutation in crop improvement (eds) by s. m. jain; d. s. brar; and b. s. ahloowalia) p. 219-231. wiame i., r. swennen, l. sagi, plas-lhw-van-der, j. j. dons, j. vanderleyden and m-de loosse. 2000. pcrbased cloning of candidate disease resistance gene from banana (musa acuminata). in: proceedings of the xxv international horticultural congress. part ii. application of biotechnology & molecular biology and breeding, genome analysis, brussels, belgium, 2-7 august, 1998. acta-horticulturae, 2000. 521,51-57. 56 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf 4. i made (lipid) revisi.cdr biotropia vol. 21 no. 2, 2014: 100 110 lipid accumulation by ath using palm oil mill effluent as substrate flavodon flavus i made sudiana , atit kanti , helbert , senlie octaviana and suprapedi received 3 april 2014/accepted 6 october 2014 large amount of palm oil mill effluent (pome) is generated annually. the waste would be potential for production of single cell oils (scos). the objective of this study was to evaluate lipid accumulation by fungi using pome as substrate. seven filamentous fungi were initially isolated from various biomes. the study results showed that out of these 7 fungi, five of them produced endoglucanase and accumulated lipid about 34.3-87.5% of their dry cell mass using pome as substrate. the five fungi were identified as ath, sp., sp., sp. 1 t30, and sp.2 t50. the highest lipid accumulation was obtained by ath. the profile of trans-esterified scos revealed high content of saturated and mono-unsaturated fatty acids i.e., palmitic (c16:0), stearic (c18:0) and oleic (c18:1) acids similar to conventional vegetable oils used for biodiesel production. the strain that was able to use organic substrates in pome indicated that they are promising strain for biofuel feedstock as well as for fulfilling effluent quality for wastewater discharge. fatty acid methyl ester, , oleaginous fungi, palm oil mill effluent (pome), single cell oil *1 1 1 1 2 1 2 research center for biology, indonesian institute of sciences, cibinong 16911, indonesia research center for physic, indonesian institute of sciences, komplek puspiptek tangerang selatan, banten, indonesia flavodon flavus aspergillus trichoderma fusarium fusarium flavodon flavus flavodon flavus abstract introduction keywords: as awareness on the limited stock of fossil fuel and concern on environmental hazard of petroleum fuel is growing, research on sustainable biofuel is gaining its popularity (richana 2010; fujita 2004; wraight & ramos 2005; jing 2010; mohammadi 2011). microbial based biofuel (micro-diesel) has become more attractive due to its environmental incentive and renewable properties (zheng et al. et al. et al. et al. * corresponding author : imadesudianalipi@gmail.com doi: 10.11598/btb.2014.21.2.4 100 2012). the merit of micro-diesel over petroleum fuel is clear, but our understanding on microbial diversity and physiology of oleaginous microbes is still incomplete to produce economically viable micro-diesel (zhao . 2012). current practice of biodiesel production is through trans-esterification of vegetable oils or animal fats with short chain alcohols. feedstock acquires more than 70% of total biofuel production cost, and this limit further expansion of biodiesel (wei . 2013). exploiting lipid accumulating microorganism would offer solution for feedstock generation (wang . 2011). some lipid accumulating fungi i.e. , sp., var. , sp., sp., sp. and sp. accumulate large amounts of lipids greater than 40% per dry cell weight (wei 2013; wynn . 2001) under n-limited cultivation conditions. oils derived from microbes have many advantages over plant oil due to having short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up (wang . 2013). these oleaginous characters would place microbial oils as potential feedstocks for biodiesel production in the future (liu . 2012). lignocellulose waste are abundant in tropical region. several microbes use lignocelluloses hydrolysate for biofuel production (tsigie . 2011). pome contains high strength organic substances with total chemical oxygen demand (cod) about 30,000-40,000 ppm and 3% lipid (wu . 2010). utilizing these wastes for biofuel production offer manifold benefits through reducing total organic in effluent, and generate biofuel feedstock via bioconversion palm oil waste into triacyl glycerol rich microbial cell (coleman 2004). fungi would be good candidate for biofuel feedstock, since some fungi produce extracellular cellulase which break down complex polysaccharide into fermentable sugar (goyal . 1991), consume it for lipid synthesis and finally accumulate it into lipid bodies which primarily consist of triacylglicerol (rossi . 2010). this study aims to evaluate the lipid accumulation by fungi using pome as substrate. this study initially isolated 7 filamentous fungi from various biomes as listed in table 1. et al et al et al cunninghamella echinulata microsphaeropsis mortierella isabellina, m. ramanniana angulispora mucor circinelloides, phomopsis cephalosporium sclerocystis nigrospora et al. et al et al et al et al et al et al et al materials and methods fungi species used table 1. fungi used in this study name of species fungi sources sp. soil of cibinong west java ath decaying wood of south east sulawesi comp insect frass sp1 t30 sludge of wastewater treatment in west java sp2 t50 sludge of wastewater treatment in west java sp soil of cibinong west java sp. soil of cibinong west java aspergillus flavodon flavus paecilomyces lilacinus. fusarium fusarium trichoderma mucor 101 lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. culture conditions determination of cellulolytic fungi endoglucanase assays biomass the fungal inoculants of the 7 filamentous fungi was prepared following mulder (1989) and kimura . (2004). briefly, fungal spores that had been incubated at 30 °c for 5 days on potato dextrose a l ×10 spores/ml was inoculated into 150 ml of the seed culture medium containing (g/l): glucose, 30; yeast extract, 5; kno , 1; kh po , 2.5; znso ·7h o, 0.01; cuso ·5h o, 0.002; mnso , 0.01; mgso ·7h o, 0.5; feso ·7h o, 0.02; and cacl , 0.1. the initial ph of the medium was adjusted to 5.5 by adding 10 m naoh in a 500 ml flask and incubated at 30 °c with shaking at 125 rpm for 24 hours. then, 10 ml of the seed culture was transferred into 250 ml of growth medium containing pome obtained from pt perkebunan nusantara, malimping, west java. prepared medium was autoclaved for 20 minutes before use. the culture was incubated at 30 °c with shaking at 125 rpm for 24 hours. the ability of fungi to hydrolyze cellulose was evaluated using cmc containing media following zhou . (2004). to evaluate the effect of temperature on the endoglucanase activity, the cultures were separately incubated at 30 °c and 50 °c with shaking at 125 rpm for 5 days. enzymatic activity was assayed following zhou . (2004). briefly, the culture were placed in 50 ml centrifuge tubes, then centrifuged at 2,500 g at 4 ºc for 30 minutes activity measurements, 2% carboxymethylcellulose (cmc, sigma) was l l l s were incubated at 50 ºc for 30 minutes. reducing sugars were determined using the 3,5-dinitrosalycilic acid (dns) assay according to dinis , (2009). l utes (after adding the supernatant to the reaction mixture) up to 45 minutes l utes and immediately cooled on ice for 5 minutes. finally, l was measured at 540 nm in a spectrophotometer (uv mini 1240 shimadzu). one enzyme unit is defined as mmol glucose produced by 1 ml enzyme per hour. biomass fungal was determined according to ogbo (2010). briefly, biomass was harvested by filtration, and fungal pellet were washed with 50 ml deionized water for removal of residual nutrients, and lyophilized at -50 °c in a vacuum of 1 mbar for 12 hours and weighed. fungal biomass was expressed as grams of dry weight per liter of culture medium. et al. et al et al et al et al. gar plates (difco laboratories, detroit, mi, usa) were harvested and suspended in seed culture medium. a spore suspension (500 μ ) containing approximately 4 . supernatants were clarified by filtration through 0.45 μm nitrocellulose filters (pall). for enzymatic dissolved in 50 mm citrate buffer ph 5. enzymatic reactions contained 200 μ of supernatant, 300 μ of 50 mm citrate buffer ph 5, plus 500 μ of each substrate solution. the reaction mixture briefly, 50 μ aliquots were taken every 5 min , then mixed with 50 μ of a dns solution, boiled for 5 min 500 μ of water were added and absorbance 6 3 2 4 4 2 4 2 4 4 2 4 2 2 biotropia vol. 21 no. 2, 2014 102 lipid concentration lipid composition analysis the lipid concentration of fungi broth was determined from a standard curve obtained by plotting absorbance against the corresponding lipid concentration determined by the conventional method of acid hydrolysis followed by solvent extraction and gravimetric estimation. forty milligrams samples were extracted with 3 ml of chloroform/methanol (1/2, v/v) by vortexing (1 minute) and centrifugating at 2,500 g for 15 minutes at room temperature. the supernatants were collected and residues were re-extracted twice with 2 ml of chloroform/methanol (1/1, v/v) by centrifugation as stated above. all the supernatants were pooled together, filtered with whatman filter no. 1 (whatman, usa), and washed with 2 ml of milli-q water, followed by centrifugation at 2,500 g for 5 minutes. the lower organic phases were collected and evaporated to dryness under nitrogen and total lipid contents were determined gravimetrically (sitepu . 2013). the total lipid concentration was determined by gas chromatographic analysis of the total fatty acids directly trans-methylesterified from dried cell (liu 2008 ; kosa & ragauskas 2011). one milliliter of 10% methanolic-hcl and 0.5 ml methylene chloride were added to the dried biomass and placed at 60 °c for 3 hours for direct methyl-esterification. the reaction was stopped by the addition of 2 ml saturated nacl solution and 1 ml hexane. the resultant methyl esters recovered in the hexane layer were then applied to a gas chromatograph (gcms-qp 2010-ultra; shimadzu, kyoto, japan) equipped with a famewax capillary column (30 m× 0.25 mm i.d., gl science, tokyo, japan) under temperature programming (150-250 °c at 5 °c/minute increments). peanut oil (nacalai tesque, kyoto, japan) was trans-methylesterified and used as the reference material. overall, all fungi studied showed cmc-ase activity (fig.1), but the strains of sp., and comp grew slowly. therefore, we omitted these strains for further study. ath produced the highest cmc-ase at 96 hours incubation. other strains having slightly lower activity were sp. and comp. maximum activity attained at 96 hours, and decreased at 144 hours. the ability of cultures to produce cmc-ase indicated these fungi produced endoglucanases, ec 3.2.1.4, and played important role in cellulose hydrolyses (hasper 2002). kitcha and cheirsilp (2014) observed newly isolated fungi as being able to hydrolyze cellulose of palm by products. to gain cellulolytic character of fungi in this study, we further evaluated their ability to hydrolyze pome at 30 °c and 50 °c. et al et al. mucor paecilomyces lilacinus flavodon flavus aspergillus paecilomyces lilacinus et al. aspergillus tubingensis results and discussion lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 103 endoglucanase activity at 30 °c overall, endoglucanase activity of fungi grown in pome is summarized in figure 2. the highest endoglucanase activity was obtained by ath and sp. grown at 30 °c after 96 hours cultivation. slightly lower activity was observed on sp.2 st50. generally lower cmc-ase was observed at 144 hours fermentation. flavodon flavus aspergillus fusarium figure 1. cmc-ase (endoglucanase) activity of fungi grown on cmc-medium figure 2. endoglucanase activity of selected isolates grown on pome at 30 °c aspergillus sp. flavodon flavus ath paecilomyces sp. comp fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. mucor sp. aspergillus sp. flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. 104 biotropia vol. 21 no. 2, 2014 endoglucanase activity at 50 °c effect of growth temperature on endoglucanase activity endoglucanase activity of selected fungi grown on pome at 50 °c varies among fungal species depending on incubation period (fig.3). maximum activity was showed by ath at 96 hours incubation. lower activity was obtained after 144 hours cultivation. in general, cmc-ase activity was higher in cultures incubated at higher temperature (fig.4). almost 50% increase of cmc-ase activity was achieved by ath. fungi have been intensively explored for hydrolyses of lignocellulose materials. ath is wood decayed fungi isolated from south east sulawesi, indonesia. appears to be widely distributed lignocellulolytic fungi isolated from decaying sea grass from a coral lagoon off the west coast of india (raghukumar 1999; mtui & nakamura 2008) and produces extracellular lignin-modifying enzymes (lmes): manganese-dependent peroxidase (mnp), lignin peroxidase (lip), and laccase when grown in n-limited media (mtui & nakamura 2008). these enzyme characters would be advantageous for producing fermentable substances for triacylglicerol synthesis using lignin containing waste such as pome (duarte 2013; lam & lee 2011). we also noticed that a culture of ath having high cmc-ase or endoglucanase activity indicated that hydrolyses and product hydrolyses consumption affected enzyme synthesis (vlasenko 2010). flavodon flavus flavodon flavus flavodon flavus flavodon flavus et al. flavodon flavus et al. figure 3. endoglucanase activity of selected isolates grown on pome at 50 °c aspergillus sp.flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 105 lipid accumulation fatty acid profiles of fungal scos pome is good substrate for growing lipid accumulating fungi (fig.5), as shown by the lipid production (75% lipid per cell dry-weight). the highest lipid accumulation was attained by ath when grown at 30 °c, but less when the culture was grown at 50 °c. incubation temperature affected lipogenesis of fungi (fig.5). the suitability of scos from oleaginous fungi was evaluated and observed contained 92% (w/w) neutral lipids in its sco (wu 2011). other lipid producer, , contained lower neutral lipid fractions (18.5%, w/w), higher amount of polar lipids (35%, w/w ), and free fatty acids (32% w/w) in its sco (wynn . 2001). fatty acid profiles compositions of selected fungi were obtained through transesterification of triacyl glycerol with methanol under alkaline condition. fatty acid methyl esters (fame) was mainly composed of methyl palmitate ( c h o ), methyl cis-10-heptadecenoate (c h o ) and methyl oleate (c h o ) (table 2), indicating that the fame is closely related to palm and soybean fatty acids (ratledge & wynn, 2002). there was slight differences in concentration of lipid species among fungi evaluated (wynn 2001; khot 2012; chan 2010). the variability in fatty acid composition of oleaginous organism could be due to culture technique (chi 2011) and species dependence (gasmi . 2011). flavodon flavus cunninghamella echinulata et al. mucor circinelloides et al et al. et al. et al. et al. et al 17 34 2 18 34 2 19 36 2 figure 4. comparison of endoglucanase activity of selected isolates grown on pome at 30 °c and 50 °c aspergillus sp. flavodon flavus fusarium sp1 fusarium sp2 trichoderma sp. aspergillus sp. flavodon flavus fusarium sp1 fusarium sp2 trichoderma sp. 106 biotropia vol. 21 no. 2, 2014 figure 5. lipid accumulation by fungi grown on pome at 30 °c and 50 °c after 6-day fermentation table 2. lipid composition of fungi grown on pome cultured at 30 °c after 6 days fermentation fatty acid methyl esters (fame) trichoderma sp. flavodon flavus ath sp. fusarium sp1 t30 fusarium sp2 t50 methyl palmitate(c17h34o2) 31.39 27.05 23.62 21.36 20.21 methyl palmitoleate(c17h32o2) 29.65 26.28 21.08 21.08 20.68 methyl cis-10heptadecenoate (c18h34o2) 3.1 5.32 4.36 5.32 4.32 methyl stearate (c19h38o2 ) 4.14 4.28 5.23 4.23 3.69 methyl linoleate (c19h34o2) 5.41 6.21 5.21 4.65 6.1 methyl butanoate 6.72 11.2 7.06 6.1 9.2 methyl oleate (c19h36o2) 6.06 6.21 7.32 7.36 6.35 methyl linolenate (c19h32o2) 4.11 3.11 9.81 9.98 8.21 methyl cisvaccenate(c19h32o2) 2.38 2.32 6.8 8.69 6.98 methyl cis-12octadecenoate 2.71 4.82 5.91 4.91 8.6 methyl myristate(c15h30o2) 4.33 3.2 3.6 6.32 5.66 aspergillus sp.flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 107 conclusions acknowledgements references fungi were able to accumulate lipid in large amount using pome as substrates, indicating that introducing fungi into pome wastewater treatment plant could be proposed for future biofuel production and pollution reduction. this research was supported by lipi competitive research grant 2013-2014, and we expressed our sincere gratitude to dian nurcahyanto, yeni yuliani for laboratory asisstance. chan yj, chong, mf, law, cl. 2010. biological treatment of anaerobically digested palm oil mill effluent (pome) using a lab-scale sequencing batch reactor (sbr). j environ manage 91(8): 1738-46. chi z zheng, y, jiang, a, chen, s. 2011. lipid production by culturing oleaginous yeast and algae with food waste and municipal wastewater in an integrated process. applied biochemistry and biotechnology 165(2): 442-53. coleman r. 2004. enzymes of triacylglycerol synthesis and their regulation. progress in lipid research 43(2): 134-76. dinis, mj, bezerra, rm. f, nunes, f, dias, a. a, guedes, c. v, ferreira, l. m., m., rodrigues, m. a m. 2009. modification of wheat straw lignin by solid state fermentation with white-rot fungi. bioresource technology 100(20): 4829-35. duarte sh, de andrade, ccp, ghiselli, g, maugeri, f. 2013. exploration of brazilian biodiversity and selection of a new oleaginous yeast strain cultivated in raw glycerol. bioresoivce technology 138: 377-81. fujita y, ito, j, ueda, m, fukuda, h, kondo, a. 2004. synergistic saccharification and direct fermentation to ethanol of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. app and environ microbiol (2): 1207-12. gasmi n, ayed, a, ammar, b, zrigui, r, nicaud, j-m, kallel, h. 2011. development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, . microbi cell fact 10(90): 1-11. goyal a, ghosh, b, eveleigh, d. 1991. characteristics of fungal cellulases. bioresovice technol 36(1): 37-50. hasper aa, dekkers, e, mil, m van, vondervoort, pj i. van de, graaff, l. h. de. 2002. a new endoglucanase from with major activity towards xyloglucan. appl environ microbio 68(4): 1556-60. jing q, spiertz, jhj, hengsdijk h, van keulen h, cao w, dai, t. 2010. adaptation and performance of rice genotypes in tropical and subtropical environments. 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transesterification oftthe lipid. bioprocess biosyst engi 35(6): 993-1004. zheng y, chi z, ahring bk, chen s. 2012. oleaginous yeast for biofuel production: ammonia's effect. biomass and bioenergy 37: 141-121. zhou x, chen h, li z. 2004. cmc-ase activity assay as a method for cellulase adsorption analysis. enzyme microbi tech 35(5): 455-59. rhodosporidium toruloides cryptococcus curvatus 110 biotropia vol. 21 no. 2, 2014 5. aurellia tatipata.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 2. topik hidayat (utility).cdr biotropia vol. 18 no. 2, 2011: 74 80 utility of k gene to assess evolutionary relationship of genus (anacardiaceae) in indonesia and thailand mat mangifera topik hidayat , adi pancoro , and diah kusumawaty maturasek ( k) gene of chloroplast dna has served as an appropriate candidate to be a dna marker in angiosperms. using this marker, 19 species of genus , one of the ecologically important crop, collected from indonesia and thailand were analyzed. phylogenetic analysis using parsimony method revealed that the gene could clasify into three major groups, namely group i, ii, and iii. moreover, the k gene can identify species originated from thailand. although this classification system is different with the previous classification system, it can provide a new information on the current status of taxonomy. further result exhibited that dna sequences of the k of two species ( dan ) are different between indonesia and thailand specimens. : dna barcode, , k gene, parsimony, phylogenetic analysis 1,2,* 3 2 1 2 3 department of biological science, faculty of bioscience and bioengineering, universiti teknologi malaysia (utm), malaysia department of biology education, indonesia university of education (upi), bandung, indonesia school of life sciences and technology, institute technology of bandung, indonesia mat mangifera mangifera mat mangifera mangifera mat mangifera m. laurina m. macrocarpa mangifera mat received 24 november 2010/accepted 28 october 2011 abstract introduction keywords the genus l., one of the most important plant groups in deciduous forest and wet tropical rainforests including mountain forests, is one of the largest genera of the family anacardiaceae to which approximately 69 species have already been described. the genus is mostly distributed in the tropical parts of asia (india, burma, sri lanka, thailand, south tropical china, malaysia, indonesia, papua new guinea, the philippines, the solomon islands) but also in the pacific islands (kostermans & bompard 1993). in spite of their economical importance, phylogenetic relationships among species within the genus have been poorly understood due to their extremely complicated vegetative and reproductive organs. mangifera 74 e-mail: topik28@yahoo.com or topik@fbb.utm.my biotropia vol. 18 no. 2, 2011 previously, marchand (1869), pierre (1897), and kostermans and bompard (1993) have revealed classification systems for the genus based upon floral characters. however, these characters were extremely complicated in the genus and subjected to parallelism (yonemori . 2002), suggesting many taxonomic and phylogenetic problems still remain unresolved. given the shortcomings of these characters, data obtained from nucleotide substitutions of appropriate molecules are preferable for clarifying phylogenetic relationships (e.g., moritz & hillis 1996). methods for clarifying relationships in species or group organisms by using dna sequences have been proposed and initiated recent years (kress . 2005). maturasek gene of chloroplast genome served as potentially usable dna regions to flowering plants. the k gene is frequently choosen by plant systematists because the region is a single copy gene and has enough variable sites of nucleotide substitution. recently, the k gene has been widely used in phylogenetic inferences of various groups of plant (e.g. ito . 1999; ferguson sang 2001; raymond . 2002; ebihara . 2005; hidayat . 2005). phylogenetic analysis to clarify phylogenetic relationships among members of genus have been carried out using dna sequences of the k gene. a total of 19 species of was collected from indonesia and thailand, plus two species of . two members of genus (m9 and m13) were used as outgroup in phylogenetic analysis based on previous research this genus was sister group to (yonemori . 2002). detailed information of the plant is summarized in table 1. dna genome was extracted from fresh materials (young leaf) using qiagen dneasy mini plant kit with slight modification. amplification was conducted using four primers as shown in figure 1. table 2 provides detailed information on sequences of primer pairs. et al et al mat mat et al et al et al et al mangifera mat mangifera bouea bouea mangifera et al & an understanding of the evolutionary relationships in this group may contribute to the field of plant systematics or ecology. materials and methods 75 matk genetrnk trnk a b c d figure 1. strategy of amplification and sequencing of the k gene. a= k-5f, b=taa-09f, c=taa09r, dan d= k-2r. two internal primers (b and c) were designed for this study. mat trn trn utility of matk gene to assess evolutionary relationship topik hidayat .et al table 1. plant materials, their geographic origins and codes used in this study species origin c ode mangifera altissima blanco var bingloe indonesia m18 mangifera applanata kosterm. indonesia m14 mangifera foetida lour. indonesia m17 mangifera gedebe miq. indonesia m10 mangifera indica l. indonesia m11 mangifera laurina bl. indonesia m7 mangifera macrocarpa bl. indonesia m3 mangifera odorata griff. indonesia m16 mangifera spp indonesia m12 mangifera rufocostata kosterm. indonesia m8 mangifera similis auct. indonesia m2 mangifera caesia jack ex wall indonesia m5 mangifera casturi kosterm. indonesia m15 mangifera macrocarpa bl. thailand s1 mangifera conchinchinensis englar thailand s6 mangifera flava evrard thailand s3 mangifera gracilipes hook.f. thailand s2 mangifera caloneura auct. thailand s5 mangifera laurina bl. thailand s7 bouea oppositifolia (roxb.) meiss indonesia m13 bouea macrophylla griff. indonesia m9 76 table 2. primers used in this study name sequences trnk-5f 5’ tgggttgctaactcatgg 3’ trnk-2r 5’ aactagtcggatggagtag 3’ taa-09f 5’ggttttcccatgagtagattatcg 3’ taa-09r 5’ cgaagtagacgaagctcttgg 3’ for amplification, we used primer pairs a and d, whereas all primers used once sequencing. pcr (polymerase chain reaction) reaction included buffer pcr (1x), mgcl (2-3mm), primers (@ 0,5 mm), enzyme taq polymerase (1 u/ul), dntps mix (1,6 mm), and dna template (100-150 ng/ul). pcr was conducted according to hidayat . (2005). pcr cycles include 1 cycle at 94 c (predenaturation) for 5 minutes; 30 cycles at 94 c (denaturation) for 30 seconds, 49 c ( ) for 30 seconds, and 72 c (extension) for 2 minutes; and ended with 1 cycle at 72 c (final extension) for 8 minutes. pcr products were cloned into pgem-t easy (promega) before sending them to macrogen (korea) for sequencing. dna sequences obtained from the k gene were aligned with clustal x (thompson . 1997) and then adjusted manually. phylogenetic analyses based on the maximum parsimony criterion was performed using paup* version 4.0b10 (swofford 1998). all characters were equally weighted and unordered (fitch 1971). all the data sets were analysed by the heuristic search method with tree bisection-reconnection (tbr) branch swapping and the multrees option on, ten replications of random addition sequences with the stepwise addition option, and all most parsimonious trees (mpts) were saved. evaluation of internal support of clades was conducted by the bootstrap analysis (felsenstein 1985) utilizing 1000 replicates with tbr branch 2 et al annealing mat et al o o o o o biotropia vol. 18 no. 2, 2011 77 swapping and the multrees option off. number of steps, consistency indices (ci) and retention indices (ri) were calculated on one of the mpts in each analysis with the tree scores command in paup*. dna extraction can be done using various types of dna sources such as leaf, stem, flower, and seed. in this research, young leaf was used for dna extraction to minimize contamination that can inhibit pcr amplification. high level of concentration (600 ng/ul in average) with good ratio ( 1.750) was obtained. size and border of k gene for were determined through comparative analysis in genebank (www.ncbi.nlm.nig.gov). the results indicated that size of k gene in is about 1500 bp. multiple alignment analysis was performed by using clustalx (thompson . 1997). the aligned k comprised 1,601 characters (fig. 2). of these, 1,429 were constant and 51 were potentially informative. reconstruction of phylogenetic tree (fig. 2) using paup resulted in 23 mpts with a length of 121 steps, ci of 0.852, and ri of 0.739. the tree (fig. 3) demonstrated that the genus was monophyletic and split into three major groups. monophyletic nature of was supported by character of stoma, anomositic (hidayat, unpublished data). the three major groups found in this study is not consistent with previous classification system by mukherjee (1953), kostermans and bompard (1993) based upon morphological characters, and even yonemori . (2002) on the basis of dna sequences of internal transcribed spacer (its) region. the number of plant materials used in this study is likely to be insufficient (only 19 out of 69 recognized species). further phylogenetic analysis is, therefore, needed using more extensive sampling. however, this study has provided new information on taxonomy of . as depicted in figure 2 , (from indonesia), and were united (group i), whereas (form thailand), and were closely related (group ii). group iii was housed by the rest of species, and species which is originated from thailand was placed within group iii (fig. 2). unfortunately, no single synapomorphic character is found to support each group. moreover, this research has revealed that there are variations of k in and which come from indonesia and thailand. as seen in figure 2, (from thailand) was separated from that of indonesia (group iii; thailand specimen in group ii). similar situation has been found in : thailand in group iii and indonesia in group i. different nature between these two countries has driven the mutation in k, but this does not lead to shift the morphology. these are related with the ability of plant to adapt to the environment changes (evans 1975). as mentioned, k gene is highly conserved (e.g. ebihara . 2005; hidayat . 2005). mutation rate in this kind of gene is very slow. this is reflected by the small number of informative characters (only 51 from a total 1,601 characters) to build the results and discussion + mat mangifera mat mangifera et al mat mangifera et al mangifera m. applanata m. macrocarpa m. altissima m. laurina m. casturi, m. odorata, m. indica mangifera mat m. laurina m. macrocarpa m. laurina m. macrocarpa mat mat et al et al 78 utility of matk gene to assess evolutionary relationship topik hidayat .et al tree. as a consequence, bootstrap value in most branches of the tree are less than 50. similar condition was found in other angiosperms (e.g. raymond . 2002; ebihara . 2005; hidayat . 2005). further analysis based on the phylogenetic scheme presented here will shed more light on overlooked characters. et al et al et al figure 2. alignment process using clustalx shows the level of homology (*) biotropia vol. 18 no. 2, 2011 79 outgroup figure 3. one of the 23 mpts of based on k gene. bootstrap value of >50 are shown above each branch. * = indonesia specimen; **= thailand specimen. species inside the box are originated from thailand. mangifera mat b. oppositifolia b. macrophylla m. applanata m. macrocarpa* m. altissima m. spp m. laurina** m. kasturi m. odorata m. indica m. caesia m. foetida m. rufacostata m. gedebe m. macrocarpa** m. similis m. laurina* m. conchinensis m. flava m. gracilipes m. caloneura group i group ii group iii 100 77 55 84 61 86 conclusions this study demonstrated that the k gene classified the into three major groups. furthermore, the k identified species that is originated from thailand. the k gene in the two species, namely and , was different between indonesia and thailand specimens. this study is subjected to be preliminary, so it is suggested that further researches employing another dna region with more extensive sampling should be conducted in the future. mat mangifera mat mangifera mat m. laurina m. macrocarpa 80 utility of matk gene to assess evolutionary relationship topik hidayat .et al acknowledgment references we gratefully acknowledge nisa, puri, and asri of institute technology bandung for their kind assistance during the completion of the study, and wichan eiadthong of kasetsart university for his kindness to provide plant materials from thailand. we would like to thank campbell webb of harvard university for fruitful discussion during preparation of this manuscript. ebihara a, h ishikawa, s matsumoto, sj lin, k iwatsuki, m takamiya, y watano y, m ito. 2005. nuclear dna, chloroplast dna, and ploidy analysis clarified biological complexity of the complex (hymenophyllaceae) in japan and adjacent areas. american journal of botany, 92:1535-1547. vans lt. 1975. the physiological basis of crop yield. cambridge university press, london, uk elsenstein j. 1985. confidence limit on phylogenies: an approach using the bootstrap. evolution, 39:783-791. ferguson d, t sang. 2001. speciation through homoploid hybridization between allotetraploids in peonies (paeonia). proceeding national academic of science, 98:3915-3919. fitch wm. 1971. toward defining the course of evolution: minimum change for a specific tree topology. systematic zoology, 20:406-416. hidayat t, t yukawa, m ito. 2005. molecular phylogenetics of subtribe aeridinae (orchidaceae): insight from plastid k and nuclear ribosomal its sequences. journal of plant research, 118:271-284. ito m, a kamawoto, y kita, t yukawa, s. kurita. 1999. phylogenetic relationships of amaryllidaceae based on k sequences data. journal of plant research, 112:207-216. kostermans ajgh, bompard jm. 1993. the mangoes: their botany, nomenclature, horticulture and utilization. ibpgr academic press, london. kress wj, wurdack kj, zimmer ea, weigt la, janzen da. 2005. use of dna barcodes to identify flowering plants. proceeding national academic of science, 102:8369-8374. marchand l. 1869. revision du groupe des anacardiacees. j.b. bailliere, paris. moritz c, hillis dm. 1996. molecular systematics: context and controversies.in: hillis dm, moritz c, mable bk (eds) molecular systematic, 2 edn. sinauer associates, sunderland, ma. mukherjee sk. 1953. origin, distribution and phylogenetic affinity of the species of l. journal of linnaues society london (bot), 55:65-83. pierre l. 1897. flore forestiere de la cochinchine. doin, paris. raymond o, piola f, sanlaville-boisson c. 2002. inference of reticulation in outcrossing allopolyploid taxa: caveats, likelihood and perspectives. trend in ecology and evolution, 17:3-6. swofford dl. 1998. paup*4.0b10. phylogenetic analysis using parsimony (*and other methods). version 4. sinauer associates, sunderland, massachussets. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clusstalx windows interface: flexible strategies for multiple sequences alignment aided by quality analysis tools. nucleat acid research, 24:4876-4882. yonemori k, c honsho, s kanzaki, w eidthong, a. sugiura. 2002. phylogenetic relationships of mangifera species revealed by its sequences of nuclear ribosomal dna and a possibility of their hybrid origin. plant systematic and evolution, 231:59-75 vandenboschia radicans mat mat mangifera e . f nd 5. sri wilarso.cdr biotropia vol. 20 no. 1, 2013: 38 49 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. : their antagonistic effects towards soilborne fungal pathogens and growth stimulation of sp. gigaspora glomus gigaspora in vitro sri wilarso budi * and nunang lamaek may recipient of biotrop research grant 2010/accepted 21 may 2012 this research was aimed at obtaining bacterial isolate from arbuscular mycorrhizal fungi (amf) spores showing antagonistic effects against fungal pathogens and yet compatible with amf. seven isolates of bacteria were isolated from surface-sterilized amf spores of sp. (gg) and five isolates of bacteria isolated from sp. (gl). all bacterial isolates were identified based on its morphological methods and biochemical reaction and revealed that 9 isolates belong to genus , while the other 3 isolates belong to genus , and respectively. the tests to the antagonists against fungal pathogens and stimulation of amf hyphal development of sp. showed that there were 3 isolates of bacteria ( gg1, gg5 and gl3) had the ability to inhibit the growth of pathogens and to enhance the development of amf hyphae sp . these three bacteria isolates potentially can be used to enhance the quality of amf inoculants as biocontrol and biofertilizers. enzymatic activity test showed that there were 7 isolates of bacteria that produced cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5. bacteria, arbuscular mycorrhizal fungi spores, antagonistic effects, stimulation effects, fungal pathogens 1 2 1 2 department of silviculture, faculty of forestry, bogor agricultural university, bogor 16680, indonesia faculty of forestry, the state university of papua, west papua, indonesia gigaspora glomus bacillus pseudomonas proteous enterobacter gigaspora in vitro bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora in vitro bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus abstract key words: * corresponding author : wilarso62@yahoo.com 38 introduction many kind of microorganisms have been observed to be associated with mycorrhizosphere of different host plants. the functional significance of the microbial associate in the ecosystem has been well documented (budi 2012) including associative n -fixing bacteria (cruz & ishii 2011), plant growth-promoting rhizobacteria (siddikee . 2010), phosphate solubilizing bacteria (khan . 2009), and antagonists of plant pathogens (artursson . 2006, bakhtiar 2010). in addition, the bacteria associated with spores of amf and (roesti . 2005), (cruz . 2008, cruz & ishii 2011, horii & ishii 2006, budi 2012), and (bharadwaj . 2008) have been reported. these amf spores associated bacteria has been reported significantly increased amf hyphal growth and suppress fungal pathogens (cruz & ishii 2011). associated microorganisms may complement mycorrhizal activities, particularly in biological control and biofertilizer in agricultural systems. nowadays, the awareness of international community to food safety has increased due to negative impacts of the use of chemical substances in crop production. the movement of “back to nature” of international community increases and organic food has become a new trend for their life style. intensive agriculture in many countries including indonesia have led to excessive inputs of agrochemicals (fertilizers, pesticides) but agricultural policies are now tending towards more organic for sustainable agricultural systems. sustainability in agriculture has been defined as the successful management of resources for agriculture to satisfy enhancing the quality of the environment and conserving resources (ladha 1992). in this respect, the government of indonesia c.q ministry of agriculture has declared “go organic in 2010”. it implies better soil management to avoid modifications that have adverse effects on chemical and biological processes supporting plant growth. one approach is the decrease in chemical inputs to rational (economic but non-polluting) levels through the development of alternative strategies to ensure acceptable yield. an increasing demand for low-input of chemical substance in agriculture has resulted in greater interest in soil microorganisms that increase soil fertility or improve plant nutrition and health including the use of amf as biofertilizer and biocontrol agents. the use of amf for sustainable agriculture has been widely used and reported significantly increased plant productivity (wu & zou 2009; siddikee . 2011), but the avaibility of high quality inoculants is very limited due to the production of this fungi as biofertilizer. up to now, biocontrol is carried out in open pot culture which is sensitive to contamination by other organism or soil borne plant pathogen. therefore, the development of amf inoculum production is needed in order to obtain high quality and purity of amf inoculum. one approach to produce a high quality and purity of amf inoculum is by using amf spore associated with bacteria. the objective of this study was to acquire bacteria from surface-sterilized spores of amf that showed antagonistic effects against fungal pathogens and yet compatible with amf which potential to improve inoculum quality. et al. et al et al et al et al. glomus constrictum glomus geosporum et al gigaspora margarita et al et al. glomus intraradices glomus mosseae et al et al 2 39 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. materials and methods isolation and identification of bacteria from the amf spores antagonistic effects of isolated bacteria towards soil borne plant pathogen stimulation effect of bacteria on hyphal growth of amf spores the spores of amf sp. and sp. originally from the collection of silviculture laboratory, faculty of forestry ipb were sieved by wet sieving according to method of gardeman and nicholson (1963). hundred spores of each species were surface sterilized according to the method described by budi . (1999a). the surface sterilized spores were crushed using sterile needles and transferred to petri dishes containing sterile agar base (sigma-aldrich) and nutrient agar (oxoid) and then incubated in the dark at 30 c for 24 h. the bacteria that grew were transferred to another petri dishes containing the same media until single colonies were obtained. a single colony was then identified based on morphological methods using biochemical reaction developed by biomerieux and carried out at laboratory of identification and determination, school of life science and technology, bandung institute of technology, bandung, indonesia. isolated bacteria were tested toward soil borne plant pathogen . the common soil borne plant pathogens like sp., sp. and sp. originally from the laboratory of pest and diseases, faculty of forestry ipb, were used in this study. the test was carried out according to the method of varese . (1996). the pathogens were grown on petri dishes containing potato dextrose agar (difco) media. colony of bacteria were placed about 1.5 cm from the pathogen. the petri dishes were then sealed with parafilm, incubated in the dark at 25 c for 7 days. the radial growth of mycelium were measured and compared to the control treatment. the experiment was carried out in a completely randomized design composed of 12 bacterial treatments and one control in triplicates for each pathogenic fungi. data were analysed by one-way anova. the amf sp. spores were collected and surface sterilized according to the method of budi . (1999a). the spores were then placed in petri dishes containing sterile zeolit medium supplemented with nutrient. ten spores were put on sterile filter paper saturated with 50 l suspension of bacteria and were then placed on each petri dish. the control sterile filter paper was saturated with sterile water. petri dishes were then sealed with parafilm, incubated in the dark at 25 c for 21 days. the hyphal growth were recorded starting from germ tube and only the main hyphae was measured (fig. 1) and compared to the control treatment. the experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control in triplicates. data were analysed by one-way anova. gigaspora glomus et al pseudomonas in vitro slerotium rhizoctonia ganoderma et al gigaspora et al o o o in vitro in vitro biotropia vol. 20 no. 1, 2013 40 plate assay for enzymatic activities test isolated bacteria from amf spores bacterial isolates were grown in erlenmeyer flasks for 24 h on a culture shaker at 30 c in nutrient broth buffered medium. ten micro liters of each bacterial suspension ±10 cfu/m/l was spotted onto plates containing the substrate of the enzyme to be tested and grown at 25 c for 48 h. cellulolytic activity was assessed as described by teather and wood (1982) using a solid medium, containing mgso 7h o (0.1 g/l), cacl 2h o (0.2 g/l), feso 7h o (0.04 g/l), nacl (0.2 g/l), kh po4 (0.3 g/l), k hpo4 (0.5 g/l), cmc (carboxymethylcellulose) (sigma) (5 g/l), yeast extract (0.1 g/l) and bacto agar (15 g/l). for visualization of -d-glucan hydrolysis, the agar medium containing cmc was flooded with an aqueous solution of congo red (1 mg/ml) for 15 min (budi . 2000). the clear zones was measured after 48 h incubation. the proteolytic activity test was performed according to the method described by dunne . (1997). the experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control with triplicates. data were analysed by one-way anova. a total of 12 bacteria isolates were isolated and identified from surface sterilized spores of am fungi. there were 7 isolates of bacteria isolated from sp. and 5 isolates isolated from sp. the morphological characteristics of each isolate of bacterial colony and its identification are shown in table 1. 0 8 0 4 2 2 2 4 2 2 2 β et al et al gigaspora glomus results and discussion figure 1. hyphal growth measurement 41 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. source of isolate isolates code morphological characteristics of bacterial colony (colour, colony surface and type) gram species name gigaspora sp. gg1 positive bacillus subtilis gg2 positive bacillus licheniformis gg3 positive bacillus cereus gg4 positive bacillus brevis gg5 negative pseudomonas diminuta gg6 positive bacillus laterosporus gg7 circular, entire, umbonate, cream, opaque circular, undulated, umbonate, cream, opaque circular, serrate, raised, opaque circular, undulated, raised, surface dried, opaque circular, entire, convex, transparent, yellowish circular, filamentous, raised, opaque circular, undulate, filamentous, raised, opaque positive bacillus pasteurii glomus sp. gl1 negative bacillus circulans gl2 negative proteus penneri gl3 negative enterobacter hormaechei gl4 positive bacillus firmus gl5 circular, entire, umbonate, translucent circular, entire, convex, translucent, yellow circular, entire, convex, transparent irregular, undulate, raised, transluent circular, serrate, raised, opaque, positive bacillus cereus table 1. species identification and morphology characteristics of isolates of bacteria isolated from amf spores of sp. and sp.gigaspora glomus antagonistic effects of isolated bacteria against fungal pathogen. the results indicated that there are 2 bacterial isolates originated from sp. spores ( gg1 and gg5) and 2 bacterial isolates from sp. spores ( gl2 and gl3) have the ability to inhibit the growth of three pathogens tested. the percentages of inhibition gigaspora bacillus subtilis pseudomonas diminuta glomus proteus penneri enterobacter hormaechei biotropia vol. 20 no. 1, 2013 42 varied among pathogens tested. the bacterial isolates of gg1 and gg5 inhibited the growth of mycelium of sp. by 7980%. while four isolates of bacteria ( gg1, gg5, gl2, and gl3) showed inhibitory effects to by 46-54%. testing against sp. showed that bacterial isolates gg1, gg5, gl2 and gl3 gave inhibitory effects of 59-90 %. (table 2). among the 12 isolates of bacteria tested, one isolate ( gg5) had a significantly higher effect than the control, while 6 other isolates ( gg1, gg2, gg3, gg4, gg6 and gl3) increased amf hyphal development higher than the control treatment but were statistically not different. of the remaining 5 isolates, 2 isolates significantly inhibited the amf hyphal growth, i.e. gg7 and gl5 while the other 3 isolates inhibited the amf hyphal growth but were not significally different from the control treatment (table 3). ten out of the 12 isolates of bacteria produced cellulase activities, i.e. gg1, gg2, gg3, gg5, gg6, gg7, gl2, bacillus subtilis pseudomonas diminuta sclerotium bacillus subtilis pseudomonas diminuta proteus penneri enterobacter hormaechei rhizoctonia ganoderma bacillus subtilis pseudomonas diminuta proteus penneri enterobacter hormaechei pseudomonas diminuta bacillus subtilis bacillus licheniformis bacillus cereus bacillus brevis bacillus laterosporus enterobacter hormaechei bacillus pasteurii bacillus cereus bacillus subtilis bacillus licheniformis bacillus cereus pseudomonas diminuta bacillus laterosporus bacillus pasteurii proteus penneri enterobacter stimulation effect of isolated bacteria on hyphal growth of amf spores sp. enzymatic activity characterization of bacteria gigaspora in vitro table 2. yradial growth of pathogenic fungal m celium in vitro no isolates radial growth of mycelium (cm)* and % inhibition sclerotium sp. % rhizoctonia sp. % ganoderma sp. % 1 control 9.00 a 0 9.00 a 0 9.00 a 0 2 gg1 1.70 d 81.11 4.17 d 53.66 1.04 c 88.44 3 gg2 9.00 a 0 9.00 a 0 9.00 a 0 4 gg3 9.00 a 0 9.00 a 0 9.00 a 0 5 gg4 9.00 a 0 9.00 a 0 9.00 a 0 6 gg5 1.88 d 79.11 4.35 d 51.66 1.12 c 87.55 7 gg6 9.00 a 0 9.00 a 0 9.00 a 0 8 gg7 9.00 a 0 9.00 a 0 9.00 a 0 9 gl1 9.00 a 0 4.65 b 48.33 9.00 a 0 10 gl2 2.99 c 66.77 4.16 d 53.77 0.92 d 89.77 11 gl3 3.86 b 57.11 4.86 c 46 3.68 b 59.11 12 gl4 9.00 a 0 9.00 a 0 9.00 a 0 13 gl5 9.00 a 0 9.00 a 0 9.00 a 0 *values in a column followed by the same letter do not differ significantly from each other at value of 0.05.p 43 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. table 3. stimulation of amf hyphae of sp. growth after 21 days incubation by bacteria gigaspora no isolates hyphal length (μm)* % increased/ decreased (-) 1 control 202.44 bc 2 bacillus subtilis gg1 286.31 ba 41.43 3 bacillus licheniformis gg2 369.84 ba 82.69 4 bacillus cereus gg3 231.79 bc 14.50 5 bacillus brevis gg4 287.37 ba 41.95 6 pseudomonas diminuta gg5 499.19 a 146.59 7 bacillus laterosporus gg6 363.89 ba 79.75 8 bacillus pasteurii gg7 19.62 e -90.31 9 bacillus circulans gl1 139.46 bcd -130 10 proteus penneri gl2 160.68 bcd -20.62 11 enterobacter hormaechei gl3 324.47 ba 60.28 12 bacillus firmus gl4 181.46 bc -11.56 13 bacillus cereus gl5 156.38 bcd -22.75 note : * values in a column followed by the same letter do not differ significantly from each other at value ofp hormaechei bacillus firmus bacillus cereus bacillus subtilis bacillus cereus bacillus brevis bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus et al. et al. et al. et al. gl3, gl4, and gl5, while 8 isolates produced protease activities, i.e. gg1, gg3, gg4, gg6, gg7, gl2, gl4, and gl5. there were 7 isolates that produced both cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5, as indicated by clear zone (halo) around the colony (table 3, fig. 2). bacteria have been observed to live in close association with amf (gopal 2012), and have been isolated from different amf spores (xavier & germida 2003; roesti 2005; horii & ishii 2006; cruz 2008; bharadwaj 2008;, figure 2. celullase activity (a) and protease activity (b) as indicated by clear zones (halo) biotropia vol. 20 no. 1, 2013 44 cruz & ishii 2011). cruz and ishii (2011) found 2 bacteria sp. and isolated from liquid present inside amf spore of , while budi (2012) found four bacteria sp. and ksc_sf9c isolated from surface sterilized amf spores . xavier and germida (2003) found 5 bacteria, , sp. , and sp. from surface sterilized amf spores of . this study demonstrated different bacteria species isolated from surface sterilized amf spores of sp. and sp. we found 7 bacteria from sp. and 5 bacteria from sp. as shown in table 1. as reported by roesti . (2005), the bacterial community associated with the amf spores was more inf luenced by the amf identity (amf species) than by the host plant. the difference in composition of the spore walls or exudates of amf species may have played a major role in the selection of bacterial populations living on the spore (roesti . 2005). bacteria associated with amf spores have been reported to play an important role in antagonistic effect of soil borne plant pathogens, amf spore germination and hyphal growth (horii & ishii 2006; cruz & ishii 2011). the interesting thing from this study was that among the 12 isolate bacteria tested, 3 bacterial isolates ( gg1, gg5 and gl3) have the ability to inhibit the growth of three pathogens and stimulate hyphal growth of sp. (table 2 and 3). this finding was in line with cruz and ishii (2011), who found that sp., and isolated from surface sterilized bacillus b. thuringiensis gigaspora margarita et al. bacillus bacillus megaterium, bacillus subtilis bacillus flexus strain g. margarita alcaligenes bacillus burkholderia flavobacterium pseudomonas glomus clarum gigaspora glomus gigaspora glomus et al et al bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora bacillus b. thuringiensis paenibacillus rhizosphaerae table 4. enzymatic activities of bacteria isolated from sp. and sp. as indicated by clear zones diameter after 48 h incubation gigaspora glomus no bacteria isolates enzymatic activities ( ø clear zones, cm) celluase protease 1 bacillus subtilis gg1 4.80 cd 4.80 ba 2 bacillus licheniformis gg2 2.83 e 0.00 c 3 bacillus cereus gg3 5.70 b 6.50 a 4 bacillus brevis gg4 0.00 f 5.77 a 5 pseudomonas diminuta gg5 5.10 cb 0.00 c 6 bacillus laterosporus gg6 5.87 b 6.20 a 7 bacillus pasteurii gg7 7.17 a 5.83 a 8 bacillus circulans gl1 0.00 f 0.00 c 9 proteus penneri gl2 5.07 cb 4.73 ba 10 enterobacter hormaechei gl3 2.97 e 0.00 c 11 bacillus firmus gl4 3.23 e 3.73 b 12 bacillus cereus gl5 4.10 d 4.73 ba * values in a column followed by the same letter do not differ significantly from each other at value of ,0.05p 45 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. amf spores had antagonistic effect to the pathogenic fungi and and stimulate hyphal growth of . many studies have reported that has the potential to be used as biocontrol agent against soil borne plant pathogens (nalisha . 2006; velmuragan . 2009). phae . (1990) found that produced , a bioactive compound that are active as antifungal agents against several plants pathogenic fungi as well as antibacterial. the production of hydrolytic enzyme protease by sp. isolated from mycorrhizosphere that caused cell wall degradation of pathogenic fungi have also been reported (budi 2000). in this experiment, also produced hydrolitic protease enzyme. we assume that combination of protease enzyme with the other active compounds produced by could inhibit pathogenic fungal growth. other bacteria such as and have been reported effectively control diseases caused by soil borne plant pathogens (thomashow . 1990; chermin . 1995, barea . 1998). these bacteria produced bioactive compound phenazine-1-carboxylic acid responsible for suppression of soil borne plant pathogenic fungi (thomashow . 1990). in this study only gg5 produced hydrolytic protease enzyme but not gl3, indicating that the role of these enzymes in controlling the growth of soil borne plant fungal pathogens is not clear and it seems that the inhibitory effect of these bacteria to soil borne plant fungal pathogen development is probably derived from more than one mechanism (budi . 2000). further investigation is necessary to isolate bioactive compound and its mode of action from these 3 bacteria isolates. the stimulation of amf hyphal growth by amf spores associated with bacteria have been reported by several researchers (budi 1999b; cruz & ishii, 2011). in this study gg1, gg5 and gl3 have the ability to stimulate hyphal growth of sp. and supressed several soil borne fungal pathogens . four others isolates only have the ability to stimulate hyphal growth of sp. (table 3). the exact mechanism of hyphal growth stimulation by spores associoated with bacteria is not known, but cruz and ishii (2011) reported that bacteria released volatile compounds and exudates involved in stimulating hyphal growth. futher study is needed to investigate the possible isolate volatile compounds and exudate released by these 3 bacteria isolates. certain arbuscular mycorrhizal fungi hyphae produce hydrolytic enzymes which hydrolyses the biopolymers such as protein, chitin and cellulose that will help the amf to degrade and infect the plant cell walls (gopal . 2012). furthermore, the hyphae and spores of amf also harbour associated bacteria that produce hydrolytic enzyme (budi . 1999b). in this experiment, 7 isolates produced both cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5, as indicated by clear zone (halo) around the colony (table 3, fig. 2). as reported by budi (2012) isolated from surface sterilized amf spores have shown to increase mycorrhizal root colonization of neem seedling in the nursery and also produced cellulase and protease activities. gigaspora margarita rosellimia necatrix, phytium ultimum, fusarium oxysporum rhizoctonia solani g. margarita b. subtilis et al et al et al b subtilis iturin paenibacillus et al. b. subtilis b. subtilis pseudomonas enterobacter et al et al et al et al pseudomonas diminuta enterobacter hormaechei et al et al. bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora in vitro gigaspora in vitro et al et al bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus et al. bacillus subtilis g. margarita biotropia vol. 20 no. 1, 2013 46 production of volatile compounds that can positively influence germination of amf spores, provision of nitrogen through nitrogen fixation, solubilization of soil phosphate sources, detoxification of the fungal microhabitat, change of ph level of siderophores are also some of the proposed mechanisms underlying stimulation of mycorrhization (bharadwaj 2012). further study is needed to test the consistency effects in the field, and to investigate the mechanisms involved in the antagonism towards fungal pathogens as well as stimulation effect to the hyphal growth of am fungi. there is a possibility that 3 isolate bacteria ( gg1, gg5 and gl3) isolated from surface sterilized spores of am fungi sp. and sp. have the ability to inhibit the growth of 3 pathogens and stimulate hyphal growth of sp. . these bacteria have a great opportunity to be used as biocontrol agents for soil borne plant pathogens as well as for improving the amf inoculum quality, since up to now the production of these fungi for biofertilizers and biocontrol is carried out in open pot culture sensitive to contamination by other organisms or pathogens. this research was supported by biotrop through the competitive research grant no 050.7/psrp/spk-pnlt/iii/10. the authors would 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growth and root nutrient status of citrus subjected to salt stress. 35: 388-91. xavier ljc, germida jj. 2003. bacteria associated with spores influence mycorrhizal activity. soil biol biochem 35: 471-8. bacillus subtilis bacillus licheniformis in vitro in vivo science asia glomus clarum 49 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. ichtyofauna at cijalu river, cilacap regency central java province, indonesia agus nuryanto, dian bhagawati, m. nadjmi abulias and indarmawan faculty of biology, jenderal soedirman university, purwokerto 53122, indonesia received 22 january 2014/accepted 21 june 2016 abstract cijalu river is located in western cilacap regency central java . the river runs through forestry, of province housing and farming areas. this condition lead to a prediction that the river has altered its physico-chemical s been on characteristics inhabited by variety of fish species. this study aim to collect data about fish causing the river to be a ed species inhabit cijalu river and its distribution. a survey method has been done with ing clustered random sampling technique the river was divided into three different areas, i.e. upper-, middle and downstreams. species diversity was . measured as the number of species, while distribution was measured as the presentation of fish species in each site . nineteen fish species of 10 families were identified from cijalu river. the 10 families were anabantidae bagridae, , balitoridae, , osphronemidae, and sisoridae cyprinidae was channidae, cichlidae, cyprinidae loricariidae, poecilidae . the family spec , followed by ( species), channidae having the highest number of species (6 ies) bagridae 3 (2 species) and osphronemidae a complex (2 species). the remaining respective families were represented by one species. pattern of fish distribution was observed during study. and were only found at the glyptothorax platypogon channa gacua the upper-streams, while was obtained in downstreams species could be anabas testudineus . the other remaining observed either middleto downstreams, or even from upperto downstreams. d pattern of species from the ifferent distribution could be caused by physico-chemical character s from upper to lower parts of the river, istic variation especially substratewater velocity and types. ci lu , , diversi , keywords: ja river distribution ty fish introduction diversity indicate the presence of species at s certain ecosystem. in macrotaxonomy, diversity measurement is a measure of number of species in an area, whereas in ecology, diversity is represented by species number and its abu ances. igh species diversity prove that an nd h s ecosystem is in an equilibrium state and play s important role n keeping ecosystem in an i equil br um condition. according to odum i i (1971) high diversity show by no dominant is n species in an ecosystem. biodiversity is an integral part of nation development after brundtland report was first published in 1987 (unep 1987). igh support of h international community on that report and followed by global consultation has led to the establishment of the convention on biological diversity in 1994, which provides guidance related to global issue on biodiversity and biodiversity initiative (nguyen de silva 2006) & . in general, biodiversity initiative focused on terre trial ecosystem. however, at present days, s aquatic ecosystem become among the most s discussed s topic , especially coral reef and about wetlands ecosystems. later on, resea cher are r s also interested to study biodiversity aspect of freshwater ecosystem including freshwater fish diversity (nguyen de silva 2006). previous & report showed that a total of 13,000 freshwater fish species ha been described and belong to ve 2,513 genera (du geon . 2006; leveque . d et al et al 2008). the majority of freshwater fish species inhabit tropical areas, mainly in asia (nguyen de & silva 2006). asia harbor approximately 121 s freshwater fish families, higher than those in freshwater ecosystems in africa and latin america which have only 50 and 55 fish families, respectively. on species level, 28 up to 32% of the 13,000 described freshwater fish species were biotropia vol. 23 no. 1, 2016: 1 9 1 doi: 10.11598/btb.2016.2 . .3623 1 * c orresponding author: yahoo.comanuryanto2003@ 2 recorded from asia with approximately 462 spesies are threatened. the number is equivalent to 17.5% of total number of threatened species in the world (l vequ . 2008). a sum of 66 e e et al species were critically end gered or end gered an an and 32 species of them belong to yprinidc . indonesia is among 12 sian ountries with the a c highest number of threatened fish species (nguyen de silva 2006).& high diversity of fish species in asia is especially found in big rivers and their floodplains (welcomme 2000; cbd 2003; coates . 2003). et al in is detail, asia's freshwater fish dominated by c ,yprinid group (±1 000 spesies) and followed by balitoridae and cobitidae (±400 species, recently, balitoridae d ivided intois balitorid ae, g a s t r o m y z o n t i d a e , e l l o p o s t o m a t i d a e , vaillantellidae, barbuccidae, serpenticobitidae and nemacheilidae s; while cobitidae become cobitidae and botiidae kottelat 2012 gobiidae ( ;) (300 species), catfishes bagridae (±100 species) and osphronemidae (85 species) (nguyen de & silva 2006). specifically, kar . (2006) also et al reported that cyprinidae is a domina t freshwater n fish in india. family previous studies reported freshwater fish diversity in africa (albaret . 2004; leveque et al 1997; harrison whitfield 2006) europe & and (collares-pereira . 2002). several studies ha et al d also been done in indonesia, however, those studies were mostly performed outside of java; such as in kalimantan (haryono 2004 sulistyarto ; et al. 2007) and sumatera (duya 2008). studies in smaller rivers in java ha also been done in ve cileumeuh and cikawung rivers. a study in cileumeuh 2 spe ies river found a total of 2 fish c within families in iver, 10 . cileumeuh r cyp inidae was the speciose family with 0r 1 species bagridae and followed by with four spe ies (nuryanto . 201 ). et al et al.c 2 nuryanto (2015) also recorded a total of 19 species and nine families from cikawung river. cijalu river is located in western cilacap regency, central java . administratively, province this river belong to district with s majenang its headwater padontelu . located in mountains cijalu river empties into cijalu cileume h river. u r siver belong to citanduy watershed together with cileumeuh and cikawung rivers. there have been no data o fish diversity d sn an it distribution at ja cijalu ci lu river. in addition to the fact that river is subjected to several impacts from forestry, farming and house waste which alter the physico-chemical characteristics of the river, cijalu river is overfished. , it is therefore important to study fish diversity at river to cijalu develop database on freshwater fish species in banyumas region which is important for further study, such as conservation strategy. this study aim collect on ed to data fish diversity and distribution cijalu river, cilacap egencyat r . materials and methods c random sampling technique was lustered performed by dividing cijalu river into three clusters i.e. upper , middle and downstream (fig. 1) . amples , based on substrate types fish s were collected at sampling sites:eleven five at upper-, three at m ddleand three at downstreams i using and nets12 volt with 0.5, electr shockeric biotropia vol. 23 no. 1, 2016 figure 1 sampling sites across ci riverjalu (108 04' and 109 30', 7 03' and 7 52') o o o o notes : 1= m11 sa pling site numbers = border among partsriver's 3 0.75 and 1 inch in mesh sizes. sampling was performed from april to july 2012. electricshocker and 0.5 inch nets were used during the sampling in the upper-stream, whereas 0.75 and 1 inch nets were used at the middleand downstreams. sampling efforts performed for one were hour netting and electroshocking each siteat . biological variables and physico-chemical characteristics were examined during the study. b were number of iological variables measured fish distribution in species (s) and fish cijalu r . physic -chemical measured iver o parameters were water depth, light intensity, water temperature, water acidity (ph), c rbon dioxide, a water velocity and substrate river . methods used in measuring physic -chemical areo parameters summarized n table 1i . fresh fish directly preserved samples were in labelled plastics bag filled with 70% ethanol. direct fixation eth nol was due using a carried out to technical difficulties obtain formalinin ing . upon arrival in the laboratory, the samples were washed running water and the used soaking under ethanol was replaced the fresh 70% ethanol. by for permanent preservation, the samples were fixed inside bottles containing fresh 70% ethanol (diluted from 100% pro-analysis ethanol). s a m p l e s we r e id e n t i f i e d a c c o r d i n g t o identification key from kottelat . (1993) and et al f b (froese pauly 2012)& .ish ase results and discussion species diversity a total of 19 fish c were spe ies of 10 families found during sampling at cijalu river (table 2) which is considered as high diversity based on guideline from ncdenr (2006). this study found lower number of species (19) compared to previous study in cimanuk river which found 40 species ( . sjafei . 2001)et al cimanuk river was chosen as compar the ison because cimanuk river is located in the same biogeographic region in the same island and is with cijalu river ifferen result between . d t s this study and study cimanuk at cijalu river the at river could be caused by different ecological characteristics river river such as length, size and annual water volumes. based on survey at cijalu river in 2012 and at cimanuk river in 2011 (nuryanto sugiharto 2011), cijalu river is & shorter and smaller than cimanuk river. cijalu river is only about 40 km in length, spanning only from orthern to outhern part of majenang n s district in cilacap regency, whereas cimanuk river has more than 100 km in length spanning from garut regency up to offshore of java the sea in indramayu regency, west java . province moreover, cijalu river is appr ximately 40 m in o width, while cimanuk river reaches 100 m in width at the lower part. in addition, cimanuk river has constant annual water volumes more than cijalu river. according to kottelat . et al (1993) longer and wider ecosystems usually are assumed to have higher microhabitat variation than smaller and shorter areas igh habitat . h variation supports high variety of inhabitants. therefore, it is reasonable that cimanuk river which is longer and wider than cijalu river has high number of species. these conditions agree with woo ton (1991) t who noted that larger streams are inhabited by higher number of species due to higher microhabitat variety than the smaller one. similar phenomenon was also reported by clavero . (2004) in 27 iberian et al mediterranean river basins, where wider rivers (tajo, guadiana and jucar rivers) support ed higher number of fish species than the r maining e twenty four smaller rivers. in addition mazeika , et al reported . (2006) that stream geomorphic, including stream size, has significant effect on fish community diversity. table 1 p o parameters mea methods hysic -chemical surement physico-chemical parameter measurement methods water depth (m) light intensity (m) water temperature (°c) water ph carbon dioxide (co2) water velocity (m/sec) substrate sechi disk sechi disk thermometer universal ph papers apha 1985 linear measurement visual ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 4 biotropia vol. 23 no. 1, 2016 species diversity comparison to the studies outside indotropic or oriental region showed complex pattern of similarit and differences. ies for example, our study obtained similar number of species with a study clavero . carried out by et al (2004) in guadalquivir rivers. however, different phenomena were observed when comparing our study to other studies. in one hand, our species record in cijalu river was lower compared to the reported by et alstudy clavero . (2004) conducted in three iberian mediterranean river basins. other hand, a slightly higher species on the number was recorded in our study compared to a study r r t epo ted by cassa ti (2005) from the morno do diabo state park, southeastern brazil. our result also recorded higher species number than the collection by . reported t al clavero e (2004). s erethe above comparison w not equal since our study and those studies from cl v ro . a e et al (2004) and cassa ti (2005) re located in t we d iff e rent biog e og raphi c r egions. t hese differences might cause different inhabitants. therefore, it is reasonable to find that results on fish species composition from our study conducted in indotropic or oriental region showed results from the studies different conducted in brazil (neotropic region) and europe (palearctic region) according to gaston . and williams (1997), each biogeographic region has their own organisms and most of the organisms are different among regions. this study at cijalu river found 10 famil ies i.e. anabantidae, bagridae, channidae, balitoridae, c i c h l i d a e , c y p r i n i d a e , l o r i c a r i i d a e , osphronemidae, d sisoridaepoeciliidae an . cyprinidae was the most speciose family with 6 species fol owed by with species, and , l bagridae 3 then channidae and osphronemidae with two species, respectively. the remaining six families only had one species, respectively (table ) 2 . comparison on family level also showed similarit and difference among studies. ies s our study at cijalu river recorded lower number of families compared to a study conducted at cimanuk river which recorded 20 families (sjafei et al. 2001). this difference could be caused by different ecological characteristics as previously discussed. however, both studies agreed on cyprinidae being the most speciose family high . number of cyprinids species was also repo ted r from musi river kejalo curup bengkulu with , , seven species (duya 2008). it seems that high number of cyprinids species is a common phenomenon in the river systems. this phenomenon was also reported in previous studies at different river systems either in indonesia or outside indonesia n . i rawa table fish species at ci l river2 and distribution ja u no species distribution upper middle lower 1 anabantidae a. anabas testudineus + 2 bagridae a. mystus gulio b. mystus micracanthus c. hemibagrus nemurus + + + + + + 3 balitoridae a. nemacheilus fasciatus + + 4 channidae a. channa striata b. channa gachua + + + 5 cichlidae a. oreochromis niloticus + 6 cyprinidae a. labiobarbus kuhlii b. mystacoleucus obtusirostris c. osteochilus vittatus d. barbodes binotatus e. barbodes microps f. rasbora argyrotaenia + + + + + + + + + + + + + + 7 loricariidae a. pterygoplichthys pardalis + + 8 osphronemidae a. osphronemus gouramy b. trichopodus trichopterus + + + 9 poeciliidae a. poecilia reticulata + + + 10 sisoridae a. glyptothorax platypogon + notes: = ; = absent + present family 5 lebak there wererungan river 19 cyprinidae species number recorded (sulistyarto . 2007). aet al of 26 cyp inids species was in bukit recordedr batikap central kalimantan (haryono 2002), , while a study in kayan mentarang national park, east kalimantan 19 c of recorded spe ies c prinidae java island, several y (haryono 2004). in stud also found number of cyp inids ies various s r species. ll stud reported that cyp inidae the a ies isr dominant family (cileumeuh river (nuryanto . et al 2012); cikawung river (nuryanto . 2015)). et al our present study and those previous studies agree with nguyen and de silva (2006) who reported that cyprinid group dominated freshwater fish species in asia. ahmad 2014 et al. ( ) also reported that cyprinid dominated fish species in sungkai wildlife reserve. the dominance of cyprinidae was also reported in the river basins throughout europe (reyjol . 2007). however, et al the trend of cyprinidae dominance was not observed in southeastern brazil where the speciose familiy were characidae followed by cichlidae and loricariidae no cyprinid ; there were species recorded (langeani in southeastern brazil et al. 2005). it was not possible to compare this study at cijalu river wi conductedth the study in the iberian mediterranean rivers at family level, because there was no family information availa le b in study (clavero the iberian mediterranean rivers et al. 2004). as it was discussed previously, the different results were caused by these studies being conducted in different biogeographic regions. however, the comparison itself is important to enrich our knowledge of other taxa (species and family) occurred in other biogeo raphic regions.g resultedanother important information from this study was that 3 19 species were of found non-native or introduced or exotic species i.e. pterygoplichthys ardalisp (page robins 2006; levin & et al poecilia reticulata o. niloticus et . 2008), and (paller al. 2011). therefore, high species diversity cijalu at river could not be used as an indicator of river health it is inhabited by three exotic because species which might become problem for native species, though maitland (2004) that if the al stated number of exotic species less than 25%, the is ecosystem is still .in healthy condition ofnevertheless, we have to be aware the presen of and in cijalu ce p. pardalis o. niloticus river it become a major problem in because may the future. it has been reported by maitland (2004) and pimentel . (2005) that exotic species might et al threaten native species through ecological alteration. moreover, introduced species has caused native species declining due to in numbers competition, disease transmi sion etc. (gozlan s et al. 2010). previous studies negative had shown impact of introduce species hermoso . d . et al (2011) reported that the declining numbers of native species in iberian streams was due to high abun ance of exotic species. a study from albins d and hixon (2008) had also a negative shown impact of exotic species on native with species 79% a aver ge value of reduction. it has been reported that nile tilapia in becomes competitor an ecosystem (cag uan 2007) or predator for a native species (morgan 2004). nile tilapia et al. win the competition because this species is mores a gre sive and voracious (morgan . 2004). a g s et al study from hoover . (2004) has also reported et al a negative impact of to native species p. pardalis in usa waters. in indonesia, yuniartiningisih (2011) has reported that nile tilapia has caused . .r argyrotaenia r lateristriata and declining to be in numbers in pelus river, purwokerto, central java province decrease. this was due to niche overlap among those species. in case of cijalu river, high awareness should be paid because introduced species have higher biological advantages than those native of species, so they can better adapt to aquatic habitat with poor water quality than that native of species. for instance, mouth sucker catfish ( ) live well in aquatic ecosystem with p. pardalis s low oxygen content because this species is equipped with arborescent organ for effective respiration in such ecological condition this . species is also well adapted to aquatic ecosystems with high organic matter content because it fe d e s on detritus and algae (page robin 2006). & whereas, nile tilapia ( ) well adapted to o. ni o icusl t is poor water quality ecosystems (figueredo & giani 2005). therefore, it is necessary to control the developmenet of and p. pardalis o. niloticus population in cijalu river to minimize the threat to native species. fish istributiond s f species was collectedive at the upperstreams se. ,the species were barbodes binotatus rasbora argyrotaenia, poecilia reticulata, channa gac ua glyptothorax platypogon. h and sampling conducted the middle part of ci river at jalu ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 6 biotropia vol. 23 no. 1, 2016 obtained a 6 fish total of 1 species the obtained . species were pterygoplichthys pardalis mystus gulio, , mystus micracanthus hemibagrus nemurus hemibagrus , , nemurus, trichopodus trichopterus, , channa striata oreoch omis niloticus, labiobarbus kuhlii, mystacoleucus r obtusirostris, barbodes binotatus barbodes microps, , osteochilus vittatus rasbora argyrotaenia poecilia , , reticulata osphronemus gouramyand . at the downstream area, total of 1 fish species a 5 was found during the study, i.e. pterygoplichthys pardalis, mystus gulio mystus micracanthus hemibagrus , , nemurus nemacheilus fasciatus, trichopodus trichopterus, , channa striata, labiobarbus kuhlii, mystacoleucus obtusirostris, barbodes binotatus barbodes microps, , osteochilus vittatus rasbora argyrotaenia poecilia , , reticulata anabas testudineus and . it seems that different species compositions were observed in each part. there was also a river tendency that some species only at occurred certain part of the river, such as in g. platypogon upper-stream and in down-streamsa. testudineus areas; while the other species were widely distributed at most part , such as river s l. kuhlii and or even along the river i.e.m. obtusirostris b. binotatus r. argyrotaenia and (table 2). this complex distr bution pattern could be i caused by some species hav specific preference to certain ing ecological condition, while the other species can adapt to wide range of ecological factors. for a example, prefer ed upstream area of g. platypogon r the river with stong current and stony substrate. g. platypogon is equip ed with ventral disc to attach p itself on the substrate in strong current upperstream area. anabas testudineus typical downstream species, were found. the finding was already assumed before sampling since s ecological everal characteristics observed at the lower part of cijalu river such as average water temperature range , d from 28 to 30 ºc, ph 5.7 to 6.8, ranged from carbon di xide range between 0.1 and 0.5 ppm o d , as well as s ,muddy and sandy sub trates fit well with the need of that species. this species adapted to poor aquatic habitat since it is equipped with arborescent organ for effective respiration in such ecological condition. this finding was congruent with tay . (2006)the finding from who et al reported sa. testudineus that inhabit swamp areas, stagnant water bodies, estuaries and ponds. the finding of along part of the b. binotatus river was not surprising since this species normally inhabits wide range of habitats from strong to weak current. a similar finding was also reported in cileumeuh river (nuryanto 2012) and et al. cikawung river (nuryanto . 2015). et al b binotatus . commonly inhabit mountain stream, river and s lake. this mean that can adapt to b. binotatuss a wide range of habitat conditions. the remaining cyprinidae species was found at middleand downstream of cijalu river, ranging from stone to sandy bottom and from high to low water velocity. this means that cyp inids species were distributed in all part of r s the river. the finding was normal since it is agreed with nikolsky (1963) who has the finding from noted that cyprinidae commonly inhabits river with either high or low velocity. g platypogon . a typical upstreams species were found during the sampling. this result was different to nuryanto . (2012; 2015) who et al find g. platypogondid not in cileumeuh and cikawung rivers. the difference could be caused by different fishing gear used to collect samples at the upstream areas in the present and previous studies. the in present study, we used electricshocker during sampling at the upstream, while nuryanto . (2012; 2015) only used et al nets. both sampling gears had different effectiveness to used atbe the areaupstream . electricshocker more effective than nets is because electricshocker can induce fish in any water velocity conditions and in any fish-hiding places to . the electricity induction caused fish become weak and easily to be caught. in contrast, strong current at upstream area washed away the nets before fishes. according to even trapping lapointe . (2006), sampling ef ectivity at each et al f part of river depend upon the fishing geara s , while the gear fish electricshocker is best for sampling upstream . at area mystus nigriceps h. nemurus bagridaeand ( ) were only found at the middle and lower part of s cijalu r conducted at serayu iiver. n another study river mystus nigriceps, it has been reported that and h. nemurus along the r were distributed iver (setijanto sulistyo 2008). the differen could & ce be due to different microhabitat of cijalu and serayu rivers. fcijalu river has many plain or lat and shallow areas with sandy substrates, while serayu river has many sites with swamp and y deep areas muddy susbtrates (nuryanto with & sugiharto 2011). these types of habitats in serayu river most likely the reason that both bagrids are species were distributed along iver.serayu r 7 physico-chemical characteristics a ver ag e valu e s o f physi c o-c he mic al parameters in cijalu rivers are presented in table 3. water velocity and substrates were the two parameters showing quite differences among parts of cijalu river; while the remaining parameters were almost similar among part of s the river. therefore, the discus ion focused on s species distribution comparison based on was those two parameters. fthe di ferences on water velocity and substrates were suggested to e responsib b le for different ish distribution along the river. f pattern it has been well known that water velocity or current is a key factor caus differences among ing river parts (odum 1971) affect distribution, ing movement and adaptive behaviour of riparian organisms (brown 1975). in this study, g platypogon c gac ua. . h were found and only at the u per part of the river with high water velocity p and stony substrate. this may be due to the adaptation capability of these species to river part which has strong current and stony substrate. according to ng and rachmatika (2005) glyptothorax is able to survive in strong water current because it is equipped with thoracic adhesive organ attach on stony , so that it can substrate. whereas, is adapted to strong c. gac uah current and stony substrate by hiding inside crevices or between stones. t he finding of ,p pardalis t trichopterus a . . . and testudineus parts of cijalu in the middle and lower river agreed with our expectation since those three species adapt to water ecosystem with can low oxygen content, high c rbon diox de, low a i acidity and muddy substrate. this finding was in agreement that of stating with pethiyagoda (1991) that well adapted to poor habitata. testudineus is quality. inhabit swamps areas and t. trichopterus s ponds with high density of freshwater vegetations low lim 2012). in addition, ( & pethiyagoda (1991) that reported p. pardalis, t. trichopterus a. testudineus are able and to live in poor habitat because they tohave the ability directly breathe oxygen from the air using additional respiratory organ. the remaining species were not specific site, so they were distributed almost along the river. the ir distribution along part the rivers is well s of supported ecological char cteristic which by a s are relatively d ra . mo e te, especially water velocity conclusions nineteen species offish 10 families were collected during the at cijalu riverstudy , which indicated that the river has high fish diversity. cyp inidae speciose family with six species. r is the some species were distributed along the river, while other species were limited at certain parts of the rivers. the d ce in species ifferen distribution could be due to various physicochemical characteristics parts of the river, along especially water velocity and substrate. acknowledgements we would like to thank jenderal soedirman university for the funding. we greatly appreciated students who involved during the sampling were and laboratory works. we also greatly appreciated fishermen and driver for their help during the study. tabl e 3 average value of physico-chemical characteristics of cijalu river physico-chemical characteristic upper middle lower depth (m) 0.66 0.48 0.60 light intensity (m) 0.47 0.35 0.39 temperature (°c) 28.0 27.0 30.0 velocity (m/s) 1.47 0.67 0.33 acidity (ph) 6.80 6.3 5.7 carbon dioxide (co2) (ppm) 0.1 0.5 0.3 substrate big to medium stones small stones to gravel sand to mud ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. references ahmad ab, fahmi-ahmad m, rizal sy. 2014. fish diversity in sm ll streams of sungkai wildlife reserve, perak, a malaysia. j wildlife and park 29:13-21 . albaret jj, simier m, darboe fs, jean-marc e, raffray j, de morais lt. 2004. fish diversity and distribution in the gambia estuary, west africa, in relation to environmental variables. aqua liv resour 17(1): t ing 35-46. albins ma, hixon ma. 2008. invasive indo-pacific lionfish pterois molitans reduce recruitment of atlantic coralreef fishes. mar ecol prog series 367:233-8. american public health association 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( our common future. geneva (ch the world ): commission on environment and development commission for the future. welcomme rl. 2000. fish biodiversity in floodplains and their associated rivers. in: gopal b, junk wj davis , ja, editor. biodiversity in wetlands: assessment, function and conservation, volume 1 leiden (nl): . backhuys publishers p 61–87.. wootton rj. 1991. ecology of teleost ishes, fish and fisheries 1.f london (uk): chapman & hall. yuniartiningsih s. 2011. analysis of ec logical impact of o introduced species on cyprinidae diversity. master thesis. purwokerto (id): postgraduate program of jenderal soedi man universityr . 9 ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 6. dyah (testing) revisi.cdr biotropia vol. 21 no. 2, 2014: 125 130 testing reliability of serum samples as a dna source on captive breeding longtailed macaques ( )macaca fascicularis dyah perwitasari-farajallah and achmad farajallah received 24 may 2013/accepted 14 september 2014 the serum reliability was tested in captive breeding conditions on the long-tailed macaques ( ). serum and buffy coat were applied to standard protocol of dna extraction following the amplification of microsatellite dna locus of d2s1777 by polymerase chain reaction (pcr) and visualized pcr products by means of polyacrylamide gel electrophoresis (page) with silver staining. the amount of genomic dna extracted from serum was sufficient for genotyping individuals at dna microsatellite locus of d2s1777 with allele size of approximately 160 bp. the study showed that serum could provide reliable alternative for obtaining dna when taking blood using anticoagulant was impracticable. serum, dna source, captive breeding, monkeys 1,2* 1 1 department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor 16680, indonesia primate research center, bogor agricultural university, bogor 16151, indonesia macaca fascicularis 2 abstract introduction keywords: primates play an essential role in biomedical research. the study of primates has also contributed to our understanding of basic biological phenomena such as reproduction and several diseases, to the development of drugs, and vaccines (nathanson & maithieson 2000; sibal & samson 2001). as in other model organisms, genetic background in primates such as long-tailed ( ) and rhesus macaques ( ) is an experimental variable that affects the response of other study variables (stevison & kohn 2008; hernandez . 2007). a standardized system for genetic testing would be beneficial for the management of breeding colonies and also facilitate the characterization of other important traits, including mhc type, resistant to simian immunodeficiency virus (siv) and the susceptibility to many human pathogens (satkoski . 2008). moreover, data on reference of hematologic and serum parameters are essential for macaca fascicularis m. mulatta et al et al * corresponding author : witafar@yahoo.com doi: 10.11598/btb.2014.21.2.6 125 evaluating results obtained from laboratory animals and this baseline biological data are needed before they are used for scientific purposes (bonfanti . 2009). in fact, to fulfill the entire criteria mentioned, the analyses relied on the availability of blood samples. upon the purposes of studies, ordinarily blood samples are taken from the animals without anticoagulant or with anticoagulant, such as heparine and sodium citrate (especially for genetic characterization). however, when obtaining blood samples with anticoagulant is impracticable for some reasons, available samples particularly serum or clot samples will be an alternative samples to be utilized. the lack of published description for detailed protocol on serum application, however, facilitated the remarkably approach for genetic analyses of captive breeding monkeys. as a result, our study aimed to examine the applicability and reliability of serum samples as a dna source. blood samples were collected in conjunction with routine health physical examinations of individuals in captive breeding population of long-tailed macaques ( ) housed at primate research center, bogor agricultural university. two kinds of blood samples were available for analyses: one using heparin as anticoagulant and the other without heparin. blood with anticoagulant were centrifuged at 3,000 rpm for 15 minutes. erythrocyte and plasma were stored at -20 c for other purposes, and buffy coat was directly applied to dna extraction protocol. along with processing blood with anticoagulant, serum was obtained after samples were allowed to clot. in total 38 sera and buffy coat were used as dna sources. dna extraction was carried out following the method of kan . (1977) with minor modification for serum and buffy coat separately to avoid potential contamination. serum or buffy coat was washed with 0.9% nacl1mm edta. centrifugation was performed at 2,000 rpm for 10 minutes. supernatant was discarded and pellet was suspended in 2.5 ml ste buffer (salted tris-edta), 200 μl 10% sds and digested with 40 microl proteinase k (5 mg/ml) for a minimum of 2 hours at 37 c. then genomic dna was extracted with standard phenol-chloroform method. as dna microsatellites are the most common neutral nuclear marker applied in genetic diversity studies, we tested microsatellite amplification to validate amplification success from the tested sources. the test was based on amplification of d2s1777 microsatellite marker, a monomorphic dna microsatellite locus on genetic study of long-tailed and pig-tailed macaques (perwitasari-farajallah . 2007; perwitasari-farajallah 2010). allele size in contemporary long-tailed macaques was 160 bp. pcr was conducted in a ptc 100 mj research inc. in 12.5 μl reaction volumes using pcr buffer (containing 25 mm mgcl l 2.5 mm dntp. each reaction contained 25 pm of each primer flanking the microsatellite region (f:5'-tccccaagtaaagcattgag-3';r:5'et al macaca fascicularis et al et al et al. materials and methods o o + 2), 1.0 u/μ taq polimerase (promega), 10xpcr buffer, 126 biotropia vol. 21 no. 2, 2014 gtatgtaggtagggaggcagg-3') and approximately 10-100 ng of dna. after 3 minutes initial denaturation at 94 c, pcr was performed in a total of 30 cycles using the following conditions: 30 seconds denaturation at 94 c, 60 seconds annealing at 48 c, and 60 seconds elongation at 70 c (perwitasari-farajallah . 2004). with a final elongation of 5 minutes at 72 c, pcr was terminated. strict precautions against pcr contamination were performed. separate area were utilized for the preparation of amplification reactions, the addition of dna template, and the carrying out of amplification reactions. negative water pcr blanks were included in every analysis. two microliters of each pcr products were electrophoresed on 6% polyacrylamide gels and visualized by silver-staining following the technique described by tegelström (1986). allele sizes were verified using dna size standard of 100 bp ladder (biorad). dna was successfully extracted from almost all the samples tested. seventy four percent of the serum samples ( = 15) and 100% of the buffy coat ( = 19) were successfully amplified and gave strong amplification products (fig.1 & 2). it revealed that dna derived from buffy coat samples have sufficient dna for repeats genotyping attempts. in contrast, insufficient dna yielded in serum samples may in part be attributed to inadequate amount of nucleated cell inside the samples. o o o o o et al n n results and discussion figure 1. amplification of the d2s1777 microsatellite locus from ten long-tailed macaques serum and buffy coat. m: size standard, 100-bp dna ladder; lanes 1-5, dna extracted from serum; lanes 6-10, dna extracted from buffy coat. 127 testing reliability of serum samples as a dna source on captive breeding dyah perwitasari– et al. dna size standard of 100 bp (biorad) and pcr products on 6% page stained with silver demonstrated that the concentration of the pcr products of isolated dna from serum samples and from buffy coat were lower and higher compared to the dna size standard, respectively. although the dna extracted from serum samples yielded low concentration of pcr product, we conceived that it revealed reliable results for further analyses. present study demonstrated that d2s1777 locus revealed to be monomorphic (fig.1&2) as detected in our previous research with the allele size of approximately 160 bp. although false alleles or amplification artefact are typically not only of low frequency, but also sporadic in occurrence, it cannot be disregarded as a potential source of error in microsatellite genotyping (taberlet 1999). despite report by fernandes . (2007) that the use of species-specific primers reduces the risk of false species assignment; the results obtained in this study consisted of apparent allele repeatedly amplified from several samples. moreover, the d2s1777 is a humanderived microsatellite locus used in our laboratory for the routine genotyping of longtailed macaques ( ) using buffy coat as dna source (perwitasari-farajallah . 2010; perwitasari-farajallah 2007). many genotyping studies depend on cross-species amplification, that is, the application of primers derived in one species for characterization of individuals in another, usually closely related species (bradley . 2008). therefore, a variety of strategies to determine dna quality and detect or reduce genotyping errors should be determined (paetkau 2003). utilization of serum samples was described previously by jiminez and tarantal (2003). they showed that fetal gender can be reliably determined in the early first trimester from maternal serum samples of rhesus macaques ( ) by real-time pcr. they adapted and applied the idea of some researchers (lo . 1997, 1998; costa . 2001; honda . 2002) regarding fetal gender determination in human by et al. et al m. fascicularis et al et al. et al m. mulatta et al et al et al figure 2. amplification of the d2s1777 microsatellite locus from ten long-tailed macaques serum and buffy coat. size standard, 100-bp dna ladder; lanes 1-4, dna extracted from serum; lanes 5-12, dna extracted from buffy coat. s = serum; bc = buffy coat 128 biotropia vol. 21 no. 2, 2014 real-time pcr analysis of maternal serum samples. even though they used serum for different purposes if compared to what we performed in our study, it appeared to be all of them obtained satisfactory results. our study provided, probably (to our knowledge), the first description of serum applicability as a source of dna for future genetic studies of long-tailed macaques captive breeding population when obtaining blood samples using anticoagulant is impracticable. we were grateful to primate research center, bogor agricultural university for making available samples and to division of animal systematic and ecology, department of biology, bogor agricultural university for laboratory assistance. drs randall c. kyes (university of washington, seattle, usa) and betsy ferguson (oregon health and science university, usa) provided important comments and suggestions. we thanked anonymous reviewers for helpful suggestions to improve this manuscript. conclusions acknowledgements references bradley b, doran-sheehy d, vigilant l. 2008. genetic identification of elusive animals: re-evaluating tracking and nesting data for wild western gorillas. j zool 275:333-40. bonfanti u, lamparelli d, colombo p, bernardi c. 2009. hematology and serum chemistry parameters in juvenile cynomolgus monkeys ( ) of mauritius origin: comparison between purpose-bred and captured animals. j med primatol 38:228-35. costa jm, benachi a, gautier e, jouannic jm, ernault p, dumez y. 2001. first-trimester fetal sex determination in maternal serum using real-time pcr. prenatal diagnostic 21:1070-4. fernandes ca, ginja c, pereira i, tenreiro r, bruford mw, santos-reis m. 2007. species-specific mitochondrial dna markers for identification of non-invasive samples from sympatric carnivores in the iberian peninsula. 9:681-90. hernandez rd, hubisz mj, wheeler da, smith dg, ferguson b, rogers j. 2007. demographic histories and patterns of linkage disequilibrium in chinese and indian rhesus macaques. science 316:240-3. honda h, miharu n, ohashi y, samura o, kinutani m, hara t, ohama k. 2002. fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal dna in maternal serum. human genetic 110:75-9. jimenez df, tarantal af. 2003. fetal gender determination in early first trimester pregnancies of rhesus monkeys ( ) by fluorescent pcr analysis of maternal serum. j med primatol 32:315-9. kan yw, dozy am, trecartin r, todd d. 1977. identification of a nondeletion defect in thalassemia. n engl j med 297:1081-4. macaca fascicularis conservation genetics macaca mulatta 129 testing reliability of serum samples as a dna source on captive breeding dyah perwitasari– et al. lo ym, corbetta n, chamberlain pf, rai v, sargent il, redman cw, wainscoat js. 1997. presence of fetal dna in maternal plasma and serum. lancet 350:485-7. lo ym, tein ms, lau tk, haines cj, leung tn, poon pm, wainscoat js, johnson pj, chang am, hjelm hm. 1998. quantitative analysis of fetal dna in maternal plasma and serum: implications for noninvasive prenatal diagnosis. am j human genetic 62:768-75. nathanson n, maithieson bj. 2000. biological consideration in the development of a human immunodeficiency virus vaccine. j infec dis 182:579-89. paetkau d. 2003. an empirical exploration of data quality in dna-based population inventories. mol ecol 12:1375-87. perwitasari-farajallah d, kyes rc, iskandar e. 2010. microsatellite dna polymorphisms for colony management of long-tailed macaques ( ) population on the tinjil island. biodiversitas 11:55-8. perwitasari-farajallah d. 2007. human short tandem repeat (str) markers for paternity testing in pig-tailed macaques. hayati 14:39-43. perwitasari-farajallah d, farajallah a, kyes rc, sajuthi d, iskandriati d, iskandar e. 2004. genetic variability in a population of long-tailed macaques ( ) introduced onto tinjil island, indonesia: microsatellite loci variations. hayati 11: 214. satkoski j, george d, smith dg, kanthaswamy s. 2008. genetic characterization of wild and captive rhesus macaques in china. j med primatol 37:67-80. sibal lr, samson kj. 2001. nonhuman primates: a critical role in current disease research. ilar journal 42:7484. stevison ls, kohn m.h. 2008. determining genetic background in captive stocks of cynomolgus macaques ( ). j med primatol 37:311-7. taberlet, p, waits lp, luikart g. 1999. noninvasive genetic sampling: look before you leap. trends in ecol evol 14 323-7. tegelström, h. 1986. mitochondrial dna in natural populations: an improved routine for the screening of genetic variation based on sensitive silver staining. electrophoresis 7:226-9. macaca fascicularis macaca fascicularis macaca fascicularis : 130 biotropia vol. 21 no. 2, 2014 microsoft word 1 biotropia no. 23, 2004 : 1 12 optimization of cellulase production with penicillium nalgiovense sll grown on pretreated wheat pollard tresnawati purwadaria1, agnes t. kumalasari2, tutiharyati1, pius p. ketaren' and arnold p. sinurat1 1 indonesian research institute for animal production, po box 221, bogor 16002, indonesia 'department of chemistry, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, indonesia abstract the cellulase production with penicillium nalgiovense s11 on wheat pollard was enhanced using substrate pretreatments, i.e.: (i) mechanic process by wiley milling, (ii) reducing sugars removal by water soaking, and (iii) chemical pretrcatment by 0.5% naoh soaking at 100°c. the enzyme production stated as enzyme activities of all prctrcated substrates were higher than the untreated substrate. although soaking with water showed significant increase in enzyme activities, the highest cmcase (ec 3.2.1.4), fpase (filter papcrase) and pglucosidase (ec 3.2.1.21) were observed on naoh pretreated pollard. the naoh prctreatment also enhanced the enzyme production by increasing substrate concentration from 2 to 4%. the optimal incubation time in the cellulase production on 4% naoh-pretrcated pollard was observed on the fifth day. addition of 250 ppm glucose also increased the enzyme activities. the optimal treatments increased the specific activities of cmcase, fpase, and |3-glucosidase into 60, 4, and 198 times, respectively, as compared to the specific activities on 2% unpretreated pollard. keywords : cellulase production/ pollard pretreatments/ water soaking/ naoh soaking introduction most poultry feedstuff such as corn and soybean meals, rye, barley, rice bran, wheat bran, coconut meal, and palm kernel cake from plants contain fibers including hemicellulose, cellulose, and lignin. in contrast with ruminants, poultry digestive track does not digest hemicellulose and cellulose. recently, it was reported that fermentation occurred in the caecum of poultry due to microbial activity (josefiak et al. 2004), the fermentation could break down cellulose in a small quantity. the addition of cellulases and hemicellulases in the poultry feed had been reported to increase body weight, efficiency of feed utilization, the apparent metabolizable energy, and dry matter digestibility (campbell and bedford 1992; friesen et al. 1992; marquardt et al. 1996). the effectiveness of the enzymes was related to the adequate activity of enzyme added. cellulase production has been studied since thrichoderma reesei was isolated by reese in 1950. however, the enzyme activity was limited by its low specific activity. penicillium nalgiovense s l l was isolated from the nest of termites glyptotermes montanus (nurbayti 2002). the mold had the ability to produce cellulase that digested high crystalline cellulose (filter paper), the most common components in natural cellulose. the enzyme could be produced in the submerged culture containing 2% wheat pollard. increasing the substrate into 3% concentration 1 biotropia no. 23, 2004 reduced the enzyme activity due to limited oxygen availability and increasing the repression effect caused by soluble carbohydrate, such as starch and reducing sugars (gong and tsao 1979). the repression effect from the pollard could be omitted by reducing the soluble sugars using water soaking. in addition, the production of enzyme could be enhanced by mechanic treatment on the substrate using milling and extrussion, and chemical treatment using phosphoric acid, hydrochloric acid, sodium hydroxide (naoh), and ammonia (klyosov 1986; marsden and gray 1986). among the chemical pretreatments, naoh was the most commonly used. the naoh treatment swells the crystalline regions of cellulose and extends the amorphous regions. the amorphous regions are more digested than the crystalline regions. recently, shin et al. (2000) found that the naoh pretreatment at room temperature extracted the short molecules in the amorphous region of tenzel, a cellulose fiber produced from wood pulp into the open-up structure. this condition enhanced the cellulase hydrolysis activity. in this experiment the cellulase production of p. nalgiovense s11 was optimized by substrate pretreatments i.e.: (i) mechanic process by wiley milling; (ii) removal of reducing sugars by water soaking; and (iii) chemical treatment by naoh soaking at 100°c. the activities of carboxymethylcellulase (cmcase) and filter paperase (fpase) in hydrolyzing the backbone of amorphous and crystalline cellulose, as well as p-glucosidase that cleaves the cellooligomers into glucose were determined as parameters for enzyme production. materials and methods pretreatment of wheat pollard wheat pollard was obtained from pt bogasari wheatmill and used as substrate for p. nalgiovense s11 (riap collection) in producing cellulase. the wheat pollard was pretreated before being used as substrate i.e.: (i) wheat pollard without pretreatment as a control referred as pollard; (ii) milling; (iii) water soaking; and (iv) naoh soaking, referred as water and naoh solution pretreated, respectively. in the wiley mill, the pollard was ground to a fine powder of 0.5 mm. for the water treatment, 25 g pollard was soaked for one hour in 500 ml of water at room temperature, then the water was squeezed out using cotton cloth, the solid material was dried in the 40°c oven, and ground with wiley mill (0.5mm). the method for naoh pretreatment was carried out following purwadaria (1988). the same amount of pollard (25g) was soaked in 500 ml of 0.5% naoh solution (w/v) for 1 hour at 100°c. then the pollard was filtered through cotton cloth and washed under running tap water until the wash water was neutral to ph paper. excess water was squeezed out, dried in the 40°c oven, and ground with wiley mill. ccllulase production of penicillium nalgiovense si 1 tresnawati purwadaria et al. enzyme production enzymes were produced by p. nalgiovense s11 in the mandels medium containing yeast extract 3 g/1, minerals in g/1 (nh4)2so4 1.4, kh2po4 2.0, mgso4 0.3, urea 0.3, and cacl2 0.3 and'in ppm feso4 5, mnso4 16, znso4 14, and cocl2 20 and pollard (purwadaria et al. 2003a). the concentration of pollard and pretreated pollard was 2, 3, or 4%. molds were cultivated in 50 ml medium in a 250 ml erlenmeyer flask at 29°c using reciprocal shaker (150 rpm), after inoculation with 2 ml of spore suspension from five day pda culture slant (15x10*'spores/ml). the incubation time was carried out for three, four, or five days. sodium azide was added at 0.2% final concentration. the culture was then centrifuged (12 000 rpm, 20 min, 4°c) and supernatant was collected for enzyme assays. enzyme activities the activity of carboxymethylcellulase (cmcase) was assayed by determining the reducing sugars produced from cmc as glucose or mannose, respectively (haggett et al. 1979). one unit was defined as amount of enzyme which liberates one umol glucose per minute. the p-dglucosidase and (3-d-cellobiosidase were assayed using p-p-d-nitrophenylglucoside and p-p-dnitrophenylcellobioside (from sigma company) as substrates and one unit was defined as enzyme which liberates one umol nitrophenol per minute (ide et al. 1983; deshpande et al. 1984). filterpaperase (fpase) was determined by using filter paper whatman no 1 (1 cm x 6 cm) as substrate following mandels and sternberg (1976). the production of 2 mg glucose in the reaction was stated as 0.185 unit of fpase. all assays considered the controls prepared as samples, but without incubation time. specific activity of all enzymes was calculated in units/mg protein (u/mg). determination of protein, reducing sugar, and fiber component concentrations protein concentration was determined by bradford method (1976) and bovine serum albumin was used as standard. reducing sugar concentration in pollards was determined after water extraction using dns method (miller 1959). the concentrations of fiber components (cellulose, hemicellulose, lignin and silica) of pollard before and after pretreatment were calculated as neutral and acid dietary fiber according to van soest and robertson (1968). results and discussions every pretreatment of the pollard enhanced the enzyme production (activities) of p. nalgiovense s11 (table 1). milling increased the ratio of surface area to volume resulting to better mold growth and enzyme production. the results are in agreement with cellulase production of bacillus subtilis cbtk 106 on banana biotropia no. 23, 2004 wastes (krishna 1999). it was reported that the increase of the bacterial enzyme activities (cmcase, fpase, and p-d-glucosidase) was parallel with particle reduction. however, the optimum reduction was observed at particle size of 0.4 mm. in the case of p. nalgiovense s11, compared with untreated pollard, milling at particle size of 0.5 mm increased fpase and p-d-glucosidase, but slightly decreased cmcase activities. in this condition, oligosaccharides and the size reduction of crystalline and amorphous celluloses might induce more fpase and p-d-glucosidase, but repressed the cmcase production. table 1. ccllulaso production of p. nalgiovense si 1 on pretrcatcd substrate pollard pretreatments [protein] (ug/ml) activity(u/ml) specific activity (u/mg protein) cmcase fpase p-gluc cmcase fpase p-gluc none (control) 322±34 0.10+0.05 0.90+0.02 0.004+0.001 0.31+0.16 2.82+0.33 0.014+0.006 milling 334±55 0.06+0.02 1.89+0.18 0.024±0.012 0.19+0.02 5.8h1.35 0.070+0.021 water soaking 181+ 4 1.02+0.02 1.77+0.07 0.091+0.004 5.61+0.03 9.78+0.50 0.504+0.017 naoh soaking 229+30 2.96+0.11 1.93+0.07 0.199+0.031 13.08+1.38 8.56+1.41 0.870+0.080 fpase was filter paperase, p-gluc was p-glucosidase milling also slightly increased extracellular (soluble) protein. therefore, the ratio of specific activities (u/mg protein) of each enzyme between milling and non-treatment was similar with the activities (u/ml). fpase and p-d-glucosidase of specific and enzyme activities were higher in milling, while cmcase was lower than those of untreated pollard. in the case of water pretreatment, soaking decreased the reducing sugar content (table 2) that reduced the repression effect. some starch and pectin were solubilised and rinsed with water resulting in the decrease of the viscosity of the medium (pawlik et al. 1990). then, the mold had better growth and enzyme production than in untreated pollard due to better oxygen transportation and substrate homogen-ization. although, all enzyme activities increased in water pretreatment, extracellular protein was decreased than in untreated pollard. these indicated that the protein in the water pretreatment process might be only related to enzyme production as the soluble protein from pollard was entirely rinsed. the increase of enzyme activities and the decrease of protein concentration resulted in the increase of the specific activities of cmcase, fpase, and p-d-glucosidase to 18, 3, and 35 times, respectively, than those in untreated substrate, while the activities were only 10, 2, and 23 times higher than those in untreated substrate, respectively. ccllulase production of penicillium nalgiovense s11 trcsnawati purwadaria et al. table 2. the fiber and reducing sugar contents of pollards before and after treatment pretreatments [reducing sugars] (%) [fiber] (%) hemiccllulosc cellulose lignin none (control) 0.16 23.0 7.4 1.5 milling 0.17 23.0 7.4 1.5 water soaking 0.13 37.8 12.0 2.0 naoh soaking 0.01 49.5 26.9 7.1 the highest enzyme activity was observed in naoh pretreatment. in this process milling, water soaking and naoh action in the fiber, all together changed the pollard composition (table 2) and altered the substrate into more digestible fibers (khrisna 1999, shin et al. 2000; and devrije et al. 2002). among pretreated pollard, naoh pollard had the highest hemicellulose, cellulose, and lignin contents (table 2) due to the solubilisation of some dry materials including amino acids, starch, pectin, and short oligosaccharides. although its fiber content was the highest, more digestible fiber in naoh treatment resulted better growth and enzyme production. since the reducing sugars of naoh pretreated pollard was very low, it was possible that s11 used only glucose or energy to grow from cellulolytic hydrolysis. the less repression effect of small amount of reducing sugars and the need for energy highly induced the enzyme production and increased the extracellular protein content (table 1). the naoh pretreatment also altered the fiber structure into more swelling materials as a result it can be more submerged in the culture. before treatment, the pollard was more floating. these conditions affected better substrate homogenization and oxygen transferred in the culture, which is important for the mold growth. the high protein content in the naoh pretreated pollard resulted in lower specific activity of fpase than of the water pretreatment. however, other enzyme activities and protein content were much higher than in the water pretreated pollard. therefore, naoh pretreatment was applied for further experiment. in the following experiment, the concentration of naoh pretreated pollard increased from 2 to 3 and 4%. increasing the substrate content changed the enzyme production in the course of incubation time (figure 1 and 2). the repression effect in the enzyme production by increasing the untreated substrate from 2 to 3% (nurbayti 2002) was not observed in this experiment. the highest production of cmcase for digesting amorphous cellulose was observed at 4% pollard. the optimum cmcase activity in 2% pollard was observed at three days incubation time and thereafter was decreasing, while in 3% pollard reached the optimum activity at four days, and in 4% pollard was still increasing even at five days incubation time (figure 1a). compared with cmcase activity, fpase showed different patterns (figure ib). for biotropia no. 23, 2004 every substrate concentration, the optimum activity was reached at 4 days incubation time and thereafter it was declining. the highest fpase activity was observed in 4% pollard that also had the highest reduction at five days incubation time. the production of hydrolytic enzymes was generally parallel with the growth curve of microorganisms, the activity was increasing until optimum incubation time and then was stable or decreasing (sachslehner et al. 1998). the optimum incubation time for enzyme production was related to substrate concentration. the higher substrate concentration of coconut meal in submerged culture of eupeniciuium javanicum resulted in longer lag phase or longer optimum incubation time for p-mannanase production (purwadaria et al. 2003b). in the case of cmcase production with p. nalgiovense sl l , the optimum activity in 2% substrate concentration was reached at three days incubation time, after that, due to limited nutrient concentration, the enzyme production ceased. in the higher substrate concentrations, cmcase reached the optimal activities after longer incubation time, four days for 3% pollard, and at least 5 days for 4% pollard. in the more concentrated substrate, the culture needs longer incubation time due to less oxygen transfer. longer optimum incubation time in the higher substrate concentration rarely occurred in the soluble substrate. increasing xylobiose from 0.05 to 1.00% for the production of pmannanase with sclerotium rolfsii resulted to similar optimum incubation time at 15 hours (sachslehner et al. 1998). in this case, concentration higher than 1% might repress the enzyme production. the reduction of fpase on 4% naoh pretreated pollard at five days incubation time showed that unlike the cmcase, the fpase was not produced anymore. it is difficult to explain why the cmcase was still produced, while fpase stopped. the reasonable explanation for the reduction of fpase was enzyme instability. nurbayti (2002) reported the reduction of fpase activity from culture filtrate of sll stored at room temperature. the highest reduction of fpase in the 4% substrate might be due to the highest reducing sugar concentration produced from the hydrolytic action of cellulase in the substrate, which inhibited the activities of the enzyme especially the fpase. the curves of specific activities for both cmcase and fpase (figure 2) were similar to curves of enzyme activities (figure 1) reflecting those which were not affected by the protein concentration. this result confirmed the data from pretreatment comparison (table 1), where the extracellular protein produced in naoh pretreated pollard was more for cellulase, but not for other proteins. the optimum substrate concentration to produce cmcase and fpase was 4%, however, the optimum incubation time for cmcase and fpase were five and four days; respectively. the high fpase is important for feed supplement application, since most natural celluloses contain crystalline structure. however, in the fifth day the increase of cmcase was much higher than the decrease of fpase. the cmcase was also needed for the synergistic hydrolytic activity on sigmacell 20, the microcrystalline cellulose (purwadaria 1995). therefore, the fifth day incubation in 4% substrate was selected for further experiment in exploring the effect of glucose addition. cellulase production of penicillium nalgiovense s l l tresnawati purwadaria et al. the addition of glucose at 250 ppm in 4% naoh pretreated pollard (containing 50 ppm reducing sugars) increased cmcase and fpase, while higher glucose concentration reduced the activities (figure 3). however, the reduction of cmcase and fpase in the addition of glucose at 1000 rpm were only 6 and 9%, respectively. p. nalgiovense s l l is quite resistant to catabolite repression. in another experiment the isolate still produced cellulases in 3% naoh pretreated pollard added with 10,000 ppm (1%) glucose, but the activity was much lower than that without glucose addition (sanjaya 2003). 250 500 750 1000 glucose concentration addition(ppm) figure 3. the relative specific cellulase activities from cultures added with glucose p-glucosidase and cellobiohydrolase activities were determined in the optimum fpase and cmcase production with the addition of glucose at 250 ppm. compared to the p-glucosidase and cellobiohydrolase produced in 4% pollard without glucose addition, those were 0.87 and 0.14 u/ml, respectively, p-glucosidase was increased to 1.10 u/ml, while cellobiohydrolase was reduced into 0.07 u/ml. similar to cmcase and fpase, p-glucosidase was enhanced by the addition of glucose at 250 ppm, but the condition repressed the cellobiohydrolase. the cellobiohydrolase might be the least resistant enzyme to catabolite repression. the cellulase production was induced by short oligosaccharides including disaccharides, such as cellobiose, xylobiose and sophorose, while pure or natural polysaccharides induced more cmcase, xylanase, and mannanase (sachslehner et al. 1998). in the low concentration of monosaccharides such as glucose, galactose, and mannose, the low constitutive cellulase could be produced (gong and tsao 1979; sachslehner et al. 1998). therefore, in the low glucose addition (250 ppm), biotropia no. 23, 2004 the cellulase production of p. nalgiovense si 1 increased, since glucose was used by the mold to start growing. the low addition of glucose (500 ppm) in 1% gum locust bean was also reported to increase the production of mannanase by e. javanicum (haryati et al. 1995). generally in the presence of high reducing sugars, the cellulase is produced after the reducing sugars are used (sachslehner et al. 1998). the same authors also reported that although one dose of cellobiose 3.0 mm (0.5%) could induce the cellulase production of s. rolfsii, the ten times incorporation at 0.3 mm at every 2.5 hours induced 2.5 times higher enzyme. in the case of our experiment, the addition of glucose at 250 ppm increased cmcase, fpase, and p-glucosidase, but reduced cellobiohydrolase. detailed experiment to observe the regulation mechanism of s11 has not yet been carried out. it could be concluded from this experiment that naoh pretreatment in wheat pollard, increasing substrate concentration from 2 to 4% and addition of glucose at 250 ppm at 5 days incubation are the optimum conditions to produce cellulase of p. nalgiovense s l l based on its specific activities of cmcase, fpase and p-glucosidase. the expense of naoh pretreatment and the gain of enzyme activities has to be economically evaluated. acknowledgment the authors appreciate drh. sulistyani phd. from chemistry department, fmipa-bogor agricultural university for sharing her comments on the result of the experiment. references bradford, m.m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochcm. 72: 248-254. campbell, l. and m.r. bedford. 1992. enzyme application for monogastric feeds: a review. can. j. anim. sci. 72: 449466. deshpande, m.v., k..e. eriksson, and l.g. pettersson. 1984. an assay for selective determination of exo-1,4-p-glucanases in a mixture of cellulolytic enzymes. anal. biochcm. 138: 481-487. devrije, t., g.g. dchaas, g.b. tan, e.r.p. reisers, and p.a.m. claassen. 2002. pretreatment of mischanthus for hydrogen production by thermotoga elfii. int. j. hydro. energy. 27: 1381-1390. friesen, o.d., w. guenter, r.r. marquardt, and b.a. rotter. 1992. the effect of enzyme supplementation on the apparent matabolizablc energy and nutrient digestibilities of wheat, barley, oats, and rye for young broiler chick. poult. sci. 71: 1710-1721. gong, c.s. and g.t. tsao. 1979. cellulase and biosynthesis regulation. ann. reps. ferment. processes. 3: 111-139. haggett, k.d., p.p. gray and n.w. dunn. 1979. crystalline cellulose degradation by a strain of cellulomonas and its mutants derivatives. eur. j. appl. microb. biotechnol. 8: 183-190. 10 cellulase production ofpenicillium nalgiovense s l l tresnawati purwadaria et al. haryati,t., t. purwadaria, j. darma, and b. tangcndjaja. 1995. pengaruh pcnambahan gula rcduksi pada produksi pmananase olch bcrbagai isolat kapang mananolitik.(effect of reducing sugar addition on the production of pmannanase with mannanolytic molds). hayati 2: 68-73. ide, j.a., j.m. daly, and p.a.d. rickard. 1983. production of glycosidase activity by cetlulomonas during growth on various carbohydrate substrate. eur. j. appl. microb. biotcchnol. 18: 100-102. josefiak, d., a. rutkowski, and s.a. martin. 2004. carbohydrate fermentation in the avian ccca: a review. anim. feed sci. technol. 113:1-15. klyosov, a.a. 1986. enzymatic conversion of ccllulosic materials to sugars and alcohol. the technology and its amplications. appl. biochem. biotcchnol. 12: 249-300. krishna, c. 1999. 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partial hydrolysis of the viscous water soluble pentosans following water soaking or fungal enzyme treatment. brt. poult. sci. 31: 525-538. purwadaria, m.b.t. 1988. purification and characterization of a cellulomonas ccllulase complex. phd thesis, university of new south wales. purwadaria, t. 1995. synergism in the hydrolysis of cellulose by endoglucanasc i and ii (endo i and endo ii) and cellobiohydrolasc i (cbh i) purified from cellulomonas csi-17. ann. bogoricnses. 3: 12-24. purwadaria, t., n. nirwana, p.p. ketarcn, d.i. pradono, and y. widyastuti. 2003a. synergistic activity of enzymes produced by eupenicillium javanicum and aspergillus niger nrrl 337 on palm oil factory. biotropia20: 1-10. purwadaria, t., t. haryati, e. frederick, and b. tangcndjaja. 2003b. optimation of p-mannanase production on submerged culture of eupenicillium javanicum as well as ph and temperature enzyme characterization. jitv. 8: 46-54. sachslehner, a., b. nidetzky, k.d. kulbe, and d. haltrich. 1998. induction of mannanasc, xylanase and endoglucanase activities in sclerotium rolfsii. appl. environ. microbiol. 64: 594-600. sanjaya, s. 2003. produksi selulase penicillium nalgiovense s l l hasil radiasi dengan ultra violet (cellulase production of penicillium nalgiovense s l l mutants by ultra violet radiation). thesis. chemistry department. fmipa pakuan university, bogor, indonesia. 11 biotropia no. 23, 2004 shin, y. k.. son, and d.i. yoo. 2000. structural changes in tencel by enzymatic hydrolysis. j. appl. polym. sci. 76: 1644-1651. van soest, p.j. and j.b. robertson. 1968. system of analysis for evaluating fibrous feeds. jn w.j. pigden ed. standardization of analytical methodology for feed. cent. canada, idrc. 134e. 12 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 4. kurniawan (melimination).cdr biotropia vol. 18 no. 2, 2011: 94 101 elimination of cvb ( ) from a range of chrysanthemum varieties by apical meristem culture following antiviral agent and heat treatments chrysanthemum virus b kurniawan budiarto*, budi marwoto, lia sanjaya, muchdar soedarjo and indijarto budi rahardjo received 06 august 2010/accepted 28 september 2011 cvb elimination for retaining healthy protocols from infected chrysanthemum plant was investigated through combined treatment of meristem culture with synthetic antiviral ribavirin or thermotherapy under conditions. the biological materials used for the experiment constituted of six commercial varieties: dewi sartika, saraswati, yellow fiji, white puma, yellow puma and white reagent. tissue culture initiation was conducted through plantlet establishment using ms supplemented with iaa. ribavirin was added in media with the concentration of 40 mg/l on cv. dewi sartika, saraswati and yellow fiji. parallel with this step, heat treatment with different durations (1, 2, and 3 weeks) was also conducted on the plantlets on white puma, yellow puma and white reagent. meristem culture was done following the chemoand thermotherapy. the experiment resumed the failure of single treatment of meristem culture in eliminating cvb from the infected chrysanthemum plantlets. under heat treatment, percentage of virus-free plantlets increased along with the duration of thermotherapy, though the survival rate of plantlets decreased in lengthened heat treatment. the best results regarding virus free plant percentage were obtained when meristem culture was applied following ribavirin or three weeks of heat treatment. (cvb), chemotherapy, heat treatment, meristem culture, virus-free indonesian ornamental crops research institute jl. raya pacet-ciheraang, po. box. 8 sdl, cianjur, west java-indonesia (43253) in vitro chrysanthemum virus-b abstract introduction key words: chrysanthemum ( kitam) is one of the major cut flowers in the world. chrysanthemum ranks the first of all cut flowers marketed dendranthema grandiflora [ramat.] * corresponding author : mbud1arto@yahoo.co 94 every year from indonesia (37.34 %) with the quantity of more than 66 million stalks in 2007 (indonesian general directorate of horticulture 2008). nowadays, however, chrysanthemum production has faced some constraints, and one of these was systemic disease attacks caused by viruses, viroids and phytoplasmic organisms. up to present, chrysanthemum virus-b (cvb) is still one of the most common viruses found in commercial growers and have caused significant economic losses. (marwoto 2004). taxonomically, cvb belongs to carlavirus wide group. genome of the virus consists of unipartite, single-stranded rna with total genome size of 7.5 kb (levay & zavriev 1991). the nucleocapsid (virions) are filamentous with no protein envelop and usually straight with a clear modal length of 685 nm (brunt . 1996). aside from chrysanthemum (compositae), cvb was also known systemically hosted in (leguminosae), nicotiana, petunia and tetragonia (megan 2001). the general symptoms of the infected plant were stunted in growth, chlorotic in leaf blades, organ structure malformation and discolored petal. degeneration in proliferation level with slower multiplication rate was also observed in the infected planlet during culture (marwoto 2004). efforts have been made to get healthy protocols by eliminating virus from the infected plants. several methods such as chemo-, thermotherapy and electrical charges have been successfully conducted for virus elimination in some crops. numerous chemicals have been tested for antiviral activity, but few were effective. the most substance is the synthetic analogue of guanosine, ribavirin (1-beta-dribofuranosyl-1-h-1,2,4-triazole-3carboxamide) added to the media in the range of 30-50 mg/l was affective against potato virus x (pvx), potato virus y (pvy), potato virus s (pvs) and potato virus m (pvm) in potato (elia . 2008), ringspot virus in citrus (sharma . 2007) and yellow leaf virus in sugarcane (parmessur & saumtally 2001). ribavirin is a member of the nucleoside anti metabolite compound that interferes with duplication of viral genetic material. though not effective against all viruses, ribavirin is remarkable as a small molecule for its wide range of activity; including important activities against both dna and rna viruses (senula 2000 . ribavirin's carboxamide group can make the native nucleoside drug resemble adenosine or guanosine, depending on its rotation. for this reason, when ribavirin is incorporated into rna, as a base analog of either adenine or guanine, it pairs equally well with either uracil or cytosine, inducing mutations in rna-dependent replication in rna viruses. such hypermutation can be lethal to rna viruses (klein & livingston 2008). heat treatment or thermotherapy was established for virus elimination related to characteristic of some viruses which showed declination in multiplication rate especially at high temperature. this method was successfully applied in the range of 35 40 c for producing virus-free potato plant (converse & tanne 1984), alstromeria (hakkaart & versluijs 1988) and apple (wang . 2006). however, both chemoand thermotherapy methods, depended on plant genotypes and viruses. varying degree of meristem cells deaths, phytotoxic causing an increase in culture time and the need for frequent transfers into fresh media coincided with the quantity of virus-free plants obtained (chen & sherwood 1991). et al. et al vicia faba et al. in vitro et al. et al et al et al. ) et al 0 95 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al the combination of both chemoand thermotherapy with meristem culture was dedicated to encounter the technical constraints in the single application of respective techniques. these methods were also reported to be more effective for virus destruction on sweet potato, nicotiana, lemon and cucumber (zaitlin & palukaitis 2000). the research was then conducted to find out the effect of ribavirin application in the media and heat treatment at different durations followed by meristem culture on the existence of cvb in infected chrysanthemum plants. the research was conducted at the indonesian ornamental crops research institute (iocri). six cvb infected chrysanthemum varieties i.e dewi sartika, saraswati, yellow fiji, white puma, yellow puma and white reagent were equally divided into two parallel experiments. the first three varieties served as chemotherapy using ribavirin, while the rest underwent the heat treatments. fifteen cutting samples from each variety were collected and replanted in 15 cm pot. these were then, maintained in growth chamber for 16 h. after 2 weeks, the plants were pinched and the new emerging lateral growths served as explants. shoot induction was conducted by inoculating apicals into ½ ms + 0.5 mg/l iaa. the shoots were then subcultured into ½ ms + 0.1 mg/l iaa to obtain uniform plantlets. after three weeks incubation, the plantlets were transferred into treatments media, consisting of ½ ms + 0.1 mg/l iaa and ½ ms + 0.1 mg/l iaa + 40 mg/l ribavirin. two weeks after incubation, meristematic apical of plantlets (< 0.2 mm) were dissected using binocular microscope and transferred into ms media for shoot induction. the dissections of meristematic apical of plantlets were repeated three times from newly emerging shoots. one week before heat treatments, the plantlets were preconditioned in the incubator with the daily temperature of 30 35 c. the temperature of the incubator was then increased up to 38 40 c with the duration of 1, 2 and 3 weeks. after the heat treatments, meristematic apical of plantlets was dissected and inoculated into ms media for shoot induction. after transferred into regeneration media, phenotypic performance of plantlet was recorded. randomly plantlet samples in every treatment were also selected for cvb rapid detection using direct elisa method (clark & adam 1977). an amount of 100 μl cvb igg (agdia, usa) was mixed with a ratio of 1 : 200 of the coating buffer (na co + nahco + nan ) and overnightly incubated at the temperature of materials and methods in vitr in vitro o treatment of ribavirin heat treatment elisa bioassay 0 0 2 3 3 3 biotropia vol. 18 no. 2, 2011 96 4 c. the microplates were then rinsed twice with pbs tween (nacl + kh po + na hpo + kcl + nan + tween 20 + h o) buffer of 3 minutes each. leaf samples of 0.2 g were extracted and buffered with 1 ml mixture of pbst + 0,02 % pvp (1 : 5). the 100 μl of leaf extracts were then, incubated for 2 h in 37 c and rinsed with pbs tween buffer. after labeled with alkaline phosphatase enzyme, 100 μl of igg cvb was pipetted into microplates and mixed with eci (pbst + 0,2 % bsa) with a ratio of 1 : 200. the mixture was incubated for 2 h in 37 c. after 2 h incubation, the microplates were then rinsed with pbs tween buffer. an amount of 100 μl substrate buffer containing 4-nitrophenylphospate was placed into microplates and incubated at room temperature. after the substrate color changed into yellow, the reactions was then ceased with 25 μl naoh 3m. color intensities of the substrate were measured using elisa reader (minireader ii dynatech) on 410 nm wavelength. virus free samples were obtained when the absorbent values were three times less than that of positive control. plantlet performances were affected by the application of antiviral ribavirin and meristem tip culture, however,there were no specific interaction between cultivars tested and antiviral treatments. the increase of regeneration capacity was significant in plant height, number of leaves and number of nodes of ribavirin-treated plantlets, when samples were supplemented with 40 ppm ribavirin compared to untreated plants, thus hastened multiplication rate of the plantlet of all varieties tested (table 1). 0 0 0 2 4 2 4 3 2 results and discussion chemotherapy by antiviral ribavirin table 1. plantlet height, number of leaves and nodes after 30 days of subculture, and multiplication rate of chrysanthemum varieties treated with ribavirin. treatments plantlet height*) (cm) number of leaves*) number of nodes*) multiplication rate*) ribavirin treatments on media ms + 0.1 mg/l iaa + ribavirin 9.5 a 12.6 a 12.7 a 10.75 a ms + 0.1 mg/l iaa 6.8 b 6.4 b 6.3 b 7.41 b chrysanthemum varieties dewi sartika 9.2 a 12.7 a 11.7 a 10.22 a saraswati 7.5 ab 10.2 ab 10.5 ab 8.75 ab yellow fiji 6.0 b 9.1 b 9.2 b 6.16 b * note : values followed by different letters in the same column differ significantly at lsd 5%. faster multiplication rate of ribavirin-treated plantlets indicated that cell totipotency was retained. the most putative concerns of these phenomena were related to viricidist effect of ribavirin. the antiviral might block virus replication and although existing virus might remain in the original stem sections, the new outgrowths would be virus-free or contain only very low amounts of the virus (simpkins 1981). ribavirin might be active in its triphosphate form, which inhibits the 5′ capping et al. 97 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al of viral rnas (dawson & lozoya saldana 1984). it inhibited virus replication at the early stage by impairing synthesis of rnadependent rna polymerase and at a later stage by impairing synthesis of the coat protein (schuster & huber 1991). with the absent of or low concentration viral particles, the interfering physiological orientation of embryonic derived cells were overruled to the maximum growth of genotype response. during the experiment, no necrotic symptom used as indication of phytotoxicity was found in all varieties. though vegetal tissues tolerance to ribavirin is genotype dependent (sidwell 2005), the chemical concentration added into the media (40 mg/l) was considered proportional for chrysanthemum. these might refer to the fact that with such concentration range, phytotoxic symptoms were observed in other crops during virus elimination thus, inhibited or decreased plant cellular metabolic activity when applied in higher concentration than 30 mg/l (verma parmessur & saumtally the survival and death rates of plantlets in every duration of thermal treatments were varied among cultivars tested, though the trend was similar. during the first week of heat treatments, 24 36 % of treated plantlets were ceased, with cv. white reagent showing the least number of death plantlets compared to the others. the number of death plantlets was slightly decreased in the following week, 12 14 % (fig. 1). a short preconditioning incubation (one week with gradual increase at 30 -35 c) before the thermotherapy was apparently not sufficient for the plantlet to make suitable adaptation on higher temperature of heat treatment. though the heat tolerance of plants was indigenously specific, the mechanical adaptation of plants was timedepended on the degree of transition in regular change. the plantlet should be provided with sufficient precondition period with small and regular increase of temperature approaching the level of heat treatment to hinder high plantlet death at the early stage (manganaris 2003). through the small gradual increase of the temperature in longer period, the plantlet could establish physiological adaptation to the temperature level of 35 to 40 c. et al. et al. 2005; 2001). et al. application of thermotherapy 0 0 heat treatment duration (weeks) 0 2 4 0 1 2 3 white reagent yellow puma white puma figure 1. plantlet death rates of three chrysanthemum cultivars after one, two and three weeks of heat treatments. 98 biotropia vol. 18 no. 2, 2011 conversely from the first and second weeks, cv. white reagent showed higher plantlet death rates in the third week compared to cv. white puma and yellow puma (fig. 1). this period was considered critical, since plantlet death rates of all cultivars tested were also highest in these week (24 38 %). wang (2006) concluded that inside the culture flask or condition, a protected environment might lead to narrower adaptation of plantlets to the extreme conditions such as high temperature. this finding suggested that at the third week the toleration limit of chrysanthemum plantlet to heat treatments was obtained. after heat treatments, apical meristematic tissue of individual plantlets was inoculated into shoot induction media. period taken for bud initiation and plantlet height after three weeks transferring into regeneration media were observed different referring to the length of thermal treatments. shortest period for bud initiation and tallest plantlets were shown by the three weeks heat treatments in all varieties tested (table 2). slower growth rate on plantlets treated by one and two weeks thermotherapy indicated that physiologically, the plantlets were still in their suboptimal potential. these growth retardations were predictably affected by virus particles remained in the tissues which systemic persistently interfered with plant metabolism (marwoto . 2004; bhatthacharyya . 1990) in vitro et al et al table 2. buds initiation and plantlet height after three weeks transferring of three chrysanthemum varieties under different period of heat treatments. cultivars period of heat treatments (weeks) 1 2 3 bud initiation (days) *) white reagent 63.8 a 64.4 a 49.8 b white puma 64.4 a 59.6 b 48.2 c yellow puma 61.6 a 59.7 a 44.4 b plantlet height alter three weeks transfering into regeneration media (cm) *) white reagent 4,32 a 5,11 a 6,34 b white puma 4,36 a 4.82 a 6.84 b yellow puma 5,02 a 6,21 b 7,13 c *) remarks : values followed by different letters in the same rows differ significantly at lsd 5 %. elisa bioassay virus detection using direct elisa method was conducted to plant samples in all treatment combinations and spectro-photometerically revealed as absorbent values (table 3). these values indicated the existence of virus particles in plant tissues. the percentage of cvb-free plantlets increased accordingly to the lengthened heat treatments and application of ribavirin within the media. three weeks thermotherapy or supplemental antiviral followed by meristem culture successfully eliminated cvb from the infected chrysanthemum plantlets. 99 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al 0 the success of chemoand thermotherapy in eliminating cvb from infected plants in our works inferred that cvb particles and their persistence was highly affected by heat treatments at 38 40 c and antiviral application (ribavirin 40 mg/l). the failure of single method of meristem culture for producing virus free plantlets as also presented in the absent of ribavirin treatment indicated that the isolation of meristematic sites was not sufficient to totally free the tissue from virus particles. due to its small size, virus particles could traffic cell to cell even through branched plasmodesmata and infected the meristemal cells (laimer 2003). consequently, these naturally high antiviral-content tissues were also difficult to be isolated, since they were mostly microscopic and very sensitive (brown 1988). thus, combination of thermoor chemotherapy with meristem culture for virus elimination would be more promising, especially when mixed infection occurred or for those more persistent strains. et al. table 3. cvb detection by direct elisa and percentage of virus-free plantlets under thermoand chemotherapy treatments. treatments absorbance values*) percentage of cvb-free plantlet *) (%) heat treatment 1 week 0.06 – 0.11 41.6 2 weeks 0.03 – 0.09 72.3 3 weeks 0.02 – 0.07 100 chemotherapy using ribavirin 40 mg/l without ribavirin 0.06 – 0.12 43.6 with ribavirin 0.01 – 0.06 100 positive control 0.22 0 negative control 0.01 100 *) remarks : values derived from 36 plantlet samples in each treatment combination batthacharyya p, dey s, das n, batthacharyya bc. 1990. rapid mass propagation of by callus derived from stem and leaf explans. in . biotechnology unit. department of chemical engineering iit. kharagpur. india. p. 22 25. chen wq, sherwood jl. 1991. evaluation of tip culture, thermotherapy and chemotherapy for elimination of peanut mottle virus from . journal of phytopathology, 132 (3) : 230 236. clark mf, adam an. 1977. characteristic of the microplate method of enzyme lingked immunosorbent assay for the detection of plant viruses. journal of genetic virology, 34: 475 483. conclusions references under heat treatment, percentage of virus-free plantlets increased along with the duration of thermotherapy, while the survival rate of plantlets decreased in lengthened duration. the procedure of meristem culture application following ribavirin application or three weeks of heat treatment may be effective in eliminating cvb from the infected plantlets. chrysanthemum morifolium plant cell reports arachis hypogaea 100 biotropia vol. 18 no. 2, 2011 converse rh, tanne e. 1984. heat therapy and stolon apex culture to eliminate mild yellow-edge virus from hood strawberry. phytopathology, 74 : 1315 1316. dawson wo, h lozoya-saldana h. 1984. examination of the mode of action of ribavirin against tobacco mosaic virus. intevirology, 22 : 77 84. elia g, belloli c, f cirone f. 2008. efficacy of ribavirin against canine distemper virus. antiviral research, 77 (2) : 108-113. hakkaart fa, versluijs jma. 1988. virus elimination by meristem tip culture from a range of alstromeria cultivars. plt dis. 68 : 216 218. klein re, livingston ch. 2008. eradication of potato virus x from potato by ribavirin treatment of cultured potato shoot tips. american journal of potato research, 59 (8) : 359-365. laimer m. 2003. detection and elimination of viruses and phytoplasmas from pome and stone fruit trees. horticultural reviews 28 :187 236. manganaris ga, economou as, boubourakas in, katis ni. 2003. elimination of ppv and pnrsv through thermotherapy and meristem-tip culture in nectarine. plant cell reproduction, 22: 195 200. marwoto bl, sanjaya, budiarto k, rahardjo ib. 2004. pengaruh antiviral dalam media kultur terhadap keberadaan chrysanthemum virus b pada 4 varietas krisan terinfeksi. jurnal hortikultura (edisi khusus) 14: 410 418. senula ae, keller rj, leseman de. 2000. elimination of viruses through meristem culture and thermotherapy for the establishment of an collection of garlic ( ). acta horticultuae. 530 : 121-128. schuster g, huber s. 1991. evidence for the inhibition of potato virus x replication at two stages dependent on the concentration of ribavirin, 5azadihydrouracil as well as 1,5diacetyl-5azahydouracil. biochemestry phytology pflanz, 187: 429 438. sharma s, singh b, rani g, zaidi aa, hallan v, nagpal a, virk gs. 2007. production of indian citrus ringspot virus free plants of kinnow employing chemotherapy coupled with shoot tip grafting. agriculture 8 (1) : 1 8. sidwell rw, bailey kw, wong mh, barnard dl, smee df. 2005. and influenza virus-inhibitory effects of viramidine. antiviral research, 68 (1) : 10 17. simpkins i, walkey dga, neely ha. 1981. chemical suppression of virus in cultured plant tissues. annals of applied biology, 99: 161 169. parmessur y, saumtally a. 2001. elimination of sugarcane yellow leaf virus and sugarcane bacilliform virus by tissue culture. j. of food and agricultural research, 7 (3): 121 133. verma n, ram r, zaidi aa. 2005. in vitro production of prunus necrotic ringspot virus free begonias through chemoand thermotherapy. science horticulture, 103: 239 247. wang i, wang g, tang nhr, deng x, zhang h. 2006. effect of thermotherapy on elimination of apple stem grooving virus and apple chlorotic leaf spot virus for in vitro-cultured pear shoot tips. journal of the american society for horticultural science, 41(3): 1327 1329. zaitlin m, palukaitis p. 2000. advances in understanding plant viruses and virus diseases. annual review of phytopathol, 38 : 117 143. in vitro in vitro allium sativum journal of central european , in vitro in vivo 101 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al 25 the quality of physic nut (jatropha curcas l.) r.l. worang et al.biotropia vol. 15 no. 1, 2008 : 25 36 the qualit y of physic nut (jatropha curcas l.) seeds packed in plastic material during storage rantje lilly worang1 okky setyawati dharmaputra2,3 rizal syarief4 and miftahudin2 1 faculty of mathematics and natural sciences, manado state university, tondano, north sulawesi, indonesia 2 department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor, indonesia 3 seameo biotrop, jl. raya tajur km. 6, po box 116, bogor, indonesia 4 department of food science and technology, faculty of agricultural technology, bogor agricultural university, darmaga campus, bogor, indonesia abstract the effect of storage duration on fungal population, moisture content, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds was investigated. physic nut seeds with initial moisture content of 7.9% were stored in plastic bags under warehouse conditions. samples of physic nut were collected before storage, and subsequently after one to six months of storage. the results showed that the moisture contents increased after one month of storage, and became relatively constant up to six months of storage. the range of moisture contents (7.9 – 8.4%) was safe for storage of physic nut seeds. sixteen fungal species were isolated from physic nut seeds during six months of storage. fungal population decreased with the increase of storage duration. at the beginning of storage, most of the fungi that infected the seeds were classified as field fungi, such as colletotrichum sp., cladosporium spp., and fusarium spp.. their populations decreased with the increase of storage duration. after three months of storage, the existence of field fungi was generally replaced by storage fungi, such as aspergillus spp., and penicillium spp. dominate the population. lipid contents, viabilities and vigors decreased with the increase of storage duration, while free fatty acids and lipase activities increased. under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted, while it can be stored up to five months for producing oil. key words : storage duration, fungi, lipid, free fatty acid, lipase, viability, vigor, physic nut, jatropha curcas l. corresponding author : okky@biotrop.org 26 biotropia vol. 15 no. 1, 2008 introduction with the increasing demand of fuel products, and the decreasing of fossil fuel sources, the world prices of petrol and diesel are increasing, including in indonesia. it is assumed, that the fossil fuel reserve will be exhausted within 10 15 years in indonesia. hence alternative sources of energy for running our generators, automobiles, etc are being considered (hambali 2006). the possibility of obtaining oil from plant resources to be a supplement or replacement of fossil fuel has aroused a great interest. in several countries including in indonesia, non-edible vegetable oil can be used as biodiesel, which is a nontoxic and biodegradable oil. physic nut (jatropha curcas l.) seeds is known to be a good source for biodiesel. the oil contents in the seeds and in the kernels are 25 – 30% and 50 – 60%, respectively. the oil contains 21% saturated fatty acids and 79% unsaturated fatty acids (gubitz et al. 1997). other uses of physic nut extracted oil are for making soap, biopesticide, and medicinal substance for skin disease treatment. the physic nut oil has the requisite potential of providing a promising and commercially viable alternative to diesel oil, since it has desirable physicochemical and performance characteristics comparable to diesel. cars could be run with physic nut without requiring much change in design (hambali et al. 2006; warsiki et al. 2007) physic nut seeds can be used as seeds and oil producer. according to adikadarsih and hartono (2006) the use of physic nut seeds for seedlings should derive from the fruits which skin is yellow up to blackish yellow in colour, because they have high percentages of viability and vigor, i.e. 89 and 81 %, respectively. wanita and hartono (2006) reported that seeds originated from physic nut fruits which skin is yellow up to blackish yellow in colour produce seeds with highest oil content, i.e. 2830 %. to obtain good quality of physic nut seeds, some factors should be taken into consideration, i.e. the degree of fruit maturity, safe seed moisture content, appropriate container and storage condition, duration of storage, good viability and vigor of seeds. consequently, appropriate postharvest handling is needed. christensen and kaufmann (1969) reported that during storage seeds or grains could be infected by fungi which cause a decrease in viability, discolouration, various biochemical changes, heating and mustiness, loss in weight, and production of toxins when it is consumed may be injurious to human and domestic animals. in many cases, fungi infecting seeds are seed-borne pathogens. they play an important role in the transmission of numerous pathogenic species to seedlings as well as to the soil (chelkowski 1991). the objective of this study was to determine the effects of storage duration on fungal population, moisture content, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds. 27 the quality of physic nut (jatropha curcas l.) r.l. worang et al. materials and methods origin of physic nut seeds physic nut seeds were obtained from fresh harvested fruits which fruit skin was yellow up to blackish yellow in colour. the fruits were obtained from plants (lampung accession) cultivated in loyang village, cikedung subdistrict, indramayu regency, west java in may 2007. fruit peeling and seed drying the fruit skins were peeled using a knife, then the seeds were air-dried on the selves in a shaded place up to moisture contents of about 8 %. packaging and storing of physic nut prior to packaging, damaged seeds were hand picked from the batch. sound seeds were packed in plastic packaging materials (2 kg/bag) under normal oxygen concentration. they were then placed randomly on wooden shelves and stored for one, two, three, four, five and six months ( june – november 2007) under warehouse conditions. the composition of the plastic packaging material was ny15/pe15/ lldpe70. according to dharmaputra et al. (2007) total fungal population of peanut kernels packed in three different types of plastic packaging materials (opp30/pe15/ lldpe80, ny15/pe15/lldpe80, and ny15/pe15/lldpe70 ) under low oxygen concentration were not significantly different. the characteristic of plastic packaging material is presented in table 1. the plastic packaging bag was produced by pt interkemas flexipack in tangerang, west java. table 1. the characteristics of plastic packaging material name of product code compositition thickness (mm) wvtr (water vapour transmission rate) (g/ m2/24 hrs) o2tr (oxygen transmission rate) (cc/m2/24 hrs) trl.vacuum bag 03 ny70 ny 15/pe 15/lldpe 70 0.0974 0.6340 1.8734 note : analyzed by test and calibration laboratory, institute for chemical and packaging, jakarta, indonesia opp : oriented polypropilene lldpe : linear low density polyethylene the experiment was arranged in completely randomized design.three replications (= bags) were used for each storage duration (including at the beginning of storage). each bag was used to pack physic nut seeds with different storage duration and 28 biotropia vol. 15 no. 1, 2008 replications. the ambient temperature and relative humidity of the storage room were recorded using a tinytag data logger. sampling method samples of physic nut seeds were collected from each bag before storage (at the beginning of storage) and subsequently every month thereafter until 6 months of storage. each sample was divided three times using a box divider to obtain working samples for moisture content, fungal population, lipid and free fatty acid contens, lipase activity, viability and vigor of seeds, and reserved sample. determination of moisture content, fungal population, lipid and free fatty acid contens, lipase activity, viability and vigor moisture contents of seeds (based on wet basis) were determined using oven method (ista 1999, with a slight modification). two replicates were used for each sample. fungi from each sample was isolated and enumerated using serial dilution method followed by pour plate method on dichloran 18% glycerol agar (dg18) (hocking and pitt 1980; pitt et al. 1992). fungal identification was conducted based on samson et al. (1996), pitt and hocking (1997) using czapek yeast extract agar (cya) and cya containing 20% sucrose (cy20s). lipid and free fatty acid contents were determined based on soxhlet and titration methods, respectively (aoac 1999). lipase activity was determined based on moore (1973, with a slight modification). viability (percentage of germination) of seeds from each sample was determined by growing 25 seeds in rectangular plastic containers containing sand (5 kg/container) 7 and 10 days after planting (dap) under green house conditions (prihandana and hendroko 2006). normal germination was observed 7 dap, while normal and abnormal germination were observed 10 dap. determination of seed vigor from each sample was conducted using rice straw paper method (sadjad 1994). vigor, less vigor, non-vigor, and death were observed 7 dap. statistical analysis standard anovas were used to analyse the results followed by duncan’s multiple range test at the 5% probability level. results and discussion moisture contents moisture content is the most important environmental factor that influence fungal growth in stored grains (christensen et al. 1992). storage duration gave very significant differences on the moisture contents of physic nut seeds. the moisture 29 the quality of physic nut (jatropha curcas l.) r.l. worang et al. contents of physic nut after one month of storage (8.41%) were higher and significantly different from those at the beginning of storage (7.89%). the moisture contents decreased after two, three, four, five, and six months of storage, i.e. 8.14, 8.12, 8.14, 8.06 and 8.16%, respectively (table 2). according to the directorate general of plantation crop (2006) the safe moisture content for storage of physic nut seeds was 7 – 9 %. the moisture content of grains is in equilibrium with the relative humidity of the storage. range of temperature and relative humidity are presented in table 3. the moisture content of the seeds was correlated with the relative humidity of the storage. the type of plastic packaging material also affects the moisture contents of physic nut seeds. plastic packaging bags used in this study are made from good material for storing peanut kernels (dharmaputra et al. 2007). according to warsiki et al. (2007) the moisture contents of physic nut seeds packed in bags were higher than those packed in polypropylene bags. table 2. moisture content, total fungal population, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds during storage duration of storage (month) moisture content (% wet basis) total fungal population (cfu/g dry basis) lipid content (% dry basis) free fatty acid content (% dry basis) lipase activity (u/ml) viability (%) vigor (%) 0 7.89 a ± 0.07 8589.0 a ± 1359.3 41.2 a ± 0.34 0.10 a ± 0.01 0.23 a ± 0.08 89.33 a ± 8.33 83.33 a ± 5.77 1 8.41 d ± 0.02 6519.6 a ± 3224.5 37.9 b ± 0.69 0.60 b ± 0.01 0.35 ab ± 0.03 74.67 b ± 4.62 70.00 b ± 0.00 2 8.14 c ± 0.04 5917.7 a ± 1026.8 37.2 bc ± 0.66 0.62 b ± 0.01 0.42 bc ± 0.04 71.33 bc ± 1.15 63.33 bc ± 5.77 3 8.12 bc ± 0.02 1405.4 b ± 1196.7 35.9 cd ± 0.56 0.67 bc ± 0.03 0.51 cd ± 0.05 68.00 bc ± 2.00 56.67 c ± 5.77 4 8.14 c ± 0.03 1297.3 b ± 377.2 35.1 de ± 0.54 0.79 c ± 0.06 0.58 d ± 0.01 64.67 cd ± 2.31 36.67 d ± 5.77 5 8.06 b ± 0.02 1145.6 b ± 761.9 33.5 ef ± 0.86 1.00 d ± 0.09 0.71 e ± 0.07 60.00 de ± 3.46 33.33 de ± 5.77 6 8.16 c ± 0.03 225.4 b ± 28.4 31.7 f ± 2.06 1.10 d ± 0.14 0.93 f ± 0.15 53.33 e ± 2.31 26.67 e ± 5.77 note : means followed by the same letter in the same column are not significantly different according to duncan’s multiple range test at the 5% level (p > 0.05). 30 biotropia vol. 15 no. 1, 2008 table 3. range of temperature and relative humidity during storage duration of storage (month) temperature (oc) relative humidity (%) range mean range mean 1 24.4 28.3 26.3 55.2 – 82.8 74.1 2 25.1 -28.7 26.5 48.2 -70.2 70.2 3 25.1 – 29.1 26.5 55.2 – 78.5 69.4 4 25.1 – 28.3 26.7 46.0 – 80.7 68.1 5 25.1 – 28.7 26.7 55.6 – 82.4 73.1 6 24.4 – 28.3 26.1 65.6 – 85.4 76.5 according to desai (2004) modern packaging materials and methods maintain seeds at their original quality from the time of their packaging to the time they are used for planting. the best way to maintain the viability and vigor of many kinds of seeds is to store them in a dry and cold place. packages designed to protect most physical qualities of seeds, such as weight, size, colour, moisture content, and purity (freedom from weeds, inert matter, disease organisms and damage), as well as their physiological aspects, like viability, vigor and dormancy, are made up of materials that have sufficient tensile strength, bursting strength, and tearing resistance to withstand normal pressures and handling procedures. polyethylene is the most extensively used thermoplastic film. conventional low-density films are generally used in seed packages. this packaging material is ideal for providing seale-storage conditions in a humid climate, giving protection against high humidity in a seed store. fungal population sixteen fungal spesies were isolated during six months of storage (table 4). they were aspergillus flavus, a. niger, a. ochraceus, a. penicillioides, a. restrictus, a. tamarii, a.wentii, cladosporium sp., c. cladosporioides, colletotricum sp., eurotium chevalieri, e .rubrum, fusarium moniliforme, f. semitectum, penicillium citrinum, and p. oxalicum. aspergillus flavus, a. niger, cladosporium sp., f. moniliforme, f. semitectum, and p. citrinum were always isolated during storage. table 4. fungal population of physic nut seeds during storage no. fungi fungal population (cfu/g dry basis) duration of storage (month) 0 1 2 3 4 5 6 1 aspergillus flavus 6.5 1.1 6.5 88.2 8.7 17.4 9.8 2 a. niger 3.3 2.2 4.4 3.3 1.1 2.2 3.3 3 a. ochraceus 0.0 0.0 1.1 0.0 0.0 0.0 0.0 4 a. penicillioides 0.0 0.0 0.0 0.0 0.0 730.9 92.6 5 a. restrictus 0.0 50.2 61.0 0.0 695.6 0.0 0.0 31 the quality of physic nut (jatropha curcas l.) r.l. worang et al. no. fungi fungal population (cfu/g dry basis) duration of storage (month) 0 1 2 3 4 5 6 6 a. tamarii 2.2 1.1 2.2 0.0 1.1 1.1 1.1 7 a. wentii 1.1 1.1 2.2 4.4 2.2 0.0 0.0 8 cladosporium sp. 427.7 462.9 90.4 37.0 20.7 44.6 17.4 9 c. cladosporioides 169.4 41.5 47.9 21.8 0.0 0.0 15.2 10 colletotrichum sp. 5609.6 1880.1 1415.2 68.6 61.0 0.0 0.0 11 eurotium chevalieri 0.0 0.0 0.0 1.1 0.0 0.0 2.2 12 e. rubrum 0.0 0.0 0.0 2.2 0.0 63.1 3.3 13 fusarium monili-forme 1652.4 3703.5 580.2 1007.8 409.3 156.6 15.2 14 f. semitectum 247.5 10.9 209.0 14.1 3.3 6.5 3.3 15 penicillium cit r i-num 276.8 291.5 3496.6 156.7 94.7 124.0 63.2 16 p. oxalicum 193.2 73.2 0.0 0.0 0.0 0.0 0.0 total 8589.7 6519.3 5916.6 1405.1 1297.6 1146.4 226.5 duration of storage gave very significant differences on total fungal population. total fungal population after one, two, three, four, five and six months of storage did not coincide with moisture contents at the same months (table 2 ). total fungal population decreased with the increase of storage duration. total fungal population at the beginning of storage (8589.0 cfu/g.db) was higher and significantly different from that after six months of storage (225.4 cfu/g.db). the highest total fungal population was found at the beginning of storage, because the high population of field fungi, such as cladosporium spp., colletotricum sp., f. moniliforme, and f. semitectum . cladosporium spp. produces abundant conidia. storage fungi found in physic nut seeds were species of aspergillus and penicillium. based on their occurrence in the seeds, christensen and kaufmann (1969) divided two groups of fungi that cause deterioration of seeds, i.e. field fungi and storage fungi. it was assumed that the decrease of fungal population during storage was due to the low of moisture contents in the seeds after one month up to six months of storage. in general the dominant fungal species during storage was field fungi. according to sauer et al. (1992) fields fungi have high water requirements for their growth, compared to storage fungi. it was also assumed that the decrease of total fungal population was due to the decrease of oxygen during storage. according to moore-landecker (1996) most fungi are obligate aerobes and require at least some free molecular oxygen in the atmosphere. physic nut seeds also need o2 found in plastic packaging bags for their life. garraway and evans (1984) concluded that the type of response by fungi to oxygen concentration depends on the species. table 4. continued 32 biotropia vol. 15 no. 1, 2008 lipid contents storage duration gave very significant differences on lipid contents of physic nut seeds. the lipid contents decreased with the increase of storage duration (table 2). after one month of storage, lipid contents (37.9% db) were lower and significantly different from those at the beginning of storage (41.2% db). after six months of storage, lipid contents (31.7% db) were lower and significantly different from those at the beginning of storage (41.2% db), after one (37.9% db), two (37.2%db), three (35.9% db), and four (35.1% db) months of storage. physic nut seeds contain high lipid content. the lipid contents in the seeds and in the kernels are 25 – 30% and 50 – 60%, respectively. the lipid contains 21% saturated fatty acids and 79% unsaturated fatty acids (gubitz et al. 1997). lipid contents of physic nut used in this study were high (37.95%), because the seeds were derived from fruits which fruit skin is yellow up to blackish yellow. although after five months of storage lipid contents (33.5% db) were significantly different from those at the beginning of storage (41.2% db), they were still able to be used for producing oil. lipid contents are affected by the degree of fruit maturities at the time of harvesting. wanita and hartono (2006) reported that the lowest (10.93 %) and the highest (29.38 %) lipid contents were found in physic nut seeds derived from fruits which fruit skins are green and yellow, respectively. free fatty acid contents and lipase activity due to hydrolysis process, fat and lipid in seeds are broken down into free fatty acids (ffa) and glycerol by lipases. rapid increases in concentrations of ffa frequently are observed in deteriorating seeds, particularly when the temperature and moisture content are high. many field and storage fungi produce lipases. seeds also produce lipases, but these commonly are produced during germination, and are not present in stored seeds (pomeranz 1992). storage duration gave very significant differences on ffa contents of physic nut seeds. ffa contents increased with the increase of storage duration (table 2). after one month of storage, ffa contents (0.60% db) was higher and significantly different from those at the beginning of storage (0.10 db %). after five (1.00 % db) and six months of storage (1.10 % db) ffa contents were higher and significantly differents from those at the beginning of storage (0.10% db), after one (0.60% db), two (0.62 % db), three (0.67% db) , and four months of storage (0.79% db). according to st. angelo and ory (1983) lipase and lipoksigenase are important enzymes to degrade lipid in seeds. storage duration gave very significant differences on lipase activity of physic nut seeds. lipase activity increased with the increase of storage duration (table 2). after two months of storage, lipase activity (0.42 u/ml) was higher and significantly different from those at beginning of storage (0.23 u/ml). after five and six months of storage lipase activity (0.71 and 0.93 u/ml, respectively) were higher and significantly different 33 the quality of physic nut (jatropha curcas l.) r.l. worang et al. from those at the beginning of storage. after six months of storage, lipase activity was higher and significantly different from those after five months of storage. as the moisture contents were considered low (table 2) and the total fungal population decreased (table 4) during storage, the lipase activity could be due to seed enzymes rather than fungal enzymes. seed viability and vigor according to pomeranz (1992) fungal infection can decrease seed viability and vigor of seeds during storage. priestley (1986) reported that the most obvious, though not the most informative, indication of the quality of a seed lot is its germinability. the speed of germination, which has long been recognized as an indicator of seed vigor, is usually a more sensitive measure of seed deterioration than loss of viability. storage duration gave very significant differences on the percentages of germination and vigor. the percentages of germination and vigor decreased with the increase of storage duration (table 2). the percentages of germination after one month of storage (74.67%) were lower and significantly different from those at the beginning of storage (89.33%). directorate general of estate crops (2006) determined the germination of physic nut seeds to be planted should be more than 80%. after one month of storage, the percentage of germination (74.67%) was significantly different from those at the beginning of storage (89.33%). nevertheless, based on statistical analyses, the percentage of germination after one month of storage was 74.67 ± 4.62%. consequently, under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted. after six months of storage, the percentages of germination (53.33 %) were lower and significantly different from those at the beginning of storage (89.33). according to copeland and mc donald (2001) seeds containing high lipid content decrease their viability faster compared to those containing high carbohydrate content. the seed germination percentage in this study was more than 80 %, because the seeds were derived from fruits which fruit skin is yellow up to blackish yellow in colour as required for good quality of physic nut seeds. the percentages of vigor after one month of storage (70.00%) were lower and significantly different from those at the beginning of storage (83.33%). after six months of storage, the percentages of vigor (26.67%) were lower and significantly different from those at the beginning of storage (83.33 %), after one (70.00%), two (63.33 %), three (56.67%) and four months (36.67%) of storage. according to adikadarsih and hartono (2006) the lowest percentages of germination (7 %) and vigor (4.33 %) were found in physic nut seeds derived from fruits which fruit skin is green in color ; while the highest percentage of germination (91.67 %) and vigor (84.67 %) were found in seeds derived from fruits with fruit skin is yellow in color. 34 biotropia vol. 15 no. 1, 2008 under conditions that favour them to grow, storage fungi invade the germs or embryos of seeds prefentially, and sometimes exclusively. very often, and especially if the moisture content of the seeds is at or just slightly above the lower limit that allow a given species of storage fungi to grow, the germs may be invaded to the point of near decay, with no evidence of molding being evident outwardly, even with microscopic examination, and little or no invasion of the endosperm immediately adjacent to the germ. the first effect of this invasion is weakening of the germ, followed by death (sauer et al. 1992). conclusions duration of storage affected the quality of physic nut seeds. after one month of storage, the moisture content of physic nut seeds increased, and became relatively constant up to six months of storage. during storage the moisture contents were still safe for storing physic nut seeds (7.89 – 8.41%). sixteen mould species were isolated from physic nut seeds during six months of storage. fungal population decreased with the increase of storage duration. at the beginning of storage most of fungi infected the seeds were field fungi (colletotrichum sp., cladosporium spp., and fusarium spp.). their populations decreased with the increase of storage duration and replaced by storage fungi (penicillium spp. and aspergillus spp.). lipid contents, viabilities and vigors decreased with the increase of storage duration, while free fatty acids and lipase activities increased. under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted, while for producing oil, they can be stored up to five months. acknowledgements the authors gratefully acknowledge dr. theresia prawitasari† for her suggestions and encouregement. we also thank to the staff member of plant pathology and service laboratory, seameo biotrop, who have in one way or another contributed to this research. references adikadarsih, s. & j. hartono. 2006. pengaruh kemasan buah terhadap mutu benih jarak pagar (jatropha curcas l). paper presented at lokakarya ii: status teknologi tanaman jarak pagar. pusat penelitian dan pengembangan perkebunan, departemen pertanian. bogor, 29 november 2006. 35 the quality of physic nut (jatropha curcas l.) r.l. worang et al. aoac. 1999. in horwits w (ed). vol. ii. official methods of analysis of food composition; additives; natural contaminants. 16th ed. association of official analytical chemist, gaithersburg. chelkowski, j. 1991. fungal pathogens influencing cereal seed quality at harvest. in chelkowski, j. (ed.). pp. 53 – 66. cereal grain; mycotoxins, fungi and quality in drying and storage. elsevier, amsterdam. christensen, c.m. and h.h. kaufmann. 1969. grain storage: the role of fungi in quality loss. university of minnesota press, minneapolis. christensen, c.m., b.s. miller and j.a. johnston. 1992. moisture and its measurement. in sauer, d.b. (ed.). pp. 39 – 54. storage of cereal grains and their products. american association of cereal chemists. inc., st. paul. copeland, l.o. and m.b. mc donald. 2001. principles of seed science and technology. 4th ed. kluwer academic publishers, boston. desai, b.b. 2004. seeds handbook; biology, production, processing, and storage. 2nd ed. marcel dekker, inc., new york. dirjenbun. 2006. pedoman mutu benih jarak pagar sistem dan prosedur pembangunan sumber benih dan peredaran benih jarak pagar. direktorat jenderal perkebunan, jakarta. dharmaputra, o.s., i. retnowati and s. ambarwati. 2007. three different types of plastic packaging materials: their effects on mould infection and aflatoxin contamination. biotropia 14(1): 9 – 23. garraway, m.o. and r.c. evans . 1984. fungal nutrition and physiology. john willey & sons, new york. gubitz, g.m, m. mittelbach and m. trabi. 1999. exploitation of the tropical oil seed plant jatropha curcas l. bioresource technology 67 : 73-82. hambali, e. 2006. proses pengembangan tanaman jarak pagar untuk biodisel dan produk turunan lainnya. paper presented at workshop pendirian kebun bibit sumber, demplot dan feasibility study untuk perkebunan jarak pagar. bogor, 16-17 may 2006. sbrc, lppm-ipb. hambali, e. et al. 2006. jarak pagar tanaman penghasil biodisel. penebar swadaya, jakarta. hocking, a.d. and j.i. pitt. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods. applied environmental microbiology 39 : 488-492. ista. 1999. international rules for seed testing. proceedings international seed testing association 31 (1), wageningen. moore, t.c. 1973. research experiences in plant physiology. a laboratory manual. sprigerverlag, new york. moore – landecker, e. 1996. fundamental of the fungi. 4th ed. prentice – hall, inc, new jersey. pitt, j.i., a.d. hocking, r.a. samson and a.d. king.1992. recommended methods for mycological examination of foods. in samson, r.a., a.d. hocking, j.i. pitt and a.d. king, eds. pp. 365-368. modern methods in food mycology. elsevier, amsterdam. 36 biotropia vol. 15 no. 1, 2008 pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic & professional, london. pomeranz, y. 1992. biochemical, functional and nutritive changes during storage. in sauer, d.b. (ed.). pp. 55 – 141. storage of cereal grains and their products. 4th ed. american association of cereal chemists, inc., st paul. priestley, d.a. 1986. seed aging; implications for seed storage and persistence in the soil. comstock publishing associates, cornell university press, ithaca. prihandana, r. and r. hendroko. 2006. petunjuk budidaya jarak pagar. pt agromedia pustaka, jakarta. sadjad, s. 1994. kuantifikasi metabolisme benih. pt gramedia widiasarana indonesia, jakarta. samson, r.a., e.s. hoeksttra, j.c. frisvad and o. filtenborg. 1996. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baarn. sauer, d.b., r.a. meronuck and c.m. christensen. 1992. microflora. in sauer, d.b. (ed.). pp. 313 – 340. storage of cereal grains and their products. 4th ed. american association of cereal chemists, minnesota. st. angelo a.j. and r.l. ory. 1983. lipid degradation during seed deterioration. phytopathology 73: 315. wanita, y.p. and j. hartono. 2006. pengaruh tingkat kemasakan buah terhadap kadar mi nyak jarak pagar (jatropha curcas l.) (the effect of degree of fruit maturity on oil content of jatropha curcas l). paper presented at lokakarya ii: status teknologi tanaman jarak pagar. pusat penelitian dan pengembangan perkebunan, departemen pertanian. bogor, 29 november 2006. warsiki, e., d. sumangat and w rismawati. 2007. pengaruh bahan dan kondisi pengemasan terhadap mutu biji jarak pagar. paper presented at konferensi jarak pagar menuju bisnis jarak pagar yang feasible. sbrc, lppm, institut pertanian bogor, bogor, 19 june 2007. 4. jani.cdr biotropia vol. 20 no. 1, 2013: 29 37 ecological impact of (l.) merrill invasion on plant diversity at bukit barisan selatan national park merremia peltata jani master *, sri s. tjitrosoedirdjo , ibnul qayim , and soekisman tjitrosoedirdjo received 25 march 2013/accepted 13 june 2013 bukit barisan selatan national park (bbsnp) is the third largest protected area in sumatra. unfortunately, the area is now invaded by or mantangan occupying about 7000 ha of the area. the aim of this study was to determine the ecological impacts of mantangan on plant species composition at bbsnp. three sites with different disturbance regimes were selected for vegetation analysis and assessment on plant species composition : primary forest representing undisturbed area, secondary forest representing burned area, and invaded forest representing forest invaded by mantangan. three line transects were constructed at each locations along 1 km, and the nested sampling plots were set up every 100 m with the following quadrant's size: 20 m x 20 m for trees, 10 m x 10 m for poles, 5 m x 5 m for sapling, and 2 m x 2 m for seedling. data collection covered the degree of mantangan invasion, trees species and diameter. results showed that invaded forest has lower plant diversity index (1.90) than the other two forests. this was caused by the invasion of mantangan as the percentage of coverage in the invaded forest reached 44% compared to secondary and the primary forest, which were 27.11% and 1.00%, respectively. percentage of mantangan coverage and diversity showed a strong negative correlation (-0.988). invasive species, impact, , mantangan, bukit barisan selatan national park 1 2 3 2 1 2 3 department of biology, faculty of mathematics and natural science, lampung university south east asian regional center for tropical biology (seameo biotrop), bogor, indonesia department of biology, faculty of mathematics and natural science, bogor agriculture university, bogor, indonesia. merremia peltata merremia peltata abstract introduction key words: bukit barisan selatan national park (bbsnp) is considered as the third largest protected area in sumatra, located at the southern tip of sumatera island covering an area of 356 800 ha, extending from the southern of lampung province up north to bengkulu province (btnbbs 1999). the park also has the distinction of being . * corresponding author : j.janter@gmail.com 29 one of the few remaining tropical rain forests that harbors rhinos, tigers, and elephants. it is also a home for over 300 bird species, sun bears, bearded pigs, tapirs, gibbons, leaf monkey, great argus pheasants, rare orchids, and . presently, it was reported that bbsnp has lost more than 20% of its original forest area and it has been facing with serious threats of deforestation (suyadi 2011). this valuable remaining forest will become highly fragmented into isolated patches if current deforestation and encroachment rates continue. beside threatened with rapid deforestation and encroachments, this area has now been invaded by or called as mantangan occupying about 7000 ha. mantangan is a local plant species native to asia. it has wide distribution, extending from indian ocean islands of pemba, madagascar, mauritius, reunion, and the seychelles, throughout malesia, northern australia and eastwards into polynesia to the society islands (smith 1991). the mantangan invasion in bbsnp has been reportedly disturbing the natural habitat of a number of wild animals. irianto and tjitrosoedirdjo (2010) reported that the wild animals migrated up to the north approaching human settlements due to declining of food resources. it was argued that habitat loss and poaching impacted badly on the population of wild animals. poaching reduced the number of tigers and rhinos in the park. tiger and human conflicts have been increasing around the park, leading to losses on both sides (irianto & tjitrosoedirdjo 2010). irianto and titrosoedirdjo (2010) reported a heavy invasion of mantangan in tambling village, an area inside the bbsnp close to the seashore; mantangan grew prolifically, the young shoot of mantangan is brown purplish in color with a leaf diameter up to 40 cm and yellow flower. trees found in the area were among others sp., pule ( ), jabon ( ) and others as well as shrubs covered heavily by the climbing , giving a view of mantangan invasion in the area. this study assessed the ecological impacts of mantangan invasion on plant diversity at bbsnp. the observation was conducted from february 2011 to january 2012. herbarium studies were conducted in the herbarium bogoriense (bo), and seameo biotrop (biot), bogor, indonesia. sampling site for vegetation analysis was divided into three categories purposively based on invasion regimes. the first location has very little invasion of mantangan, an average of only 1%. the second location has a moderate invasion (5% 50% with an average value of 27%). the third location is the location with the highest invasion, up to 100% with an average of 44%. invasion of mantangan was calculated by estimating the percentage of the plot covered by mantangan. the second location was a post-fire forest in 1997, fires that occur mostly on the forest floor which affect the lining of plant down and open up some of the forest canopy (wcs-ip 2001). the third location lies close to the village enclave way haru, bandar dalam and tampang. based on land cover maps, this site has been decreasing forest cover an area of 2565.54 ha from 2000 to 2009. presumably these changes are rafflessia amorphophallus titanum merremia peltata ficus alstonia scholaris anthocepalus cadamba m. peltata materials and methods 30 biotropia vol. 20 no. 1, 2013 the result of forest clearing intended for plantations (prasetyo . 2011), while the first location is never disturbed. accordingly, the first location is referred as primary forest, the second as secondary forest and the third location as invaded forest (fig.1). line transects were constructed and replicated three times at each location. each line transect was drawn from the edge of the forest up to the forest along 2 km in distance, and square plots were made every 100 m. the nested sampling plots were constructed with the following measurement: 2 x 2 m for seedling, 5 x 5 m for sapling, 10 x 10 m for poles, and 20 x 20 m for trees (fig. 2). the data collected were diameter of the trees, trees species (include poles, sapling and seedling). computation on density, dominance, frequency and importance value indices were made from trees, pole, sapling and seedlings. diversity index of shannon-wiener diversity, evenness index and similarity index were calculated. et al figure 1. map of study site figure 2. plot design of the vegetation analysis 31 2 x 2 m : plot size for seedling (height < 1,5 m) 5 x 5 m : plot size for seedling (height > 1,5 m and dbh< 10 cm) 10 x 10 m : plot size for poles (dbh 10 – 20 cm) 20 x 20 m : plot size for trees (dbh > 20 cm) 100 m ecological impact of invasion jani mastermerremia peltata – et al. results and discussion based on vegetation analysis that was conducted on three sampling sites of 1 ha sample area, the primary forest had 153 species of trees with diameter above 10 cm. in the secondary forest, there were 103 species of trees with diameter above 10 cm, while the invaded forest had 116 species of trees with diameter above 10 cm. based on the observation, species composition within the three sample sites was relatively high compared to a number of research findings taken from other lowland tropical rainforests. by comparison, trees with diameter above 10 cm within 1 ha sample plot in kayan mentarang national park, east kalimantan had 106 species of plants (purwaningsih 2009); in pasoh malaysia, there were 683 species of plants recorded from a sample area of 50 ha; in baro colorado island (bci) panama, there were 229 plant species in a sample area of 50 ha (condit 1996); in costa rica, there were 561 plant species in a sample area of 23.4 ha (lieberman . 1996); and in uppangala, ghats, india, there were 103 plant species in a sample area of 28 ha (pascal 1996). the tree density in primary forest indicated that the forest was still in good condition than the secondary forest and invaded forest. tree density with diameter > 10 cm in primary forest was 403 trees per hectare, while in secondary and invaded forests; there were 365 and 305 trees per hectare, respectively. according to campbell (1992), tree density with diameter >10 cm in tropical areas was about 245 859 trees per hectare. meanwhile, according to sheil . (2002), tree density with diameter over 10 cm in tropical rainforests was about 400 600 trees per hectare. invaded forests had fewer species than primary and secondary forests due to mantangan invasion. the highest percentage of mantangan coverage was in the invaded forest with an average of 44.00%, followed by the secondary forest with an average of 27.11% and the primary forest as the lowest coverage with an average of 1.00%. in line with the number of species' data and mantangan coverage, it can be obviously seen that the more the forest was invaded, the less the plants lived there (fig. 3). not only the number of species, but also the number of individuals in the invaded forest was less than in primary forest. et al. et al et al. et al 32 biotropia vol. 20 no. 1, 2013 figure 3. the number of individuals on three research sites 33 tropical rainforests had vegetation composition that illustrated the dynamics of regeneration which occurred naturally, in which the vegetation at seedling stage had the highest number of species. furthermore, the number of species started to decrease at sapling and timber stages. however, it does not occur in the invaded forest. figure 3 shows that the number of seedlings was significantly less than the number of saplings. besides, the number of seedlings on this invaded forest was less than those of two other sites. each research site was dominated by different species, either seedling, sapling, pole, or tree. trees of the primary forest was dominated by and . meanwhile, the secondary forest was dominated by , while the invaded forest was dominated by that was a plant with the second-highest ivi on the secondary forest (table 1). the dominant trees on the secondary and invaded forests were fast growing trees, such as and it indicated that the forests were ever in a disturbed condition. moreover, another indication was vegetation of the family datiscaceae such as that could survive on burned land (goldammer 1996). in the invaded forest, there were still some vegetation of the family dipterocapaceae. this was demonstrated by the presence of as the second-highest ivi in that site. this tree showed high capability to withstand againts invasion of mantangan. in addition, there were other five members of dipterocapaceae, for instance , and . pole stage at the primary forest was dominated by . the species composition at this stage was similar to the tree stage. meanwhile, secondary forest was dominated by , different species with tree stage in this location. in the invaded forest, dominate this stage. the highest ivi in the secondary forest was and in the invaded forest. furthermore, at sapling stage, the primary forest was dominated by , while the secondary and invaded forests were dominated by the domination of at sapling stage in the primary forest also occurred at seedling stage. at seedling stage dominated the secondary forest, while dominated the invaded forest (table 1). research findings showed that plant community in the primary forest was more steady than secondary and invaded forests. species domination at tree stage at secondary and invaded forest was not followed by other stages. this condition enables structure and species composition revolution in the future. if the dominant trees on both forests were fallen, so it would be substituted by other dominant species from the stages underneath. in terms of shannon-wiener's diversity index, tree, pole and sapling in three forest types did not differ one from the other (table 2). however, when all stages were considered, diversity in invaded forests was less than primary and secondary forests. the low diversity is negatively correlated with invasion of mantangan (-0.988, p value = 0.097), high invasion of mantangan led to a low diversity of plant. shanon-wiener diversity index is calculated from numbers of species, without considering if the species which make up the number is different , when the species making the number is considered and compared each other using index of similarity, strombosia javanica dipterocarpus palembanicus tetrameles nudifora cananga odorata c. odorata, t. nudiflora glochidion arborescens. t. nudiflora d. palembanicus dipterocarpus retusus, shorea javanica, anisoptera costata vatica obovata strombosia javanica t. nudiflora dillinea exelsa bridelia monoica croton argyratus popowia bancana dillenia excelsa. p. bancana cleisthantus myrianthus aglaia macrophyla ecological impact of invasion jani mastermerremia peltata – et al. 34 biotropia vol. 20 no. 1, 2013 table 1. the three highest ivi at each research site family spesies ivi primary forest secondary forest invaded forest tree sterculiaceae strombosia javanica 31.70 dipterocarpaceae dipterocarpus palembanicus 25.11 22’,90 anacardiaceae dracontomelon dao 23.80 datiscaceae tetrameles nudiflora 43.75 annonaceae cananga odorata 35.16 36.14 euphorbiaceae glochidion arborescens 14.31 17.14 pole sterculiaceae strombosia javanica 33.64 euphorbiaceae croton argyratus 17.25 37.82 meliaceae aphanamixis humile 15.77 euphorbiaceae bridelia monoica 34.55 27.59 dilleniaceae dillenia excelsa 32.60 annonaceae cananga odorata 30.50 dipterocarpaceae dipterocarpus palembanicus 22.48 sapling annonaceae popowia bancana 41.07 13.12 euphorbiaceae glycosmis pentaphylla 24.90 annonaceae popowia pisocarpa 21.26 dilleniaceae dillenia excelsa 28.41 39.25 lauraceae endiandra rubescen 15.47 euphorbiaceae croton argyratus 25.81 euphorbiaceae cleisthantus myrianthus 19.84 seedling annonaceae popowia bancana 24.61 euphorbiaceae glycosmis pentaphylla 22.58 sterculiaceae pterospermum javanicum 9.49 euphorbiaceae bridelia monoica 10.76 euphorbiaceae cleisthantus myrianthus 35.20 sapindaceae xerospermum noronhianum 10.72 meliaceae aglaia macrophyla 19.39 euphorbiaceae bridelia monoica 15.59 dilleniaceae dillenia excelsa 14.42 they are different one from the other (dyke 2003). according to those arguments, similarity index (is) was calculated by using steinhaus coefficient. its result was low index value (close to 0) that means those three sites at all stages had different species composition (table 3). mantangan devastated the habitat by covering other plants so that it reduced the availability of light for other plants. 35 table 2. shannon-wiener diversity index (h') and the average percentage of covermantangan location shannon-wiener’s diversity index (h’) a) % average of coverage mantangan tree pole sapling (g) seedling (g) all stage primary forest 3.84a 3.61a 3.51a 3.51a 2.49a 1.00 secondary forest 3.31a 3.12a 3.74a 2.75b 2.21b 27.11 invaded forest 3.46a 3.23a 3.46a 2.66b 1.90c 44.00 note : a) numbers followed by same letter on the same column are not obviously different based on t-test at 5% significance level table 3. steinhaus similarity index on three research sites phase of growth location secondary forest invaded forest tree primary forest 0.35 0.29 secondary forest 0.43 pole primary forest 0.24 0.19 secondary forest 0.41 sapling primary forest 0.36 0.36 secondary forest 0.35 seedling primary forest 0.24 0.13 secondary forest 0.10 erythrina variegata coffea arabica mantangan climbed up the canopy for sunlight, then rapidly grew and covered the entire canopy. the covered trees did not receive enough sunlight and hold the mantangan's weight as well. this condition caused those covered trees gradually died. another case was that mantangan would twist the trunk so the growing process of the twisted tree would be devastated. however, this case was rarely found. mantangan invasion was calculated by percentage estimation of its cover. in the primary forests, the coverage of mantangan was very little with an average of only 1% and the highest value of coverage was 15%. in the secondary forests, the average coveriage was 27% and the highest value of coverage was 55%. in addition, the invaded forests were the forests with the highest percentage of mantangan coverage. it reached an average of 44% coverage. in some locations, they were fully covered by mantangan (100%) (figure 2). mantangan invasion made possible because of the forest devastation caused by forest fragmentation. forest fragmentation might occur due to several activities such as illegal logging, farming, and land clearing for road construction. in several invaded forests, there were remnants types of crops that was abandoned for a long time such as dadap ( ) and coffee ( ). moreover, some logged trees were found. it was assumed that this location was a farming area. ecological impact of invasion jani mastermerremia peltata – et al. 36 biotropia vol. 20 no. 1, 2013 forest devastation in bbsnp already happened since 1960s. according to satellite image analysis from 1972 to 2006, bbsnp had lost 22% of its forest cover and left about 67.225 ha of total area 356.800 ha of national parks (suyadi 2011). forest devastation in bbsnp was mostly done by the farmers who live in and around the forest (suyadi 2011). on an invaded forest, there was an enclave that was a district in the administrative area of way haru village, west lampung regency. furthermore, on this location there was a four-meter-dirt road for nature tourism activities and the conservation of sumatran tigers. the existence of enclave and dirt road in this national park became the causes of forest fragmentation. forest fragmentation might cause some impacts, one of which was the edge effect (watson 2004). edge effects on forest ecosystem occurred in a form of microclimate changes, species composition changes, the decline of plants diversity, the abundance and density of species. this might cause local extinction of species because they were unable to adapt to the changing of condition there (meffe 1994). these edge effects became worse with the presence of invasive species, such as mantangan. it was alleged that at the edges of forest fragments, mantangan was able to live well and start invading into the forest. at the edges of open forests, soil moisture would decrease (sizer 1999). in this condition, a liana like mantangan was able to live better than other trees (londre 2006). invasion of mantangan was feared to have an effect on forest regeneration in the future, because in the invaded forest, the number of seedlings was significantly less than the number of saplings, pole and tree. furthermore, the diversity and number of seedlings was lower than in the two other forests. hence, it is necessary to do efforts to control the invasion in bbsnp. this study was part of the master thesis of the first author financially supported by seameo-biotrop dipa 2011 through dr. sri s. tjitrosoedirdjo. the author wishes to thank artha graha peduli and wildlife conservation society indonesia program for their facilities in conducting the field works. et al. m. peltata conclusions acknowledgments references [btnbbs] balai taman nasional bukit barisan selatan. 1999. rencana pengelolaan taman nasional bukit barisan selatan, buku ii. kota agung, lampung 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[wcs-ip] wildlife conservation society indonesia program. 2001. taman nasional bukit barisan selatan dalam ruang dan waktu. laporan hasil penelitian 2000 2001. bogor : phka/wcs-ip. merremia peltata ecological impact of invasion jani mastermerremia peltata – et al. 1. francis (high).cdr biotropia vol. 21 no. 2, 2014: 71 81 high turbidity affects filtration rate and pseudofaeces production of the mud clam (solander 1786) (bivalvia: corbiculidae) polymesoda erosa francis albert t. argente *, senona a. cesar and danilo t. dy received 18 october 2012/accepted 1 october 2014 is an economically and ecologically important bivalve which thrives in brackish water mangroves or zones. unpredictable weather conditions and unregulated anthropogenic activities in mangrove area can lead to high turbidity conditions and possibly affect the filtering capacity of . this study will aid in the development of culture technique of this species in the region. results could also be used to facilitate adequate stock management for potential commercial exploitation in central philippines. a laboratory experiment was conducted to determine the effects of turbidity concentration and body size on the filtration rate and pseudofaeces production of . filtration rates significantly increased with higher turbidity concentration up to 750 mg/l ( < 0.05). pseudofaeces production also increased with increasing turbidity concentration ( < 0.05). body size did not affect the filtration and pseudofaeces production of . results suggested that was resilient to highly disturbed turbid environments and therefore, could be a potential candidate species for aquaculture. biodeposit, bohol, , pseudofaces production, silt, suspension filter-feeders ,1,3 2,3 3 1 2 3 pangasinan state university-binmaley campus, binmaley, pangasinan, philippines visayas state university, baybay city, leyte, philippines university of san carlos, cebu city, philippines abstract introduction polymesoda erosa nypa p. erosa p. erosa p p p. erosa p. erosa nypa polymesoda erosa,keywords: development in a country is coupled with increasing demand for food that could be sourced out from aquatic ecosystems. among many aquatic species, bivalves are now heavily cultured to meet such demand, yet there are still some fisheries that heavily rely on the wild stock, but with high potential for mariculture (nuryanto & susanto 2010). understanding the physiological and ecological attributes of a species is crucial in deciding its mariculture feasibility. such species include the mangrove or * corresponding author : faargente@gmail.com doi: 10.11598/btb.2014.21.2.1 71 mud clam, (solander 1786), synonymously known as (morton 1984). is widely distributed in the indo-pacific region (morton 1984, nuryanto & susanto 2010). it is also found in northern australia as component of earth mounds (gimin 2004, brookwell 2006). possesses some characteristics that make it both an economically and ecologically important species. this clam is a potential source of bioactive compounds exhibiting antiviral (chatterji 2002) and antimicrobial (sharma 2009) properties. its congeneric species, is found in 16 estuaries in mexico and is part of mexico's commercial fishery (wakida-kusunoki & mackenzie 2004) while is also heavily exploited in the artisanal fishery in india (clemente & ingole 2009), malaysia (hamli 2012), indonesia (hardinsyah 2006), northern australia (gimin 2005) and philippines (germano 2003, laureta 2008). is noted to be harvested and traded in bohol, central philippines (argente 2012). biological studies showed that is dioecious and hermaphrodite (morton 1988), with no external dimorphism (clemente & ingole 2009) but with high polymorphism (nuryanto & susanto 2010). it can attain a size of 110 mm shell length (morton 1988). in goa, india where it is an exploited fishery product, adult shell density is 9 ind./m , described to be of non-random, patchy distribution (clemente & ingole 2011). clam harvest is still done by hand gleaning between thickets. thrives in extreme adverse environmental conditions like low ph (morton 1976); in waters with high heavy metal concentration (modassir 2000), and high turbidity (yap & mohd azri 2009). it is an oligohaline species (morton & chan 1986). field studies have reported that thrives in brackish water environments with salinity ranges of 7-31 ppt (clemente & ingole 2009, yap & mohd azri 2009) but probably prefer higher salinity range since the low tide mark does not support adult population (clemente & ingole 2011). it can be found in areas only inundated during very high spring tides (clemente & ingole 2011), with ability to rapidly resume filter feeding activity when inundated (yap & mohd azri 2009). thrives well in true mangrove dominated area or in dominated estuarine area. is a filter feeder, referred as suspensivore by wakida-kusunoki and mackenzie (2004), with ability to remove particles from water current by bio-filter mechanism accomplished by the gills (ruppert & barnes 1994). this form of nutrient acquisition significantly contributes to bentho-pelagic coupling (barnes 2006, jones 2011). the removal of suspended particles in the water column by filter feeding results in the production of biodeposits (clavier & chauvaud 2010); particles coated with mucus which are processed in the mantle cavity and ejected through the inhalant siphon or along the ventral mantle margin (berg 1996). in loay-loboc river, bohol, central philippines, natural population of may experience high turbidity condition due to unregulated anthropogenic activities and erratic weather disturbances. currently, it is not yet known up to what extent can tolerate the turbidity concentrations that are normally encountered in natural as well as in disturbed mangrove or environments. there are evidences that variations in body size and concentration of suspended particles influence their physiological responses (rajesh 2001, rueda & smaal 2002, hatton 2005). thus, a laboratory experiment was conducted to determine the effect of body size and polymesoda erosa geloina coaxans p. erosa et al. p. erosa et al. et al. p. caroliniana p. erosa et al. et al. et al. et al. polymesoda p. erosa nypa p. erosa p. erosa p. erosa nypa fruticans p. erosa et al. et al. p. erosa p. erosa nypa et al. et al. 2 72 biotropia vol. 21 no. 2, 2014 turbidity concentrations on the filtration rate and pseudofaeces production of collected in loay-loboc river, bohol, central philippines. this study will aid in the development of culture techniques of this species in the region. results could also be used to facilitate adequate stock management for potential future commercial exploitation in central philippines. at the moment, the populations in central philippines are harvested and considered as low level artisanal fishery for sustenance consumption (laureta 2008, argente 2012). the clams were collected from a zone in masayon, calvario, loay-loboc river, bohol (9.60853° n, 124.01265° e) (fig. 1). the estuarine river is known for different anthropogenic activities such as fishing, gastropod and bivalve gleaning, recreational boat cruise and sand quarry. water samples were taken from the collection site to determine the prevailing salinity and total suspended solids (tss) condition. substrate samples were also collected for use as suspended particles during the experiment. the clams were brought to the laboratory (university of san carlos marine station, mactan island) within 24 hours after collection and acclimated in a basin filled with filtered brackish water (7 ppt) for 12 hours prior to the experiment. the clams were not fed during the acclimation period. two size classes of , smaller-sized clams (47.4±4.3 mm) and larger-sized clams (58.0±2.4 mm) were used during the experiment. substrate samples mostly composed of sand and mud were wet sieved to separate the silt (< 63 µm diameter). the silt were air-dried, pounded and oven-dried (75 °c) to constant dry weight. experimental concentrations of suspended silt were based on the prevailing tss condition at the collection site. a range finding test was conducted to determine the turbidity concentrations used in the experiment. consequently, four higher turbidity concentrations (250, 500, 750 and 1,000 mg/l) and a control (40 mg/l) were used. the experiment employed a randomized complete block design (rcbd) with the experimental replications as the blocking variable. an experimental unit (fig. 2) included an individual clam glued on a bamboo stick using aquatic epoxy and placed in a plastic container filled with 1 l filtered brackish water (7 ppt) and silt particles of the established experimental turbidity concentrations. the container was aerated from the bottom to ensure suspension of silt particles throughout the experiment. a piece of plastic was placed above the air source to avoid clam distress which may affect filtration activity. six replications were conducted for each experimental turbidity concentration per size class. each clam was randomly allocated to an experimental unit and a wait-period of approximately five minutes was observed before the actual timing of the incubation experiment which lasted for 120 minutes (2 hours). after the experiment, the clams were removed from the experimental units. pseudofaeces clinging from the bamboo p. erosa p. erosa nypa p. erosa materials and methods 73 filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. sticks, clam shells and walls of the containers were collected and placed in pre-labeled petri dishes and oven dried (75 °c) to constant dry weight. the brackish water in the experimental units were filtered using pre-weighed (constant dry weight) whatmann™ gfc 47 mm filters. the filters with residues were air-dried for 24 hours and oven-dried at 75 c to constant dry weight o . 74 biotropia vol. 21 no. 2, 2014 figure 1. map of loay-loboc river, bohol, central philippines. collection site is shown with a marker (star). inset map a is the philippines, b is bohol figure 2. experimental unit of the study bamboo stick clam plastic container piece of plastic air stone air hose the filtration rate (fr; mg/minute/ind) and pseudofaeces production (pp; mg/minute/ind) of the were determined using these equations: fr = [tss (filter filter )] / time elapsed (1) pp = wt of pseudofaeces / time elapsed (2) where: tss = initial quantity (mg) of total silt in the experimental unit filter = initial weight (mg) of the filter filter = weight(mg) of filter with residue time elapsed = 120 minutes a two-way anova (analysis of variance) with replications was used to determine the effects of body size and turbidity concentration on the filtration rate and pseudofaeces production of the experimental clam. raw data were logtransformed (log+1) to satisfy parametric statistical assumptions. in cases of significant differences, the tukey hsd test was used as post hoc test. the significance level was set at 95% ( = 0.05). in this study, the prevailing salinity in the sampling area was 7 ppt, while the prevailing total suspended solids (tss) was 40 mg/l. total organic matter content of substrate was 30%. table 1 shows the results of two-way anova on the effects of turbidity concentration (tc) and body size (bs) to the log-transformed fr and pp of . tc significantly influenced fr and pp ( < 0.05). however, bs showed no significant effect on fr and pp. likewise, no interaction effect between tc and bs was observed. the log-transformed data showed that the fr of significantly increased with higher tc up to 750 mg/l, beyond which no significant increase was observe (fig. 3). similarly, the log-transformed pp of also showed significant increased with higher tc (fig. 4). however, the variations in pp within each tc were wider compared to fr. bivalves tend to escalate their filter feeding behavior with higher turbidity conditions (bayne 1993, rajesh 2001). in highly turbid waters, these filter feeders consume much energy to filter suspended particles (hibbert 1977). consequently, bivalves have different threshold limits in their filter feeding activities (berg 1996, rajesh 2001). exceeding beyond the threshold may have negative effects in the physiological responses of these species within their environment. in the work of morillo-manalo and del norte-campos (2010) on a burrower bivalve, , the initial increase in filtration rate followed by a decrease at higher p. erosa p p. erosa p p. erosa p. erosa et al. et al. et al. et al. paphia undulata i r i i i r results and discussion water quality measurement results filtration rate (fr) and pseudofaeces production (pp) of p. erosa 75 filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. 76 biotropia vol. 21 no. 2, 2014 effect ss df ms f p-value log-transformed fr tc 14.114 4 3.528 501.100 0.000* bs 0.004 1 0.004 0.580 0.448 bs*tc 0.031 4 0.008 1.100 0.369 error 0.352 50 0.007 log-transformed pp tc 17.064 4 4.266 130.495 0.000* bs 0.021 1 0.021 0.640 0.427 bs*tc 0.079 4 0.020 0.604 0.661 error 1.635 50 0.033 table 1. anova results on the effects of turbidity concentration (tc) and body size (bs) to the log-transformed filtration rates (fr) and pseudofaeces production (pp) of p. erosa figure 3. log-transformed filtration rates of larger-sized (a) and smaller-sized (b) at different turbidity concentrations p. erosa (bars indicate standard deviation) l o g -t r a n sf o r m e d f il tr a ti o n r a te s (m g /m in u te /i n d .) turbidity concentration (mg/l) 1.8 40 2.0 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 a 250 500 750 1,000 1.8 2.0 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 40 250 500 750 1,000 b concentration, was attributed to the inherent nature of the clam to stimulate valve opening and filtration activity when exposed to lower concentration. then, at higher concentration, this may result to overloading of the ctenedia, the filter apparatus that led to closing of the valve, thus reduction in fr. however, in this experiment, the plateau of fr was not yet reached. ecologically, it could mean that can still withstand higher turbidity level, as in the case during heavy rains of which the loayloboc river receives water from tributaries as far as carmen (the interior part of bohol). the unpredictable weather disturbances and the increasing human perturbations can result in turbid aquatic environments. bivalves with higher threshold of filtration would be more resilient in disturbed waters. in this study, showed potential in adapting to such situation. the fr of the clam significantly increased in turbidity conditions of up to 750 mg/l. this was a remarkable feat as compared to , and which could filter in turbidity concentrations ranging from 10 to 570 mg/l (bayne 1993, navarro & widdows 1997, rueda & smaal 2002). p. erosa p. erosa mytilus edulis cerastoderma edule spisula subtruncata et al. 77 figure 4. log-transformed pseudofaeces production of larger-sized (a) and smaller-sized (b) at different turbidity concentrationsp. erosa (bars indicate standard deviation) filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. l o g -t r a n sf o r m e d p se u d o fa c c e s p r o d u c ti o n (m g /m in u te /i n d .) turbidity concentration (mg/l) a 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 2.0 40 250 500 750 1,000 40 250 500 750 1,000 b 78 biotropia vol. 21 no. 2, 2014 p. erosa p. erosa et al dreissena polymorpha spisula subtruncata paphia undulata et al. p. erosa p. erosa perna canaliculus perna viridis crassostrea madrasensis et al. paphia undulata paphia undulata p. erosa et al. et al. et al. et al. p. erosa et al p. erosa p. erosa the pp of showed increasing rate at higher tc. morillo-manalo and del norte-campos (2010) stated that the production of pseudofaeces implies maximum energy gain for the bivalves during feeding. produced more pseudofaeces in higher tc to optimize energy gain instead of utilizing more to metabolize excess particles. this is also a mechanism to regulate ingestion rate as well as preventing saturation of the gills. iglesias . (1998) equated fr to ingestion rate, when all filtered material is ingested with no pseudofaeces production. similar condition was encountered with other bivalves such as , and (bayne 1993, rueda & smaal 2002, morillo-manalo & del norte-campos 2010). the bs of did not affect the filtration and pseudofaeces production. during the experiment, the valves of opened all throughout the incubation period indicating that they were actively filtering regardless of size. in other bivalves such as , and , these physiological responses tend to increase with larger body size (rajesh 2001, hatton 2005). smaller individuals of these species may have little chance of survival in highly turbid waters. on the other hand, the short-necked clam showed a reduction of fr with increasing bs (morillo-manalo & del norte-campos 2010). the higher fr by smaller is supposedly needed to support its fast growth and development (del norte-campos & villarta, 2010), compared to which is considered to be a long lived species (morton 1988). another important consideration here was the range of shell length between the two treatment levels used in the study. a wider range (i.e. juvenile size vs. mature size) in future experiment might reveal important information on the effect of clam size. in this study, we experienced difficulty in obtaining small clams in the collection site. ecologically, the filtration of suspended particles and pseudofaeces production are physiological functions of bivalves (berg 1996, higano 2004). the processing of suspended materials in the water column carried out by suspension filter feeders was significant in assessing trophic relationships in freshwater, estuarine and marine ecosystems (prins 1996, espinosa 2008, manganaro 2009, sarikhani & javanshir 2010, mamun & khan 2011). this study suggested that natural population of in loay-loboc river was resilient in their environment, which agreed to experiments conducted by yap and mohd azri (2009) and by bayen . (2005). in this time of climate change and rising human population, the search for new and better adapted species for aquaculture is deemed necessary (de silva & soto 2009). the resiliency of to adverse environmental conditions makes it a potential candidate species for aquaculture population in loay-loboc river was resilient with the ability to respond to highly turbid waters. the filtering activity and pseudofaeces production in the population tended to escalate at higher turbidity concentrations up to 750 mg/l. size is a non-factor in its physiological responses suggesting high survivability in disturbed, conclusions 79 turbid waters compared to other bivalves. as a result of its resiliency to adverse environmental conditions, would be considered as potential candidate species for aquaculture in central philippines. we thanked the university of san carlos, marine biology section for logistic support. we also thanked mr. henry palaca for collecting the clams used in this study. the scholarship grants were given by the department of science and technologyphilippine council for agriculture, aquatic and natural resources research and development (dost-pcaarrd) to fata and sac were hereby acknowledged. this was a marine science contribution of psu, vsu and usc. p. erosa acknowledgements references argente fat. 2012. commercially important mangrove bivalves of the visayas, philippines: diversity and fishery. in: 21 annual fr. heinrich schoenig biology: symposium: 2012 march 3. philippines. university of san carlos talamban campus, cebu city. barnes p. 2006. . vancouver(ca): centre for shellfish research, malaspina university-college, british columbia. bayen s, wurl o, karuppiah s, sivasothi n, lee hk, obbard jp. 2005. persistent organic pollutants in mangrove food webs in singapore. chemosphere 61: 303-13. bayne bl, iglesias jip, hawkins ajs, navarro e, heral m, deslous-paoli jm. 1993. feeding behaviour of the mussel, : responses to variations in quantity and organic 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sharma s, chatterji a, das p. 2009. effect of different extraction procedures on antimicrobial activity of marine bivalves: a comparison. pertanika j trop agric sci 32(1): 77-83. wakida-kusunoki at, mackenzie cl. 2004. rangia and marsh clams, and , in eastern mexico: distribution, biology and ecology, and historical fisheries. mar fish rev 66(3): 13-20. yap ck, mohd azri a. 2009. heavy metal concentration (cd, cu, fe, ni, pb and zn) in clam, , collected from intertidal area of tok bali and kuala kemasin, kelantan. malaysian appl biol 31(1): 81-4. th spisula subtruncata anodonta cygnea rangia cuneata, r. flexuosa, polymesoda caroliniana polymesoda erosa filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. 81 3. vincentius (primary).cdr biotropia vol. 20 no. 2, 2013: 89 103 primary productivity of jakarta bay in a changing environment: anthropogenic and climate change impacts vincentius siregar *, and alan. f. koropitan received 12 june 2013/accepted 3 december 2013 jakarta bay receives direct impact from the rapid development of infrastructure and landbased industries which contributed to the increase in pollution and nutrient, and at the same time facing climate change. this condition influenced growth of chlorophylland primary production. to investigate changes of primary production in jakarta bay due to anthropogenic and climate change impacts, a field measurement, laboratory experiment and collection of several data sets have been conducted. the study showed that impact of anthropogenic, particularly sediment load from the land to primary production is important. the intensification of primary production occurs in the middle region of jakarta bay, while the chlorophyllconcentration is high in the river mouth area. the anthropogenic impact is indicated by the land use change that has increased to 73% during the last ten years. the laboratory experiments by injecting co in the waters, as a global warming simulation, have shown a decrease in chlorophylland primary production. therefore, the combination of anthropogenic and climate change may have a double impact on the jakarta bay ecosystem. primary production, jakarta bay, anthropogenic, climate change, impact 1,2 1,3 1. 2. 3. department of marine science and technology, faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia, laboratory of remote sensing and ecology, seameo biotrop, bogor, indonesia center for oceanography and marine technology surya, university, tangerang, indonesia a a a abstract introduction key words: 2 as a part of the java sea, the mass transport of water and material in jakarta bay are controlled by ocean dynamics which is generally influenced by monsoon system and tidal mixing. wyrtki (1961) showed that there are two distinct seasons over the indonesia seas: southeast monsoon and northwest monsoon. associated with the east wind from australia, southeast monsoon brings warm dried air during boreal summer (june-august). in contrast, the west wind from asia or northwest monsoon brings warm moist air during the boreal winter (december-february). thus, * corresponding author : vincents@biotrop.org doi: 10.11598/btb.2013.20.2.5 89 monsoon systems also control the dry season (northwest monsoon) and wet season (southeast monsoon) in indonesian seas where the monsoon wind patterns also affect those of surface flow. in jakarta bay, the distribution of nutrients is strongly affected by monsoon system along the year, but its spatial distribution is more pronounced, high near the coast and low at seaward (ilahude 1995). although coastal waters is an area relatively small in global seas, but it plays an important role in biogeochemical cycles. gattuso . (1998) reported that coastal water contributed 14-30% of primary productivity, 80% of organic matter decomposition, and 90% mineralized sediments. in the sea, half of the production of organic matter was provided by phytoplankton (boyce . 2010). as many shelf seas in the world, ecosystems of the java sea are under pressure due to human activities in the coastal region. the java sea covers an area of 450 000 km where some cities and industries are located along northern coast of java island. talaue mcmanus (2000) reported that most of the pollution in the java sea is caused by domestic sewage, agricultural, industrial and waste as well as solid waste that goes to the coastal region. rapid development of infrastructure and land-based industries also contributed to the pollution increase of the coast, for example: jakarta bay and its surrounding waters are influenced by human activities of the jakarta metropolitan area (jma) which contribute to the pollution of the jakarta bay (suwandana . 2011). population in jma has been increasing year by year, as reported by the indonesian statistics that the population has increased to 30.2 % from 7.6 million in 2007 to 9.8 million in 2012 (provinsi dki 2012). this has an implication to land use change of the coastal area and its vicinities, hence affected jakarta bay. therefore, the population of jma may have direct impact (exploitation and human settlements) and indirect impact (via watersheds) to jakarta bay. anthropogenic impact due to human activities has caused an increase in the concentration of nitrates and phosphates in jakarta bay, to 5 and 15 times, respectively, in the last three decades (arifin 2005). in addition, the eutrophication condition in jakarta bay can be categorized as hyper-eutrophication, especially in estuarine waters or region near river mouth as reported by damar (2003). on the other hand, the java sea ecosystem is facing climate change impact, particularly sea surface temperature (sst) rise and marine acidification. according to the ipcc's predictions (christensen . 2007), increase of sst in the southeast asia waters will be about 1.5 3.7 c during the last 21st century. marine acidification is related with decline of ph as a result of absorbing atmospheric co by the global ocean. according to orr . (2005) marine ph has decreased to 0.1 since post-industrial era. regarding to the recent condition of jakarta bay and climate change issue, hence, this research aim was to investigate changes of primary production in jakarta bay due to anthropogenic and climate change impacts. the field measurement of some water quality parameters at 21 stations and laboratory experiments had been carried out during the period of 9 14 july 2012. in et al et al et al et al et al 2 o 2 materials and method 90 biotropia vol. 20 no. 2, 2013 addition, landsat satellite data as well as secondary data had been collected and analyzed. water samples parameters were taken including water quality, primary production and chlorophyll. water quality parameters measured were surface temperature, salinity, turbidity, using water quality checker at a depth of 0 10 m. primary production and chlorophyllwater samples were collected using water sample at a depth of 1 2 m, and the transparency was measured using a secchi disk. secondary data for nutrient concentrations analysis was obtained from damar (2003). the field stations are shown in fig. 1. laboratory experiments were set up in untung jawa island, northern part of jakarta bay using seawater collected directly in real time from the sampling area to measure chlorophylland primary production influenced by increasing temperature and co injection as treatments. two different treatments have been performed to increase the seawater temperature 1 c and 2 c of the initial temperature (29 -30 c) using tube lamps, while c gas was injected to seawater until 450 ppm and 560 ppm, respectively. gross primary production (gpp) and net primary production (npp) were measured based on dissolved oxygen in the light and dark bottles before and after incubation (3 hours incubation using halogen lamp) in field laboratory with ambient condition. dissolved oxygen was measured based on modified winkler method (strickland & parsons, 1972; apha, 1980). chlorophyll-a parameter was measured using fluorometric method. in addition, the landsat satellite data were downloaded from source : ( ) to analyze land use coverage change during 10 years (2002-2011 using color composite, total suspended solid (tss) applying formula of ambarwulan (2002) and chlorophylldistributions in jakarta bay using an algorithm (wouthuyzen 1991). a a niskin a a 2 2 0 0 0 0 o http://earthexplorer.usgs.gov/ 91 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. figure 1. sampling stations and its clustering in jakarta bay results and discussion general condition of jakarta bay water jakarta bay receives material and water discharges from several rivers that cover jam and its surrounding area, such as cisadane in the western part and citarum in the eastern part. in addition, there are several rivers that drain in the central part of jakarta bay, such as: muara gembong, cikarang, marunda, muara angke, and kamal. all rivers are responsible in carrying sediment in the bay and affect tss and transparency of the water. considering the sediment distribution regime, four clusters of sampling stations were based on their distance from the coast-line as shown in table 1. 92 biotropia vol. 20 no. 2, 2013 table 1. clustering of sampling stations cluster station description 1 15, 12, 11, 4, 5 near estuary and coastline 2 16, 13, 10, 3 45 km from coastline 3 17, 14, 6, 2 8-10 km from costline 4 19, 18, 9, 1, 8, 7, 20 >10 km from coastline sea surface temperature depends on several factors such as precipitation, evaporation, wind speed, intensity of sunlight, and freshwater input from the river. in jakarta bay, distribution of the sea surface temperature horizontally decreased from near the coast to seaward (fig. 2). the vertical distribution of sea temperature showed the same pattern as those of horizontal distribution (fig. 3). figure 2. horizontal distribution of sea surface temperature july 2012 93 figure 3. vertical distribution of sea temperature of jakarta bay waters the role of river discharges in jakarta bay was represented by sea surface salinity distribution. in jakarta bay, the river discharges were relatively low as shown in figure 4 where the observed salinity distribution tended to decrease slightly from cluster 4 to cluster 1.this condition was actually influenced by level of rainfall during dry season (july 2012) at which the sampling period was carried out. in general, the sea surface salinity in jakarta bay is typical of coastal water varying between 30.4 to 32.1 psu. figure 4. sea surface salinity distribution in jakarta bay july 2012 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. cluster 1 cluster 2 cluster 3 cluster 4 94 biotropia vol. 20 no. 2, 2013 the river discharges were relatively low, although the effect of human activities in the coastal region has affected the turbidity of jakarta bay, particularly in custer 1 and cluster 2 regions. turbidity consisted of colloidal particles and suspended pollutants, such as: organic materials and inorganic waste from industry, households and aquaculture. turbidity could be caused by the presence of organic materials produced by the waste industry. the turbidity in jakarta bay varied between 2.43 7.00 ntu (fig. 5) and the high turbid water was located in st.15 (muara angke), st. 11 (priok), and st. 3 and 4 (cikarang). so, the turbidity was high at the coastline due to land runoff and gradually decreases seawards. similar to the turbidity, total dissolved substance (tds) distribution had the same pattern. the dissolved substance could be carbonate and bicarbonate, chloride, sulfate, phosphate, nitrate, calcium, magnesium, sodium, organic ions, and more ions. the tds ranges between 31 32 g/l, where the highest concentration occurs in st. 15 (muara angke). the observed secchi disk depth distribution in jakarta bay showed that cluster 1 and cluster 2 were lower than cluster 3 and cluster 4. it is emphasized that the transparency of jakarta bay close to the estuary and coastline were poor. the secchi depth for each cluster was varied between 1.3 and 3.5 m (cluster 1), 1.6 and 3 m (cluster 2), 4 and 4.5 m (cluster 3), 3 and 6.2 m (cluster 4). figure 5. turbidity distributions in jakarta bay based on field measurement on july 2012 95 a a a chlorophyllis a necessary pigment of phytoplankton for photosynthesis. phytoplankton serves as primary producers in the chain of life in the sea, so its existence is very important as the basis of life on the sea. the chlorophyllconcentration in jakarta bay varied between 0.78-15.74 mg/l (figure 6), while the annual chlorophyllconcentration derived from the satellite data (2002-2012) varied from 1.09 mg/l to 6.07 mg/l and showed a stable trend , except in 2011-2012 the trend was increasing significantly close to the coastal area (cluster1,2) (fig. 7). figure 6. chlorophylldistribution in jakarta bay based on field measurement on july 2012 a figure 7. annual chlorophyllconcentration derived from satellite data(2002-2012)a primary productivity of jakarta bay in a changing environment vincentius siregar– et al. c h lo ro p h y ll (m g /l ) 96 biotropia vol. 20 no. 2, 2013 increasing chlorophyllmainly occurred in the near river mouth of st. 11 (tanjung priok) and st. 15 (muara angke) due to runoff from the jma. however, low primary production was observed surrounding the river mouth, whereas high production occurred in the centre of jakarta bay (fig. 8). this could indicate that little support of the photosynthesis in the river mouth occurred and was probably due to the high turbidity. a figure 8. npp distributions in jakarta bay based on field measurement on july 2012 anthropogenic impact the studies of anthropogenic impact and primary productivity seas were analyzed based on the ratio of chlorophylland npp. chlorophyllas major component in phytoplankton is responsible for primary production activities. increasing of chlorophyll-a concentration in the waters resulted high primary productivities. ratio of chlorophylland npp has relation to an individual's capacity to produce phytoplankton primary productivity. the higher the ratio of chlorophylland npp, the lower the capacity of phytoplankton result in the primary productivity. this indicated that abundance of chlorophylldoes not mean ability of primary productivity to increase in proportion to the amount of chlorophyll. in this case, the impact of anthropogenic pollution, can affect capacity of chlorophyll-a in generating primer productivity. to analyze the impact of land use change on primary production in jakarta bay, figure 9 shows ratio of chlorophyll/npp. the high ratio means more chlorophyllto produce primary production or the high ratio the less efficiency in photosynthesis the ratio indicates the efficiency in photosynthesis processes by phytoplankton. the average value of ratio 79.83 in cluster 1, 60.1 in cluster 2, 38.05 in cluster 3 and 20.39 in cluster 4 . thus, anthropogenic pressures were high in near river mouths or coastal region (cluster 1) and gradually decrease to the seaward (cluster 4). a a a a a a a a figure 9. distribution of ratio between chlorophylland npp in all stationsa the analysis of the land cover change is only carried out by grouping some particular class settlements, pond/swamp, rice field, and vegetation corresponding with the anthropogenic impacts of the waters. the maps of the land cover change during the last ten years are shown in figure 10. the variation of land use change in jma concluded that settlement class has increased approximately 73% in the past 10 years. it implies that the anthropogenic contribution from residential waste (household and industry) has increased year by year. as the rice fields are likely to decrease, pond or swamp tends to remain unchanged. meanwhile, vegetation cover increased due to the development of the green city program of jakarta, which stated that every area should leave at least 30% of the open green area. the land cover change from 2002 to 2011 is shown in figure 11. the total suspended sediment concentration (tss) extracted from satellite data during 2002-2011 in jakarta bay waters increased and showed higher concentration in the mouth of rivers (cluster 1-2). tss concentrations showed positive correlation with the turbidity. this similar pattern was also shown during the observation period and the concentration varied from 0.11 mg/l to> 135 mg/l. the spatial distribution of concentration of the tss tends to develop year by year in the coast seaward. this is probably due to the increase of sediment load from land as a result of significant land cover change in jma and surrounding cities. in addition, figure 12 shows ratio of compensation depth (dk) and attenuation depth (k). the compensation depth (dk) is a depth of water column where photosynthesis rate is the same as respiration rate. the compensation depth characterizes the occurrence of primary productivity capacity. attenuation depth (k) is a maximum depth for sunlight intensity in water column, to allow photosynthesis processes. in turbid water such as jakarta bay, depth attenuation can be a limiting factor in photosynthesis process. thus, ratio of compensation depth and attenuation depth characterize capacity of primary productivity. the higher ratio of dk and k, the greater capacity of primary production. the ratio of dk/k is generally high in cluster 4 and low in cluster 1. this is consistent with the high sediment load from land as shown previously in turbidity distribution (fig. 5). therefore, the land use change and sediment supply have influenced significantly to the primary production in jakarta bay. of 97 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. legend cloud rice field settlements vegetation pond / swamp figure 10. land cover changes during the last ten years (2002-2011) biotropia vol. 20 no. 2, 2013 98 figure 11. land cover change from 2002 to 2011 figure 12. ratio between compensation depth(dk) and attenuation depth (k) in all stations. climate change impact the annual average of minimum surface air temperature in jakarta bay have increased during 2002-2011 (fig. 13). the lowest and the highest of minimum surface air temperature occurred in 2008 and 2011 were 25.33 c and 25.76 c, respectively. at the same period, the maximum surface air temperature tended to decrease, where the highest and the lowest occurred in 2002 and 2008, respectively. those surface air temperatures could be used as a primary measurement of climate change in jakarta bay and its surrounding areas (hansen . 2010). 0 0 et al 99 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. the laboratory experiments as simulation of the effects of climate change was set up streamlined in nature, where the created conditions are closed systems. however, to achieve conditions similar to the process in nature, the location of the experiment was placed close to the environmental conditions with a relatively recent natural changes. in general, the experiment concluded that there has been a decline in chlorophyll-a and primary productivity (npp) with increasing temperature 1 c and 2 c and co gas injection (450 and 560 ppm) into the water sample, after the incubation process. in contrast, hashioka and yamanaka (2007) reported that chlorophylland npp in the sub-tropical and temperate waters will be increasing under global warming scenario, based on numerical modeling. this contradiction may suggest that marine ecosystem in tropical region has different response with those in sub-tropical and temperate regions. the summary of laboratory experiments related with climate change scenarios is presented in table 2. 0 0 2 a 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 tm 25.40 25.44 25.42 25.55 25.60 25.53 25.33 25.79 25.85 25.87 y = 0.050x + 25.30 r² = 0.599 25.00 25.10 25.20 25.30 25.40 25.50 25.60 25.70 25.80 25.90 26.00 °c figure 13. annual average of minimum (left) and maximum (right) of surface air temperature in jakarta bay during 2002-2011 (source : bmkg, jakarta, 2012) table 2. the increased influence of temperature and co towards primary productivity and chlorophyll2 a type of experiment gpp respiration npp chlorophyll-a (mgc/m3/day) (mgc/m3/day) (mgc/m3/day) (mg/m3) control before incubation 1.916 control after incubation 2.634 3.088 0.061 2.394 temperature 1 oc after incubation 2.593 3.108 0.003 1.373 temperature 2 oc after incubation 2.557 3.065 0.003 1.075 injection co2: 450 ppm 2.441 3.086 -0.131 1.391 injection co2: 560 ppm 2.392 3.257 -0.323 1.073 note :gpp = gross primary productivity/photosynthesis npp = net primary productivity biotropia vol. 20 no. 2, 2013 100 in order to evaluate the dominant parameters that affect npp in jakarta bay, principal component analysis (pca) has been performed. selected parameters for pca are nutrients (nitrite, nitrate, ammonium and phosphate) and chlorophyllas shown in figure 14. in this analysis, nutrient concentrations was obtained from damar (2003) where the chlorophyllis based on the present study. a a figure 14. pca of chlorophyll, ammonium, phosphate and nppa figure 15. ammonia concentration on the tide and low tide in estuary of jakarta bay . pca results showed that chlorophyll, ammonium, phosphate and npp have positive correlation. in this case, chlorophylland ammonium are closely associated. this linkage suggests that ammonium influence significantly on phytoplankton growth (chlorophyllconcentrations) in jakarta bay. ammonium is a kind of nutrient nitrogen species, which generally come from sources of organic nitrogen remineralization processes in form of particles, as well as dissolved. in addition, ammonium sources are also coming from industrial activities. nitrogen species such as nitrate and nitrite are generally derived from agriculture due to the use of chemical fertilizers. according to data from the environmental status report of the jma (bplhd jakarta 2011), the ammonium supply in jakarta bay is mainly by rivers where the high contribution is muara cakung and muara sunter, particularly during ebb tide (fig. 15). both river mouths are located at tanjung priok, an international marine port of jma a a a 101 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. tide low tide bm 8.00 7.00 6.00 5 4 3 2 1 .00 .00 .00 .00 .00 m k am al c en gk ar en g m a ng ke m k ar an g m a nc ol m s un te r m c ak un g m m ar un da m g em bo ng tide low tide bm assessment of anthropogenic impacts and climate change on primary production in jakarta bay, indicated that the role of human activities in jakarta is more dominant, especially in the waters close to coastal or estuarine river. for example, the efficiency in photosynthesis is controlled by sediment load, not only containing clay and sand, but also organic particles or contaminants that enter through the rivers. this condition influences the change of water quality, resource supply , habitat availability and climate (lotze . 2002). the impact of climate change in this case was significant and may have a double impact in combination with the anthropogenic change. the anthropogenic impact was clearly shown by less efficiency in primary production in jakarta bay, particularly close to the river mouths or coastline due to sediment load from jakarta metropolitan area. the anthropogenic impact was mainly influenced by domestic waste and land use change that had increased to 73% during the last ten years. the climate change impacts based on laboratory experiments by simulation of increasing temperature and c0 gas have shown a decrease in chlorophylland primary production. therefore, the anthropogenic and climate change may have a double impact to the jakarta bay ecosystem. the financial support for this research was provided by seameo biotrop under their dipa 2012. we would like to thank nasa land processes distributed active archive center (lp daac) for providing us with landsat data. et al a conclusions acknowledgements references 2 ambarwulan, w. 2002. mapping of tsm concentrations from spot and landsat tm satellite images for integrated coastal zone management in teluk banten, indonesia. msc thesis. international institute for geo-information science and earth observation.131 p. arifin, z. 2005. pollution prevention and reduction strategies and implementation in the jakarta bay. paper presented in the . pemsea-gef-ministry of maritime affairs and fisheries (momaf) republic of kore. masan city, june 1-3, 2005. boyce, d.g., m.r. lewis and b. worm. 2010. global phytoplankton decline over the past century. 466:591596 christensen, j.h., b. hewitson, a. busuioc, a. chen, x. gao, i. held, r. jones, r.k. kolli, w.-t. kwon, r. laprise, v. magaña rueda, l. mearns, c.g. menéndez, j. räisänen, a. rinke, a. sarr and p. whetton, 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g.-k. plattner. 2004. the role of coastal zones in global biogeochemical cycles. , , 85(45), 469. strickland.j.d.h, and t. r. parsons.1972: a practical handbook of seawater analysis. fish.res.board can. bull., 167:1-326. suwandana, e., k. kawamura, k.tanaka, y. sakuno, and p. raharjo, 2011. and biophysicochemical relationships of seawater and water pollution index in the jakarta bay. american journal of environmental sciences 7 (3): 183-194, 2011 talaue-mcmanus, l. 2000. transboundary diagnostic analysis for the south china sea. no. 14, unep, bangkok, 105 p. wouthuyzen, s. 1991. desertation, nagasaki university. jepang. wyrtki, k., 1961: physical oceanography of the southeast asian waters, , scripps institution of oceanography, university of california, la jolla, california, 195 p. phd dissertation annual review of ecology and systematics, ecological modelling in limnol. oceanogr. nature eos transactions american geophysical union escherichia coli . eas/rcu technical report series analysis of potential utility of remote sensing data acquired from earth observation satelites for monitoring in coastal zone environment. naga report volume 2 103 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. 1. etti sartina (the liverwor... biotropia vol. 20 no. 2, 2013: 73 80 the liverwort genus (marchantiaceae) of mount sibayak north sumatra, indonesia marchantia etti sartina siregar , nunik s. ariyanti , and sri s.tjitrosoedirdjo received 21 january 2013/accepted 02 july 2013 knowledge on the liverworts (marchantiophyta) flora of sumatra is very scanty including that of genus (marchantiaceae). this study was conducted to explore the diversity of in mount sibayak north sumatra, indonesia. altogether, seven species of are found in mount sibayak north sumatra, of which five are previously known ( , , , , and ), while one is as new species record ( ) for sumatra, and one species has not been identified ( sp ). an identification key to the species of from sumatra is provided. liverwort, , marchantiaceae, mount sibayak, north sumatra 1,2 3 3,4 1 2 3 4 plant biology graduate program, graduate school, bogor agricultural university, ipb-campus darmaga, bogor, indonesia university of sumatra utara, medan, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, ipb-campus darmaga, bogor indonesia seameo biotrop, jl. raya tajur km 6, bogor, indonesia marchantia marchantia marchantia marchantia acaulis m. emarginata m. geminata m. paleacea m. treubii m. polymorpha marchantia . marchantia marchantia abstract introduction key words: marchantia marchantia marchantia, m. emarginata m. mucilaginosa m. nitida marchantia m. acaulis, m. emarginata, m. geminata, m. miqueliana, m. paleaceae, m. treubii m. mucilaginosa m. acaulis m. nitida m. paleaceae l. is one of the largest genera in the liverworts order marchantiales. this genus is represented by 36 species found in the world (bischler-causse 1998). in indonesia especially sumatra, the floristic work on is still very scarce. herzog (1943) in his study of liverworts from sumatra, recorded three species of namely , and . a monograph of from asiatic and oceanic regions was by bischler-causse (1989), who listed six species from sumatra, namely and ( is a synonym of and is a synonym of ). * corresponding author : ettisartina@yahoo.com doi: 10.11598/btb.2013.20.2.3 73 it is well known that the species of liverworts from sumatra, especially north sumatra is very much undercollected. so it is assumed that this study will improve the knowledge on in sumatra especially north sumatra. this study is conducted to explore and improve our understanding of the diversity of in mount sibayak, north sumatra, indonesia. the study area is located in mount sibayak, north sumatra indonesia, approxi mately 60 km from medan city. the locality has an altitude of about 700 2050 m asl, annual rainfall of 2400 2800 mm/year, and relative humidity of at least 80 90 %. exploration was carried out along the tracks of mount sibayak from lowland to the peak. eighty newly collected material specimens of from mount sibayak and three dried specimens deposited in bo (collected from north sumatra) were used for the study. the specimens collected were classified and identified based on morphological characters, using bishler-causse (1989) and gradstein (2011) in concept and species delimitation. all newly acquired specimens are deposited in bo herbarium. an identification key to the species of known from sumatra is provided. there are seven species of found in mount sibayak, north sumatra, , , , and sp. is reported as a new species record from sumatra. and are the most common species in mount sibayak, found from lowland to high level altitude. was found from lowland to near the peak and numerous around the dwi warna waterfall. is common species in lowland altitude (less than 1000 m altitude). rare species of was found in mount sibayak: (only at high level altitude ± 1500 m) and sp. (only around the dwi warna waterfall). the populations of these two species were limited . was found in certain places from 1150-1250 m altitude, but densely populated. in this study, we did not find the species of that has been reported by bischler-causse (1989) from sumatra. var. was reported by burgeff (1943) from brastagi, north sumatra. however, this species was not confirmed by bischler-causse (1989) since the specimen type was unknown. it might belong to or to (bischler-causse 1989). marchantia marchantia marchantia marchantia marchantia m. acaulis m. emarginata m. geminata m. paleacea, m. polymorpha, m. treubii marchantia marchantia polymorpha marchantia emarginata, m. geminata m. treubii marchantia geminata marchantia acaulis marchantia m. polymorpha marchantia marchantia paleacea m. miqueliana marchantia grisea sumatrana m. acaulis m. wallisii materials and method results and discussion 74 biotropia vol. 20 no. 2, 2013 key to species of in mount sibayak north sumatra and the species of recorded from sumatra (modified from bischlercausse 1989): marchantia , marchantia 1. thallus margin crenulate; scales over almost entire ventral surface of thallus, reaching the thallus margin, sometimes visible at the margin in dorsal view; lobes of female receptacle terete.....................................................................5. 1. thallus margin entire; scales only along thallus midline of ventral surface, invisible in dorsal view; lobes of female receptacle flat apically.................................................2 2. cupules with ciliate lobes and densely papillose on outer surface...........4. 2. cupules ciliate, no or few papillae on outer surface..................................................... 3 3. thallus more than 5 mm wide. involucres ciliate .................................. 3. thallus as wide as or less than 5 mm wide. involucres entire or crenulate................ 4 4. compact ventral tisssue of thallus with numerous mucilage cavities per section. involuces are almost as long as or longer than lobes....................................................5 4. compact ventral tisssue of thallus without or with 1-3 mucilage cavities per section. involucres are shorter than lobes ....................................................................6 5. appendage of median scales of length : ratio width = 1.1-1.5 : 1; margins sharply toothed. male receptacles sessile or nearly so..............................................1. 5 appendage of median scales of length : ratio width= 1.5-2.4 : 1; margins nearly entire to bluntly toothed. male receptacle distinctly stalked....................... 6 appendages of median scales acute or acuminate with row of 2-3 cells apically...............................................................................................................................7 6 appendages of median scales rounded or obtuse with single cell apically ................................................................................................................. 7. sp. 7 appendage of median scales toothed, the teeth mostly consisting of 2-3 cells and often recurved towards base of appendage. lobe of female receptacle emarginate or truncate apically ................................................................................. 2. 7 appendage of median scales entire or toothed, the teeth mostly unicellular and oriented obliquely towards apex of appendages. lobes of female receptacle split apically...............................................................................................................................8 8 appendage of median scales yellow with orange or purplish borders, ovate. number of female lobes usually 4...................................................... ......3. 8 appendage of median scales purple, narrowly triangular. number of female lobes variable 3-6......................................................................................................6. 1. steph. (fig.1 a-b). thallus always with dark median band on dorsal surface, however bischlercausse-causse (1989) found sometimes specimens without median band on dorsal surface. antheridium and archegonium sometimes are found on the same thallus (monoecious) but male rays are never found on the female receptacles as the description by bischler-causse (1989). lobes of female receptacle sometimes curved up when mature. m. polymorpha m. paleacea m. miqueliana* m. acaulis m. wallisii* marchantia m. emarginata . m. geminata m. treubii marchantia acaulis 75 the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. * the species was reported from sumatra, but it was not found and described in this study note: habitat: distribution: specimens examined: note: habitat distribution: specimens examined: note: habitat distribution: specimen examined: is similar to but the latter species has a distinctly stalked male receptacles, thallus without large cavities, and longer appendage of median scales. is easily recognized by the male receptacle without stalk, the large and numerous mucilage cavities in compact ventral tissue in the thallus and the yellowish appendage of median scales. found abundant on soil or rocks in open place or semi shaded place from 870 to 900 m altitude. indonesia (sumatra, java, kalimantan, sulawesi), borneo (sabah, sarawak), malaysia, singapore, philippines, sri lanka (burgeff 1943; bischler-caussecausse 1989; piippo 2002; chuah-petiot 2011). mt. sibayak, etti siregar 02, 03, 33, 102, 587, 589, 592, 594, 914, 1426, 1428; etti siregar & nunik s ariyanti 1517, 1519, 1526, 1753. 2. reinw., blume et nees. (fig.1c-d). this species shows wide variations in thallus and female receptacles. thallus can be without or with median band on dorsal side, small to medium size, with margins purplish, reddish, sometimes hyaline. female receptacles are often curved toward stalk, sometimes straight; lobes varies from (5-13); dorsal surface flat or with indistinct to distinct rounded median projection, slightly asymmetric to symmetric. apex of receptacle lobes varies from emarginate, truncate or sometimes rounded. in bischlercausse (1989), all of have a rounded median projection on dorsal side of female receptacles, but we found some specimens with indistinct to distinct median pojection on dorsal surface of female receptacles. : soil, rocks (moist, damp or wet, shaded, semi-exposed places, riversides, creeks) from 870 to 1450 m altitude. japan, korea, china, india, sri lanka, andaman and nicobar island, thailand, malaysia, indonesia (sumatra, java, lesser sunda island, bali, moluccas, irian jaya), borneo (sabah, sarawak), philippines, marianas, guam, new guinea, new britain, solomon island (bischler-causse 1989; bischler-causse & piippo 1991; song 2006; lai 2008; chuah-petiot 2011; singh & singh 2012). mt. sibayak, etti siregar 31, 47, 70, 112, 134, 219, 367, 374, 375, 380, 460, 462, 463, 465, 467, 468, 473, 475, 476, 593, 599, 608, 757, 760, 863, 868, 1427; etti siregar & nunik s ariyanti 1445, 1447, 1520, 1524, 1794, 1826. sibolangitnorth sumatra, lorzing 12707 (bo). 3. reinw., blume et nees (fig.1e). this species is easily recognized by the appendages of median scales yellow with orange-red borders; ovate and acuminate apically. : soil, rocks (shady or open places) from 870-1975 m altitude. india, andaman island, indonesia (sumatra, java, kalimantan), malaysia, philippines (tan & engel 1986; bischler-causse 1989; chuah-petiot 2011; singh & singh 2012). mt. sibayak, etti siregar 426, 427, 517, 745, 762, 821, 912, 915; etti siregar & nunik s ariyanti 1522, 1603, 1607, 1610. brastagi-north sumatra, meijer 15609 (bo). marchantia acaulis m. geminata marchantia acaulis et al. marchantia emarginata m. emarginata et al. marchantia geminata 76 biotropia vol. 20 no. 2, 2013 77 figure 1. a-b. with female and male plants, b. shows monoicous plant; cd. . with female and male plants; e. with female plants; f. (gc: gemmae cup); g. with female plants; h. with female and male plants. (photographed by: etti s siregar). marchantia acaulis m emarginata m. geminata m. paleacea m. polymorpha m. treubii the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. 78 biotropia vol. 20 no. 2, 2013 4. bertol. (fig. 1f). is easily recognized vegetatively by the large thallus without median band on dorsal surface and cupules with ciliate lobes. soil (wet place, waterfall); rocks of creek wall in semi shaded place, from 1150 to 1250 m altitude. turkey, lebanon, iran, yemen, russia, ussr, afghanistan, pakistan, india, bhutan, china, taiwan, korea, japan, thailand, vietnam, indonesia (sumatra, java, irian jaya), borneo, philippines, new guinea (lai . 2008), (bischler-caussecausse 1989; piippo 1990; bischler-causse & piippo 1991; konstantinova . 2009; daniels 2010; dandotiya . 2011; wang . 2011; singh & singh 2012). mt. sibayak, etti siregar 185, 763; etti siregar & nunik s ariyanti 1611. 5. l. (fig. 1g). is easily recognized by its large thallus with scales extending over the entire ventral surface, reaching the thallus margin and ± visible at the margin in dorsal view. its female receptacles are also distinctive with terete rays. rocks of creek wall in exposed places, 1500 m altitude. : turkey, syria, lebanon, israel, iraq, iran, russia, ussr, uzbekistan, tadzhikistan, afghanistan, pakistan, india, sri lanka, nepal, bhutan, china, taiwan, korea, japan, thailand, vietnam, malaysia, indonesia (java, sumatra new record based on present study, irian jaya), philippines, new guinea, new zealand, tasmania (bischler-causse 1989; söderström . 2010; singh & singh 2012). mt. sibayak, etti siregar 373, 377, 378, 466, 470, 759; etti siregar & nunik s ariyanti 1808. 6. schiffn. (fig. 1h). is similar to , but the latter species has a longer apical split in the lobes of female receptacle, receptacle usually constantly 4-lobed (in receptacles vary from 3-5 lobed), appendage of median scales are ovate and acuminate apically, epidermis of thallus are without papilla. soil or rock in open place and semi shaded place, altitude from 870-1610 m. indonesia (sumatra, java, lesser sunda island), borneo, malaysia (bischler-causse 1989; chuah-petiot 2011). mt. sibayak, etti siregar 28, 588, 743, 746, 608, 744. brastaginorth sumatra, breedveld gjf 5 (bo). 7. sp. (fig.2). thallus with dark median band on dorsal surface. epidermal pores 50-112 µm in diameter. appendages orange or purplish with dark colour in borders or apically (rarely entire orange or purplish); apex vary from rounded, obtuse to acuminate (figure 2. c-e). cupules with cilia 1-9 cells long and 2-4 cells basally, arranged in uniseriate, biseriate or sometimes triseriate cell. female receptacle alway dissected into 5 shallowly lobes; lobes apex emarginate; involucres purplish, margin entire. this species is closely related to . however, in , thallus without median band on dorsal surface, the cilia of cupules are shorter (3-4 cells long) and the basal cell of cupules are smaller (1-2 cells); apex of appendage of median scale marchantia paleacea m. paleaceae et al et al et al et al marchantia polymorpha marchantia polymorpha et al marchantia treubii marchantia treubii m. geminata m. treubii marchantia m. rubribarba m. rubribarba note: habitat: distribution: specimens examined: note: habitat: distribution specimens examined: note: habitat: distribution: specimens examined: note: 79 figure 2. m . archantia sp. : a. female plants; bd. appendage of median scales; e. margin of thallus; f. cupule; gh. involucre; i. scale of female receptacle; all from etti siregar 1972 (bo). scale bar = 200 µm. (photographed by: etti s siregar) obtuses, rounded or bluntly acute. the female receptacle dissected into 5-9 lobed; involucre hyaline. we have not seen any specimen of . so we could not decide wether it belongs to only from description and drawings. more data are needed to decide wether it belongs to or as a new species. sp. is also different from the species of from sumatra has been reported by bischler-causse (1989). the latter species has larger thallus with no distinct median band on dorsal surface; the cilia of cupules are shorter and arranged in uniseriate cell; appendage of median scales acute or apiculate apically with row of 13 cells (in sp. with single apical cell); lobes of female receptacle truncate or rounded apically; involucrum ciliate with row of 3-9 cells long (bischler-causse 1989). found on soil in wet place, only around the dwi warna waterfall at 1100 m altitude. mt. sibayak, etti siregar 1972; etti siregar & nunik s. ariyanti 1612, 1613, 1616. m. rubribarba m. rubribarba m. rubribarba marchantia m. miqueliana, marchantia marchantia habitat: specimen examined: the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. 80 biotropia vol. 20 no. 2, 2013 conclusions acknowledgments references there are seven species of found in mount sibayak, north sumatra, , , , and sp. is reported as a new species record from sumatra. this research is part of the dissertation of the first author, funded by bpps project of the indonesian ministry of education (dikti) and biotrop dipa project 2012 (research fellowship for phd student). we would like also to thank the reviewers of this manuscript. marchantia marchantia acaulis m. emarginata m. geminata m. paleacea, m. polymorpha, m. treubii marchantia marchantia polymorpha bischler-causse h. 1989. l. the asiatic and oceanic taxa. 38: 1-317. bischler-causse h, piippo s. 1991. bryophyte flora of huon peninsula, papua new guinea. l. (marchantiaceae, hepaticae). 28: 277 301. bischler-causse h. 1998. systematics and evolution of the genera of the marchantiales. 51: 1201. burgeff h.1943. genetische studien an . jena verlag von gustav fischer. chuah-petiot ms. 2011. a checklist of hepaticae and anthocerotae of malaysia. 56(1): 1 44. dandotiya d, govindapyari h, suman s, uniyal pl. 2011. checklist of the bryophytes of india. 88: 1 126. daniels aed. 2010. checklist of the bryophytes of tamil nadu, india. 65: 1 117. gradstein sr. 2011. guide to the liverworts and hornworts of java. bogor: seameo-biotrop. herzog t. 1943. lebermoose aus sumatra. 53: 358-373. konstantinova na, bakalin va, andrejeva en, bezgodov ag, borovichev ea, dulin mv, mamontov yu s. 2009. checklist of liverworts (marchantiophyta) of russia. 18: 1 64. lai mj, zhu rl, chantanaorrapint s. 2008. liverworts and hornworts of thailand: an updated checklist and bryofloristic accounts. 45:321-341. piippo s. 1990. annotated catalogue of chinese hepaticae and anthocerotae. 68:1-192 piippo s, he xl, juslén a, tan bc, murphy dh, pócs t. 2002. hepatic and hornwort flora of singapore. . 26: 101-127. singh d, singh dk. 2012. an appraisal of the genus in india with a note on subspecies in indian himalayan region. 83(1): 15 26. söderström l, gradstein sr, hagborg a. 2010. checklist of the hornworts and liverworts of java. 9: 53-149. song js, yamada k. 2006. hepatic flora from jeju (cheju) island, korea 100:443 450. tan bc, engel jj 1986 an annotated checklist of philippine hepaticae. 1986, 60:283-355. wang j, lai mj, zhu rl.2011. liverworts and hornworts of taiwan: an updated checklist and floristic accounts. 48:369 395. marchantia bryophyt biblioth marchantia ann bot fenn bryophyt biblioth marchantia pol bot j arch bryol arch bryol ann naturhist mus wien arctoa ann bot fenn j hattori bot lab ann bot fenn marchantia marchantia emarginata emarginata proc natl acad sci., india sect b biol sci phytotaxa j hattori bot lab j hattori bot lab ann bot fenn . 1. nur ain izza etal page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 4. rini (dengue).cdr biotropia vol. 19 no. 1, 2012: 30 41 dengue early warning model using development stages of mosquito and climate information aedes aegypti rini hidayati , rizaldi boer , yonny koesmaryono , upik kesumawati , and sjafrida manuwoto received 10 january 2012/accepted 15 may 2012 this study aims at developing a dengue early warning model using climate information. the model was developed through three steps of analyses. the first step was to determine the length of periods used in prediction and optimal time for eradicating mosquito's breeding sites. the second step was to identify the best prediction model of dengue incidence rate (ir). the third step was to develop an early warning model using stochastic spreadsheet. it was found that the best predictor for predicting dengue incidence rate at week-n (ir ) was (1) rainfall index with two weeks lead time (ich ). the rainfall index of week-n is a function of three week moving averages rainfall (ch3), . (ch3 -1.155*ch3 +0.702*chn-2), and (2) ir with one week lead time (ir ). the ir model prediction was ir = 0.795*ir +0.067*ich with r =76.6%. these models (models resulted from the first and third steps) can be used to provide early warning on optimum time for controlling the mosquito's breeding sites and the need for fogging action in order to prevent the dengue incidence rate beyond the critical limit as defined by the ministry of health. , early warning model, breeding site, time control, climate variability, climate change 1* 2 1 3 4 th 2 1. 2. 3. 4. departement of geophysics and meteorology, bogor agricultural university (ipb), darmaga, bogor, 16680, indonesia centre for climate risk and opportunity management in southeast asia and pacific (ccrom-seap), bogor, 16143, indonesia veterinary health entomology laboratory, veterinary faculty, ipb, bogor, 16680, indonesia entomology laboratory, dept. of plant protection, ipb, bogor, 16680, indonesia. aedes aegypti i.e aedes aegypti abstract introduction n n-2 n n-1 n-1 n n-1 n-2 key words: since mid 20 century (christopher 1960), until the beginning of the 21 century, the disease of dengue haemorragic fever (dhf) has been given attention worldwide. data of 2007 indicated that the number of cases and death rate of the th st * corresponding author : rinihidayatigfm@gmail.com 30 patients, as well as the number of cities infected by this disease keeps on increasing. according to ipcc (2007), the attack will increase due to climate changes at high confidence level. the use of early warning system and epidemic prediction is one potential of adaptation options to reduce the impact of weather and climate in the field of health (mc michael . in kovats . 2000). the life of vector and the transmission process of dhf disease are closely related with climate conditions before disease incidence; thus, studies related to climate have been increasing. the current study, however, has been directed to the relation between this disease and climate information related to climate change issues (hales . 2002; reiter 2001) and climate variability in order to anticipate both seasonal and annual incidence of such a disease (focks . 1995; schreiber 2001; peterson . 2005; sasmito 2006; sintorini 2006). the models have been developed consisting of a mechanistic models (fock . 1993, focks . 1995, reiter 2001, and sintorini 2006), as well as empirical models (hales . 2002, schreiber 2001; peterson . 2005; sasmito . 2006). however, there has not been any prediction model on the number of dhf incidence used by the department of health in indonesia. this is due to the consideration that the model is inaccurate, complicated in application, relatively costly in obtaining input parameter, or related to a specific location only. model to be developed is a simple model that will be easy to apply. the model will be a combination of mechanistic and empirical models. input variables are easily obtained. the prediction model of dhf based on climate information is expected to be used as materials for an early warning system that is beneficial to develop strategies for anticipation and control for such disease; particularly the one in indonesia. a number of reasons for this are, among others: (1) there is a climate observation station, particularly for rainfall, in every city/regency (2) climate observation has been carried out routinely and institutionally, and (3) climate determines the breeding places, activities of the vector ( ) and pathogen ( virus) so that the incidence of dhf needs climate pre-condition. in general, this study was aimed at developing dengue early warning model as the basis for determining the anticipation and control step upon dengue incidence which is related to climate occurrence. this research was carried out through three stages. the first stage was determining the length of mosquito's life cycle period. the second was developing a prediction model on dengue incidence rate by using climate variables. while, the third stage was an early warning model by using stochastic spreadsheet. et al et al et al et al et al et al. et al et al et al et al et al aedes aegypti dengue materials and method 31 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. determining the length of development stages period for mosquito and dengue virus developing a prediction model for dengue incidence rate (ir) developing early warning model using stochastic spreadsheet psn ir aedes aegypti the prediction on the length of periods for mosquito's development stages at one particular location was determined based on heat unit. the period length of one stage in mosquito's development (n days) was counted from heat unit value (hu c days), divided by the difference between the daily mean air temperature (t c) in the study area and basic temperature t by using the following formula: such length of period was used as: (1) basis for determining the period for controlling mosquito's breeding sites (psn) which consisted of the eradication of immature mosquito breeding sites (tpn, days) and mature mosquito's resting places (snd, days), and (2) basis for determinating of predictor's length period based on climate variables to predict ir. incidence rate of dengue (ir) data availability was weekly. therefore, the analysis of climate variables to predict ir were also used in weekly period. predictors of climate variables were grouped into wetness and thermal data. the length of wetness period was determined from immature stage (pd) mosquito phase length in weekly (n /7), whereas thermal length period was determined by the length of virus extrinsic incubation period (eip), also in weekly period (n /7). weekly wetness data (in cm/week) was obtained from the sum of daily rainfall data. weekly thermal factors that consist of mean, maximum and minimum temperature (tr, tn and tx, in c), were obtained by averaging daily data. further analysis was conducted to calculate the data in the form of moving averages. determining the period for moving averages analysis were based on pd for wetness data and eip for thermal data. further analysis was also conducted to construct predictive models in the form of principal component regression of the best predictors obtained from the previous analysis. the data used were the ones from indramayu regency (2002 2006). meanwhile, the data used in validation process were taken from indramayu (2007), bogor (2003 2006), north jakarta (1998 2002), and padang (2003 -2005). the early warning model discussed here was the prediction of the optimum time model for implementation, dengue incidence rate ( ), and the need for fogging. model was developed in the form of stochastic spreadsheet by using crystall ball program package. the prediction of the optimum time for psn implementation refered to optimum time for the implementation of cleaning water tanks where immature mosquito (tpn) lives, and optimum time for the implementation of cleaning adult mosquito's breeding sites (snd). each of tpn and snd was predicted based on the number of days in the period of pd mosquito and eip virus using the daily mean air temperature as an input. in the case of pd and eip prediction, the average daily temperature in a week was 0 0 o a b pd eip n = hu/(t -t ).a b 32 biotropia vol. 19 no. 1, 2012 considered to represent the average daily temperature during the period of pd or eip, so the prediction for the next few days could be done. prediction of dengue incidence rate was conducted by using the best prediction model. the need for fogging was determined based on the critical boundary of ir figure determined by the minister of health namely, 20 patients per 100 000 inhabitants per year for an endemic regency. focks . (1993) have shown that the higher the air temperature, the faster the gonotropic cycle and virus extrinsic incubation. in accordance to heat unit concept, hidayati . (2007) revealed that the higher the air temperature, the faster the egg hatches and grows into adult mosquito, or, the faster the mosquito complete all of their life cycle stages, including their age. furthermore, the heat unit of each development phase was relatively constant, thus, the higher the air temperature, the faster the heat unit that needs to be reached. pd and eip were determined based on the following formulation: results and discussions the length of development stages period for mosquito and dengue virus aedes aegypti et al et al � �ba tt hu n � � hu nd tb were heat unit and basic temperature which are suitable for immature stage and extrinsic incubation period. t was the daily mean air temperature in the study region. basic temperature and heat unit value in mosquito's various development phases and their relation to dengue virus transmission are presented in table 1 a . a table 1. basic temperature and heat unit at various stages of mosquito's life phase tb (0c) heat unit (hu in c0 days) average cv(%) immature 15. 0 224 27.4 eip virus 17. 0 128 5.0 gonothropic 17. 5 36 6.4 age 10. 0 544 24.6 cv : variation coefficient (source: hidayati 2007).et al. d . hf endemic regions are generally located at low altitude or city with dense population and relatively high temperature. data on temperature in 4 research locations; namely, indramayu, north jakarta, bogor and padang were considered to represent data on air temperature of several big cities /regencies with endemic areas of dhf in indonesia. in the weekly period, the average length of pd and eip in four locations were nearly the same (table 2); that is, 3 weeks for the phase of mosquito immature stage (pd) and 2 weeks for virus extrinsic incubation period (eip) 33 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. table 2. t s he average length of the phase of mosquito immature stage and virus extrinsic incubation period in four research location t . t . a he most influential climate variable in pd period of mosquito was the existence of wetness (rain), while the dominant influence in eip was temperature. accordingly, to develop a relation model between dengue incidence rate and climate variables, there is a need to have 3 weeks moving average analysis, like the length of pd mosquito period for wetness variable, and 2 weeks moving average analysis, like the length of eip for thermal variable he interview with the head of disease eradication and prevention department, health office of indramayu regency (august 2006) revealed that the shortest period for about 2 weeks was required to be able to utilize the result of early warning in determining operational steps for controlling the diseases. therefore, in selecting the best climate predictor variable, an interval of two weeks between climate occurence and diseases incidence was used results of the analysis of all available climate variables, showd that the best subset of predictive models predictors in dhf incidence rate of two weeks after the climate incidence were rainfall (ch) and temperature (t) in the form of , , , , , and . number 2 or 3 attached to variable ch (rainfall) and temperature including tr (average temperature), tx (maximum temperature), and tn (minimum temperature) were weekly periods for moving averages analysis. index indicates the time of occurrence during week before the ir occurrence that has been predicted. actually, the rainfall and air temperature data were the easiest climate variable to be obtained. ll climate information consisting of those rainfall and temperature were calculated in terms of the relation between climate variable with dengue incidence rate of dhf by using the principle component regression analysis. by using this method, multikolinieriti among the climate data could be ignored. the result of the principle component regression analysis was: (equation 1) (r adjusted = 52.1%; dws = 0.634). p s only p )rediction model for dengue incidence rate (ir rediction based on climate variable i ch3 ch3 ch3 tr2 tx2 tn2 ir = 0.920 + 0.157*ch3 0.052*ch3 + 0.066*ch3 + 0.826*tr2 0.387*tx2 0.492* tn2 n-2 n-4 n-5 n-2 n-2 n-2 n n-2 n-4 n-5 n-2 n-2 n-2 n-i 2 district/city mean air temperature (oc) immature stage(pd) extrinsic incubation period (eip) days weeks days weeks indramayu 27.3 21 3 12 2 jakarta utara 28.0 20 3 12 2 bogor 27.0 21 3 13 2 padang 27.1 21 3 13 2 34 biotropia vol. 19 no. 1, 2012 these results indicate that rainfall from 7 until 2 weeks before the week of disease incidence and air temperature at 3 and 2 weeks before the period of the disease incidents would determine the amount of ir. ir will be high if the rainfall in week 7, 3, and 2 is high, while in contrary low at week 6, 5 and 4. redictors comprised the combination of climate variables of two weeks before the dengue occurrance period, and ir one week before the disease incidence. by including ir aside from the climate variables consisting of rainfall, average temperature, maximum temperature, and minimum temperature, the equation of relation model obtained was reasonably good with the following form: r the next analysis result obtained by ignoring air temperature data did not reduce the model quality. if only rainfall was used as predictor, the equation produced would also be good. the form of its equation model is: , where (equation 3) if the model is developed based on predictor that consists of the combination of climate and ir at two weeks before the incidence, temperature has to be calculated together with rainfall in order to obtain a good equation model: (r : ; . (equation 4) (r adjusted = 78.0% ; dws = 2.202 ; ~ n(0; 0.33; p:0.064) et al the equation model obtained was not good enough with a determination coefficient only 52%, the dws value stayed far below 2.0, that means that residual model still contained an obvious autocorrelation value at level 1 so that the model was not adequately stable when used for prediction (selvanathan . 2004). the obstacle on the low accuracy of prediction model which only includes climate variables was also found by not only schreiber (2001) for the weekly-scale model, but also sasmito (2006) and sintorini (2006) for the monthly-scale model. a better prediction model can be obtained by including non climate factor; that is, ir before prediction period. ir was chosen as a predictor variable by considering that ir was relatively easy to be obtained by the health office in regency/city, and able to describe the level of inhibitant's susceptibility. p . p d rediction based on the combination between climate variables and ir before predictionperio ir = 0.795*ir + 0.067*ich3 ir = 0.612*ir + 0.111*ich3 + 0.315*it2 ir = 0.744*ir + 0.070*ch3 0.073*ch3 + 0.042*ch3 + 0.199*tr2 0.115*tx2 0.079*tn2 . ( ich3 = ch3 1.155*ch3 + 0.702*ch3 ich3 = ch3 0.715*ch3 + 0.244*ch3 , and it2 = tr2 0.577*tx2 0.397*tn2 n n-1 n-2 n-4 n-5 n-2 n-2 n-2 n-2 n-2 n-4 n-5 n-2 n-2 n-4 n-5 n-2 n-2 n-2 n-2 2 2 2 adjusted = 78.4%; dws = 2.151; ε ~ n(0; 0.33; p:0.094). corrected = 70.3%; dws : 1.182; ε ~ n(0; 0.38; p:0.040). the form of the equation model is ε n n-1 n-2 n n-2 n-2 n-2 wnwnwnwnwnwnwn-wnch3n ch3n ch3n t2 nirn-1 ir note: w: week, ch: rainfall period, t:temperature period, ir: incidence rate period figure 1. time frame period of ir prediction, climate variables and ir as predictors 35 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. t . t . he rainfall in indonesia is, in fact, the most fluctuative climate variable which influences other climate variables including temperature, relative humidity, and incoming radiation. in developing ir model with a climate variable, the most obvious predictor was the rain. hidayati . (2008) found that the pattern of monthly dengue proneness index in major parts of indonesia was the same as the pattern of their monthly rainfall, except in some big cities where their maximum index experienced an interval two or three months after the maximum monthly rainfall. the temperature, indeed has an obvious impact, however, the data on temperature was relatively difficult to obtain. based on the equations of prediction models on dengue incidence rate, equation 3 was the most potential to be used as a model for early warning since the information on rain together with ir could be used to predict the incidence of diseases for the next period he result of model validation showed that the model from equation 3 resulted in good ir prediction, not only in indramayu, but also in bogor, north jakarta and padang. rmse relative value was in average less than 1, and the correlation value between prediction result and actual data was significantly high (table 3). furthermore, actual ir plot and ir resulted from the prediction did not show significant difference. however, this model has been underestimated in predicting the high ir et al n-1 table 3. result of prediction model validation based on equation 3 in cities where the altitude was higher (> 500 m asl), or where mean air temperature was significantly lower (< 24 c), the length of period for moving average calculation of climate variable needs to be adjusted to the heat unit concept. furthermore, wetness variable also has to be adjusted to the length of pdn period, and thermal variable has to be tailored to the length of eip. as an example, a place with an air temperature average of 21 c, or at the height of about 1000 m asl, the moving average for wetness variable was approximately 5 weeks, and its thermal period was 4 weeks. thus, the rainfall variables that can be used in developing predictive models of ir are five weekly rainfalls ( ). if we include the temperature in the model, then the form of temperature variables are 4 weekly temperature or (modification of fig. 1). however, the prediction model equations cannot be presented here in detail, because it is not sufficient supporting data are available. in general, a city located at an elevation > 500 m asl is not an endemic area. 0 0 ch5 t4 n n regency/ city year rmse rmse/average correlation coefficient indramayu 2007 1.272 0.601 0.75 bogor 03 – 06 0.930 0.496 0.83 north jakarta 98-02 1.693 0.603 0.90 padang 03 – 05 0.724 0.601 0.75 36 biotropia vol. 19 no. 1, 2012 d teveloping early warning model using stochastic spreadshee early warning dengue model . i stochastic spreadsheet model was developed using weekly time frame, consisting of input cell, assumption cell, formula cell, and prediction cell. model inputs comprised the number of dengue cases one week before prediction period, the number of population, average weekly air temperature (tr ) one week before prediction period (mm). the assumptions used in ir model were error model which was distributed normally with mean value of 0.00 and standard deviation of 0.33. the heat unit of immature mosquito stage was distributed normally with an average of 224 and with a standard deviation of 61. likewise, the eip heat unit was normally distributed with an average of 128.4 and standard deviation of 6.6. this assumption is built from the analysis of the mosquito life cycle heat units (table 1) and the distribution of the ir. thus, this assumption can be applied to most of the dengue endemic areas. the formula cell was used to define ir at the present week, rainfall index, ir the week after, and the averages of pd mosquito and eip virus periods n addition, input, assumption and formula previously defined were then used to conduct a simulation in the dengue early warning model. simulations which were carried out for several times would produce optimum prediction period for tpn and snd, ir value for the following week, and decision whether or not fogging at various n-1 confidence levels should be conducted. each of the optimum period for psn implementation was determined based on the length of both pd mosquito and eip with confidence level (exceeded probability) of 90% or 75% in accordance with level of interest. such value was obtained by filling out the certainty column with the display of simulation result. fogging was suggested if the ir in this week was > 0.5 and predicted to raise in the following week; or if ir prediction for the following week was > 1 table 4. an example of the number of input for early warning dengue model . input parameter value average daily air temperature in the last one week 27.5 number of patients in the last one week 15 number of population 1.500.000 weekly rainfall (mm) in … week before (wb) 7 wb 6 wb 5 wb 4 wb 3 wb 2 wb 0 mm 20 mm 75 mm 100 mm 80 mm 150 mm a . s an illustration, the number of input as shown in table 4, produced prediction values as presented in figure 2. with confidence level of 90%, optimum period of tpn and snd cleaning were conducted in 7 days and 4 days after simulation time, or 14 and 11 days after previous cleaning, and ir prediction value in the following week would exceed the number of patients i.e. 1.6 persons per 100 000 population. consequentrly, fogging is highly recommended 37 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. figure 2. output of dengue early warning model from 1000 times simulations anticipative steps and control of dhf disease incidence i . b . t . n order to control dhf disease, the decree of minister of health (kepmenkes) no: 581 year 1992 suggested to prioritize activities on prevention and society empowerment by performing what is called: psn 3 m plus (eradicating mosquito's breeding site drying, covering, burying, plus adding larvaside, raising fish, using mosquito net, and spraying). in wet tropical regions where the vegetation and fauna are varied, it was relatively difficult to eradicate mosquito. in order to suppress the number of dhf patients, therefore, psn has to be carried out regularly and entirely. so that, mosquito's life cycle can be totally cut. as an effort to control mosquito, the optimum time for psn implementation as resulted from the simulation can be used as guidance ased on the decree of the minister of health (menkes) 581/92 fogging was carried out when there was an outbreak, that is, when the number of cases doubled from the previous period, or when a case occurred in the region where previously none of such case was found. in order to suppress the number of patients not exceeding the critical limit as determined by the ministry of health, fogging is suggested to be carried out when the ir value early in the week is > 0.5. based on rainfall data, it is predicted that ir will increase; and/or ir prediction in the following week is > 1 he model operationalization should be conducted every week, particularly during rainy season, so that the ir prediction value and information whether or not fogging was performed will be available in each of the previous week. dhf disease season generally occurs from the beginning to the end of rainy season. unlike the ir prediction and fogging, psn simulation has to be performed one week after psn was carried out aedes aedes 38 biotropia vol. 19 no. 1, 2012 t . i . he step for anticipation and control of the disease could make use of the result of ir value simulation one week after. such a superficial model can be utilized by personnels who work in hospitals and city/regency health offices as the basic for calculating both structure and infrastructure that need to be provided for nursing and treating the patients in the following week n the beginning of rainy season, where the number of dhf patients starts to rise, it is suggested to carry out psn at time period in accordance with exceeded probability of 90%. in the season where the ir is relatively low, psn period at probability level of 75% can be chosen (fig. 3) 2928272625 14 12 10 8 6 4 2 temperature (cdeg) d a y a ft e r a w e e k t e m p m e a su re m e n t tpn75 tpn90 s nd75 s nd90 va ria b le scatterplot of tpn75; tpn90; snd75; snd90 vs t ( c)o figure .3 period (days) of controlling immature mosquito breeding sites (tpn) and mature mosquito breeding sites (snd) recommendation for the dry season (75) and rainy season (90) t . he result of ir value prediction simulation is needed as the base of calculation upon structure and infrastructure for controlling dhf diseases. since treating dhf patients has to be immediately carried out, both supporting structure and infrastructure should be available in an adequate number, or even, higher number. to accommodate such needs, ir prediction is suggested to be below the exceeding probability levels; namely 10%, or 25% at the highest the implementation of fogging is highly suggested if the probability level to gain value of 1 in the simulation is higher . each of the value, either 1 or 0 described the need to carry out fogging. from such description, it can be identified that the higher the ich, the higher confidence value upon the suggestion to carry out fogging. conversely, fogging will not be suggested if, naturally, climate supports the decrease of ir cases. accordingly, although ir prediction is high , but naturally there will be a decrease untill less than 1, fogging is not really suggested. the fogging of superficial model result (fig. 4) has not been able to describe the characteristics of fogging: focused (on the selected/limited area), or mass (on an extensive area). fogging can be carried out by the community or funds provided by 39 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. figure 4. level of fogging requirement the local government. such a built model can only predict the incidence of one week ago (for the next week). as a result, fogging activities are suggested to be prioritized by the community independently under supervision or as a program of city/regency's local health office. conclusions early warning models can be developed based on (1) the weekly ir model prediction and the optimum time for controlling mosquito's breeding sites (psn implementation) models using heat unit concept, (2) for controlling immature mosquito's breeding site, and (3) for controlling moquito's resting places. the rainfall index of week-n , is a function of three weeks moving averages rainfall (ch3), . (ch3 -1.155*ch3 + 0.702*chn-2), and ir is ir one week lead time. model was developed in the form of the stochastic spreadsheet by using crystall ball program package, so that the confidence level of the model outputs can be demonstrated. rom the beginning of rainy season to the beginning of dry season, optimum period for psn implementation of model superficial result is used with 90% exceeded probability, but from the middle to the end of dry season, simulation result with 75% exceeded probability can be used. the provision of both structure and infrastructure is recommended to be in line with ir prediction value at high estimated value with 10% exceeded probability, minimal at low estimated value in accordance with ir prediction value with 25% exceeded probability. fogging activity is suggested if the simulation result of exceeded probability is more than or the same as 90%. a routine simulation model in the endemic region in the low altitude should be conducted every week, particularly in endemic regions. in mid and high altitude, models need to be evaluated ir = 0.795*ir +0.067*ich , n (days) = 256 cdays/((ta-15) c) n (days) = 128 cdays/(ta-17) c ich n n-1 n-2 n i.e. i.e 0 0 0 0 th n n-1 n-1 f . 40 contour plot of fogging vs irn-1, ich3n-2 biotropia vol. 19 no. 1, 2012 references christophers sr. 1960. 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of vulnerable region of dengue fever disease based on climate and non-climate condition). j agromet xxii (1) : 61-9 ipcc 2007: climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the 4 assessment report of the intergovernmental panel on climate change, m.l. parry, o.f. canziani, j.p. palutikof, p.j. van der linden and c.e. hanson, eds., cambridge university press, cambridge, uk, 976pp. kovats, r.s., menne, b., mcmichael, a.j., corvalan, c., bertollini, r. 2000. climate change and human health: impact and adaptation. pp:28 kusriastuti r. 2006. kebijaksanaan penanggulangan demam berdarah dengue di indonesia. makalah disampaikan pada pelatihan klinis, lampung, 19 april 2006 peterson at, martinez-campos c, nakazawa y, martinez-meyer e. 2005. time-specific ecological niche modeling predicts spatial dynamics of vector insects and human dengue cases. transactions of the royal society of tropical medicine and hygiene (2005) 99, 647 – 55. reiter p. 2001. climate change and mosquito-born disease. environ health perspect, 109(1): 141-61 sasmito a, tim bmg. 2006. protipe model kewaspadaan dini bahaya demam bedarah dengue (dbd) di wilayah dki jakarta. makalah disampaikan pada seminar hasil penelitian pengembangan meteorologi dan geofisika tahun 2005. badan meteorologi dan geofisika. jakarta schreiber kv. 2001. an investigation of relationship between climate and dengue using a water budgeting technique. int j biometeorol (2001) 45: 81-9 selvanathan a, selvanathan s, keller g, warrack b. 2004. australian business statistic-abridged 3rd edition. thomson, australia. sintoroni mm. 2006. model dinamika sistim penularan demam berdarah dengue dalam kaitan dengan pola variabilitas iklim di jakarta. disertasi ilmu kesehatan masyarakat, ui. jakarta (unpublished) wmo. 1981. guide to agricultural meteorology practices (wmo-no:134). secret of wmo. geneva. switzerland aedes aegypti aedes aegypti aedes aegypti h . th 41 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. doi: 10.11598/btb.2015.22.2.450 diversity of epiphytic orchids and host trees (phorophytes) in secondary forest of coban trisula, malang regency, east java, indonesia siti nurfadilah purwodadi botanic garden – indonesian institute of sciences, j surabaya-malang km. 65 purwodadi pasuruan east java 67163alan , , , indonesia received 19 december 2014/accepted 18 november 2015 abstract epiphytic orchids are an integral component of forest ecosystems that contribute to a high proportion of plant diversity. the aim of this study was to investigate the diversity of orchids and their host trees (phorophytes) in a secondary forest of coban trisula (the trisula waterfall) of bromo tengger semeru national park javain east province . e fifteen, indonesia. two line transects were established ach transect was 150 m long and composed of 10 x 10 m plots, resulting in the total number of 30 sampling plots at the study site. the following data were recorded in each plot: species name and individual numbers of epiphytic orchids, species name and individual numbers of the phorophytes and vertical distribution of the orchids on the phorophyte. 15 epiphytic orchid species there were found from 13 genera in the secondary forest of coban trisula. was the most abundant appendicula angustifolia epiphytic orchid species (relative abundance = 52 4%), followed by (29 9%). all recorded orchids . .trichotosia annulata grew on 21 individuals from nine phorophyte species. and castanopsis javanica (mean = 589.5 individuals/tree) engelhardia spicata (mean = . )425 67 orchid individuals/tree were phorophytes hosting the largest number of individual orchids, respectively. the greatest abundance of epiphytic orchids was on the basal and the middle part of phorophyte branches (zone 3 and zone 4). this study indicated that orchid conservation management is required in the coban trisula to protect the survival of orchids in this area from potential human disturbances, as coban trisula is one of tourist destination. keywords: coban trisula diversity, epiphytic orchid, phorophyte, secondary forest, introduction epiphytes are known as one of important components in forest ecosystems, contributing to a high proportion to floral diversity (wolf 2005). the role of epiphytes is vital as the habitat for canopy invertebrates and as nutrient sources in the forest canopies (nadkarni 2004; cardelus et al. & mack 2010). contribution of epiphytes is also important to the total biomass and nutrient pools in the forest ecosystems (nadkarni 2004). et al. the family orchidaceae is among the most dominant groups of vascular epiphytes (johansson 1974; gentry dodson 1987; & annaselvam parthasarathy 2001; kromer & et al. 2005; zotz schultz 2008)& . orchidaceae is one of the biggest families containing around 25 000-35 000 species , , (dressler 1981; 1993), representing 1/10 of the tot al vasc ular plan t sp ec ies. h owever, orchidaceae is also one of the threatened plant families (iucn/ssc orchid specialist group 1996; mondragon elliott 2013), due to & overexploitation, overcollection, deforestation and fire (koopowitz dixon 2003). orchids & are h ly eigh sensitiv to environmental changes (newman 2007) and high dependen on et al. ly t other organisms (mycorrhizal fungi and insects as pollinators) for their survival (swarts dixon & 2009). orchid conservation efforts need to be done by considering , and the biology ecology the nature of threats . towards the orchids * corresponding author : siti.nurfadilah@lipi.go.id; biotropia vol. 22 no. 2, 2015: 120 128 120 mailto:siti.nurfadilah@lipi.go.id; coban trisula is a waterfall in bromo tengger semeru national park (btsnp), which is administratively located in ngadas village, malang regency, east java . this province waterfall is a frequent tourist destination in east java, usually visited as part of regular tours to mount bromo and mount semeru. coban trisula can be reached a pathway a secondary through in forest lined by epiphytic orchids growing on trees (host trees or phorophytes) the orchids in , making this area vulnerable to human disturbances due to illegal orchid collection by visitors/tourists. some studies showed the negative impact of tourism and recreation on vascular plants, various especially on orchids family (orchidaceae) (pickering hill 2007; ballantyne pickering & & 2013; rankin 2015). rankin (2015) et al et al. . reported that more than 45 plant families have species listed as orchids were threatened, in which the most common species listed as at risk from threats. the most common threat plant is collection by visitors in protected areas. the aim of this study was to investigate the diversity of epiphytic orchid and the phorophytes along the s pathway to coban trisula to develop conservation management of orchids in that area. materials and methods study site this study was conducted along the pathway to coban trisula located in secondary forestthe , village ngadas, malang regency, east java province, indonesia ( s and 08 00'213"o 112 8 e) with elevation of , m above o 7'82" , 1 475 sea level (asl). the site under the management is of bromo tengger semeru national park (btsnp). the dominant trees in this forest are macropanax dispermus lithocar pus (pampung), sundaicus engelhardia spicata(pasang) and (danglu). data ollectionc records of diversity of epiphytic orchids and the phorophytes were in two line-conducted transects. each transect was 150 m long and composed of 15 plots (each plot 10 x 10 's size is m), resulting in the total number of 30 sampling plots at the study site (annaselvam & parthasarathy 2001; focho 2010). species et al. name and individual number of the epiphytic orchids and the phorophytes were recorded. further, the vertical distribution of epiphytic orchids on the phorophytes within the five zones determined by johansson (1974) was recorded (fig 1). . biotropia vol. 22 no. 2, 2015 vertical distribution of epiphytic orchids on the host tree in five zones: zone 1 : the bottom part (1/3) of the main stem zone 2: the upper part (2/3) of the main stem zone 3: the bottom part of the branches zone 4: the middle part of the branches zone 5: the outer part of the branches. (johansson 1974) figure 1 division of the phorophyte into five ones johansson (1974) z determined by 121 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – data nalysisa parameters z weremeasured and analy ed relative frequency of phorophyte (% ft), relative abundance of orchids (% fo), the average number of individuals of orchids of each phorophyte species (ji/jt), the average number of epip ytic h orchid species on a phorophyte species (js/jt), and the vertical distribution of the orchids on the phorophytes (yulia budiharta 2012a; 2012b).& a. relative requency of phorophyte (% ft)f nt % ft = x 100% total number of all phorophytes where: nt = the number of trees in the plot hosting a particular orchid species b a. relative bundance of orchid (% fo) no % fo = x 100% total number of all orchid species where: sno = the number of individual of a particular orchid species within the plot c . the average number of orchid individuals on a phorophyte species = ji jt where: orchid ji = the number of individuals sjt = the number of individual of each phorophyte species d orchid . the average number of species on a phorophyte = species js jt where: speciesjs = the number of orchid jt = the number of individual of each phorophyte species e. vertical distribution of epiphytic orchids on the phorophyte s specie were determined by mapping vertical distribution of each epiphytic orchid species on the phorophyte, from the trunk to outer branches in five zones (zone 1, zone 2, zone 3, zone 4 and zone 5) and by calculating the average number of individuals of epiphytic orchids in each zone. results and discussion the ccur ence of piphytic rchids on the o r e o phorophytes there were 15 epiphytic orchid species and 9 phorophyte species (table 1). the recorded results of the present study showed that the number of phorophyte species each hosting epiphytic orchid varied 1 5 phorophyte from to species (table 1). some epiphytic orchid species occu red on a single phorophyte species; such as r appendicula elegans (100 individuals) found were exclusively on phorophyte ; lithocar pus sundaicus bryobium hyacinthoides (10 individuals) were recorded only on phorophyte ; engelhardia spicata and (50 individuals) dendrobium luxurians were only observed on phorophyte castanopsis javanica (table 1). other epiphytic orchid species occurred on multiple phorophyte species; such as appendicula angustifolia gr on four phorophyte ew species ( , macr opanax dispermus lithocar pus sundaicus dr ypetes sumatrana castanopsis , and javanica parapteroceras odoratissimum). was hosted by 5 in the study site i.e. phorophyte species engelhardia spicata ficus syzygium , sp , sp ,. . lithocarpus sundaicus drypetes sumatrana (table and 1). the results of the present study were similar to other studies showed that the number of which phorophyte species epiphytic orchids hosting varied from a single to multiple phorophyte species. adhikari (2012) reported that the et al . orchid occur ed on one rdendrobium nobile phorophyte species, while the orchid rhynchostylis retusa was found on many different phorophyte species. rosa-manzano (2014) also reported et al . that most epiphytic orchids in tropical dry forests of yucatan, mexico occu red on a single tree r species. tremblay (1998) also showed a et al . puerto rican orchid, lepanthes caritensis hosted by one phorophyte species ther epiphytic , while o orchids were reported to occur in many p h o r o p hy t e s p e c i e s. a n n a s e l va m a n d parthasarathy (2001) reported that epiphytic orchids in tropical evergreen forest at varagalaiar, western ghats, india g w on many phorophyte re species. trapnell and hamrick (2006) also showed 33 hosting phorophyte species epiphytic orchid laelia rubescens at one site. 122 epiphytic rchidso the present study showed the secondary that forest of coban trisula contained 15 epiphytic orchid species lower , in which the diversity was compared to the diversity of epiphytic orchids in other areas within the bromo tengger semeru national park; such as in resort senduro that had 42 epiphytic orchid species (utama 2005). the lower diversity of epiphytic orchids in coban trisula compared to resort senduro may have been caused by the in difference ecosystem types in which coban trisula is secondary forest, while resort senduro is a primary forest. the difference of primary forest and secondary forest in terms of their plant diversity has been widely studied. primary forest ha higher plant diversity than s that of secondary forest, including epiphytic orchid diversity (barthlott 2001 kubota 2005). et al et al . .; studies in the venezuelan andes and japan comparing the diversity of vascular epiphytes in biotropia vol. 22 no. 2, 2015 table 1 the epiphytic orchid species and the phorophytes in the secondary forest of coban trisula no epiphytic orchids number of phorophyte species phorophyte species 1 appendicula angustifolia blume 4 macropanax dispermus (blume) kuntze lithocarpus sundaicus (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm castanopsis javanica (blume) a.dc. 2 appendicula elegans rchb.f 1 lithocarpus sundaicus (blume) rehder 3 bryobium hyacinthoides (blume) y.p.ng & p.j.cribb 1 engelhardia spicata var. colebrookeana (lindl. ex wall. ) koord. & valeton 4 bulbophyllum odoratissimum (sm.) lindl. ex wall. 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 5 ceratostylis brevibrachiata j.j. sm . 2 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton actinodaphne procera 6 cymbidium sp. 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 7 dendrobium luxurians j.j.sm. 1 castanopsis javanica (blume) a.dc. 8 dendrobium spathilingue j.j.sm. 2 lithocarpus sundaicus (blume) rehder castanopsis javanica (blume) a.dc. 9 dendrochilum abbreviatum blume 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 10 mycaranthes oblitterata blume 1 castanopsis javanica (blume) a.dc. 11 parapteroceras odoratissimum (j.j.sm.) j.j. wood 5 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton ficus sp. syzygium sp. lithocarpus sundaicus (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm 12 schoenorchis juncifolia reinw. ex blume 3 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton lithocarpus sundaicus (blume) rehder ficus grossularioides burm.f. 13 thrixspermum subulatum (blume) rchb.f 1 castanopsis javanica (blume) a.dc. 14 trichotosia annulata blume 4 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton lithocarpus sundaicus, (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm castanopsis javanica (blume) a.dc. 15 vanda tricolor lindl. 2 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton castanopsis javanica (blume) a.dc. 123 primary forests and secondary forests showed that primary forests had higher diversity of epiphytic orchids compared to secondary forests (barthlott et al et al . . ; 2001 kubota 2005). the present study also showed that the most abundant epiphytic orchid was appendicula angustifolia with r (% fo)elative abundance of 52 41%, followed by % . with trichotosia annulata fo 29 9% (table 2). the most abundant of . orchid in this study site, is a appendicula angustifolia sympodial orchid, that ha continuous s lateral growth of the the stems through rhizome, which is an effective vegetative reproduction to grow a large number of individuals. appendicula angustifolia also ha many flowers along the stems, s with each rachis bear 10–15 flowers (comber s 1990; 2001). the large number of a. angustifolia flowers and seedincrease the chance for the fruit set to produce large population. all these s characters of support the a. angustifolia high abundance of at the study appendicula angustifolia site. comber (1990) also reported a tree covered by high density in indonesia,of a. angustifolia. appendicula angustifoli a is i and widely distr buted found all over java and sumat ra, from 800e to 1 700 m asl (comber 1990), elevation . host ree (phorophyte) peciest s the results of the present study also showed that there were 9 phorophyte species in coban trisula, which the diversity is lower than that in resort senduro located within the same national park ( ) bromo tengger semeru national park . utama (2005) reported 16 phorophyte species in resort senduro. the lower diversity of phorophyte in coban trisula compared to resort senduro caused by different may have been ecosystem types (secondary forest in coban trisula and primary forest in senduro). resort other studies also showed similar results. t able 2 the epiphytic orchid species in coban trisula and the parameter values no epiphytic orchid species nt no % ft % fo 1 appendicula angustifolia blume 6 2 ,056 15 52 .4 2 appendicula elegans rchb.f 1 100 2 .5 2 .55 3 bryobium hyacinthoides (blume) y.p.ng & p.j.cribb 1 10 2 .5 0 .25 4 bulbophyllum odoratissimum (sm.) lindl. ex wall. 1 120 2 .5 3 .06 5 ceratostylis brevibrachiata j.j. sm. 2 50 5 1 .27 6 cymbidium sp. 1 2 2 .5 0 .05 7 dendrobium luxurians j.j.sm. 1 50 2 .5 1 .27 8 dendrobium spathilingue j.j.sm. 2 16 5 0 .41 9 dendrochilum abbreviatum blume 1 75 2 .5 1 .91 10 mycaranthes oblitterata blume 1 100 2 .5 2 .55 11 parapteroceras odoratissimum (j.j.sm.) j.j. wood 9 74 22 .5 1 .89 12 schoenorchis juncifolia reinw. ex blume 4 41 10 1 .05 13 thrixspermum subulatum (blume) rchb.f 1 50 2 .5 1 .27 14 trichotosia annulata blume 5 1 ,172 12 .5 29 .9 15 vanda tricolor lindl. 4 7 10 0 .18 notes: nt = the number of trees in the plot hosting a particular orchid species no = the number of individuals of a particular orchid species within the plot s% ft = relative frequency of phorophyte % fo = relative abundance of orchids 124 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – biotropia vol. 22 no. 2, 2015 barthlott (2001) reported that the diversity et al . of phorophytes in the secondary forest was lower than that of the primary forest in the venezuelan andes. the individual number of orchids growing on a phorophyte ranged from to 589.5 6.5 individuals/tree (ji/jt) (table 3). the largest individual numbers of epiphytic orchids were found on (ji/jt = 589.5 castanopsis javanica individuals of orchids/tree), followed by engelhardia spicata (ji/jt = 425.6 individuals of orchids/tree). and castanopsis javanica engelhardia spicata not only had a large individual number of epiphytic orchids, also had the highest species but richness of epiphytic orchids. eight orchid species ha been recorded growing on the phorophyte d engelhardia spicata and seven species on the p h or o p hy t e . o t h e r c a st a no p s i s j a v an i c a phorophytes such as and actinodaphne procera macropanax dispermus only hosted one orchid species. were all recorded phorophytes specialized on sub-montane and montane areas (hardyanti & hakim 2014). most of them such as castanopsis javanica, engelhardia spicata, macropanax dispermus and ha rough or fissured lithocarpus sundaicus d bark. this bark structure might support litter accumulation and therefore build a nutrient and , humidity reservoir providing a comfort habitat for epiphytic orchids (annaselvam pathasarathy & 2001). the thickness of organic substrates layer on the tree bark varied within the vertical distribution of a single tree species from bare bark (thin substrates < 1 cm) to 5 cm thick substrates. the variety of substrat thickness covering the e phorophyte was also observed by johansson (1974) in west african rainforests and barthlott et al. (2001) in the venezuelan andes. in our study the highest abundance of orchids occur ed on thick substrates. this is consistent r with the results of the study conducted by annaselvam and parthasarathy (2001) showing that most thick branches densely covered were with vascular epiphytes, accumulating substantial amounts of humus, nutrients and moisture. rosamanzano (2014) also reported that bark et al . roughness and substrate were the most area important phorophyte characteristics affecting the epiphytic orchids abundance. vertical istribution of piphytic rchids on d e o p specieshorophyte the present study showed a range of vertical distribution of epi hytic orchid species from p zone 2 to zone 5 (table 4). the most abundant orchid, was found to have appendicula angustifolia, the widest vertical distribution ranging from zone 2 to zone 5. the ability of to occupy a. angustifolia a large area and different zones supported population growth resulting in the abundance of table 3 the phorophyte species in coban trisula and the parameter values no phorophyte species jt js ji js/jt ji/jt 1 actinodaphne procera nees 1 15 2 castanopsis javanica (blume) a.dc. 3 drypetes sumatrana (miq.) pax & k.hoffm. 4 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 5 ficus grossularioides burm.f. 6 ficus sp. 7 lithocarpus sundaicus (blume) rehder 8 macropanax dispermus (blume) kuntze 9 syzygium sp. 1 4 2 3 2 1 5 2 1 7 3 8 1 1 6 1 1 2,358 13 1,277 30 10 175 35 10 1 1.75 1.5 2.67 0.5 1 1.2 0.5 1 15 589.5 6.5 425.67 15 10 35 17.5 10 notes: jt = the individual number of each phorophyte species js = number of orchid species orchidji = number of individuals js/jt = the average number of epiphytic orchid species on a phorophyte species epiphytic orchid phorophyte ji/jt = the average number of individuals on a species 125 the orchids. sp. and cymbidium dendrobium luxurians were observed to occur in zone 5 only (table 4). most epiphytic orchid species grew on zone 3 and zone 4. a small number of orchid species occu red on the trunk (only two orchid r species on zone 2 and no orchid species grew grew on zone 1. the pattern of vertical distribution of epiphytic orchids in the forest of coban trisula was similar to that of other regions africa, i.e. in south america and mexico where vascular epiphyte abundan were higher in zone 3-5 tree ce ( crown than in zone 1-2 along the trunk ) ( ) due to better light the crown intensity nearby tree (johansson 1974 kromer 2005 rosa-; ; et al . manzano 2014)et al. . implications for onservationc coban trisula is one of tourist destination within area of bromo tengger semeru the national park. the tourist number bromo to tengger semeru national park very high was (table 5). the number of visitors increased sharply from 2011 to 2014, with the peak of tourist number reached 551 644 visitors in 2013 , (table 5). table 4 zones of the occur ence of epiphytic orchids on their phorophytes and the number of rchid individuals in each r o zone no epiphytic orchid species zone 1 zone 2 zone 3 zone 4 zone 5 1 appendicula angustifolia blume 2 appendicula elegans rchb.f 3 bryobium hyacinthoides (blume) y.p.ng & p.j. cribb 4 bulbophyllum odoratissimum (sm.) lindl. ex. wall. 5 ceratostylis brevibrachiata j.j. sm. 6 cymbidium sp. 7 dendrobium luxurians j. j. sm. 8 dendrobium spathilingue j. j. sm. 9 dendrochilum abbreviatum blume 10 mycaranthes oblitterata blume 11 parapteroceras odoratissimum (j. j. sm.) j.j. wood 20 5 12 schoenorchis juncifolia reinw. ex blume 13 thrixspermum subulatum (blume) rchb.f 14 trichotosia annulata blume 15 vanda tricolor lindl. 796 10 20 45 11 33 34 25 152 3 800 100 10 100 5 5 30 33 35 6 50 1,020 77 total number of orchid individuals 0 25 1,129 2,271 200 2 50 33 10 2 297 table 5 the number of visitors bromo tengger semeru national park to visitors year 2011 2012 2013 2014 domestic foreign total visitors 103,091 22,380 125,471 249,577 26,297 275,874 518,746 32,898 551,644 512,887 23,451 536,338 source: balai besar taman nasional bromo tengger semeru 126 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – biotropia vol. 22 no. 2, 2015 management of orchid conservation is required to protect the orchids in coban trisula and other places within bromo tengger semeru national park rom population and loss f decrease of diversity. this is related to the impact of tourism and recreation the survival of activities on plants, especially orchids (pickering hill 2007; & rankin 2015). he most common type of et al. t threat in area isthe tourism and recreation plant collection by visitors which can decrease plant species diversity . (rankin 2015; calderon-et al aguilera 2012). a recommendation to et al . protect the epiphytic orchids in this area from human disturbances is required. conclusions fifteen epiphytic orchid species from 13 genera were found in the secondary forest of coban trisula, bromo tengger semeru national park. nine phorophyte species were found to be the host the epiphytic orchid species in coban of trisula. the most abundant orchid species was appendicula angustifolia and the phorophyte species hosting the largest number of orchids was castanopsis javanica. management of orchid conservation is required to protect the survival of orchids in coban trisula. acknowledgements the present study was funded by dipa. my sincere thanks to pak tarmudji and pak went tatang (purwodadi botanic garden) and pak sukiyono (bromo tengger semeru national park) for the assistance in the field. references adhikari yp, fischer hs, fischer a. 2012. host tree utilization by epiphytic orchids in different land-use 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in the highlands of chiapas, mexico. forest ecol manag 212: 376–93. yulia nd budiharta s. 2012a. epiphytic orchids and , phorophytes diversity at gunung manyutan forest reserve, wilis mountain,ponorogo, east java. biodiversitas 12(1): 22-7. yulia nd budiharta s. 2012b. the diversity of epiphytic , orchid and its phorophyte along cemoro sewu hiking pathway, lawu mountain, district of magetan, east java, indonesia. j nat stud 10(2): 26 31. zotz g, schultz s. 2008. the vascular epiphytes of a lowland forest in panama—species composition and spatial structure. plant ecol 195:131–41. 128 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – 3. loukrakpam (two) revisi.cdr biotropia vol. 21 no. 2, 2014: 91 98 two new fischer, 1894 (nematoda: tylenchina) from manipur, northeast india aphelenchoides loukrakpam bina chanu and naorem mohilal received 25 february 2014/accepted 1 october 2014 two new nematode species of discovered from different localities of manipur, north east india had been described in the present study. sp. nov. has a long stylet and four lateral lines which are gradually merged into two at the tail region. sp. nov. has a small body and short tail with a stylet length of 8.6 μm. sp. nov., sp. nov., manipur, northeast india * parasitology section, department of life sciences, manipur university canchipur 795003, manipur aphelenchoides aphelenchoides longistylus aphelenchoides neominoris aphelenchoides longistylus aphelenchoides neominoris abstract introduction keywords: aphelenchoides aphelenchoides aphelenchoides spp. are soil dwelling nematodes and often found in decaying plant materials. their characteristic features include slender stylet with narrow lumen and usually with small basal knobs or swellings with presence of post uterine sac. female tails are of medium length, conoid, with a tip pointed or rounded and often mucronate terminus. most important characteristics of males include paired spicule, separate, rose thornshaped or derived from them with absence of gubernaculums. a number of spp. have been reported from india and from throughout the world. but description of new species of the genus is increasing due to intensive survey from different parts of the world including the biodiversity hot spot areas of north east india. the present manuscript deals with descriptions of two new species of spp. from manipur, north east india. the discovery of the specimens is an added asset to the knowledge of the rich biodiversity of the region. * corresponding author : bina.chanu@gmail.com doi: 10.11598/btb.2014.21.2.3 93 materials and methods results and discussion nematodes were extracted from collected soil samples by baermann funnel technique. collected nematodes were fixed in taf (triethanolamine, formaldehyde and water) and processed by glycerol-ethanol method of seinhorst (1959). specimens were mounted in dehydrated glycerine. after slide preparation, measurements were taken using an ocular micrometer fixed on nikon, trinocular research microscope, model eclipse e200 and diagrams were drawn under a drawing tube attached to the same microscope. sp. nov.aphelenchoides longistylus 94 biotropia vol. 21 no. 2, 2014 table 1 morphometric data of species of sp. nov.. aphelenchoides longistylus characters holotype ♀ paratypes ♀ s paratype ♂s n 1 12 4 stylet 24.22 24.22 24.22 length 0.59 0.59 – 0.66 (0.625±27.79) 0.562 – 0.620 (0.59 ±0.02) a 34.4 34.4 35.85 (35.02±0.61) 38.2 – 42.02 (40.11±1.91) b 8.6 8.33 – 9.81(9.08±0.52) 7.24 –8.33 (7.78±0.54) b´ 6.03 6.03 – 6.49 (6.27±0.18) 3.25 – 5.603 (4.42±1.17) c 14.33 13.67 – 14.58 (14.19±0.38) 13.54 – 17.45 (15.22±1.43) c´ 6.00 5.6 – 6.14 (5.91±0.22) 4 – 6.2 (4.92±0.86) g1 40.69 39.39 – 43.08(41.05±1.52) v 69.76 67.78 – 69.76 (69.08±0.92) pvs/ v-a% 11.25 11.25 – 12.25(11.71±0.41) t 92.61 – 102.84 (97.56±3.66) max. body width 17.3 17.3 – 19.03(17.87±0.815) 13.38 lip diameter 5.19 5.19 5.13 lip height 1.73 1.73 1.73 oesophagus 98.61 98.61 – 102.07 (99.76±1.63) median bulb length 13.84 13.84 13.84 – 15.57 (14.55±0.73) median bulb diam. 10.38 8.65 10.38 (9.51±0.86) 8.65 –10.38 (9.53±0.70) ovary length 242.2 242.2285.45 (257.35±19.88) testis 335.62 – 342.45 (339.14±1.91) sperm theca 48.44 48.44 pus 15.57 8.65 – 18.65 (16.74±1.35) pus/vbd 0.9 0.9 – 1.07 (0.96±0.07) rectum 6.92 6.92 spicule 24.22 tail 41.52 41.52 – 48.44 (44.16±3.05) 41.52 – 46.23 (43.89±1.91) abd 6.92 6.92 – 8.65 (7.49±0.81) 10.32 note: all measurements are in μm except length is in mm description female: male: type habitat and locality: type material: differential diagnosis: body is straight to curved ventrally upon fixation, 0.59 0.66 (0.62±27.79) mm in length and about 34.4 35.85 (35.02±0.61) times greatest body width long. cuticle is finely annulated which is about 0.7 μm wide. lateral fields with 4 longitudinal lines which merge into 2 lines at around tail regions. cephalic region is indistinctly set off from body, appearing smooth and with 6 equal lips. stylet is 24.22 μm long with indistinct basal knobs. cylindrical portion of the stylet is slightly longer than anterior conical section. procorpus is wider anteriorly, gradually narrowing at posterior part, 48.44 59.1 (52.48±4.15) μm long. median bulb is spherical to pyriform in shape and occupying four-fifth of body width, 13.84 μm in length and 8.65 10.38 (9.51±0.86) μm in diameter. oesophageal glands forms a non overlapping lobe with intestine, about 34.6 38.06 (36.60±1.28) μm in length. nerve ring is about one-half of body width behind median bulb, encircling the anterior end of oesophageal glands. excretory pore is at base of median bulb, 74.39 83.04 (78.33±3.22) μm long from anterior body region forming a lobe at dorsal side of nerve ring. hemizonid, deirids and phasmids were not seen. vulva is a transverse slit, two-fifth of body width long and is located approximately at one-half body length from anterior end. genital tract is monoprodelphic, outstretched, usually extending up to the oesophageal gland lobe. oocytes are arranged in a single row, post uterine sac about 2.15 2.31 (2.21±0.07) μm times anal body diameter long. tail is 5.6 6.2 (5.9±0.22) μm times anal body diameter long, tapering gradually into a cylindrical tube terminating in a ventral prong tip. found in abundance as is female, is slightly smaller than female and is more straight upon fixation. cephalic region, stylet and oesophagus are as described for females. tail is slender with a single terminal mucro. spicules are about 24.22 μm long. testis is single, 335.62 342.45 (339.14±2.51) μm long. collected in december 2012 from soil around rhizospheric regions of coconut plant, linn. at ninghsing khul, jiri, imphal west district, manipur, india. holotype female on the slide fsb a -1/♀ sp. nov., paratype females on the slides fsb a 212/♀ sp. nov., paratype males on the slides fsb a 14/ ♂ sp. nov. and deposited in the nematode collection center of parasitology section, department of life sciences, manipur university, canchipur 795003, manipur, india. sp. nov. differs from all other species of fischer, 1894 in possession of the longest stylet and 4 lateral lines which gradually merged into 2 at the tail regions. cocos nucifera aphelenchoides longistylus aphelenchopides longistylus aphelenchoides longistylus aphelenchoides longistylus aphelenchoides 3 3 3 95 two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. however, it comes close to (hussain & khan 1967); (chawla & khan 1979); (tandon & singh 1974) and (hooper 1958). sp. nov. differs from (hussain & khan 1967) in having 4 lateral lines throughout body length, longer body length, larger values of a, b, c´, stylet and longer spicule; smaller values of c, v and indistinct stylet knobs (lateral lines =3, body length = 0.39 0.45, a= 30 33, b= 4 4.5, c´=3, stylet = 11 13, spicule = 15 19, c= 16 20, v = 69 77 and presence of stylet knobs in (hussain & khan 1967). sp. nov. comes close to (chawla & khan 1979) in having similar range of a, b, c and v values. but, it differs from (chawla & khan 1979) in having 4 lateral lines gradually merging into 2, larger values of body length, c value, stylet length and absence of stylet knobs (lateral lines= 3, body length = 0.4 0.5 mm, stylet = 10 -11 μm and presence of stylet knobs in (chawla & khan 1979). sp. nov. also comes close to (tandon & singh 1974) in having similar range of body length, a, b, c values and spicule length. but, it differs from tandon & singh, 1974 in having larger value of c´, smaller value of v, longer stylet and 4 lateral lines which merged into 2 and indistinct stylet knobs (c´= 2.9, v = 71 73, stylet = 11 14 μm, 4 lateral lines and presence of stylet knobs in (tandon & singh 1974). similarly, sp. nov. comes close to (hooper 1958) in having similar range of body length, a, b, c and v values. but it differs from (hooper 1958) in having 4 lateral lines, smaller values of b and longer stylet with indistinct stylet knobs and larger values of spicules (lateral lines = 3, b= 10 14, stylet = 11 μm, spicules = 15 20 μm and presence of stylet knobs in (hooper 1958). sp. nov. body is finely annulated, tapering towards both extremities, straight and slightly curved ventrally on the tail region upon relaxation. lateral fields have 4 incisures. cephalic framework is smooth, set off from the body, about 5.19 μm wide and 2.53 μm high. stylet is prominent with distinct stylet guards, 8.65 μm in length with distinct stylet knobs. oesophagus has zig-zagly coiled procorpus, has strongly developed and rounded corpus with median sclerotised plates and elongated gland lobe overlying the intestine dorsally at a distance behind the bulb which is equal to about 3 4 times body width long. neither deirids nor phasmids were seen. excretory pore is close behind the nerve ring. vulva is prominent, protruding both lips with vagina inclined. large and elongated spermatheca, about 65.74 70.24 (67.99±2.25) μm long, oocytes are arranged in single row reaching up to the oesophageal bulb. post-vulval uterine sac is about one-half vulval body width and is empty. anterior lip of anus is protruding, tail is bluntly rounded, 12.11 25.95 (19.03±6.92) μm in length with a small hair like mucro. aphelenchoides absari aphelenchoides chalonus aphelenchoides lanceolatus aphelenchoides sacchari aphelenchoides longistylus a. absari a. absari aphelenchoides longistylus a. chalonus a. chalonus a. chalonus aphelenchoides longistylus a. lanceolatus a. lanceolatus a. lanceolatus aphelenchoides longistylus a. sacchari a. sacchari a. sacchari aphelenchoides neominoris description female: 96 biotropia vol. 21 no. 2, 2014 97 male: type habitat and locality: type material: differential diagnosis: not found. collected in september 2013 from soil around the rhizospheric regions of orange plant, from sibilong, chandel district, manipur, india. holotype female on the slide fsb-a 2/ ♀ sp. nov., paratype females on the slides fsb a 1, 3 10/♀ sp. nov. and deposited at the nematode collection center of parasitology section, department of life sciences, manipur university, canchipur 795003, manipur, india. sp. nov. differs from all other species of fischer, 1894 in having the shortest body length. but, it comes close to (ebsary 1991); (hussain & khan 1967) and (masleen 1979) in several other characters. 4 4 aphelenchoides neominoris aphelenchoides neominoris aphelenchoides neominoris aphelenchoides aphelenchoides minoris aphelenchoides absari aphelenchoides vaughani two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. characters holotype paratypes n 1 10 stylet 8.65 8.65 length 0.439 0.351 – 0.439 (383.48 ± 39.71) a 36.29 36.29 – 40.6 (38.44± 2.16) b 6.51 4.23 – 6.51 (5.37± 1. 14) b´ 6.03 6.03 – 6.49 (6.27±0.18) c 36.29 13.53 – 36.29 (24.90± 11.37) c´ 1.75 1.75 – 3.75 (2.75±1) g1 50 41.87 – 50.0 (45.93±4.06) v 71.65 69.95 – 71.65 (70.8±0.85) lip width 5.19 5.19 lip height 2.53 2.53 oesophagus 67.47 67.47 – 83.04 (75.25±7.78) median bulb width 8.65 6.92 – 8.63 (7.49±0.81) ovary 219.71 147.05 – 219.71 (183.38±36.33) spermatheca 65.74 65.74 – 70.24 (67.99±2.25) pus 15.57 12.11 – 15.57 (13.84±1.73) pus/vbd 1.28 1.28 – 1.4 (1.34±0.06) rectum 5.19 5.19 tail 12.11 12.11 – 25.95 (19.03±6.92) abd 6.92 6.92 table 2. morphometric data of female species of sp. nov.aphelenchoides neominoris note: all measurements are in μm except length is in mm 98 biotropia vol. 21 no. 2, 2014 legends to figures a female anterior body b male anterior body c female entire body d female reproductive system e female lateral lines f female tail region g male entire body h male tail region i l. sp. nov. i female anterior body j female reproductive system k female lateral lines and l female tail region. aphelenchoides neominoris figure 1: a h. sp.nov.aphelenchoides longistylus 99 aphelenchoides neominoris aphelenchoides minoris aphelenchoides minoris a. minoris aphelenchoides neominoris a. absari a. absari a. absari aphelenchoides neominoris aphelenchoides vaughani a. vaughani a. vaughani no. sb/ft/ls-113/2013 sp. nov. comes close to (ebsary 1991) in having smaller body length and a ventral mucro. but, it differs from (ebsary 1991) in having 4 lateral lines, larger values of a, c; smaller values of b, pus, stylet and indistinct stylet knobs (lateral lines = 3, a= 26 29, b= 89, c= 15, pus= 5 vbd, stylet= 10 μm and presence of stylet knobs in (ebsary 1991). sp. nov. comes close to (hussain & khan 1967) in having almost similar body length, 4 lateral lines and presence of ventral mucro. but, it differs from hussain & khan, 1967 in having larger values of a, b and c but smaller values of c´, stylet length, presence of post uterine sac and indistinct stylet knobs (a= 30 33, b= 4.0 4.5, c= 16 20, c´=3, stylet = 1113 μm, absence of post uterine sac and presence of stylet knobs in (hussain & khan 1967). sp. nov. is similar to (masleen 1979) in having almost similar body length, presence of 4 lateral lines and ventral mucros. but, the species differs from (masleen 1979) in having larger values of a, c and g and smaller values of b, stylet length, tail length and in having a simple ventral mucro (a= 2431, b= 6.3 9.2, c= 12.1 16.9, g = 32 44, stylet = 9.5 11 μm, tail = 23.5 38.5 μm with a ventral mucro minutely multi-papillate at its tip in (masleen 1979). the authors thanked the head, department of life sciences, manipur university, canchipur for providing necessary laboratory facilities. the corresponding author also acknowledged the science and engineering research board, department of science & technology, govt. of india for financial support in the form of fast track young scientist fellowship (serb ). 1; 1 acknowledgements references aebsary ba. 1991. . canada: agriculture canada. iv. chawla ml, khan e.1979. two nomenclatorial corrections. indian j nematol 7: 100. fisher m. 1894. über eine clematis -krankheit. bericht aus dem physiolischen laboratorium des landwirthschaftlichen, instituts der universitat halle (11)3: 1 11. hooper dj. 1958. n. sp. and n. sp. (nematoda: aphelenchoidea). nematologica 3: 228 35. hussain si, khan am.1967. on the status of the genera of the superfamily aphelenchoidea (fuchs, 1937). proc helm soc wash 34: 167 74. maslen nr. 1979. six new nematode species from maritime antarctic. nematologica 25: 288 308. tandon rs, singh sp. 1974. nematode parasites of the common sponge gourd, from lucknow geobios 1: 24 7. seinhorst jw.1959. a rapid method for the transfer of nematodes from fixative to anhydrous glycerine. nematologica 4: 67 9. catalog of the order tylenchida (nematoda) aphelenchoides dactylocercus a. sacchari luffa cylindfrica . two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. 3. nunik s (contrasting).cdr biotropia vol. 18 no. 2, 2011: 81 93 81 contrasting arboreal and terrestrial bryophytes communities of the mount halimun salak national park, west java nunik s. ariyanti and sulistijorini bryophytes are frequently neglected due to its small size and their economical value is not much known. however, a recent study (harris 2008) listed about 150 ethnobotanical species of bryophytes; about 27 percent of those species are used in traditional chinese medicine including which is used for treating nervous disorder and cardiovascular disease. this species is also found at mount gede pangrango national park (hasan & ariyanti 2004). bryophytes occupy a wide range of habitats, colonizing various terrestrial substrates, tree trunks and tree canopies. bryophytes represent important components of forest floor and epiphyte communities in many ecosystems, contributing to forest diversity, structure and ecosystem-level processes. even bryophytes especially liverworts are abundant and dominant in “cloud” or “mossy” forest. department of biology, faculty of mathematics and natural sciences, bogor agricultural university recipient of biotrop research grant 2009 / accepted 30 april 2011 bryophyte communities were compared between arboreal (trunk bases) and terrestrial habitats in primary forest mount halimun salak national park, west java. the communities were analyzed based on species diversity, abundance, and biomass. a total of 150 bryophytes species were identified, including 67 species of mosses (bryopsida) and 83 of liverworts (hepaticopsida). both bryophyte groups varied in diversity and abundance between arboreal and terrestrial communities as well as among different elevations. species diversity of arboreal habitats (116 species) was higher than that of terrestrial habitats (64 species). moss species were more abundant in terms of coverage in terrestrial habitats whereas liverworts species were more abundant in arboreal habitats. species richness in both terrestrial and arboreal habitats decreased towards higher elevation, whereas the abundance increased. : bryophytes, mosses, liverworts, terrestrial habitat, arboreal habitat abstract introduction key words rhododendron giganteum corresponding author : nuniksa@gmail.com biotropia vol. 18 no. 2, 2011 82 despite of their traditional medicinal use, bryophytes have a range of important roles to play in the environment. bryophytes may serve as substrate for other plants and offer shelter to small animals. changes in epiphytic bryophyte assemblages affect other canopy dwelling biota, such as vascular epiphytes, invertebrates, and especially foraging birds (sillet 1994; andrew . 2003). the moist environment created by the bryophytes is also quite favorable to the establishment and growth of important groups of microorganisms, such as the nitrogen-fixing blue green algae (gradstein 2001). the abundance of liverworts in “cloud” or “mossy” forest is considered an important factor in eliminating the deteriorating effect of heavy rains, including helping to prevent soil erosion and adding to hill stability (pócs 1980). bryophytes may serve as potential indicators of climate changes since they are closely assosiated with climatically sensitive habitat and ecosystem (gignac 2001). in addition, bryophytes have important contributions to forest nutrient cycling, particularly to n-cycling (longton 1984; turetsky 2003). structure and floristic composition in tropical rain forests vary considerable. the bryophyte flora of tropical rain forest changes significantly with elevation, different taxa often occur in the different forest belts. bryophytes are very useful indicators of life zones and forest types in tropical mountain regions, since they have relatively modest number of species and genera, very wide geographical ranges, and great variation in biomass (frahm & gradstein 1991). studies dealing with diversity and abundance of bryophytes are more frequently referred to temperate region and tropical america region (e.g. mcgee & kimmerer 2002; sillet . 1995; acebey . 2003; nöske . 2008) than to tropical asia. among a few ecological studies of bryophytes in tropical asia (frahm 1990; sporn . 2009; gradstein & culmsee 2010) is the diversity and abundance of epiphytic bryophytes assessed in primary and secondary submontane rain forest and cacao agroforestry in sulawesi (ariyanti . 2008). however, the study did not simultaneously compare the community structures and contributions of both arboreal (epiphytic) and terrestrial bryophyte species in the same ecosystem. although java was considered well inventoried, recent publications dealing with the bryophytes of java are very few and a modern checklist for mosses is lacking. in contrast, checklists of both mosses and liverworts of others major islands in malesian region (borneo, sulawesi, philippines) have been provided (ariyanti & gradstein 2007; ariyanti . 2009; gradstein . 2005; menzel 1988; tan & engel 1986; tan & iwatsuki 1991; suleiman . 2006). söderström . (2010) published a first modern checklist of the liverworts and hornworts of java and reported more than 600 species for the island. of these, 479 are accepted species as currently understood based on revision and monographs; the remaining species are little known or of doubtful status, or invalid names. this research examines bryophyte communities in understory forest of mount halimun, west java, indonesia. mosses of mount halimun have recently been inventoried (tan ., reinwardtia 12(3): 205-214. 2006) but liverworts and hornworts of the mountain have not yet been reported. there are two primary aims: to assess and compare bryophyte communities in two different substrate type (arboreal and terrestrial) and different altitudes of study sites. et al et al. et al et al et al et al et al et al et al et al et al et al 83 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al materials and methods results study area terrestrial sampling arboreal sampling data analysis species diversity the study took place at mount halimun which is located in mount halimun salak national park, west java province, indonesia 106º12’ 106º45’ east, 06º32 06º55 south. the four study sites were established at different altitudes: at 1730 m (site 1), 1250 m (site 2), 1100.m (site 3) and 1000. m (site 4). at each of the four study sites, a plot of 30 x 30 m was established. at each plot, terrestrial and arboreal bryophyte diversity were estimated based on species richness and abundance (cover cm and frequency %); the total biomass of the several most dominant species were surveyed; the forest stand and vascular plant characteristics were inventoried. field samplings were done between july and august 2009. methods for terrestrial diversity sampling referred to (botting . 2008) with few modifications. at each plot of 30 x 30 m, two parallel 30 m transects were established 10 m apart, along which the bryophytes were surveyed in 1 x 1 m subplot placed at five equi-distant points on the transect. each species present was recorded and collected for future identification; the coverage (cm of cover) and frequency (%) of each species occurred in the subplots were recorded based on the quadrat 20 x 30 cm. arboreal bryophytes were sampled on five selected trees per plot of 30 x 30 cm following ariyanti . (2008) and botting . (2008) with modifications. selected arboreal sampling trees supporting bryophytes are the trees of more than 20 cm of diameter breast high. five quadrats of 20 x 30 cm were placed at different directions of 0-200 m high of tree trunk. each species present on the quadrat was recorded or collected for future identification. the coverage (cm cover) and frequency (%) of the species in a quadrat of 20 x 30 cm were also recorded. species identification was done using the following literature: eddy (1988; 1990; 1996) and bartram (1039) for the mosses, draft version of gradstein (2011) for the liverworts. an analysis of variance (anova) with the main fixed effects of altitude (plots at different altitude) and substrates type (arboreal and terrestrial) were used to evaluate bryophyte communities in the understorey forest with respect to species richness, coverage. in total, 150 bryophytes species were identified across the terrestrial and arboreal (trunk bases) habitats in the primary forest of the mount halimun salak national park. they consist of 67 species of mosses (bryopsida) and 83 species of ’ ’ 2 2 2 2 2 et al et al et al 84 liverwort (hepaticopsida). in this research we did not found any hornwort species (antocerotopsida) in the plots. hornwort commonly occurs in humid, non-forest terrestrial habitats such as river bunk or the open slope of forest margins. arboreal bryophytes with 116 species found on the base of tree trunks seems more diverse than those of terrestrial bryophytes with 64 species, identified from various substrates such as rotten log, root of trees, rock, and humus. thirty three species were identified from both terrestrial and arboreal substrates. the lists of species found in the study sites is presented in appendix 1. the total number of species found in the plots of 30 x 30 m ranged from 46 species in plot iii to 79 species in plot iv. species richness of arboreal bryophytes in the plots was higher than that of terrestrial bryophytes and varied from 38 to 64 (fig. 1). among the arboreal bryophytes, the number of liverworts species in each plot mostly was higher than those of mosses species. on the other hand, the terrestrial bryophytes composed of more mosses than liverworts species. species richness and abundance biotropia vol. 18 no. 2, 2011 26 9 32 21 9 27 22 7 25 41 7 47 12 15 22 18 9 24 16 9 21 23 16 32 0 10 20 30 40 50 60 70 80 a t a+t a t a+t a t a+t a t a+t plot i plot ii plot iii plot iv figure 1. the species number of liverworts ( ) and mosses ( ) on arboreal (a) and terrestrial (t) substrates in plot i, ii, iii, and iv (30 x 30 m) that located at different altitudes � � s p ec ie s ri ch n es in th e p lo ts o f 3 0 x 3 0 m 0 3 6 9 12 15 18 21 plot i plot ii plot iii plot iv figure 2. the average of species number of liverworts ( ) and mosses ( ) of arboreal bryophytes in each tree trunk base in plot (30 x 30 m) i, ii, iii, and iv � � t h e av er ag e o f sp ec ie s n u m b er 85 terrestrial bryophytes arboreal bryophytes plot / species coverage cm 2 plot / species coverage cm 2 plot i plot i hypnodendron sp. 1 60.1 ± 17.8 syrrhopodon tristichus 119.7 ± 61.2 trismegistia regida 48 ± 33.2 plagiochila frondescens 95.5 ± 52.4 bazzania sp. 1 24.8 ± 15.5 schistochila sciurea 95.3 ± 50.2 pogonatum macrophyllum 24 ± 24.0 bazz ania tridens 94.4 ± 52.6 trichosteleum elegantissimum 19 ± 14 .0 plagiochila dendroides 85.3 ± 49.5 bazzania vittata 18.0 ± 12.4 bazzania vittata 81.2 ± 43.7 plot ii plot ii callyscostella papillata 58.6 ± 29.3 spruceanthus polymorphus 21.4 ± 10.7 heteroscyphus argutus 26.9 ±15.66 plagiochila sciophyla 18.8 ± 12.4 lejeunea anisophylla 18.6 ± 18.6 heteroscyphus argutus 17.7 ± 9.6 telanarea neesii 15.8 ± 10.9 exostratum blumei 14.2 ± 13.5 vesicularia reticulata 15.5 ± 9.2 leucophanes massartii 14.2 ± 10.9 distichophyllum schmidtii 11.8 ± 8.3 plagiochila javanica 12.2 ± 8.5 plot iii plot iii callyscostella papillata 65.8 ± 57.4 lejeunea anisophylla 36.0 ± 18.2 heteroscyphus argutus 30.8 ± 26.4 mitth yridium junquilianum 28.9 ± 15.9 achanthorrinchium papillatum 30.5 ± 18.4 radula javanica 20.0 ± 9.6 vesicularia reticulata 24.3 ± 16.3 mitthyridium flavum 19.4 ± 10.1 lejeunea anisophylla 23.3 ± 18.4 acanthorrhyncium papillatum 10.4 ± 7.0 telanarea neesii 18.6 ± 15.5 lepidozia wallichiana 10.2 ± 10.2 plot iv plot iv trichosteleum boschii 33.1 ± 19.0 syrrhopodon muelleri 19.6 ± 13.3 achanthorrynchium papillatum 31.7 ± 31.7 plagiochila propingua 14.5 ± 10.8 isopterygium albesce ns 17.6 ± 12.5 thysananthus retusus 13.9 ± 8.8 heteroscyphus argutus 14.0 ± 7.1 radula javanica 13.1 ± 8.8 isopterygium bancanum 12.1 ± 9.0 pyrrobryum spiniforme 12.7 ± 12.7 chaetomitrium lanceolatum 6.5 ± 6.5 acroporium lamprophyllum 9.4 ± 9.0 table 1. the six highest coverage (cm , ± standard error) of terrestrial and arboreal bryophytes species in quadrates of quadrat (20 x 30 cm) in plot i, ii, iii, and iv. see also appendix 1. 2 bryophytes found at trunk bases (0-2 m) ranged from 11 to 28 species. each tree trunk base mostly had more liverworts (6 13 species) than mosses (5 6 species) (fig. 2). though the difference was not significant (anova, n=5, p=0.05), the number of liverworts and mosses in plot iv was slightly higher than those in other plots. on the contrary, plot ii has lower species richness than the other plots. species abundance of bryophytes that were estimated based on bryophytes coverage on the substrate showed that the most dominant species of terrestrial bryophytes (indicated by high coverage) is mostly included in the group of mosses, whereas most of arboreal bryophytes having high coverage are the liverworts species. in common, coverage of arboreal species is higher than that of terrestrial species (table 1). though species richness in the plot iv was higher than that of other plots, the coverage of species in plots iv was lower than those in other plots. the arboreal species with the highest coverage in plot iv is which covered aboutsyrrhopodon muelleri arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 86 biotropia vol. 18 no. 2, 2011 table 2. the species of arboreal and terrestrial bryophytes occurred at least 20% of sampled quadrates (n = 25) in plot i, ii, iii, and iv. see also appendix 1. arboreal bryophytes terrestrial bryophytes plot / species frekuency of occurance (%) plot / species frekuency of occurance (%) plot i plot i syrrhopodon tristichus 80 hypnodendron sp. 1 40 leucobryum javense 40 trichosteleum elegantissimum 24 bazzania vittata 40 plagiochila frondescens 36 plagiochilion oppositum 36 schistochila sci urea 28 plagiochila dendroides 24 radula javanica 20 plot ii plot ii lejeunea anisophyla 28 callyscostella papillata 20 lopidium trichocladon 24 heteroscyphus argutus 20 heteroscyphus argutus 24 spruceanthus polymorphus 24 pinnatella sp. 1 20 radula multiflora 20 plot iii plot iii lejeunea anisophyla 44 trichosteleum boschii 24 mitthyridium junquilianum 24 harpalejeunea filicuspis 24 lejeunea discreta 24 radula javanica 24 leucophanes octoblepharoides 20 mitthyri dium flavum 20 mitthyridium wallisii 20 thysananthus spatulistipus 20 plot iv plot iv leucophanes octoblepharoides 28 heteroscyphus argutus 20 heteroscyphus argutus 28 schistochila aligera 28 cheilolejeunea ceylanica 24 acroporium lam prophyllum 20 leucophanes massartii 20 plagiochila bantamensis 20 radula javanica 20 20 cm (3.3%) of the quadrats. the arboreal species with highest coverage is in plot i that covered 120 cm (20%) of the quadrates. the six most abundant species in the plot i covered more than 80 cm (13.3%) area of quadrate, whereas the six most abundant species in others plot cover less than 40 cm (6.7%) of the quadrates (table 1). distributions of species in the plots are mostly rare. most species were found in only one of 25 sampled quadrates. some species are found only twice and others more often in the quadrates (appendix 1). only two species were found in more than 10 quadrates. the common species found in at least 20% of the sampled quadrates are 2 2 2 2 syrrhopodon tristichus 87 listed in table 2. liverwort species are more numerous in the arboreal plots (16 species, of 26 species in total), whereas mosses are more numerous in the terrestrial plots (4 of 5 in total). among the species which have high frequency of occurrence is the epiphytic mosses in plot i which were found at twenty quadrates. the frequency of occurrence of the species is about 80 %. other species were often found in the plot are , , (both species are epiphytic species in plot i) sp. 1, and (both are terrestrial species in plot i and ii respectively), all of those species have 40 % of the frequency of occurrence (table 2). the bryophyte diversity at natural primary forest of the mount halimun salak national park hutan is quite high, it comprises no less than 150 species found across the arboreal (trunk bases) and terestrial habitates. the arboreal diversity is almost as high as in the submontane forest area of lore lindu national park, central sulawesi, where ariyanti . (2008) found about 169 bryophytes species on trunk bases in twelve plots. the slightly higher figure for central sulawesi is perhaps because the research in central sulawesi was conducted in three forest types (natural primary forest, secondary forest, cacao agroforest), while the present research was conducted in primary forest only. the result of arboreal species (116 species) that were inventoried from 20 sample trees was higher compared with the number of species collected from fewer tree samples and reported for primary submontane (1100 m), lower montane (1400 m) and upper montane (3250 m) forest in lore lindu park, central sulawesi, by gradstein & culmsee (2010). having the same number of tree samples, ariyanti . 2008 collected 112 species from primary submontane (1000 m) forest in lore lindu national park. the arboreal diversity would have been much higher when the whole trees would have been inventoried. sporn . (2009) collected more epiphitic species than ariyanti . (2008) from the same locations studied by ariyanti . (2008). however, sporn . (2009) sampled the whole trees (the tree trunk and canopy). when species richness of arboreal and terrestrial bryophyte are compared, the richness of terrestrial bryophytes is lower than that of arboreal species in all the plots. it is not in accordance with the richness of terrestrial bryophytes at the temperate spruce forest which species richness of terrestrial bryophyte exceeds those of arboreal bryophytes (botting . 2008). tropical rain forests are characterized by high diversity of big tree plants which offers various habitats for epiphytic bryophytes, it perhaps relates to the high diversity of arboreal bryophytes at tropical forest than those at temperate forest in which commonly dominated by several tree species. on the other hand, understorey habitat are covered by canopy forest that determined light intensity penetrating to the ground for bryophytes photosynthesis. more over, the low rate of litter decomposition in the tropical forest may affect the substrates availability for terrestrial bryophytes. the terrestrial substrates in the understorey of primary syrrhopodon tristichus leucobryum javense bazzania vittata hypnodendron taxithelium isocladum et al et al et al et al et al et al et al discussion arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 88 biotropia vol. 18 no. 2, 2011 forest in the mount halimun salak national park were restricted, since the ground was mostly covered by leaf litters, herbs and scrub plants. terrestrial bryophytes were found growing in mats or clusters at patched substrates such as rotten log, humus, or rock at slightly open area in the forest understorey. the greater richness of the arboreal habitat than the terrestrial habitat has been discussed by numerous authors (richards 1984; gradstein & pocs 1989; frahm 1990; frahm & gradstein 1991; gradstein . ; gradstein & culmsee 2010). richards (1984) mentioned that bryophytes may be absent on undisturbed soil in lowland rain forests and only locally common in montane forests. gradstein & culmsee (2010) also reported that for the arboreal species: liverworts species richness increased toward higher elevation, whereas moss richness decreased. bryophytes may represent important components of forest floor and epiphytes communities in the forest ecosystem, contributing high number of species to forest diversity. vegetation analysis of corridors forest at the mount halimun salak national park conducted by sambas and purwaningsih yield about 27 36 species of tree plants in the plot of 0.2 ha (http://www.tnhalimun.go.id/document.php/ document/article/49/38/). based on this research it appeared that the richness of bryophytes may exceed that of the tree plant. based on the species number in the plots, it is estimated that bryophytes may contribute about 46 79 species to the plants community in the forest of the mount halimun salak national park. though the difference was not statistically significant (anova, n=25, p=0.05), species richness in the plot at higher altitude (plot i, 1730 m) tend tobe lower than those at the lower altitudes (plot iv, 1000 m). the differences in species richness was also reported by gradstein and culmsee (2010) that occurred between lower montane and upper montane rain forest of lore lindu national park in central sulawesi. on the other hand, species abundance of bryophytes at higher altitude was higher than those at lower altitudes. the result is in accordance with that of tropical american forest that the coverage of bryophytes increased with the increase of the altitude (gradstein & pocs 1989) and also observed between lower montane and upper montane rain forest of lore lindu national park (gradstein & culmsee 2010) some terrestrial species that have highest coverage in the plots were the mosses species of , , dan . those three species were common species, they were often found in the plots (with the frequency of occurrence of about 20 % and they are included in the family hypnodendraceae, hookeriaceae, and sematophyllaceae, respectively. the most dominant arboreal species in terms of coverage are , (calymperaceae), , (lejeuneaceae), , , and (plagiochilaceae). the moss species of and the liverwort species of were also the most common species which occurred in 40 % of the 25 quadrate samples. this result is relevant to the bryophytes commonly found in oligophotic habitat such as the understorey forest consisting of many dendroid, feather or bracket-type of mosses and liverworts which are specific of this type habitats. these bryophytes belong to the family of hookeriaceae, pterobryaceae, neckeraceae, plagiochilaceae, and lejeuneaceae (gradstein & pocs 1989). et al 2001 hypnodendron calicostella papillata trichosteleum boschii syrrhopodon tristichus syrrhopodon muelleri, mitthyridium junquilianum spruceanthus polymor phus lejeunea anisophylla plagiochila frondescens plagiochila sciophyla plagiochila frondescens syrrhopodon tristichus lejeunea anisophyla 89 conclusions acknowledgments references bryophytes communities at primary forest of the mount halimun salak national park comprised about 150 species of liverworts and mosses found across various terrestrial and arboreal substrates. the species diversity of arboreal bryophytes at 900 1730 m is higher than those of terrestrial bryophytes. forest at the higher elevation tends to have lower species richness than that at lower elevation. in the contrary, the bryophytes abundances are higher at high elevation compared to that at lower elevation. we would like to thank prof. s. robbert gradstein (museum national d'histoire naturale, department systematique et evolution) for the valuable suggestions and review of this manuscript. indah wahyuni, dian apriani, marinda sari sofiyana, and saiful bachri assisted us with collecting samples in the field. funding for this study was provided by research project of dipa-biotrop dikti 2009. a a . a cebey c, gradstein sr, kromer t. 2003. species richness and habitat diversivication of bryophytes in submontane rain forest and fallows in bolivia. journal of tropical ecology, 18: 1-16. ndrew nr, rodgerson l, dunlop m. 2003. variation in invertebrate bryophyte communitiy structure at different spatial scales along altitudinal gradients. journal of biogeography, 30: 731-746 riyanti ns, bos mm, kartawinata k, tjitrosoedirdjo ss, guhardja e, gradstein sr. 2008. bryophytes on tree trunks in natural forests, selectively logged forests and cacao agroforests in central sulawesi, indonesia. biological conservation, 141: 2516-2527. aryanti ns, gradstein sr, sporn sg, angelika e, tan bc. 2009. catalogue of the bryophytes of sulawesi, suppplement 1. blumea, 54: 287-289. aryanti ns, gradstein sr. 2007. wallace's line and the distribution of the liverworts of sulawesi. cryptog. bryol. 28: 3-14. bartram eb. 1939. . vol. 68. manila: bureau of printing. botting rs, campbell j, fredeen al. 2008. contrasting arboreal and terrestrial macrolichen and bryophyte communities in old-growth sub-boreal spruce forests of central british columbia. the bryologist, 111(4): 607-619. eddy a. 1988. vol. 1, sphagnales to dicranales. london: british museum. eddy a. 1990. vol. 2, leucobryaceae to buxbaumiaceae. london: the natural history museum. eddy a. 1996. vol. 3, splachnobryaceae to leptostomataceae. london: the natural history museum. frahm jp, gradstein sr. 1991. an altitudinal zonation of tropical rain forest using bryophytes. journal of bryogeography. 18: 75-78. frahm jp. 1990. the ecology of epiphytic bryophytes of mt. kinabalu, sabah (malaysia). nova hedwigia, 51: 121-132. the philippine journal of science a handbook of malesian mosses. a handbook of malesian mosses. a handbook of malesian mosses. arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 90 biotropia vol. 18 no. 2, 2011 gignac ld. 2001. bryophytes as indicators of climate change. the bryologist, 104: 410 420. gradstein sr, churchill sp, salazar an 2001. guide to 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of the hattori botanical laboratory, 55: 147-163. mcgee gg, kimmerer rw. 2002. forest age and management effect on epiphytic bryophyte communities in adirondack northern hardwood forest, new york, usa. canadian journal of forest research 32: 1562-1576. menzel m. 1988. annotated catalogue of the hepaticae and anthocerotae of borneo. journal of the hattori botanical laboratory 65: 145-206. nöske n, hilt n, werner f, brehm g, fiedler k, sipman hj, gradstein sr. 2008. disturbance effects on diversity in montane forest of ecuador: sessile epiphytes vs. mobile moths. basic and applied ecology. pócs t. 1980. the epiphytic biomass and its effect on the water balance of two rain forest types in the uluguru mountains (tanzania, east africa). acta botanica academiae scientiarum hungaricae, 26:143-167. richards pw. 1984. the ecology of tropical forest bryophytes. : schuster rm. (ed.) new manual of bryology, nichinan: hattori botanical laboratory, p. 1233-1270. sillet ts. 1994. foraging ecology of epiphyte-searching insectivorous birds in costa rica. condor, 96: 863-877. sillett s, gradstein sr, griffin d. 1995. bryophyte diversity of tree crowns from cloud forest and pasture in costa rica. 98: 251-260. söderström l, gradstein sr, hagborg a. 2010. a checklist of the hornworts and liverworts of java. phytotaxa 9: 53-149. sporn sg, bos mm, keßler m, gradstein sr. 2009. vertical distribution of epiphytic bryophytes in an indonesian rainforest. biodiversity and conservation, 19: 745-760. suleiman m, akiyama m, tan bc. 2006. a revised catalogue of mosses reported from borneo. journal of the hattori botanical laboratory, 99:107-183. tan bc . 2006. mosses of gunung halimun national park, west java, indonesia. reindwartia, (12): 205-214. tan bc, engel jj. 1986. an annotated checklist of philippine hepaticae. journal of the hattori botanical laboratory 60: 283-355. tan bc, iwatsuki z. 1991. a new annotated philippine moss checklist. harvard papers botany, 3:1-64. turetsky mr. 2003. the role of bryophytes in carbon and nitrogen cycling. the bryologist, 106(3):395-409 in in ficus the bryologist, et al , , , , , . appendix 1. alphabetical list of arboreal and terrestrial bryophyte species collected in the plots. a = arboreal; t = terrestrial; + = present; = absent. species habitat plot i plot ii plot iii plot iv mosses: acanthorrhyncium papillatum a, t + acroporium diminutum a, t + + + acroporium hamulatum t + acroporium lamprophyllum a, t + + + + acroporium rufum a, t + + + acroporium sec undum a + acroporium sigmatodontium a, t + + callyscostella papillata t + + chaetomitrium lanceolatum t + dicranoloma blumii t + dicranoloma braunii a, t + + dicranoloma brevisetum a, t + + + distichophyllum schmidtii t + distichophyllum sp. 1 t + + ectropothecium sp. 1 t + + exostratum blumei a,t + + fissiden crassinervis var. laxus t + fissidens gedehensis a + fissidens hollianus a, t + floribundaria floribunda a, t + himantocladium plumula a + homaliodendron flabellatum a + homaliodendron javanicum a + hypnaceae sp. 1 t + hypnodendron sp. 1 t + isopterygium albescens a, t + + + isopterygium bancanum a, t + isopterygium minu tirameum a, t + + leucobryum bowringii a + leucobryum candidum t + leucobryum javense a, t + + + + leucobryum juniperoideum a + leucophanes massartii a, t + + + leucophanes octoblepharoides a, t + + + lopidium sp. 1 a + lopidium struthiopteris a + lopidium trichocladon a + mitthyridium flavum a + mitthyridium junquilianum a, t + mitthyridium wallisii a, t + neckera tjibodensis a + pelekium velatum a, t + pinnatella anac amptolepis a + pinnatella cf. ambigua a + pinnatella microptera a + pinnatella sp. 1 a + pogonatum macrophylum t + pterobryopsis gedehensis a + pyrrobryum medium a + pyrrobryum spiniforme a + + + 91 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al species habitat plot i plot ii plot iii plot iv symphyso dontella attenuatula a + symphysodontella cylindrica a + syrrhopodon albovaginatus a + syrrhopodon muelleri a + + syrrhopodon prolifer a + syrrhopodon tristichus a, t + + taxithelium instratum a + taxithelium is ocladum t + trichosteleum boschii t + + trichosteleum elegantissimum a, t + + trimegistia calderensis a + liverworts: acromastigum divaricatum a + + aneura pinguis t + archilejeunea planiuscula a + bazzania intermedia a + + + bazzania sp. 1 a + bazzania sp. 2 a + bazzania sp. 3 a + bazzania sp. 4 a + bazzania tridens a + + + bazzania vittata a, t + + + bazzania wallisiana a + calypogaea goebelii a + ceratolej eunea belangeriana a + cheilolejeunea ceylanica a, t + + cheilolejeunea imbricata a + + cheilolejeunea longiloba a + + chiloscyphus minor a + chiloscyphus muricatus a + cololejeunea sp. 1 t + drepanolejeunea angus tifolia a + drepanolejeunea ternatensis a + + frullania apiculata a + + harpalejeunea filicuspis a + harpalejeunea filicuspis t + herbertus dricanus a + heteroscyphus argutus a, t + + + heteroscyphus coalitus t + heteroscyphus succulenthus a, t + + heteroscyphus zollingeri t + + jubula sp. 1 t + kurzia gonyotricha a + lejeunea anisophyla a, t + + + + lejeunea discreta a + + + lejeunea eifigri a + lejeunea exilis a + lejeunea obscura a, t + + + lejeunea punctiformis a + lejeunea sordida a + + lejeunea sp. 1 a + + lepidolejeunea bidentula a + appendix 1. continued 92 biotropia vol. 18 no. 2, 2011 appendix 1. continued species habitat plot i plot ii plot iii plot iv lepidolejeunea integristipula a + lepidozia hasskarliana a + lepidozia trichodes a + lepidozia wallichiana a, t + + leujenea anisophyla a + lophocolea bidentata a, t + + lophocolea minor t + lopholejeunea eulopha a + lopholejeunea horticola a + lopholejeunea nigricans a + lopholejeunea subfus ca a + lopholejeunea zollingeri a + + mastigolejeunea auriculata a + metalejeunea cuculata a + metzgeria leptoneura a + metzgeria sp. 1 a + plagiochila bantamensis a + + plagiochila dendroides a + + plagio chila frondescens a + + + plagiochila javanica a + + plagiochila junghunhiana a, t + + + plagiochila obtusa a + plagiochila parvifolia t + plagiochila pleurata a + plagiochila propingua a + + plagiochila sciophyla a, t + + + plagiochilion oppositum a + + pticanthus striatus a + radula javanica a + + + radula multiflora a + + radula retroflexa a + + + riccardia sp. 1 t + schistochila aligera a + schistochila doriae a + schistochila sciurea a + spruceanthus polymorphus a + telenarea neesii a, t + + + thysananthus retusus a + thysananthus spatulistipus a + + trichocolea tomentella a, t + + zoopsis liukiuensis t + 93 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al biotropia book juni revisi 14 juli 09.indd 1 biotropia vol. 16 no. 1, 2009: 1 10 *corresponding author: jtmasagca@dasma.dlsu.edu.ph, jmasagca@yahoo.com. feeding ecology of tree-climbing mangrove sesarmid crabs from luzon, philippines jimmy tevar masagca de la salle university-dasmariñas cavite 4115, th e philippines abstract despite the large ecological study of tree-climbing mangrove sesarmid crabs in other countries, the philippine representatives appear to have not been investigated extensively. this paper presents the feeding ecology as to dependence on mangrove trees of sesarmids in different mangrove areas of southern luzon. this is biased on the nature of the crab habitats, arboreal climbing skills and burrowing behavior of the sesarmids: selatium elongatum and episesarma versicolor − exclusive mangrove tree climbers (emtc); sarmatium germaini − occasional mangrove tree climber (omtc); and the non-mangrove tree-climbing (nmtc) sesarmidsneosarmatium smithii, perisesarma bidens and perisesarma eumolpe. key words: mangrove crabs, sesarmid crabs, climbing skills, burrowing skills, arboreal climbing crabs, catanduanes, philippines introduction the crustaceans (e.g. brachyurans) are always a prominent and diverse element of the fauna in mangals (morton 1990). crabs are diverse in mangrove habitats and abundant in many mangals (jones 1984). several authors (e.g. jones 1984, lee 1998) have attested that brachyurans are important in the mangrove ecosystem structure and function. unfortunately, it appears that there is still a dearth of detailed published information on the occurrence of several families of this group of crustaceans in the mangrove areas of the philippines. moreover, there seems to be limited published data on the ways in which these faunal elements use the mangrove resources. the sesarmids are less documented in the philippines unlike in other countries, wherein several reports are available from singapore (sivasothi 2000; sivasothi et al. 1993; tan & ng 1994; ng & liu 1999) malaysia (tan & ng 1994; leh & sasekumar 1985), indonesia (soemodihardhjo & soerianegara 1989; rahayu & davie 2002), hong kong (poovatchiranon 1986; lee & leung 1993; lee 1998; lee 1997; kwok 1995; kwok & tang 2005; and ashton 2002). crabs have different functions and impacts on the mangrove ecosystem. firstly, they can process as much as 70% of the leaf litter, and leaf processing can turn over a litter at a rate in excess of 75 minutes (see studies of leh & sasekumar, 1985; slim 2 biotropia vol. 16 no. 1, 2009 et al. 1997; dahdouh-guebas et al. 1999; ashton 2002). secondly, most mangrove crabs feed on vascular plant materials, but they also feed on green leaves (lee 1998; skov & hartnoll 2002), including seedlings. mangrove plant seedlings grazing, particularly in the genus sesarma may also slow regeneration of mangroves. on the other hand, grapsoid crabs are the primary seed operators. emmerson & mcgwynne (1992) described the feeding and assimilation of mangrove leaves by the crab sesarma meinerti. moreover, the feeding ecology of neosarmatium smithii was studied by giddins et al. (1986) and new records from taiwan on this sesarmid crab was reported by naruse et al. (2006). the crabs of the family sesarmidae are known by various workers to have significant ecological role in mangals (lee 1998, gillikin 2000; gillikin & schubart 2004, gillikin et al. 2004). many species of these mangrove crabs do not assimilate much carbon from the mangrove leaves but rely on the sediments (bouillon et al. 2002, skov & hartnoll 2002). some species of the genus perisesarma supplement their diet with leaves (leh & sasekumar 1985, slim et al. 1997). hence, this aspect on the feeding ecology of sesarmid crabs helps to trap the energy stored in these leaves within the mangal before the tide can carry them away ( lee 1998; skov & hartnoll 2002). despite the large ecological study of mangrove sesarmids in other countries (bright & hogue 1972; hartnoll 1975; micheli, gherardi & vannini 1991; cannicci et al. 1996; slim et al. 1997; dahdouh-guebas et al. 1999; fratini, cannicci & vannini 2000, flores et al. 2003) the philippine representatives appear to have not been investigated extensively. this study on the patterns of biodiversity in aquatic systems of the philippines attempted to determine the feeding ecology of selected taxa of sesarmids as to (a) the nature of the crab habitats in the mangrove forest with reliance to the mangrove trees, trunks, roots, and leaves that offer a wide variety of ecological niches for the species under consideration; and (b) the behavior of the sesarmid crabs as revealed by their burrowing abilities and tree-climbing skills needed to explain the reliance of these crabs on the mangrove forest. materials and methods study sites the collection and observation sites of the study include: ipalsabangon mangrove area in pagbilao, quezon, philippines; ii maqueda channel in the bicol region, agojo inlet mangrove reserve area, san andres and palnab-pajo mangrove area, catanduanes, philippines. feeding ecology as to dependence on mangroves, arboreal climbing skills and burrowing behavior specimens of mangrove crabs were collected by handpicking and with the use of scoop nets and locally made traps. collections were made both in the morning and in the evening from the mangrove areas along rivers, creeks, inlets and the buffer zones or marginal strips of the coastline in one site each for the study areas from june 2005 to february 2006. 3 ecological studies on feeding behavior took place in the purposively selected study plots in palnab-pajo mangrove swamp, catanduanes during the months of october and december in 2005, while in the mangrove areas of palsabangon in pagbilao, quezon studies were done from january to february 2006. ecological studies of sesarmids were carried out in each of the mangrove areas following the methods of gillikin (2000). in the selected mangrove forests, presence or absence of crab species were determined by visual inspection in 10 m diameter plots along a transect perpendicular to the coastline, covering the full width of the forest. the study investigated at least 10 plots along a 100 to 200 m long transect in the areas under study. emergence and re-emergence of the sesarmids during fieldwork, each plot was first inspected using binoculars from a distance of approximately 6 m for 20 min. and the crab species recognized were noted and recorded. afterwards, naked eye observations were undertaken with two field workers (observers) sitting on opposite sides of the plot for at least 30 min, following hartnoll et al. (2002) and skov et al. (2002). as a modification, the researcher and field assistants were positioned in two separate paddled boats (“banca”) when the plots were observed. the researcher approached the plot and a disturbance was created (either by throwing some mud, twigs or branches of mangrove plants) so that the sesarmids, under study take refuge in the burrows, or holes, and other materials to take cover (e.g. leaves, wood, twigs etc.). the time at which the first individual of the sesarmids will emerge or re-emerge was recorded. observations for this portion of the study were undertaken during the day at low tides. diurnal and lunar cycles were not used in the distinction of the observation. the aforementioned procedures are modifications of the works of gillikin (2000), hartnoll et al. (2002) and skov et al. (2002). results and discussions reliance on the mangroves as habitats table 1 shows the summary of the observations made on the habitats of various grapsoid sesarmid crabs in the mangals of catanduanes island and pagbilao, quezon. the nature of their habitat, climbing abilities and burrowing skills could explain the feeding ecology of the mangrove crabs under investigation. table 1. summary of the feeding ecology of grapsoid sesarmid crabs from different mangals in luzon grapsoid sesarmid species behavior/nature of habitat related to feeding remarks on fi eld observations episesarma versicolor burrower and exclusive mangrove tree climbing (emtc) species migrating from burrows to the tree stems and some at the canopies; constructs burrows around soft sediments of mangrove roots and tree trunks; seen feeding on calyx and leaves; seen cropping on leaf litters and bringing fragments to the burrows. feeding ecology of tree climbing mangrove sesarmid crabs – jimmy t. masagca 4 selatium germaini occasional mangrove tree climber (omtc) species climbs on the branches of r. apiculata and r. mucronata in the mangroves of catanduanes. neosarmatium smithi burrower and non-mangrove tree climbing (nmtc) species constructing burrows on mudfl ats; not seen climbing on stems of mangrove trees in quezon and catanduanes perisesarma bidens burrower and non-mangrove tree climbing (nmtc) species seen resting on rhizophora tree (just within the water lining) during high tide apparently for avoidance and possibly for feeding purposes; also seen hiding on rocks, crevices of boulders and tree falls. perisesarma eumolpe burrower and non-mangrove tree climbing (nmtc) species on rare occasions, some crab samples were caught on the trunks possibly carried by water current during high tides. selatium elongatum arboreal climber or emtc in mangrove trunks, branches and canopies seen on main branches of rhizophora; on aerial roots; also on crevices of trunks. episesarma versicolor feed on calyx and leaves and crop on leaf litters, then bring fragments to the burrows. the grapsoid sesarmid crabs, perisesarma spp. and episesarma sp. were found to be herbivores and omnivore/deposit feeders, eating mangrove litter and water plants. moreover, the sesarmid crabs sarmatium germaini (figure 1), perisesarma eumolpe and neosarmatium smithi feed on the mangrove litter, composed of fallen mangrove leaves of rhizophora, seedlings, calyx and twigs that fall from the trees on the forest floor and into the water. this study confirmed that the leaves of the mangroves, r. marina, r. apiculata, and ceriops tagal were devoured by the generally observed herbivorous/omnivorous crabs such as perisesarma spp. the feeding habits of mangrove crabs have been divided into seven groups by jones (1984): herbivore, carnivore, omnivore, deposit feeder, omnivore/deposit feeder, specialized filterer, and filterer/omnivore. in the study of islam & uehara (2005), stomach content analysis showed that p. bidens diet consists mainly of mangrove leaves fragments, with small amounts of animal, algae and sediment matters, indicating that this sesarmid is primarily detritivorous. in chiromanthes onychophorum, malley (1998) noted that this sesarmid crab consumes fallen leaves or their fragments, incompletely digests them, and returns them to the environment as fecal matter in a more finely-divided state than when they were ingested. of recent, ya et al. (2008) reported that both perisesarma eumolpe and p. indiarum are mainly sediment grazers, but also feed on mangrove leaves and roots and occasionally animal matter. these crab species prefer avicennia alba leaves to other mangrove species, i.e., a. officinalis, a. rumphiana, r. apiculata and bruguiera gymnorhiza. dahdouhguebas (1997), ashton (2002), buck et al. (2003), thongtham & kristensen (2003) and schwamborn et al. (2006) analyzed the diets of sesarmid crabs showed that their diet mainly consisted of mangrove leaves and in addition to bustle animal matter. biotropia vol. 16 no. 1, 2009 table 1. continued 5 a. b. figure 1. sarmatium germaini from palsabangon mangrove area in pagbilao, quezon (a) and palnabpajo mangrove swamp, maqueda channel, in bicol region (catanduanes) (b). burrowing and arboreal climbing skills of sesarmids similar with other sesarmids, p. bidens are burrowers, but observations made in quezon and catanduanes indicate that some individuals tend to hide in natural refuges found in the mangals investigated. these hiding places are the crevices of rocks and boulders and in between portions of root buttresses of mangrove trees. field observation in quezon, indicates that these crabs are also having preferences in arboreal environments wherein selatium elongatum and episesarma versicolor were seen clinging on mangrove stems or rush to the mangrove stems when disturbed, while the boat is cruising in the waterways. these tree climbing crabs appear to be less antagonized during the investigation and remained to be motionless for about 1 hour. when tides are rising or lowering, these crabs appear to be unaware as observed for at least an hour. however, when the rising tide reaches their location in any of the protruding branches of the mangrove trees in the embankments, these tree-climbing crabs tend to adjust or move slowly with their heads upside down. researches indicate the possibility that these crabs cling to the mangrove stems not to find food but as avoidance from very long immersion in the water during high tides and due to the presence of predators. this could not be confirmed in the study. during field observation, it was rather easy to find p. bidens in the open sand or mudflats. behavioral differences between the male and female samples of p. bidens was observed. on the sexes, results of initial experiment suggest that there is no difference as to the aggressiveness. however, the males are more receptive to antagonistic encounters, especially when disturbed inside the aquarium prior to feeding observation. it was difficult for the researcher to catch this sesarmid due to its swiftness and alertness. one striking character of this crab is its fast action in retreating from external stimuli such as touch or other mechanical disturbance. this could be the reason why the feeding experiment did not proceed because the individuals of p. bidens placed in the glass aquaria did not feed on the pieces of r. apiculata, r. mucronata, a. marina and other associated mangrove plants. however, the report of ólafsson et al. (2002) cited that male and female crabs are likely to have different energy requirements, due to differences in the energetic costs of producing eggs or sperm (michell 1993). a study of kyomo (1992) conducted on the sesarmid crab sesarma intermedia showed that females were more specialized in their feeding habits and had higher assimilation rates than males. feeding ecology of tree climbing mangrove sesarmid crabs – jimmy t. masagca 6 n. smithii form burrows around r. mucronata and r. apiculata roots but rarely in avicennia marina found in the mangals of catanduanes. observations indicate that n. smithi feed on leaf litter but also some shrimps, specifically palaemonetes sp. this was observed during the afternoon hours when tides start to rise. observation on emergence and re-emergence of sesarmids in the burrows in general, p. bidens appears to be the most active of the three sesarmids observed during the day. as shown in table 2, the red-clawed grapsoid sesarmid crab, p. bidens emerged and re-emerged from the burrows much faster (mean of 3.48 mins) than the sesarmids n. smithi (mean of 4.15 mins) and e. versicolor (3.93 mins). all three sesarmid crab species display similar behavior of picking up the leaf fragments by first standing still in motionless for about 7 to 10 seconds. some sesarmids, e.g e. versicolor has to consume the leaf fragment where they found it, then migrate for at leas 0.5 meters and to be continued at the mouth of the burrows. some crabs move away from the areas where they got the leaf fragments and continue feeding near the burrows. some bring these fragments inside the burrows but mostly will have the tendency to leave behind when disturbance was made. there is much to be learned as to the sesarmid crab’s feeding ecology in the philippines since there are various similarities and differences in the findings of other authors. some researchers report on the unusual presence or occurrence as to their level of dependence in the different habitats, arboreal or burrows. climbing skills and burrowing abilities also vary depending on the species. while others focus on how sesarmids rush to the mangrove tree and and cling to the branches, it is still unknown whether they are apparently resting or catching some insects or invertebrates. other researchers put premium on their occurrence in the mudflats or soft sediments of mangals for feeding on some leaf litter. moreover, the sesarmids under study have been observed cropping and bustling on sand sediments and other fragments of leaf litter that some crab ecologists might have ignored and do not give emphasis. further studies can be carried out in the future in terms of the feeding ecology of these vital species of mangrove crabs in relation to morphological variation of the feeding apparatus. lastly, deeper reflections on the notions of convergent evolution (fratinni et al. 2005) and the timing of development in relation to arboreal climbing skills and burrowing abilities of sesarmids deserve further attention. table 2. data on the time of re-emergence of sesarmid crabs (p. bidens, n. smithi and e. versicolor) in burrows after disturbance sesarmid crab species time of re-emergence mean time of re-emergence (in minutes) trials (each consists of 3 repeats) (in minutes) 1 2 3 perisesarma bidens 3.35 3.65 3.45 3.4833 neosarmatium smithi 3.95 4.05 4.45 4.1500 episesarma versicolor 2.78 4.25 3.75 3.933 biotropia vol. 16 no. 1, 2009 7 conclusions based on the results presented, selatium elongatum and episesarma versicolor are exclusive mangrove tree climbers (emtc); sarmatium germaini as occasional mangrove tree climber or omtc; and the sesarmid species − neosarmatium smithii, perisesarma bidens and perisesarma eumolpe as non-mangrove tree climbers or nmtc. the sesarmid species, e. versicolor, n. smithii, p. bidens and p. eumolpe posses burrowing skills, while p. bidens appeared to be the most active of the three sesarmids investigated. acknowledgments the author acknowledges with thanks to the administration of de la salle university-dasmariñas (dlsu −d) university faculty research office (ufro) director dr. j. morta; education dean, dr. o. legaspi ; oic vvice chancellor, dr. c. cervillon; and dlsu−d br. president gus boquer for funding this research project on the mangrove crabs. sincere thanks is also given to dr. david gillikin, vrije universiteit, t brussel, belgium and dr. peter k.l. ng of the national university of singapore for sending reprints on the mangrove crabs and ms. marivene manuel, philippine national museum (pnm) for evaluating the earlier drafts of the report on this study. my special thanks to elver sison, while finalizing this paper. the assistance of my brother (rico masagca ); 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(eds.), proceedings of a symposium on mangrove management: its ecological and economic considerations held in bogor, indonesia, august 9-11, 1988. biotrop special publication, no. 37. tan c.g.s. and p.k.l. ng. 1994. an annotated checklist of mangrove brachyuran crabs from malaysia and singapore. hydrobiologia, 285: 75-84. vannini m., oluoch a. and r.k. ruwa. 1997. the tree-climbing crabs of kenyan mangroves. in mangrove ecosystems studies in latin america and africa (b. kjerfve, b.l. de lacerda and e.s. diop, eds.), pp. 325–338. unesco technical papers in marine sciences. new york: unesco. ya b.p., darren c. j. yeo, and peter a. todd. 2008. feeding ecology of two species of perisesarma (crustacea: decapoda: brachyura: sesarmidae) in mandai mangroves, singapore. journal of crustacean biology, 28(3):480-484. biotropia vol. 16 no. 1, 2009 thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html diversity of actinomycetes from eka karya botanical garden bali**, shanti ratnakomala , puspita lisdiyanti , ,1 1 1 2* nita r. prayitno , evi triana yulin lestari , ratih d. hastuti , , misa otoguro3 4 1 5yantyati widyastuti , katsuhiko ando5 4 and endang sukara1, 1research center for biotechnology cibinong 16911, indonesia, indonesian institute of sciences, 2research center for biology cibinong 16911, indonesia, indonesian institute of sciences, 3 aculty of mathematics and natural sciences, i p b ,680department of biology, f nstitut ertanian ogor, bogor 16 indonesia 4soil research institute, bogor 16002, indonesia 5 inite biolog cal resource center (nbrc), 2-5-8 kasuzakamatari, kisarazu, chiba, japan received 1 july 2015/accepted 6 may 2016 abstract a total of 2 actinomycetes were isolated 29 strains of and identified by full sequence of 16s rrna gene analysis. samples ed from , bali island,consist of 18 soil and 20 leaf litter were collected eka karya botanical garden indonesia. two isolation methods, sdseast xtract (sy) and rehydration-centrifugation (rc) used . i.e. y e were in this study based on 16s rrna gene analysis, isolated actinomycetes may be grouped into 2 genera. b 8 ased on molecular analysis of 16s rrna gene similarities showed that eka karya botanical garden . isolated actinomycetes of origin diverse is analysis on from soil samples, resulted in is most 144 isolates 24 genera and more than 87 species. the streptomyces dominant gen where or from isolated actinomycetes belong to this genus. it was followed by us 65 isolates 45% actinoplanes (25 isolates = 17%). from leaf-litter 85 isolates 9 genera samples, the total number of may be group into ed and more than 41 species. the most dominated genus is (42 isolates = 49%) (16 actinoplanes catenuloplanesfollowed by isolates = 19%). eka karyakeywords: 16s rrna gene analysis, actinomycetes, biodiversity, botanical garden introduction actinomycetes are microorganisms belong to gram positive bacteria which are often saprophytic while some of them produce spores and mycelium. they play t roles n degrading importan i and decomposing organic compounds in the soil. they also produc secondary metabolites may e such as antibiotics, enzymes and other bioactive compounds for human welfare. actinomycetes constitute a significant component of the microbial population in most soils and counted of over 1 million per gram of soil. oil is, cells s therefore this , the most prolific source of particular group. s oil represents the most intensively studied habitat of actinomycetes. actinomycetes are thought to be most also the significant in the degradation of relatively group complex, recalcitrant polymers naturally in found plant litter and soil (hopwood 2007; baskaran et al. 2011). the preliminary characterization of the actinomycetes isolates was colony appearance, observing of zoospore bearing isolates, and dap analysis. dap analysis is a biochemical itself analysis of cell walls to observe the dap isomers of g the cell wall of actinomycetes. most of ram positive bacteria have lysine instead of dap in the ir c ell wall , . t hro ugh thi s met hod actinomycetes could be into 3 groupsgrouped . according miyadoh (2004), actinomycetes that to have ll-dap in their cell wall generally belong to genus and ; while those streptomyces streptacidiphilus with -dap type in their cell wall generally meso belong to the non or so called rare streptomyces actinomycetes. actinomycetes that have both biotropia vol. 23 no. 1, 2016: 42 51 doi: 10.11598/btb.2016.2 . .5043 1 * c orresponding author: yahoo.comshanti_ratna@ ** this paper was presented at symposium on recent advances on microbiological researches and its application (8-9 november 2011). serpong, indonesia. 42 ll-dap and meso-dap usually belong to genus kitasatospora. therefore, the dap analysis could differentiate those three types of actinomycetes. some actinomycetes share the same ability to release flagellated zoospores at a certain stage in their life cycle (cross 1986). current classification of motile-spored actinomycetes can identify at least six suborders containing zoosporic genera, including micr omonosporinae, micr ococcinae, frankinae, pseudonocardinae, kineosporiineae and streptosporanginae et al (stackebrand . 1997). these t zoospores bearing actinomycetes have been associated with river, lake and fresh water, river sediments, desert soil (garrity . 1996 bredholt et al ; et al. et al 2008 sibanda . 2010), decaying plant ; materials submerged in streams and cast up on lake shores (kudo 1998 tamura . 2010), et al. et al; blades of grass inhabiting streams and soils (hasegawa 1991). it has become increasingly apparent that motile actinomycetes can produce a variety of antibiotics and other bioactive metabolites or be used for biochemical conversion of complex compounds (hasegawa 1991 garrity . 1996 khamna . 2010 ; ; ;et al et al khanna . 2011).et al according to hayakawa (2000), et al. actinomycetes could be divided into two types based on spores produced by the non-motile and motile. actinomycetes which bear non-motile spores that are not generally form flagella, for example, are , , , streptomyces nocardia micromonospora and so on. and are actinoplanes catenuloplanes motile zoospores bearing actinomycetes and the zoospores can move. several actinomycetes genera such as actinoplanes, amycolatopsis, catenuloplanes, dactylosporangium, kineospor a, i microbispora, micromonospora nonomuraeaand are often very difficult to isolate and cultivate due to their slow growth and those rare belong to the actinomycetes (hayakawa 2008). eka karya botanical garden, bedugul bali in , island is a unique plant , indonesia ex situ conservation site for plant species of high elevated eastern tropical rain forest of indonesia, adjoining with the tropical forest of batukahu nature reserve. this garden is located at 1,2501,450 m above sea level, with area of 157.5 hectares (389 acres). temperature is about 17 25 c in daytime and is dropped to 10-15 c at o o night with 70-90% humidity (mukaromah & suparta 2007). based on the uniq ness of the ue above location, we stud about the diversity of ied actinomycetes this location. this study wasin intended to be done as pioneer research in eka karya botanical garden. several stud on ies diversity of actinomycetes were to be done in indonesia, such as from lombok island (lisdiyanti et al. 2012) and cibinong science center (widyastuti et al. 2013). to obtain new strains that poten ially produce new can t metabolites, it is still necessary to conduct exploration and examination of obtained samples from diverse habitats and environments. few parts of the research had been orally presented in 2011 during symposium on recent advances on microbiological researches and its application, c o n d u c t e d by i n d o n e s ia n s o c i e t y f o r microbiology (permi) in serpong. materials and methods sampling ethodsm soil samples were obtained from eka karya botanical garden located at 1,250-1,450 m above sea level, 115 9'0-58” e and 8 15-17'0-59” n, o o with 5-10 cm depth from soil surfacesoil ph of . the soil samples was between 6.0-6.5. the samples were put in plastic bag ecaying immediately to . d leaf-litter samples were collected from soil surface were put in. the samples immediately to paper bag. all samples were air dried at room temperature for 1-2 weeks, ground using blender and filtered with 200 m mesh filter paperµ . sds-yeast extract (sy) solation ethodi m sy isolation method described by was widyastuti (201 ) a combination of 0.05% et al. 3 . sds (sodium dodecyl sulphate) as a germicide to eliminate soil bacteria, 6% yeast extract as spore activating agents and heating at 40 c for 20 o minutes increase the recovery of could also s actinomycetes from various soil samples. this method was used for isolating general actinomycetes. rehydration and centrifugation (rc) i msolation ethod the rc isolation method was used for isolati . the sample is ng motile actinomycetes rehydrated by air-dried container in 10 mm phosphate buffer containing 10% soil extract, at 43 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala 30 c for 90 minutes, followed by centrifugation at o 1,500 x g for 20 minutes (hayakawa . 2000 et al ; otoguro 2001 widyastuti . 201 )et al. et al; 3 . humic acid with vitamins (hv) ediumm hv medium contained (in liter) 1 g humic was acid, 0.02 g caco , 0.01 g feso .7h o, 1.71 g 3 4 2 kcl, 0.05 g mgso .7h o, 0.5 g nahpo , 5 ml of 4 2 4 vitamins solution, 50 mg cycloheximide, 18 g agar, ph 7.2. the composition of vitamins solution was 0.5 mg thiamine hcl, 0.5 mg riboflavin, 0.5 mg niacin, 0.5 mg pyridoxine hcl, 0.5 mg inositol, 0.5 mg ca-panthotenate, 0.5 mg p-aminobenzoic acid and 0.25 mg biotin in 5 ml water and sterilized by 0.22 m filtration (hayakawa & nonomura 1987). this vitamin solution was added after autoclave sterilization. analysis of diaminopimelic acid (dap) preliminary biochemical test performed is the dap determination using a method of thin layer chromatography (tlc) on cellulose to separate isomers of dap (hasegawa . 1983). three et al loops of the cells was put in screw cap plastic tube, added with three drops of 6n hcl, autoclaved at temperature of 121 c, 1 atm for 15 minutes, and o then applied to cellulose chromatography plate. tlc eluent solution used was mixture of methanol: water: 6n hcl: pyridine (80: 26: 4: 10 v/v), and eluted for 12 hours. after that, the spots were sprayed with ninhydrin solution (0.3 g ninhydrin in 100 ml of butanol + 3 ml of acetic acid), and heated at 100 c for 3 minutes.o preparation of emplate dna and pcr t a gmplification of 16s rrna ene chromosomal dna was extracted as described by saito & miura (1963) from 14-dayold cell cultures grown on yg agar medium by using dneasy plant maxi kit (qiagen). 16s rrna gene replication reaction was performed using primer pair, 9f (forward: 5'-gagtttgatcctggctcag-3' positions 9-27) and 1541r (reverse: 5'-aaggaggtgatccagcc-3' position 1541-1525) of numbering escherichia coli system (brosius 1978). pcr amplification et al. was performed used taka a ex taq with total r volume of 50 l, consisting of 0.4 mm of each primer, 1 ng of dna template, 2.5 mm of dntp, 1 of ta a a pcr buffer, and 5u of taq k r polymerase in final volume. pcr conditions was 95 c for 3 minutes to denaturate the target dna, o then by 30 cycles at 95 c for 3 seconds for o denaturation again, 55 c for 15 minutes for o primer annealing, and 72 c for 1 minute for o primer extension, and subsequently, 1 cycle at 72 oc for 5 minutes to complete the process of amplification. pcr reaction was conducted using a geneamp pcr system 9700 (applied biosystem). pcr products were examined by electrophoresis on agarose 2%, to assure that the target dna had been amplified. pcr products were then purified using the gfx-96 pcr purification kit (amersham pharmacia biotech), with reference to the protocol. 16s rrna ene equencingg s pcr products that had been purified were cycle sequenced using the bigdye terminator sequence with version 3.1 cycle sequencing kit. this reaction used 6 primers to amplify 1,500 bp of 16s rrna gene, which is 9f, 515f (5'gtgccaagcagccgcggt-3' position 515531), 1099f (5'-gcaacgagcgcaaccc-3' position 1099-1114), 536r (5'-gtattaccgcggctgcttg-3' positions 536-519), 1115r (5'agggttgcgtcgttg-3' position 1115 1100), and 1541r of numbering escherichia coli system (brosius . 1978). in total 10 l of et al reaction sequence containing 2.0 l of big dye terminator premix, 1.0 l of 5 big dye sequencing buffer, 0.8 l of each primer (1 pmol/l), and 0.5 l of template dna were synthesized of the chain by using a geneamp pcr system 9700 (applied biosystem) with the following conditions predenaturation at 96 c for 1 minute, 45 cycles at a o temperature of 96 c for 10 seconds for o denaturation, 50 c for 5 seconds for primer o annealing, and 60 c for 90 seconds for primer o extension, and subsequent to storage at 16 c. the o product was purified using dyeex 96 kit (qiagen) and sequenced using abi prism 3700 (applied biosystem) dna sequencer. sequence ata nalysis and lignment d a a search 16s rdna sequence was translated from the 16s rrna gene by using atgc sequencing analysis software version 7.3 (abi prism) and corrected manually. nucleotide sequence data of the isolates was searched the closest homology 44 biotropia vol. 23 no. 1, 2016 with other strains in the 16s rrna gene data base using blast (http://www.ncbi.nlm.nih. gov) . results and discussion from t 38 he total number of samples consist of ed 18 soil samples and 20 leaf-litter samples 409 actinomycetes were isolated, . a total of 229 isolates based on the colony appearance, were selected and used study. in this from 229 isolates, 144 were isolated from soil samples and 85 were isolated from leaf-litter samples. from soil samples, 60 and 84 actinomycetes were isolated by sy and rc isolation method, respectively; and 85 from leaf-litter samples by using rc were isolated isolation method ( ). the dap analysis table 1 showed that within the actinomycetes isolated from soil source by sy isolation method, 18 isolates had ll-dap, 31 isolates had /ll-meso meso/oh dap, 11 isolates not but the rest d id have dap polymers containing . actinomycetes isolated by rc isolation method showed that 24 isolates had ll-dap, 48 isolates had -meso dap/ll/oh in their cell wall, and the rest meso 12 isolates d not have dap id containing polymers using. from leaf litter source rc isolation method, 3 isolates had ll-dap, 66 isolates had dap/lloh on their cell meso meso/ wall, and 16 isolates not dap containing d have id polymers. no suborder no family no genus blast result >99 % 98 % 97 % <96 % total 1 corynebacterineae 1 nocardiaceae 1 nocardia 2 5 2 9 2 rhodococcus 1 1 2 frankineae 2 cryptosporangiaceae 3 cr yptosporangium 1 1 3 kineosporiaceae 4 kineosporia* 7 3 10 3 micrococcineae 4 intrasporangiaceae 5 lapilicoccus 1 1 5 promicromonosporaceae 6 pr omicromonospora 1 1 4 micromonosporineae 6 micromonosporaceae 7 actinoplanes* 8 33 22 4 67 8 catellatospora 1 1 9 catenuloplanes* 12 4 16 10 dactylosporangium* 3 3 11 krasilnikovia* 3 3 12 micromonospora 3 4 1 8 13 verrucosispora 1 1 5 propionibacterineae 7 nocardioidaceae 14 kribbella 2 2 15 nocardioides 1 1 2 6 pseudonocarnineae 8 actinosynnemataceae 16 actinokineospora* 1 1 17 saccharothrix 1 1 9 pseudonocardiaceae 18 amycolatopsis 1 2 3 19 pseudonocardia 3 1 4 20 saccharomonospora 1 1 7 streptomycineae 10 streptomycetaceae 21 kitasatospora 5 2 7 22 streptomyces 42 24 4 3 73 8 streptosporangineae 11 nocardiopsaceae 23 nocardiopsis 4 4 12 streptosporangiaceae 24 acrocar pospora 1 1 25 nonomuraea 1 2 2 5 26 streptosporangium 1 1 13 thermomonosporaceae 27 actinocoraliia 1 1 28 actinomadura 1 1 73 98 41 17 229 45 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala table 1 number of isolated and selected actinomycetes from eka karya botanical garden, indonesia sampling site source no. of samples isolation method selected isolates dap isomer ll m/ll-m/oh nd eka karya botanical garden soil 18 sy 60 18 31 11 18 rc 84 24 48 12 leaflitter 20 rc 85 3 66 16 38 229 45 145 39 table 2 actinomycetes isolated from eka karya botanical garden, bali, 2003 indonesia, note: * = zoospore-bearing actinomycetes http://www.ncbi.nlm.nih. identification of 229 isolates based on 16s rrna gene sequencing showed that the isolates belong to 8 suborders, 13 families and 28 genera of the lass actinomycetales (table 2). the largest c g r oup of ac tino my ce te s f ou nd be long .to ( ) genus 73 isolates the second streptomyces largest group genus 67 belong to (actinoplanes isolates he third largest group ) belong to . t genus 16 isolates . about 58 catenuloplanes ( ) isolates (25%) may be new species or new genus, because it has <98% of 16s rrna gene similarity compared to the known strains in the database. and cultures sy isolation method incubation on hv agar plates containing nalidixic acid introduced by hayakawa and nonomura (1989) improve the possibilities of isolating d actinomycetes while decreasing the number of bacterial . this method proved to be an colonies effective tool for isolating act nomycete . i s rc isolation method described hayakawa . by et al ( ) and ( found to et al2000 otoguro . 2001) was also be an effect ve tool for the ion ofi isolat zoospore from the genera of , , actinoplanes actinokineospora actinosynnema catenuloplanes, dactylosporangium, , geodermatophylus kineospor a.iand the phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material, and the centrifugation eliminated streptomyces and other non-motile actinomycetes. in general actinomycetes isolated , using sy were by many non-method dominated motile actinomycetes, while those isolated using the rc method we motile re dominated by actinomycetes. rc is an isolation method developed isolat motile zoospore for ing (hayakawa . 2000).et al weall 229 selected isolates re identified using m procedure olecular identification based on full sequence of 16s rrna gene (±1,500 bp). the isolates were identified into genus and further species level by blast and phylogenetic tree construction. currently, actinomycetes consisted of 24 families, 80 genera and 500 species (liu . et al 2009). in study, identify 8 suborders, our we could 13 families and 28 genera ( )table 3 . we predicted that there more than were 109 species. this is the first comprehensive study of actinomycetes conducted in eka karya botanical garden, bali island, indonesia. table 3 diversity of actinomycetes in soil samples note: * = zoospore bearing actinomycetes 46 biotropia vol. 23 no. 1, 2016 no suborder no family no genus no of species rc sy total 1 corynebacterineae 1 nocardiaceae 1 nocardia 4 1 8 9 2 rhodococcus 1 1 1 2 frankineae 2 kineosporiaceae 3 kineosporia* 1 1 1 3 micrococcineae 3 promicromonosporaceae 4 promicromonospora 1 1 1 4 micromonosporineae 4 micromonosporaceae 5 actinoplanes* 12 25 25 6 catellatospora 1 1 1 7 dactylosporangium* 1 3 3 8 krasilnikovia* 1 3 3 9 micromonospora 1 2 2 10 verrucosispora 1 1 1 5 propionibacterineae 5 nocardioidaceae 11 kribbella 2 2 212 nocardioides 2 2 2 6 pseudonocarnineae 6 actinosynnemataceae 13 actinokineospora 1 1 1 14 saccharothrix 1 1 1 7 pseudonocardiaceae 15 amycolatopsis 2 1 2 3 16 pseudonocardia 3 2 1 3 7 streptomycineae 8 streptomycetaceae 17 kitasatospora 5 6 1 7 18 streptomyces 36 33 32 65 8 streptosporangineae 9 nocardiopsaceae 19 nocardiopsis 4 2 2 4 10 streptosporangiaceae 20 acrocarpospora 1 1 1 21 nonomuraea 3 2 3 5 22 streptosporangium 1 1 1 11 thermomonosporaceae 23 actinocoraliia 1 1 1 24 actinomadura 1 1 1 87 84 60 144 diversity of ctinomycetes on oil amplesa s s from soil samples, we obtained 144 isolates of actinomycetes that had been identified by 16s rrna gene analysis and preserved well in liophilized form. the isolates contained 24 genera and more than 87 species. the most dominated genera in the soil samples was (65 streptomyces isolates = 45%) and the next was (25 actinoplanes isolates = 17%). based on the isolation methods, 15 genera (60 isolates) were by sy isolation successfully isolated method and 16 genera (84 isolates) were isolated by rc isolation method. , genera of nocardia rhodococcus catelatospora micromonospora kribbella, , , , nocardioides streptosporangium actinocoralia, , and actinomadura were easily isolated using sy isolation method; while rc method was useful for isolating genera , , actinoplanes krasilnikovia dactylosporangium verrucosispora actinokineospora, , and . most of soil actinomycetes saccharothrix isolated by rc isolation method belong to zoospore bearing actinomycetes. by using different isolation method, the dominant species of actinomycetes were also differe . in isolated nt this study, we proved that actinomycetes isolated using the rc method were dominated by groups of zoospore bearing actinomycetes. this result is similar to that described by et al.hayakawa (2000) and otoguro (2001).et al. several ecological factors that played a role in the distribution of genera actinomycetes included humus content and ph of the soil (nonomura & hayakawa 1988), climate may influence the specific type of soil-inhabiting actinomycetes (hayakawa . 2010). soil of eka karya et al botanical garden a humus-rich soil with ph is range from 6 to 6.5. this soil type is suitable for the growth of actinomycetes. some of actinomycetes are distributed in iverse plant species plant rhizosphere soils. d found in the garden should also support the growth of actinomycetes . actinomycetes have been found to play an important role in rhizosphere soil (suzuki . 2000 el-tarabily & et al ; sivasithamparam 2006). there is a possibility that these microorganisms can protect plant roots from plant pathogen and promote plant growth. figure 1 phylogenetic position based on 16s rrna sequences of several isolates under the genera from eka karya nocardia botanical garden. bar, 1 substitutions per 200 nucleotides 47 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala for plant root protection, the modes of action of actinomycetes include antibiosis, parasitism, the production of extracellular hydrolytic enzymes and competition for iron (getha . 2005 ;et al errakhi . 2007). sy isolation method was et al particularly successful for isolating common actinomycetes such as spp. in natural streptomyces habitats, streptomycetes are common and are usually a major component of the total actinomycetes population. kim (1984) reported that within population in the soil, actinomycetes are dominated by (95 43%).streptomyces . d indicate identification by molecular approach that actinomycetes obtained from eka karya botanical garden have potential should value as a source find newto new species or genus. based on the analysis of 16s rrna gene, <97% sequence were in homology with the closest species on blast searching compared to the current database. n ew species and new genus among the strains studied re obviouswe . the16s rrna gene sequence of strain id03-0848 and id03-0856 were aligned with those of the type species of the major and other actinomycete lineages. nocardia the resulting phylogenetic tree is shown in figure 1. strain id03-0848 and id03-0856 formed a coherent clade within the lineage, clearly nocardia distinguished from other described strains with highly bootstrap value. this was suspected to be new genus or new species in the lineage.nocardia diversity of ctinomycetes on eaf-litter a l samples meanwhile, from the leaf-litter as a source material, we obtained 85 isolates of actinomycetes that had been identified by 16s rrna gene analysis and preserved well in liophilized form. the isolates contained 9 genera ( ) and table 4 more than 41 species. the most dominated actinoplanesgenus was (42 isolates = 49%) and catenuloplanesthe next was (16 isolates = 19%) and (9 isolates = 10%). the same as in kineosporia soil samples, most of the leaf-litter actinomycetes isolated by rc method belong to the zoospore bearing actinomycetes. this finding is in agreement with other reports which mentioned that actinomycetes belonging to genera actinoplanes, catenuloplanes kineosporia and were frequently isolated from leaf-litter samples (pagani 1978 kudo . 1998 & parenti ; ;et al hayakawa . 2000 ratnakomala 2011).et al et al.; they showed very similar characteristics such as possession of motility, absence or rarity of hydrophobic aerial hyphae and formation of orange colonies, similar to the color of fallen leaves (van hop 2011).et al. et al. et alxu (1996) and meliani . (2012) reported that there was a positive correlation between diversity of actinomycetes with vegetation. land of primary forest has higher diversity of actinomycetes compared with land of secondary forest and agricultural land. on dry, barren and cold land, there are less actinomycetes found (xu 1996 garrity . 1996). search et al. et al; of new active compounds, especially from actinomycetes requires a large number of isolates. it would be more promising if sampling and isolation techniques more specific (lo . are et al 2002). therefore, it is essential to look for unique types of vegetation where the soil sample will be taken new taxonomical for finding ly important actinomycetes. it is also to find important table 4 diversity of actinomycetes from leaf-litter samples note: * = zoospore bearing actinomycetes 48 biotropia vol. 23 no. 1, 2016 no suborder no family no genus no of species rc 1 frankineae 1 cryptosporangiaceae 1 cryptosporangium 1 1 2 kineosporiaceae 2 kineosporia* 1 9 2 micrococcineae 3 intrasporangiaceae 3 lapilicoccus 1 1 3 micromonosporineae 4 micromonosporaceae 4 actinoplanes* 21 42 5 catenuloplanes* 2 16 6 micromonospora 6 6 4 pseudonocarnineae 5 pseudonocardiaceae 7 pseudonocardia 1 1 8 saccharomonospora 1 1 5 streptomycineae 6 streptomycetaceae 9 streptomyces 7 8 41 85 a metaboli . cti omycete with new c propertiesn s t find a new actimomycete here is a possibility to species for the tion of newproduc antibiotics or other secondary metabolites. these microbes will specifically generate new secondary metabolites which allow them to degrade toxic compounds from these plants (park 1999 ho et al. ; et al . 2000). eka karya botanical garden is one place for conservation of tropical ex situ plant forests in indonesia understood that high . it is diversity of actinomycetes will likely to be found in place.such selection of proper method of isolation is crucial to obtain new actinomycetes species. wasit obvious from our study that the use of rc method was useful to isolate new significantly species from leaf-litter samples, especially from genus kineosporia . the 16s rrna gene sequences of 10 strains (id03-0578, id03-0677, id03-0678, id03-0683, id03-0684, id03-0714, id03-0716, id03-0722, id03-0739 and id03-0760) were aligned with those of type species of the major kineosporia and other actinomycete lineages. as shown in , strain id03-0739 and id03-figure 2 0714 were moderately related to the type strain k. succinea ab003932. strain id03-0683 and id03-0760 were related to type strain k. rhizophila ab003933. strain id03-0684 was closely related to type strain d86937. strain id03-k. aurantiaca 0677 and id03-0678 shared the same branching position and formed a single clade with id030716 and id03-0722. these four strains were clearly distinguished from other described strains with highly bootstrap value. this was suspected to be new genus or new species in the kineosporia lineage. this study is significantly important to describe the diversity of actinomycete in s indonesia. there are ample space to use isolated s actinomycete for the benefit of society. further s research on several important taxa including proposing new species or genus is mandatory. more d ata on phenoty pe, bioche mical characterization, dna-dna hybridization and chemotaxonomic are required. conclusions selection of proper isolation method is crucial to obtain a new actinomycetes species. using sy isolation method, this research was successfully isolated 2 new species of actinomycetes from eka k ar ya botanical garden. this study is significantly important to describe the diversity of actinomycete in indonesia. there are ample s space to use isolated actinomycete for the benefit s of society. f on important urther research some taxa including for proposing new species or genus is mandatory more. data on phenotypic, biochemical characterization, dna hybridization and chemotaxonomic data are required to describe the other actinomycetes candidates as new species. figure 2 phylogenetic position based on 16s rrna sequences of several isolates under the genera from eka kineosporia karya botanical garden. bar, 1 substitution per 100 nucleotides 49 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala 0.01 kax 1000 1000 1000 1000 1000 665 641 938 995 644 532 438 540 567 536 1000 861 709 999 629 id03-0739 id03-0714 id03-0760 id03-0683 id03-0684 id03-0678 id03-0722 id03-0716 id03-0677 id03-0578 kineosporia succinea (ab003932) kineosporia rhizophila (ab003933) kineosporia aurantiaca (d86937) kineosporia aurantiaca (x87110) kineosporia aurantiaca (ab003931) kineosporia aurantiaca (af095336) kineosporia rhamnosa (ab003935) kineosporia rhamnosa (ab003934) kineospria radiotolerans (af247813) kineosporia aurantiacus (ab007420) kineococcus mikuniensis (x92618) cryptosporangium japonicum (d85466) cryptosporangium sp. (ab006168) cryptosporangium arvum (d85465) cryptosporangium aura (ab047490) cryptosporangium minu (ab037007) streptomyces lavendulae (d85116) acknowledgements this study was conducted under the joint research project between department of biotechnology, national institute of technology and evaluation, japan and the indonesian institute of sciences (lipi) representing indonesian government research institutes. the authors thanked eka karya botanical garden, lipi and technician in nite and research s center for biotechnology lipi for their assistance. references baskaran r, vijayakumar r, mohan pm. 2011. enrichment method for the isolation of bioactive actinomycetes from mangrove sediments of andaman islands, india. malay j microbiol 7(1):26-32. bredholt h, fjærvik e, johnsen g zotchev sb. 2008. , actinomycetes from ediments in the trondheim s fjord, norway: iversity and iological ctivity. d b a mar drugs 6(1):12 24. brosius j, palmer ml, kennedy pj, noller hf. 1978. complete nucleotide sequence of a 16s ribosomal rna gene from . proc natl acad sci escherichia coli 75(10):4801-5. cross t. 1986. the occurrence and role of actinoplanetes and motile actinomycetes in natural ecosystems. in: megusar f, gantar m, . perspectives in (eds) microbial ecolog y. proceedings of the iv international symposium on microbial ecology. p 265 70. el-tarabily ka, sivasithamparam k. 2006. nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. soil biol biochem 38: 1505-20. errakhi r, bouteau f, lebrihi a, barakate ml. 2007. evidences of biological control capacities of streptomyces sclerotium rolfsi i spp. against responsible for damping disease in sugar beet ( l.). beta vulgaris world j microbiol biotechnol 23:1503-9. garrity gm, heimbuch bk gagliardi m. 1996. isolation of , zoosporogenous actinomycetes from desert soils. j ind microbiol 17:260-7. getha k, vikineswary s, wong wh, seki t, ward a, goodfellow m. 2005. evaluation of sp. streptomyces strain g10 for suppression of wilt and fusarium rhizosphere colonization in pot-grown banana plantlets. j indian microbiol biotechnol 32:24-32. hasegawa t, takizawa m, tanida s. 1983. a rapid analysis for chemical grouping of aerobic actinomycetes. j gen appl microbiol 29:319 22.hasegawa t. 1991. studies on motile arthrospore-bearing rare actinomycetes. actinomycetol 5(2):64-71. hayakawa m, nonomura h. 1987. efficacy of artificial h a s n aumic cid as a elective utrient in hv gar used for the solation of oil ctinomycetes. j ferment i s a technol 65(6):609-16. hayakawa m, nonomura h. 1989. a new method for the intensive isolation of actinomycetes from soil. actinomycetol 3(2):95-104. hayakawa m, otoguro m, takeuchi t, yamazaki t, iimura y. 2000. application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. antonie van leeuwenhoek 78:171-85. hayakawa m. 2008. studies on the isolation and distribution of rare actinomycetes in soil. actinomycetolo 22:12 9.hayakawa m, yamamura h, sakuraki y, ishida y, hamada m, otoguro m, tamura t. 2010. diversity analysis of actinomycetes assemblages isolated from soils in cool-temperate and subtropical areas of japan. actinomycetol 24:1-11. ho cc, tan gya, seow i, ajam n, tan ei, goodfellow m, ward ac, brown r, wong nk, lo cw, cheah hy, lai ns, suzuki ki. 2000. isolation, characterization and biological activities of actinomycetes isolated from dipterocarp rain forest soils in malaysia. in: nnga bh, tan hm, suzuki k-i, editor. microbiology diversity in asia. singapore: world scientific. hopwood da. 2007. , streptomyces in nature and medicine the antibiotic makers. uk: oxford university press, inc. khamna s, yokota a, peberdy jf, lumyong s. 2010. indole3-acetic acid production by sp. isolated streptomyces from some thai medicinal plant rhizosphere soils. eurasia j biosci 4:23-32. khanna m, solanki r, lal r. 2011. selective isolation of rare actinomycetes producing novel antimicrobial compounds. int j adv biotechnol res 2(3):357-75. kim cj. 1984. isolation and screening of actinomycetes from natural environments. swed n: genetic e engineering research institute, kist. kudo t, matsushima k, itoh t, sasaki j, suzuki k. 1998. description of four new species of the genus kineosporia kineosporia succinea kineosporia : sp. nov., rhizophila kineosporia mikuniensis sp. nov., sp. nov. and sp. nov., isolated from kineosporia rhamnosa plant samples, and amended description of the genus . int j syst bacteriol 48:1245 55.kineosporia liu n, wang h, liu m, gu q, zheng w, huang y. 2009. streptomyces alni sp. nov., a daidzein-producing endophyte isolated from a root of alnus nepalensis d.don. int j syst evol microbiol 59:254-58. lo cw, lai ns, cheah hy, wong nki, ho cc. 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transforming deoxyribonucleic acid by phenol treatment. biochimica et biophysica acta 72:619-29. sibanda t, mabinya lv, mazomba d, akinpelu da, bernard k, olaniran ao, okoh ai. 2010. antibiotic producing potentials of three freshwater actinomycetes isolated from the eastern cape province of south africa. int j mol sci 11:2612-23. stackebrandt e, rainey fa, ward-rainey nl. 1997. proposal for a new hierarchic classification system, actinobacteria classis nov. int j syst bacteriol 47: -479 91. suzuki s, yamamoto k, okuda t, nishio m, nakanishi n, komatsubara s. 2000. selective isolation and distribution of strains in actinomadura rugatobispora soil. actinomycetol 14:27 33.tamura t, ishida y, otoguro m, suzuki k. 2010. amycolatopsis helveola amycolatopsis sp. nov. and pigmentata sp. nov. isolated from soil. intl j syst evol microbiol 60:2629 33.vanhop d, sakiyama y, binh ctt, otoguro m, hang d t, miyadoh s, luong dt, ando k. 2011. taxonomic and ecological studies of actinomycetes from vietnam: isolation and genus-level diversity. j antibiot 64:599 606.widyastuti y, lisdiyanti p, ratnakomala s, kartina g, ridwan r, rohmatussolihat r, ando k. 2013. genus diversity of actinomycetes in cibinong science center, west java, indonesia. microbiology indonesia 6(4):165. xu lh, li qr, jiang cl. 1996. diversity of soil actinomycetes in yunnan, china. appl environ microbiol 62(1):244-8. 51 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala http://www.arbec. the expansion of (l.) merrill in merremia peltata fragmented forest of bukit barisan selatan national park enhanced by its ecophysiological attributes yansen*, wiryono, deselina, muhammad f. hidayat efratenta k. depari and department of forestry, university of bengkulu, bengkulu, indonesia r 4eceived 28 november 2013/accepted 28 september 201 abstract forest opening and fragmentation may affect the composition of vegetation by permitting the expansion or domination of one or two species in the area. this study found that , an invasive climbing creeping merremia peltata / species, has dominated some area of bukit barisan selatan national park (bbsnp). domination of this species was favoured by forest fragmentation, indicated by the significantly higher number of individuals of in the open m. peltata area than in intact forest. the ecophysiological characters of support the ability of this species to become a m. peltata strong invader. as the expansion of this species may negatively affect the biodiversity and vegetation health, the application of appropriate ecological approaches to control the domination of in the conservation area is m. peltata necessary. keywords: ecophysiology, forest fragmentation , population , merremia peltata size introduction the abundance of species in an area is a fundamental ecological parameter and a critical consideration in decision making for conservation (he gaston 2000). on a geographic scale, one & species might be abundant in one region but rare in other regions (brown 1984). unlike temperate forests, where a single factor may drive abundance and distribution of species (he 1997), many et al. underlying factors control these patterns in tropical rainforest ecosystems. some of those factors are intra and interspecific competition, predation, niche differentiation, disturbances and stochastic recruitment (janzen 1970 connell ; 1978; denslow 1987). disturbances may stimulate the regeneration in the tropical rainforest. the falling of large individual or group of trees is the most common cause of natural gap formation. this will create an open area inside the forest, which has different microclimate, particularly light intensity, from the surrounding shaded areas. this opening will allow more light reaching the forest floor stimulating seed germination and seedling establishment. many rainforest species of seedlings need more light to grow faster. however, large scale disturbances may negatively affect plant communities. ecosystem disturbances are important in changing the dynamic of species competition and may increase the opportunity of certain species to invade the ecosystem. however, the success of alien or local species to dominate one area is influenced by their biological attributes, environmental characteristic of the invaded area and biotic interaction with earlier communities, including interspecific competition (vila weiner 2004).& in the forest area, ecosystem disturbances may negatively affect the composition of vegetation by permitting the expansion or domination of one or two species in the area. biological invasion by invasive species is one of the major environmental problems which could threaten biodiversity, affect forest vegetation health and in the long term it could reduce the ability of forest to absorb carbon (vila weiner 2004 clavero& ; & garcia-berthou 2005; master . 2013). et al* corresponding author : y.yansen@ymail.com biotropia vol. 22 no. 1, 2015: 25 32 doi: 10.11598/btb.2015.22.1.353 25 mailto:y.yansen@ymail.com biotropia vol. 22 no. 1, 2015 26 thus, if invasive species invade a large area of forests, the ability of forest ecosystem to mitigate climate change may also be affected. on the other hand, invasive species may be better adapted to the temperature rise (hellman 2008).et al. merremia peltata (l.) merrill (convolvilaceae) is a climbing creeping plant that originally comes / from the pacific. this species has been reported to become invasive in many areas of the pacific. it grows very quickly in the open area and dominates degraded ecosystems (kirkham 2004). the development of this species has become a major threat for plant and animal biodiversity in national parks. bukit barisan selatan national park (bbsnp) is the third-largest area conservation in sumatera covering about . it is l3,568 km ocated in 2 southwest sumatera (4 31 5 57 s, 103 34 104 43 0 0 0 0'' '' e) 150 km along the barisan , which extends bukit range. the area of bbsnp spans across two provincial administrations, lampung and bengkulu. t contains some of his national park the largest tracts of lowland forest remaining on sumatera (o'bri n . 2004)e .et al the expansion of in relation to forest m. peltata fragmentation has been happening in bukit barisan selatan national park (bbsnp). the development of has dominated about m. peltata 7,000 ha of bbsnp area (master . 2013). this et al species grows very quick y in the open area and l forest edges. the invasion of this species results in significant ecological consequences. m. peltata creeps and covers the area and block the s development of seedling. it also climbs up the host trees or shrubs and may eventually kill them. master . (2013) found that area dominated byet al m. peltata has lower diversity index than area where m. peltata is not dominant. mega faunas, such as elephant, are also found to avoid area where this liana is dominant. in the long run, the expansion of this type of invasive species may contribute to the extinction of other species (clavero garcia & berthou 2005). existence of in an area affects species m. peltata conservation. therefore, ecological aspects of this species need to be studied. to date, there is lack of information on the ecological aspects and ecophysiological characters of this species. available information on this species so far includes the record on the invasive plants of the pacific (josekutty . 2002), its natural et al distribution in certain area in the pacific (kirkham 2004) and its impact on the plant biodiversity in the invaded area (master . 2013). the et al information on the ecological and ecophysiological characters may provide scientific foundation in future management of this species. therefore, this research investigates the population of in open density and size m. peltata and intact forests and its ecophysiological characters which may contribute to this species ability to invade a certain areaforest . materials and methods this study was conducted in pemerihan resort of bbsnp in the lampung province . (fig. 1) pemerihan resort is about 19,009 ha. this area was chosen as has started dominating m. peltata several sites. however, compared with other areas in the bbsnp, such as tampang way haru and resorts, the development of in the m. peltata pemerihan resort has not been as massive as in those two resorts. therefore, this research may provide insight into earlier stage of the invasion of this species. early obser vation indicated that the proliferation of might be enhanced by m. peltata forest opening; therefore, ecological aspects of this species were studied by comparing its population structure in open/fragmented and intact forests. intact forest is dominated by large trees, while open area is dominated by shrubs and smaller trees. these two areas have distinct microclimates. light intensity in the two areas was observed by using solar radiation meter. fifty (50) 5 x 5 m plots were established in the open area where this species was dominant. other 50 plots were placed in the intact forest next to the open area. the number of was counted and m. peltata the stem diameter was measured at the 0.5 m from the plant base. in case that plant individual has climbed and covered shrub or tree canopy, the canopy projection of the affected shrubs or trees was quantified. of light intensity the differences and the population attributes between the two sites were analysed by employing student's t-test . the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen observation of coverage to its host m. peltata when it climbs was approached by measuring the light intensity under the occupied canopy and above the canopy. the percentage of canopy coverage by to host's canopy was then m. peltata counted by employing formula: percentage of canopy coverage = 100 – (liuc/liac) x 100 where: liuc= light intensity under the canopy liac= light intensity above the canopy obse r ve d e cophy siological characters included pecific eaf rea (sla), wood density s l a and xylem anatomy. fifty (50) healthy leaves were taken from 50 individuals. leaf area was then measured by analyzing the leaf images using a computer program called imagej (national health institute usa). those leaves were then oven dried at 70 c for two days and were 0 measured for their dry weight. sla is the ratio of leaf area (cm ) and dry weight (g). wood density 2 was observed for 50 wood fractions from 50 individuals. after the volume of wood pieces was quantified, the wood samples were oven dried at 70 c for two days and were measured for their dry 0 weight. the wood density is the ratio between wood dry weight (g) and wood volume (cm ). 3 fifty wood samples from 50 individuals above were also taken for xylem observation. before they were observed, the wood samples were put in 70% alcohol. in the laboratory, the samples were transversally sectioned and then were observed under microscope. pictures of the samples were taken. diameter of the xylem vessels was measured using imagej software (national health institute usa). vessel diameters of stems were divided into 10 μm size classes. to determine the theoretical contribution of each vessel size class to the hydraulic conductance of the stem, vessel diameters were raised to the fourth power (hagen-poiseuille law) (tyree & zimmerman 2002). the hagen-poiseuille law is based on the experiments by hagen in 1839 and poiseuille in 1840 which found that water molecules are stationary on the capillary wall and move faster in the centre of the capillary. therefore, flow rate is proportional to the fourth power of the radius of the capillary (tyree & zimmerman 2002). the relative contribution of each diameter class is the proportion of the sum of all the conduits raised to the fourth power. this sum reflects the capacity of stem to conduct water, which will consequently affect the ability of plants to grow faster. figure 1. the location of bukit barisan selatan national park in southwest sumatera (inset). the study site was at the pemerihan resort ( ) 27 results and discussion the expansion of has become a real m. peltata problem for species conservation in the bbsnp area. since it established in early 1970s, was m. peltata has dominated approximately 7,000 ha of bbsnp area (master 2013). satellite image et al. showed that in 2002, dominated area of m. peltata about 6,393 ha. this area had become about 7,008 ha in 2008 (tnbbs 2011). area of bbsnp which will be dominated by to be wider m. peltata is likely in the future. in the pemerihan resort, where this study was conducted, the expansion of m. peltata has just been occurring for the last years. this few showed that this species keeps spreading to all areas of bbsnp. light intensity between the two study sites differed significantly ( = 10.571; <0.01). in the t p intact area, the light which went through the canopy and reached forest floor was about 171.26 lux. in the open area, the average light intensity was about 293.30 lux. this difference reflected the gradient of energy available for vegetation in these two different sites. the average number of in both m. peltata study sites differed significantly ( = 4.907; t p <0.01) (fig. 2). in the open area, the average number of per area was 4.46 individuals m. peltata per 25 m . in the intact area it was only 0.20 2 m. peltata m. plants per 25 m . the figure shows that 2 peltata preferred an open and fragmented area. this research indicated that forest opening enhanced the expansion of . as a fast m. peltata growing species quickly dominated the m. peltata landscape and form a dense population. the number of in the open area was 20 times m. peltata as many as that the in shaded area. the area openness provides a full sunlight, in which this condition is favoured by pioneer species (whitmore 1989). the domination of one species can also be indicated by the total basal area. this study found that total basal area of in the open m. peltata area, i.e. 8.84 cm per 25 m , was higher than 2 2 m. peltata plants in the shaded area, i.e. 0.58 per 25 m 2 ( = 4.435; <0.01) (fig. 3). this indicated that t p total accumulation of biomass of was m. peltata much higher in the open area than in the shaded area. disturbances, in this case forest clearing/ fragmentation, change the ecosystem dramatically (hooper 2004) by affecting the et al. availability of crucial resources, such as light, water and nutrients, in the area. consequently, plants may respond differently to this gradient and ultimately this will determine which species will coexist (brokaw busing 2000). the & differences in performance involve establishment, growth and survival. the success of m. peltata to dominate the area depended on their ability to exploit resources and adapt with the available environmental niches. as the development of this species was much better in the open area, the more forest fragments created more chances this species had to invade the forest area. biotropia vol. 22 no. 1, 2015 open forest intact forest figure 2. the number of individuals of per 25 m in the open and intact forest areas. error bar indicates 95% m. peltata 2 confidence intervals 28 open forest intact forest canopy of the host plants occupied by m. peltata had high canopy density. the average percentage of canopy coverage of m. peltata occupied trees/shrubs was 76.19% in the open area and 77.63% in the shaded area (table 1). in comparison, surrounding trees/shrubs which were not occupied by had only about m. peltata 30–50% canopy coverage. this showed that m. peltata had covered the host canopy severely, which subsequently might affect the growth of those trees or shrubs. figure 3. total basal area of per 25 m in the open and intact forest areas. error bar indicates 95% confidence m. peltata 2 intervals table 1. the percentage of canopy density of trees/shrubs occupied by in the two sites of observationm. peltata ind open forest intact forest canopy diameter (m) percentage of canopy coverage canopy diameter (m) percentage of canopy coverage 1 4 86.79 3 69.20 2 3 77.47 7 58.37 3 4 72.48 4 86.40 4 4 80.96 7 86.25 5 3 74.92 4 82.71 6 4 74.13 2 86.75 7 3 68.25 3 79.45 8 4 75.58 5 81.58 9 3 74.25 7 58.80 10 4 77.03 4 86.85 average 76.19 77.63 wood density of was 0.6 g/cm m. peltata 3 (table 2). in comparison, the value of water density as a standard at 4 c f was 1 g/cm . 0 3 hence, the value of wood density of m. peltata was categorized low. the value of sla of m. peltata was 160.33 cm /g (table 2). compared 2 with other climbing species, this value was very high. observation on and s freycinetia excelsa raphidophora australasica showed that their sla values erew 9.74 cm /g and 16.36 cm /g, 2 2 respectively (yansen 2012). 29 the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen sla is commonly found to have a significant negative correlation with leaf thickness (vile . et al 2005). leaves with high sla (i.e. low leaf mass area/lma), are usually thinner and have low tissue density and low leaf construction cost (osunkoya . 2010). on the other hand, tet al hicker leaves (i.e. lower sla/higher lma) may correlate to longevity of leaf life span (chabot hicks & 1982; putz . 1995 mediavilla . 2001), high et al et al; construction cost (westoby . 2002) and more et al drought tolerance (salleo lo gullo 1990& ; mediavilla . 2001). the leaf turn-over of et al m. peltata is fast; hence it can grow very quickly in a short time. the average diameter of the xylem m. peltata vessels was 292 µm (fig. 4). the vessel size class distribution showed that about 40% of vessels in m. peltata stem had a diameter greater than 300 μm. however, over 300 μm vessels in fact provid ed about 70% of the hydraulic conductance (fig. 5). the average vessel diameters of are also higher than several m. peltata climbing plants studied by fisher . (2002) et al and yansen (2012) (table 3). these findings indicated that the ability of stem to m. peltata conduct water was high which significantly contributed to the ability of this species to grow faster. the ecophysiological characters of m. peltata show that this species do not invest too much es on structural strength tend to grow faster. and it s this indicates that this species is categorized as ruderal (grime 2002) which dominates the disturbed areas but it will not persist in the long term when the forest area becomes more intact and shaded. therefore, an ecological engineering is needed to reduce the domination of this species in the area. biotropia vol. 22 no. 1, 2015 table 2. specific leaf area and wood density (with standard errors) of m. peltata no observed characters value + se 1 specific leaf area 160.30 + 4.80 cm2/g 2 wood density 0.60 + 0.03 g/cm3 figure 4. the transverse section through stem xylem tissue of showing its vessels (v)m. peltata 30 conclusions the differences in population attributes of m. peltata in the open and intact forests indicated that the expansion of this species was enhanced by forest fragmentation. it grew quickly in open area. the ecophysiological characters of also m. peltata supported the ability of this species to become a strong invader and competitor in a short-time. if more forest fragments were created, this species had more chances to invade the forest area. as the domination of this species had significant ecological consequences, a long term observation on population and growth dynamics essential. was therefore, the application of appropriate ecological approaches to overcome the domination of this species could be applied. acknowledgements this research was fully funded by the directorate of higher education, the ministry of education and culture, republic of indonesia, in which the authors thank the institution. ed the authors also thanked candra lumbagaol and ahmad ritonga for their assistance in the field. this project was conducted in bukit barisan selatan national park and we thank the bbsnp office that ha granted ed d permit to access the conservation area. size class (µm) figure 5. distributions of stem vessel diameters of in 10 μm size classes. the graphs show the number of vessels m. peltata of each size class as a percentage of the total vessel number (%n) and the theoretical contribution of each size class to hydraulic conductance (σd ) of the stem. the hypothetical contribution to conductance was calculated by 4 summing the diameters of vessels in each size class to the fourth power (hagen-poiseuille law) (tyree & zimmerman 2002). table 3 average diameter of vessels of several climbing rattans (1-6), climbing pandan (7) and climbing aroid (8) . us compared to current stud species (9) (fisher . 2002; yansen 2012).ly ied et al no. species average diameter of vessels (µm) 1 2 3 4 5 6 7 8 9 calamus insignis daemono ropshystrix korthalsia echinometra korthalsia rigida korthalsia rostrata plectocomia elongate freycinetia excelsa rhaphidophora australasica merremia peltata 213 243 380 532 205 458 150 170 292 31 the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen references brokaw n, busing rt. 2000. niche versus chance and tree diversity in forest gaps. tree 15: 183-88. brown jh. 1984. on the relationship between abundance and distribution of species. am nat 124: 255-79. bchabot bf, hicks dj. 1982. the ecology of leaf life spans. ann rev eco sys 13: 229-60. clavero m, garcıa-berthou e. 2005. invasive species are a leading cause of animal extinctions. trends eco evo 20: 110-15. connell, jh. 1978. diversity in tropical rainforest and coral reefs. science 199: 1302-10. denslow js. 1987. tropical rainforest gaps and tree species diversity. ann rev eco sys 18: 431-52. fisher jb, tan htw, toh lpl. 2002. xylem of attans: r vessel dimensions in climbing palms. am j bot 89: 196-202. grime jp. 2002. . new plant strategies and 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ecophysiology of secondary hemi-epiphytic vines. phd hesist . townsville (au): james cook university . biotropia vol. 22 no. 1, 2015 32 biotropia book juni revisi 14 juli 09.indd 11 biotropia vol. 16 no. 1, 2009: 11 20 corresponding author: atriw@ipc.ac.id or aristri2003@yahoo.com cloning of a gene encoding protein belonging to abc transporter involved in bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 aris tri wahyudi department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract magnetospirillum magneticum amb-1 synthesizes intracellular magnetic particles, magnetite (fe3o4), enveloped by membrane called magnetosome under micro-aerobic conditions. initial study of random transposon-based mutagenesis generated 62 nonmagnetic mutants of amb-1 in a mini-tn5 library. in order to identify a gene involved in bacterial magnetic particle (bmp) synthesis in the magnetic bacterium m. magneticum amb-1, a nonmagnetic mutant from the library designated as nma38-4, was analyzed. th e amino acid sequence deduced from the gene directly interrupted by transposon, orf4 (1482 bp), showed homology to atp binding cassette (abc) transporter of mesorhizobium loti with 62 % identity and 74 % similarity. it was strongly indicated by the occurrence of putative consensus sequence of atp-binding motifs (atpbinding protein). th e orf4 was subsequently cloned in pet-15b and the recombinant orf4histag fusion protein was heterologously expressed in escherichia coli bl21 (de3) plyss. a 55 kda protein corresponding to the orf4-histag fusion protein was obtained after purifi cation using ni-nta column. th is is the fi rst report describing a gene cluster containing gene encoding protein belonging to abc transporter organized in an operon which is involved in bmp synthesis. key words: magnetospirillum magneticum amb-1, bacterial magnetic particle (bmp), atp binding cassette (abc) transporter, transposon mutagenesis. introduction magnetospirillum magneticum amb-1 isolated from fresh water sediment (matsunaga 1991) synthesizes bacterial magnetic particles (bmps) of the iron mineral magnetite (fe3o4) enveloped by membrane called magnetosome, under microaerobic conditions. these intracellular single domain magnetic particles are aligned in chains with each individual crystal having a diameter of 50-100 nm enveloped by an organic membrane (gorby et al. 1988; matsunaga 1991). it is postulated that bmps confer sensitivity of the bacterium to the earth’s magnetic field lines for migration along oxygen and iron gradients. 12 biotropia vol. 16 no. 1, 2009 so far, only few magnetic bacterial strains can be cultivated under laboratory conditions in pure culture, these include m. magnetotacticum ms-1 (blakemore et al. 1979) and m. magneticum amb-1 (matsunaga et al. 1991), and m. gryphiswaldense (schleifer et al. 1991). these strains are usually used as model systems for the analysis of bmp synthesis. the capability of m. magneticum amb-1 to grow on agar plate and liquid medium both under microaerobic and aerobic conditions (matsunaga et al. 1992) makes the bacterium amenable for genetic manipulations to elucidate important metabolic mechanisms most especially for the complex process of bmp synthesis. since magnetic bacteria were fi rst discovered (blakemore 1975), only few genes were isolated namely reca (berson et al. 1989), arod (berson et al. 1991), and mam22 (okuda et al. 1996) from m. magneticum ms-1. in the previous studies, maga (nakamura et al. 1995a) and mms16 (okamura et al. 2001) genes were isolated from m. magneticum amb-1. th e maga gene functions for the iron transport across the bmp membrane. mutation of this gene rendered the cells defective in iron uptake. th e mms16 protein has a gtpase activity for the invagination of the cytoplasmic membrane for the formation of the bmp membrane. cells with inhibited gtpase activity showed that they produced disrupted bmps. previously, a non-magnetic mutant of m. magneticum amb-1, designated nma38-4, was generated by mini-tn5 transposon mutagenesis (wahyudi et al. 2001). in the present study, we have identifi ed a gene (orf4) involved in bmp synthesis in m. magneticum amb-1. th is gene encoding protein which belongs to abc transporter was cloned and heterologously expressed in e. coli as an orf4-histag fusion polypeptide. mutation within this gene rendered m. magneticum amb-1 was unable to synthesize bmps. th e aim of this study was to isolate and clone a gene (orf4) from m. magneticum amb-1 genome and express it in escherichia coli bl21 (de3) plyss. materials and methods bacterial strains and culture conditions escherichia coli dh5α was routinely cultured in luria broth (lb) (tryptone 5.0 g l-1, nacl 10 g l-1, yeast extract 5.0 g l-1) and e. coli bl21 (de3) plyss was cultured in lb supplemented with chloramphenicol 34 μg ml-1 at 37ºc. magnetospirillum magneticum amb-1 (atcc 700264) was cultured micro-aerobically in msgm at 25oc (blakemore et al. 1979). a nonmagnetic mutant, nma38-4 (wahyudi et al. 2001), was micro-aerobically cultured in msgm supplemented with kanamycin (5 μg ml-1). observation of nma38-4 a nonmagnetic mutant, nma38-4 cells (wahyudi et al. 2001) was cultured until logarithmic phase and cells of this phase were observed under light microscopy (olymphus bh2, tokyo, japan). samarium-cobalt magnet was moved in different directions near the glass slide to determine the cells magnetic response. 13 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. isolation of flanking dna and sequence analysis mutant dna fragments flanking transposon in this work was isolated by inverse pcr after ecorv digestion and circularization, using primers designed from the mini-tn5km1 sequence (primer 1: 5’-gta ccg agc tcg aat tc-3’ and primer 2: 5’-gat cct cta gag tcg ac-3’). the primers were directed outward from the transposon. a 1.3 kb inverse pcr product (fig. 1) was purified from the gel by gene clean iii kit (bio-101, carlsbad, ca.) and sub-cloned in pcr2.1 (ta cloning, invitrogen, usa), designed pcr2.1-38.4. this recombinant plasmid was transformed into e. coli dh5α. the recombinant plasmid was subsequently isolated from e. coli by qiaprep miniprep (qiagen, gmbh, germany) and used as a template for dna sequencing. the dna sequencing was performed using an automatic dna sequencer abi 377 (perkin elmer, usa). the dna sequence was subsequently aligned against the complete genome sequence of m. magneticum amb-1 (matsunaga et al. 2005). a computer software package, lasergene (dnastar, madison, wi) was used for dna and protein sequence analysis. the sequence was further analyzed by performing homology searches using program of blast (altchul et al. 1997) against the genbank and embl dna databases. gene cloning and expression in escherichia coli based on the sequence of orf4 (fig. 1), two oligonucleotide primers (primer p1: 5’-ggg gga cat atg agc gac gtc gtc gaa-3’ and primer p2: 5’-ggg gga tcc aaa tca cgt gtc gtc ccc cca-3’) were designed (underlined nucleotides indicate ndei and bamhi sites, respectively). the recombinant plasmid pet15b-orf4 was constructed by cloning of the pcr product amplified from orf4 into the ndei/bamhi site of expression vector pet15b (novagen, usa). to amplify orf4, a primer p1 with ndei site introduced at a start codon atg, and primer p2 with the bamhi site introduced downstream of the stop codon (tga) of the gene were used. the 50 μl pcr reaction mixture contained 100 ng m. magneticum amb-1 genomic dna, 2.5 u la taq (takara, tokyo, japan), 400 μm dntps, 2.5 mm mgcl2, and 0.2 μm of each of the two primers. the temperature program for pcr was one cycle of 3 min at 95 oc, 30 cycles of 1 min at 95 oc, 60 oc, and 72 oc, respectively, and one cycle for 10 min at 72 oc. the amplified fragment was subsequently isolated from the gel and purified by gene clean iii kit (bio 101). a 1.5 kb purified fragment was sub-cloned in pcr2.1 vector (invitrogen, usa), designed pcr2.1-orf4, and was subsequently transformed to e. coli dh5α. the recombinant plasmid was isolated and digested with ndei and bamhi. the fragment corresponding to the orf4 was ligated into pet15b expression vector (novagen, madison, wi) linierized with ndei and bamhi, to yield a recombinant plasmid, designed as pet15b-orf4 (fig. 1). this recombinant plasmid was introduced into e. coli dh5α and subsequently isolated and then transformed into e. coli bl21 (de3) plyss as a host strain for gene expression. transformants were plated on lb plate containing ampicillin (50 μg ml-1) and chloramphenicol (25 μg ml-1). 14 biotropia vol. 16 no. 1, 2009 bamh xho nde lac a orf4 (1482 pet15b (5.7 a or or lacpet15b-orf4 nde bamh ndebamh figure 1. construction of a recombinant plasmid pet15b-orf4 (~7.2 kb). purifi cation of orf4-histag fusion protein ten milliliter culture of e. coli bl21 (de3) plyss carrying pet15-orf4 was induced by 0.1 mm iptg at od600 of 0.6 for 3 h. by shaking at 37 oc. the culture was then centrifuged, and pellet was frozen at –70 oc until use. the protein was purified under denatured condition using ni-nta column (qiagen, gmbh, germany). sds-page and western blotting pellets of uninduced cells (1 ml) and induced cells (0.5 ml), solubilized lysate, or purified -orf4-histag fusion protein, were mixed with 2 x sample buffer (tris. hcl, glycerol, sds, 2-mercaptoethanol, and bromophenol blue) and denatured by boiling for 5 minutes. sds-page was performed at 12.5 % (w/v) acrylamide gel, and protein was stained with commassie brilliant blue. for western blotting, the orf4histag fusion protein polyacrylamide gel was blotted onto a pvdf membrane by electroblotting. the western blot was stained using monoclonal mouse anti-histag antibody at 1:5000 dilution. a secondary goat anti-mouse igg antibody conjugated to alkaline phosphatase was used for imaging (zymed laboratories inc.). 15 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. results observation of nma38-4 cells and colony colony of this mutant, designated as nma38-4, was grown on msgm plate. the color was white indicating the bmp was not synthesized. whereas the color of the amb-1 wild type grown on the same media was brown-black. the brown-black color indicated that bmp was synthesized. observation of nma38-4 cells under light microscopy showed that cells did not respond to the magnetic fields applied. this indicates bmp may have not been synthesized due presumably to transposon insertion into the genome, especially in the gene involved in bmp synthesis. to confirm this result, nma38-4 cells were observed by transmission electron microscopy which showed that they did not contain bmps in the cell (data not shown), indicating that bmps were not synthesized completely. dna sequence analysis of orf4 identification of the gene interrupted by mini-tn5 transposon in nma38-4 genome was accomplished by isolation of flanking dna by inverse pcr, sequencing of the flanking dna, and sequence analysis through homologous searches of major databases. figure 2 shows a gel electrophoresis of 1.3 kb inverse pcr product amplified from dna flanking the transposon of nma38-4 genome. to characterize this locus, we used sequence of dna flanking the transposon aligned against the whole genome sequence of m. magneticum amb-1 (matsunaga et al. 2005) and assembled one contig which contained mini-tn5-interrupted gene, orf4. homology search of orf4 sequence by blastx program revealed its homology with abc transporter from mesorhizobium loti (62% identity, 74% similarity). an orf directly interrupted by transposon, orf4, with location of transposon insertion site, is shown in fig. 3. putative ribosomal binding site (rbs) was found at the position of 7 bp upstream of start codon (atg). amino acid sequence analysis deduced from the genes the orf4 of m. magneticum amb-1 encodes a protein of 494 amino acids with molecular mass of 52.8 kda, as shown in fig. 4. examination of the deduced amino acid sequence revealed a high degree of homology with abc transporter. further analysis indicated that the sequence contains atp-binding sites in the orf4 and the presence of two segment walker motifs, walker a and b was identified (walker et al. 1982). the two sequences, gskkegkltcdtmlal (walker a) and kvakpghrllmvs (walker b) were localized in orf4 at position 170-185 and 309-321, respectively (fig. 2). the multi alignment of the amino acid sequences revealed a highly conserved region in segment a, whereas the consensus in segment b was less restrictive, permitting various amino acid substitutions. analysis of hydrophilicity plot of the protein deduced from orf4 using an algorithm kitedoolittle showed that orf4 protein possessed 10 putative hydrophobic transmembrane α helixes (data not shown). 16 biotropia vol. 16 no. 1, 2009 figure 2. 1.3 kb dna fragment flanking transposon mini-tn5km1 amplified by inverse pcr method (1) and dna marker 1 kb ladder (m). expression and purifi cation of histag fusion protein after recombinant plasmid pet15b-orf4 was transformed to e. coli bl21 (de3) plyss, expression of the orf4 was induced by addition of 0.1 mm iptg and under the control of promoter t7 lac. th e total protein profi le of the whole cells was analyzed by sds-page to express the histag-orf4 fusion protein. as shown in figure 4a, the protein band ~55 kda corresponding to the molecular mass of the histag-orf4 fusion protein was highly expressed in e. coli, and pure protein was obtained after denatured purifi cation. it was confi rmed to be the histag-orf4 fusion protein by western blot analysis using anti-histag antibody (fig. 4b). discussions in this study, we demonstrated that the gene encoding protein which belongs to atp binding protein is linked to bmps synthesis. mutation of this gene in nma384 generated non-magnetic cell. th e mutant did not respond to the magnetic fi elds and bmps were not completely synthesized. inverse pcr method allowed us to amplify 1.3 kb genomic fl anking dna from nma38-4 genome and align the sequence against the whole genome sequence of m. magneticum amb-1. th e contig containing the sequence of the gene directly interrupted by transposon comprised 1482 bp (fig. 3). although this gene did not contain a native promoter, it could be highly expressed in e. coli under t7 lac promoter (fig. 4). th is indicates orf4 isolated from m. magneticum amb-1 genome involved in bmps synthesis could be expressed in escherichia coli. eff ect of the transposon, not only inactivated the gene directly interrupted, but also the genes located downstream of the transposon insertion (de bruijn & lupski 1984; 17 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. kleckner et al. 1977). th erefore, orf4 may most probably have important roles during bmp synthesis. interestingly, orf encodes majority of the protein with functions which is related to atp-binding cassette (abc) transporter. involvement of abc transporter in iron transport has been reported in gram negative and gram positive bacteria such as yersinia pestis (gong et al. 2001; fetherston et al. 1999; bearden et al. 1998), streptococcus pneumoniae (brown et al. 2001a; brown et al. 2001b), neissiria meningitides (khun et al. 1998), s. pyogenes (janulczyk et al. 1999), and brachyspira hyodysenteriae (dugourd et al. 1999). taken these all together, it is therefore possible that genes with close homology to atp-binding protein identifi ed in this study may have a signifi cant role in iron transport during bmps synthesis after the iron passed across the outer cell membrane. despite the signifi cant impact of bmps to the physiological functions of the cell, information on the mechanisms and the factors aff ecting their formation is still very limited. our data suggests that a gene for atp binding cassette protein and the other genes within the same cluster may most probably be linked to bmps synthesis in m. magneticum amb-1. th is fi nding may signifi cantly contribute to the complete elucidation of the complex process of bmp synthesis, especially in m. magneticum amb-1. figure 3. dna sequence of orf4 (boxed) started with atg (start codon) and ended by tga (stop codon). 9 bases boxed show mini-tn5 insertion site. walker a (ggskkegkltcdtmlal), walker b (pkvakpghrllmvs), and ribosomal binding site (rbs) are also indicated by underline. 18 biotropia vol. 16 no. 1, 2009 figure 4. (a). sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of protein profile of orf4 expressed in escherichia coli. lane m: marker protein. lane 1: total cell proteins from uninduced-cells of e. coli bl 21 (de3) plyss harboring pet15-orf4. lane 2: total cell proteins from induced-cells of e. coli bl 21 (de3) harboring pet15-orf4 induced by 0.1 mm iptg. lane 3: total protein cell lysate (15 μg). lane 4: purified histag-orf4 protein (6 μg). (b) lane rm: rainbow marker protein. lane 5: western blot analysis of purified histagorf4 fusion protein (6 μg) corresponding to lane 4. conclusion a gene involved in bacterial magnetic particle synthesis has been isolated from m. magneticum amb-1 genome, cloned and over-expressed in e. coli bl21 (de3) plyss. th is gene encoded protein had homology with abc transporter. a 55 kda protein corresponding to the gene (orf4)-histag fusion protein resulted from the gene expression was detected by sds-page. th e purifi ed protein was confi rmed to be orf4-histag fusion protein by western blot analysis. mutation within this gene (orf4) rendered m. magneticum amb-1 defective in bacterial magnetic particle synthesis. acknowledgments part of this work was conducted at tokyo university of agriculture and technology, japan, especially at department of biotechnology (matsunaga-takeyama laboratory). th erefore, i am grateful to prof tadashi matsunaga and prof haruko takeyama for the laboratory facilities and valuable supports. references altchul sf, madden tl, scaff er aa, zhang j, zhang z, miller w, and dj. lipman. 1997. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids research, 25:3389-3402. blakemore rp. 1975. magnetotactic bacteria. science, 190: 377-379. 19 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. blakemore rp, maratea d, and rs. wolf. 1979. isolationand pure culture of a fresh water magnetic spirillum in defi ned growth medium. journal of bacteriology, 140: 720-729. bearden sw, stagg tm, and rd. perry. 1998. an abc transporter system of yersinia pestis allows utilization of chelated iron by escherichia coli sab11. journal of bacteriology, 180: 1135-1147. berson ae, hudson dv, and ns. waleh. 1991. cloning of a sequence of aquaspirillum magnetotacticum that complements the arod gene of escherichia coli. molecular microbiology, 5: 2261-2264. berson ae, hudson dv, and ns. waleh. 1989. cloning and ccharacterization of the reca gene of aquaspirillum magnetotacticum. archives of microbiology, 152: 567-571. brown js, ogunniyi ad, woodrow mc, holden dw, and jc. paton. 2001a. immunization with components of two iron uptake abc transporters protects mice against systemic streptococcus pneumoniae infection. infection and immunity, 69: 6702-6706. brown js, gilliland sm, and dw. holden. 2001. a streptococcus pneumoniae pathogenicity island encoding an abc transporter involved in iron uptake and virulence. molecular microbiology, 40: 572-585. debruijn fj, and lupski jr. 1984. th e use of transposon tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of dna segments cloned into multi copy plasmidsa review. gene, 27: 131-149. dugourd d, martin c, rioux cr, m. jacques m, and j. harel. 1999. characterization of a periplasmic atpbinding cassette iron import system of bracyspira (serpulina) hyodysentriae. journal of bacteriology, 181: 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you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html 478 (kumala dewi effect).cdr effects of blue light and paclobutrazol on seed germination, vegetative growth and yield ** of black rice (oryza sativa l. 'cempo ireng') 1* 2 2 kumala dewi , rizkika zakka agustina and farida nurmalika 1 faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2 alumni of faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia received: 8 april 2015/accepted: 8 august 2016 abstract black rice (oryza sativa l. “cempo ireng”) is one of local rice varieties in sleman regency, yogyakarta. the black color is caused by high anthocyanin content which is important source of antioxidant. the cultivation of black rice is still limited due to its tall phenotype, long vegetative stage and low productivity compared to white rice. paclobutrazol is a growth retardant causing dwarfing in several crop plants and reducing lodging. blue light can improve plant quality. this research was aimed to evaluate the effect of blue light and paclobutrazol on seed germination, vegetative growth and yield of black rice. the results showed that the average of seed germination as well as the activity of α-amylase of seeds subjected to blue light were lower compared to those subjected to sunlight; however, paclobutrazol concentrations did not affect seed germination percentage. the height of rice plants treated with paclobutrazol decreased in accordance with the increase of paclobutrazol concentration. chlorophyll content and tiller numbers increased by paclobutrazol treatment of 12.5 ppm. nitrate reductase activity was higher in rice seedlings subjected to blue light compared to those subjected to sunlight. iron (fe) content of rice plants treated with 25 or 50 ppm paclobutrazol increased compared to control. it was concluded that paclobutrazol application of 12.5 ppm already reduced plant height. the higher concentration of paclobutrazol applied the greater reduction on plant height was observed. blue light treatment applied during black rice seed germination slightly reduced germination percentage and α-amylase activity in the germinated seeds. however, blue light treatment combined with paclobutrazol application during black rice seed germination increased chlorophyll content, tiller numbers and fe content in black rice grain. keywords: α-amylase activity, anthocyanin, blue light, fe content, paclobutrazol introduction rice is among staple food having many varieties. besides white rice, there are brown rice, red rice and black rice varieties. the price of black rice is relatively more expensive than that of white rice. the higher price of black rice may have been caused by the increasing awareness of people to consume black rice which contains more vitamins, minerals and anthocyanin. anthocyanin content in black rice was reported to be beneficial for human health as it can lower cholesterol level in the blood and reduce the risk of arteriosclerosis (ling et al. 2001). however, not many farmers choose to cultivate black rice because the vegetative phase of black rice is longer than that of white rice. in addition, birds as well as the rice earhead bug (leptocorisa oratorius) like to feed on developing black rice caryopsis which cause severe loss in black rice yield. the phenotype of black rice is also taller compared to other rice hybrids causing problems in cultivation. paclobutrazol is a synthetic growth inhibitor that acts by blocking gibberellin biosynthesis in plant (rademacher 2000) resulting to semidwarf or dwarf plants. semidwarf character is one of important traits, as semidwarf plants are lodging resistance. the productivity of semidwarf plant also increased because assimilates translocation is more directed to develop seeds rather than to develop new leaves or buds (evans 1993). dwarfisme can be due to either deficiency in biotropia 3 2 6 84 95 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 478 * corresponding author: kumala.dewi@ugm.ac.id nd ** this paper was presented in the 2 international conference on advances in plant sciences (icaps 2014) organized by v sivaram research foundation and universiti teknologi mara on 18 22 november 2014 in kuching, sarawak, malaysia 84 gibberellin biosynthesis or inhibition in gibberellin signalling (spielmeyer et al. 2004). application of paclobutrazol to create semidwarf plants was reported in rice subspecies “keng”, in which 22.5 g/ml paclobutrazol reduced stem height and increased tiller numbers (minzi 1993). it was also reported that application of paclobutrazol effectively reduced vegetative growth of rice plants and increased chlorophyll content. rice seedlings treated with paclobutrazol allocated more photosynthates for seed development compared to control plants or those plants treated with gibberellin (yim et al. 1997). in corn (zea mays l.) under drought stress, application of 50 ppm paclobutrazol increased yield and average weight of 1,000 seeds (bayat & sepehri 2012). blue light is light spectrum having wave length of 320 490 nm (dougher & bugbee 2004). receptor of blue light is known as blue light absorbing photoreceptor (bap) (meijer 1968; gaba & black 1979; thomas 1981). plants respond differently to blue light irradiation (dougher & bugbee 2004). according to guerra et al. (1985), wheat plants subjected to blue light showed a decrease in the activity of enzymes regulating lignification which promoted shoot elongation. in phaseolus vulgaris, blue light increased cell expansion (van volkenburgh et al. 1990). in rice seedlings, respiratory activity of plants subjected to blue light increased to about 1.5 folds compared to those treated with white light. this was due to an increase in the activity of piruvat kinase and malat dehidrogenase enzymes regulating cell respiration which caused the increase of atp production and blue light irradiation (yu & pan 1996). in addition, activity of nitrate reductase, nitrite reductase, glutamate synthetase and glutamin synthetase were higher in rice seedlings subjected to blue light for 5 7 days compared to those subjected to white light. however, if the seedlings were subjected to blue light for more than 10 days, the activity of those enzymes declined (deng et al. 2000). nitrate reductase is an enzyme that catalyzes reduction of nitrate to nitrite. several studies indicated that the level of nitrate reductase in plant tissues is altered by exogenous application of phytohormones (mackintosh 1998). the activity of nitrate reductase in rice increases with the application of cytokinin and 2-chloro ethyl trimethyl ammonium chloride, which are gibberellins biosynthesis inhibitor (hemalatha 2 0 0 2 ) . g l u t a m i n e 2 o x o g l u t a r a t e aminotransferase (glutamate synthase, gogat) and glutamine synthetase (gs) are the key enzymes in primary assimilation and reassimilation of nitrogen in plants. these enzymes catalyze coupled reactions known as the gs/gogat cycle, through which inorganic nitrogen is transformed into organic nitrogen (lu et al. 2011). improving iron content and its bioavailability in rice have been applied to alleviate or to solve micronutrient deficiency in human. iron content in black rice is higher than that in other rice types (meng et al. 2005). several methods to improve iron content and its bioavailability in rice seeds have been conducted, such as breeding, genetic e n g i n e e r i n g , b i o ch e m i c a l a n d p hy s i c a l approaches. the objective of this research was to evaluate the effect of blue light and paclobutrazol on growth, α-amylase activity, nitrate reductase, iron content and yield in black rice. materials and methods 'cempo ireng' black rice seeds were selected and soaked in aquadest for 24 hours. plastic trays of 18 x 24 cm were prepared and filled with a mixture of top soil and compost (3 : 1 = v/v) as growth media. one hundred seeds were spread and germinated in each plastic tray. the media was then wetted with either tap water (control) or paclobutrazol solution of 12.5, 25, 50 or 100 ppm. seeds were then placed under natural light in the green house or in a growth chamber installed with blue led (light emitting diode). the average daily temperature and relative o humidity inside the green house were 30 c and 70%, respectively. the seeds were subjected to blue light for 12 hours per day from 06:00 am to 06:00 pm until the seedlings were 3 weeks old. the photosynthesis photon flux level for natural 2 light and blue light was 300 µmol/m /s and 30 2 µmol/m /s, respectively (li-250a light meter, ® li-cor usa). for each combination treatment, five replications were made. germination percentage was determined on day 7. at the same time, the α-amylase activity in germinated seeds was determined in 10 mg whole grain samples. the ceralpha kit (megazyme international i r e l a n d l t d ) wa s u s e d b a s e d o n t h e 85 effect of blue light and paclobutrazol on black rice – dewi et al. manufacturer's protocol and with appropriate dilutions. data were expressed in cu (ceralpha unit) per g flour. at day 14, several parameters were determined such as seedling height, chlorophyll content and activity of nitrate reductase. chlorophyll content was determined spectrophotometrically according to harborne (1973). nitrate reductase activity was determined spectrophotometrically according to hartiko et al. (1984). at day 21, seedlings treated with paclobutrazol showing dwarf phenotype were selected and then transplanted in plastic pots containing a mixture of top soil and compost (3 : 1 = v/v). in each plastic pot, two seedlings were planted. each treatment combination had five replications. all plants were watered regularly and organic liquid fertilizer was applied every two weeks. at week twelve, plant height was measured and tiller numbers were determined. the plants were harvested at week 20, followed by the determination of 100 harvested seeds weight and iron content. the iron content was determined using atomic absorption spectrometry (aas). one gram of black rice grain (two samples per treatment) were weighed and placed in a furnace at 500 – 600 °c for 4 hours. samples were then cooled and subjected to wet digestion with 10 ml of hcl 20%. samples were filtered with whatman no.3 filter paper and placed in 50 ml volumetric flask and filled up to the mark with doubledistilled water. total fe content of the grain samples were determined using perkin elmer 3110 aas (welz & sperling 2007). data were analyzed using anova and dmrt (duncan's multiple range test) to determine the significant difference between treatments. results and discussion the average of seed germination percentage of black rice seeds treated with paclobutrazol and blue light is presented in figure 1. under sunlight, germination percentage of black rice seeds was 100% for control seeds. germination percentage of seeds treated with 12.5 ppm paclobutrazol was 98%. germination percentage of seeds treated with 25, 50 or 100 ppm reached 90%. subjected to blue light, germination percentage of black rice seeds was 80 85% and there were no significant differences between control seeds and seeds treated with paclobutrazol. this result indicated that primary dormancy of black rice seeds was possibly influenced by blue light. seed germination can be influenced by dormancy level and light quality. in arabidopsis, white light and red light tend to increase seed germination, while in cerealia, such as barley and wheat, white light and blue light maintain dormancy level in the seeds (gubler et al. 2008). in barley, blue light increased the expression of genes encoding enzymes that catalyze the biosynthesis of abscisic acid. as a consequence, the abscisic acid content was high, but on the other hand, the expression of genes encoding enzymes that catalyze gibberellins biosynthesis decreased (gubler et al. 2008). inhibition in barley seed germination subjected to blue light was due to an increase in abscisic acid level to about 3.5 folds compared to control (hoang et al. 2013). the increase in abscisic acid level was a result of an increase in the expression of genes encoding of the enzyme that catalyzed abscisic acid biosynthesis namely hvnced1 and figure 1 effect of sunlight and blue light against paclobutrazol on average of germination percentage±se of black rice 'cempo ireng' (n = 5) a ve ra g e o f g er m in at io n p er ce n ta g e 120 100 80 60 40 20 0 control 12.5 ppm 25 ppm 50 ppm 100 ppm paclobutrazol treatments sunlight blue light biotropia vol. 23 no. 2, 2016 86 hvnced2, as well as an increase in the embryo sensitivity to abscisic acid. blue light also decreased the expression of gene encoding of an enzyme that catalyzed gibberellin biosynthesis (hvga3ox2), but increased the expression of gene encoding of an enzyme that catabolized gibberellin. in wheat, seed ger mination inhibition subjected to blue light can be prevented by the presence of methyl jasmonate or nitrite oxide. it was also proven that methyl jasmonate application reduced the level of abscisic acid, and it was due to a decrease in the expression of gene encoding of an enzyme that catalyzed abscisic acid biosynthesis (tanced1) and a reduction the expression of taaba'oh-1 which encoded an enzyme that catalyzed degradation of abscisic acid (jacobsen et al. 2013). the mechanism by which seed germination in black rice was inhibited by blue light was probably due to an increase in abscisic acid level and reduction in gibberellin biosynthesis. this assumption warrants further examination. α-amylase activity germination processes in cereals are also determined by the activity of enzymes that degrade carbohydrate in the endosperm to its sugar component. α-amylase is one of enzymes that plays a role during cereals seed germination. in this experiment, the activity of α-amylase was determined according to procedure mentioned in ceralpha kit (megazyme international ireland ltd). α-amylase activity in germinated rice grains without paclobutrazol application was slightly lower in grains subjected to blue light compared to those subjected to sunlight (table 1). this finding is related to the average of germination percentage which also decreased in black rice seeds subjected to blue light. in barley, blue light increased abscisic acid level and significantly increased embryo sensitivity to abscisic acid (hoang et al. 2014; barrero et al. 2014). the result of this study indicated that a slight inhibition in seed germination of black rice seeds subjected to blue light was due to either higher level of abscisic acid in the seeds or the sensitivity of embryo to abscisic acid. it was known that abscisic acid repressed the activity and signalling of gibberellin that induced α-amylase during seed germination in cereals (ishibashi et al. 2012). in arabidopsis, the level of endogenous abscisic acid increased about two folds as compared to control by application of uniconazol (saito et al. 2008). in this study, interaction between paclobutrazol and blue light on α-amylase activity was evaluated in the germinated black rice seeds. the activity of αamylase in black rice seeds treated with 12.5 ppm paclobutrazol and germinated under sunlight decreased around 13% compared to control. this could be due to lower level of gibberellin produced during seed germination which led to reduction in α-amylase activity. nevertheless, αamylase activity in black rice seeds treated with 25 to 100 ppm paclobutrazol was relatively similar to control. these results were probably due to the feedback mechanism in which gibberellins biosynthesis was repressed by paclobutrazol. however, the expression of genes encoding of enzymes that catalyzed gibberellins biosynthesis was changed and it might also alter the expression of genes encoding α-amylase and/or α-amylase activity. lenton et al. (1994) showed that paclobutrazol application in wheat caused a decrease in the endogenous gibberellin in the scutellum, but it did not influence the expression of α-amylase mrna. however, the decline in endogenous gibberellin in wheat endosperm was related with downregulation of α-amylase mrna. from that report, it was suggested that the initiation of α-amylase gene expression in the table 1 average±se of α-amylase activity in black rice treated with blue light and paclobutrazol (n=5) treatment α-amylase activity (ceralpha units/g) subjected to sunlight blue light paclobutrazol 0 ppm 204.00±10.29 d 165.88±2.63 a paclobutrazol 12.5 ppm 176.02±7.98 ab 196.47±6.37 bcd paclobutrazol 25 ppm 201.96±11.53cd 198.07±5.72 bcd paclobutrazol 50 ppm 204.60±4.19 d 193.54±7.15 bcd paclobutrazol 100 ppm 197.51±6.67 bcd 178.71±5.14 abc effect of blue light and paclobutrazol on black rice – dewi et al. 87 scutellum did not depend on a new gibberellin. however, the expression of α-amylase gene in the endosperm depended on gibberellin biosynthesis in the embryo. this suggestion is in accordance with the fact that germination still occurred in black rice seeds treated with 100 ppm paclobutrazol, which might be caused by sufficient gibberellin in the scutellum. therefore, initiation of α-amylase gene expression could also be carried out. it should be noted that there was still an inhibition on shoot growth of black rice seeds treated with paclobutrazol. this may be related to inhibition of new gibberellin biosynthesis in the shoot. in arabidopsis, application of uniconazole, which is also a gibberellins inhibitor, caused shoot stunting, but with enhanced elongation of the primary roots (bidadi et al. 2009). in semidwarf rice (sd1) obtained from a mutation of the ga20ox-2 gene, the level of endogenous gibberellin was low, however, the normal height of the semidwarf rice is restored by exogenous application of ga (ashikari et al. 2002; sasaki et al. 2002). this gene is expressed mainly in stems and is an important regulatory step on the pathway to synthesize active ga (reviewed in hedden 2003). semidwarf rice was also obtained from suppression of osga20ox1 gene which suggested to be involved in the regulation of shoot stature in rice (oikawa et al. 2004). in addition, different alleles of the garesponsive dwarf rice (d18) are associated with mutations in the osga3ox2 gene that is also expressed highly in stems (itoh et al. 2001). the probability of phototropin involvement in the biosynthesis of gibberellin and activity of αamylase also warrant further evaluation. the activity of amylase in black rice seeds treated with paclobutrazol and subjected to blue light tend to increase compared to those of control. the endogenous gibberellin decreased rapidly in rice seedlings subjected to blue light, due to a decrease in expression of genes that involved in gibberellin biosynthesis (osga20ox2 and osga20ox4) and an increase in expression of genes encoding the enzyme that catabolized gibberellin (osga2ox4 and osga2ox7). in addition, analysis in blue light receptor mutants (cry 1a dan cry 1b) also showed that the mechanism of blue light in decreasing the biosynthesis of gibberellin was through an increase in the expression of genes that involved in gibberellin catabolism (hirose et al. 2012). commonly, there are several isozymes that catalyze the biosynthesis of gibberellins and it could be that some of those isozymes were upregulated as a feedback mechanism when the level of endogenous gibberellins was lowered by paclobutrazol treatment. the upregulation of some isozymes that involved in gibberellins biosynthesis may also influence the activity of αamylase. mustard (sinapis alba l.) seedlings experienced increase of β-amylase activity in the cotyledon after being subjected to continuous blue light for 42 – 96 hours from sowing. after that, the β-amylase activity abruptly declined (manga & sharma 1988). in this research, the αamylase activity was only determined in 3 days old germinated seeds. further experiment is still required to observe the pattern of amylase activity until seedlings were established. average of plant height and tiller numbers application of paclobutrazol can cause semidwarf or dwarf phenotype and it depends on the concentration applied (rademacher 2000). paclobutrazol concentrations of 200 mg/l to 600 mg/l decreased gibberellin content in the leaves compared to that of control when applied to rice plant during (syahputra et al. 2013). the average of plant height in black rice treated with blue light and paclobutrazol at seedling stage 3 weeks old and at heading date is presented in figure 2 and figure 3. paclobutrazol application reduced plant height (fig. 2 & 5). the measurement that was carried out on 3 weeks old black rice seedlings showed that 12.5 ppm paclobutrazol applied during seed germination stage caused plant height reduction to about 25% compared to control, both in seedlings subjected to blue light or sunlight. greater concentration of paclobutrazol caused severe dwarfism. seeds that still germinated under 100 ppm paclobutrazol were so small and it was impossible to replant the seeds in bigger plastic pots. thus, further growth evaluation only used seedlings treated with either control or 12.5, 25 or 50 ppm paclobutrazol. this study showed that after black rice seedlings were being grown in open area for 12 weeks, those plants treated with 12.5 ppm paclobutrazol with or without blue light during germination still experienced 16 20% reduction in the average of plant height compared to control. black rice plants that previously treated with either 25 or 50 ppm biotropia vol. 23 no. 2, 2016 88 figure 2 average±se height of seedlings treated with blue light and paclobutrazol (n = 18) notes: a – e: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 3 growth performance of black rice seedlings grown under (a) sunlight and (b) blue light control pbz 12.5 ppm pbz 25 ppm pbz 50 ppm figure 4 growth of black rice plant treated with paclobutrazol (pbz) at 6 weeks after planting figure 5 average±se plant height at heading date of black rice plant that previously treated with blue light and paclobutrazol (n = 8) notes: a – g: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra n g e h ei gh t o f s ee d li n g (c m ) 20 18 16 14 12 10 8 6 4 2 0 control 12.5 ppm 12.5 ppm 25 ppm 25 ppm 50 ppm 50 ppm control sunlight blue light paclobutrazol treatments a b c d e b c c a ve ra g e + s e p la n t h ei gh t at h ea d in g d at e (c m ) 70 60 50 40 30 20 10 0 sunlight blue light control 12.5 ppm 25 ppm 50 ppm paclobuttrazol treatments a e b f c g d g effect of blue light and paclobutrazol on black rice – dewi et al. 89 paclobutrazol still showed dwarfism (about 50% reduction in plant height compared to control). this result indicated that paclobutrazol treatment during seed germination inhibited biosynthesis of gibberellin. however, tiller numbers in plants treated with 50 ppm paclobutrazol increased significantly compared to control. the average of tiller numbers in black rice plants treated with paclobutrazol is presented in figure 6. tiller numbers of black rice seeds subjected to blue light and 12.5 or 25 ppm paclobutrazol increased compared to control (fig. 6). in rice plant, auxin inhibited the biosynthesis of cytokinin and limited tiller growth (liu et al. 2011). application of paclobutrazol on mango plants (mangifera indica l.) inhibited biosynthesis of gibberellin and auxin (adil et al. 2011). thus, it is possible that paclobutrazol treatment in black rice plants reduced biosynthesis of gibberellin as well as auxin. on the other hand, cytokinin biosynthesis increased causing the increase of tiller numbers. the mechanism of paclobutrazol in inducing tiller growth is interesting to be further evaluated. the dwarfism caused by paclobutrazol may also increase the resistance of plants towards waterlogging as well as pests and diseases attacks (bridgemohan & bridgemohan 2014; cohen et al. 1987). chlorophyll content figure 7 shows the average of chlorophyll content in 3 months old black rice plant. the average of total chlorophyll was higher on plants treated with paclobutrazol compared to control (fig.7). this could be due to triazol applied during seed germination increased the biosynthesis of chlorophyll precursor, so that figure 6 average±se tiller numbers of black rice plants subjected to paclobutrazol and blue light (n = 8) notes: a – f : bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 7 average±se total chlorophyll content of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – g: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra g e + s e t il le r n u m b er 18 16 14 12 10 8 6 4 2 0 control 12.5 ppm paclobutrazol treatments a 25 ppm 50 ppm sunlight blue lightd b e ab e c f a ve ra g e+ s e c h lo rr o p h yl l co n te n t (m g/ g f w ) paclobutrazol treatments sunlight blue light control 12.5 ppm 25 ppm 50 ppm 3 2.5 2 1.5 1 0.5 0 a e b f c g d g biotropia vol. 23 no. 2, 2016 90 plants treated with paclobutrazol usually have higher chlorophyll content compared to control (chaney 2005). paclobutrazol application in camelina sativa l. crantz also increased chlorophyll content which led to greater rate in photosynthesis and higher yield (kumar et al. 2012). paclobutrazol also increased the activity of oxidative enzymes and enhanced plant resistence to water lodging in ipomoea batatas l. (lam) (lin et al. 2008). the results of this experiment showed that black rice plants treated with either 25 or 50 ppm paclobutrazol have greener leaves compared to control; the leaves also experienced late senescence. this could be due to an increase in the activity of oxidative enzymes which prevented cell maturation. slow senescence in leaves can prolong the phase of seed development and maturation. as a consequence, the yield can be increased, but the harvest time become late (pan et al. 2013). activity of nitrate reductase nitrate is a source of nitrogen for plant's cell. nitrate is reduced to ammonium in roots which then be stored in the vacuole or transported to leaves. reduction of nitrate to ammonium is catalyzed by nitrate reductase. the activity and transcript level of nitrate reductase in rice plant was suggested to be influenced by g-protein and protein kinase (ali et al. 2007). the average of nitrate reductase activity in black rice plants treated with paclobutrazol and blue light is presented in figure 8. activity of nitrate reductase increased in black rice plant treated with paclobutrazol and subjected to blue light during seed germination (fig. 8). similar finding was reported in arabidopsis, in which application of ga in 3 light-grown col-0 seedlings decreased nitrate reductase activity, but exogenous paclobutrazol enhanced nitrate reductase activity (zhang et al. 2011). it was suggested that paclobutrazol application may also increase several oxidative enzymes (lin et al. 2008). the increase in oxidative enzymes such as guaiacol peroxidase (pod), superoxide dismutases (sod) and catalase (cat) were related to an increase in nitrate reductase activity in ginger plant -5 (zingiber officinale rosco) treated with 10 m s a l y c y l i c a c i d ( g h a s e m z a d e h & ja a f a r 2013). average weight of 100 seeds the average weight of 100 seeds in black rice plants treated with blue light and paclobutrazol of 25 ppm is presented in figure 9. paclobutrazol treatment did not influence the average weight of 100 seeds obtained from black rice plants subjected to sunlight (fig. 9). however, in plants previously subjected to blue light, application of 25 ppm paclobutrazol increased figure 8 average±se nitrate reductase activity of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – d: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra g e + s e n it ra te r ed u ct as e ac ti v it y m m n it ri t/ g l ea f f re sh w ei gh t/ h o u r paclobutrazol treatments sunlight blue light control 12.5 ppm 25 ppm 50 ppm 25.000 20/000 15.000 10.000 5.00 0.00 a c a c a b d d effect of blue light and paclobutrazol on black rice – dewi et al. 91 the average weight of 100 seeds compared to control or other treatments. this result was probably due to the fact that when plants were treated with blue light and 25 ppm paclobutrazol, the leaves were greener and the chlorophyll content was higher compared to those of control. in terms of productivity, those plants treated with paclobutrazol of 25 or 50 ppm had higher tiller numbers. eventhough the weight of 100 seeds was similar to control, the total harvest of the plants treated with paclobutrazol was still relatively higher. fe content in the seeds fe content in the seeds was determined for plants treated with blue light and paclobutrazol (fig. 10). t here was no significant effect of paclobutrazol on fe content in black rice seeds harvested from plants without blue light treatment during seed germination (fig. 10). on the other hand, when the seeds were germinated under blue light, the plants produced seeds containing higher fe content. the increase of fe content in potato tuber following treatment with paclobutrazol was also reported by tekalign (2005). delaat et al. (2014) reported that ferritin is an essential iron homeostasis regulator in plant. their studies showed that the expression of three ferritin gene in phaseolus vulgaris l (pvfer1, pvfer2 and pvfer3) increased under iron excess and water deficit conditions. it has been reported that application of paclobutrazol could ameliorate abiotic stress condition, such as chilling or drought stress figure 9 average±se weight of 100 seeds of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – b: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 10 average fe content in black rice seed treated with blue light and paclobutrazol (n = 2) a ve ra g e w ei gh t o f 1 0 0 s ee d s (g ) 30 25 20 15 10 5 0 control 12.5 ppm paclobutrazol treatments a 25 ppm 50 ppm sunlight blue light a a a a a a b a ve ra g e f e co n te n t in b la ck r ic e se ed 45 40 35 30 25 20 15 10 5 0 (m g/ k g) control 12.5 ppm paclobutrazol treatments (ppm) 25 ppm 50 ppm sunlight blue light biotropia vol. 23 no. 2, 2016 92 through the ability of the plant to increase the activity of several antioxidant enzymes (pan et al. 2013). in sweet potato, application of triazole increased the content of ascorbic acid, αtocopherol, riboflavin, anthocyanin, and xanthophylls and activities of ascorbate peroxidase, superoxide dismutase, and catalase activities (sivakumar et al. 2010). iron serves as a component of many vital enzymes such as cytochromes of the electron transport chain, thus required for a wide range of biological functions (rout & sahoo 2015). it seems that there is an interaction between the increasing enzymatic activity triggered by triazole and iron uptake and accumulation in plant. the mechanism by which iron was accumulated in rice seeds of those plants treated with blue light and paclobutrazol is important to be further evaluated, since the bioavailability of iron in grains is important for human health. in addition, a simple and environmentally friendly technique for iron biofortification in grains is also important to be developed. conclusions blue light slightly reduced percentage of seed germination. paclobutrazol application up to 100 ppm did not inhibit seed ger mination. paclobutrazol reduced seedlings height. tiller numbers, total chlorophyll content, nitrate reductase activity and fe content in black rice increased in plants treated with blue light and 25 ppm paclobutrazol. acknowledgements we wish to thank ms. dewi kusumawati, and mr. martono for technical assistance and the “boptn fakultas biologi ugm tahun anggaran 2013” for financial support. references adil os, rahim a, elamin om, bangerth fk. 2011. effects of paclobutrazol (pbz) on floral induction and associated hormonal and metabolic changes of biennialy bearing mango (mangifera indica l.) cultivars during off year. arpn j ag & bio sci 6(2):55–67. ali a, sivakami s, raghuram n. 2007. regulation of activity and 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light induced nitrate reductase activity in arabidopsis seedlings. j plant physiol 168(8):2161–8. effect of blue light and paclobutrazol on black rice – dewi et al. 95 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 6. arida (genetic) revisi daf... biotropia vol. 20 no. 2, 2013: 122 133 genetic variation, heritability and correlation between resin production character of high resin yielder (hry) pinus merkusii arida susilowati *, iskandar z. siregar , supriyanto , imam wahyudi and corryanti (kopertis wilayah i sumut-nad) recipient of biotrop research grant 2010/accepted 18 april 2013 tree improvement programs for high resin yielder were started in 2006 through a series of survey and morphological identification of candidate trees with high resin production. specific information about genetic parameter of resin yielder candidates in cijambu seedling seed orchard is still not determined yet, although based on resin distribution trend this sso has the highest mean of resin production. in this research, individual and family heritability, coefficient genetic variation and genetic-phenotypic correlation were estimated for resin production and growth data from 15 open polinated families of planted in 1982 (set 1) 1983 (set 2) in cijambu sso. the results showed high value of coefficient genetic variation (cvg: 14.5-28.43%), individual narrow sense heritability values for resin production character (0.58-0.77) which resemble with previous researches. this indicates that genetic factor was dominant for resin production and selection activities were effective to get high yielder superior candidate. phenotypic and genotypic correlation found that bark thickness, crown length and stem diameter character was positively significant correlated to resin production, whereas severity attack level of pests-diseases and number of branches were negatively significant correlated to resin production. selection, high resin yielder, , heritability, phenotypic 1 2 2 3 4 1 2 3 4 coordinatior of private higher education region i north sumatera-aceh, indonesia silviculture department, faculty of forestry, bogor agricultural university, bogor, indonesia forest product department, faculty of forestry bogor agricultural university, bogor, indonesia research and development center of perum perhutani, bogor, indonesia p. merkusii pinus merkusii p. merkusii, variation abstract key words: * corresponding author : arida_iswanto@yahoo.co.id doi: 10.11598/btb.2013.20.2.1 122 introduction pinus merkusii et al p. merkusii p. merkusii pinus merkusii is known as an important industrial species for pulp and paper and sawn wood, gum rosin production, as well as considerable species for reforestation and land rehabilitation in indonesia (suhardi . 1994). one valuable product of resin and very demanded by international market is gondorukem (gum rosin). gondorukem is a potential product, grouped under pine chemical products and it plays an important role as non timber forest product in indonesia because it provides high national income about us$ 50 million/year as well as more job opportunities (fachrodji 2010). problems faced in gondorukem export was lower productivity so that indonesia still ranks as third position after china and brazil as gum rosin producer (cunningham 2006). in order to sustain indonesian gondorukem export, several activities have been undertaken to increase resin production through tree breeding activities, application of improved silvicultural techniques, improvement of tapping techniques and management (fahrodji 2010). breeding activities focusing on resin production is the most prospective way to be developed since early studies conducted in 2006, have resulted to several plus trees producing high yield of resin (high resin yielder). high resin yielder is the term given for pine genotype producing over 50 g/tree/3 days (fakultas kehutanan ugm 2006) higher than the current production (21 g/tree/3 days). breeding activities in indonesia were started since 1976 through a series of progenies test and descendants of plus tree selection which was focused on stem straightness character (soeseno 1988), while resin production is only a side product. in considering high value of resin products, pine breeding activities were recently conducted to obtain plus trees for resin production (soeseno 2001). for breeding activities concentrating on resin yielder, it is important to collect information on genetic parameters such as coefficient of genetic variation, heritability and association of characters related to resin production for directing efficient selection. several researches on conducted in early establishment of seedling seed orchard (sso) in java and other pine species showed that resin production character has high heritability value, but specific information of cijambu sso is not determined yet. therefore, research on "genetic variation, heritability and correlation between resin production characters of high resin yielder (hry) in cijambu seedling seed orchard (sso)" is needed. the result will be used to provide best possible trees for further genetic improvement as well as to establish plantations using improved genetic materials. experiment was carried out in cijambu sso, sumedang. data for this research were obtained from progenies test plantation established in 1982 (set 1) and 1983 (set 2). at early plantation the research focused on stem character, where 200 families materials and methods 123 genetic variation, heritability and correlation between resin production character arida susilowati– et al. were planted/year designed in completely randomized block design, 5-tree line plot with ten blocks as replication at a spacing of 3 x 3 m. together with selective thinning leaving only 2-3 trees per plot. second selection focused on resin production conducted in 2006 found 96 families with 110 plus trees in progenies trial planted in 1978-1983. planting year 1982 involved 20 open pollinated families and in 1983 involved 25 open pollinated families of resin yielder candidates spread in 2 different blocks. for cvg and heritability estimation, 15 selected open polinated families were used. in this research two assumption and 2 types of estimation were used, those were: 1) resin production was not different at the age 29 and 30 years, for these reason we combined 2 set of families (1982 and 1983) into one estimation and 2). resin production was different at the age of 29 and 30 years so we used separate estimation using set 1 and set 2. characters studied in this research were resin production and other quantitative characters which assume to be related to resin production those were: total height (th), clear bole height (cbh), diameter, crown length (cl), crown width (cw) number of branches (nb), first branch angle (fba), bark thickness (bt), severity attack level from pest and disease (sl), clear bole volume (vbc) and total volume (vtot). all characters were measured based on previous methods for forest trees developed by bacilieri (1996); cantini (1999); kremer (2002); ginwal (2004); weber and montes (2005); baliuckas (2005) and devagiri (2007). resin production was measured by calculating resin tapping weight for 3 days. analyses of variance (anova) were carried out on single tree plot design according to isik (2008) using individual tree basis for each progeny test with following linear model: y : μ+ b + f +e where: y : individual tree observation; μ: the overall mean; b : the effect of i block; f :the effect of j family; e : within plot error. only 2 replications were included in the analyses. variance and covariance component were calculated by equating the means square or mean cross product to their expectation. as progeny trial assumed to be half-sib, heritabilities and standar error (s.e) were estimated by using single site analyses formula: heritability of family means: h f/ + /nb+ ), with s.e h f: (s.e f)/ + /nb+ ), individual tree heritability: h : 4 + ), with s.e. h : (4s.e f)/ ( + ) where: h f: family mean heritability; h :individual tree heritability f: the component due to family means : the component of block : the component of variance within plot. the standard error of family component variance (s.e f) was calculated by anderson & bancroft (1952) hardiyanto (1996): s.e f: [2/k (ms ) / (df +2)] , et al. et al. et al. et al. et al. et al. ( ( in data analysis ijk i j ijk ijk i j ijk f b e f b e f b+ e f b+ e b e i i i th th 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0.5 f : σ σ σ σ σ σ σ σ f/ (σ σ σ σ σ σ ; σ ; σ ; σ σ σ δ δ biotropia vol. 20 no. 2, 2013 124 i: i. where: k is the coefficient of family means square; ms the i means square used to estimate n component; df:the number of degree of freedom for ms criteria for heritability f) was calculated according to cotteril and dean (1990). estimates of genetic variation coefficient performed using cornelius (1994) formula as follows: genetic and phenotypic correlation between resin production character and growth character estimated using statistical formula as follows: estimates of variances and allied statistics for contributing sources of variability are presented for each trait in table 1. based on cornelius (1994) classification, genetic variation in cijambu sso showed variation value with total character contribution ranging from 4.05% (total height character) to 28.70% (severity attack level from pest and disease). this result indicated that not all of the characters observed in cijambu sso were influenced by genetic factors. resin production and severity attack level from pest-disease character have high value of coefficient genetic variations. diameter, crown, bark thickness, number of branches, clear bole volume, total volume, have moderate value of coefficient genetic variations, while total height, clear bole height and branching angle in set 2 have lower value of coefficient genetic variations. high cvg value represents resin production character (14.5-28.43%) and severity attack level from pest-disease (24.028.70%). it indicates that genetic factors will have a big impact on the appearance of characters. high value for resin production character was obtained compared to previous research conducted by roberds (2003) in sso (cvg:13.7%). high value for severity attack level from pestdisease was also compared to bastein & alia (2000) in (cvg:26.8%). in order to tree improvement focused on resin production in cijambu sso, these characters are important to be studied in detail because of the influence of genetic factors. cvg value for total height, clear bole height, crown width, crown length and number of branches character has lower value. it indicated that these characters belong to vegetative characters which is influenced by environmental factors. th th 2 (h et al. p.taeda p. sylvestris coefficient genetic variation (cvg) results and discussion genetic variation, heritability and correlation between resin production character arida susilowati– et al. 125 heritability based on cotteril and dean (1990) classification, heritability value for resin production character at cijambu sso was high (h2f:0.700.09-0.820.08 and h2:0.580.08-0.770.08), severity attack level from pest-disease also has high heritability value (h2f: 0.640.02-0.800.16 and h2:0.640.13-0.690.04). stem diameter, bark thickness, crown length, number of branches and volume have lower to moderate heritability value (table 1). characters σ2f σ2b σ2e cvg (%) h 2f se h2 se 29 and 30 years old resin production days (g/3) 581.8 338.1 86.1 21.6 0.70 0.09 0.58 0.08 total height (m) 1.94 2.58 11.07 6.0 0.14 0.05 0.12 0.05 clear bole height (m) 3.40 7.0 3.39 6.6 0.33 0.23 0.25 0.17 diameter (m) 0.002 0.007 0.002 10.1 0.29 0.72 0.20 0.52 bark thickness (cm) 0.36 0.89 0.26 8.8 0.34 0..83 0.24 0.58 number of branches 55.92 69.15 33.17 12.8 0.45 0.10 0.35 0.08 crown length (m) 1.507 3.122 1.74 10.6 0.31 0.30 0.24 0.23 crown width (m) 1.272 2.96 1.55 12.5 0.30 0.30 0.22 0.21 first branch angle 105.97 34.32 58.12 11.2 0.58 0.10 0.53 0.09 clear bole volume (m3) 0.002 0.009 0.04 13.9 0.35 0.22 0.33 0.20 total volume (m3) 0.007 0.34 0.14 9.4 0.18 0.60 0.12 0.42 severity attack level from pest and disease 139.91 15.75 49.86 28.2 0.71 0.13 0.68 0.12 set 1 (29 years old) resin production days (g/3) 200.70 53.94 46.82 14.5 0.72 0.13 0.67 0.19 total height (m) 0.67 3.5 21.83 3.3 0.04 0.01 0.03 0.03 clear bole height (m) 1.69 3.5 6.08 4.89 0.21 0.11 0.15 0.13 diameter (m) 0.001 0.0005 0.002 7.9 0.47 0.15 0.36 0.04 bark thickness (cm) 0.27 0.36 0.12 6.5 0.39 0.15 0.36 0.54 number of branches 15.73 77.16 40.13 13.2 0.14 0.03 0.12 0.09 crown length (m) 2.42 3.02 1.13 13.0 0.40 0.48 0.37 0.18 crown width (m) 1.11 1.56 3.26 10.3 0.26 0.18 0.19 0.05 first branch angle 6.4 36.15 106.2 2.8 0.07 0.01 0.04 0.02 clear bole volume (m3) 0.02 0.002 0.07 13.1 0.31 0.16 0.19 0.06 total volume (m3) 0.06 0.07 0.23 13.8 0.25 0.15 0.17 0.37 severity attack level from pest and disease 95.59 5.29 37.83 24.0 0.80 0.16 0.69 0.04 table 1. family f), block variance b), within plot error e), coefficient of genetic variation (cvg), family heritability (h f) and individual tree heritability for resin production and growth characters variance (σ (σ variance (σ 2 2 2 2 biotropia vol. 20 no. 2, 2013 126 p. merkusii et al. p. eliotii et al. p. pinaster et al. p. taeda et al. et al. et al. et al. et al. et al. et al. heritability for resin production shows high value (0.580.08-0.770.08). it seems to be slightly different than previous research results (0.69) conducted by leksono (1990) at cijambu and sempolan on 12 years old progenies test plantation. this result was higher than reported by zhang (2010) on (0.37), tadesse (2001) on (0.5) and roberds (2003) on (0.44 to 0.59), and similar value was also found at stem diameter and branching quality. higher value in this research compared to previous researches at early sso establishment suggested that the materials used for this research originated from second selection focused for resin production. for high resin production heritability values, wenger (1984); burczyk (1998); kassuth (1984); mergen 1955, gill (1998) explained that resin production character is controlled by gene. this indicates that improvement program for resin production character through genetic selection would provide higher genetic gain not affected by other interaction factors. heritability value for height and stem diameter were slightly different from previous researches conducted by hardiyanto (1996) who focused on stem straightness, and leksono (1996) for resin yield at 12 years old progenies test (h2f: 0.40 dan h2f: 0.43). heritability value for branching trait character is lowe in set 1 (0.12 and 0.07) but in set 2 the heritability value is high (0.44 dan 0.71). the changes of heritability value in long rotation crops such as a tree is not surprising since genes involved in growth may change with age (namkoong 1980; monteuis 2011), and these changes also may be related to different growth phases (franklin 1979). change of heritability value for diameter, tree height and bark thickness character at different age probably is influenced by silvicultural practices such as thinning and other management practices (gwaze 1997 & lopez-upton 1999). result from cvg and heritability estimation in cijambu sso showed high value for resin production, it indicated that more dominant genetic factors determine this character. based on this value, tree improvement program for high resin yielder can be initially conducted by mass selection of individual trees with high resin production. characters σ2f σ2b σ2e cvg (%) h 2f se h2 se set 2 (30 years old) resin production (g/3 days) 1012.5 151.2 5 151.3 28.43 0.82 0.084 0.77 0.08 total height (m) 1.15 1.02 0.86 4.64 0.46 0.06 0.38 0.52 clear bole height (m) 3.08 6.75 0.33 4.91 0.45 0.09 0.30 0.06 diameter (m) 0.002 0.01 0.001 8.49 0.19 0.07 0.12 0.411 bark thickness (cm) 0.09 0.19 0.38 10.93 0.16 0.01 0.14 0.29 number of branches 66.50 14.09 5.61 8.12 0.84 0.05 0.70 0.41 crown length (m) 0.82 0.05 1.007 7.81 0.44 0.06 0.44 0.55 crown width (m) 0.50 0 0.48 7.84 0.51 0.09 0.51 0.01 first branch angle 201.95 18.75 75 5.26 0.71 0.10 0.68 0.10 clear bole volume (m3) 0.0090.04 0.03 12.04 0.62 0.09 0.50 0.06 total volume (m3) 0.14 0.33 0.009 13.06 0.38 0.08 0.26 0.07 table 1. continued genetic variation, heritability and correlation between resin production character arida susilowati– et al. 127 biotropia vol. 20 no. 2, 2013 this condition agrees with tadesse . (2001) who stated that when a population has high heritability value for a character, mass selection method would be more efficient for improving the character. furthermore, white . (2007) stated that mass selection was appropriate to be implemented in early selection and for high heritability value characters because the phenotypes of individual trees describe its genetic ability. phenotypic and genotypic correlation (table 2) showed low to moderate coefficient value, indicated that not overall phenotypic appearance describing genetic expression because interaction between environment and genetic factor also influencing phenotypic expression of trees. phenotypic correlation showed resin production character was positively significant correlated with stem diameter, bark thickness and crown length. furthermore, number of branches and severity attack level from pest -disease character was negatively significant correlated with resin production. positive correlation between resin productions with some characters, indicated that resin production will increase equally with the increase of character value. on the other hand, a negative correlation between resin production with some characters indicates that resin production will decrease with high value of character components. genetic correlation between resin production and stem diameter, bark thickness, and crown length indicated that resin production will increasedequally with the increase of these characters. on the contrary resin production will decrease equally with the increase of branching number and severity attack level of pest-disease. different results were obtained in set 2 (30 years old progenies trial), in set 2 we found negative correlation between resin production and tree height, it indicates that resin production will decrase with the increase of tree height. correlation between resin production with stem diameter and crown length are in accordance with previous research results conducted by pswaray (1996); coppen (1984) on westbork (2011) on and tadesse (2001) on . panshin & de zeeuw (1984) also explained that a good tree is characterized by large diameter and large crown size because trees need more wider light absorbance for photosynthesis process. correlation between resin production and stem diameter have been reported also by coppen 1984 who stated that wider diameter trees have wider annual ring and giving great chance to have more resin ducts and produce higher resin yield than smaller diameter trees. number of branches and severity attack level from pest and disease have negatively significant effect to resin production meaning that resin production decreased with the increasing number of branches and severity attack level from pest. papajiannopoulos (2002) in also found that tree with canopy openes (lower branch number) have higher resin yield compared with higher branch number which assume to be related to photosyntetic process, resin viscocity and accumulation. correlation between resin production and severity attack level from pest-disease have been deeply studied in other pine species and conifers. previous researches conducted et al et al et al. et al. p.elliottii; p.taeda et al. p.pinaster naval store et al. p.halepensis phenotypic and genetic correlation between growth character and resin production 128 c ar ac te r p ro d t h c b h d b t c l c w n b f b a s l v b c v to t s et 1 d an se t 2 p ro d 1 0 .0 1 4 -0 .1 1 2 0 .5 8 4 ** 0 .2 9 9 0 .1 7 6 0 .0 9 6 -0 .4 9 6 * -0 .0 2 3 -0 .5 1 7 * -0 .2 8 4 -0 .2 5 4 t h 0 .1 2 1 1 0 .2 0 4 -0 .0 4 3 -0 .2 8 3 0 .3 1 6 0 .7 1 6 * 0 .1 5 5 0 .2 5 2 0 .0 4 7 0 .1 6 3 0 .4 1 1 c b l 0 .1 0 6 0 .4 4 5 1 -0 .3 4 4 -0 .4 3 5 -0 .1 8 7 0 .0 3 3 0 .2 4 0 -0 .2 0 0 0 .3 0 6 0 .4 9 0 * -0 .0 .8 1 d 0 .6 5 0 * 0 .2 7 2 0 .2 3 6 1 0 .6 0 2 ** 0 .1 2 8 -0 .1 0 5 -0 .0 9 6 -0 .1 4 3 -0 .2 4 1 0 .1 8 3 0 .3 8 7 b r 0 .1 8 6 0 .7 7 4 0 .6 7 2 0 .4 1 5 1 -0 .0 6 3 -0 .1 4 4 -0 .1 4 5 -0 .0 1 1 -0 .1 1 9 0 .0 6 5 0 .2 4 5 c l 0 .1 2 9 0 .5 4 0 0 .6 4 9 0 .2 9 0 0 .2 4 8 1 0 .3 2 3 -0 .1 5 0 0 .0 5 3 -0 .2 0 4 -0 .1 5 9 0 .0 6 9 c w 0 .1 3 5 0 .1 7 4 0 .4 9 0 0 .3 0 2 0 .2 5 4 0 .5 0 0 1 0 .1 4 1 0 .2 8 1 0 .1 9 8 0 .0 3 0 0 .2 8 6 n b -0 .0 5 2 0 .2 1 9 0 .1 9 0 0 .1 1 7 0 .3 3 4 0 .2 3 3 0 .2 4 3 1 0 .0 4 7 0 .3 5 7 0 .3 2 1 0 .2 5 6 f b a 0 .0 4 4 0 .1 8 6 0 .1 6 2 0 .1 0 0 0 .2 8 4 0 .1 9 8 0 .2 0 7 0 .0 8 1 0 .1 1 4 0 .0 3 8 0 .2 3 3 s l 0 .0 4 1 0 .1 7 4 0 .1 5 1 0 .2 3 6 0 .2 6 5 0 .1 6 2 0 .1 9 3 0 .0 7 0 .0 6 1 0 .3 1 8 0 .2 5 4 v b c 0 .3 6 6 0 .1 5 2 0 .1 3 2 0 .4 7 2 0 .2 3 2 0 .1 6 2 0 .1 6 9 0 .2 5 4 0 .3 0 5 0 .3 2 2 1 0 .7 5 1 ** v to t 0 .2 8 0 0 .1 1 6 0 .1 0 1 0 .6 2 3 0 .1 7 8 0 .1 2 4 0 .1 2 4 0 .2 0 4 0 .4 0 9 0 .4 0 0 0 .3 5 6 1 s et 1 (3 0 ye ar s o ld ) p ro d t t b t b c d t k b p t l t c p t s c p h p v b c v to t p ro d 1 0 .3 2 9 0 .2 9 7 0 .4 2 9 * 0 .4 0 3 * 0 .4 2 6 * 0 .1 2 2 -0 .3 5 5 * 0 .2 3 8 -0 .3 2 1 * 0 .5 6 4 0 .4 6 9 t h 0 .2 0 8 1 0 .1 9 8 0 .5 7 5 0 .0 7 0 0 .1 9 5 0 .1 5 6 0 .2 8 5 0 .6 4 2 -0 .1 7 9 0 .4 9 8 0 .8 3 8 * c b l 0 .1 6 4 0 .6 8 1 1 0 .0 5 9 -0 .3 3 9 0 .3 6 9 0 .4 4 4 -0 .1 8 8 -0 .1 2 4 -0 .0 8 6 0 .7 2 0 * 0 .0 8 5 d 0 .5 6 1 * 0 .4 2 4 0 .3 3 6 1 0 .4 6 9 0 .0 8 2 0 .1 6 0 -0 .2 6 8 -0 .4 2 8 -0 .1 9 2 0 .7 3 1 * 0 .9 3 3 ** b r 0 .3 4 5 0 .1 0 9 0 .6 6 4 0 .5 3 3 1 -0 .5 0 6 -0 .0 9 7 0 .1 9 0 0 .5 6 0 0 .3 4 9 0 .0 9 7 0 .3 3 8 c l 0 .1 5 0 0 .6 2 7 0 .3 1 1 0 .3 0 7 0 .6 8 9 1 0 .2 8 2 -0 .2 9 0 -0 .0 7 1 -0 .4 5 1 0 .0 5 8 0 .2 8 1 c w 0 .1 8 3 0 .7 6 2 0 .4 9 7 0 .3 7 3 0 .8 5 1 0 .5 5 1 1 0 .2 0 4 0 .1 4 5 -0 .0 4 7 0 .1 5 7 0 .5 4 9 n b -0 .0 9 4 0 .3 1 1 0 .3 1 1 0 .1 9 2 0 .6 1 9 0 .2 8 4 0 .3 4 5 1 0 .1 7 4 -0 .1 9 0 0 .1 4 1 0 .0 0 4 f b a 0 .1 1 8 0 .3 9 3 0 .3 9 0 0 .2 4 0 0 .4 9 4 0 .3 5 6 0 .5 5 1 0 .2 2 3 1 0 .0 4 5 0 .1 8 0 -0 .0 5 9 s l -0 .0 6 0 0 .2 5 4 0 .1 9 8 0 .3 1 5 0 .3 1 5 0 .1 8 1 0 .2 2 0 0 .1 1 3 0 .1 4 2 1 -0 .1 7 9 0 .1 7 4 v b c 0 .2 5 2 0 .2 1 9 0 .1 7 4 0 .5 0 5 0 .2 7 6 0 .1 5 9 0 .1 9 3 0 .6 6 7 0 .1 2 4 0 .6 3 1 0 .6 8 3 v to t 0 .3 7 8 0 .1 5 7 0 .1 2 5 0 .5 5 2 0 .1 9 8 0 .1 1 4 0 .1 3 8 0 .5 7 6 0 .6 8 9 0 .4 5 5 0 .4 0 0 1 t ab le 2 .p h en o ty p ic (a b o v e d ia g o n al ) an d g en et ic co re la ti o n (b el o w d ia g o n al ) b et w ee n re si n p ro d u ct io n ch ar ac te r an d o th er co m p o n en t genetic variation, heritability and correlation between resin production character arida susilowati– et al. 129 biotropia vol. 20 no. 2, 2013 c ar ac te r p ro d t h c b h d b t c l c w n b f b a s l v b c v to t t ab le 2 .c o n ti n u ed s et 2 (2 9 ye ar s o ld ) p ro d t t b t b c d t k b p t l t c p t s c p h p v b c v to t p ro d 1 0 .0 4 5 -0 .1 2 6 0 .5 5 5 * 0 .0 9 4 0 .3 9 7 * 0 .0 6 0 -0 .7 1 4 * -0 .1 6 2 -0 .6 6 6 * 0 .4 7 4 0 .4 5 6 t h -0 .0 5 6 1 0 .1 7 8 -0 .4 9 1 0 .3 5 6 * 0 .3 4 9 0 .3 0 0 * -0 .0 2 9 0 .5 5 0 -0 .1 1 5 0 .4 0 0 0 .4 4 2 * c b l -0 .0 7 6 0 .2 3 5 1 -0 .4 2 3 -0 .0 9 7 -0 .5 1 6 -0 .4 2 8 0 .0 1 4 -0 .6 4 1 -0 .0 9 4 0 .0 3 1 0 .0 4 2 d 0 .4 9 9 * 0 .1 0 7 0 .2 9 6 1 0 .6 5 3 -0 .1 8 7 -0 .2 1 8 -0 .1 8 7 -0 .2 5 9 -0 .0 8 4 0 .2 2 7 0 .3 3 3 b r 0 .3 5 5 0 .0 5 1 0 .5 1 9 0 .1 1 6 1 -0 .2 8 3 -0 .6 4 7 0 .2 9 8 -0 .1 8 1 0 .4 2 8 0 .6 6 4 0 .5 8 0 c l 0 .2 6 5 0 .2 6 7 0 .1 9 1 0 .3 4 5 0 .1 0 5 1 0 .4 0 5 0 .0 8 0 0 .1 5 2 -0 .1 2 5 -0 .5 7 9 -0 .2 5 4 c w 0 .0 6 5 0 .2 9 1 0 .2 9 1 0 .4 0 6 0 .1 3 5 0 .3 2 0 1 -0 .2 0 5 0 .6 3 0 0 .1 1 5 -0 .1 4 3 0 .2 3 9 n b -0 .4 8 9 0 .0 9 1 0 .4 7 3 0 .4 3 5 0 .0 4 3 0 .1 3 3 0 .3 3 3 1 0 .0 9 6 0 .5 3 2 * 0 .3 0 1 0 .3 2 6 f b a 0 .0 7 5 0 .0 8 7 0 .0 8 7 0 .3 3 5 0 .1 3 2 0 .0 9 4 0 .2 0 1 0 .0 1 9 2 1 0 .1 1 5 -0 .1 4 3 0 .2 3 9 s l -0 .4 9 6 0 .0 6 5 0 .1 3 5 0 .0 8 5 0 .2 3 5 0 .1 9 2 0 .4 3 4 0 .4 5 3 0 .1 9 8 1 0 .1 9 3 0 .1 6 9 v b c 0 .1 2 6 0 .0 3 5 0 .0 7 9 0 .4 2 1 0 .2 5 6 0 .1 3 2 0 .1 7 7 0 .1 5 6 0 .2 5 3 0 .0 6 7 1 0 .6 8 7 v to t 0 .2 7 8 0 .2 3 0 0 .2 1 7 0 .4 3 5 0 .2 9 9 0 .3 4 3 0 .1 9 2 0 .2 5 7 0 .1 9 8 0 .0 9 9 0 .1 8 8 1 n o te : ** : si g n if ic an t d if fe re n t at 9 9 % * : si g n if ic an t d if fe re n t at 9 5 % 130 in temperate regions by kleinhentz . (1998); blada (2000); kim . (2003); rafael . (2005) concluded that pest and disease caused significant decrease of resin production quantities. furthermore, raffa & berryman (1982) in also found severity attack level which caused loss of resin yield quantities. however, specific studies about correlation between resin production and severity attack level in cijambu sso have not been conducted yet. from this research we found that both resin production and severity attack level in cijambu sso have high cvg value, it indicated that selection activities focused for resin production also can escorted together with resistance for pest and disease. although some characters have a correlation to resin production, further research still need to be conducted because phenotypic observation is influenced by growth phase and environment, so it could not differentiate recessive genotype with resemble morphological appearance such as secondary metabolites (finkeldey 2005). to overcome this problem molecular marker such as rflp, rapd, aflp and microsatellite can be used. results from genetic variation and heritability estimation of resin yielder candidates character in cijambu sso showed high coefficient of genetic variation value (cvg: 14.5-28.43%) and heritability value (h2:0.580.08-0.770.08) for resin production character. it indicated that genetic factor strongly affected resin production character. genetic and phenotypic correlation found that stem diameter, bark thickness and crown length character were positively significant correlated to the resin production, whereas severity attack level from pest-disease and number of branches was negatively significant correlated to resin production. it indicated t genetically improvement on stem diameter, bark thickness and crown length increasing resin production. whereas, higher severity attack level from pest-disease and number of branches decreased resin production. this research was part of arida susilowati's studies toward doctorate degree from bogor agricultural university (ipb). i would like to express my sincerest thank to seameo-biotrop for supporting my research with and development center of the perum perhutani for the access to their experimental plot in cijambu sso and in providing samples as well as for technical assistance during the fieldwork. et al et al et al p.taeda phd research grant. my sincerest appreciation also goes to research conclusions acknowledgments genetic variation, heritability and correlation between resin production character arida susilowati– et al. 131 biotropia vol. 20 no. 2, 2013 references baliuckas vt, lagerstrom i, norell and g eriksson 2005. genetic variation among and within populations in swedish species of l. and l. assessed in a nursery trial. , 54: 1-8. bastein c, alia r 2000. what might be useful measures of genetic variability for adaptive characters within populations of scots pine . burczykj, lewandowskai, chojnackib 1998. resin production of scots pine trees may be associated with multilocus allozyme 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(eds.), 349-357 pp. prosea bogor indonesia. 610 pp. tadesse w, nanos n, aunon fj, alia r, gill l. 2001. evaluation high resin yielder of ait. , 8 (suppl 4): 271-278. weber jc, sotelo montes c. 2005. variation and correlations among stem growth and wood characters of calycophyllum spruceanum benth. from the peruvian amazon. , 54:31-40. westbrook j. 2011. quantitative genetics of constitutive oleoresin defense in loblolly pine. southern forest tree improvement conference. white tl, adam wt, neale db. 2007. . cromwell press group. trowbridge. zhang jh, shen f, jiang j , luan q, yang q, xu y, liu z. 2010. heritability estimates for real resin capacity and growth characters in high-gum-yielding slash pine. cnki. 03-010. dioryctria sylvestrella (lepidoptera:pyralidae) pinus pinaster forest genetics usda. fol: sew. general tech. report, northeastern sta et al. quercus robur quercus petraea annals of forest sciences pinus merkusii buletin penelitian kehutanan silvae genet forest science tectona grandis commonw. for rev pinus elliottii elliottii silvae genetica pinus pinaster hylobius abietis ann. for. sci . environ. entomol can. j. for. res pinus merkusii et in pinus l. in plant resources of south-east asia 5(1) pinus pinaster forest genetic silvae genet forest genetic genetic variation, heritability and correlation between resin production character arida susilowati– et al. 133 516 isrok (eimeria species).cdr eimeria species composition and factors influencing oocysts shedding in dairy farm, bandung, indonesia isrok malikus sufi , umi cahyaningsih nd etih sudarnika 1* 2 2 a 1 disease investigation center subang, ministry of agriculture, subang 41212, indonesia 2 department of animal disease sciences and veterinary public health, faculty of veterinary medicine, institut pertanian bogor a, bogor 16680, indonesi received 15 july 2015/accepted 25 may 2017 abstract coccidiosis is one of the most widely distributed parasitic diseases of cattle throughout the world. coccidiosis infection in ruminants was caused by eimeria spp. the objective of this study was to determine eimeria species composition and various factors influencing eimeria oocysts shedding in dairy farm. this study was conducted with a cross-sectional study design in dairy farm in south bandung district from july 2014 to january 2015. samples were obtained from 400 dairy cattle (196 cattle at age < 6 months, 37 cattle at age 6 12 months and 167 cattle at age > 12 months). fecal samples were collected, examined and counted for eimeria species composition and numbers of oocysts per gram of feces (opg) using mcmaster technique. a questionnaire was completed for individual dairy cattle farmer to record information about cattle's health and husbandry. the effect of cattle's sex, age and type of pen flooring to opg values were analyzed using mann-whitney and kruskal-wallis tests. the kruskal-wallis test was performed followed by dunn test as a multiple comparison test. ten species of eimeria were identified in all infected cattle. among the eimeria identified species, eimeria bovis was found to have the highest prevalence (42.5%), followed subsequently by eimeria wyomingensis (39.1%), eimeria bukidnonensis (32.4%), eimeria pellita (26.3%), eimeria auburnensis (19.6%), eimeria zuernii (17.3%), eimeria cylindrica (3.9%), eimeria canadensis (3.9%), eimeria brasiliensis (3.4%) and eimeria alabamensis (1.1%). the numbers of oocysts shed was correlated significantly (p < 0.05) with cattle's sex and age as well type of pen flooring which influenced the infection pressure. younger calves aged less than 6 months shed the highest amount of eimeria oocysts than older cattle. many factors may cause the increasing number of opg in fecal samples. therefore, it is important to keep good sanitation and control of eimeria among dairy cattle in the kpbs pangalengan dairy farm. keywords: coccidiosis, cross-sectional, dairy farm, eimeria, opg introduction coccidiosis is a protozoic disease in cattle caused by eimeria spp more than twelve different . species of eimeria have been described in cattle ; most of which are considered harmless. of these many species, e. bovis and e. zuernii are highly pathogenic causing mortality and morbidity by disturbing absorption mechanisms (rehman et al. 2011). infection calves large occurs on caused by numbers of oocyst of e. bovis or e. zuernii may s , result in severe diarrhea with feces containing blood, fibrin and intestinal tissue. however, coccidiosis in cattle commonly occurs as subclinical disease without signs of the disease and involving great economical losses due to reduced appetite, reduced body weight, impaired feed conversion, unthriftness, diarrhea, dysentery, anemia and increased susceptibility to other diseases (abebe et al. 2008). a poorer body condition score (bcs), potentially as the result of a disease, can be connected to a lower milk production in dairy cattle (lassen 2009). c is estimated to cause annualoccidiosis economic loss in excess of us 400 million in the us d (bruhn et al. 2011). the development of clinical coccidiosis in cattle mainly depends on factors such as species * corresponding author: isrok.sufi@gmail.com 104 biotropia 4 2 7 104 113 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 516 of , age of infected animal, number of eimeria oocysts ingested, presence of concurrent infections, as well as type of production system and management practices (abebe . 2008). et al the aim of this present study was to determine the species composition and factors eimeria influencing the incidence of oocysts eimeria shedding in cooperation of south bandung dairy farm (kpbs) pangelangan, district of bandung, indonesia. thus, determination of species composition and evaluation of factors influencing the shedding of oocysts per eimeria gram (opg) is very useful in designing efficient control strategies. materials and methods study area t he study was conducted in kpbs pangalengan, south bandung district, west java province from july 2014 to january 2015. kpbs pangalengan bandung covers 27.3 ha of highland area with an altitude of 1,000 to 1,420 m asl, 12 – o 28 c mean annual temperature and 60 – 70% relative humidity. the average annual rainfall was > 2,000 mm during the study period. study design c applied based ross-sectional study design was on thrusfield (2007). a total of 400 dairy cattle (n) from a total population of 14 000 dairy cattle , distributed on 37 cooperation service area (tpk) and 225 farmers group were selected for the s study with 95% confidence interval, 50% expected prevalence (p) and 5% accepted error (l). the relevant formula for a 95% confidence interval is thrusfield 2007) based on ( : n = 4pq/l 2 where: n = total of cattle p = expected prevalence q = 1 – p l = accepted error samples obtained from dairy cattle farmers covered all age groups i.e. calves (age < 6 months); weaners (6 12 months); and adults (age > 12 months). from the total of 225 farmers groups, 10 farmers groups consisted of 80 farmers were randomly selected because these farmers had complete cattle age groups. from each of these farmers, five fecal cattle samples were collected including all calves in the selected farmers, while the remainder fecal samples were taken from weaners and adults. sample collection fecal samples of 20 50 g were collected from each age group. the feces was directly obtained from cattle's rectum or immediately collected after cattle defecation. fecal samples were stored in respective plastic bag and preserved in o refrigerator at 4 c before being tested in the laboratory for the occurrence of eimeria spp. oocysts. fecal consistency was assessed immediately after sampling and classified as normal or diarrheal without any additional differentiations (bangoura et al. 2011). the plastic bags containing fecal samples were labeled with sampling information such as sample number, sampling date, sampling location, cattle's age group, and farmer's name. practices of animal health management were recorded by the participating farmers in a questionnaire. in addition, record of cattle's sex and age (in months) were documented for each cattle from which a sample was taken. counting of poocysts er gram of feces (opg) fecal samples were examined and oocysts numbers were counted using the mcmaster method. two grams of fecal material was mixed thoroughly with 28 ml of sugar-salt solution and filtered through a 200 µm mesh wire sieve or tea strainer. the suspension was equally poured into two mcmaster counting chambers (2 x 0.15 ml) and was let still for 5 minutes. the counting was carried out using light microscope with 100x magnification. the numbers of oocysts in the two chambers were multiplied by dilution factor (50) to obtain the number of oocysts per gram of feces (opg). this protocol was modified from dong et al. (2012), lucas et al. (2006) and soulsby (1986). eimeria species identification the remainder of each positive samples were centrifuged at 1,500 rpm for 5 minutes at room temperature of 25 c for identification, a o . eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. 105 data analysis the effect of cattle's sex and age as well as type of pen flooring to eimeria opg counts were analyzed using mann-whitney and kruskalwallis tests. the kruskal-wallis test was performed followed by dunn test as multiple comparison test. results and discussion species identification and composition of eimeria. results of this study showed that the shedding of eimeria oocysts were commonly occurred in kpbs pangalengan dairy farm in bandung. from flotation of the oocysts in sugar-salt eimeria solution was performed. oocysts were measured under ocular eye piece that was calibrated with micrometer under 40x objective lense of a light microscope. identification of species was eimeria based on the morphological features of the oocysts (size, index, shape, color and texture of oocyst's wall, presence or absence of micropyle and polar cap) with the aid of taxonomic keys (soulsby 1986; daugschies & najdrowski 2005). measurement of oocysts index value were performed by dividing the length and width of oocysts. the shape of oocysts eimeria were examined for each sample and classified as round (length/width = 1), ovoid (length/width between 1 1.5) and ellips (length/width > 1.5) (cahyaningsih & supriyanto 2007). 106 biotropia vol. 24 no. 2, 2017 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) 10 µm10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm figure 1 the results of eimeria species oocysts in dairy cattle in kpbs pangalengan, bandung (a) eimeria bovis; (b) eimeria wyomingensis; (c) eimeria bukidnonensis; (d) eimeria pellita; (e) eimeria auburnensis; (f) eimeria zuernii; (g) eimeria cylindrica; (h) eimeria canadensis; (i) eimeria brasiliensis; (j) eimeria alabamensis 400 fecal samples, there were 179 fecal samples (44.8% prevalence; confidence interval (ci) 95%; 40.0 49.6 ) contained eimeria oocysts. a total of 10 eimeria species i.e. e. bovis, e. wyomingensis, e. bukidnonensis, e. pellita, e. auburnensis, e. zuernii, e. cylindrica, e. canadensis, e. brasiliensis and e. alabamensis were identified from the 179 positive fecal samples collected from kpbs pangalengan dairy farm (fig. 1, table 1, table 2). our study showed that e. bovis was the most prevalent species (42.5%; ci 95%: 37.1 47.8) and had the opg highest mean opg number (538.2 ( ocysts er ram of feces); ci 95%: 191.5 -o p g 884.8) followed by (39.1; ci 95%: e. wyomingensis 33.8 44.4 and 285.0; ci 95%: 136.0 434.0) and e. bukidnonensis (32.4; ci 95%: 27.3 37.5 and 237.1; ci 95%: 128.3 345.9) the . e. zuernii had second highest mean opg counts (509.7; ci 95%: 46.1 973.3). the lowest prevalent species was e. alabamensis (1.1; ci 95%: 0.0 2.3 and 75.0; ci 95%: 0.0 392.7) (table 3). a number of authors reported that e. bovis was the most prevalent species in cattle, but clinical coccidiosis was not observed in the calves or in adults cattle (kennedy & kralka 1987; lucas et al. 2006; heidari & gharekhani 2014). the results of those studies were in close agreement with the results of our study. according to waruiru et al. (2000), the mere present of pathogenic eimeria spp. did not necessarily indicate clinical disease. eimeria species were classified as highly pathogenic (e. bovis and e. zuernii), low pathogenic (e. ellipsoidalis, e. alabamensis, e auburnensis and e. subspherica) and non-pathogenic (e. brasiliensis, e. bukidnonensis, e. canadensis, e. cylindrica, e. pellita dan e. wyomingensis) (lassen & jarvis 2009). in other tropical area such as brazil, s several species of eimeria in cattle found were also ( floriao et al. 2015). e. bovis was recorded as the highest prevalen coccidian species which s in t wa accordance with reports of arslan and tuzer (1998), lucas et al. (2006) and abebe et al. (2008). 107 table 1 oocyst morphology of eimeria species of cattle has been identified in fecal samples obtained from kpbs pangalengan, bandung length (µm) width (µm) index form color other characteristics eimeria species 26.25-33.00 18.75-22.50 1.17-1.48 ovoid greenish-brown bilayered wall, micropyle e. bovis *29.77±1.37 *21.58±0.73 *1.38±0.05 37.50-43.50 26.25-30.75 1.22-1.49 ovoid greenish-brown single layered wall, micropyle e. wyomingensis *42.67±2.04 *29.58±1.20 *1.44±0.04 47.25-49.50 33.75-37.50 1.26-1.47 ovoid yellowish -brown bilayered wall, micropyle e. bukidnonensis *48.70±0.68 *35.72±1.88 *1.37±0.08 36.00-37.50 26.25-29.25 1.26-1.37 ovoid dark brown thick wall, micropyle e. pellita *37.43±0.28 *29.16±0.51 *1.28±0.02 37.50-45.75 22.50 1.67-2.03 ellips yellowish -brown bilayered wall, micropyle e. auburnensis *38.74±2.73 *22.50±0.00 *1.72±0.12 15.75-20.25 15.00-17.25 1.05-1.35 ovoid pale yellow thin wall, no micropyle e. zuernii *19.87±1.19 *16.65±1.01 *1.20±0.08 25.50-27.00 15.00 1.70-1.80 ellips pale yellow single layered wall, no micropyle e. cylindrica *25.93±0.73 *15.00±0.00 *1.73±0.05 30.00-33.75 25.50-26.25 1.14-1.32 ovoid yellowish bilayered wall, micropyle e. canadensis *30.42±1.25 *26.00±0.38 *1.17±0.06 41.25-42.75 25.50-26.25 1.60-1.63 ellips brownish-yellow bilayered wall, micropyle e. brasiliensis *41.50±0.53 *25.67±0.33 *1.62±0.007 15.00-18.75 11.25-15.00 1.25-1.33 ovoid pale yellow smooth wall, no micropyle e. alabamensis *16.88±2.65 *13.13±2.65 *1.29±0.06 note: * mean; ± standard deviation = = eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. 108 biotropia vol. 24 no. 2, 2017 t ab le o o cy st m o rp h o lo g y o f s p ec ie s o f c at tl e b as ed o n r ef er en ce s 2 e im er ia s p ec ie s a cc o rd in g to s o u ls b y (1 9 8 6 ) a cc o rd in g to d au gs ch ie s an d n aj d ro w sk i (2 0 0 5 ) l en gt h (µ m ) w id th (µ m ) in d ex f o rm c o lo r o th er ch ar ac te ri st ic s l en gt h (µ m ) w id th (µ m ) in d ex f o rm c o lo r o th er ch ar ac te ri st ic s e . bo vi s 2 3 -3 4 1 7 -2 3 1 .3 5 -1 .4 8 o v o id g re en is h b ro w n s m o o th w al l, m ic ro p yl e 2 3 -3 4 1 7 -2 3 1 .3 5 -1 .4 8 o v o id b ro w n is h ye ll o w b il ay er ed w al l, m ic ro p yl e e . w yo m in ge ns is 3 7 -4 4 .9 2 6 .4 -3 0 .8 1 .4 0 -1 .4 6 o v o id g re en is h b ro w n m ic ro p yl e 3 7 -4 5 2 6 -3 1 1 .4 2 -1 .4 5 o v o id y el lo w is h b ro w n s in gl e la ye re d w al l, m ic ro p yl e e . bu k id no ne ns is 4 7 -5 0 3 3 -3 8 1 .3 2 -1 .4 2 o v al y el lo w is h b ro w n b il ay er ed w al l, m ic ro p yl e 4 7 -5 0 3 3 -3 8 1 .3 2 -1 .4 2 p ea rsh ap ed b ro w n is h ye ll o w b il ay er ed w al l, m ic ro p yl e e . pe lli ta 3 6 .1 -4 0 .9 2 6 .5 -3 0 .2 1 .3 5 -1 .3 6 e g gsh ap ed d ar k b ro w n t h ic k w al l, m ic ro p yl e 3 6 -4 1 2 6 -3 0 1 .3 7 -1 .3 8 o v o id d ar k b ro w n t h ic k w al l, m ic ro p yl e e . au bu rn en si s 3 2 -4 6 2 0 -2 5 1 .6 0 -1 .8 4 e ll ip s y el lo w is h b ro w n s m o o th w al l, m ic ro p yl e 3 2 -4 6 2 0 -2 5 1 .6 0 -1 .8 4 e ll ip so id y el lo w is h b ro w n b il ay er ed w al l, m ic ro p yl e e . zu er ni i 1 5 -2 2 1 3 -1 8 1 .1 5 -1 .2 2 s u b sp h er ic al p al e ye ll o w t h in w al l, n o m ic ro p yl e 1 5 -2 2 1 3 -1 8 1 .1 5 -1 .2 2 s u b o vo id c o lo rl es s s in gl e la ye re d w al l, n o m ic ro p yl e e . cy lin dr ic a 1 6 -2 7 1 2 -1 5 1 .6 7 -1 .8 0 c yl in d ri ca l c o lo rl es s t h in w al l, n o m ic ro p yl e 1 6 -2 7 1 2 -1 5 1 .6 7 -1 .8 0 e li p so id c o lo rl es s s in gl e la ye re d w al l, n o m ic ro p yl e e . ca na de ns is 2 8 -3 7 2 0 -2 7 1 .3 7 -1 .4 0 o v o id / el li p s y el lo w is h b ro w n s m o o th w al l, m ic ro p yl e 2 8 -3 7 2 0 -2 7 1 .3 7 -1 .4 0 o v o id / el li p so id y el lo w is h b il ay er ed w al l, m ic ro p yl e e . br as ili en si s 3 4 .2 -4 2 .7 2 4 .2 -2 9 .9 1 .6 4 -1 .6 5 e ll ip s y el lo w s m o o th w al l, m ic ro p yl e 3 4 -4 3 2 4 -3 0 1 .6 5 -1 .6 7 e ll ip so id b ro w n is h ye ll o w b il ay er ed w al l, m ic ro p yl e e . al ab am en si s 1 3 -2 4 1 1 -1 6 1 .1 8 -1 .4 4 p ea rsh ap ed c o lo rl es s t h in w al l, n o m ic ro p yl e 1 3 -2 4 1 1 -1 6 1 .1 8 -1 .4 4 o v o id c o lo rl es s / p al e ye ll o w t h in w al l, n o m ic ro p yl e according to daugschies najdrowski (2005), and e. bovis and e. zuernii are the most pathogenic of the bovine coccidia urthermore, e. alabamensis . f causes disease at extremely large infective doses (> 10 million oocysts) only. e. alabamensis usually induces water y diar rhea without blood, dehydration, depression and reduced growth , whereas infection remains subclinical under moderate infection pressure. single or mixed infection -species caused by different pecieseimeria s in this study, mixed-species infection experienced by a single cattle hosting several eimeria species were commonly observed. the number of eimeria species in single and mixedspecies infection per examined fecal sample ranged from 1 to 5. mixed-species infection caused by 2 to 5 eimeria species were found in 55.9% of cases (ci 95%; 50.5 61.3) the . remainder fecal samples had been infected with single infection of eimeria species (44.1%; ci 95%: 38.7 49.5) (table 4). thethis finding is similar to works of kennedy kralka (1987) in canada, which and reported 5 species. the results of mixedeimeria species we infection re lower than the observation of arslan and tuzer (1998) in turkey who reported 6 species, abebe . (2008) in eimeria et al ethiopia who reported 7 species yu eimeria , et al. (2011) in china who reported 10 eimeria species and dong (2012) who recorded et al. also in china 8 species. many previous studies indicated eimeria that under natural conditions, mixed-species infection re much more common than cases we single species infection (yu 2011; dong et al. et al. 2012). the level of pathogenicity of eimeria species increased higher level due to mixedto a species eimeria, infection with other pathogenic causing mortality in cattle (daughschies & najdrowski 2005). 109 table 3 eimeria species identified, the prevalence and mean opg counts in dairy cattle in kpbs pangalengan dairy farm, bandung species dairy cattle (n = 179) mean opg (ci 95%)positive no. mean prevalence (%) (ci 95%) e. bovis 76 42.5 (37.1 47.8) 538.2 (191.5 884.8) e. wyomingensis 70 39.1 (33.8 44.4) 285.0 (136.0 434.0) e. bukidnonensis 58 32.4 (27.3 37.5) 237.1 (128.3 345.9) e. pellita 47 26.3 (21.5 31.1) 177.7 (124.7 230.7) e. auburnensis 35 19.6 (15.2 23.9) 397.1 (0.0 820.9) e. zuernii 31 17.3 (13.2 21.4) 509.7 (46.1 973.3) e. cylindrica 7 3.9 (1.8 6.02) 78.6 (42.2 115.0) e. canadensis 7 3.9 (1.8 6.02) 121.4 (51.5 191.3) e. brasiliensis 6 3.4 (1.4 5.3) 125.0 (45.4 204.6) e. alabamensis 2 1.1 (0.0 2.3) 75.0 (0.0 392.7) table 4 single and mixed infection of eimeria species no. of eimeria spp. dairy cattle (n = 179) positive no. % of positive samples (ci 95%) 1 79 44.1 (38.7 49.5) 2 59 33.0 (27.8 38.1) 3 24 13.4 (9.7 17.1) 4 15 8.4 (5.4 11.4) 5 2 1.1 (0.0 2.3) total 179 100.0 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. the effect of sex, age and type of cattle's pen flooring to opg countseimeria the effect of various categorical factors to opg counts were first analyzed with the mannwhitney and kruskal-wallis tests. a mannwhitney test was used to compare mean opg between 2 groups, whereas kruskal-wallis test was used to compare mean opg among 3 or more groups. the kruskal-wallis test was performed followed by dunn test as a multiple comparison test. a p value < 0.05 was required to indicate significance. several categorical factors (cattle's sex, age and type of pen flooring) and mean opg counts are presented in table 5. a highly significant ( = 0.000) effect was p observed between different sexes of cattle . intensity of infections were shown by eimeria mean opg found in cattle. this study showed that higher opg counts were found in male cattle (855.6 opg; ci 95%: 150.9 – 1 560.4) compared , with females (144.5 opg; ci 95%: 77.7 211.4). in this study, male cattle harbored more coccidia than female cattle. this situation might be caused by less care given to the male cattle as compared to the female cattle that were deemed to produce future cows. this result was consistent with reports from other researchers showing significant correlation ( < 0.05) between cattle's p sex and coccidiosis infection (heidari & gharekhani 2014). previous studies done on cattle reported higher prevalence of in female eimeria than in male cattle. nevertheless, this could be attributed to the greater physiological stress experienced by female cattle in relation to pregnancies and breeding as compared to male cattle (rehman 2011; dawid 2012; et al. et al. alemayehu 2013; heidari 2014).et al. et al. r is shed esults of th study showed that calves much higher numbers of oocyst than s those of weaners and adults. for instance, the mean opg for the calves was 537.0 (ci 95%; 231.8 842.2), whereas the weaners and adults mean opg for were 154.1 (ci 95%; 79.8 228.3) and 22.5 (ci 95%; 11.0 33.9), respectively. analysis showed that there were statistically significant differences in opg levels among different age groups ( <p 0.05). subsequently, dunn test as a multiple comparison test showed that the opg values for adult cattle were found to be significantly correlate ( < 0.05) the weaners and calves, d p to respectively. however, weaners the did not show significant effect ( > 0.05) the calves in opg p to levels caused by coccidiosis (table ).5 age is a major risk factor in coccidiosis spreading orbidity and risk of infection , while m s s are gr ater in calves (abebe 2008)e et al. compared to other age groups. calves had significantly higher oocyst counts ( < 0.05) than adultss p . these results were et in agreement with waruiru al. et al. (2000) in kenya and dong (2012) in shanghai, china who demonstrated that age strongly influence the intensity of opg d eimeria counts in cattle. coccidiosis is a self-limiting disease. spontaneus recovery without specific treatment is common when multiplication stage of the coccidia has passed, which suggests that previous exposure may have contributed to the 110 biotropia vol. 24 no. 2, 2017 table 5 results of mann-whitney and kruskal-wallis tests followed by dunn test toward several factors and mean opg counts categorical factors no. dairy cattle (n) mean opg (ci 95%) p value sex male 80 855.6 (150.9 – 1,560.4) 0.000* female 320 144.5 (77.7 211.4) age calves (< 6 months) 196 537.0a (231.8 842.2) 0.000* weaners (6 -12 months) 37 154.1a (79.8 228.3) adults (> 12 months) 167 22.5b (11.0 33.9) type of pen flooring straw 13 53.9a (0.0 162.4) 0.000* wood 70 703.6b (0.0 – 1,450.2) rubber 96 405.7b (116.9 694.6) cement 221 116.7a (52.4 181.1) development of a certain immunity level in the older cattle as compared to the younger ones that did not have previous exposure (dong 2012; et al. heidari 2014; kocis 2015).et al. et al. this study showed that w eimeriaeaners shed oocysts opg counts of 154.1 (ci 95%; 79.8with 228.3). ean opg the weaners re lower m for we than the calves ( > 0.05). in tudy, that for p this s range of were 0 opg in calves and weaners 24 450 and 0 900, respectively. opg values over , 5 000 indicate a clinical case (arslan tuzer , & 1998). esults of oocysts counts calves r eimeria for and weaners in were not different from this study previous bruhn (2012) wh study by et al. ich reported that no difference was observed ( >p 0.05) in epg and opg counts between calves in the pre-weaning and post-weaning phases. most cattle examined during this study had low opg counts, suggesting that the infections were usually subclinical. this result concurred with other cross-sectional observational studies on eimeria spp. in iran, which also did not observe cases of clinical cases of clinical coccidiosis among infected cattle, probably due to low quantities of oocysts eliminated in the cattle's feces (heidari 2014; heidari & gharekhani et al. 2014). l eimeriaevel of oocyst shedding was significant depende on different types of pen ly d flooring ( < 0.05). when the types of flooring p were compared, on straw floorcattle kept ing exhibited lower opg values (53.9; ci 95%: 0.0 162.4) than on wood those kept on flooring (703.6; ci 95%: 0.0 – 1 450.2) and rubber , on floor (405.7; ci 95%: 116.9 694.6) ( < 0.05). ing p however, straw floor significanting did not show difference on p compared to opg counts ( > 0.05) cement floor . moreover, wood floor ing ing did not show difference on significant opg counts compared to ing p rubber floor ( > 0.05). yet, eimeria p on opg counts was higher ( < 0.05) wood floor compared to cement floor . ing ing higher oocysts count was recorded in eimeria cattle ing reared on rubber floor compared to cement floor ( < 0.05) (table ).ing p 5 this finding was in agreement with a study by bangoura et al. (2011) who described a significant association between different flooring types used for rearing cattle and the level of eimeria oocysts shedding ( < 0.05). rehman et al. (2011) stated p that eimeria infection was more prevalent ( < 0.05) in non-cement flooring type compared p to partially cement flooring type. lower eimeria prevalence in cattle kept on cement flooring may be caused by the easiness of cleaning and disinfecting cement flooring, which resulted to less contaminated floor compared to non-cement floor. in this study the eimeria, lowest level of oocysts in cattle kept on straw occurred flooring than those kept on other flooring types, because solid floor (wood, rubber and cement ing types ) are ier the solid flooring dirt than straw floors if types are s not scraped and cleaned properly. traw floor type usually used by calves ing is temporarily aged 0 to 1 month to keep warm. the calves daily r al ement of ingemov and replac straw floor with a new straw resulted in lower opg set of s eimeria counts occurence of and reduced the eimeria oocyst shedding. revious studies concluded that p cleaning s an important factor in preventing wa high of oocysts (bangoura counts eimeria et al. 2011; rehman 2011).et al. was cattle clinical coccidiosis observed in pens having leadinginsufficient aeration to high concentration of ammonia, co and moisture. 2 also, clinical coccidiosis occurred in cattle pens where feces accumulated on the ground due to unsuited slatted floors (daughschies & najdrowski 2005). local temperature, humidity, ammonia concentration and ph on pen flooring affected ' et al. oocysts survival (gulliksen 2009; bangoura 2011).et al. in having intensive rearing high population density, disease transmission more readilyis occurred and oocysts are highly available within the environment. thus, in dairy calves, coccidiosis occurs more frequently and appears with greater severity (bruhn 2011). it can be et al. assumed that the in a group will be cattle living infected at the same time, or at around the same time, under field condition. moreover, continuous oocysts shed from subclinical ding infected calves contaminate the environment and the caus severe coccidiosis in cattle's hair ing highly susceptible new calves that are kept in these areas (abebe 2008; alemayehu 2013). et al. et al. different hygiene conditions and far m management, breeding, study design and animal s methods, climates and different geographical regions may be the main cause of varied results (yu 2011)et al. . 111 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. conclusions e. bovis was the highest prevalent eimeria species and had the highest level of mean opg compared to other eimeria spesies. mixed infections in single cattle with 2 – 5 eimeria species were commonly observed. several species of eimeria in cattle are found in other tropical areas. various categorical factors (cattle's sex, age and type of pen flooring) influenced the number of opg found in fecal samples. eimeria has significant pathogenic potential. it is important to control the occurrence of eimeria. acknowledgements t he authors thank all members of protozoology laboratory, faculty of veterinary medicine, institut pertanian bogor for facilitating this study with the necessary equipment. references abebe r, wossene a, kumsa b. 2008. epidemiology of eimeria infections in calves in addis ababa and debre zeit dairy farms, ethiopia. intern j appl res vet med 6(1):24-30. 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[retrieved on 15 february 2015]. 113 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 50 biotropia vol. 15 no. 1, 2008 *corresponding author : tatikkhusni@yahoo.com the preservation of milk with the addition of antibacterial and aromatic supplements produced in indonesia tatik khusniati1* and yantyati widyastuti2 1microbiology division, research center for biology, indonesian institute of sciences jl. raya bogor-jakarta km 46 cibinong 16911. indonesia 2research center for biotechnology, indonesian institute of sciences abstract the preservation of milk with additional antibacterial and aromatic supplements, produced in indonesia, was investigated. organoleptic performances of milk with the addition of 10% supplements, made as juices, were tested by panellists, and the total bacteria, protease activities, lipase activities and acidities, were detected by total plate counts, azocasein method, modified dole extraction and base-acid titration, respectively. out of the 27 supplemented skim and whole milk samples, 15 whole milk samples and 10 skim milk samples were selected as acceptable, based on their better organoleptic performances, their lower bacterial counts, protease and lipase activities, and their acidities percentages which were not significantly different, compared to that of control, at 5 days after the expiry date (p<0.05). these 15 whole milk samples contained honey, cinnamon, citronella, ginger, turmeric, galingale, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf, garlic leaf and aloe vera; and the 10 skim milk samples contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera. key words: milk, preservation, antibacterial and aromatic supplements, protease, lipase introduction pasteurised milk spoils at refrigerated temperatures due to the activities of psychotropic bacteria, pseudomonas spp., and the main species is pseudomonas fluorescens (deeth et al. 2002; chandler et al. 1990; craven and macauley 1992). to ensure the quality of the milk during storage, the milk is stored at a temperature of 4-7oc (chandler et al. 1990), and the average shelf life of stored milk is approximately 7 days (khusniati et al. 2006). however, heo (1989) examined commercial milk samples stored at 7.20c and found that after 10 days of storage 91% of the milk was acceptable, whereas after 14 days, 82% of the whole milk was still acceptable. the qualities of pasteurised milk in refrigerated storage were affected by the growth of psychrotrophic bacteria, especially pseudomonas spp., and to keep the quality biotropia vol. 15 no. 1, 2008 : 50 64 51 the preservation of milk t. khusniati & y. widyastuti of the milk at storage, the milk were stored at temperature of 4-7oc (chandler et al. 1990). the psychrotrophic bacteria began to grow at the expiry date, and times after the expiry date. the bacteria, especially pseudomonas spp grew rapidly in the stored milk. furthermore, the different qualities of milk may have resulted from the different types of milk in storage. the quality of whole milk was higher than that of skim in the refrigerated storage, due to the higher lipid content in whole milk than that in skim ( janzen et al. 1982). the main problem with milk, after the pasteurisation process, was recontamination. during the pasteurisation process in factories, psychotrophic bacteria are killed by the process. however, after pasteurisation, particularly if hygiene within the processing factory is poor, these psychotrophic bacteria can re-contaminate milk (craven and macauley 1992). during storage after the expiry date, the psychotrophic bacteria in the milk grew rapidly, and the longer the time of storage, the higher the growth and enzyme activities of psychotrophic bacteria within the milk (chandler et al. 1990; deeth et al. 2002) the spoilage of pasteurised milk may result in microbial and chemical changes in the milk (reinheimer et al. 1993). the biochemical changes of the milk at spoilage may have resulted from the activities of extracellular enzymes, especially protease, which degrades protein (deeth et al. 2002; janzen et al. 1982); and lipase, which degrades lipid (deeth et al. 2002; bucky et al. 1986). the addition of antibacterial and aromatic supplements, to reduce the spoilage, can be added to the milk. it is widely known that there are a lot of commercial antibacterial and aromatic plant materials produced in indonesia, such as: honey, cinnamon, citronella (sweet), ginger, radish, turmeric, galingale, zingiber (roots), wild ginger, nutmeg, cardamom, cumin, pepper (seeds), garlic, clove, javanoni, galangale (bubs); green tea, laurellike (dried leaves); bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine, celery, garlic leaf, aloe vera (fresh leaves). these twenty-seven plant materials contain antibacterial and aromatic compounds, which may inhibit the growth and enzymatic activities of the psychotrophic bacteria within the stored milk. honey contains ”inhibine”, hydrogen peroxide and flavonoid (lusby et al. 2005); cinnamon contains cinamic acid, cinnamon oil, ethanol, methylene chloride, eugenol and benzyl benzoate (tabak et al. 1999). the root materials of ginger contains “gingerin”, gingerols, α-zingiberene, shorgaols and ginger oil (kikuzaki. 2000); radish has some glucosinolates and hexenyl acetate (tirranen et al. 2001); turmeric has turmeric oil (lantz et al. 2005); galingale contains aromatic galingale (anon 1999). nutmeg contains diphenylpropanoids and ethyl acetate (sherry et al. 1982); cardamon contains aromatic cardamom (mummenhoff and hurka 1991); and pepper, adipic acid, piperine and oleorecine (chitwood et al. 2003). garlic contains ‘allicin’, free phenolics, p-coumaric, ferulic, p-hydroxybenzoic, garlic oil and vanillic acid, and specific garlic aromatic (tirranen et al. 2001); and javanoni contains anthraquinon and aromatic javanoni (anon 2001). 52 biotropia vol. 15 no. 1, 2008 citronella contains citronella oil and kingisidic acid; zingiber contains zingiber oil, acyclic oxygenated and monoterpenes; wild ginger roots contain wild ginger oil; clove contains clove oil, isobiflorin,biflorin, eugenol, phenolic compound and ferulic acid; cumin contains glycosides of 2-c-methyl-d-erythritol and cumin oil ;and galangale contains galangale oil (cineol, α-pinene, eugenol, camphor, methyl cinnamate and sesquiterpenes) (deans and ritchie 1987). the dried leaves of green tea contains polyphenol, phenolic acid, catechins, caffeine, flavonoid, epicatechin and ascorbic acid (an et al. 2004); and laurellike that has aromatic laurellike (anon 2005). the fresh leaves of bamboo contains cinnamic derivatives (tachibana et al. 1992); banana has starch phosphorylase, polycyclic aromatic hydrocarbon and non-volatile organic acid (palmer and wyman 1965); guava contains aromatic guava leaf (settheeworrarit et al. 2005); and avocado contains (r)-2-hydroxy4-oxohenicosan-1-yl acetat, aldehydes, ketones, alcohols, terpenoides, estragole and 2-hexenal (carman and duffield 1995). the fresh leaves of celery contain apiole, 3-butylphthalide, sedanenolide, monoterpene hydrocarbon, phthalates and limonene (macleod and ames1989); garlic containing garlic leaf lectin (tirranen et al. 2001); aloe vera has glucomannans, uronic acid, glycoproteins, anthraquinon, saccharrides and phenolic compounds (ni et al. 2004); pandan contains pandan oil, monoterpene hydrocarbon and sesquiterpene hydrocarbon, while betel vine contains betel vine oil, eugenol, 1, 3 benzodioxol (5)-2-propenyl and anethole (deans and ritchie 1987). the differences in the structure of the antibacterial compounds may have caused variation in the effectiveness of antibacterial substances and the aromatic materials in inhibiting spoilage bacteria and/or pathogenic bacteria. in the indonesian dairy market, the supplements of strawberry and chocolate were added into the commercially pasteurised milk, and stored at refrigerated temperatures in cartons or plastic packages. the addition of these supplements was mainly to create more tastes and flavours for the consumers. these milk are produced in factories, located mainly in java, and distributed not only to local areas but also outside java. the shelf life of these milk is known to be around 7 days as is the shelf life of fresh milk. this may be due to the lack of antibacterial compounds within the supplements. to make longer the shelf life of the commercial milk distributed both in the local area and outside the factories locations, antibacterial and aromatic materials may be used. it is expected that these materials, when added to milk in storage can suppress the growth and enzymatic activities of psychotrophic bacteria, thus increasing the quality and shelf life (the time started after completion of the pasteurisation process up to the time of expiry date), of pasteurised milk. this paper reports on the preservation of milk with the addition of antibacterial and aromatic supplements produced in indonesia. 53 the preservation of milk t. khusniati & y. widyastuti materials and methods milk samples the same types, of commercially batched pasteurised milk, at a temperature of 85oc for 30 minutes, in 1 and 2 litre cartons were used. the milk samples, stored at 4oc until use, were transported on ice to the laboratory, and a 100-ml aliquot of each sample was then transferred aseptically into 200-ml sterile bottles. preparation of antibacterial and aromatic materials the collection of the 27 supplements from supermarkets and traditional markets was carried out in the bogor area, indonesia. all the materials selected were clean and in good condition . juice supplements extracted from antibacterial and aromatic materials juice supplements were made by the extraction of juices from the antibacterial and aromatic materials. fifty (50)-grams of the materials were then made into juice by the addition of 500 ml of boiled water. the materials were then homogenized by using a blender. the juices produced were subsequently filtered through a sterilized stainless steel filter with ø (diameter) 0.5 ml, and the cleared juices were kept at a refrigerated temperature until ready for use as supplements. pasteurised milk with the addition of supplements one and two litre cartons of commercially pasteurised milk were used. the addition of extract after pasteurisation instead of before was because of: (1) the total bacterial counts after pasteurisation were lower than that of before. (2) the pathogenic bacteria were killed after pasteurisation, but the bacteria weren’t killed before pasteurisation (3) materials extracted were used for inhibiting psychotrophic bacteria, which caused the spoilage of the milk at storage. the milk was then transferred aseptically from the big bottles into 200 ml sterile bottles. one representative of each of the twenty-seven different liquid supplements was segregated into separate 200-ml aliquots of the batch of pasteurised milk. the samples, with the addition of the supplements, together with milk without supplements (controls), were incubated at 4oc for up to 5 days after the expiry date. at 5 days before the expiry date, samples of controls and the supplemented twenty-seven milk samples were organoleptically assessed. at 5 days after the expiry date, all the milk samples were investigated for total aerobic counts, acidities, and used for production of supernatants to be assayed for protease and lipase activities. the results presented for bacterial counts, protease activities, lipase activities and acidities are mean values for the three replicates. 54 biotropia vol. 15 no. 1, 2008 sensory assessments the sensory evaluation was conducted by 18 panellists, and assessed by a modified ranking test. the ranking test was as follows; the tastes, colours, flavours and homogeneity, of the milk’ samples, were organoleptically evaluated with scores of: 1.00<2.00 (unacceptable), 2.00-<2.50 (less acceptable), 2.50-<2.65 (acceptable), and >2.65 (more acceptable). the characteristics of organoleptic assessments were tastes (acidic, sweet, plain, milky or others), colours (white, green, red or others), flavours (plain, acidic, sweet, milky or others), and homogeneity (homogeneous, non-homogeneous or others). the equation between the scores and the acceptability ranking was based on the results of the scores and the comments of the sensory evaluation of the milk samples. total aerobic bacterial counts ten-fold serial dilutions of the milk samples were made and spread plate counts performed according to australian standard as 1766.1.4 using nutrient agar. the plates were incubated for 2-3 days at 30oc. acidity measurement the acidities of the milk’ samples were measured by base-acid titration. 10 ml of each milk sample was poured into erlenmeyer 100 ml and several drops of phenolphthalein were added. the solutions were titrated by naoh 0.1n un-till the colours changed to pink. the formula of acidity (based on lactic acid) was a.b. 90.1000/c [a: volume of na oh used (ml); b: concentration of naoh standardized (n); c: volume of milk titrated (ml); d: be lactic acid (g/equivalent)]. preparation of the supernatants (crude enzymes) bacterial cells were removed from the incubated milk samples (with and without the additional supplements) by centrifugation at 24 000 g for 10 min. at 40c. the resulting supernatants (crude enzymes) were collected and stored at -200c in sterile bottles until assayed for enzyme activities protease assay proteolytic activity was then assayed by the use of the azocasein method, using sulphanilamide-azocasein (sigma chemical co., usa) as the substrate, according to the method of christen & marshall (1984), with some modifications. the reaction mixture contained 2 ml of azocasein (10g/l, dissolved by heating 0.1 m-tris-hcl buffer ph 7.4 (sterile) containing 2mm-cacl2 at 63 0c for 30 minutes) and 0.5 ml crude enzyme solution (supernatant) was incubated for 1 h at 370c. one unit of proteolytic activity was defined as the volume of enzyme solution (ml) required for producing an absorbance increase at 345 nm of 0.01 a.u. in an hour under the assay conditions. 55 the preservation of milk t. khusniati & y. widyastuti lipase assay the lipase activity assay was carried out based on the assay procedure of a modified dole extraction procedure (deeth et al. 1975). the supernatants (crude enzyme; 0.5 ml) , 0.25 ml buffer (2 m-diethanolamine-hcl, ph 8.5), 3 ml uht cream, and 1 ml sterile water (in a stoppered test tube) were incubated at 40oc for 2 hours in a shaking (100-rpm) water-bath, and lipase activities were then measured by titration. the reaction mixture was added with 10 ml of extraction mixture (isopropanol: petroleum ether: 4n h2so4)(40:10:1), 6 ml of petroleum ether, and 4 ml of water. the mixture was then shaken for 15 sec., and an-aliquot (8 ml) of the upper layer was transferred to a 50 ml conical flask and 0.5 ml of 0.02% bromothymol blue indicator was added. the free fatty acid (f.f.a) was titrated with 0.02n methanolic koh. the activities of the supernatants are expressed as µequiv.ml-1h-1 using the formulae: n (ttest/ptest – tcontrol/pcontrol) x10 3 ___________________________ v x h where n is the normality of the methanolic koh, ttest and tcontrol are the titration volumes for the test and the control respectively, ptest and pcontrol are the proportions of the upper layers titrated, v is the volume of enzyme solution and h is the incubation time in hours. statistical analysis all treatments of all milk samples, with and without the addition of supplements, were statistically analysed by anova with factorial complete randomised design (snedecor and cochran 1989) using general linear model with three replications. results and discussion the organoleptic performances of all supplemented milk were generally better than that of the controls (without supplements), although the panellists classed some of them as organoleptically un-acceptable. of the 27 supplemented whole milk samples 20, 4 and 3 samples at 5 days before the expiry date were classed as acceptable, less acceptable and unacceptable by panellists, while of the 27 supplemented skim milk samples 14, 8 and 5 samples were classed as acceptable, less acceptable and unacceptable by panellists at the same date, respectively (table 1). the 20 supplemented whole milk samples contained honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, laurellike, bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine and aloe vera. 56 biotropia vol. 15 no. 1, 2008 the 4 supplemented whole milk samples contained cumin, garlic, celery and garlic leaf, while the rest i.e. 3 supplemented whole milk samples contained radish, cardamom and javanoni. the 14 supplemented skim milk samples contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine and aloe vera. the 8 supplemented skim milk samples contained turmeric, zingiber, wild ginger, nutmeg, garlic, clove, laurellike and celery, while the 5 supplemented skim milk samples contained radish, cardamom, cumin, javanoni and garlic leaf. therefore, the 20 supplemented whole milk samples and the 14 supplemented skim milk samples were classed as acceptable, based on organoleptic assessments. to further classify their quality and shelf life, these acceptable milk, were then counted for total bacteria and measured for enzymatic activities and acidities the preservation of the whole milk (table 2) and skim (table 3) can be explained by the lower total bacterial counts, proteolysis and lipolysis, and the acidities percentages which were not significantly different, than those of controls. this is caused by the lower production of psychotrophic bacteria within these milk. the total bacterial counts, protease and lipase activities, of the 20 acceptable whole milk, and the 14 acceptable skim, were significantly lower than that of the controls (without supplements), at the time of storage 5 days after the expiry date (p<0.05). table 1. the organoleptic performances of skim and whole milk with the addition of antibacterial and aromatic supplements, at 5 days before the expiry date no. antibacterial and aromaticsupplements whole milk skim milk 1 honey > acceptable > acceptable 2 cinnamon > acceptable acceptable 3 citronella > acceptable > acceptable 4 ginger > acceptable > acceptable 5 radish unacceptable unacceptable 6 turmeric > acceptable < acceptable 7 galingale > acceptable acceptable 8 zingiber acceptable < acceptable 9 wild ginger acceptable < acceptable 10 nutmeg acceptable < acceptable 11 cardamon unacceptable unacceptable 12 cumin < acceptable unacceptable 13 pepper > acceptable acceptable 14 garlic < acceptable < acceptable 15 clove acceptable < acceptable 57 the preservation of milk t. khusniati & y. widyastuti no. antibacterial and aromaticsupplements whole milk skim milk 16 javanoni unacceptable unacceptable 17 galangale > acceptable acceptable 18 green tea acceptable acceptable 19 laurellike > acceptable < acceptable 20 bamboo leaf > acceptable acceptable 21 banana leaf > acceptable acceptable 22 guava leaf > acceptable acceptable 23 avocado leaf > acceptable acceptable 24 betel vine acceptable acceptable 25 celery < acceptable < acceptable 26 garlic leaf < acceptable unacceptable 27 aloe vera > acceptable > acceptable 28 milk (control) > acceptable > acceptable notes: > acceptable (>2.65); acceptable (≥ 2.50); < acceptable (≥2.00); unacceptable (<2.00) the total bacterial counts of the 27 supplemented milk at storage 5 days after the expiry date, were in the range of 6.0x102-8.4x104 cfu/ml (whole milk) and 8x102-9.4x105 cfu/ml (skim milk), while the total bacterial counts of milk without supplements (controls) were 8.5x105 cfu/ml (whole milk) and 9.7x106 cfu/ml (skim milk) at the same times, respectively (table 2-3). the total bacterial counts of the twenty (20) acceptable whole milk, and the fourteen (14) acceptable skim, were in the range of 6.0x102-8.5x103 cfu/ml and 8.0x102-9.3x104 cfu/ml, respectively. therefore, the 20 whole milk and the 14 skim milk categorized as acceptable were based not only on organoleptic performances, but also on total bacterial counts. the protease activities of the 27 supplemented milk were in the range of 0.200.40 u/ml (whole milk) and 0.30-0.50 u/ml (skim milk), at storage 5 days after the expiry date, while that of without supplements (controls) were 0.50 u/ml (whole milk) and 0.60 u/ml (skim milk), at the same times, respectively (table 2-3). the protease activities of the 20 acceptable whole milk, and the 14 acceptable skim milk, were in the range of 0.20-0.35 u/ml and 0.30 0.45 u/ml, respectively. furthermore, the protease activities of the 15 out of the 20 acceptable whole milk were lower than that of the other 5, while the protease activities of the 10 out of the 14 acceptable skim milk were lower than that of the other 4 (p<0.05). the protease activities of these 15 acceptable whole milk (added with honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera) were in the range of 0.20-0.30 u/ ml, at 5 days after the expiry date, while the protease activities of these 10 acceptable table 1. continued 58 biotropia vol. 15 no. 1, 2008 skim milk (added with honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera) were 0.30-0.40 u/ml. thus, the 15 whole milk and the 10-skim milk categorized as acceptable were based not only on organoleptic performances and total bacterial counts, but also on protease activities. the lipase activities of the 27 supplemented skim and whole milk were significantly lower than those without supplements (controls), and the lipase activities in supplemented whole milk were significantly higher than that in skim, at the storage 5 days after the expiry date (p<0.05)(tables 2-3). the lipase activities of the supplemented milk were in the range of 0.17-0.27 µequiv. ml-1 h-1 (whole milk) and 0.11-0.22 µequiv. ml-1 h-1 (skim milk), at storage 5 days after the expiry date, while the lipase activities of the milk without supplements (controls) was 0.29 µequiv. ml-1 h-1 (whole milk) and 0.24µequiv. ml-1 h-1 (skim milk), at the same times, respectively. the lipase activities of the 20 acceptable whole milk and the 14 acceptable skim were in the range of 0.170.24 µequiv. ml-1 h-1 and 0.12-0.20 µequiv. ml-1 h-1, respectively. furthermore, the lipase activities of the 15 out of the 20 acceptable whole milk were lower than that of the other 5, and that of the 10 out of the 14 acceptable skim were lower than that of the other four (p<0.05). the lipase activities of the 15 acceptable whole milk (added with honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera) were 0.16-0.22 µequiv. ml-1 h-1, at 5 days after the expiry date, while the 10 acceptable skim (additional honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera) were 0.11-0.17 µequiv. ml-1 h-1. thus, the 15 whole milk and the 10 skim classified as acceptable were based not only on organoleptic assessments, total bacterial counts and protease activities, but also on lipase activities. the acidities of the 27 supplemented milk, at storage 5 days after the expiry date, were in the range of 0.27-0.36% (whole milk) and 0.29-0.39% (skim milk), while the acidities of milk without supplements (controls) were 0.26% (whole milk) and 0.28% (skim milk), at the same times, respectively (tables 2-3). the acidities of the 20 acceptable whole milk and the 14 acceptable skim at 5 days after the expiry date were in the range of 0.27-0.34% and 0.29-0.37%, respectively. furthermore, the acidities of the 15 out of the 20 acceptable whole milk, and the 10 out of the 14 acceptable skim, at the same date, were in the range of 0.27-0.38% and 0.29-0.36%, respectively. the acidities of the 15 acceptable whole milk, and the 10 acceptable skim milk, at 5 days after the expiry date, were not significantly different to that of the controls (p<0.05). 59 the preservation of milk t. khusniati & y. widyastuti table 2. preservation of whole milk with the addition of antibacterial and aromatic supplements at 5 days after the expiry date no. antibacterial and aromatic supplements total bacterial counts (cfu/ml) protease activities (u/ml) lipase activities (µequiv. ml-1h-1) acidities (%) 1 honey 6x102 (k) 0.20i 0.17 klj 0.27k 2 cinnamon 7x103 (fg) 0.25 h 0.18 ijk 0.28jk 3 citronella 6.3x103 (gh) 0.20i 0.17 klj 0.30hij 4 ginger 5x103 (i) 0.20 i 0.17 klj 0.27k 5 radish 8.3x104 (c) 0.40 d 0.26 bc 0.35cde 6 turmeric 7.2x10 3 (ef ) 0.20i 0.19 hij 0.29ij 7 galingale 7.2x103 (ef ) 0.20i 0.19 hij 0.29ij 8 zingiber 8.4x103 (d) 0.30g 0.21 fgh 0.32fgh 9 wild ginger 8.3x103(d) 0.30g 0.20 ghi 0.32fgh 10 nutmeg 8.3x103 (d) 0.30g 0.20 ghi 0.32fgh 11 cardamon 8.4x104 (c) 0.40 d 0.27 ab 0.36bcd 12 cumin 8.3x104 (c) 0.40 d 0.26 bc 0.27bcd 13 pepper 7x103 (f ) 0.25h 0.19 hij 0.28jk 14 garlic 6x103 (h) 0.20 i 0.19 hij 0.27k 15 clove 8.3x103 (d) 0.30g 0.20 ghi 0.32fgh 16 javanoni 8.4x104(c) 0.40 d 0.26 bc 0.33efg 17 galangale 8.3x103 (d) 0.30 g 0.22 efg 0.28jk 18 green tea 8.3x103 (d) 0.30 g 0.21 fgh 0.31ghi 19 laurellike 8.3x103 (d) 0.35 e 0.24 cde 0.33efg 20 bamboo leaf 8.3x103 (d) 0.30 g 0.21 fgh 0.31ghi 21 banana leaf 8.4x103 (d) 0.35 e 0.24 cde 0.34def 22 guava leaf 8.5x103 (d) 0.35 e 0.24 cde 0.34def 23 avocado leaf 8.4x103 (d) 0.35 e 0.24 cde 0.33efg 24 betel vine 8.4x103(d) 0.35 e 0.24 cde 0.33efg 25 celery 8.4x104 (c) 0.40 d 0.25 bcd 0.35cde 26 garlic leaf 8.3x103 (d) 0.30 g 0.22 efg 0.32fgh 27 aloe vera 6x103 (h) 0.20i 0.16 klm 0.27k 28 whole milk (control) 8.5x10 5 (b) 0.50 b 0.29a 0.26k note: numbers followed by different letters were significantly different (p<0.05) 60 biotropia vol. 15 no. 1, 2008 table 3. preservation of skim milk with the addition of antibacterial and aromatic supplements at 5 days after the expiry date no. antibacterial and aromatic supplements total bacterial counts (cfu/ ml) protease activities (u/ml) lipase activities (µequiv. ml-1h-1) acidities (%) 1 honey 8x102 (j) 0.30 g 0.12 op 0.30hij 2 cinnamon 8x103 (de) 0.35e 0.13 nop 0.31ghi 3 citronella 7.2x103 (ef ) 0.30 g 0.12 op 0.33efg 4 ginger 6x103 (h) 0.30g 0.12 op 0.29jk 5 radish 9.4x105 (ab) 0.50 b 0.21 fgh 0.38ab 6 turmeric 8.1x103 (c) 0.40 d 0.15 lmn 0.34def 7 galingale 8x 103 (d) 0.30g 0.14 mno 0.32fgh 8 zingiber 8.6x104 (d) 0.40 d 0.16 klm 0.36bcd 9 wild ginger 8.4x104(c) 0.40 d 0.15 lmn 0.36bcd 10 nutmeg 8.5x104 (c) 0.40 d 0.16 klm 0.36bcd 11 cardamon 9.4x105(ab) 0.50 b 0.22 efg 0.39a 12 cumin 9.3x105 (ab) 0.50 b 0.21 fgh 0.39a 13 pepper 8x103 (d) 0.35 e 0.14 mno 0.31ghi 14 garlic 7.1x103 (f ) 0.30g 0.14 mno 0.29jk 15 clove 8.5x104 (c) 0.40 d 0.15 lmn 0.36bcd 16 javanoni 8.6x105 (b) 0.45 c 0.19 hij 0.37abc 17 galangale 8.1x103 (c) 0.40d 0.17 klj 0.31ghi 18 green tea 8.4x104 (c) 0.40 d 0.16 klm 0.36bcd 19 laurellike 8.7x104 (c) 0.45 c 0.19 hij 0.37abc 20 bamboo leaf 8.4x104 (c) 0.40 d 0.17 klj 0.36bcd 21 banana leaf 9.0x104 (c) 0.45 c 0.18 ijk 0.37abc 22 guava leaf 9.3x104 (c) 0.45 c 0.19 hij 0.37abc 23 avocado leaf 8.9x104 (c) 0.45 c 0.19 hij 0.36bcd 24 betel vine 9.2x104(c) 0.45 c 0.20 ghi 0.36bcd 25 celery 9.4x105 (ab) 0.50 b 0.20 ghi 0.38ab 26 garlic leaf 8.4x104(c) 0.40 d 0.17 klj 0.36bcd 27 aloe vera 7x103 (fg) 0.30g 0.11p 0.29jk 28 skim milk (control) 9.7x106 (a) 0.60 a 0.24 cde 0.28k note: numbers followed by different letters were significantly different (p<0.05) 61 the preservation of milk t. khusniati & y. widyastuti the differences in the acceptability of the 15 acceptable whole milk and the 10 acceptable skim, may be due to the antibacterial and aromatic compounds of these supplements which were more effective in inhibiting bacterial growth and enzymatic activities of psychrotrophic bacteria in whole milk than that in skim. the better organoleptic performances of the 15 acceptable whole milk than that of skim, at 5 days before the expiry date (table 1), may be due to the higher lipid contents of the acceptable whole milk than that of the skim which may result in the better organoleptic performances of acceptable whole milk than that of skim. it has been reported that at 5 days before the expiry date (the designated expiry date), the tastes, flavours, colours and homogeneity of pasteurised milk were fresh due to, at that time, psychotrophic bacteria of pseudomonas spp. hadn’t grown yet (bishop and white 1986; chandler et al. 1990; craven and macauley 1992). the lipid contents of the whole milk were higher than that of the skim and the shelf life of the whole milk was higher than that of the skim, as supported by chandler et al. (1990) and deeth et al. (2002). at storage 5 days after the expiry date, the antibacterial and aromatic compounds in the 15 acceptable whole milk, and the 10 acceptable skim, can suppress the bacterial counts, protease and lipase activities of psychrotrophic bacteria in skim and whole milk (table 2-3). the differences in the total bacterial counts, protease and lipase activities between these 15 acceptable whole milk and 10 acceptable skim may be due to the differences in the type of the antibacterial and aromatic compounds, and the nutritional and organic acid compounds, of these acceptable skim and whole milk. it is known that the various antibacterial and aromatic compounds, nutritional compounds and acidities, of aromatic supplements, may have resulted in the different bacterial counts, protease and lipase activities between the supplemented skim and whole milk, at 5 days after the expiry date. after the expiry date, the longer the time of storage may result in the higher total bacterial counts in refrigerated, pasteurised milk (bishop and white. 1986; chandler et al. 1990; craven and macauley 1992), and the longer the time of storage, the higher the growth of psychotrophic bacteria, especially pseudomonas spp. in refrigerated milk (bishop and white 1986; chandler et al. 1990; craven and macauley 1992). furthermore, the protease activities of refrigerated pasteurized milk increased at times of storage, and the longer the time of storage the higher the protease activities of refrigerated milk ( janzen et al. 1982), and the lipase activities in refrigerated milk increased at the time of storage, also the higher the times of the storage the higher the lipase activities of refrigerated milk (bucky et al. 1986). for the aromatic supplements, there are various examples, with different ph, which may affect the acidities of the skim and whole milk. the lower bacterial growth of the 15 acceptable whole milk, at storage 5 days after expiry date, than that of the skim may be due to the increased effectiveness of the 62 biotropia vol. 15 no. 1, 2008 antibacterial compounds, in the acceptable whole milk, in inhibiting psychrotrophic bacteria, than that of in the skim. the higher lipid contents of the supplemented whole milk than that of the skim may have resulted in the stronger protection of bacterial attack, in the whole milk than that in skim. bacterial growth of whole milk, in refrigerated storage, was lower than that in the skim (chandler et al., 1990). the lower protease activities of the 15 acceptable whole milk at storage, than that of the skim (tables 2-3), may be due to the effectiveness of the antibacterial compounds, in the acceptable whole milk, in inhibiting protease activities of the psychrotrophic bacteria, than that in the skim. the higher lipid contents of the supplemented whole milk, compared to the skim, may have resulted in the stronger inhibition of the protease activities of psychrotrophic bacteria, on the supplemented whole milk than that on the skim. the protease activities of psychrotrophic bacteria in the whole milk at refrigerated storage were lower than that of the skim (deeth et al. 2002; janzen et al. 1982). on the contrary, the higher lipase activities of the 15 acceptable whole milk in storage than that of the skim may be due to the higher lipid contents of the supplemented whole milk than that of the skim. the lipase activities in the whole milk, at refrigerated storage, were higher than that in the skim (deeth et al. 2002; bucky et al. 1986). the differences in the acidities between the 15 acceptable whole milk and the 10 acceptable skim in the storage, may be due to the differences in the organic acidic compounds between these acceptable skim and whole milk. conclusions the study on the preservation of milk with an additional 10% of antibacterial and aromatic supplements, produced in indonesia showed that 15 out of the 27 supplemented whole milk, and 10 out of the 27 skim (preserved milk) were tested and classified as acceptable. this classification was based on their better organoleptic performances, their lower bacterial counts, protease and lipase activities, and their acidities percentages which were not significantly different, compared to that of milk without supplements (unpreserved milk), at 5 days after the expiry date (p<0.05). these 15 acceptable whole milk contained honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera, and the 10 acceptable skim milk contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera. acknowledgement the authors would like to acknowledge (1) jsps-ronpaku for providing the scholarship and (2) biodiversity projects of research centre for biology, indonesian institute for sciences for partly providing the funds for this research. 63 the preservation of milk t. khusniati & y. widyastuti references an, bj., kwak, jh., son, jh., park, jm., lee, jy., jo, c. & m.w. byun. 2004. biological and antimicrobial activity of irradiated green tea polyphenols. food chem., 88, 549-555 anonymous. 1999. greater galangale (alpinia galangal [l.] willd). spice pages: greater galangale (galangal, khaa, laos). anonymous. 2001. javanoni, bad flavours which contain medicinal substances. kompas (newspaper). june. 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114°44'00.8" e) having ultisols soil type with soil ph (h o) of 5.5; exchangeable al and h of 2 0.67 me.100/g and 1.53 me.100/g, respectively. other soil properties are shown in table 1. plant materials ten promising lines of sc2p2.99.5.4.5-1-6-1 (g1), sc2p2.151.3.5.1-10 (g2), sc5p2p3.5.4.1-5 ( g 3 ) , s c 5 p 2 p 3 . 2 3 . 4 . 1 3 2 8 3 ( g 4 ) , sc5p2p3.23.4.1-5 (g5), sc5p2p3.48.31.1-10 (g6), sj-5/msr.99.5.4.5-1-6-1 (g7), msr/sj5.21.3.7-3-27-1 (g8), msr/sj-5.23.4.1-3-28-3 (g9) and msr/sj-5.23.4.1-5(g10) and two check varieties tanggamus (g11) and wilis (g12) were used in this experiment. the ten promising lines were derived from crossing of mansuria and sj-5 genotypes. tanggamus is an acid-adaptive soybean variety, while wilis is a broadly-adaptive soybean variety. soil preparation to obtain optimal soil structure condition for ideal growth of the soybean, soil tillage was carried out by plowing the soil twice until 20 cm depth and then flatting by harrowing the soil. drainage canals were made every 4.5 m to prevent water logging in case of hard rainfall. design and planting the experimental design applied was randomized completely block design with four replications. plot size was 2.8 × 4.5 m and plant spacing of 40 × 15 cm with two plants per hill. fertilizers applied were 75 kg urea, 125 kg sp36 and 75 kg kcl per ha, which were spread before planting. control of weeds, pests and diseases were performed optimally by monitoring scheme. observation and data analysis observations were conducted on days to 50% flowering (then stated as days to flowering) and days to 95% maturing (then stated as days to maturing), plant height, number of reproductive nodes per plant, number of filled and unfilled pods per plant, 100 grains weight and grain yield. data were analyzed using analysis of variance (anova) and followed by least significant difference test (lsd) at = 0.05.α table 1 soil properties soil properties value ph (h2o) 5.05 ph (kcl) 4.15 n (%) 0.13 co (ppm) 1.48 p2o5 (ppm) 8.08 so4 (ppm) 42.30 fe (ppm) 48.90 mn (ppm) 245.00 cu (ppm) 6.13 zn (ppm) 4.20 k (ppm) 0.48 na (me/100g) 0.21 ca (me/100g) 5.11 mg (me/100g) 2.06 ktk (me/100g) 34.00 aldd (me/100g) 0.67 hdd (me/100g) 1.53 53 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – results and discussion in this study, there were three genotypes having early days to f lowering namely sc5p2p3.5.4.1-5, msr/sj-5.23.4.1-3-28-3 and wilis with days to flowering of 34 days. on the other hand, there were four genotypes having days to flowering of 40 days or higher namely sc5p2p3.23.4.1-5, sc5p2p3.48.31.1-10, sj5/msr.99.5.4.5-1-6-1 and msr/sj-5.23.4.1-5 (fig. 1). different days to flowering are affected by the genotypes, where they are affected by genes that repressing (cao . 2015) or inducing et al (na . 2013) as the response to the environ-et al mental parameters such as temperature (lee . et al 2005; xia . 2012), photoperiod and other et al environmental stimuli (xia . 2012).et al days to maturing of the tested genotypes varied. trend of days to maturing is similar to trend of days to flowering. genotype having longer flowering days also showed longer maturing days and vice versa. however, there is one genotype (wilis) having early flowering day and longer maturing days. days to maturing is important in soybean because different maturing day response to the yield. soybean is a photoperiod-sensitive and self-pollinated species. days to flowering (dtf) and maturing (dtm), duration from flowering-to-maturing (dftm) and plant height (ph) are crucial for soybean adaptability and yield (zhang . 2015). et al therefore, temperature is related to the maturity of soybean (kumagaia & sameshima 2014). plant height varied among the tested genotypes. the highest plant height was sc5p2p3.23.4.1-5 and the lowest plant was sc5p2p3.5.4.1-5. the check varieties tanggamus and wilis were included as having medium plant height in this study. plant height is influenced by the genotypes and environment. genes in the genotypes works to perform plant height supported by the environment. in rainy season soybean plant height is higher than in dry season (kuswantoro & zen 2013). in physiological point of view, plant height is also affected by solar radiation. zhang . (2014) stated that solar et al ultraviolet radiation exclusion increases soybean plant height due to the internodes elongation. plant height has relationship to grain yield as stated by many authors (lee . 2015; liu 2013).et al the lowest number of reproductive nodes was achieved by sc5p2p3.5.4.1-5. the highest number of reproductive nodes was achieved by sc2p2.99.5.4.5-1-6-1, in which sc2p2.99.5.4.51-6-1 was also included as having higher plant height. number of reproductive nodes had similar pattern to plant height (fig. 2). higher plant height leads to higher number of reproductive nodes, because higher plant allows more branches to grow than lower plant. in this case, the length of the main stem is the most important factor for plant height. at the r5 growth stage, nodes development on the main stem and on the branches reach the maximum (egli . 1985). number of reproductive nodes et al is affected by number of branches. h branch igher figure 1 days to flowering and days to maturing of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) 54 biotropia vol. 23 no. 1, 2016 dry matter per plant affecting more branch nodes and branch reproductive nodes indicates that number of branches and number of reproductive nodes have a close relationship (carpenter & board 1997). the highest number of filled pods was achieved by sc5p2p3.23.4.1-5, msr/sj-5.23.4.13-28-3 and tanggamus, while the lowest was achieved by sc5p2p3.5.4.1-5. this indicated that the tested soybean lines varied in pods number trait. numbers of filled pods in this study were lower than other study with the same soybean lines, although the soil ph is higher. this phenomenon might be due to the lower water availability (kuswantoro & zen 2013). low water availability at pod filling period may accelerate senescence and shorten pod filling period (de sousa 1997).et al. the pattern of number of filled pods was not similar to the pattern of number of unfilled pods (fig 3). sc5p2p3.5.4.1-5 had the highest number of unfilled pods and had the lowest number of filled pods. tanggamus was the soybean variety that achieved the highest number of filled pods and unfilled pods. this difference indicated that there is no relationship between thes e two characters. genetic constitution has important role in expressing the number of unfilled pods against environmental effects. it is indicated by the broad-sense heritability of unfilled pods achieving 93.1% (sahay 2005).et al. figure 2 plant height and number of reproductive nodes of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) figure 3 number of filled pods and number of unfilled pods of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) 55 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – the largest grain size were shown by sc5p2p3.23.4.1-5 and sc5p2p3.48.31.1-10, while the smallest were shown by sj5/msr.99.5.4.5-1-6-1 and msr/sj-5.23.4.1-3-28-3 (fig. 4). grain size of the check varieties were in the middle rank of the tested genotypes. grain size is one of the main components affecting grain yield. grain size can decrease (kuswantoro . et al 2014) or increase (kuswantoro 2015) grain yield through modification of environmental factors such as soil acidity. therefore, grain size is more affected by environmental factors rather than by genetic constitution (liu 2010). this is et al. supported by hakim . (2014) who found et al medium heritability with value of 46.3%. the line of sc5p2p3.23.4.1-3-28-3 achieved production of 1.51 t/ha which was higher than that of wilis and tanggamus varieties which reached production of 1.41 t/ha and 1.13 t/ha, respectively. other line that achieved relatively higher grain yield was sc5p2p3.23.4.1-5 with grain yield of 1.48 t/ha. tanggamus is the check variety for wide adaptation variety. in this study wilis had lower grain yield than that of tanggamus, the soybean check variety for adaptability to acid soil. grain yield is a complex character and affected by other components. the most important characters in supporting grain yield are grain size and number of grain yield. some authors reported that there were significant correlation (malik 2011) and genetic et al. correlation (arshad . 2006) between grain yield et al and 100 grains weight; and a positive direct effect between 100 grains weight and grain yield (elbadawy 2012). similar to number of grain size, some authors also reported the relationship between grain yield and number of pods (arslan et al. 2005), and positive direct effect with number of pods per plant (valencia-ramírez & ligarretomoreno 2012). conclusions the promising lines of sc5p2p3.5.4.1-5 and sc5p2p3.23.4.1-5 achieved production of 1.51 t/ha and 1.48 t/ha, respectively, which were higher than the production of tanggamus and wilis varieties. the yield of these two p r o m i s i n g l i n e s w a s a l s o s u p p o r t e d by number of filled pods. promising lines of sc5p2p3.5.4.1-5 and sc5p2p3.23.4.1-5 has potency to be developed in tanah laut regency, south kalimantan or other areas with similar soil characteristics. acknowledgements this study was funded by the korean gover n ment thr ough asian foo d and agriculture cooperation initiative (afaci) project 2012. references arshad m, ali n, ghafoor a. 2006. character correlation and path coefficient in soybean (l.) glycine max merrill. pakistan j bot 38:121-30. 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bmc genomics 16:217. zhang l, allen jr. lh, vaughan mm, hauser ba, boote kj. 2014. solar ultraviolet radiation exclusion increases soybean internode lengths and plant height. agr forest meteorol 184:170 8. 57 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – biotropia book final.indd 87 occurrence and distributional range of mangrove vascular flora of catanduanes island, luzon, philippines jimmy tevar masagca* professional education department, college of education de la salle university-dasmariñas cavite 4115, the philippines abstract mangroves play very significant roles not only on the economic aspects but also on the ecological aspects as biobelting for tidal surges and tsunamis. the loss of human lives due to the deadly tsunamis in east asia and the unabated destruction of coastal vegetation have resulted in a renewed focus on the mangrove resources. the purpose of this paper is to report the existing mangrove database of the typhoon-prone island province of catanduanes in luzon, philippines which will be used as bases in determining the appropriate educational management initiatives of various sectors for mangrove rehabilitation and regeneration. a total of 37 species of mangrove vascular flora (13 species of major mangrove elements, 10 species of minor mangrove elements and 14 associated mangrove species) were identified in the island under study. two species of the genus avicennia (a. marina and a. officinalis) were noted in the island. the other genera (bruguiera, ceriops, sonneratia and rhizophora) are well distributed in the designated eco-zones. nypa fruticans is the most important mangrove plant species and a member of the screwpine family (pandanaceae), pandanus tectorius which is an associated mangrove species is well-distributed all throughout the island. key words: mangroves, vascular flora, catanduanes, philippines, occurrence and distribution. introduction in recent years, there had been an increased interest on the status of mangrove environment and the degradation of mangrove ecosystems in the south east asian (sea) region. the rapid depletion of mangrove cover in the philippines was due to the over-all exploitation by coastal dwellers and conversion to agriculture and aquaculture industries (zamora 1989a, 1991b; bennagen & cabahug 1991; primavera 2001). the coastal-dwelling communities of many countries of the region have been utilizing the mangrove resources in many different ways. they depend on wood and non-wood products as well as the economically important species of mangrovebiotropia vol. 15 no. 2, 2008: 87 94 *corresponding author: jtmasagca@dasma.dlsu.edu.ph 88 associated fauna. on the utilization and conservation of mangroves, masagca (2007) observed the general agreement of science teachers and coastal dwellers on their perceptions as to the ecological values such as protection from floods, erosion and other climatic factors. japar (1994) compiled the information on plant species associated with mangals in the country based on the papers of gomez (1980), zamora (1987), and calumpong (1994). there are 31 to 34 exclusive mangroves species and 40 to 65 non-exclusive categories. these numbers vary slightly from country to country. studies on mangroves in the philippines are numerous, in bukidnon for instance, quimpang et al. (1987) described the mangrove forest structure in gingoog city. the team of vega et al. (1991) assessed the mangroves of san miguel bay in camarines sur using the transect plot method in their fieldwork as outlined by dartnall & jones (1986). de leon (1992) in negros oriental noted that avicennia dominates talabong mangrove forest with 251-stems/10 sq m on the average. in guimaras, babaran & ingles (1997) assessed the mangrove forests and nipa swamps. a total of 26 mangrove plant species were identified in the island. this paper reports the mangrove-associated flora of catanduanes island, luzon, philippines. the said typhoon-prone island is located in the bicol peninsula wherein mangroves play very significant roles not only on the economic aspects but also on the ecological aspects as biobelting for tidal surges and tsunamis. materials and methods location of the study catanduanes is an island province (total land area of 1,483 sq km) of the eastern part of the philippine archipelago (13.5˚ to 14.1˚n lat. and 124˚ to 125˚ e long.). highest elevation of the island is 803 m above sea level. the monsoonal climate of the province consists of two distinct dry season and wet season. typical diurnal range of temperatures is 25˚ to 32˚c with minimal variations throughout the year. relative humidity varies from 75 to 89%. the most likely current that affects the island is the north equatorial current moving westward across the pacific. research methods, sources of data and sampling procedures heywood (1995) propounded that characterizing biodiversity involves the observation and characterization of the main units of variation (e.g. genus and species), as well as the quantification of variation within and between them (e.g. taxonomic relatedness). ocular surveys in the present study cover mangrove areas in river mouths, creeks and buffer zones or marginal strips of the coastline found in the different study zones (see table 1). the transect surveys were employed in gathering data and collection of voucher specimens for the mangrove floristic components of the mangroves in the island. primary data were from direct systematic surveys or observation of the mangrove areas in the island. laboratory observations were done to identify floral specimens not identified in situ. biotropia vol. 15 no. 2, 2008 89 the study sites cover the whole island, except for a landlocked town and a coastal town without a distinct mangrove area suited for scientific observation. there were 6 study zones established in the province for the purpose of showing the whole province as an ecological unit for analysis as shown in table 1. table 1. arbitrary ecological zones on catanduanes island, luzon zone zonal location municipality* code i northern pandan pan-06 ii northwestern caramoran car-04 iii northeastern panganiban viga bagamanoc pag-07 vig-09 bag-01 southern virac vrc-10 v southeastern bato baras gigmoto bat-03 bar-o2 gig-05 *also termed as town study stations for the transect surveys were selected based on the availability of mangrove forests stands suitable for the sampling method, i.e. pcqm (pointcentered quarter method). two to three transect lines were established from the forest margin at right angles to the edges of the mangrove forest. these were made landward to seaward direction with the use of magnetic compass. data gathering procedures the predominant plants in the mangrove ecosystem are the trees or shrubs and these species were generally observed in the mangrove sites. some plant specimens not identified in the site were collected from the mangrove sites and were plant pressed. some samples of leaves, flowers and fruits were also obtained and photographed in the laboratory. taxonomic diagnoses were noted following the reports of ding hou (1960), tomlinson (1986) and santisuk (1985). the works of morton (1990) and calumpong & meñez (1997) were used as guides. tomlinson (1986) who arbitrarily categorized mangrove flora into major, minor, associated and specialized mangal elements used fairly rigid criteria to distinguish the mangrove floral species. since there is no standard way of categorizing the mangroves, tomlinson’s categories were employed in the present work. taxonomic identification and counting of the different species of flora were accomplished using the aforementioned guides. some specimens were dispatched to the philippine national museum (pnm) in manila for confirmation of the identity. herbal collections of pnm were also used as reference materials to identify the taxa of plant specimens. results and discussion tables 2, 3 and 4 present the checklist of the major, minor and associated mangrove flora, respectively found in catanduanes island. occurrence and distributional range of mangrove – jimmy tevar masagca 90 a total of 13 species of major mangal elements (table 2), 10 species of minor mangal elements (table 3), and 14 associated mangrove species (table 4). three species of specialized mangrove plants were also identified as caesalpinia crista, derris trifoliate and asplenium sp. table 2. list of major mangal elements recorded in catanduanes island, luzon, philippines family genus species code used avicenniaceae avicennia avicennia marina am avicennia offi cinalis ao combretaceae lumnitzera lumnitzera racemosa lr lumnitzera littorea ll arecaceae nypa nypa fruticans nf rhizophoraceae bruguiera bruguiera gymnorhiza bg bruguiera sexangula bs ceriops ceriops decandra cd ceriops tagal ct rhizophora rhizophora apiculata ra rhizophora mucronata rm sonneratiaceae sonneratia sonneratia alba sa total: 5 families 7 genera 13 species table 3. list of minor mangal elements recorded in catanduanes island, luzon, philippines family genus species code bombacaceae camptostemon camptostemon philippinensis cp euphorbiaceae excoecaria excoecaria agallocha er lythraceae pemphis pemphis acidula pa meliaceae xylocarpus xylocarpus moluccensis xm xylocarpus granatum xg myrsinaceae aegiceras aegiceras corniculatum ac aegiceras fl oridum af pteridaceae acrostichum acrostichum aureum aa acrostichum speciosum as sterculiaceae heritiera heritiera littoralis hl total: 7 families 7 genera 10 species table 4. list of associated mangal elements recorded in catanduanes island, luzon, philippines family genus species code acanthaceae acanthus acanthus ebracteatus acanthus illicifolius ae ai apogonaceae cerbera cerbera manghas cm bignoniaceae dolichandrone dolichandrone spathacea ds combretaceae terminalia terminalia catappa tc cyperaceae cyperus cyperus malaccensis cm lecythidaceae barringtonia barringtonia racemosa br biotropia vol. 15 no. 2, 2008 91 family genus species code leguminoceae aganope ipomoea pongania aganope heptaphylla ipomea pes-caprae pongania pinnata ah ip pp malvaceae hibiscus hibiscus tiliaceus ht th espesia th espesia populnea tp palmae corypha corypha elata ce pandanaceae pandanus pandanus tectorius pt total: 10 families 13 genera 14 species 90 there are 94 species of vascular flora in mangals and associated coastal communities of the philippines (zamora 1995). tomlinson (1986) noted that there are 18 species of major mangal elements, 12 species of minor elements, and 24 specialized mangal elements. the number of species obtained in catanduanes is comparatively lower than the reports from other areas of the country, but slightly higher than the number of species obtained from guimaras island. the categorization of mangrove flora by tomlinson (1986) indicates that there are 34 species of major mangrove species worldwide; 20 species of minor element; and 46 mangal associates. considering this available reference data, catanduanes island shares 38% of the major elements (13 out of 34 species) and 72% (13 out of 18 from zamora’s list). for the minor elements, the province shares 50% (10 out of 20 species of tomlinson’s list) and 83 % (10 out of the 12 species from zamora’s list). table 5 shows the comparison of the various listings. table 5. category and number of mangrove floral species recorded in catanduanes island, philippines compared to other reports/listings. group of mangroves tomlinson (1986) zamora (1995) this study elements world-wide philippines catanduanes island major 34 17 13 minor 20 12 10 associated 46 40 14 total 100 69 37 distributional range of mangroves in catanduanes island globally, mangrove ecosystems are thought to contain about 60 species of trees and shrubs and more than 20 additional species frequently associated with the mangrove flora but not necessarily restricted to it (barth 1982; hamilton & snedaker 1984). some 70 species of the mangrove plants are recognized from various regions of the world, with the highest concentrations of species being found in sea and australia (spalding et al. 1997). according to fortes (1989), the most diverse single stand of mangrove forest is found in pagbilao, quezon (luzon, philippines) with a total of 29 species. the report of cadiz & de leon (1994, cited by calumpong & meñez 1997) revealed that the mangrove forest of misom in baliangao, misamis occidental consisted of 21 species, while in bais bay in negros island (visayas, philippines) a total of 14 species were noted by calumpong (1992). babaran & ingles (1997) table 4. continued occurrence and distributional range of mangrove – jimmy tevar masagca 92 indicated that not all species of major as well as minor elements are simultaneously present in all municipalities of guimaras, philippines. in the genus avicennia, 2 species (a. marina and a. officinalis) are found in catanduanes. the species, a. eucalyptifolia as reported by zamora (1991) is not found in this province. the other genera, e.g. bruguiera, ceriops, sonneratia and rhizophora are also well distributed in this island. the most important major mangal species in the province is the palm, nypa fruticans. a member of the screwpine family (pandanaceae), pandanus tectorius (an associated mangrove species) is distributed all throughout the province. however, this is not true in other places of the philippines. in guimaras, babaran & ingles (1997) made no report about its presence there. likewise, calumpong & menes (1997) did not also mention pandanus in their report. among the members of family pandanaceae, p. tectorius is the most well known species. this is the commonest and the most widespread (gruezo & zamora 2000; stone 1976). moreover, terminalia catappa (locally known as “tarisoy”) was described by calumpong and meñez (1997), and earlier included by dr. p. zamora in his previous papers. this species of the combretaceae family is well distributed in back mangals of the southern and western portions of island under study. among the minor mangrove elements, the genus acanthus, represented by the species, acanthus ebracteatus and a. illicifolius are well distributed in the different zones of the island from the northern to southern zones and eastern to western zones. of the major elements, sonneratia and avicennia are well distributed all throughout the island. among the minor elements, excoecaria agallocha and aegicera corniculatum are distributed in almost all zones of the island. conclusions a total of 37 species of mangrove vascular flora (13 species of major mangal elements, 10 species of minor mangal elements and 14 associated mangrove species) were identified in the island under study. two species of the genus avicennia (a. marina and a. officinalis) are present in the study sites. the other genera (bruguiera, ceriops, sonneratia and rhizophora) are well distributed in the designated eco-zones. the palm, nypa fruticans is the most important mangrove plant species in the island under study and a member of the screwpine family pandanaceae , pandanus tectorius which is an associated mangrove species is well-distributed all throughout the island. acknowledgment the author expresses his sincere thanks to the administration of de la salle university-dasmariñas for giving the impetus in carrying out the study on the mangroves while investigating the mangrove brachyurans of catanduanes and quezon. earlier studies on the mangroves of the island were carried out by the author while teaching in the premier state college. this work was completed with the assistance of various individuals. likewise, profound thanks is also given to raul evasco, ma joie estopin, biotropia vol. 15 no. 2, 2008 93 elver sison, macoy leyba, igo de castro and others from molino, cavite for the assistance provided during the writing of the final drafts of the report. references babaran, r.p. and j.a. ingles. 1997. philippine marine coastal habitats at risk: a case study of guimaras island. institute of marine fisheries and oceanology, university of the philippines in the visayas institute of integrative and development studies-marine affairs program, up press. barth, h. 1982. the biogeography of mangroves. in: d.n. sen and k.s. rajpurohit (eds.). tasks for vegetation science: contributions to the ecology of halophytes. the hague: dr. w. junk. bennagen, m.e.c. and d. cabahug jr. 1991. natural resources accounting: mangroves. technical report no. 2. natural resources accounting project final workshop. national institute of geological sciences, 15 oct. 1991. university of the philippines, diliman, quezon city. calumpong, h.p. 1992. systematic studies on the red algal genus laurencia (ceremiales: rhodomelaceae) in the philippines. phd dissertation, university of california, berkeley, california, usa. calumpong, h.p. 1994. status of mangrove resources in the philippines. in: c.r. wilins, s. sudara and c.l. ming (eds.) proceedings of the 3rd asean-australian symposium on living coastal resources, chulalongkorn university, thailand held on 16-20 may, 1994. calumpong, h.p. and e. g. meñez. 1997. field guide to the common mangroves, seagrasses and algae of the philippines. bookmark, inc.: makati city, philippines. dartnall, a.j. and m. jones (eds.). 1986. a manual of survey methods: living methods in coastal areas. asean-australia cooperation program on marine science handbook. townsville, australia: aims. de leon,r.o.d., j.a.u. nuque and r.j. raymundo.1992.leaf litter production and tidal export of rhizophora apiculata, r.mucronata, avicennia marina and sonneratia alba from talabong mangrove forest in bais, negros oriental. philippine council for agriculture, forestry and development. los banos, laguna. pccard series no. 127, pp. 84-90. ding hou, 1958. rhizophoraceae. flora malesiana. series 1, 5:429-493. fortes, m. 1989. mangrove and seagrass study. living resources in coastal areas: phase i terminal report. university of the philippines, quezon city, philippines. gruezo, w.sm. and p.m. zamora. 2000. conservation status of philippine biota. 2. sararanga philippinensis merr., family pandanaceae. asia life sciences, the asian international journal of life sciences, 9(2):189=204. hamilton, l.s. and s.c. snedaker (eds.). 1984. handbook for mangrove area management. unesco and east west center, environment and policy institute, hawaii. japar, s.b. 1994. mangrove plant resources in the asean region. in: c.r. wilins, s. sudara and c.l. ming (eds.) proceedings of the 3rd asean-australian symposium on living coastal resources, chulalongkorn university, thailand held on 16-20 may, 1994. masagca, j.t.2007. perceptiones de los maestros de ciencia y habitants costeros sobre conservacion de la biodiversidad de manglar (science teachers and coastal dwellers perceptions on mangrove biodiversity conservation). revista de educacion en ciencias, journal of science education, 7(1):24-29. morton, j. 1990. the shore ecology of the tropical pacific. jakarta: unesco rotsea. primavera, j. 2001. mangroves. haring ibon, 3(2):16-21. occurrence and distributional range of mangrove – jimmy tevar masagca 94 quimpang, v.t., h.c. porquiz, i. gurea, l.a. samaniego. 1987. the community structure of the mangrove forest in pangasihan, gingoog city. cmu journal of agriculture, food and nutrition, 9(1):84-102. tomlinson, p.b. 1986. the botany of mangroves. cambridge university press, cambridge. santisuk , t.1985. explanatory keys to terrestrial trees and shrubs recorded from the mangrove formations in thailand. proceedings of a training course in life history of selected species of flora and fauna in mangrove ecosystems. thailand, vol. 1. spalding, m.f., m.f blasco and c. field (eds.). 1997. world mangrove atlas. international society for mangrove ecosystems. the world monitoring center and international tropical timber organization. stone, b.c. 1976. studies in malesian pandanaceae, xiv. notes on philippine taxa. kalikasan, the philippine journal of biology, 5(1):1936. vega, m.j.m., l.r. garces, q.p. sia iii and r.g.g. ledesma. 1994. assessment of mangrove resources in san miguel bay. in: g. silvestre, c. luna and j. padilla (eds.) multidisciplinary assessment of the fisheries in san miguel bay, philippines (1992-1993). iclarm technical report. zamora, p.m.1984. philippine mangroves: assessment of status, environmental problems, conservation and management strategies. in: e. soepadmo, a.n. rao and d.j. macintosh (eds.), pages 606-707. asian symposium on mangrove environment research and management. unversity of malaya, kuala lumpur, malaysia. zamora, p.m. 1989. mangroves of the philippines. biotrop special publication (bogor, indonesia), 37:43-65. zamora, p.m. 1991. managing the philippine mangal for long-term human survival. transactions of the national academy of science and technology (nast), 12:89-125. biotropia vol. 15 no. 2, 2008 678 irang wahyu (an snp).cdr an snp marker potentially linked to somatic embryogenesis of oil palm ( )elaeis guineensis irang wahyunanto , diana e waturangi , nurita toruan-mathius and 1,2* 1 2 adi yulandi 1 1 faculty of biotechnology, atma jaya catholic university of indonesia, jakarta 12930, indonesia 2 plant production and biotechnology division, pt smart tbk., jakarta 10350, indonesia received 28 december 2016/accepted 14 april 2017 abstract oil palm (elaeis guineensis) is one of the most important oil-bearing crop in the world. this crop can be vegetatively propagated only using tissue culture technique. oil palm tissue culture technique has low efficiency, with callogenesis and embryogenesis stages as the limiting factors. genetic factor has a major role in determining the success rate of these two stages. the use of molecular markers which represent the rate of embryogenesis or callogenesis has the potential to improve the efficiency of oil palm tissue culture process. in this study, snp mining was conducted on embryogenesis transcriptome data, oil palm cdna database, oil palm genome database, and oil palm snps marker database in ncbi. the objective of this study was to obtain snp marker which represents the embryogenesis potential, to be further used in marker assisted selection of oil palm ortets. one snp (emb6) showed significant association with embryogenesis rate. this snp was found in one of auxin response factor (arf) family gene. th nucleotide replacement from adenine to guanine changed the 307 amino acid from isoleucine to methionine. oil palms with adenine homozygote (a/a) pattern on the emb6 showed 8-fold higher chance to produce significantly higher embryogenesis rate than adenine-guanine heterozygote (a/g). keywords: callogenesis, elaeis guineensis, snp, somatic embryogenesis introduction oil palm (elaeis guineensis) is among the most important crop which produces vegetable oil. vegetative propagation for oil palm can only be conducted using tissue culture methods to obtain highly productive oil palm seeds (wong et al. 1997). tissue culture technique is beneficial for individual propagation of oil palm having high productivity and/or certain trait of interest. the tissue culture technique can increase oil palm production by 30% compared to the commercial seed dura x pisifera (dxp) on a large scale field trials (cochard et al. 1999; wahid et al. 2005). tissue explants of oil palm resulted from in vitro culture are developed through callus formation or normally termed as indirect embryogenesis (te-chato & hilae 2007). however, not all of the cultured explants have the potential to be developed into embryogenic callus. those explants which are not successfully developed into embryogenic callus usually form a nonembryogenic callus, that will remain in the form of callus without any possibility to develop into ramet (rohani et al. 2000). corley and tinker (2003) reported that the level of callogenesis in oil palm is still low (around 19%), while the ability to form somatic embryoid is only 3 6% (wooi 1995). one of the main factors that determine the ability of oil palm tissue culture is the genetic factor, which is indicated by the fact that some genotypes are more productive than the others (wooi 1995). i d e n t i f i c a t i o n o f s i n g l e n u c l e o t i d e polymorphism (snp) in oil palm genome had been attempted in several studies. identification of quantitative trait loci (qtls) associated with callogenesis and embryogenesis in oil palm with broad range of markers from afrlps, rflps, and ssrs has been done (ting et al. 2013), but studies to determine the process of de-differentiation and embryogenesis with * corresponding author: irangwahyunanto@hotmail.com biotropia 4 2 7 153 160 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 678 153 specific snp markers in plant genomes are limited. in this study, the identification of candidate snp associated with somatic embryogenesis in oil palm were done in silico, which was continued to the validation process based on the phenotype data of oil palm tissue culture. this study was aimed to obtain snp marker candidates which represents the embryogenesis potential, to be further used in marker assisted selection of oil palm ortets, and thus increase the tissue culture process efficiency. materials and methods in silico selection of snps marker target genes were derived from expressed sequence tags (ests) from low et al. (2008), and lin et al. (2009) which were deposited in genbank database ey396120-ey413718 and gh635901gh637767. ests were subsequently assembled using cap3 program (huang & madan 1999). contigs and singletons were annotated to oil palm cdna database from mpob's research (genomsawit.mpob.gov.my) (singh et al. 2013), using the blast tool (altschul et al. 1990) to obtain a list of genes that was expressed during the process of embryogenesis. the candidate genes were further aligned to the oil palm genome sequence (genomsawit. mpob.gov.my) (singh et al. 2013) to get the fulllength gene sequence. the alignment process was done using sim4db program (walenz & florea 2011). the full-length gene sequence was generated using getfastabed tool in galaxy platform (quinlan & hall 2010). then, the fulllength gene sequences were used as the database for blast alignment of oil palm snp sequences obtained from mpob (genomsawit.mpob.gov. my) (ting et al. 2014), and ncbi (teh et al. 2016) as the queries (fig. 1). ten candidate snp markers were selected based on the function, expression and sensitivity of the mutation position. dna fragment sequences were aligned using unipro ugene (okonechnikov 2012). primers of each snp et al. marker were designed using primer-blast tool from ncbi, which combined the algorithm of primer3 (untergrasser 2012) and ncbi et al. blast. primer quality was analyzed and selected using netprimer tools from premier biosoft. snps marker validation thirty ortets were kindly provided by the tissue culture laboratory of pt smart tbk. these ortets were selected based on the tissue culture productivity data, including callogenesis and embryogenesis rate. genomic dna were isolated and purified from leaf tissue using nucleospin plant ii kit (macherey-nagel gmbh & co kg, duren, germany). the quality of extracted dna was measured using 1% agarose gel electrophoresis and nanodrop s p e c t r o p h o t o m e t e r ( t h e r m o s c i e n t i f i c , massachusetts, usa). the dna was amplified by figure 1 oil palm embryogenesis snp mining bioinformatics workflow 154 biotropia vol. 24 no. 2, 2017 contigs and singletons from assembly process were aligned to two sets of mpob's cdna database (genomsawit.mpob.gov.my), namely v1 and v2 (table 2). the genes were selected based on its correlation to embryogenesis function via text mining methods. selection of the embryogenesis related gene candidates' selection was according to elhiti et al. (2010), resulting in 423 embryogenesis related genes. these genes only work as reference for this study. there might still be other genes involved in embryogenesis due to the complexity of embryogenesis process. the sequences were aligned to mpob's genome database using sim4db program to locate the index position of the intact gene (intron+exon). the index position in gff3 format was then used as input to generate the intact gene's fasta sequence. the whole genomic sequence of candidate genes was used as the database to align the oil palm snp markers. from mpob's 1,766 snp positions, there were 12 snps with positive hit to target genes, 10 snps were in introns, while the other two were synonymous codon variant in exons. in ncbi, which has 112,360 snp positions, there were 1,575 positive hits to target designed primers using pcr. the pcr products were confirmed using agarose gel electrophoresis. pcr products were purified using qiaquick pcr purification kit (catalog no.28104, qiagen, hilden, germany). purified pcr products were sequenced, then statistically analyzed and compared with the callogenesis and embryogenesis productivities data using spss 20.0 (spss inc., chicago, usa). for odds ratio analysis, tissue culture productivity data (callogenesis and embryogenesis rate) were converted into categorical data (low callogenic, high callogenic, low embryogenic and high embryogenic), then compared with nucleotide variation using n cochran's and mantel-haenszel cross tabulation statistics in spss. the tissue culture productivity data variation was analyzed with one-way anova. results and discussion a total of 19,471 embryogenesis related ests were obtained from low et al. (2008) and lin et al. (2009). the ests libraries were assembled using cap3 program resulting to 13,020 sequences of contigs and singletons (table 1). table 1 ests assembly results source subjects ests contigs + singletons low et al. (2008) nec 6,498 3,760 ec 2,717 2,130 lin et al. (2009) emb 8,389 5,456 initiation 949 854 proliferation 918 820 note: source of ests: non-embryonic callus (nec), embryogenic callus (ec), embryoid (emb), embryoid initiation and embryoid proliferation table 2 embryogenesis related candidate genes number subjects contigs + singletons v1 (genes) v2 (genes) nec 3,760 2,269 1,841 ec 2,130 1,085 821 emb 5,456 3,640 3,074 initiation 854 530 437 proliferation 820 561 506 note: source of ests: non-embryonic callus (nec), embryogenic callus (ec), embryoid (emb), embryoid initiation and embryoid proliferation 155 an snp marker potentially linked to somatic embryogenesis of oil palm – wahyunanto et al. table 3 snps with positive hits to target genes snps database functional consequence amount (hits) mpob intron variant 10 mpob synonymous codon 2 ncbi intron variant 19 ncbi downstream variant 61 ncbi upstream variant 99 ncbi 3’utr variant 36 ncbi 5’utr variant 11 ncbi splice-donor variant 1 ncbi synonymous codon 34 ncbi missense variant 53 ncbi undefined 1,261 table 4 snps marker for oil palm embryogenesis snps gene amino acid variation used for further sequencing emb1 elaeis guineensis e3 ubiquitin-protein ligase ring1-like, transcript variant x2, mrna leu → ser yes emb2 elaeis guineensis auxin-responsive protein iaa10-like, mrna arg → ser yes emb3 elaeis guineensis ethylene-responsive transcription factor 1 like, transcript variant x2, misc_rna ile → val yes emb4 elaeis guineensis protein phosphatase 2c and cyclic nucleotide-binding/kinase domain-containing protein, transcript variant x5, misc_rna thr → ala yes emb5 elaeis guineensis probable wrky transcription factor 70, mrna glu → gly yes emb6 elaeis guineensis auxin response factor-like, mrna ile → met yes emb7 elaeis guineensis cytochrome p450 85a1-like, transcript variant x3, mrna pro → ser yes emb8 elaeis guineensis coatomer subunit beta-1-like, mrna ile → leu → val no emb9 elaeis guineensis coatomer subunit beta-1-like, mrna pro → ala → ser no emb10 elaeis guineensis dna-binding protein bin4, transcript variant x4, mrna thr → pro → ala yes emb11 elaeis guineensis auxin response factor-like, transcript variant x1, mrna met → val → leu yes emb12 elaeis guineensis probable cellulose synthase a catalytic subunit 1 [udp-forming], mrna ile → val no emb13 elaeis guineensis glutathione s-transferase zeta class-like, mrna asp → his yes 156 biotropia vol. 24 no. 2, 2017 genes, which includes 1,261 undefined variants, 61 downstream variants, 19 intron variants, 1 splicedonor variants, 99 upstream variants, 36 3'utr variants, 11 5'utr variants, 34 synonymous codon variants and 53 missense variants (table 3). in this study, we focused on missense snps. however, the selection of snps marker had functional consequence only as a priority scale adjusting to the scope and research resources. snp variations in other functional positions remain a potential determinant of embryogenesis rate (ting . 2013). from 53 missense variant et al snps, 40 snps were eliminated because the cdna annotation of the ests did not match with the gene locus defined in the snp information. thirteen snps position used for further process were described in table 4. from all thirteen selected snps, only 10 were selected for further dna sequencing process. from dna sequencing process, we obtained data of snp variations, which were further statistically analyzed using cross tab chi square, odds ratio analysis and one-way anova (spss 20.0) (table 5). cross tab chi square was used to analyze the degree of dependency between snp variations with embryogenesis rate. one-way anova was used to observe the difference in embryogenesis rate between snp variations. odds ratio analysis was used to compare the relative odds of the snp variations to the occurrence of high embryogenesis. from 10 snp positions, only one snp, in auxin response factor family (emb6), showed significant result in cross tab chi-square and in one-way anova. odds ratio analysis showed 8 times chance of higher embryogenesis when the snp at emb6 is adenine homozygote (a/a) compared to adenine-guanine heterozygote (a/g). sample size of population in this study is still relatively small. further observation with larger population is needed for marker revalidation. there is also a possibility of other genes influencing embryogenesis of oil palm, which needs further investigation. table 5 statistical result of snp variation and embryogenesis rate gene code snp*) indels**) cross tab chi square one way anova odds ratio e3 ubiquitin protein ligase ring 1 emb1 0 no independent not significant n/a auxin responsive protein iaa 10 emb2 8 no independent not significant n/a ethylene responsive transcription factor emb3 1 no independent not significant n/a pp2c emb4 1 yes independent not significant n/a wrky transcription factor emb5 4 yes independent not significant n/a auxin response factor a emb6 3 yes dependent (p = 0.01) significant (p = 0.015) aa = 8x ag (p = 0.014) cytochrome p450 85 a 1 like emb7 0 no independent not significant n/a dna binding protein bin 4 emb10 1 no dependent (p = 0.04) not significant n/a auxin response factor b emb11 1 no independent not significant n/a glutathione s transferase emb13 1 yes independent not significant n/a 157 an snp marker potentially linked to somatic embryogenesis of oil palm – wahyunanto et al. auxin response factors (arf) family has been suggested to play a key role in regulating the expression of auxin response genes (liscum & reed 2002). gliwicka et al. (2013) found that the expression of over half of aux/iaa and arf g e n e s w e r e c h a n g e d d u r i n g s o m a t i c embryogenesis in arabidopsis. auxins play critical roles in most of the major growth responses throughout different developmental stages of plants; such as organogenesis, vascular tissue differentiation, apical dominance, root initiation, and tropism; as well as cellular level processes including extension, division, and differentiation (guilfoyle & hagen, 2007; mockaitis & estelle, 2008; su et al. 2014). a large number of potentially auxin regulated candidate genes, which function in growth and developmental processes, have been identified in arabidopsis and other plant species (rosado et al. 2012; liu et al. 2014; di et al. 2015; guilfoyle 2015). arf regulation is well-studied (salehin et al. 2015). at low auxin levels, aux/iaa proteins form dimers with arfs to inhibit arf activity resulting in the repression of auxin-responsive genes. at high auxin levels, aux/iaas bind to the tir1/afb scf complex and subsequently become ubiquitinated and degraded by the 26s proteasome. the arf is then released and regulate the transcription of its target auxin response genes (wang & estelle 2014) (fig. 2). most of the arf proteins consist of an nterminal b3-type dna binding domain (dbd), a variable middle region that functions as an activation domain (ad) or repression domain auxin response geneauxin response element auxin response factor aux/iaa auxin (low auxin) auxin response geneauxin response element auxin response factor aux/iaa auxin (high auxin) off on auxin auxin auxin auxin tir1/afb scf complex ub ub ub ub proteasome ub ub auxin response geneauxin response element auxin response factor aux/iaa auxin (low auxin) auxin response geneauxin response element auxin response factor aux/iaa auxin (high auxin) off on auxin auxin auxin auxin tir1/afb scf complex ub ub ub ub proteasome ub ub figure 2 auxin signalling pathway (adopted from da costa . 2013; salehin 2015; wang & estelle 2014)et al et al. figure 3 emb6 amino acid variation position 158 biotropia vol. 24 no. 2, 2017 (rd) and a carboxy-terminal dimerization domain (ctd:domain iii/iv), which is involved in protein– protein interactions by dimerizing with auxin/indole-3-acetic acid (aux/iaa) family genes or between arfs (kim et al. 1997; guilfoyle & hagen 2007; piya et al. 2014). emb6 th was located at the 307 amino acid, inside the conserved domain of arf family (fig. 3). the position was in the middle region which is critical in determining arf function. the variations found in this study were a/a and a/g, which change the amino acid from isoleucine to methionine. although both amino acids are hydrophobic and have similar size, methionine has a unique structure as a sulphur-containing amino acid, which might change the protein folding due to its sulphuric bond. conclusions in this study, bioinformatic analysis of snp markers was performed by comparing the snp database with the genes related to embryogenesis. one snp located in an auxin respone factor family gene showed a significant association with the embryogenesis rate of oil palm tissue culture. this snp may be used as a marker to select sample source to increase the efficiency of oil palm tissue culture process in the near future. further observation with a larger population is needed for marker revalidation. acknowledgements this research was conducted at biotechnology department laboratory; the samples and tissue culture productivity data were obtained from the tissue culture department. both places are part of the plant production and biotechnology division of pt smart tbk. references altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j mol biol 215:40310. cochard b, durand-gasselin t, amblard p, konan ek, gogor s. 1999. performance of adult oil palm clones. in ariffin d, chan kw. sharifah, editors. emerging technologies and opportunities in the next 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seaweed ( (doty) collected from natuna islands, riau islands province kappaphycus alvarezii erina sulistiani , dinar tri soelistyowati , alimuddin , samsul ahmad yani recipient of biotrop research grant 2010/accepted 28 juni 2012 the objective of this study was to obtain the optimal medium for callus induction from thallus explants of doty and to regenerate filamentous callus from induced callus. before cultured cottonii seaweeds collected from the natuna islands (riau islands province) were acclimatized in greenhouse and in semi-sterile culture in the laboratory. sterilized explants were cultured on pes and conwy media solidified with 0.8% bacto agar. in each of these media two combinations of plant growth regulators i.e. ba+iaa and ba+naa were added. the concentrations of ba used were 0, 0.5, 1 mg/l, the concentrations of iaa were 0, 2.5, 5 mg/l, whereas the concentration of naa were 0, 0.5, 1 mg/l. the result indicated that the optimal medium for callus induction was pes solidified medium supplemented with ba 1 mg/l. types of callus formed were (a) white compact callus, (b) white filamentous callus, (c) greenish/brownish callus. regeneration of callus into clumps of filament had been done by subculturing the callus into pes solidified medium supplemented with ba 1 mg/l + iaa 2.5 mg/l. tissue culture, callus induction, filamentous callus, seaweed, 1* 2 2 1 1) 2) tissue culture-services laboratory-seameo biotrop, bogor, indonesia faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia kappaphycus alvarezii ( ) , kappaphycus alvarezii abstract introduction key words: cottonii seaweed or is one type of carrageenan-producing seaweed massively cultivated in indonesia. the ministry of marine affairs and fisheries of republic of indonesia continues to attempt the development of seaweed farming and cottonii seaweed processing industry. to support the efforts to increase the production of this seaweed, continuous availability of quality seeds is required. kappaphycus alvarezii * corresponding author : esulistiani@biotrop.org 103 since cultivated in the 1970s, seeds of cottonii seaweed are obtained from vegetative propagation. repeated clonal propagation caused a decrease in genetic variability resulting in decreased growth rate, carrageenan yield and gel strength. in addition, the decrease in variability also causes increased susceptibility to disease (hurtado & cheney 2003). application of tissue culture techniques in seaweed is expected to help produce a superior clone, thereby increasing the provision of seeds required for cultivation and to produce uniform seedlings in large quantities but in a short time. generally, seaweed tissue culture stages include preparation of axenic explants, callus induction and regeneration of callus into thallus and young plantlets of seaweed. the objective of this study was to obtain the optimal medium for callus induction from thallus explants of collected from natuna islands, riau islands province, indonesia. to regenerate filamentous callus from induced callus. seaweeds were collected twice from the natuna islands, first on april 20 and the second on may 20, 2010. to keep it fresh during the journey to bogor, seaweed was inserted into cardboard boxes in a quite wet condition. every two clumps of seaweeds were placed separately from each other on the boxes, while old newspapers were used as partition.then the cardboard boxes were closed and inserted into a sack tied using raffia. to reduce mortality at the time of semi-sterile culture in the laboratory, new seaweed brought from the sea need to be acclimatized and maintained in a clean and controlled environment in greenhouse before the semi-sterile culture in the laboratory. the purpose of acclimatization was to adapt the seaweed from the sea to the new environment of the aquarium placed in greenhouse. seaweeds were maintained in the aquarium containing seawater as much as 7/8 part of the aquarium sized 30 cm wide x 35 cm high x 90 cm long. using recirculation system, seawater in the aquarium was flown into the container filter consisting layers of fiber glass wool, activated carbon and coral. seawater that had been filtered was reflown using a pump and pvc pipe to the aquarium. the aquaria also were equipped with an aerator, and shaded by 65% shading net. after 1 month, the seaweed with light green color, clean from dirt and epiphyte were acclimatized in semi-sterile culture in the laboratory. seaweed was washed using seawater which was sterilized by autoclave. the dirt was cleaned gently using a soft brush. mucus attached to the seaweed was cleaned by rinsing 2-3 times with sterilized seawater. subsequently, the seaweeds were cut into pieces ± 10 cm long and cultured in 10-liter glass jar, which already contains conwy (cw) liquid medium (liao . 1983). before use , the culture media were sterilized by autoclave at temperature of 121ºc for 1 hour. the experiment was repeated in 3 jars, each jar contained 4 liters of media and 150 g of seaweed. after 3 weeks, the weight of still alive seaweeds was k. alvarezii et al materials and methods seaweed acclimatization. 104 biotropia vol. 19 no. 2, 2012 measured again to obtain the survival rate of seaweed (%) in the semi-sterile culture system. the cultures were placed on culture rack, illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. the room temperature was set between 22-25ºc. in the first 2 weeks, the media was replaced 2 times a week with fresh media. after 2 weeks, the media was replaced once a week, for another 3 weeks. preparations of axenic explants in this study were mostly obtained according to methodology described by reddy . (2003). the apical parts of thallus were selected from semi-sterile culture to be used as explants for callus induction. explants were taken from the cultures age 5 weeks and 1 week. thalli were cut into pieces with a length of 4-5 cm. subsequently, the explants were soaked in tween solution (5 drops in 200 ml) for 10 minutes while occasionally shaken, and then the explants were rinsed twice with sterilized seawater. explants then were soaked in 0.5% povidone iodine (betadine) (0.5 ml in 100 ml of sterilized seawater) for 3 minutes. after that, the explants were rinsed 3-4 times with sterilized seawater. explants were then dried with sterilized tissue papers and immersed in cw medium containing 3% antibiotics mixture (penicillin g 1 g, streptomycin sulphate 2 g, kanamycin 1 g, nystatin 25 mg, neomycin 200 mg in 100 ml distilled water). the cultures were shaken with a shaker for 60 hours, at room temperature of 22-25ºc and illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. then the explants were washed with sterilized seawater, and dried with sterilized tissue papers then planted in the cw medium solidified with 0.8% bacto agar. observations were carried out to record the condition of explants and the percentage of contaminated explants. explants that had been observed for 2 weeks and did not contaminate were used as explants for callus induction. explants were cut into pieces with length of 4-5 mm. each explant then was wiped gently with sterile tissue papers to remove moisture and any mucilaginous substances exuded from the cut ends. then explants were planted on treatment media for callus induction. to investigate the optimal basic culture media, explants were cultured on provasoli enriched seawater/pes (provasoli 1968) and conwy/cw (liao . 1983) media solidified with 0.8% bacto agar. in each of these media two combinations of plant growth regulators i.e. ba+iaa and ba+naa were added. the concentrations of ba were 0, 0.5, 1 mg/l, the concentrations of iaa were 0, 2.5, 5 mg/l, whereas the concentrations of naa were 0, 0.5, 1 mg/l. each treatment was done with 10 replications. the cultures were stored in a culture room, with room temperature between 22-25ºc, and 60-70% of relative humidity. the cultures were illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. preparation of axenic explants callus induction et al et al 105 callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. regeneration of filamentous callus seaweed acclimatization observations on callus growth were done by giving a rating (score). quantity of callus growth was determined in 6 values representing 6 categories of callus growth, namely: 0 = no callus induction, the explants were bleached ; 1 = quantity of callus growth was very low; 2 = quantity of callus growth was low; 3 = quantity of callus growth was medium, 4 = quantity of callus growth was high; and 5 = quantity of callus growth was very high. data on callus growth were analyzed with non-parametric statistical test of kruskal-wallis using spss 16.0 software. the dun's multiple comparison test was used to distinguish treatments with significant differences. after 6 weeks in treatment media of callus induction, all explants were subcultured in pes solid medium containing 1 mg/l ba + 2.5 mg/l iaa for 2 months to enhance callus growth. subsequently, all callus outgrowth were excised from the explants and subcultured separately in pes solid medium containing 1 mg/l ba + 2.5 mg/l iaa for another 2 months to regenerate filamentous callus. installation of seaweed maintenance equipment in the greenhouse, was good enough to maintain the life of seaweeds brought from the sea. cottonii seaweed harvested from natuna islands, on 20 of may 2010 had survived up to one month, with 20% of mortality rate. the highest mortality occurred in the first two weeks, where the seaweed suffered stress during travel distance from the sea to the laboratory and the influence of packaging. after 3 weeks maintained in greenhouse, the tip of thallus began to grow. it was characterized by the formation of light green buds at the tip of thallus. installation of seaweed maintenance equipment in the greenhouse is useful to adapt the seaweed to the new environment (acclimatization). it also can be used to store seaweed stock for a long time as source of explants for seaweed tissue culture studies and genetic improvement to produce quality seeds of seaweed. observations after three weeks maintained in semi-sterile culture showed that survival rate of seaweeds that came from greenhouse was 72%. in our first experiment, seaweeds brought directly from the sea were acclimatized in the semisterile culture. as a result, the survival rate of seaweeds was 0-25%. this indicated that the acclimatization of seaweed in greenhouse, succeeded to increase the survival rate of seaweed in acclimatization phase in semi-sterile culture in the laboratory. due to long distance transportation from natuna islands to bogor, which took two days one night and humid condition of packaging, cottonii seaweed produced a lot of mucus. the mucus caused the seaweed thallus to bleach and die.. in addition, the high concentration of mucus in the static culture may also be a factor causing the death of seaweed. results and discussions th k. alvarezii 106 biotropia vol. 19 no. 2, 2012 based on these results, the new seaweeds brought from the sea need to be adapted first and maintained in a cleaner and controlled environment in the greenhouse before semi-sterile culture in the laboratory. acclimatization in a greenhouse with a recirculation system could refresh the seaweed conditions which suffered stress during delivery. in addition, the mucus contained in the seaweed could be reduced by recirculation system of water management. seawater in the aquarium was flown into the filter container to clean the dirt and slime carrying bacteria, then the filtered seawater was reflown to the aquarium. acclimatization stages in laboratory was very important to be optimized, because the sterilization of explants to obtain axenic explants could be more easily obtained from explants that had been acclimatized first in laboratory than seaweed explants collected directly from the sea. in addition, rate of callus induction would be higher when thallus explants were used that had been acclimatized first in the laboratory (reddy . 2003). the number of explants that had been sterilized was 283 explants. observation data 2 weeks after planting indicated that explants which were acclimatized 5 weeks in semi sterile culture produced higher sterile explants (64.5%) than the explants that has been acclimatized 1 week (43.4%). all contaminations were caused by bacteria. however, the explants remained green during the entire section, where for 1 week old explants (44%) the percentage was higher than for 5-week old explants (27.4%). the rest changed color to brown (browning) or white (bleaching) as a sign that explants were dead, because 5-week-old explants had more young thallus with a diameter <4 mm. these thalli were the result of seaweed growth during acclimatization on semi-sterile culture which had smaller diameter than thallus of seaweed which grows in the sea (fig. 1). while for 1 week old explants grown on semi-sterile culture, thalli were originally derived from the sea, and had larger diameter between 4-5 mm. these thalli had a higher resistance to the sterilization process than <4 mm diameter of thallus explants (muñoz . 2006). et al et al preparation of axenic explants 107 figure 1. cottonii seaweed on cw medium has survived and grown up to 5 weeks after planting in semi-sterile culture. callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. concentration of pgr time required for callus induction (days) in basic medium : cw pes without pgr 16 15 bap 0 mg/l + iaa 2.5 mg/l 19 14 bap 0 mg/l + iaa 5 mg/l nc 16 bap 0.5 mg/l + iaa 0 mg/l 22 18 bap 0.5 mg/l + iaa 2.5 mg/l 24 19 bap 0.5 mg/l + iaa 5 mg/l 25 nc bap 1 mg/l + iaa 0 mg/l 26 22 bap 1 mg/l + iaa 2.5 mg/l 26 15 bap 1 mg/l + iaa 5 mg/l 25 15 bap 0 mg/l + naa 0.5 mg/l 25 15 bap 0 mg/l + naa 1 mg/l 20 25 bap 0.5 mg/l + naa 0 mg/l 22 18 bap 0.5 mg/l + naa 0.5 mg/l 15 15 bap 0.5 mg/l + naa 1 mg/l 21 21 bap 1 mg/l + naa 0 mg/l 26 22 bap 1 mg/l + naa 0.5 mg/l 19 17 bap 1 mg/l + naa 1 mg/l 16 nc average time (days) 22 18 callus induction the most rapid callus induction occurred at 14 days after planting in the media treatment, while the slowest occurred at 28 days after planting. explants which did not show the growth of callus over 28 days after planting become bleached. the growth of callus usually occurred in medullar region (fig. 2a), but also on cortical regions in some explants i.e. the skin surface of the thallus and the tip of apical thallus (fig. 2b). most explants in pes medium formed callus faster than in cw medium. the average time required for callus induction on pes media was 18 days, while for cw medium it was 22 days (table 1). 108 a b figure 2. the location where the callus began to grow on thallus explants namely: (a) in the medullar region; (b) in the cortical region and apical thallus tip. tabel 1. average time required for callus induction in pes and cw media supplemented with combination of ba + iaa and ba + naa. note: nc = no callus induction and explants bleached biotropia vol. 19 no. 2, 2012 six weeks after planting, pes medium generated an average rate of callus induction higher (35.6%) than in cw medium (31.7%) (table 2). this result was different from the research of suryati and mulyaningrum (2009) which indicated that the cw medium had a better rate of callus induction than pes medium. this difference could be due to different sources of seaweed explants used, because according to george (1993) in addition to the culture medium, there are three factors that could affect growth and morphogenesis in tissue culture, namely genotype of explants source, environment of culture and tissue-dependent factor. pes medium had been widely reported to successfully establish and regenerate callus into plantlets of young seaweeds (reddy 2003; and munoz . 2006). in addition to , pes medium had also succeeded to induce and regenerate callus on other species of seaweed such as sp, sp, sp (huang & fujita 1997), sp (collantes 2004; kumar . 2007) , and (kumar . 2007). in addition to pes medium, research of hayashi (2008) showed that the induction of callus had succeeded either by using the f/2 50 medium (seawater enriched with guillard & ryther 50% solution) and vs 50 medium (seawater enriched with 50% von stosch's solution). hurtado and biter (2007) had done plantlet regeneration of var. adik-adik by using ess (erd schreibers seawater) medium. while hurtado . (2009) and yunque (2010) had established procedures for production of tissue culture seedlings of with media ampep (acadian marine plant extract powder). the highest rate of callus induction was found in pes medium + ba 1 mg/l (70%) (table 2). explants that did not form callus generally bleached on the third week after planting. explants which mostly bleached were thallus explants with diameter less than 3 mm. the addition of iaa or naa produced a reduction on the average rate of callus induction, both on cw and pes media, as shown on most media added with auxins iaa or naa had an average rate of callus induction lower than in media without auxins. according to bradley (1991) and yokoya . (2010), some types of plant hormone naturally occur in seaweed tissue both auxin and cytokinin (endogenous auxin/cytokinin), such as iaa, aba (absisic acid), paa (phenyl acetic acid), ip (isopentenyladenin), and cz (cis-zeatin). the decrease rate of callus induction (%) due to the addition of auxin iaa/naa was probably due to hormones content in thallus explants was quite optimal to form callus, so that the addition of plant growth regulator caused in excessive concentrations resulting in decreased rate of callus induction. negative influence of iaa on callus growth was probably caused by the use of too high concentration , while suryati and mulyaningrum (2009) found that the optimal concentration was 0.4 mg/l. concentrations higher than 0.4 mg/l caused a decrease rate of callus induction. observations on callus growth on each treatment media was done by giving a rating (score). quantity of callus growth was determined in 6 values representing 6 categories of callus growth. examples of callus growth in each category were shown in figure 3. k. alvarezii et al. et al k. alvarezii grateloupi carpopeltis ptilophora gracilaria et al. et al hypnea musciformis turbinaria conoides et al et al. k. alvarezii k. alvarezii et al et al. k. alvarezii et al 109 callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. ~ table 2. callus induction rate (%) on pes and cw media supplemented with combination of ba + iaa and ba + naa. 110 concentration of pgr average rate of callus induction (%) in basic medium cw pes without pgr 40.00 20.00 bap 0 mg/l + iaa 2.5 mg/l 20.00 50.00 bap 0 mg/l + iaa 5 mg/l 0.00 40.00 bap 0.5 mg/l + iaa 0 mg/l 40.00 40.00 bap 0.5 mg/l + iaa 2.5 mg/l 30.00 20.00 bap 0.5 mg/l + iaa 5 mg/l 40.00 0.00 bap 1 mg/l + iaa 0 mg/l 30.00 70.00 bap 1 mg/l + iaa 2.5 mg/l 30.00 50.00 bap 1 mg/l + iaa 5 mg/l 30.00 30.00 bap 0 mg/l + naa 0.5 mg/l 20.00 30.00 bap 0 mg/l + naa 1 mg/l 30.00 50.00 bap 0.5 mg/l + naa 0 mg/l 40.00 40.00 bap 0.5 mg/l + naa 0.5 mg/l 10.00 50.00 bap 0.5 mg/l + naa 1 mg/l 40.00 50.00 bap 1 mg/l + naa 0 mg/l 30.00 70.00 bap 1 mg/l + naa 0.5 mg/l 60.00 10.00 bap 1 mg/l + naa 1 mg/l 40.00 0.00 average rate (%) 31.18 36.47 a cb d e f figure 3 . callus represented by 6 values of callus growth, namely: (a) 0 = no callus induction, the explants become bleached; (b) 1 = quantity of callus growth was very low; (c) 2 = quantity of callus growth was low; (d) 3 = quantity of callus growth was medium, (e) 4 = quantity of callus growth was high; and (f) 5 = quantity of callus growth was very high ( scale bar = 3 mm). biotropia vol. 19 no. 2, 2012 the data of callus growth after 6 weeks in treatment media were analyzed with non-parametric statistical test of kruskal-wallis using spss 16.0 software. the results showed that the media types, the addition of plant growth regulator ba, iaa and naa did not significantly affect quantity of callus growth of cottonii seaweed. similarly, the interaction between these factors had no significant effect on quantity of callus growth (p < 0.05). the data on pes mediated callus growth were re-tested for statistical analysis of krukall wallis for two factors, namely the concentration factor of ba and iaa, and concentration factor of ba and naa. statistical test of data on callus growth in pes medium with ba and iaa treatment indicated that both of these factors and their interactions had no significant effect on callus growth (p < 0.05). meanwhile, in pes medium with ba and naa treatment, the results of statistical tests showed the two factors also had no significant effect, but the interaction of both factors significantly affected callus growth (p < 0.05). the highest mean rank of callus growth was pes medium + ba 1 mg/l without the addition of naa. based on the results of multiple range test of dunn, the treatment was significantly different from other treatments (p < 0.05) (table 3). the addition of plant growth regulator was often used to increase the rate of callus induction (%) and callus growth in seaweeds (dawes & koch 1991; huang & fujita 1997; yokoya & handro 2004; yokoya . 2004; munoz 2006; hayashi . 2008). plant growth regulators of auxin and cytokinin were used either individually or in combination. in this study, the addition of plant growth regulator ba + naa and bap + iaa in the medium did not significantly result in increased rate of callus induction and growth of seaweed . similar results of this study were also reported by reddy . (2003). et al et al et al k. alvarezii et al table 3. effect of naa and ba concentration on the growth of callus 6 weeks after planting 111 naa concentration (mg/l) ba concentration (mg/l) 0 0.5 1 0 37.85 abc 51.00 abc 58.50 a 0.5 42.50 abc 55.45 abc 32.60 bc 1 51.35 abc 51.25 abc 29.00 c a b c figure 4 . types of callus formed after 6 weeks in callus induction media : (a) white compact callus; (b) white filamentous callus; (c) greenish/brownish callus (scale bar = 4 mm). note: numbers followed by same letter are not significantly different based on dunn's multiple range test (p < 0.05). callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. ~ the role of plant growth regulators (pgrs) on callus induction in multi-cellular algae such as seaweed is still debatable and showed no definite trend (bradley 1991; evan & trewasvas 1991; baweja . 2009; yokoya 2010). the inconsistency in responses to pgrs by seaweeds could be mostly due to lack of understanding of the physiological role of these substances in their growth and differentiation (reddy 2008). based on the observations after 6 weeks on induction medium, there were several types of callus, namely: (a) white compact callus, (b) white filamentous callus, (c) greenish/brownish callus (fig. 4). filaments are rows of cells which form long chains. generally in seaweed, the long chain of cells are not branched (unbranched filaments) except of oscillatoriaceae species (lobban & harrison 1994). after 6 weeks in the treatment media for callus induction, all of formed callus were sub-cultured into pes medium + ba 1 mg/l + iaa 2.5 mg/l in order to enhance callus growth. after 2 months in the media, the appearance of callus changed. the et al et al. et al. regeneration of filamentous callus figure 5. changes of white compact callus to greenish/brownish callus after 2 months subcultured in pes medium added with ba 1 mg/l + iaa 2.5 mg/l (scale bar = 4 mm). f f figure 6 . growth of filaments clumps (f) on greenish/brownish callus 8 weeks after subcultured in pes medium +ba 1 mg/l + iaa 2.5 mg/ (scale bar = 2 mm). biotropia vol. 19 no. 2, 2012 112 white compact callus turned into greenish/ brownish friable callus with filaments that grow on it (fig. 5). the greenish/brownish callus was isolated from thallus explants then subcultured in pes medium + ba 1 mg/l + iaa 2.5 mg/l. after 2 months, greenish/brownish filaments in large quantities grown on the callus. growth of these filaments aside from growing on the surface of the callus also grow at the bottom of the callus penetrating the culture medium (fig. 6). pes medium supplemented with ba and iaa had successfully regenerated callus into more clumps of filaments. filament formation is the initial phase of young seaweed formation (shao 2004). addition of ba and iaa also had been reported to regenerate callus into young seaweed as in the study of huang and fujita (1997) and hayashi . (2008). the procedures of callus induction and filaments regeneration from callus of cottonii seaweed ( derived from natuna islands (riau islands province) were as follows: 1. due to long distance transportation, seaweed brought from the sea has to be acclimatized in greenhouse for 4 weeks, then 1 week in laboratory before cultured in medium of callus induction 2. the most effective medium for callus induction of seaweed was pes medium supplemented with ba 1 mg/l. 3. regeneration of callus into clumps of filaments could be done in pes medium supplemented with ba 1 mg/l and iaa 2.5 mg/l. this research was funded by seameo biotrop awarded to erina sulistiani, tissue culture laboratory, services laboratory, seameo biotrop, bogor indonesia. et al. et al kappaphycus alvarezii) k. alvarezii conclusions acknowledgments references baweja pd, sahoo p, garcia-jimenez, robaina rr. 2009. seaweed tissue culture as applied to biotechnology: problems, achievements and prospects. phycol res 57: 45-58. bradley pm. 1991. plant hormones do have a role in controlling growth and development of algae. j phycol 27: 317-21. collantes g, melo c, candia a. 2004. micropropagation by explant of bird, mclachlan and oliveira. j appl phycol 16: 203-13. dawes cj, koch ew. 1991. branch micropropagule and tissue culture of the red alga and rezii farmed in the philippines. j appl phycol. 6: 21-4. gracilaria chilensis eucheuma denticulatum kappaphycus alva callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. 113 evan lv, trewavas aj. 1991. is algal development controlled by plant growth substances? j phycol 27 : 322-26. george ef. 1993. plant propagation by tissue culture, part 1, in practice. 2 edition. exegetics ltd. england. 573 p. hayashi l, yokoya ns, kikuchi dm, oliveira e c. 2008. callus induction and micropropagation improved by colchicines and phytoregulator in (rhodophyta, solieriaceae). j appl phycol 20: 653-59. huang w, fujita y. 1997. callus induction and thallus regeneration in some species of red algae. phycol res 45: 105-11. hurtado aq, cheney dp. 2003. propagule production of (burman) collins et harvey by tissue culture. bot mar 46: 338-41. hurtado aq, biter ab. 2007. plantlet regeneration of var. adik-adik by tissue culture. j appl phycol. 19:783-6. hurtado aq, yunque da, tibubos k, critchley at. 2009. use of acadian marine plant extract powder from in tissue culture of . j appl phycol 21:633 9. kumar gr, reddy crk, jha b. 2007. callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the indian coast. j appl phycol 19: 15-25. liao ic, su hm, lin jh. 1983. larval foods for penaeid prawn. in: mc vey jp, editor. crc handbook of mariculture volume 1: crustacean aquaculture. crc press, inc. boca raton, florida. p 29-60. lobban cs, harrison pj. 1994. seaweed ecology and physiology. cambridge university press. cambridge uk. 367 p. muñoz j, cahue-lópez ac, patiño r, robledo d. 2006. use of plant growth regulators in micropropagation of (doty) in airlift bioreactors. j appl phycol 18: 209-18. provasoli l, 1968. media and prospects for the cultivation of marine algae. : watanabe a, hattori a, editors. cultures and collections of algae. proc u.s.-japan conf. hakone, japan, september 1966. jap soc plant physiol. p 63-75. reddy crk, kumar grk, siddhanta ak, tewari a. 2003. somatic embryogenesis and regeneration of somatic embryos from pigmented callus of (doty) doty (rhodophyta, gigartinales). j phycol 39: 610-6. reddy crk, jha b, fujita y, ohno m. 2008. seaweed micropropagation techniques and their potentials: an overview. j appl phycol 20: 609-17. shao k, wang j, zhou b. 2004. production and application of s of (halymeniaceae, rhodophyta). j appl phycol 16: 431-7. suryati e, mulyaningrum srh. 2009. regenerasi rumput laut (doty) melalui induksi kalus dan embrio dengan penambahan hormon perangsang tumbuh secara . jurnal riset akuakultur 4(1): 39-45. yokoya ns, handro w. 2004. effects of plant growth regulators and culture medium on morphogenesis of (rhodophyta) cultured . j appl phycol 14: 97-102. yokoya ns, west ja, luchi ae. 2004. effect of plant growth regulator on callus formation, growth and regeneration in axenic tissue culture of and (gracilariales, rhodophyta). phycol res 52: 244-54. yokoya j phycol. 46: 1198-205. yunque d, tibubos k, hurtado aq, critchley at. 2010. optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte . j appl phycol. doi 10.1007/s10811010-9594-7. nd kappaphycus alvarezii eucheuma denticulatum kappaphycus alvarezii ascophyllum nodosum kappaphycus varieties kappaphycus alvarezii in in vitro kappaphycus alvarezii filament grateloupia turuturu kappaphycus alvarezii in vitro solieria filiformis in vitro gracilaria tenuistipitata gracilaria perplexa kappaphycus ns, stirk wa, van staden j, nova´k, o, turecˇkova´, v, peˇncˇı´k a., strnad m. 2010. endogenous cytokinins, auxins, and abscisic acid in red algae from brazil. biotropia vol. 19 no. 2, 2012 114 cellulase production by m1 using bacillus subtilis pretreated groundnut shell based liquid state fermentation ashish vyas *, chayanika putatunda , joginder singh and deepak vyas1 1 1 2 1department of microbiology, school of biosciences and biotechnology, lovely professional university, phagwara, punjab, india 2laboratory of microbial technology and plant pathology, department of botany, dr hs gour university (a central university), sagar, madhya pradesh 470003, india received 22 january 2014/accepted 21 june 2016 abstract groundnut shell which is rich in natural cellulose was assessed as a substrate for production of cellulase enzyme by cellulolytic bacteria. in the present investigation the bacterial isolate m1 was found to be capable of bacillus subtilis producing high amount of endoglucanase and exoglucanase on alkali treated groundnut shell. the effect of some nitrogen sources, amino acids and ca ions in the medium containing pretreated groundnut shell were also evaluated. ++ it was observed that 2% substrate concentration, 1 mm calcium concentration were optimum for cellulase production. ammonium nitrate was found to be the best among nitrogen sources tested. asparagine, tryptophan and methionine were found to be stimulatory for cellulase activity. : , cellulase, endoglucanase, exoglucanase, groundnut shellkeywords bacillus subtilis introduction microbial cellulases consist of three types of hydrolytic enzymes: 1. endo-(1, 4) -d-glucanase β (also known as endocellulase, carboxymethyl cellulose and endoglucanase) which makes random cleavages at the b-glycosidic linkages of cellulose; 2. exo-(1, 4) -d glucanase (also called β cellobiohydrolases and exocellulase) which hydrolyses cellobiose units from the termini of cellulose chain; and 3. -glucosidase (synonym β cellobiase ) which releases glucose from cellobiose and short chain oligosaccharides (kim . 2008). et al the cellulases form of a very important group of enzymes that find applications in a variety of industries including textile industries, paper and pulp industries, food processing industries, wine and brewery industry etc. (kuhad . 2011). et al however, the cost of enzymes poses a problem to their large scale utilization, especially for the environmentally crucial processes like production of biofuels (brijwani . 2010). so, there is a et al need to search cheaper methods for producing these enzymes. one of the possible methods for achieving this, is by using lignocellulosic wastes for the production of cellulases. lignocellulosics offer a promising solution, since they are abundantly available and are produced in large amount as agricultural by-products. organic waste from renewable forest and agricultural residues are rich source of cellulose, hemicellulose and lignin (brauns & brauns 1960) and therefore, can be possibly used in fermentation process for the production of cellulases. groundnut ( l) is an arachis hypogea important oil seed crop of india. the pod or dry pericarp contains about 25-40% shell (dey et al. 2002). the compositional analysis of groundnut shell indicates that the shell contains cellulose (65.7%), carbohydrates (21.2%), protein (7.3%), minerals (4.5%) and lipids (1.2%) (masenda 2004). groundnut shell is used as manure (rao et al. 2009), used in mushroom cultivation (jain & vyas 2002), and in production of extracellular enzymes (vyas 2005).et al. the capacity of selected to bacillus subtilis produce and secrete large quantities of biotropia vol. 23 no. 1, 2016: 28 34 doi: 10.11598/btb.201 .2 . .6 3 1 472 * c orresponding author: ashish.vyas@lpu.co.in 28 mailto:ashish.vyas@lpu.co.in extracellular enzymes has placed them among the most important industrial enzymes producers (schallmey 2004). this study reports et al. bacillus subtilis m1 as potent producer of cellulase enzymes on pretreated groundnut shell. materials and methods organism bacillus subtilis m1 was isolated from decomposing organic waste containing cellulose. bacteria was isolated by enrichment method (hans & srinivasan 1968) for which 1 g mixture of rotting cellulosic substrates was inoculated into the isolation medium consisting of mineral salts solution supplemented with 0.1% yeast extract and strip of filter paper. after 3 to 7 days of incubation at 30 c on reciprocal shaker, a patch o of a yellow pigmented material appeared at the liquid air interface on the filter paper. as soon as the pigmented material appeared, a portion of filter paper was transferred with sterile wire and inoculated into fresh medium. the process was repeated several times to enrich aerobic and mesophilic cellulose utilizing organisms. the filter paper from the enriched culture was removed, macerated in a small amount of sterile water and streaked on the plates containing carboxy methyl cellulose agar. the bacterial colony was purified and maintained on nutrient agar medium slants at 4 c.o substrate gs (groundnut shell) was collected from local suppliers in and around sagar district, madhya pradesh, india. shell (1 kg) was dipped in water (5 l) to remove any amount of soluble sugar present in the substrate and then dried at 80 c for o 36 hours in electric oven and then chopped into small pieces (krishna 1999), which were then ground in an electric grinder and sieved through 100 µm mesh sieve and kept at room temperature. pretreatment of gs powdered groundnut shell was treated separately with alkali (naoh) and designated as sample atrt (alkali treated), while acid (hcl) treated shells were labeled as sample actrt (acid treated). dried and powdered shell (100 g) was treated for 24 hours with 0.25n naoh (500 ml) (koijam 2000) and with 0.25n hcl (500 ml). et al. after treatment the powdered shell was repeatedly washed with distilled water until it reached neutral ph 7 and then dried over night at 60 c.o inoculum preparation bacterial inoculum consisted of 2 ml of 24 hours old culture grown on nutrient broth giving absorbance of 1.2 at 660 nm. fermentation medium (submerged) mandels and reese medium (mandels & reese 1957)was used for cellulase production. chemical composition of medium was proteose peptone, 1.0; (nh ) so , 1.4; kh po 2.0; nh -co-nh 4 2 4 2 4, 2 2, 0.3; mgso .7h o, 0.3; cacl , 0.3; feso .7h o, 4 2 2 4 2 0.005; mnso .h o, 0.0016; zncl , 0.0017; and 4 2 2 pretreated lignocellulosic substrate (gs), 10 g/l; 1,000 ml distilled water; ph 5.3. cellulase production the medium (25 ml) was dispensed in 150 ml conical flask and autoclaved at 15 psi for 15 minutes. the flasks were inoculated with bacterial culture and incubated at 37 1 c under stationary + o conditions. the samples (10 ml) were withdrawn aseptically after the 6 day of incubation and th supernatant was obtained after centrifugation at 9,500 rpm for 20 minutes. the supernatant obtained was used for enzymatic activity. the control consisting of untreated gs was also run simultaneously. all tests were performed in duplicate. enzyme assay endoglucanase and exoglucanase activities were measured (mandels 1974) in terms of international unit (iu), which is micromoles (µm) of glucose released/minute/ml. results and discussion cellulases are a group of industrially important enzymes and their demand is increasing day by day with the growth of enzyme industry. groundnut shell was assessed as cheap alternative lignocellulosic substrate for producing 29 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. cellulase enzymes since it is known to possess high percentage of cellulose (masenda 2004). however, the lignin present in the groundnut shell can impede the cellulase enzymes. so, delignification was performed through alkali (naoh) and acid (hcl) pretreatment. in the present investigation, the isolate m1 was found to be potent producer of cellulase enzymes on pretreated groundnut shell. it was isolated from decomposing organic waste containing cellulose as per the enrichment method described by hans and srinivasan (1968). microorganism found was gram positive, spore producing, catalase positive rod shaped bacterium. it was identified as bacillus subtilis by imtech (institute of microbial technology), chandigarh, india. under the conditions of ph 5, temperature 50 c and an incubation period of 6 days, the o bacterial isolate m1's endoglucanase b. subtilis activity was found to be 2 fold (0.314 iu/ml) and exoglucanase activity was found to be 3.3 fold (0.043 fpu/ml) in atrt gs as compared to untreated gs. in actrt gs, 1.5 fold (0.219 iu/ml) increased endoglucanase and 1.5 fold (0.020 fpu/ml) exoglucanase activity was obtained as compared with the untreated. so, the pretreatment resulted in higher enzyme production and the alkali treatment was found to be more efficient than the acid treatment (fig. 1). alkali (naoh) treatment of gs was found to be mor superior to acid (hcl) e treatment as it modifies lignocellulosic substrate by increasing the pore size and solubilizing lignin and hemicellulose. it increases the surface area of cellulose, reducing its crystallinity (ming . et al 20 ).08 figure 1 effect of alkali and acid treatments of groundnut shell on production of cellulase by m1bacillus subtilis figure 2 effect of alkali pretreatment of different concentrations on groundnut shell on cellulase production by bacillus subtilis m1 endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 30 biotropia vol. 23 no. 1, 2016 control alkali treated (0.25n naoh) acid treated (0.25n hcl) control 0.25n naoh 0.50n naoh 1n naoh 2n naoh pretreatment of powdered gs at different alkali concentrations (0.25n, 0.5n, 1n, 2n naoh) showed that m1 produced the b. subtilis highest endoglucanase (0.381 iu/ml) and exoglucanase (0.087 fpu/ml) activities in case of 1n naoh treated gs. endoglucanase activity was increased by 2.7 fold and exoglucanase activity by 6.9 fold higher compared to untreated gs (fig. 2). the increased cellulase activity at 1n naoh concentration might be due to increased solubilization of lignin and increase in swelling of cellulose i to cellulose ii. aguiar (2001) also r e p o r t e d 1 n n a o h p r e t r e a t m e n t o f lignocellulosic substrates as best source for cellulase production. chemical pretreatment of lignocellulosic material followed by its hydrolysis suggested that accessible surface area is a key determinant for enhancing cellulase yield. however, sarkar and aikat (2012) reported the highest activity of cellulases by aspergillus fumigatus nitdgpka3 on rice straw pretreated with 0.5 m naoh. the amount of nutrients present in the production medium is one of the major factors impacting the microbial enzyme production. the effect of five nitrogen sources (ammonium sulfate, ammonium nitrate, potassium nitrate, peptone, urea) were tested separately (fig. 3). each nitrogen source was added in equivalent amount to the total nitrogen present in the medium (587 mg nitrogen/l medium) keeping the available nitrogen constant. ammonium nitrate was found to be the best for endoglucanase activity (0.507 iu/ml) and exoglucanase activity (0.091 fpu/ml) followed by ammonium , sulfate potassium nitrate, peptone and urea. nitrogen is the major constituent of protoplasm and building block of enzymes (proteins). sethi (2013) et al. also reported ammonium salt to be excellent source of nitrogen for cellulase production and concluded that this may be due to their direct entry of ammonium ion in protein synthesis. similarly, amino acids are nitrogen containing organic compound and therefore, have influence on cellulase synthesis, but the specific side groups present on the amino acids can also play some role in cellulase synthesis. among various amino acids added to the medium (0.2 w/v), tryphtophan (0.675 iu/ml), asparagine (0.629 iu/ml) and methionine (0.564 iu/ml) were found to be the better suited for endoglucanase activity (fig. 4). on the other hand, alanine, arginine and threonine were found to be suppressive in action. b. subtilis m1 showed maximum exoglucanase activity in the presence of asparagine (0.126 fpu/ml) followed by metheonine (0.108 fpu/ml) and tryptophan (0.095 fpu/ml). amino acids, being the building blocks of protein, have a profound influence in the cellulase synthesis of bacteria. the increased production might be due to the enhanced synthesis of cellulolytic enzymes in the presence of amino acids. asparagine and metheonine were found to be stimulatory for cellulase activity in av49 by vyas . aspergillus terreus et al (2005). figure 3 effect of nitrogen sources on cellulase production on alkali treated groundnut shell by m1bacillus subtilis endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 31 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. control ammonium sulfate ammonium nitrate potassium nitrate peptone urea the activity and stability of various enzymes are known to be impacted by various ions. different concentrations of calcium (0.5-5 mm) had differential impact on the enzymatic activity (fig. 5). low (1 mm) concentration produced maximum endoglucanase activity (0.564 iu/ml). however, at higher concentration (2-5 mm) endoglucanase activity was almost static, yet remained higher than control. exoglucanase activity was maximum (0.085 fpu/ml) at 1 mm concentration and thereafter, it decreased up to 5 mm. metal ions are generally cellulase stimulators at low concentration, whereas inhibitory at very high concentration. ca was found to positively ++ i n f l u e n c e t h e a c t iv i t y of c e l l u l as e at concentrations less than 1 mmol/l by wang et al. (2012). in the present study, ca (1 mm) was ++ found to be stimulatory and higher concentration showed static endoand exoglucanase activities by . ca (10 mm) is reported (kim b. subtilis et al.++ 20 01) t o be s t imula tor y f or c el lul as e (exoglucanase) activity in trichoderma reesei. strong affinity and tightness of the enzymes would disrupt the hydrogen bonding network in the crystal lattice, ultimately collapsing the ordered structure of cellulose particles and preventing adhesion. kotchoni (2003), et al. however, reported enhanced cellulase activity at low calcium concentration (1 mm) in bacillus pumilus bpcri6. the effect of substrate concentration is also known to impact the enzyme activity. in the present investigation it was observed that with figure 4 effect of different amino acids on cellulase production on alkali treated groundnut shell by m1bacillus subtilis figure 5 effect of calcium concentration on cellulase production on alkali treated groundnut shell by bacillus subtilis m1 endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) control alanine methionine threonine asparagine arginine tryptophan 32 biotropia vol. 23 no. 1, 2016 0.5 mm ca 1 mm ca 2 mm ca 3 mm ca 4 mm ca 5 mm ca i nc r e as e d su b st r at e c o nc e n tr a ti on , th e endoglucanase activity showed increasing trend up to 2% substrate concentration (0.517 iu/ml), thereafter, no further increase in activity was observed (fig. 6). maximum exoglucanase activity was obser ved at 1% substrate concentration (0.090 fpu/ml). okonkwo (2014) also observed maximum activity at 1% substrate concentration in case of cellulase produced by aspergillus flavus. conclusions the present findings raise the possibility of using groundnut shell as a substrate for production of cellulase enzymes by bacillus subtilis m1. the cultural conditions as well as the type of pretreatment also seem to have profound impact on the enzyme activity. there is a need to assess the production at pilot plant scale, so that it can be used subsequently at the commercial scale in the long run. acknowledgements the first author thanked the head of botany department, dr hs gour university for providing necessary laboratory facilities during research tenure. thanks are also due to prof m.k. bhat, institute of food research, colney, norwich, england for providing relevant literature references aguiar cl. 2001. biodegradation of the cellulose from sugarcane bagasse by fungal cellulase. cienc tecnol aliment 3:117-21. brauns fe, brauns da. 1960. the chemistry of lignin covering the literature for the 1949-1958. san diego (usa): academic press san diego. brijwani k, oberoi hs, vadlani pv. 2010. production of a cellulolytic enzyme system in mixed-culture solidstate fermentation of soybean hulls supplemented with wheat bran. process biochem 45:120–8. dey r, pal kk, chauhan sm, bhatt dm, misra jb. 2002. groundnut shell decomposition potential of some cellulolytic microorganisms. ind j microbiol 42: 165-7. hans yw, srinivasan vr. 1968. isolation and characterization of cellulose utilizing bacterium. app microbiol 16:1140-5. jain ak, vyas d. 2002. yield response of on pleurotus florida wheat straw in combination with other substrates. mushroom res 11:19-20. kim dw, jang yh, kim cs, lee ns. 2001. effect of metals ions on the degradation and adsorption of two cellobiohydrolases on microcrystalline cellulose. bull korean chem soc 22:716-20. kim sj, lee cm, han br, kim my, yeo ys, yoon sh, koo bs, jun hk. 2008. characterization of a gene encoding cellulase from uncultured soil bacteria. fems microbiol lett 282:45-51. koijam b, sharma nc, gupta s. 2000. production and characterization of fungal cellulase from cellulosic waste. asian j microbiol biotechnol environ sci 4:113-20. kotchoni os, shonukan oo, gachomo we. 2003. bacillus pumilus bpcri 6, a promising candidate for cellulase production under condition of catabolite repression. afr j biotechnol 2:140-6. figure 6 effect of substrate concentration on cellulase production on alkali treated groundnut shell by m1bacillus subtilis endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 33 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. 1% gs (w/v) 2% gs (w/v) 3% gs (w/v) 4% gs (w/v) 5% gs (w/v) krishna c. 1999. production of bacterial cellulases by solid state bioprocessing of banana wastes. biores technol 69:231-9. kuhad rc, gupta r, singh a. 2011. microbial cellulases and their industrial applications. enzyme research [internet]. [cited 2015]; article id 280696. available from: http://www.hindawi.com/journals/er/ 2011/280696/ doi:10.4061/2011/280696. mandels m, reese et. 1957. induction of cellulases in fungi in as influenced by carbon source. j trichoderma viride bacteriol 37:269-78. mandels m. [internet]. 1974. laboratory procedures, hand book. usa: us army natick laboratories. available from: http://calvin.biotech.wisc.edu/ jeffries/cellulases/mandels.html. masenda e. [internet]. 2004. groundnut shells, substrate. mushroom growers handbook i. south korea: mu sh wo rl d. p 1 20 12 2 . avai l ab l e fr om : www.mushworld.com/service/handbook/2004/ch apter-5-8.pdf. ming c, jing z, liming x. 2008. enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. carbohydr polym 71:411–5. okonkwo if. 2014. effect of substrate concentration on the activity of cellulase produced by . aspergillus flavus ind j app res 7: 32-4. rao cs, wani sp, sahrawat kl, rajasekharao b. 2009. nutrient management strategies in participatory watersheds in semi arid tropical india. ind j fert 5(12):113-28. sarkar n, aikat k. 2012. alkali pretreatment of rice straw and enhanced cellulase production by a locally isolated fungus nitdgpka3. aspergillus fumigatus j microbiol biotech res 2(5):717-26. schallmey m, singh a, ward op. 2004. developments in the use of species for industrial production.bacillus canadian j microbio 50(1):1-17. sethi s, datta a, gupta bl, gupta s. 2013. optimization of cellulase production from bacteria isolated from soil. isrn biotechnology [internet]. 2013:985685. available from: http://www.hindawi.com/ journals/isrn/2013/985685/ doi: 10.5402/2013/ 985685. vyas a, vyas d, vyas km. 2005. production and optimization of cellulases on pretreated groundnut shell by av49. j sci ind aspergillus terreus res 64:281-6. wang g, zhang x, wang li, wang k, peng f, wang l. 2012. the activity and kinetic properties of cellulases in substrates containing metal ions and acid radicals. adv biol chem 2:390-5. 34 biotropia vol. 23 no. 1, 2016 http://www.hindawi.com/journals/er/ http://calvin.biotech.wisc.edu/ http://www.mushworld.com/service/handbook/2004/ch http://www.hindawi.com/ doi: 10.11598/btb.2015.22.2.422 rapd analysis to detect somaclonal variation of pineapple cultures during in vitro micropropagation ika roostika , nurul khumaida and sintho wahyuning ardie1* 2 2 1indonesian center for agricultural biotechnology and genetic resources research and development (icabiograd) bogor 16111, indonesia 2department of agronomy and horticulture, institut pertanian bogor, bogor 16680, indonesia received 7 september 2014/ accepted 20 januari 2015 abstract plant off-type becomes a concern in pineapple micropropagation. reliable methods are needed to detect and to reduce plant off-type. this research was conducted to confirm the occurrence of somaclonal variation during micropropagation of pineapple clone simadu. the culture period (longand short-period) and the regeneration methods (direct organogenesis, indirect organogenesis and somatic embryogenesis) were studied to know their contribution in bringing out somaclonal variation. rapd analysis using 10 primers was performed to confirm genetic variation. the result showed that rapd assay could be applied as early detection of somaclonal variation of pineapple, where opa primers were better to be used than opj primers. the phenotypic variation occurred in the four year-old pineapple plants and plantlets were due to genetic variation. it has proved that the long-period of cultures is the main contributor of somaclonal variation, while the regeneration method and plant growth regulator could also induce genetic variation. the new cultures showed higher level of similarity. therefore, it needs a correct strategy to apply micropropagation method of pineapple to minimize plant off-type. it was recommended to avoid the use of long-period culture as mother stock and to apply direct organogenesis method for micropropagation rather than indirect organogenesis and somatic embryogenesis. : keywords ananas comosus (l.) merr., molecular markers, plant off-type introduction conventionally, pineapple can be propagated from various plant propagules, namely crown, sucker, butt or stump, hapas, ratoons and slip. however, their reproductive time is not uniform (international board for plant genetic resources 1991; coppens d'eeckenbrugge & leal 2003). conventional propagation techniques were not appropriate for rapid mass propagation, such as in cultivar smooth cayenne, where availability of vegetative propagules are commonly limited both in the type (only crown and sucker) and the number. culture techniques may be able to in vitro solve the problem, since it can provide alternative methods for mass propagation plants with higher uniformity and at shorter times. according to smith (2003), micropropagation has been et al. used for establishment of multiplication blocks, which then provide conventional planting material for larger production blocks, because the micropropagated plants are more expensive and the growers concern about the genetic off-type. it was reported that variation may be resulted from the pre-existing variation of the explants (wakasa 1979; nwauzoma & jaja 2013), the et al. long period of the culture (koornneef 1991; masoud & hamta 2008), the high level of plant growth regulator (bairu 2006), the frequent et al. subculture (eeuwens 2002), the genotype et al. dependence (zucchi . 2002), activation of et al transposable elements (bairu 2011), and et al. hypoor hypermethylation of dna (abdellatif et al. 2012). to avoid the high level of variation and to streamline the efficiency of micropropagation method, it needs a strategy to produce uniform * corresponding author : ikatambunan@yahoo.com biotropia 2 109 119 vol. 22 no. , 2015: 109 mailto:ikatambunan@yahoo.com 110 genetic identity in the seedlings. therefore, variations in the mother stocks and regenerants need to be characterized. based on the morphological characterization of cultures in vitro and phenotypic evaluation of the seedlings in the previous research, it was suggested that those variations were caused by somaclonal variation (roostika 2012a). in order to confirm this suggestion, molecular analysis needs to be conducted. molecular markers are widely used to detect somaclonal variation and genetic fidelity during micropropagation (tawar 2008; rout et al. et al. 2009; khoddamzadeh 2010; shahid . et al. et al 2014). randomly amplified polymorphism dna (rapd) is applied because it is useful for analysis of variation at many loci requiring small quantities of dna, but not prior knowledge of dna sequence nor involvement of radioactivity (williams 1990). in the case of pineapple, et al. rapd analysis has been performed to detect genetic fidelity and variants of vara. comosus queen (soneji 2002), var et al. a. comosus amarelinho (feuser 2003), var et al. a. comosus bracteatus et al.(santos 2008) and the promising clone golden pineapple (suminar 2010). the objectives of this study were: (1) to confirm the occurrence of somaclonal variation by rapd method; (2) to know the level of variation of regenerants derived from four regeneration methods (axillaries shoot proliferation, direct organogenesis, indirect organogenesis, and somatic embryogenesis); and (3) to detect the variation and to reduce the level of variation. materials and methods plant materials the plant materials were four-year-old pineapple cultures of simadu (smooth cayenne) from subang, west java, indonesia. there were three populations used in this research. preparation of population i the cultures were established from axillary bud explants, excised from the crown of pineapple plants grown in the field. the explants were sterilized using 70% ethanol and 10.5% sodium hypochlorite. the basal medium was ms salt (murashige & skoog 1962), 3% (w/v) sucrose, 0.8% (w/v) agar, 100 mg/l myo-inositol 0.1 , mg/l thiamine-hcl, 0.5 mg/l pyridoxine-hcl, 0.5 mg/l nicotinic acid with addition of 8.84 μm benzyl adenine (ba) and 9.3 kinetin (kin). μm prior to autoclaving the medium, its ph was adjusted to 5.7±0.1. the cultures were illuminated 16 hours per day with 20 μmol p h o t o n s / m / s i r r a d i a n c e p r o v i d e d b y 2 fluorescence lights in a culture room at 25±2 c. 0 after a four-year period and more than 10 in vitro times subculture, the cultures were maintained on ms basal medium with addition of 2.21 m ba μ and 4.65 m kin. the root induction was μ conducted by planting the shoots onto the ms medium containing of 15 indole butyric acid μm (iba) and 5.37 naphthalene acetic acid μm (naa) for 3 weeks. preparation of population ii the cultures that grew normally were selected. six hundred and fifty leaf bases were isolated as sources of explants that comprised approximately 1 cm of basal portion of the leaves. the explants were regenerated through direct organogenesis (roostika 2012a) and indirect et al. organogenesis regeneration (roostika et al. 2012b). the other cultures were prepared through indirect somatic embryogenesis as described by roostika . (2012c). the root et al induction medium was the same as mentioned above. preparation of population iii population iii consisted of plants collected from field (clone simadu and non simadu), the new cultures of both clones derived from shoot pr oli fe r atio n a nd d ir ec t or g anog e ne s is regeneration. the axillary buds of crown were sterilized and the shoots were initiated on in vitro ms media supplemented with 8.84 ba and μm 9.3 kin for shoot proliferation. after 6 μm months of culture period without subculture, they were maintained on ms media with addition of 2.21 μm ba and 4.65 μm kin prior to rapd analysis. rapd assay to detect somaclonal variation the leaves of acclimated variants were used as the samples in population i. about 10 shoots of cultures were pooled to make bulk samples in population ii and iii. rapd assay was performed biotropia vol. 22 no. 2, 2015 rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro using a modification method of soneji (2002). et al. the dna samples were isolated using cetyl trimethyl ammonium bromide (ctab). subsequently, they were amplified by polymerase chain reaction (pcr) machine. each reaction mixture contained 1 µl 25 ng/l genomic dna, 1 µl primer as listed at table 1 (operon tech. alameda, usa) at 10 ng/µl, 11 µl ddh o and 12 2 µl pcr mix, in a 25 µl total reaction. the pcr consisted of pre-denaturation at 94 c for 4 0 minutes, 40 cycles at 94 c for 30 seconds 0 (denaturation), 36 c for 1 minute (annealing) and 0 1 minute at 72 c (extension), post-extension at 0 72 c for 5 minutes and a final extension at 40 c 0 0 for 4 minutes. the pcr products were resolved on 1.2% agarose (promega) and electrophoresed at a constant voltage of 50 v for 50 minutes and then the bands were stained with ethidium bromide (etbr) and visualized under a uv transiluminator. a 1 kb dna ladder was used as a molecular standard. the samples were scored based on presence (coded as 1) or absence (coded as 0) of the same size bands. to select the primers, the mother plant dna and one variant ( ) needle leaf were used. the primers that showed polymorphic bands and/or scorable bands were selected. the scored data (binary data) were analyzed using the simqual, sahn and tree programs from the numerical taxonomy and multivariate analysis system version 2.02 (ntsys-pc 2.02). the similarity degrees were calculated according to the dice coefficient. groupings were carried out using the unweighted pair group method and arithmetic average (upgma) cluster analysis. results and discussion genetic variations occurred in the longperiod cultures of pineapplein vitro in order to determine the source of phe noty pic variations occ ur r ed in the micropropagated cultures and plants, molecular analysis using rapd markers was conducted. plants with normal phenotype and exhibited several phenotypic variations (dwarf posture, albino stripe leaf, narrow leaf, spines spot leaf, erect posture, wider leaf, powdery leaf, climb posture, needle leaf and ultra dwarf posture) were subjected to rapd analysis using 10 primers. opa and opj have been used to characterize the variation in orchid (chen 1998), phalaenopsis et al. a. comosus et al. var. queen (soneji 2002) and var. bracteatus et al. (santos 2008). the result showed that the primers of opa were better than opj in generating amplified bands. the two primers generated the highest number of amplified bands, namely opa13 and opa16, whereas the primer of opj11 produced the lowest number of amplified bands. the result showed that the phenotypic variation occurred in the population i (the four-year-old pineapple cultures were due to genetic variation as the rapd method revealed a high polymorphism of dna bands (fig. 1). a total of 73 bands were amplified, 70 (95.9%) of which were polymorphic (table 2), indicated the high level of variation. the ntsys analysis yielded a dendrogram where the similarity coefficient ranged from 0.32 to 0.91 with high rohlf matrix correlation value (r=0.97, very 111 table 1 the list of primers and their sequences used in the rapd assay no primer sequence 3’ to 5’ 1 opa2 tgccgagctg 2 opa3 agtcagccac 3 opa7 gaaacgggtg 4 opa9 gggtaacgcc 5 opa13 cagcacccac 6 opa16 agccagcgaa 7 opa18 aggtgaccgt 8 opa19 caaacgtcgg 9 opj11 actcctgcga 10 opj13 ccacactacc biotropia vol. 22 no. 2, 2015 112 suitable). it separated the variants at the similarity coefficient of 0.32-0.61 to the control plant (fig. 2). there were 82.3% plants with altered phenotype in the population i (roostika et al. 2012a) and all of those variants were genetically different from the control plants (plants with normal phenotype) based on rapd analysis. this present study clearly showed that the use of long-period cultures open a high risk of somaclonal variation for pineapple micropropagation. regeneration method affects the somaclonal variation of pineapple based on table 3, it seems that the opa primers generated higher number of amplified bands than those produced by opj primers. the highest amplified bands were yielded from opa2 and opa3, whereas opj primers generated the lowest amplified bands. there was also a high polymorphism obtained from population ii (fig. 3; table 3). from a total 65 amplified products, 60 bands (92.3%) of which were polymorphic (table 3). the ntsys analysis yielded a dendrogram where similarity coefficient ranged from 0.34-0.99 (r=0.98). it separated the variants at the similarity coefficient of 0.34-0.65 to the control plant (fig. 4). the percentage of polymorphism was only slightly lower than population i. this result showed that the regeneration of normal phenotype-selected cultures could only reduce a small variation. it seems that chimera might have been existed in those explants sources (population i). figure 4 also showed that picloram-derived cultures had the lowest similarity (0.34). picloram is an auxinic herbicide belong to the pyridine group and commonly used at low concentration as an inducer for callus formation during somatic embryogenesis regeneration (firoozabady & moy 2004; ahmed et al. et al. 2011; noormi 2012). beside regenerated through callus formation phase, it needs a longer time to conduct somatic embryogenesis, so that the probability of genetic changes would increase. callus is an undifferentiated structure and actively doing cell division. according to miguel and marum (2011), tissue culture systems that involve acquisition of competence for extensive cell division are usually regarded as more risky of genome instability. rani and raina (2000) reported that there was a genetic instability of seedlings derived from micropropagation via callus formation. a high polymorphism (35/84 bands) was also reported by soniya (2001) on et al. tomato, cultured by picloram containing media. s e ve ral p re vi ous stu d ie s r e p or te d t he contribution of plant growth regulator in somaclonal variation, but they put less consideration in period of cultures (feuser et al. 2003; santos 2008). our study showed that et al. the long-period of cultures is the main contributor of somaclonal variation, the regeneration method and plant growth regulator could also induce genetic variation in the in vitro culture of pineapple. table 2 the number of amplified product and polymorphic bands in population i, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 8 8 2 opa3 7 6 3 opa7 8 8 4 opa9 7 6 5 opa13 10 10 6 opa16 13 13 7 opa18 8 8 8 opa19 6 6 9 opj11 2 1 10 opj13 4 4 total number of bands 73 70 percentage (%) 100 95.9 note: population i was derived from the four-year cultures regenerated through shoot proliferation methodin vitro 113 figure 1 profiles of dna variants of pineapple cultures in population i (regenerated through shoot proliferation method), amplified by pcr using 10 rapd markers. m = kb ladder; n = control plant from field; v13 = dwarf posture; v10 = albino stripe leaf; v8 = narrow leaf; v7 = spiness spot leaf; v21 = erect posture; v12 = wider leaf; v14 = powdery leaf; v4 = climb posture; v2 = needle leaf; and v15 = ultra dwarf posture rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro opa2 opa3 opa9opa7 opa13 opa16 opa18 opa19 opj11 opj13 114 biotropia vol. 22 no. 2, 2015 figure 2 variability of variants derived from four year-old cultures of pineapple cultivar smooth cayenne regenerated through shoot proliferation method (population i), performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.97). n = control plant from field; v13 = dwarf posture; v10 = albino stripe leaf; v8 = narrow leaf; v7 = spiness spot leaf; v21 = erect posture; v12 = wider leaf; v14 = powdery leaf; v4 = climb posture; v2 = needle leaf; and v15 = ultra dwarf posture coefficient of similarity 0.32 0.47 0.62 0.77 0.91 n v13 v10 v8 v7 v4 v15 v21 v2 v12 v14 figure 3 profiles of dna variants of pineapple cultures in population ii, amplified by pcr using 10 rapd markers. the arrows show polymorphic bands. kb = kb ladder; 1 = control plant; 2 = cultures derived from direct organogenesis; 3 = cultures derived from indirect organogenesis induced by 2,4-d 21 µm; 4 = cultures derived from indirect organogenesis induced by 2,4-d 41 µm; 5 = cultures derived from indirect organogenesis induced by 2,4-d 62 µm; 6 = cultures derived from somatic embryogenesis induced by 2,4-d; 7 = cultures derived from somatic embryogenesis induced by picloram 115 shorter period of culture could suppress the somaclonal variation of pineapple in order to further confirm our finding that culture period is the main contributor of genetic variation occurred in the culture of in vitro pineapple, the new population (population iii) was generated through shoot proliferation and direct organogenesis regeneration methods. the use of opa primers was also better than opj. it can be concluded that opa primers were more suitable for detecting pineapple off-type than opj primers. interestingly, the new population revealed a higher level of similarity. the level of polymorphism in the population iii was lower than that in population i (fig. 5; table 4). the ntsys analysis yielded a dendrogram where similarity coefficient ranged from 0.65-1.0. it separated the regenerants at the similarity coefficient of 0.65-0.85 (r=0.96) to the control plant. it showed that the grouping is shifting to right, indicated the increase of similarity degree. it can be concluded that the use of the new population was better than the old population. table 3 the number of amplified product and polymorphic bands of population ii, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 9 9 2 opa3 9 9 3 opa7 5 5 4 opa9 8 8 5 opa13 6 6 6 opa16 5 5 7 opa18 8 8 8 opa19 5 3 9 opj11 5 4 10 opj13 5 3 total number of bands 65 60 percentage (%) 100 92.3 note: population ii was derived from the normal phenotype-selected plantlets of four-year cultures, regenerated through direct in vitro organogenesis, indirect organogenesis and somatic embryogenesis figure 4 variability of cultures of pineapple cultivar smooth cayenne in population ii, performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.98). n = control plant; bn = cultures derived from direct organogenesis; d21 = cultures derived from indirect organogenesis induced by 21 µm 2,4-d; d41 = cultures derived from indirect organogenesis induced by 41 µm 2,4-d; d62 = cultures derived from indirect organogenesis induced by 62 µm 2,4-d; esd = cultures derived from somatic embryogenesis induced by 2,4-d; esp = cultures derived from somatic embryogenesis induced by picloram coefficient of similarity 0.34 0.50 0.66 0.82 0.99 s bn d21 d41 d62 esd esp rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro table 4 the number of amplified product and polymorphic bands of population iii, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 4 4 2 opa3 3 2 3 opa7 5 2 4 opa9 4 3 5 opa13 5 1 6 opa16 6 6 7 opa18 6 3 8 opa19 8 8 9 opj11 3 3 10 opj13 4 4 total number of bands 48 36 percentage (%) 100 75 note: population iii was derived from the new cultures, regenerated by shoot proliferation and direct organogenesis in vitro method figure 5 profiles of variants dna of pineapple cultures in population iii, amplified by pcr using 10 rapd markers. kb = kb ladder; (1) clone simadu from field; (2) clone non-simadu from field; (3) normal phenotype plant derived from shoot proliferation method; (4) cultures of clone simadu derived from shoot proliferation; (5) cultures of clone nonsimadu derived from shoot proliferation; (6) cultures of clone simadu derived from direct organogenesis; and (7) cultures of clone non-simadu derived from direct organogenesis. the arrows showed several polymorphic bands biotropia vol. 22 no. 2, 2015 116 figure 6 variability of cultures of pineapple cultivar smooth cayenne in the population iii, performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.96). s = clone simadu from field; ns = clone non-simadu from field; ni = normal phenotype plant derived from shoot proliferation method; ps = cultures of clone simadu derived from shoot proliferation; pns = cultures of clone non-simadu derived from shoot proliferation; os = cultures of clone simadu derived from direct organogenesis; and ons = cultures of clone non-simadu derived from direct organogenesis coefficient of similarity 0.32 0.49 0.66 0.83 1.00 s ni ns pns ps os ons 117 monitoring the identity of cultures is important to establish the stability of a particular trait (soneji . 2002). our result showed that the et al molecular pattern of the normal phenotype plant derived from shoot proliferation method was identical with the control plant of clone simadu from field. therefore, phenotypic screening in the individual cultures is considered to be applied as early detection of plant off-type during micropropagation. rapd assay can also be applied as early detection of somaclonal variation in the population. according to bairu (2011), et al. molecular technique enables detection of variants at the juvenile stage. furthermore, getting rid of variants also could be performed in nurseries to avoid the occurrence of off-type in mass seedlings production (mohamed 2007). generally, it can be concluded that the longperiod of cultures is the main contributor of somaclonal variation of pineapple, while the regeneration method and plant growth regulator could induce genetic variation. according to leva et al. (2012), the system by which the regeneration is induced, type of tissue, explant source, media components and the duration of the culture cycle are some of the factors that are involved in inducing variation during culture. in vitro conclusions rapd assay could detect genetic changes in micropropagated pineapple so that it can be used as early detection of off-type plant. the use opa primers were better than opj primers. the longperiod of pineapple cultures was the main contributor of somaclonal variation. the use of direct organogenesis method was more recommended than the application of the indirect organogenesis and somatic embryogenesis since it does not undergo callus formation. acknowledgements the authors acknowledged the financial support by indonesia agency for agricultural research and development, ministry of agriculture of the republic of indonesia, through kkp3n program 2011. the authors also rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro thanked prof dr g.a. wattimena and prof dr ika mariska for their supervision and critical suggestions. references abdellatif kf, hegazy ae, aboshama hm, emara ha, elshahed aa. 2012. morphological and molecular characterization of somaclonal variations in tissue culture-derived banana plants. j genet eng biotechnol 10:47–53. ahmed aba, rao as, rao mv, taha m. 2011. effect of picloram, additives and plant growth regulators on somatic embryogenesis of (l.) phyla nodiflora greene. braz arch biol techn 63: 147-73. b the effect of airu mw, fennell cw, staden j van. 2006. plant growth regulators on somaclonal variation in cavendish banana ( aaa cv. 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of pineapple cultures roostika . – et alin vitro 4. decky (exotic).cdr biotropia vol. 21 no. 1, 2014: 38 52 exotic plants in halimun salak corridor:micro-environment, detection and risk analysis of invasive plants decky indrawan junaedi , dodo received 18 june 2013/accepted 2 june 2014 research of exotic plant species detection and risk analysis of invasive plant was conducted in halimun-salak corridor area. this study aimed to conduct inventory of exotic plant species in this area and to perform risk analysis of invasive to the exotic plants found. the invasion risk assessment of detected exotic plants was analyzed using weed risk assessment procedure (wrap) method. moreover, analysis of multi-dimensional scaling (mds) based on inequality was performed on relative humidity, light intensity, and soil ph. there are eleven exotic plant species consisted of three tree species and eight species of herbs/shrubs. and are the exotic species with the highest and the lowest wrap score, respectively. mds analysis shows that exotic tree species have similar environmental variables. moreover, environmental variables of are relatively different from other exotic species found in the halimun-salak corridor. recommendations for the management of invasive exotic plant species in the area are: immediate management implementation, priority of eradication to exotic plants that have not been abundant but have high risk score, two management options (gradual eradication or containment) should be considered for exotic plants with very high wrap score, such as and . plant ecology, halimun-salak corridor, multi-dimensional scaling, weed risk assessment, invasive plant, conservation 1 2 1 2 cibodas botanical garden, indonesian institute of sciences (lipi), po. box 19 sdl sindanglaya, cipanas, cianjur 43253, centre for plant conservation, bogor botanical garden, indonesian institute of sciences (lipi) austroeupatorium inulifolium camellia sinensis clidemia hirta ageratina riparia, austroeupatorium inulifolium chromolaena odorata abstract key words: * corresponding author : deqee82@gmail.com doi: 10.11598/btb.2014.21.1.4 38 introduction halimun-salak corridor ecosystem is important because it is not only a mountain forest ecosystem, but also acts as a connecting corridor between mount salak and mount halimun. moreover, halimun-salak corridor has several risk factors associated with the threat to ecosystem integrity. along with habitat degradation, fragmentation and disturbance from human activities, invasive alien species (ias) (including invasive alien plants) being one of the factors that poses a threat to the integrity of the halimun-salak corridor ecosystem (jica 2005). within an 11 years period (19902001), the width of the halimun-salak corridor has decreased from 1.4 km (1990) to 0.7 km (2001) (cahyadi 2003). on the other hand, there are lack of studies on invasive species in south east asia, where the biodiversity hotspot lies in (peh 2010). considering invasive species issues, detection and risk analysis of invasive plants are needed in the management of invasive plants. upon the detection, alien species which potentially become invasive could be detected and managed earlier so that management cost becomes cheaper. by doing risk assessment, existed exotic plants, either invasive or not invasive can be estimated and ranked due to its invasiveness rates or invasiveness possibilities. therefore, management prioritization of exotic invasive plants will be accomplished easier with risk assessment. risk assessment reflects the chances of an exotic species to become invasive in the future or reflects the rate of invasiveness itself. weed risk assessment procedure (wrap) (virtue 2008) is a risk assessment system used to assess the risk of invasive plant species from an exotic species that are already existed in a new host area. the objective of this study was to conduct inventory of exotic plants (either invasive or not invasive (yet)) that are already existed in the halimun-salak corridor. this study also aimed to look at the risk of exotic plants to become invasive in the halimun-salak corridor. in addition, this study also aimed to analyze the diversity of habitat environment (micro-environment) variables (ph, light intensity and air humidity) of areas where exotic species were detected. halimun-salak corridor is the area that connects mount salak and mount halimun. halimun-salak corridor included as a part of halimun-salak national park based on minister of forestry decree no. 175/kpts-ii/2003 dated june 10, 2003. northern part of the corridor is part of administrative region of bogor covering area as large as 1662.72 hectares. southern part of the corridor is included in the district of sukabumi which covered 2533 hectares. the altitude range of halimun-salak corridor is from 1000 m above sea level to 1400 m above sea level. annual rainfall in the halimun-salak corridor ranges from 4000 mm to 5000 mm per year (jica 2005). et al. materials and methods study site 39 exotic plants in halimun salak corridor decky indrawan junaedi– et al. exotic plants detection exotic species inventory was conducted at 27 observation points in 7 different locations in the halimun-salak corridor. sampling areas covered by the detection sampling are: bivak, legok buluh, cigorowek-3, palahlar, pasir panjang, pasir bedil and kubang (table 1). tabel 1. detection sampling location in halimun-salak corridor location coordinates altitude (m asl) sampling number ar/fi s e bivak 06044'59.8" 106036'47.9" 1100 6 ar and fi legok buluh 06044'53.0" 106037'07.0" 1068 6 ar and fi cigorowek-3 06044'42.1" 106038'01.3" 1015 4 ar and fi palahlar 06044'53.1" 106037'22.6" 1021 2 ar pasir panjang 06045'11.2" 106037'12.6" 1061 3 ar and fi pasir bedil 06045'06.9" 106036'54.6" 1069 2 ar kubang 06045'17.6" 106037'52.2" 1042 4 ar and fi notes: ar= adjacent to footpath, fi= forest interior detection samplings were conducted in two types of locations. the first location type is the area adjacent to forest access road into the woods with the assumption that the exotic species introduction pathway is via humans (carried away to the forest by humans). the second location type is the area inside the forest interior with the assumption that in addition to human pathways, exotic species can also be dispersed by the help of animals, wind and other vectors. at each sampling point, exploratory observation of the exotic plant species was conducted within a radius of 3 meters from the point of observation. exotic plant species were observed and recorded. invasiveness risk assessment exotic plants that have been detected were then assessed by using australia weed risk assessment procedure (wrap) (virtue 2008) to assess the risk of invasiveness. wrap is a framework used to analyze the risk of an invasive exotic plant that are already existed in an invaded area. wrap consists of questions that should be answered and then scoring was performed based on the answers. the scoring decision was conducted fully based on available scientific information provided from online global invasive plant database such as pacific island ecosystem at risk (pier) and invasive species specialist group (issg), iucn database. total accumulated value of all wrap scoring answer was expressed as a risk score. the risk scores are quantitative and can be compared between one species and another in the analysis. wrap used in this study were adapted and modified to match the conditions and circumstances in et al. biotropia vol. 21 no. 1, 2014 40 the study locations as provided in annex 1. accuracy of the risk assessment results is measured using logistic regression analysis to generate the roc curve (receiver operating characteristic, using mysystat 13) that describes how accurate risk analysis model used (quinn & keough 2002). logistic regression follows the equation: e / (1 + e )...........................................................................................................1) whe p score) ants obtained (real data) was defined as the relative frequency (fr) more than 10%. on every detection sampling plot, ecological (micro-environment) data were recorded for analysis of ecological habitats. habitat ecological data recorded include: air relative humidity, light intensity, and soil ph. analysis of multi-dimensional scaling (mds) was based on inequality using xlstat software (microsoft excel add-in) and was conducted based n micro-environment data. mds analysis was used to examine the degree of differences among detected exotic species based on its microenvironment/ micro-habitat variables measured. based on the sampling detection of exotic plants in 27 locations of the study site, species area curve is presented in figure 1. the curve indicates the relation between the numbers of sampling plots which was taken with the addition of new exotic species detected. π (x) = re: g (x) = β0 + β1 (wra and π (x) is the opportunity of exotic species (i) will become invasive. definition of invasive exotic pl g (x) g (x) i micro-environment results and discussion detected exotic plants in the halimun-salak corridor based on modified wrap scoring results are presented in table 2. figure 1. species-sampling relation curve that describes the relation between the number of sampling (x axis) and new exotic species counted (y axis) 41 exotic plants in halimun salak corridor decky indrawan junaedi– et al. t ab le 2 . d et ec te d ex o ti c p la n t sp ec ie s in h al im u n -s al ak co rr id o r, o cc u rr en ce fr eq u en cy in al lt o ta ls am p li n g s an d ri sk as se ss m en t sc o re b as ed o n m o d if ie d w ee d r is k n o s p ec ie s n am e a u th o r o cc u re n ce f re q u en cy f k f r r el at iv e o cc u r re n ce f re q u en cy (% ) in v as iv e (i ) / n o t (n ) m o d if ie d w r a p sc o re 1 a ge ra ti n a ri pa ri a (r eg el ) 1 0 .0 4 0 .0 1 1 .2 8 n 2 3 2 a u st ro eu pa to ri u m in u li fo li u m (k u n th ) r .m .k in g & h .r o b . 1 9 0 .7 0 .2 1 2 6 .9 2 i 2 4 3 b el lu ci a pe n ta m er a n au d in 1 1 0 .4 1 0 .1 2 1 5 .3 8 i 1 8 4 c al li an dr a ca lo th yr su s m ei sn . 9 0 .3 3 0 .1 1 2 .8 2 i 1 6 5 c am el li a si n en si s (l .) k u n tz e 1 0 .0 4 0 .0 1 1 .2 8 n 9 6 c h ro m ol ae n a od or at a (l .) r .m .k in g & h .r o b . 9 0 .3 3 0 .1 1 2 .8 2 i 1 9 7 c in ch on a pu be sc en s v ah l 1 0 .0 4 0 .0 1 1 .2 8 n 1 6 8 c li de m ia h ir ta (l .) d .d o n 8 0 .3 0 .0 9 1 1 .5 4 i 1 7 9 l an ta n a ca m ar a l . 2 0 .0 7 0 .0 2 2 .5 6 n 1 6 1 0 m ae so ps is em in ii e n g l. 4 0 .1 5 0 .0 4 5 .1 3 n 1 4 1 1 s pe rm ac oc e al at a a u b l. 6 0 .2 2 0 .0 7 8 .9 7 n 1 6 42 biotropia vol. 21 no. 1, 2014 roc curves that illustrate the accuracy of the risk analysis model are presented in figure 2. the value of area under the roc curve is 0.817.this value showed that the accuracy of the logistic regression model is approximately 81.7% figure 2. roc ( ) curve that determines the accuracy of the logistic regression conducted in the risk assessment receiver operating characteristic figure 3. mds configuration of three environment variables of detected exotic plants: soil ph, light intensity and air humidity. dimension 2 was used in the configuration. detected exotic species are, ai: , bp: , cc: , co: , ch: , lc: , me: and sa: austroeupatorium inulifolium bellucia pentamera calliandra calothyrsus chromolaena odorata clidemia hirta lantana camara maesopsis eminii spermacoce alata 43 exotic plants in halimun salak corridor decky indrawan junaedi– et al. mds analysis results based on three environmental variables (ph, light intensity and air humidity) are presented in figure 3. kruskal stress value of 0.002 indicates that the model configuration presented in figure 3 is valid. the valid kruskal stress value is smaller than 0.1 (quinn & keough 2002). shepard diagram of mds analysis is presented in figure 4. shepard diagram indicates the reliability of the data used in the mds analysis. based on the detection results, from totally 11 exotic species detected, four of them are tree species: , , and . the rest of other seven species are herbaceous/shrubs. based on detection sampling data, two exotic tree species having fr value above 10% are and . is native to south america (backer & backhuizen 1963). this plant is likely to spread rapidly in the halimunsalak corridor because of rapid spread through the animal's assistance that eats the fruit. exotic species can become invasive when it got past the “barrier” that exists in the invaded locations (theoharides & dukes 2007). in general, exotic plants have become invasive when it is said to have had offspring and form new colonies that are relatively numerous and have an adverse impact to invaded ecosystems (richardson 2000). however, not all exotic species detected are able to reach interior part of the forest where the forest canopy cover is still relatively intact. most of the exotic plants of the asteraceae found in the exterior forest areas or in the forest gaps (forest openings). mds analysis showed that two asteraceae species ( and ) have different positions relative to the other exotic species due to different types that formed on the configuration. the different position in the mds maesopsis eminii cinchona pubescens calliandra calothyrsus bellucia pentamera bellucia pentamera calliandra calothyrsus bellucia pentamera et al. c. odorata a. inulifolium figure 4. shepard diagram of mds analysis of three environment variables: soil ph, light intensity and air humidity 44 biotropia vol. 21 no. 1, 2014 configuration refers to different micro-environment preferences. therefore, and have relatively different micro-environment preferences compared to other exotic plants that are clustered in different quadrants (fig. 3). even though has the highest wrap value and refers to high risk to become invasive, but the environmental constraints (the intensity of light/canopy shade availability) becomes the limiting factor for these two species to become very invasive. however, if there is a gap occurred in the forest area, this species will be able to establish and invade. another interesting point is the grouping of exotic tree species in the same quadrant of the mds analysis result ( and ) (fig. 3). habitat preference (relative humidity, light intensity, and soil ph) were similar among these three exotic tree species in halimun-salak corridor. these three exotic tree species were mostly found in the interior forest, not in the forest edges. indeed, these three exotic tree species have not reached invasive stage. however, after a period of adaptation time (lag time), these species will quickly become invasive and will have an impact on the halimun-salak corridor ecosystem. time lag determines exotic plant species to become invasive (hobbs & humpries 1995). ias management in halimun-salak corridor should be focused on species that are already existed in the forest interior. effective and efficient management can minimize the cost of subsequent management (radosevich 2007). 's micro-environment preference (three variables: soil ph, light intensity and relative humidity) is very different from almost all exotic species recorded. backer and backhuizen (1963) mentioned that naturalized in west java, especially around bogor. in its native range in south america and the caribbean, dominates open areas, forest edges and forest openings/ disturbed forest and is rarely found under the shade-covered forests floor (dewalt ., 2004). but dewalt . (2004) also have indicated that is more shade tolerant in the invasion region than in the native range. included as one of the 100 worst invasive species in the world (issg 2006). and is an interesting case in this study. wrap values of and are relatively high, while the results of the detection sampling populations is relatively low (table 2). there are several factors that may explain the low number of sampling populations of and . first, the number of sampling may be relatively small compared to the pattern of spread of this species in the halimun-salak corridor. with the equal number of sampling units, the representation of the types of populations obtained with the clumped distribution pattern is more adequate than diffuse or random dispersal patterns. secondly, is not (or not yet?) penetrating the ecological constraints that exist in the halimun-salak corridor forest to reach the invasive stage. is still in the process of establishment/early naturalization stage. naturally occurs in open areas and the seed germination is inhibited by low light intensity (gentle & duin 1997, 1998). c. odorata a. inulifolium a. inulifolium maesopsis eminii, calliandra calothyrsus bellucia pentamera clidemia hirta c. hirta c. hirta et al et al c. hirta c. hirta ageratina riparia lantana camara a. riparia l. camara a. riparia l. camara l. camara l. camara l. camara 45 exotic plants in halimun salak corridor decky indrawan junaedi– et al. conclusions acknowledgements references there are 11 species of exotic plants in the halimun-salak corridor area consisted of three woody tree species and eight herbs/shrubs species. is an exotic species that has the largest wrap value (24) and is an exotic species with the lowest wrap value (-4). exotic tree species have similar environmental preferences. is an exotic species that has relatively different environment variables compared to other exotic species found in the halimun-salak corridor. based on the risk assessment of invasiveness and field observation conducted, some recommendations for the management of invasive exotic plant species in the halimun-salak corridor are: (1) management of invasive species can be done with several options, but the more immediate the implementation, the cheaper the management cost and the simpler the management methods will be, (2) eradication can be implemented to exotic plant species that have not been abundant in population but have relatively high wrap value such as and . whenever possible, eradication should start from the individual which exist in the forest interior and continue toward the outside of the forest, (3) gradual eradication or control (containment) are two possible management options for exotic plant species that have a very high wrap score such as , and . future study should be focused on how to conduct ias management implementation that gives the smallest impact to the ecosystem of halimun-salak corridor. it is important to conduct ecological study of exotic species micro-habitat as well as investigating the impact of these exotic species to halimun-salak corridor ecosystem. authors thank soekisman tjitrosoedirdjo for significant recommendations and review on the methodology section of this work. authors also thank sri astutik for valuable discussion during field data collection, megawati for dedicated plant identification and to pak uci and pak atma for their field assistance and guidance. this study funded by grant “program prioritas nasional 9 program penelitian, penguasaan, dan pemanfaatan iptek: pembangunan kebun raya daerah” (national priority program, 9 programs for research, control, and utilization of science and technology on the development of district botanical garden). lantana camara camellia sinensis clidemia hirta bellucia pentamera cinchona pubescens ageratina riparia chromolaena odorata lantana camara backer ca, backhuizen rcvdb. 1963. flora of java (spermatophytes only) volume i. groningen: n.v.p. nordhoff. 46 biotropia vol. 21 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2006. global species invasive database: clidemia hirta. accessed 10 january 2013 from jica team, endangered species. 2005 ecological study halimun-salak corridor mount halimun-salak national park. accessed 10 september 2011 from . peh ksh. 2010. invasive species in southeast asia: the knowledge so far. biodiversity and conservation 19: 10831099. quinn gp, keough, mj. 2002. experimental design and data analysis for biologist. cambridge: cambridge university press. richardson dm, pysek p, rejmanek m, barbour mg, panetta fd, west, cj. 2000. naturalization and invasion of alien plants: concepts and definitions. diversity and distributions 6: 93-107. radosevich, sr 2007, plant invasions and their management, chapter 3 in cipm (ed.), invasive plant management: cipm online textbook, bozeman, mt: center for invasive plant management. accessed 2 may 2011 from . theoharides ka, dukes js. 2007. plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. new phytologist 176: 256-273. virtue s, spencer, rd, weiss, je, reichard, se. 2008. australia's botanic gardens weed risk assessment procedure. plant protection quarterly 23: 166-178. clidemia hirta lantana camara choricarpia leptopetala lantana camara . http://www.issg.org/database/species/ecology.asp?si=53&fr=1&sts=sss& lang =en. http://sci.kagoshima-u.ac.jp http://www.weedcenter.org/textbook/index.html 47 exotic plants in halimun salak corridor decky indrawan junaedi– et al. annex 1. modified weed risk assessment procedure used in the study (virtue 2008)et al impacts score 1. weed history – what is the weed history of the plant? it is a weed in your region 4 it is a weed elsewhere in indonesia 3 it is a weed overseas 2 it is not known to be weedy, but other forms of the plant and/or plants in the same genus are weeds in indonesia and/or overseas 1 the genus is not known to be weedy 0 2. competition – how well does the plant out-compete other types of plants? if not controlled it can grow to dominate the following three size classes of plants: 4 trees (or emergent aquatics) shrubs (or surface aquatics) ground covers (or submerged aquatics) if not controlled, it can grow to dominate one of the above size classes of plants. or 2 at certain times of the year it can dominate two size classes if not controlled, it can dominate one of the above size classes at certain times of the year 1 it is not competitive, and is readily dominated by most other plants if they are not controlled 0 3. health – is the plant a health risk to people and/or animals? is it highly toxic and has caused deaths 3 it can cause significant physical injuries or illness 2 it can cause slight physical injury or mild illness with no long-lasting effects 1 it is not a health risk to animals or humans 0 4. movement – if the plant escapes does it have the potential to block the movement of people, animals, vehicles or water? a group of plants is very tall, thorny, tangled and/or dense & impenetrable year-long 3 a group of plants is rarely impenetrable, but does significantly undergo slow physical movement year-long 2 a group of plants is never impenetrable but can significantly undergo slow movement for part of the year 1 plant has no significant effect on movement 0 48 biotropia vol. 21 no. 1, 2014 impacts score 5. environmental effects – does the plant have attributes that, at high density, can cause detrimental changes to the environment? the plant has two or more of the following attributes: 2 highly flammable leaves fixes nitrogen salty leaves habitat or food source for pest animal invasion high water use removes habitat or food source for native animals it has one of the above attributes 1 it has none of the above attributes 0 6. ease of control how easy is the plant to kill? hard. it readily tolerates or reshoots after herbicide application, cutting, cultivation, grazing or fire 3 medium. one herbicide application or cultivation kills the plant, but not cutting, grazing or fire 2 simple. plants are killed by hand-pulling, cutting, grazing or fire 1 potential distribution 7. hardiness – how well is the plant adapted to the local climate? in the garden it needs no maintenance to establish, grow and flower, if planted at the right time of year. the localclimate is similar to other places where it grows in the world. or, it is a weed of drier, exposed land in the region or in other parts of indonesia or overseas with similar climate types 3 it needs some maintenance to establish when planted in the garden, but will then grow and flower well without further maintenance. the local climate is harsher in some aspects to what the plant is originally adapted to. or, it is a weed of wetter, sheltered land in the region or in other parts of indonesia or overseas with similar climate types 2 it needs some maintenance to establish when planted in the garden, but can then survive unassisted. however, it grows poorly in the local climate and needs occasional maintenance during stressful periods (e.g. watering in dry months). or, it has naturalised occasionally in the region in specialist habitats (i.e. it has a narrow ecological amplitude) but hasn’t formed high-density infestations. it grows in distinctly different climates elsewhere in indonesia or overseas 1 it needs frequent maintenance and/or special conditions to grow and flower in the garden. or, the local climate isvery different to where the plant grows elsewhere in the world and it has not been recorded as naturalised in the region or in other parts of indonesia or overseas with similar climate types 0 annex 1. continued 49 exotic plants in halimun salak corridor decky indrawan junaedi– et al. annex 1. continued invasiveness 8. reproduction – how well does the plant reproduce? the plant has all three of the following attributes: 4 short time to seeding (within 3 years from planting for herbaceous plants, 5 years for woody plants) ‘volunteer’ seedlings commonly come up in the garden, or as a weed, seedlings are commonly seen nearby mature plants. mature plants produce at least 5 new plants by vegetative means per year it has two of the above attributes 3 it has one of the above attributes 2 it has none of the above attributes, but is able to reproduce by itself 1 it sets no seed in any natural circumstances and has no vegetative spread. the plant can only be propagated with human assistance (eg. shoot cuttings). it will not naturalise. (low weed risk) 9. natural spread – are the plant’s propagules (seed or vegetative) likely to spread long distances to susceptible habitats by natural means? propagules are likely to be dispersed by two or more of the following: 4 flying animals ground animals water wind and susceptible habitats (e.g., farmland, native vegetation, road and rail corridors, waterways) are within 1 km of the gardens propagules are likely to be dispersed by two or more of the above means and susceptible habitats are within 5 km of the gardens, or, propagules are likely to be dispersed by one of the above means and susceptible habitats are within 1 km of the gardens 3 propagules are likely to be dispersed by one of the above means and susceptible habitats are within 5 km of the gardens 2 propagules are not normally spread long distances from parent plants but the botanical garden abuts susceptible habitats 1 propagules are not normally spread long distances from parent plants and the botanical garden is from susceptible habitats by urban developmentbuffered 1 10. human spread – are the plant’s propagules (seed or vegetative) likely to spread long distances due to human activities? the plant is appealing and easily to propagate. visitors to the gardens may intentionally take propagules (e.g., seeds, cuttings, fruit etc.) to grow elsewhere 3 propagules are likely (or known) to be dispersed by two or more of the following means: 2 people (accidentally) vehicles/machinery contaminated produce in soil propagules are likely (or known) to be dispersed by one of the above means 1 propagules are not normally spread long distances from the parent plant 0 score lwr 50 biotropia vol. 21 no. 1, 2014 annex 2. detection sampling data from 27 plots. date plot number species name altitude (m asl) soil ph air relative humidity (%) light intensity (x 2000 lux) 20/06/2012 1 austroeupatorium inulifolium 1110 6 50 322 2 austroeupatorium inulifolium 1103 6.2 55 219 3 austroeupatorium inulifolium 1100 6.8 56 1270 4 austroeupatorium inulifolium 1100 6.8 56 1270 calliandra callothyrsus 5 calliandra callothyrsus austroeupatorium inulifolium 6 bellucia pentamera 21/06/2012 7 austroeupatorium inulifolium 1050 6 43 62 bellucia pentamera 8 spermacoce alata 1068 6.8 52 594 austroeupatorium inulifolium bellucia pentamera 9 austroeupatorium inulifolium 1019 6.5 47 364 calliandra callothyrsus 10 maesopsis eminii 1056 6 51 433 maesopsis eminii 12 austroeupatorium inulifolium 1099 6.8 37 1010 spermacoce alata maesopsis eminii bellucia pentamera austroeupatorium inulifolium 23/06/2012 13 chromolaena odorata 1046 6 49 209 bellucia pentamera 14 bellucia pentamera 1014 6.2 49 219 calliandra callothyrsus 15 calliandra callothyrsus 1041 6 51 697 bellucia pentamera 16 none 1003 6.5 55 267 24/06/2012 17 austroeupatorium inulifolium 1005 6.5 50 3390 chromolaena odorata bellucia pentamera 18 austroeupatorium inulifolium 1049 6 51 1627 lantana camara clidemia hirta 51 exotic plants in halimun salak corridor decky indrawan junaedi– et al. date plot number species name altitude (m asl) soil ph air relative humidity (%) light intensity (x 2000 lux) chromolaena odorata spermacoce alata 25/06/2012 19 lantana camara 1036 6.2 57 90 clidemia hirta spermacoce alata austroeupatorium inulifolium chromolaena odorata 20 cinchona succirubra 1028 6.5 57 8.6 bellucia pentamera 21 austroeupatorium inulifolium 1010 6 58 6.1 chromolaena odorata bellucia pentamera 26/06/2012 22 austroeupatorium inulifolium 1061 6 47 29 calliandra callothyrsus clidemia hirta chromolaena odorata spermacoce alata 23 austroeupatorium inulifolium 1020 6.4 50 1311 clidemia hirta bellucia pentamera 27/06/2012 24 calliandra callothyrsus 1028 6.8 45 1590 austroeupatorium inulifolium camellia sinensis clidemia hirta spermacoce alata chromolaena odorata 25 maesopsis eminii austroeupatorium inulifolium clidemia hirta 26 calliandra callothyrsus 1028 6.8 45 1590 clidemia hirta 27 austroeupatorium inulifolium 1028 6.8 45 1590 calliandra callothyrsus clidemia hirta chromolaena odorata ageratina riparia annex 2. continued 52 biotropia vol. 21 no. 1, 2014 biotropia book final.indd 129 pollination effectiveness of apis cerana fabricus and apis mellifera linnaeus (hymenoptera: apidae) in jatropha curcas l. (euphorbiaceae) tri atmowidi*, puji riyanti, andeng sutrisna department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia abstract pollinators are well known to provide key ecosystem. animal pollinators are thought to contribute between 15 and 30% of global food production and bees are recognized to be the most important taxon. th e pollination eff ectiveness of two species of bees, apis cerana and a. mellifera (hymenoptera: apidae) in jatropha curcas (euphorbiaceae) was studied. th ree cages, made of insect screen were set up. each cage contains three individual plants. one colony of a. mellifera and a. cerana were placed in the fi rst and second cage, while the third cage was used for control plants. bee colonies were placed during one month in the fi rst and second cages. fruit set of plants pollinated by a. mellifera, a. cerana, control plants, and open plants were counted and compared. pollination by honeybees increased the number of fruits per plant and fruits per raceme of j. curcas. based on measuring of fruit produced by plants, pollination eff ectiveness of a. mellifera was higher than a. carana. key words: pollination, eff ectiveness, apis cerana, a. mellifera, seed set, jatropha curcas. introduction th e honeybees are the principal species used for crop pollination worldwide (free 1993). th e true honeybees (tribe apini, genus apis) consist of nine species i.e. a. mellifera linnaeus, a. cerana fabricus, a. dorsata fabricus, a. laboriosa smith, a. fl orea fabricus, a. andreniformis smith, a. koschevnikovi buttel-reepen, a. nigrocincta, dan a. nuluensis. genus apis primarily tropical was restricted to the old world until a. mellifera was introduced worldwide. both species, a. cerana and a. mellifera consist of medium -sized (10-11 mm) species with multiple combs in cavities, dances on vertical surfaces of combs in the dark (michener 2000). th e combs are built under the ceiling of the cavity and attached to the cavity’s walls. th e arrangement and distribution on the combs are similar in all races of each species (koeniger 1995). colonies of a. cerana are relatively small (6000-7000 workers), but a. mellifera colonies consist of 100, 000 or more individuals (winston 1987). * corresponding author: atmowidi@ipb.ac.id biotropia vol. 15 no. 2, 2008 : 129 134 130 comparative foraging behaviour of a. cerana and a. mellifera on apple orchard had been reported by verma (1995). worker bees of a. cerana started their foraging activity signifi cantly earlier in the morning (06.03 h) than a. mellifera (06.27 h), but duration of foraging trip of a. mellifera (17.92 minutes) was signifi cantly longer than that of a. cerana (11.85 minutes). th e peak of foraging activities of a. cerana was between 09.00 h and 13.00 h with a temperature range of 15.5-21oc, and that of a. mellifera was between 11.00 h-13.20 h with a temperature range from 21 to 25oc. worker bees of a. mellifera carried signifi cantly heavier pollen loads than a. cerana throughout the day. a. cerana contacted the stigma on average 3.09 stigmas per visit and spent 5.90 seconds on each fl ower, whereas these values for a. mellifera were 3.33 stigmas per visit and 6.63 seconds on each fl ower, respectively (verma 1995). selection for eff ective pollen transfer and receipt has been considered to be the principal force in the evolution of the angiosperm fl ower. most fl owers, however, allow access to a variety of visitors (faegri & van der pijl 1979). diff erences among visitors in morphology, physiology, and foraging behavior may result in diff erences in their eff ectiveness as pollinators (young 1988). wallace et al. (2002) also reported that the natural pollinators were extremely effi cient in producing fertilization and fruit set of jeff ersonian virgata. in male-sterile oilseed rape (brassica napus), steff an-dewenter (2003) reported that insect pollination can positively aff ect several yield components. however, these eff ects are dependent on cultivar and growing conditions. total yields are often not increased due to the considerable compensatory capacity of oilseed rape (westcott & nelson 2001). jatropha curcas (euphorbiaceae) is a perennial, deciduous shrub or treelet. th e plant produces fl owers in racemose infl orescences, with dichasial cyme pattern. th e fl owers are unisexual, and male and female fl owers are produced in the same infl orescence. normally, the infl orescences produce a central female fl ower surrounded by a group of male fl owers. in some cases the female fl owers are substituted by male fl owers. numerically, 1–5 female fl owers and 25–93 male fl owers are produced per infl orescence. th e average male to female fl ower ratio is 29 : 1. each infl orescence, once it begins fl owering, fl owers daily, and the fl owering lasts for 11 days. th e fl owering pattern showed that the male fl owers are produced earlier (compared to the female fl owers) and will produce fl owers daily until the male buds are exhausted. th e female fl owers bloom between the second and the sixth day. th e fl oral base contains nectar in trace amount, which is 0.3 ml per fl ower. th e fl owers open daily during 0530–0630 h. th e unpollinated fl owers fall off on the fourth day, while the pollinated ones remain in place. th e sepals and petals gradually enlarge and protect the growing fruit until the latter reaches its full size (raju & ezradanam 2002). here, we studied the pollination eff ectiveness of two species of honeybees, apis cerana and a. mellifera in j. curcas. th e eff ectiveness of the pollinators was measured by the number of seeds set, and seeds weight. materials and methods study site th e study was conducted in jatropha plantation located at indramayu district, west java from may to september 2007. biotropia vol. 15 no. 2, 2008 131 measuring of the pollination eff ectiveness of honeybees th e plants selected for measuring pollination eff ectiveness were about three years old. we set up three cages made by of insect screen. th e size of each cage was 9x3x2.5 m3 and each cage contains three plants of j.curcas. one colony of a. cerana and a. mellifera were placed in the fi rst and second cages for approximately 1 month (june 22 to july 25, 2007) to pollinate the fl owers. th ird cage was used as a control (no bee application). at this time, bees were fed with honey that placed in a cup. each infl orescence observed was tagged by color ribbon. after the end of the fruiting period, the number of fruits per plant, fruits per raceme, seeds per fruit, and seed weight were counted. observation of fl ower visitors species composition of fl oral visitors was observed visually at jatropha plantation using scan sampling method (martin & bateson 1993) for a few days. after observation, insect visitors were caught by sweep netting for species identifi cation in the laboratory. data analysis fruits set of j. curcas pollinated by a. cerana and a. mellifera were compared with control plants and open plants (plants pollinated by natural pollinators). diff erences in fruit numbers of j. curcas pollinated by bees, control plants, and open plants were analysed by analysis of variance (anova) and scheff e test at the 95% level by using systat 10 for windows. results and discussion pollination eff ectiveness of honeybees in j. curcas pollination eff ectiveness of two species of honeybees in j. curcas varied. pollination eff ectiveness was shown by the number of fruits produced by plants. result showed that the mean number of fruits produced per plant as a result of pollination by a. cerana (17 fruits), a. mellifera (19 fruits), and open plants (16 fruits) were higher than that of control plants (5 fruits) (table 1). although, based on statistical tests, they were not signifi cantly diff erent. table 1. reproductive success of j. curcas pollinated by a. cerana, a. mellifera, open plants, and control plants plant reproductive success numbers (+st.dev) control plants a. cerana a. mellifera open plants number of fruits per plant 17a (+7.94) 19a (+11.93) 16a (+12.15) 5a (+5.13) number of fruits per raceme 2.04a(+1.65) 3.05a (+1.75) 2.45a (+1.47) 2.29a (+1.25) number of seeds per fruit 2.76a (+0.51) 2.61a (+0.50) 2.47a (+0.63) 2.63a (+0.52) seed weight (g) 0.47ac (+0.17) 0.39b (+0.14) 0.51a (+0.16) 0.41cb(+0.11) note: different lower case letters in the same row indicate different values based on analysis of variance (anova) and scheffe test at 95% level (p<0.05). eff ectiveness of bees to pollination of jatropha – t. atmowidi et al. 132 th e number of fruits per raceme of plants pollinated by a. cerana was lowest (2.04 fruits) compared to plants pollinated by a. mellifera (3.05 fruits), open plants (2.45 fruits), and control plants (2.29 fruits). pollination by bees increased the number of fruits per plant and fruits per raceme of j. curcas. mean number of seeds per fruit of plants pollinated by a. cerana, a. mellifera, open plants, and control plants were 2.76, 2.61, 2.47, and 2.63 seeds, respectively. th e number of seeds per fruit ranged from 1 to 3 seeds (table 1). results showed that a. mellifera was the superior pollinator compared to a. cerana. in addition, the number of fruits per plant and fruits per raceme of plants pollinated by a. mellifera were higher than that pollinated by a. cerana. verma (1995) stated that some characteristics of a. mellifera were better than a. cerana, i.e. duration of foraging trip was signifi cantly longer and worker bees carried signifi cantly heavier pollen loads throughout the day. th e increased yields of j. curcas pollinated by bees was probably caused by cross pollination. as stated by westcott & nelson (2001), insect pollination leads to earlier cessation of fl owering and more synchronous pod and seed ripening, thereby possibly increasing the weight of seed harvest. th e natural pollinators were extremely effi cient in producing fertilization and fruit set of p. virgata (wallace et al. 2002). in male-sterile oilseed rape (b. napus), steff an-dewenter (2003) reported insect pollination could positively aff ect several yield components. however, these eff ects are dependent on cultivar and growing conditions and total yields are often not increased due to the considerable compensatory capacity of oilseed rape (westcott & nelson, 2001). similarly, atmowidi et al. (2007) reported that mustard (brassica rapa) pollinated by insects (mostly by bees) increased the number of pods, seeds per pod, and seeds per plant. lower fruit set of j. curcas plants caged with insect screen could be caused by other factors, such as the number of plants observed, size of plants, and screen eff ect. th e number of plants observed (three plants for each application) very limited. size of plants observed was quite uniform, but individual variation in size occurred. th e screen may be aff ected to plant photosynthetic rate, but most probably this eff ect is small, because the screen transmitted the light intensity easily. th e natural fruit set rate indicates that j. curcas does not suff er seriously if there are no other pollination agents. flowers of the species are unisexual, and male and female fl owers are produced in the same infl orescence. th e production of female fl owers in small number, surrounded by a large number of male fl owers seems to be a strategy to ensure pollination to the maximum extent. th e stigma receptivity lasting three days also additionally provides opportunities for pollination, if not pollinated on the fi rst and second day. however, the plant with predominant xenogamy requires mostly xenogamous pollen for more fruit set, after selective elimination of growing fruit. th erefore, pollen transfer between conspecifi c has a great infl uence on the net percentage of natural fruit set (raju & ezradanam 2002). diversity of fl ower visitors based on fi eld observations, the fl ower-visitors of j. curcas belong to bees and ants (hymenoptera), butterfl ies and moths (lepidoptera), beetles (coleoptera), thrips (th ysanoptera), and fl ies (diptera). bee species belonging to a. cerana, ceratina sp, trigona sp., and hylaeus sp. were found visiting the fl owers. at least, three species of biotropia vol. 15 no. 2, 2008 133 butterfl ies (nyctemera sp., eurema hecabe, and neptis hylas) also visited the fl owers. snout beetle and some species of diptera (syrphus balteatus, sarcophaga sp., and musca domestica), and thrips were also found visiting the fl owers. in general, insect pollinators visit the fl owers in the morning. on brassica rapa (brassicaceae), the peak abundance of insect pollinators occurred between 08.30-09.30 h (atmowidi et al. 2007). similar visitation was shown by two species of pollinators (trigona carbonari: apidae and leiopcoctus speculiferus: colletidae) visited persoonia virgata (proteaceae) occurred before 12.00 h (wallace et al. 2002). foraging time of insect pollinators related to plant resources, especially pollen and nectar. th e increase of plant resources such as during mass fl owering showed an increase in available resources so that the foragers could collect more rewards per unit time. if resources are limited, the foragers require longer searches and travel time (wesphal et al. 2006). generally, both pollen and nectar content of most plants are higher in the morning. conclusions pollination by honeybees, a. cerana and a. mellifera increased the number of fruits per plant and fruits per raceme of j. curcas. in j. curcas, pollination eff ectiveness of a. mellifera was higher than that a. cerana. naturally, fl owers of j. curcas are visited by bees and ants (hymenoptera), butterfl ies and moth (lepidoptera), beetles (coleoptera), thrips (th ysanoptera), and fl ies (diptera). acknowledgments th is project was funded by biotrop dipa 2007. we greatly acknowledge dr. sih kahono, dr. rosichon ubaidillah, dr. yayuk rahayuningsih, woro nurjito,m.sc. and pudji aswari, m.sc. of zoological museum, indonesian institute of science, cibinong for their help in identifi cation of insect pollinators. we are also grateful to dian sari and sunaryo for the assistance in collecting the data. finally, the authors are grateful to the two anonymous referees for their comments of this manuscript. references atmowidi t, buchori d, manuwoto s, suryobroto b, hidayat p. 2007. diversity of insect pollinators and seed set of mustard (brassica rapa: brassicaceae). hayati 14:155-161. faegry k, van der pijl l. 1971. th e principles of pollination ecology. ed ke-2. braunschweig: pergamon press. free jb. 1993. insect pollination of crops. san diego: academic press. koeniger n. 1995. biology of the eastern honeybee apis cerana (fabricus 1773). in: kevan pg, editor. th e asiatic hive bee: apiculture, biology, and role in sustainable development in tropical and subtropical asia. ontario: enviroquest ltd. pages 29-39. eff ectiveness of bees to pollination of jatropha – t. atmowidi et al. 134 martin p, bateson p. 1993. measuring behaviour: an introductory guide. ed ke2. cambrige: cambrige univ. press. michener dm. 2000. th e bees of the world. baltimore: johns hopkins univ. press. raju ajs, ezradanam v. 2002. pollination ecology and fruiting behaviour in a monoecious species, jatropha curcas l. (euphorbiaceae). current science 83:1395-1398. steff an-dewenter i. 2003. seed set of male-sterile and male-fertile oilseed rape (brassica napus) in relation to pollinator density. apidologie 34:227–235. verma lr. 1995. apis cerana: biometric, genetic, and behavioural aspects. in: kevan pg, editor. th e asiatic hive bee: apiculture, biology, and role in sustainable development in tropical and subtropical asia. ontario: enviroquest ltd. hlm 41-53. wallace hm, maynard gv, trueman sj. 2002. insect fl ower visitors, foraging behaviour and their eff ectiveness as pollinators of persoonia virgata r. br. (proteaceae). australian journal of entomology 41: 55– 59. wesphal c, steff an-dewenter i, tscharntke t. 2006. foraging trip duration of bumblebees in relation to landscape-wide resource availability. ecological entomology 31:389–394. westcott l, nelson d. 2001. canola pollination: an update, bee world 82:115–129. winston ml. 1987. th e biology of the honey bee. cambridge: harvard univ. press. young hj. 1988. diff erential importance of beetle species pollinating dieff enbachia longispatha (araceae). ecology 69:832-844. biotropia vol. 15 no. 2, 2008 introduction of the serine green fluorescent protein (sgfp) pyricularia grisea gene nto ra dc4 i ce isolated from using digitaria ciliaris agrobacterium tumefaciens-mediated genetic transformation stephanie , utut widyastuti and suryo wiyono1 2,3* 4 1program study of graduate school, institut pertanian bogor; bogor 16680, indonesiabiotechnology, 2research center for bioresources and biotechnology, institut pertanian bogor; bogor 16680, indonesia 3department , faculty of mathematics and natural science , of biology s institut pertanian bogor; bogor 16680, indonesia 4department of , faculty of agriculture, institut pertanian bogor, bogor 16680, indonesiaplant protection r 4eceived 24 july 2013/accepted 12 september 201 abstract blast disease (caused by ) has long been known as a serious problem for up rice. now, it also pyricularia grisea land attacks lowland rice. however, the mechanism facilitating this range expansion is still unknown. one option is to insert a marker into so that it can be used to monitor the spread of infection. (p. grisea p. grisea s sgfp erin green fluorescent protein ) gene of the study of has been used to monitor gene expression specific tagged proteins for fungal cell. genome of dc 4 from in this study, the gene has been integrated into the the sgfp igitaria c . p. grisea d iliaris p using thelasmid was introduced into triparental mating method (tpm). genetic transformation sgfp a. tumefaciens was performed by co-cultivating spore dc4 with lba4404–pcamb-s . dc4 s of the p. grisea a. tumefaciens gfp p grisea. transformant was selected by using selection medium 300 µg/m hygromycin. the integration of a containing l of the the the c thesgfp sgfp sgfp gene into genome was confirmed by pcr using 's spe ific primer pair, -nos terminator primer pair and -tubulin primer pair as internal control. expression of from the an the gene the β sgfp p. grisea dc4 transformant w detected with blue light fluorescent microscopas y. keywords: agrobacterium tumefaciens, digitaria ciliaris, pyricularia grisea , erine green mediated transformation dc4 s fluorescent protein introduction pyricularia grisea magnaporthe grisea (teleomorph ), synonymous with cav, is a rice pyricularia oryzae plant pathogen in many countries and is known as the agent causing last disease (rho 2001). et al.b the disease is estimated to be responsible for 30% of annual yield loss, the equivalent in food volume to meet the needs of 60 million people (dagdas 2012). in indonesia, blast infestation et al. reached 36,727 ha of the total 13,153,080 ha area of rice cultivation in 2012 (ministry of agriculture 2013). blast disease has been known as a serious problem for up rice, however, land recent results showed that the last pathogen also b attacks rice planted in low s or irrigated land land (sobir 2003). to date, the mechanism of blast et al. disease transmission from upland rice to lowland land has not been examined. p griseayricularia is a pathogen on more than 50 species of wild grass in the vicinity of rice field (couch & khon 2002 ). has a wide variety p. grisea of hosts besides rice plant; among them are triticum aestivum zea mays eleusine coracana, , (finger millet), (cultivated grass), setaria italica brachiaria mutica et al (couch & khon 2002; listiyowati . 2011). because of this wide host range, cereal grasses and wild grasses around the area of cultivation are also prone to infection and p. grisea therefore, could become alternative hosts as well as alternative inocula sources for spreading disease.* corresponding author : ututsuharsono2002@yahoo.com biotropia vol. 22 no. 1, 2015: 73 79 doi: 10.11598/btb.2015.22.1.329 73 mailto:ututsuharsono2002@yahoo.com biotropia vol. 22 no. 1, 2015 74 research showed that grasses growing around rice field could become temporary hosts for fungi that cause last disease (listiyowati 2011). et alb . those fungi were capable of infecting rice plants and showed some genetic structure alterations from the original grass isolate. isolated p. grisea from grass (dc4) is able to infect rice d. ciliaris plants that are moderately resistant and susceptible plants. this isolate of dc4 that switches host undergoes genetic alterations, which are marked by changes in scar marker for cut1, pwl2, erg2 as well as physiological race based on the ability to infect differential rice varieties in indonesia (listiyowati 2011). this et al. is presumed as a genomic adaptation response of the fungi isolate to the new host. however, the mechanism that causes such change is still unknown. therefore, as an initial step it is necessary to insert a marker into to p. grisea monitor infection.p. grisea according to lorang (2001), the et al. gfp gene could be used as a tool in studying interactions between fungi and plants. has gfp also been used to monitor the virulence of blastcausing fungi. gene has several advantages as gfp a marker. gene detection does not require any gfp substrate addition to obtain the visualization; it does not require special treatment of the tissue and its presence in the cell is not harmful to the cell itself. these advantages make a great gfp marker in gene transformation and expression (lorang 2001). today, the gene has been et al gfp. shown to be expressed in 16 species of 12 genera of fungi including (lorang 2001). m. grisea et al . the gene has been successfully used as gfp important marker for several fungi that cause diseases and is among them (balhadere & p. grisea talbot 2001). sgfp gene transfer into fungi genome requires a biological vector. has agrobacterium tumefaciens been long used as a biological vector for gene transfer in plants. besides transferring genes to its host plant, is also able to transfer a. tumefaciens dna to yeasts and filamentous fungi (combier 2003). the objective of this research was to introduce gene into dc4 sgfp p. grisea isolated from using d. ciliaris a. tumefaciens-mediated genetic transformation. subsequently, there would be a gene introduction into the fungi sgfp genome. the success of this gene introduction could be developed to facilitate molecular studies of pathogen-host interactions. materials and methods triparental mating the introduction of binary plasmids that contain gene into was donesgfp a. tumefaciens with the tpm assay described by hanum (2011). three bacteria: dh donor ing e. coli 10b contain pcamb plasmids, dh1 ing sgfp e. coli contain helper prk2013 plasmids and a. tumefaciens lba4404 as the recipient strain were used in tpm assay. the three bacteria were grown in solid lb to prevent conjugation. a. tumefaciens sgfp transformants were then identified using colony pcr with specific primers -f and -r.sgfp sgfp fungal strains and culture conditions p. grisea d. ciliaris race dc4 isolated from was provide by d mycology laboratory, department of biology, faculty of mathematics and natural s c i e n c e s , i n s t i t u t p e r t a n i a n b o g o r . fungal cultures were grown on oatmeal agar medium (oma: 30 g of oatmeal for 1 l). fungi were grown in oa medium and incubated for 710 days at 28 ºc. for production of conidia, fungal cultures were grown under light of a nearuv lamp for 5 6 days to using sterilized water ( ).munandhar 1998et al. transformation one colony of containing a. tumefaciens pcamb plasmids was grown on 2 ml of sgfp minimal medium (hooykaas 1979) et al. sup lemented withp 100 µg/ml streptomycin, 60 µg/ml kanamycin, and 50 µg/ml hygromycin at 28 c for 48 hours with 250 rpm rotation ino no light condition. the bacterial culture was diluted with 5 ml induction medium (im) containing 200 µm acetos ringone toy an optical density of 0.15 at a600. the bacterial culture of a. tumefacien -sgfps was grown for 4-6 an additional hours at 28 c with 250 rpm rotation o to reach an a600 . (betts 2007) of 0 5 . . approximately et al 100 µl of dc4 spores (10 spores/ml) was p. grisea 6 mixed with 100 µl of cells a. tumefaciens-sgfp (a600 = 0.5) then added with a etosyringon 200c e µm, and incubated for 30 minutes with no light. the co-cultivated cultures were then into plated im medium and incubated at 28 c for 48 hours. o the co-cultivation result was transferred into complete medium containing 300 µl/ml introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. hygromycin and 200 µg/ml cefotaxime and incubated at 28 c for 5-7 days. pore o single s selection was done by spreading 100 l µ conidia suspension on oa medium that contained 300 µg/ml h gromycin and incubated for 5-7 days y until dc4 appeared (rho p. grisea et al single spores . 2001 modified by betts 2007).et al. analysis of transformant genomic dna of mycelium from p. grisea transformant and non-transformant were p. grisea isolated for verification using pcr. dna isolation was done with the method described by listiyowati (2011) using 2% et al etyl rimethyl . c t a b mmonium romide (ctab). the isolated dna sgfp gene as then w amplified through pcr using -f and -r primers as well as sgfp sgfp combined primers of f and nos-r. the sgfppcr mixture used consisted of 1 µl (100 ng) genomic dna, 0.5 mm forward primers, 0.5 mm reverse primers, 5 µl pcr mix (fermentas) and added with ddh o up to 10 l of total volume. 2 µ the pcr program to amplify fragments sgfp consisted of: pre-denaturation at 94 ºc for 1 minute, denaturation at 94 c for 1 minute, o annealing at 53 c for 30 seconds, elongation at o 72 c for 1 minute and final elongation at 72 c o o for 5 minutes; this process was run in 35 cycles. the pcr results were through visualized electrophoresis 1% (b/v) agarose gel at 100 on volt for 30 minutes in tae 1 and continued with gel immersion in 0.5 mg/l etbr for 20 minutes and were visualized under uv transluminator (shanti 2008). m yicroscop to visualize , p. grisea transformants they were subcultured in oa medium containing 300 µg/ml hygromycin. the culture was incubated at room temperature for 6 days. the transformants were grown under light of the a near-uv lamp for 5 6 days. the acquired spores were then to observed using an olympus bh2-rfch microscope on the fluorescent setting with a 515 bandpass emission fillter (blue light) . results and discussion triparental mating (tpm) the plasmid camb was introduced p sgfp into using the tpm method (fig. 1). a. tumefaciens plasmids in dh10b were contained e. coli moved into lba4404 through a. tumefaciens conjugation with the help of prk2013. e. coli triparental mating generated several colonies growing in a medium containing 50 µg/ml kanamycin, 50 µg/ml streptomycin and 50 µg/ml hygromycin. the only bacterium able to grow in that selective medium was a. tumefaciens lba4404 containing pcamb plasmids. sgfp colonies from tpm that were able to obtained grow in the selective medium were then analyzed with pcr. the results of the pcr analysis showed that colonies of with -f and -a. tumefaciens sgfp sgfp r primers produced amplicon with the size of 643 bp (fig. ). the results of this amplification 1 had the same size as the positive control (fig. ). 1 this showed that pcamb plasmid w sgfp as successfully introduced into a. tumefaciens through the tpm method. this method was previously used by hanum (2011) to move the binary plasmid pmsh into mmcuzn-sod a. tumefaciens et al.. according to wise (2006) tpm is a quite efficient method for transferring gene in a non-conjugated plasmid into a. tumefaciens. 1000 bp 750 bp 500 bp 250 bp 643bp figure . pcr identification of the gene introduced into lba4404 colon using tpm1 ies . m: marker 1 kb; sgfp a. tumefaciens k+: plasmid pcamb (positive control); k-: ddh o (negative control); 1-5: transformant sgfp a. tumefaciens2 sgfp 75 transformation p. griseagenetic transformation of dc4 using a. tumefaciens w s achieved using the method a described in rho (2001) and betts (2007). et al et al. . one hundred microliter (100 µl) of spores (10 6 per ml) were co-cultivated with 100 µl of bacterial cells in an induction medium that had been added to 200 m acetosyringone for 48 µ hours. an acetosyringon concentration of e 200 µm is important in fungal transformation to induce vira genes of so that t-a. tumefaciens dna transfer may take place (knight . 2009). et al an acetosyringon concentration of 200 µm is e commonly used in fungal transformation with a. tumefaciens as a biological vector and it has been shown to have consistent results (covert . et al 2001; dos reis . 2004; knight . 2009; et al et al xiaoran . 2012)et al . p. grisea dc4 grown in hyg omycin and r cefotaxime selective medium was then cultured to produce spores. the spores were then spread in oa medium containing 300 µg/ml hygromycin and incubated for 5-7 days until some single spores that were transformed were obtained (fig. 2). selection of spore was done to single s eliminate false transformant fungi (betts et al. 2007). mycelium growth on hygromycin medium indicated the success of transformation (tucker & orbach 2007). some s that single spore were not able to grow on hygromycin medium because the genes having important roles in growth might undergo some damage or their activities might be interrupted by the presence of foreign genes. p. griseasingle s s pore of dc4 transformant and p. grisea dc4 non-transformant were cultured in a medium containing 300 µg/ml hygromycin then the growth diameter was observed at day 7, 14, and 21. the growth of transformant p. grisea increased rapidly which was 14.8 mm at day 7 became 28.7 mm at day 14 and became 37.3 mm at day 21. non-transformant growth was p. grisea slower which was 6.5 mm at day 7, 12.55 mm at day 14 and 17.3 mm at day 21 (fig ). . 3 hygromycin added into the oa medium was able to inhibit the growth of non-p. grisea transformants compared to dc4 p. grisea transformant (fig 4). this was caused by sgfp . the inhibiting activity of hygromycin on the p. grisea dc4 non-transformant. the use of an appropriate selection marker is essential for the success of transformation. selection using an antibiotic is performed to eliminate nontransformant cells as well as to ensure the resistance level carried by the transformant (frandsen 2011). in this research dc4 p. grisea transformants were resistant to hyg o-sgfp r mycin because of the addition of the hygromycin-resistance gene contained in the pcamb plasmid. betts (2007) used sgfp et al. the concentration of 300 µg/ml for the selection of both transformant and non-m. grisea transformant. in contrast, shanti (2008) used a concentration of 225 µg/ml hygromycin to inhibit 173 (originated from rice plant) p. grisea non-transformant and transformant generated through speroplas. the difference in growth rate between the transformant on hygromycin medium and non-hygromycin medium (fig. 4) was likely due to the influence of the number of gene copies integrated, however, further evidence for this is required. f 2 single spore race igure . of dc4 at day 5 grown in oa medium containing 300 µg/ml hygromycinp. grisea biotropia vol. 22 no. 1, 2015 76 figure . average growth rate of dc4 non-transformant and transformant in oa selective medium 3 p. grisea sgfp containing 300 µg/ml hygromycin figure 4. growth of dc4 non-transformant (nt) and transformant (t) in oa medium containing 300 µg/ml p. grisea hygromycin (hgr+) and without hygromycin (hgr-) analysis of ransformantt putative transgenic dc4 from grass that p. grisea had been selected in hygromycin medium was then analyzed using pcr with specific primers sgfpsgfpsgfp-f and r as well as f and nos-r primers to detect the presence of gene. sgfp pcr molecular analysis generated amplicons of 643 bp and 899 bp which were in alignment with the positive control amplicon (fig. ). pcr 5 of non-transformant dna did not generate sgfp gene amplicons. this result showed that the sgfp gene was inserted into the fungal genome. the gene was used as an internal b-tubulin control to ensure the amplified dna was in good condition. the pcr using bt1af and b-tubulin bt1ar primers generated amplicons of 550 bp (fig ). 5 . p. grisea sgfp dc4 with gene insertion was the bright green fluorescence observed using a fluorescence microscope (fig. 6). this showed that the gene was well integrated in the sgfp fungal genome and constitutively expressed in p. grisea sgfpdc4. the use of requires a promoter which is needed for expression. the sgfp pcamb plasmid receiving gene sgfp sgfp insertion from the pct74 plasmid was able to express the gene under the control of the sgfp toxa promoter (lorang 2001).et al. 77 introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. conclusions pathogenic dc4 from had been p. grisea d. ciliaris successfully transformed with the gene sgfp using -a. tumefaciens-mediated genetic trans formation. the gene had been integrated sgfp into the genome. mycelia had p. grisea fluorescence been observed under a fluorescence microscope. acknowledgements thanks to dr. osbourn from john innes institute, england who provided plasmid pcamb. thanks to margaret cargill and sgfp pattric o'connor for early reading this manuscript. this research was funded by imhere b2c titled “biological role of rice blast disease to develop rice plant tolerance to rice blast” under the name of dr utut widyastuti, ag reement letter no : 12it3.24.4/spp-i-mhere/2012. references balhadere pv, talbot nj. 2001. pde1 encodes a p-type atpase involved in appressorium-mediated plant biotropia vol. 22 no. 1, 2015 78 figure . pcr results of dc from transformation5 4 . m: marker 1 kb; k+ dan : plasmid pcamb-p.grisea sgfp sgfp-nos sgfp -tubulin p. grisea p. grisea , k+ : ras 173 (positive control); k-: ddh2o (negative control); 1: dna dc4 β nontransformant; 2-3: dna dc4 transformant p. grisea sgfp figure . dc4 transformant mycelium 7 hours after spore harvest observed under microscope without and with 6 p. grisea sgfp fluorescent infection by the rice blast fungus . magnaporthe oryzae science direc 44:1035-49t . betts mf, tucker sl, galadima n, meng y, patel g, li l, donofrio n, floyd a, nolin s, brown d .et al 2007. development f igh hroughput ransformation o a h t t s f i m iystem or nsertional utagenesis n magnaporthe oryzae. science direct 44:1035 49. . combier jp, melayah d, raffier c, gay g, marmeisse r. 2 0 0 3 . m e d i a t e d a g r o b a c t e r i u m t um e f a c i e n s transformation as a tool for insertional mutagenesis in the symbiotic ectomycorhizal fungus hebeloma cylindrosporum. microbiol lett 220:141-48. couch bc, khon lm. 2002. a multilocus gene genealogy concordant with host preference indicates segregation of a new species, , magnaporthe oryzae from mycologia 94(4):683-93.m. grisea. couch bc, fudal i, lebrun mh, tharreau d, valent b, van kim p, notteghem jl, kohn lm. 2005. origins of host-specific populations of the blast pathogen magnaporthe oryzea in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice. genetics 170:613-30. covert sf, kapoor p, lee mh, goh j, yoo sy, park j, jung k, kim h, park sy, rho hs . 2001. et al agrobacterium tumefaciensfusarium mediated transformation of circinatum. myco res 105:703-9. dos reis mc, pelegrinelli fungaro mh, delgado duarte rt, furlaneto l, furlaneto mc. 2004. agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus . j beauveria bassiana microbiol methods 58:197-202. dagdas yf, yoshino k, dagdas g, ryder ls, bielska e, steinberg g, talbot nj. 2012. septin-mediated plant cell invasion by the rice blast fungus magnaporthe grisea. science. 336:1590-5. departemen pertanian, direktorat jenderal tanaman pangan. 2012. evaluasi rakiraan erangan tama opt p s u utama tanaman padi, jagung, kedelai mt 2011/2012. jakarta (id): deptan. frandsen rjn. 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(id): institut pertanian bogor. hao x, ji y, liu s, bi j, bi q, pan j, zhu x. 2012. optimized integration of t-dna in the taxol-producing fungus . african j biotec pestaliopsis maliocola h 11(4):771-6. hooykaas pjj, roobol c, schilperroot ra. 1978. regulation of the transfer of ti-plasmid of agrobacterium tumefaciens . microbiol 110:99-109. knight cj, bailey am, foster gd. 2009. -agrobacterium mediated transformation of the plant pathogenic fungus plant pathol 91(3):745-verticillium albo-atrum. 50. listiyowati s, utut w, rahayu g, hartana a, jusuf m. 2011. diversity of scar markers of raced pyricularia grisea from following cross infection to digitaria ciliaris rice. microbiol indones 5(1):1-8. lorang jm, tuori rp, martinez jp, sawyer tl, redman rs, rollins ja, wolpert tj, johnson kb, rodriguez rj, dickman mb . 2001. green f u rescent protein l oet al is lighting up fungal biology. appl environ microbiol 67:1987-94. munandhar hk, jorgensen hjl, smedegaard-petersen v, mathur sb. 1998. seedborne infection of rice by pyricularia grisea and it's transmission to seedling. plant dis 82:1093-99. olmedo-monfil v, cortes-penagos c, herrera-estrella a. 2004. three decades of fungal transformation. balbas p, lorence a, editor. recombinant gene expression. reviews and protocols volume 267 methods in : molecular biology. ess ptotowa (us): human pr . 297309. rho hs, kang s lee yh. 2001. -, agrobacterium tumefaciens mediated ransformation of he lant athogenic t t p p f .ungus, . mol cells 12:407-11magnaporthe grisea sesma a, osbourn ae. 2004. the rice leaf blast pathogen undergoes developmental processes typical of rootinfecting fungi. natur 431:582-86.e. shanti nr. 2008. introduksi gen ( ke dalam serin gfp sgfp) genom . kripsi. bogor : institut pyricularia grisea s (id) pertanian bogor. sobir, andrianyta h, amir m. 2003. scar (sequence characterized amplified region) analysis for blast resistant evaluation on 12 genotypes of rice. bul agron 31(1):21-5. tucker sl, orbach mj. 2007. -mediated agrobacterium transformation to create an insertion library in magnaporthe grisea. ronald pc, lorence a, editor . plant-pathogens interaction methods and protocols; methods and protocols volume 354 methods in olecular iology . : m b totowa (us): human pr . 57-67.ess p wise aa, liu zz, binns aw. 2006. three methods for introduction of foreign dna into . agrobacterium methods in mol iol 43-54b . 79 introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. preliminary study of herbal topical lotion repellent made of betel leaves ( ) and piper betle patchouli oil ( ) mixture against pogostemon cablin yellow fever mosquito ( )aedes aegypti mutiara widawati , and m. umar riandi1* 1 1ciamis unit of vector borne diseases research, national institute of health research and development, indonesian inistry of healthm , pangandaran, ciamis, indonesia r 4eceived 25 february 2014/accepted 7 december 201 abstract exploration of plants as natural repellent against yellow fever mosquito ( ) that transmits dengue virus aedes aegypti to human is still under way betel leaves ( ) could be used as repellent material. the study was aimed to test the . piper betle repellency and safety etel and patchouli oil , potency, of topical lotion repellent of b mixture against yellow fever mosquito. this study used nulliparous of 3-5 days old female yellow fever mosquito. irritation test before the study, was conducted as . five treatments with five replications was implemented to seek the most effective safety test repellent substance i.e. deet lotion for positive control, betel leaves and patchouli oil mixture lotion, betel leaves only lotion, patchouli oil only lotion and base lotion only. the substance effectiveness was determined based on the rejection of yellow fever mosquito to bite human's arms and was analyzed using protective percentage. the irritation test showed no safety concerns. protective percentage analysis showed that modified betel leaves lotion had mean protective power of over 90% for 6 hours the modified lotions had the same protective . the data showed that percentage as the deet (ratio: pp (betel+patchouli)/pp (deet) =0.98; 95% confidence interval 0.93, 1.04; -p value=0.50). this indicated that betel leaves mixed with patchouli oil is potential to be used as safe repellent against yellow fever mosquito. keywords: aedes aegypti, betel leaves, dhf, formulations, lotion, repellent, yellow fever mosquito introduction utilization of synthetic insecticide can cause a resistance of the exposed mosquitoes towards the insecticide compounds when used continuously . insecticides commonly used are available in spray, burnt, electric and lotion forms. spray, burnt, and electric insecticides have dangerous effects to health through inhalation which vapor may easily enter the respiratory system and may even enter the blood stream. various effects including nerve, liver, and respiratory disorders may happen. long-term use may cause cancer (zaim & guillet 2002). lotion repellent is one of alternative insecticides that do not harm the respiratory system because it is directly applied to the skin. the lotion is used to discourage mosquitoes from biting human's skin. generally, the marketed lotion contains active chemical substance called diethyl toluamide (deet). this substance is only allowed in indonesia at the concentration of 15%. deet has negative effects i.e. skin irritation, hives (urticaria), and even brain malfunction (encephalopathy) the use of essential oils as . direct repellents is less effective because of its volatile nature. therefore, alternative repellent form may be developed to lengthen the repellent's durability. in this study, essential oil used as fixative agent in developing mosquito repellent lotion was patchouli oil extracted from patchouli (pogostemon cablin) . lotion is a clear preparation that does not leave stain on its user. in addition, lotion is a liquid form, thus facilitating the active ingredient inside the lotion to be evenly absorbed through the skin providing protection to its user. adding fixative biotropia vol. 22 no. 1, 2015: 45 51 doi: 10.11598/btb.2015.22.1.378 45 * corresponding author : mutiara_61@yahoo.com mailto:mutiara_61@yahoo.com biotropia vol. 22 no. 1, 2015 46 agent to repellent substance increases the potency of repellent lotion to function as long as deet lotion. patchouli oil has 100% protection against yellow fever mosquito ( )a aegypti . for 2 hours under betel leaves ( laboratory condition. piper betle) are among therapeutic plants growing in indonesia. the leaves contain saponin, phenolic, alkaloid, and substance that can be used as mosquito repellent . eugenol is essential oil extracted from clove leaves proven to control mosquito larvae and adult mosquitoes or repel mosquitoes. betel leaves contain lower concentration of eugenol (30%) compared to eugenol concentration in clove leaves (71%). the objective of this study was to test the repellency, potency of topical and safety lotion repellent of betel and patchouli oil mixture against yellow fever mosquito. materials and methods rearing nulliparous mosquitoes colonies of yellow fever mosquito ( ) a. aegypti were reared in the insectarium at laboratory of health research, ciamis unit of vector borne diseases research, national institute of health research and development, indonesian inistry m of health at pangandaran, ciamis, indonesia. the hatched larvae were held in plastic trays and larval diet was added to each tray. newly emerged pupae were transferred to screen cage (size 30x30x30 cm ) and emerged as adult mosquitoes. 3 these adult mosquitoes were kept in the insectarium with ambient temperature (25-30 c) o and were provided with soaked cotton balls containing 5% multivitamin solution. production of betel+patchouli mixture lotion the first lotion base solution was produced by weighing carbopol ultrez 10, adding it to 3 g of 10 ml dispersedwater until it . stir the solution completely until there were no trapped air bubbles. finally, add trietanolamine into the 0.50 g solution. this was called the first lotion base solution. the next step was adding lycerine 2 g of g to the first lotion base while being stirred solution to achieve homogenous substance. then, the remaining trietanolamine was added. 0.25 g of this was called the second lotion base solution. the next step was diluting methyl paraben 0.10 g in water which was then added to the 10 ml of second lotion base solution and stirred to achieve homogenous mixture. this was called the final lotion base solution. afterwards, betel oil 2 ml of in 95% ethanol 0.2 ml was mixed with patchouli oil and then was added to the final in 95% ethanol lotion base solution while being stirred until the solution became homogenous to obtain the formula of anti-mosquito lotion with optimal concentration. after all of ingredients were homogenously mixed, ml distilled water was 80 added to the mixture subsequently, ph was . measured and verified at the range of 4-7. test on human subject for lotion safety tests, 20 adult male and female volunteers 25-45 years with age range of old weight 50-70 kg and body range of were recruited for the efficacy tests, other f. ive adult male volunteers with age range of 25-45 years old and body weight range of 50-70 kg who had no history of allergy to arthropod bites were recruited. before signing an informed consent form, the volunteers were interviewed and explain about the methodology, probable ed discomforts and remedial arrangements. testing the biological stability s and afety lotions were stored in closed vials for up to six months and stability of the fraction was determined in conditions at varied room temperatures. testing was lotion safety conducted on 20 volunteers. their skin was applied with 4 treatments for 4 days, one treatment each day. s (betel + the treatment were deet, modified patchouli mixture) betel lotion, lotion (2% betel mixed with lotion), patchouli lotion (0.2% patchouli mixed with lotion). lotion was applied and the reaction was observed for 6 hours after application. laboratory repellent bioassay the lotions were tested for repellency against yellow fever mosquito ( ) by a. aegypti following indonesian pesticide commission guidelines under laboratory conditions. six hundred 3 to 5 days old female nulliparrous mosquitoes were taken from a stock cage using an preliminary study of herbal topical lotion repellent made of betel leaves ( ) and mutiara widawatipiper betle – et al. aspirator and placed inside six cages. each cage had a length of 50 cm, width 35 cm and height 40 cm and was made of nylon netting, iron wire framed, with 2 holes on the front to insert hands. each cage consisted of 100 mosquitoes. the night before being exposed to the blood meal they were deprived of the multivitamin solution and supplied with water pads only. six hours prior to the blood feeding, the water soaked cotton pads were removed. he assays were done in a well-t ventilated room that is equipped with 6 tables and 6 chairs, with temperature of 26-30 c and relative o humidity around 60-80%. thirty minutes before starting the test, the left forearm of a human subject was treated with repellent there were 5 test repellents i.e. lotion . base, deet, betel+patchouli mixture, betel 2%, and patchouli 0.2%. each repellent was tested on one subject. one gram of repellent was equally applied on 650 cm of skin surface area2 between the left elbow and wrist. the area between the elbow and wrist served as negative control by applying water on the skin surface. a latex glove was worn over the hand to protect from mosquito bites. tests were conducted by placing the repellent-treated (left) forearm into a test cage for 5 minutes, and any mosquitoes that had taken a blood meal were replaced. the control (right) forearm was placed into the test cage for 5 minutes, immediately after placing the repellenttreated (left) forearm into the test cage. these tests were conducted at 60 minutes intervals for 6 hours starting from the moment when the treatment applied (hour 0: 08.00 am) was , and the number of landing mosquitoes was recorded. landing mosquitoes were blown from the arm, in an effort to stop any blood from being taken. repellency data were as protective presented percentage (pp). the whole procedure was repeated same volunteers n on the i 5 separate days. during testing, body temperature of the human as well as ambient temperature, subject, humidity and lighting were checked. each test repellent was tested on 35 separate occasions (each hour for hours, repeated in 5 0-6 days). for each test repellent, the protection percentage at each of the 35 occasions was calculated using the following formula: where: c = the number of mosquito landings on the control (right) forearm t = the number of mosquito landing on the repellent-treated (left) forearm statistical analysis to calculate confidence intervals for the pp for each hour, the percentile of confidence interval were set on a parametric bootstrap with 10,000 replications. it was assumed that the number of landings on each arm is in accordance with poisson distribution with mean determined by the sample mean from the 5 days. there was no over dispersed data in the data distribution. the pp for different test repellents was calculated using a ratio. the 95% confidence interval was determined using the percentile parametric bootstrap with 10,000 replications using similar assumptions as with the pp confidence intervals. results and discussion safety and preference subjectbased on human safety test, 10 s thought betel+patchouli did not that the lotions give any effect , 6 s s subject thought that the betel+patchouli lotion was good subject, and 4 s thought that the betel+patchouli lotion was very good. deet and lotion did not betel+patchouli show irritation . n atchouli lotion test, 1 any s i the p out of 20 skin rash subjects experienced . in the betel subjects lotion test, 2 out of 20 showed skin ras 2h (table ). efficacy experiment subjects had body temperature d range between c environmental temperature 36-36.5 , 0 was 27-28 was 89-95%, and lighting c, humidity0 was 12-87 lux. environment the conditions were considered as confounding variables because they might tamper with the condition of the mosquitoes and human subjects, and it was hard to control. efficacy experiment showed that human subjects applied with deet and lotion had stable body temperature during experiment. 47 pp = 100 × c t c according to the results, deet and modified betel lotion had a protection n estimated percentage of more than 90% from yellow fever mosquito ( ) for the 6-hour duration. a. aegypti although the number of mosquito landings decreased quite drastically during the fourth hour, since a similar finding occurred in the control group, it did not clearly influence the magnitude of protection. during the fourth hour, when the test performed after 12:00, the activity of the mosquitoes started to decrease. this might be explained by the cycle of mosquito's life, where their resting period lied after 12:00. thus, the mosquito perching started to decrease (chadee 2013). betel leave contains various chemical compounds, including saphonine, phenolic, alkaloids, and substance which can be used as repellents . betel leave (paluch . 2010)et al commonly contains 30% eugenol (bhalerao . et al 2013) which has been proven for controlling biotropia vol. 22 no. 1, 2015 48 table 1 overall impression of lotion from 20 s. betel+patchouli mixture subject subject overall impression not good regular good very good 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 + 16 + 17 + 18 + 19 + 20 + table . number of adverse events by treatment out of 20 subjects tested (with percent).2 treatment skin rash itch swollen no adverse events deet 0 0 0 20 (100%) modified (betel+patchouli mixture) lotion 0 0 0 20 (100%) betel lotion 2 (10%) 0 0 18 (90%) patchouli lotion 1 (5%) 0 0 19 (95%) 49 larvae and adult mosquitoes (eliningaya 2008). other study also reported that eugenol derived from clove leaves (70-93%) has a potential to repel mosquitoes. although the composition of betel leaves were not as eugenol-rich as clove leaves, it also has the potential to repel yellow fever mosquito ( ) after some modifications.a. aegypti betel lotion mosquitoes repellent produced in this study is targeting and antennae of palpi mosquitoes because these two structures on mosquito body are most sensitive to the aroma of modified lotions. the aroma of plant extracts could cover the scent of the human body and thus impairing the mosquito's ability to detect the presence of humans (stella et al. 2010) . based on the safety test, this preliminary test showed that our modified repellent potentially safe to be is used in repellency test. the control arms were comparable landing between the 4 subjects who got the test repellents, while the control arm of subject who got the base only had substantially fewer mosquito landings. figure 1. number of mosquito landings for the control arm (dotted lines) and the repellent-treated arm (solid lines) for each of the 5 days (different colors) preliminary study of herbal topical lotion repellent made of betel leaves ( ) mutiara widawatipiper betle – et al. biotropia vol. 22 no. 1, 2015 50 the mean protection percentage (pp) for each test repellent was presented with 95% confidence intervals. mixture of betel+patchouli lotion performed very similarly to deet at every hour, while the betel 2% only matched the deet until hour 3, by hour 4 its pp dropped substantially. at 6 hours, deet and betel+patchouli had pp around 90%. deet had pp = 92.2% (95% ci 88.1; 95.2), while betel+patchouli had pp = 90.4% (95% ci 86.7; 93.4). both deet ( = 0.29) and p betel+patchouli ( = 0.85) were not significantly p different from 90%. the “base only lotion” provided no protection, and the patchouli 0.2% provided poorer protection compared to other active repellents. the betel+patchouli had about the same protection power as the deet (ratio: pp (betel+patchouli)/pp (deet) = 0.98; 95% confidence interval 0.93; 1.04; -value = 0.50), p while betel 2% alone had significantly less protection (ratio: pp (betel)/pp (deet) = 0.78; 95% ci 0.71; 0.86; < 0.001).p ata analysis was expected to be the d uninterrupted for the by environmental aspect. comparison of pp between different test repellents, each test repellent was tested on only one subject. this is a limitation of the design. however, each subject was acting as his or her own control o systematic biases on the landing , s counts (e.g. caused by personal odor of a subject) might affect both the control and the repellenttreated arm. these biases might cancel out when pp was used. moreover, the systematic bias caused by always starting the test with the control arm at each hour. for comparing pp for different test repellents, both test repellents will have that same bias, and it may be canceled out in the comparison. deet performed well. he lotion t modified could be considered as a repellent due to potential its 90% protective power for hours and lack of 6 safety concerns. although deet protective power was high, betel lotion might work as an alternative for deet. the main problem of natural repellent is durability. several studies have shown that natural ingredients rarely fulfill the requirements of repellent effectiveness. however, the modification of formulation by adding patchouli oil as a fixative increased the potential of natural ingredients to be used as repellent. conclusions modified betel (betel+patchouli mixture) lotion had shown repellent potential for yellow fever mosquito ( ) mosquitoes with aedes aegypti estimated mean protection power of 90. % 6 4 at hours. this study has opened up the possibility for further research to assess the effects of this repellent formulation, and modifications of other natural repellent plants. figure 2. mean protection power with 95% confidence intervals (some cis were hidden, since they were not as large as the mean symbol). the points were slightly shifted at each hour so that the different test repellent points do not overlap 51 acknowledgements authors would like to thank to michael fay for statistical assistance. the author would also like to thank to infectious disease ina responds group mentors, ibu emil, ibu inge, and mr. joshi for their suggestions, member of loka litbang ciamis for their contribution in this research assays and ina respond for their assistance s in making manuscript writing workshop. this research was funded by balitbangkes as one of risbinkes project. references bhalerao sa, verma dr, gavankar rv, teli mc, rane yy, drawana vs, tri kanned a. 2013. phytochemistry, pharmacological profile and therapeutic uses of piper betle linn. an overview. j pharmacogn phytochem 1(2): 10 9.bhuiyan mni. 2012. constituents of the essential oil from leaves and buds of clove ( (l.) syzigium caryophyllatum alston). ajpp 6(16): 1260-3. badan pengawas obat dan makanan. 1985. ormularium f kosmetik indonesia. jakarta. chadee dd. 2013. resting behaviour of in aedes aegypti trinidad: with evidence for the re-introduction of indoor residual spraying (irs) for dengue control. parasites & vectors[internet].[cited 2014 01 15]; 6(1): 255. available at:http://www.pubmedcentral. nih.gov/articlerender.fcgi?artid=3847653&tool=p mcentrez&rendertype=abstract. eliningaya njk. 2008. etnobotanical study of some of mosquito repellent plants in north eastern tanzania . malar j 7:152. hemingway j. . 2004. hawkes mj, mc carrao l, ranson h the molecular basis of insecticide resistance in mosquitoes. j insect biochem molec biol 34(7): 653 65.kardinan a. 2010. potensi adas ( ) sebagai foeniculum vulgare bahan aktif lotion anti nyamuk demam berdarah ( ). bul littro 21(1): 61 8aedes aegypti . komisi pestisida departemen pertanian. 1995. metoda standar pengujian efikasi pestisida. jakarta. koreng g, matsui d, bailey b. 2003. deet based insect repellents safety implications for children, pregnant and lactating women. can med assoc j 169: 209 12.loka litbang p2b2 ciamis. 2010. standard operational procedure rearing nyamuk. ciamis. paluch g, bartholomay l, coats j. 2010. mosquito repellents: a review of chemical structure diversity and olfaction. pest management science 66(9): -925 35. stella l, olivero-verbel j, stashenko e. 2010. repellent activity of essential oils: a review. bioresour technol j [internet]. [cited 2013 12 26]; 101(1): 372 78. available at: http://dx.doi.org/10.1016/ j.biortech.2009.07.048. tawatsin a, thavara u, chansang u, cahavittumrung p, thidarat b, wongsin kongman p, bansidhi j, mulla ms. 2006. field evaluation of deet, repel care, and three plant based essential oil repellents against mosquitoes, black flies (diptera: simuliidae) and land leeches (arhynchobdellida: haemadipsidae) in thailand. j am mosq control assoc 22(2): 30613. trongtokit y, rongsriyam y, kolamisran m, apiwath nasurn c. 2005. comparative repellency of 38 essential oils against mosquito bites. phytother res 19(4): 3039. zaim m, guillet p. 2002. alternative insecticides: an urgent need. trends parasitol 18(4): 1613. preliminary study of herbal topical lotion repellent made of betel leaves ( ) mutiara widawatipiper betle – et al. http://www.pubmedcentral. http://dx.doi.org/10.1016/ 702 sujan balami (effect of invasive) revisi tambah gambar.cdr effect of invasive on species ageratina adenophora richness and composition of saprotrophic and pathogenic soil fungi sujan balami, lal b. thapa and sanjay kumar jha * central department of botany, tribhuvan university, kirtipur 44618, nepal received 29 september 2016/accepted 3 september 2017 abstract belowground modification of soil microbial community by invasive plants is well evident. similar instances of ageratina adenophora invasion have been reported. this study was aimed to determine the effect of a. adenophora invasion on species richness, species or community composition and occurrence frequency of soil fungi. these parameters were analyzed using culture method on invaded and uninvaded soils. species richness of soil fungi was lower in the a. adenophora invaded soil compared to the uninvaded soil. the occurrence frequency of particular fungi was different for those two soil conditions. a. adenophora also altered soil fungi species composition in the invaded soil by replacing saprophytic fungi and accumulating pathogenic fungi. thus, a. adenophora is associated to lower species richness of saprophytic soil fungi and high occurrence frequency of pathogenic soil fungi. this study concluded that the invasive a. adenophora modifies belowground soil fungi communities as one of the mechanisms involved in the successful invasion of a. adenophora. keywords: belowground modification, soil fungi, species composition, species richness introduction uncoupling of ecosystem dynamics and consequent threat to biodiversity loss are attributed to invasion of alien plants (iaps) (webster et al. 2006; boy & witt 2013). the invasive alien species have adopted multiple strategies to proliferate (holzmueller & jose 2009) in an introduced range and able to compete with native plants for resources and space (mack & d' antonio 2003). various experimental evidences showed that modification in soil biota is one of strategies involved in the invasion of iaps (xiao et al. 2014a). the iaps release myriads of chemicals from their above and belowground parts into soils by leaching process and exudation (dayakar et al. 2009). these chemicals are capable of altering soil biophysical properties and ultimately, plant diversity (garbeva 2008; doornbos 2012; thapa et al. 2016a). in response to this, aboveground biota sharing the same niche with the iaps invariably regulates the changes (darrah 1993) by applying a feedback system (van der putten et al. 1993). soil microbial community has either negative or positive feedback to the plants (bonanomi et al. 2005). for example, negative feedback may involve pathogenic effect, production of bioactive compounds and production of allelochemicals, while positive feedback involves root fungus mutualism and secretion of plant growth promoting substances (bias 2006). et al. on the other hand, regulation in nutrient cycle is a result from plant-soil feedback system (johnson et al. 1997). such a plant-soil feedback system determines diversity and relative abundance of the above and belowground organisms (van der putten 1993).et al. plant soil microbe studies in iaps shows that they can modify soil microbial communities (kourtev et al. 2002; van der putten et al. 2007) for successful invasion (reinhart & callaway 2004; li et al. 2006; boudiaf et al. 2013). negative feedback mechanisms due to iaps may result to the decrease in competitive abilities of native species (ehrenfeld et al. 2005). the negative feedbacks are also recognized in biomass and growth of native * corresponding author: lal_thapa25@yahoo.com biotropia 4 3 7 212 219 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 702 212 plants (stinson et al. 2006), which might be hostspecific, leading to a decline of native plants population (didham et al. 2007). ageratina adenophora (spreng.) r. m. king and h. robinson, an invasive alien plant of asteraceae family, is a perennial, erect or decumbent subshrub (tiwari 2005), natively found in et al. mexico and costa rica and spread worldwide (niu 2007; xue 2010; inderjit 2011). et al. et al. et al. this species contains an array of bioactive constituents such as terpenoids, flavonoids, phenyl propanoids and their derivatives (kundu et al. 2013). several mechanisms have been suggested as the reasons of successful invasion of iaps including in their novel range. most a. adenophora studies on iaps focused on aboveground vegetation changes (levine 2003). a wide et al. range of belowground biotic interactions can play important role in determining plant interactions and ecosystem function (callaway 2001). et al. therefore, it is important to understand the interactions between iaps and soil biotic community and their interactions (wolfe & klironomos 2005). moreover, an integrated understanding of how aboveground and belowground biota interact with iaps is necessary to manage and restore alien invaded native communities (wolfe & klironomos 2005). there are some studies on modifications in the belowground biotic community associated with a. adenophora invasion, for example mangla and callaway (2008) and niu (2007). in nepal, et al. severe invasion of occurred from a. adenophora tropical to subtropical regions affecting native schima-alnus and other types of vegetation (tiwari 2005; thapa 2015; 2016b; 2017). et al. et al. hence, this study hypothesized that a. adenophora is responsible for changing species richness, species or community composition and occurrence frequency of the native soil fungi as invasion mechanisms of in a. adenophora nepalese forests. materials and methods the experiment was designed for comparing species richness of soil fungi, species composition and occurrence frequency between a. adenophora invaded and uninvaded soil. the invaded soil consisted of invaded non-rhizosphere soil and rhizosphere-contained invaded soil. the experiments were conducted at the central department of botany, tribhuvan university, kathmandu, nepal in march and august 2014. sampling site and method of soil sampling soil samples were collected from champadevi community forest located at southwest of kathmandu valley, nepal. altitude of the location varied from 1,400 – 2,300 m asl with annual mean o temperature of 18 c and annual precipitation of 1,343 mm. schima wallichii, alnus nepalensis, myrsine capitelata, maesa chisia, castanopsis indica and quercus sp. were common native trees in the forest. the forest was invaded by a. adenophora for 40 50 years and the invasion has become a serious challenge for the native diversity of the area due to its severe colonization (thapa et al. 2016b). uninvaded soil was collected from the forest site located at 27˚42ʹ06″ n and 85˚19ʹ14″ e with altitude of 1,600 m. rhizosphere-contained invaded soil was collected by uprooting a. adenophora. a total of 30 plants were uprooted from three sampling sites of the invaded patches nearby the uninvaded site, 10 plants from each sampling site (distance between each sampling site was at least 50 m). the soil around root surfaces was collected in sterile plastic bags and a composite sample was prepared. invaded non-rhizosphere and uninvaded soil samples were collected from 10 random points at the invaded and uninvaded patches of the forest. soil samples were collected from soil depth of 10 cm below soil surface. composite sample of each invaded non-rhizosphere and uninvaded soil was prepared. freshly collected soils were sieved separately through sterile mesh (2 mm o size), and stored in a refrigerator at 4 c for 5 days, until use. fungi culture, isolation and identification serial dilution method (benson 2002) followed by pour plate technique were adopted for culturing and isolating fungi from all types of -3 -5 composite soil samples. dilutions of 10 and 10 were used for plating (aneja 2003). czapek dox agar [with 30 mg/l (amoxicillin)] and potato dextrose agar were used for culture, isolation and pure culture. the culture plates were o incubated at 25±5 c (gallenkamp economy incubator size 1) for 15 days. there were 90 replication plates for each soil sample, where the presence or absence (1/0) of particular fungi was 213 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. followed by three species from class zygomycota, one species from basidiomycota and one actinomycetes. species richness of soil fungi in the uninvaded soil was greater (28 species) than that in the invaded soil ( < 0.05). twenty soil fungi species p were recorded in the rhizosphere-contained invaded soil. twenty two soil fungi species were recorded in the invaded non-rhizosphere soil (table 1). these results indicated that a. adenophora reduced species richness of soil fungi. these findings also clarified the interactions between soil fungi and invasion of , a. adenophora which was still contradictory (mangla callaway & 2008). czapek dox agar medium was used to culture and isolate soil fungi targeting saprophytic or pathogenic soil fungi, as this media was proven to be appropriate for culturing and isolating common saprophytic and pathogenic fungi (abildgren et al. 1987). this study proved that soil fungi enumeration favored czapek dox agar medium. other studies showed that a. adenophora increased the abundance of mycorrhizal soil fungi which sug gested that mycorrhizal recorded in each replication plates. fungi grown in the plates were observed at day 3, 7 and 15 and identified based on standard literature (barnett & hunter 1960; gilman 1975; watanabe 2010). a data matrix of species was prepared statistical analysis species richness of soil fungi in different soil samples was compared using one-way analysis of variance (anova). frequency rank curve was used to compare the occurrence frequency of fungi found in different soil samples. species composition of soil fungi was analyzed using n o n m e t r i c m u l t i d i m e n s i o n a l ( n m d s ) technique. the analyses were carried out using r software (version 2.15.3) (r core team 2015). the acceptable significance level was p < 0.05. results and discussion species richness a total of 34 soil fungi species were found in soil samples (table 1). four species twenty-nine were reported from division ascomycota biotropia vol. 24 no. 3, 2017 214 table 1 total numbers of soil fungi species found in different soil type soil type total species uninvaded soil 28a a. adenophora invaded non-rhizosphere soil 22b a. adenophora invaded rhizosphere soil 20 b figure 1 species composition of soil fungi in the invaded soil, rhizosphere-contained invaded soil and uninvaded soil based on nmds analysis (final stress = 0.0002163944, k = 2, distance measure = “jaccard” distance, trymax = 1,000) colonization induced positive feedback to enhance the invasiveness of a. adenophora (niu et al. 2007; yu et al. 2011; and xiao et al. 2014b). comparing the results of those studies with the results of present study, it is suggested that a. adenophora can replace certain groups of soil fungi commonly found in the uninvaded soil. analysis of soil fungi species composition (fig. 1; table 2) and frequency ranking curve (fig. 3) showed the response of soil fungi species toward rhizosphere-contained soil, invaded soil and uninvaded soil. species composition and occur rence frequency nmds analysis showed that species composition of soil fungi varied in accordance with soil types. species compositions recorded in the invaded non-rhizosphere soil and the rhizosphere-contained invaded soil were different from that in the uninvaded soil. soil fungi species such as curvularia sp., alternaria alternata, colletotrichum sp., acremonium sp., aspergillus fumigates, verticillium sp. and fusarium moniliforme occurred more frequently in the rhizosphere-contained invaded and the invaded non-rhizosphere soils. other soil fungi species such as absidia sp. and chaetomium funicola were exclusively occurred in the uninvaded soil (fig. 1 & 2). species belonging to genera alternaria, colletotrichum, fusarium, verticillium, acremonium 215 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. table 2 soil fungi species and respective species codes depicted on nmds space in figure 1 sn fungi name abbreviation 1 absidia sp. abs 2 acremonium sp. acrs 3 alternaria alternata alta 4 alternaria sp. alts 5 aspergillus a aspa 6 aspergillus b aspb 7 aspergillus fumigates aspf 8 aspergillus niger aspn 9 aspergillus fumigates aspf 10 bipolaris sp. bips 11 curvularia sp. curs 12 cladosporium sp. clas 13 melanospora sp. mels 14 cunninghamella sp. cuns 15 fusarium oxysporum fuso 16 fusarium moniliforme fusm 17 fusarium ciliatum fusc 18 gliocladium sp. glis 19 mucor sp. mucs 20 penicillum a pena 21 penicillium b penb 22 penicillium c penc 23 penicillium d pend 24 penicillium e pene 25 rhizophus sp. rhis 26 rhizoctonia sp. rhizs 27 staphylotrichum sp. stap 28 streptomyces sp. stre 29 trichoderma harzianum trih 30 trichoderma koningii trik 31 verticillium sp. verts 32 gonytrichum sp. gons 33 chaetomium funicola chaf 34 colletotrichum sp. cols note: a, b, c, d and e are arbitrarily given specific names for unidentified fungi (consis ting more than one species) to species level and curvularia were contributors in species composition of soil fungi in the invaded soil, whereas the species belonging to genera aspergillus, penicillium, absidia, chaetomium were major contributors in forming species composition of soil fungi in the uninvaded soil (fig. 1 & 3). these results supported hypothesis of soil biota alteration as proposed by various researchers (wolfe & klironomos 2005; si et al. 2013). goodness of fit for environmental factors (represented by soil types) in figure 1 was 2 significant in ordination space (r = 0.29, p < 0.01). permutational anova (permanova) showed that species composition of soil fungi significantly varied in accordance with soil types (p < 0.05). nmds analysis showed that species composition of soil fungi was changed by the invasion of a. adenophora (fig. 1). alteration of species composition could be caused by differences in species richness in the invaded and uninvaded soils. species composition of soil fungi in the rhizosphere-contained invaded soil and invaded non-rhizosphere soil was not different (fig. 1). species richness of soil fungi in those two soil types was similar (table 1). frequency rank analysis showed that a. alternata, penicillium sp., curvularia and fusarium oxysporum were the frequently occurred soil fungi in the rhizosphere-contained invaded soil (fig. 3). similarly, f. oxysporum frequently occurred in the invaded non-rhizosphere soil followed by curvualria sp., penicillium sp. and a. alternata (fig. 3). rhizophus sp. and cunninghamella sp. were the least frequent in rhizospherecontained soil. rhizoctonia sp., rhizophus sp., verticillium sp. were the least occurred soil fungi species in the invaded non-rhizosphere soil. trichoderma harzianum was the most frequently found soil fungi in the uninvaded soil followed by penicillium t. knoningii 3 sp. and (fig. ). the least 216 biotropia vol. 24 no. 3, 2017 figure 3 frequency rank curve of (a) rhizosphere-contained invaded soil; (b) invaded non-rhizosphere soil; and (c) uninvaded soil figure 2 fungal plate of chaetomium funicola frequently found soil fungi species in the uninvaded soil were sp. sp.curvularia , alternaria , staphylotrichum aspergillus .sp. and sp occurrence frequency in this study showed a similar tendency of accumulation of pathogenic soil fungi in the invaded soil. nmds and frequency analysis showed that fusarium, colletotrichum alternariaand species were very frequently found in the invaded soil (fig. 1, 3 & ) 4 and these genera are common pathogens (chalermpongse 1987). previous studies in warm tropical humid monsoonal climate of india also reported that and a. adenophora chromolaena odorata accumulated pathogenic fungi (mangla & callaway 2008; mei 2014).et al. on the other hand, soil fungi belonging to genera and were penicillium, aspergillus chaetomium common decomposers (fu-qiang 2004) et al. found in the uninvaded soil (fig. 1 & ). this 3 indicated that saprophytic soil fungi in the invaded soil might be reduced by where a. adenophora pathogenic soil fungi increased. the reduction of saprophytic soil fungi might be important mechanism behind the invasion and affliction of native species by .a. adenophora plant species could determine rhizosphere microbes (both bacteria and fungi) via root exudates, phytoanticipins, phytoalexins or allelochemicals secreted by the plants (bever et al. 2010). presence of allelochemicals in root exudates or leachates from aerial parts such as leaf and litter of (wan 2011; zhang a. adenophora et al. et al. 2013) could be responsible for the decrease of species richness, the alteration of soil fungi species composition and the alteration of occurrence frequency of saprophytic fungi. inderjit (2011) found that is et al. a. adenophora responsible for higher mortality of native species in china and india. similarly, thapa (2017) et al. reported that reduced the growth a. adenophora and development of native seedlings nepal. this study suggested that changes in species richness and species composition of soil fungi might also be responsible in seedling mortality, growth and development of aboveground native plant. in the field, is found in thick a. adenophora stands in the invaded areas of nepal. a. adenophora deposits litter in the soil and leaches substances from aerial parts, including green leaves, during rainy days (thapa 2017). litter et al. accumulation, decomposition in soil and leached substances from aerial parts may have antifungal properties against certain fungi (broeckling et al. 2007). various allelochemicals in aerial parts of a. adenophora such as phenolics, sesquiterpens (zheng 2012) might affect soil microflora or et al. alter soil quality (katherine 2006). et al. additionally, might alter the cycle of a. adenophora soil nutrient which could have affected the abundance or distribution of particular soil fungi species or community in the soil. plant-soil feedback mechanism is important to explain vegetation dynamics and ecosystem function including plant invasiveness. positive feedback is evident through colonization of mycorrhizal fungi by (niu 2007; a. adenophora et al. yu 2011; xiao 2014 ). there might be et al. et al. b negative feedback to the native species through accumulation of pathogenic soil fungi and decrease of saprophytic soil fungi in the invaded soil by a. adenophora. conclusions a. adenophora invasion led to the decrease of soil fungi species richness and altered soil fungi species composition. the invasion facilitated the 217 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. figure 4 fungal plates of fusarium (a) and alternaria (b) (a) (b) 218 biotropia vol. 24 no. 3, 2017 occurrence of saprophytic fungi as well as 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18(11):14096-104. zheng g, zhao x, zhang f, luo s, li s, li w. 2012. ocoumaric acid from invasive eupatorium adenophorum is a potent phytotoxin. chemoecology 22(2):131-8. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 sri-13 mei 2017-550 (mohd khalizan isolation and characteriz).cdr isolation and characterization of a molybdenum-reducing and phenolicand catechol-degrading enterobacter sp. strain saw-2 mohd khalizan sabullah 1,2* 2 2 , mohd fadhil rahman , siti aqlima ahmad , mohd rosni sulaiman , mohd shukri shukor , azlan jualang gansau , 3 1 1 nor aripin shamaan and mohd yunus shukor 5 2 1 faculty of science and natural resources, universiti malaysia sabah, kota kinabalu 88400, sabah, malaysia 2 department of biochemistry, faculty of biotechnology and biomolecular sciences, universiti putra malaysia, serdang 43400, selangor, malaysia 3 faculty of food science and nutrition, universiti malaysia sabah, kota kinabalu 88400, sabah, malaysia 4 snoc international sdn bhd, lot 343, inland port 71800, negeri sembilan, malaysia 5 faculty of medicine and health sciences, islamic science university of malaysia, kuala lumpur 55100, malaysia received 21 october 2015/accepted 10 august 2016 abstract molybdenum is an emerging pollutant worldwide. the objective of this study is to isolate molybdenum-reducing bacterium with the ability to grow on phenolic compounds (phenol and catechol). the screening process was carried out on a microplate. the bacterium reduced molybdenum in the form of sodium molybdate to molybdenum blue (mo-blue). the bacterium required a narrow ph range for optimal reduction of molybdenum, i.e. between ph 6.3 and o 6.8, with temperature between 34 and 37 c. molybdate reduction to mo-blue was best supported by glucose as the carbon source. however, both phenol and catechol could not support molybdate reduction. other requirements for molybdate reduction included sodium molybdate concentrations between 15 and 30 mm, and phosphate concentration of 5.0 mm. the bacterium exhibited a mo-blue absorption spectrum with a shoulder at 700 nm and a maximum peak near the infrared region at 865 nm. the mo-reducing bacterium was partially identified as enterobacter sp. strain saw-2. the capability of this bacterium to grow on toxic phenolic compounds and to detoxify molybdenum made it a significant agent for bioremediation. keywords: catechol, enterobacter sp., molybdenum, molybdenum blue, phenol introduction heavy metals are toxic to organisms. some microorganisms are able to detoxify heavy metals by employing mechanisms such as biosorption, sequestration or chelation, active pumping and reduction (batta et al. 2013; oves et al. 2013; banerjee et al. 2016). microorganisms having capability to reduce heavy metals such as chromium, molybdenum mercury into less toxic forms were documented. molybdenum (mo) is essential to organisms as it is a cofactor to many important enzymes such as nitrogenase, sulfite oxidase, aldehyde oxidase and xanthine oxidoreductase (daniels et al. 2008; leimkühler et al. 2011). molybdenum is not generally toxic to human. however, molybdenum is very toxic to ruminants, such as sheep and cattle at concentrations as low as several parts per million (ppm). an elevated level of molybdenum in r uminants causes a disease known as hypercuprosis (ward 1978). recent data showed that molybdenum is toxic to spermatogenesis and embryogenesis in animals, including catfish and mice (meeker et al. 2008; bi et al. 2013; zhai et al. 2013; zhang et al. 2013). toxic effect of molybdenum to these animals warrants removal of molybdenum from the environment. heavy usage of molybdenum in various steel products and lubricants resulted in high concentration of molybdenum reaching hundreds of ppm in the water bodies of the black sea and tokyo bay biotropia 4 1 7 47 58 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 550 * corresponding author: ejanz_sart2@yahoo.com 47 (davis 1991; neunhäuserer et al. 2001). terrestrially, in the mine tailings of a molybdenum mine in new mexico, as high as 2,000 ppm of molybdenum concentration were documented (runnells et al. 1976). together with heavy metals, oil, grease and phenolics are hydrocarbon which are reported to be the number one scheduled waste (yadzir et al. 2016). phenol and phenolic compounds (fig. 1) are not only toxic to human, but also to many other organisms (shukor et al. 2008a; shukor et al. 2008b; rahman et al. 2009; yunus et al. 2009). the presence of multiple pollutants requires microorganisms with multiple detoxification ability. thus, the objective of this study is to isolate such a microorganism. in this study, we reported on the isolation of a molybdenumreducing bacterium with the ability to grow on phenolic compounds (phenol and catechol). materials and methods chemicals c h e m i c a l s s u c h a s n a m o o . 2 h o, 2 4 2 mgso .7h o, (nh ) .so nacl and na hpo 4 2 4 2 4, 2 4 were purchased from sigma aldrich (st. louis, mo, usa) and were of analytical grade while glucose and yeast extract were purchased from fisher (malaysia). i s o l a t i o n o f m o l y b d e n u m r e d u c i n g bacterium molybdenum-reducing bacterium was isolated using two kinds of media, i.e. low phosphate molybdate (lpm) and high phosphate molybdate (hpm) media. lpm medium consists of the following composition: mgso .7h o (0.05% 4 2 w/v), (0.206% or 10 mm), yeast na moo .2h o2 4 2 extract (0.05% w/v), (nh ) .so (0.3% w/v), 4 2 4 glucose (1% w/v), nacl (0.5% w/v) and na hpo (0.071% w/v or 5 mm). the lpm 2 4 medium was adjusted to ph 7.0. agar having concentration of 1.5% (w/v) was added to the lpm solid media. bacterial cell harvesting from the lpm medium could not be carried out, as blue aggregates formed in the lpm liquid media. hence, the phosphate concentration was increased to 100 mm which made the medium became hpm (ghani 1993). soil materials et al. for the isolation of molybdenum-reducing bacteria were taken from topsoil depth of 5 cm in a polluted area in kuching, sarawak (1.6077° n, 110.3785° e) in april, 2011. a cell suspension containing 1 g of soil and 10 ml of sterile distilled water was prepared. soil suspension of 0.1 ml aliquot was immediately spread on an agar plate containing lpm media. the plates were incubated for 48 hours at room temperature (27 c). several white and blue colonies appeared o afterwards. colony with the strongest blue intensity was restreaked on lpm agar until a pure culture was obtained. hpm media was utilized to prepare for the resting cells of the molybdenumreducing bacterium. growth of the bacterium was carried out in a 1 l culture volume, shaken using orbital shaker, set at 120 rpm, and incubated for 48 hours at room temperature (27 c). cells o were harvested via centrifugation (10,000 xg, for 10 minutes). distilled water was utilized to wash the bacterial pellets after centrifugation twice. the pellets were then resuspended in 10 ml of lpm media (5 mm) with the glucose omitted. preparation of resting cells preparation and use of resting cells in a microtiter format were carried out based on shukor and shukor (2014). cell suspension prepared previously (180 ml) was transferred to each well of a sterile microplate. sterile glucose (20 ml) from a 10% (w/v) stock solution was 48 biotropia vol. 24 no. 1, 2017 (a) phenol (b) catechol (c) 2-chlorophenol figure 1 chemical structure of some toxic phenolic compounds results and discussion bacterial molybdenum reduction to mo-blue is a phenomenon that has been described for more than one hundred years and is a prospective bioremediation tool (shukor et al. 2014). this phenomenon was initially observed in 1896 in e. coli bacterium (levine 1925). comprehensive research about this phenomenon was only started in 1985 in the e. coli k12 bacterium (campbell et al. 1985). later on, sodium molybdate reduction into mo-blue by the thiobacillus ferrooxidans bacterium, a chemolitotroph, was reported by sugio et al. (1988) without mentioning the works of campbell et al. (1985). this suggested the scarcity of publications on this phenomenon. enterobacter cloacae strain 48 (ec 48) is the first bacterium isolated from the malaysian soils with the capacity to reduce molybdate (ghani et al. 1993). t he first molybdenum-reducing bacterium reported with the ability to detoxify other xenobiotics is the sds-degrading klebsiella oxtoca (halmi et al. 2013). isolation of more xenobiotics-detoxifying molybdenum-reducing bacterium can expand the capability of existing isolates for bioremediation of sites containing molybdenum, together with other xenobiotics. partial identification of the molybdenumreducing bacterium the bacterium was gram-negative, rodshaped and motile. the biochemical test results using the abis online software showed that bacterium having the highest homology (90%) and the highest accuracy (97%) was enterobacter cloacae (table 1). the bacterium is tentatively named enterobacter sp. strain saw-2. identification of the bacterium up to the species level can only be carried out through the addition of several more polyphasic methods, such as16s rrna gene sequencing, dna–dna hybridization determination of genomic dna g+c content and fatty acid profile (ahmad et al. 2013). two molybdenumreducing bacteria from this genus, i.e. enterobacter cloacae strain 48 (ghani et al. 1993) and enterobacter sp. strain dr.y13 (shukor et al. 2009) were previously reported. a bacterium from this genus has been reported for having ability to tolerate high concentrations of heavy metals such as nickel, cadmium and lead at levels of up to 700, mixed with the cell suspension. the microplate was covered with a sterile sealing tape (corning® microplate). the microplate was incubated at o room temperature (27 c) for 48 hours. mo-blue production was determined at 750 nm in a microtiter plate reader model no. 680, (biorad, richmond, ca). the specific extinction -1 -1 coefficient of 11.69 mm cm was utilized to determine mo-blue concentration (shukor et al. 2003). heavy metals such as lead (ii), arsenic (v), mercury (ii), silver (i), chromium (vi) copper (ii) and cadmium (ii) on mo-blue production were obtained from atomic absorption spectrometry (aas) calibration solutions (merck chemical co., germany). the capability of phenolics to act as electron donors was tested using the microplate format. the phenolics tested were phenol, 2,4d i n i t r o p h e n o l , p e n t a c h l o r o p h e n o l , 2 chlorophenol, 4-chlorophenol, catechol, salicylic acid, 4-nonylphenol, p-hydroxybenzoic acid, benzoate and 2-napthol. the phenolics replaced glucose and were tested at 200 mg/l, but in a volume of 50 ml (arif et al. 2013). if the phenolics could be used as electron donors, moblue production will increase. on the other hand, ability of the above phenolics to support the growth of this bacterium independently from molybdenum-reduction was carried out in hpm media minus molybdenum. the increase of bacterial growth after 48 hours of incubation at room temperature, which was an indication of phenolics assimilation, was measured at 600 nm. identification of the molybdenum-reducing bacterium standard biochemical tests according to the bergey's manual (holt et al. 1994) was used to identify the molybdenum-reducing bacterium. in addition, a software-based method (abis online system) was utilized to interpret the results (costin & ionut 2015). briefly. briefly, the standard methods included gram staining, detection of motility via the hanging drop method, and various biochemical tests. statistical analysis data analyses were carried out using graphpad prism v 6.0 (www.graphpad.com). the student's t-test or anova with tukey's test as the post hoc analysis was carried out to compare means of groups. 49 isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. 900 and 1,100 ppm, respectively (banerjee et al. 2015). this phenomenon suggested that being heavy metal tolerant and conducting heavy metal reduction are part of the strategies employed by bacteria from this genus to combat heavy metal toxicity. nickel was detected to be more toxic (700 ppm), followed by cadmium (900 ppm) and lead (1,100 ppm). optimal ph that supported molybdenum reduction was between 6.5 and 6.8 (fig. 2), in temperature range of 34 37 °c (fig. 3). 50 biotropia vol. 24 no. 1, 2017 table 1 biochemical tests for enterobacter sp. strain saw-2 notes: + = positive result; − = negative result; d = indeterminate result 0.0 0.5 1.0 1.5 2.0 5.5 6.0 6.5 7.0 7.5 8.0 ph a b s 7 5 0 n m figure 2 effect of initial ph on molybdenum reduction by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) high-throughput method using a microplate format was employed in this study. this method can accelerate characterization works, while acquiring more information as opposed to the regular shake-flask method (iyamu et al. 2008; shukor & shukor 2014). the use of resting cells for studying mo-blue production was initiated in ec 48 (ghani et al. 1993). resting cells were also used in studying heavy metals reduction such as in chromate (llovera et al. 1993), selenate (losi & jr 1997) and xenobiotics biodegradation such as diesel (auffret et al. 2015) and phenol (sedighi & vahabzadeh 2014). molybdenum absorbance spectrum the scanning absorption spectrum of the resultant mo-blue from enterobacter sp. strain saw-2 showed a maximum peak at 865 nm, and a characteristic shoulder at about 700 nm (fig. 4). 51 0.0 0.5 1.0 1.5 2.0 2.5 20 30 40 50 60 temperature ( o c) a b s 7 5 0 n m figure 3 effect of temperature on molybdenum reduction by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) 0.0 0.5 1.0 1.5 2.0 600 650 700 750 800 850 900 950 wavelength (nm) a b s 8 h 24 h 32 h 48 h figure 4 scanning absorption spectrum of mo-blue from enterobacter sp. strain saw-2 at different time intervals isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. it was observed that the resulting spectrum resembled the spectrum of the mo-blue produced by the phosphate determination method (clesceri et al. 1989). spectrum produced by phosphate determination exhibited a maximum absorption around 880 to 890 nm. a characteristic shoulder was seen around 700 to 720 nm (hori et al. 1988). all of the mo-blue spectra from previously isolated molybdenum-reducing bacteria showed similar spectra. based on this knowledge, a hypothesis was developed for this study i.e. molybdenum reduction should proceed via the intermediate phosphomolybdate (shukor et al. 2007). another basis for the hypothesis was the confirmation of the presence of phosphomolybdate species through spectrophotometric analysis of the resultant absorption spectrum (sims 1961; yoshimura et al. 1986; hori et al. 1988). aside from the demonstration of the unique spectroscopic profile of the blue product, the presence of mo-blue by itself can visually prove that molybdenum reduction has taken place. reduced phosphomolybdate or mo-blue has a fractional oxidation state between 6+ and 5+ (sidgwick 1951; kazansky & fedotov 1980). other metals, such as mercury and chromate, show no visible color when experiencing reduction through bacterial process (suzuki et al. 1992). therefore, confirmation of reduction has to be carried out through epr-electron paramagnetic resonance (suzuki et al. 1992). the presence of an intermediate species is not unique to mo-blue production, as this is also reported in bacterial chromate reduction, such as bacteria pseudomonas ambigua (suzuki et al. 1992) and shewanella putrefaciens (now known as s. oneidensis) (myers et al. 2000). in these bacterial reduction processes, an intermediate species cr 5+ was observed (myers et al. 2000). effect of electron donor on molybdate reduction molybdate reduction to mo-blue was best supported by the sugar glucose. this was followed by sucrose, maltose, l-rhamnose, raffinose, d-mannose, lactose, mucate, cellobiose, d-mannitol, d-adonitol, melibiose, glycerol, d-sorbitol and l-arabinose in descending order (fig. 5). mo-blue production by other sugars such as dulcitol, salicin and myo-inositol were found to be not significantly different ( > 0.05) from p control based on anova . with tukey's test previous works demonstrated that molybdenumreducing bacteria prefer simple assimilable sugars, such as sucrose, glucose and fructose (campbell et al. et al et al 1985; ghani . 1993; rahman . 2009; yunus . 2009; et al shukor et al. 2009; ahmad et al. 2013; othman et al. 2013; abo-shakeer et al. 2013). the metabolic pathways glycolysis, kreb's cycle and electron transport chain were used to convert these sugars to nadh and nadph. both reducing equivalents are electron donating substrates for the molybdenum reducing-enzyme (shukor et al. 2014). 52 biotropia vol. 24 no. 1, 2017 0.0 0.5 1.0 1.5 2.0 2.5 d -a do ni to l la ra bi no se c el lo bi os e d ul ci to l g ly ce ro l d -g lu co se m yo -in os ito l la ct os e m al to se d -m an ni to l d -m an no se m el ib io se m uc at e r af fin os e lr ha m no se s al ic in d -s or bi to l s uc ro se c on tro l a b s 7 5 0 n m figure 5 effect of different electron donor sources (1% w/v) on molybdenum reduction note: error bars represent mean±standard deviation (n = 3) ef fect of phosphate and molybdate concentrations to molybdate reduction the optimal concentration of phosphate supporting optimal molybdenum reduction occurred at 5 mm. concentrations higher than this dramatically ceased production of mo-blue (fig. 6). high phosphate concentrations inhibit the stability of phosphomolybdate, of which upon reduction, it is converted to mo-blue glenn & crane 1956; sims 1961; shukor et al. 2000). molybdenum-reducing bacteria isolated previously are also strongly inhibited by phosphate concentration higher than 5 mm (shukor & syed 2010; lim et al. 2012; ahmad et al. 2013; halmi et al. 2013; othman et al. 2013; aboshakeer et al. 2013; khan et al. 2014; shukor et al. 2014). enterobacter sp. strain saw-2 can tolerate and reduce molybdenum at sodium molybdate concentrations as high as 60 mm, but the production of mo-blue was severely inhibited. the optimal concentrations of molybdate which supported the reduction, were between 15 and 30 mm (fig. 7). the other molybdenum-reducing 53 0.0 1.0 2.0 3.0 0 10 20 30 40 50 phosphate (mm) a b s 7 5 0 n m figure 6 molybdenum reduction by enterobacter sp. strain saw-2 at various phosphate concentrations note: error bars represent mean±standard deviation (n = 3) 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 molybdate (mm) a b s 7 5 0 n m figure 7 molybdenum reduction by enterobacter sp. strain saw-2 at various sodium molybdate concentrations note: error bars represent mean±standard deviation (n = 3) isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. bacteria isolated to date, require optimal molybdate concentration between 10 and 80 mm (shukor, rahman . 2010; shukor & syed 2010; et al lim . 2012; ahmad . 2013; halmi . et al et al et al 2013; othman . 2013; abo-shakeer . et al et al 2013; shukor . 2014). reduction at these very et al high concentrations is an advantage for molybdenum bioremediation in an area having high concentration of this metal. for instance, in new mexico where molybdenum concentration as high as 2,000 ppm or about 20 mm was reported (jacobs et al. 2014). effect of heavy metals bacterial molybdate reduction to mo-blue was inhibited by other heavy metals, which included cadmium (ii), mercury (ii), copper (ii) and silver (i) at 2 ppm with inhibition levels of 45.5, 36.8, 28.4 and 24.4%, respectively. these inhibition levels were compared to control, which was assigned as having 100% activity (fig. 8). molybdate reduction to mo-blue in many of the previously isolated molybdenum-reducing bacteria were inhibited by similar toxic heavy metals (shukor & syed 2010; lim et al. 2012; othman et al. 2013; shukor et al. 2014). in hexavalent chromate reduction to the trivalent state by bacillus sp. (arutchelvan et al. 2006) and enterobacter cloacae strain h01 (rege et al. 1997), the metal ions of mercury and copper were strong inhibitors. the supposed target of these metals was chromate reductase. this enzyme utilized the electron donors nadh and nadph to convert soluble toxic chromium 6+ to the insoluble less toxic chromium 3+. mercury binds strongly to sulfhydryl groups. additionally, mercury also binds, with variable strength, to carboxyl, amide, phosphoryl and amine groups of protein. this is the reason why mercury is one of the most toxic metal ions known to human. silver also binds to the sulfhydryl group of enzymes. copper preferentially binds to cysteine, histidine and methionine residues of enzymes (camakaris et al. 1999). the toxicity of these metal ions can be remedied through the addition of chemical additives including phosphate, calcium carbonate, manganese oxide and magnesium hydroxide (hettiarachchi et al. 2000; deeb & altalhi 2009). p h e n o l i c s a s c a r b o n s o u r c e s f o r molybdenum reduction and independent growth preliminary screening works on phenolics as carbon sources supporting molybdenum reduction failed to give positive results (data not shown). however, the bacterium was able to grow on the phenolic compounds (phenol and catechol) (fig. 9). 54 biotropia vol. 24 no. 1, 2017 0 20 40 60 80 figure 8 the effect of other heavy metals on mo-blue production by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) biodegradation of phenol and phenolic compounds by microorganisms has long been an object of research. bacteria that could degrade phenol and phenolic compounds include pseudomonas species (folsom et al. 1990; tomasi et al. 1995; aravindhan et al. 2014; hasan & jabeen 2015), bacillus brevis (arutchelvan et al. 2006), alcaligenes sp. (bai et al. 2007), ochrobactrum sp. (kiliç 2009), acinetobacter sp. (ahmad et al. 2011; yadzir et al. 2016) and rhodococcus species (arif et al. 2013). conclusions enterobacter sp. strain saw-2 showed the novel ability to reduce heavy metal molybdenum to mo-blue. this bacterium can also grow on the phenolic compounds (phenol and catechol). the identity of this bacterium is not completely robust; therefore, molecular identification is needed to further identify this species. this bacterium demonstrated a narrow ph and temperature ranges for optimal reduction. glucose was the most effective electron donor for optimal molybdenum reduction. the bacterium needed a critical phosphate concentration of 5.0 mm. the optimal molybdate concentrations were between 15 and 30 mm. the absorption spectrum of the mo-blue generated indicated that it was a reduced phosphomolybdate. cadmium (ii), mercury (ii), copper (ii) and silver (i) inhibited the process of molybdenum reduction to mo-blue. presently, efforts are ongoing to purify the molybdenumreducing enzyme and to characterize phenolics biodegradation studies in greater detail. from bioremediation point of view, this reported bacterium might be an important bio-tool in reducing environmental pollutants like molybdenum. acknowledgements this project was funded by the research university grant scheme (rugs), project no. 0501-09-0750ru/f1. references abo-shakeer lka, ahmad sa, shukor my, shamaan na, syed ma. 2013. isolation and characterization of a molybdenum-reducing bacillus pumilus strain lbna. j environ microbiol toxicol 1:9–14. ahmad sa, shukor my, shamaan na, mac cormack wp, syed ma. 2013. molybdate reduction to molybdenum blue by an antarctic bacterium. bio med res int 2013, a r t i c l e n u m b e r 8 7 1 9 4 1 . doi:10.1155/2013/871941. ahmad sa, syed ma, arif nm, shukor mya, shamaan n a . 2 0 1 1 . i s o l a t i o n , i d e n t i f i c a t i o n a n d characterization of elevated phenol degrading acinetobacter sp. strain aq5nol1. aust j basic appl sci 5:1035–45. 55 0.0 0.2 0.4 0.6 0.8 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baumannii strain serdang 1 in a packed-bed column reactor. desalination water treat 57(28):13307–17. doi:10.1080/19443994. 2015.1063459. yoshimura k, ishii m, tarutani t. 1986. microdetermination of phosphate in water by gel-phase colorimetry with molybdenum blue. anal chem 58:591–4. 58 biotropia vol. 24 no. 1, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 2. sepus (tropical).cdr biotropia vol. 19 no. 2, 2012: 64 79 tropical forest types in west papua, the presence of forest wallaby ( ) and human disturbancedorcopsis muelleri sepus fatem & karle v. sykora received 20 march 2012/accepted 08 october 2012 the vegetation in the nuni watershed area, part of a tropical lowland forest area in the northern part of manokwari, west papua, was classified with twinspan. the area is important as a natural habitat of the forest wallaby. four habitat types comprising 6 plant communities could be distinguished belonging to grassland, four different types of open forest and undisturbed primary closed forest. a vegetation table is presented and species composition is described. each vegetation plot the presence of trails, wallaby droppings, food remains and signs of human disturbance, i.e. logging, hunting and gardening activity, and distance to settlement areas was noted. the presence of wallabies could only be noted in grassland, open forest with only little logging activity, and in undisturbed closed forest. it is strongly correlated to distance from villages and negatively correlated to logging and hunting. the relation with food plant availability appears to be only low. the results indicate that vegetation structure, vegetation composition and food plant availability are less important than human disturbance. regulations reducing the disturbance by logging and hunting are urgently needed. wallaby, plant communities, vegetation analysis, wildlife, tropical forest, papua 1* 2 1 2 environmental and forest conservation laboratory,forestry faculty, papua state university, jl. gunung salju amban manokwari, papua barat-indonesia, 98314 nature conservation and plant science group, environmental dept, wageningen university and research centre, the netherlands abstract introduction key words: the island of new guinea (papua new guinea and west papua) occupies a phytogeographically important position between asia and west melanesia on one hand, and australia and the pacific on the other. in west papua, many ecosystems occur in a range from the coastal to the alpine zone of which the tropical forest is the dominant ecosystem. mammalian species are numerous in the tropical forest ecosystems of west papua. new guinea hosts a unique fauna of mammals due to its geological history (petocz 1989; muller 2005), * corresponding author : sepus_fatem@yahoo.com 64 differing from other areas in indonesia, but also other regions in the world (petocz 1994; flannery 1995). however, forest fragmentation, changes the environment, i.e. the physical conditions, tree species richness or tree structure (carlot 2009) and plant demography (borhidi 1998). changes in the size, shape or configuration of a habitat as a result of fragmentation has an effect on populations of various animals, such as small mammals), bird sand arboreal marsupials (connel 1978; chave 2008). menzies (1991), petocz (1994), flannery (1995) and westerman 2001) suggest that the diversity in endemic marsupials of new guinea is higher than is reflected by current classifications. their population status, habitat, vegetation structure and food composition should urgently be studied in order to better understand their ecological needs. this knowledge can be used as guidelines for ecological planning and management to avoid habitat destruction and decline of endemic marsupials. the current status and ecology of the forest wallaby ( , macropodidae) is insufficiently known. our study site manokwari (west papua) is part of its distribution area. the vegetation of manokwari has hardly been described. in this research we describe the vegetation composition of the vegetation types in the nuni watershed area in the northern part of manokwari regency, west papua. besides, we investigated the presence of the forest wallaby and its relation with vegetation, including food plant availability and human influence. our research is based on the following questions: 1. what plant communities occur in the area and how are they characterised by species composition, soil, human influence and structure? 2. what is the presence of the forest wallaby in the different plant communities? 3. how can differences in forest wallaby presence be explained?. the vegetation in the nuni watershed area, part of tropical lowland forest area in the northern part of manokwari regency, west papua provinces was studied between february 4 and april 6, 2010. for a map showing the location see appendix 1 (internet appendix). the vegetation composition and the wallaby presence was studied in 8 transects of 4 vegetation plots or relevés sized 50 x 50 m and one extra plot of 70 x 70 m, i.e. 33 plots in total. in order to have a wider range of forest types and possible densities of forest wallabies, one extra plot of 70x70 was made. the bigger size did not, however, influence the plant species composition and wallaby presence. due to the purpose for comparing, which plot more presence and covered macropodidae present. the transects were selected based on differences in forest type, hunting and logging intensity and distance from villages. average distance between the plots was 50-70 m. the survey was done in primary forest, open forest and grassland, from the river bank upward to the top of the mountain. vegetation composition was recorded using the braun-blanquet method. cover was estimated using the scale as modified by barkman, doing and segal (1964). this scale was transformed into an ordinal 9 scale (sykora 2009). species, were identified et al. ( dorcopsis muelleri materials and methods 65 tropical forest types in west papua – sepus fatem & karle v. sykora according to jhon (1997), whitmore (1997); paijmans (1976); van steenis c.g.g.j (2005); lekitto 2008); c.g.g. j van steenis (1989). most of the identification was done directly in the field and only few species were identified in herbarium of papua state university, manokwari-indonesia. the vegetation was classified using twinspan (hill & gaugh 1980; sýkora 2009; de boer . 2009). the plant communities distinguished were named using two different and/or dominant species. for each plot the presence of trails, wallaby droppings, food remains and signs of human disturbance, i.e. logging (number of tree stumps), hunting and gardening activity, and distance to settlement areas was noted. hunting frequency per week/ month/year was determined as well as the number of animals shot per hunting trip. the distance between settlement areas and the hunting areas was measured. the extent of the conversion of forests into gardens was registered by measuring the total garden area (m ) and the distance to settlement area (m). the presence of macropodidae species was recorded visually in each plot in the night, evening and morning. all strips were observed at the same time with two people per strip. for each plot the presence and cover (%) of food plant species was recorded. the relation between vegetation composition and external variables like food avalability, anthropogenenic pressure and wallaby presence was studied using multivariate gradient analysis (detrended correspondence analysis). significance was tested by monte carlo permutation test. in this research we studied the habitat of forest wallaby ( ) and its relation to vegetation composition. the plant communities in the different habitats were described. so far the habitat of the macropodidae species has only been described in general terms (menzies 1991; petocz 1994; flannery 1995). according to these authors macropodidae, especially forest wallaby occur from sea level up to an altitude of 400 m. their habitats vary from flood plains, few gravel, bushy areas, rocky river banks to forested hills optimally to an altitude of 200 meter asl (flannery 1995). however, habitat use and territory of this species has not yet been studied in more detail. the ecology of has not been studied recently. most research only focuses on tree kangaroos. the habitat used of forest wallaby is described. a total of 258 plant species was recorded (245 angiosperms, 10 ferns allies, 3 gymnosperms, see appendix 1). after twinspan analysis, six plant communities were distinguished. only four of these plant communities appeared to be used by as habitat. an overview is given of the habitat types and plant communities in which was found. the two plant communities where no activity of could be detected are described as well. et al. et al. ( et al dorcopsis muelleri d. muelleri darcopsis muelleri, dorcopsis muelleri d. muelleri d. muelleri d. muelleri 2 results and discussions vegetation biotropia vol. 19 no. 2, 2012 66 habitats, plant communities and activityd. muelleri . grassland this habitat type consists of a small thicket with grass species in combination with small herbs, woody herbs, shrubs and trees. the canopy is formed by small trees only and covers only 10 %. the herb layer is 90 cm high and there is no moss layer. the depth of the litter layer is only 1 cm. it is represented by the and community which is characterised by 17 different species for habitat and food of forest wallaby. this habitat is characteristic of open sites: sp, sp. and sp . , sp. , and provide food for forest wallabies. mostly the grass species such as and are up to 1 m high. in papua new guinea, , and common grass species of floodplains can grow up to 1-2 m high (harkink 1987). grasslands grow all along the river basins and in small valleys, where they occur due to the fluctuating water levels resulting in temporary flooding, followed by drainage of the river banks. this grassland is presently the most common natural grassland of lowland tropical vegetation area in west papua. this community is generally found on relatively flat (< 3°), temporarily flooded, sandy soil mixed with some gravel. as it occurs close to the river the sandy soil structure is crumbly, drainage is high and organic matter is lacking (brookfield 1971; petocz 1987; bps 2009). this habitat (fig. 1) is mainly used by the forest wallaby as feeding area, but also for shelter and to drink and play. trails and food remains were found of two individuals. imperata cylindrica ipomea aquatica imperata ipomea batatas, sacharum spontaneum, neprolepsis biserata, jussiaee octavilis, malotus philipinensis, piper aduncum, macaranga mappa, mimosa pudica, muntingia callabura, mucuna novaeguinesis, puararai javanica, spondias cytherea, micania zingiber ipomea batatas zingiber muntingia callabura mimosa pudica imperata cylindrica sacharum spontaneum imperata cylindrica, sacharum spontaneum sorghum nitidum phragmites karka, 2 2 figure 1. typical river bank grassland consisting of sparse thicket, grasses and herbs. (photo: sf sepus fatem) this natural grassland, which can be very extensive (128 665 meters in one research site), is also used by animals like deer. seventeen food plants were found with a mean cover of 2.1%. tropical forest types in west papua – sepus fatem & karle v. sykora 67 open forest two communities, the sp and community, were distinguished in the open forest habitat, depending on the absence or presence of some logging activity. this habitat type, a transition between grassland and tall forest, is characterised by shrubs, climbing vines and woody vines, lianas and pioneer species combined with small trees (fig. 2). the canopy cover is 47 % and the tree diameter is medium; besides some ferns and lianas occur. the herb layer is 51 cm high. it is commonly found surrounding the closed forest. it is represented by the community and sp which is differentiated by , , , , , , , , sp, , sp. 1, , , , , , sp, sp., , , , , sp., and . furthermore, , , and were food plant wallaby in this habitat. this forest type grows on hills at an altitude of approximately 40 meter above sea level. it occurs on inceptisols i.e. new immature, still developing soils with hardly any soil horizon. the depth of the solum is less than 2 m. the rock material in these sites mostly consists of sandstone and mudstone (brookfield 1971; petocz 1989; bps 2009). the soil is covered with 2 cm of litter. the distance to the nearest village is about 6.5 km and anthropogenic pressure is low. also the hunting frequency is very low (in average 1 time/month). this forest type appears to be one of the main habitat types of due to the low human pressure and its importance as a feeding habitat. more than 5 individuals of forest wallaby were spotted and their dung and trails were observed. in average 12 (7-20) food plant species were found with a mean cover of 2.5 %. ficus robusta dendrochide community musa paradisiaca callamus longipina of ficus robusta dendrochide hornstendia scottiana pandanus dubius derris alba sterculia shillinglawi ficus japonica carica papaya orioconide nitida endospernum moluccanum rhapidophora spathodea campanulata ficus neolaleba atra durio zibethinus lancium domesticum cyatea molucanna mangifera indica dendrobium prinium callamus warbugii ananas comosus macaranga gigantea drymopholeus litigiosus cleytances ficus variegata cyatea molucanna ficus variegata, mangifera indica, sphatodea campanulata carica papaya, ficus japonica ananas commosus lancium domesticum d. muelleri open forest transitional to grassland. figure 2. transition between river bank grassland and real forest (photo : sf-sepus fatem) biotropia vol. 19 no. 2, 2012 68 open forest after logging (successional forest) figure 3. open forest after logging (photo: sf-sepus fatem) in this habitat type the process of secondary succession is clearly visible. due to selective logging several trees with big diameter are still present. it is represented by three plant communities, the community of and the community of and the (fig. 3). the community of and is differentiated by sp, and . it is characterised by plants often seen some years after logging. is a pioneer species, and are light demanding and prefer open areas. the different species group is represented by plants indicative of high anthropogenic disturbance. even though food plants like , and are present no forest wallabies were registered. trees left over after logging have a height of 10-12 m, while tree of the regrowth reach 3-7 m. as the logging intensity was only low and the number of tree species selected to be cut was limited, the vegetation already started to restore after three years time. the average canopy cover is 48 %. the undergrowth is dominated by a species rich herb layer which is 90 cm tall, and by shrubs. it grows on flat areas (average slope 7°) with an altitude between 38-45 m. asl, at a distance of about 4 km from the nearest village. the soil consits of an inceptisol, i.e. a new, still developing soil with a hardly developed soil horizon. the solum is not more than 1 m deep (brookfield 1971; petocz 1989; bps 2009). the soil surface is covered by an average litter layer of 2 cm. due to the flatness of the area, logging is easy and the forest on this site was logged some years ago. this habitat type is used as feeding area by species. four individuals of the forest wallaby and their trails, food remains and dung were noted.in average 14 (11-17) food plant species were registered with a mean cover of 2.7 %. the community of is differentiated by sp, sp., sp sp., musa paradisiaca callamus longipina, diospyros hebecarpa-lepinopsis ternatensis smilax malacensispandanus tectorius communit y musa paradisiaca callamus longipina musa paradisiaca, callamus longipina, palaquium lobbianum, planconella obofata, archidendron bogoriensis, machinlaya celebia, toona coleynea sperata, fagraia rasemosa, macaranga tesylata, pigafetta filaris, ficus tingtoria, haplolobus selebica, podocarpus blumei canarium indicum musa paradisiaca callamus longipina palaquium lobbianum musa paradisiaca canarium indicum ficus tingtoria d. muelleri diospyros hebecarpa-lepinopsis ternatensis diospyros hebecarpa, lepinopsis ternatensis, spatiostemon javensis, clerodendron gluta premna corymbosa, tetrameles nudiflora, prunus arborea, amorphopalus , corimborchis tropical forest types in west papua – sepus fatem & karle v. sykora 69 aglaia spectabilis, bambusa alectrion davallia solida, harpulia branchin redgea, lindsea repens, streblus elongate, rhapidophora duabanga molucanna, giowa horsfieldia laevigata, apostasia odorata, ficus nodosa, callamus ficus anulata, ochrosia barbonica pangium edula. d. muelleri smilax malacensis-pandanus tectorius smilax malacensis smilax malacensis, pandanus tectorius, garcinia picrorrhiza, cayratia trifoliate, mangivera minor, endiandra ficus septica, litsea ladermanii, sterculia parkinsonii, arenga microcarpa, policyas nodosa, cananga odorata, adina nerifolius, rhus taitensis, lea acualeata, pandanus polycarpa, dianella ensifolia, elaeocarpus angustifolius, syzigium versteegi, horsfeldia sylvestris, ficus simisfera, cerbera floribunda, myristica gigantea, actinodaphne nitida, pterocimbium beccari, syzigium archidendron parviflorum, ficus pubescens, ligodium circinatum, davallia hymenophy, disoxylum cyclopeltis crenata, smilax malabatricum, phacomeria speciosa, alocasia zebrine, aglaia simisifera, sononia krasipen, ficus aurantiaceae, micania micantha, nastus holtumianus, gramatophylum papuana, aserantium opositifolium, terminalia complanata, calocasia mastixiodendron pachyclados. ficus aurantiaceae, ficus nodosa, ficus septica, ficus anulata, ficus semisfera, myristica gigantea, horsfeldia laevigata, premna corymbosa, rhapidophora terminalia complanata, syzigium ficus pubescens horsfeldia sylvestris mangivera minor syzigium versteegii smilax malacensis sp., sp., sp., sp., sp., sp., and this vegetation is 10-40 m tall and mainly consists of species that remained after logging besides of newly settled trees and of some pioneer species. canopy cover ranges between 40-50 %. unlike other communities a moss layer is present. mosses are growing on the rocks present under the canopy. the average height of the herb layer is 15 cm. the soil is covered with an average litter layer of 1 cm. it is characteristically developing 15-30 years after logging and grows at an altitude of 123 m at a distance of about 400 m from village. it is frequently found on mid slopes of moderately rocky sites (6-30%) consisting of limestone outcrops. recently, this forest was intensively logged (>10 lumberjacks/day, 20 times/week). although on average 11 (5-19) food plants were registered with a mean cover of 2.4%, no trails, dung or food remains of could be observed. the community is dominated by and is further differentiated by sp., sp., sp., sp and in the last two communities about 15 species of food plant were found: sp., sp., , , and . this forest has a canopy cover of 55 % and is characterized by climbing species and lianas like . the herb layer is 40 cm high and the moss layer is 1 cm. because of logging and the nearness of a logging road, this open forest is characterised by the presence of pioneer species. it grows at an altitude of 167 meter asl on hills with slopes of 8-20°. the landscape consists of undulating plateaus with humus or karstic mounds (bps 2009). the soil is classified as an inceptisol on limestone (brookfield 1971; petocz 1989; bps 2009) and covered by 1 cm of litter. the high calcium content of the soil indicates a ph which is sufficiently high to support the nutrient availability for the plants. the nearest distance to a village is about 700 m. although on average 12 (10-15) food plant species were registered with a mean cover of 2.7 % no trail, dung and other indications of forest wallaby presence could be observed. biotropia vol. 19 no. 2, 2012 70 undisturbed (“primary”) closed forest this habitat type is dominated by trees with big diameter. the size of the trees is variable, both small and big trees occur. the vegetation is further characterized by many lianas and other climbing species. vegetation height is ranging from 5 to 40 m. the herb layer is 38 cm high. as canopy cover is high (80%), the undergrowth consists only of few small shrubs and herbs (fig. 4). this habitat is represented by the community of which is differentiated by , sp., , , sp., , sp., , , , , , , sp., , sp., sp., , sp., , and sommeria leucophylaa-paraltropis glabra sommieria leucophylla alpinia paraltropis glabra buchanania arborescens adina garcinia latisima garcinia orania palindan pterocarpus indicus ficus benyamina intsia bijuga anthocepalus chinensis paracroton pendulous licuala parasarianthes falcataria baringtonia eudia hernandia ovigera popowia ficus pincorhiza alleuritis molucanna gymnacantera farcuhariana. figure 4. undisturbed(“primary'') forest, one of habitat of wallaby (photo: sf-sepus fatem) it grows on flat valley floors with meandering rivers, at an altitude between 40-102 m asl. it is composed of plant species frequent on moderate slopes (15°) of stabile shaded ecosystems with flat topography. the soil can be classified as an inceptisol (brookfield 1971; petocz 1987; bps 2009). are food plants for the forest wallaby. eight individuals of the forest wallaby and its dung and food remains were observed. in average 10 (6-12) food plant species were counted with a mean cover of 2.34 %. plants common for open to closed forest have a wide amplitude concerning light conditions and can grow both in light open forests and below the canopy of tall trees. some have their optimum in shade while other species grow better in the presence of light. species indicative of more shady conditions below taller vegetation, include , , sp., and , (alhamid 1988; maturbongs 2001; asri 2005; arijani 2006). , sp. and and are characteristic of open woody vegetation with and without partial shade; (maturbongs 2001 and asri 2005). this represents the ecotone between open forest and closed ficus benyamina, ficus pincorhizza, intsia bijuga selaginella martensii scindapsus pietus scindapsus euscuarius, phylodendron meremia peltata asplenium nidus, korthalzia zippelii amomum aculeatum octomeles sumatrana pandanus arthocarpus altilis homalium foetidium common differentiating plants tropical forest types in west papua – sepus fatem & karle v. sykora 71 forest. this is supported by asri (2005) in west papua; harkink (1987) in papua new guinea; and meijaard . (2005) in kalimantan. sp., is the most dominant species in this habitat type, and has been found in our research to co-occur with some early pioneer species. , , , , sp. and are differentiating the primary forest, where human interference is only low or even absent. some species are commonly occurring in open to closed forest and in forest with former low logging intensity. the logged forest is restoring to later forest stages by succession. in this forest type some plant species facilitate the growth of other species by providing shade and protection. here some species like; and provide food for herbivores, like macropodidae animals (maturbongs 2001; fatem 2008). also open forest and logged forest have species in common. in these habitat types species grow fast in order to catch light, like , sp., . are food source for animals like . one of the lianas, occurs in all distinguished plant communities. it suppresses tree regeneration and increases tree mortality. lianas also influence competition between trees and thus they effect forest composition. lianas are also a valuable food source for some animals as well as for local population of people. they enable canopy to canopy access for arboreal species (bongers 2002). other species are common differentials for closed forest regenerated after logging. also in this vegetation several lianas grow as pioneer species. the vegetation is further characterized by tall trees (10-40 m), like , , , , , and this vegetation is found in the lowland forest area and close to villages and the coastal area. besides, , , and are species typical of lowland tropical vegetation as reported by jhon (1997); maturbongs (2001); meijerd (2005); kartikasari (2012). wallaby presence was observed in 4 of the 6 distinguished plant communities belonging to grassland, open forest with only little logging activity, and in undisturbed closed forest. it was however not registered from forests with clear influence of logging. appears to be very sensitive to human disturbance. according to detrended correspondence analysis (fig. 5) the presence of the forest wallaby (mac pre, tra) is strongly correlated to distance from villages (r respectively 0.69, 0.54) and negatively correlated to logging (log perr -0.79 and -0.60, cut int r -0.75 and 0.65, amostu r -0.75 and -0.65) and hunting (hunt int r -0.32 and -0.28). et al clomarippsidacae sommeria leuchophylla garcinia latisima pterocarpus indicus buchaninia arborenscens adina orania palindan musa paradisiaca ficus tingtoria et al. callamus aruensis poliyalthia freycinetia scandens arthocarpus vresianus, pometia corriacea d. muelleri meremia peltata, alstonia scholaris gnetum gnemon canarium dekamanum callamus cayensis pommetia acuminata syzigium malacensis prainea limpato. gnetum gnemon alstonia scholaris pometia acuminata canarium dekamanun et al. d. muelleri vegetation, disturbance and wallaby presence 2 2 2 2 2 biotropia vol. 19 no. 2, 2012 72 figure 5. ordination diagram showing the first two axes of a dca analysis. the arrows represent the correlation with external variables, showing both the direction and the strength of the correlation (length of the arrow). arrows in the same direction are positively correlated, opposite arrows are negatively correlated. note; hunting intensity (hunt int), altitude (alt), cutting intensity (cut int), amount of stump (amostu), logging period (log per), slope (slo), moss layer (ml), dung (dun), encountered (macrenc), feeding remnant (fee rem), present (mac pre), distance (dis), trail (tra), litter layer (ll), herb layer (hl), food plant species (foo pla), food plant cover (fp cov), canopy cover % (can). d. muelleri d. muelleri the relation between wallaby presence and food plant availability appears to be only low (r 0,14 and 0,02). as the structure and species composition of the 4 wallaby plant communities differs considerably, and food plants are present in all plant communities and as wallaby presence is highly correlated to human disturbance and has not been found in vegetation with human disturbance, our results indicate that vegetation structure, vegetation composition and food plant availability are less important than human disturbance. four plant communities are used by the forest wallaby for foraging and as their territory i.e. the community, the community of sp, the community of and and the community of representing grassland, open and closed forest. it was not detected in the community of and the community representing logged forests. even the presence of food plants did not guarantee the presence of forest wallabies. species appear to be very sensitive to human disturbance. there is a strong negative relation between the presence of this species and on the other hand logging, distance to villages and hunting. therefore, the habitat of this species 2 conclusions imperata cylindrica, ipomea aquatica ficus robusta, dendrocnide musa paradisiaca callamus longipina sommeria leucophylaa-paraltropis glabra diospyros hebecarpa-lepinopsis ternatensis smilax malacensis-pandanus tectorius d. muelleri should tropical forest types in west papua – sepus fatem & karle v. sykora 73 be protected and conserved by government regulations reducing the disturbance by logging and hunting. other stake holders should be involved to create public support. comunity based wildlife management can be used to reduce anthropogenic pressure. although this study gives a good description of the habitat and vegetation in which the wallaby has been found to forage, further more detailed research is needed to better understand the relation between wallaby presence, food preferences and human influence. our study is descriptive and consequently only shows correlations. although it clearly indicates the sensitivity of the forest wallaby for anthropogenic disturbance even if food plants are present, it is recommended to prove this relation experimentally for instance by reducing the anthropogenic influence in certain areas. references arijani, setiadi d, edi g, ibnul q. 2006.vegetation analysis of the up-stream cianjur watershed in mount gedepangrango national park's. biodiversitas. volume 7. alhamid h. 1988. studi habitat dan populasi burung cenderawasih kecil ( , shaw) di areal bekas tebangan pt. inhutani ii dalam kawasan cagar alam pegunungan arfak manokwari. 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f -2 0 b f -2 1 w f -2 4 w f -2 5 s f -3 3 s f -3 0 s f -3 1 s f -3 2 a 1 a 2 a 2 a 2 a 2 a 2 a 2 a 2 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 4 a 4 a 4 a 4 a 5 a 5 a 5 a 5 a 5 a 5 a 6 a 6 a 6 a 6 a 6 a 6 m o s s la y e r (c m ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 1 1 1 0 1 0 0 1 h e rb la y e r (c m ) 9 0 5 0 6 0 4 0 4 0 9 0 3 0 5 0 5 0 4 0 5 0 5 0 3 0 5 0 1 0 4 0 2 0 9 0 9 0 9 0 9 0 1 0 2 0 2 0 1 0 2 0 2 0 1 5 2 0 5 0 5 0 5 0 5 0 l it te r la y e r (c m ) 1 2 2 2 2 1 2 2 4 4 4 2 4 4 4 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 c a n o p y (% ) 1 0 5 0 4 0 5 0 5 0 4 0 5 0 5 0 9 0 9 0 7 0 8 0 9 0 9 0 9 0 6 0 7 0 5 0 5 0 4 0 5 0 5 0 5 0 4 0 4 0 5 0 5 0 5 0 5 0 5 0 6 0 6 0 6 0 a lt it u d e (m .a .s .l .) 2 9 4 7 3 8 4 2 3 0 5 0 3 9 4 6 5 1 4 9 7 1 5 4 7 1 5 7 1 0 2 4 4 8 2 4 5 4 5 3 8 4 3 1 0 9 1 3 8 6 2 9 8 1 5 5 1 7 8 1 5 5 1 7 8 1 7 9 1 3 0 1 6 9 1 9 4 s lo p e (° ) 3 .4 3 5 .7 1 9 .0 9 5 .7 1 5 .7 1 1 2 .4 1 5 .7 1 1 4 .5 7 1 3 .5 0 1 .1 5 3 2 .6 2 1 3 .5 0 1 6 .7 0 1 1 .3 1 7 .9 7 6 .8 4 2 3 .7 5 5 .7 1 7 .9 7 4 .5 7 1 0 .2 0 1 2 .4 1 2 2 .7 8 1 9 .8 0 5 .7 1 3 2 .6 2 1 6 .7 0 1 0 .2 0 1 6 .7 0 1 6 .7 0 1 4 .5 7 1 9 .8 0 5 .7 1 d is ta n c e (m ) 5 1 4 3 7 2 6 4 5 1 2 0 6 7 2 3 6 7 3 4 6 0 0 2 6 7 4 6 6 7 5 1 8 0 8 4 8 0 5 6 6 0 1 5 6 0 4 2 6 0 6 7 8 0 9 3 5 1 2 0 8 0 3 5 5 1 2 0 4 3 2 6 4 3 4 9 4 2 3 0 4 2 6 5 4 9 9 6 1 7 4 8 2 6 0 8 7 3 7 8 9 0 7 5 5 8 9 1 8 7 6 4 1 8 5 0 8 6 7 8 a m o u n t s tu m p (i n d /c u tt in g ti m e s ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 5 5 6 5 5 5 5 5 5 5 5 5 h u n ti n g in te n s it y (t im e s /w e e k ) 0 2 1 1 1 2 1 1 1 1 2 1 2 1 0 1 1 0 0 1 0 3 3 1 1 1 1 3 3 3 3 3 3 c u tt in g in te n s it y (t im e s /w e e k ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 l o g g in g p e ri o d (y e a r) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 m a c ro p o d id a e e n c o u n te r (i n d ) 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 f e e d in g re m n a n t (g r) 3 0 0 0 3 0 3 0 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 t ra il (i n d ) 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 d u n g (g r) 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 1 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 m a c ro p o d id a e p re s e n t (i n d ) 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 f o o d p la n t (i n d ) 1 7 2 1 8 1 0 7 1 5 9 1 3 7 6 1 6 1 1 1 3 9 1 4 8 8 1 1 1 2 1 7 1 6 1 3 9 1 9 1 1 5 1 0 1 5 1 3 1 2 1 2 1 1 1 0 f o o d p la n t c o v e r (% ) 2 1 2 0 2 9 5 9 1 9 3 9 2 6 1 5 2 1 0 7 2 2 8 7 2 6 8 5 2 5 5 8 2 5 6 9 2 3 9 7 2 5 5 0 2 6 0 3 2 4 2 6 2 3 5 6 2 3 7 8 2 4 9 1 2 1 0 3 2 5 7 9 2 7 3 3 2 5 3 2 2 7 6 0 2 4 3 7 2 1 5 5 2 5 0 9 2 4 0 6 2 0 1 7 2 3 1 5 2 5 0 6 2 4 0 1 2 8 8 4 2 5 4 2 2 6 1 7 2 2 2 0 c o m m u n it y d iv is io n a ft e r t w in s p a n 1 r e le v e n u m b e r a ft e r t w in s p a n 1 2 3 4 5 6 7 8 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 9 1 0 1 1 1 2 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3 r e l e v e c o d e p f -a 1 s f -b 1 3 p f -a 2 s f -a 9 s f -a 1 0 h f -1 4 s f -a 1 1 s f -a 1 2 p f -b 7 p f -b 6 h f -1 5 h f -1 6 h f -1 7 p f -b 8 p f -a 4 p f -b 5 p f -a 3 l a -2 8 l a -2 9 l a -2 6 l a -2 7 w f -2 2 w f -2 3 b f -1 8 b f -1 9 b f -2 0 b f -2 1 w f -2 4 w f -2 5 s f -3 3 s f -3 0 s f -3 1 s f -3 2 d if fe re n ti a l s p e c g ro u p 1 . im p e ra ta c y li n d ri c a ip o m o e a a q u a ti c a g ra s s la n d im p e ra ta s p 9 1 0 0 9 n e p h ro le p s is b is e ra ta 9 1 0 0 9 9 1 1 4 3 4 1 1 1 1 3 2 5 3 8 1 7 8 im p e ra ta c y li n d ri c a 9 1 0 0 9 9 1 4 9 ip o m e a a q u a ti c a 8 1 0 0 8 s a c c h a ru m s p o n ta n e u m 8 1 0 0 8 8 1 4 8 9 1 1 9 m a lo tu s p h il ip in e n s is 6 1 0 0 6 1 1 3 3 6 7 2 3 1 2 5 0 2 ip o m e a b a ta ta s 5 1 0 0 5 j u s s ia e e o c ta v a li fi s 4 1 0 0 4 5 3 2 4 5 7 4 3 5 4 3 3 4 p ip e r a d u n c u m 1 1 0 0 1 5 5 3 3 4 7 1 4 4 1 1 4 2 2 5 0 2 4 1 3 3 3 6 1 7 6 m a c a ra n g a m a p p a 1 1 0 0 1 9 8 2 4 3 6 1 2 5 1 1 1 3 3 1 3 7 3 3 5 m im o s a p u d ic a 1 1 0 0 1 m u n ti g ia c a ll a b u ra 1 1 0 0 1 m u c u n a n o v a e g u in e s is 1 1 0 0 1 1 4 3 8 5 7 4 2 2 5 2 p u e ra ri a ja v a n ic a 1 1 0 0 1 1 1 4 1 1 1 1 1 s p o n d ia s c y th e re a 1 1 0 0 1 1 2 5 1 3 6 3 5 0 4 7 1 7 7 m ic a n ia s p 1 1 0 0 1 3 3 3 4 3 3 7 8 5 0 8 1 1 7 1 z in g ib e r s p 2 1 1 0 0 1 2 2 2 2 1 5 6 2 3 1 7 3 d if fe re n ti a l s p e c . g ro u p 2 : f ic u s ro b u s ta -d e n d ro c n id e s p , g ra s s la n d -f o re s t tr a n s it io n f ic u s ro b u s ta 8 2 9 3 2 7 3 1 0 0 5 1 1 1 3 3 1 1 1 5 0 1 2 1 7 2 1 1 3 4 6 7 2 d o n a x c a n if o rm is 5 2 9 8 8 9 9 1 0 0 7 3 3 4 2 3 5 6 3 2 5 5 0 4 2 1 3 3 2 1 1 7 5 0 3 d e n d ro c h id e s p 9 1 7 3 5 7 1 5 1 1 1 1 2 2 5 2 f ic u s tr a c ip is o n 5 1 5 2 3 7 1 3 3 2 5 3 1 1 7 1 h o rn s te n d ia s c o tt ia n a 7 4 1 9 4 7 1 5 2 3 3 3 3 3 7 2 5 7 1 1 3 3 1 p a n d a n u s d u b iu s 4 2 2 1 5 7 2 d e rr is a lb a 5 5 7 1 5 7 5 1 1 7 1 s te rc u li a s h il li n g la w i 1 2 2 4 3 2 1 1 1 1 1 1 7 1 1 1 7 1 f ic u s ja p o n ic a 5 1 4 4 3 3 1 2 5 1 1 1 3 3 1 c a ri y a p a p a y a 1 1 1 4 3 1 1 1 3 3 1 o ri o c n id e n it id a 1 1 2 9 1 1 1 7 1 e n d o s p e rn u m m o lu c c a n u m 1 2 2 9 2 1 2 5 1 1 1 3 3 1 1 1 7 1 r h a p id o p h o ra s p 1 3 2 9 2 s p a th o d e a c a m p a n u la ta 7 1 2 9 4 n e o la le b a a tr a 1 2 2 9 2 3 1 7 3 f ic u s s p 1 9 1 4 9 v it te x p in n a ta 8 1 4 8 1 1 1 3 3 1 1 1 7 1 1 2 3 3 2 d u ri o z ib e th in u s 7 1 4 7 l a n c iu m d o m e s ti c u m 7 1 4 7 c y a te a m o lu c a n n a 6 1 4 6 1 1 1 1 f ic u s v a ri e g a ta 3 1 4 3 1 2 5 1 c le y ta n c e s s p 3 1 4 3 1 1 1 1 p a n d a n u s c o n o id e u s 2 1 4 2 d ry m o p h o le u s li ti g io s u s 2 1 4 2 7 1 1 7 t h e o b ro m a c a c a o 2 1 4 2 m a c a ra n g a g ig a n te a 1 1 4 1 m a n g if e ra in d ic a 1 1 4 1 a n a n a s c o m o s u s 1 1 4 1 c a ll a m u s w a rb u g ii 1 1 4 1 c o c o s n u c if e ra 1 1 4 1 p ri n iu m s p 1 1 4 1 s a ll a c a e d u li s 1 1 4 1 k o k o o n a o c h ra c e a 1 1 4 1 1 1 1 1 d e n d ro b iu m s p 1 1 4 1 m u s a p a ra d is ia c a -c a ll a m u s lo n g ip in a , lo g g e d fo re s t d io s p y ro s h e b e c a rp a -l e p in o p s is te rn a te n s is , lo g g e d fo re s t o n s it e s w it h la rg e c o v e r o f ro c k s . s m il a x m a la c e n s is -p a n d a n u s te c to ri u s , l o g g e d fo re s t w it h ro c k s 6 im p e ra ta c y li n d ri c a ip o m o e a a q u a ti c a g ra s s la n d c o m m u n it y 2 3 4 5 f ic u s ro b u s ta -d e n d ro c n id e s p , g ra s s la n d -f o re s t tr a n s it io n s o m e ri a le u c h o p h il la -p a ra tr o p is g la b ra tropical forest types n west papua, the presence of forest wallaby sepus fatemi – et al. 76 a p p en d ix 2 . d if fe re n ti al v eg et at io n ta b le w it h b o th se p ar at e re le v és an d sy n o p ti c in fo rm at io n . f o r th e se p ar at e re le v és th e co v er -a b u n d an ce is re p re se n te d b y th e o rd in al 9 -s ca le ,s yn o p ti c in fo rm at io n co n si st s o f % p re se n ce an d ch ar ac te ri st ic co v er (m ea n co v er o f th e n u m b er o f o cc u rr en ce s o f a sp ec ie s in a ce rt ai n cl u st er ). d if fe re n ti a l s p e c ie s g ro u p 3 : s o m e ri a le u c h o p h il la -p a ra tr o p is g la b ra s o m m ie ri a le u c o p h y ll a 7 7 9 8 9 1 6 7 7 a lp in ia s p 2 4 3 1 3 5 6 3 1 1 3 3 1 p a ra lt ro p is g la b ra 4 1 3 1 1 5 6 2 8 2 5 8 b u c h a n a n ia a rb o re s c e n s 4 4 2 3 3 3 a d in a s p 2 1 1 3 3 1 g a rc in ia la ti s im a 1 1 1 3 3 1 1 2 5 1 g a rc in ia s p 1 1 1 3 3 1 o ra n ia p a li n d a n 5 1 6 3 3 4 p te ro c a rp u s in d ic u s 1 1 4 1 3 1 1 3 3 2 f ic u s b e n y a m in a 3 1 1 3 3 2 in ts ia b ij u g a 5 1 2 2 3 a n th o c e p a lu s c h in e n s is 3 1 4 3 1 3 2 2 2 p a ra c ro to n p e n d u lo u s 2 2 2 2 2 l ic u a la s p 5 1 4 5 5 1 2 2 3 p a ra s a ri a n th e s fa lc a ta ri a 1 1 4 1 2 1 2 2 2 2 1 7 2 b a ri n g to n ia s p 9 1 1 9 e u d ia s p 4 1 1 4 h e rn a n d ia o v ig e ra 2 1 1 2 p o p o w ia s p 1 1 1 1 f ic u s p ic ro rr h iz a 1 1 1 1 a le u ri ti s m o lu c a n n a 1 1 1 1 g y m n a c a n te ra fa rc u h a ri a n a 1 1 1 1 d if f e r e n t ia l s p e c ie s . d 1 s e la g in e ll a m a rt e n s i 9 3 9 7 9 9 9 1 0 0 8 8 9 9 5 7 8 7 7 9 1 0 0 8 9 9 5 0 9 s c in d a p s u s p ie tu s 4 4 2 3 3 7 1 3 2 3 2 3 4 2 4 4 3 1 0 0 3 t a b e rn a e m o n ta n a s p 1 4 6 1 4 7 8 6 4 2 3 4 6 7 5 6 4 s c in d a p u s e u s c u a ri u s 1 4 4 3 5 7 3 2 4 1 3 4 4 3 7 8 3 h o m a li u m fo e ti d iu m 6 8 4 2 5 7 5 3 1 2 4 2 8 6 7 3 4 2 5 7 5 4 1 1 7 1 2 1 3 3 2 f la g e ri a in d ic a 1 3 5 2 5 7 3 1 1 1 3 3 1 2 2 5 0 2 p le o m e le a n g u s ti fo li a 3 1 2 9 2 4 3 2 2 4 3 2 5 3 b a lb it is rh y z o p h y ll a 4 2 4 4 3 3 4 7 5 4 6 5 4 7 8 5 p ip e r in te rr u p tu m 1 2 1 4 3 1 2 1 2 2 1 5 6 2 1 2 2 3 6 7 2 c lo m a ri p p s id a c a e s p 2 4 2 3 5 7 3 4 2 6 1 3 6 6 7 4 1 1 7 1 d if fe re n ti a l s p e c . g ro u p 4 : m u s a p a ra d is ia c a -c a ll a m u s lo n g ip in a , lo g g e d fo re s t m u s a p a ra d is ia c a 5 1 1 4 3 2 1 1 1 1 5 2 3 8 1 0 0 5 1 1 3 5 0 2 1 1 7 1 c a ll a m u s lo n g ip in a 1 5 2 9 3 2 7 4 3 3 4 4 2 3 3 1 0 0 3 p a la q u iu m lo b b ia n u m 3 1 4 3 6 9 2 4 1 5 6 4 9 8 4 7 1 0 0 7 7 5 3 3 6 1 2 3 3 2 p la n c o n e ll a o b o fa ta 1 1 4 1 8 1 1 8 7 3 4 6 1 0 0 5 2 2 7 3 6 7 4 2 1 7 2 a rc h id e n d ro n b o g o ri e n s is 0 3 4 5 0 4 2 1 3 3 2 m a c h in la y a c e le b ia 0 1 1 5 0 1 1 1 7 1 t o o n a s p 1 1 1 1 7 2 5 7 c o le y n e a s p e ra ta 0 1 2 5 1 f a g ra ia ra s e m o s a 0 1 2 5 1 m a c a ra n g a te s y la ta 0 1 2 5 1 p ig a fe tt a fi la ri s 0 1 2 5 1 f ic u s ti n g to ri a 0 1 2 5 1 h a p lo lo b u s s e le b ic a 0 1 2 5 1 6 1 7 6 p o d o c a rp u s b lu m e i 1 2 2 2 2 1 2 5 1 1 1 7 1 c a n a ri u m in d ic u m 1 1 1 1 1 2 5 1 d if f e r e n t ia l s p e c ie s . d 2 s e m e c a rp u s p a p u a n a 8 1 1 3 3 3 6 3 1 7 5 3 1 1 3 3 1 1 1 7 1 d ry m o p h lo u e s o li v o rm is 2 1 4 2 4 3 7 3 3 5 2 6 3 7 5 4 1 1 7 1 f re y n e ti a s p 3 1 4 3 5 7 1 1 1 1 6 7 3 4 7 5 0 6 2 1 7 2 b u b ia s p 1 2 2 4 1 5 6 2 3 2 5 3 l a p o rt e a c a n d e n s is 1 1 2 9 1 3 8 4 2 1 5 6 4 6 2 5 6 g o n o c a ri u m li to ra ll e 3 1 3 1 4 4 2 6 6 5 0 6 1 1 1 5 0 1 c y n o m e tr a s p 2 1 1 3 3 1 5 1 5 0 3 1 1 7 1 p in a n g a ru m p h ia n a 1 7 4 1 4 4 3 2 5 5 0 4 c a n a ri u m h ir s u tu m 1 6 7 3 3 5 2 2 5 2 1 1 7 1 c a m p n o s p e rm a b re v ip e ti o la ta 4 1 4 3 3 3 1 2 5 1 g ir in o p s s p 7 1 1 7 8 2 5 8 d if fe re n ti a l s p e c . g ro u p 5 : d io s p y ro s h e b e c a rp a -l e p in o p s is te rn a te n s is , lo g g e d fo re s t o n s it e s w it h la rg e c o v e r o f ro c k s . d io s p y ro s h e b e c a rp a 3 8 1 7 2 8 3 4 l e p in o p s is te rn a te n s is 1 1 1 1 3 2 5 3 1 1 2 2 6 7 2 s p a ti o s te m o n ja v e n s is 2 6 8 9 6 7 6 4 1 7 4 c le ro d e n d ro n s p 1 1 3 5 0 2 g lu ta s p 2 8 6 5 0 5 p re m n a c o ry m b o s a 7 1 1 5 0 3 t e tr a m e le s n u d if lo ra 1 1 1 5 0 1 p ru n u s a rb o re a 1 2 3 3 2 a m o rp h o p a lu s s p 3 1 3 3 2 c o ri m b o rc h is s p 1 1 2 2 1 1 1 3 3 1 a g la ia s p e c ta b il is 1 1 3 3 1 b a m b u s a s p 1 1 4 1 9 1 7 9 a le c tr io n s p 3 1 7 3 d a v a ll ia s o li d a 3 1 7 3 h a rp u li a s p 2 1 7 2 1 1 7 1 b ra n c h in re d g e a 1 1 1 1 2 1 7 2 l in d s e a re p e n s 2 1 7 2 s tr e b lu s e lo n g a ta 1 1 7 1 r h a p id o p h o ra s p 1 1 7 1 d u a b a n g a m o lu c a n n a 1 1 7 1 g io w a s p 1 1 7 1 h o rs fi e ld ia la e v ig a ta 1 1 7 1 a p o s ta s ia o d o ra ta 1 1 7 1 f ic u s n o d o s a 2 1 4 2 1 1 1 1 1 1 7 1 c a ll a m u s s p 1 1 4 1 1 1 7 1 f ic u s a n u la ta 1 1 1 1 1 1 7 1 o c h ro s ia b a rb o n ic a 1 1 7 1 p a n g iu m e d u la 1 1 7 1 biotropia vol. 19 no. 2, 2012 77 tropical forest types n west papua, the presence of forest wallaby sepus fatemi – et al. a p p en d ix 2 . c o n ti n u ed d if fe re n ti a l s p e c . g ro u p 6 : s m il a x m a la c e n s is -p a n d a n u s te c to ri u s , l o g g e d fo re s t w it h ro c k s s m il a x m a la c e n s is 1 2 2 9 2 1 1 1 1 1 5 3 3 3 2 6 1 3 7 9 1 0 0 5 p a n d a n u s te c to ri u s 1 1 7 1 2 1 1 4 6 7 2 g a rc in ia p ic ro rr h iz a 1 1 4 1 1 1 1 1 1 1 3 3 1 1 1 1 1 6 7 1 c a y ra ti a tr if o li a ta 2 6 5 5 0 4 m a n g iv e ra m in o r 1 1 4 1 2 1 6 5 0 3 e n d ia n d ra s p 2 1 7 2 1 3 2 5 0 2 f ic u s s e p ti c a 4 1 2 9 3 1 1 4 5 0 2 l it s e a la d re m a n ii 5 1 1 5 1 1 7 1 1 2 2 5 0 2 s te rc u li a p a rk in s o n ii 1 1 7 2 1 4 5 0 2 a re n g a m ic ro c a rp a 2 1 4 2 1 1 1 1 1 3 1 5 0 2 p o li c y a s n o d o s a 1 1 3 5 0 2 c a n a n g a o d o ra ta 1 1 2 9 1 1 2 5 1 1 1 7 1 4 7 3 3 6 a d in a n e ri fo li u s 1 1 3 3 1 2 9 3 3 6 t e y s m a n io d e n d ro n b o g o ri e n s is 2 1 1 2 9 2 5 9 2 7 3 3 5 r h u s ta it e n s is 1 1 3 3 1 3 5 3 3 4 d ia n e ll a e n s if o li a 1 1 4 1 1 6 3 3 4 e la e o c a rp u s a n g u s ti fo li u s 1 1 1 1 1 6 3 3 4 s y z ig iu m v e rs te e g i 1 1 1 5 0 1 2 3 3 3 3 h o rs fe ld ia s y lv e s tr is 1 1 1 1 2 1 7 2 1 3 3 3 2 f ic u s s e m is e fe ra 1 4 3 3 3 c e rb e ra fl o ri b u n d a 6 1 1 6 1 1 7 1 1 2 3 3 2 m y ri s ti c a g ig a n te a 1 1 7 1 1 1 3 3 1 a c ti n o d a p h n e n it id a 1 1 7 1 1 1 3 3 1 p te ro c im b iu m b e c a ri 2 1 2 2 2 1 1 7 1 1 1 3 3 1 s y z ig iu m s p 5 1 2 2 3 1 1 3 3 1 a rc h id e n d ro n p a rv if lo ru m 1 1 7 1 1 1 3 3 1 f ic u s p u b e s c e n s 9 1 7 9 l ig o d iu m c ir c in a tu m 7 1 7 7 d a v a ll ia h y m e n o p h y 1 1 4 1 6 1 7 6 d is o x y lu m s p 2 1 7 2 5 1 7 5 c y c lo p e lt is c re n a ta 1 1 1 1 4 1 7 4 s m il a x m a la b a tr ic u m 4 1 7 4 p h a c o m e ri a s p e c io s a 4 1 7 4 a lo c a s ia z e b ri n a 3 1 7 3 a g la ia s im is if e ra 3 1 7 3 s o n o n ia k ra s ip e n 2 1 7 2 f ic u s a u ra n ti a c e a e 2 1 7 2 m ic a n ia m ic a n th a 2 1 7 2 n a s tu s h o lt u m ia n u s 2 1 7 2 g ra m a to p h y lu m p a p u a n a 1 1 7 1 a s e ra n ti u m o p o s it if o li u m 1 1 7 1 t e rm in a li a c o m p la n a ta 1 1 1 1 1 1 7 1 c a lo c a s ia s p 1 1 7 1 m a s ti x io d e n d ro n p a c h y c la d o s 4 1 4 4 2 1 1 2 1 1 7 1 l e a a c u a le a ta 5 1 1 5 1 1 7 1 1 1 7 1 p a n d a n u s p o ly c a rp a 7 1 1 7 1 1 7 1 d if f e r e n t ia l s p e c ie s .d 3 k o o rd e rs io d e n d ro n p in n a tu m 2 2 4 2 7 3 1 0 0 3 8 3 9 5 6 7 6 s te rc u li a m a c ro p h y ll a 1 1 1 1 4 1 2 4 7 6 1 0 0 4 8 1 7 6 6 7 6 p o m e ti a c o rr ia c e a 1 2 2 2 2 8 3 3 8 9 8 3 6 8 8 9 9 9 8 1 0 0 9 p te ri g o ta h o rs fe ld ia 1 1 2 9 1 1 1 1 1 7 4 3 2 1 8 3 3 7 3 4 7 6 7 5 c ry p to c a ry a s p 6 1 4 6 1 1 1 1 1 7 1 5 0 3 1 5 7 7 2 8 3 4 l u n a s ia a m a ra 7 9 2 8 9 8 3 7 3 1 9 5 0 4 s is ip u s ju ju b a 4 1 1 4 1 2 5 1 3 1 1 5 0 2 2 3 6 2 7 8 3 4 p a la q u iu m a m b o in e n s is 1 1 1 1 1 8 9 9 9 8 3 7 7 4 3 6 6 7 5 d ra c o n tu m e lu m d a o 1 1 1 4 3 1 2 1 1 2 2 2 5 2 2 2 4 1 6 7 2 2 7 2 5 0 4 a re a n g il is a fl a v a 2 1 2 2 2 3 3 2 7 6 7 4 6 2 3 3 4 d is o x y lu m m o ll is im u s 2 1 3 5 0 2 1 1 3 9 6 7 4 f re y n e ti a s c a n d e n s 3 1 3 3 2 1 1 2 1 6 7 1 78 m y ri s ti c a fa tu a 1 1 4 1 1 5 3 3 3 1 1 2 3 6 7 2 c a ll a m u s a ru e n s is 1 2 1 5 0 1 1 1 3 5 6 7 3 g lo s id io n s p 1 1 1 5 0 1 2 2 3 3 2 p o li y a lt h ia s p 1 1 2 2 1 3 3 4 5 0 3 2 7 3 3 5 n u c le a o ri e n ta li s 4 1 3 3 3 2 7 3 5 0 4 p o li y a lt h ia s u m a tr a n a 5 2 5 5 4 3 3 3 4 2 3 3 3 3 in o c a rp u s fa g iv e ru s 1 1 1 1 1 1 7 1 2 1 3 3 2 d io s p y ro s p a p u a n a 1 1 7 1 1 1 7 1 p la ti c e ri u m b if fu rc a tu m 1 1 4 1 1 1 7 1 1 1 7 1 p y s o n ia u m b e li fe ra 1 1 7 1 1 1 7 1 r a p a n e a s p 1 1 1 1 1 1 7 1 2 1 7 2 t e rm in a la c a n ic u la ta 1 1 7 1 1 1 7 1 m o ri n d a c it ri fo ll ia 1 1 4 1 1 1 7 1 1 1 7 1 p o ly a lt h ia g la u c a 4 1 7 4 4 1 7 4 a rt h o c a rp u s v re s ia n u s 1 1 7 1 1 1 7 1 d if f e r e n t ia l s p e c ie s .d 4 a ls to n ia s c h o la ri s 3 1 1 1 4 4 2 1 2 2 7 5 2 1 1 1 3 1 8 3 1 4 1 7 4 g n e tu m g n e m o d e is 7 3 1 1 2 5 6 3 2 2 5 0 2 1 2 3 3 2 1 1 5 1 1 8 3 2 c a n a ri u m d e k a m a n u m 2 1 8 7 4 4 5 7 7 1 7 5 5 4 2 3 3 3 4 3 3 3 4 l ic u a la te li v e ra 7 6 2 1 7 1 6 7 4 1 7 8 7 5 5 2 3 3 3 3 4 1 2 5 0 2 c a ll a m u s c a y e n s is 1 1 1 3 3 1 2 1 5 0 2 1 1 7 5 0 3 9 1 7 9 p o m e ti a a c u m in a ta 1 1 1 1 1 2 5 1 1 1 3 3 1 3 1 7 3 s y z ig iu m m a la c e n s is 1 4 1 3 3 2 1 2 5 1 1 1 7 1 3 1 7 3 p ra in e a li m p a to 2 1 4 2 0 2 2 5 2 1 1 7 1 c o m m u n it y c r e m a in in g s p e c ie s m e re m ia p e lt a ta 2 1 0 0 2 7 5 9 4 3 8 8 6 6 4 5 5 6 2 3 6 7 4 8 9 7 7 5 8 6 3 8 4 7 8 1 0 0 6 2 2 7 2 6 7 3 g n e tu m g n e m o 4 1 1 1 5 7 2 3 3 2 1 3 6 1 1 8 9 3 2 5 1 2 1 0 0 3 1 1 3 3 1 1 2 4 4 3 3 1 0 0 3 c e lt is l a ti fo li a 1 1 2 9 1 9 1 1 6 1 5 6 4 8 3 2 5 1 0 0 5 7 8 5 5 0 7 9 9 9 8 8 3 1 0 0 8 a m o m u m a c u le a tu m 8 1 3 4 7 9 1 1 0 0 5 1 4 3 4 4 4 3 3 5 4 7 5 4 2 4 3 3 3 1 1 1 5 0 1 k o rt h a lz ia z ip p e li i 1 1 9 8 9 7 8 6 6 7 7 9 7 3 1 1 7 8 5 5 4 3 7 1 0 0 5 1 1 7 1 h o rs fe ld ia p a rv if lo ra 5 2 9 8 5 7 6 5 6 7 7 2 2 1 7 8 9 5 8 6 3 5 1 0 0 6 8 3 2 5 0 4 3 2 3 5 0 3 m e d u s a n th e ra la x if lo ra 1 1 1 4 3 1 1 7 5 3 1 5 6 3 4 1 6 8 1 0 0 5 5 7 7 5 0 6 2 1 7 2 p im e le o d e n d ro m a m b o in ic u m 1 1 2 9 1 5 2 1 1 2 1 6 7 2 4 6 3 7 1 0 0 5 8 4 8 8 8 8 3 7 1 3 7 3 7 8 3 4 p h y lo d e n d ro n s p 1 1 2 4 3 1 6 2 5 8 2 1 1 1 8 9 3 4 6 5 8 1 0 0 6 2 5 1 5 0 3 2 7 7 5 0 5 h o rs fe ld ia ir y a 1 1 4 1 2 2 1 3 3 2 3 1 3 6 1 0 0 3 2 8 3 5 0 4 1 1 3 3 1 p ip e r g ib b il im b u m 2 1 3 7 4 7 1 3 4 3 4 5 6 9 6 7 5 5 8 5 0 7 3 3 6 4 6 7 4 6 5 3 8 8 1 1 0 0 5 b a u h in ia to m e n to s a 3 1 1 1 6 7 1 2 5 5 7 1 8 5 6 5 7 3 5 0 5 3 3 2 7 6 7 4 6 1 7 6 7 2 1 0 0 5 p o m e ti a p in n a ta 6 4 2 8 5 7 5 8 9 7 9 9 6 5 9 9 1 0 0 8 9 5 8 7 5 7 1 3 3 3 2 1 1 2 5 0 1 m a n il to a b ro w n o id e s 1 1 4 1 5 2 5 1 4 4 3 6 7 7 7 5 7 4 5 3 2 1 2 1 0 0 3 7 7 7 5 0 7 a rt h o c a rp u s a lt il is 4 1 5 9 5 7 5 4 7 1 1 1 1 1 1 8 9 2 6 5 3 7 5 5 1 1 2 5 0 1 6 1 2 2 6 7 3 a g la ia o d o ra ta 1 1 1 4 3 1 3 5 5 1 7 6 1 7 8 4 4 4 5 0 4 5 1 8 7 6 7 5 2 1 2 4 6 7 2 l it s e a ti m o ri a n a 3 7 2 9 5 2 1 4 1 4 4 2 5 1 2 7 5 3 3 7 3 3 5 1 1 7 1 h y d ri a s te le rh o p a lo c a rp a 1 7 5 4 3 4 6 6 2 2 6 2 2 6 7 5 3 1 4 3 3 3 h a p lo lo b u s la n c e o tu s 1 1 4 1 2 7 6 3 3 5 4 7 2 7 5 4 1 1 2 5 0 1 1 3 3 3 2 a s p le n iu m n id u s 1 1 4 1 2 1 1 2 3 2 2 7 5 2 1 1 1 5 0 1 a re c a m a c ro c a ly x 1 2 2 9 2 3 3 5 1 1 5 6 3 3 2 8 7 5 4 1 1 7 1 1 1 7 1 z in g ib e r s p 1 4 1 2 2 1 7 1 2 1 2 2 2 2 2 2 1 7 5 2 5 2 3 3 4 2 1 1 5 0 1 p a n d a n u s s p 5 6 9 6 2 7 1 6 1 1 5 4 4 4 3 3 2 5 3 7 1 2 5 0 3 g o n io ta la m u s s p 1 1 4 1 5 1 2 2 3 1 6 7 2 7 2 5 0 5 2 3 3 3 3 2 1 7 2 c a ry o th a ru m p h ia n a 1 1 1 1 5 7 1 1 1 1 1 1 2 5 1 1 2 1 5 0 1 1 1 1 2 1 8 3 1 c h is o c h e to n s e ra m ic u s 1 4 3 9 5 7 4 9 9 6 8 7 5 6 8 8 6 5 0 7 2 1 7 2 r h o p a lo b a s te la d e rm a n ii 4 3 2 9 4 9 1 5 3 3 5 3 1 5 0 2 1 1 1 5 0 1 1 2 1 5 0 1 e u o d ia e le ry a n a 1 2 2 9 2 1 1 1 1 5 3 5 0 4 2 2 3 3 2 p a lm e ri a s c a n d e n s 2 1 4 2 3 3 5 3 3 4 3 2 5 0 3 7 1 7 7 o s m o x y lu n g lo b u la re 1 1 2 9 1 1 1 1 1 1 2 5 0 2 1 1 7 1 1 1 7 1 l ic u a la la u te rb a c h ii 3 1 2 9 2 6 7 8 3 3 7 1 7 5 0 4 1 1 7 1 3 7 3 3 5 d ry p e te s s p 1 1 4 1 6 2 2 2 4 3 1 1 5 0 2 3 1 7 3 o c to m e le s s u m a tr a n a 5 1 1 4 3 2 1 1 1 1 1 2 5 1 2 1 7 2 2 1 3 3 2 g o m p a n d ra g la b o s a 1 1 4 1 5 6 7 3 3 6 2 1 7 2 2 2 3 3 2 a n ti a ri s to x ic a ri a 1 1 4 1 1 1 1 3 3 1 3 1 7 3 2 1 3 3 2 c h is o c h e to n c e ra m ic u s 9 1 4 9 7 9 6 3 3 7 8 2 5 8 1 1 3 3 1 a g la ia s p 1 4 2 9 3 1 9 2 2 5 1 1 7 1 1 1 3 3 1 m y ri s ti c a p s e d o a rg e n te a 7 8 2 9 8 1 1 2 2 1 7 2 5 7 1 1 7 1 2 1 7 2 biotropia vol. 19 no. 2, 2012 79 biological remediation of cyanide: a review karamba kabiru ibrahim, mohd arif syed, mohd yunus shukor and siti aqlima ahmad* faculty of biotechnology and biomolecular sciences, universiti putra malaysia, 43400 upm serdang, selangor, malaysia received 8 april 2014/accepted 9 november 2015 abstract cyanide and its complexes are produced by industries all over the world as waste or effluents. biodegradation is considered to be the cheapest and the most effective method to clean-up cyanide from the environment. several studies on different types of microorganisms that can degrade cyanide in the environment have been carried out. hydrolytic, oxidative, reductive and substitutive/transfer reactions are some of the common pathways used by microorganisms in cyanide degradation. biodegradation of cyanide can occur aerobically or anaerobically depending on the environmental conditions. immobilized enzymes or microorganisms prove to be very effective method of degradation. microorganisms such as , , , klebsiella oxytoca corynebacterium nitrophilous brevibacterium nitrophilous bacillus spp., spp. and ukmp-5m have been reported to be very effective in biodegradation of pseudomonas rhodococcus cyanide. : keywords biodegradation, cyanide, environment, microorganisms introduction carbon and nitrogen elements form cyanide. they are ubiquitously found in the environment. amygdalin, which is found in vegetables, fruits, seeds, cashew nuts, cherries, apricots and bean sprouts is a natural source of hydrogen cyanide (hcn) (mark . 1999). chemical treatment is et al the most widely used method in the degradation of industrial effluents containing cyanide. but, this treatment is very expensive and sometimes inefficient in the process and thus requires an alternative means of treatment (patil & paknikar 2000). researches on microorganisms have been carried out to see if they can be an alternative means of cyanide degradation. bioremoval has been reported to be less expensive than physical and chemical methods of cyanide degradation and faster than natural oxidation (ozel . 2010; et al dash . 2009). destruction of cyanide in et al wastewaters and tailing solutions by capable microorganisms has been proved to be an alternative to the long-practiced chemical methods for the removal of cyanide. biological methods of treating industrial effluents have been reported to have high capital cost but low operating cost. therefore, these methods are more profitable than the traditional process. biological process that could satiate the need for extraction and environmental control is now practiced in some countries that understood the process (dash . 2009).et al cyanide degrading microorganisms biological treatment process facilitates growth of microorganisms that are essential for treatment (akcil 2003). it has been discovered that cyanide naturally occurs in the environment via the degradation of plant cyanogenic glycosides. a lot of microorganisms are able to detoxify simple forms of cyanide (gadd 2001). pseudomonas fluorescens ncimb 11764 has been reported to be able to utilize potassium cyanide (kcn) in fed-batch culture (kunz . 1998). et al pseudomonas fluorescence has also been reported to degrade ferrocyanide (arzu & zumriye 2000). the growth of ncimb pseudomonas fluorescens 11764 on medium containing potassium cyanide (kcn) has also been reported (kunz . 1998). et al cyanide degradation by , alcaligenes, escherichia coli * corresponding author : aqlima@upm.edu.my biotropia vol. 22 no. 2, 2015: 151 163 151 doi:10.11598/btb.2015.22.2.393 mailto:aqlima@upm.edu.my 152 acinetobacter bacillus and species have also been reported in a number of studies. it has been reported that strains of bacteria from genus are very effective in the klebsiella bioremediation of cyanide and thiocyanate. k. oxytoca isolated from an industrial waste containing high level of cyanide proliferates well with the cyanide as the only nitrogen source (kao et al k. oxytoca. 2003). it has been reported that is effective in biodegradation even at concentrations higher than 1 mm of cyanide (chena . 1999). et al the microorganism biodegrades cyanide to products that are nontoxic using cyanide as the only nitrogen source aerobically or anaerobically (kao . 2003; stephen 2004). seven bacterial et al strains isolated from gold mine environment in korea have been found to be very effective in the biodegradation of thiocyanate with the initial concentration of 150 mg/l within sixteen days of incubation (lee . 2003). four strains from the et al genus , one strain of bacillus corynebacterium nitrophilus brevibacterium and two from have been found to effectively degrade cyanide. pseudomonas pseudoalcaligenes cect5344 is an alkaliphilic strain of bacteria that is reported to be capable of biodegrading cyanide. the organism was isolated from sludge of guadalquivir (cordoba, spain). it proliferates at an optimum ph of 9.5 and utilizes 2 mm of cyanide as the only hydrogen source (luque-almagro . 200 ; et al 5 huertas . 2010). the organism can also utilize et al complex of cyano-metal, cyanate and residue from jewellery industry as the sole nitrogen source (luque-almagro . 200 ). this strain has been et al 5 described to possess a cyanide-insensitive respiration system, which includes cytochrome bdtype alternate oxidase (aox) that replaces the cytochrome c oxidase (quesada . 2007)et al . general pathway reaction for biodegradation of free cyanide there are four common pathways involved in the bioremoval of cyanide i.e. hydrolytic reaction, oxidative reaction, reductive reaction and substitution/transfer reaction. more than one pathway can be used for cyanide degradation by certain microorganisms (raybuck 1992; ezzimufaddal & lynch 2005). the pathway to be used depends on various factors such as oxygen availability, ph level of the environment, concentrations of the cyanide and cyanide bioavailability and solubility in the soil water system (aronstein . 1994).et al hydrolytic reaction the following equations show the reactions that occur during hydrolytic pathway for cyanide degradation (david . 2006):et al hydrolytic reactions contain nitriles with r denotes either analiphatic or aromatic group. hydrolytic reactions are catalyzed by cyanide hydratase, founding a formamide or cyanidase and yields formate and ammonia. cyanide hydratase is principally a fungal enzyme and is extremely preserved between species (barclay et al. 2002). cyanide dihydratase (cyanidase) is produced chiefly by bacteria. cyanide hydratase and cyanidase have, in recent times, been presented to have certain resemblances at both the amino acid and structural stages to nitrilase and nitrile hydratase enzymes (reilly & turner 2003). enzymes that utilize nitrile have been established in a wide variety of fungal, plant and bacterial species. nitrilases and nitrile hydratases modify both aliphatic and aromatic nitriles to the equivalent acid or amide, respectively, but indicate less substrate specificity than cyanide hydratase and cyanide dihydratase. for instance, conversion of with the cyanide hydratase gene from e. coli fusarium lateritium permits the proliferation of nitriles as the only source of nitrogen. sitedirected mutagenesis of this gene stops the activity of both cyanide hydratase and nitrilase, signifying that cyanide hydratase possesses nitrilase activity, as well (nolan . 2003). the et al variety of enzymes in this fantastic family and their diverse catalytic act and substrate specificities grant significant chance for biotechnological improvement, encompassing the bioremediation of industrial nitrile waste (rezende . 2000; dias . 2001).et al et al oxidative reaction in the oxidative reaction, the cyanate formed by the enzyme cyanide monoxygenase is changed to ammonia and carbon dioxide by the same pathway as cyanate and thiocyanate (david . et al 2006). biotropia vol. 22 no. 2, 2015 substitution/transfer reaction this is referred to as assimilatory pathway. several genera of bacteria are said to assimilate cyanide. this reaction is more extensively researched in . other chromobacterium violaceum bacteria that utilize this reaction include escherichia coli, bacillus megaterium, citrobacter freundii and (david . 2006). the enterobacter aerogenes et al substitution/transfer reaction uses cyanoalanine synthase enzyme as the catalyst using oacetylserine (oas) as substrate. t h e c y a n a t e p r o d u c e d b y c y a n i d e monoxygenase is changed to nh and co by 4 2 + very similar pathway as the cyanate from thiocyanate (stephen 2004). thiocyanate biodegradation reactions of cyanide with pyretic materials in effluents lead to the production of thiocyanate. the enzyme that is responsible for the production of thiocyanate is sulfurtransferase via the in vivo action of thiosulfate-cyanide. its biodegradation can be achieved by at least two pathways namely, cyanate and carbonyl pathways (david . 2006; et al kwon . 2002; plessis . 2001; sorokin . et al et al et al 2001; yamasaki . 2002).et al three species of thiocyanate-degrading bacteria obtained from very high alkaline soda lagoon soils and sediments produce high amounts of cyanate when grown at ph 10 with thiocyanate as the only source of nitrogen. the activity of cyanase that converts cyanate to carbon dioxide and ammonia is also high (sorokin . 2001). et al the fungus produces sulphate acremonium strictum and ammonia from thiocyanate devoid of the production of cyanate (kwon . 2002). in the et al cyanide is converted to cyanate by cyanide monoxygenase with cyanate standing as the catalyst for conversion of cyanate to ammonia and carbon dioxide dependent on bicarbonate. cyanases have been identified in several bacteria, fungi, plants and animals (guilloton . 2002).et al the assumed task of cyanase has, for a long time, been as a defense against poisoning by cyanate (raybuck 1992). as cyanate is not a familiar metabolite, new essential roles in favor of cyanases in nitrogen and bicarbonate/carbon dioxide metabolism have been suggested. more proposed roles for plant cyanases comprise ammonia absorption as a result of cyanate bioremediation and a task in the concentration and deliverance of carbon dioxide for photosynthesis (guilloton . et al 2002). another oxidative pathway makes use of cyanide dioxygenase to produce ammonia and carbon dioxide directly (stephen 2004). moreover, in strain bcn6 and ncimb e. coli p. fluorescens 11764, the production of cyanohydrin complexes is reportedly essential for oxygenase-mediated cyanide biodegradation (kunz . 1998; figueira et al et al. 1996). reductive reaction the reductive pathway results from the act of nitrogenase enzyme and the products ensuing from transfer of pair of electrons (stephen 2004; david . 2006).et al studies have been carried out in two bioreactors in cassava wastewaters and synthetic wastewater. this indicates a clear reductive process on biodegradation (paixao . 2000; annachhatre & et al amornkaew 2000). the studies indicate that the anaerobic bioreactors prop up the proliferation of methanogenes which can result in the production of biogas. the increase in the cyanide concentration inhibits methanogenesis from anaerobic biogranules. this effect could be a result of high cyanide concentration in the feed stock (annachhatre & amornkaew 2000). biological remediation of cyanide: a review ibrahim – et al. 153 carbonyl pathway, the thiocyanate is transformed to ammonia and carbonyl sulphide. the confirmation for this pathway is as a result of the identification of thiocyanate hydrolase that is responsible for the transformation in the chemolithotroph and the thiobacillus thioparus genes encoding this enzyme have been acknowledged in other thiocyanate-degrading bacterial cultures (yamasaki . 2002)et al . aerobic biodegradation of cyanide the process of biodegradation that requires the use of oxygen is termed as aerobic system of biodegradation. under aerobic conditions, cyanide is broken down by cyanide-oxidizing bacteria into harmless compound. the process degrades hydrogen cyanide and produces hydrogen cyanate, which then undergoes hydrolysis to form ammonia and carbon dioxide as shown in the equation below: reports have indicated that aerobic process of biodegradation is many times faster and better than anaerobic degradation (stephen 2004). algae such as ,arthrospira maxima scenedesmus obliquus, and spp. have also been reported in the chlorella detoxification of cyanide (dwivedi . 2011; et al gurbuz . 2009).et al anaerobic biodegradation of cyanide anaerobic system of biodegradation is the process that occurs in the absence of oxygen (dwivedi . 2011) fedorak and hurudey in et al . 1989 was the first to report biotreatment of cyanide anaerobically using semi continuous batch cultures and hydraulic retention time (hrt) of 25 days for one liquid volume replacement (fallon . 1991). anaerobic biodegrada-et al tion can only occur in the presence of hs or h s 2 2 and is restricted to reduce portion of the heap environment. the sulphur species that is present depends on the ph. at ph ≥ 7, hs is the species 2 that is dominant and at ph ≤ 7 although h s is the 2 more dominant species of sulphur in other phs (dwivedi . 2011).et al the hcns will undergo hydrolyses to produce nh , h s and co .2 2 2 anaerobic cyanide biodegradation has been proven to be a concomitant method of biogas generation that is of economic benefit (stephen 2004). under anaerobic condition, biodegradation results in the formation of nitrogen as the end product. furthermore, anaerobic treatment is not fast and it is more vulnerable to toxic upsets ensuing from exposure to other elements present in the solution undergoing the treatment. in addition, cyanide toxicity threshold for anaerobic bacteria is only 2 mg/l, while aerobic bacteria have 200 mg/l threshold. therefore, anaerobic bioremoval is considered to be less effective method of bioremoval mechanism (stephen 2004). effect of immobilization on cyanide biodegradation immobilization has been reported to be a very effective and efficient tool of biodegradation. this tool offers many economic and technical advantages over the free cell type as it offers the possibility of maintaining the cells in a stable and viable condition with high specific surface area for microbial proliferation (dursun & aksu 2002). immobilized cells are less vulnerable to compounds that are toxic and exhibit high tolerance towards distress in the reaction environment (chen . 2008) moreover, et al . immobilized cells are more advantageous when compared with the use of free cells because of the ability to support higher concentration of cell density and eliminate the difficult, timeconsuming and expensive process of cell recovery and recycling (zhou . 2007). et al bioremediation by cct candida guilliermondii 7207 was very effective on immobilized cells (dias . 2001). this is a remark that reiterates et al previous researches indicating that immobilized microorganisms or enzymes make available an active stage for bioremediation (stephen 2004). effective biodegradation of cyanide compounds by immobilized on zeolite has been p. fluorescens reported (suh . 1994). m. graca campos in et al 2005 reported effective, feasible and efficient detoxification of cyanide by immobilized f u s a r i u m o x y s p o r u m c c m i 8 7 6 a n d methylobacterium sp. rxm ccmi 908 using a packed bed reactor for integrated biodegradation biotropia vol. 22 no. 2, 2015 154 155 of cyanide (campos . 2006) maegala et al et al.. (2012) reported effective biodegradation of cyanide by immobilized cells of rhodococcus ukmp. oxygenation in immobilized cell culture oxygenation is a major challenge in immobilization. researchers have reported critical challenges in supplying sufficient and adequate concentration of oxygen in order to keep high amount of viable and productive cells throughout a culture period (meuwly . 2005). in packed et al bed reactors it was observed that if there is low supply of oxygen, there will be decrease in viability of culture, metabolic activity and productivity until optimal supply of oxygen is achieved. this is because oxygen has a very poor solubility in cell culture medium (fassnacht & portner 1999). factors affecting cyanide biodegradation in environment the presence of microorganisms that have the physical and metabolic abilities to degrade the contaminants in the polluted environment ensures the success of biodegradation. cyanide compounds are found broadly in natural surroundings and the metabolic degradation of these compounds by microorganisms is thus possible. however, the following factors affect the process (kao . 2006; baxter & cummings et al 2006; dash . 2009):et al  cyanide concentration in the environment can have significant effect in the treatment. for instance, high concentration of acetonitrile has been proved toxic to by causing klebsiella oxytoca damage to nitrile hydratase, which is the nitriledegrading enzyme and by preventing bioremoval of the compound by the microorganism.  biodegradation of cyanide compounds can be affected by the availability of nutrients. carbon has been recognized as the restrictive factor in the biodegradation of cyanide compounds, which may make the biodegradation of industrially polluted soils not feasible.  a e r a t i o n i s ve r y i m p o r t a n t i n t h e biodegradation of cyanide as oxygen is required during the degradation pathways.  cyanide toxicity can be paramount to anaerobic bacteria principally methanogens. other contaminants present at the polluted areas may also affect bioremoval.  existence of high concentration of other pollutants can have negative effect on degradation of cyanide by swaying the native population and possibly hindering the proliferation of specific organism. biodegradation of free/complex cyanide and thiocyanate in the biotreatment of cyanide, bacteria have the capacity to change free and metal cyanides to bicarbonate and ammonia, whereas the free metals are adsorbed within the biofilm or unconfined as precipitates in solution. alkali metal cyanides such as potassium cyanide (kcn) and sodium cyanide (nacn ) are easily being 2 degraded by different forms of bacteria across the globe. in 1969 in usa, free cells of bacillus megaterium was reported to degrade potassium cyanide (castric & strobel 1969). in the same year in canada, free cells was reported bacillus pumilus to degrade 2.5 mg/l of potassium cyanide at the ph of 8.5 9.0 and temperature of 40 – °c (skowronski & strobel 1969). in 1972, free cells of which is a pathogenic fungus stemphylium loti, of the cyanogenic plant 'bird's foot-trefoil' ( l.) (fry & mills 1972) was lotus corniculatus reported to degrade 0.97 m of potassium cyanide at ph of 6.5 7.5 and temperature of 25 with – °c the removal efficiency of 77 nm (fry & mills 1972). several reports have been reviewed across the world up to the year 2012. in malaysia rhodococcus ukmp-5m in both free and immobilized forms was reported to degrade potassium cyanide at 30 temperature °c condition with the removal efficiency of 64 and 96%, respectively (maegala . 2011; maegala et al et al. 2012). this removal capacity is not only limited to bacteria but other fungal organisms such as fusarium solani, which in 1997 its free cells were re ported to degrade potassium cyanide concentration of 0.5 0.8 mm at the ph of – 9.2 10.7 and temperature condition of 30 in – °c france (dumestre . 1997). in 1975, free cells et al of nca 1503 was reported to b. stearothermophilus degrade nacn and nahso with initial 2 3 concentration of 5 mm and 50 mm at a ph of 7.8 and temperature of 27±2 with the removal °c efficiency of 5 8 g/l/hour (atkinson 1975). the – simplicity within which the complexes of metal biological remediation of cyanide: a review ibrahim – et al. 156 biotropia vol. 22 no. 2, 2015 cyanide are being degraded commonly follows the sort of general stability, with free cyanides being the readily degradable while cyanide of iron being the least degradable. zinc (zn), nickel (ni) and copper (cu) are moderate in terms of degradability of their metal cyanides (young & jordan 1995; botz 2001). a study conducted in korea in 1994 reveals that p. fluorescens immobilized on zeolite was able to degrade tetracyano nickelate ii at 30 (suh . 1994). °c et al studies in turkey in 1999 showed that immobilized was able to degrade p. fluorescens ferrous (ii) cyanide complex (dursun . 1999) et al while free cells of was able to degrade p. fluorescens 100 mg/l ferrous (ii) cyanide complex at ph 5 and temperature of 25 at degradation rate °c of 30 mg/g/hour (dursun . 1999). free cells et al of spp. and spp. were citrobacter pseudomonas reported in india to degrade 52 mg/l metal cyanide complexes such as copper cyanide and zinc cyanide with the removal efficiency of > 99.9% (patil & panikar 2000). in the united kingdom in 2005, free cells of sp.trichoderma and sp. were reported to degrade 2,000 fusarium mg/l metallo cyanide at ph 6.5 and temperature of 25 , with the removal efficiency of 2,000 °c p p m ( e z z i m u f a d d a l & l y n ch 2 0 0 2 ) . degradation of complex cyanide is not limited to only bacteria because in the united kingdom in 1998, mix cultures of ,fusarium solani, t. polysporum f. oxysporum scytalidium thermophilum , and penicillium miczynski were reported to degrade k ni(cn) and k fe(cn) at a concentration 2 4 4 6 of 0.75 mm and 0.25 1 mm concentrations and – ph 4.5 7.0 and temperature conditions of 25 – °c with the removal efficiency of 50 56%, – respectively (barclay . 1998). in 2001 in south et al africa, some free cells of microorganisms were reported to degrade thiocyanate at the concentration of 500 mg/l with the removal efficiency of 0.5 mg/l/h (sorokin . 2001). in et al the same year in russia, free cells of alkaliphilic bacteria were reported to degrade 40 mm initial concentration of thiocyanate at a ph 10 and temperature of 28 with removal efficiency 4 °c mm (plessis . 2001). in 2002 in korea, et al acremonium strictum was reported to degrade 7.4 g/l concentration of thiocyanate at a ph 6 with 100% removal efficiency in 85 hours (kwon . et al 2002). in 2002 in tokyo, japan, free cells of thiobacillus thioparus thi115 was reported to degrade thiocyanate with initial concentration of 0.1 g/l at a temperature of 30 (yamasaki . °c et al 2002). there is no doubt from this review that pseudomonas sp. has an upper hand in the degradation of both free and complex cyanides in quite a number of countries across the globe. table 1 summarizes the degradation potentials of some microorganisms on free and complex cyanides and thiocyanate. 157 biological remediation of cyanide: a review ibrahim – et al. biotropia vol. 22 no. 2, 2015 158 159 biological remediation of cyanide: a review ibrahim – et al. biotropia vol. 22 no. 2, 2015 160 conclusions certain economic and physical factors chiefly limit the use of biotechnologies in the degradation of cyanide. biodegradation is potentially the cheapest means to get rid of cyanide but factors such as ph, temperature and nutrients concentration of the cyanide affect the process. bioremoval can be achieved under aerobic and anaerobic conditions. free and immobilized cells have proven to be very effective and efficient methods of biodegradation. the microbes potentially have certain enzymes that can change cyanide into naturally occurring compounds. four types of pathways are used by the microorganisms to biodegrade and one or combination of two pathways can be employed in the process by microorganisms (raybuck 1992; ezzi-mufaddal & lynch 2002). spp. shows great pseudomonas capability for cyanide removal. in 2011 and 2012, rhodococcus ukmp-5m obtained from culture collection unit, institute of bio-it selangor was reported to be used to degrade potassium cyanide in free and immobilized forms and it has been proven to be efficient in the degradation (maegala et al et al. 2011; maegala . 2012). table 1 indicates that from 1969 to date, researches have been carried out on the bioremoval of cyanide. however, despite all the investigations that have proved microorganisms can degrade cyanide in the laboratory; it has not been accomplished in a large scale (fatma . 2009).et al references akcil a. 2003. destruction of cyanide in gold mill effluents: biological versus chemical treatments. biotechnol adv 21: 501–11. akcil a, karahan ag, ciftci h, sagdic o. 2003. biological treatment of cyanide by natural isolated bacteria ( sp.). miner eng 16: 643–9pseudomonas . annachhatre ap, amornkaew a. 2000. toxicity and degradation of cyanide in batch 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niversity of queensland, st. lucia campus, brisbane australia received /accepted 21 may 2014 6 june 2015 abstract begomovirus was identified as one of the causal agents associated with yellow mosaic disease on yard long bean ( subsp. l.) in java. previous study reported that has infected several vigna unguiculata sesquipedalis begomovirus leguminosae in south asia. several have been reported to infect important crops in indonesia. those begomoviruses begomoviruses were characterized based on nucleotide sequences. this study was conducted to identify and characterize taken from yard long bean samples based on specific genome property of common begomovirus in java region. three main activities were conducted: i) sample collection in yard long bean fields located in central java (tegal, magelang, and klaten), yogyakarta (sleman) and west java (bogor and subang) provinces; ii) virus detection using i-elisa, pcr and sequencing; iii) molecular characterization of using software bioedit v.7.0.5 and begomovirus mega 6.06. yellow mosaic disease was found in almost all fields. infection of and was detected potyvirus begomovirus using i-elisa and pcr, respectively. both viruses were detected as either single or mixed infection samples . collected from tegal, klaten, magelang, subang and bogor were positively infected by based on specific begomovirus viral dna amplification. sequence analysis indicated that infecting yard long bean is mungbean yellow begomovirus mosaic india virus (mymiv) and it belongs to the same group with mymiv from bangladesh, india, pakistan and nepal. further analysis showed the conserved region of around common region, i.e. “tata box” begomovirus sequence, hair pin loop structure, repetitive sequence and the conserved nonanucleotide sequence taatattac were also determined. this is the first report of mymiv infection in indonesia. keywords: begomovirus, common region, dna sequencing, i-elisa, mymiv, pcr, yard long bean introduction a outbreak yellow mosaic disease was reported in yard long bean ( subsp. vigna unguiculata sesquipedalis l.) growing area in java since 2008 (damayanti 2009). disease was asso-et al. the ciat bean common mosaic ed with infection of virus (bcmv), cucumber mosaic virus (cmv) and (damayanti 2009; hidayat begomovirus et al. 2011, ; tsai . 2013). the personal communication et al at same time, similar yellow mosaic disease caused by begomovirus was first reported from pakistan (ilyas et al. et al. . 2010) and nepal (shahid 2012) begomovirus geminivirus is a member of group which is transmitted in nature by whitefly bemisia tabaci gen. (hemiptera: aleyrodidae). molecular character of its genome is very unique, istics having one or two circular single stranded dna (2.6–2.7 kb) and represented as monopartite or bipartite, respectively (hull 2002). serious crop diseases caused by infection of have begomovirus been reported in indonesia. pepper yellow leaf curl virus (pepylcv), tomato yellow leaf curl virus (tylcv), and tobacco leaf curl virus (tlcv) are considered as the major factor causing yield loss on chilli pepper, tomato and tobacco, respectively (aidawati 2005; hidayat et al. et al. 2006; 2008). damage and potential yield loss in yard long bean caused by should begomovirus be anticipated by establishing detection method and disease control strategy. to do so, basic information regarding molecular and biological characters of the virus is required.* corresponding author : srihendrastutihidayat@gmail.com biotropia vol. 22 no. 1, 2015: 53 60 doi: 10.11598/btb.2015.22.1.401 53 mailto:srihendrastutihidayat@gmail.com biotropia vol. 22 no. 1, 2015 54 m begomovirusethod for detection of is commonly based on polymerase chain reaction (pcr) technique using universal primers. degenarate primers pal1v1978/ par1c715 was first used by rojas (1993) as universal primer et al. to detect several infectedgeminiviruses from plants belong to solanaceae, leguminosae, euphorbiaceae and malvaceae from south america. since then, the primer pair has been used to identify and characterize various members of . begomovirus fragment of dna amplified by this begomovirus pair of primers covers part of al1 region (replicase gene), common region and intergenic region and part of ar1 region (coat protein gene). molecular characters of can be begomovirus determined by analyzing this dna fragment sequences due to its conserved and diversed region. common region for each is begomovirus different, except loop region on hairpin structure, 5'-taatattac-3' which is known as the conserved nonanucleotide region (lazarowitz 1987). >92% similarity on begomovirus posse sings their hairpin loop structure can be as one grouped species (fauquet 2005).et al. this paper explained the attempts to identify and characterize from yard long bean begomovirus samples based on specific genome in java property of common region. materials and methods collection of field samples samples collection was conducted in west java (bogor and subang), central java (tegal, klaten and magelang) and yogyakarta (sleman). leaf samples were collected from the plants age 6–9 wap using purposive sampling method based on yellow mosaic symptom described by damayanti et al. et al. (2009) and ilyas (2010). fresh tissue was directly subjected for virus detection and the remaining were stored as isolate collection in the laboratory -80 c.at o virus detection by indirect enzyme-linked immunosorbent assay (i-elisa) two major viruses on yard long bean i.e. potyvirus and cmv were detected from leaf samples using i-elisa following commercial kit manufacturer (protocol leibniz-institut dsmz gmbh germany) infected leaves was ground in , . coating buffer (1/10, v/v). coating buffer (ph 9.6) contain 15 mm na co (1.59 g), 2.38 mm 2 3ing nahco (2.93 g), 3.08 mm nan (0.20 g) and 3 3 h o was added to final volume of 1 l. aliquots 2 of each sample (100 µl) was dispensed to each microtitters' well, then incubated overnight at 4 c. microtitter plate was washed 8 times using o pbs-tween (0.137 m nacl (8.0 g), 1.47 mm kh po (0.2 g), 8.1 mm na hpo (1.15 g), 2.68 2 4 2 4 mm kcl (0.2 g), 3.08 mm nan (0.2 g), add h o 3 2 up to 1 l ph 7.4 and add 0.5 ml tween 20/ l). plates were dried up by tapping upside down on tissue paper. blocking solution (2% skim milk diluted in pbs-tween) was added 100 µl to each well then incubated at 37 c for 30 minutes. o blocking solution was removed and plate was tapped dry. first antibody (igg) was diluted in conjugate buffer (pbs-tween containing 2% pvp (serva pvp-15 polyvinyl pyrrolidon) and 0.2% egg albumin) according to manufacturer recommendation, then 100 µl conjugate buffer contains igg was added to each well, incubated at 37 c for 2-4 hours, then washed by pbs-tween. o second antibody (igg-ap) was diluted in conjugate buffer according to manufacturer recommendation, then 100 µl conjugate buffer containing igg-ap was added to each well, incubated at 37 c for 2 hours then washed using o pbs-tween. substrate (10 mg p-nitrophenyl phosphate) was dissolved in substrate buffer (97 ml diethanolamine, 600 ml h o, 3.08 mm nan 2 3 (0.2 g), adjust to ph 9.8 with hcl and make up to 1 l with h o) added 100 µl to each well. plate 2 was incubated in the room temperature under low light intensity and reaction was evaluated. positive reaction was qualitatively indicated by appearance of yellow color and quantitatively determined by measuring absorbance value at 405 nm wave length, i.e. two times absorbance value of negative control (healthy plant), using elisa reader (biorad 550). virus detection by pcr total viral dna was isolated from infected leaf following a procedure described by doyle and doyle (1987) with modification. fresh minor tissue (0.1 g) was ground with liquid itrogen to n powder, 500 µl of ctab buffer (10% cetyltrimethyl-ammonium (100 ml), 0.1 m bromide tris-hcl ph 8 (100 ml), 0.05 m edta (50 ml), 0.5 m nacl (126 ml), 1% β-mercapto-ethanol molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. (10 ml and added h o up to 1 l was added and ) 2 the sap was transferred to 1.5 ml clean tube. the sap was incubated in water bath at 65 c for 1 hour, o shaked every 10 minutes to separate and lipid p r o t e i n . f i v e h u n d r e d m i c r o l i t e r o f chloroform/iso-amyl alcohol (24:1, v/v) was added o the liquid, then the tube was vortexed for t 5 minutes and centrifuged at 14,000 rpm for 15 minutes. the supernatant was pipetted to 1.5 ml clean tube, 1/10 volume of 3 m ammonium acetate and 2/3 volume of isopropanol was added, respectively. the liquid was mixed gently then incubated overnight at -20 c or 4 hours at o room temperature. after incubation, the liquid was centrifuged at 12,000 rpm for 10 minutes to precipitate dna and then was discarded flowthrough. the pellets were washed with 500 µl of 70% ethanol, centrifuged at 8,000 rpm for 5 minutes and dried in room temperature after discarding the flow through. the dried pellets containing total dna were dissolved in 50 to 100 µl of nuclease free water or te buffer, ph 8 and the dna was ready for pcr. amplification of viral dna was conducted following method described by rojas (1993) et al. to confirm geminivirus infection. pcr reaction contained 10xpcr buffer, 25mm mgc , 2.5mm l2 dntps, 10µm each of primer pal1v1978 and par1c715, polymerase (5u/µl), 1 µl of taq dna and the reaction was adjusted to 25 µl with nuclease free water. amplifications was performed in geneamp pcr system 9700 machine with 5 minutes at 94 c for pre-heating, o followed by 30 cycles of denaturation (1 minute at 94 c), annealing (1 minute at 50 c) and extension o o (3 minutes at 72 c). the last cycle was followed by o 72 c for 3 minutes and decreased at 4 c. agarose o o gel electrophoresis was used to visualize pcr products. dna sequencing viral dna fragments obtained from direct pcr amplification were sent to pt genetika science, indonesia and australian genome research facility, australia, respectively for dna sequencing. sequence data were compared with other sequences from genbank (ncbi 2013) and analysed using software programs bioedit v.7.0.5, clc sequence viewer 7, and mega 6.06. results and discussion identification of from field begomovirus samples incidence of yellow mosaic disease was very high i.e. 80% to 100% in most growing areas. infected plants were easily recognized in the field based on visual symptoms. three main symptoms of yellow mosaic disease in the field were observed i.e. 1) yellowing; 2) yellowing with green spot; and 3) mosaic vein b nding (fig. 1 and a table 1). the most common symptom found in every field was yellowing. yellowing with green spot was thought as early symptom before it developed into yellowing. further severe infection caused smaller pods and leaves. these type of symptom had also been reported as s typical symptoms of yellow mosaic disease of yard long bean in south asia (ilyas 2010). et al. begomovirus was detected in 11 infected plant samples (fig. 2) showing yellowing and yellowing with green spot symptoms from all locations. however, fragment was not begomovirus successfully amplified from samples showing mosaic vein banding from subang viral detection using i-elisa revealed the infection of potyvirus 55 figure 1. symptoms of yellow mosaic disease on yard long bean: (a) yellowing; (b) yellowing with green spot; (c) mosaic vein banding biotropia vol. 22 no. 1, 2015 56 table 1. detection of cucumber mosaic virus, and from leaf samples potyvirus begomovirus collected in wet season 2012 from various locations using i-elisa and pcra location code of isolates symptoms description i-elisab pcr cmv potyvirus begomovirus tegal tegal 1 yellowing + + tegal 2 yellowing mosaic + klaten klaten 1 yellowing + klaten 2 yellowing mosaic sleman sleman 1 yellowing + + sleman2 yellowing mosaic + + magelang magelang 1 yellowing mosaic + magelang 2 yellowing + + magelang 3 yellowing mosaic + subang subang 1 mosaic vein banding + subang 2 mosaic vein banding + subang 3 mosaic vein banding subang 4 yellowing + bogor bogor 1 yellowing + bogor 2 yellowing + figure 2. visualization of amplification from leaf samples using universal primers pal1v1978/ par1c715 on begomovirus 1 % agarose gel. m, 1 kb marker dna (thermo scientific, us); k+, dna of pepylciv; 1-15 leaf samples from fields (1, tegal 1; 2, tegal 2; 3, klaten 1; 4, klaten 2; 5, sleman 1; 6, sleman 2; 7, magelang 1; 8, magelang 2; 9, magelang 3; 10, subang 1; 11, subang 2; 12, subang 3; 13, subang 4; 14, bogor 1; 15, bogor 2) a most plants were in generative stage when samples was collected = not detected; + = detectedb but not of cucumber mosaic virus (cmv). mosaic vein banding symptom caused by potyvirus infection had been described previously by damayanti (2009). this result indicated the et al. association of with yellow mosaic begomovirus disease of yard long bean in java. analysis of sequence identity of begomovirus infecting yard long bean nucleotide sequences were obtained for begomovirus isolates from tegal, klaten, magelang, subang and bogor. the sequence of isolate from sleman was not good due to unreadable chromatogram, therefore it was not included in the further sequence analysis. analysis of their identity by comparing to sequences on the genebank showed their highest homology with mungbean yellow mosaic india virus (mymiv) from brebes and purwakarta, i.e. >92%, followed by mymiv from bangladesh, nepal, pakistan and india, i.e. >87% (table 2). their homology to mymv, another virus causing yellow mosaic disease in south asia, was only 71-76% and to other reported from indonesia was begomovirus even lower i.e. 5154%. further dendogram analysis to study their relationship showed that all 57 table 2. nucleotide sequence homology (%) of infecting yard long bean in java with other reported begomovirus begomoviruses earlier in genebank begomovirus infecting yard long bean begomovirus isolates from genebank a) 1 2 3 4 5 6 7 8 9 10 11 12 ptgl1 99.7 97.9 92.5 91.1 92.2 92.1 76.0 55.8 53.8 51.4 51.5 16.6 tegal 2 95.7 94.7 89.5 88.3 89.4 89.3 73.1 54.6 53.9 52.5 52.0 17.4 klaten 96.0 93.2 88.0 86.8 87.9 87.9 71.9 54.1 54.2 52.9 51.9 17.2 magelang 95.9 92.8 87.7 86.4 87.5 87.5 71.7 53.5 53.8 52.1 51.6 16.8 magelang 2 95.3 92.8 87.7 86.4 87.5 87.5 71.7 53.5 53.8 52.1 51.6 16.8 subang 95.5 94.7 89.5 88.3 89.4 89.3 73.0 54.6 54.0 52.5 52.0 17.4 bogor 1 95.4 94.8 89.6 88.4 89.5 89.4 73.0 54.7 54.1 52.5 52.2 17.4 bogor 2 95.1 93.8 88.6 87.4 88.5 88.4 72.6 53.7 54.1 52.7 52.1 17.5 a)1= mymiv brebes (jn368436); 2= mymiv purwakarta (jn368434); 3= mymiv india (kc852204); 4= mymiv bangladesh (af314145); 5= mymiv nepal (ay271895); 6= mymiv pakistan (am992618); 7= mymv india (kc911271); 8= pepylciv (ab246170); 9= tlciv (ab241671), 10= tolcndv (data unpublished); 11= tolcjv (189848); 12= bcmv bogor (fj653916) figure 3. phylogenetic analysis of mungbean yellow mosaic india virus based on alignment of partial nucleotide sequences of the dna-a of using mega 6.06 (algorithm neighbor joining with 1,000 bootstraps replicates)begomoviruses begomovirus infecting mungbean (mymiv and mymv) belonged to similar cluster and they were separated from infecting other crops begomoviruses (pepylciv, tlciv, tylcndv, tolcjv) (fig. 3). it indicated that mymiv from java had closer genetic relationship to mymiv from south asia than other from begomoviruses indonesia. analysis of common region sequences of begomovirus infecting yard long bean molecular characters of could be begomovirus determined by analyzing the top region of its genome covering common region and intergenic region representing the conserved and diversed region, respectively. common region for each begomovirus was different, except loop region on molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. biotropia vol. 22 no. 1, 2015 58 figure 4. alignment of nucleotide sequences of the cr of mungbean yellow mosaic india virus (mvmiv) isolates from java with other reported mymiv in genebank. the alignment showing repetitive sequences (grey shadow), invert repeat (green shadow), tata sequences (bold letter with purple shadow) and the hairpin loop region (underlined letter with yellow shadow) with nonanucleotide sequence (bold and underlined letter with turquoise shadow) 59 hairpin structure, 5'-taatattac-3' which is known as the conserved nonanucleotide region (lazarowitz 1987). comparison of iteron and protein products variations have been used to determine of mymv and mymiv from several regionals in india (varma & malathi 2003; usharani . 2004) and et al tylcv isolates from israel, sardinia and thailand (argüelo-astorga . 1994). however, et al exception was reported on several cases, for example potato yellow mosaic virus with tylcv-sardinia and bgmv-brazil with tylcv-israel have identical iterons although they have a rather far distance relationship (argüelo-astorga 1994).et al. our analysis showed that common region of begomovirus isolates infecting yard long bean in java consists of repetitive sequence (atcggtgt), s tata box, and hair pin loop structure (fig 4). . three direct repeats (two of them are tandem repeat sequences) can be found before tata .box in all mymiv and mymv isolates (fig 4). iteron and tata box sequence always present together in cr of each begomovirus species (lazarowitz 1987). the function of iteron and tata box was described by argüelo-astorga et al. (1994) as initiator for rolling circle replication which was known as rap-specific binding sites. similar characteristics were reported for eastern hemisphere geminivirus except acmv and icmv, western hemisphere geminivirus, et al.and sqlcv-e and –r (argüelo-astorga 1994), and mymiv on soybean (usharani . et al 2004). hair pin loop structure of each isolates was identical to mymiv from bangladesh, pakistan, india, nepal, and one isolate of pepylciv, but they were different from tlciv and pepylciv (fig. 5). >92% similarity begomovirus posse sings on their hairpin loop structure could be grouped as one species (fauquet 2005; hidayat . et al. et al 2008). therefore, all isolates from this begomovirus study was the same species with mymiv from bangladesh, india, pakistan, and nepal. conclusions molecular detection and characterization confirmed the association of mymiv in yellow mosaic disease of yard long bean in java. virus isolates from java had the highest similarity (>85%) with mymiv isolates from bangladesh, pakistan, india, and nepal. further study on characters of the virus, such as host range, insect transmission and disease spread, are very important to understand disease development and control. acknowledgements this research was funded by collaboration b e t w e e n au s a i d f u n d e d e c o n o m i c cooperation work program of the asean– australia–new zealand free trade agreement and department of plant protection, faculty of agriculture, institut pertanian bogor. figure 5. hairpin loop structures of several isolates. a. mymiv tegal; b. mymiv klaten; c. mymiv begomovirus magelang; d. mymiv subang; e. mymiv purwakarta; f. mymiv bogor; g. mymiv bangladesh; h. mymiv pakistan; i. mymiv india; j. mymiv nepal; k. tlciv indonesia; l. pepylciv indonesia molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. biotropia vol. 22 no. 1, 2015 60 references aidawati n, hidayat sh, suseno r, hidayat p, sujiprihati s. 2005. identifikasi yang menginfeksi tomat geminivirus berdasarkan pada teknik polymerase chain reactionrestriction fragment length polymorphism. j mikrobiol indon 10(1): 29-32. argüelo-astorga gr, guevara-gonzález rg, herreraestrella lr, riverabustamante rf. 1994. geminivirus replication origins have a groupspecific organization of iterative elements: a model for replication. virology 203:90100. damayanti ta, alabi oj, rauf a, naidu ra. 2009. severe outbreak of yellow mosaic disease on the yard long bean in bogor, west java. hayati j of biosci 16(2):78-82. doyle jj, doyle jj. 1987. a rapid dna isolation of procedure for small quantities of fresh leaf tissue. phytochem bull 19:11-9. fauquet cm, mayo ma, maniloff j, desselberger u, ball la, editor. 2005. . eight report of virus taxonomy the international committee on taxonomy of viruses. san diego (us): viroldivint union of microb soc. hidayat sh, chatchawankanpanich o, rusli e, aidawati n. 2006. associated with pepper yellow leaf begomovirus curl disease in west java, indonesia. j mikrobiol indon 11:87-90. hidayat sh, chatchawankapanich o, aidawati n. 2008. molecular identification and sequence analysis of tobacco leaf curl virus from jember, east java, indonesia. hayati j of biosci 15(1):13-7. hull r. 2002. . london (gb): matthew's plant virology 4 edth academic press. ilyas m, qazi j, mansoor s, briddon rw. 2010. genetic diversity and phylogeography of begomoviruses infecting legumes in pakistan. j gen virol [internet]. [cited 2012 apr 24]; 91:2091-2101. available from: http://www.pubfacts.com/detail/20375225/gene t i c d i v e r s i t y a n d p h y l o g e o g r a p h y o f beg omoviruses-infecting-legumes-in-pakistan. doi: 10.1099/vir.002 0404-0. lazarowitz sg.1987. the molecular characterization of geminiviruses. plant mol biol rep 4: 177-92. national centre for biotechnology information. 2013. [ c i t e d 2 0 1 3 j u n 2 0 ] ; av a i l a b l e f r o m http://www.ncbi.nlm.nih.gov. rojas mr, gilbertson rl, russel dr, maxwell dp. 1993. use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. plant dis [internet]. [cited 2012 apr 24]; 77(4): 340-47. available from: http://www. apsnet.org/publications/plantdisease/backissues/ documents/1993articles/plantdisease77n04_340 .pdf. shahid ms, pudashini bj, khatri-chhetri gb, ikegami m, natsuaki kt. 2012. first report of kidney bean in nepal. new dis rep [internet]. [cited 2013 oct 22]; 25:30. doi:10.5197/j.2044-0588.2012.025.030. available on: http://www.ndrs.org.uk/article.php ?id=025030. tsai ws, shih sl, rauf a, safitri r, hidayati n, huyen btt, kenyon l. 2013. genetic diversity of legume yellow mosaic in indonesia and begomoviruses vietnam. ann appl biol 163:367-77. usharani ks, surendranath b, haq qmr, malathi vg. 2004. yellow mosaic virus infecting soybean in northern india is distinct from the species infecting soybean in southern and western india. current sci. [internet]. [cited 2012 may 8]; 86(6):845-850. av a i l a b l e o n : h t t p : / / w w w. i i s c. e r n e t . i n / currsci/mar252004/845.pdf. varma a, malatthi vg. 2003. emerging geminivirus problems: a serious threat to crop production. ann appl biol. [internet]. [cited 2014 apr 3]; 142:145164. available on: http://onlinelibrary.wiley.com/ doi/10.1111/j.17447348.2003.tb00240.x/pdf. http://www.pubfacts.com/detail/20375225/gene http://www.ncbi.nlm.nih.gov. http://www. http://www.ndrs.org.uk/article.php http://onlinelibrary.wiley.com/ 6. c hanny wijaya.cdr biotropia vol. 20 no. 1, 2013: 50 71 flavour of papaya ( l.) fruitcarica papaya c. hanny wijaya * and feng chen received 28 august 2012/accepted 18 december 2012 papaya is included in five major tropical fruits of the world after banana, mango, and pineapple. indonesia is one of the leading countries of papaya production after india and brazil. the centre of tropical fruits study (pkbt) at bogor agricultural university (ipb), indonesia, has started a longterm breeding program since 2003 in order to improve the quality of local papayas. fruit exports are being targeted at more specialized consumers, who are either seeking products either raised in more environmentally and health-conscious ways or those with a significant outstanding flavour. flavour continues to be the predominant quality characteristic important for a successful international marketing. to understand the biosynthesis pathways are becoming more important for the flavour industry in recent years, as this could aid in the production of the flavour volatiles in the same manner as the natural biosynthesis. it is also necessary to understand the aroma active compounds and their changes during processing because unexpected changes in aroma may cause a product unmarketable even if other quality factors are acceptable. understanding the changes of aroma active compounds in the papaya fruit, such as loss of desirable flavour and development of offflavour during processing will be very helpful for determining proper processing condition. the flavour composition of papaya fruit is reviewed in this paper. this paper presents an overview of important publications regarding the characteristic features of the biology of the fruits, consumption worldwide, commercial application in food processing, though the review of biogenesis of volatiles is still the main focus. papaya ( l.), flavour, review, volatiles, tropical fruit, biogenesis 1 2 1 2 department of food science and technology , faculty of agricultural technology bogor agricultural university (ipb), bogor 16680, indonesia department of food, nutrition, and packaging sciences, clemson university, clemson, sc 29634, usa carica papaya abstract introduction key words: excellent in vitamin c, pro-vitamin a, minerals (wall 2006) as well as rich in dietary fibre, papaya ( l.) is emerging as a popular fresh fruit which offers health benefiting properties. by the mid-19 century, papaya had spread over the tropical region, from florida, hawaii, india, sri lanka, malaysia, indonesia, to africa (morton & macleod 1990). carica papaya th * corresponding author : hazemi@indo.net.id 50 et al et al et al et al et al et al et al et al wide variability is shown by papaya grown in various countries. colour, texture, size, and particularly, flavour continue to be the predominant quality characteristics important for a successful international marketing of horticultural crops (picha 2006). fruit exports are being targeted at more specialized consumers, who are either those seeking products either raised in more environmentally and health-conscious ways or those who are prepared to pay a significant price for outstanding flavour (zoe 2006). there are a number of numerous literatures which discussed the volatiles present in the papaya fruits (flath & forrey 1977; idstein . 1985 ; mohammed . 2001; pino . 2003; almora . 2004). papaya possesses a characteristic aroma, which is due to several volatile components, such as alcohols, esters, aldehydes, and sulphur compounds (marostica & pastore 2007). like many other climacteric fruits, papaya undergoes a variety of physical and chemical changes after harvest (shiota 1991). the biogenesis of flavours in fruits has been found to take place mostly during the ripening stage. the understanding of the biosynthesis pathways have been of increasing importance in the flavour industry in recent years, as this could aid in the production of the flavour volatiles in the same manner as the natural biosynthesis. this is crucial in allowing the flavours to be labelled as natural. pino . (2003) reported that based on more than 40 years of intensive research toward the volatile profiles of various papaya cultivars, almost 400 volatiles have been identified. the identified volatile profiles were various depending on the various methods used to extract and isolate the compounds and the species of the papaya used in the analysis (pino . 2003; devitt . 2006). furthermore, as mentioned before, the flavour of papaya fruit is a result of a complex interactions among sugars, organic acids, minerals, and aroma volatile compounds, which may vary with cultivar and production location. when the papaya fruits are subject to processing, there would be qualitative and quantitative alterations in the overall composition of volatile compounds (mohammed . 2001). the processing of the papaya fruits involves the major structural changes in the fruit (e.g. slicing, pulping, heating and freezing) that can result in a significant change in the sensory characteristics. it is also necessary and crucial to understand the aroma active compounds and their changes during processing because unexpected/unpleasant changes in aroma may make a product unmarketable even if the other quality factors are acceptable. therefore, understanding the changes of aroma active compounds in the papaya fruit, such as loss of desirable flavour and development of off-flavour during processing will be very helpful for determining proper processing condition. in this paper, the flavour composition of papaya fruit is reviewed. however, the information is not limited to a compilation of reported volatile compounds. this review will also present an overview of important publications and a description of the characteristic features of the biology of the fruits, consumption worldwide, commercial application in food processing, though review of volatile compounds will still be the main focus which that includes two aspects, i.e., the analytical methodology used for the determination of the volatile compounds and the contribution of individual components to the characteristic flavour. future researches will be also highlighted. b 51 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. biology and worldwide consumption the papaya, l., is a member of the small family caricaceae with genus and species l. according to its taxonomy classification, papaya is under the superkingdom , kingdom , phylum , sub phylum , division , sub division , super class class , order , and the family (trace 2012; paull & duarte 2011). however, a recent taxonomic revision proposed that some species formerly assigned to were more appropriately classified in the genus (badillo 2002). papaya is a fast growing tree like herb that can grow at the rate of 6 to 10 ft (1.8-3 m) in the first year and reaching 20 or even 30 ft (6-9m) in height, with a hollow green or deep-purple stem becoming 12 to 16 inch (30-40 cm) or more thick at the base and roughened by leave scars. the leaves emerge directly from the upper parts of the stem in a spiral on nearly horizontal petioles 1 to 3 ½ ft (30-105 cm) long, hollow, succulent, green or more less dark purple. the blade, deeply divided into 5 to 9 main segments, each irregularly subdivided, varies from 1 to 2 ft (30-60 cm) in width and has prominent yellowish ribs and vein. the life of a leaf is 4 to 6 months. both the stem and leaves contain copious white milky latex (morton 1987). the five-petal led flowers are fleshy, waxy and slightly fragrant. papaya flowers are born on inflorescences which appear in the axils of the leaves. female flowers are held close against the stem as single flowers or in clusters of 2-3 (chay-prove . 2000). male flowers are smaller and more numerous and are born on 60-90 cm long pendulous inflorescences (nakasone & paull 1998). bisexual flowers (hermaphrodite) are intermediate between the two unisexual forms (nakasone & paull 1998). the functional gender of flowers can be altered or reversed, depending on environmental conditions, particularly temperature. some trees bear only short-stalked female flowers or bisexual (perfect) flowers also on short stalks, while other may bear only male flowers, clustered on panicles 5 or 6 ft long. some trees have both male and female flowers. at certain seasons the trees produce short-stalked male flowers, while at other times perfect flowers. certain varieties have a propensity for producing certain types of flowers. male or bisexual trees may change completely to female trees after being beheaded. only hermaphrodite (bisexual flowered) papaya tree can bear fruit in a single tree by self-pollinating. the female papaya tree can also produce fruit from cross pollination by either bisexual or male trees. although male tree may sometimes bear fruit, it is not edible (fao 1992). the fruit is melon-like with an oblong or elliptic shape. fruits from female trees are spherical, whereas the shape of fruit from bisexual trees is affected by environmental factors, particularly temperature that modifies floral morphology during early development of the inflorescence (nakasone & paull 1998). fruits are ready to be harvested five to six months after flowering, which occurs five to eight months after seed germination (chay-prove . 2000). ripe papaya fruits have smoothed, thin green-yellow-orange coloured skin. depending on the cultivar, flesh thickness varies from 1.5 to 4 cm (nakasone & paull 1998) and flesh colour may be pale yellowish-orange to red (villegas 1991; nakasone carica papaya carica carica papaya eukaryota virdiplantae streptophyta embryophyta tracheophyta spermatophyta magnoliophyta, rosidae brassicales caricaceae carica vasconcella et al et al 52 biotropia vol. 20 no. 1, 2013 & paull 1998). the central cavity of mature fruits is containing numerous grey-black spherical seeds of 5 mm in diameter (villegas 1991). the fruits range in size from 7-30 cm long and vary in mass from about 250 to 3000g (oecd 2003). according to rivera (2005), papaya varieties generally can be divided into two groups, the small fruits weighing 500 grams below and big fruits weighing from 500 g up to 10 k. whereas, cosidine and cosidine (1982) divided papaya into four categories according to its size, i.e., small size weighing 0.3-.4 kg, medium size weighing 0.4-0.45 kg, big size weighing 0.45-0.9 kg, and very big size weighing more than 0.9 kg. in order to improve the quality of local papayas, the centre of tropical fruits study (pkbt) at bogor agricultural university (ipb), indonesia, has started a longterm breeding program since 2003. this program targets at developing new cultivars which will have outstanding specifications and characteristics that will be desirable for international market demand, resistant to biotic and abiotic stress and have high yield (anonymous 2003). until 2005, the program has screened 75 genotypes of papaya collected from several areas in indonesia and those introduced from abroad. this papaya genetic bank collection has been divided into two categories based on size: small papaya and medium-big papaya, or on its specific utilization purpose as vegetable/fruit papaya or papain produced papaya (anonymous 2005). among the 24 genotypes that have been morphologically characterized, 'eksotika', 'sunrise solo', 'bangkok', 'red king' and 'california' were recommended by pkbt as outstanding varieties of vegetable/fruit papaya. there are also other three outstanding genotypes obtained from open pollinated breeding released by pkbt named 'papaya arum bogor' (small-type), 'papaya prima bogor (medium-type) and 'papaya wulung bogor (papain producer). some other superior genotype candidates such as ipb 3, ipb 8, ipb 6, ipb 9 are still under evaluation (anonymous 2005; sriani 2008-personal communication). healthy, low in calories and a rich source of vitamins, calcium and phosphorous, the papaya is one of the most easily digested fruits. the ripe papaya fruit is consumed as fresh table fruit. sometimes it is cut in wedges and served with lime or lemon juice, also in few cases, a few seeds (just a few) are left attached to a peppery flavour. the firm-ripe flesh is often cubed or shaped into balls and served in fruit salad or fruit cup as well as seasoned and baked for consumption (morton 1987). however, in many asian countries, especially japan, malaysia, vietnam, indonesia and thailand, the fruit is consumed as grated vegetable while still in the green stage. papaya cuisine is now becoming popular in european society. there is also an array of processed papaya products available prepared by minimal processing, drying, canning, pickling and freezing. ripe flesh is commonly made into sauce for shortcake or ice-cream sundaes, or is added to ice cream just before freezing. it can also be cooked as pie, pickled, or preserved as marmalade or jam, papaya cubes with other fruits, covered with sugar syrup, may be quick-frozen for later serving as dessert. papaya juice and nectar may be prepared from the fruit and are sold fresh in bottled or canned. half-ripe fruits are sliced and crystallized as a sweetmeat (morton 1987). green papaya puree can also be utilized as chilly-sauce thickener. young leaves of papaya can be cooked and eaten as vegetables in several countries in asia. papaya leaves contain the bitter alkaloids, carpaine and pseudeocarpaine, 53 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. which can be destroyed by heat. sprays of male flowers are sold in asian including indonesian and new guinea markets after being boiled with several changes of water to remove the bitterness and eaten as a vegetable. in africa, the young stems are cooked and served. papaya seeds are sometimes found as an adulterant of whole black pepper (morton 1987). according to philippine council for agricultural, forestry and natural resources research and development (pcarrd-dost 2004), papaya is grouped in five major tropical fruits after banana, mango, pineapple which shared 6 percent of on estimated world production of tropical fruits in 2004. evans and ballen (2012) reported that based on fao statistical division data 2012, the global papaya production in 2010 was estimated 11.22 metric tons or 15.36 percent of the total tropical fruit production, ranked third. however, other sources said that it is impossible to obtain reliable estimates of total world papaya production, since the home-grown crops are unregistered. this phenomenon clearly occurs in indonesia, although it is believed that the annual production must be several million metric tons. the leading global papaya producing countries for period 2002-2010 were india (36.6%), followed by brazil (17.5%) and indonesia (6.9%). while sidhu (2006) reported the leading country of papaya production is brazil, followed by nigeria, india, mexico and indonesia. according to the national agricultural statistics service (nass 2007), only hawaii in the united states produces 45.9 million pounds of papaya fruit on about 2,320 acres in 2002, however, the production is declining every year. the production was only 28.7 million pounds on about 2,095 acres in year 2006 (nass 2007). most of the papaya grown in hawaii is consumed as fresh fruit (93%), only small amounts were processed into juices or other processed foods. the determination of aroma substances by instrumental technique generally consists of two stages. the first phase is the isolation of analytes from the complicated food matrix based on two primary principles-volatility and/or solubility. the second phase is the identification of the analytes. it is essential to isolate the desired volatile compounds in order to eliminate interfering signals coming from the complex food matrix. hence, it is important to select an appropriate sample preparation method so that the isolated product can be possibly representative. currently, there are many isolation techniques that can be used depending on the properties of the food product. generally, there are two common extraction techniques used in extracting volatile compounds from papaya, namely the simultaneous distillation/solvent extraction (sde) (macleod & pieris 1983; morales & duque 1987; almora . 2004) and the dynamic headspace analysis (mohammed . 2001; flath . 1990). these two flavor in papaya ( )carica papaya methods of extraction, isolation and identification of papaya aroma compounds et al et al et al 54 biotropia vol. 20 no. 1, 2013 methods are also commonly used for the study of volatiles compounds of other fruits such as apples (argenta . 2004; shashirekha . 2008), mangoes (sakho . 1998; torres . 2007), mangosteens (macleod & pieris 1982), peaches and nectarines (lavilla . 2002). the principal of sde technique is based on the differences in volatility and polarities among the analytes and other non-volatile components present in the food matrix (teixeira . 2007). an appropriate solvent also should be chosen to facilitate the extraction of the important volatile components, while excluding/limiting the components that could interfere with the analysis. in sde method, the sample is simultaneously distilled and extracted from steam condensate by organic solvents reflux, such as diethyl ether (almora . 2004) and ch cl (morales & duque 1987) in a likens and nickerson apparatus. the concentrated extracts will then be analyzed by gco and/or gc-ms. the advantages of sde analysis of volatile compounds are obvious that only two single main operations (extraction and concentration) are required and yet give a relatively wide spectrum of chemical compounds detected (peng . 2004), and due to the continuous recycling, a relatively small quantity of organic solvent is used. in addition, it minimizes the possibility of artefact introduction from this source (schultz . 1977). dynamic headspace technique uses a ”purge and trap” method involving the passing of carrier gas through a liquid sample, followed by trapping of the volatile analytes on an absorbent material, and the analytes are flushed onto the column for analysis by gc or gc-ms (snow 2002). mohammed . (2001) had collected the volatiles by using tenax as the absorber, and used purified nitrogen gas to flush sample onto the tenax trap. the aliquots collected were then injected into gc-ms. recent study on the papaya volatiles has been reported by ulrich and wijaya (2010). two different sample preparation methods which are liquid-liquid extraction and stir bar sorptive extraction (sbse) have been utilized in the analysis. there were clear different results obtained using these two methods. it has been known that the utilization of sbse as well as solid phase microextraction (spme), although they are more rapid isolation compared to liquid-liquid or sde, only strong substances will be discriminated. the sample preparation using sbse is effective and usable in the aroma comparison research topic. however, it is not a proper approach for aroma identification since it will not cover all character impact compounds responsible for the wholesome aroma of the sample. generally, selecting different extraction techniques might lead to the identification of different volatile compounds. this could be due to inadequate sensitivity (i.e., only the most abundant volatiles can be detected and/or unable to detect trace compounds) and different in selectivity of the trap or solvent used (i.e., non-polar compounds can hardly be extracted by polar solvents/trap absorbent). for instance, larrayoz (2001) reported that the dynamic headspace technique combined with a purge & trap device could extract more highly volatile compounds than the sde method. in contrast, the sde is more efficient for extracting low-volatile components such as phenols, free fatty acids, lactones and longer-chain aldehydes, ketones, alcohols and esters. et al et al et al et al et al et al et al et al et al et al et al. 2 2 55 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. aroma active compound synonym (s) odor descriptors molecular mr (g/mol) b.p. (oc) cas registry no. butanoic acid butyric acid, octyl butyrate pungent unpleasant odor, acrid-taste c4h8o2 88.106 163.5 107-92-6 4-methyloctane 4-methyloctane somewhat pungent, citrus-like c9h20 128.255 142.4 2216-34-4 hexanoic acid caproic acid; pentane 1carboxylic acid; hexylic acid; hexoic acid strong fruity odor, pineapplelike c6h12o2 116.16 205 142-62-1 benzenemethanol phenyl carbinol; alphahyroxytoluene; benzoly alcohol; phenyl methanol mild-sweet aroma like c7h8o/ c6h5ch2o h 108.1 205 100-51-6 translinalool oxide (furanoid) (2r,5r)-5 isopropyl 2-methyl-2vinyltetrahydrofuran earthy-leafy cislinalool oxide (furanoid) (2r,5r)-5 isopropyl 2-methyl-2vinyltetrahydrofuran sweet, floral,creamy, fruity benzylacetate acetic acid benzyl ester; acetic acid phenylmethyl ester; alphaacetoxytoluene; benzyl ethanoate; phenylmethyl acetate fruity, apple and pear-like ch3cooc h2c6h5 150.18 215 140-11-4 linalool cispyranic oxide na citrus-like cyclohexane hexamethylene; hexanaphthene; hexahydro-benzene; benzenehexahydride; sweet-fruity c6h12 84.16 80.7 695-06-7;57129-701 (±) form; 6335795-9 (r) -form; 41035-07-8 (s) form: (£)-form gammahexalactone 4-hexanolactone; 4-caprolactone; 4-ethyl butrolactone;4hydroxyhexanoic acid lactone;4hexanolide sweet, creamy, lactonoic, fruity, coumarin-like with green coconut nuances c6h10o2 114.44 216 gammaoctalctone 5-butyldihydroxy -2 (3 h)furanone;4octanolide;4buthyy butyrolactone coconut-like odor, sweet, lactonoic, fruity creamy, coumarin c8h14o2 142.197 117 108943-45-9 (r) form deltaoctalactone 5-octanolide;5hydroxyoctanoic acid lactone sweet, creamy, fatty with tropical and dairy nuances, fruity c8h14o3 142.197 140 108943-46-0 (s) form 80 104426-32-6 (± ) form 126 table 1: aroma active compounds as reported by different researchers found in papaya 56 biotropia vol. 20 no. 1, 2013 c10h18o2 170.2487 222.6°c at 34995-77-2 c10h18o2 170.2487 na 60047-17-8 c15h24o3 252.3493 356.1°c at 14009-71-3 760 mmhg 760mmhg et al et al et al carica papaya et al approximately 150-200 volatile components of ripe papaya pulp have been reported in various papers (flath & forrey 1977; macleod & pieris 1983; idstein & schreier 1985; schreier . 1985). these volatiles are released by harvested intact fruit in small amount per unit time, and some of the reported volatile compounds are only generated in quantity from non-volatile precursors due to the disruption of fruit tissue (flath . 1990). volatile constituents from papaya extracted by both the simultaneous distillation/solvent extraction method and dynamic headspace analysis are listed in table 1. volatiles identified in these studies included heterocyclic compounds, terpenoids, aromatic hydrocarbon, alcohol, acids, esters and ketones. a fairly wide range of different types of compounds have been identified in different studies (as shown in table 1), partly due to papaya species aside from the type of extraction and analysis technique used. for instance, the volatiles of sri lanka papaya were dominated by esters (macleod & pieris 1983), whereas terpenoids (mainly linalool and linalool oxides) provided the most abundant group of volatiles for hawaiian papaya (flath & forrrey 1977). on the other hand, different method approaches might be able to show the same tendencies. for instance, the volatile components of papaya (solo variety) which were recovered by four different methods: e.g., vacuum trapping train (distillation under low temperatures with liquid nitrogen traps), co-distillation-extraction (vacuum), distillation, and co-distillation-extraction (1 atm.), showed that, in spite of great variations due to the recover method, linalool was always the major compound detected in all the methods (flath & forrey 1977). mohammed . (2001) had identified a total of 26 aroma volatiles emanating from fresh cut papaya ( l. cv. solo) using the dynamic headspace technique followed by gc-ms, where some had not been reported by schreier and winterhalter (1986). however, some of these newly identified volatiles such as 4methyl-1-octane, tetrahydro-3-furfuryl-furan and 2,4-dimethylhexane may have arisen due to the mechanical damage sustained by the tissue during preparation (mohammed . 2001). in addition, 11 out of these 26 aroma volatiles were identified as aroma active compounds, namely, butanoic acid, 4-methyloctane, hexanoic acid, benzenemethanol, trans-linalool oxide, linalool, benzyl acetate, linalool cis-pyranic oxide, cyclohexane and benzaldehyde. it was also shown in the study that the volatile extract obtained from papaya slices changed from a distinct fresh sweet and flowery odour to slightly less evident, then to an unpleasantly dry, pungent and fruity odour which could be explained by the changes in the distribution of these aroma active compounds during storage. this in turn illustrated the aroma of papaya due to a complex and dynamic integration of compounds which changed with time as well. papaya is a climacteric fruit that undergoes the ripening process upon harvesting, until the stage where a fully ripened papaya gives fully developed flavours and odours which then perceived and recognized by human as an indication that the papaya is ripened. hence, the papaya fruit gives its characteristic fruity, balsamic, sweet odours aroma-active compounds of papaya 57 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. after undergoing a variety of physical and chemical changes during the ripening process. herein, aroma components that give the aroma characteristic of a food are called aroma active compounds or character impact compounds. mohammed (2001) reported that the dominating aroma-active compounds in papaya were linalool and benzaldehyde. this claim is also supported by chan (1973) who reported that the major odorous compounds from fresh, ripened papaya ( ) were linalool, with smaller amount of ethyl acetate, 1-butanol, two configurational isomers of the linalool oxides (2-methy-2-vinyl-2-(hydroxyl-2-propyl) tetrahydrofuran), and benzyl isothiocyanate. also, gamma-hexalactone, gamma octalactones and delta octalactones were reported in other literatures (sidhu, 2006) as aroma-active compounds contributing to the overall perceived characteristic aroma of the papaya. other prominent volatiles like butanol, 3-methylbutnol, benzyl alcohol an terpineol in papaya ( l., maradol roja) at its mature stage were also reported by almora . (2004), who also mentioned a decrease in benzyl isothiocyante, and increase in linalool, terpinen-4-ol, esters (methyl butonoate, ethyl hexanoate and ethyl dodecanoate). the combination of the different esters is associated with the detected fruit concentrations during ripening (almora . 2004). the major aroma-active compounds present in cut papaya was identified to be linalool, benzaldehyde and benzyl isothiocyanite (bitc). linalool is the most dominant aroma-active compound present in the papaya ( l.). the chemical name is 3,7-dimethyl-1,6-octdine-3-ol. (±)-linalool (22564-99-4), like the individual enantiomers, a colourless liquid. it is an acylic terpene with a flowery and fresh odour reminiscent of lily of the valley (bauer . 1997). however, the enantiomers differ slightly in odour (klein & ohloff 1962). the aroma threshold for linalool was found to be within 4 to 10 ppb. at 30 ppm, it has a taste characteristic of floral, woody, and sweet, with a green spicy tropical nuance. hence, the presence of linalool at different concentrations in a wide range of foods might give a slightly different characteristic sweet-flowery aroma in the foods. in the , whilst hydrogenation of linalool yields tetrahydrolinalool, which is often used as a fragrance compound. also, linalool can be converted into linalyl acetate by reacting with ketone or excess of boiling acetic anhydride. benzaldehyde is also the dominating aroma-active compound in papaya, having a characteristic of bitter almond oil and imparting a bitter-nutty and almond odour (mohammed . 2001). it is also named benzene carboxaldehyde and almond artificial essential oil. benzaldehyde undergoes auto oxidation in the absence of inhibitors to perbenzoic acid, which then react with a second molecule of benzaldehyde to benzoic acid. upon hydrogenation of benzaldehyde, benzyl alcohol will be formed. benzyl isothiocyanate (bitc) is found in enzymic hydrolysates of extracts from various plant families: namely, , , , , and (ettlinger & hodgkins 1956; ettlinger & et al. et al. carica papaya carica papaya et al et al carica papaya et al et al cruciferae moringaceae capparidaceae tropaeolacea, caricaceae gyrostemonaceae salvadoraceae d α y note (arctander 1969), whilst balsamic note could be attributed to benzyl alcohol and α-terpineol presence of acids, linalool isomerizes readily to geraniol, nerol and αterpineol. it is oxidized to citral by chromic acid. oxidation with peracetic acid gives linalool oxides 58 biotropia vol. 20 no. 1, 2013 kjaer 1968), or can be synthesized in bruised or injured papaya. it possesses pungent, off-flavour aroma. as revealed by tang (1971), benzyl isothiocyanate is a natural metabolite emanating from intact green papaya. also, he mentioned that the concentration of benzyl isothiocyanate (or its glucosinolate precursor) decreased in the flesh of papaya whilst it increased in the seeds with maturity. despite the decreasing concentration of bitc along with maturation, it can make significant sensory impression since it has a very obnoxious nature of low threshold. on top of the elaborated aroma-active compounds, other aroma active compounds that are present in smaller amounts (shown in table 1) also contribute to the overall perceived aroma of ripe papaya. hence, each individual aroma active compounds has its own vital role in giving our sensory a full picture on the papaya aroma. in another investigation, linalool was detected in relatively low concentration in the solvent-extracted volatiles of fresh papaya pulp from sri lanka, while esters represented the majority. the authors attributed the characteristic sweaty note of this papaya fruit mainly to methyl butanoate. phenylacetonitrile was also found in high amounts (17.7%) combined with lower concentration of benzyl isothiocyanate (1.5%), which played a critical role in the aroma of papaya (macleod & pieris 1983). oxygenated terpenoids derived from linalool might play an important role in brazilian papaya aroma. several oxygenated derivatives of linalool were identified in the solvent-extracted samples, such as the two diasteroisomers of 6,7epoxylinalool:2,6-dimethyl-octa-1,7-diene-3,6-diol and 2,6-dimethyl-octa-3,7-diene2,6,-diol (winterhalter 1986). fifty-one volatile components from intact hawaiian papayas of different ripeness were recovered by tenax using the “trap and purge” method. as expected, the largest number of components was found in the fully ripe fruits. linalool, followed by linalool oxide a, linalool oxide b, and ethyl acetate were the major components in the fully ripe fruits. it is worth noting that several compounds, e.g., linalool and all aldehydes, exist in all four ripeness stages (flath . 1990). another investigation reported the esters as the predominant volatile components of the maradol variety (about 41% w/w of the total volatiles) (pino . 2003). the major representative compounds in the simultaneous steam distillationsolvent extraction were methyl butanoate and ethyl butanoate. previous work described the esters as the predominant compounds among the volatiles; papaya, for example from sri lanka and colombia had 52 and 63% of esters in the total volatiles, respectively (macleod & pieris 1983; morales & duque 1987). studies on the diversity of papaya volatiles in different cultivars and new breeding lines have been conducted by ulrich and wijaya (2010). character impact compounds such as hexanal, cis-2-pentenol, nonanal, cis-linalool oxide, linalool, butanoic acid, verbenone, phenyl methyl of butanoic acid and others have been detected. the volatiles patterns differ significantly among the genotypes resulting in very different sensory quality. the gained obtained knowledge will contribute to the breeders' ability in developing new papaya varieties with acceptable flavour properties (ulrich & wijaya 2010). et al et al 59 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. despite the significant diversity in aroma compounds of papaya fruit, the volatiles in papaya are mainly derived from three major metabolism pathways: the catabolism of fatty acid, amino acid, and carbohydrates. the biosynthesis pathways are usually interlinked, with products from one pathway serving as the precursors for another pathway. these degradation reactions account for many aroma compounds, as they result in the formation of a host of low molecular weight products which has significant sensory properties. this catabolism is associated with fruit ripening, during the climacteric rise in respiration, and the rate of flavour formation reaches a maximum during the post-climacteric ripening phase (reineccius 2006). the metabolism pathways will be elaborated in the following sections. aroma compounds may be formed from lipids via several different pathways. the primary pathways involved in the aroma of papaya include the enzymatic conversion of acyl lipids through n/lipoxygenase pathways (reineccius, 2006). these pathways generate short branched-chain aldehydes, alcohol, ester and ketones. fatty acid derived volatiles play important regulatory roles in defence d tissues can form volatiles via the oxylipin biosynthesis pathways (devitt . 2006). in papaya, methyl and ethyl ester derivates of lipid catabolism have been identified as strong contributors to aroma (macleod & pieris 1983; morales & duque 1987), examples include ethyl butyrate, ethyl acetate, methyl butyl acetate, ethy butanoate and methyl butanoate. significant amount of butanol has also been reported (morales & duque 1987). cules, one atp, one fad, one water molecule, and one nad to form an acetyl coa molecule and an acyl coa molecule. using acetyl coa as precursor, several other compounds such as esters, sterols, and most terpenes can be synthesized (reineccius 2006). the acetyl coa molecule can also undergo reaction to reform into fatty acid such as butyric acid which contributes to the papaya aroma. as mentioned above, the biogenesis of certain volatiles in papaya can be further explained by a process known as the oxylipin pathway, which in brief, is the metabolism of polyunsaturated fatty acids (pufas) by lipoxygenase (linoleate oxygen oxidoreductase; lox) and the subsequent reactions after it. stumpe (2005) and chehab (2007) reported that: “ this pathway is initiated by the action of lipases on complex membrane lipids in the fruit, causing the release of unesterified fatty acids. the free polyunsaturated fatty acids which contain a ( )-1,4-pentadiene structure then act as substrates for the enzyme lox introducing molecular oxygen to them to form their corresponding hydroperoxy derivatives. these derivatives then undergo further enzymatic reactions to give rise to fatty acid hydroperoxides, hydroxyl fatty acids, epoxy fatty acids, keto fatty acids, volatile aldehydes and cyclic compounds, which are collectively known as oxylipins. some of these products are responsible for the unique aroma of the papaya fruit papaya flavour biogenesis βoxidation and oxylipi and plant development. fatty acid volatiles are formed in intact fruit via the βoxidation pathway while cut or damage in the papaya plant, β-oxidation occurs in the peroxisomes, organelles which contain enormous amounts of enzymes. the substrate of the reaction is an acyl. the acyl lipid reacts with two coa mole et al et al. et al. cis, cis . 60 biotropia vol. 20 no. 1, 2013 et al et al et al cis et al unstable allene oxides are formed from the activity of allene oxide cyclase (aos). they can either undergo nonenzymatic hydrolysis leading to alpha-and gamma-ketols, or be metabolized to 12-oxophytodienoic acid (opda) by aoc. subsequently, opda is transported to the peroxisomes where its cyclopentenone ring is reduced by opda reductase to form opc:8. finally, the opc:8 is subjected to three rounds of beta-oxidation to yield jasmonic acid (ja) (chehab . 2007). ja and its precursor opda are signalling compounds that play a role in plant development and response to injury (stumpe . 2005). ja is also converted to a variety of derivatives like its ester, methyl jasmonate, which contributes to the aroma of the papaya fruit. the other major metabolic route that is dominant for the direct production of important compounds of the characteristic aromas of many fruits including papaya is hydroperoxide lyase (hpl) pathway (yilmaz 2001). hpl catalyzes the oxidative cleavage of fatty acid hydroperoxides, producing volatile aldehydes and oxoacids. the hpl enzyme can yield c-6 aldehydes and c-12 omega-oxoacids from the 13hydroperoxide derivatives (kalua . 2007). the c-6 aldehydes include saturated aldehyde, hexanal from linoleic acid and the unsaturated aldehyde, -3-hexenal from linolenic acid. this unsaturated aldehyde is unstable and undergoes rapid isomerisation to a stable compound, trans-2-hexenal. the c-6 aldehydes formed through the hpl activity can also be further reduced by alcohol dehydrogenase to form the corresponding calcohol, like cis-3-hexen-1-01 (matsui 2006), which is one of the constituents of papaya's volatiles. furthermore, alcohol acetate transferase catalyses the formation of acetate esters through acetyl coa derivatives together with alcohol. acetate ester, esters of alcohols with other fatty acids, as well as the aldehydes and alcohols produced in this pathway are important constituents of papaya fruit which give it a distinct aroma by virtue of the volatiles. many of the aliphatic esters, alcohols, acids, and carbonyls found in the fruit are derived from the oxidative degradation of linoleic and linolenic acids. amino acid metabolism generates aromatic, aliphatic, and branched chain alcohol, acids, carbonyls, and esters that are important to the flavour of papaya (reineccius 2006). variations of the free amino acid content in fruits have been known to occur during ripening, corresponding to the period of maximum production of aroma compounds. this is evidence that amino acids are precursors of aroma compounds generated during fruit ripening, and is an important source of volatile compounds contributing to their aroma (tomas-barberan . 1997). the amino acids present in plants in general are classified under two categories, namely the non-aromatic (absence of benzene ring) amino acids (cysteine, methionine, leucine, isoleucine and valine) and the aromatic amino acids (phenylalanine, tryptophan, tyrosine). phenylalanine and tyrosine are believed to be the chief precursors of aromatic volatile compounds in plants, synthesized by the shikimic acid pathway. following the shikimic acid pathway, phenylalnine can enter other two pathways, one of which produces benzyl glucosinolate, the precursor of benzyl isohiocyanate (one of the major compounds in papaya); the other is the cinnamic acid metabolism pathway which produces aromatic alcohols, acids, esters and carbonyls. aroma compounds can also be synthesized from non-aromatic amino acids precursors (eg. cysteine, methionine, leucine, isoleucineand valine). 61 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. the aromatic amino acids are not naturally present in plants and have to be synthesized by a metabolic pathway, namely the shikimic acid pathway. the shikimic acid pathway converts simple carbohydrate precursors derived from glycolysis and the pentose phosphate pathway to the aromatic amino acids. in the initial steps, d-glucose is phosphorylated. after a series of enzymatic transformations, it is subsequently converted into phenolic compounds, ultimately yielding the active precursor amino acids. following the shikimic acid pathway, the other main pathway which phenylalanine enters is the cinnamic acid metabolism (phenyl propanoid pathway). in this pathway, the amino acids are further transformed into aromatic alcohols, acids, esters and carbonyls. benzaldehyde, 4-methylbenzaldehyde, acetophenone, benzophenone, bdamascone, b-ionone (trace), and benzyl alcohol and phenylacetonitrile (10-50 ppb), which are aromatic compounds found in papaya (idstein . 1985a) are hypothesized to be generated in this manner. out of the three aromatic amino acids generated through shikimic pathway, phenylalanine has been stipulated as the chief precursor of aromatic aroma compounds derived from amino acid metabolism (lamikanra 2002). there are two main pathways which phenylalanine can react to form other compounds. the first of these two main pathways is responsible for forming benzyl glucosinates in the papaya plant. the synthesis of volatile aroma compounds with non-aromatic amino acids as precursors takes place through a completely different pathway. the pathway follows the citric cycle instead of the shikimic acid pathway. the first step of transamination occurs when glutamic acid is produced from 2-oxoglutarate. the next step is the decarboxylation of 2oxoacid formed after amino acid transamination. this produces only aliphatic and its branched aroma compounds particularly methyl-branched (aliphatic) alcohols, acids, esters and carbonyls. as mentioned above, esters are very important contributors to the aroma of papaya (macleod & pieris 1983; morales & duque 1987). they are formed by the reaction between alcohols and acyl coa's derived from fatty acid and amino acid metabolism. this reaction is catalyzed by acyl alcohol transferase (aat) (sanz . 1997; perez . 1996; ueda . 1992). according to a study done by morales and duque (1987), the papaya aroma extract's main constituents are carboxylic esters, dominated by the ethyl and butyl esters of the c2-c8 saturated-chain carbocylic acids, with the exception of c5. saturated normal-chain c4, c6, c7, and c8 methyl esters were also found, as well as other saturated aliphatic and aromatic esters. some unsaturated esters were also detected: ethyl but-2-enoate, n-butyl but-2-enoate, ethyl oct-2-enoate, and n-butyl oct2s, i.e., ethyl-3-hydroxybutyrate and n-butyl 3-hydroxybutyrate. alcohols, including ethanol, butanol, hexanol, and octanol, were the another group of important components observed in the papaya fruit. these alcohols corresponded to the alcohol part of the identified ester. there are few flavour constituents that come directly from carbohydrate metabolism. however, terpenes for example, which arise both from carbohydrate and lipid metabolism, also play very important roles in plant's aroma. et al et al et al et al enoate together with two βhydroxy ester 62 biotropia vol. 20 no. 1, 2013 terpenes are classified by the number of isoprene units (c5). in one particular study by flath and forrey (1977), terpenoids constituted 81% of the total volatiles detected, with the monoterpene alcohol, heidlas . 1984; flath . 1990). of particular interest are linalool and its pyranoid and furanoid oxides due to their prominence as flavour components of papaya (schreier & winterhalter 1986; flath . 1990). linalool and its oxides are biosynthesized through geranyl diphospate which produce linalyl cation and linalyl pyrophosphate. linalyl pyrophosphate are the amount of free linalool is relatively small in papaya, instead, it often occurs in ripe papaya fruits in a glycosidic bound form, from which it is released through cell disruption (heidlas 1984). linalool glycoside is synthesized by the enzyme glycosyltransferse, and its concentration versus time increased with the maturity of the plant organ. this accumulation is generally coupled with the production of the corresponding free forms. the degradation of carotenoids, particulary the oxygenated xanthophylls, contributes . 2003; flath & forrey 1997). the co-oxidative cleavage of carotenoid polyene chains to form ionones and damascenones occurs enzymatically through the action of oxidases including lipoxygenases, peroxidases, and dioxygenases, released during fruit ripening. glucosinolates are thioglucosides mainly found in the botanical family cruciferae, although rare occurrence outside this family is also documented. papaya is one of the best examples of a nonplant which contains benzyl glucosinolates, and hence produces benzyl isothiocyanates among its volatiles, by enzyme actions (macleod & pieris 1983). flath and forrey (1977) confirmed the presence of benzylglucosinolate in papaya fruit based on their discovery of relatively large amounts of both benzylthiocyanate and phenylacetonitrile (another product of benzyl glucosinolate degradation) among the volatile components. benzyl glucosinate is a compound that is continually formed during the growth of plant, from seed to fruit. it is present in a high concentration in the young papaya plant, especially in the leaves and roots. as the papaya plant grows, the compound is gradually transported to the fruit (bennet . 1997). from phenylalanine, benzyl glucosinolate is synthesized via three stages: (1) amino acid chain elongation, (2) synthesis of the glucosinolate from the amino acid, and (3) chain modifications. although it does not contribute much to the flavour of the papaya fruit, it is crucial to the formation of benzyl isothiocyanate (phytochemical containing sulphur and linalool, representing 68% of the total emissions. other terpene hydrocarbons like myrcene, ocimene, limonene, sabinene and neoalloocimeme, and terpene alcohols like αterpineol, nerol, and geraniol also contribute to the major volatiles in papaya ( also formed other monoterpenes such as α-terpineol, limonene, and pinene. e.g. endogeneous βglucosidase activity during to the aroma profiles of many fruits and vegetables as well. trace amounts of the ketone β-ionone have been identified previously as papaya volatile component (pino et al et al et al et al. et al cruciferae et al synthesis of benzyl glucosinolate 63 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. nitrogen) of which is a major flavour volatile of the papaya fruit with specific sensory) (flath & forrey 1977; flath . 1990). benzyl isothiocyanate has a protective function in the papaya fruit as well. it is produced by the enzymatic hydrolysis of benzyl glucosinolate during fruit injury (patil . 1973). the formation of benzyl isothiocyanate, which are formed from the aglycone by a lossen re-arrangement involving the migration of the side chain from the oxime carbon to the adjacent nitrogen, is catalysed by myrosinases (halkier &and gershenzon 2006). in fact, even intact papaya fruit releases traces of benzyl isothiocyanate vapour (patil & tang 1974). like the other soft pulp fruits, papaya is highly perishable & susceptible to fungal attack during storage. most of the volatiles formed after storage are mainly due to the mechanical damage sustained by the tissue during preparation such as cutting or fungal attack. the cellular disruption allows enzymes and substrates that are previously sequestered separately within the cells to be able to be interacted, mediated qualitatively and quantitatively and altered, which results in the synthesizing of volatile compounds including 4-methyloctane, tetrahydro-3-furfur yl-furan, 2,4dimethylhexane, benzene-methanol, isopelletierins, 5-6-dimethydecane, cyclohexylazide, undecane, n-tridecane, butyl ketone, 7-tridecanone, 5-9-undecadien2-one & benzyl tiglate (mohammed . 2001). schreier & winterhalter (1986) has also reported the presence of 6,7-epoxy-linalool, 2,6-dimethyl-1,7-octadiene-3,6-diol, 2,6-dimethyl-3,7-octadiene-2,6-diol, 2,6-dimethyl-3,7-octadiene-2,6-diol, cis and trans-2,6-dimethyl-2,7-octadiene-1,6-diol and 2,6-dimethyl-7-octene-2,3,6-triol as well as four diastereoisomeric epoxy-linalool oxides in their furanoid and pyranoid forms as well. although low temperature is used to maintain the freshness of the ripen papaya, continuous changes of the volatile compounds composition were observed during this storage. on the third day of storage, there were major changes in the odour perceived in the ripen papaya (mohammed . 2001). the freshly cut papaya initially has a mixture of flowery, fresh, bergamot-like, fruity, bitter, nutty, almond odour, mainly due to the presence of the dominant odouractive compounds, linalool, which gives flowery and fresh odour reminiscent of lily and benzaldehyde which contributes to bitter nutty and almond odour (bauer . 1997). there are also other trace volatiles such as and -linalool oxides, cyclohexane, hexanoic acid and benzenemethanol which contribute to the fruity flavour (bauer . 1997). furthermore, the presence of cyclic ester, -and linalool also contribute to the earthy and slightly bergamot-like odour in papaya (bauer . 1997). these flavours perceived from the freshly cut ripen papaya would last for the first two days of storage and change to the different volatiles. the sweet flowery odour perceived initially was lost on the third day of storage due to a significant decrease of about 50% of linalool, while benzaldehhyde, linalool cispyranic oxide and cyclohexanebeing vanished (mohammed . 2001). on the other et al et al et al et al et al trans cis et al cis trans et al et al flavor changes during storage and processing 64 biotropia vol. 20 no. 1, 2013 hand, benzyl acetate was the dominant aroma volatile at the storage interval and together with un-pleasant earthy flowery odour of trans-linalool and un-pleasant coffee like aroma taking place. the significant increase of butanoic acid present on day 3, also causes the impartment of dry-flowery lily-like odour, thus intensifies the non pleasant odour of the papaya after 3 days storage. upon realizing the undesirable changes of the aroma active compounds during storage of the sliced ripen papaya, it would be advisable that ripe papaya should be consumed immediately after the cutting of the papaya. otherwise, the papaya should be left without processing which would prevent the formation of non-pleasant volatile compounds due to the cellular disruption. papaya puree is the major semi-processed product that is used in juices, nectars, fruits cocktails, jams, jellies and fruit leather (salunkhe & kadam 1995; somogyi . 1996). commercially, papaya puree is produced by pushing the whole fruit into a pulpier fitted with a 0.033 inch screen. the puree is not heated to inactive the enzymes and even contain pulverized peel and & seeds. the next step is subjected to subsequent thawing and freezing to dispatch and preserved the puree. as mentioned above, squeezing of the whole fruit into the pulpier will cause the cellular disruption that allows enzymes and substrates to be able to interact, resulting in a similar sweet flowery and fruity odour due to the release of a large amount of linalool, benzaldehyde and other trace compounds. however, using the commercial method on producing papaya puree cannot maintain the sweet flowery and fruity odour, and in turn resulted in the off-flavour perceived in the papaya puree. the formation of off flavour was mainly due to the reaction induced by naturally occurring enzymes in the papaya during processing and frozen storage. it is also due to the microbial action in the centre of the puree mass prior to the freezing process and at the periphery of the mass during the thawing process (chan . 1973). both enzymatic and microbial action causes the formation of butyric, hexanoic, and octanoic acid and their methyl esters. the formation of these free acids which mainly has pungent-rancid odour might contribute to the off-flavour. furthermore, during the puree process, the content of benzyl isothiocyanate increases significantly higher than the linalool content due to the hydrolysis of glucosinolate by enzyme. this proportion will suppress the sweet flowery odour and intensify the pungent cabbagelike odour as well. to minimize the off-flavour formation, an improved method for processing puree, by acidification to ph 3.55 and thermal in-activation of enzyme, has been developed (chan . 1973). aside from being made into puree, as has been mentioned above, papaya can be processed into other products to meet consumer's needs. in processing of all these products, thermal treatment or thermal processing is ubiquitously applied as an essential part, for the purposes of in-activating enzymes, softening of texture, concentration or drying of product, and sterilization. however, the thermal treatment also brings detrimental effects to the product quality, especially with regard to the aroma profile of papaya. the volatile aroma active compounds present in papaya will be inevitably lost to different extent during the thermal treatment. in a previous summary of the processed papaya products and their processing conditions (salunkhe & kadam 1995; somogyi . 1996), it seems that the et al et al et al et al 65 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. temperatures involved in the thermal treatments of various processing's are below the boiling points of most aroma active compounds (table 2). it indicates that during the thermal treatments, cycloxehane will probably be the one which loses most due to its low boiling point, while other compounds will be partly lost due to evaporation. the loss of cyclohexane and other aroma active compounds will lead to the deterioration of olfactory quality of papaya. therefore, the processed products are often perceived as less sweet or fruity in aroma than the fresh-cut, non-processed fruits. based on the boiling point and likelihood of evaporation, the aroma active compounds listed in table 2 can also be classified roughly into three categories. cyclohexane which has the lowest boiling point and thus most easily evaporated; and the second group are benzaldehyde, butonoic acid and 4-methyloctane, with the medium boiling point, evaporate at a moderate rate; while other aroma compounds, including linalool, bezyle isothicynate, hexanoic acid, benzenemethonal, cis-linalool oxide, benzyl acetate and gamma-hexalactone, with relatively high boiling points, evaporate at a slow rate. during the heat treatment, besides loss due to evaporation, some of the aroma active compounds may be oxidized with the presence of oxygen. among the 15 aroma active compounds identified in papaya, linalool and benzaldehyde, the two major aroma compounds in fresh papaya, are the most susceptible to oxidation. linalool undergoes autoxidation with o to form hydroperoxides, of which 7-hydroperoxy3,7-dimethyl-octa-1,5-diene-3-ol is the most important one (skold 2006; backtrop . 2006) this would result in reduction of linalool and in turn the sweet smell of the papaya aroma in the processed product. benzadehyde also undergoes autoxidation to perbenzoic acid and converts to benzyl alcohol upon hydrogenation (bauer . 1997). as a result, the bitter almond aroma contributed by benzaldehyde will decrease in the processed papaya product. to reduce loss of aroma in processed papaya products, irradiation is proposed as an alternative to heat treatment when the purpose is to kill fruit flies, prevent fungal growth or inactivate enzymes. irradiation is able to achieve the same or better et al et al 2 table 2. summary of boiling points of several aroma active compounds in papaya aroma compounds boiling point (oc) aroma compounds boiling point (oc) linalool 198 cis-linalool oxide 188 benzaldehyde 178 benzyl acetate 214 benzyle isothicynate 243 linalool cis -pyranic oxide not available butonoic acid 164 cyclohexane 81 4-methyloctane 142 gamma hexalactone 220 hexanoic acid 223 gamma octalactones not available benzenementhonal 205 delta octalactones not available trans-linalool oxide not available 66 biotropia vol. 20 no. 1, 2013 outcomes as heat treatment, with much less damage to the sensory qualities of papaya (moy 1993). however, irradiation cannot replace heat treatment if the product requires be softening or dehydrating. in addition, the resistance to irradiated foods of consumers is still strong in the current context, which poses big challenge to the promotion of irradiation in food products. papaya is a climacteric fruit that undergoes the ripening process upon harvesting, until the stage where a fully ripened papaya gives fully developed flavours which then is perceived and recognized by human as ripening indication. the flavour of papaya fruit is a result from a complex interactions between sugars, organic acids, minerals, and aroma volatile compounds, which may vary with cultivar and production location. when the papaya fruits are subject to processing, there would be qualitative and quantitative alterations in the overall composition of volatile compounds due to the processing conditions. volatiles identified in these studies included heterocyclic compounds, terpenoids, aromatic hydrocarbon, alcohol, acids, esters and ketones. a fairly wide range of different types of compounds was identified in different studies. these volatiles are released by harvested intact fruit in small amount per unit time, and some of the reported volatiles are only generated in quantity from non-volatile precursors due to the disruption of fruit tissue. despite the significant diversity in aroma compounds of papaya fruit, the volatiles in papaya are mainly derived from three major metabolism pathways: the catabolism of fatty acid, amino acid, and carbohydrates. the biosynthesis pathways are usually interlinked, with products from one pathway serving as the precursors for another pathway. these degradation reactions account for many aroma compounds, as they result in the formation of a host of low molecular weight products which has significant sensory properties. moreover, papaya is one of the best examples of a nonplant which contains benzyl glucosinolates. although this compound does not contribute much to the flavour of the papaya fruit, it is crucial to the formation of benzyl isothiocyanate (phytochemical containing sulphur and nitrogen with specific sensory), which is a major flavour volatile of the papaya fruit. benzyl isothiocyanate has a protective function in the papaya fruit. this manuscript has been accomplished during the same program 2012 funded by dghe-ministry of education and culture, ri. thanks should be also addressed to mr. i kadek putra yudha prawira for his technical support. conclusions acknowledgments cruciferae 67 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. references almora k, pino ja, hernandez m, duarte c, gonzalez j, roncal e. 2004. evaluation of volatiles from ripening papaya ( l., var maradol roja). food chem 86(1): 127-30. anonymous. 2003. riset unggulan buah tropis indonesia. 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mantangan ( ) invasion in bukit barisan selatan merremia peltata national park jani master , soekisman tjitrosoedirdjo and ibnul qayim1* 2 3 1department of biology, faculty of mathematics and natural sciences, universitas lampung, bandar lampung 35145, indonesia 2weed science society of indonesia 3department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia received 20 january 2015/accepted 27 january 2016 abstract bukit barisan selatan national park merremia peltatasome areas in have serious problems related to (family convolvulaceae) invasion. the abiotic factors influencing invasion were investigated in this study. this m. peltata research compared abiotic factors in three forest locations with different invasion levels i.e. severe, moderate and mild invasions. abiotic factors measured were percentage of tree canopy coverage, micro climate factors (temperature and humidity), elevation above sea level and physical and chemical properties of the soil surface. in this study, canopy coverage is classified as abiotic factors because it influences the intensity of light that can reach the forest floor. a posthoc duncan's multiple range test (dmrt) was employed to determine significant differences ( <0.05) among p abiotic factors. in addition, a test of correlation and multiple linear regression were conducted to determine the relationships between abiotic factors and invasion. regression testing showed that canopy coverage multiplem. peltata significantly ( <0.05) influenced invasion. based on the generated model, a 1% addition of canopy coverage p m. peltata would decrease invasion by 2.8% multiple linear regression analysis was used to predict the relationship m. peltata . between soil nutrients and invasion. the c/n ratio and p o , ca, mg and na concentration significantly ( <0.05) 2 5 p influenced invasion level. again, based on the generated model, the increase in the c/n ratio as well as in p o and ca 2 5 concentrations were correlated with the of invasion; while the increase of mg and na reduction m. peltata concentrations were correlated with the increase of invasion. invasion in bukit barisan selatan m. peltata m. peltata national park was influenced by opened forest canopy and was correlated with high concentration of na and mg in the soil of the invaded forest areas. : bukit barisan selatan national park, invasive species, mantangan, keywords merremia peltata introduction indonesia is a country with abundant flora and fauna, some of which are conserved and protected in conservation areas such as national parks. existing national park areas in indonesia are threatened by alien plant species that invade and threaten flora and fauna biodiversity. alien species that have the ability to grow quickly so as to impact biodiversity in invaded areas can be referred to as invasive alien species (ias) (pyšek & richardson 2010). broadly, an ias is a species that has the potential to harm the economy, environment and/or cause damage to human, animal or plant health (national invasive species council 2006). merremia peltata mantangan or in the local language, is an invasive species of conservation areas. this species causes severe problems in bukit barisan selatan national park (bbsnp) with more than 7,000 ha covered by m. peltata (master 2013). infestation of the weed is et al. suspected to be the cause of animal migrations, such as tiger, elephant and sumateran rhinoceros, to rural areas up north (irianto & tjitrosoedirdjo 2010). analysis of landsat and advanced land observing satellite (alos) images indicated that m. peltata invasion is found from south part of biotropia vol. 23 no. 1, 2016: 21 27 21 doi: 10.11598/btb.2016.2 . .3 1 457 * c orresponding author: gmail.comj.janter@ 22 bbsnp running 33 km to the north of the national park. it is also found in small outbreaks as far as 50 km north of bbsnp (master 2013).et al. the bbsnp is the third biggest conservation area in sumatera island covering an area of 356,800 ha. administratively, this area belongs to lampung barat, pesisir barat and tanggamus districts of lampung province and the bengkulu district of bengkulu province, (4 31' – 5 57' s; 0 0 103 34' – 104 43' e) (gaveaua 2007).0 0 et al. m. peltata is a liana (climbing plant) of the convolvulaceae et al. family (paynter 2006), and it is distributed throughout madagascar, the mescarenes, seychelles, malay peninsula, malaya islands, philippines, new guinea, northern a u s t r a l i a a n d t h r o u g h o u t p o l y n e s i a (ooststroom & hoogland 1953). it is similar to sweet potato in morphology, with a wide heart shaped leaf connected by peltate petiole, in contrast to the petiole connection of sweet potato. the hairless plant stem grows to 20 m in length and excretes white liquid when injured. it may grow over other plants (stone 1970). the corolla is white or yellow, 5–6 cm in length and forms a bell-shape corolla (fosberg & sachet 1977). spreading of this plant occurs generatively, both by seeds or vegetatively from roots along its stems which touch the ground. egetative growth v rates reach 16 cm/weekcan (pengembara 2014). environmental factors, both biotic and abiotic, determine the presence of a species in a particular location (huang 2003). et al. accordingly, not all alien species introduced to an area will become invasive; this can be influenced by factors including environmental fitness and the presence or absence of predators or competitors (theoharides & dukes 2007). the rapid growth of in bbsnp is probably caused by a m. peltata number of abiotic factors. the objective of this research was to determine abiotic factors influencing invasion in bbsnpm. peltata . materials and methods erremiam peltata is found mostly in southern regions of bbsnp, but not all of in this m. peltata region grows densely. to determine abiotic factors influencing plant invasion, environmental factors were recorded in three forest locations with different invasion levels, i.e. severely invaded, moderately invaded and mildly invaded forest (fig. 1). first location (severely invaded forest) is close to the village enclave way haru, bandar dalam and tampang. based on land cover maps, this site has a decreasing forest cover for an area of 2,565.54 ha from 2000 to 2009. presumably these changes were the result of forest clearing biotropia vol. 23 no. 1, 2016 figure 1 map of research location of invasion in bukit barisan selatan national park, lampung, sumatera m. peltata (master 2013)et al. abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. 23 for plantations (prasetyo 2011). the second et al. location (moderately invaded forest) is a post-fire forest in 1997; fires that occurred mostly on the forest floor affecting the lining of plant and open up some forest canopy (wcs-ip 2001). the third location (mildly invaded forest) is a primary forest. selection and classification of these invasion levels were based on coverage of from m. peltata landsat and alos image analyses in 2002 and 2008 (master 2013). the final selection was et al. conducted by recording the percentage of m. peltata canopy coverage by using plot of 20 x 20 m area. three line transects of 1,000 m each, with minimum of 200 m interval distance between each line transect, were made in each forest location. plots of 20 x 20 m were sampled along each line transect, at 100 m intervals, thus totaling 30 plots in each location. invasion was m. peltata estimated based on the percentage of plot that was covered by . environmental factors m. peltata recorded for each plot were: 1. the percentage of tree canopy coverage (tree canopies that were more than 4 m in height) which was estimated using a concave densiometer; 2. microclimate (temperature and humidity) were measured at noon, 1 m above the ground using thermometer and hygrometer on each plot; 3. elevation above sea level; and 4. physical and chemical properties of top soil using composite method (crozier . et al 1998). soil physical and chemical properties observed were soil texture, ph (h o), kcl, c-2 organic, n-kjedahl, cation exchange capacity (cec) and exchangeable cations (k, na, ca, mg). soil texture fraction comparisons were separated as follows: 1. sand fraction having particles with diameter of 2 mm 50µ; 2. loam fraction having particles with diameter of 50µ 2µ; and 3. clay fraction having particles with diameter of <2µ) (hardjowigeno 2010). data for each environmental factor from each of the three locations were analyzed using anova and the mean differences were tested using the duncan's multiple range test (dmrt) at <0.05). tests of correlation and linear p regression were carried out using spss 12 program to determine any relationships existed between abiotic factors and invasion.m. peltata results and discussion percentage cover of invading m. peltata forests m. peltata the mean coverage percentage of in the mildly invaded forest ranged from 1 to 15%. in comparison, the mean coverage percentage in the moderate invaded forest ranged from 27 to 55%. in the severe invaded forest, the mean coverage percentage was 44%, while in some location the percentage was up to 100%. abiotic factors (non-soil) and m. peltata invasion levels of the six abiotic factors observed i.e. air temperature, air humidity, ground temperature, soil humidity, soil ph and percentage of tree canopy coverage (canopy above 4 m), only the percentage of tree canopy coverage was significantly varied ( <0.05) among the three p locations (table 1). forest canopy coverage had significant negative correlation to m. peltata invasion. based on a generated model, every 1% addition of canopy coverage would decrease m. peltata invasion by 2.8% (table 2). the severely invaded forest locations had more open tree canopy coverage. the open canopies facilitate access of to solar m. peltata radiation such that the weed dominated these table 1 summary of abiotic factors at the three research locations location air temperature (oc) air humidity (%) ground temperature (oc) soil humidity (%) soil ph canopy coverage (%) mildly invaded forest 26.41 a 90.72 a 25.65 a 47.93 a 6.75 a 92.31 a moderately invaded forest 27.93 b 83.79 b 26.10 b 47.65 a 6.65 a 81.51 b severely invaded forest 27.15 ab 88.84 a 26.00 ab 54.05 a 6.51 a 69.89 c note: numbers followed by the same letters in same columns are not significantly different ( <0.05)p 24 biotropia vol. 23 no. 1, 2016 areas. in the mildly invaded forest, mean canopy coverage was denser (92.31%). denser canopy coverage prevented extensive growth m. peltata and development such that it only grew through gaps of canopy where there were collapsed trees. this result is consistent with other findings which reported that grew on the edges of m. peltata forest, especially in degraded land in fiji (kirkham 2005), and that this plant grew densely in logged forest and open forest in other parts of sumatera (irianto & tjitrosoedirdjo 2010). air temperature, air humidity and ground temperatures were all significantly ( <0.05) higher p in moderately invaded forest than in mildly invaded forest (table 1). the increase in these factors was most likely related to tree canopy coverage, such that more open canopies would increase solar radiation, air and ground temperatures, as well as rainfall reaching the forest floor (sasaki & mori 1981). severely invaded forest had more open tree canopy coverage, but air temperature, air humidity and ground temperatures in that location were not different to mildly invaded forest. this is probably because the ground coverage of m. peltata in both levels of invaded forest produced similar microclimate. there were no relationship between elevation above sea level and the infestation size of m. peltata. m. peltata this is despite the fact that is known to occupy lowland habitats to 300-400 m above sea level in samoa and fiji (meyer 2000, kirkham 2005). soil abiotic factors and invasion m. peltata levels beside the aforementioned environmental factors, the physical and chemical properties of the soil were also analyzed in the three research locations. the mildly invaded forest was dominated with a sandy-clay-loam soil texture, while the moderately and severely invaded forest had clay and clay-loam soil texture (table 3). soil in the secondary and invaded forests had higher clay content than soil in the primary forest. the clay fraction is the most important table 2 results of multiple linear regression between percentages of coverage and abiotic factorsm. peltata coefficientsa model unstandardized coefficients standardized coefficients t sig. collinearity statistics b std. error beta tolerance vif 1 (constant) 89.020 53.253 1.672 0.099 air temperature -2.841 3.189 0.148 -0.891 0.376 0.356 2.812 air humidity -2.782 1.668 0.281 -1.668 0.100 0.345 2.897 ground temperature 6.209 7.892 0.119 0.787 0.434 0.432 2.317 soil humidity -0.603 0.386 0.236 -1.562 0.123 0.431 2.320 soil ph -23.790 8.175 0.448 -2.910 0.005 0.414 2.418 canopy coverage -1.218 0.301 0.427 -4.047 0.000 0.882 1.134 note: a = dependent variable: percentage of m. peltata coverage table 3 soil texture samples in the three research locations location texture percentage (%) mildly invaded forest sandy clay loam 33.33 clay 55.56 clay loam 11.11 moderately invaded forest clay 44.44 clay loam 55.56 severely invaded forest clay 55.56 clay loam 44.44 25 component because its particles have more surface area than sand particles. soil with higher clay content has higher cation exchange capacity (cec) than sandy soil due to greater overall surface area of clay particles (munawar 2011). the cec value was higher in the severely invaded forest (25.67 cmol /kg), followed by moderately c invaded forest (19.07 cmol /kg), with the lowest in c the mildly invaded forest (13.51 cmol /kg) (table c 4). the cec is a chemical property closely related to soil fertility. soil with higher organic and clay contents had higher cec values than soil with low o r g a n i c c o n t e n t a n d / o r s a n d y s o i l s (hardjowigeno 2010). further, soil with higher cec values is able to better retain and provides nutrients (cations and anions) than soil with low cec values. the lack of cation exchange capacity in low cec soil results in the nutrients being easily washed through the soil with underground water movement (infiltration, percolation) such that these nutrients are no longer available for plant growth. the severely invaded forest had a higher alkaline density (base saturation) value (71.11%) than the other two forest locations. contributing to this higher alkaline density were the alkaline cations ca, mg, k and na. these alkaline cations are important soil nutrients required by plants. the presence of organic substances in the soil can improve soil fertility chemically, physically and biologically. the amount of organic carbon in the severely invaded forest was higher (3.46%) than that in the mildly invaded forest. based on soil fertility criteria, c-organic in the severely invaded forest belonged to high category. nitrogen (n) is a crucial soil nutrient, often found with low availability. total nitrogen in the severely invaded forest was significantly ( <0.05) p higher (0.35%) than that in the other two locations. there was no significant difference ( >0.05) of total nitrogen between the mildly p invaded forest (0.17%) and the moderately invaded forest (0.21%). higher nitrogen values contribute to low c/n ratios such that more nitrogen is available to microorganisms to decompose carbon containing material, recycling the nitrogen quickly (munawar 2011). higher nitrogen contents in plants will result in faster vegetative growth. was rarely found to m. peltata flower in severely invaded forest, and this may have been caused by high nitrogen contents in the soil. regression analyses between soil factors and invasionm. peltata double linear regression analyses were conducted to predict whether soil nutrient status was correlated with invasion. c/n ratio and concentration of p o , ca, mg and na 2 5 significantly ( <0.05) contributed to p m. peltata invasion (table 5). based on a generated model, the increase of c/n ratio and concentration of p o and ca may reduce invasion; while 2 5 m. peltata the increase of mg and na concentration may increase invasion. invasion in m. peltata m. peltata the severely invaded forest was correlated with table 4 average soil chemical properties in three research locations soil chemical property mildly invaded for est moderately invaded forest severely invaded forest value level value level value level c-organic (%) 2.07 a middle 2.62 ab middle 3.46 b high n (%) 0.17 a low 0.21 a middle 0.35 b middle c/n ratio 11.66 a middle 11.88 a middle 9.88 b low p2o5 (mg/100 g) 24.22 a middle 18.44 b low 24.88 a middle k2o (mg/100 g) 28.33 a middle 31.11 a middle 39.77 a middle p-bray (ppm) 4.00 a very low 2.76 a very low 6.53 b very low ca (cmolc/kg) 4.59 a low 5.50 a low 11.11 b middle mg (cmolc/kg) 3.04 a high 3.96 a high 6.28 b high k (cmolc/kg) 0.57 a middle 0.50 a middle 0.67 a high na (cmolc/kg) 0.41 a middle 0.46 b middle 0.51 c middle cec* (cmolc/kg) 13.51 a low 19.07 a middle 25.67 b high base saturation (%) 62.55 a high 55.11 ab high 71.11 b high notes: cec = cation exchange capacity numbers followed by the same letters in the same row are not significantly different ( <0.05)p abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. 26 biotropia vol. 23 no. 1, 2016 low c/n ratios and high concentration of mg and na in the soil. sodium has an important role in determining soil characteristics and plant growth, especially in areas close to a beach. the severely invaded forest in this study is located close to a beach. this is a suspected cause of the high na content in the soil in this location. excessive na content can also be toxic for plants (hanafiah 2007), influencing cell membrane and organelle functions, as well as disrupting plant metabolism sequences (hamim 2008). soil sodium concentrations significantly ( <0.05) increased as invasion levels of p m. peltata increased in this study. this weed is suspected of being a halophyte, being able to grow in a land with high salt content. another soil nutrient correlated with m. peltata invasion was mg. soil samples from the severely invaded forest had significant ( <0.05) higher p content of mg (6.28 cmol /kg) than either mildly c invaded forest (3.04 cmol /kg) or moderately c invaded forest (3.96 cmol /kg) (table 4). c magnesium is a crucial element used in chlorophyll formation. like other soil nutrients, magnesium deficiency results in a typical color change to leaf material. premature leaf fall is also caused by magnesium deficiency (hanafiah 2007). magnesium is a macro element in soil when found in its organic form (sutcliffe & baker 1975) and has important role in phosphate availability (agustina 2004). conclusions abiotic factors influencing invasion m. peltata in bukit barisan selatan national park are opened forest canopy coverage and high content of na and mg in the soil. acknowledgements this study was part of master thesis of the first author, financially supported by seameobiotrop dipa 2011. the author thanked artha graha peduli and wildlife conservation society indonesia program for their assistance in conducting the field work. references agustina l. 2004. . jakarta dasar-dasar nutrisi tanaman (id): pt rineka cipta. crozier cr, heiniger rw. 1998. soil facts: soil sampling for precision farming systems. north carolina (usa): north carolina state university. fosberg fr, sachet mh. 1977. flora of micronesia, part 3, convolvulaceae. contributii botanice 36:1-34. gaveaua dla, wandono h, setiabudi f. 2007. three decades of deforestation in southwest sumatera: have protected areas halted forest loss and logging, and promoted re-growth biol conserv 134:495-. 504. table 5 result of multiple linear regression between the percentage of coverage and soil nutrient factorsm. peltata coefficientsa model unstandardized coefficients standardized coefficients t sig. collinearity statistics b std. error beta tol erance vif 1 (constant) 50.768 144.015 0.353 0.729 loam 0.797 1.181 0.117 0.675 0.509 0.315 3.173 clay -0.569 0.905 0.140 -0.629 0.538 0.190 5.277 h2o -11.183 23.641 0.128 -0.473 0.642 0.128 7.818 cn ratio -8.535 2.739 0.370 -3.116 0.006 0.668 1.498 p2o5 -2.721 1.197 0.377 -2.273 0.036 0.342 2.924 ca -5.902 1.694 0.888 -3.485 0.003 0.145 6.899 mg 18.726 4.721 0.970 3.966 0.001 0.157 6.351 na 210.264 74.615 0.390 2.818 0.012 0.493 2.029 base saturation (%) 0.735 0.459 0.292 1.601 0.128 0.284 3.522 note: a = dependent variable: coverage of merremia peltata 27 hamim. 2008. jakarta (id): universitas fisiologi tumbuhan. terbuka. hanafiah ka. 2007. . jakarta (id): dasar-dasar ilmu tanah pt. raja grafindo persada. hardjowigeno s. 2010. jakarta (id): akademika ilmu tanah. pressindo. huang w, pohjonen v, johansson s, nashanda m, katigula mil, luukkanen o. 2003. species diversity, forest structure and species composition in tanzania tropical forest. for ecol manage 173:11-24. irianto r, tjitrosoedirdjo s. 2010. invasi (l.) merremia peltata merr., convolvulaceae di taman nasional bukit barisan selatan, indonesia. journal gulma dan tumbuhan invasi tropika 1:65-70. kirkham ws. 2005. valuing invasives: understanding the merremia peltata invasion in post-colonial samoa. dissertation. austin (usa): the university of texas at austin. master j, tjitrosoedirdjo ss, qayim i, tjitrosoedirdjo s. 2013. ecological impact of (l.) merremia peltata merrill invasion on plant diversity at ukit barisan b selatan national park. biotropia 20(1):29-37. meyer jy. 2000. preliminary review of the invasive plants in the pacific islands. in: sherley g, editor. invasive species in the pacific: a technical review and draft regional strategy. samoa (ws): south pacific regional environment programme. p 85-114. munawar a. 2011. . kesuburan tanah dan nutrisi tanaman bogor (id): ipb press. national invasive species council. 2006. invasive species definition clarification and guidance white paper. https://www.invasivespeciesinfo.gov/docs/council / isacdef.pdf (accessed october 20 , 2014).th ooststroom van sj, hoogland rd. 1953. convolvulaceae. flora malesiana i 4:452-3. paynter q, harman h, waipara n. 2006. prospects for biological control of . . new merremia peltata report zealand: conservation international. pyšek p, richardson dm. 2010. invasive species, environmental change and management and health. ann rev environ resour 35: 25-55. pengembara t, master j, yulianty, rustiati el, subiakto a. 2014. laju ertumbuhan antangan (merremia p m peltata l. merr.) ang umbuh elalui egenerasi y t m r vegetatif. proceedings of seminar nasional pengembangan teknologi pertanian. lampung (id): politeknik negeri unila. prasetyo a, hikmat a, prasetyo lb. 2011. pendugaan perubahan cadangan karbon di tambling wildlife conservation taman nasional bukit arisan b selatan. media konservasi 16: 87 – 91. sasaki s, mori t. 1981. growth responses of dipterocarp seedlings to light. malaysian forester 44: 319-45. stone bc. 1970. the flora of guam. micronesica 6:1-659. sutcliffe jf, baker da. 1975. . london plant and mineral salts (uk): edward arnold publishing. theoharides ka, dukes jf. 2007. plant invasion across space and time: factors affecting non indigenous species success during four stages of invasion. j new phytol 176:256-73. wildlife conservation society – indonesia program (wcsip). 2001. taman nasional bukit barisan selatan dalam ruang dan waktu. . bogor (id): research report phka/wcs-ip. abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. https://www.invasivespeciesinfo.gov/docs/council 712 luluk s (jabon).cdr jabon (anthocephalus cadamba potency for roxb) remediating lead (pb) toxicity under nutrient culture condition luluk setyaningsih , yadi setiadi , sri wilarso budi , hamim 1* 2 2 3 and didy sopandie 4 1 department of forestry, universitas nusa bangsa, bogor 16166, indonesia 2 department of silviculture, faculty of forestry, institut pertanian bogor, bogor 16680, indonesia 3 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia 4 department of agronomy, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia received 25 october 2016/accepted 18 september 2017 abstract information on metal adaptation of plants is necessary to understand the mechanism of heavy metal, including lead (pb), remediation mediated by forest plants in contaminated land. this study aimed to find out the adaptation mechanism of jabon (anthocephalus cadamba roxb) seedlings to excessive lead level based on the tolerance index of growth performance and lead transport to plant tissue. the seedlings were exposed to lead (pb(no ) ) with the 3 2 concentrations of 0, 0.5, 1, 1.5, 5 and 10 mm in nutrient culture for 15 days. the result showed that the tolerance index (ti) of the seedlings was significantly decreased by pb exposure up to 1.5 mm, but the ti values were more than 75%. all seedlings died at pb concentration of 5 mm and up. pb accumulated in all parts of the seedlings, with the highest concentration found in the leaves (735.9 ppm) under pb concentration of 0.5 mm. the pb was found to be transported to the top portion of the seedlings indicated by transport factor (tf) that was more than 1. the results suggest that jabon can adapt to excessive pb exposure up to 1.5 mm and has the potential as a remediator plant. keywords: accumulation, jabon, lead, nutrient culture, tolerance introduction lead (pb) is considered as the second most dangerous heavy metal after arsenic and it is among the biggest pollutants in water and terrestrial ecosystems generated from a variety of activities including household and industrial works (ebrahimpour & mushrifah 2008; tangahu et al. 2011; alaribe & agamuthu 2015). in a certain concentration, pb is toxic to plants, animals, and microorganisms. when absorbed by the human body, it will produce various deleterious effects on the hematopoietic, renal, reproductive and central nervous system, mainly through increased oxidative stress (flora et al. 2012). heavy metal contamination in the soil can reduce soil fertility for long-term. high pb levels in the soil need remedial action to enable the soil to re its normal function ( . 2011). gain tangahu et al phytoremediation has become an effective, environmentally friendly and affordable technological solution through the extraction or removal inactive metals and metal pollutants of from contaminated soil (henry . 2013; et al sharma & pandey 2014). phytoremediation is based on the fact that plants have membrane active pumps that work solar energy to extract through certain heavy metals from the environment to proceed with the translocation, bioaccumulation, or contaminant degradation depend on the ing abilities of the entire plant body (rascioa & navari-izzo. 2011; tangahu . 2011).et al jabon (anthocephalus cadamba) is known as a tropical forest species with high tolerance to marginal land, and high shoot and roots biomass productions (krisnawati et al. 2011; setyaningsih et al. 2012). forest plants with deep roots * corresponding author: luluk.setya@gmail.com biotropia 5 1 8 64 71 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.712 64 character and great biomass have a great potential to be used as remediator plant (capuana 2010) in contaminated forest land. however, there is still limited information about the tolerance character of tropical forest plants to heavy metals and their potentcy as remediators. each plant may have a tolerant level and pattern of different adaptations to heavy metal exposure conditions. there is still little information related to tolerant level of jabon to lead, even though many other species have been extensively evaluated such as sunflower (niu et al. 2010; kim et al. 2009), canola, mustard and vetiver grass (kim et al. 2009), lantana camara (alaribe & agamuthu 2015) and pinus radiata (javris & leung 2002). therefore, the effort to learn about the tolerance and remediation capability of jabon to heavy metal pb is necessary and important. this research was conducted in nutrient culture media to determine (1) the tolerant level of jabon seedling to by observing root growth s pb and biomass, and (2) the degree jabon by which seedlings absorb pb. materials and methods s aged one month old ninety jabon seedling with 5 cm height were the obtained from tissue culture laborator of seameo y the regional centre for tropical biology ( ) in biotrop bogor, indonesia, transferred into culture and media in plastic box container equippeds with aerator tube for lead exposure experiment. these s seedlings were grown in aerator tube containing 1 s litre of liquid culture media (sopandie nutrient 1990) adapted for 4 days and then subjected to a treatment using pb for 15 days. adaptation media nutrient was 1/5 dosage of nutrient culture without pb. media treatment was 1/3 dosage of nutrient culture treated by pb (no3) with 6 2 different pb concentrations i.e. 0, 0.5, 1, 1.5, 5 and 10 mm respectively (setyaningsih 2012). , et al. during the experiment the media culture was , set to have ph 4.5-5 using naoh and hcl. composition of nutrient culture refers to the “ n s ”sopandie utrient olution (sopandie 1990) consisting of distilled water and nutrient solution w ich contained 1.5 mm ca(no ) 4h o; 1.0 mm h 3 2. 2 nh no ; 1.0 mm kcl; 0.4 mm mgso ; 1.0 mm 4 3 4 kh po ; 0.50 ppm mnso ; 0.02 ppm 2 4 4 cuso .5h o; 0.05 ppm znso .7h o; 0.05 ppm 4 2 4 2 h bo ; 0.01 ppm (nh ) mo o .4h o; 68 mm 3 3 4 6 7 24 2 feedta. during the adaptation and treatment, the aeration was supplied using electrical aerator. parameters analysed in the experiment were root and shoot growth and plant biomass to verify the response of plants to all pb treatments. pb content was analyzed only from samples of control plants (without pb), the plants with lowest pb treatment (0.5 mm), and the plant with highest pb treatment by which the plants were still alive until the end period of the treatment, to compare the accumulation capacity of plants under lower and higher pb concentration in the media. seedling growth was observed by measuring the length of root and stem , and biomass s s dry weight harvest . when the plant samples were ed the number of dead seedlings was also counted. the tolerance index (ti) was calculated at different pb concentrations using the following equation: ti (%) = 100 x (root length under metal treatment)/(root length in the control solution) (wang . 2011). if ti <50 = not tolerant; et al 50≤ti <100 = medium tolerant; ti ≥100 = tolerant (professional judgment). pb accumulation in seedling tissue was calculated by measuring the concentration of pb in the roots, stems and leaves of control plants, the plants with lowest pb treatment (0.5 mm), and the highest treatment that were still alive. the seedlings were harvested and dried in the oven with a temperature of 45 ± 2°c until its weight was constant. for seedling special preparation roots, before being dried, considted of soaking them in 1.0 mm h-edta for 30 minutes to remove pb attached on the root surface and rinsed three times in h o (javris & leung 2002). 2 pb concentration ined using atomic was determ absor ption spectroscopy (aas) which previously had been calibrated with a standard solution of pb. the obtained pb concentration was then used for calculating the value of bioconcentration factor (bcf), transport factor (tf) and bioaccumulation (b) (mun et al. 2008), using the following sthe equation : bcf = c / c root soil where rootc is the concentration of pb in roots and c is pb concentration in soil soil / media t aerial rootf = c / c where aerialc is pb concentration in the shoots b = c x dw dw is (g pb/plant) where aerial tissue dry weight 65 jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. unguiculata (kopitte et al. 2007) and paraserianthes falcataria (setyaningsih et al. 2012). chlorosis and n e c r o s i s o c c u r r e d p r e s u m a b l y b e c a u s e photosynthetic pigment was damage as a result of decreased production of chlorophyll a and b when pb exposure increased up to 1500 ppm (wang et al. 2011). the declined production of chlorophyll occurred as a result of heavy metal disturbance that replaced magnesium (mg), an important ion in the central of chlorophyll molecules, and inhibit the synthesis of chlorophyll by inhibition of enzyme activity (wang et al. 2011; xiao et al. 2008). root damage was very likely caused by the rapid inhibition of root growth. inhibition of root growth could be caused by inhibition of cell division in root tip due to immobilization of pb ion in the cell wall (jiang & liu 2010). roots b e c a m e b l a ck i s h , s h o r t a n d f a t . t h i s phenomenon has also been reported no the roots of v. unguiculata which became shorter and had more secondary roots ( ), and hadi & aziz 2015 also the root of in response to pb on s p. falcataria exposure (setyaningsih . 2012).et al pb treatment for 15 days caused significant decrease (p< 0.05) root length, dry weight and in tolerance index (ti) of jabon seedling . root s length value range from 8.3 11 cm which d to g r a d u a l l y d e c r e a s e d by i n c r e a s i n g p b concentration exposure (table 1). dry weight of seedling had value range of 1.28 1.63 g which s decreased following the pattern of open polynomial curve of y = 0.48x 0.94x + 1.63 (r² 2 = 1, p <0.05). ti of jabon seedlings rangethe d from % to 75 100% which decreased significantly with the increase pb exposure, in concentration and the maximum reduction was 25% (table 1). e the there was no significant chang on growth performance of jabon seedling exposed to s when pb up to 1.5 mm, with value concentration ti of 75% or higher. this data showed that jabon seedlings categorized as can be having intermediate ce pb the ti value of toleran to . jabon seedling is apparently smaller than to pb p. falcataria with ti value of seedling 90% when exposed to the same level of pb (setyaningsih et al. chlorophytum 2012), and also smaller than comosum value with ti of 73% even though it was -1 exposed to 1500 mg kg (1500 ppm) of pb for 90 days (wang . 2011). moreover, et al the ti of jabon was also even smaller compared to acacia farnesiana pb , known as bioaccumulator, which the study was carried out in completely randomized design for treatments of 6 different pb concentrations, i.e. p0 = without pb (0 mm), p1 = 0.5 mm of pb, p2 = 1.0 mm of pb, p3 = 1.5 mm of pb, p4 = 5 mm of pb and p5 = 10 mm of pb. each treatment was repeated 3 times. three plants were used per unit of treatment, so that the total plants were 54 plants. all the data were analysed using the microsoft excel and spss 16 to calculate the analysis of variance and to compare the means of the treatments by dmrt analysis. the analysis of regression was also carried out to recognize the typical pattern of the treatment effects (exposure of pb). results and discussion pb tolerance of jabon seedling all jabon seedlings exposed to pb up to 1.5 mm were able to survive until 15 days. however, seedlings exposed to pb of 5 mm and 10 mm showed toxicity symptoms during the second day and completely died after 5 days suggesting that 5 mm is a toxic concentration for jabon seedling. this supports the findings of hadi and aziz (2015) and wang et al. (2011). the symptom started with chlorosis and necrosis on the tip of the leaves (the leaves turned to yellow and dried nd up) on the 2 day, followed by the appearance of rd th brown spots on the 3 day. on the 4 day, most of th the leaves rolled up and finally dried on 5 day followed by drying of the petiole and branch in the following days. toxic symptoms also appeared in the roots of the seedlings. by this reason, the measurement of pb concentration of the highest treatment was carried out from the plants treated by 1.5 mm of pb. the seedlings grown in the media without pb had fibrous root with white and transparent colour. the seedlings exposed to 0.5 1.5 mm of pb had white fibrous roots with some dark gray spost (about 1 cm) while the whole roots of seedlings exposed to 5 and 10 mm of pb had became dark indicating cell death. symptoms of toxicity due to pb began with yellowing leaves, the appearance of brown spots in almost all the leaves, leaf curling and leaf drying as well as stem drying on fol owing days with a l grayish black color of roots. these symptoms had also been reported in other plants such as chlorophytum comosum (wang et al. 2011), vigna biotropia vol. 25 no. 1, 2018 66 the ti decreased only 8% exposure to 1000 when -1 mg l pb for 60 days (amalia . 2011). ti et al the level of jabon to is better than cuttings of pb jatropha curcas has a value which ti less than 80% when exposed to 0.5 mm of pb and became only 39.2% when it was exposed to 4 mm pb (shu . et al 2011), or even lower if it was compared to the growth of annual plant such as cowpea (vigna unguiculata was recorded to be ) which hampered by only being exposed to 1 μm of pb (kopitte . et al 2007). pb concentration, distribution and bioaccumulation in jabon seedlings exposure of jabon seeldings to pb for 15 days in liquid culture caused an increase of pb concentration in seedling tissue up to 735 ppm or approximately 12.6 times higher in seedlings under control treatment. the largest average of pb concentration was found in the leaves (735.93 ppm), then in the roots (459.04 ppm) and the in the stems (438.96 ppm) (table 2). the pattern of pb concentration in response to the increasing pb exposure followed the trend of open downward polynomial curve of y = -626.79x2 + 1077.9x + 76.81 (r ² = 1, p <0.05) (figure 1). comparison of pb concentration in seedling tissues to that in media indicates the value of bioconcentration factor (bcf). bcf value of jabon was found to seedlings range from 0.5 to 2.9, and the biggest average of bcf was found in the leaves (0.6 4.6). the bcf value of jabon seedling exposed to 0.5 mm of pb was the s highest as compared to that of abon exposed to j 1.5 mm of pb or lower concentration (figure 2). scomparison of pb content in the stem and leaf tissue to that of root seedling indicates s the s the value of the transport factor (tf). the tf value of pb in jabon seedling range s was found to from to 0.4 1.63. the tf value of leaves was greater than that of stem. the tf value increased by pb exposure to 0.5 and 1.5 mm, with the highest tf value seedling exposed to observed in s 0.5 mm of pb (figure 3). in s was pb bioaccumulation jabon seedling -2 observed to from to range 1.73 36.55 x 10 mg/plant. pb accumulationthe highest was -2 recorded at to in roots 3.74 36.55 x 10 mg/plant. the seedling exposed to 0.5 mm of pb had the s highest bioaccumulation value ranging from 10.41 36.55 x 10 mg/plants, followed by the -2 to seedling exposed 1.5 mm of pb (table 3). the s highest value of bioaccumulation occurred in the root treated by 0.5 mm of pb that was almost 10 s times higher than control, i.e. 36.55 x that of the 10 mg/plant (table 3) -2 . 67 table 1 growth of jabon seedling exposured to pb on nutrient culture during 15 days pb (mm) root length (cm) dry biomass (gram) ti (%) pb 0 11.0 ± 1.0 b* 1.63 ± 0.34 b 100 bc pb 0.5 8.8 ± 0.2 a 1.28 ± 0.14 a 80 ab pb 1 8.3 ± 0.6 a 1.38 ± 0.33 a 75 a pb 1.5 8.5 ± 0.5 a 1.30 ± 0.13 a 77 ab * note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test table 2 pb concentrations on parts of jabon seedlings after 15-days exposure in nutrient culture pb exposure (mm) pb concentration per g dry weight (ppm) roots stem leaves pb 0 76.81 ± 18.60 b * 27.61 ± 15.98 a 58.38 ± 34.97 a pb 0.5 459.04 ± 94.76 d 438.96 ± 206.63 c 735.93 ± 205.78 d pb 1.5 283.32 ± 1.06 c 164.34 ± 24.44 ab 360.06 ± 56.35 c * note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. 68 biotropia vol. 25 no. 1, 2018 figure 2 bioconcentration factor of pb on roots, stem and leaves of jabon seedlings exposed to pb during 15 days (note: the dashed line showed the threshold of plant categorized as pb extractor (tangahu et al. 2011)) figure 3 transport factor of pb on stems, and leaves of jabon seedlings exposed to pb during 15 days figure 1 relation of pb concentration (▲) in root and dry biomass (ᴏ) of jabon seedling on pb exposure 69 jabon seedlings exposed to pb for 15 days in a nutrient culture led to an increase of pb concentration in seedling tissues. based on the bcf value which ranged between 0.4 4.6, and the pb exposure in the media culture led to the increase of pb concentration in the tissues of the seedlings recorded with the highest concentration at that of the 735 ppm or 12.6 times higher than control (table 2). the leaves of the seedlings exposed to 0.5 mm of pb were found to have the largest pb concentration followed by the roots and then stems, with the tf values was ranging from 0.9 1.59 (table 2, fig. 1 and 2). to this result showed that pb concentration in jabon seedlings was greater as compared to that of (390 ppm) , pinus radiata (javris & leung 2002) but lower than that in kenaf (4281 ppm) (mun . et al 2008), p. (1773 ppm) (setyaningsih . falcataria et al 2012) and (12.7 ppm) (monfared platanus orientalis et al. 2013). the data also showed that bcf value of seedlings exposed 1.5 mm of pb was lower than to that exposed pb 0.5 mm. the exposure of 1.5 to mm of pb inhibited the plants more and caused more damage than 0.5 mm , the exposure to of pb which resulted in the reduction of pb accumulation from media to the seedling tissues. another possibility was the leakage or efflux of pb and exit back to the media due to damaging effect of pb. concentration seedling roots pb in followed the polimonial pattern of open downwards, while seedling biomass (dry weight) followed the polimonial pattern of open up in response to the increased pb concentration (fig. 3). in other words, exposure 0.5 mm to pb caused the highest pb concentration in the seedling tissue and resulted in the lowest dry biomass. this suggests that the growth declining of jabon seedling is highly correlated with the levels of pb in the tissue, the higher level of pb caused decreasing of growth more indicated by lower dry biomass. such conditions was also reported by other investigator in c. comosum (wang . 2011).et al the higher tf value of seedling due to pb exposure as compared to control suggested that the transfer of pb into the shoot of seedling occurred efficiently. tf value of jabon seedlings was likely to exceed one (tf> 1) indicating that jabon seedling prefer to keep pb in the shootsed which could classify ajabon as metal accumulator (tangahu . 2011).et al pb accumulation in jabon roots was the largest which was almost 3 times of that in the stem (2.82 – 10.41 x 10 mg/plant) and 2 times of that in the -2 leaves (1.73 24.58 x 10 mg/plant). therefore, -2 the sequence of pb accumulation in jabon seedling was the roots > leaves >stem. this phenomenon has also been reported in some other plants such as kenaf which has been reported to accumulate pb up to 97% of the total bioaccumulation (mun . 2008) and the indian et al mustard which accumulated pb more than 95% (reisinger . 2008)et al . however, the pb accumulation capacity of jabon is still lower compared with some other species that have been characterized as pb accumulators. these . pb accumulators include brassica campestris l, brassica carinata a. br., brassica juncea (l.) czern. and brassica nigra (l.) koch which could accumulate -1 more than 100 mg pb g dry weight (tangahu et al. 2011). pb although the concentration in the tissue was relatively small, because this plant produced high biomass with high bcf and tf , the values total pb accumulation of jabon seedling was relatively high. based on the concept that remediation effectiveness depends on the amount of contaminant accumulated by plant biomass and/or by contaminant level in plant tissues table 3 pb bioaccumulation in jabon seedlings exposed to pb during 15 days in nutrient culture pb (mm) bioaccumulation (10-2 mg/plant) roots stem leaves pb 0 3.74 ± 1.06 a* 2.82 ± 0.11 a 1.73 ± 0.56 a pb 0.5 36.55 ± 8.90 c 10.41 ± 1.15 c 24.58 ± 0.76 c pb 1.5 18.12 ± 1.98 b 7.18 ± 1.27 b 8.98 ± 0.82 b * note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. 70 biotropia vol. 25 no. 1, 2018 (rodriguez . 2005) we conclude that jabon et al , may have potential function as pb remediator. the efficiency of metal translocation from roots to shoots is one of the key factors to distinguish whether the plant is categorized as metal hyper-accumulator or not. metal accumulation in the shoot are often limited by the binding of metal cations that is being exchanged in the cell walls of xylem (kim . 2009). et al however, the hyper-accumulator plant may have the ability to overcome this limitation by forming a negative charge or neutral formation of organicmetal complex. some aspects such as plant species, properties of medium, the root zone, vegetative uptake and addition of chelating agent are several factors which can affect the uptake mechanism of heavy metals (rodriguez et al. 2005; tangahu et al. 2011). formation of metal-organic acid complex also can be an important mechanism to increase the uptake by plants although free pb ions of metal are easier to be accessible. conclusions j a b o n s e e d l i n g s e x h i b i t e d t o l e r a n t characteristic at least up to 1.5 mm (450 ppm) of pb, based on some indicators of tolerant index. the pb was distributed to all part of the seedlings, with the highest concentration found in the leaves (735.9 ppm). however, because of higher biomass, the highest bioaccumulation was found -2 in the roots (36.55x10 mg per plant). jabon seedling can be classified as metal accumulator and may have great potential as remediator in the lead-contaminated areas. acknowledgements the author acknowledges the government of republic of indonesia through the 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lead setyaningsih et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 5. dwi retno (arbuscular).cdr biotropia vol. 20 no. 2, 2013: 112 121 arbuscular mycorrhizal fungi associated with ( )bisbul diospyros blancoi dwi retno ningsih , kartini kramadibrata , and agustin wydia gunawan received 15 september 2013/accepted 10 december 2013 ( ) is a kind of edible fruit and could be beneficial as inhibitor for skin ageing process. majority of root plants have symbiotic associations with arbuscular mycorrhizal fungi (amf); however, amf in has never been reported. the objective of this study was to observe amf colonies and to identify amf spores in bisbul tree rhizospheres and in potcultured . roots and soil samples from rhizospheres were collected from three locations in bogor area. roots were stained using trypan blue 0.05%. soil samples were air dried, then parts of them were used for spore isolation and the rest were used for pot cultures. spores were isolated by wet sieving and decanting method and then continued with centrifugation. the results showed that the structures of amf colonies in roots were arbuscules, vesicles, coiled hyphae, and internal hyphae. isolated amf spores were identified as , . and . this is the first report of mycorrhizal infection in the root of and amf association with root. am fungi, symbiosis, 1 2 1,3* 1 2 3 department of biology, faculty of mathemathics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia herbarium bogoriense, botany division, research center for biology lipi, jalan raya jakarta-bogor km 46, cibinong 16199, indonesia faculty of biotechnology, atma jaya catholic university of indonesia, jalan jendral sudirman 51, jakarta 12930, indonesia bisbul diospyros blancoi bisbul pueraria javanica bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, claroideoglomus geosporum, funneliformis etunicatum, gigaspora candida, g. ramisporophora, glomus albidum g glomerulatum, scutellospora calospora bisbul bisbul diospyros blancoi abstract introduction key words: at present, researches on the diversity of arbuscular mycorrhizal fungi (amf), which have mutualistic symbiotic associations with fruit trees, are starting to gain attention. one of the fruit plant considered to have economic value is . ( ) is a kind of red fruit indigenous to the philippines and was bisbul bisbul diospyros blancoi * corresponding author : agustinwydiagunawan@yahoo.com doi: 10.11598/btb.2013.20.2.2 112 introduced to bogor botanical garden in 1881. the wood from trees can be used as material for the roof and handicraft. the fruit contains vitamint beneficial to make the skin smooth, maintain healthy eyes, and prevent constipation (coronel 1992). symbioses of amf with fruit plants in indonesia have been reported in salak ( ) (retnaningsih 1998); persimmon ( ), mango ( ), papaya ( ), and soursop ( ) (septyarini 1999); durian ( ) (chairani 2002); rambutan ( ) (muliawan 2002); mangosteen ( ) (lucia 2005); banana ( ) and tomato ( ) (duaja & jasminarni 2008); and jambu-jambuan ( sp.) (setiadi & setiawan 2011). however, there has been no report on amf in tree yet. therefore, the first step to examine the symbiosis of amf in roots is to observe their amf colonies and to identify amf spores in rhizospheres as well as in pot cultures with as the host. the samples of roots and soil were collected from three locations in bogor, i.e. ipb darmaga campus, cibinong, and ciampea. three trees were selected from every location, and sampling was replicated two times for every tree. for each replication ± 500 g sample was taken from each position, which was 100 cm from the tree and at a depth of 15-20 cm. the samples were then mixed to make one composite sample. root samples were immediately cleaned and immersed in 70% alcohol solution, while soil samples were air-dried for spore isolation and pot cultures. amf colonies were observed using trypan blue stain (phillips & hayman 1970) with a modification in cell clearing with 10% koh for 120 min. the structures of amf colonies were observed in 20 randomly picked root cuts. the roots that showed the structures of arbuscules, vesicles, coiled hyphae, and internal hyphae indicated the occurrence of symbiosis between the roots and amf. these mycorrhizal roots were examined to calculate the percentage of am in roots using the following formula : pot cultures with as the host were prepared with planting media as follow: 50 g sterile zeolite, 100 g soil sample from rhizosphere, and then 50 g sterile zeolite as the cover layer. three pot cultures were prepared from each rhizosphere soil sample of every tree, so there were 18 pots. then, the plants bisbul salacca edulis diospyros kaki mangifera indica carica papaya annona muricata durio zibethinus et al. nephelium lappaceum et al. garcinia mangostana musa paradisiaca solanum lycopersicum syzygium bisbul bisbul bisbul pueraria javanica bisbul bisbul p. javanica bisbul bisbul material and methods sampling of roots and soil amf colonies in roots pot cultures bisbul number of fields-of-view containing mycorrhizae total observed fields-of-view × 100% 113 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. were maintained for three months in order to produce amf spores. each pot was watered daily with sterile water. fertilizer containing 5% p was used; the amount of water for each plant was 100 ml. fertilizing was done when the plants were two weeks old, and then repeated every week until they were three months old. after three months, the plants were left to dry for three weeks. . amf spores were isolated from 100 g rhizosphere soil sample for every tree, so the total amount of soil sample for every location was 300 g. likewise for the pot cultures, 100 g was isolated from every pot culture so there was 300 g soil sample for each tree, or 900 g for each location. amf spores were isolated employing wetsieving and decanting method and centrifugation (walker 1982). collected spores were preserved by mounting them on slides in as media. identification was conducted based on morphology, while the nomenclature followed schü & walker (2010). roots are pigmented, so it took up to 120 min to clear the cells using 10% koh in order to observe the internal structures of arbuscular mycorrhizae clearly. the structures of the observed roots were found to be arbuscules, vesicles, coiled hyphae, and internal hyphae (fig. 1) which indicated that the roots had symbiotic associations with amf. the average of colonization was 41% in roots collected from ipb darmaga campus, 49% from cibinong, and 64% from ciampea. bisbul bisbul et al. polyvinyl alcohol lacto glyserol bisbul bisbul bisbul bisbul spore isolation and identification amf colonies in roots βler results and discussion bisbul vesicles coiled hyphae coiled hyphae arbuscules figure 1. arbuscular mycorrhizal structures (arrow) in rootsbisbul . biotropia vol. 20 no. 2, 2013 114 the occurrence of the structures proved that roots had symbiotic association with amf, and that amf spores could be found in the soil of rhizospheres. its close relative, (septyarini 1999), (hawley and dames 2004), and (delvian 2010) were also reported to have symbiotic association with amf. the arbuscular mycorrhizae observed in roots were classified as arum-paris type. different number of spores were isolated from the soil of rhizospheres collected from three locations in bogor (table 1). amf spores collected from rhizosphere soil in ipb darmaga campus had the highest quantity as well as diversity, 17 isolated spores consisted of 7 amf spesies: and . spores in rhizospheres from cibinong were and 2 other species different from those found in darmaga, they were and amf spores found in rhizospheres in ciampea were and bisbul bisbul diospyros kaki d. nigrocartex (zhao et al. 2001), d. scabrida d. pendula bisbul bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, gigaspora ramisporophora, funneliformis etunicatum, claroideoglomus geosporum, glomus glomerulatum bisbul c. geosporum, g. glomerulatum, glomus albidum scutellospora calospora. bisbul acaulospora longula, a. scrobiculata, c. geosporum. amf spores table 1. total spores of arbuscular mycorrhizal fungi (amf) obtained from rhizospheres and pot cultures with as the host bisbul pueraria javanica amf total amf spores darmagaa cibinonga ciampeaa brb pcc brb pcc brb pcc acaulospora longula 1 13 0 16 4 16 acaulospora scrobiculata 4 8 0 16 1 7 acaulospora tuberculata 3 2 0 3 0 4 claroideoglomus geosporum 3 85 2 34 1 48 funneliformis etunicatum 2 3 0 2 0 9 glomus albidum 0 7 3 17 0 24 glomus glomerulatum 2 4 1 6 0 9 scutellospora calospora 0 2 1 6 0 2 total 15 124 7 105 6 123 when the soil from bisbul rhizospheres was used in pot cultures with as the host, the amf species observed in rhizospheres were also found in their pot cultures. this study produced a high quantity of amf spores, so it was possible to conduct the identification in order to confirm the identity of the amf obtained directly from bisbul rhizospheres, and also to trap amf propagules which spores could not be isolated directly from the soil of rhizospheres. p. javanica bisbul gigaspora candida bisbul notes: br: bisbul rhizospheres; pc: pot culture soil ph: darmaga (5.8-6.0), cibinong (5.8-6.0), and ciampea (6.2-6.6) amf spores collected per 300 g soil of br, amf spores collected from 300 g pc media, which was an average of 3 pot culture replications of each bisbul rhizosphere soil in 1 location. a b c 115 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. 1. spain & n.c. shenck the spores were globose to subglobose, yellow to brown in color, measured 90132 × 93-150 μm. spore wall was yellow to brown and without ornamentation. total wall thickness was < 6-9 μm. was successfully obtained from darmaga (ph 5.7-5.9) and ciampea (ph 6.2-6.6). chairani (2002) reported that this species was found in durian in bogor with ph 4.2-6.3 and fahriny (2013) in rhizospheres at ph 5.9. it seemed that this species could survive in a wide range of acidic ph. had similarities in color and form compared with those first described by schenck . (1984). the spore size in this study was bigger compared to that reported by schenck . (1984), (60-)70fahriny (2013), 126-14 × 96-117 μm. in this study, the sporiferous saccule of was not found. schenck . (1984) reported that sporiferous saccule was found at the terminal hyphae and after the spores matured, the sporiferous saccule would be left empty. specimens examined: drn 6, drn 18, drn 19, drn 20, drn 25, drn 26, drn 36, drn 38, drn 39, drn 47, drn 50, drn 51, drn 52, drn 68, drn 70, drn 76, drn 77, drn 80, drn 82, drn 97, drn 98, drn 103, drn 105, drn 109, drn 111, drn 112, drn 113, drn 114, drn 118, drn 119, drn 120, drn 121, drn 122, drn 123, drn 127, drn 132, drn 136, drn 139, drn 144, drn 146, drn 160, drn 171, drn 173, drn 175, drn 176, drn 178, drn 179, drn 182, drn 184, drn 187, drn 191, drn 195, drn 193, drn 194, drn 196 and drn 199. 2. trappe the spores were globose to subglobose, yellow in color, and measured 90-115 × 93-112 μm. spore surface had ornamentation of evenly spaced, linear elliptical crescent-like forms. spore wall was hyaline to yellow. total wall thickness was 6-9 μm. was obtained in darmaga soil at ph 5.7-5.9 and ciampea (ph 6.2-6.6). this species had been reported by chairani (2002) from durian rhizospheres in bogor at ph 4.2-6.5 and fahriny (2013) from rhizospheres at ph 6.7. had the same color, form, and ornamentation as those described by trappe (1977). this spore size was smaller compared with that reported by trappe (1977), 100-240 × 100-220 μm; widiastuti and kramadibrata (1992) 100-200 × 100200 μm; septyarini (1999) 105.6-211.2 × 96-220.8 μm; as well as haerida and kramadibrata (2002) 108.9-144 × 108.9-144 μm; kramadibrata (2009) 90-(130)-250 × 100-(120)-250 μm. however, it was bigger compared to that reported by lucia (2005), i.e. 82-125 × . specimens examined: drn 3, drn 10, drn 20, drn 29, drn 30, drn 33, drn 34, drn 35, drn 37, drn 41, drn 43, drn 44, drn 45, drn 56, drn 65, drn 72, drn 85, drn 99, drn 111, drn 118, drn 119, drn 124, drn 125, drn 127, drn 130, drn 131, drn 132, drn 136, drn 137, drn 140, drn 142, drn 143, drn 144, drn 146, drn 148, drn 149, drn 150, drn 152, drn 156, drn 157, drn 158, drn 159, drn 167, drn 176, drn 178, drn 181and drn 182. acaulospora longula acaulospora longula et al. areca a. longula et al et al a. longula et al acaulospora scrobiculata acaulospora scrobiculata et al. areca a. scrobiculata 90(-110) μm, and 67-120 μm 116 biotropia vol. 20 no. 2, 2013 3. janos & trappe. the spores were globose to subglobose, yellow to brown in color, measured 114272 × 111-272 μm. spore surface had ornamentation of smooth beads, dense, and uniform. spore wall was yellow to brown. total wall thickness was 9-12 μm. was successfully obtained from darmaga (ph 5.7-5.9). chairani (2002) reported the presence of this species in durian in bogor with ph range of 4.2-6.5 and it had the same form, color, and ornamentation as those described by janos and trappe (1982). the spore size was relatively smaller compared to that reported by janos and trappe (1982), i.e. 255-327 × 255-340 μm. likewise, spores reported by septyarini (1999), chairani (2002), lucia (2005) and kramadibrata (2009) were smaller than that of janos and trappe (1982). specimens examined: drn 2, drn 44, drn 55, drn 70, drn 80, drn 101, drn 103, drn 122, drn 124, drn 126, drn 145, drn 150, drn 155, drn 177, drn 180, drn 186, drn 190, drn 192, drn 193 and drn 199. 4. (w.n. becker & gerd.) c. walker & a. schüßler the spores were globose to subglobose, yellow to brown in color, and measured 81-108 × 81-111 μm. spore wall was yellow to brown. total wall thickness was 3-9 μm. (syn./formerly: ) was observed in darmaga at ph range of 5.7-5.9. chairani (2002) was successful in obtaining this species at ph range of 4.2-5.0. this species had the same color and form as those described by becker dan gerdemann (1977). the spore size in this study was relatively the same or bigger compared to that reported by becker dan gerdemann (1977) which was 68144( × kramadibrata . (2007) 48-77(-156) × 48-77(-156) μm and kramadibrata (2009) 150 × 150 . specimens examined: drn 8, drn 10, drn 15, drn 46, drn 56, drn 77, drn 86, drn 87, drn 97, drn 103, drn 107, drn 121, drn 123, drn 153, drn 159, drn 169, drn 170, drn 175, drn 176, drn 178, drn 179, drn 181, drn 184, drn 187, drn 190, drn 192, drn 193, drn 194 and drn 197. 5. (t.h. nicolson & gerd.) c. walker & a. schüßler the spores were globose to ellipsoid, brown to red in color, and measured 75-158 × 72-132 μm. spore wall was brown. total wall thickness was 6-15 μm. was found in all study area, at ph range of 5.7-6.6. these spores had the same color and form as those described by walker (1982). the spore size in this study was smaller than what was reported by walker (1982), which measured 110-190 μm (syn./formerly ). however, the spore size was relatively the same or bigger than lucia (2005) which measured 99-124 × and smaller than kramadibrata (2009) which measured 100 × 280 . specimens examined: drn 4, drn 9, drn 11, drn 12, drn 17, drn 22, drn 23, drn 24, drn 25, drn 26, drn 27, drn 28, drn 29, drn 30, drn 31, drn 32, drn 33, drn 34, drn 35, drn 36, drn 37, drn 38, drn 39, drn 40, drn 42, drn 43, drn 47, drn 48, drn 49, drn 52, drn 53, drn 54, drn 55, drn 56, drn 57, drn 59, drn 60, drn 61, drn 62, drn 63, drn 64, drn 66, drn 67, drn 69, drn 71, drn 72, drn 73, drn 74, drn 75, drn 76, drn 79, drn 81, drn 82, drn 83, drn 86, drn 87, drn 88, acaulospora tuberculata acaulospora tuberculata et al. a. tuberculata et al. claroideoglomus etunicatum claroideoglomus etunicatum glomus etunicatum et al. et al funneliformis geosporum funneliformis geosporum glomus geosporum -162) μm, lucia (2005) 47-107 43-99 μm, μm 92-107 μm μm 117 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. drn 89, drn 90, drn 91, drn 92, drn 93, drn 94, drn 95, drn 96, drn 97, drn 100, drn 101, drn 103, drn 104, drn 106, drn 107, drn 108, drn 109, drn 110, drn 113, drn 114, drn 115, drn 116, drn 122, drn 123, drn 125, drn 128, drn 130, drn 131, drn 132, drn 133, drn 136, drn 137, drn 138, drn 139, drn 140, drn 141, drn 149, drn 150, drn 151, drn 152, drn 153, drn 155, drn 157, drn 159, drn 161, drn 162, drn 165, drn 166, drn 168, drn 169, drn 172, drn 174, drn 175, drn 174 , drn 178, drn 179, drn 180, drn 183, drn 184, drn 187, drn 188, drn 190, drn 191, drn 192, drn 193, drn 194, drn 195, drn 196, drn 197, drn 198, drn 199 and drn 200. 6. bhattacharjee, murkeji, j.p.tewari & skoropad the spores were globose to subglobose, greenish white in color, and 90-225 × 90228 μm in size. spore wall was white. total wall thickness 3-9 μm. bulbous suspensor was white to yellow and 27-42 × 21-39 μm in size. had the same color and form as those described by bhattacharjee and mukerji (1982). the obtained spores were 90-225 × 90-228 μm in size, this is smaller compared to that described by bhattacharjee and mukerji (1982), i.e. 200-300 μm. the suspensor was also smaller in size, compared to that reported by bhattacharjee and mukerji (1982), i.e. 30-50 μm. specimens examined: drn 150, drn 155, drn 170 and drn 171. 7. spain, sieverd. & n.c. schenck the spores were globose to subglobose, brown to reddish brown in color, and 150372 × 165-372 μm in size. spore wall was brown. total wall thickness 6-9 μm. bulbous suspensor was yellow to brown and 27-36 × 21-39 μm in size. only 2 spores of were found in darmaga at soil these spores had the same color and form as those described spain (1989). the spore size was 150-372 × 165-372 μm, still in the same range as that described by spain (1989), i.e. (143-)150-400 × 200-450(-501) × 288-595 μm, and lucia (2005) 182-317 × (1989) sized (32-)40-60(72) × . specimens examined: drn 1, drn 2, drn 126, drn 128, drn 132, drn 133, drn 136, drn 138, drn 139, drn 144, drn 148, drn 149, drn 170, drn 181and drn 185. 8. c. walker & l.h. rhodes the spores were globose to subglobose, hyaline to yellowish white in color, and 69120 × 66-135 μm in size. spore wall was hyaline to yellow. total wall thickness was < 39 μm. was found in cibinong soil with ph range of 5.9-6.0. fahriny (2013) reported that this species associated with at ph 6.9. had the same color and form as those described by walker and rhodes (1981). in this study, the spore size was smaller compared to that reported by walker and rhodes (1981) which measured (85-) 95-168(-198) × (85-)95-168(-177) μm and fahriny (2013) which measured 48-144 × 66-171 μm and kramadibrata (2009) which measured 85-160 × 85-160 μm. gigaspora candida gigaspora candida gigaspora ramisporophora gigaspora ramisporophora ph 5.7-5.9. by et al. et al. et al. glomus albidum glomus albidum areca g. albidum μm, septyarini (1999) 230-576 172-317 μm. the obtained suspensor relatively smaller than that reported by spain (50-) 60-83 μm 118 biotropia vol. 20 no. 2, 2013 glomus glomerulatum glomus glomerulatum scutellospora calospora s. calospora et al bisbul a. scrobiculata a. tuberculata, g. ramisporophora specimens examined: drn 13, drn 14, drn 29, drn 41, drn 43, drn 44, drn 45, drn 58, drn 61, drn 62, drn 84, drn 91, drn 99, drn 101, drn 111, drn 117, drn 118, drn 119, drn 121, drn 123, drn 125, drn 126, drn 127, drn 128, drn 129, drn 135, drn 136, drn 137, drn 141, drn 143, drn 144, drn 145, drn 146, drn 148, drn 150, drn 152, drn 153, drn 154, drn 155, drn 156, drn 158, drn 159, drn 160, drn 161, drn 163, drn 164, drn 166, drn 168, drn 169, drn 171, drn 175, drn 176, drn 177, drn 179, drn 190, drn 192, drn 193, drn 196, drn 197 and drn 199. 9. sieverd the spores were globose to subglobose, yellow to brown in color, and 70-117 × 70123 μm in size. spore wall was yellow to brown. total wall thickness was 6-9 μm. was successfully obtained from darmaga (ph 5.7-5.9) and cibinong (ph 5.9-6.0). the specimens had the same color and form as those described by sieverding (1987). the size of these spores was bigger compared to that reported by sieverding (1987) which measured 40-70 μm, while in this study the spores were 70-117 × 70-123 μm in size. specimens examined: drn 7, drn 9, drn 46, 52, drn 61, drn 73, drn 77, drn 97, drn 98, drn 99, drn 103, drn 105, drn 107, drn 114, drn 117, drn 118, drn 121, drn 122, drn 123, drn 136, drn 139, drn 140, drn 163, drn 164, drn 170, drn 175, drn 176, drn 177, drn 179, drn 180, drn 181, drn 186, drn 190, drn 194 and drn 196. 10. (t.h. nicolson & gerd.) c. walker & f.e. sanders the spore was globose to subglobose, greenish yellow to brown in color, and 135234 × 141-210 μm in size. spore wall was hyaline to yellow. total wall thickness was 618 μm. bulbous suspensor was hyaline to brown and 27-45 × 24-39 μm in size. the quantity of amf spores was the lowest, only 1 spore was found from cibinong at ph (5.9-6.0). it had the same color and form as those described by koske and walker (1986). the spore size was same range as that described by koske and walker (1986), which was 114-285(-511) × 110-412(-511) μm, but this spore was bigger than that reported by kramadibrata . (2007), which measured 86-134 × , kramadibrata (2009) 150 × 150 um (globose) and 100-160 × 165-250 μm (oblong). the suspensor was 27-45 × 24-39 μm specimens examined: drn 16, drn 83, drn 84, drn 85, drn 119, drn 126, drn 133, drn 134, drn 138 , drn 139, drn 144, drn 146, drn 159, drn 164, drn 185 and drn 192. ten species of amf spores were found in the rhizosphere of in bogor, also eleven species of amf were found in persimmon rhizosphere in cibinong (septyarini 1999), but only 3 same species of amf were recorded from both plants i.e. , and . the quantity and diversity of amf species obtained from every location were different from each other. it could be influenced by different locations and soil types. ipb darmaga campus and cibinong soil belong to ultisol (dominated by clay), while in 86134 μm in size, relatively smaller than what was reported by koske and walker (1986), which measured 33-48 μm. 119 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. ciampea is alfisol (dominated by fine clay) (soil survey staff 2010). widiastuti and kramadibarata (1992) suspected that the soil dominated by clay fraction was suitable for the development and growth of spp. spores, while koske (1987) reported that the spores from genera and were found in large quantity in sandy soil. amf species diversity can also be influenced by the soil ph which was different in each location (moreira-souza 2003). was the dominant genus in rhizospheres and pot cultures. carvalho (2001) reported that this species (formerly . ) was the dominant fungi in saline soil. furthermore, gai (2006) also reported that the spread of (including syn. and ) was wider in tropical and subtropical regions compared to other genera. this study is the first report on mycorrhiza and it showed that amf was associated with trees in the form of arum-paris type. the spores found in tree rhizospheres were identified as , . and . glomus gigaspora scutellospora et al. funneliformis geosporum bisbul et al. g geosporum et al. glomus claroideoglomus funneliformis bisbul bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, claroideoglomus geosporum, funneliformis etunicatum, gigaspora candida, g. ramisporophora, glomus albidum g glomerulatum, scutellospora calospora conclusions references becker wn, gedermann jw. 1997. sp. nov. . 6:29-32. bhattacharjee m, mukerji kg. 1982. structure and hyperparasitism of a new species of . . 78(1):184-188. carvalho lm, cacador i, loucao mam. 2001. temporal and spatial variation of arbuscular mycorrhizas in salt marsh plants of the tagus estuary (portugal). 11:303-309. doi:10.1007/s00572-001-0137-6. chairani, gunawan aw, kramadibrata k. 2002. mikoriza durian di bogor dan sekitarnya. 7(2):44-46. coronel re. 1992. edible fruits and nuts. in plant resources of south-east asia 2. edited by verheij ewm, coronel re bogor (id): prosea foundation. p 151-152. delvian. 2010. keberadaan cendawan mikoriza arbuskula di hutan pantai berdasarkan gradien salinitas. j ilmu dasar. 11(2):133-142. duaja md, jasminarni. 2008. isolasi dan karakterisasi cendawan mikoriza arbuskular di rhizosfer beberapa jenis tanaman di kebun percobaan fakultas pertanian, universitas jambi. . 12(2):34-38. fahriny ra. 2013. ragam cendawan mikoriza arbuskula pada di kebun raya bogor. 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arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. biotropia book juni revisi 14 juli 09.indd 21 viability and plasma membrane integrity of the spotted buffalo epididymal spermatozoa after thawing with the addition of dextrose into the extender yulnawati 1, h. maheshwari 2, herdis 3, m. rizal4 1* rc. biotechnology, lipi, jl. raya bogor km. 46, cibinong, 16911, 2 dept of anatomy, physiology and pharmacology, faculty of veterinary medicine ipb, jl. agatis ipb campus, darmaga, bogor, 16680 3 bppt , jl. mh. th amrin kav. 8, jakarta 4dept. of animal husbandary, faculty of agriculture, pattimura university, jl. ir. m. putuhena, kampus pokka, ambon abstract th e objective of this study was to obtain the viability and plasma membrane integrity of the spotted buff alo epididymal sperm after addition of dextrose into andromed® extender. spermatozoa that have been collected from cauda epididymis were diluted with andromed® extender as control (k) and andromed® + 0.2% dextrose (p1) and andromed® + 0.4% dextrose (p2) as treatments. th e results showed that the quality of epididymal spermatozoa decreased during cryopreservation process. th e percentage of motility after thawing in p1 (46%) and p2 (46.67%) were signifi cantly higher (p<0.05) compared to k (41%) as well as the percentage of live sperm in p1 (58.8%) and p2 (60%) compared to k (52.2%). th e percentage of membrane integrity in p1, p2 and k were 67.4; 66.8 and 68 %, respectively. in conclusion, the addition of 0.2 and 0.4% of dextrose into andromed® acted as an extra cellular cryoprotectant and could maintain the viability and membrane integrity of the spotted buff alo epididymal spermatozoa after thawing. key words: epididymal sperm, cryopreservation, dextrose, spotted buffalo introduction indonesia is worldwide known to have high biodiversity that need to be considered for the conservation of the flora and fauna specific to this country. spotted buffalo is one of the indonesian biodiversity that need a particular attention as its population tends to decrease every year. this animal is thought to bring fortune and is usually used for the veneration in traditional ceremony in tana toraja, south sulawesi. according to the local people believe, spotted buffalo can only survive in its natural habitat, which is in tana toraja. the farmer put a special treatment for the spotted buffalo; particularly the male is maintained separated from the female biotropia vol. 16 no. 1, 2009: 21 27 corresponding author : yulnawati@yahoo.com 22 to prevent it from reproduction activities. this condition is, therefore, thought to make the conservation effort of this animal become more difficult. one of the alternative attempts to prevent this animal from extinction is to utilize the gonad, which are the testis and the epididymis of the male that is sacrificed in the traditional ceremony. the epididymis can be used as a source of potential spermatozoa that can conserve the genetic material. the fertility of the spermatozoa taken from the cauda epididymis is more or less the same as the spermatozoa from the ejaculate (hafez and hafez 2000). collection of the epididymal spermatozoa has been done in various species such as primate (feradis et al. 2001), sheep (rizal 2006), cattle (graham 1994), cat (tsutsui et al. 2003, yulnawati and setiadi 2005), pig (kikuchi et al. 1998), rhinocerous (lubbe 1999), equine (squires et al. 2000; papa et. al. 2008), deer (soler et al. 2003), dog (setiadi et al. 2007, hori et al. 2004) and african buffalo (herold et al. 2004, herold et al. 2006). spermatozoa collected from cauda epididymis is then stored in the form of liquid and/or frozen sperm. furthermore, epididymal sperm could be used for assisted reproductive technology such as artificial insemination (ai), in vitro embryo production (ivep) or intra cytoplasmic sperm injection (icsi). in order to maintain the quality of the spermatozoa during storage and after thawing for further process, the extender with a certain composition is needed. during the freezing process, the important material in the extender is the cryoprotectant that can protect sperm from coldshock. cryoprotectant can directly prevent the formation of ice crystal in the cell, as well as working extracellularly as a membrane protector. both the intracellular and extracellular cryoprotectants can be added at the same time in a certain ratio into the extender. sugar is used in the research as one of the extracellular cryoprotectant that should be added to the glycerol-as intracellular cryoprotectant-contained andromed® extender. it is hoped that the addition of dextrose sugar can maintain the viability dan integrity of plasma membrane of the spotted buffalo’s epididymal spermatozoa after thawing. materials and methods cauda epididymis of the spotted buffalo was collected during the rambu solo’ funeral ceremony in the pangli village, sesean, north toraja. the cauda epididymis was flushed and stored in 0.9% of nacl before the collection of spermatozoa. epididymal spermatozoa was collected using combination technique, slicing/ flushing and pressing all parts of the cauda’s tissues (rizal et al. 2004) using andromed® solution as an extender. evaluation of the quality of fresh spermatozoa observed was percentage of progresive motility, percentage of live spermatozoa, concentration, percentage of abnormality and percentage of plasma membrane integrity (mpu). collected spermatozoa was centrifuged at 500 g for 20 min. at room temperature. the supernatant was discharged and the pellet that contained spermatozoa was re-diluted with andromed® (minitub, germany) extender. the volume of the extender used is based on the concentration previously calculated. basic extender used in this research was andromed®. andromed® is a commercial extender that contains glycerol as an intracellular cryoprotectant. andromed® without biotropia vol. 16 no. 1, 2009 23 any addition of substance was grouped as control, whereas the addition of 0.2 and 0.4% w/v of dextrose (merck, germany) into andromed® were grouped as treatments. the diluted epididymal spermatozoa were placed in 0.25 ml of mini straw with 60 million of motile spermatozoa per straw and equilibrated in the refrigerator at 5°c for 3 hours. freezing of the epididymal spermatozoa was started by arranging the straw 10 cm above the surface of the liquid nitrogen with the temperature of -130°c for 15 min. afterwards, the straw was plugged into the liquid nitrogen with the temperature of -196°c and stored in the container. later, each frozen epididymal spermatozoa sample was thawed for quality evaluation. thawing process was carried out by placing the straw into the water bath at 37oc for 30 seconds. the percentage of sperm motility, live sperm, and plasma membrane integrity of post diluted, equilibrated and thawed epididymal spermatozoa were recorded. the percentage of progresive motility (move forward) of the sperm was counted subjectively at eight different visual fields, using light microscope with the magnitude of 400x. the percentage of live sperm was counted using eosin staining. live spermatozoa were characterized by white colour of the head, whereas the dead sperm had red head (toelihere 1993). plasma membrane integrity (mpu) was characterized by circular tail or inflated, whereas the broken one was characterized by by straight tail when the sperm was exposed to the hypoosmotic solution and incubated at 37oc for 60 min (revell and mrode 1994). for each parameter observed, a sum of minimum 200 sperma was evaluated using light microscope with the magnitude of 400x. data obtained were analyzed using random analysis of variance (anova) with three treatments and five replications. difference between treatments was tested using duncan multiple range test (steel and torrie 1993). results and discussions the quality of fresh epididymal spermatozoa of the spotted buffalo obtained from this research is shown in table 1. the concentration of spermatozoa collected from the cauda epididymis using flushing technique and pressure, was 10445x106 spermatozoa/ml. theoretically, this was reliable because cauda epididymis functions as a spermatozoa storage before ejaculation and in the cauda and the spermatozoa has not have seminal plasma addition (garner and hafez 2000). the sperm concentration in bull cauda epididymal was 3593 4406,7 x 106 sperm/ml (suhendra 2002) and 6,26 x 109 sperm/ml in buffalo cauda epididymal tissue (toelihere 1993). table 1. the avarage quality of fresh spotted buffalo epididymal spermatozoa parameters mean ± st.dev concentration (x 106 sperma/ml) 10445 ± 43.62 volume (ml) 0.45 progressive motility (%) 65.0 ± 0.00 live (%) 79.3 ± 1.30 abnormality (%) 15.0 ± 2.24 plasma membrane integrity (%) 80.8 ± 0.43 epididymal spermatozoa of the spotted buff alo – yulnawati et al. 24 the percentage of the epididymal spermatozoa motility obtained from the research was 65 % and this value is still in the range of the requirements for using it in the artificial insemination (ai) application. the percentage of the abnormality of this epididymal spermatozoa was also high (15 %), however this value is still in the normal range and reliable to be used for fertilization process, since the normal fertile semen contains not more than 20 % abnormal spermatozoa (ax et al. 2000). the percentage of abnormal morphology of 8-10 % did not give any significant effect to the fertility, however, if the abnormality form one ejaculate which is more than 25 %, the decrease of the fertility could not be anticipated (bearden and fuquay 1997). generally, after undergoing three steps of freezing process, the research indicated that the percentage of the motility, viability and plasma membrane integrity of the treatment groups were better compared to the control group. the percentage of motility after thawing in the control group, dextrose 0.2 and 0.4 %, were 41, 46 and 46.67 %, respectively. there was a significant difference (p<0.05) between treatment groups which had dextrose addition and the control group. on the other hand, there was no significant difference (p>0.05) between treatment groups and the control group of the percentage of plasma membrane integrity (table 2). it could be concluded that the value of post thawing epididymal sperm still met the requirements for ai, ivf or icsi application. table 2. the mean (±stdev) percentage of motility and viability of epididymal spermatozoa of spotted buffalo at different stages of freezing processes frozen stage parameters control dextrose 0,2% dextrose 0,4% dilution % m 65,00 ± 0,00a 65,00 ± 0,00a 65,00 ± 0,00a % live 76,00 ± 2,83a 82,00 ± 1,41b 82,67 ± 1,25b % mpu 78,67 ± 0,47a 77,33 ± 2,87a 80,00 ± 0,82a equilibration % m 50,00 ± 0,00a 56,67 ± 4,71a 56,67 ± 4,71a % live 70,33 ± 0,47a 73,00 ± 1,89a 72,67 ± 1,25a % mpu 72,00 ± 0,82a 71,33 ± 0,94a 73,00 ± 0,82a th awing % m 41,00 ± 2,00a 46,00 ± 2,00b 46,67 ± 2,36b % live 52,20 ± 2,48a 58,80 ± 1,83b 60,00 ± 0,82b % mpu 68,00 ± 1,10a 67,40 ± 1,36a 66,80 ± 1,47a note: m: motility, mpu: plasma membrane integrity, a,b different superscript in the different column showed significantly different (p<0,05). dextrose is produced by the conversion enzyme found in cornstarch and undergoes demineralization ion exchange process. dextrose is the form of d-glucose that can be metabolized by the cell through biochemical process called glycolysis to be an energy source. in general, the addition of the sugar into the spermatozoa extender can affect the motility, viability and plasma membrane integrity of the spermatozoa. the addition of sugar into the extender can diminish the damage of the spermatozoa acrosome (yildiz et al. 2000). sugar is known as an extracellular cryoprotectant. the mechanism of how dextrose works in this research is assumed as a protector for the plasma membrane rather than to prevent the formation of the ice crystal in the cell. biotropia vol. 16 no. 1, 2009 25 whereas, glycerol already contained in the andromed®, play a role as intracellular cryoprotectant,to prevent the formation of ice crystal during freezing process. there are no reports of experiments on the epididymal sperm of spotted buffalo freezing and thawing process. therefore, none of the publications reported on the use of sugar that was added into the basic extenders in spotted buffalo sperm cryopreservation process. for the references, the use of sugar as cryoprotectant in different animal species were reported, such as sheep (aisen et al. 2002; molinia et al. 1994), dog (yildiz et al. 2000; rigau et al. 2001), pig (de los reyes 2000) and bull (woelders et al. 1997). generally, sugars play a role as the capacity agent of cryoprotectant and works as the spermatozoa plasma membrane protector during freezing process. it was also known that the sugars have dehydration activity and be able to interact with the cell membrane (aisen et al. 2000). conclusions in conclusion, recovered spermatozoa from cauda epididymis of spotted buffalos that undergo freezing processes using andromed with the addition of 0.2 or 0.4% dextrose are suitable for ai. acknowledgments this research was finacially supported by dipa biotrop 2008, no: 047.1/ psrp-sp/iii/2008. special thank goes to the head of animal division in north toraja, dr. yulius and mr. slamet sumitro for supplying the cauda epididymis samples and using the laboratory facilities. references aisen e.g., alvarez h.l., venturino a. and j.j. garde. 2000. effect of trehalose and edta oncryoprotective action of ram semen diluents. theriogenology, 53(5): 1053-1061. aisen e.g., medina v.h. and a. venturino. 2002. cryopreservation and post-thawed fertility of ram frozen semen in different trehalose concentrations. theriogenology, 57:1801-1808. ax r.l., dally m., didion b.a., lenz r.w., love c.c., varner d.d., hafez b. and m.e. bellin. 2000. semen evaluation. in: hafez b and hafez ese. reproduction in farm animals.7th ed. philadelphia: lea and febiger. p. 365-375 axner e., sormholst b. and c. linde-forsberg. 1998. morphology of spermatozoa in the cauda epididymis before and after electroejaculation and comparison with ejaculated spermatozoa in the domestic cat. theriogenology, 50 (6): 973-979. epididymal spermatozoa of the spotted buff alo – yulnawati et al. 26 bearden h.j. and j.w. fuquay. 1997. applied animal reproduction. 4thed. new jersey: prentice hall, upper saddle, p. 133-177. de los reyes m., saenz l., lapiere l., crosby j., and c. barros. 2000. in vitro evaluation of boar spermatozoa frozen with permeable and non permeable cryoprotectant. proceeding 14th international congress on animal reproduction. stockholm 2-6 july 2000. p. 161. abstract vol. 2. feradis a.h., pawitri d., suatha i.k., amin m.r., yusuf t.l., sajuthi d., budiarsa i.n. and e.s.hayes. 2001. cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (macaca fascicularis). j. med. primatol., 30: 100-106. garner d.l. and e.s.e. hafez. 2000. spermatozoa and seminal plasma. in: hafez b and hafez ese. reproduction in farm animals. 7th ed. philadelphia: lea and febiger, p. 96-109 graham j.k. 1994. effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during cryoprservation process. theriogenology, 46: 1151-1162. hafez e.s.e. and b. hafez. 2000. reproduction in farm animals. 7th ed. baltimore: lippincott williams & wilkins. herold f.c., aurich j.e. and d. gerber. 2004. epididymal sperm from the african buffalo (syncerus caffer) can be frozen successfully with andromed® and triladyl™ but the addition of bovine seminal plasma is detrimental. theriogenology, 61: 715-724. herold f.c., de haas k., colenbrander b. and d. gerber. 2006. comparison of equilibration times when freezing epididymal sperm from african buffalo (syncerus caffer) using triladyl™ or andromed®. theriogenology, 66: 1123-1130. hori t., ichikawa m., kawakami e. and t. tsutsui. 2004. artificial insemination with frozen epididymal sperm beagle dogs. j. vet. med. sci. ; 66(1): 37-41. kikuchi k., nagai t., kashiwazaki n., ikeda h., noguchi j., shimada a., soloy e. and h. kaneko. 1998. cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4°c. theriogenology, 50:615-623. lubbe k., smith r.l., bartels p. and r.a. godke. 1999. freezing epididymal sperm from white rhinoceros (ceratotherium simum) treated with different diluents. theriogenology, 51:288. abstract. molinia f.c., evans g., quintana casares p.i. and w.m.c. maxwell. 1994. effect of monosaccharidaes and dissacharides in tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. anim. reprod. sci., 36(1-2): 113-122. papa f. o., melo c. m., fioratti e. g., dell’aqua jr. j. a., zahn f. s. and m. a. alvarenga. 2008. freezing of stallion epididymal sperm. anim. reprod sci., 107(3-4): 293-301. revell s.g. and r.a. mrode. 1994. an osmotic resistance test for bovine semen. anim. reprod. sci. , 36:77-86. rigau t., farre m., ballester j., mogas t., pena a. and j.e. rodriquez-gil. 2001. effect of glucose and fructose on motility patterns of dog spermatozoa from fresh ejaculate. theriogenology, 56(5): 801-815. rizal m. 2006. the fertility of frozen-thawed ejaculate vs epididiymal garut ram sperm. veterinary sains j., 24: 49-57. biotropia vol. 16 no. 1, 2009 27 rizal m., herdis and a. boediono . 2004. viability of ram sperm epididymal after storage at 5°c. j. anim. prod. , 6(1): 30-36. setiadi m.a., yulnawati and a. suprayogi. 2007. the quality of dog epididymal sperm during storage at 4°c. jitv., 12 (2): 134-138. soler a.j., perez-gusman m.d. and j.j. garde. 2003. storage of red deer epididymides for four days at 5oc: effects on sperm motility, viability, and morphology integrity. j. exp. zool., 295a: 188-199. steel r.g.d. and j.h. torrie. 1993. principles and procedures of statistic. london: mc graw-hill book co. inc. pub. ltd. squires e.l., gomez-cuetara c. and j.k. graham. 2000. effect of seminal plasma on cryopreserving epididymal and ejaculated stallion spermatozoa. proceedings 14th international congress on animal reproduction. stockholm, 2-6 july 2000; 2, p.166. suhendra. 2002. the study about some parameters of bull sperm quality in different part of epididymis. bachelor thesis. bogor: faculty of veterinary medicine, bogor agricultural university. toelihere m.r. 1993. artificial insemination in animals. bandung: angkasa. tsutsui t., wada m., anzai m. and t. hori. 2003. artificial insemination with frozen epididymal sperm in cats. j. vet. med. sci. , 65 (3): 397-399. woelders h., matthij a. and b. engel. 1997. effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. cryobiology, 35:93-105. yildiz c., kaya a., aksoy m. and t. tekeli. 2000. influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. theriogenology, 54(4): 579-585. yulnawati and m.a. setiadi . 2005. motility and membrane integrity of cat epididymal sperm during storage at 4°c. med. vet j., 21(3): 100-104. epididymal spermatozoa of the spotted buff alo – yulnawati et al. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html sri-13 mei 2017-507 (anjana evaluation).cdr evaluation of cytotoxicity of the aqueous leaf extract of benth.pogostemon heyneanus (java patchouli) anjana sheela* and john ernest thoppil cell and molecular biology division, department of botany, university of calicut, malappuram 673635, kerala, india received 4 july 2015/accepted 27 january 2017 abstract several plants have been recently reported to possess anticancer potential. allium cepa root tip assay is a preliminary study to assess the cytotoxic effect of plant extracts. the cytotoxic activity of plants can be correlated to their anticancer potential. cytotoxic potential of pogostemon heyneanus (lamiaceae) was evaluated using a. cepa root meristematic cells. this study was aimed at analyzing cytotoxic potential of p. heyneanus using allium cepa root tip assay. four different concentrations of the aqueous leaf extracts at three different durations were examined. distilled water was used as control. the extract was found to be cytotoxic at all tested concentrations, when compared to control. mitotic index was found to be decreasing with the increase in extract concentrations and treatment durations. the aqueous extract of p. heyneanus was found to be an effective cytotoxic agent, inducing various clastogenic and nonclastogenic aberrations such as chromosome gaps, bridges, multipolar anaphase, fragments, nuclear budding and lesions, hyperchromasia, laggards and mitotic pairing. keywords: allium cepa assay, anti-mitotic, chromosomal aberrations, cytotoxicity, pogostemon heyneanus introduction recent years have witnessed the importance of plant-derived herbal medicines against many dreadful diseases. the cytotoxic potential of plant extracts can be tested preliminarily for their anticancer activity using assay. allium cepa p. heyneanus, commonly called as java patchouli, is an aromatic species of lamiaceae family. the plant is used to make the pungent, dark amber colored patchouli oil, containing patchoulene, patchouli alcohol and eugenol as the major components. the oil possesses antibacterial activity against , , escherichia coli staphylococcus aureus streptococcus pyogenes bacterium coli bacterium typhosum, , and . it is also used in mycobacterium tuberculosis insect-repellent preparations. essential oil extracted from the leaf was reported to possess remarkable larvicidal potential against aedes albopictus (anjana & thoppil 2013). another species of the genus, has been reported to p. cablin, be having anticancer properties (jeong . 2013).et al a wide variety of secondary metabolites obtained from medicinal plants possesses the ability to treat cancer. they exhibit cytotoxic effect by interfering with cell-cycle kinetics (galimuhtasib & bakkar 2002). these medications are effective against proliferating cells. these medications produce cytotoxic effect either by damaging the cell dna during the s-phase of the cell cycle or by blocking the formation of mitotic spindle (sehgal . 2006). plant et al cytotoxic bioassays have a good correlation with mammalian test systems, validating the application of plant-based medications for genotoxic assays (jovtchev . 2002; yi & meng et al 2003; çelik & äslanturk 2006, 2007). toxicological tests using was found to allium cepa have high correlation with other test systems (mit-217 cell test with mice, rats or humans in vivo) and could be used in toxicological research (lubini . 2008).et al there are no previous reports on the cytotoxic activities of and therefore, the present p. heyneanus study was aimed at evaluating the cytotoxic and antimitotic properties of p. heyneanus. biotropia 4 1 7 28 34 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 507 * corresponding author: anjana.s586@gmail.com 28 materials and methods collection of plant materials plant materials of p. heyneanus was obtained from the centre of medicinal plants research, arya vaidya sala, kottakkal, kerala, india. the collected plant materials were maintained in the greenhouse and authenticated by dr a.k. pradeep, assistant professor, department of botany, university of calicut, kerala india. a voucher specimen was deposited at the calicut university herbarium (cali 123733). preparation of leaf extracts fresh leaves of p. heyneanus were collected from the plants grown in the greenhouse and were rinsed thoroughly, shade dried and then ground into powder. the crude extract was prepared by boiling the dried powdered material in distilled water for 30 minutes. the extracts were prepared in four different concentrations (0.5, 0.1, 0.05 and 0.01%). the extracts were then filtered to remove particulate matter. allium cepa root tip assay fresh and healthy bulbs of allium cepa were obtained from the local market. the bulbs were germinated over water before being transferred to the test plant extracts. when the roots were about 5 mm long, the bulbs were placed on beakers containing p. heyneanus leaf extracts of four different concentrations (0.5, 0.1, 0.05 and 0.01%), such that the roots were immersed in the extracts. the roots were immersed for 3 different durations, i.e. 2, 1 and ½ hours. the sprouted roots were also treated with distilled water, which served as control. the experimental set up had five replicates. the root tips were fixed in carnoy's fluid (1 part of glacial acetic acid: 2 parts of absolute alcohol) after the treatment. these roots were then hydrolyzed in 1n hcl for 5 minutes. after hydrolysis, the roots were washed in distilled water. root tips were cut into 1 2 mm long pieces and placed on a glass slide. a small drop of 2% aceto-orcein was placed on the root tip and the root tip was squashed. the slides were then scanned under leica dm 1000 trinocular research microscope and photomicrographs were taken. the numbers of cells, dividing and nondividing, were recorded. the degree of chromosomal aberration was expressed as the percentage of aberrant cells, which was calculated as the number of aberrant cells to the total number of cells examined. mitotic index (mi) was calculated by expressing the numbers of dividing cells as a percentage of total cells counted for each treatment and the control. numbers of aberrant cells _____________________percentage aberration = x 100 total number of cells numbers of dividing cells ____________________________mitotic index = x 100 total number of cells statistical analysis the data were analyzed using one way analysis of variance (one-way anova). means of the mitotic indices and chromosome aberrations with standard errors for each concentration of the extracts were calculated. duncan's multiple range test was performed to determine the significant differences between the different treatments made. statistical significance was accepted for p < 0.05 (auti et al. 2010). all statistical analyses were carried out using spss 17.0 statistical package. results and discussion mitosis was normal in the root tips kept as control, whereas the mitotic indices of all a. cepa roots treated with p. heyneanus leaf extracts were significantly lower than the mitotic index of control (table 1). the mitotic index rates were found to be decreasing with the increasing concentrations of the aqueous extract compared with the control (table 1). chromosome aberrations were observed in all stages of mitosis. major clastogenic abnormalities observed were nuclear budding and lesions, chromosome fragments and gaps, ring chromosomes, chromosome bridges, stickiness, hyperchromasia and non-clastogenic aberrations including stellate anaphase, ball t e l o p h a s e , e q u a t o r i a l s e p a r a t i o n o f c h r o m o s o m e s, c h r o m o s o m e l a g g a r d s, misorientation at metaphase, hyperploids and hypoploid cells, double metaphase, early ball metaphase, mitotic pairing at metaphase, cytotoxicity analysis of pogostemon heyneanus benth. – sheela and thoppil 29 biotropia vol. 24 no. 1, 2017 30 table 1 percentage of mitotic indices and chromosomal aberrations observed in allium cepa root meristems treated with p. heyneanus leaf extracts concentration mitotic index (+ se) % of aberrant cells (+ se) ½ hour 1 hour 2 hours ½ hour 1 hour 2 hours control 26.00+ 0.86 a 23.14+ 0.94 a 25.47 + 0.09 a 0 a 0 a 0 a 0.01% 17.11 + 3.00b 16.37 + 1.50b 16.23 + 1.39b 36.03+0.75b 41.81+0.56b 46.07+1.30b 0.05% 15.67 + 0.34b 15.10+ 0.93b 15.09 + 0.80b 39.16+1.32bc 43.64+1.82bc 48.14+0.93bc 0.1% 14.32 + 1.31b 14.30 + 1.31b 13.83 +0.50bc 39.50+1.86bc 47.98+3.07c 51.60+2.61c 0.5 % 13.39+ 0.62b 12.89+ 1.12b 12.14 + 0.52c 42.88+2.88c 48.32+1.61c 62.85+1.91d note: numbers followed by the same letters in the same column are not significantly different at p < 0.05 action on dna biosynthesis (majewska et al. 2003). nuclear buds were observed to originate from the nuclear envelope in situ at certain regions of the interphase nucleus of the treated cells and these might be a result of the excessive production of nucleic acids and proteins, induced by cytotoxicants (akaneme & iyioke 2008). stickiness (fig. 1t) might result from improper folding of chromosome fibers which made the chromatids connected by means of subchromatid bridges (hellgren & morre 1992). chromosome gaps (fig. 1d), which were distributed at random on the chromosomes, were formed by the action of mutagenic substances (mcgill et al. 1974). chromosome bridges (fig. 1b & 1t) and fragments (fig. 1c) were clastogenic effects, resulting from chromosome and chromatid breaks (fiskesjo 1997). ring chromosomes (fig. 1r) might arise d u e t o t h e s p o n t a n e o u s b r e a k a g e o f chromosomal ends, followed by the joining of the raw ends of the chromosomes (singh 2003). ball metaphase (fig. 1g) might be due to the localized activity of spindle apparatus at the centre, so that the centromeres remained at the equator and arms radiated in different directions, in the form of a ball. in ball telophase (fig. 1a), the sister chromatids separated into a hollow ball of chromosomes that resulted from the early cleavage divisions in some aberrant cells (morgan 2006). separation of daughter chromosomes parallel to the equator rather than towards the poles (fig. 1h) was an acute aberrant condition that arose as multipolar anaphase, pole to pole arrangement at metaphase and vagrant chromosomes at metaphase (fig. 1). there were significant differences (p < 0.05) between treated groups and control group in mitotic index. low mitotic index observed in the treated root tips might be due to direct genotoxic effect of p. heyneanus extract. the percentage of mitotic indices and chromosomal aberrations induced by different concentrations of the extract at different treatment durations are summarized in table 1. decrease in mitotic index rates explained cytotoxicity of the aqueous extract. this might be due to the obstruction of the onset of prophase, the arrest of one or more mitotic phases, or the slowing of the rate of cell progression through mitosis (fiskesjo & levan 1993). reduction in the mitotic activity could also be due to inhibition of dna synthesis or a blocking in the g2 phase of the cell cycle, preventing the cell from entering mitosis (christopher & kapoor 1988). chromosomal aberrations are changes in chromosome structure resulting from a break or exchange of chromosomal material. most of chromosomal aberrations observed in cells are lethal, but there are many corresponding aberrations that are viable and can cause genetic effects, either somatic or inherited (sudhakar et al. 2001; akinboro & bakare 2007). aberrations observed at the interphase stage included nuclear lesions (fig. 1f) and nuclear budding (fig. 1p). the presence of nuclear lesions offers cytological evidence for the inhibitory 31 figure 1 chromosomal aberrations induced by different concentrations of aqueous leaf extracts of p. heyneanus notes: a) ball telophase; (b) chromosome bridge at anaphase; (c) chromosome fragment at metaphase; (d) chromosome gaps at metaphase; (e) double metaphase; (f) double nuclear lesion; (g) early ball metaphase; (h) equatorial separation at anaphase; (i) hyperchromasia; (j) hyperploid cell; (k) hypoploid cell; (l) laggards at anaphase; (m) misorientation at metaphase; (n) mitotic pairing at metaphase; (o) multipolar anaphase; (p) nuclear budding at interphase; (q) pole to pole arrangement at metaphase; (r) ring and vagrant chromosomes at metaphase; (s) stellate anaphase; (t) sticky double chromosome bridge at anaphase cytotoxicity analysis of pogostemon heyneanus benth. – sheela and thoppil a result of errors in the mitotic spindle assembly and dynamics (ford & correll 1992). stellate arrangement (fig. 1s) of chromosomes might be due to the clumping of daughter chromosome groups in a star like manner and might be occurring as a result of the complete disturbance of spindle (raj & rao 1972). hyperchromasia (fig. 1i) was an extremely condensed and deeply staining state of interphase nucleus induced by toxic environmental chemicals or incompatible conditions, which might be caused by progressive heterochromatinization (gernand . 2005). misorientation of et al chromosomes (fig. 1m) could be due to a distortion of the spindle apparatus or a tilt in the e q u a t o r i a l o r g a n i z a t i o n o f m e t a p h a s e chromosomes (selim . 1981). the occurrence et al of hyperploid (fig. 1j) cells might be attributed to spindle inhibition, lack of anaphase movement or failure of cell plate formation (meske & hartmann 1995). abnormal pole to pole orientation of chromosomes (fig. 1q) at metaphase leading to equatorial separation of chromosomes at anaphase was an acute aberrant condition that arose as a result of irregular pathways of spindle assembly and abnormal spindle activity (waters & salmon 1997). multipolar anaphases (fig. 1o) were consequences of weak c-mitosis and could lead to aneuploidy (fiskesjo 1988). the induction of vagrant chromosomes (fig. 1r) led to the separation of unequal number of chromosomes in the daughter nuclei and subsequently formation of daughter cells with unequal sized or irregularly shaped nuclei at interphase (el-ghamery . et al 2003). laggards were whole chromosomes that failed to migrate to either pole at anaphase because of possible damage to the kinetochore (asita & mokhobo 2013). presence of hypoploid cells (fig. 1k) might be due to the occurrence of multipolar mitosis or lagging chromosomes (seoana . 2000). mitotic pairing (fig. 1n) was a et al rare abnormality which might be due to an increase in rna content (wen . 1989).et al mitotic index (mi) measured the proportion of cells in the m-phase of the cell cycle. the inhibition of m-phase could be considered as cellular death or a delay in the cell proliferation kinetics (nagpal & grover 1994; ribeiro . et al 2016). the decrease in mitotic index and the presence of these chromosomal aberrations in root a. cepa tips treated with extract when p. heyneanus compared with the control might be attributed to the presence of phytochemical constituents in the crude extract of . is rich in p. heyneanus p. heyneanus volatile oils containing compounds like patchouli alcohol, nerolidol, -pinene (murugan . β etc. et al 2010). patchouli alcohol, isolated from p. cablin was found to exhibit antitumor effect against human lung cancer cell lines (lu . 2016)et al . conclusions the aqueous extract of p. heyneanus possesses a dose dependent inhibitory effect on cell division of allium cepa. further studies are required to characterize and isolate the cytotoxic agents present in p. heyneanus. acknowledgements the first author is thankful to the council for scientific and industrial research (csir), india for funding this study. references akaneme fi, iyioke iv. 2008. mutagenic potentials of the sterilizing fluid – purital on root tip mitosis of allium cepa. bio res 6:293-7. akinboro a, bakare aa. 2007. cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on allium cepa linn. j ethnopharmacol 112:470 -5. anjana s, thoppil je. 2013. chemical composition of the essential oils of four spp. and their pogostemon larvicidal activity against skuse aedes albopictus (diptera: culicidae). int j environ biol 3:26-31. asita ao, mokhobo mm. 2013. clastogenic and cytotoxic effects of four pesticides used to control insect pests of stored products on root meristems of . allium cepa environ nat resour res 3:133-45. auti s, pagare r, ahire d, sawale v. 2010. cytogenetical studies on the effect of omnacortil on root tip cells of l. j cell tissue res 10:2331-5.allium cepa çelik ta, äslanturk ös. 2006. anti-mitotic and antigenotoxic effects of aqueous plantago lanceolata extract on root tip meristem cells.allium cepa biologia 61:693-7. 32 biotropia vol. 24 no. 1, 2017 çelik ta, äslanturk ös. 2007. cytotoxic and genotoxic effects of lavandula stoechas aqueous extracts. biologia 62:292-6. christopher hb, kapoor mb. 1988. the cytogenetic effects of sodium salicylate on the root meristem cells of allium sativa l. cytologia 54:203-9. el-ghamery aa, el-kholy ma, abou el-yousser ma. 2+ 2003. evaluation of cytological effects of zn in relation to germination and root growth of nigella sativa l. and triticum aestivum l. mutat res. 537:29-41. fiskesjo g. 1988. the allium test-an alternative in environmental studies: the 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vitro. protoplasma 167:238-42. jeong jb, choi j, lou z, jiang x, lee sh. 2013. patchouli alcohol, an essential oil of pogostemon cablin, exhibits anti-tumorigenic activity in human colorectal cancer cells. int immunopharmacol 16:184-90. jovtchev g, stergios m, schubert i. 2002. a comparison of n-methyl-n-nitrosourea-induced chroma tid aberrations and micronuclei in barley meristems using fish techniques. mutat res 517:47 -51. lu x, yang l, lu c, xu z, qiu h, wu j, wang j, tong j, zhu y, shen j. 2016. molecular role of egfr-mapk pathway in patchouli alcoholinduced apoptosis and cell cycle arrest on a549 cells in vitro and in vivo. biomed res int article id4567580. 12 p. lubini g, fachinetto jm, laughinghouse hd, paranhos jt, silva acf, tedesco sb. 2008. extracts affecting mitotic division in root-tip meristematic cells. biologia 63:647-51. majewska ae, wolska e, sliwinska m, furmanowa n, urbanska a, pietrosiuk a, zobel a, kuras m. 2003. a n t i m i t o t i c e f f e c t , g 2 / m a c c 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of allium cepa. cytologia 66:235-9. waters jc, salmon ed. 1997. pathways of spindle assembly. curr opin cell biol 9:37-43. 33 cytotoxicity analysis of pogostemon heyneanus benth. – sheela and thoppil wen f, barnett fl, liang gh. 1989. somatic pairing and separation of chromosomes in roottip cells of regenerated sorghum plants. j hered 80:159-60. yi h, meng z. 2003. genotoxicity of hydrated sulfur dioxide on root tips of and . allium sativum vicia faba mutat res 537:109-14. 34 biotropia vol. 24 no. 1, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 microsoft word 1 biotropia vol. 13 no. 1, 2006 : 1 10 transposition and expression of gfp gene in the genome of vibrio harveyi to monitor its adherence in shrimp larvae wldanarni0, antonius suwanto^sukenda1', and blbiana wldiati lay3) '' faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia '' faculty of science and mathematics, bogor agricultural university, bogor 16680. indonesia ''faculty of veterinary science, bogor agricultural university, bogor 16680, indonesia abstract expression of green fluorescent protein encoded by gfp gene in vibrio harveyi was investigated to understand the ability of the gene as a molecular marker for adherence of this pathogenic vibrio in shrimp larvae. the gfp gene was inserted into pl/clsnot and putmini-tn.') to generate a recombinant plasmid pwg02 and p\vg03, respectively; which was transferred into the three isolates of v. han'eyi employing diparental mating. recombinant e. coli carrying p\vg02 and pwg03 resulted in green-fluorescent colonies and cells due to the production of gfp. however, all of mini-tn.j, including mini-tn.5-gfp were not successfully transferred to v. harveyi. therefore, we used mini-tn/fl (plofkm-gfp) for inserting of gfp gene into v. han'eyi genome. although we could obtain relatively high (10's) transconjugans employing tn/rt, only one of tnjo derived isolate of v. harveyi g3 (g3-tn/flgfp) showed gfp expression and was further employed for adherence assay. fluorescent g3-tn70gfp cells could be observed inside the digestive tract of shrimp larvae and could be distinguished from vibrio that naturally exist in shrimp larvae. key words: gfp gene, vibrio han'eyi, gene expression, shrimp larvae, molecular marker introduction vibrio harveyi was identified as a causative agent of mass mortalities of shrimp larvae and were frequently associated with luminous vibrio (lavillapitogo et al. \ 990; karunasagar et al. 1994; ruangpan 1998, suwanto et al. 1998). luminescent vibriosis in shrimp larvae is characterized by lethargy, anorexia, muscle opacity, bacterial masses in the hemocoel, and luminosity of the larvae (lavilla-pitogo et al. 1990). electron microscopy observation has revealed that bacteria colonize the feeding apparatus, forming bacterial plaques in heavily infected larvae, therefore, it is highly probable that the mouth is the main entrance for colonization of inner tissues (lavilla-pitogo et al. 1990). pathogenicity assays based on koch's postulates (madigan et al. 2003) were practically difficult to be conducted in shrimp larvae due to their relatively small size and lack of availability of vibrio-free larvae (widanarni and suwanto 2000). investigations into adherence and pathogenicity processes of this disease might ' corresponding author: e-mail: asuvvanto@indo.net.id biotropia vol. 13 no. 1, 2006 greatly facilitate if a visible marker could be introduced into the bacterial cells. pathogenic v. harveyi have observed their attachment to crustacean larvae by epifluorescence microscopy using 5-(4,6-dichorotriazin-2-yl) aminofluorescein (5-dtaf, d-16) as a marker (soto-rodriguez et al. 2003). one visible molecular marker which has extensively been used for studying bacterial activity in the environment is gfp, i.e. a gene encoding green fluorescent protein (gfp) from a jellyfish (aequorea victoria) (manning 1997). as a molecular gene marker, gfp has some advantages, such as no requirement for exogenous substrate or energy source for their visualization, sensitivity of detection, high stability, lack of toxicity, and no disturbance in cell function and growth (josenhans et al. 1998; ling et al. 2000). gfp as a molecular marker has been used to demonstrate the mechanism of edwardsiella tarda infection on epithelial cells of giant gouramy (ling et al. 2000); and pseudomonas plecoglosicida infection in ayu (plecoglossus altivelis) (sukenda and wakabayashi 2001). gfp was also successfully used as a marker in lactic acid bacteria (lactobacillus plantarum and l. lactis) to study the possibility of using the bacteria as live vaccine carriers (geoffroy et al. 2000). a broad host-range plasmid expressing gfp gene (pwgol) was constructed and has been successful to tag k harveyi (widanarni et al. 2005). however, under non selective longterm experiments without antibiotic pressure, pwgol was highly unstable. therefore, transposon insertion may provide an alternative method to insert gfp gene directly into the genomic dna of v. harveyi in order to yield stable recombinants. in this report, we describe the construction in tn vector and expression of v. harveyi carrying gfp gene in the genomic dnas to monitor its adherence in shrimp larvae. materials and methods bacterial strains and plasmids bacterial strains and plasmids used in this study and their relevant characteristics are described in table 1. escherichia coli and v. harveyi were grown in luria bertani (lb) medium at 37°c and seawater complete (swc) medium at 28°c, respectively. lb medium was made as previously described (sambrook et al. 1989) and swc medium contained 5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water. plasmid construction and molecular techniques recombinant plasmid pwg02 which has the lac promoter was constructed and used to drive the expression of gfp. the promoter and gfp gene were isolated from psklol using £cori sites and ligated into puclsnot linearized with £cori. the recombinant plasmid vector (pwg02) (figure 1) was digested with notl and then promoter and gfp gene were ligated into putmini-tnj linearized with noil resulting transposition and expression of gfp gene widanami et al. in recombinant plasmid (pwgos) (figure 2). the recombinant plasmid vector was transformed into e. colt dh5a using a standard heat shock transformation (sambrook et al. 1989) and the colonies carrying pwg02 and pwg03 were examined for green fluorescence under uv-transilluminator at 260 nm (biometra ti 1, gottingen). plasmid extraction, restriction enzyme digestions, agarose gel electrophoresis, gel isolated dna fragment purification, and ligation were carried out using standard methods (sambrook et al. 1989), and following the manufacturer's instructions. restriction endonucleases and other enzymes were obtained from new england biolabs inc (beverly, ma, usa). bacterial mating to transfer recombinant plasmid pwg03 harboring mini-tn5gfp into v. harveyi we used diparental mating (suwanto and kaplan 1992). escherichia coli dh5a (pwgos) donors were grown overnight in lb medium supplemented with spectinomycin and streptomycin (sp/sm) 50 ugmt1 at 37°c; whereas v. harveyi recipients were grown in swc medium at 28°c. each 1.5 ml of the donor and recipient were pelleted in a micro-centrifuge at maximum speed for 1 min, and then the cells were washed with 1.0 ml 0.85% nacl, re-centrifuged, and suspended in 40 ul of lb medium before being spotted onto a filter (1 cm diameter; pore size 0.45 urn; millipore) on lb medium agar. the bacteria were allowed to conjugate at 28°c for 16 to 18 hours. at the end of the mating period, the filter containing the bacterial transposition and expression of gfp gene — widanarni et al. mixture was transferred into 1.5 ml microfuge tube containing 0.8 ml of 0.85% nacl. the bacterial cells were suspended thoroughly by agitation on a vortex mixer. the transconjugants were selected on thiosulphate citrate bile salt (tcbs, oxoid) medium supplemented with sp/sm (50 jugm!"1). the selective medium tcbs was used to inhibit the growth of e. coli, while allowing v. harveyi transconjugants harboring tnjgfp (resistant to spectinomycin and streptomycin) to grow. the same method was conducted to transfer recombinant plasmid plofkm-gfp (resistant to kanamycin) into v. harveyi and the transconjugants were selected on tcbs medium supplemented with kanamycin (100 (agml"1). some of v. harveyi transconjugants were analyzed by pulsed-field gel electrophoresis (pfge) with notl restriction enzyme (suwanto and kaplan 1992; widanarni and suwanto 2000) to show the place of tnlogfp inserted in the genomic of v. harveyi. gfp stability and pathogenicity assay vibrio harveyi strains harboring g/p both in the plasmid pwgol (widanarni et al. 2005) and in the genomic's dna were grown overnight in swc broth supplemented with kanamycin. sequential propagation under non selective conditions were performed by inoculating with 1:100 (v/v) to assess gfp existence by comparing duplicate colony counts on selective and non selective plates. for pathogenicity assay, two groups with three duplicates of shrimp post-larvae (pl4) were immersed for 30 min in 106 cfuml"1 of gfp recombinants and wild type of v. harveyi (final concentration), respectively, and then placed in a 2 l shrimp rearing tank. a control group was immersed in sterile scawater. daily survival rate of shrimp larvae for 5 days were recorded and compared with the control group. adherence assay samples of shrimp larvae from control and treatment groups were directly observed under a fluorescence microscope. samples from dead shrimp larvae were also inoculated onto swc plates containing kanamycin (100 figrnl"1) to show that the dead shrimp larvae were infected by recombinant v. harveyi. results and discussion gfp-containing plasmid construction the gfp-plasmid vector was constructed for molecular marker in v. harveyi. two gfp vectors i.e. pwg02 and pwgos, were constructed with lac promoter to drive the expression of gfp. recombinant e. coli carrying pwg02 or pwg03 (mini-tnjgfp) resulted in green-fluorescent colonies and cells due to the production of gfp (figure 3). however, all of mini-tn5, including mini-tnj-gfp was not successfully transferred to v. harveyi. the same results were observed by stretton e? biotropia vol. 13 no. i, 2006 a b figure 3. fluorescence micrograph of (a) e. coli dh5a (pwg02) and (b) e. coli dhsct (pwg03) al. (1998). therefore, we used mini-tn70 (plofkm-gfp) that has been constructed by stretton et al. (1998) for inserting ofgfp gene into v. harveyi genome. construction of v. harveyi gfp+ employing mini-tn/0 (plofkm-gfp) we could obtain relatively high transconjugants employing tnlo (table 2). pulsed-field gel electrophoresis (pfge) analysis of some v. harveyi transconjugants demonstrated that tn70gfp was randomly inserted in the gcnomic v. harveyi g3 (figure 4). however, only one of tnlo derived isolate of v. harveyi g3 (g3-tn70gfp) resulted in green-fluorescent colonies and cells due to the expression of gfp and that fluorescence levels qualitatively was almost the same with g3 (pwgol) (figure 5). this result occurred due to the fact that gfp in plofkmg/p was constructed promoterless, so its expression depended on promoter strength in the insertion site within v. harveyi genome. gfp stability and pathogenicity assay colonies of v, harveyi g3 (g3-tn70gfp) that were grown on media with or without antibiotic exhibited uniform fluorescence appearance. this was not the case transposition and expression of gfp gene widanarni et al. m figure 4. pfge profiles of genomic dna of v. harveyi g3 (lane m: axel-digested genotnic dna of r. sphaeroides 2.4.1 as a molecular size marker (suwanto and kaplan 19 89). lane 1 and 8: g3 wild type, lane 2: g3 tn/flgfp (kmr and showed gfp expression), lane 3 -7: mutants 03 (kmk) figure 5. fluorescence micrograph of (a) v. harveyi g3 (pwgoi) and (b) g3-tn/ogfp for v. harveyi g3 (pwgol). their colonies grown on antibiotic-containing media exhibited uniform fluorescence appearance, whereas those grown on media without antibiotic showed mixture of fluorescent and non fluorescent colonies which might indicate plasmid loss. the stability of the gfp on v. harveyi g3 both in plasmid and in the genomic's dna were investigated during sequential propagation in the absence of antibiotic selection for five successive days. under nonselective long-term experiments without antibiotic pressure, pwgol was highly unstable but tn/0gfp was stably maintained in v. harveyi g3 strain (figure 6). insertion of tn70gfp also did not show alteration in g3 pathogenicity to shrimp larvae (figure 7), so that it was further employed for adherence assay. adherence assay sample from dead shrimp larvae showed that the dead larvae were infected by v. harveyi. vibrio harveyi g3-tn/0gfp could be isolated from dead shrimp larvae placed on tcbs+km media and fluorescent g3-tn70gfp could also be observed directly in the carcasses of dead larvae. fluorescent g3-tnjogfp cells were observed in the oral region at 15-30 min after inoculation and could be observed inside the digestive tract at 2-3 h (figure 8v the concentration of v. harveyi g3-tnlogfp used in this study was 10 cfuml". soto-rodriquez et al. (2003) reported that 105 cfuml"1 v. harveyi labeled with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (5-dtaf, d-16) could be observed in the oral region of litopenaeus vannamei mysis at 0 and 2 h after ingestion. after 4 h inoculation, individual cells could already be seen inside the middle intestine, and at 18 or 24h, the fluorescent v. harveyi were observed throughout the intestinal tract. when presented at higher densities of bacterial cells (exclusively), fluorescent v. harveyi could be easily observed along all regions of zoea digestive tracts after 30 min (soto-rodriquez et al. 2003). 8 conclusions recombinant v. harveyi harboring gfp both in plasmid pwgol and in the genomic dnas produced green-fluorescent cells. however, tn7(?gfp was stably maintained in the genomic v. harveyi so that it could be used to monitor adherence and pathogenicity of v. harveyi in shrimp larvae. acknowledgments this work was supported by dip-funded research of seameo-b1otrop (2000-2002) and 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sugiyama 4 1 department of agronomy, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia 2 asian natural environmental science center, university of tokyo, tokyo 188-0002, japan 3 faculty of agriculture, tokyo university of agriculture, kanagawa 243-0034, japan 4 graduate school of agricultural and life sciences, university of tokyo, tokyo 113-8657, japan; present address: faculty of agriculture, tokyo university of agriculture, kanagawa 243-0034, japan received 06 may 2016/accepted 17 july 2017 abstract amorphophallus variabilis blume, a member of araceae, is a fleshy perennial tuber crop endemic in java island, indonesia. the plant produces white edible corm; and it was used as food during famine time before 1960s. rapid ecological changes and land fragmentations in java in recent times threaten populations of a. variabilis. here, compound microsatellite markers were developed in order to develop conservation strategies in the populations. twelve primers pairs produced high polymorphism ranging from 5 to 22 alleles per locus. the observed and expected heterozygosities ranged from 0.191 to 0.851 and 0.380 to 0.943, respectively. this high allelic diversity indicates that these markers are suitable for the study on population genetic structure. cross-amplification on related and nonrelated species was performed. application of the markers on populations from dramaga conservation forest revealed high allelic richness, high diversity within and among populations. genetic distance among populations increased with an increase of geographic distance. present study suggested that, it is important to study population of a. variabilis in java in order to understand the population genetic structure and develop effective in situ conservation programs. keywords: araceae, genetic population, ssr, tuber crop, white iles-iles introduction amor phophallus variabilis blume, synonym brachyspatha variabilis (blume) schott, an araceae, is a diploid endemic perennial tuber crop native to java. its distribution in indonesia is exclusively in java, kangean and madura islands (jansen . et al 1996; yuzammi 2000). the plant naturally grows in less disturbed land under partial trees shading and at gap of forest trees at low altitude up to 700 to 900 m above sea level (jansen . 1996; et al sugiyama & santosa 2008). is locally a. variabilis called white (safii 1981; wiyani 1988) iles-iles reflecting the white color of the tuber. it is easily d i s t i n g u i s h e d m o r p h o l o g i c a l l y f r o m amorphophallus muelleri blume (sugiyama & santosa 2008) known as yellow iles-iles. the plant produces a single underground corm with several cormlets; corms and cormlets exhibit dormancy during dry season (jansen . et al 1996). the corm was utilized as staple food in java particularly during famine situation before 1960s. corm of contains high glucomannan a. variabilis (ca. 35% on a dry weight basis) (ohtsuki 1968; safii 1981; wiyani 1988). glucomannan is used as an important material in beverage, food and pharmaceutical industries (jansen . 1996; et al alonso-sande . 2009).et al botanically, a large genetic diversity has been observed within a. variabilis plants, including inflorescence (santosa et al. 2004) and leaf morphologies (sugiyama & santosa 2008). according to yuzammi (2000), the petioles vary from pure green to dark brown with different color and pattern of spots. therefore, a. variabilis * corresponding author: edisang@gmail.com biotropia 5 1 8 22 32 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.652 22 germplasm could be used as breeding materials (sugiyama & santosa 2008). according to zhang et al. (1998), several amorphophallus species could be hybridized. the monoecious unisexual inflorescence produces unpleasant odor during anthesis (kite & hetterschieid 1997) to attract pollinators. red bright mature berries develop after crosspollination by nitidulidae insects (santosa . et al 2004). seeds are dispersed long distance by birds and clonally propagules i.e. cormlets were dispersed nearby mother plants (sugiyama & santosa 2008). thus, in an undisturbed population, both ramets and genets co-exist. recently, the sliced corms of are a. variabilis used as feeds for swine (santosa . 2004; et al sugiyama & santosa 2008). people collect the corms without considering the conservation. on the other hand, land fragmentations and agricultural intensifications in agroforestry system for cash crops (santosa . 2005) disturbed the et al populations. consequently, the populations of a. variabilis in java are in danger. high exploitation has been known causing population decline and loss of genetic diversity in many species (leimena et al. et al. 2007; nuryanto & susanto 2010; santosa 2010). therefore, in order to develop effective program on genetic conservation of , it a. variabilis is important to evaluate population size using reliable genetic markers, such as microsatellite. microsatellite or simple sequence repeat (ssr) is widely used to develop conservation program in many species (abdul-muneer 2014; nachimuthu et al. 2015). in this study, we developed microsatellite of using compound a. variabilis isolation method for the first time. many methods have been developed for m i c r o s a t e l l i t e i s o l a t i o n s e. g. b a s e d o n sequence data (lian . 2001; fatimah & sukma et al 2011), cloning, enrichment library and dualsuppression methods (lian & hogetsu 2002; lian . 2006). in the present study, et al compound microsatellites are isolated according to procedure of lian (2006). the objective et al. of this study was to develop microsatellite markers from and its application for a. variabilis genetic population study. materials and methods plant material plant materials were collected separately for microsatellite development and population study i.e. from bogor botanical garden (bbg) and d r a m a g a c o n s e r va t i o n fo r e s t ( d c f ) , respectively with permission from the authorities. according to yuzammi (2000), bogor area including the sampling sites, is the center of a. variabilis diversity. to develop microsatellite markers, 2 5 g fresh and healthy leaves of were collected a. variabilis during rainy season in 2005 at the bbg, bogor, west java province, indonesia. the leaves were cleaned using moist tissue paper and put into a plastic bag containing silica gel, then stored at -30 o c until being used in genomic dna extraction. microsatellite development was conducted in the laborator y of forestr y, asian natural environmental science center, university of tokyo, japan from 2009 to 2010. for population study, accessions a. variabilis were sampled and characterized in the dcf (244 m asl), bogor, west java province, indonesia from 2010 to 2011. dcf is managed by the indonesia ministry of environment and forestry. around 40 of the 60 ha dcf area is dedicated for conservation forest, locally known as the cifor forest (cifor = center for international forestry research). dcf is located about 8 km northwest of bogor botanical garden, 1 km west of situ gede lake and 1 km north of cisadane river. three populations were selected for this study, i.e. a (-6.5509798, 106.7497507,17z), b (6 . 5 5 1 9 3 9 1 , 1 0 6 . 7 4 9 4 9 3 2 , 1 7 z ) a n d c ( 6.5548596,106.7507271,17z) (fig.1). in each population, plants were sampled from an area of about 0.5 to 1 ha. all plants having pseudo stems thicker than 2 cm at 10 cm above soil surface and were spaced more than 1 m apart were collected. plants spaced less than 1 m apart were considered as a ramet (similar genet) and not sampled. petiole colors of accessions were characterized (table 1). initially, 35, 25 and 38 samples were collected, and after optimizing the dna results a set of 11, 21 and 15 23 isolating microsatellite from amorphophallus variabilis and its application santosa et al. (novagen, usa) according to the manufacturer's instructions. the positive clones were amplified using the u19 and m13 reverse primers, and then sequenced using a thermo sequenase pre-mixed cycle sequencing kit (amersham biosciences) plus the t7 or u19 primer labeled with texas red (sigma-aldrich) on sq-5500e sequencer (hitachi, tokyo). a primer (ip1) was designed from the sequenced region flanking to the ssr. the ip1 and corresponding microsatellite primers were used as the marker. in addition to microsatellites developed from a. variabilis, twenty microsatellite markers which were developed from by santosa a. paeoniifolius et al. (2007) and stored in genbank were evaluated for its suitability toward genotyping. a. variabilis in order to extend usefulness of the microsatellite markers, the developed markers from a. variabilis were evaluated on several araceae members and other root crops species. pcr amplification and detection pcr reaction was carried out in a reaction mixture (10 µl) containing 5 10 ng dna, 0.2 mm of each dntp, 1 pcr buffer (mg free, 2+ accessions were used for further analysis from a, b and c populations, respectively. the other samples were excluded from analysis because they failed to amplify, or produced multiple and unclear bands. microsatellite development a modified cetytrimethyl ammonium bromide (ctab) method was adopted for conducting genomic dna extraction (zhou . 1999). et al dna from 1 g dry leaves was dissolved in a final volume of 200 µl water and stored in -30 c until o use. isolation of codominant compound microsatellite markers was performed according to the method of lian . (2006). in brief, the et al hae iii blunt-end restriction enzyme was used to digest dna sample. the digested dna was then ligated to an adaptor using a dna ligation kit (takara shuzo, japan). fragments flanked by a microsatellite region at one end were amplified from the constructed dna library using ssr primers, (ac) (ag) or (tc) (ac) and an adaptor 6 5 6 5 primer, ap2 (5´–ctatagggcacgcgt ggt–3´). the pcr products were subcloned using a pt7 blue perfectly blunt cloning kit biotropia vol. 25 no. 1, 2018 24 a10 a9 a8 a12 a11 a1 a3 a4 a6 a5 a2 pop. a b2 b3 b6 b7 b8 b4 b5 b9 b10 b11 b15 b18 b17 b19 b25 b26b27 b21 b20 b22 b24 b23 b28 b16 b12 b13 b14 pop. b c1 c13 c14 c15 c16 c18 c12 c7 c11 c8 c10 c6 c4 c20 c21 c22 c17 c19 pop. c indonesia java n cluster i cluster ii cluster iii mt salak bbg dcf bogor area 5 m 5 m 5 m road 2 km figure 1 site and accession positions within populations in dramaga conservation forest (dcf), bogor, indonesia (note: lines represent distance and visibility between two accessions; accessions without lines represent blockage by dense trees; color represents cluster membership; bar represents 5 meter) applied biosystems), 2.5 mm mgcl , 0.25 u of 2 ampli gold (applied biosystems, usa), 5 taq 0. μm of each ip1 primer and the corresponding ssr primer (labeled with texas red). the pcr thermal cycler (applied biosystems) were used with cycling profile: 9 minutes at 94 ºc, followed by 40 cycles of 30 seconds at 94 ºc, 30 seconds at the locus-specific annealing temperature (table 2) and 1 minute at 72 ºc, and finally a 5 minutes extension at 72 ºc. the pcr products were electrophoresed on a 6% polyacrylamide gel using an sq-5500e sequencer, and then analyzed with fraglys ver. 3 software (hitachi, tokyo). data analysis characteristics of microsatellite markers developed in the present study were tested on a. variabilis across all populations (n = 47). two bands from an individual were considered as different alleles if the differences in molecular weight were bigger than 3 base pair (bp) for the microsatellite containing trinucleotide repeat and 2 bp for the microsatellite containing dinucleotide repeat; these bp different are matter of technical procedure not related to nucleiotide repeat motif (santosa 2017). the numbers of alleles, observed (h ) and expected heterozygosities (h ) and o e polymorphic information content (pic) were calculated using cervus ver. 3.0.3 software (marshall et al. 1998). hardy-weinberg equilibrium (hwe) and linkage disequilibria between loci were tested using genepop version 4.0 software on the web (rousset 2008). presence of null allele was estimated using cervus ver. 3.0.3 software (marshall et al. 1998). number of migration (nm) was estimated using genalex software. population code petiole color population code petiole color a a1 pure green b b20 brown with small white spot a a2 pure green b b21 brown with large white and green spots a a3 brown with white spot b b22 brown with large light green spot a a4 green with brown spot b b23 brown with white spot a a5 green with white spot b b25 green with large white spot a a6 green with brown spot b b26 dark brown-black with white spot a a8 pure green b b27 brown with small white spot a a9 brown with black spot b b28 brown with small dark brown spot a a10 brown with dark brown spot c c1 pure green a a11 brown with black spot c c4 brown with white spot a a12 dark green with brown spot c c6 green with grey spot b b2 grey with brown spot c c7 light green with brown spot b b3 brown with green spot c c8 brown with pink spot b b4 grey with green spot c c10 pure green b b5 brown with green spot c c11 light green with grey spot b b6 brown with white spot c c12 pure green b b7 green with small white spot c c13 brown with white and black spot b b8 light green with black and white spot c c14 brown with light brown spot b b9 light green c c16 brown b b10 brown with brown and black spot c c17 dark brown with pink spot b b11 pure green c c20 pure green b b14 light brown with white spot c c21 dark brown with pink and black spo t b b16 dark green with white spot c c22 light green with black spot b b18 brown with black spot table 1 characterization of a. variabilis accessions obtained from dramaga conservation forest, bogor, indonesia note: large spot = spot width 10mm> small spot = spot width 2.5mm< otherwise mentioned = spot size was medium spot was measured from 10 cm above soil surface to middle petiole length 25 isolating microsatellite from amorphophallus variabilis and its application santosa et al. analysis of molecular variation (amova) within (f ) and among population (f ) was is st determined from a, b and c populations separately using genalex software in 999 permutations. cluster analysis was performed using ntsyst spc 2.11p (exeter software, setauket usa). presence of allele at each locus was coded in binary form 1 (presence) or 0 (absence). individual across populations was clustered in upgma dendrogram using jaccard similarity coefficient. results and discussion polymorphism test from twenty-two microsatellite loci isolated from a. variabilis, twelve loci were polymorphic and codominant. polymorphic information content (pic) ranged from 0.351 to 0.932 (table 2 ) . t h e o b s e r ve d ( h ) a n d e x p e c t e d o heterozygosities (h ) ranged from 0.191 to 0.851 e and from 0.380 to 0.946, respectively. the full length of amplified region from a. variabilis microsatellite had been deposited at genbank for public access (www.ncbi.nlm.nih.gov/genbank). two of 12 loci isolated from a. variabilis, i.e. avar01 and avar07, could deviate from hwe proportion (p 0.001) by having an excess of heterozygosities (table 2). linkage disequilibria analysis indicated that the loci had high linkages (p ≤ 0.001), i.e. between avar02 and avar04, avar04 and avar08, avar07 and avar10, and avar07 and ampa10. excluding loci avar04 and avar07, the other 10 loci could be used in population genetic study. according to li et al. (2009), a set of 10 15 or more loci was desirable for genetic population study. furthermore, two microsatellite primers from a. paeoniifolius, i.e. ampa10 and ampa15, produced clear bands in a. variabilis accessions. therefore, in total, fourteen polymorphic microsatellite loci could be used for a. variabilis genotyping. this study is the first work on the development of polymorphic codominant microsatellite markers in . microsatellite markers a. variabilis developed in the present study could be used to identify the polymorphism based on the standards proposed by lian . (2006), i.e. clearness of et al bands, common annealing temperature and different allele sizes. in the present study, annealing temperature was set at 58 and 62 c. o allele sizes among several loci had large differences in base pair size (table 2). therefore, loci avar01 and avar09, avar05 and avar06, and avar03 and avar10 could be mixed together in pcr reaction, to speed up pcr preparation and allele analysis. the loci developed from cross-a. variabilis amplified in other species (table 3), indicating that the loci could provide a useful tool for g e n o t y p i n g i n o t h e r s p e c i e s. i n d e e d , microsatellite primers are well known for its species-specific (lian & hogetsu 2002; csencsics et al. 2010), however, cross-amplifications sometimes exist (santosa . 2007; fatimah & et al sukma 2011). successful cross-amplification is probably due to the presence of homologous loci among them. santosa (2007) have reported et al. that microsatellites isolated by dual-suppression from amplified in related species, a. paeoniifolius while fatimah sukma (2011) reported that and microsatellite markers obtained from sequence data could amplify across genus. phalaenopsis however, amplification pcr products did not mean produce polymorphic allele in present study. it needs further investigation on the usefulness of the developed loci for genotyping other speciesa. variabilis . population and genetic diversity of dcf the average numbers of alleles per locus was 6.5 for 14 loci. the numbers of alleles per locus varied from 3 to 11 in population a, from 3 to 16 in population b and from 3 to 14 in population c (table 4). locus ampa15 produced the smallest number of alleles, whereas locus ampa10 produced the largest number of alleles across dcf populations. several accessions failed to amplify, i.e. locus avar02 for a12 and c20 accessions, locus avar04 for c6 and c10 accessions, locus avar06 for b9 and b11 accessions, locus avar11 for b23 and c17 accessions, locus ampa10 for c13 accession and locus ampa15 for a8 and c7 accessions. these accessions probably had null allele for particular loci. point mutation in the primer annealing sites may lead to the occurrence of null alleles causing the primer failed to amplify (lian et al. 2006). 26 biotropia vol. 25 no. 1, 2018 t ab le 2 c h ar ac te ri st ic s o f t w el ve m ic ro sa te ll it e m ar k er s is o la te d f ro m a . va ri ab ili s l o cu s g en b an k z r ep ea t m o ti f p ri m er s eq u en ce 5 ’ to 3 ’ t a ( °c ) n a a ll el e (b p ) h o h e p ic a va r0 1 * m f 5 2 7 2 5 0 (c t ) 6 (g t ) 7 f : c t t g t t c g g a c c a c c t t c t t g a c a a t c r : (a c )7 (a g )3 6 2 5 1 2 0 -1 3 0 0 .4 8 9 0 .5 4 8 0 .4 9 2 a va r0 2 m f 5 2 7 2 5 1 (c t ) 1 2 (g t ) 7 f : c t c a a a a t c g a a t c t t c t c c a t t t t a c r : (a c )7 (a g )3 6 2 1 4 1 2 4 -1 5 4 0 .3 3 3 0 .8 7 7 0 .8 5 4 a va r0 3 m f 5 2 7 2 5 2 (c t ) 8 ..( c t t ) 1 1 ..( c t ) 5 (g t ) 7 f : c a c t c t t c c a c a c t c c c c c t g t t a c a c r : (a c )7 (a g )3 6 2 5 1 2 8 -1 4 0 0 .2 1 3 0 .3 8 0 0 .3 5 1 a va r0 4 m f 5 2 7 2 5 3 (c t ) 1 3 ... .( c t ) 4 (g t ) 7 f : c t t c c c t a t g c a g g t g a g t c r : (a c )7 (a g )3 5 8 8 1 1 3 -1 5 5 0 .3 3 3 0 .4 2 7 0 .3 9 6 a va r0 5 m f 5 2 7 2 5 4 (g t ) 5 (g a ) 7 f : g t g a a g g a g g t g g g c g t t t t g r : (t c )7 (a c )3 5 8 5 1 7 7 -1 8 7 0 .4 6 8 0 .5 2 0 0 .4 6 0 a va r0 6 m f 5 2 7 2 5 5 (g t ) 1 0 (g a ) 7 f : c t a a c g a c t a a g g a c t t a a g c r : (t c )7 (a c )3 5 8 2 2 6 3 -1 4 1 0 .5 1 1 0 .9 4 3 0 .9 2 8 a va r0 7 * m f 5 2 7 2 5 6 (g t ) 1 0 (g a ) 7 f : c g c t g a t g t a c t t g t t g a c a t t g r : (t c )7 (a c )3 5 8 1 1 1 3 9 -1 7 1 0 .7 2 3 0 .7 6 1 0 .7 1 9 a va r0 8 m f 5 2 7 2 5 7 (g t ) 8 (g a ) 7 f : c a t g g t c c a t g g g t t t a g c t t t g c r : (t c )7 (a c )3 6 2 2 0 1 6 6 -2 3 6 0 .7 4 5 0 .9 3 3 0 .9 1 8 a va r0 9 m f 5 2 7 2 5 8 (g t ) 5 (g a ) 7 f : g g t g t a c g a c t a g a g t t t t g t c g r : (t c )7 (a c )3 6 2 7 1 7 5 -1 8 3 0 .4 2 6 0 .6 7 6 0 .6 2 0 a va r1 0 m f 5 2 7 2 5 9 (g t ) 8 (g a ) 7 f : g a t g t c a t t c t c c g c c a c c g a g t a g r : (t c )7 (a c )3 6 2 1 0 1 9 6 -2 2 8 0 .8 5 1 0 .7 6 9 0 .7 2 6 a va r1 1 m f 5 2 7 2 6 0 (g t ) 6 (g a ) 7 f : g a t a c t g c t a t a c c g a g t g t c c t a t g r : (t c )7 (a c )3 6 2 7 1 5 3 -1 8 5 0 .2 0 0 0 .6 5 5 0 .6 0 7 a va r1 2 m f 5 2 7 2 6 1 (t c ) 8 g (c t ) 2 (g t ) 5 (c t ) 5 (g t ) 7 f : g g a a c a c t a g c g a g t a c a a t g t a t c r : (a c )7 (a g )3 6 2 5 1 3 5 -1 4 9 0 .1 9 1 0 .7 8 6 0 .7 4 1 n o te : = g en b an k r ep o si to ry c o d e o f p ri m er s an d f ra gm en ts s eq u en ce z * = s ig n if ic an t d ev ia ti o n f ro m h ar d y– w ei n b er g e q u il ib ri u m ( < 0 .0 1 ) p t = an n ea li n g te m p er at u re a n a = n u m b er o f a ll el e h o = o b se rv ed h et er o zy g o si ty h e = ex p ec te d h et er o zy g o si ty p ic = p o ly m o rp h ic i n fo rm at io n c o n te n t e ac h r ev er se p ri m er ( r ) w as t ai le d w it h a n a d d it io n al 1 9 n u cl eo ti d e (u 1 9 ) to t h e 5 ' e n d 27 isolating microsatellite from amorphophallus variabilis and its application santosa et al. 28 t ab le 3 c ro ss -s p ec ie s am p li fi ca ti o n o f t w el ve m ic ro sa te ll it e p ri m er s d ev el o p ed f ro m a . va ri ab ili s n o s p ec ie sz f am il y o ri gi n o f t h e sp ec im en l o ci a v a r 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 1 1 1 2 1 a gl ao ne m a pi ct um ( r o x b .) k u n th . a ra ce ae b b g + + + + + + + + + + + + + + + + + + 2 a lo ca si a al ba s ch o tt . a ra ce ae b b g + + + + + + + + + + + + + + + + + + 3 a nt hu ri um s p p . a ra ce ae b b g + + + + + + + + + + + + + + + 4 a m or ph op ha llu s k on ja c k . k o ch a ra ce ae t an as h i f ar m , u t + + + + + + + + + + + + + + + + + + + + 5 a m or ph op ha llu s m ue lle ri b lu m e a ra ce ae ip b + + + + + + + + + + + + + + + + + 6 a m or ph op ha llu s pa eo ni ifo liu s (d en n st .) n ic o ls o n a ra ce ae k u n in g an , in d o n es ia + + + + + + + + + + + + + + + + + + + + + + + + 7 a m or ph op ha llu s ti ta nu m ( b ec c. ) b ec c. e x . a rc an g a ra ce ae b b g + + + + + + + + + 8 c al ad iu m b ic ol or ( a it o n ) v en t. a ra ce ae ip b + + + + + + + + + + + + + + + + + + 9 c ol oc as ia e sc ul en ta ( l .) s ch o tt a ra ce ae ip b + + + + + + + + + + + + + + + 1 0 d ie ff en ba ch ia f ou rn ie ri n .e . b r. a ra ce ae b b g + + + + + + + + + + + + + + 1 1 d ra co nt iu m g ig as ( s ee m .) e n gl . a ra ce ae b b g + + + + + + + + + + + + + + 1 2 h om al om en a pe nd ul a (b lu m e) b ak h . f. a ra ce ae b b g + + + + + + + + + + + + + + + + + + + 1 3 ip om oe a ba ta ta s (l ) l am . c o n vo lv u la ce ae b b g + + + + + + + + + + + + + + + 1 4 m an ih ot e sc ul en ta c ra n tz e u p h o rb ia ce ae b b g + + + + + + + + + 1 5 n el um bo s p p . a ra ce ae t an as h i f ar m , u t + + + + + + + + + + + + + + + + 1 6 s ch is m at og lo ti s ca ly pt ra te ( r o x b .) z o ll . & m o ri tz i a ra ce ae b b g + + + + + + + + + + + + + + + + + + + + 1 7 s pa th ip hy llu m c an na ef ol iu m s ch o tt a ra ce ae b b g + + + + + + + + + + + + + + + + + + + 1 8 s ol an um l yc op er si cu m l . s o la n ac ea e t an as h i f ar m , u t + + + + + + + + + + + + + + 1 9 s ol an um t ub er os um l . s o la n ac ea e c ia n ju r, i n d o n es ia + + + + + + + + + + + + + + + + + + + 2 0 t ac ca s p p . a ra ce ae b b g + + + + + + + + + + + + + + + 2 1 x an th os om a sa gi ti fo liu m ( l .) s ch o tt . a ra ce ae b b g + + + + + + + + + + + + + + + + + + + + + 2 2 z am io cu lc as z am ii fo lia e n gl . a ra ce ae b b g + + + + + + + + + + + + + + + + + z n o te : = o n e in d iv id u al p er s p ec ie s b b g = b o g o r b o ta n ic al g ar d en u t = u n iv er si ty o f t o k yo -j ap an ip b = in st it u t p er ta n ia n b o g o r '– ' = n o a m p li fi ca ti o n '+ ' = am p li fi ca ti o n s in gl e b an d '+ + ' = m u lt ip le b an d s a m p li fi ca ti o n b an d d id n o t a lw ay s p ro d u ce m ic ro sa te ll it e al le le biotropia vol. 25 no. 1, 2018 number of effective alleles of a, b and c populations were 5.43, 7.36 and 6.71, respectively. b population showed higher allelic richness as compared to a and c populations. more than one specific allele per locus existed in each population. out of 147 alleles generated by 14 loci across populations, 61 alleles were specific to particular population. a, b and c populations had 16 (26.2%), 27 (44.3%) and 18 (29.5%) specific alleles, respectively. h value was the highest in a o population, while h value was the highest in c e population. fixation indices (f ) averaged for is each population across all loci had high values ranging from 0.240 to 0.294 (table 4). amova analysis showed low f values both st within and among populations. f value across all st populations was 0.026, whereas f values among st a-b, a-c and b-c populations were 0.022 ( = p 0.009), 0.034 ( = 0.003) and 0.025 ( = 0.001), p p respectively, suggesting that each population was slightly conserved. on the other hand, variation among individuals within population was high (64.0%). according to jansen (1996) and et al. yuzammi (2000), has large phenotypic a. variabilis variations in leaf size, petiole and inflorescence color. the populations exhibited low genetic distance based on nei (1972), i.e. 0.151 for population a to population b, 0.210 for population a to population c and 0.149 for population b to population c. individuals across all populations were clustered in three groups (fig. 2). group i composed of accessions from a and c populations. group ii composed of accessions from a, b and c populations. group iii contained only one accession from population c, i.e. c22. population b was exclusively clustered under subgroup ii. in each population, with exception for b28, c1, c4, c6 and c7, accessions clustered in the same group were generally located close to each other (fig. 1). accessions a10, a11 and a12 from a population were clustered in subgroup ii with those from b population. these accessions were geographically close to each other (fig. 1). this suggests that genetic exchange via pollen occurred between accessions located at the peripheral zone of a population, as stated by pasquet . (2008). sugiyama dan santosa (2008) et al stated that nitidulidae insect becomes important pollinator in .a. variabilis morphological variations in petiole (table 1) was unlikely correlated with microsatellite profiles. accessions with green petiole without spot, i.e. a1, a2, a8, b11, c1, c10, c12 and c20 were clustered into two different groups, i.e. group i and group ii. this finding was in disagreement with the results of santosa et al. (2012), where flower size, leaf size and the presence of petiole spot were tightly linked with aflp grouping. the discrepancy between the result of santosa et al. (2012) and this study could be due to the fact that aflp is a dominant marker. in a. paeoniifolius, petiole color, shape and color of spot and petiole roughness are affected by soil fertility (sugiyama & santosa 2008). in the previous work, santosa et al. (2004) grouped a. variabilis accessions from west java province, indonesia based on inflorescence morphology into four groups. therefore, it is important to conduct further study to investigate whether petiole and peduncle colors in a. variabilis are also affected by soil fertility. table 4 description for three amorphophallus variabilis populations collected from dramaga conservation forest, bogor, indonesia population number of allele ho he fis effective richness specific a (n=11) 5.43 3.56 1.14 0.481 0.645 0.240 b (n=21) 7.36 4.58 1.93 0.464 0.667 0.282 c (n=15) 6.71 4.20 1.29 0.465 0.681 0.294 note: f = fixation index calculated using formula: (h h ) / h or 1 (h / h )is e o e o e n = number of sample, evaluated using 10 microsatellite loci from a. variabilis and two loci from a. paeoniifolius 29 isolating microsatellite from amorphophallus variabilis and its application santosa et al. conservation strategy three populations evaluated in dcf exhibited unique genetic feature. according to an interview with administrative staff members of dcf, there was no intercropping program, harvesting or any man-made disturbance on the a. variabilis populations. unexpectedly, during field survey, several a. paeoniifolius plants were found at dcf. according to local officer, the plants were introduced from yogyakarta by farm laborers in year 2000s. many bushes, weeds and tree seedlings grew densely inside dcf, forming shady condition and blocking more than 75% of sunshine. according to sugiyama and santosa (2008), amorphophallus species grow well under shading up to 75% shading level. it was possible that heavy shading disturbed the growth of , resulting to a. variabilis low density of the studied-population in dcf. sugiyama and santosa (2008) also stated that single mother corms of produced 14.3 a. variabilis cormlets per year. cormlets were detached from mother corm after dormancy release. two to seven cormlets usually grew close (0 13 cm) to mother corms, unlike seedlings from seeds which usually grew apart (> 60 cm) from the mother plant. considering that mature plant of a. variabilis is able to produce 25 280 seeds (sugiyama & santosa 2008), the population density could increase steadily yearly in undisturbed condition. it is interesting to conduct further study on population dynamic of a. variabilis in isolated condition, such as in dcf. in the present study, several accessions failed to amplify resulting to low number of sampling size in each population (table 4) that might cause an underestimation of population genetic. according to li (2009), the numbers of et al. effective alleles were determined by the number of sampling size and the numbers of loci used. a set of 40 samples per population is necessary for population study. during dna extraction, several leaf samples contained large amount of glucomannan. thus, it is important to improve 30 figure 2 dendrogram of upgma based on jaccard similarity index of a. variabilis constructed using microsatellite data obtained (note: a, b, and c codes represent a, b and c populations, respectively) biotropia vol. 25 no. 1, 2018 dna extraction method in the near future, to enhance the accuracy of population genetic evaluation. genetic distance between a and c populations was significantly larger than those of other population pairs. however, it was still unclear why these populations were distantly separated in terms of genetic profile. geographically, a and c populations were separated by about 200 meters. the large genetic distance suggested that exchange of genetic materials among a and c populations was restricted. interestingly, within c population, c22 seemed to be out of group (fig. 2). this might be caused by the fact that c22 was an introduced seed from other distant populations by birds as primary agent for long-distance seeds dispersal of (sugiyama & santosa a. variabilis 2008). the number of estimated genetic exchange or migration among a-b, a-c and b-c populations were 11.23, 7.21 and 9.81, respectively, and were considered as high. average number of estimated migrant (nm) based on the number of specific alleles across all populations was high, i.e. 9.39. smaller degree of genetic differentiation (f ) st among a and b, and b and c populations, indicated populations located closely to each other mostly underwent intense genetic exchange through cross-breeding. finding in dcf indicated that the a. variabilis existed as a meta-population. santosa (2012) et al. has suggested to conserve single large population in conservation strategy of , however, a. variabilis it should be further clarified by evaluating accessions from distant populations across java island using codominant microsatellite primers. many researchers, students and professional workers stated that a large number of a. variabilis plants existed in various agroecological condition in west java and yogyakarta provinces. therefore, it is interesting to evaluate population a. variabilis from different sites in java island to develop conservation strategy for a. variabilis. conclusions microsatellite primers developed in the present study were applicable for the population study of a. variabilis. three a. variabilis populations in dramaga conservation forest exhibited low genetic diversity among populations, but high genetic distance within a population. increasing number of specific allele with the increasing geographical distances among populations implies the importance to study genetic population from the larger geographical range in java island to determine the best conservation strategy for a. variabilis. acknowledgements part of this work was supported by grant-inaid from the japan society for the promotion of science (jsps), university of tokyo, japan and institut pertanian bogor (ipb), indonesia. we thank mr yuzammi from bogor botanical garden, indonesia, dr kazuhike nara and dr kimura megumi from laboratory of forest science, and dr o new lee from laboratory of horticulture sciences, university of tokyo, japan, for their kind technical assistances. we thank bogor botanical garden and center for international forestry research (cifor), bogor, indonesia for providing plant materials. references abdul-muneer pm. 2014. application of microsatellite markers in conservation genetics and fisheries management: recent advance in population structure analysis and conservation strategies. g e n e t re s i n t e r n a t 2 0 1 4 : i d 6 9 1 7 5 9 . doi:10.1155/2014/ 691759 alonso-sande m, teijeiro-osorio d, remuñán-lópez c, alonso mj. 2009. glucomannan, a promising polysaccharide for biopharmaceutical purposes. eur j pharm biopharm 72:453–62. csencsics d, brodbeck s, holderegger r. 2010. coste f f e c t i ve , s p e c i e s s p e c i f i c m i c r o s a t e l l i t e development for the endangered dwarf bulrush ( ) using next-generation sequencing typia minima t e c h n o l o g y. j h e r e d 1 0 1 ( 6 ) : 7 8 9 – 9 3 . 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(in indonesian) yuzammi. 2000. a taxonomic revision of the terrestrial and aquatic (araceae) in java [thesis]. retrieved aroid from school of biological science, faculty of life science, university of new south wales. 359 p. zhang sl, liu py, sun ym. 1998. artificial adjustment of flower period and hybridizing techniques of amor phophalus konjac. acta bot yunn 0. suppl(5):62–6. zhou z, miwa m, hogetsu t. 1999. analysis of genetic structure of a population in a suillus grevillei larix kaempferi stand by polymorphism of inter-simple sequence repeat (issr). new phytol 144:55–63. 32 biotropia vol. 25 no. 1, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 fiber disruption of betung bamboo ( ) by combined fungal and dendrocalamus asper microwave pretreatment widya fatriasari *, wasrin syafii , nyoman wistara , khaswar syamsu ,1 2 2 3 bambang prasetya , s. heris anita and lucky risanto4 1 1 1research center for biomaterials, indonesian institute of sciences (lipi), jalan raya bogor km 46 cibinong, bogor 16911, indonesia 2department of forest product technology, faculty of forestry, institut pertanian bogor, bogor 16680, indonesia 3department of agro-industrial technology, faculty of agricultural engineering and technology, institut pertanian bogor, bogor 16680, indonesia 4 thnational standardization agency, manggala wanabakti building blok iv, 4 floor, jalan gatot subroto, senayan, jakarta, indonesia received 22 january 2014/accepted 3 december 2015 abstract combined microwave pretreatment is an attractive method to alter carbohydrate and lignin structure of fungal and lignocellulosic materials for improving hydrolysis process to convert these lignocellulosic materials to bioethanol. this study was conducted to obtain information on the and lignin characteristic changes after carbohydrate combined biological microwave pretreatment of amboo. based on our previous research, incubation for 30 days and betung b using 5 and 10% (w/v) inoculum loading of white rot fungi, which has better delignification trametes versicolor selectivity compared to the other incubation time, was chosen as the pretreatment prior to microwave fungal pretreatment for 5, 10 and 12.5 minutes at . the evaluation of characteristic changes after pretreatment was 330 w performed using the analysis of ftir spectroscopy, x-ray diffraction and sem. ftir spectra demonstrated that the combined change pretreatment only affected the of intensity bands of ftir spectra, without any changes in the functional groups. r unconjugated bonds of carbohydrate peaked at 1,736 cm this band intensity decrease occu red on -1 (c = 0 in xylan), 1,373 cm (c-h deformation in cellulose and hemicellulose), 1,165 cm (c-o-c vibration in cellulose -1 -1 and hemicellulose) 895 cm (c-h deformation or c-o-c stretching at β-glicosidic linkage characteristic in and -1 cellulose) the pretreatment decreased the hydrogen bond stretching of cellulose and the linkage between lignin and . carbohydrate associated with crystallinity of bamboo cellulose l. this decrease of hydrogen bond was , i lustrated by occurring structural changes. the crystallinity tended to increase slightly due to the cleavage of the amorphous index fraction. sem image illustrated that the pretreatment disrupted the fiber structure. the longer duration of microwave s irradiation, the greater the degradation level of fiber. : keywords betung bamboo, and lignin changes, biological microwave pretreatment, carbohydrate combined and ftir, sem, xrd introduction increasing concern of greenhouse gas emission and the depletion of fossil fuels have been considered as the main driving force in exploring renewable energy sources (hu & wen 2008; zhang 2008). abundant lignocellulosic materials are potential bioresources to produce liquid biofuel, such as bioethanol. however, the recalcitrance nature of biomass due to the presence of lignin and cellulose crystalline structure prevents optimum enzyme penetration during hydrolysis. effective pretreatment prior to hydrolysis stage is required to improve biomass digestibility. pretreatments are emphasized mainly to increase feedstock surface area and porosity, as well as to reduce cellulose crystallinity, lignin content and hemicellulose content (mosier . 2005; galbe & et al zacchi 200 ; wyman . 2007; cara . 2008). 7 et al et al an effective pretreatment of biomass is indicated * corresponding author : widya_fatriasari@yahoo.com biotropia vol. 22 no. 2, 2015: 81 94 81 doi: 10.11598/btb.2015.22.2.363 mailto:widya_fatriasari@yahoo.com 82 by sug ar release improvement, reduced carbohydrate degradation and the lack of inhibitor y by-products such as furfural, hydroxymethyl furfural (hmf) and organic acids formation ( ) kuhnel . 2011; agbor . 2011et al et al and also be cost-effective ( &yang wyman 2008; agbor . 2011et al ). bamboos are versatile fast growing species of c plant type with very efficient photosynthesis 4 ability. theoretical value of c photosynthesis is 4 approximately 8%. biomass productivity of bamboo is about 20-40 ton s/ha/year. it is ne approximately 7-30% higher than that of woody plants (kant 2010) and other energy crops such as poplar, switch grass, miscanthus, common reed and bagasse (sathitsuksanoh . 20 ; zhang et al 10 2008). bamboos are distributed in the tropics, subtropics and temperate zones ( . lobovikov et al 2007) and cover 1% of the world's forest area (kant 2010). most of bamboo population (65%) grows in asia, especially in indonesia with 160 bamboo species (widjaja 2001). this ranks third (5%) in world bamboo's population after china (14%) and india (30%) (lobovikov . 2007). et al betung amboo is considered among the most b important species in indonesia (dransfield & widjaja 1995). previous study of six indonesia's bamboo species demonstrated that fiber morphology, physical and chemical properties of betung bamboo were better than those of kuning, tali, andong, ampel and black bamboos (fatriasari & hermiati 2008). after single pretreatment, enzymatic hydrolysis in simultaneous saccharification and fermentation (ssf) can be applied to produce bioethanol. however, single pretreatment tends to produce low sugar yield, consume time and require high production cost due to enzyme requirement in saccharif ication process. combined biological-microwave pretreatment could improve ethanol yield of biomass via ssf method. white-rot fungi used in biological pretreatment degrade lignin polymer by secreting ligninolytic enzyme nazarpour (zhang 2007; et al. et al. 2013). an appropriate fungal strain is needed to obtain a satisfying delignification selectivity and enzymatic hydrolysis yield. delignification selectivity of betung bamboo with trametes versicolor was found to be better than that with and pleur otus ostr eatus phaner ochaete chrysosporium et al (fatriasari . 2011; falah . et al 2011). microwave radiation of lignocellulosic material in aqueous environment is also found promising (kheswani . 2007). this et al pretreatment method has been applied for switch grass, bagasse, rice straw, woody plants, oil palm empty fruit bunch, oil palm trunk and frond (hu & wen 2008 keshwani 2007; anita . ; et al 2012; risanto . 2012; lai & idris 2013). the et al advantages of the method include short processing time and high product yield and quality (hermiati . 2011). microwave et al pretreatment supplies direct internal heat to biomass resulted from polar bond vibration as they align with the magnetic field (kheswani .et al 2007). microwave pretreatment can increase ion production, solubilize non-polar material and hydrolyze biomass without catalyst (tsubaki & azuma 2011). in a study on biological and microwave pretreatment of etung amboo, incubation for b b 30 days in biological pretreatment resulted in high lignin removal and less cellulose loss (fatriasari et al. 2014a). furthermore, microwave pretreatment of biomass at utes330 w for 5, 10 and 12.5 min resulted in lower of the aweight loss cellulose and hemicellulose other compared to that of pretreatment conditions (fatriasari 2014b)et al. . to the best of our knowledge, no study has been reported on the changes lignin and of carbohydrate structure after combined biologicalmicrowave pretreatment. this study was conducted to obtain information on the carbohydrate and lignin characteristic changes after biological microwave combined and pretreatment of amboobetung b . materials and methods material preparation bet ung b amboo ( dendr o calamus asper (schult.f.)) of less than 2 years old was collected from bamboo plantation of the research center for biomaterials lipi, cibinong, indonesia. the bark of the collected bamboo was removed and the barkless bamboo was chipped before being ground into fine powder. the powder was sieved to obtain 40-60 mesh bamboo meal and then stored in a sealed plastic bag at room temperature. betung bamboo meal was subjected to biological pretreatment before microwave irradiation. biotropia vol. 22 no. 2, 2015 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. bamboo sample preparation bamboo meal was watered with ratio of 1:4 and then manually stirred until completely mixed. the wet bamboo-meal was then put in a jar and steamed for 30 minutes at approximately 100 c o and finally in an for 20 minutes sterilized autoclave at 121 c.o inoculum stock preparation fungus inoculum was cultured on t. versicolor malt extract agar (mea) slant (10.65 g of mea were diluted in 300 ml distilled ) for water 7-14 days. 5 ml at the end of incubation period, of jis (japan industrial standard) broth medium 3 g kh po , 2 g mgso .7h o, 25 g ( 2 4 4 2 glucose, 5 g pepton and 10 g malt extract diluted into 1 l of distilled water) was injected to the then scratched with loop to release slant and the mycelium from the slant agar. resulting as much as of previously prepared 5 ml fungi suspension was then poured into the remaining 95 ml of the jis broth medium and stationery incubated at 27 c for 7 days. after incubation, o 8 10 g of corn steep liquor was poured into the 100 ml inoculum and homogenized twice with a high speed waring blender (each homogenization was conducted for 20 seconds). inoculation method bamboo-meal 15 g ied ) having ( oven dr weight 7.46% moisture content was inoculated with 5 and 10% (w/v) iedinoculum of dr bamboo and incubated at 27 c for 30 dayso . microwave pretreatment microwave pretreatment was carried out in an oven microwave sharp p-360j (s) with 2,450 mhz frequency and power output of 1,100 w. as much as 1 g of oven-dried pretreated sample was inserted into a teflon tube (vessel), added with distilled water to obtain a solid-toliquid ratio (slr) of 1:30 (w/v) and stirred for 15 minutes. subsequently, the sample was exposed to microwave irradiation at 330 w for 5, 10 and 12.5 minutes. after microwaving, the pulp was removed from the oven and immediately put into iced water for 15-20 minutes to cool the pulp. the residue (solid fraction) was separated from the hydrolysate (liquid fraction) by filtration. the changes of content, morphological, cellulose and lignin characteristics chemical component determination p rior to d eter mination of chemic al component of control and pretreated bamboo, the moisture content samples were measured of following the procedures of tappi t12 os-75 . free extractive bamboo-meal was prepared with ethanol-benzene (1:2) extraction for chemical component analysis. acid-insoluble lignin, acid soluble lignin, holocellulose, a-cellulose and ash content were determined in accordance with the tappi t13 os-54, , tappi tappi um 250 t9m-54, and t 15 os-tappi t17m-55 tappi 58 standards, respectively. the calculation of weight loss following method of was done the pandey pitman (2003), while selectivity value & was calculated as ratio of lignin loss to the cellulose loss (yu . 20 )et al 09 . cellulose crystallinity index determination the crystallinity was determined using index diffraction intensity data of x-ray diffraction (xrd) according to the formulation of zhou et al. (200 ). measurement was carried out with 5 shimadzu xrd-700 maximax series. ni radiation was filtered by cuk at 0.15406 nm wave α number. x-ray was operated at 40 kv of voltage, 30 ma of electrical current and scanned 2 theta ( )0 of 10-40 in 2 minute.o o per allomorphic structure of cellulose z-discriminate function 200 ) was (hult . 3et al used to differentiate allomorphic properties of cellulose crystalline structure. the function was built up by separating cellulose i and i using d-α β spaces obtained from x-ray analysis, i.e. two equatorial d-spacing: 0.59-0.62 (d ) and 0.52-0.55 1 nm (d ). z > 0 indicates bacteria algae type (i , 2 α rich triclinic structure) and z < 0 indicates cotton and flax types (predominantly i structure/ β monoclinic). crysta lite size of cellulosel the cr ystallite size of cellulose was determined using diffraction pattern obtained from 101 , 10-1 , 002 and 040 lattice planes ( ) ( ) ( ) ( ) of bamboo ).(zhao . 2007et al morphological structure analysis morphological structure of pretreated bamboo was analyzed through sem micrograph obtained by a jeoul/eo sem. bamboo 83 sample was installed in the sample holder (stub) using sputter canter and then scanned at 15 kv with 10 mm of working distance with 750x and 10,000x of magnification. biodegradation pattern biodegradation pattern was analyzed through fourier transform infrared spectrometry (ftir) spectrograph. to obtain ftir spectra, 4 mg of bamboo-meal was embedded in 200 mg of kbr (potassium bromide) spectroscopy grade and then pelletized at 5,000 psi. the diameter and thickness of the pellet were approximately 1.3 cm and 0.5 cm, respectively. infrared spectrum patterns (peak height and area) were analyzed by using ftir abb mb 3000. all of the spectra were recorded at a spectral resolution of 16 cm -1 with the accumulation of 5 scans per sample with absorption mode in the range of 4,000-500 cm . -1 the characteristic of carbohydrates and lignin was analyzed based on the relative band intensity change referring to the method of pandey (pandey & pitman 2003). peak height and area values of lignin associated bands were rationed compared to carbohydrate reference peaks at 1,720; 1,366; 1,180 and 879 cm to provide relative -1 changes in the composition of the structural components relative to each other determined using horizon mb software. statistical analysis all experiments were performed in triplicate. sample preparation for sem, ftir and xrd analyses has been conducted by manually mixing all triplicate treated and untreated samples. the pretreatment combination effects on chemical component changes and losses were analyzed by anova (analysis of variance) using minitab release 13.2 software. significant differences among treatment combinations were evaluated using tukey's multiple range comparisons at < p 0.05. results and discussion chemical content of pretreated bamboo the chemical component composition change of pretreated bamboo is depicted in figure 1 which shows that bamboo has high a-cellulose content. cellulose is the main source of c-6 sugar convertible to ethanol. in this study, combined biological-microwave pretreatment was utilized to reduce lignin content. the pretreatment was expected to degrade lignin and hemicellulose. removal of the c-5 hemicellulose could increase sugar fermentation by saccharomyces cerevisiae considering that the c-5 sugar of hemicellulose cannot be efficiently fermented by the yeast. 84 ds : 2.91 0.46 1.08 0.77 0.59 1.14 wl asl ail hc ac e 100% 80% 60% 40% 20% 0% control biological-microwave pretreatment 5% il for 5 min 5% il for 10 min 5% il for 12,5 min 10% il for 5 min 10% il for 10 min 10% il for 12,5 min figure 1 chemical component composition change of bamboo after biological-microwave pretreatment. components: s il (inoculum loading); wl (weight loss); asl (acid soluble lignin); ail (acid insoluble lignin); hc (hemicellulose); ac (alpha cellulose); e (ethanol-benzene extractive); ds (delignification selectivity) biotropia vol. 22 no. 2, 2015 h e m i c r o w a v e t c o m b i n e d f u n g a l pretreatment changed the compositionchemical of pretreated bamboo of (fig. 1). pretreatment 10% inoculum loading show lower weight loss ed or higher yield inoculum loading. than that of 5% longer irradiation time tended to increase weight loss. might be related more intensive this to a lignin degradation activity than carbohydrate removal in higher inoculum loading. total weight loss was approximately of 5.47-19.88% tat stical . s i analysis indicated that inoculum loading gave only significant effect to alpha cellulose and hemicellulose content, weight loss and hemicellulose loss only . on the other hand, irradiation time gave significant effect on weight loss and lignin loss . based on ukey's ( < 0.05) tp pairwise comparison, weight loss due to irradiation time differen . were significantly t i nteraction between inoculum loading and irradition time weight loss. significantly affected however, no interaction between inoculum effect loading and irradition time on the alpha cellulose loss . prolonging irradiation time for 10 was found min affected decrease of apha cellulose utes the loss for both inoculum loading. even though, the irradiation time for 12.5 min caused higher utes lignin degradation, also caused higher alphait cellulose loss. thus, greater extend of irradiation time was not required. the highest selectivity value (up to 2) was found after bamboo pretreated 5% was with inoculum loading and then irradiated for 5 was min . a higher selectivity value indicate that utes s lignin polymer is more effective than cleavage cellulose degradation. tatistical analysis s of the present results dindicate that the inoculum loading and irradiation time did not significant ly affect selectivity value ( < 0.05).p degradation of carbohydrate (alpha cellulose) also occurred during delignification activity (fig. 1). it might be due to partial hydrogen bond disruption of the lcc (lignin carbohydrate complex) (li . 2010). bet al iological pretreatment caused opening complex of the lignocellulose structure through depolymerization of lignin and brought about carbohydrates increasing accesibility. icrowave pretreatment after m biological pretreatment also help toed alter the ultrastructure of cellulose and degrade lignin and hemicellulose in lignocellulosic materials that bring about increasing susceptibility of lignocellulosic materials (binod . 2012). et al microwave heating transfers and induces heat directly into bamboo substrate, causing the depolymerization of sugar building block into oligosaccharides (ebringerova 2006). under acid pretreatment at high pressure and temperature, sugar monomer such as glucose and xylose can be further degraded in hydroxymethyl furfural and furfural (hmf) behera . 2014) ( . et al the degradation product can be released during acid pretreatment condition. more severe of microwave pretreatment condition led to decrease hemicellulosic monosaccarides in hydrolyzate and increase the formation of sugar degradation product (kuhnel 2011). variouset al. potential s edcompound in hydrolyzate consist acetic acid, formic acid, furan derivatives of (5-hmf and furfural) and phenolic compounds might be wasgenerated. the inhibitor presence not excepted the effect of limationdue to efficient process (zhang . 2011; fermentation et al talebnia . 2010). however, due to our research et al focus to observe the change of chemical was component and cellulose structure of pretreated samples, this potential inhibitor on hydrolyzate was d not observe . cellulose structure changes of pretreated bamboo ftir spectroscopy was used to investigate changes in the chemical structure of pretreated samples (fig. 2 3). slight changes occurred in and spectrum peaks of biomass were treated with both 5 and 10% inoculum loading, but no changes appeared in the functional groups during pretreatment. it might be caused by uncompleted disruption of lignin that encapsulated cellulose. the broad absorption was observed at the wave number of around 3,340 cm . this wave number -1 was assigned to hydrogen bond (o-h) stretching absorption. o-h stretching region at the wave number of 3,000-3,600 cm of pretreated -1 bamboo spectra was more identical to the o-h stretching region from cellulose i. the band at 2,700-2,901 cm is related to the c-h stretching -1 (pandey & pitman 2003). biological-microwave pretreatment affected the peak area and bandheight of 3,340 cm wave number (o-h -1 stretching) (fig. 2 and 3). it indicated a weak intra and inter molecular bond of o-h group (goshadrou . 2011).et al 85 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. ftir spectra with frequencies in the region of 1,600 and 1,510 (aromatic ring vibration), 1,470 and 1,460 cm (c-h deformations and aromatic -1 ring vibrations) can be found in the lignin structure (fengel & wegener 1992). lignin of bamboo consisting of guaiacyl (g) and syringyl (s) propane units containing one and two metoxyl groups can be clearly observed in all treatments at wave number of 1,327 cm for syringyl propane -1 units and 1,257 cm for guaiacyl propane units. -1 the higher absorbencies in the finger print of pretreatment of both 5 and 10% inoculum loading of pretreated samples compared to control was found at irradiation of 10 minutes. the higher lignin and cellulose content in this condition (fig. 2) which can be confirmed by this spectra (fig. 3). the absorbance of syringyl (1,327 cm ) was lower than that of guaiacyl -1 86 figure 2 ftir spectra of bamboo after fungal pretreatment (5% inoculum loading for 30 days) subjected to microwave pretreatment figure 3 ftir spectra of bamboo after fungal pretreatment (10% inoculum loading for 30 days) subjected to microwave pretreatment biotropia vol. 22 no. 2, 2015 (1,257 cm ) indicating a higher syringyl content in -1 control and treatments. the typical infrared band frequencies and ftir spectras of bamboo components in units of wave numbers are listed in table 1. the sharp bands around 895 cm is attributed -1 to β-glicosidic linkage between the sugar units in cellulose (nelson & o’connor 1964) which can be clearly seen in all spectra. the increasing microwave irradiation reduced band intensity of functional group (c=o) in hemicellulose (3), c-h in cellulose and hemicellulose (9), and c-o-c in hemicellulose (12). it might be attributed to the decrease of hemicellulose content after pretreatment along with lignin. sixty functional groups can be observed in six pretreatment conditions. each identified functional group can be found in all treatments, although a slight shift in wave number occurred. the treatments decreased functional groups intensity without changing functional group types. effect of combined fungal and microwave pretreatment on bamboo morphology sem micrograph of pretreated samples (fig. 4 and 5) was used to observe morphological features and surface characteristics of pretreated bamboo with increasing microwave irradiation. 87 table 1 assignments of ir band of bamboo after biological-microwave pretreatment no control* biological pretreatment assignments 5% of inoculum loading 10% of inoculum loading microwave pretreatment 5 minutes 10 minutes 12.5 minutes 5 minutes 10 minutes 12.5 minutes wave number (cm-1) 1 3,394 3,340 3,333 3,418 3,418 3,425 3,364 a strong and broad hydrogen bond (o-h) stretching absorption1 2 2,901 2,901 2,901 2,901 2,901 2,901 2,901 a prominent c-h stretching absorption1 3 1,736 1,728 1,736 1,736 1,728 1,728 1,713 unconjugated c=o in xylans 1 4 1,643 1,643 1,651 1,651 1,643 1,643 1,643 absorbed o-h and conjugated c-o 1 aromatic skeletal1 5 1,605 1,605 1,605 1,605 1,605 1,605 1,605 aromatic skeletal1 6 1,512 1,512 1,512 1,512 1,512 1,512 1,512 7 1,458 1,458 1,458 1,458 1,458 1,458 1,458 c-h deformation 1 8 1,427 1,427 1,427 1,427 1,427 1,427 1,427 c-h2 scissoring motion1 9 1,373 1,373 1,373 1,373 1,373 1,373 1,373 c-h deformation1 10 1,335 1,327 1,335 1,335 1,327 1,327 1,327 c-h vibration1 c1-o vibration in syringyl derivates 1 11 1,257 1,257 1,257 1,250 1,257 1,257 1,250 guaiacyl ring1 c-o stretch1 12 1,165 1,165 1,165 1,165 1,165 1,165 1,165 c-o-c vibration1 13 1,111 1,111 1,111 1,111 1,111 1,111 1,111 aromatic skeletal and c-o stretch1 14 1,049 1,041 1,034 1,041 1,041 1,041 1,041 c-o stretch1 15 895 895 895 895 895 895 895 c-o-c stretching at β-glicosidic linkage or c-h deformation in cellulose2 16 833 833 833 833 833 833 833 c-h vibration3 notes: control* = data has been used in other paper (fatriasari . 2014b)et al 1 = pandey and pitman (2003) 2 = chen (2011)et al. 3 = cheng . (2013)et al fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. sem images of samples treated with both 5 and 10% inoculum loading show that partial disruption occurred on the fiber structure. degraded lignin polymer and removal of hemicellulose in cell wall might responsible to be this disorganized morphology. longer microwave irradiation caused greater fiber degradation level. the cell wall morphology changes caused by lignin removal resulted in greater deconstruction of fiber surface, providing better cellulose penetration. partial degradation of lignin and hemicellulose destroyed some ether bonds in lignin and lignin-carbohydrate complex leading to the disruption of the hydrogen bond between cellulose, thus fibrillation occurred (li . 2010). et al the cellulose digestibility can be potentially enhanced by preferential cleavage of lignin (nazarpour . 2013). treatments of 5% et al 88 10 min utes (330 w) 12.5 min utes (330 w) 5 minutes (330 w) a b 10 minutes (330 w) 12.5 min utes (330 w) 5 minutes (330 w) a b figure 4 sem micrograph pretreated bamboo (5% inoculum loading for 30 days) subjected to microwave pretreatment of with (a) 750x and (b) 10,000x magnification figure 5 sem micrograph pretreated bamboo (10% inoculum loading for 30 days) subjected to microwave pretreatment of with (a) 750x and (b) 10,000x magnification biotropia vol. 22 no. 2, 2015 inoculum loading for 10 and 12.5 minutes microwave irradiation resulted in more openedup sponge-like structures providing wider surface area to increase the rate of subsequent hydrolysis reactions. cellulose structure allomorph crystalline allomorph of pretreated bamboo as result of mixing triplicate samples a observed by xrd is presented in table . in general, 2 cellulose consists of i (one-chain triclinic) and i α β (two-chain monoclinic cells), which can be determined by z-discriminant. z < 0 and z > 0 indicate the i and i allomorph types, respectively β α . monoclinic (i ) cellulose is more stable than β triclinic (i ), and tends to be the final product in α annealing treatment credou of all celluloses ( & barthelot . 2014) all treatments except for the 5% inoculum loading for 10 minutes had monoclinic structure. however, the cause of this phenomenon has not yet understood the presence of the i phase been . α was expected to improve cellulose digestibility due to its higher degradation than that of i . in β addition, this structure is meta-stable and more reactive than i ( ).β moon . 2011et al bio of ombined degradation patter n c biological-microwave p tretreatmen biodegradation patterns of bamboo during combined biological-microwave pretreatment were evaluated by ftir analysis 6 7 . (figure and ) a detailed ftir spectroscopic analysis based on pandey's analysis method was performed to calculate relative intensities of aromatic skeletal vibration against typical bands of carbohydrate on pretreated bamboo (pandey & pitman 2003). 89 table 2 crystalline allomorph of pretreated bamboo biological pretreatment microwave pretreatment crystallite allomorph crystal allomorphinoculum loading (%) power loading (w) microwave irradiation (minutes) d (101) nm d (10-1) nm z control* 0.5824 0.5349 -45.47 iβ 5 330 5 0.5987 0.5466 -28.49 iβ 10 0.6110 0.5230 13.69 iα 12.5 0.5955 0.5473 -34.50 iβ 10 5 0.5520 0.5163 -80.11 iβ 10 0.5583 0.5134 -66.92 iβ 12.5 0.5740 0.5181 -44.48 iβ note: control* = data have been used in other paper (fatriasari . 2014 )et al b fig 6 ftir spectra of bamboo after biological pretreatment (5% inoculum loading for 30 days) subjected to microwave ure pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. relative changes in the intensities of aromatic skeletal in lignin peaks at 1,512 cm against four -1 unconjugated bonds of carbohydrate peaks at 1,736 cm (c=o in xylan), 1,373 cm (c-h -1 -1 deformation in cellulose and hemicellulose), 1,165 cm (c-o-c vibration in cellulose and 1 hemicellulose), 895 cm (c-h deformation or c--1 o -c str e tch ing at β -g lic os id ic l in kag e characteristics in cellulose) calculated by peak heights and areas are summarized in table .3 in 5% inoculum loading, increasing microwave irradiation period tended to increase the ratio of lignin/carbohydrate. it indicated that prolonged microwave irradiation decreased its lignin degrading ability of pretreated bamboo. carbohydrate degradation after pretreatment contributed to this phenomenon. crystallinity index (ci) and crystallite size of cellulose crystalline and amorphous structure of cellulose can be identified from primary peak of xrd pattern ranging from 22-23 and 0 secondary peak in the range of 16-18 (lai & idris 0 2013; liu . 2012). these peaks can be et al determined within the mentioned range for all treatments, which indicates that the crystalline and amorphous region of cellulose. the crystallinity index of pretreated bamboo has been used to interpret changes of cellulose after pretreatment (table 4). intensity transformation in hydrogen bonding of cellulose can be reflected from width variation of crystallization peak. 90 fig 7 ftir spectra of bamboo after biological pretreatment (10% inoculum loading for 30 days) subjected to microwave ure pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1 table ratio of intensity of lignin-associated band with carbohydrate bands of pretreated 3 bamboo biological pretreatment microwave pretreatment relative intensities a of aromatic skeletal vibration (i1,512) againts typical bands for carbohydrates inoculum loading (%) power loading (w) irradiation time (minutes) i1,512/i1,736 i1,512/i1,373 i1,512/i1,165 i1,512/i897 control* 1.04(1.06) 1.02(1.06) 0.98(0.95) 1.28(1.29) 5 30 5 1.24(0.83) 0.86(0.6) 0.67(0.33) 1.74(3.75) 10 1.49(1.19) 0.88(0.57) 0.64(0.34) 1.59(3.57) 12.5 1.74(1.88) 0.94(0.81) 0.58(0.48) 3.26(2.5) 10 5 1.42(1.37) 0.83(0.74) 0.59(0.48) 1.55(1.51) 10 1.28(0.75) 0.78(0.48) 0.56(0.29) 1.58(4.0) 12.5 1.36(1.33) 0.84(0.77) 0.57(0.47) 1.73(1.69) note: control* = data have been used in other paper (fatriasari . 2014 )et al b biotropia vol. 22 no. 2, 2015 the crystallinity index tended to rise along with the increasing microwave irradiation. in biomass, cellulose contains crystalline area, while lignin and hemicellulose are amorphous in nature (o'dowyer . 2007). the increasing of et al cr ystallinity index might be caused by solubilization of amorphous component from the fibers under pretreatment condition (kim & holtzapple 2006). this phenomenon was supported by the occurring component loss in lignin as presented in figure 1. the increase of crystallinity index after pretreatment was also described in previous research (singh 2014; et al. bak . 2009).et al crystallinity index of cellulose is among the most important properties of lignocelluloses that can be measured by ftir spectroscopy. crystallinity index changes can be studied from loi (lateral order index), defined as the absorbance ratio of a to a (oh . 2005), 1,427 895 et al obtained from ftir spectra data. increasing of microwave irradiation time tended to increase loi. it might be attributed to higher amorphous region of cellulose compared to crystalline region. this phenomenon was in line with crystallinity index measured by xrd analysis. crystallite size of cellulose in bamboo varied at lattice planes of (101), (10-1) and (002). the crystallite size ranged from 5.19 to 10.68 (table 5) . the highest crystallite size of cellulose at (002) lattice plane was found in pretreatment of 5% inoculum loading for 12.5 minutes. crystallite size at lattice planes (101) and (10-1) of several pretreated bamboo can not be calculated because s there no value of full width at half were maximum (fwhm) in this peak. the microwave irradiation duration increaed crytallite size at (002) lattice plane. the highest crystalline length 91 table 4 crystallinity index (ci) and lateral order index (loi) of pretreated bamboo biological pretreatment microwave pretreatment crystallinity index (ci) lateral order index (loi) inoculum loading (%) power loading (w) irradiation time (minutes) fc (crystaline) fa (amorf) ci a1,427 (crystalline) a897 (amorf) loi control* 0.69 2.13 24.58 0.50 0.40 1.25 5 330 5 0.99 1.48 40.19 0.98 0.51 1.92 10 1.23 1.72 41.76 1.43 0.84 1.70 12.5 1.12 1.54 42.04 0.74 0.23 3.22 10 5 1.16 1.74 39.98 1.14 0.65 1.75 10 1.13 1.63 40.96 0.75 0.40 1.88 12.5 0.93 1.34 40.84 1.20 0.63 1.91 note: control* = data have been used in other paper (fatriasari . 2014 )et al b table 5 crystallite size of pretreated bamboo biological pretreatment microwave pretreatment crystallite size (nm) inoculum loading (%) power loading (w) irradiation time (minutes) d (101) d (10-1) d (002) d (040) controla 5.46 8.71 5.59 16.52 5 330 5 ndb 6.57 5.17 20.99 10 14.95 7.07 5.74 38.80 12.5 8.69 4.86 6.19 16.51 10 5 ndb 5.32 5.74 86.33 10 5.20 5.36 5.82 22.85 12.5 10.68 nd b 5.47 36.98 notes: control = data have been used in other paper (fatriasari . 2014 )a et al b = b not detected fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. at (040) lattice plane of cellulose was found in the 10% inoculum loading for 5 minutes. there was no similar trend in crystalline length changes caused by increasing of microwave irradiation between 5 and 10% of inoculum loading. conclusions the characteristic changes of lignin and carbohydrate combined of betung bamboo with biological-microwave pretreatment was evaluated. the pretreatment caused the chemical loss of the component. the 5% inoculum loading irradiated for 5 min demonstrated the highest selectivity utes value (up to 2). based on ftir spectra, there was no ch ang e i n f unc ti ona l g ro ups af t er pretreatment also . moreover, ftir spectra demonstrated the presence of hydrogen bond stretching along with microwave irradiation exposure indicating the structural changes occur ed the r after pretreatment. cleavage of the amorphous d to the component contribute increasing crystallinity index disruption of fiber . structure due to pretreatments was confirmed by sem the longer duration of microwave , in which irradiation, the greater the degradation level of fiber. acknowledgements the authors would like to thank seameo biotrop for providing financial support as part of phd thesis of the first author through dipa 2013. moreover, the authors expressed their gratitude to dwi h restuningsih, st. and raden budi l.permana in for their support financial report and technical assistance. references agbor vb, cicek n, sparling r, berlin a, levin db. 2011. biomass pretreatment: fundamentals toward application. biotechno adv 29:675-85l . anita sh, risanto l, hermiati e, fatriasari w. 2012. pretreatment of oil palm empty fruit bunch (opefb) using microwave irradiation. in: the 3 rd international symposium of iwors (indonesia wood research society). proceedings: 2015 november 4; 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(gong & shun 2008; siregar 2009; isnaini . 2009; mohamed . 2010). in the interaction process between the fungus with its host, the fungus pathogenicity greatly affect the response given by the plant (mendgen & deising 1993). the response is a plant defense mechanism that serves as a physical biochemistry of plant barriers in cells and tissues that would kill or inhibit the growth of pathogens (groenewald 2005). * corresponding author : rhsiburian@yahoo.com doi: 10.11598/btb.2013.20.2.4 104 biochemical resilience is a series of biochemical reactions that occur in cells and tissues of plants in order to produce toxic substances to pathogens or create conditions that inhibit the growth of pathogens in the plant (agrios 1996). biochemical changes that may occur including the synthesis and accumulation of salicylic acid (wobbe & klessig 1996) or fitoaleksin (beynon 1997), the compound result of secondary metabolites toxic to viruses, bacteria, and fungus-like fatty acid (lowton . 1992), and issuance of oligosaccharide elicitor by the plant (nothnagel . 1983). these compounds may not only protect plants against pathogens comprehensively but can also suppress the development of pathogens without reducing production. in addition, the plant can defend itself without producing metabolites that are required by the pathogen in order to inhibit the developmental process of pathogen. research on the interaction of plant with sp. has been conducted by examining young plants/seedlings (rahayu 2009; putri 2009) but the extent of the difference between the anatomical characteristics and its content at inoculated and uninoculated plants have not been studied and reported. therefore, this study aimed to identify differences in the anatomical characteristics and content of chemical compounds between fusarium-inoculated and uninoculated of plants. plant material used was that had been inoculated three years prior to the research initiation and formed tree aloes compound at tree id# 5. samples were also selected from un-inoculated plants at the same approximate age and grown in location close to inoculated plants with id# 22. sampling was carried out by drilling trunk of wood at four cardinal directions (east, west, north and south). chemicals used were technical alcohol, technical glycerine, faa solution (37% formaldehyde: glacial acetic acid: 70% alcohol = 5:5:90), a solution of n-butanol, gifford solution (glacial acetic acid: 60% ethanol: technical glycerin = 20:80:5), 4% formaldehyde, 100 mm k hpo , 2% safranin, 1% i ki, and a solution of copper acetate [cu (ch coo) 2 h o] 50%. all of these chemicals were used to make preparative slides. the tools used were foto-microscope (nikon obtiphot 2), microscope (nikon afx-dx labophot -2), rotary microtome (yamato rv-240), sliding microtome (hm 400 microm r) and frozen microtome (yamato rv-240). observations were conducted through the microtome specimens after being stained by safranin to identify the differences in anatomical characteristics between inoculated and un-inoculated trees. wood samples were then evaluated by gas chromatography using gas chromatography mass spectrometry (gcms). wood samples from inoculated and uninoculated plants were used for anatomical observation. the samples were soaked into 70% ethanol immediately, infiltrated with 20% polyethylene glycol 2000 in ethanol, dried for 3-4 days in the oven of 60ºc, and et al et al a. microcarpa fusarium a. microcarpa a. microcarpa materials and method 2 4 2 3 2 microtome specimen preparation 105 identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. then sliced to a thickness of 12-20 in transverse, radial and tangential directions. the thin specimen obtained was stained using 2% safranin for 1-2 hours, washed and then dehydrated with ethanol 30%, 50%, 70%, 90%, and absolute consecutively for 10 minutes each. furthermore, the specimens were immersed in xylol for about 1 minute and then mounted on the object glass. after mounting, the specimen was put on a slide warmer at 40-50ºc for 1 day and ready to be observed. the procedure was based on richter (1990) after modification. characteristics of both inoculated and un-inoculated wood were observed by using photomicroscope (nikon obtiphot 2) and microscope (nikon labophot afxdx -2). the observed characteristics consist of wood color and odor, growth ring, vessel elements (shape, distribution, diameter, length, arrangement and type of perforation plate), ray parenchyma (type, width, height and frequency), axial parenchyma, intercellular canals, sediment or tyloses and mineral inclusions as well as the frequency of included phloem. results obtained in each sample were described qualitatively. encountered exudates were further tested by using gcms to determine the chemical compounds. analysis was only performed on wood sample containing sediment, using pyrolysis gcms of shimadzu gcms-qp2010. helium (0.8 ml/min.) was used as a carrier, equipped with capillary column db-5 ms (60 mm x 0.25 mm, film thickness 0.25 ), operated by electron impact (ei) mode at 70 ev and ion source temperature of 2000ºc. identification of chemical compounds was conducted on retention time and ms analysis. it was observed that anatomical characters of wood from the inoculated and uninoculated of were partly different. the difference was found in wood color, odor, deposits in vessel-element and the frequency of included phloem. in case of inoculated plants, the color of wood especially around the injection area is slightly darker than that of inoculated plants. wood odor was also somewhat different: the inoculated wood has a little bit distinct odor compared to that of un-inoculated wood. it was also observed that inoculated wood has a gold-plated resin inside their lumen. such kind of resin was not observed in the uninoculated wood. some similarities and differences in anatomical features of inoculated and un-inoculated of wood were presented in table 1. resin produced by tree was not exudated out of wood, but deposited and infiltrated inside the tissues. it then contributes to the changes in tissue color from white to dark brown in general. according to rao . (1992), a large amount of resin in case of was accumulated in included phloem, but in small amount in other parts such as trachea xylem elements, fiber cells and ray parenchyma. included phloem is a secondary phloem located in the secondary xylem (mauseth 1988). network in which the resin accumulated was well known, but the networking μm μm observation results and discussion a. microcarpa a. microcarpa a. microcarpa et al a. agalocha aquilaria biotropia vol. 20 no. 2, 2013 106 for its secretion was still unknown (mandang & wiyono 2002). it was assumed that secreting tissue of parenchyma phloem was composed by the living cells capable to store starch, fat, organic compounds, and some secondary metabolite materials such as tannins and resins (fahn 1991). table 1. observation of anatomical characters of wooda. microcarpa characters observed a. microcarpa inoculated uninoculated wood color wood odor growth ring vessel elements distribution perforation plate deposit in pores frequency of included phloem wood scent light to dark brown distinct odor unclear mostly in radial multiple and partly solitaire simple present higher scented white (light) regular unclear mostly in radial multiple and partly solitaire simple none lower unscented identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 107 aquilaria aquilaria aquilaria pinus et al et al et al fusarium included phloem was produced during secondary growth. according to blanchette (2005), included phloem is a dedicated networking capable to secrete resin. in addition to the , the similar structure was also found in nyctaginaceae and amaranthaceae. included phloem on has the same function as the resin glands on sp. it was referred as a place to resin being accumulated (nagy . 2000). included phloem that located in the secondary xylem (mauseth 1988) is a complex structure formed by the cambium to the inside, made up of filter elements, companion cells, parenchyma tissues, and fibers (rao . 1992). cell wall of included phloem components is very thin, less of lignin content so it was not stained by the safranin, and has a plate-type sieve foraminate (rao . 1992; nobuchi & siripatanadilok 1991). if close to or surrounded by the included phloem, parenchyma cells will die and function as the resin storage (fahn 1991). response of inoculated plant to the infection of sp. occurred at cellular level, tissues or organs. it was observed that in case of inoculated plant there was a large number of included phloem in which the terpenoid compound and others were accumulated as the deposit but has no plate-gold resin inside the lumen of vessel elements (figs. 1 & 2). biotropia vol. 20 no. 2, 2013 108 figure 1 cross section of (a) uninoculated and (b) inoculated. a. microcarpa figure 2 included phloem islands in (a) uninoculated (b) inoculated. a. microcarpa chemical components of a. microcarpa . the chemical compounds detected from by using gcms analysis are listed on table 2. table 3 shows similarity and also variation in the chemical composition of several agarwood oil samples. generally, agarwood oils are mixtures of sesquiterpenes, sesquiterpene alcohols, oxgyenated compounds, chromone derivatives and resins. some important compounds are agarospirol, jinkohol-eremol, jinkohol and kesenol that may contribute to aroma of agarwood (nakanishi 1984; ishihara 1993). yuan (1995) found differences in chemical components between high and low quality of gaharu. agarol was the first sesquiterpene compound isolated from aloe (yuan 1995), and oxoagarospirol, an aloe fragrance components resulting from infection of fungus on the wood. the oxoagarolspirol content in increased two months after inoculation. according to ishihara . (1991), kusunol, dihidrokanon, karanon, and oxo-agarospirol were a sesquiterpene compound isolated from low-quality gaharu. benzilaseton is a constituent of aloes identified by yang and cheng in (burfield 2005). these compounds and the compounds of kromone (also known as a constituent of aloes (yagura . 2005; konishi . 2002) start to be detected since the third day of inoculation. agarwood is a type of tree defense fitoaleksin compounds triggered its formation after the attack, then the formation of kromone benzilaseton compounds. this prompts the induction of defense responses against tree. a. microcarpa a. sinensis a. sinensis et al a. sinensis et al et al f bulbigenum table 2 agarwood components from inoculated a. microcarpa identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 109 table 3 similarity and variation in chemical compounds of several spp.aquilaria compounds authors elemol baimuxinal 3-phenyl-2-butanone chromen-4-one chen h 2011 (. et al. aquilaria sinensis lour.) chen h 2011 (. et al. aquilaria sinensis lour.) faridah s. 2009 (aquilaria maleccencis) faridah s. 2009 (aquilaria maleccencis), dai h 2009 (. et al. aquilaria sinensis lour.) biotropia vol. 20 no. 2, 2013 110 conclusions references the difference in anatomical features of plants that interacted with sp., was distinctively found in wood color, deposited material deposition in pore, the smell/aroma of wood and sedimentation in included phloem. further, examination of deposited material in pores by using gcms in inoculated plants interacting with sp., its aloe contains several compounds that forming the basic structure of seskuiterpenoid such as baimuxinal, elemol, 6-methoxy-2-(2-phenylethyl)-4hcromen-4-one and 3-phenyl-2-butanone. fusarium fusarium burfield t. 2005. agarwood chemistry. www.cropwatch.org. [2 february 2010]. chen h, yang y, xue jian, wei j, zhang z and chen h 2011.comparison of compositions and antimicrobial activities of essential oils from chemically stimulated agarwood, wild agarwood and healthy (lour.) gilg trees. 2011, , 4884-4896; doi:10.3390/molecules16064884. dai hao f, liu j, zeng y, han z, wang h, mei w. 2009. a new 2-(2-phenylethyl) chromone from chinese eaglewood. , , 5165-5168; doi:10.3390/molecules14125165 fahn a. 1991. 4th ed.. oxford: butterworth-heinemann. faridah s. 2009. analysis of agarwood oil composition via preparative thin layer chromatography. [thesis] universiti malaysia pahang. groenewald s. 2005. biology, pathogenecity and diversity of fusarium oxysporum f.sp. cubense [thesis]. universitas of pretoria; pretoria. hou d, 1960. thymelaeaceae. in: van steenis, c.g.g.j. (ed), flora malesiana series 1, volume 6. wolternoordhoff publishing, groningen, the netherlands, p 1-15. ishihara m, tomoyuki t, tsuneya, kenji u. 1991. fragrant sesquiterpenes from agarwood. phytochem 33: 11471155 konishi t, konoshima t, shimada y, and kiyosawa s. 2002. six new 2-(2-phenylethyl) chromones from agarwood. 50(3) 419-422 kunoh h 1995. cytological approaches in understanding host-parasite interaction signal transduction mandang yi, wiyono b. 2002. anatomi kayu gaharu ( lamk.) dan beberapa jenis sekerabat. 20: 107-126. aquilaria sinensis molecules 16 molecules 14 . plant anatomy. . chem. pharm. bull. japan. . aquilaria malaccensis buletin penelitian hasil hutan identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 111 mauseth j d. 1988. plant anatomy. pearson education. glenview il. mendgen k, h deising. 1993. infection structures of fungal plant pathogena cytological and physiological evaluation. 124: 193-213. mohamed r, jong pl, s zali. 2010. fungal diversity in wounded stems of fungal divers 43:67-74 nagy ne, franceschi vr, solheim h, krekling t, christiansen e. 2000. wound-induced traumatic resin duct development in stems of norway spruce (pinaceae): anatomy and cytochemical traits. american journal of botany 87: 302-313. nakanishi, t., yamagata, e., yoneda, k., nagashima, t., kawasaki, i. yoshida, t., mori, h. and muira, i. 1984. three fragrant sesquiterpenes of agarwood. 23(9):2066-2067 nobuchi t, siripatanadilok s. 1991. preliminary observation of wood associated with the formation of aloewood 63:226 -235. nofryanti e. santoso e, sitepu i, turjaman m. 2010. kajian kimia gaharu hasil inokulasi fusarium sp pada hutan vol. vii no. 2 : 175-188. pojanagaroon s, kaewrak. 2002. mechanical methods to stimulate aloewood formation in (kritsana) trees [abstract]. acta horticul 676:161-166 qi shu-yuan. 1995. spesies: culture and production of eaglewood (agarwood). in: bajaj yps, editor. , : springer. p 36-46. rahayu g, santoso e, wulandari e. 2009. efektifitas dan interaksi antara sp dan sp dalam pembentukan gubal gaharu pada . workshop pengembangan teknologi produksi gaharu berbasis pada pemberdayaan masyarakat di sekitar hutan. bogor. rao, k.r., dayal, r. and ramesh-rao, k 1992. the secondary xylem of (thymeleaceae) and the formation of agar. iawa bull. 13(2): 163-172 richter hg. 1990. wood and bark anatomy of lauraceae iii. aspidostemon rohwer & richter. iawa bull. (n.s.). 11. 47-56. wong, t.m. 1977. wood structure of the lesser known timbers of peninsular malaysia. malayan forest record no. 28. forest research institute. kepong. yuan qs 1995. species: culture and the production of eaglewood (agarwood) dalam bajaj yps editor. biotecnology in agriculture and forestry 33: medicinal and aromatic plants viii. berlin. springer. p. 36-46 yagura t, shibayama n, ito m, kiuchi f, honda g. 2005. three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding. 46 : 4395-4. new phytol aquilaria malaccensis. . phytochemistry . aquilaria crassna . bulletin of the kyoto university forest aquilaria microcarpa. info aquilaria crassna . aquilaria in vitro biotechnology in agriculture and forestry 33 medicinal and aromaticplant viii acremonium fusarium aquilaria microcarpa aquilaria agalocha . aquilaria in vitro . tetrahedron letters potential of mangrove seedlings for utilization in the maintenance of environmental quality within silvofishery ponds endah dwi hastuti* and rini budihastuti faculty of science and mathematics, diponegoro university, semarang , indonesi 50275 a received 10 january 2016/accepted 24 june 2016 abstract silvofishery system ha been applied to aquaculture activities and it has been developed in the coastal area of s semarang city, indonesia. however, information on the initial development of silvofishery ponds concerning the functionality of mangrove seedlings on environmental quality of fishponds had not been studied. this experiment aimed to determine the environmental conditions of silvofishery ponds and to analyze the effect of seedling stands of mangrove on environmental quality control. he presence of mangrove seedlings temperature t caused the decrease of and salinity. anova showed that mangrove species significantly affected water salinity, while canal the increase of width and mangrove species significantly affected turbidity and ph regression analysis showed . that the height of rhizophora mucronata partially significant effect on otal uspended lids (tss), rganic atter (om), had t s so o m as well as n p concentrations. d a ed . the height and itrogen (n) and hosphorus (p) iameter of ffect temperaturer. mucronata diameter of a ed dissolved oxygen (do) savicennia marina a. marina r. mucronata ffect . mixed population of and had an effect on water turbidity, while population of only had a partial effect on water salinity. a. marina r. mucronata seedlings had dominant effect on the environmental quality. mangrove seedlings can be used as environmental quality control within silvofishery ponds to maintain optimal conditions for fish growth. the application of silvofishery more abundant in early stage of mangrove seedlings should consider the plantation of compared to r. mucronata a. marina. : anal width environmental quality seedling silvofishery species compositionkeywords c , , , , introduction the unsustainable utilization pattern of coastal areas has caused environmental damage (primyastanto 2010), and the most affected et al. sectors by the damage is pond aquaculture activities (pramudyanto 2014). however, the development of beach and upland areas also ha s contributed to degradation of environmental quality (vatria 2010). one of the applied methods to maintain the sustainability of pond culture activity is the silvofishery culture system (surtida 2000). silvofishery is an aquaculture system which combines mangrove trees with shrimp/fish ponds. the integration of mangrove stands within silvofishery ponds are expected to improve the environmental quality and increase the carrying capacity of the system (wibowo & handayani 2006). according to suwarto et al. (2015), the existence of mangrove vegetation in ponds will improve primary productivity as well as assimilation capacity of pond effluents. several silvofishery pond models have been applied in many regions including , komplangan empang parit empang parit and enhanced (bengen 200 ). all three models integrate the mangrove 2 community into the pond area (inlet/outlet). the function of mangroves within the ecosystem is to provide nutrients through nutrient trapping and litter production and to absorb pollutants. mangrove should grow in both the inlet area as pollutant absorbers and in the outlet area to neutralize the pond effluent. however, the biotropia 3 1 6 63 vol. 2 no. , 201 : 58 doi: 10.11598/btb.2016.2 . .3 1 606 * c orresponding author: endah_pdil@yahoo.com 58 mailto:endah_pdil@yahoo.com application of a mangrove community in both inlet and outlet of silvofishery systems had not been developed. the potential utilization of mangrove in environmental quality control within aquaculture ponds through the application of silvofishery is expected to decrease the risk of aquaculture activity as well as to improve pond productivity (lewis iii gilmore 2007). however, the & optimal function of mangrove stands to the environment may not be achieved until trees are mature. in the meanwhile, the growth of mangrove from seedling stage to tree require a s long period of time. the influences of mangrove seedlings planted in plantations on the quality of pond seedling environment ha received very little attention. ve although the effect of mangrove seedlings on the control of environmental quality is not hig ly h significant, the existence of mangrove seedlings should provide certain effects on water circulation pattern as well as absorption of nutrients or pollutants. thus the role of mangrove seedlings , in silvofishery ponds, especially in early plantations needs to be studied. semarang city is a region in indonesia that has experienced ecological disturbance from unsustainable development activities in the past as well as regional development to leading environmental stress in coastal areas. increase pond culture occupying silvofishery system had been applied in semarang city, but it is not optimized to support the productivity of aquaculture. aimed to determine this experiment the environmental conditions of silvofishery ponds and to analyze the effect of seedling stands of mangrove on environmental quality control. materials and methods t h e e x p e r i m e n t wa s c o n d u c t e d i n mangunharjo village, tugu district, semarang city , central java province, indonesia from march to september 2015 by planting mangrove seedlings in silvofishery pond at the inlet and s outlet canals. aterials m used were height and diameter of as well as water and seedling stands sediment quality parameters representing environmental quality i.e. temperature, turbidity, salinity, ph, issolved xygen (do), otal d o t s so o m as uspended lids (tss), rganic atter (om), well as n p itrogen (n) and hosphorus (p) concentrations. experimental design involved treatments of canal widths and mangrove compositions. canal widths were 1 2 nd 3 m anal length was 5 m , a . c for all treatments he size of culture ponds were . t 5 x 5 m with 1.5 m depth. the canals were build 2 on both sides of the ponds as inlet and outlet canals. composition of the mangrove s seedling were (a) : 1. ; 2.avicennia marina rhizophora mucronata ; 3. mangrove (r) or a mixture of the two species among (m). the plantation space seedling stands was 1 x 1 m , so the number of mangrove 2 stands for each treatments were 5 stands (1 m – l1); 10 stands (2 m l2) and 15 stands (3 m – l3). the experiment was conducted with 3 replications. design is the of the experiment shown in table 1 and the diagram of the experiment is shown in figure 1. and observations data collections were conducted every 3 months i.e. in march, june and september 2015. d ed were: 1. ata collect water quality temperature, turbidity, parameters i.e. salinity, ph, do issolved xygen and tss (d o ) table 1 design of the experiment seedling composition canal width 1 m 2 m 3 m avicennia marina l1-a l2-a l3-a rhizophora mucronata l1-r l2-r l3-r mixture of both mangrove species l1-m l2-m l3-m 59 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds (t s so ); and 2.otal uspended lids sediment quality om rganic atter , parameters i.e. (o m ) as well as (n ) (p ) n itrogen and p hosphorus concentrations. data processing was conducted to determine the impact caused by mangrove stands on the environmental quality represented by water and sediment quality parameters value changes f in the inlet and outlet canals. actorial anova and regression multiple analysis were used to analyze the collected data. factorial anova was conducted to analyze the impact of treatment involving the combination of canal s widths and mangrove composition on water and sediment quality parameters. multiple regression analysis was conducted to analyze the influence of seedling variations (including the combination of population, measurements and species compositions) on the changes of value environmental quality parameter . multiple s regression involved independent s analysis : 1. variables for respective mangrove compositions i.e. seedling height, seedling diameter and population of mangrove seedling particularly for a. marina r. mucronata; and 2. and dependent variables changes of temperature, turbidity, i.e. salinity, ph, do, tss, om, n and p concentrations. results and discussion data collected showed changes in water and sediment quality parameters in the silvofishery ponds (table 2). consistently water temperature decreased from first to the third observation the s, while salinity consistently increased other parameters . did not show any specific pattern of changes. the observed water and sediment quality parameters within the inlet and outlet canals of silvofishery pond showed the decrease and increase of parameters values, before and after passing the canal with mangrove stands. the changes of water and sediment quality parameters value within the canals varied among treatments. this experiment showed the value changes on water and sediment quality parameters within the pond canals i.e. changes ranged temperature from (-)5.5 (+)6.4 °c; turbidity to ranged from to changes ranged (-)710 (+)769 ntu; salinity from to ranged from to (-)6.3 (+)6.1‰; ph (-)6.1 (+)6.2; do (-)4.1 (+)4.5 mg/ ; ranged from to l tss (-)343.2 (+)509.4 mg/ ; om ranged from to l ranged from to concentration (-)2.7 (+)2.5%; n ranged from to (-)0.7 (+)0.7%; and p concentration ranged from to (-)50.9 (+)52.8 figure 1 xperiment diagram of the e ponds mangrove silvofishery widths (1 m; 2 m; 3 m) compositions (avicennia marina; rhizophora mucronata; mixture) experiment environment quality (entrance) inlet environment quality (inside) outlet environment quality (exit) maintenance / enhancement capability 60 biotropia vol. 23 no. 1, 2016 ppm. the changes indicated that water and sediment quality changed during the 3 observation periods. the range of parameter value varied among periods.s observation height and diameter measurements on mangrove seedling stands also showed variations among the 3 observation periods. height of a. marina stand ranged from 41 106 cm; 43 to from to 101 cm; and 39 106 cm with from to stand diameter range of 0.20 – 0.89 cm; 0.32 – 0.88 cm; and 0.15 – 1.94 cm for the first, second and third observations . height of , respectively r. ucronata m stand ranged from 28 60 cm; 24 76 cm; to from to and 38 78 cm with stand diameter range from to of 0.22 – 1.90 cm; 0.30 – 1.32 cm; and 0.55 – 2.36 cm , for the first, second and third observations respectively decreased height and . seedling diameter f each mangrove species was caused by o the mortality of seedlings, hence seedlings replacements were conducted several times. data analysis with anova to measure the effect of canal width and mangrove compositions (treatments) on water and sediment quality parameters that several parameters were showed significantly affected by the treatments, such as mangrove composition affected salinity, canal width and mangrove composition affected turbidity and ph. s effectignificant on water salinity was achieved from silvofishery pond canals with and . while a. marina r. mucronata significant water turbidity w achieved effect on as from different canal widths and from different mangrove seedling species. si effectgnificant on ph was achieved from combination of canal width and mangrove seedling species. multiple regression a to determinenalysis the effect of mangrove stands on the water and sediment parametersquality of silvofishery pond showed there were significant effect of that s mangrove stand on several observed parameters (table 3) . data analysis was conducted partially for each mangrove composition structure as well as water and sediment quality . parameters parameterschanges in environmental quality within silvofishery pond canals were dominantly influenced by seedlings of (table ). r. mucronata 3 parameters influenced by partially r. mucronata stands included change in tss concentration, s temperature, om concentration, n concentration and p seedlings concentration. combination of and r. mucronata a. marina influenced water turbidity and do concentration. a. marina seedlings influenced water salinity. r. mucronatathere was significant effect of on tss concentration. according to furukawa and eric (1996), mangrove stands can function as sediment trap. sediment trapping processes by mangrove stands begins with the slowing down of water current, and this leads to the accumulation of tss which finally gravitate . s also, sediment trapping is influenced by the tide condition (kathiresan 2003). smangrove stand ha negative effect on the change of temperature, which means that as the height and diameter of mangrove stands increase, water temperature decrease . according to s hadikusumah (2008), mangrove vegetation is capable of bsorbing heat. the photosynthetic a capability of mangrove seedling of rhizophora occur in the leaves and green stems, hence as the s table 2 changes in water and sediment quality parameters observed during the experiment value no. parameter observation i observation ii observation iii 1. temperature (°c) 34.2 31.4 30.6 2. turbidity (ntu) 379.7 313.0 353.7 3. salinity (‰) 22.0 31.0 39.8 4. ph 7.49 9.26 8.11 5. do (mg/l) 6.56 6.99 5.72 6. tss (mg/l) 411.37 492.04 218.00 7. om (%) 1.64 1.74 1.62 8. n (%) 0.53 0.55 0.55 9. p (ppm) 33.86 39.68 34.44 61 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds stand height and diameter increase the amount of photosynthetic surface increases. water turbidity was affected significantly by both and . eedling stands a. marina r. mucronata s of inhibited water flow which r. mucronata increased the concentration of suspended sediment (yang 2013). , et al. on the contrary a. marina seedlings had negative effect on the change of water turbidity. according to weiffen et al. (2006), turbidity could be formed by the increasing population of plankton caused by the accumulation of sediment within the canal. the consumption rate of dissolved nutrient by a. marina probably was the cause of its negative effect on the water turbidity. a water salinity was ffected significantly by a. marina r. mucronata by but not , due to higher evaporation capacity of than a. marina r. mucronata. evapotranspiration within a pond is able to decrease water concentration, while salt is excreted back to the environment through the leaves (ball . 1988). hence, the concentration et al of salt (salinity) increase .d concentration of do was negatively affected by height of both and , but a. marina r. mucronata was positively affected by diameter of . a. marina stand height of mangroves were suggested to affect the canopy coverage which leads to the decreased light penetration (kennedy 2002). et al. as the mangrove stand increase , so does height s canopy coverage. , mangrove on the contrary seedlings still have chlorophyll in the stem, which means they are able to conduct photosynthesis. photosynthesis has positive effect o seedling n diameter and on the increas dissolved oxygen e of concentration. nutrient concentration, including om, n and p within the sediment was significantly affected by the height of , but not . r. mucronata a. marina nielsen and andersen (2003) stated that nutrient accumulation by is higher than that r. mucronata of . in the seedling stage, the capability a. marina to accumulate nutrients is related to diameter size of mangrove stand which is generally larger for r. mucronata a. marinafor herefore, than stands. t r. mucronata has higher capacity to inhibit water flow and influence the accumulation rate of sediment which binds nutrients than more a. marina. conclusions silvofishery pond canals random showed pattern of parameter environmental quality s value changes of turbidity, ph, do, tss, om, n and p emperature consistently decreased . t was table effect of angrove tands on hanges of uality within anals of ilvofishery 3 m s c water and sediment q parameters c s p sond no. mangrove composition independent variable dependent variable equation 1. single height of r. mucronata (x1) tss y = 524.574 – 8.483(x1) 2. mixed diameter of r. mucronata (x1); height of r. mucronata (x2) temperature y = -1.091 + 0.057(x1) – 1.799(x2) 3. mixed population of a. marina (x1); population of r. mucronata (x2) turbidity y = -81.627 – 209.753(x1) + 213.887(x2) 4. mixed population of a. marina (x1) salinity y = -0.432 + 0.103(x1) 5. mixed height of a. marina (x1); diameter of a. marina (x2); height of r. mucronata (x3) do y = 2.127 – 0.046(x1) + 7.410(x2) – 0.050(x3) 6. mixed height of r. mucronata (x1) om y = -1.796 + 0.050(x1) 7. mixed height of r. mucronata (x1) n y = -0.587 + 0.017(x1) 8. mixed height of r. mucronata (x1) p y = -41.303 + 1.142(x1) 9. mixed height of r. mucronata (x1) tss y = -248.833 + 6.794(x1) 62 biotropia vol. 23 no. 1, 2016 and salinity consisten ly increased. mangrove was t seedling stands for or had a. marina r. mucronata sign if ic ant e f f ec ts on t he chan g es of environmental quality involving parameters vari combinations on temperature, turbidity, ous salinity, do, tss, om, n and p. pecies that had s most effect on environmental quality parameters value changes was r. mucronata. acknowledgements t d thehe authors acknowledge director of research and community services (ditlitabmas), general directorate of higher education (ditjen dikti), ministry of education and culture, government of indonesia for financial support. references ball mc, cowan ir, farquhar gd. 1988. maintenance of leaf temperature and the optimisation of carbon gain in relation to water loss in a tropical mangrove forest. aust j plant physiol 15:263 76. bengen dg. 2002. technical guide on the ntroduction and i m m eanagement of angrove cosystem. center bogor (id): of coastal and marine resources studies, ipb. furukawa k, eric w. 1996. sedimentation in mangrove forest. mangrove salt marshes 1(1):3 10. hadikusumah. 2008. characteristics of physical parameters and chlorophyll-a concentration in java sea. j ilmu kelautan 13(2):103-12. kathiresan k. 2003. how do mangrove forests induce sedimentation? revista de biologia tropical 51:35560. kennedy vs, kleypas ja, cowan jr jh, hare sr. 2002. coastal and arine ecosystems & lobal climate changem g . us: pew center on global climate change. lewis iii rr, gilmore rg. 2007. important considerations to achieve successful mangrove forest restoration with optimum fish habitat. bull mar sci 80(3): -823 37. nielsen t, andersen fo. 2003. phosp orus dynamics h during decomposition of mangrove (rhizophora apiculata) leaves in sediments. j exp mar biol ecol 293:73 88.pramudyanto b. 2014. pollution and degradation control in coastal areas. jurnal lingkar widyaiswara 1(4):2140. primyastanto m, dewi rp, susilo e. 2010. the environment destructive habit by coastal society in slamic i perspective (case study on the fishermen and fish traders in tambak beach area, tambakrejo village, wonotirto district, blitar region east java). jurnal pembangunan dan alam lestari 1(1):1-11. surtida mb. 2000. silvofisheries in indonesia. seafdec asian aquacult 22(6):20-1. suwarto, lahjie am, ruchaemi a, simorangkir bdas, mulyadi f. 2015. ecological aspect of non productive fishponds at mahakam delta area: revitalization with silvofishery system. g jar 3(1):27 37.vatria b. 2010. various human activities which lead to coastal ecosystem degradation and its impacts. journal of berlian 9(1):47-54. weiffen m, moller b, mauck b, dehnhardt g. 2006. effect of water turbidity on the visual acuity of harbor seals ( ). vision research phoca vitulina [internet]. 46:1777–83. doi :10.1016/ available from: j.visres.2005.08.015. wibowo k, handayani t. 2006. conservation of mangrove forest through silviculture (silvofishery). jurnal teknik lingkungan 7(3):227-33. yang j, gao j, cheu a, liu b, schwendenmann l, costello mj. 2013. vegetation and sediment characteristics in an expanding mangrove forest in new zealand. estuarine, coastal and shelf science [internet]. 134:1–18. http://dx.doi.org/available from: 10.1016/j.ecss.2013.09.017. 63 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds http://dx.doi.org/available mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty, endang suhendang , i nengah surati jaya2 2 and cecep kusmana3 1researcher on dipterocarps research center, samarinda 75119, indonesia 2department of forest management, institut pertanian bogor (ipb), bogor 16680, indonesia 3department of silviculture, institut pertanian bogor (ipb), bogor 16680, indonesia received /accepted 3 may 2013 24 november 2014 abstract in primary and logged-over natural forest trees conditions tree structure, mortality and ingrowth rates , the such as will vary according to the species characteristic. quantitative management variables become very important to support yield regulation tools for achieving sustainable forest management. the objective was to determine study mortality and ingrowth rates to formulate biometric characteristic variability dipterocarps forest in logged-over of forests based on time series the was labanan, east kalimantan province. permanent data. study site located in measurement within -over were located to represent three , i.e plots logged forest different logging techniques . a) reduced impact logging with diameter limit 50 cm (ril 50); b ril 60; c conventional logging; and d) primary forest ) ) as control total plot permanent area was 48 ha and was measured periodically every 2 years within 17 years after . about logging. for data analysis purpose, trees were divided into dipterocarps and non-dipterocarps. two major groups, i.e. range of mortality rates for all species in logged-over forest were 2.5-29.3% per ha per 2 years which was close very to primary forest at year-5 after logging. while range of ingrowth rate for all species in logged-over forest were 1.3-21.3% per ha per 2 years which were higher than those for the primary forest within 17 years. the mortality and ingrowth rates fluctuation of dipterocarps species group were different from those of non-dipterocarps. : keywords dipterocarps, ingrowth, logged-over forest, mortality introduction lowland tropical rain forest is a natural forest with trees having typical characteristics, harboring the greatest species diversity in the world (whitmore 1990; richards 1996) and having numerous variations of tree's dimensions (prodan 1968). lowland tropical rain forests in southeast asia are dominated by dipterocarpaceae (ashton 1982), therefore, it is often referred to as the dipterocarp forests. dipterocarp forest is a tropical rainforest inhabiting type a and type b climate types are a, cove ring sumatera, kalimantan, sulawesi, north maluku and papua with the highest layer of forest canopy filled with family dipterocarpaceae, especially genus shorea, dipterocarpus, dryobalanops hopea and (ashton 1982). dipterocarp forest in west malesia region is the most productive tropical forest types based on timber value (fao 2001). in indonesia, dipterocarpaceae is the largest contributor (over 25%) to commercial timber forests in decades, with volume of 50-100 m per ha, especially in 3 kalimantan (sist . 2003)et al . in primary and logged-over natural forest, the stand stand conditions having differences in structure, species composition, tree density, canopy structure, mortality and ingrowth, will have varied growth rates depending on tree the age after logging (silva . 1995; lewis . 2004; et al et al ishida . 2005). recovery of a logged-over et al forest happens in a long period after logging (smith & nichols 2005), which varies depending on deforestation rate and environmental carrying capacity (muhdin . 2008). natural production et al forests in indonesia have more than 50% of logged-over forests (ditjen planologi kehutanan 2011). therefore, it is very important to know * corresponding author : fhsusanty@gmail.com biotropia vol. 22 no. 1, 2015: 11 23 doi: 10.11598/btb.2015.22.1.297 11 mailto:fhsusanty@gmail.com biotropia vol. 22 no. 1, 2015 12 about the variation of tree characteristics in a log ged-over forest. biology and ecology information of dipterocarps is needed as scientific basis for developing effective forest management policies (naito . 2008).et al forest biometric characteristics is among quantitative approaches to study the properties or characteristics of forest trees in size (metric) for a specific biological dimension as the user identification by ratio and interval scale (prodan 1968). input variables to determine quantitative tools are mostly provided by classical forest inventories on plots (vanclay 2003; gourletfleury . 2005). most of the early biometric et al research are found in studies on plantations and temperate forests that do not have such complexity as tropical forests. the heterogeneity and complexity of obstacles occur in the forms of diversity and variation of conditions as well as limitations or lack of long term data observation. average rate of mortality and its correlations to several reliable and measurable variables in size or site characteristics as input factors mostly determine the mortality model (keister 1972; hamilton & edwards 1976; monserud 1976; hamilton 1994; monserud & sterba 1999; cited flewelling & monserud 2002). according to chertov . (2005), a new paradigm in achieving et al sustainable f orest management requires prediction of effective growth forest tree dynamics involving aspects of ecological characteristics. to achieve sustainable forest management, preparation of quantitative management tools such as yield regulation models becomes very important. important variables needed to build the models are mortality and ingrowth rates of forest trees. this aimed to determine study mortality and ingrowth rates of dipterocarps and non-dipterocarps spesies groups for 17 years after being logged, which rates will be used to formulat biometric characteristics variability e of dipterocarp forest in logged-over forests based on time series data. materials and methods study site this study was carried out at labanan research forest station (1˚49'-2˚10' n and 116˚7'-117˚27' e) located in berau region, east kalimantan province. according to schmidt and ferguson climate classification (1951), the study site was within type b climate (q = 14.3–33.3%). based on koppen system classification the study site was within type afa climate with many rainy days over in a year with mean annual precipitation about 1,800-3,000 mm/year. the highest monthly mean precipitation happened in january (242.5 mm) and the lowest is in august (90.9 mm). maximum temperature rate happens in september and november (35 ºc) and the lowest was in february and august (21 ºc), with average temperature of 26 ºc. the study site was located at 500 m above sea level (asl) and it was a relatively hilly forest. soil type in labanan research forest station consisted of ultisol (87.3%), entisol (10.7%) and incenptisol (2.0%). labanan research forest station as a low land tropical forest is dominated by family dipterocarpaceae, consisted of 7 genera i.e. anisoptera, cotylelobium, dipterocarpus, dryobalanops, parashorea, shorea vatica and . besides family dipterocarpaceae, other dominant genera are also present in labanan research forest station such as sapotaceae, meliaceae, moraceae, ebenaceae, sapindaceae leguminaceae and . among landscapes at the labanan research forest station was a swamp forest dominated by and lophopetalum shorea balangeran (saridan and susanty 2005. figure 1. study site-labanan research forest station, berau, east kalimantan province mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. data collecting permanent plots were set up in the logged-over forest as well as in the primary forest. the size of each plot was 200x200 m (4 ha) which was divided into 4 square subplots with size of 100x100 m (1 ha). the permanent plots were built in 4 condition variations with total area of 48 ha. measurements were carried out by census method for all species with limit diameter of 10 cm including number of trees, tree species, stem diameter (diameter at breast height or 20 cm above buttresses) and number of dead trees. repeated measurements were performed every two years. reduced impact logging can be defined as logging technique to minimize environment impact on forest trees and soils (dykstra 2008). this technique is needed to preserve ecological aspect of forest trees and to ensure sustainable yield of production forest in the future. data used in this study were data collected from 1990 to 2008. treatments applied on research plots were as follows: a) ril 50: reduced impact logging techniques with limit diameter of 50 cm, skid trail planning was based on contour maps and tree position as well as supervision of tree felling and skidding (3 plots). b) ril 60: reduced impact logging techniques with limit diameter of 60 cm, skid trail planning was based on contour maps and tree position as well as supervision of tree felling and skidding (3 plots). c) cnv: conventional logging techniques with limit diameter of 60 cm, no skid trail planning, conducted without considering contour line maps or tree position, felling was done by loggers experiences (3 plots). d) pf: primary forest as controls (3 plots). data analysis data organization was carried out using database software microsoft visual foxpro 9.0, while data analysis was performed by using spreadsheet and spss 15.0. data were analyzed based on tree density and tree structure (stems per ha) as well as basal area (m per ha) with two major 2 species groupings e.g. dipterocarps and nondipterocarps species. calculations of mortality and ingrowth rates were performed every 2 years. residual tree characteristics assessment was done by comparing variations in forest conditions by using different test mean values (t-test), analysis of variance (anova) and regression analysis. regression equation tested was linear equations, polynomials, exponential and logarithmic. criteria used for selecting the best equation were based on the regression coefficient (r), determination coefficient (r ) and the highest 2 value of the smallest standard error (se) (steel & torrie 1995) results and discussion tree density the dynamics of logged-over forest within 17 years were represented by number of trees per hectare and basal area per hectare against tree fluctuations after-log ging using different logging techniques (fig. 2). mean values of tree density at the initial conditions (pre-harvest) were compared using t-test and the results showed no significant differences in all study plots (t <t ). fluctuations of tree density calc tab(0:05; 6) and basal area of logged-over forest would increase up to the initial conditions before logging (primary forest). logged-over dipterocarp forest recovery reached the conditions close to the primary forest at 9 years after-logging based on tree density and at 11 years after-logging based on basal area. according to gourlet-fleury . et al (2005) and kao and iida (2006), tree density extremely increases at 5 to 7 years after-logging, which was explained by the increasing growth of open canopy in after-logging periods. range of tree density in the studied loggedover forest was 461-647 stems per ha with average value of 531 stems per ha. sianturi and kanninen (2005) stated that at the period of 2 years afterlogging in jambi forest, the tree density reached 90% compared with tree density at the same forest before logging. tree density in a primary forest in sangai, central kalimantan (478-738 stems per ; ha average of 583 stems per ) was similar to tree ha density in labanan forest (susanty 2006). this study results were also similar to the condition in eastern amazon forest showing average tree density of 480±96.6 stems per ha (sist & ferreira 2007). while gourlet-fleury . (2005) et al presented higher value of tree density for french guiana forest (625 stems per ha). 13 range of basal area value in the studied logged-over forest was 19.35-31.84 m per ha. as 2 comparisons, basal area value in a several years after-logging forest in central kalimantan ranged from 16.4 to 26.7 m per (krisnawati 2001). a 2 study conducted by setiawan (2013) showed that basal area value in a logged-over forest in muara wahau, east kalimantan ranged from 12.63 to 32.57 m per ha, while basal area value in a 2 primary forest in muara wahau, east kalimantan ranged from 27.80 to 32.57 m per ha. basal area 2 value in amazon forest had average of 28±4 m 2 per ha (sist & ferreira 2007). in logged-over forests, tree density was about 93-102% and basal area were about 81.0-88.8% compared with initial condition or before logging condition. based on this result, the recovery pattern of forest seemed to have positive trend with similar variations. the tree density afterlogging variable is important to know ecological sustainability (sist . 2003; smith & nichols et al 2005; muhdin 2012). this variable may indicate the level of all logged-over forest which have well recovered, with assumption no interference or disturbance, thereafter. different recovery patterns of logged-over forest influenced ecological factor and forest tree condition. growth monitoring on a 24 ha permanent sample plots in silviculture experiments in amazônia (1980-1989) indicated that even in the same block of concession area, forest recovery has different characteristics and patterns shown by fluctuations of tree dynamic (silva 1995). initial et al. succession process in logged-over forests started from the end of logging operational, followed by growing process in a time series function. mortality rates the mortality level in different logging techniques is influenced by logging intensity based on number of trees and volume per hectare which were felled by logging. logging intensity in ril 50 had felled trees of 10.7 4.9 stems per ha + with volume of 96.8 66.5 m ha per; ril 60 had + 3 felled trees of 7.0 3.0 stems per ha with volume + of 56.5 23.3 m per ha and cnv had felled + 3 trees of 10.1 4.2 stems per ha with volume of + 107.2 59.6 m per ha (sist & bertault 1998). + 3 these results indicated that logging intensity had a tendency to increase from ril 60, ril 50 and cnv logging techniques, respectively. in the logged-over forest, the mortality rates for all species ranged from 2.5 to 29.3% per ha per 2 years (table 1). the highest mortality rates occurred in year-1 and year-3 after logging then declined or was similar to mortality rates at the primary forest in year-5 after logging. the mortality rates in this study were higher than those in some studies; for example mortality rate in year-2 after logging in east kalimantan forest was 2.5% per year (primack . 1985; nguyen-et al the . 1998); mortality rate in a logged-over et al forest in papua new guinea was 2.5% per ha per year (mex 2005); mortality rate in a mixed dipterocarp forests in asia was 1.5% per year (nguyen-the . 1998). compared to other et al forest types, dipterocarp forest has lower mortality rate than that of peat swamp forest which was 6.13% per year and of heath forests which was 4.26% per year (nishimua . 2006).et al mortality rates in forest with different logging techniques indicated higher logging intensity for both dipterocarps and non-dipterocarps (fig. 3). biotropia vol. 22 no. 1, 2015 14 figure . forest trees for all species with different logging techniques based on (a) average tree density (stems per ha) and (b) 2 average basal area (m per ha)2 non-dipterocarps species have a tendency to have higher mortality rate than dipterocarps. mortality rate for both dipterocarps and nondipterocarps in primary forest ranged from 2.0 to 6.0% per ha per 2 years with average of 3.29% per ha per 2 years. the increasing logging intensity was negatively correlated with stem densities, species abundance and biodiversity, where prior changes in the logged-over forest for the first 5–10 years after logging were gradually occurred in successive samples (kariuki . 2006). the et al mortality rates of trees in a forest is relatively high within 7 years, indicating that damaging effects of logging would permanently influence trees, either directly or indirectly. in other words, within 7 years after logging, the remaining trees will begin to recover. fluctuations of mortality rate within 17 years after logging indicated that the mortality rates decreased over years for both dipterocarps and non-dipterocarps. a theoretical frame for ingrowth limitations, climatic variation or many factors became the constraints to predict treelevel change (harcombe . 2002). several et al variables causing differences of mortality rates were logging intensity, initial structure and dominant species composition. the mortality degree is different for every tree's stage in the forests. ingrowth rates ingrowth rates in logged-over forest have the same tendency with mortality rates that correlate with level of logging intensity (table 2). in this case, conventional logging technique can still control the total felled trees to only cut down trees having limit diameter of 60 cm. the ingrowth rates in logged-over forest (1.3-21.3% per ha per 2 years) were higher than those in primary forest (0.6-4.7% per ha per 2 years). the highest 15 table . tree mortality rates (% per ha per 2 years) for all species1 forest condition pal 1 pal 3 pal 5 pal 7 pal 9 pal 11 pal 13 pal 15 pal 17 (% per ha per 2 years) ril50 mean 23.7 10.9 4.3 5.8 3.0 2.9 2.7 3.4 3.3 sd 10.6 6.2 1.4 1.2 0.7 1.1 1.1 1.7 1.5 ril60 mean 22.3 8.3 4.4 5.5 3.3 2.5 2.5 2.5 3.5 sd 8.1 3.5 1.1 1.3 0.9 0.8 1.1 1.3 2.0 cnv mean 29.3 12.8 2.8 6.8 3.3 3.6 2.8 2.9 4.3 sd 9.2 8.0 1.3 2.3 0.9 2.1 1.0 1.9 0.4 p1 p3 p5 p7 p9 p11 p13 p15 p17 pf mean 2.9 3.2 4.7 6.0 3.2 3.4 2.7 2.0 3.2 sd 1.0 1.3 5.1 1.8 1.2 1.5 1.5 1.4 2.0 notes : pal = period after logging (years) sd = standard deviation p = monitoring period (years) figure . mortality rates (stems per ha per 2 years) of trees with different logging techniques for species group (a) 3 dipterocarps and (b) non-dipterocarps mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. ingrowth rate for all species occurred at year-5 to year-7 (table 2). after logging, the ingrowth rates increased substantially during the first 5 years. during the next five years, there was a sharp decrease. hardiansyah . (2005) stated that at et al year-1 after logging in jambi, ingrowth rate was 0.19-2.89% per year with average 2.1% per year. meanwhile, in papua new guinea mex (2005) stated that at year-13 after logging the trees had smaller ingrowth rate with mean of 41 stems per ha. in the primary forest, ingrowth rate fluctuation were relatively low i.e. 2.0-4.7% per ha per 2 years with average of 2.5% per ha per 2 years. fluctuations of ingrowth rates for dipterocarps and non-dipterocarps species in logged-over forest with different logging techniques had different patterns (fig. 4). in this study 68 tree families had been identified (appendix 1), in which there were 8 genera of dipterocarps (appendix 2) namely anisoptera, cotylelobium, dipterocarpus, dryobalanops, hopea, parashorea, shorea, vatica and consisted of 92 species. non-dipterocarpaceae trees dominating the logged-over forest were especially pioneer species such as sp. this result was macaranga similar to the research results in brazilian amazon forest which showed that the ingrowth rate increased in the first 8 years after logging provided there were more light in the under storey which stimulated ingrowth (silva . et al 1995). ingrowth can soar through the canopy opening after logging provided there were more growing space and ingrowth would decrease in line with the competition among trees (gourlet-fleury . 2005). logged-over forests et al would have good recovery with high growth rate at year-3 after logging (kao & iida 2006). the first highest ingrowth rate occurred in year-7 indicating high growth regeneration which in line with the beginning of mortality rate decline in year-7. the second highest ingrowth rate occurred in year-15 which might be caused by recovery process of forest trees after being logged-over. biotropia vol. 22 no. 1, 2015 16 table . tree ingrowth rates (% per ha per 2 years) for all species2 forest condition pal 1 pal 3 pal5 pal 7 pal 9 pal11 pal 13 pal 15 pal17 (% per ha per 2 years1) ril50 mean 1.8 9.0 21.3 14.5 8.8 2.8 3.4 6.5 0.6 sd 0.6 4.9 25.3 8.9 4.4 2.4 1.7 2.7 0.2 ril60 mean 2.1 6.3 9.0 10.5 7.9 1.6 3.5 9.8 0.7 sd 1.0 2.3 2.3 3.3 3.8 1.0 1.4 5.7 0.1 cnv mean 1.3 12.7 19.1 19.6 7.8 3.5 5.2 5.4 0.7 sd 0.6 8.5 13.4 8.2 4.8 1.2 2.6 1.9 0.2 p1 p3 p5 p7 p9 p11 p13 p15 p17 pf mean 2.1 3.8 3.7 4.7 2.0 1.1 1.6 4.5 0.7 sd 1.0 1.9 1.9 2.2 1.1 1.1 0.9 2.7 0.6 figure . ingrowth rates (stems per ha per 2 years) of trees with different logging techniques for species group (a) 4 dipterocarps and (b) non-dipterocarps notes : pal = period after logging (years) sd = standard deviation p = monitoring period (years) species grouping regression equation r r2 se d lof y(m) = -0.039x3 + 1.295x2 13.177x + 43.501 0.8955 0.8020 4.855 y(i) = 0.011x3 0.3676x2 + 3.2295x + 0.2946 0.5326 0.2837 3.552 pf y(m) = 0.0073x3 0.2272x2 + 1.8366x + 0.8951 0.6448 0.4158 1.577 y(i) = 5.107e -0.133x 0.4106 0.1686 0.371 nd lof y(m) = -0.0779x3 + 2.6842x2 28.4x + 101.3 0.9071 0.8229 9.985 y(i) = 0.0667x 3 2.1667x 2 + 18.792x 11.094 0.6317 0.3990 14.765 pf y(m) = 0.0196x 3 0.5982x 2 + 4.8012x + 4.3734 0.6118 0.3743 4.142 y(i) = 13.873e-0.082x 0.4900 0.2401 0.339 all species lof y(m) = -0.1174x3 + 3.9793x2 41.577x + 144.81 0.9160 0.8391 13.686 y(i) = 0.0778x3 2.5343x2 + 22.022x 10.799 0.6363 0.4049 17.288 pf y(m) = 0.0269x 3 0.8254x 2 + 6.6378x + 5.2685 0.6375 0.4064 5.474 y(i) = 17.636e-0.078x 0.4775 0.2280 0.3359 canopy gap occurred in a logged-over forest can function as a catalyst for tree ingrowth until certain condition. this phenomenon would then decrease due to competition in population (gourlet-fleury . 2005; kao & iida 2006), et al which would explain why natural ingrowth formed in the period of 11-17 years in the forest recovery. different species group will be affected variably to competition for light based on respective tolerable range (harcombe . 2002). et al research on forest tree in peninsular malaysia showed variations in regenerated tree by class diameter of trees in the logged-over forest area (seng . 2004).et al period after logging to mortality and ingrowth pattern results of analysis of variance (anova) showed that different harvesting techniques had no significant effect on mortality rate and ingrowth rate for all species. however, descriptively, the mortality and ingrowth rates were higher at year-1 and year-3 after logging (tables 1 and 2). from this study it was indicated that ril 50 seemed to be the best technique to increase ingrowth rate, but ril 60 was the selected technique to decrease the mortality rate after logging. regression equations were developed to show relationship between after-logging period to mortality and ingrowth rates for each species group (table 3). these equations showed that after-logging period correlated with mortality rate in logged-over forest for both dipterocarps and non-dipterocarps. recovery period after logging will affect the level of mortality and ingrowth rates for dipterocarps, non-dipterocarps and all species groups (fig. 4). all species group in logged-over forest had polynomial regression pattern for mortality and ingrowth rates, but ingrowth rates on primary forest had negative exponential pattern. high mortality rate was shown to happen in dipterocarps in early years of logging and seemed to be caused by dipterocarps being a major commercial species. meanwhile, high mortality rate for non-dipterocarps seemed to be due to forest area clearing related to skidding trails and logging yard as well as to logging operation. mortality rates for dipterocarps and nondipterocarps as either direct or indirect effect of logging was continued until the third year. ingrowth rates of non-dipterocarps species group was higher than that of dipterocarps. this was correlated with general growth or increment characteristics of total tree basal area increment of non-dipterocarps which was higher than that of dipterocarps species groups (silva . 2002; susanty & suhendang et al 2013). 17 table . regression equations between after-logging period (x) to mortality rates (m) and ingrowth rates (i) for dipterocarps 3 (d), non-dipterocarps (nd) and all species notes: lof : logged-over forest; pf : primary forest; x : after-logging period (years); y(m) : mortality rates (stems per ha per 2 years); y (i) : ingrowth rates (stems per ha per 2 years) mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. results of regression analysis (table 3; fig. 5) showed that after-logging period was significantly cor r elated to mor tality rate s for both dipterocarps and non-dipterocarps. the function of recovery time after logging would reduce mortality rates from logging effect into natural mortality rates. ingrowth rates after logging for both dipterocarps and nondipterocarps could not be explained only from recovery time after logging. opportunity for seedlings and saplings to grow is influenced by biocharacteristics of each species and by distribution pattern (husch 2003; adam & et al. kolbs 2005; gersonde & o'hara 2005). natural characteristics of mortality and ingrowth rates shown by primary forest conditions indicated that mortality rate tended to be higher than ingrowth rate within the 17 years of measurements for both dipterocarps and non-dipterocarps. in natural forest, growth will increase the dimension of individual trees while reducing the number of trees due to competition of growing space. results from this research provided the first quantitative information for management planning in labanan forest. to achieve higher ingrowth rate and minimize mortality rate until year-3 after-logging, the reduced impact logging technique (ril 50 and ril 60) was better than conventional logging technique. these data serve as basic information needed to start forest management planning and provide useful guide for choosing suitable planning system. this study highlighted the need to consider silviculture treatment to stimulate biotropia vol. 22 no. 1, 2015 18 figure . patterns of mortality and ingrowth rates (stems per ha per 2 years) on logged-over forest (lof) and primary forest 5 (pf) for species groups (a) dipterocarps; (b) non-dipterocarps; (c) all species growth rates in forest areas, especially to increase wood production. the methods described in this study can be enhanced to involve a more detail species grouping for increasing model accuracy and to accommodate high species diversity (phillips . 2002; valle . 2006). in et al et al dipterocarp forest, valuation of tree recovery could be assessed by tree structure model, mortality rate and ingrowth rate, involving species characteristic by species grouping. conclusions residual trees in dipterocarp forest in year-17 after-logging based on had been well recovered density and basal area value that reached more than 85% close to the initial condition. mortality rates in logged-over forest would be similar to that in the primary forest at year-7 after-logging, while ingrowth rate in logged-over forest would be similar to that in the primary forest at year-17 afterlogging. fluctuations in mortality and ingrowth rates in logged-over forest reached equilibrium conditions at year-7 after-logging. in the subsequent years, ingrowth rate was higher than mortality rate. fluctuations of mortality and ingrowth rates in the primary forests were relatively low. recovery period for trees in logged-over forest had an important effect to decrease mortality rates for both dipterocarps and non-dipterocarps. different types of logging techniques (ril 50, ril 60 and conventional logging) with variations in logging intensity 7-14 stems per hectare did not have significant influence on recovery of density and basal area value, as well as on mortality and ingrowth rates. the reduced impact logging technique was better than conventional logging technique to achieve higher ingrowth rate and minimize mortality rate until year-3 after-logging. quantitative dimensions of forest trees at 17 years after logging will be close with the primary forests, but still dominated by the non-dipterocarps species. acknowledgements the authors thanked the forest research and d e v e l o p m e n t a g e n c y ( f o r da ) a n d dipterocarps research center (direc) for permitting the use of data measurement of permanent sample plots in the labanan forest station. references adams hd, kolbs te. 2005. tree growth response to drought and temperature in a mountain landscape in northern arizona, usa. j biogeogr 32:1629-40. ashton ps. 1982. dipterocarpaceae. flora malesiana the netherlands 1(9): 237-552. chertov o, komarov a, mikhailov a, andrienko g, andrienko n, gatalsky p. 2005. geovisualization of forest simulation modeling results: a case study of carbon sequestration and biodiversity. comput electron agr 49: 175-91. davis ls, johnson kn, bettinger ps, howard te. 2001. forest management: to sustain ecological, economic and 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the contribution of strek project. jakarta(id): cirad-forêt-forda-pt inhutani i. p 139-61. sist p, fimbel r, sheil d, nasi r, chevallier mh. 2003. towards sustainable management of mixed dipterocarp orests of south-east asia: oving f m beyond minimum diameter cutting limits. environ conserv . 30(4): 364 74 sist p, ferreira fn. 2007. sustainability of reduced-impact logging in the eastern amazon. forest ecol manag 243: 199-209. smith rgb, nichols jd. 2005. patterns of basal area increment, mortality and recruitment were related to logging intensity in subtropical rainforest in australia over 35 years. forest ecol manag 218: 31928. steel rgd, torrie jh. 1995. prinsip dan prosedur statistika: suatu pendekatan biometrik. sumantri b, penerjemah. jakarta (id): pt gramedia pustaka utama. terjemahan dari: principles and procedures of statistics. edisi ke-2. susanty fh, suhendang e. 2013. riap individu dan tegakan periodik hutan dipterocarpaceae setelah penebangan. in: ngatiman, wahyudi a, editor. restorasi ekosistem dipterokarpa dalam rangka peningkatan produktivitas hutan. prosiding: 2013 oktober 22; samarinda (id): kementerian kehutanan. balai besar penelitian dipterokarpa. p156-67. vanclay jk. 2003. growth modelling and yield prediction for sustainable forest management. the malaysian forester 66(1):58-69. valle d, mark s, edson v, james g, marcio s. 2006. identifying bias in stand-level growth and yield estimations : a case study in eastern brazilian amazonia. forest ecol manag 236: 127-35. whitmore tc. 1990. tropical rain forest dynamics and its implications for management. in: gomez-pompa a, whitmore tc , hadley m, editor. rain forest regeneration and management: man and the biosphere series volume 6. paris(fr): parthenon publishing group. p 67-89. 21 mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. appendix 1. list of species by family in labanan forest family famil y famil y famil y actinidiaceae dilleniaceae moraceae saxifragaceae alangiaceae dipterocarpaceae myristicaceae simaroubaceae amonaceae ebenaceae myrsinaceae sonneratiaceae anacardiaceae elaeocarpaceae myrtaceae sterculiaceae annonaceae euphorbiaceae ochnaceae symplocaceae apocynaceae fagaceae olacaceae theaceae aquifoliaceae flacourtiaceae oleaceae thymelaeaceae araucariaceae clusiaceae oxalidaceae tiliaceae bignoniaceae hypericaceae polygalaceae ulmaceae bombacaceae icacinaceae proteaceae urticaceae burseraceae juglandaceae rhamnaceae verbenaceae caesalpiniaceae lauraceae rhizophoraceae celastraceae lecythidaceae rosaceae chrysobalanaceae fabaceae rubiaceae combretaceae loganiaceae rutaceae connaraceae lythraceae sapindaceae convolvulaceae magnoliaceae sapotaceae crypteroniceae melastomataceae sapuidaceae datiscaceae meliaceae sarcotheca biotropia vol. 22 no. 1, 2015 22 47 shorea bentongenensis 48 shorea confusa 49 shorea exelliptica 50 shorea faguetiana 51 shorea falciferoides 52 shorea fallax 53 shorea guiso 54 shorea hopeifolia 55 shorea inappendiculata 56 shorea johorensis 57 shorea laevis 58 shorea lamellata 59 shorea leprosula 60 shorea leptoderma 61 shorea longisperma 62 shorea macrophylla 63 shorea macroptera 64 shorea maxwelliana 65 shorea mecistopteryx 66 shorea multiflora 67 shorea ochracea 68 shorea ovalis ssp ovalis 69 shorea parvifolia 70 shorea parvistipulata appendix 2. list of dipterocarps species no species no species 1 anisoptera costata 2 anisoptera laevis 3 anisoptera sp 4 cotylelobium melanoxylon 5 cotylelobium sp 6 dipterocarpus acutangulus 7 dipterocarpus caudiferus 8 dipterocarpus confertus 9 dipterocarpus conformis 10 dipterocarpus costulatus 11 dipterocarpus elongatus 12 dipterocarpus fusiformis 13 dipterocarpus glabrigemmatus 14 dipterocarpus gracilis 15 dipterocarpus grandiflorus 16 dipterocarpus hasseltii 17 dipterocarpus humeratus 18 dipterocarpus mundus 19 dipterocarpus pachyphyllus 20 dipterocarpus palembanica 21 dipterocarpus stellatus 22 dipterocarpus tempehes 23 dipterocarpus verrucosus 24 dipterocarpus sp 25 dryobalanops beccarii 26 dryobalanops lanceolata 27 dryobalanops sp 28 hopea bracteata 29 hopea cernua 30 hopea dryobalanoides 31 hopea ferruginea 32 hopea mengarawan 33 hopea nervosa 34 hopea pachycarpa 35 hopea rudiformis 82 shorea sp 83 vatica albiramis 84 vatica micrantha 85 vatica nitens 86 vatica oblongifolia 87 vatica odorata 88 vatic a rassak 89 vatica sarawakensis 90 vatica umbonata 91 vatica vinosa 92 vatica sp 36 hopea sangal 37 hopea semicuneata 38 hopea sp 39 parashorea malaanonan 40 parashorea smythiesii 41 parashorea sp 42 shorea agamii ssp agamii 43 shorea almon 44 shorea angustifolia 45 shorea atrinervosa 46 shorea beccariana 71 shorea patoiensis 72 shorea pauciflora 73 shorea pinanga 74 shorea scrobiculata 75 shorea semicuneata 76 shorea seminis 77 shorea smithiana 78 shorea superba 79 shorea symingtonii 80 shorea virescens 81 shorea xanthophylla 23 mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. biotropia vol. 1 no. 1, july-december 1987 multidisciplinary research on shorea javanica foreword it was april 1985, under the leadership of dr. ir. z. goto, then tropical forest biology program manager, that it was decided to launch in biotrop a multidisciplinary research on shorea javanica. this followed the publication in 1984 by e.f. torquebiau of a paper describing the traditional planting of this tree for resin production by farmers near the small town of krui, in lampung province, southern sumatra (man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127). a proposal was subsequently made to develop in biotrop different research topics around t his species in order to promote it for plantation forestry. the choice of this species was justified by t he important knowledge from its traditional uses and planting, while in the long term, it is hoped that the development of plantations of this species will promote the use of other dipterocarps and native trees for plantation forestry. 41 biotropia 1 (1) 1987: 42-45 i. introduction e.f. torquebiau* tropical forest biology program, biotrop, bogor, indonesia the plantations of shorea javanica k.&v. (dipterocarpaceae) in the district of krui (lampung province, sumatra; see fig. 1 for situation map and main climatic data) are remarkable examples of successful land development after deforestation and shifting cultivation which was mentioned in the indonesian forestry literature as far back as 1937 (rappard 1937). this tree is a white meranti which is locally found in the natural forest and tapped for its beautiful, crystalline resin, or "damar". the local name of the tree is "damar mata kucing", which means "cat's eye resin". one of the traditional cultivation systems in the area is shifting cultivation ("ladang"): rain-fed rice is grown during one or two years and then coffee, other crops, and damar trees are planted to convert the ladang into a permanent agricultural field. the damar trees close their canopies above the other crops after some years and can be tapped for resin after about 15 years and during a rotation of approximately 50 years. they constitute dense stands of 40—50 m high trees called "kebun damar" (damar gardens) which look like a natural rain forest. seeds for planting stock were formerly obtained from the surrounding natural forest but nowadays they come from the pre-existing plantations which cover an area of approximately 1000 ha (scholz 1983). fruiting seasons are occasional and irregular, often several years spaces, so that the farmers manage large nurseries of seedlings which can be maintained for several years and transplanted to the plantations when needed. transplantation of bare-rooted seedlings is easy. other useful trees (e.g. clove trees), are simultaneously planted in the ladang at the time of planting the damar trees, so that, although the latter largely dominate, the resulting stand is a multi-layered, mixed one, comprising of different useful plants (fruits, vegetables, medicinal plants, etc.). the whole cultivation system, from the shifting cultivation stage to the establishment of a permanent tree plantation, constitutes an efficient agroforestry system which is extensively described by michon 1984, 1985; michon et al 1984; and torquebiau 1984. the resin of shorea javanica is traditionally used for torches, caulking boats, batik coloring, etc., and is now exported to industrial countries where there is market for uses such as food additives, cosmetics, paints, varnishes and * present address: icraf, p.o. box 30677, nairobi, kenya. 42 i. introduction e.f. torquebiau figure 1. situation map and main climatic data (torquebiau 1984). 43 43 biotropia vol. 1 no. 1, july-december 1987 medications. however, the resin is exported as raw material and there is no industrial unit yet for resin processing in indonesia. although the only significantly large area of damar mata kucing plantation in sumatra is in the surroundings of krui, some other localities were found in southern sumatra, indicating that at one time, the damar business was probably a very attractive one. these localities are: — bakauheni, at the very southern tip of sumatra, 10 km from bakauheni, to the north of the trans-sumatra road, is a small stand of fairly old, disused damar trees. other damar trees can be recognized here and there in the landscape, and there are probably other plantations in this area. — sukadana, near way kambas nature reserve. this locality was reported by meijer (1975), but only one [huge!] tree is now left in the village and is disused. people said the other damar trees have been felled for timber! — wana (sribowhono), some 25 km east from the above. approximately 1 ha stand of fairly old damar trees is present. other stands are said to exist in the surroundings. — hujan mas, near muaraenim. some damar trees in home gardens not far from the trans-sumatra road were noted. — batu raja, some km to the north of the town. damar trees in home gardens can be seen from the trans-sumatra road. — localities near semangka bay and kota agung, mentioned in meijer (1975), could not be visited. some localities are probably also to be found in southern bengkulu province (rappard 1937). it is important to collect seeds or seedlings when a survey is made of these different localities, so that the gene pool of damar trees can be diversified. resin quality may differ between localities (and between trees!). tapping techniques are also different according to the zone. the best tool to scrape the resin from the trees as said by rappard (1937) is the "damar axe" of krui, while further north in lais, a different tool causing more damage to the trees is used. the aim of biotrop's multidiciplinary research project on shurea javanica is to gather sufficient information on the silviculture of this tree to serve as basis for proposing it as a species for plantation forestry and agroforestry. up to now, only some of these topics could be dealt with and the first results of the studies are presented in the following chapters. a sub-project on tissue culture and water culture is also going on, but has not produced any significant result yet. aseptic germination of embryo has been successfully achieved and will be used as basis for new vegetative propagation experiments. data and material gathering in the field were conducted in the surroundings of krui during field trips attended by different staff members from 44 i. introduction — e.f. torquebiau biotrop in june, september and october 1985 (ripe seeds could be collected then!), and february 1986. although much research work is still to be done, it is thought that there is sufficient knowledge on the silvicultural management of this species to warrant the establishment of experimental plantations now. executive proposals have been submitted to the directorate of reforestation and land rehabilitation (rrl) and to p.t. kutai timber indonesia with the hope that such trial plantations could be started as soon as possible, with the scientific collaboration of biotrop. however, these proposals have not received any answer so far. references meuer, w. 1975. indonesian forests and land-use planning. thomas hunt morgan school of biological sciences, univ. of kentucky, lexington, kentucky 40506/missouri botanical garden. mimeogr. 113 pp. michon, g. 1984. prospects for the use of agroforestry systems in regional forest management: examples from indonesia. in: biotrop symposium on forest regeneration in south east asia. may 1984, biotrop, bogor, indonesia. biotrop special publication no. 25. michon, g. 1985. de phomme de la foret au paysan de i'arbre: agroforesteries indonesiennes. thesis, university of montpellier, france, 273 pp + figs. michon, g., f. mary, j.m. bompard & p. lombion, 1984. traditional agroforestry in indonesia. lipi, jakarta, mimeogr. rappard, f.w. 1937. de damar van bengkoelen. tectona, dl. xxx, pp. 697-915. scholz, u. 1983. the natural regions of sumatra and their agricultural production pattern. vol. 1, text: crifc, bogor, indonesia, vol. 2, maps: sarif, padang, indonesia. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 45 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 564 (analyn tiger).cdr tiger beetles (coleoptera: carabidae: cicindelinae) of compostela valley province, mindanao island, philippines 1* 2* analyn anzano cabras , milton norman dejadena medina and 3 jürgen wiesner 1 college of arts and sciences, university of mindanao, davao city, philippines 2 research and publication center, university of mindanao, davao city, philippines 3 dresdener ring 11, d-38444 wolfsburg, germany received: 25 november 2015/accepted: 16 september 2016 abstract the first faunistic record of tiger beetles (coleoptera: carabidae: cicindelinae) in compostela valley province, mindanao with notes on their habitat is presented. opportunistic sampling, standard light trapping and photo documentation were conducted in selected areas from january to april 2013 and in september 2015. one hundred four tiger beetles belonging to twelve species and seven genera were recorded. an astonishing (100%) endemicity was observed identified to subspecies level. thopeutica and calomera species, specifically thopeutica anichtchenkoi wiesner, 2015 and calomera mindaoensis cassola, 2000 were observed to be abundant in the samples mostly found in the mountainous and forested open riparian ecosystem of cagan, new bataan. cylindera (ifasina) discreta elaphroides (dokhtouroff 1882) was the most common species and was found in different habitat types, but the majority of the species showed a narrow and specialized habitat preference. protection and conservation of remaining forest areas in cagan is especially important in preserving endemic species. keywords: compostela, conservation, endemics, fauna, tiger beetle introduction the philippine tiger beetle fauna is one of the most unique in the world that despite its small land area, philippines ranks fifth in terms of tiger beetle richness and third on the level of endemism after madagascar and australia (cassola & pearson 2000; cassola & ward 2004; cassola 2011). based on endemism per unit area, philippines ranks number one. with very limited entomological expeditions, the tiger beetle fauna of the philippines is poorly known. early collections date back to 1859 1865 by c.g. semper and 1913-1919 by g. boettcher. these tiger beetles were made known by european entomologist, schaum (1860, 1862, 1863) and horn (1907, 1908, 1909, 1923, 1924, 1937). recent publications on the tiger beetle fauna of the philippines were made by bogenberger (1988), cassola (2000, 2011), cassola and ward (2004), cassola and zettel (2006), deuve (2015), dheurle (2015), naviaux (1992, 2002) and wiesner (1980, 1988a, 1988b, 1989, 1992a, 1992b, 2015). since the early 1990's, new tiger beetle species have been described from the different, but poorly explored islands such as palawan, leyte, mindoro and mindanao. to date, there are 139 tiger beetle species known from the philippines, of which 111 (86.3%) are endemic to this country with a limited geographic range (santos 2014). in 1998, an international workshop on the natural history of the philippines held at berlin, germany was conducted where insect studies were greatly emphasized hurdling the concern on increasing loss of philippines forest through deforestation (cassola 2000). since the majority of the species are forest dependent, loss of the forest has caused a tremendous threat to different species and others may have gone extinct without being known to science. biotropia 3 2 6 133 139 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 564 * corresponding author: mnd_medina@umindanao.edu.ph; ann.cabras24@umindanao.edu.ph 133 tiger beetles are often used as good bioindicators of the microhabitat changes of an ecosystem. since many faunal elements including several tiger beetles are associated with forest ecosystems and have a limited habitat range, they are most vulnerable to deforestation (cassola & ward 2004). the philippines had already lost 75% of its forest in the past century, so many tiger beetles may have disappeared without being described. compostela valley province belongs to the eastern biodiversity corridor which is known as one of the remaining biodiversity hotspot of the philippines. however, there is a great concern over its biodiversity, as the province is experiencing major anthropogenic disturbances including illegal logging and mining. this paper presents the baseline data of tiger beetle fauna of compostela valley province with notes on the habitat preference of tiger beetles. this information is important in drafting conservation efforts for this insect group. materials and methods opportunistic sampling, light trapping and photo documentation were conducted for diurnal and nocturnal tiger beetles in selected areas in compostela valley province from january to april 2013 and in september 2015. seven areas were sampled: (1) cagan, new bataan (07°41'n; o o 126°08'e), (2) sampao (07 42' n, 125 59' e), (3) mayaon, montevista (07°48'n; 126°02'e), (4) o o barangay andili, mawab (07 30' n, 125 56' e), (5) pasian, monkayo (07°56'n; 125°58'e), (6) san o o isidro (07 36' n, 125 58' e) and (7) cabalinan, nabunturan (07°36' n; 125°57' e). specimens were obtained using insect net during the day and light trapping during the night. the specimens were killed using ethyl acetate, dried and stored in the first authors' collection. results and discussion a total of 104 individuals belonging to 12 species and seven genera of tiger beetles were recorded. three species were recorded for each of the genera therates and thopeutica, while two species were recorded for the genera calomera and cylindera. prothyma, tricondyla and neocollyris were represented only by one species. seven species (58%) widely distributed from luzon to mindanao are philippine endemics (table 1, while five species have no known record except in mindanao and at the moment they are recognized as mindanao endemics (table 1, fig. 2) unless new records say otherwise. two species out of three calomera present in mindanao were recorded in compostela valley province namely calomera lacrymosa (dejean 1825) and calomera mindaoensis (cassola 2000). majority of the species were collected close to the riverbanks using light trapping method. species of the genera thopeutica, prothyma, 134 biotropia vol. 23 no. 2, 2016 figure 1 map of sampling sites in compostela valley province, philippines 135 – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. t ab le 1 s p ec ie s d is tr ib u ti o n , h ab it at a n d a b u n d an ce o f t ig er b ee tl es i n t h e c o m p o st el a v al le y p ro v in ce s p ec ie s h ab it at t yp e g eo g ra p h ic d is tr ib u ti o n c g c b s i s m m a a n p a n p er ce n ta g e c al om er a m in da na oe ns is (c as so la 2 0 0 0 ) o p en , ri ve ri n e sa n d y ar ea s in m o u n ta in o u s se co n d ar y fo re st m in d an ao e n d em ic 1 3 1 7 2 1 2 0 .1 9 c al om er a la cr ym os a (d ej ea n 1 8 2 5 ) o p en , ri ve ri n e, l o w la n d s an d y ar ea s m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 3 3 2 .8 8 c yl in de ra ( if as in a) m ou th ie z i (d h eu rl e 2 0 1 5 ) b o th f o u n d i n t h e o p en , ri ve ri n e sa n d y ar ea s o f a s ec o n d ar y fo re st in m o u n ta in o u s ar ea an d l o w la n d m ix ed ag ri cu lt u ra l ec o sy st em m in d an ao e n d em ic 1 4 1 6 5 .7 7 c yl in de ra ( if as in a) d is cr et a el ap hr oi de s (d o k h to u ro ff 1 8 8 2 ) b o th f o u n d i n t h e o p en , ri ve ri n e sa n d y ar ea s o f a lo w la n d s ec o n d ar y fo re st a n d m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 4 1 1 6 6 2 7 2 5 .9 6 n eo co lly ri s (h et er oc ol ly ri s) si m ili or (h o rn 1 8 9 3 ) s h ad ed a re as a n d b ra n ch es o f s h ru b s in th e m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 3 3 2 .8 8 p ro th ym a (s ym pl ec th ym a) he te ro m al lic ol lis h et er om al lic ol lis (h o rn 1 9 0 9 ) o p en , ri ve ri n e sa n d y ar ea s in a m ix ed ag ri cu lt u ra l ec o sy st em m in d an ao e n d em ic 1 1 0 .9 6 t he ra te s co ra ci nu s co ra ci nu s (e ri ch so n 1 8 3 4 ) s h ad ed r iv er in e sa n d y ar ea s an d s h ru b s in m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 1 1 2 1 .9 2 t he ra te s fa sc ia tu s fa sc ia tu s (f ab ri ci u s 1 8 0 1 ) s h ad ed r iv er in e sa n d y ar ea s an d s h ru b s in m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 1 2 3 6 5 .7 6 t he ra te s fu lv ip en ni s ev er et ti (b at es 1 8 7 8 ) s h ad ed r iv er in e sa n d y ar ea s an d s h ru b s in m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 3 1 4 3 .8 5 t ho pe ut ic a ( t ho pe ut ic a) a ni ch tc he nk oi (w ie sn er 2 0 1 5 ) o p en , ri ve ri n e sa n d y ar ea s in s ec o n d ar y fo re st in m o u n ta in o u s ar ea m in d an ao e n d em ic 2 4 2 4 2 3 .0 7 t ho pe ut ic a (t ho pe ut ic a) r ol an dm ue lle ri (c as so la 2 0 0 0 ) o p en , ri ve ri n e sa n d y ar ea s in s ec o n d ar y fo re st i n m o u n ta in o u s ar ea a n d l o w la n d m ix ed a g ri cu lt u ra l ec o sy st em m in d an ao e n d em ic 3 1 4 3 .8 4 t ri co nd yl a (t ri co nd yl a) e lo ng at a (h o rn 1 9 0 6 ) s h ad ed a re as a n d b ra n ch es o f s h ru b s in th e m ix ed a g ri cu lt u ra l ec o sy st em p h il ip p in e en d em ic 3 3 2 .8 8 t o ta l 1 0 4 n o te s: c g = c ag an ; c b = c ab al in an ; s i = s an i si d ro ; s m = s am p ao ; m a = m ay ao n ; a n = a n d il i; p a = p as ia n 136 biotropia vol. 23 no. 2, 2016 cylindera and calomera were all found in the open and sandy riverbanks and caught using both light trapping and hand netting methods. these four genera were found to inhabit open and sandy riverine ecosystems, agreeing with dangalle et al. (2012). species of the genera therates, neocollyris and tricondyla were often found perching or running in the shrubs and trees near rivers preferring shade rather than exposed open areas. these species were easily caught using a hand net or by hand. species from the genus therates were especially found in close proximity to river banks while the species from the genera neocollyris and tricondyla were found along the roads and some were near creeks and rivers. the most common species found to be widespread in compostela valley were cylindera (ifasina) discreta elaphroides, therates fasciatus fasciatus and calomera mindanaoensis (table 1). cylindera (ifasina) discreta elaphroides) was the most dispersed species found in four municipalities, while therates fasciatus fasciatus and calomera mindanaoensis were found in three municipalities indicating the wide distribution of these species in the province. thopeutica anichtchenkoi was abundant but exclusively found in a single locality in cagan which is characterized by mountainous secondary forest. other species from the genera therates, tricondyla, neocollyris and prothyma were exclusively distributed in the lowland riparian ecosystems with mixed agricultural vegetation, while species from the genera calomera, cylindera and theopeutica were found either in riverine ecosystems with secondary forest in mountainous areas as well as in lowland open riverine ecosystem with mixed agricultural vegetation. among the areas sampled, mayaon had the highest species richness. the lowland riparian ecosystem with mixed agricultural vegetation with patches of remaining secondary forests was the most preferred habitats by eight species of tiger beetles including calomera mindanaoensis, calomera lacyrmosa, cylindera (ifasina) mouthiezi, cylindera (ifasina) discreta elaphroides, therates coracinus coracinus erichson, 1834, therates fasciatus fasciatus, therates fulvipennis everetti and thopeutica rollandmulleri. cagan which is a mountainous ecosystem was represented by four species of tiger beetles namely calomera mindanaoensis, thopeutica anichtchenkoi, thopeutica rollandmuelleri and cylindera (ifasina) mouthiezi. among the species mentioned only thopeutica anichtchenkoi was exclusively found in cagan which inferred the high habitat preference of this species to forested riverine ecosystems in mountainous areas. the species of the genera calomera, cylindera and thopeutica showed a high preference to mountainous and forested riverine ecosystems as indicated by their high abundance in cagan, whereas the species of the genera therates, neocollyris, tricondyla and prothyma showed high preference to lowland riparian ecosystem with a b figure 2 endemic species (a) calomera mindanaoensis; (b) prothyma (symplecthyma) heteromallicollis heteromallicollis 137 mixed agricultural vegetation. the arboreal nature of the species of the genera therates, neocollyris and tricondyla agreed with cassola (2000) and dangalle et al. (2014) about the habit of these beetles. the preference of the tiger beetles to live in close proximity to the water enables them to be near water and food and provides a good habitat for their reproduction (bhargav & uniyal 2008). the high abundance of cylindera (ifasina) discreta elaphroides being found in five sampling sites as compared to other species could indicate the high adaptability of the species to habitat modification since it was found inhabiting the mountainous forested riverine ecosystem of cagan as well as the low land river banks with mixed agricultural ecosystems in mayaon, san isidro, cabalinan and pasian. on the contrary, thopeutica anichtchenkoi was abundant but exclusively found in a single locality in cagan which was characterized by mountainous secondary forest. the exclusive distribution and high abundance of this species could indicate its high preference to mountainous forested riparian ecosystem. many species of tiger beetles are known for their association with forest habitats and limited distributional range as well as narrow habitat preferences as related to temperature, exposure to sunlight, ph and soil moisture and salinity (dangalle et al. 2014). the other species recorded in cagan such as calomera mindanaoensis, thopeutica rollandmulleri and cylindera (ifasina) mouthiezi were also found in the lowland riverine ecosystems with mixed secondary forest and agricultural vegetation indicating the species association with forested habitats. this also implied the species ability to adapt and thrive in habitats with human encroachment brought by conversion of land to farming. santos (2014) mentioned that further studies should be conducted in tiger beetles' ability to adapt to the changing environmental conditions brought by human disturbances and non-forested habitats. cagan which is a mountainous forested ecosystem had the highest abundance of tiger beetles with 41 (39.2%) individuals belonging to four species and three genera followed by mayaon with 29 (27.88%) individuals recorded belonging to eight species and four genera. pasian had the third highest abundance of tiger beetles with seventeen individuals recorded belonging to five species and three genera. the high abundance of tiger beetles in cagan may indicate the preference of the two most abundant species i.e. calomera mindaoenses and thopeutica anichtchenkoi for riparian ecosystems in mountainous and forested ecosystem due to its vegetation, soil moisture and ph which are important factors that determine tiger beetles' habitat preference (dangalle et al. 2014). the high abundance could be due to the ample availability of food resources which is a key factor in most insect taxon abundance. although mayaon had the highest species richness, species abundance was quite low indicating the high competition of food among the different species of tiger beetles. species coexisting in a locality had been observed. calomera lacrymosa and calomera mindaoensis as mentioned by cassola (2000) can occur sympatrically. both were found occurring in mayaon. calomera lacrymosa was most common in mayaon, while calomera mindaoensis was most abundant in cagan and pasian. these two species can be easily mistaken as one species. however, upon closer inspection it could be found that calomera lacrymosa was smaller at 10 11 mm with paler and dull elytra with the two discal dots connected to each other by a narrow lineole, while calomera mindaoensis was bigger with a size that ranged about 13 14 mm and had velvety and darker elytra with elytral punctuation poorly or not visible at all and the two discal dots not connected. in cagan calomera mindanaoensis and thopeutica anichtchenkoi were the most dominant. however, thopeutica rolandmulleri and cylindera (ifasina) mouthiezi were also found, but in very low number. mayaon which was inhabited by seven species of tiger beetles was dominated by cylindera (ifasina) discreta elaphroides with 16 recorded individuals, while the other species were only represented by low abundance of around 1 4 individuals. the high species richness in mayaon could be attributed to the optimum sunlight and brightness, which were necessary in the predatory activities as well as thermoregulation of the different species of tiger beetles (knisley 1984). areas sampled with the lowest species richness and abundance were cabalinan, barangay. andili and sampao with only one to two species recorded. these riparian areas have a mixed agricultural ecosystem with few standing forest and high anthropogenic disturbances which can inhibit the diversity by reducing the habitat. – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. 138 biotropia vol. 23 no. 2, 2016 conclusions most philippine species of tiger beetles showed a consistent habitat preference. species of the genera tricondyla, neocollyris and therates were arboreal species found in shrubs and trees near the rivers and roads, while other species of the tiger beetles were found in open riparian ecosystems. a narrow distribution of thopeutica anichtchenkoi was observed and found exclusively in cagan. cylindera (ifasina) discreta elaphroides was the most dispersed species found in almost all areas sampled indicating its high adaptability. immediate conservation efforts should be conducted for the tiger beetles thopeutica anichtchenkoi, cylindera (ifasina) mouthiezi and thopeutica rollandmulleri which were highly associated with forested riparian ecosystems and had narrow distribution. references bhargav vk, uniyal vp. 2008. communal roosting of tiger beetles (cicindelidae: coleoptera) in the shivalik hills, himachal pradesh, india. cicindela 40(1-2):112. bogenberger jm. 1988. two new species of tiger beetles from palawan (coleoptera, cicindelidae). mitteilungen der münchner entomologischen gesellschaft 78:109-14. cassola f. 2000. studies on tiger beetles. cii. the cicindelidae collected by roland a. müller in the philippine islands, with description of three new species (coleoptera: cicindelidae). zool med leiden 73(33):491-509. cassola f. 2011. studies of tiger beetles clxxxix. a new calomera species from mindanao, philippines. spixiana 34(1):129-31. cassola f, pearson dl. 2000. global patterns of tiger beetles species richness (coleoptera: cicindelidae): their use in conservation planning. biol cons 95:197208. cassola f, ward rd. 2004. systematics and zoogeography of the philippine species of the genus thopeutica chaudoir, 1861. ann mus civ stor nat genova 96:132. cassola f, zettel h. 2006. a new species and a new record of thopeutica chaudoir, 1861 (coleoptera: cicindelidae) from polillo island, quezon province, the philippines. z arb gem österr entomol 58:45-52. dangalle c, pallewatta n, vogler a. 2012. tiger beetles (coleoptera: cicindelidae) of ancient reservoir ecosystems of sri lanka. j threat taxa 4(4):2490-8. dangalle c, pallewatta n, vogler a. 2014. distribution and habitat preferences of tiger beetles (coleoptera: cicindelidae) of the riverine ecosystems of sri lanka. j threat taxa 6(9):6195-203. deuve t. 2015. deux nouvelles cicindèles des philippines et du mozambique (coleoptera, caraboidea). coléoptères 21(8):99-104. dheurle c. 2015. cylindera (ifasina) mouthiezi, nouvelle espèce des philippines (coleoptera cicindelidae). l'entomologiste 71(2):123-4. horn w. 1907. a new subspecies of philippine cicindelidae. philipp j sci 2(1):77-8. horn w. 1908. prothyma schultzei, a new species of philippine cicindelidae. philipp j sci 3:273-4. horn w. 1909. zwei neue philippinen-prothymae. dtsch. entomol. z. 311-4. horn w. 1923. philippine species of the genus prothyma and other cicindelidae. philipp j sci 22: 357-63. horn w. 1924. three new cicindelidae from the philippines. philipp j sci 24:87-9. horn w. 1937. drei neue orientalische cicindeliden aus dem nat.-mus. in washington. entomol bl biol syst kaefer 33(1):55-7. knisley b. 1984. ecological distribution of tiger beetles (coleoptera: cicindelidae) in colfax county, new mexico. the southwest nat 29(1):93-104. naviaux r. 1992. diagnose de quatre neocollyris nouveaux des philippines et du vietnam (col. cicindelidae). bull soc entomol fr 97(1):42. naviaux r. 2002. les tricondylina (coleoptera, cicindelidae). revision des genres tricondyla latreille et derocrania chaudoir et descriptions de nouveaux taxons. memoires de la sef 5:1-106. santos bs. 2014. distribution patterns of tiger beetle species in the philippines and southeast asia. j entomol zool stud 2 (4): 271-5. schaum h.1860. beiträge zur kenntnis einiger laufkäfergattungen. berliner entomologische zeitschrift 4:180-203, tafel 3. schaum h.1862. die cicindeliden der philippinischen inseln. berliner entomologische zeitschrift 6:17284. schaum h. 1863. beiträge zur kenntnis einiger carabicinen-gattungen. berliner entomologische zeitschrift 7:67-92, tafel 3. wiesner j. 1980. beiträge zur kenntnis der philippinischen cicindelidae (coleoptera). mitteilungen der münchner entomologischen gesellschaft 70:11927. wiesner j. 1988a. die gattung therates latr. und ihre arten. 15. beitrag zur kenntnis der cicindelidae (coleoptera). mitteilung en der münchner entomologischen gesellschaft 78:5-107. 139 wiesner j. 1988b. eine neue cylindera von den philippinen (coleoptera: cicindelidae). 16. beitrag zur kenntnis der cicindelidae. entomologische zeitschrift 98:153-5. wiesner j. 1989. beiträge zur kenntnis der philippinischen cicindelidae (ii) (coleoptera). 22. beitrag zur kenntnis der cicindelidae. entomologische zeitschrift 99:237-8. wiesner j. 1992a. eine neue thopeutica von den philippinen (coleoptera: cicindelidae). 26.beitrag zur kenntnis der cicindelidae. entomologische zeitschrift 102:128-30. wiesner j. 1992b. verzeichnis der sandlaufkäfer der welt. checklist of the tiger beetles of the world. verlag erna bauer, keltern, 1-364. wiesner j. 2015. two new thopeutica species from the philippines (coleoptera: carabidae: cicindelinae). 122. contribution towards the knowledge of cicindelinae. mitteilungen des internationalen entomologischen vereins 40(1/2):1-8. – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 sri-13 mei 2017-524 (sunardi invasion).cdr invasion of willd. after eruptionacacia decurrens of mount merapi, indonesia sunardi sulistijorini and titiek setyawati 1*, 1 2 1 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680 indonesia, 2 rresearch center of conservation and ehabilitation, ministry of environment and forestry, bogor 16610 i, ndonesia received 12 august 2015/accepted 12 november 2016 abstract eruption of mount merapi in 2010 caused a dense cover of acacia decurrens willd., which is an invasive alien plant species (iaps). the dense cover happened in all areas of mount merapi national park (mmnp) in java, indonesia. this study was aimed to describe the relationship between major natural disturbance from volcanic eruption in triggering the invasion of a. decurrens in mount merapi national park. vegetation data were collected using line transect in two different sites. the first site was cangkringan which was affected by pyroclastic flow and the second site was selo which was not affected by pyroclastic flow. distribution patterns and association of a. decurrens with other species in each location was analyzed using ordination analysis of the non-metric multidimensional scaling (nmds). microclimate such as temperature, humidity, light density and soil humidity was recorded in each location. correlation between species abundance and microclimate data was assessed using canonical correspondence analysis (cca). the results showed that the population of a. decurrens was more dominant in cangkringan than in selo site. cangkringan site was impacted with pyroclastic flow during mount merapi eruption in 2010, while selo site was not affected. in cangkringan, a. decurrens was distributed in clump, while in selo the plant was randomly distributed. ordination analysis using nmds showed that there was positive association between a. decurrens and herbaceous plant. negative association was observed between a. decurrens and other tree species. cca analysis showed that temperature and light density was positively correlated with a. decurrens abundance. this study showed that the iaps invasion in mmnp was correlated with the eruption of mount merapi. keywords: acacia decurrens, autecology, eruption, invasive introduction mount merapi national park (mmnp) is a protected forest, rich with various flora and fauna. mount merapi, located in the mmnp, is the most active volcano in indonesia. the eruption of mount merapi from 26 october to 6 november 2010 was recorded as the worst disaster since its eruption in 1870 (bnpb 2011). mount merapi eruption in 2010 was characterized by pyroclastic flow (a fast moving flow containing high-density mixture of hot lava blocks, pumice, ash and volcanic gas) which reached temperature of o 400 – 600 c and speed of 130 km/h, causing vegetation destruction, among others. vegetation succession after the eruption showed a decline in the diversity of alpine vegetation. the native plant was replaced by invasive alien plant species (iaps) which became dominant in several locations affected by the eruption of mount merapi. dadap ( and pine (erythrina longifolia) pinus merkusii), which were initially the most common plant species in this area, were not recorded after the eruption. pine is not a local species in this mountain area, however, this plant might have been escaped from the neighboring plantation forest managed by state forest enterprises. invasive alien species (ias) is a combination of alien species and their invasive characteristics. an alien species is a species (at the level of species, subspecies, varieties) that had been intentionally or unintentionally introduced (as whole organism, part of the body, gametes, seeds, eggs or propagules that are able to live and reproduce in biotropia 4 1 7 35 46 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 524 * corresponding author: sunardi.mansyur@gmail.com 35 the new habitat) into areas outside of its natural distribution range. invasive species, either native or introduced species, become densely established and broadly affect the habitat and land uses resulting to serious environmental, social and economic impacts (cbd-unep 2014). acacia decurrens is one of serious invasive alien plant species (iaps) in mmnp. the plant became dominant in the areas affected by pyroclastic flow. this plant, originated from australia, belongs to family of fabaceae, subfamily of mimosoidae and genus of . acacia within their native distribution range, acacia decurrens is considered as fast growing pioneer species and often causes serious concern due to its potential invasion to their new habitat. iaps can establish themselves in a disturbance area and begin to dominate native vegetation. invasion by an alien species can occur in an area if the native species are unable to quickly adapt to the change of environmental conditions. iaps can significantly suppress the native plant, invertebrate and vertebrate population and communities. for example, lantana camara invaded large areas of tropical asia and australia as well as contributed to the declines of several endangered species in australia (coutts-smith & downey 2006). the invasion of in acacia nilotica baluran national park has changed the savanna into a shrub of and contributed to the acacia decreasing number of banteng ( ) bos javanicus population (setiabudi et al. 2013). plant invasion can occur due to natural disasters, climate and environment changes. the invasion can also happen in disturbed habitats. there are only a few studies focused on the impact of natural disturbance from volcano eruption to the invasion of iaps. therefore, studying biological invasions in mountains having natural disturbance is scientifically important for nature conservation. this study was aimed at determining correlation between mount merapi eruption and the invasion of in a. decurrens mmnp and at determining the autecology of a. decurrens as basic information for managing plant invasion in mmnp. it was assumed that the eruption of mount merapi with the pyroclastic flow has positive correlation with the abundance population of in mount merapi. the a. decurrens autecology study was performed to explain the characteristic, dynamic population of individual species, life cycle and ecological factors, including physical, biological and environmental factors. materials and methods study site the research was conducted from march to december 2014. the research was conducted in mount merapi national park (mmnp). the research location was divided into two sites. the first site was cangkringan located at o o 7 35.771” – 110 26.375” (954 m asl). this site was affected by pyroclastic flow during the eruption in 2010. the second site was selo located o o at 7 30.843” – 100 27.188” (1,044 m asl). this site was not affected by the pyroclastic flow. the study at each site was conducted at three different altitude zones i.e. high zone (1,400 – 1,700 m asl), middle zone (1,100 – 1,400 m asl) and lower zone (800 – 1,100 m asl). sampling design and data analysis vegetation analysis was performed using a systematic sampling with transect line along 100 m for each zone in the two study sites. each transect line consisted of 10 quadrats of 10 x 10 m (for tree species), 10 quadrats of 5 x 5 m (for sapling species) and 10 quadrats of 2 x 2 m (for herb species) (cropper 1993; krebs 2002). height and diameter data of a. decurrens trees were collected at each site. these data were collected from 15 trees that were inside the 10 quadrat plots of 10 × 10 m. environmental data collected at each site were soil physical and chemical properties, air temperature, humidity, light intensity and wind speed. importance value index (ivi) was calculated to determine the overall importance of each species in the community structure. in calculating this index, the percentage values of the relative frequency, relative density and relative dominance were summed up together . (kent & paddy 1992) species diversity was determined using shannonwiener diversity index (h), while species dominance was determined using simpson's dominance index (d). shannon-wiener diversity index (h) is an index that is commonly used to characterize species diversity in a community. shannon36 biotropia vol. 24 no. 1, 2017 ∑ = sum of the quadrat countxi ∑ = sum of the quadrat count from the total of 2 xi species in the community the deviation from random expectation can be tested using critical values of the chi-square distribution with n-1 degrees of freedom. confidence interval around 1 can be calculated by the uniform (mu) and clumped (mc) indices (krebs 2002). where: 2 x 0.0975 = value of chi-square from the table with (n-1), degree of freedom that has 97.5 % of the area to right 2 x 0.0025 = value of chi-square from the table with (n-1), degree of freedom that has 2.5 % of the area to right xi = number of individual of species in a set of quadrat n = number of quadrat standardized morisita index was calculated using the following four formulas: standardized morisita index of dispersion (ip) ranges from -1.0 to +1.0 with 95% confidence limit at +0.5. ip = 0 means that the plants are in random dispersion, ip > 0 means that the plants are in clumped dispersion and ip < 0 means that the plants are in uniform dispersion. correlation between environmental factors and abundance of a. decurrens was analyzed using canonical correspondence analysis (cca) of the past v2. software (legendre & legendre 1998; hammer et al. 2005). distribution and association between a. decurrens and other species in the community were determined using species ordination of the non-metric multidimensional scaling and calculated using primer v5. software (clarke & gorley 2015). wiener diversity index (h) accounts for both abundance and evenness of the existing species, as is shown in this formula: where: h = shannon-wiener diversity index pi = proportion of species n = number of speciesi n = number of quadrat simpson's dominance index (d) is a simple mathematical measure that characterizes species diversity in a community. the proportion of species (ni) relative to the total number of species (n) is calculated and squared. the squared proportions for all the species are summed, and the reciprocal is taken: where: d = simpson's dominance index n = number of speciesi n = number of quadrat the simpson's index ranges: 1. if d = 0 – 0.5 this means that none species were dominant 2. if d = 0.5 – 1 this means that there is a dominant species evenness index (e) can be calculated by dividing h by h (where h = ln ). equitability smax max assumes a value between 0 and 1 with 1 being complete evenness. where: e = evenness index h = shannon-wiener value s = total number of species in the community plant distribution pattern was analyzed using morisita index of dispersion (ið), as is shown by this formula: where: ið = morisita index of dispersion n = sample size 37 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. results and discussion species composition of the vegetation at cangkringan and selo sites in terms of importance value index (ivi) and morisita index of dispersion for herbs, sapling and tree for each altitude zone are shown in table 1 and 2. vegetation at cangkringan site was dominated by herbs species of imperata cylindrica, centella asiatica, impatiens balsamina and by sapling and tree species of a. decurrens (table 1). vegetation at selo site was dominated by species of , , c. asiatica eupatorium odoratum eupatorium riparium pennisetum pur pureum, , pogonatum sp., and by sapling and tree species of a. decurrens vaccinium varingiaefolium albizia , and lophantha (table 2). 38 biotropia vol. 24 no. 1, 2017 table 1 vegetation observed at cangkringan site that were affected by pyroclastic flow no. species family importance value index morisita index zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 herbs 1 centella asiatica apiaceae 14.46 19.46 13.59 c c c 2 ageratum conyzoides l.* asteraceae 4.59 7.88 11.88 c c c 3 bidens biternata asteraceae 10.64 6.32 8.85 c c u 4 emilia sonchifolia asteraceae 4.30 10.92 7.11 c c c 5 eupatorium odoratum* asteraceae 8.54 1.18 7.00 c c c 6 eupatorium inulifolium* asteraceae 5.46 13.70 4.37 c c c 7 eupatorium triplinerve* asteraceae 4.20 5.39 3.35 c c c 8 gynura crepidioides asteraceae 1.35 7.60 4.83 c c 9 mimosa pudica* asteraceae 10.14 3.64 8.23 c c c 10 pennisetum macrostachyum* asteraceae 6.85 4.77 5.68 c c c 11 polygala paniculata* asteraceae 11.87 1.28 10.11 c c c 12 sida rhombifolia asteraceae 1.07 5.29 0.86 c c c 13 stachytarpheta jamaicensis asteraceae 2.51 2.46 2.04 c c c 14 wedelia trilobata* asteraceae 9.84 3.45 8.23 c c u 15 impatiens platypetala balsaminaceae 6.97 10.92 6.14 c r c 16 cyperus rotundus* cyperaceae 3.86 3.73 3.14 c c c 17 erigeron sumatrensis retz gleicheniaceae 10.33 8.39 c u 18 melastoma malabathricum* melastomaceae 1.97 6.76 c c c 19 selaginella kraussiana mimosaceae 3.93 3.11 r c r 20 synedrella nodiflora oxalidaceae 10.07 8.26 c u c 21 imperata cylindrica* poaceae 31.19 59.85 26.59 c c 22 tithonia diversifolia* poaceae 1.07 1.09 0.86 c c c 23 tridax procumbens poaceae 3.15 2.08 11.24 c c c 24 gleichenia longissima polygalaceae 5.39 3.73 4.40 c c 25 oxalis corniculata* silaginellaceae 7.13 4.67 5.93 c c c 26 lantana camara* verbenaceae 5.48 6.53 4.48 c c c 27 rubus chrysophyllus miq. verbenaceae 1.97 1.99 1.56 c c c sapling 1 acacia decurrens* fabaceae 80.84 81.84 84.00 c c c 2 erythrina variegata fabaceae 57.82 35.05 c c 3 paraserianthes falcataria fabaceae 23.19 79.62 c u 4 schima wallichii theaceae 59.82 c pole 1 acacia decurrens* fabaceae 157.68 158.68 146.09 r c c 2 erythrina variegata fabaceae 28.93 30.93 21.30 c c c 3 paraserianthes falcataria fabaceae 9.64 12.64 32.61 c c c notes: = not found in the site; c = clump; r = random; u = uniform * = invasive plant based on information from global ias database (www.issg.org) herbs at cangkringan site were dominated by asteraceae family, while at selo site were dominated by asteraceae and verbenaceae families. the domination of asteraceae family occurred because this family can well adapt to the mountain slope conditions. this family also has role in ecosystem functions, such as preventing erosion and enriching soil organic matter 39 table 2 vegetation observed at selo site that were not affected by pyroclastic flow no. species family importance value index morisita index zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 herbs 1 centella asiatica apiaceae 34.16 20.43 5.18 r c c 2 foeniculum vulgare apiaceae 5.05 0.0 0.00 c 3 anaphalis javanica asteraceae 6.32 10.50 16.21 u c c 4 anaphalis longifolia asteraceae 3.79 11.31 9.96 u c c 5 lactuca sativa asteraceae 4.25 6.07 19.20 c c c 6 athyrium sp. athyriaceae 7.34 6.07 6.62 c c c 7 impatiens platypetala balsaminaceae 12.16 12.16 0.00 c c 8 erigeron sumatrensis retz* compositae 5.51 9.84 0.00 c c 9 indigofera cassioides fabaceae 4.40 1.97 0.00 c c 10 eupatorium odoratum* malvaceae 5.93 9.03 27.02 c c r 11 eupatorium riparium* malvaceae 21.34 9.36 8.49 c c c 12 melastoma malabathricum * melastomaceae 5.97 3.44 5.15 c c c 13 oxalis corniculata oxalidaceae 12.38 59.80 0.00 c c 14 pennisetum purpureum* poaceae 42.09 25.40 50.56 c r r 15 pogonatum sp. poaceae 10.70 0.0 31.00 c c 16 brugmansia candida solanaceae 4.10 0.0 0.00 c 17 lantana camara* verbenaceae 7.08 5.90 11.43 c c c 18 rubus chrysophyllus miq. verbenaceae 2.83 3.44 4.78 c c c 19 rubus plicatus verbenaceae 4.55 5.24 7.72 c c r sapling 1 acacia decurrens* fabaceae 33.67 42.51 58.67 r r r 2 vaccinium varingiaefolium ericaceae 39.29 64.64 104.53 c c m 3 albizia lophantha* fabaceae 37.90 30.38 36.00 c c m 4 erythrina lithosperma fabaceae 27.29 30.95 0.00 c u 5 toona sureni meliaceae 16.52 8.13 0.00 c c 6 cinchona succirubra rubiaceae 22.90 11.69 0.00 c c 7 dodonaea viscosa sapindaceae 14.52 12.69 0.00 a 8 schima wallichii theaceae 21.90 42.51 0.00 c c pole 1 casuarina junghuhniana casuarinaceae 17.44 18.44 23.81 c c c 2 cupressus montana cupressaceae 13.04 15.04 10.48 c c c 3 vaccinium varingiaefolium ericaceae 19.60 22.60 47.86 c c c 4 acacia decurrens* fabaceae 41.94 45.94 107.38 c c r 5 albizia lophantha fabaceae 37.77 42.77 11.19 c c c 6 erythrina lithosperma fabaceae 18.39 24.39 0.00 c c 7 toona sureni meliaceae 6.52 0.00 0.00 c 8 myrica javanica myricaceae 8.06 0.00 0.00 c 9 cinchona succirubra rubiaceae 9.01 13.52 0.00 c c 10 dodonaea viscosa sapindaceae 4.98 0.00 0.00 c 11 schima wallichii theaceae 24.69 16.06 0.00 c r _notes: = not found in the site; c = clump; r = random; u = uniform * = invasive plant based on information from global ias database (www.issg.org) invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. (kumolo & utami 2011). they are often classified as weed on agricultural land. one species member i.e. was reported by sunaryo e. odoratum et al. (2012) and uji (2010) for invading several locations in the mount salak and mount gede pangrango national park. based on database from invasive species specialist group (issg 2015), numbers of herbs and tree species in the two study sites were listed as invasive alien plant species (iaps). only few numbers of native plants were found after the eruption. native plants can adapt to a very low frequency of disturbance and take a long time to recover into the same population size as before the disturbance (smith & tunison 1992). a. decurrens is the most dominant and has high risk to the environment in mount merapi national park. this species densely covered the vegetation at cangkringan site. morisita index of dispersion showed that the a. decurrens was dispersed into clumped pattern (table 1 & 2). in natural ecosystem, plant distribution or dispersion was correlated with environmental condition and competition with other species in the community. random dispersion of plant in the community is rarely found in the tropic ecosystem (call & nilsen 2003). clump distribution of the plant was also caused by high seed reproduction of mature plants which seeds fall surrounding the parent plant. environmental factors such as climate, wind, soil nutrients may influence plant distribution. clump distribution of plant species affected species diversity and evenness of plant community (soerianegara & indrawan 1998). the results showed that at cangkringan site population of was clump distribution a. decurrens (table 1) with lower diversity index compared to the index at selo site (table 3). a. decurrens at selo site was randomly distributed (table 2). species diversity in a plant community was affected by density of the individual species, the larger number of the species and the spread of each species. at cangkringan site the evenness index showed that the herbs were more stable than the pole or tree plant due to the invasion of which a. decurrens densely distributed in clumps. simpson's dominance index (d) showed that some species dominated plant community both at cangkringan and selo sites. pole and tree vegetations at cangkringan site had low dominance indices than those at selo site. smaller dominance index indicated that there was a species spread and dominated the coverage of an area (krebs 2002). 40 biotropia vol. 24 no. 1, 2017 table 3 shannon-wiener diversity index (h), evenness index (e) and simpson's dominance index (d) of vegetations at cangkringan and selo sites location zone vegetation category h e d cangkringan 1 herb 2.80 0.84 0.91 sapling 1.39 0.86 0.73 pole 0.39 0.35 0.19 2 herb 2.80 0.88 0.72 sapling 0.55 0.79 0.86 pole 0.17 0.15 0.87 3 herb 2.89 0.87 0.92 sapling 1.00 1.44 0.60 pole 0.15 0.14 0.96 selo 1 herb 2.15 0.65 0.82 sapling 2.66 1.65 0.85 pole 2.02 1.84 0.53 2 herb 1.89 0.57 0.75 sapling 1.84 1.14 0.37 pole 2.02 1.84 0.76 3 herb 1.95 0.59 0.80 sapling 0.98 0.61 0.44 pole 0.99 0.90 0.52 invasion of a. decurrens, forming monoculture thickets, may reduce the abundance of native plant species. few native species found at the cangkringan site was highly invaded by a. decurrens. reduction of species diversity may be caused by competition for soil nutrients between native species and iaps. other possible cause was the ability of a. decurrens (legume family) to increase soil nitrogen content (nitrogen fixing). changes in vegetation composition and loss of diversity are considered to negatively affect wildlife through quality and quantity of ecosystem services (mc. donald et al. 2012). autecology perhutani (state forest enterprise) introduced a. decurrens into mmnp around 1980s, before the region was declared as a conservation area. a. decurrens was used as divider between plantation area with agriculture land. over time, was acacia widespread in several regions around the national park. significant high number of a. decurrens population was recorded in 2011, a few months after the eruption of mount merapi which occurred from october to november 2010. acacia has hard and thick seed coat to remain dormant in certain circumstances. also, the seed structure makes it possible for to germinate under acacia u n s u i t a b l e c o n d i t i o n c a u s e d by m a j o r disturbances. invasive species tend to have generalist pollination, mass seed production, efficient seed dispersal and persistent seed bank which can endure heat caused by fire or disturbance that triggered seed germination (gibson 2011). this study indicated that the et al. pyroclastic flow broke the dormancy of seed stored in the soil (soil seed bank). invasion acacia in mmnp was caused by major disturbances resulted from mount merapi eruption. the invasion occurred in several areas impacted by the pyroclastic flow. the pyroclastic flow contained materials that can stimulate biological production in the impacted environment. the materials can be used as fertilizer and contain many elements that can increase soil fertility. there is positive correlation between a. decurrens invasion and mount merapi eruption, accompanied by pyroclastic flow. fire is one of natural disturbances and important processes for maintaining species diversity in forest ecosystem (walker & del moral 2003). non-native plant species with an established seed bank tend to respond positively to post-fire conditions, such as increased soil temperatures, increased light levels, reduced competitions and increased available nitrogen (wagner & fraterrigo 2015). a. decurrens is known for their high flammability and has tendency to quickly recover from disturbance compared to native species (brooks et al. 2004). a. decur r enshigh population of at cangkringan site was an example showing correlation between area impacted by pyroclastic flow and invasion in the area. selo site had acacia low population of because selo site was acacia not impacted by pyroclastic flow (table 1 & 2). invasive species responded quicker to disturbance than non-invasive species (gibson et al. 2011). invasive alien plant species can widely spread, have high growth rate and have high tolerance to physical conditions such as fire, flood, drought and other natural disturbances. these physical conditions are the major factors for alien plant species to invade a new area (velde et al. 2006). there are four factors influencing the success of species invasion i.e. disturbances occurred in ecosystem (fire or natural disaster), ability to compete, availability of resources and pressure of propagules (moser 2009). et al. species invasion is the result of interactions between habitat suitability and pressure of propagules (rejmànek 2005). changes in et al. environmental conditions may affect the level of species invasion. changes in environmental conditions interfere with the balance of competition between native plants and foreign plants. among changes in the environment are limited metabolism, high temperature and presence of toxin (alpert 2000).et al. a. decurrenscluster analysis showed that has positive correlation only with herbs. high numbers of correlation were observed between a. decurrens c. asiatica e. riparium impatiens and , , platypetala pennisetum pur pureum and (fig.1). negative cor relations occur red between a. decurrens and native species. the results also showed that at cangkringan site there was a decrease in the number of native tree species due to high numbers of (fig. 2)a. decurrens . 41 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. being tree and woody species, a. decurrens is a competitor to other tree species. the dominance of a. decurrens presents an extreme challenge for the native plant communities because the rapid canopies growth of a. decurrens hindered other plants from obtaining light. it is very important to understand that invasion process varies along biotic and abiotic gradients within a local environment. abiotic variable is the most important factor to study the plant autecology (swamy et al. 2000). the study of abiotic gradient analysis showed that the invasion of a. decurrens correlated with available light and weather temperature (fig. 3). changes in available light, wind speed, humidity, temperature and soil moisture induced by fragmentation of forest ecosystems often add to competitive advantages of invasive species over native species (emily et al. 2004). mount merapi eruption has burned down all of the native vegetation and transform it into the open area. as the open area, automatically the light density was increased light intensity and also the increase of the temperature after the eruption. 42 biotropia vol. 24 no. 1, 2017 figure 1 nmds ordination result (2d stress = 0.01, measured from relationship between actual dissimilarities and distances) which shows species composition difference in each sample plot and change in abundance of a. decurrens figure 2 nmds ordination result (2d stress = 0.11, measured from relationship between actual dissimilarities and distances) which shows association clusters of plant community including a. decurrens at sampling locations s1 s3 c3 c2 s2 c1 figure 3 the influence of environmental factors in the two study sites towards a. decurrens population using canonical correspondence analysis (cca) notes: c = cangkringan; s = selo; 1 = zone 1; 2 = zone 2; 3 = zone 3 the increase of temperature and the light density was triggered the seed bank to germinate. iaps can endure extreme temperature which makes them competitor to the native vegetation (hellmann et al. 2008; ordonez et al. 2010; van kleunen et al. 2010). song et al. (2010) studied the effect of extreme high temperatures for the invasive wedelia trilobata, and found out that in the extreme high temperature condition, w. trilobata experienced less inhibition of relative growth rate (rgr) and biomass production than the native plant wedelia chinensis. our study compared diameter and height of a. decurrens located in area affected by pyroclastic flow (cangkringan) and those located in area not affected by pyroclastic flow (selo). diameter of a. decurrens at cangkringan site was lower than that at selo site. inverse effect observed for the height of a. decurrens (fig. 4 & 5). tree height or diameter was also affected by species composition of understory (suryanto et al. 2010). iaps also produce large amount of litter under the canopies, which may influence understory species (williams & wardle 2007). results of this figure 4 diameter of a. decurrens at cangkringan site (invaded) and selo site (uninvaded) 43 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. a x is 2 -1.8 -1.5 -1.2 -0.9 -0.6 -0.3 0.3 0.6 0.9 1.2 1.5 1.8 0.3 0.6 0.9 1.2 1.5 1.8 -0.3 -0.6 -0.9 -1.2 -1.5 -1.8 axis 1 light temperature soil humidity soil ph wind humidity xs1+c2 xs2 xs3 +c3 +c1 figure 5 height of a. decurrens at cangkringan site (invaded) and selo site (uninvaded) study revealed that there were different sizes of diameters as well as dissimilarity in native understory plant composition between those located at cangkringan site (invaded by a. decurrens) and those located at selo site (not invaded by a. decurrens). dense population at cangkringan (invaded site) would lead to intraspecific competition in obtaining resources from the environment. a. decurrens invaded from low elevation to higher elevation; therefore, it was adapted to the climate of mount merapi. the management of mmnp has to be aware of the invasive ability of a. decurrens in order to control the spreading of this plant species. among effective treatments to eradicate population are cut stump a. decurrens method, herbicide treatment and seed production decrease. we suggest conducting a study for determining the most feasible and most economical management strategy to eradicate this invasive species. among characteristics of are a. decurrens having mass seed production, seed germination triggered by high temperature, high potential to spread and ability to produce root sucker. in managing the invasion, it is not immediately apparent whether the eradication method used will be cost effective or not. therefore, it is very important to have a good understanding of the invaded site, the invasive plant species characteristics, the biological impacts and the management strategy to control invasive plant species (moore 2011).et al. conclusions mount merapi eruption triggered the spread of a. decurrens in mmnp. the importance value index showed that the invasion of a. decurrens was more dominant in cangkringan site (affected by the eruption) than in selo site (not affected by the eruption). nmds ordination showed the differences in the composition and abundance of a. decurrens between cangkringan site and selo site. a. decurrens showed a clump dispersal pattern at cangkringan site and a random dispersal patterns at selo site. environmental factors positively correlated with the abundance of a. decurrens were temperature and light intensity. acknowledgements we are thankful to unep/gef project “removing barriers to invasive species management in production and protection forest in south east asia (foris) – indonesia who funded this research. many thanks to the management of mount merapi national park for assistance in collecting field data. references alpert p, bone e, holzapfel c. 2000. invasiveness, invisibility and the role of environmental stress in the spread of non-native plants. perspect plant ecol evol syst 3(1):52-66. 44 biotropia vol. 24 no. 1, 2017 badan nasional penanggulangan bencana [bnpb]. 2011. impact of mount merapi eruption. gema bnpb. 2(1):17-20. brooks ml, d'antonio cm, richadson dm, grace jb, keeley je, ditomasso jm, hobss rj, pellant m, pyke d. 2004. effect of invasive alien plant on fire gerimes. bioscience 54: 677-88. call lj, nilsen et. 2003. analysis of spatial patterns and spatial association between the invasive tree-ofheaven ( ) and the native black ailanthus altissima locust ( ). am midl nat. 150:1-4.robinia pseudoacacia clarke kr, gorley rn. 200 . primer: plymouth routines 1 in multivariate ecological research. plymouth (uk): primer-e ltd. 129 p. convention on biological diversity-united nations environment program cbd 2014. 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1-10. 46 biotropia vol. 24 no. 1, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 684 goutam banerjee (effect of culture).cdr effect of culture parameters on protease and cellulase production by two bacterial strains, corynebacterium alkanolyticum bacillus licheniformisath3 and cbh7 isolated from fish gut goutam banerjee , ankita nandi and arun kumar ray 1,2 1 1,* 1 department of zoology, visva-bharati university, santiniketan 731235, india 2 department of biochemistry, university of calcutta, kolkata 700019, india received 7 august 2016/accepted 22 july 2017 abstract microbial protease and cellulase are in high demand by different industries due to their minimal cost and availability. this study was aimed to maximize the production of protease and cellulase using two bacteria, corynebacterium alkanolyticum ath3 and bacillus licheniformis cbh7, isolated from fish gut. this study demonstrated the effect of different culture parameters in protease and cellulase production using two different bacterial strains. results of this study clearly indicated the importance of different parameters such as moisture content, ph, incubation temperature, incubation period, inoculum size, carbon sources and nitrogen sources in enzyme production. the most critical parameters affecting the enzymes production were ph, temperature, carbon and nitrogen sources. further investigations are required to enhance the enzymes production using genetic engineering. keywords: bacterial strains, culture parameters, product maximization, protease and cellulase introduction protease catalyzes the hydrolysis of protein and cellulose forming amino acids, while cellulase catalyzes glucose (ray et al. 2012a). protease and cellulase are biotechnologically very important and mainly isolated from different living organisms such as plants, bacteria and fungi (saddler 1993; gupta et al. 2002; banerjee et al. 2015b). gut bacteria are proven to produce various substances such as vitamin k, riboflavin, and various enzymes (e.g. protease, amylase, cellulase, lipase, phytase) (ray et al. 2012a). proteases isolated from microbial source are preferred over the enzymes isolated from plant and animal sources because they possess all the characteristics required for their biotechnological application (rao et al. 1998; beg & gupta 2003). proteases constitute 50 – 65% of the global industrial enzymes market (banerjee et al. 2015b). proteases from microbial sources are widely used in different industries such as food, textile and beverages (pastor et al. 2001). proteases are useful for preparing high quality functional feeds through bioconversion of low-cost feed materials (esakkiraj et al. 2007). among several bacterial strains, genus bacillus is considered to be the most important commercial enzyme producers (ray et al. 2012a; banerjee & ray 2016). cellulose, a polymer of glucose, is the primary structural component of most plant cell walls. although this polysaccharide is the most common carbohydrate on earth, relatively few animals are able to utilize this resource efficiently (goodenough & goodenough 1993; he et al. 2015). utilization of cellulose as a nutrient source requires enzymes that cleave beta-1, 4 glycosidic bonds between constituent sugars. the enzyme referred to as cellulases, are required to split beta1, 4 glycosidic bonds in the polymer to release glucose units (barr et al. 1996). although a large number of microorganisms are capable of degrading cellulose, only a few of these microorganisms produce significant quantities of extracellular enzymes capable of completely hydrolyzing crystalline cellulose in vitro. fungi are * corresponding author: aray51@yahoo.com biotropia 4 3 7 192 201 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 684 192 the main cellulase-producing microorganisms, though a few bacteria and actinomycetes are also recently reported to be good producer of cellulase (ray et al. 2012a; lugani et al. 2015; banerjee & ray 2016). microbial enzymes have the advantage of large scale production using established fermentation techniques. a successful fermentation process requires favorable environmental and nutritional conditions for the microorganisms (ray . et al 2007). information on the optimum conditions for protease and cellulase production using fish gut bacteria is limited (esakkiraj 2007; ray . et al. et al 2007, 2012b). this study was aimed to optimize the environmental and nutritional parameters to enhance protease and cellulase production using two bacterial strains, corynebacterium alkanolyticum ath3 and cbh7, isolated bacillus licheniformis from the gut of climbing perch, anabas testudineus and walking catfish, , respectively.clarias batrachus materials and methods bacterial strains and culture medium two bacterial strains cor ynebacterium alkanolyticum ath3 (genbank accession no. jx656749) and bacillus licheniformis cbh7 (genbank accession no. hq005269) were isolated from the gut of two species of indian airbreathing fish, i.e. climbing perch, anabas testudineus and walking catfish, clarias batrachus, respectively (banerjee & ray 2013; banerjee et al. 2015a). both isolates were cultured in tryptone soya agar broth for 24 hours at 30 oc to increase the viable count. the bacterial culture medium was prepared according to ray et al. (2012b). enzyme assay extracellular protease activity was assayed following the method of walter (1984). the amount of tyrosine released in the assay medium was measured at 273 nm. cellulase activity was estimated according to the method of denison and kohen (1977). the production of reducing sugar (glucose) from cmc substrate through cellulolytic activity was measured at 540 nm. one unit of specific activity is defined as amount of product released per milligram protein per m i l l i l i t e r e n z y m e p e r m i nu t e. p r o t e i n concentration was determined according to lowry . (1951) using bovine serum albumin as et al a standard. effect of different physical parameters on enzyme production moisture content maximization in order to determine the optimum moisture content, bacteria were cultured in protease ( p e p t o n e g e l a t i n ) a n d c e l l u l a s e m e d i a (carboxymethylcellulose), which were prepared by moistening the media with basal salt (elaborate basal salt) solution. moisture content of the fermentation medium ranged from 5 to75%. effect of ph of the medium optimization of suitable ph for enzymes production was determined by culturing the bacterial strains in tryptone soya agar broth at different ph ranging from 5.0 10.0. incubation temperature maximization optimum temperature of incubation for protease and cellulase production was determined by culturing the bacterial strains at increasing temperature ranging from 20 to 50 c with 10 c o o intervals. incubation period maximization incubation period is one of the most important parameter in fermentation process. to determine the optimum incubation period, enzymes activity was measured up to 120 hours at an interval of 24 hours. effect of inoculum size for this purpose, fermentation media were seeded with 1%, 2%, 3%, 4%, 5% and 6% seed culture in tryptone soya agar broth. effect of different carbon sources in order to optimize the suitable carbon source for enzyme production, fermentation media were prepared with different carbon sources such as glucose, maltose, sucrose, lactose, arabinose, fructose and starch. these alternative carbon sources at 0.2% level were used to replace the original carbon source in the media. effect of different nitrogen sources to optimize the appropriate nitrogen source, the fermentation media were supplemented with both organic (arginine, l-asparagine, tryoptone, beef extract, tyrosine, gelatin) and inorganic 193 effect of culture parameters on enzyme production banerjee et al. metabolic process requirements to supply sufficient energ y for maximum biomass production (ray et al. 2012b). cellulase production reached the maximum level using two bacterial strains bacillus subtilis and bacillus circulans at 10% moisture content (ray et al. 2007). protease production reached the optimum level using bacillus cereus at at 120% moisture content (vijayaraghavan et al. 2014). moisture content was a crucial factor for producing bacterial extracellular enzyme, because water was required for growth, metabolic activities and biochemical process. thus, optimum moisture content was required during fermentation process. ph of the culture medium was another important factor controlling enzyme production and activity. in the present study, protease production exhibited by . ath3 c alkanolyticum was maximum at ph 7.5 (3.81±0.096 u), whereas b. licheniformis cbh7 showed the highest production at ph 8.0 (2.22±0.204 u (table 2). on the other hand, cellulase production exhibited by these two bacterial strains was recorded to be the highest at ph 7.5 (8.41±0.83 u in c. alkanolyticum ath3 and 6.27±0.216 u in b. licheniformis cbh7, table 2) and thereafter declined rapidly. cellulase production using bacillus subtilis cy5 isolated from the gastrointestinal (gi) tracts of common carp, cyprinus carpio and using b. circulans tp3 isolated from the gi tracts of tilapia, oreochromis (ammonium chloride, ammonium sulphate, sodium nitrate and potassium nitrate) nitrogen sources at 0.2% level replacing the original nitrogen source in the media. statistical analysis the analysis of variance for different values was carried out in microsoft excel 2007. one way anova followed by duncan's multiple range test (duncan 1955) was conducted at significance level of p < 0.05. results and discussion protease and cellulase production using the two bacterial strains at different moisture content (5 75%) are tabulated in table 1. protease production using the two bacterial strains was the highest at 10% moisture content (3.79± 0.155 u in corynebacterium alkanolyticum ath3 and 2.16±0.132 u in bacillus licheniformis cbh7). maximum cellulase activity was recorded at 15% moisture content (8.34±0.144 u in c. alkanolyticum ath3 and 6.17±0.115 u in b. licheniformis cbh7). medium maximization was the first and foremost step to enhance enzymes production in laboratory experiments and in fermentation industries. each and every element should be in a proper amount to fulfill the biotropia vol. 24 no. 3, 2017 194 table 1 effect of moisture content on enzyme production moisture content (%) specific activity of protease (u )1 specific activity of cellulase (u 2) 5 10 15 20 25 30 50 75 ath3 cbh7 ath3 cbh7 2.79± 0.110b 3.79± 0.155a 2.63± 0.098c 1.22± 0.045d 0.71± 0.051e 0.42± 0.06f 0.33± 0.036f 0.29± 0.018g 1.24± 0.045c 2.16± 0.132a 1.71± 0.055b 0.63± 0.08d 0.58± 0.043d 0.49± 0.034e 0.66± 0.036d 0.42± 0.062e 6.91± 0.183c 7.56± 0.266b 8.34± 0.144a 4.25± 0.158d 2.45± 0.062e 2.29± 0.034e 2.04± 0.055f 1.38± 0.045g 4.18± 0.183d 5.13± 0.098b 6.17± 0.115a 4.69± 0.238c 4.31± 0.091 d 3.45± 0.111f 3.88± 0.135e 2.77± 0.078g note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. mossambicus was higher in optimum ph range of 7.0 to 7.5 (ray et al. 2007). protease activity was maximum at ph range of 6.0 to 6.5 using two bacterial strains, bacillus licheniformis bf2 isolated from the foregut of bata fish (labeo bata) and bacillus subtilis bh4 isolated from the hindgut of bata fish (labeo bata) (ray et al. 2012b). increasing ph beyond optimum level could interfere with amino acid composition of the enzyme leading to the significant decrease of the enzyme activity (esakkiraj et al. 2007). optimum protease production using b. subtilis and the highest protease activity were reached at ph 10.0 (pant et al. 2015). sethi et al. (2013) reported optimum ph required for producing cellulase using several soil bacterial species. in a detailed investigation, fagade and bamigboye (2012) also optimized culture media parameters for cellulase production. the results of their study showed that ph 7.0 was optimum for bacterial strains pseudomonas putida, bacillus subtilis and bacillus licheniformis i, however, ph 5.5 was recorded to be the best for cellulase production using bacterial isolate bacillus licheniformis ii. incubation period is an important parameter for enzyme production by the microorganisms. some microorganisms produce maximally in their exponential growth phase, while other 195 effect of culture parameters on enzyme production banerjee et al. table 2 effect of ph on protease and celluase production ph specific activity of protease (u1) specific activity of cellulase (u2) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 ath3 cbh7 ath3 cbh7 0.49± 0.055f 0.58± 0.043f 1.74± 0.13d 2.78± 0.193c 3.45± 0.16b 3.81± 0.096a 1.61± 0.052d 0.86± 0.021e 0.31± 0.01f 0.28± 0.052f 0.71± 0.07e 1.13± 0.052d 1.56± 0.085c 1.73± 0.088b 1.94± 0.134b 2.22± 0.204a 1.77± 0.160b 0.95± 0.052d 2.09± 0.075f 3.82 ± 0.17 e 4.90± 0.262d 5.77± 0.145c 7.55± 0.124b 8.41 ± 0.83 a 4.13± 0.410e 2.68± 0.461f 1.43± 0.104g 2.39± 0.249e 2.95± 0.301d 3.48± 0.153d 4.16± 0.202c 5.20± 0.215b 6.27± 0.216a 6.09± 0.705a 5.10± 0.306b 4.49± 0.514c note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. table 3 effect of fermentation period on enzymes production fermentation period (hours) specific activity of protease (u )1 specific activity of cellulase (u 2) 24 48 72 96 120 ath3 cbh7 ath3 cbh7 1.98± 0.121d 2.72± 0.141b 3.06± 0.206b 3.95± 0.206a 2.32± 0.225c 0.36± 0.062d 0.88± 0.141c 1.57± 0.262b 2.32± 0.113a 1.61± 0.347b 3.87± 0.326d 5.23± 0.248c 8.53± 0.655a 8.07± 0.370a 6.29± 0.266b 2.14± 0.141e 2.99± 0.283d 4.58± 0.643c 6.42± 0.471a 5.47± 0.481b note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. microorganisms exhibited maximum production in their stationary growth phase. bacterial strains, c alkanolyticum b. licheniformis. ath3 and cbh7 exhibited the highest protease activity (3.95±0.206 u and 2.32±0.113 u, respectively) at 96 hours fermentation period (table 3). cellulase production exhibited by .c alkanolyticum ath3 was maximum at 72 hours of incubation period (8.53±0.655 u). b. licheniformis cbh7 exhibited maximum cellulase production at 96 hours (6.42±0.471 u) (table 3). singh and kaur (2012) optimized the culture condition for cellulase production using js1 bacillus sphaericus and recorded maximum cellulase production at 48 hours of incubation period. in the year 2013, suganthi . (2013) also optimized the culture et al condition of and stated that the b. licheniformis maximum protease production occurred at 24 hours of incubation period. the optimization of incubation period is crucial for obtaining maximum product and thus, several investigations have been conducted in this direction (shafee . et al 2005; ray . 2007; sethi . 2013). bacterial et al et al growth physiology (lag, log, stationary and decline phase) in liquid medium depends on incubation period. in general, maximum enzymes are produced during the log phase and then decreased rapidly due to interference of secondary metabolites. inoculum size is a cr ucial factor in fermentation process, as the duration of bacterial lag phase is highly dependent on inoculums quantity. the culture used to inoculate the fermentation medium must be in a healthy condition. in this present experiment, a wide range of inoculums size (1 to 5%) was used to get the maximum yield. protease production by .c alkanolyticum b. licheniformis ath3 and cbh7 was the highest when the inoculum size was 2% (4.04±0.132 u) and 3% (2.39±0.243 u), respectively (table 4). cellulase activities exhibited by . c alkanolyticum ath3 (8.58±0.340 u) and cbh7 b. licheniformis (6.51±0.473 u) were found to be the highest at 3% inoculum size (table 4). inoculum quantity normally used is between 3% and 10% of the medium volume (lincoln 1960; meyrath & suchanek 1972; hunt & stieber 1986). lincoln (1960) stressed that bacterial inocula should be transferred in the logarithmic growth phase when the cells were still metabolically active. shafee et al. (2005), however, reported that 4% is the optimum inoculum size for protease production in b. cereus strain 146. similarly, goyal . (2014) obtained et al the highest cellulase activity produced by bacillus sp. at 1% inoculum size using rice straw as substrate. in recent years, the effects of inoculum sizes on protease and cellulase production have been investigated and published by several researchers (suganthi . 2013; agarwal . et al et al 2014; lugani . 2015).et al temperature plays a critical role in maintaining normal body physiology in all living organisms, including bacteria. maximum protease yield by b. licheniformis cbh7 was observed when incubation temperature was 40 c (2.47±0.461 u), while . o c alkanolyticum ath3 exhibited the highest protease production at 45 c (4.18±0.57 u) (table 5) o . 196 biotropia vol. 24 no. 3, 2017 table 4 effect of inoculum size on enzymes production inoculum size (%) specific activity of protease (u1) specific activity of cellulase (u2) 1 2 3 4 5 ath3 cbh7 ath3 cbh7 2.98± 0.208b 4.04± 0.132a 3.10± 0.384b 0.87± 0.079c 0.21± 0.069d 0.82± 0.06c 1.43± 0.2b 2.39± 0.243a 0.98± 0.124c 0.27± 0.036d 6.23± 0.317c 7.16± 0.416b 8.58± 0.340a 3.23± 0.245d 2.01± 0.13e 3.58± 0.26c 5.63± 0.70b 6.51± 0.473a 3.42± 0.205c 2.22± 0.246d note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. on the other hand, the highest cellulase activity exhibited by these strains was observed at 40 c o (8.66±0.762 u for . ath3 and c alkanolyticum 6.63±0.34 u for cbh7) (table 5). b. licheniformis in this direction, ray . (2007) observed and et al stated that 40 c is the most effective o temperature for cellulase production using b. subtilis b. circulans cy5 and tp3. transport of enzymes to extracellular medium is lower at low temperature compared to that at high temperature (rajoka 2004). however, at higher temperature, the high maintenance of energy requirement for cellular growth is needed due to thermal denaturation of enzymes of the metabolic pathway (aiba . 1973), resulting in maximum et al production. recently, pant . (2015) reported 44 et al o c as the most effective temperature for protease production using . on the other bacillus subtilis hand, sethi . (2013) optimized the cellulase et al production using different soil bacterial strains and reported that 40 c as the optimum o temperature. rasul . (2015) also isolated et al different bacterial strains from soil and sugar industries and optimized their cellulase production. microorganisms grow slowly at a temperature below or above the normal growth temperature due to a reduced rate of cellular production (ray . 2007). therefore, it is et al important to maintain ambient temperature to obtain maximum production. effect of various carbon sources on protease and cellulase production were presented in figure 1a and 1b, respectively. among seven carbon sources, fructose and sucrose were detected to be the most suitable carbon sources for protease production using .c alkanolyticum b. ath3 (4.25±0.52 u) and licheniformis cbh7 (2.53±0.28 u). on the other hand, glucose was recorded to be more effective for cellulase production using . c alkanolyticum ath3 (8.71±0.78 u) and cbh7 b. licheniformis (6.66±0.61 u). it is well established that carbohydrates induce most of the cellulolytic enzymes in bacteria and fungi (morikawa . et al 1995). rajoka and malik (1997) used cellulose, cellulosic residues, xylan, cellobiose and cmc as carbon sources for the enhanced production of cellulases using strain. juhász . cellulomonas et al (2005) reported that high cellulolytic activity could be enhanced using steam-pretreated corn stover as carbon source. carbon sources are very important for bacterial protease and cellulase production (sethi . 2013; suganthi . 2013; et al et al pant . 2015).et al nitrogen sources are the building block of amino acids and considered to be the most important parameter in enzyme production. among various inorganic and organic nitrogen sources, tryptone was detected to be the most important nitrogen source in both protease (4.33±0.388 u and 2.64±0.17 u) and cellulase (8.77±0.66 u and 6.72±0.38 u) production using c alkanolyticum b. licheniformis. ath3 and cbh7 (fig. 2a & 2b). recently, lugani et al. (2015) tested the effect of different nitrogen sources (peptone, yeast 197 effect of culture parameters on enzyme production banerjee et al. table 5 effect of different temperature on enzymes activity different temperature (oc) specific activity of protease (u1) specific activity of cellulase (u2) 20 30 35 40 45 50 ath3 cbh7 ath3 cbh7 0.73± 0.081e 1.45± 0.105d 2.23± 0.246c 3.19± 0.42b 4.18± 0.42a 1.99± 0.19c 0.46± 0.043c 0.79± 0.19c 1.51± 0.173b 2.47± 0.158a 0.88± 0.131c 0.45± 0.072c 2.87± 0.182d 5.13± 0.228c 7.18± 0.55b 8.66± 0.762a 5.03± 0232c 1.89± 0.122e 2.72± 0.25d 3.79± 0.255c 4.92± 0.56b 6.63± 0.34a 4.42± 0.482b 2.34± 0.381d note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. 198 biotropia vol. 24 no. 3, 2017 figure 1 effect of different carbon sources on enzyme production (note: glu = glucose; mal = maltose; sucro = sucrose; lac = lactose; arabi = arabinose; fruc = fructose; star = starch. data 1 2 are presented as mean±sem. a = protease activity; b = cellulase activity. u = µg of tyrosine liberated/mg protein/minute; u = µg of glucose liberated/mg protein/minute) figure 2 effect of nitrogen sources on protease and cellulase production (note: 1 = arginine; 2 = beef; 3 = tryptone; 4 = aspergine; 5 = tyrosine; 6 = nh cl; 7 = nano 8 = (nh ) so ; 9 = kno 4 3; 4 2 4 3; 1 10 = gelatin. a = protease activity; b = cellulase activity. data are presented as mean±sem. u = µg of tyrosine liberated/mg 2 protein/minute; u = µg of glucose liberated/mg protein/minute) 199 effect of culture parameters on enzyme production banerjee et al. extract, ammonium sulphate, ammonium chloride and ammonium nitrate) on cellulase production from bacterial source. results of their investigation reported peptone as the effective nitrogen source in culture medium of bacillus sp. y3. in this direction, ray et al. (2007) stated that organic nitrogen sources exert better result compared to inorganic nitrogen sources in cellulase production using bacillus subtilis cy5 and b. circulans tp3. in recent years, the effects of nitrogen sources on bacterial protease production have also been reported by several researchers (ray et al. 2012b; suganthi et al. 2013; nisha & divakaran 2014; pant et al. 2015). in the present study, inorganic nitrogen sources and other amino acids such as l-aspergine and tyrosine were the poor nitrogen sources for cellulase production. on the contrary, spiridonov and wilson (1998) found that nh compounds are the most 4 favorable nitrogen sources for protein and cellulase synthesis. bacterial flora existing in digestive tract represents a very important and diversified enzymatic potential (ray . 2012a; banerjee . et al et al 2013; banerjee & ray 2016). extracellular enzymes production using bacterial flora not only helps in digestion, but also has great application in different industries (saha . 2006; roy . et al et al 2009; banerjee & ray 2016). ringø et al. 2012; among several enzymes, microbial protease and cellulase have high demand in different biotechnological industries such as food, leather, beverage and detergent (banerjee . 2015b). et al production of these different types of extracellular enzymes largely depends on medium compositions and different physical factors (ray et al. 2012b). medium optimization is the most important step to enhance the production in laboratory experiments and in fermentation industries. until now, reports on optimization of enzymes produced by gut bacteria are scanty, and thus, extensive research should be conducted to fulfill the demand. conclusions this study emphasized on production maximization of bacterial protease and cellulase due to their important contribution in several biotechnological industries. bacterial strains corynebacterium alkanolyticum bacillus ath3 and licheniformis cbh7 as active producer of extracellular protease and cellulase, might be useful in different industries like bakery, dairy, meat, like textile, food, laundry, paper, biofuel and brewing sectors. acknowledgements we are very much thankful to university grants commission (ugc), new delhi, for financial support. we are also grateful to de par tment of zoolog y, visva-bharati university, india, for providing such vast support. references agarwal t, saxena mk, chandrawat mps. 2014. production and optimization of cellulase enzyme by pseudomonas aeruginosa mtcc 4643 using sawdust as a substrate. int j sci res 4:1-3. aiba s, humphrey ae, millis nf. 1973. biochemical engineering. second edition. new york (us): academic press. p. 92127. banerjee g, ray ak, askarian f, ringø e. 2013. characterization and identification of enzymeproducing autochthonous bacteria from the gastrointestinal tract of two indian air-breathing fish. benef microbes 4:277–84. banerjee g, dan sk, nandi a, ghosh p, ray ak. 2015a. autochthonous gut 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anbalagan m, sivakumar a, gothandam km. 2013. screening and optimization of protease production from a halotolerant isolated from saltern bacillus licheniformis sediment. j genet eng biotechnol 11:47-52. vijayaraghavan p, lazarus s, vincent sgp. 2014. de-hairing protease production by an isolated bacillus cereus strain at under solid-state fermentation using cow dung: biosynthesis and properties. saudi j biol sci 21:27–34. walter he. 1984. methods of enzymatic analysis. weinheim (de): verlag chemie. p. 238. 201 optimal bacterial density and fertilizer dosage for bioremediation banerjee et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 spatial pattern in beta diversity of echinoidea and asteroidea communities from the coastal area of tomia island, wakatobi marine national park, indonesia kangkuso analuddin , nasaruddin , andi septiana , wa ode sarliyana ,1 2 1 1 1, * agus nurlyati , wa masa nd saban rahim1 1 2a 1department of biology, faculty of mathematics and natural sciences, halu oleo university, kendari 93232 indonesia, 2the museum and research center of wallacea, halu oleo university, kendari 93232 indonesi, a received 15 december 2013/accepted 16 january 2015 abstract t e purpose of this was elucidate the spatial pattern in the beta diversity of marine benthic echinoidea h study to and asteroid that inhabit the coastal area of island wakatobi marine national park, indonesia wo ea tomia , . t transect placed lines of 460 and 260 m in length with small quadrats of 1 m were at the open and protected beaches 2 perpendicular to the coastlines. similarity ind ( )ly the importance value index and ex si of organisms on these taxa were calculated along each transect. was the most dominant echinoid at open and protected echinometra mathaei beaches, while was the most dominant asteroid both areas. most si values of echinoidea the protoreaster nodusus in at open beach were estimated less than 50%, which was lower than si values of organisms protected beach. to be at the on the other hand, most of si values of asteroidea at both areas were estimated more than 70% reflecting high degree of similarity of its species composition among sites. the dis imilarity index of organisms taxa of s in the echinoidea and asteroid increased significantly with increasing distance between sites, which suggested the ea that pattern in beta diversity of these taxa was associated with spatial heterogen ity e . keywords: asteroidea eta diversity, echinoidea, similarity ndex, wakatobi marine national park, b i introduction now days, biodiversity of marine organisms a is declin global scale (gatson 2000; roberts ing on et al. 2002), and therefore, attention more should be directed to conservation of marine biodiversity. many scientists use the term beta diversity as a key component of biodiversity survey (gray 2000). beta diversity defined as the changes in composition of organi ms or species diversity s between habitats (whittaker 1960 . beta diversity ) is known to provide useful information on marine area relationship or connectivity, which reflects the processes operating in those areas, s and ha been considered to be essential in environmental and conservation-based censuses and establishment of nature reserves (purvis & hector 2000; cleary 2003; tuomitso 2003; et al. koleff 2003). marine organisms play very et al. important roles of coastal for stabilizing ecology ecosystem (menge 1999), stabili inshore et al. zing environments (jie 2001), regulat et al. ing atmospheric processes (murphy duffus 1996), & providing forfood and pharmaceuticals human (hunt vincent 2006), as well as & functioning as recreational and aesthetic aspects (ponder et al. 2002). however, human activities such as over fishing (jackson 2008), bottom trawling and dredging (pauly 2005), as well pollution of et al. as coastal waters (halpern 2008) are known to et al. have negative impact on bent ic ecosystems h sustainability. wakatobi marine national park often called an underwater paradise is among the orld's most w popular marine parks. this marine park has very high resource potential, in terms of biodiversity* corresponding author : zanzarafli@gmail.com biotropia vol. 22 no. 1, 2015: 33 43 doi: 10.11598/btb.2015.22.1.355 33 mailto:zanzarafli@gmail.com biotropia vol. 22 no. 1, 2015 34 both species and uniqueness, and has become a leading t urist destination and focus of research o activities. however, most studies in this region have been done for ecological asse sment on coral s reef and fishes only, while few data are available s regarding ecological organization for marine be thic echinoidea and asteroidea, which are n fundamental for sustainable conservation of this marine park becau e of their significant role in the s coastal food chain. these taxa also have high aesthetic value for tourism. therefore, analyses of the spatial pattern in the beta diversity of these taxa may be useful for the potential ecotourism and conservation of biodiversity and the coastal environment of wakatobi marine national park, indonesia. echinoids are important food source for human aily exploitation of these organisms . d may threaten some species by over exploitation as well as habitat destruc ion. most people living at t the coastal area at the wakatobi marine national park capture fish and also some species of marine benthic organisms. therefore, understanding the spatial trends in beta diversity for echinoidea and aster idea taxa may help to preserve their habitat o and to ensure their future sustainability. many ecologists realize that good understanding of the marine organisms in relation to enviro mental n condition essential to generate effective is conservation schemes and guidelines for the sustainable exploitation of natural resources. in this effort determination of beta diversity of marine organisms plays an essential role (gaston 2000; lubchenco 2003; tuomitso 2003). et al. et al. understanding the distribution and complexity of benthic habitats will provide important information for management goals (kendall et al. 2005) and conservation strategy (fortin et al. 2005), while identifying habitat characteristics for particular species are being increasingly taken up by marine ecologists to describe patterns of benthic diversity (barrett 2001).et al. in the present study the spatial pattern in the , beta diversity of marine benthic organisms of echinoidea and asteroidea inhabiting the open and protected beaches of tomia island, wakatobi marine national park . he spatial was elucidated t trend in abundance, similarity and di similarity s index and ordination pattern of each taxon es according to their distribution across the seashore gradient . the spatial patterns of was described beta diversity on these taxa were elucidated in relation to environmental condition open and s at protected beaches and distance from the beach. to know whether distance between or sites patches affects the similarity index in each taxon, the relationship between distance and disimilarity index among sites . th analyses were analyzed ese were estimating performed by interval ordination from randomly selected paired , and then sites generating regression model between interval ordination and dissimilarity index. materials and methods study ites the present study was carried out along the coast of tomia island, wakatobi marine national park located at the 5 46' s and 123 55' e of 0 0 s province . .outheast sulawesi , indonesia (fig 1) the study was december 2010. conducted in meteorological data taken from kendari station showed that annual temperature range was c24-33 , while the minimum and maximum 0 temperature during study period were 24 c 0 c s ,and 32 , re pectively. annual rainfall was 2 000 0 mm at mountain, while it was 200 mm at the the coastal area. in addition, annual relative moisture ranged 75% to 84%, whereas from relative moisture was 76% at the study period with six days. the beach condition of tomia rainy island provides an excellent site for ecological study of marine invertebrates. the beach provides a unique panorama as it is surrounded by some small islands, which protect the beach from heavy wave action of banda ea, while other the s areas are exposed to direct heavy wave action. the substratum is composed of rock and sand. however, some species of seagrasses occur at the study site, such as cymodocea rotundata, cymodocea serulata, halophila minorenhalus acoroides and . spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso sampling method the two transe ct lines we re placed perpendicularly to the costal area of tomia island at the wakatobi marine national park. transect i with a length of 460 m was placed open beach at and divided into 13 sites or patches (400 m wide 2 each), while transect ii with length of 260 m was placed protected beach and divided into 7 sites at (400 m wide each). furthe more, five small r2 quadrats (1 m each) were placed purposively wide 2 in each site both open and protected beaches, at respectively. the name of species and their individual number for echinoidea and s asteroidea in each small quadrat were directly observed in the field using shirai (1997), while unknown species was brought to the aboratory l for identification. for determining the organic content, the sediment samples were taken from both transects and brought to the laboratory of ecology and taxonomy, halu oleo university, kendari. the sediment samples were air dried for seven days hen 10 g . t of the air-dried sediment samples were taken cand oven dried at 105 for 0 24 hours and weighed. the samples then dryashed c at 700 for 2 hours and weighed. the 0 percentage of organic matter content of sediment samples was estimated according to brower 1997et al. ( ). data analysis the similarity and dissimilarity indexes of organisms among s s for echinoidea and ite asteroidea both transects open and in at protected beaches were calculated using formula of bray and curtis (1957). in addition, ordination analyses were applied for elucidation the of multidimensional pattern. the spatial pattern in the beta diversity of each taxon was elucidated in figure 1. study sites of open and protected beaches ( ) in the coastal areas of tomia island, wakatobi marine national circled park, indonesia 35 protected beach open beach relation to the condition of open and protected beaches, distance from shore and substrate types of the beach. to know whether interval among sites affects the similarity for each taxon, the index relationship between distance among sites and di similarity each taxon . s index of were analyzed th analys s performed by estimating the ese e were interval ordination from randomly selected paired sites, and then regression model generated between ordination and dissimilarity for the index both taxa. the coefficient correlation of regression line was estimated, and a statistical test was done for determining whether there s any wa or no correlation between distance among sites and dis imilarity index for each taxon.s results and discussion the present study showed a little variation of organic matter content of sediment among habitats: 1 sandy areas with the seagrasses ) estimated at 14.62%; 2 rocky habitat at 14.43%; ) and 3 mixed rocky and sand habitat at 13.78%. ) however, diversity of the marine benthic for echinoid and asteroid communities inhabited the coastal area of tomia island, wakatobi marine national park varied at open and protected beaches. species of echinoids occurred on nine open beach, while eight species were found only at protected beach in the coastal area of tomia island (table 1) . was the dominant echinoid echinometra mathaei both open and protected beaches of tomia at island, indicating its high important role for susta nability of coastal ecosystem. on the other i hand, was the rarest echinoid in diadema setosum this coastal area, which might be less adaptable with environmental condition (fig 2) . . meanwhile, few aster ds species were found oi in this coastal area with only four species on both open and protected beaches (table 2). protoreaster nodusus was the dominant steroid a in both areas, while was the rarest archaster typicus a .steroid (fig 3). differences in species compostion of marine benthic at present study might be determined by habitat characteristics and organic matter content. beaman (2005) et al. found that habitat characteristic affect marine ed benthic composition. rocky reef habitats influence the behavior of benthic organisms, d and edin turn influenc higher level processes of population dynamics and community structure (knight morris 1996). sandy habitats & are important for burrowing organisms and their predators and provide foraging areas for species which may also use nearby firm substrate areas for sheltering purposes (ross 2007). et al. therefore, spatial pattern in the community composition of echinoidea and asteroidea might be associated with substrate types at small patches in particular the coastal area of tomia island. biotropia vol. 22 no. 1, 2015 table 1 species composition of open and protected beaches in the coastal area of tomia island, wakatobi . echinoidea at marine national park, indonesia no. family species open beach (transect i) protected beach (transect ii) 1 echinometridae echinostrephus aciculatus echinometra mathaei echinometra sp. a echinometra sp. b 2 temnopleuridae salmacis sphaeroides temnopleurus alexandrii 3 diadematidae astropyga radiate diadema setosum echinotrix calamaris 4 toxopneustidae toxopneustes pilieolus tripneustes gratilla 5 brissidae brissus latecarinatus note (present), (absent)s : √ 36 the similarity indexes of echinoid community both open and protected beaches are described at in tables 3 and 4, respectively. as shown in table 3 the similarity indexes of echinoids varied among sites, while three values of imilarity ndexes (si) were estimated more s i than 85%, i.e. the si values of paired sites i-ii, vvii and of paired sites i-xii. however, twentythree si values paired sites of echinoids were of estimated more than 50%, while more than half of the si values of echinoids were less than 50%, even five si values paired sites were zero. these of results suggested that there was a lower degree of similarity species of echinoids among sites at open beaches of tomia island. on the other hand, table 4 shows that more than half the si values of of echino ds protected beaches were estimated i at to be more than 50%, indicating a high degree of similarity among sites for echinoids protected at beaches, although two si values were estimated as zero or completely different. these trends demonstrate the change in the echino ds species i composition across open beaches, which might be due to heavy wave action. there was only small change in species composition of echinoids across protected beach from heavy wave action. 0 50 100 150 200 0 50 100 150 200 250 300 protected beach echinometra mathaei echinometra sp a echinotrix calamaris echinostrephus aciculatus temnopleurus alexandrii echinometra sp b brissus latecarinatus diadema setosum distance from the land (m) figure 2. spatial trends in the mportance alue ndex ivi of echin idea community at open beach (upper) and protected i v i ( ) o beach (below) in the coastal area of tomia island, wakatobi marine national park, indonesia table 2 species composition of astero open and protected beaches in the coastal area of tomia island, wakatobi . idea at marine national park, indonesia no. family species open beach (transect i) protected beach (transect ii) 1 ophidlasteridae protoreaster nodusus linckia laevigata 2 oreasteridae culcita novaeguineae 3 archasteridae archaster typicus 4 astropectenidae astropecten scoparius note (present), (absent)s : √ 37 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso biotropia vol. 22 no. 1, 2015 0 50 100 150 200 0 50 100 150 200 250 300 350 400 open beach linckia laevigata protoreaster nodusus archaster typicus culcita novaeguineae distance from the land (m) 0 50 100 150 200 0 50 100 150 200 250 300 protected beach linckia laevigata protoreaster nodusus archaster typicus astropecten scoparius distance from the land (m) figure 3. spatial trends in the mportance alue ndex ivi of asteroidea community at open beach (upper) and protected i v i ( ) beach (below) in the coastal area of tomia island, wakatobi marine national park, indonesia table 3. similarity and i similarity of chinoids open beaches in the coastal area of tomia island, wakatobi d s indexes e at marine national park, indonesia si\di i ii iii iv v vi vii viii ix x xi xii xiii 1 12.5 61.46 28.27 62.57 56.25 53.33 46.96 100 54.58 70.83 62.5 14.89 2 87.5 61.46 21.19 67.98 61.67 58.75 52.38 100 60 70.83 50 26.36 3 38.55 38.54 66.47 61.46 58,33 61.46 53.91 67.71 38.55 67.71 61.46 52.78 4 71.73 78.81 33.53 46.79 44.44 37.55 38.10 100 63.41 84.52 50 41.8 5 37.43 32.05 38.54 53.20 36.23 13.44 42.58 63.19 57.78 89.01 47.83 62.23 6 43.75 38.33 41.67 55.56 63.77 47.22 25.24 63.89 51.11 61.11 58.33 55.91 7 46.67 41.25 38.55 62.45 86.56 52.78 53.57 76.62 57.78 100 49.58 57.27 8 53.04 47.62 46.09 61.91 57.42 74.77 46.43 75.71 49.60 58.57 70.71 46.62 9 0 0 32.29 0 36.81 36.11 23.38 24.29 63.33 45.83 55 100 10 45.42 40 61.46 36.59 42.22 48.89 42.22 50.40 36.67 57.78 78.89 39.7 11 29.17 29.17 32.29 15.48 10.99 38.89 0 41.43 54.17 42.22 79.17 70.83 12 37.5 50 38.54 50 52.17 41.67 50,42 29.29 45 21.11 20.83 76.36 13 85.11 73.65 47.22 58.20 37.77 44.09 42.73 53.38 0 60.30 29.17 23.65 38 s indexes ( )table 5 hows the imilarity si for a atsteroids open beaches. the si values of asteroids were less variable with only a few si values of steroids estimated less than 50%, a to be while many si values were estimated more to be than 80%, and even some si values estimated to be indicatednearly 100%. these results that there was high similarity of species composition of a atsteroids open beaches of tomia island. a similar trend in the si values of steroids was a found protected beaches.at as shown in table 6 there only three si is values of steroids protected beaches were a at estimated less than 50%, while more than to be half of si values of steroidea were estimated a to be above 70%, indicating the high degree of similarity in species of steroids protected a at beaches of tomia island. multidimensional patterns of the benthic community of chinoids and steroids at the e a study site were elucidated by ordination analyses. the ordination pattern of chinoids at open e beach (fig 4, upper) showed four groups: 1) . group a consisted of four sites or patches including sites i, ii, iv and sites xiii; 2) group b consisted of five sites (sites iii, vi, viii, x and sites xii); 3) group c consisted of sites v and vii; and 4) group d consisted of sites ix and xi. the sites located at the same groups indicated that chinoids might have e species similar or matching resource requirement and enviro mental preferences. the ordination n pattern of chinoids along transect ii or e protected beaches (fig , below) could be also . 4 divided into 4 groups, though the sites in each group differed as compared to the ordination pattern of chinoids open beaches. these e at wefour groups re as follows: 1) group a consisted of sites iii and v; 2) group b consisted of sites i, ii, and iv; 3) group c consisted of site viii only; and 4) group d included sites vi and vii. table 4. similarity and is imilarity of chinoids protected beaches in the coastal area of tomia island, d s indexes e at wakatobi marine national park, indonesia si \ di i ii iii iv v vi vii viii 1 24.78 47.69 36.52 59,83 47.81 35.31 76.97 2 75.22 46.43 11.74 58,57 35.42 35.42 64.58 3 52.31 53.57 46.43 24,29 58.57 58.57 70.71 4 63.48 88.26 53.57 58,57 43.18 43.18 58.33 5 40.17 41.43 75.71 41.43 58.57 58.57 82.86 6 52.19 64.58 41.43 56.82 41.43 20.83 100 7 64.69 64.58 41.43 56.82 41.43 79.17 100 8 23.03 35.42 29.29 41.67 17.14 0 0 table 5. similarity and is imilarity of steroids open beaches in the coastal area of tomia island, wakatobi d s indexes a at marine national park, indonesia si \ di i ii iii iv v vi vii viii ix x xi xii 1 22.5 24.28 34.16 14.16 14.16 20.83 18.33 22.12 18.75 36.66 36.67 2 77.5 19.82 25.41 22.5 22.5 25.41 38.75 24.20 27.5 38.75 38.75 3 75.71 80.17 7.38 36.07 36.07 37.73 24.28 44.02 33.57 33.57 38.75 4 65.83 74.58 92.61 19.16 19.16 20.83 18.33 27.12 18.75 33.57 38.75 5 85.83 77.5 63.92 80.83 19.16 20.83 18.33 27.12 18.75 41.66 41.67 6 85.83 77.5 63.92 80.83 100 20.83 32.5 85.45 21.25 31.11 22.5 7 79.16 74.58 62.26 79.16 79.16 79.16 32.5 19.09 21.25 31.11 22.5 8 81.66 61.25 75.71 81.66 67.5 67.5 84.16 8.03 4.166 20.83 100 9 77.87 75.79 55.97 72.87 14.54 80.90 91.96 77.87 11.66 14.54 38.75 10 81.25 72.5 66.42 81.25 78.75 78.75 95.83 88.33 89.54 14.54 17.36 11 63.33 61.25 66.42 58.33 68.88 68.88 79.16 63.33 85.45 75 27.5 12 63.33 61.25 61.25 58.33 77.5 77.5 74.58 61.25 82.61 72.5 85.13 39 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso biotropia vol. 22 no. 1, 2015 table 6. similarity and i similarity of steroids protected beaches in the coastal area of tomia island, wakatobi d s indexes a at marine national park, indonesia si \ di 1 2 3 4 5 6 7 1 57.38 45 40 31.66 55.53 58.33 2 42.61 20.79 24.28 26.07 17.14 6.19 3 55 79.20 44.72 36.08586 36.08 15.55 4 60 75.71 55.27 50 40 29.16 5 68.33 73.92 86.66 50 41.36 26.67 6 44.46 82.85 63.91 60 58.63 23.33 7 41.66 93.80 84.44 70.83 73.33 76.66 figure 4. ordination pattern of community at open beach ( ) and protected beach ( ) in the coastal area of echinoidea left right tomia katobi island, wa marine national park, indonesia statistical analysis regression model using d s(fig. 5) showed that the i similarity indexes (di) of echinoids among sites both open and at protected beaches increased significantly with increasing interval ordination or separation of sites or patches ( < 0.001). this means that the p degree of similarity of echinoids both open at and protected beaches decreased with increasing distance sites.among the ordination pattern of asteroids on open beach (fig. 6) could also divided into 4 group: (1) group a consisted of site xii only; (2) group b consisted of sites iii, iv, v, vi, vii, ix and site xi; (3) group c included sites i and ii; and (4) group d consisted of site viii only. the same ordination pattern of asteroids was found at the transect ii (figure 6), although the sites in esch froup were different as compared to the grouping in the transect i. these groups at transect ii are: (1) group a contained sites, ii, iv and vi; (2) group b consisted of site iv only; (3) group c contained site i only; and (4) group d included sites iii and v. statistical analysis a regression model using d i(fig. 7) showed that issimilarity ndexes (di) atof asteroids among sites both open and protected beaches also increased significantly with increasing interval ordination or separation of sites ( < 0.01). this mean that the degree of p s similarity of asteroids among sites open and at protected beaches decreased with increasing distance sites both open and protected among at beaches. these spatial heterogeneities might be correlated to the environmental condition of the beach, such as substrate types and distance from the offshore. this because the happened echinoids and asteroids communites showed high similarity among sites in general both open at and protected beaches. the degree of similarity in some sites for both taxa were completely different although they responded to the same sets of substrate types, which might influence the community structure of marine benthic community in the tomia island. the community composition of echinoids and asteroids in each site might be significantly associated with substrate types in small patches. for species that have limited dispersal abilities, distance or spatial component expected to be is 40 0 20 40 60 80 100 0 20 40 60 80 100 open beach interval ordination 0 20 40 60 80 100 0 20 40 60 80 100 protected beach interval ordination figure 5 relationship of i o tion to the i similarity ndex of echinoidea community at the oastal area of . nterval rdina d s i c tomia island, indonesia figure 6 ordination pattern of asteroidea community at open beach ( ) and protected beach ( ) in the coastal area of . left right tomia island, wakatobi marine national park, indonesia 41 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso important structuring factor in community similarity, which would result in assemblages of sites close together being more similar which are than assemblages further of those which are apart. many previous studies revealed that distance between sites can be a function of differences in spatially explicit environmental variables (borcard . 1992; harrison . 1992; et al et al ohmann spies 1998). the present result clearly & found a significant association between distance of sites and community similarity for echinoids and asteroids both at open and protected beaches, because they showed a highly significant relationship between interval ordination and di similarity indexes for those two taxa. many s previous studies that pattern in beta indicated diversity can differ among taxa due to communitywide differences in dispersal ability and other taxon-specific factor (gatson 2000; reyers . et al 2000). beta diversity of organisms in many taxa ha been affected by not only distance between s sample stands but also enviro mental variables n (ellingsen gray 2002; clearly . 2004). in & et al addition, barros . (2004) found that et al sedimentary bedforms such as ripples, sand and waves are known to affect the abundance and distribution of benthic organisms. abiotic factors such as human disturb nce might be factor the individual number, a reducing and even the species number of these benthic communities. it is known that sea urchins are frequently collected by people for consumption, which might have strong effects on their abundance and distribution. on the other hand, open beach protected beach 42 biotropia vol. 22 no. 1, 2015 interaction wit in benthic community itself s h become biological control for the community stability. meanwhile, steroids act as predator for a s sea urchin in general, and might reduce the number of sea urchin in the coastal of tomia island. the association with distance itself may also be due to distance-dependent environmental variable. several studies showed that the distribution of chinoderms french polynesia e at beach was primarily inf luenced by the concentration of carbonates (adjeroud 1997). however, ellingsen and gray (2002) also found in a norwegian coastal area that sea urchins were , more restricted in their distribution than other macrobenthos, and the beta-diversity of echinoderms had the weakest association with environmental variables of all the observed taxa. conclusions spatial patterns of the benthic community of echinoidea and asteroidea that inhabit the coastal area of tomia island, wakatobi marine national park varied habitat. ultidimensional pattern by m of each taxon created four groups in general , although the sites or patches in each group were quite different. the trend in beta diversity of echinoidea and asteroidea seem toed be associated with spatial heterogen ity because e there was a significant correlation between separation of sites and community similarity of benthic community. this infor mation is fundamental for conser vation wit in the h wakatobi marine national park. acknowledgements edwe thank halu oleo university for financial support this research. the for we also thanked staff of wakatobi marine national park for coopera-tion and support our . we of field work would also like to thank the student and general s volunteers who had provided assistance during the field work. references adjeroud m 1997 factors influencing spatial patterns on . . coral reefs around moorea, french polynesia. mar ecol progr ser 159: 105 19. beaman rj, daniell jj, harris pt 2005 geology-benthos . . relationships on a temperate rocky bank, eastern bass strait, australia. mar fresh res 56 (7) 943-58. : barros f, underwood aj, archambault p 2004 the . . influence of troughs and crests of ripple marks on the structure of subtidal benthic assemblages around rocky reefs. estuar coast shelf sci 60 (4) : 781-90. barrett n, sanderson jc, lawler m, halley v, jordan a. 2001 mapping of inshore marine habitats in south . eastern tasmania for marine protected area planning and marine management. technical 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ecological encyclopedia of the marine animals of the ryukyus island.. okinawa(jp): shinsei tosho tuomisto h, ruokolainen k, yli-halla m 2003 dispersal, . . environment, and floristic variation of western amazonian forests. science 299 241 4.: whittaker rh 1960 vegetation of the siskiyou mountains, . . oregon and california. ecol monog 30 279 338: . biotropia vol. 29 no. 1, 2022: 1 6 doi: 10.11598/btb.2022.29.1.834 1 biological characteristics of indonesian gayo horse juli melia1*, amrozi amrozi2, muhammad agil2 and iman supriatna2 1faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia 2faculty of veterinary medicine, institut pertanian bogor, bogor 16680, indonesia received 5 april 2017 / accepted 16 july 2019 abstract this study aimed to find out the characteristics of gayo horses and to identify the population of gayo horses. data collection was conducted through a selection process from more than 100 local horses distributed in central aceh subdistricts, gayo lues and bener meriah. the selection process resulted in 30 gayo horses having varying ages. detail observation was performed on six gayo horses consisting of 3 male horses and 3 female horses. the observation comprised morphological observation and morphometry. data collection of gayo horse population was based on the annual report from the local animal husbandry and fishery office of central aceh subdistrict over the last 5 years. results of this study showed that gayo horses have a straight cranial shape with smaller size, thick and stiff mane, and ears akin to donkey’s ears. the study also showed that gayo horse’s body height ranged between 113-120 cm with a body weight range of 215-280 kg. gayo horse is agile despite living in mountainous areas and able to carry heavy loads. the population of gayo horses have been declining since 20102014. gayo horse is included in a large pony category. conservation efforts are essential to save gayo horse from extinction. keywords: characteristics, gayo horse, hallmarks, population introduction indonesian local horses are among livestock commodities supporting national development, especially in the animal husbandry subsector. gayo horse is one of the indonesian native horse germplasms (soehardjono 1990). this has been written in the ministry of agriculture decree no. 1054/kpts/sr.120/10/2014 which stated that the gayo horse is an indonesian native horse strain. gayo horse population is spread all over gayo highland in the central aceh subdistrict, including its autonomous regions called gayo lues and bener meriah. central aceh subdistrict is known by various other names such as “nation above the clouds”, “gayo highland” and “negeri antara”. in this region, the annual gayo traditional horse race was held to commemorate the proclamation of independence of the republic of indonesia and the anniversary of the central aceh subdistrict. many owners of gayo horses have the intention to breed gayo horse with a thoroughbred horse, creating a new type of horse known as “astaga horse” (australia-gayo). nowadays, pure gayo horse is hard to find, which indicated that there might have been a very drastic decline of the gayo horse population. the attempt to conserve the gayo horse as one of the indonesian native horse germplasms must be done immediately to save the gayo horse from extinction. the objective of this study is to describe gayo horses’ characteristics and to identify the number of gayo horses’ population, which is crucial for future research concerning the reproduction of gayo horses. materials and methods research procedure data collection was carried out by means of direct identification at the gayo horses’ habitat. additionally, the past and present information on gayo horses was obtained through direct *corresponding author, email: julimelia@unsyiah.ac.id biotropia vol. 29 no. 1, 2022 2 interviews with several owners of gayo horses and with officials of the local animal husbandry and fisheries office of the central aceh subdistrict. one hundred local horses were selected from areas surrounding the central aceh subdistrict and its autonomous regions called gayo lues and bener meriah. in the initial phase, the identification process was conducted according to the criteria written in the ministry of agriculture decree no. 1054/ kpts/sr.120/10/2014. of the 100 selected local horses, 30 horses were identified as gayo horses having varying ages. a more thorough identification process was conducted on 6 gayo horses consisting of 3 male horses and 3 female horses. gayo horse morphology and morphometry the morphological examination was carried out visually and documented by slr camera (nikon d3200, af-s dx zoom-nikkor 1855mmf/3,5-5,6g ed ii). the morphological examination included observation on body shape, eye shape, mane condition, head shape, ear shape, neck shape, nape and tail. morphometry examination included measurement on body height, girth and body length. body height was measured from the tip of the front leg up to its withers. the girth was measured from the lower abdomen from the fossa olecranon perpendicular with os sternum until above os vertebrae. body length was measured from the connection of cartilago and os scapula horizontally until behind the horse’s os pelvis. body weight measurement is predicted by using the schoorl formula: (body weight = (chest circumference + 22)2/100) and measured in kilograms (kg). population data data on gayo horses’ population were collected from the annual reports of the years 2010-2014 published by the local animal husbandry and fisheries office of central aceh subdistrict and its autonomous regions, i.e., gayo lues and bener meriah. data analysis gayo horses’ morphology and morphometry data were descriptively analyzed. the population data were analyzed and depicted in graphs. the prediction of gayo horses’ population extinction was analyzed exponentially by using excel 2016 software. results and discussion gayo horses are included into large ponies for having a body height of not more than 1.47 m. on the other hand, miniature ponies have body height ranging from ≤ 86 cm (a type) up to 88.6 cm (b type) (campbell 1992). according to ensminger (1962), a pony’s height is less than 1.45 m while standing, with 250-450 kg body weight, and usually are descendants of lightweight horses. being included as large ponies, gayo horses have different body sizes compared to foreign horses. the size differences are often influenced by environmental factors, such as topography and climate (ohsawa et al. 2008; steinheim et al. 2008; kosoma & purzyc 2009). ponies are often used for recreation purposes (rogers et al. 2006), and for therapy programs (burke 2002). generally, gayo horses have similar characteristics to other local horse types in indonesia. however, gayo horses have several uniqueness, such as having straight head shape with smaller body size, thick and stiff mane, and having ears similar to donkey’s ears (table 1; fig. 1). table 1 the morphology of gayo horses morphology shape color body short black, dun, chestnut, grey, white eye small black gaze sharp mane thick and stiff head straight ear similar to donkey nape long throatlatch wide tail medium to long similar to body color, mix biological characteristics of indonesian gayo horse – juli melia et al. 3 figure 1 gayo horse appearance notes: a. female; b. male. gayo horses have body height of 113-120 cm, chest circumference of 136-139 cm, body length of 102-105 cm, and body weight of 215280 kg (table 2). a pictured guide for easy identification of gayo horse in the field is presented in figure 2. table 2 the morphometry of gayo horses morphometry height (cm) chest circumference (cm) body length (cm) weight (kg) male 118-120 (119.00±1.00) 137-139 (138.00±1.00) 103-105 (104.33±1.15) 225-280 (256.67±28.43) female 113-115 (114.33±1.15) 136-138 (137.00±1.00) 102-105 (103.67±1.53) 215-250 (233.33±17.56) figure 2 a pictured guide for easy identification of gayo horses in the field a b biotropia vol. 29 no. 1, 2022 4 several distinctive marks indicating the temperament and intelligence of local indonesian horses, which also exist in gayo horses, are the condition and shape of hair whorl, i.e., 1. pusar cekak; hair whorl under the jaw, indicating that the horse is always being preyed upon by tiger; 2. pusar terbang; hair whorl by the front feet around the left and right knees area, indicating that the horse is suitable as a racehorse due to its fast speed; 3. pusar dada; hair whorl by the chest, indicating that the horse can spin fast and not fall in a sharp turn during a race; 4. pusar gedung; hair whorl by the flank or below the flank, indicating a mild or tame temperament and is suitable as brood; 5. pusar turun tangis; hair whorl under both eyes, indicating temperamental personality, sometimes being tame and sometimes being wild; 6. pusar ruk; hair whorl by the hind legs behind the knees, indicating a wild and hard to control personality; 7. pusar lipan; hair whorl around the throat and under the ear, indicating a hard to control or wild personality. gayo horses have several positive characteristics, such as power and agility, having capabilities to carry heavy loads, having good temperament, having good endurance, having high adaptivity toward various environmental conditions, having survival capabilities with minimal feed availability, and having easy maintenance, which makes gayo horses very economical for its owner. judging from various aspects, gayo horses have considerably high strategic values. based on cultural aspects, the gayo horse symbolizes social status. usually, a boy’s manhood is determined from the moment a boy can ride a gayo horse without a saddle in a race, which custom is still practiced nowadays. from an economical aspect, the gayo horse is used as a source of animal protein for the local people who consume horse meat. gayo horse is often sold to other regions such as north sumatra to be consumed. from the utilization aspect, the gayo horse is used as a plow puller in rice fields and as a freight horse to transport agricultural products. based on data obtained from the local animal husbandry and fisheries office of the central aceh subdistrict, the population of gayo horses drastically decrease in 2010-2014 (fig. 3). prediction analysis indicated that gayo horse will extinct in 2037 (fig. 4). main reasons that might cause the decrease of the gayo horse population: 1. crossbreeding; 2. uncontrolled culling; 3. existence of modern machinery. figure 3 gayo horses population at central aceh subdistrict biological characteristics of indonesian gayo horse – juli melia et al. 5 figure 4 prediction of gayo horse extinction the upkeeping of gayo horses is usually done conventionally. after the agricultural harvest season, the gayo horses are released to the mountainous area and rice fields. the mating system of the gayo horse is akin to that of primates. only superior stallions may mate with the female horses around the area where the stallion is kept. if a stallion is defeated in a fight before mating, the defeated stallion will be moved to the other group of horses, which causes inbreeding leading to the decrease of body size, height, and weight of gayo horses. nowadays, the upkeeping system of gayo horses has separated the stallion from the mare gayo horses, even though it is still conventionally conducted. the mare horse is brought to the stallion only at the breeding time. the body maturity of gayo horses occurs at 12-15 months of age, while sexual maturity starts at 12-18 months of age (personal communication with owners). usually, the mare of the gayo horse has 21 days of the estrus cycle. all information obtained from owners was proven through complete research on the reproduction status of gayo horses. determination of reproduction status is among the most important factor of animal nurturing and breeding management, including for horses, because reproduction status has a close link with animal’s basic reproductive physiology. a number of techniques can be utilized, such as ovarium dynamic observation by using ultrasonography (amrozi et al. 2004; cuervoarango & newcombe 2008; derar & hussein 2011; melia et al. 2014), hormonal analysis (agil et al. 2008) and histology examination on reproductive organs. hormonal analysis in a horse is generally conducted using the horse’s blood samples (bollwein et al. 2002; utt et al. 2007; ginther et al. 2010). all of these data can be used as the basic reproductive physiology data of gayo horses, which will support the application of reproductive technology as one of the means to save endangered the population of gayo horses from extinction. conclusion gayo horse is included in a large pony category. conservation efforts are essential to save gayo horses from extinction. acknowledgments the authors thank the directorate of research and community services, directorate general of research development, technology, and higher education, the ministry of research technology and higher education for providing research funding through doctor dissertation research scheme number: 025/sp2h/lt/ drpm/ii/2016 dated 17 february 2016. special gratitudes are also delivered to the central aceh office of animal husbandry and biotropia vol. 29 no. 1, 2022 6 fishery for assisting the authors during data collection process. references agil m, supriatna i, purwantara b, candra d. 2008. assessment of fertility status in the male sumatran rhino at the sumatran rhino sanctuary, way kambas national park, lampung. hayati journal of bioscience 15:39-48. amrozi, kamimura s, ando t, hamana k. 2004. distribution of estrogen receptor alpha in the dominant follicles and corpus luteum at the three stages of the estrous cycle in japanese black cows. j vet med sci 66(10):1183-8. bollwein h, mayer r, weber f, stolla r. 2002. luteal blood flow during the estrous 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[accessed 2013 april 06]. available at: http:// burkesbackyard.com.au/2002/archives/2002/road tests/horse_breeds/australian_miniature_ponies campbell me. 1992. selected aspects of miniature horse reproduction. proceeding of the annual meeting of the society for theriogenology. p. 89-96. cuervo-arango j newcombe jr. 2008. repeatability of preovulatory follicular diameter and uterine edema pattern in two consecutive cycles in the mare and how they are influenced by ovulation inductors. theriogenology 69:681-7. derar ri, hussein ha. 2011. ovarian follicular dynamics during the estrous cycle in jennies in upper egypt. veterinary medicine international article id 860518, 6 p. https://doi:10.4061/2011 /860518. ensminger me. 1962. animal science. animal agriculture series. 5th ed. danville (us): printers & publishers. inc. ginther oj, almamun, shahiduzzaman akm, beg ma. 2010. disruption of the periovulatory lh surge by a transient increase in circulating 17β-estradiol at the time of ovulation in mares. j anim r sci 117:178-82. kosoma m, purzyc h. 2009. konik and hucul horses: a comparative study of exterior measurements. j anim sci 87:2245-54. melia j, amrozi, tumbelaka li. 2014. ovarian dynamics of endometritis cows treated with a combination of intrauterine infusion of gentamicin, flumequine, and pgf2α analog. j ked hewan 8(2):111-5. ohsawa t, saito y, sawada h, ide y. 2008. impact of altitude and topography on the genetic diversity of quercus serrata populations in the chichibu mountain central japan. j anim sci 203(3):187-96. rogers cw, gee ek, hangoor e, firth ec. 2006. preliminary survey of congenital and reproductive disorders in the new zealand miniature horse population. proceedings of the new zealand society of animal production 66:274-8. soehardjono o. 1990. kuda. jakarta (id): yayasan pamulang. steinheim g, odegard j, ådnoy t, klemetsdal g. 2008. genotype by environment interaction for lamb weaning weight in two norwegian sheep breeds. j anim sci 86:33-9. utt md, acosta tj, wiltbank mc, ginther oj. 2007. acute effects of prostaglandin f2α on systemic oxytocin and progesterone concentrations during the mid-or late-luteal phase in mares. j anim r sci 97:63-73. 2. jimmy t. masagca page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 antibreast cancer activity of nanopropolis indonesia induced mammary gland tumor by on dmba in virgin sprague-dawley rats akhmad endang zainal hasan jumali1,2 2*, d mangunwidjaja , t c unarti , o suparno a setiyonoiti andra s no gus2 2 3and 1 institut pertanian , bogor 16680, indonesiadepartment of biochemistry, faculty of mathematics and sciences, bogor 2 epartmen engineering and td t of agroindustrial technology, faculty of agricultural echnology, institut pertanian bogor bogor 16680, , indonesia 3d t of clinic, reproduction and pathology, faculty of veterinary, epartmen institut pertanian bogor, bogor 16680, indonesia received 15 march 2015/accepted 15 may 2016 abstract the objective of this study was to determine the effect of nanopropolis induced rat mammary to cure cancer on tumor . after the first tumors appearance, twenty rats were using 7,12-dimethylbenz(a)anthracene (dmba) eight divided into seven groups. group 1, 2 and 3 served as , 32 and 56 recipient of nanopropolis dosages 8 µg/ml treatments served recipient of dosage µg/ml treatment served recipient of ; group 4 as propolis of 233 ; group 5 as do orubi in treatment served recipient of treatment (control) x c ; group 6 as dmba and group 7 as normal group . the effect of nanopropolis of 32 µg m and propolis 233 µg m were similar in reducing umor dosage / l dosage of / l t size, healing the wounds caused by the tumor and it turns out that there is a relationship eliminating cancer cells. between particle size absorbent material . the study suggest that nanopropolis was very s ed with small concentration effective treat mammary gland tumors and breast cancersto rat . keywords: breast cancer nanopropolis, propolis, prague-dawley rat, s introduction cancer is one of the leading causes of death in the world, especially in developed countries and the second killer in developing countries. based on data from the hospital information system (sirs) in 2007 breast cancer ranks first until 2013, in hospitalized patients in all hospitals in indonesia (16.85%), followed by cervical cancer (11.78%) (ministry of health 2013). in accordance with opinion of , tet almuir . (2003) he cause of this disease a cover of estrogen-are dependent pathway, circulating androgen, estrogen and exposure to carcinogenic materials such as smoke, ultraviolet radiation, improper diet and stress; except for age and race that cannot be changed (bates american cancer society 2016). in the early stage, breast cancer is only in the form of a lump in the mammary tissue and in an advanced stage resulting an injury to the mammary tissue. t this disease he ways to treat are the removal of tissue, radiation therapy and c h e m o t h e r a p y. r a d i a t i o n t h e r a p y a n d chemotherapy aim to destroy cancer cells are ed and control the disease so will the main tumor be shr nk, tumor growth will be slowed and u spread of cancer cells to other tissues will be prevented. now been a lot of research esadays, there have to obtain cancer , either directly medications for the treatment of cancer to reduce theor side effects of chemotherapy. the use of medications such as doxorubicin is one to inhibit cancer mean growth and reduce the occurrence of new cancers. however, the negative impact of doxorubicin treatment is the occurrence of hair loss, heart rhythm disorders and decreased white blood cell count. there is a challenge to find cancer treatment is effective and minim which ize bad effects of the main treatment. ne effort is to o biotropia vol. 23 no. 1, 2016: 35 41 35 doi: 10.11598/btb.201 .2 . .6 3 1 473 * c orresponding author: , zainalhasan@ipb.ac.id pakzainalhasan@gmail.com mailto:zainalhasan@ipb.ac.id mailto:pakzainalhasan@gmail.com 36 find and develop from medication herbs and to do on herbal medication. medication clinical test originated from herbs must be tested through a series of studies prior to its usage in cancer therapy. the tests should not involve animals. a b b a s a l i p o u r k a b i r . ( 2 0 1 0 ) a n d e t a l purushothaman . (2012) investigated the et al incidence of breast cancer using virgin rats of sprague-dawley strain as test animals. in-vitro showed th studies at the origin of propolis locations in indonesia from five and nanopropolis from pandeglang ere le w ab to inhibit the growth of mcf-7 cell line. the nanopropolis inhibit the growth of mcf-7 cells ed at very low concentrations compared propolis to instead of nanoparticles . the (hasan . 2014)et al purpose of this study was examineto the in-vivo antibreastcancer activity of nanopropolis on the dmba induced of virgin sprague-dawley rats. materials and methods preparation of nanopropolis nanopropolis were prepared in three stages of homogenization using high speed homogenizer . some modifications were done for preparing of nanopropolis particle as previously mentioned by aimi . (2009), bhaskar . (2009), hasan . et al et al et al (2012), chen (2006) and kim . (2008)et al. et al . mammary umor nductiont i twenty eight rats were intraperitoneally injected using a m with ixture of dmba ( concentration of /25 mg kgbody weight), olive oil and physiological saline. experimental esignd after the first tumors appearance, twenty eight rats were divided into seven groups, with three animals each group. group 1, 2 and 3 served asfor recipient of nanopropolis dosages of 8, 32 and 56 µg/ml treatments served ; group 4 as recipient of dosage of 233 µg/ml propolis treatment served recipient of ; group 5 as do orubi in treatment; served x c group 6 as recipient of treatmentdmba ; and group 7 as (control)a normal group . tumor stwo dimen ional tumor areas were calculated as an ellips (abbasalipourkabir . 2010). the e et al volume of tumor was calculated by the formula of: 2v = ab /2 w here a = the longest diameter = b the shortest diameter of tumor at sacrifice, the tumor were removed for histopathological . examination hi topathology analysiss siaafter euthana , at the end of the study, the mammary tumor masses of rats were removed. sample of tissues were fixed immediately in 10% formalin overnight, embedded in paraffin, cut into 4 m sections and stained with hematoxylin-μ eosin (he). results and discussion the results of body weight rats measurements after injection and treatment were presented in figure 1. een in figure 1 normal rat body as s , weight continued to increase with time , addition as well as other treatments. the dmba treatment showed a decrease in body weight. according to cordeiro d kaliwal (2011), an effect of dmba can reduce body weight rat caused by the of nature of its toxicity despite an increase in tumor volume. tumor progress can be determined by measuring the volume test animal tumor of (abbasalipourkabir 2010; purushothaman et al. et al et al in the study of. 2012; martic . 2011). abbasalipourkabir . (2010) volume et al , tumor was calculated by measuring the length and width of the swelling, while et al.purushotaman (2012) an calcu d the tumor et ald martic . (2011) late volume by measuring the length, width and height of the tumor section. esults in mammary tissue r tumor volume of research data using the method of abbasalipourkabir (2010) can be seen in et al. figure een in figure 2 increasing tumor 2. as s , volume in dmba treatment happened only without propolis or nanopropolis treatments. biotropia vol. 23 no. 1, 2016 weeks after treatment antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . 37 the propolis or nanopropolis treatments performed in rats decreased tumor volume. even in the 32 and 56 nanopropolis µg/ml treatment and 233 µg/ml propolis treatment there were decline after an increase in tumor volume in the fi th week after being given treatment. when f viewed from the point of decline, the injection of 56 µg/ml treatment hadnanopropolis sharp corners the tumor size reduction process , o red rccur very quickly. this occur ence may be due to the relatively high dose of the 50(6 x ic ) ingredients in nanopropolis sufficient to that is eliminate cancer cells. influence the amount of of the active ingredient component in nanopropolis ) was in trumental in (32 and 56 µg/ml s comparison with the influence of the size particle of the repair the component nanopropolis for tissue from tumor to compare propolis at 56 µg/ml. n a n o p r o p o l i s t r e a t m e n t w i t h t h e concentration 8 ha been done to heal of µg/ml d cell and tissue, although there are tumor to repair figure 1 dy weight of rats after induction of dmba and prior necropsy (1 = group nanopropolis 8 µg, 2 = bo s of group of nanopropolis 32 µg, 3 = group nanopropolis 56 µg, 4 = group propolis µg, 5s s of s of 233 = doxorubicin s of group , 6 = group dmba and 7 = normal control weeks after treatment figure 2 tumor volume of mice after induction by dmba prior to necropsy (1 = group nanopropolis 8 µg , of /ml 2 = group of nanopropolis 32 µg , 3 = group nanopropolis 56 µ , 4 = group propolis /ml of g/ml of 233 /ml doxorubicin of group µg , 5 = group, 6 = group dmba and 7 = normal control ) 38 biotropia vol. 23 no. 1, 2016 cells. on tre tment with a concentration of 32 a µg/ml, had occurredformation of intact tissue to washeal and subcutaneous fat formed and there were e rpr sence of intact epide mal glands and many gl bular epithelium as well as o s reversal of in ,tact tumor draining wounds. similarly nanopropolis treatment at a concentration 56 of µg/ml r to lower occu red heal much better than concentration of result nanopropolis. this show thated the amount of active ingredient content affected more to the healing rate of cancer. figure 3 showed the result of administration of nanopropolis on rat mammary tissue (32 and 56 ) which ha repair skin µg/ml d ed tissue after induced by dmba being . t issue conditions were better in he t nanopropolis concentration of fig 456 µg/ml ( . ), the formation of new tissue was much more than giving nanopropolis at concentration of and 8 32 µg/ml. d of this prove that the administration nanopropolis increasingly play a role in ed mammary tissue wounds caused by dmba induced tumor results. this result showed that the amount of active ingredient affected the healing rate of cancer. the greater the concentration nanopropolis used, the greater of the effect tumor tissue healing wounds. the on influence of different concentrations and time of administration of propolis against different cancer cells ha also been studied by d bufalo . et al (2007). presence of a tumor as a result of wound healing propolis administration s the using wa formation of epithelial tissues been that had studied by de moura . (2011), although using et al different propolis. this condition was supported by the presence of components of propolis (hasan . 2014) or mineral contents (hasanet al et al. 2013). at a concentration of the 56 µg/ml mammary were healthy and aesthetically alveoli clean in figure 4a seen the contain . it is ingalveoli blood plasma (blue arrows pointing as result of ) a mammary tissue repair. on other rats the groups (dmba or negative control) in which group nanopropolis or propolis injection was not p , erformed mammary tissue condition was still going on angiogenesis and cancer cells enters blood vessels. in this condition, cancer cell figure 3 the mammary tissue of virgin rat after induced by mba and received treatment of being d injection a. nanopropolis (32 µg/ml) b. nanopropolis (56 µg/ml) ( = skin epi = and blue arrow thelium, yellow arrow normal hair licles white arrow capillary epidermis he stainingfol , = ) ( , 200x) figure 4 a) the ammary tissue of rats sd induced by dmba and nanopropolis every seven days within m 56 mg/ml two months, b) the mammary tissue of rats sd induced by dmba and no treatment within two months after induction (black arrow blue arrow = filled blood plasma white arrow = cancer cells, = , with , alveole alveole red arrow = connective tissue) he staining, ( 200x) 39 spread in so much connective tissue and almost eve y cancer cells r tissue as is shown in figure 4b ( (white arrows) and connective tissue (red arrows) .) the physical condition of the mammary tissue healing drying can be seen when condition occur in an area that has suffered injury due to red the tumor fig 5a while the mammary tissue ( . ). was swollen , caused by the tumor the effect of dmba induced in virgin rats were large swell (fig. 5b). even one rat in this group had inflammation of the mammary tissue. his condition can be t refe red to as stage iv breast cancerr . tested activities of propolis curing cancer proved dosage 233 µg/ml c that a of an heal damage by the tumor tissue compared s caused with the positive control treatment without induction by dmba treatment. positive control (without induction by dmba) does not cure cancer develop accumulation of and even ed cancer cells and tissue damage fig 6( . ). inoue et al. (2008) showed that the inhibition of cancer growth occur ed after concentration of r propolis 320 µg/ml. , bermúdez (2006) similarly et al. reported that 10%wound healing with propolis only occur as much as only the 60% for occurrence . in breast of re-epithelialization tissue repair seen around the damaged can be tissue with (blue arrow) and re-epithelialization hair l ( for the follic es yellow arrow) in figure 6a. dmba research, induced mammary wounds were formed in the mammary tissue and there were many cancer cells fig ( . 6b). f shows drying igure 7 cancer cells condition with black spots on the new blood vessels, but there are still cancer cells expected to remain active. this is because the treatment required quite long , so that the healing process a time was still running and still not finished on was . treatment with propolis 233 µgdosage of /ml, the drying process indicated the healing in tissues affected by cancer. dosage µg/ml at of 32 nanopropolis has the ability to cure cancer which to has the same effect a s mu ch as 2 3 3 o f p r o p o l is µ g/ m l administration. the particle size in nanopropolis is very small so active ingredient remaining in , nanopropolis can log into the network with ease, while the of propolis with 233 dosage µg/ml which was active against tumor growth is caused figure 5 a) the physical condition of the mammary tissue healing can be experienced with drying occur in an area that has s suffered injury due to tumor after injection of b) non treatment or only induction of nanopropolis 32 µg/ml, dmba c) for ( = , and condition before treatment after induction of dmba 90 days yellow arrow dry wound, white arrow mammary swelling bec use of tumor = a ) figure 6 rat mammary tumor tissue after being induced by dmba and treated with injection of (a) propolis (233 virgin μg/ml) and (b) without treatment (positive control) (blue arrow = skin epithelium, yellow arrow = normal hair follicle, white arrow = inflammation) (he staining, 200x) antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . 40 biotropia vol. 23 no. 1, 2016 by the presence of the active components in propolis prevent tumor progression. this fact ing was caused by the presence of compounds in either nanopropolis or propolis, such as organic acids like firulic acid and acid, polyphenols caffeic and flavonoids in propolis inhibit the which proliferation of cancer cells ole of . the r flavonoids and ca fe acid is to inhibit the f ic formation of protein kinases that are used for cell proliferation result is going cell which to inhibit formation process and apoptosis induced occur (madeo . 2004). in accordance rence et al with the results of de moura (2011), the et al. healing of wounds caused by the tumor may occur due to administration of propolis. meanwhile, according to sun . (2012) crysin components et al present in propolis can decrease the volume of tumor that occurs in the mammary tissue of dmba induced . the research conducted by mice bhattacharjee . (2012) and lim . (2011) et al et al states that there is a relationship between particle size absorbent materials on the healing of cancer. conclusions the effect of nanopropolis of 32 s with dosage µ /ml with dosage of µ /ml g and propolis 233 g w the same in ere reducing tumor size, healing the wounds caused by the tumor and eliminat ing cancer cells. it turn out that there is a ed relationship between particle ize absorbent s material . the study s on the healing of cancer sug gest that nanopropolis ed with small concentration (dosage of 32 µg/ml) was very effective for the treatment of rat mammary gland tumors and breast cancers. acknowledgements the authors to thank bpps fellowship wish of indonesian ministry of education and culture, and seameo biotrop for their financial supports and institut pertanian bogor for its facilities for conducting this study. references abbasalipourkabir r, salehzadeh a, abdullah r. 2010. antitumor activity of tamoxifen loaded solid lipid nanoparticles on induced mammary tumor gland in sprague-dawley rats. afr j biotech 9(43):733745. aimi m, nemori r, ogiwara k .. 2009. casien nanoparticle inventor. bates american cancer society [internet]. 2016. causes, risk factors, and prevention topics. usa: american cancer society; [cited 2016 may 31]. available from: http://www.cancer.org/cancer/ breastcancer/detailedguide/breast-cancer-riskfactors. bermúdez ic, garcía gs, piloto aa, pérez yf, valdivieso ag. 2006. effect of the cuban propolis collected in manzanillo area on the wounds healing in rats. pharmacol 3:416-21. bhaskar k, anbu , ravichandiran , venkateswarlu , rao j v v ym. 2009. lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in-vitro, ex-vivo and studies. lipids health disease 8(6):758-in vivo 76. bhattacharjee s, erchov d, fytianos k, van der gucht j, alink gm, rietjens imcm, marcelis atm, zuilhof h. 2012. cytotoxicity and cellular uptake of triblock copolymer nanoparticles with different size and surface characteristics. particle fibre toxicol 9(11):1-19. figure 7 mammary tissue (a) and skin tissue (b) dmba-induced sd rats treated with propolis at a dosage of 233 g/ml µ every 7 days within 2 months (red arrows = vein, white arrow = dead cancer cells, black arrow = hair follicle) (he staining, 200x) http://www.cancer.org/cancer/ 41 bufalo mc, candeias mgj, sforcin jm. 2007. in vitro cytotoxic effect of brazillian green propolis on human laryngeal epidemoid carcinoma (hep-2) cells. advance access pub 6(4):483-7. chen m, diao g, zhang e. 2006. study of inclusion complex of beta-cyclodextrin and nitrobenzene. chem 63:522-9. cordeiro mc, kaliwal bb. 2011. antioxidant activity of bark extract if spreng on dmba bridelia retusa induced mammary carcinogenesis in female sprague dawley rats. j ph rm 2(1):14-20.a de moura sal, negri g, salatino a, lima ldc, dourado lpa, mendes jb, andrade sp, ferreira mand, cara dc. 2011. aqueous extract of brazilian green propolis: primary components, evaluation of inflammation and wound healing by using subcutaneous implanted sponges. evid based complement alternat med 2011:1-8. hasan aez, ambarsari l, artika im, julistiono h, tarunasari d. 2013. induction resistance of candida sp. y390 to ethanol stress by kopyor coconut and virgin coconut oil. emir j food agric 25(10):790-7. hasan aez, artika im, fahri vr, sari n. 2012. potency of nanopropolis from stingless bee spp. as trigona antibacteria gents. chem progress 5(1):1-7.a hasan aez, mangunwidjaja d, sunarti tc, suparno o, setiyono a. 2014. investigating the antioxidant and anticytotoxic activities of propolis collected from five regions of indonesia and their abilities to induce apoptosis. emir j food agric 26(5):390-8. inoue k, saito m, kanai t, kawata t, shigematsu n, uno t, isobe k, liu ch, ito h. 2008. antitumor effects of water soluble propolis on a mouse sarcoma cell line and . am j chin med 36(3):625-34.in vivo in vitro kim dm, lee , aum , kim 2008. preparation of gd sh hj. propolis nanofood and application to human cancer. biol pharm bull 31(9):1704 10. lim hn, nurzulaikha r, harrison i, lim ss, tan wt, yeo mc. 2011. spherical tin oxide, sno particles 2 fabricated via facile hydrothermal method for detection of mercury (ii) ions. int j electrochem sci 6:4329-40. madeo f, herker , wissing , jungwirth , eisenber , e s h t frohlich . 2004. apoptosis in yeast. current ku opinion microbiol 7:655–60. martic k, vlacic z, rudman f, lambasa s, tomasovicloncaric c, stanec z. tumor and reast 2011. b volume ratio as a predictive factor for axillary lymph node metastases in t1c ductal invasive breast cancer: prospective obser vational clinicopathological study. jpn j clin oncol 41(12):1322-6. ministry of health . 2013. [internet] indonesia: ministry of health; [cited 2013 june 14]. available from: http://www.depkes.go.id/index.php/berita/pressrelease/2233-seminar-sehari -dalam-rangkamemperingati-hari-kanker-sedunia-2013.html. muir d, kanthan r, kanthan sc. 2003. male versus female breast cancers. arch pathol lab med 127(1):36-41. purushothaman a, nandhakumar e, sachdananram p. 201 . anticancer effect of shemamrithaa (a 2 phytochemical formulation) on 7,12-dimethyl benz(a)anthracene induced mammary carcinoma in rats. asian j pharm clinical res 5(1):101-7 . sun lp, chen al, hung ac, chien yh, huang js, huang cy, chen yw, chen cn. 2012. chrysin: a histone deacetylase 8 inhibitor with anticancer activity and a suitable candidate for the standardization of chinese propolis. j agric food chem 60:11748-58. antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . http://www.depkes.go.id/index.php/berita/presssri-13 mei 2017-621 (romnick ecological) rev layout.cdr ecological services of agroforestry landscapes in selected watershed areas in the philippines and indonesia romnick s. baliton , christine wulandari , leila d. landicho , rowena 1 2 3* esperanza d. cabahug , roselyn f. paelmo , reynaldo a. comia , roberto g. 3 4 3 visco , pitojo budiono , susni herwanti , rusita and arnold karl sa. castillo 1 5 6 6 3 1 institute of renewable natural resources, college of forestry and natural resources, university of the philippines los banos, college, laguna 4031, philippines 2 graduate program of forestry, universitas lampung, bandar lampung 35141, indonesia 3 institute of agroforestry, college of forestry and natural resources, university of the philippines los banos, college, laguna 4031, philippines 4 institute of crop science, college of agriculture and food science, university of the philippines los banos, college, laguna 4031, philippines 5 faculty of social science and politic, universitas lampung, bandar lampung 35141, indonesia 6 forestry department, faculty of agriculture, universitas lampung, bandar lampung 35141, indonesia received 7 february 2016/accepted 18 january 2017 abstract this article argues that the practice of agroforestry provides ecological contributions to the smallholder farmers cultivating in the watershed areas. specifically, this farming system provides contribution to carbon sequestration potential of the woody perennials and the biodiversity conservation of the other components of the system. this argument is based on the research conducted in molawin-dampalit sub-watershed, mt. makiling forest reserve in the philippines and way betung watershed in indonesia. the research involved an interview session of 106 and 261 smallholder farmers and an assessment of 27 and 14 agroforesty plots for carbon stock assessment and biodiversity assessment, respectively. results indicated that the total carbon found among the crop components was 52.32 mgc/ha in molawin-dampalit sub-watershed and 244.26 mgc/ha in way betung watershed, which suggested the high carbon sequestration potential of the woody perennials and understory crops in an agroforestry system. the farm lots being cultivated by the smallholder farmers were found to contribute to biodiversity conservation having a moderate biodiversity index of 2.59 and 2.53, respectively. with these findings, promotion of desired agroforestry systems in suitable portions of the watershed areas should be intensified and heightened to contribute to ecological balance across the landscape. agroforestry should always be an integral part of all initiatives toward ecological restoration with the cultivators/smallholder farmers as potential partners. the agroforestry system should consider all the technical and socioeconomic considerations toward having diverse components and ensure food security among the smallholder farmers throughout the year. keywords: agroforestry, biodiversity index, carbon stock, molawin-dampalit sub-watershed, way betung watershed biotropia 4 1 7 71 84 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 621 * corresponding author: ldlandicho@gmail.com 71 introduction southeast asia is among regions enlisted as biologically rich and diverse. three countries, including malaysia, philippines and indonesia, are in fact cited as mega-diverse countries in the region, being the homes of a number of plant and animal species. at present, however, southeast asia's biodiversity is highly threatened. sodhi et al. (2004) highlighted that the international union for the conservation of nature and natural resources (iucn) listed three plant and eight animal species as already extinct in the region. furthermore, the authors emphasized that the number of threatened species in southeast asia including in the iucn categories of critically endangered (ce), endangered (en) and vulnerable (vu) ranges from 20 (ce) to 686 (vu) 72 biotropia vol. 24 no. 1, 2017 crops, woody perennials and/or animals or aquatic resources for the twin purpose of production and conservation. tolentino et al. (2010) highlighted that the diversity of plants used in agroforestry provides multiple benefits at different times of the year. these diverse combinations can help buffer its practitioners from the risk of income loss due to price variability, crop failure and other unanticipated problems. ecologically, agroforestry helps enhance biodiversity of the environment. it is expected that as the diversity of agroforestry farms increases, farmers would have the opportunity to make use of the flora-fauna interaction to control pests and diseases, improve microbiology and nutrient cycling. all these are requisites for survival and improved plant growth. this article highlights the respective biodiversity and carbon sequestration potentials of selected agroforestry landscapes in the molawin-dampalit sub-watershed in the philippines and way betung watershed in indonesia. materials and methods study site way betung watershed has an area of 5,260 ha, 51% of which is classified as the forest park area and the remaining 49% is classified as agricultural areas (fig. 1). most of the upland farmers are engaged in agroforestry, particularly species for vascular plants, 6 91 species for fish, 0 23 amphibian species, 4 28 reptile species, 7 116 bird species and 5 147 mammal species (sodhi et al. 2004). biodiversity loss in southeast asia is attributed to a number of factors, including deforestation or clearing of the forest cover; conversion of agricultural lands to other economic purposes; natural calamities such as el niño, extreme weather events, climate change, forest fires; continued dependence on forest resources as livelihood of the growing population; and, invasive species. these all boil down to the rapid ecological, social and economic changes that the world faces. most often, the forest dwellers are accused as culprits of the biodiversity loss because of their continued dependence on the forest resources for economic and livelihood activities. two of the most popular watersheds in southeast asia are the molawin-dampalit subwatershed located inside the mt. makiling forest reserve (mmfr) in the philippines and the way betung watershed in indonesia. besides being the habitat of different flora and fauna, both watersheds have similar conditions such that they are both forest reserves, and therefore, should be free from human occupancy. while policies for the protection, preservation and conservation of these two watersheds are being imposed by their respective governments, these areas continue to be the homes of a number of upland farmers and migrants. agroforestry is a land use management system which combines the production of agricultural figure 1 map of way betung watershed, lampung, indonesia 73 ecological services of agroforestry landscapes in watershed areas – baliton et al. figure 2 map of the mmfr highlighting the molawin-dampalit sub-watershed as the study site in the philippines combining the major fruit tree species such as durian, mangosteen, jackfruit, integrated with cacao, coffee, rubber and other forest trees such as mahogany, alstoria and other forest species. the mmfr in the philippines, on the other hand, is a 4,244-ha multiple use forest reservation area (fig. 2). according to sargento (1995), mmfr is a protection forest and a watershed reserve, specifically used for training and research laboratory, water source for surrounding communities, biological sanctuary and gene pool of many plant and animal species. this reserve, however, has become a home to a number of farmers and migrants who are engaged in farming. these farmers practice agroforestry, particularly the multi-storey system which combines the production of coconut, fruit trees and other agricultural cash crops. socioeconomic characterization the characterization was carried out using a p r e t e s t e d s u r v e y q u e s t i o n n a i r e . t h i s q u e s t i o n n a i r e c a p t u r e d s o c i o e c o n o m i c information, views and perceptions about agroforestry practices. a total of 106 respondents in molawin-dampalit sub-watershed and 261 respondents in way betung watershed were selected using random sampling. results of the socioeconomic survey were analyzed using descriptive statistics, such as frequency counts, percentages and weighted scores. biodiversity assessment the assessment was conducted by measuring the following parameters of biodiversity, i.e. population density or the number of individual species per unit area; frequency of species distribution; dominance value based on frequency, diameter or biomass; relative and importance values based on density and frequency; and diversity and evenness indices based on the relative and importance values. importance value (iv) was computed to determine the dominant species for each site. the iv is the sum of the relative density, relative frequency and relative coverage. these values were computed using the following formula: total area sampled total number of tree individuals counted per species density = 100* relative density total number of all species total number of tree individuals counted per species = species dominance 2 (dbh)*(0.7854)= relative dominance 100* dominance of a species total dominance of all species = 100* total number of plots number of plots species occur =species frequency 100* total frequency of all species frequency of a species relative frequency = importance value =�relative density + relative coverage +�relative frequency 74 biotropia vol. 24 no. 1, 2017 s h= ∑ (pi * ln p i) i=1 h j = ______ ln s the measures of biodiversity were obtained using the shannon-wiener diversity index (h) (magurran 2004) calculated using formula as follows: the pielou's evenness index (j) was calculated using formula: where: h = shannon-wiener diversity index j = pielou's evenness index p = fraction of the entire population made up of i species i s = total numbers of species encountered ∑ = sum from species 1 to species s note: the power to which the base e (e = 2.718281828.......) must be raised to obtain a number is called the natural logarithm (ln) of the number the index was calculated by dividing the number of individuals of each species found in the sample by the total number of all species (represented by p), multiplied by the fraction of its natural log (p * ln p ). this procedure was 1 1 repeated for all of the different species. the sum of all the (p * ln p ) represents the value of h. 1 1 physical evidences of the movement of wildlife in the agroforestry matrix were noted in terms of frequency, duration and kind of species. carbon stock assessment the carbon stock of different agroforestry systems was measured using the biomass estimation method. tree biomass was calculated using the allometric equation of brown (1997) (equation 1). this was done by measuring the standing aboveground biomass of the woody perennials or live trees with diameter at breast height (dbh) of 5 cm and above. the total tree biomass density and carbon stored in various agroforestry systems were calculated using equation 2. carbon stock of herbaceous (living non-perennial crops) and litter found in the soil surface was calculated to get the total aboveground biomass of each agroforestry system (equation 3). belowground biomass of trees and other perennials was obtained using the default value proposed by delaney (1999) i.e. 15% of the aboveground biomass. equation 1: tagb= exp(2.134+2.530 ln(dbh)) where: tagb = total aboveground biomass in kg/tree exp {…} = “raised to the power of ” ln = natural log of {…} dbh = diameter at breast height in cm equation 2: c stored (mgc/ha) = tree biomass density *c content tree biomass density = tree biomass (mg)/sample area in hectare equation 3: total fresh weight (kg) * subsample dry weight (g) 2 ______________________________________total dry weight (kg/m ) = 2 subsample fresh weight (g) * sample area (m ) c stored (mgc/ha) = tree biomass density *c content results and discussion socioeconomic and biophysical characteristics of the study sites way betung watershed and molawindampalit sub-watershed are considered as watershed and forest reserves, which are inhabited by a number of people. results of the socioeconomic characterization indicated that most of the farmers in the two study sites were male as represented by 73% of the total number of respondents (table 1). this finding validated previous research which concluded that, in general, farming had become a male-dominated activity (landicho et al. 2014; landicho, 2015). majority (86%) of them were married with an average household size of 5. majority (51%) of the participants had family members ranging from 4 6 members. concurrent with other research, this finding implied the availability of family labor and that the farm households in the upland communities were mostly big. table 1 also highlights that the age of farmers in the two study sites were entirely different. the age range of the farmers in molawin-dampalit watershed was from 51 to 60. this data suggested that despite their age, the farmers were able to maintain their current far ming systems. however, this finding also presented threat on the sustainability of their farming system, especially considering that not all family members 75 table 1 socioeconomic characteristics of the farmer-respondents in molawin-dampalit sub-watershed and way betung watershed socioeconomic characteristic study site total % molawin-dampalit way betung frequency % frequency % sex male 63 59 204 78 267 73 female 43 41 57 22 100 27 subtotal 106 100 261 100 367 100 civil status single 9 9 5 2 14 4 married 80 75 237 90 317 86 separated 2 2 1 1 3 1 widow/er 14 13 18 7 32 9 no answer 1 1 0 0 1 0 subtotal 106 100 261 100 367 100 household size 1-3 40 38 87 33 127 35 4-6 41 39 147 56 188 51 > 6 23 22 27 10 50 14 no answer 2 1 0 0 0 total 106 100 261 100 367 100 average 5 5 age < 30 8 32 40 11 30-40 12 93 105 29 41-50 24 83 107 29 51-60 29 42 71 19 > 60 33 11 44 12 subtotal 106 100 261 100 367 100 average 54 43 number of household members involved in farming 1-3 101 96 164 63 265 72 4-6 4 4 37 37 41 27 > 6 1 1 0 0 1 00.27 subtotal 106 261 367 100 income source farming 44 41 214 82 258 70 off-farm 0 0 27 10 27 7 non-farm 2 2 20 8 20 5 farming+off-farm 2 2 0 0 2 1 farming+non-farm 52 49 0 0 52 14 farming +off-farm+ non-farm 5 5 0 0 5 2 subtotal 106 100 261 100 367 100 ecological services of agroforestry landscapes in watershed areas – baliton et al. table 2 biophysical characteristics of the farms being cultivated by the farmer-respondents in molawin-dampalit subwatershed and way betung watershed biophysical characteristic study site total % molawin-dampalit way betung frequency % frequency % farm size < one hectare 51 44 149 57 200 54 1-3 45 46 111 42 156 43 3.1-5 6 6 1 1 7 2 > 5 4 4 0 0 4 1 total 106 100 261 100 367 100 status of farm ownership owned 9 8 0 0 9 2 tenant 19 18 17 7 36 10 rented 1 1 0 0 1 1 in public lands 70 66 244 93 314 85 no answer 7 7 0 0 7 2 total 106 100 261 100 367 100 farm topography rolling 48 45 132 51 180 49 steep 10 9 57 22 67 18 flat 31 29 72 27 103 27 flat to rolling 16 15 0 0 16 15 no answer 1 1 0 0 1 1 total 106 100 261 100 367 100 source of water for crop irrigation spring 12 11 60 23 72 19 river/creek 5 5 35 13 40 11 rainfed 85 79 166 640 251 68 others (e.g. irrigation) 6 5 0 6 2 total 108 100 261 100 369 100 76 biotropia vol. 24 no. 1, 2017 were trained to develop and maintain their farms. on the other hand, the farmers in way betung were still young and most probably in their productive age, as majority of them fell within the age range of 31 40 years old. this finding suggested that these farmers could already be the second-line farmers. these young farmers might have already been trained by the older farmers, and/or farming may have just started recently in way betung watershed. furthermore, this data indicated that these young farmers would have higher opportunities for improving their farms. there were only 1 3 members of the family that were engaged in farm development activities. in most cases, though, only the husband and the wife concentrated in farming. it could be that their children were still young; busy in their schooling, or not interested in farming at all. while farming was the major source of income of most (70%) of the farmer-respondents, there were also households whose members were engaged in non-farm activities as an additional source of income. in general, farm income was relatively low with most of the respondents having an estimated farm income of less than usd 200 and usd 200 500 in the philippines and indonesia, respectively. the low farm income could be attributed to the biophysical conditions of their farm as well as the scope and orientation of their agricultural production. in general, farm lots in the two study sites were cultivated by smallholder farmers. this was because majority (54%) of the far merrespondents in the two study sites cultivated lands which were less than a hectare (table 2). this farming practice provided them with an estimated annual income of less than usd 200 (table 1). table 3 agricultural production systems being employed by the farmer-respondents in molawin-dampalit subwatershed and way betung watershed production system frequency total % molawin-dampalit way betung frequency % frequency % cropping system monocropping 7 7 1 0.40 7 2 crop rotation 3 3 0 0 3 1 relay cropping 4 4 0 0 4 1 multiple cropping 43 42 31 12 74 20 agroforestry 45 43 229 87.6 274 77 forest plantation 2 1 0 0 2 1 total 106 100 261 100 367 100 crop components vegetables 54 16 0 0 52 9 rice 1 0.30 0 0 1 0.17 corn 14 4 0 0 14 2 root crops 56 16.7 0 0 56 9 fruit trees 101 30 192 73.56 293 49 herbs 2 12 0 0 2 0.33 ornamentals 40 12 0 0 40 7 forest trees 65 19 69 26.44 134 23 total 333 100 261 100 594 100 77 understandably, these farmers could not cultivate big farm sizes primarily because they were cultivating in the public/state lands. thus, they were bound with certain rules and policies in their agricultural production. most (85%) of the farmer-respondents did not own the lands that they cultivated, and therefore, agricultural expansion was not possible. smallholder farmers are described as those who cultivate less than three hectares of land area (esfim ). by this definition, farmers in 2017 the two study sites are categorized as smallholder farmers. while their production orientation was for subsistence, the surpluses were sold in the market for their additional household income. besides being smallholder far mers, the biophysical characteristics of their farms were characterized as marginal. the topography was generally rolling (49%) and some with steep slopes (18%) and, therefore, the risk of soil erosion was high. however, the risk was being controlled with the practice of sustainable farming system, such as agroforestry. crops are generally dependent on rainfall as the main source of water/irrigation. thus, any drastic changes in rainfall and temperature patterns greatly affect their agricultural production. agroforestry practices in the two study sites with the prevailing biophysical and socioeconomic conditions, the far merrespondents were observed to maximize the land use of their farms. most of the farmerrespondents were engaged in agroforestry (77%) and multiple cropping (20%) across the landscapes in the two study sites (table 3). the practice of agroforestry was noted in the high-elevation areas, while multiple cropping was highly observed in relatively lower elevation across the two landscapes. this was because the study sites were mostly dominated by forest and fruit trees. thus, opening of areas to give way for the production of agricultural crops was not permitted. farmers whose farms were located within the upper stream of the reserves/ watershed planted other woody perennials with smaller canopy, root crops and other shadeecological services of agroforestry landscapes in watershed areas – baliton et al. 78 biotropia vol. 24 no. 1, 2017 tolerant crops as understory. on the other hand, farmers cultivating in open areas having lower elevation had higher opportunities for raising short-term and medium-term crop species. farmer-respondents in molawin-dampalit sub-watershed planted a variety of crop components compared with the far merrespondents in way betung watershed who planted only fruit trees (table 3). among crop components included vegetable crops (16%), fruit trees (30%), root crops (17%), cereals like rice and corn (4.34%), forest trees (19%) and ornamentals (19%). farmers in the molawin-dampalit subwatershed might have enough open spaces where they could plant short-term crops, while farmers in way betung watershed might have shaded spaces, which might not be suitable for cultivating short-term agricultural crops. furthermore, almost 100% of the farmlands in way betung watershed were considered as public lands, which made the farmers bound with policies and regulations on crop cultivation (table 2). farmlands in molawin-dampalit sub-watershed were bound for forest reserve. farmlands located in the lowland ecosystems were still suitable for appropriate agricultural production. biodiversity assessment species composition in the two watersheds a total of 35 tree species with at least 5 cm dbh were found across the 27 sampling plots in molawin-dampalit sub-watershed (table 4). these identified species consisted of 333 individuals belong to 19 tree families. the data revealed that fabaceae had the highest number of species (6), followed by moraceae (5) and meliaceae with three (3) species. annonaceae, th malvaceae and sapindaceae ranked 4 having two (2) species each, while the remaining families had one (1) species each. in terms of the total number of individuals, the dominant families recorded were meliaceae with a total of 90, followed by musaceae (84), sapindaceae (50) and fabaceae (30). four families namely euphorbiacea, lamiaceae, oxalidaceae and sapotaceae ranked the least with only one individual recorded across the sampling areas. in way betung watershed, there were 14 families and 26 species, with 548 individuals found (table 5). the highest number of species belongs to family fabaceae having six (6) species, followed by myrtaceae, meliaceae and arecaceae. family table 4 summary of existing tree families with corresponding number of species and individuals in molawin-dampalit sub-watershed family name number of species number of individuals rank # of species # of individual anacardiaceae 1 8 5 8 annonaceae 2 7 4 9 arecaceae 1 9 5 7 bignoniaceae 1 2 5 12 caricaceae 1 6 5 10 euphorbiaceae 1 1 5 13 fabaceae 6 30 1 4 fagaceae 1 2 5 12 lamiaceae 1 1 5 13 lauraceae 1 2 5 12 malvaceae 2 16 4 5 meliaceae 3 90 3 1 moraceae 5 13 2 6 musaceae 1 84 5 2 oxalidaceae 1 1 5 13 rubiaceae 1 5 5 11 rutaceae 3 5 3 11 sapindaceae 2 50 4 3 sapotaceae 1 1 5 13 total 35 333 --79 table 5 summary of existing tree families with corresponding number of species and individuals in way betung watershed family name number of species number of individuals rank # of species # of individual anacardiaceae 1 2 4 10 arecaceae 3 9 2 9 euphorbiaceae 1 161 4 1 fabaceae 6 27 1 5 gnetaceae 1 35 4 4 lauraceae 1 16 4 7 malyaceae 2 80 3 3 malvaceae 1 154 4 2 meliaceae 3 12 2 8 myrtaceae 3 27 2 5 rubiaceae 1 21 4 6 rhamnaceae 1 2 4 10 sapindaceae 1 1 4 11 sapotaceae 1 1 4 11 total 26 548 - - euphorbiaceae had the highest number of individuals (161), followed by malvaceae and malyaceae. these findings indicated a higher general species composition in molawin-dampalit subwatershed compared to that in way betung watershed. however, the number of individual species in molawin-dampalit sub-watershed was lower compared to that in way betung watershed. this could be explained by the fact that the molawin-dampalit sub-watershed was one of the area severely hit by typhoon glenda (international name rammasun) in 2014. this could explain why, at the time of the study, the floral components were still on their regeneration stage, mostly below 5 cm dbh. importance value of identified plant species in agroforestry landscape across the sampling plots of molawindampalit sub-watershed, banana (musa sapientum) was found to be the most dominant having the highest importance value (iv) of 62.07%. it was followed by rambutan (nephelium lappaceum) and lanzones (lansium domesticum) with iv of 36.47% and 32.59%, respectively. other species having high iv were chico (manilkara sapota) – 25.66%, mangga (mangifera indica) – 21.58% and mahogany (swietenia macrophylla) – 18.79% . table 6 shows the summary of seven (7) dominant plant species with the highest iv. the dominance of banana, rambutan and lanzones in molawin-dampalit sub-watershed table 6 top seven dominant species across sampling plots in molawin-dampalit sub-watershed, mt. makiling forest reserve, philippines species name scientific name importance value (iv) (%) saging musa sapientum 62.07 rambutan nephelium lappaceum 36.47 lanzones lansium domesticum 32.59 chico manilkara sapota 25.66 mangga mangifera indica 21.58 mahogany swietenia macrophylla 18.79 durian durio zibethinus 12.00 ecological services of agroforestry landscapes in watershed areas – baliton et al. 80 biotropia vol. 24 no. 1, 2017 indicated the farmers' preference for cultivating these crops, primarily because of their economic value. meanwhile, this research found out that the dominant tree species in way betung was rubber tree (hevea brasiliensis) with iv of 70.36%, followed by durian, cacao, melinjo, petai, avocado and coffee. based on the iv, the dominance level of a species in a community can be known (indriyanto 2006). table 7 shows the top seven dominant species across the sampling plots in way betung watershed. rubber tree is one of the major high value crop that is being cultivated in indonesia, malaysia and thailand because of its economic potential. this explains why this species was frequently found in way betung watershed. shannon-wiener diversity index (h) and pielou's evenness index (j) across sampling plots in molawin-dampalit sub-watershed was 2.59 and 0.45, respectively (table 8). sampling plots in way betung watershed recorded shannon-wiener diversity index (h) of 2.53 and pielou's evenness index (j) of 0.41. based on the h value, diversity of the agroforestry landscape in molawin-dampalit sub-watershed and way betung watershed was considered to be moderate (fernando et al. 1998) (table 9). the computed shannon-wiener diversity index (h) indicated that employing an agroforestry practice/system in a landscape may increase the diversity of the landscape compared table 7 top seven dominant species across sampling plots in way betung watershed, indonesia species name scientific name importance value (iv) (%) rubber hevea brasiliensis 70.36 durian durio zibethinus 59.72 cacao theobroma cacao 48.07 melinjo gnetum gnemon 22.54 petai parkia speciose 12.46 avocado persea americana 12.14 coffee coffea robusta 8.87 table 8 shannon-wiener diversity index and pielou's evenness index across sampling plots in the two study sites main plot molawin-dampalit sub-watershed way betung watershed circular plot: 8.9 m radius h 2.59 2.53 j 0.45 0.41 table 9 classification scheme of shannon-wiener diversity index (fernando et al. 1998) relative value shannon-wiener diversity index (h) very high 3.50 and above high 3.00 – 3.49 moderate 2.50 – 2.99 low 2.0 – 2.49 very low 1.99 and below 81 to employing monoculture type of farming system. meanwhile, low value of computed pielou's evenness index (j) indicated that the number of individual per species in the agroforestry landscape was not evenly distributed. this finding was validated by noble and dirzon (1997) who highlighted that agroforestry is increasingly being identified as an integrated land use that can directly enhance plant diversity while reducing habitat loss and fragmentation (brent et al. 2006). khanal (2011) also contends that traditional agroforestry practices contribute to the conservation of biodiversity in the western hills of nepal through in-situ conservation of tree species on farms, reduction of pressure on remaining forests, and the provision of suitable habitat for a number of plants on farmland. this contention was supported by meta-analysis on the effects of agroforestry, biodiversity levels and ecosystems services conducted by torralba et al. (2016). torralba et al. (2016) argued that agroforestry can enhance biodiversity and ecosystem service provisions related to conventional agriculture and forestry in europe. furthermore, agroforestry can help the flow of wild plants’ genes as well as increase fauna population size and diversity in protected area corridors, if it is tried as a buffer to connect patches of natural forests to facilitate habitat interconnectivity on a larger scale (baguinon et al. 2007). carbon stock assessment biomass density of agroforestry landscape a mean total of 116.26 mg/ha was recorded in the agroforestry landscape of molawin-dampalit sub-watershed (table 10). the computed mean total biomass density in this study site was higher than the overall mean of agroforestry (102.80 mg/ha) in the philippines (lasco & pulhin 2003). another study of zamora (1999) on biomass density of narra (pterocarpus indicus) + cacao agroforestry system in makiling (191.6 mg/ha) was also comparable with the results obtained in this study. it can be noted that sampling plots in way betung watershed were mostly forest and fruit trees which in turn contributed much on the biomass density with a mean total value of 542.80 mg/ha. this was comprised mostly of 86.12% table 10 biomass density (mg/ha) of agroforestry landscape in molawin-dampalit sub-watershed and way betung watershed item biomass density (mg/ha) aboveground belowground mean total trees and other perennial herbaceous litter trees and other perennial molawin-dampalit minimum 1.98 0.27 0.34 0.30 116.26 maximum 401.83 2.31 7.45 60.27 mean (µ) 97.87 (84%) 1.33 (1%) 2.38 (2%) 14.68 (13%) standard deviation (±) 91.76 0.73 2.07 13.76 # of sample plots (n) 27 27 27 27 way betung minimum 168.74 1.58 1.78 25.31 542.80 maximum 1,160.17 2.31 4.22 174.03 mean (µ) 467.47 (86.12%) 1.81 (0.33%) 3.40 (0.63%) 70.12 (12.92%) standard deviation (±) 242.10 0.17 0.59 36.31 # of sample plots (n) 14 14 14 14 note: values shown inside the parenthesis are the percentage compositions of different carbon pools ecological services of agroforestry landscapes in watershed areas – baliton et al. 82 biotropia vol. 24 no. 1, 2017 item carbon density (mgc/ha) aboveground belowground mean total trees and other perennials herbaceous litter trees and other perennials molawin-dampalit minimum 0.89 0.12 0.15 0.13 52.32 maximum 180.82 1.04 3.35 27.12 mean (µ) 44.04 (84%) 0.60 (1%) 1.07 (2%) 6.61 (13%) standard deviation (±) 41.29 0.33 0.93 6.19 # of sample plots (n) 27 27 27 27 way betung minimum 75.93 0.71 0.80 11.39 244.26 maximum 522.08 0.96 1.90 78.31 mean (µ) 210.36 (86.12%) 0.82 (0.33%) 1.53 (0.63%) 31.55 (12.92%) standard deviation (±) 108.94 0.08 0.27 16.34 # of sample plots (n) 14 14 14 14 table 11 carbon stored in agroforestry landscape of molawin-dampalit sub-watershed and way betung watershed note: values shown inside the parenthesis are the percentage compositions of different carbon pools from the trees and other perennials, while the lowest was recorded in herbaceous and litter with less than 1%. these results were consistent with the previous study conducted by wulandari (2013) in watershed areas in indonesia. biomass density of the agroforestry landscape varied considerably in all carbon pools measured as indicated by high values of standard deviation. huge variation in the biomass density could be attributed to the differences of the components of sampled agroforestry or farming systems. based on the characterization of the farms, there were some farmers who cultivated fruit trees as their main crop which contributed much to the biomass density. other farmers planted only few fruit trees. in addition, farming practices influenced the amount of biomass density of the herbaceous and litter pool, such as weeding and composting. these practices reduced the amount of herbaceous/undergrowth biomass in the area. some farmers were doing these practices, while others were not. carbon stock of agroforestry landscape the aboveground tree and other perennial crops (84%) ranked first in terms of percentage contribution to mean total carbon density of the area. it is followed by belowground tree and other perennial with 13%, then litter (2%) and herbaceous plants provided the least percentage contribution with 1% (table 11). the computed mean total carbon stock (52.32 mgc/ha) was comparable to the overall mean carbon density of secondary forests in the philippines (59.0 mgc/ha) as reported by lasco and pulhin (2003). a study of palma and carandang (2014) reported a higher mean carbon stock (92.78 mgc/ha) of an agroforestry system in misamis oriental. results of these studies already included soil carbon content in the analysis, while this research only focused on the total above and belowground biomass. inclusion of the soil carbon pool could significantly increase the carbon stock due to high concentration of carbon in the soil. expectedly, the computed mean total carbon density of way betung watershed was higher than that of the molawin-dampalit sub-watershed. carbon density in way betung was 244.26 mgc/ha, which was almost four (4) times bigger than that in molawin-dampalit (52.32 mgc/ha). the huge difference could be directly attributed 83 to the abundance of tree in agroforestry systems in indonesia. therefore, this research also validates the argument that agroforestry is a costeffective strategy for climate change mitigation, particularly the tree-based farming systems. conclusions agroforestry farms and practices contribute to the conservation and protection of the way betung watershed and molawin-dampalit subwatershed. diverse crop components in the agroforestry farms, including their interaction, promote biodiversity conservation in these watershed areas, both yielding a moderate level of diversity index. woody perennials, herbaceous crops and litter components of the agroforestry farms contribute to carbon sequestration by having carbon stock of 244.26 mgc/ha in way betung watershed and 52.32 mgc/hain molawindampalit sub-watershed. these ecological ser vices are significant contributions of agroforestry to climate change mitigation. implications and recommendations agroforestry farming practices provide ecological contributions, particularly in carbon sequestration and biodiversity conservation. these contributions of agroforestry practices a l r e a d y o f f e r p o t e n t i a l s i n a d d r e s s i n g environmental degradation in many upland communities in southeast asia. it is necessary to promote the use of agroforestry as a production technology of the government and/or non-government programs on sustainable forest management and upland development. such programs or policies should put emphasis on the use of fruit tree-based agroforestry system to avoid further opening or clearing of forested areas in higher and midelevation areas. the use of fruit tree-based system can enhance the use of soil and water conservation measures and other supportive technologies to control soil erosion and degradation particularly in high-elevation areas. results of carbon stock assessment of various agroforestry systems can provide information on national green house gas (ghg) inventory which is mandated to the committed parties including the philippines in the united nations framework convention on climate change (unfccc). future research should dwell on analyzing the effects and contribution of each of the different types and variants of agroforestry systems to b i o d i ve r s i t y c o n s e r va t i o n a n d c a r b o n sequestration. this kind of research would generate empirical data that would guide researchers, extension workers and policy makers the most appropriate type of agroforestry system that should be scaled-up among the upland farming communities. references baguinon nt, lasco rd, macandog dm, pasicolan pn, villancio vt. 2007. agroforestry and land use in the philippines. bogor (id): world agro-forestry centre. 236 p. brent s, boffa jm, scherr sj. 2006. 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biodiversity and ecosystem services? a meta-analysis. agric ecosyst environ volume 230:150-61. available from: http://www. sciencedirect.com/science/article/pii/s016788091 6303097. retrieved on 3 february 2017. wulandari c. 2013. pendugaan karbon di lahan agroforestry register 19 (carbon prediction on agroforestry area in register 19). bandarlampung (id): lampung university. zamora d. 1999. carbon dioxide (co ) storage potential of 2 multistory agroforestry systems in mt. makiling. msc thesis. laguna (ph): university of the philippines. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 795 tri ratna (identification) revisi.cdr identification of endophytic bacteria from curcuma zedoaria based on protein profile using maldi-tof mass spectrometry 1* 2 tri ratna sulistiyani and puspita lisdiyanti 1 (lipi) bogorresearch center for biology, indonesian institute of sciences , cibinong science center, 16911, indonesia 2 (lipi) aresearch center for biotechnology, indonesian institute of sciences , cibinong science center, bogor 16911, indonesi received 30 december 2016 / accepted 06 october 2017 abstract valid identification of microorganisms is a vital information to establish culture collections. currently, molecular approach based on 16s rdna is widely used for bacterial identification. this approach is however, time consuming and expensive. matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms) allows the identification of microorganisms directly from colonies and it only takes some few minutes. the interest of this study was to identify endophytic bacteria associated with curcuma zedoaria based on protein profile using maldi-tof ms system and compare with 16s rdna sequence results. endophytic bacteria were isolated from part of medicinal plant c. zedoaria collected from bogor, west java indonesia. the identification of selected bacteria was performed by protein profile using maldi-tof ms. a total of 66 endophytic bacteria from c. zedoaria plant, were selected for identification. the result of maldi-tof ms analysis showed that eleven isolates (16.67%) were correctly identified to the species level and 23 isolates (34.85%) matched on genus level of molecular approach. these results demonstrates that the maldi-tof system is suitable and feasible approach for the bacterial identification, mainly for screening and grouping of large number isolates. keywords: curcuma zedoaria, endophytic bacteria, identification, maldi-tof ms introduction rapid and accurate identification of bacterial isolates has crucial role in the culture collections. several methods of identification have been d e v e l o p e d . b a c t e r i a l i d e n t i f i c a t i o n i s p r e d o m i n a n t l y b a s e d o n p h e n o t y p i c , morphological (gram-positive and negative) and biochemical properties testing analysis. however, classification and identification by these methods can be difficult because of variations in phenotypic characteristics, time-consuming and laborius (seng et al. 2009; rychert et al. 2013). several years ago, molecular identification based on nucleotide sequencing of ribosomal dna (rdna) sequence analysis could replace the phenotypic, morphological and biochemical c h a r a c t e r i s t i c s f o r i d e n t i f i c a t i o n o f microorganisms to genus level (rajendhran & gunasekaran 2011). molecular approach based on rdna have widely been used for sequence identification of procaryotic taxa. the 16s rdna sequences has confirmed the representativeness of the in bacterial phylogeny (woo et al. sequence 2008). however this method is not suitable for a preliminary screening and grouping of large numbers of isolates in environmental and clinical microbiology laboratories. analysis based on protein profile present in bacterial cells using matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms) is becoming feasible and precise for bacterial identification (bizzini et al. 2011; santos et al. 2013). malditof ms allows rapid identification of bacteria, less expensive on operational cost, accurate and low sample volume requirements compared to molecular approach (wunschel et al. 2005; singhal et al. 2015). it can be performed as soon as colonies are isolated and can be done just in a few minutes (eigner et al. 2009; seng et al. 2009; neville et al. 2011; rychert et al. 2013; jamal et al. 2014).* corresponding author: trilisty01@gmail.com biotropia 5 2 8 112 120 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.795 112 maldi-tof ms analysis was done by comparing the maldi-tof ms spectra or protein profile “fingerprints” obtained from bacterial cells to a library from proteomic database (martiny et al. 2012; guo et al. 2014). protein profile are significantly different for each bacteria and it contain certain unique mass marker even over the small mass range detected. each bacteria shows signature characteristic peaks that are distinct for species level (cain et al. 1994). the identification of microorganisms by malditof ms is based on the detection of mass signals from biomarkers that are specific at genus, species or sub-group level (ferroni et al. 2010; benagli et al. 2012), therefore changes in protein profiles would be easily identified. the protein profile from each bacteria can serve as unique information for bacteria chemotaxonomic marker, for example to differentiate closely related species, also could enriche the information contained in procaryotic protein sequence databases. kudirkiene et al. (2015) reported that maldi-tof ms was succesfully used in sub-species identification of streptococcus equi. using maldi-tof ms, routine bacterial identification can be obtained much earlier than identification by molecular and conventional methods. therefore, this technique can be used as quality control management of cultures deposited in culture collections. the main objective of the study was to evaluate the identification of endophytic bacteria associated with c. zedoaria based on protein profile using maldi-tof ms system compared with the results based on 16s rdna sequence from previous study (sulistiyani et al. 2014). materials and methods bacterial strains a total of 66 endophytic bacteria isolates from b i o s y s t e m a t i c a n d c u l t u r e c o l l e c t i o n s laboratory-research center for biology, lipi, was used to evaluate the maldi-tof ms analysis were recovered from previous study (sulistiyani et al. 2014). the isolates were opreserved in 10% glycerol stock at -80 c. the bacteria were grown using nutrient agar (na) media. the bacteria isolates were from c. zedoaria (white turmeric) planted in 3 areas in bogor, indonesia, in 2013. the plants materials collected from private garden in bojong gede (bg), experiment garden of research center for biology, indonesian institute of sciences, cibinong (cbn) and garden of medicinal plants collection of biopharmaca research center, bog or ag ricultural university, dramaga (drmg). all bacteria were previously identified by molecular identification based on 16s r na d sequence. the bacteria represented 23 genera and 46 species (sulistiyani et al. 2014). preparation of acterial ell xtractsb c e bacterial isolates were cultured from glycerol stock on nutrient agar (na) and incubated under standard conditions at 35-37 c for 48 o hours and subcultured twice to ensure all isolates were in the same physiological state. cell extract was prepared by picking a single colony of a fresh culture and suspended in 20 µl formic acid of 25% concentration and then mixed until complete suspension is formed by vortex. the extract was sonicated for 10 min and spinned to get the supernatant. finally, the supernatant was used for maldi-tof analysis. maldi-tof analysis the bacterial cell extract of 0.5 µl was placed in duplicates onto a steel target plate and allowed to dry at room temperature. each sample was overlaid with 1 µl matrix of α-cyano-4hydroxycinnamic acid (chca) in ethanol : acetonitrile : water : trifluoroacetic acid. the sample and matrix were mixed thoroughly and air dried at room temperature. measurements were taken using a maldi-tof ms axima system (microbiology laboratory of the mulhouse hospital, france). the laser frequency was 50 hz, the acceleration voltage was 20 kv, and the extraction delay time was 200 ns. each spectrum resulted from 5 laser shots at 100 random positions within measuring spot. the spectra were recorded in the linear positive mode within a mass range of 2 to 20 kda. all mass fingerprints were analyzed by the saramis software, which first compares them to the superspectra and in a second step to the individual spectra of the database using anagnostec version 4.07 (axima system manual). 113 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti such as morphologic, phenotypic, physiologic and molecular methods. however, as mentioned earlier, these methods are not suitable for a preliminary screening and grouping of large numbers of isolates from environmental and clinical samples and also, for controlling the identity and purity of microbial cultures preserved in culture collections. to overcome these problems, the present study evaluated the capability of maldi-tof ms for species identification of microorganisms such as bacteria isolates. validity of maldi-tof ms results was compared with the results of molecular identification based on 16s rdna sequences as a calibration and validation analysis the instrument was calibrated and validated using a control strain of escherichia coli k12 inacc b5. several strain were also used for quality control including of bacillus substilis inacc b1, pseudomonas aeruginosa inacc b3 and staphylococcus aureus inacc b4. results and discussion accurate identification of bacteria could be obtained from several identification methods, figure 1 spectral profiles of three isolates k. pneumoniae obtained from different part of white turmeric plant. these mass signals spectra (3,853; 4,365; 6,292; 7,245; 7,384; 7,705; 9,140; and 9,479 m/z) are specific to k. pneumoniae. biotropia vol. 25 no. 2, 2018 114 reference method. the results of maldi-tof ms analysis are indicated in percentages of similarity compared to spectra profiles of known s t r a i n i n s a r a m i s d a t a a s e s y s t e mb . superspectra contain common peaks to different strains of the same species. iate individual spectra correspond to the spectra of ed each strain cultivated under specific conditions. the manufacturer recommends validation of superspectra identifications with the confidence level between 85.00 and 99.9%. accuracy of the identification strongly relies upon the robustness of the database and the choice of reference isolates. this is especially important when the genera involving species of environmental and clinical a high genetic diversity samples represents (benagli 2012)et al. . the spectra were analysed in a mass range of 2 to 20 kda and this mass range representing ribosomal proteins were obtained from bacterial extract. these proteins are numerous in the cell and are positively charged. fig. 1 shows the representative results of spectral profiles from klebsiella pneumonia based on the mass signals obtained. isolated bacteria from different part of plant (rhizome, stem, leaves) and locations (dar mag a and bojong) showed similar identification due to their identical protein profiles which were indicated by mass spectra value. three isolates of k. pneumoniae isolated from rhizome, stem and leaves of c. zedoaria plant showed almost the same spectral profiles. it had several specific mass signals 3,853; 4,365; 6,292; 7,245; 7,384; 7,705; 9,140; and 9,479 m/z. each mass signal is specific at species and family level. mass signal of 3,853; 6,292; 7,384; 7,705; and 9,479 m/z were specific for species level, while 4,365; 7,245; 9,140 m/z were specific for family level. according to these results, it is clear that each species of bacteria has specific mass spectra, and can be used as a taxonomic marker identification. compared with the data presented in table 1, these mass signals correspond to the superspectrum database of k. pneumoniae. the identification results obtained by maldi-tof ms and 16s rrna gene sequencing shown in table 2. of 66 isolates are forty-three (43) of the 66 isolates (65.15%) showed the intepretable maldi-tof ms spectra by sequence comparison analysis, . maldi-tof ms spectra allowed good identification to the species, genus and family level total of 43 endophytic bacteria with a covering 16 genus and 21 species. an additional 11 isolates (16 67%) with the . gave similar results sequencing results, correctly identified to the and mass (m/z) identification level 3853.5 species level 4155.3 species level 4365.1 family level 5381.6 family level 6292.8 species level 6384.1 family level 6857.4 family level 7165.8 family level 7245.2 family level 7272.7 family level 7319.6 family level 7384.9 species level 7705.3 species level 7735.2 species level 8310.1 family level 8852.1 family level 9093.2 species level 9140.1 family level 9479.5 species level 9852.3 species level table 1 superspectrum database of k. pneumoniae 115 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti level of id and isolates 16s rdna sequencing maldi-tof ms species id level of id species id reference database correctly identified into species level di.p.3 alcaligenes faecalis species alcaligenes faecalis a rb.p.1 bacillus subtilis species bacillus subtilis a rl.s.2 enterobacter aerogenes species enterobacter aerogenes a ri.p.2 enterobacter cloacae species enterobacter cloacae a bb.p.3 klebsiella pneumoniae species klebsiella pneumoniae a ri.s.2 klebsiella pneumoniae species klebsiella pneumoniae a ri.s.9 klebsiella pneumoniae species klebsiella pneumoniae a ri.p.3 pantoea dispersa species pantoea dispersa a db.s.1 pseudomonas stutzeri species pseudomonas stutzeri a rb.p.4 pseudomonas stutzeri species pseudomonas stutzeri a ri.p.7 stenotrophomonas maltophilia species stenotrophomonas maltophilia a correctly identified into genus level ri.s.3 acinetobacter calcoaceticus genus acinetobacter baumannii a dl.p.5 bacillus safensis genus bacillus pumilus na ri.p.5 bacillus thuringiensis genus bacillus cereus/ mycoides/thuringiensis a ri.p.1 burkholderia cenocepacia genus burkholderia sp. a di.p.1 enterobacter cancerogenus genus enterobacter sp. a dl.p.4 enterobacter cancerogenus genus enterobacter cloacae a ri.p.8 enterobacter ludwigii genus enterobacter sp. na bi.p.3 enterobacter ludwigii genus enterobacter sp. na db.p.1 klebsiella pneumoniae genus enterobacteriaceae a rb.p.2 klebsiella pneumoniae genus enterobacteriaceae a di.p.4 klebsiella variicola genus klebsiella pneumoniae na ri.s.8 klebsiella variicola genus klebsiella pneumoniae na rb.s.3 klebsiella variicola genus enterobacteriaceae na bi.s.2 klebsiella variicola genus klebsiella pneumoniae na ri.p.4 microbacterium trichothecenolyticum genus microbacterium arborescens na db.s.4 micrococcus yunnanensis genus micrococcus luteus na ri.s.7 pantoea agglomerans genus pantoea dispersa a ri.s.6 pseudomonas azotoformans genus pseudomonas fluorescens na rb.p.3 pseudomonas denitrificans genus pseudomonasnitroreducens na dl.p.3 pseudomonas denitrificans genus pseudomonas nitroreducens na db.s.3 pseudomonas gessardii genus pseudomonas fluorescens na di.s.1 pseudomonas korensis genus pseudomonas aeruginosa na di.s.6 pseudomonas korensis genus pseudomonas sp. na not precisely identified bb.s.8 bacillus subtilis lysinibacillus sphaericus a bi.s.3 citrobacter freundii enterobacter sp. a di.s.4 microbacterium laevaniformans klebsiella pneumoniae a bi.s.6 microbacterium laevaniformans arthrobacter russicus a bi.p.1 microbacterium resistens pseudomonas aeruginosa a di.s.7 microbacterium testaceum oligella urethralis a bb.s.3 microbacterium trichothecenolyticum gordonia alkanivorans na ri.p.6 pseudomonas geniculata stenotrophomonas maltophilia na bi.s.1 ralstonia mannitolilytica rhizobium radiobacter a not identified yet db.p.2 agrobacterium larrymoorei no id na dl.p.6 bacillus subtilis no id a bl.p.2 bacillus subtilis no id a rl.s.1 bacillus safensis no id na rb.s.5 bosea thiooxidans no id na rb.s.2 enterobacter ludwigii no id na bb.p.4 erwinia chrysanthemi no id na dl.s.1 methylobacterium organophilum no id na bb.s.5 microbacterium hominis no id na table 2 identification results for the 66 isolates obtained by maldi-tof ms in comparison to those obtained by 16s rdna 116 biotropia vol. 25 no. 2, 2018 species level; 23 isolates (34 85%) matched genus . level of molecular approach, and among them 3 isolates were identified to the family level. for three isolates (db.p.1, rb.p.2, rb.s.3), malditof ms correctly identified to the family level, whereas 16s r na sequencing gave a species d identification. ine isolates (13 64%) were not n . accurately identified and 23 isolates (34 85%) were . rated as n3 .no identifiable (table ) this study has proved that maldi-tof ms is useful for identification of microorganisms into species level in a relatively short time. of the 66 isolates analyzed, 43 isolates (65.15%) were identified and 23 isolates (34.85%) were not identified (table 3). eleven isolates (16.67%) of 43 isolates showed good result, concordant with the 16s rdna sequencing result. however, twenty-three isolates (34.85%) could not be identified. these included 12 isolates of microbacterium genus, mycobacterium cosmeticum, mycobacterium simiae, agrobacterium larrymoorei, erwinia chrysanthemi, xanthobacter flavus, enterobacter ludwigii, bosea thiooxidans, stenotrophomonas maltophilia, methylobacterium organophilum, providencia vermicola, phenylobacterium koreense, bacillus safensis, roseomonas mucosa, rhizobium tarimense. among them, 14 isolates could not be identified due to the absence of reference spectra database. non-identifiable isolates for maldi-tof was as a result of an incomplete database, and can be resolved with the addition of appropriate reference. supplementation of the maldi-tof database, can reduce the rate of non-identifiable results. among 66 isolates of endophytic bacteria, 30 isolates had no reference spectra database (table 3). currently, a total of 1,309 references spectra are contained in saramis database system. this is inadequate to identify indigenous microbes that have been abundant in indonesia. maldi-tof ms library can be enriched with protein profile of strain or species of indigenous microbes from indonesia, and will be important for future studies. maldi-tof ms generates protein mass spectra which can be used for grouping and identification of bacteria. these mass spectra contain mainly peaks corresponding to ribosomal protein that are in abundance in the bacterial cell (rhyzhov & fenselau 2001). protein profile of mass spectra will help in characterizing the level of id and isolates 16s rdna sequencing maldi-tof ms species id level of id species id reference database correctly identified into species level table 2 continued rl.p.4 phenylobacterium koreense no id na rl.p.1 providencia vermicola no id na di.s.5 rhizobium tarimense no id na rl.s.3 roseomonas mucosa no id na bl.p.1 stenotrophomonas maltophila no id a dl.p.2 stenotrophomonas maltophila no id a bb.s.7 xanthobacter flavus no id na db.s.2 microbacterium laevaniformans no id a bi.p.6 microbacterium laevaniformans no id a di.s.2 microbacterium resistens no id a di.s.3 microbacterium testacneum no id a dl.p.1 microbacterium testaceum no id a bl.s.2 mycobacterium cosmeticum no id na bb.s.6 mycobacterium simiae no id a note: a: available; na: not available table 3 distribution of the discrepancies observed in the maldi-tof ms axima-saramis level of identification by maldi-tof ms no. (%) of reference database available not available total correctly identified into species level 11 (16.67) 0 11 (16.67) correctly identified into genus level 8 (12.12) 15 (22.73) 23 (34.85) not precisely identified 7(10.60) 2 (3.03) 9 (13.64) not identified yet 10 (15.15) 13 (19.70) 23 (34.85) total 36(54.54) 30(45.46) 66 117 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti incorrectly identified microbes by comparing its protein profile spectra to those in the reference spectra database. these spectra can generate patterns that provide unbiased identification of particular species and even genotypes within species. data presented in table 3, shows that 9 isolates were incorrectly identified. as stated earlier, each species of bacteria has specific protein profile mass spectra, therefore the identification result from maldi-tof ms should be same to the identification result of 16s rdna. the result will be incorrect when experimental factors occur, such as sample contamination and/or sample preparation. maldi-tof ms analysis is affected by several experimental factors, such as matrix p r e p a r a t i o n , s p e c t r a l r e p r o d u c i b i l i t y, contaminants, sample preparation, mass range and measurement accuracy on the database search (demirev et al. 1999). a total of 12 isolates of microbacterium genus were incorectly identified, might be due to the complex structure of their cell walls. specific sample extraction procedures to breakdown the cell wall are required before maldi-tof ms analysis. the process of sample preparation for identification of microbes depends upon the source of isolated microbe, or on chemical structure of the constituents of its cell wall. different group of microbes has different sample preparation (singhal et al. 2015). alatoom et al. (2011) reported that sample extraction was needed for identification of grampositive bacteria. furthermore, the lower score values, may be caused by the incomplete separation of protein. the protein interfered with the sample and disrupt the mass spectrum (reich et al. 2013). gram-positive bacteria like mycobacterium sp., also require specific extraction procedure. the extraction was done by lysed cells in boiling water, followed by ethanol precipitation of proteins. the precipitated proteins were dried, resuspended in 70% formic acid and acetonitrile, and analyzed by maldi-tof ms (verroken et al. 2010). to investigate the reproducibility of the instrument during the study we included inacc , strains in every analysis of identifications as positive controls and reference isolates. the instruments correctly identified the control strains. the incorrect identification by malditof ms was to sample preparation attributed failure such as sample volume and the large also, amount of matrix was not sufficient. consequently the sample and matrix were not completely mix and only a few small crystal ed were obtained. a sufficient number of bacterial cells (typically ~10 cells per well) are required to 4 generate detectable maldi-tof ms ion signals (chiu 2014). lohman et al. (2013) reported that malditof system succesfully identified 312 isolates. furthermore 2,860 of 2,900 (99%) samples identified by maldi-tof ms matched with the identification results obtained using other methods (standard and high end microbiological identification methods including automated b i o c h e m i c a l a n a l y s e s a n d m o l e c u l a r identification) (reich et al. 2013). study of guo et al. (2014) informed that using maldi-tof ms for 1,025 isolates, 1,021 (99.60%) isolates were accurately identified at the genus level, and 957 (93.37%) isolates at the species level. theel et al. (2012) reported that from 90 yeast and 78 corynebacterium species isolates, were obtained 95.6% and 81.1% of yeast, also 96.1% and 92.3% of cor ynebacterium isolates were correctly identified to the genus and species levels, respectively. as compared to other studies, the result of this study showed small percentage of correctly identified to the genus and species levels, however this method provide reliable results. therefore maldi-tof ms could be used for screening and grouping of large numbers o f b a c t e r i a l i s o l a t e s. m i c r o o r g a n i s m identification by mass spectrometry is already considered as a revolution of bacteriology, offering many advantages compared with the conventional biochemical identification of microorganisms. within the next few years maldi-tof-ms based identification of microorganisms will replace conventional methods. conclusion among the 66 selected isolates, 43 isolates were identified. eleven isolates (16.67%) that were tested matched on species level of molecular approach. spectral analysis of microbial diversity from indonesia is one way to build up the maldi-tof ms library. rotein profile spectra p of each species be used as ataxonomic could marker. maldi-tof ms systems for bacterial 118 biotropia vol. 25 no. 2, 2018 identification was good for grouping large number of isolates. however molecular analysis also must be done as a standard reference. combination of molecular analysis and protein profile using maldi-tof ms slightly accelerated the bacterial identification. acknowledgements this project was fully supported by dipa tematik 2013, research center for biology lipi and jst-jica. special thanks to sulistiani and all members of the biosystematic laboratory, research center for biology, lipi. references alatoom aa, cunningham sa, ihde sm, mandrekar j, patel r. 2011. comparison of direct colony method versus extraction method for identification of grampositive cocci by use of bruker biotyper matrixassisted laser 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(11) .species j clin microbiol 48 :4015-21 woo pcy, lau skp, teng jll, tse h, yuen ky 2008 then . . and now: se of 16s rdna gene sequencing for u bacterial identification and discovery of novel bacteria in clinical microbiology laboratories clin . microbiol infect 14 :908-34(10) . wunschel sc, jarman kh, petersen ce, valentine nb, wahl kl, schauki d, white ve 2005 bacterial … . . analysis by maldi-tof mass spectrometry: an inter-laboratory comparison. j am soc mass spectrom 16(4):456-62. doi: org/10.1016/j.jasms. 2004.12.004 120 biotropia vol. 25 no. 2, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 74 biotropia vol. 15 no. 1, 2008 recovery of more than 10 years-drying monascus cultures and its purification methods from fungal and bacterial contamination nandang suharna microbiology division, research center for biology, indonesian institute of sciences (lipi), cibinong, jakarta, indonesia abstract this study was carried out to understand the recovery capability of more than 10 years drying monascus cultures. a new simple purification technique from fungal contamination using ethanol-soaking treatment was also reported as a part of this study. the result showed that all drying cultures were recovered well and retained their characters such as good growth, pigmentation and production of fruit bodies (ascomata), sexual spores (ascospores) and asexual spores. several cultures showed its good growth in 20% ethanol medium. this study also reported successful purification of cultures from fungal contamination using ethanol-soaking treatment. this self-drying method, therefore, could be suggested as a good long-term preservation method for monascus cultures. moreover, purification method from fungal contamination soaked in ethanol 70% or 95% was successfully effective. key words: recovery, preservation, monascus, drying, purification introduction monascus becomes recently popular as this fungus is used in food, specially chinese red rice (angkak), red wine, rice wine, kaoliang beer, soya cheese and pigment, food colorant in asia, mainly china, philipines, japan, thailand and indonesia (steinkraus 1983). the major mold that plays this important role is m. purpureus. this fungus is mainly used in the production of angkak. this product mostly is known by its value as food or drink colorant since it can be used as an alternative for synthetic chemical based on its health concern. monascus purpureus is also known as a producer of monacolin, a statin substance which can inhibit 3-hydroxyi-3-methylglutaryl coenzyme a (hmg-coa) reductase in cholesterol biosynthesis (endo 1979). hence, monacolin production by this fungus is being extensively studied recently. corresponding author: nsuharna@yahoo.com biotropia vol. 15 no. 1, 2008 : 74 85 75 recovery of more than 10 years-drying monascus culture – nandang suharna. accordingly, on account of its highly benefits, maintenance and preservation of living monascus cultures become extremely important so as to ensure its continuous use without risk of loss of its capability. currently, three primary methods of culture preservation are already known such as continuous growth, drying, and freezing. continuous growth methods, in which cultures are grown on agar medium, generally are used for short-term storage. such cultures are stored at temperatures of 5 °c-20 °c, or they may be frozen to increase the interval between subcultures. the methods are simple and inexpensive because specialized equipment is not required (smith 1993; smith and onion 1994; nakasone et al. 2004). in short-term storage, routine maintenance is usually not preferable, since it is laborious and has higher risk of loss of capability due to frequent transfer. whilst, long term storage much reduces transfer frequency and risk loss of capability. therefore, the long-term storage will supply stock culture more safely with good quality. drying is the most useful method of preservation for cultures that produce spores or other resting structures. drying methods are technically simple and also do not require expensive equipment (smith 1993; smith and onion 1994; nakasone 2004). freezing methods, including cryopreservation, are versatile and widely applicable. with or without cryoprotectants, most fungi can be preserved in liquid nitrogen or in standard home freezers. with freeze-drying, or lyophilization, the fungal cultures are frozen and subsequently dried under vacuum condition. the method is highly successful with cultures that produce mitospores. freeze-drying and freezing below -135°c are excellent methods for permanent preservation, and we highly recommend them. however, both methods require specialized and expensive equipment, as described in the next section. the choice of preservation method depends on the species concerned, the resources available and the goal of the project (nakasone et al. 2004). we experienced with yearly storage of monascus cultures preserved by grown on agar slant but then let dry naturally at room temperature. these cultures were tested for its recovery from years of incubation period. preservation of monascus culture using liquid drying started last year. however, our findings facilitate many things including handling of the microorganism. the objective of this study is to know the recovery of monascus cultures after drying for more than 10 years. this study also covered purification of drying monascus cultures from microbial contamination. in this study, we also report our experience with purification of monascus cultures from fungal contamination besides bacterial contamination. this report also covers a new simple method to purify monascus cultures from fungal contamination. 76 biotropia vol. 15 no. 1, 2008 materials and methods monascus strains seventeen strains of monascus were used in this study (table 1). these strains were preserved on slope agar and underwent drying during storage at room temperature for 10 years. monascus cultures are maintained on taoge (germinates of phaseolus radiatus l.) extract agar 6% (ta) slope at room temperature. ta medium preparation referred to saono et al. (1969). this medium contained extract of germinating beans (phaseolus radiatus l.), 6% sucrose and 15% agar bacto. cultivation medium media used for cultivation was malt extract agar (mea) 2% (difco ltd.) which composed of malt extract (2%), peptone (10%), glucose (10%) and agar bacto (15%). water agar (wa) media used for cultivation of bacterial-contaminated culture of monascus contained agar bacto (15%) and tap water; and ethanol liquid medium which was composed of ethanol 20%, peptone 10%, glucose 20% and yeast extract 20% (pgy ethanol 20%) were used for cultivation in high concentration of alcohol. water soaking treatment prior to re-cultivation of drying monascus cultures water soaking treatment is to soak dry cultures in ta medium by adding sterile distilled water three hours before re-cultivated on a new fresh ta medium. recultivation of drying monascus cultures re-cultivation was carried out by transferring a small part of culture after treated by water soaking treatment on to mea 2% plate. this plate is then incubated at room temperature. purification from bacterial contamination cultivation on water agar (wa) medium is very effective method to separate fungal colony from bacteria (watanabe 2004). therefore, this method was applied to purify monascus culture f rom bacterial contamination. before cultivation the contaminated culture was pre-treated by soaking in distilled water for three hours in order to soften and to re-hydrate the agar. subsequently, a small part of the culture was removed and transferred into wa medium. after few days of incubation, the separation of fungi and bacteria can be observed. isolation of monascus colony free from bacterial colony was carried out and monascus culture was re-cultivated onto a new fresh mea 2% medium. 77 recovery of more than 10 years-drying monascus culture – nandang suharna. purification from fungal contamination by ethanol soaking treatment to eliminate fungal contamination, a small part of the contaminated dry culture was soaked into ethanol 70% or 95% for one minute prior to cultivation on mea 2%. incubation was done at room temperature. after three days incubation period, monascus culture which was free from the fungal contaminant was transferred under aseptic condition to a new fresh medium. the effect of one minute-ethanol soaking treatment on monascus growth the effect of one minute-ethanol-soaking treatment on monascus growth was studied. each sample of 14 drying monascus cultures was soaked into ethanol before cultivation in agar medium. prior to this treatment, the sample was soaked in water for three hours for re-hydration and to soften the agar so as to facilitate the fungus to grow. the cultures were then ready to be soaked in ethanol for one minute before cultivation on new fresh agar medium without rinsing using water. observation on retained important characters of monascus a. observation on colony growth was determined by its growth intensity as + (good growth) or – (poor growth). b. observation on pigmentation is carried out by observing the occurrence of pigmentation visually. c. production of cleistothecium (ascomata/fruit bodies), ascospores (sexual/generative spore) and aleurispores (asexual/vegetative spore). after making slide preparation, microscopic observation was carried out and each production determined as + (good production) or (no or poor production). d. growth in 20% ethanol medium. this is a test to know its growth capability of monascus strains in high concentration of ethanol medium. table 1. list of monascus collection maintained on slope agar that underwent drying during ten years storage. strain code fungus name year cultured & stored source of strains a70.1.2 monascus sp. 1994 shrimp specimen preserved in ethanol coel monascus sp. 1994 coelenterata specimen preserved in ethanol ka15.1(i) monascus sp. 1994 shrimp specimen preserved in ethanol ka15.2 monascus sp. 1994 shrimp specimen preserved in ethanol ka15.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.1 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.2 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.1 monascus sp. 1994 shrimp specimen preserved in ethanol 78 biotropia vol. 15 no. 1, 2008 strain code fungus name year cultured & stored source of strains ka70.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.4 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.4 nr4 monascus sp. 1995 shrimp specimen preserved in ethanol ka70.5 monascus sp. 1994 shrimp specimen preserved in ethanol ktb monascus sp. 1993 coelenterata specimen preserved in ethanol myom monascus sp. 1994 maibua fasciata specimen preserved in ethanol myot monascus sp. 1994 myotis ultisima specimen preserved in ethanol ngk m. purpureus 1993 chinese red rice results and discussion after pre-treated by soaking in sterile water for three hours and subsequently recultivated on new fresh agar medium, 17 drying monascus cultures successfully recovered. one culture was contaminated by bacteria, three were contaminated by molds (aspergillus and penicillium), and 13 cultures were in pure condition (table 2). recovery capability (table 2) indicated that 15 cultures were in good and the other two in bad condition. however, poor growth of these two cultures might be affected by the growth of the contaminant. after purification (table 4) these cultures could grow well. all monascus growth could be observed after three days incubation at room temperature (25-31°c). the purpose of purification work was to get rid of bacterial contamination from culture with code strain myot, by re-cultivation the monascus colony on water agar medium and the other four monascus such as m. purpureus srb6.1, m. purpureus slc, m. purpureus mlgb, and m. purpureus bdg 2.1 as well (table 3). those four cultures were also known to suffer from bacterial contamination. this additional work was intended to enrich the data. the result indicated that this purification was much possible since the bacteria grew very restricted, but monascus grew rapidly leaving bacterial colony. after isolation and re-cultivation on the new medium this monascus colony grew well (table 3). as the three monascus cultures were contaminated by other molds, purification attempt was carried out using ethanol-soaking treatment before its cultivation on agar medium. this technique could eliminate mold contamination from the three cultures (table 4). however, this technique did not work for bacterial contamination. the purification of culture with code strain myot contaminated by bacteria could not be carried out, as the bacteria were not effectively inhibited by ethanol. therefore, this technique can be used for purification from fungal contamination. table 1. continued 79 recovery of more than 10 years-drying monascus culture – nandang suharna. after pre-treated by one minute-ethanol (70% or 95%) soaking on monascus drying cultures before cultivation on mea 2% showed that all cultures could grow well (table 5). visually, there was obviously no effect on the monascus growth. figure 1 shows the growth of monascus sp. a70.4 on mea 2%. without pre-treated by soaking in ethanol, the fungal contaminant grew well and no growth of the monascus was observed when this contaminated culture was cultivated in mea 2%. when this contaminated culture was pre-treated by soaking in ethanol at 70% or 95%, the monascus culture grew well and there was no growth of fungal contaminant observed. therefore, this treatment enables to purify monascus cultures from fungal contamination. hence, it is recommended to use this simple technique to deal with fungal contamination of monascus cultures especially m. purpureus, m. ruber or maybe other osmophilic monascus. observation under light microscope of all monascus strains indicated that all strains still produced ascomata abundantly, ascospores and aleuriospores (table 6). the color of monascus colony was whitish shade, except ngk strain was blood red. based on these results, at least drying more than 10 years did not change the above characters observed. cultivation in 20% ethanol medium showed that 11 monascus strains were able to grow but not the other nine strains (figure 2). the growth of these 11 strains was observed at various days (7-12 days) of incubation on pgy ethanol 20% (table 6). this treatment aimed at the capability of growing monascus strains in ethanol at extreme concentration showed that the 11 strains which originated from degraded ethanol still retained their viability. table 2. recovery of monascus drying culture after 10 years of storage. strain code viability purity recovery capability(colony growth) a70.1.2 viable pure good coel viable pure good ka15.1 (i) viable pure good ka15.2 viable pure good ka15.3 viable pure good ka30.1 viable pure good ka30.2 viable contaminated by aspergil-lus (overgrowth) good ka30.3 viable pure good ka70.1 viable pure good ka70.3 viable pure good ka70.4 viable contaminated by penicil-lium (overgrowth) good 80 biotropia vol. 15 no. 1, 2008 strain code viability purity recovery capability(colony growth) ka70.4 nr4 viable pure good ka70.5 viable contaminated by asper-gillus (overgrowth) poor ktb viable pure good myom viable pure good myot viable contaminated by bacteria (overgrowth) poor ngk viable pure good table 3. recultivation of monascus colony contaminated by bacteria. strain code growth (3 days old) mea 2% wa monascus sp. myot m r m. purpureus srb6.1 m r m. purpureus slc m r m. purpureus mlgb m r m. purpureus bdg 2.1 m r notes : m: bacterial and monascus growth mixed; direct monascus isolation was not possible. r: bacterial growth was very restricted, direct monascus isolation was possible. table 4. purification from fungal contamination using ethanol-soaking treatment strain code ethanol treatment 0% 70% 95% growth condition, purity ka30.2 poor, aspergillus good, pure good, pure ka70.4 poor, penicillium good, pure good, pure ka70.5 poor, aspergillus good, pure good, pure myot poor, bacteria poor, bacterial contamination poor, bacterial contamination table 2. continued 81 recovery of more than 10 years-drying monascus culture – nandang suharna. table 5. the effect of 1 minute-ethanol soaking treatment on monascus grown on malt extract agar 2% after three days incubation at room temperature. strain code ethanol concentration 0% 70% 95% monascus sp. a70.1.2 + + + monascus sp. coel + + + monascus sp. ka15.1 (i) + + + monascus sp. ka15.2 + + + monascus sp. ka15.3 + + + monascus sp. ka30.1 + + + monascus sp. ka30.2 + + + monascus sp. ka30.3 + + + monascus sp. ka70.1 + + + monascus sp. ka70.3 + + + monascus sp. ka70.4 + + + monascus sp. ka70.4 nr4 + + + monascus sp. ka70.5 + + + monascus sp. ktb + + + monascus sp. myom + + + monascus sp. myot + + + m. purpureus ngk + + + note : + = good growth figure 1. monascus sp. a70.4 pre-treatment with and without soaking in ethanol for one minute before cultivation on mea 2% and incubated for 7 days at room temperature. the green colony is penicillium growing from inoculation point of monascus without pre-treatment; no monascus colony observed (a). two whitish colonies are monascus, no fungal contaminant colony observed after the pre-treatment (the upper right was pre-treated by 70% ethanol) (b); below one was pre-treated by 95% ethanol (c). 82 biotropia vol. 15 no. 1, 2008 table 6. the retained cultural and morphological properties of monascus drying cultures and their resistance to 20% ethanol after stored more than 10 years. strain code growth on mea 2% (room temperature) grown on pgy ethanol 20% (room temperature) recovery (day) production colony pigmentatiom recovery (day)ascmt ascpr aleu a70.1.1 3 + + + white yellowish 7 a70.1.2 3 + + + white yellowish no growth coel 3 + + + white yellowish 10 ka15.1 3 + + + white yellowish no growth ka15.2 3 + + + white yellowish no growth ka15.3 3 + + + white yellowish 10 ka30.1 3 + + + white yellowish 10 ka30.2 3 + + + white yellowish 10 ka30.3 3 + + + white yellowish no growth ka70.1 3 + + + white yellowish no growth ka70.3 3 + + + white yellowish 7 ka70.4 3 + + + white yellowish 7 ka70.4 nr4 3 + + + white yellowish no growth ka70.5 3 + + + white yellowish no growth kns15.1 3 + + + white yellowish 10 ktb 3 + + + white reddish orange 12 mm 3 + + + white reddish orange no growth myom 3 + + + white yellowish 10 myot 3 + + + white yellowish 12 ngk 3 + + + red blood no growth note : ascmt= ascomata, ascpr= ascospores, aleu= aleurispores, + = good production the above test obviously showed good stability of the drying monascus cultures after ten years storage at room temperature, although from the growth tests in 20% ethanol only 55% were able to grow well (table 6). figure 2 shows 5 monascus strains growing well in 20% ethanol medium. this study clearly showed that all drying monascus cultures over 10 years could recover well and still retained its production of ascomata, ascospores and aleurispores, but not all monascus tested including one m. purpureus strain showed its capability to grow in liquid medium containing ethanol at high concentration. 83 recovery of more than 10 years-drying monascus culture – nandang suharna. good recovery of monascus after 10 years storage in dry condition is in fact caused by the presence of ascospores produced by monascus. these ascospores can germinate rapidly after one-day incubation period (suharna 1999). furthermore, these typical spores are abundantly produced (hawksworth and pitt. 1983; suharna 1999). without these spores, particularly m. purpureus, the fungus cannot survive in dry condition for years. we ever came across with failure of one drying collection of m. purpureus to grow after incubation for 5 years because of lacking of ascospores. this fact showed that the presence of ascospores is very vital for monascus survival in dry condition for long time. the result showed that all drying monascus cultures for 10 years could recover well with 100% viability. however, without prior treatment by soaking in water for three hours before cultivation these recoveries would not succeed. it is known that monascus is commonly found in substrate with low water activity. the monascus collection tested here originated from dry substrate. twenty monascus isolates were isolated from deteriorated ethanol. this indicated that the 20 isolates were typical fungi with their survival capability in dry condition. this result indicated important information at least an alternative for the maintenance of monascus not only cheap and simple but also long-term storage (10 years) in particular. therefore, it is recommended to use this method for the maintenance of m. purpureus or m. rubber in fermentation industry. it is well known that one of the very simple methods to preserve mold is to use agar medium which comprises serial sub-culturing from poor medium to rich medium. figure 2. five monascus strains show its growth in liquid medium containing ethanol 20% after incubated for two weeks at room temperature. the growth indicated that the five monascus strains still have the capability to grow in medium containing alcohol at very high concentration. 84 biotropia vol. 15 no. 1, 2008 the use of medium depended on the fungal strain (smith 1993; smith and onion 1994). these methods need routine maintenance because the fungal cultures could only be stored for a short-term period before subculture. this storage period ought to be taken into account in maintaining fungal cultures to avoid late handling causing the death of the cultures. several molds are known having its storage period between 2 and 4 weeks before sub-culturing. whereas, the majority of fungi have the storage period between 2 and 4 months before re-cultured and others can be maintained until 12 months (smith 1993; smith and onion 1994). therefore, preservation method using agar causes routine maintenance and take much time. during storage cultures kept on agar slope usually underwent drying because of evaporation. the nutrient content of the medium is already low and the fungal growth of cultures is in late phase (linear phase). in this condition most microbes including bacteria and fungi usually produce resistant spores. these spores are engineered for survival of its life. it is suggested that this kind of spores can survive for tens or hundreds or may be thousands years under dry condition. drying cultures can also be obtained by letting cultures kept on agar medium to dry in room temperatures. monascus purpureus cultures kept on slope agar will dry quickly several weeks at room temperature. during its growth this fungus produces ascospores masses abundantly. this typical spore has a great function for survival of its life. this spore can survive from agony condition such as very low nutrient or drought of water. however, it is of interest using more monascus species in this study to know recovery capability of the monascus originated from wet habitat such as m. sanguneus and m. pallens from dry condition compared to the other monascus species which originated from dry habitat such as m. purpureus and m. rubber. monascus sanguneus and m. pallens were firstly described by cannon et al. (1995). these 2 species were isolated from the surface sediment of a river in iraq and showed a non-osmophilic nature (cannon et al. (1995). most monascus species are showing osmophilic affinity (pitt & hocking 1997). conclusions ten-year drying cultures of 20 monascus strains were well recovered (the viability is 100%) with good retained characters such as growth, pigmentation and production of ascomata, ascospores and aleurispores. the capability to grow in alcohol at high concentration was shown by 60% of monascus strains. the purification method from fungal contamination by soaking in ethanol 70% or 95% was successfully effective. the study on monascus species showed long survival in dried agar medium, while there was no change in their cultural and morphological characteristics. 85 recovery of more than 10 years-drying monascus culture – nandang suharna. references cannon, p.f., s.k. abdullah, and b.a. abbas. 1995. two new species of monascus from iraq, with a key to known species of the genus. mycol. res., 99(6): 659 662 endo, a. 1979. monacolin k; a new hypocholererolemic agent produced by a monascus species. j. antibiotic, 32(8), 852-854) hawksworth, d.l. and j.i. pitt. 1983. a new taxonomy for monascus species based on cultural and microscopical characters. aust. j. bot., 31:51 61 nakasone kk, sw. peterson, and s jong, 2004 preservation and distribution of fungal cultures in biodiversity of fungi: inventory and monitoring methods.. elsevier academic press, amsterdam. p: 37-47 pitt ji, hocking ad, 1997. fungi and food spoilage. 2nd ed. balnckie academic & professional, london, united kingdom saono s., i. gandjar, t. basuki, and h. karsono. 1969. mycoflora of ragi and some other traditional fermented food of indonesia annales bogorienses 5, part iv : 187 204 smith d. 1993. notes on the preservation of fungi for small culture collections. international mycological institute. egham, surrey, united kingdom. 36 p. smith d. and a.h.s. onions. 1994. the preservation and maintenance of living fungi. imi technical handbooks no.2. international mycological institute. egham, surrey, united kingdom. 120 p. steinkraus, k.h. 1983. handbook of indigenous fermented foods. marcel dekker, inc. new york : 547 553 stchigel am, jf canon, sk abdullah and j guarro. 2004. new and interesting species of monascus from soil, with a key to the known species. studies in mycology 50: 299-306 suharna n. 1999. pengaruh perendaman di dalam air sebelum pemindahan terhadap pemulihan biak-biak monascus spp. yang mengering. jurnal mikrobiologi tropika, 74 80 watanabe, t. 2004. personal communication. 6. iwan saskiawan page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia (2) 1988/1989: 8-11 critical period of mungbean (phaseolus radiatus l.) to weed competition i.h. utomo* tropical agricultural pest biology program, biotrop, bogor, indonesia abstract a field experiment was conducted to study the critical period of weed control on the crop of mungbean (phaseolus radiatus l.). the studies were done in the field of biotrop experimental station with the natural existing weed population. it was found that the critical period of mungbean to weed competition was from 3-6 weeks after planting. introduction competition constitutes the major noxious aspect of weeds in crop cultivation. since weeds and crops largerly use the same resources for their growth, they will compete when these resources are limited (zimdahl 1980). part of these resources is continuously available in limited amounts, for instance light, or in some extent, nutrient that becomes available by mineralization. part of these resources, however, is only available as a limited stock at a certain period during crop development. environmental factors determine competition by changing space conditions and by affecting the phenotypes of the crops and the weed concerned. when weeds emerge in a newly establishing crop, the amount of resources already utilized by the weeds or crop before competition starts is determined by their seed weight, time of emergence, relative growth rate and population density. a newly emerged crop seedling relying for food stored in the seed would experience little competition from weed seedlings at a similar growth stage, if only because at that time total demands on water and minerals are small. likewise a crop towards the end of its growth cycle, especially if it is senescing and ripening seed will often be unharmed by the presence of weeds. between these situations, there would be a period when crops are most susceptible and weed competition would be more severe. this period is known as the critical period for weed competition. this experiment was aimed at studying the critical period of an annual crop, mungbean (phaseolus radiatus l.) as basic information on when to control the weeds in such mungbean cultivation. * present address: department of agronomy, faculty of agriculture, bogor agricultural university, bogor, indonesia. 8 critical period of mungbean to weed competition-1.h. utomo materials and method the experiment was conducted at biotrop field experiment, tajur, bogor from july september 1985. the seed of mungbean (var. pr 74) was planted after land preparation in 30 plots ( 3 x 4 m 2 ), fertilized with 50 kg urea/ha, 100 kg tsp/ha and kcl 50 kg/ha. the experiment was arranged in a randomized block design with 3 replications. the treatments of the experiment were as follows (figure 1) figure 1. the treatment of the experiment the serial of periods consisting of weed-free and weedy conditions were introduced at 1, 2, 4, 6 and 8 weeks after planting. the growth and yield performance parameters of mungbean were recorded as follows: (1) biomass at 2, 4, 6 and 8 weeks after planting (2) plant height at 2, 4, 6 and 8 weeks after planting (3) yield of mungbean, expressed as dry weight of seed (g/m 2 ). 9 biotropia no. 2, 1988/1989 results and discussion the highest yield of mungbean was obtained with treatment g (weed present only 1 wap) which was not significantly different with treatment e which was weed-free during the entire mungbean growth period. weed competition which commenced during the first week was not considered severe (table 1). table 1. yield of mungbean. treatment g/m 2 *) a 63.75 de b 75.84 cde c 106.74 ab d 95.60 bc e 112.11 ab f 51.16e o 125.84 a h 88.42 bcd i 98.77 bc j 61.58e *) means followed by the same letter are not significantly different at p≤0.05. data in table 1 show that mungbean yield decreased from 112.11 to 51.16 g/m 2 when weeds were allowed to grow unchecked throughout the growing period. the competition effect of weed was evident at each stage of the growing period and the longer weed was present in the cultivation, the lower yield of mungbean. the same results were also observed in relation to the duration of weeding application, i.e. the longer the duration of weeding application, the higher the yield of crop. the highest yield (g) was not significantly different from treatments c, d, e (for respective weed-free treatments) and h, i (for respective weedy treatments). the grain yield of mungbean therefore started to decrease if weeds occurred in crop cultivation before 6 weeks after planting. thus, 4 weeks after planting is the indicated time for weeding so that the crop will produce yield comparable to plots free of weed throughout the growing period. the period of weeding time seemed not to influence the other measurements like biomass (except at 6 wap) and plant height of mungbean (table 2). 10 critical period of mungbean to weed competition-1.h. utomo table 2. plant height and biomass of mungbean. references nieto, j.h., m.a. brondo and j.t. gonzales. 1968. critical periods of the growth cycle for competition from weeds. pans (c) 14: 159-166. zimdahl, l. 1979. crop-weeds competition. international plant protection centre. 11 8.pdf 9.pdf 10.pdf 11.pdf biotropia book final.indd 95 compatibility studies of interspecific in vitro micrografting of agarwood (aquilaria malaccensis lamk.) nurita toruan-mathius*, jonner situmorang, dewi rachmawati & anida biotechnology and plant breeding, seameo biotrop, bogor, indonesia abstract aquilaria spp produced agarwood as nonwood forest production, and has high economic value. a. malaccensis is susceptible to white rot diseases and termites. on the other hand most of the community plantations are a mixed culture with rubber trees, oil palms and with high risk of contamination causing white root diseases. besides that, vegetative propagation by cuttings, stumping or air layering are still very diffi cult with low percentage of growth. th e objectives of this research were to analyze the best suitable micrograft type, changes of sds-page protein band patterns of compatible and incompatible micrografts with several combinations of gaharu planlets in in vitro condition, and histology of union area between rootstocks and scion. th e results showed that wedge or v type was the best of the micrografs. ms medium with the addition of 3 mg/l iba was the best medium for gaharu planlet growth after micrografting. acclimatization was conducted in husk chacoal and top soil (1:1) medium and grown under plastic house of 70% shading with paranet. compatible combination (ac/am) of micrografting showed that anatomy structure of union area is the same as anatomy structure of non micrograftd planlet. while incompatible (gv/am) micrografting produced necrotic layer growth from pith and parenchymateous tissues of the wood in union area along the middle of radial shoot. recovery period of union area between stocks and scion is initiated by callus formation from the pith and parenchymatous tissues of the wood. callus will diff erentiate into mature cells or tissue and become combined phloem and xylem vessels between rootstocks and scion. sds-page protein band pattern on compatible combination was the same as plants originated from seedlings. while, incompatible combination produced new protein bands with molecular weight around 21 and 30 kd. key words : agarwood, aquilaria spp, micrografting in vitro, incompatible micrografting, sdspage protein, incompatible histology. introduction a. malaccensis, is a famous species for production of good-quality agarwood. agarwood is an inducible secondary metabolite caused by interaction between defence materials like phytoalexin of gaharu trees against compatible pathogen of cells in woody biotropia vol. 15 no. 2, 2008 : 95 109 *corresponding author: nurita-toruan@smart-tbk.com 96 tissues (van der plank 1978; kunoh 1990; rahman & basak 1980; parman et al. 1996). th is secondary metabolite resin is not secreted from trees like other resins or gum, but accumulated in infected stems or branches. th is phenomenon causes the white and smooth part of wood turns to be dark and hard. th is wood becomes heavier and fragrant if it is burned (hou 1960; anonim 2002; subansenee et al. 1985). gaharu is of high economic importance in asia due to its use for the production of incense, perfumes and traditional medicines. a. malaccensis is recognized on the iucn red list as critically endangered and considered to be at risk from overexploitation. all species of aquilaria were included on the appendix ii list of the convention on international trade in endangered species of wild fauna and flora (cites) in 1994 (cites 2003) to improve control of commercial gaharu trade in all participating countries. to prevent a. malaccensis to become an endangered species, indonesia has compiled a national program for sustainable agarwood production by planting of this species in several provinces through government project and community participation (suparman 2006). th ese programs faced problems because a. malaccensis is susceptible to white root diseases and termites. on the other hand, most of the community plantations are a mixed culture with rubber trees, oil palms and with high risk of soil contamination causing white rot diseases; a. malaccensis is a relatively slow-growing plant compared with other aquilaria species; recalcitrant seed with low viability, hand–sown seeds have germination rate (67% for those planted immediately, decreasing to 47% for those sown after 1 week). to solve these problems in vitro micrografting between a. malaccensis as a scion and other species of aquilaria or gyrinops as a rootstock, or interspecifi c micrografting could be used for propagation. toruan-mathius et al. (2006 b) reported that seameo biotrop has succeeded to develop tissue culture technique for propagation of a. malaccensis, a. crassna, a. becariana and gyrinosp verstigii from selected mother plants, acclimatized and planted under fi eld conditions. th ese techniques are used as a basis for in vitro micrografting. in vitro micrografting : shoot-tip grafting in vitro (stg) consists of grafting, under aseptic conditions, a small shoot tip onto a young seedling or planlet root stocks growing in vitro. th e technique has the following steps: rootstock preparation, scion preparation, grafting procedure, growing grafted plants in vitro, and transferring to soil. possibility of micrograft less diff erentiates shoot tip tissue may also help in reducing compatibility problems between scion and stock (jonart 1986). several reports have described the application of in vitro and in vivo micrografting that may provide several advantages such as rejuvenation of mature tissues, year round plant production, enhanced compatibility studies and correlative relations between root stocks and scions, make specifi c genotypic combinations to increase productivity, and extend ecological limits of a particular plant species or cultivar to tolerate edaphic conditions (richardson et al. 1996; hartmann et al. 1997; estrada-luna et al. 2002; sobhana et al. 2001; sanjaya et al. 2006). toruan-mathius et al. (2006a) have succeeded to use in vitro and in vivo interspecifi c micrografting of cinchona ledgeriana as a scion onto c. succirbura as a rootstock to avoid white root disease. under fi eld condition, in vitro micrografting grows faster than that of in vivo grafting. da costa et al. (1991) observed that production of plants grafted on robusta coff ee was three to four times higher than that of nongrafted plants. in addition it is biotropia vol. 15 no. 2, 2008 97 also used to increase resistance to soilborne parasites or soil-borne diseases caused by pathogens such as fusarium oxysporum. grafting is also commonly used for fruit trees and in silviculture (i) to improve tree adaptation to unfavorable soil or climatic conditions, (ii) to enhance water and nutrient uptake, (iii) to increase plant vigour and extend duration of economic harvest time, and (iv) to shorten the breeding period by limiting the breeding objective for resistance to soil-borne diseases and nematodes in root stock. th ese fi ndings suggest that the existence of this technique is an eff ective means of rapid and true-to-type multiplication of desired a. malaccensis genotypes. th e objectives of this study were: (i) to determine the appropriate and reliable micrografting techniques for agarwood planlets; (ii) to determine survival, growth, rootstock suckering, and short-term compatibility of a. malaccensis scion on several species of rootstocks under in vitro conditions, and (iii) to characterize graft incompatibility based on histological and biochemical aspects of grafting. materials and methods th e study consisted of rootstock preparation, scion preparation, (a) in vitro micrografting, (b) analysis of compatibility by histology and (c) electrophoresis sds page protein of stem from scion. plant materials a. malaccensis b23 line used as scions were micropropagated following techniques established by situmorang (2000). axillary buds excised from young planlets (3-5 cm long) were initially used as explants and induced to develop shoots using murashige and skoog (1962) basal salt formulation medium adjusted at ph 5.7 and supplemented with 5.0% sucrose and 0.6% bacto agar. ba (6-benzyl aminopurine) of 1.0 mg/l was added for shoot induction and proliferation cultures. adventitious roots developed in ms medium with the addition of 3 mg/l iba (indole butyric acids). planlets of vigorous a. crassna, a. fi laria and girynops verstigii as stocks were propagated by tissue culture the same as for planlet scions. all cultures were grown in a culture room with light of 100 μmol m-2 s -1 photosynthetic photon fl ux density (ppfd) cool white fl uorescent lamps at planlet level and photoperiod of 16 h. temperature was maintained at 27 ± 2o c. micrografting under aseptic conditions, shoots of uniform length and diameter were selected from in vitro culture and used as rootstocks and scions in micrografting. stainless silver blades mounted on a handle were used for cutting the plant material. freshly picked shoot tips and in vitro regenerated shoot tips were used as microscions. to prepare the initial scions of the micrografts, the leaves from those portions were eliminated, and four types of grafting were made in each of their basal parts. for micrografting, the following method was used. when the stem axis of the plantlets reached a length of about 2 cm the upper 1.0 cm of the tips were excised and used as scion, while remaining portion was used as rootstock. in the fi rst experiment four types of grafting viz., a) wedge (v), (b) horizontal cut grafting, (c) slant-cut grafting (/) interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 98 and (d) cleft grafting (i) (fig.1) were made in the rootstock and scion. th e scions were placed in the incisions made in the rootstock with both the cut surfaces in good physical contact (fig. 1). th e stock and scion were held together at the point of graft with sterile (1x1 cm) light aluminum foil. for optimizing the method of micrografting in vitro plant materials and as scion only b 23 and a. crassna as a rootstock were used. on the other hand, for compatibility studies three clones of a. malaccensis as scion and three species as rootstocks were used, with the best method of micrografting, and as a control the same species of scion and rootstocks were used. th e percentage of successful and unsuccessful grafts was determined 30 days after grafting and in vitro culture. all planlets of graft treatments were cultured in ms medium + 3 mg/l iba for rooting. after three months the growth of scion as a result of graft union was observed. for incompatibilities studies, the combination of scion-stock i.e. a. malaccensis as scion and a. fi larial, a. crassna, gyrinops verstigii as stocks could be used, while as control the same species of scions and rootstocks were used. cultures were incubated in light culture room (16 hrs/days) with rh 70-80%. a b c d (i) (ii) (iii) (iv) biotropia vol. 15 no. 2, 2008 figure 1. in vitro micrografting between scion of a. malaccensis planlets with rootstocks of several species of aquilaria sp and g. verstigii, using (i) wedge, (ii) horizontal, (iii) slant, and (iv) cleft grafts. a. micropropagated shoots, b. rootstock, c. scion, and d. methods of grafts. 99 growth analysis th e in vitro micrografting success rate was determined 6 weeks after grafting by recording the number of scions still alive out of all individual treatments. number of leaves were also recorded and measured for each sample, 2 months after the date of micrografting, and recorded continuously for every month during 8 months observations. at the end of the experiment, all plants were carefully removed to study their root systems. for fi ve plants from each graft combination and the control, the stem was cut crosswise for macroscopic observation of graft union or analysis of compatibility. experimental design combination of stock-scion viz.: a.malaccensis/a.malaccensis (control 1), a. crassna/ a. crassna (control 2) , a. fi laria/ a. fi laria (control 3); a. malaccensis/ a. crassna; a. malaccensis/ a. fi laria was used. experiments used completely randomized design of 1x3 units with 15 replicates. analysis of compatibility analysis of compatibility between scion and rootstocks studies by histology and electrophoresis sds page protein of graft union, were conducted for in vitro micrografting. histology of graft a histological technique was used to observe the graft union of every combination of root stock/scion. th e graft union was cut into segment of 2 cm long and microtome was used to cut 25 μm slices from the segments. th e samples were fi xed for 24 h in solution of faa and then dehydrated in an ethanol series (50-70-80-90-95-100-100%) for 30 min. each. safranin (2%) was added in 70% ethanol and fast green (1%) in 100% ethanol for 15 seconds. th e slices were placed in a solution of 1 absolute ethanol: 1 xylene for 30 seconds and then in pure xylene (2x for 30 min. each), before being examined under the microscope (esau 1965). electrophoresis sds-page protein total protein was extracted from scion shoot (2 cm above graft line) by frozing in liquid nitrogen and stored at -40o c. plant tissues were homogenized in a mortar in the presence of liquid nitrogen and extracted using buff er containing 5 mm tris hcl, ph 8, 500 mm nacl, 2 mm ascorbic acid, 0.5 mm phenylmethylsulphonyl fl uoride and 1 mm dithiotreitol (laemmli 1970). th e homogenate was heated at 80o c for 10 min. and centrifuged at 17.000 g. supernatant were thawed and heat stable proteins were quantifi ed by the binding-dye assay (bradford 1976). protein was separated by 10-15% polyacrilamide gradient gels. heat stable proteins 5-15μg were loaded in each well with discontinuous buff er systems (laemmli 1970) using a mini protean 3-electrophoresis cell (bio-rad, hercules, ca, usa). gel lanes were loaded with equal amounts of perceptible counts; low molecular weight protein standards (bio-rad) were run as size markers. electrophoresis run with constant current of 75 ma was applied per gel. during electrophoresis the voltage increased from about 300 to 600 v. a 0.025 m tris-glycine buff er (ph 8.6) was used as the electrode buff er. when the tracking dye had migrated to the bottom of gel (after approximately 3.5 hrs) electrophoresis was stopped. gels were interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 100 stained with commasie brilliant blue. after staining gels were fi xed in solution containing methanol, glacial acetic acid and distilled water with the ratio of 4:1;15 (v/v/v) for 15 min. and photographed, then gels were wrapped with cellophane for documentation. th e eff ect of stock on scion was studied by the formation of new protein band or disappeared protein band compared with control (non micrografting and micrografting scion-stock with the same species). statistical analysis all data were subjected to one-way analysis of variance (anova) using sas version 6.12 (sas institute 1996). for categorical data, the normality of residuals and homoscedasticity were verifi ed to fulfi ll the requirements for anova. duncan’s multiple range test was used for mean separation. th e diff erences were considered signifi cant at p ≤ 0.05. results and discussion in vitro micrografting th e type of graft and nature of support signifi cantly infl uenced the success of micrograft. wedge or v graft gave the most successful micrografts followed by cleft graft method (tabel 1). v graft may provide a better surface for the contact of stock and scion providing good cambial joining at the graft union. it was shown that v type of micrograft also gave highly signifi cant diff erence on grafting success, except on the fi rst week of incubation because of callus formation (table 2). a good result was obtained with micrograft v type, which gave faster growth compared to cleft graft, caused by v graft which have high levels of graft union (table 2). micrografting of gaharu planlet with v graft in combination between rootstocks and scion ac/am and am/am showed the same vigour (fig. 2). (a) (b) figure 2. micrografting of gaharu planlet with v type in combination between rootstocks and scion: (a) ac/am, and (b) am/am. ac – aquilaria crassna; am – aquilaria malaccensis biotropia vol. 15 no. 2, 2008 101 table 1. effect of micrografting techniques on union and growth of scion method of grafting number of grafts grafting success (%) wedge (v graft) 25 98,5 a horizontal 20 25.6 c slant 25 28,6 c cleft grafts 25 80,7 b estrada-luna et al. (2002) reported that the best method for in vitro micrografting of prickle cactus (opuntia spp) were horizontal and wedge grafts. th e fi rm contact between rootstock and scion is extremely important at the graft junction to get fusing between them and callus formation. lukman et al. (2005) found that micrografting of two diff erent species (mundo and manggis) with v type, gave 76% succesful graft rate when the shoot tips were used directly as microscions in in vitro studies. oda (1995) said that micrografting with v type will gave a fi rm and faster union between stocks and scion. tirtawinata (2003) explained that the success rates of micrograft are highly dependent upon the cambium union of rootstocks and scion. partial contact of rootstock and scion cambium will contribute to unsuccesful joining. th ese cases are described as “translocated” graft incompatibilities (estrada–luna et al. 2002) according to hartmann et al. (1997) callus formation occurred as parenchym cells in a process of wound recovery. th ese callus will diff erentiate into cambium, xylem and phloem. th ese vessels are used for transportation of nutrition and photosyntate. kala et al. (2002) reported that in vitro micrografts rubber begin to grow weeks after micrografting. rifa’i (2003) found callus formation up to three months after micrografting. tabel 2. the effect of micrograft types on leaf number of 1-8 weekold planlets after micrografting. combination and type of micrograft on weeks .... 1 2 3 4 5 6 7 8 ac/am–v 0 0.4 a 1.56 a 2.28 a 3.00a 3.88 a 4.56a 5.08a ac/ac-l 0 0.71b 1.20b 1.58b 1.72b 2.95b 2.14b 2.23b am/am-v 0 1.27a 2.24a 3.00b 3.84 a 4.08a 4.92 a 5.48a am/gv-l 0 0.85b 1.23b 2.08b 2.84b 3.52 b 4.20b 4.84b ac/ac-v 0 1.14a 2.00 a 2.60a 3.,52a 4.,00a 4.68,a 5.32a cl/cl-l 0 0.71b 1.16b 2.00b 2.40b 3.24b 4.00b 4.48b respons tn * * * * * * * explanation ns : not significantly ; *.significantly different. numbers followed by the same letter in the same column are not significantly different according to duncan test p < 0,05 interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 102 acclimatization th e average of planlets survived after acclimatization in husk charcoal : sterile top soil (1 : 1) after one month incubation was 90% (tabel 3). gunawan (1992) reported that planlet from in vitro culture i.e. has wax layer on undeveloped cuticula, limited shoot lignifi cation, number of leaves palisade cells very low, undeveloped xylem, disfunction of stomata caused high transpiration it caused the scion become sensitive to evapotranspiration, attack of fungi and soil bacteria, and also to high light intensity. th at is why planlets should be acclimatized before transfering into the fi eld. table 3. survival percentage of gaharu planlet after acclimatization two months grown in plastic house. combination and graft type % of survived planlet ac/am -v 92 b ac/ac -v 90 b am/am -v 90 b ac/am -cleft 60 a ac/ac -cleft 66 a am/am -cleft 65 a explanation : numbers followed by the same letter in the same column are not significantly different according to duncan test p < 0.05 histology of micrografts th e results showed that anatomy structure of gaharu shoot is the same as anatomy structure of plantlet shoot of another dicotyledone plant which consisted of phloem, cambium and xylem. cambium was radially in between of phloem and xylem vessels. th e inner part of cambium develops into xylem vessels which function as a transportation of water and nutrition from roots to upper parts. outside the cambium is phloem which functions as a transportation of photosyntate to ground parts of plant. our observation revealed fi ve development stages during graft union formation: development of necrotic layer, proliferation of callus bridge at the graft interface, diff erentiation of new vascular cambium, restoration of new vascular tissue, and restoration of the continuity of the epidermis at the graft union. unions between parenchymatous tissues have been confi rmed 2-3 weeks after grafting. fifteen days after grafting parenchyma unions occur in all micrografts that have prospects of further development. only cells newly formed after grafting are able to unite. th e fi rst unions occur between cells originating from tissues outside the cambial region. in side-slit grafts with radial incision in the bark of the rootstocks, the fi rst union developed between callus from the rootstock cambial region in the barks fl ap, and callus from cortex, phloem rays and pith of the scion. trial incision on the rootstock unions also occurs at the incision face in a way similar to that of veneer side grafts. union of newly formed cells may develop independently of the kind of originial tissues (fig. 3a & 3b). biotropia vol. 15 no. 2, 2008 103 figure 3. histology union area of compatible and incompatible micrograft (a, b & c) radial cross section of stem at union area. (a). non micrografted stem, (b) union area of compatible micrograft, (c) union area of incompatible micrograft (d & e) longitudinal cross section of stem union area. (d) union area of compatible micrograft, (e) union area of incompatible micrograft. (a). 1cambium, 2pericycle, 3cortex (e) 1necrotic cells ; 2callus; — bars (40 μm) histological investigations concluded that callus bridging between scion and the rootstock was completed three weeks after micrografting and that both partners took part in the development of the callus. diff erentiating xylem elements within the callus bridge were observed by the fourth week but no bridging of the graft union was achieved at this stage. noticeably, each partner established new vascular system outside the callus bridge, directed towards the others. hartmann et al. (1997) explained that callus is a group of parenchym cells which has developed around wounded tissues. growth of parenchym cells is a initial phase of callus formation in union area of stocks and scion. parenchym cell has high ability to divide and develop into mature cells. cell development and anatomy of planlet will change, and its function of each tissue/cells one to another is also diff erent. undiff erentiated tissues have diff erent functions compared to diff erentiated tissues. callus will diff erentiate into mature cells or tissue and become a combined tissue between rootstocks and scion. callus growth was found from the combination of gv/am and gv/amc (fig. 3 b, c & d) but diff erent with the seedling or am/am (fig. 3). it was shown that the rootstock aff ected the growth of the scion, changes the morphology and interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 104 anatomical structure of the scion. in contrast, the scions have little eff ect on rootstock growth. mismatch of phloem fi ber bundles has an important eff ect on graft success (fig 3e). on the whole, the rootstocks produce a large volume of callus before union than the scions but leaf traces adjacent to cut surfaces of the scions cause locally larger formation of callus. rays that have been cut far from the phloem usually form more callus than those which have been cut close to the cambium. th e boundary between phloem and cortex is a very active zone of growth. th e cambial region plays a subordinate role as a callus producer in veneer side grafts in the rootsocks of v graft. however, the loose bark from the wood in the cambial region and the cells produce vigorous callus formation (erea et al. 2001). th is process is initiated by ray cells, but soon the major portion of the cambial cells participate if they are undamaged by grafting. callus formation from the pith and from parenchymatous tissues of the wood occurs only in union area . th e pith of the scion is particularly active when cut. since the cambium rootstocks had already entered an active stage at grafting and the rootstocks have water and nutrients supply higher than that of the scion, its cambium will be able to deposit several new rows of tracheids up at the time when union is possible. th is means that the cambium is forced to move from scion cambium which has been placed in front, but which is catching another cambium that has been placed outside (fig 3). th e fi rst unions of vascular tissues in veneer side grafts are often found between the stock fl ap and tissues situated on the short cut surface of the scion leaf traces which appear to play an important role in the establishment of vascular connections. th e cambial unions in the innermost corner of side slit grafts become complex depending on the mutual position of the tissues of the grafts components. it is not necessary for the space between the wood surfaces of the graft components to be fi lled with callus tissues, above the uppermost point of contact with the scion. th us the wound caused is callused over from all sides in the same manner as in cut branch. rifa’i (2003) found that callus formation at union area still continues up to three months after micrograft. tirtawinata (2003) reported diff erentiation process of new cambium occurred after three months of micrografting. after fourty days to 6 months of union area will make apple micrografts become fi rm (richardson 1996). estrada-luna et al. (2002) explained that compatible micrograft growth of scion planlet is very vigourous, while the incompatible one is dwarfi sh. in walnut (junglans regia) union of vascular tissues have been confi rmed after about three weeks in wellmatched grafts. when the matching has not been well done, union may require 5-6 weeks. in well-matched grafts it is common that divisions in the cambial region are able to serve in the union of vascular tissues almost at once. only a small number of short, irregular cells are then formed. when the cambial region is located further apart, they spread through intermediate parenchyma tissues toward each other by joining from one cell to the next. heteroplastic or interspecifi c micrografting is used in fruit tree breeding to produce early and abundant fl owering small trees. th e same eff ect also occurs when grafting forest trees for seed orchards. in order to avoid nematode damage to roots of coff ea arabica l. in latin america, a common practice is to apply interspecifi c grafting on c. canephora var. biotropia vol. 15 no. 2, 2008 105 robusta (pierre) rootstocks. villain et al. (1996) observed that with heavy pratylenchus sp, infestations, production of arabica plants grafted on a c. canephora var. robusta (2n=2x=44) rootstock was four times higher compared to nongrafted plants. electrophoresis sds page protein th e results showed that leaf protein of scion tested has molecular weight of 14 45 kd. generally sds-page protein band patterns of scion are the same with planlet control or non micrografting, except from am/amc and am/gv. two of the small protein molecules, 21 and 30 kd were found only in these combinations (fig. 4). th ere are several basic strategies for identifying stocks/scion interactions at the biochemical level. 2 0 0 1 1 6 9 7 2 0 0 1 1 6 9 7 k k figure 4. electrophoregram leaf protein of scion as a result of micrografting in vitro. m. marker; 1. am; 2. amc; 3. gv; 4. am/am; 5. amc/amc; 6. gv/gv; .am/amc; 8. am/gv; 9. amc/ gv th e most important step in identifying a biochemical basis for stocks/scion interaction is verifying that there is causedandeff ectrelationship between the present particular proteins. new protein formation in incompatible rootstocks/scion may be regulated by proteins because these molecules constitute the machinary of cell metabolism. when examining a development change of varietal diff erence in a metabolic process, the cause of the change or diff erence is most commonly a change in the presence, stability, or activity of particular proteins. ji-zhong et al. (2002) reported that incompatible micrografting of apple caused an increase in pod and iod enzymes activities. th ese enzymes controlled auxin translocation from root to upper parts of plant. holbrook et al. (2002) found that on compatible micrografts there was a positive relationship between rootstocks and scion. th ese conditions have been correlated with nutritions distributions, translocation of water and nutrition, and regulation of hormon transportation. raghothama (1999) explained that in interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. m 1 2 3 4 5 6 106 compatible micrograft of arabidopsis the relationship of source and sink between stocks and scion is normal. incompatible micrografts of peach and plum planlet aff ected protein and amino acids content of the whole tissues. bertrand and etienne (2001) said that the synthesis of a conserved set of proteins, heat shock proteins, in response to water stress caused inhibition of translocation of nutrient and water. toruan-mathius et al. (1999) found that variations of protein band patterns in union area of micrograft rubber plant are caused by diff erent interactions between micrograft combinations. erea et al. (2001) explained that micrograft can be used as early detection of compatible and incompatible combination of rootstocks and scion in plants propagated by micrografts. it was clearly distinguishable that protein banding patterns signifi cantly have a relationship with the success or failure of grafting. in vitro micrografts usually fail due to incompatibility between stock and scion, oxidative browning of cut surfaces, poor contact or poor development of the root system (toruan-mathius et al. 2006a; moor 1991). sobhana et al. (2001) studied about physiological and biochemical aspects of stock-scion interaction in hevea brasiliensis and found that assimilation rate of scion is being infl uenced by the rootstock. th e considerable cv observed between the individual plants within clone in total soluble sugars, reducing sugars, phenol and amino acid contents, also indicated the existence of stock-scion interaction. toruan-mathius et al. (2006a) reported that incompatibility in interspecifi c micrografting between cinchona ledgeriana and c. succirubra showed formation of 21 and 30 kd protein and formation of stone cells in a union area. conclusions wedge or v graft was the best micrograft method for gaharu planlets, cultured in ms medium with the addition of 3 mg/l iba, acclimatized in medium of husk chacoal : top soil (1:1) and incubated in plastic house. histology structure of compatible micrografting showed that shoot union area was the same as structure anatomy of seedlings, while histology structure of incompatible micrografting has a union area and necrotic layers along radial parts of shoot. union of vascular tissues have been confi rmed after about three weeks in wellmatched grafts, and able to serve in the union of vascular tissues almost at once. only a small number of short, irregular cells are then formed. compatible combination (ac/am) of micrografting showed that histology structure of union area is the same as histology structure of nonmicrografted planlet. while incompatible (gv/am) micrografting produced necrotic layer growth from pith and parenchymateous tissues of the wood in union area along the middle of radial shoot. protein pattern in compatible combination is the same as in seedlings. while protein pattern of incompatible combination produced a new protein band with molecular weight around 21 and 30 kd. biotropia vol. 15 no. 2, 2008 107 references anonim. 2002. cultivating the world’s most expensive incense. enews 25 july 2002: 2-3. http://www.umn. edu/systemwids/enews /072502.html. 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[disertasi] bogor : program doktor institut pertanian bogor. biotropia vol. 15 no. 2, 2008 109 toruan-mathius n., sa adimihadja and i boerhendhy. 1999. rootstock-scion interaction in havea : bark protein patterns and anatomy in correlation with genetic similarities. menara perkebunan 67(l): 1-2. toruan-mathius n., lukman, agus-purwito. 2006a. in vitro micrografting technique of chincona succirubra and c. ledgeriana. menara perkebunan 74:1-10. toruan-mathius n., j situmorang, yupi-isnaini and dewi-rachmawati . 2006b. database and the diversity of aquilaria spp, gyrinops spp and fungi associated with agarwood in indonesia. laporan hasil penelitian, dipa, seameo biotrop 2006. villain l., jl. sarah, b decazy, a molina and s sierra. 1996. evaluation of grafting on coff ea canephora var. robusta, and chemical treatment for control of pratylenchus sp in c. arabica cropping systems, p.407-422. in: proc. 3rd intl. nematol. congr. gosier, guadeloupe, 7-12 july, 1996. van der plank je. 1978. genetic and molecular basis of plant pathogenesis. springer. verlag, berlin heidelberg. new york. interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. biotropia 1 (1) 1987: 53-57 iii. mycorrhizae in agroforestry: a case-study s.t. nuhamara* tropical forest biology program, biotrop, bogor, indonesia keywords: agroforestry, mycorrhiza, resin, shorea javanica, sumatra abstract census of mycorrhizae in shorea javanica agroforests has been made periodically in the district of krui, lampung, sumatra. amanita hemibapha (amanitaceae), cantharellus cibarius (cantharella-ceae), lactarius spp., russula spp. (russulaceae) and scleroderma sp. (sclerodermataceae) were commonly encountered on the agroforest floor. these mycorrhizal fungi are naturally associated with the planted trees. the significance of mycorrhizae for the maximization of growth and sustained productivity of resin is discussed as well as the need to design well defined agroforestry systems to facilitate growth and to improve production management techniques. introduction torquebiau (1984) reported for the first time that the "kebun damar" or "resin garden" of shorea javanica k & v (dipterocarpaceae) in krui (sumatra) was a typically good example of an agroforestry system for resin production. to obtain the maximum sustained yield of resin, all the growth factors should be optimally maintained. ideally, a tree is expected to perform active and continuous chemo-ecophysiological processes for long possible periods of time. mycorrhizae play an important role at root level by dynamizing the nutrients and water uptakes. in the case of an agroforestry system, two groups of mycorrhizae can be considered: those which are directly associated with the main crop and those which are not, and/or are associated with the other component species of the system. in this paper, the author presents information related to the first group. nevertheless, new ideas proposed about the second group and the specific case of mycorrhizae in agroforestry especially the inter-relationships between the above-ground organs, e.g. leaves and the mycorrhizal roots of the system, is discussed. material and methods census of the occurrence of fruiting bodies of mycorrhizal fungi having connections with the roots of s. javanica has been made periodically at krui, * present address: faculty of forestry, bogor agricultural university, darmaga campus, bogor, indonesia. 53 biotropia vol. 1 no. 1, july-december 1987 lampung, sumatra in june and october 1985 and february of 1986. the study sites were at ngaras, gunung kemala and pahmongan, all in the surroundings of krui. soil analysis corresponding to these sites was described by sheehy skeffington in chapter 2. taxonomic identifications were made at the biotrop laboratory. this simple procedure was adopted from bakshi (1974) and based on experience with other dipterocarp species either in haurbentes, jasinga, west java or in lempake, east kalimantan. interviews with the local farmers were also conducted especially relating to seedling preparation. results and discussion based on the periodical census made so far, the following, well-known, ectomycorrhizal fungi have been recorded: amanita hemibapha amanitaceae cantharellus cibarius cantharellaceae lactarius spp. russulacea russula spp. russulaceae scleroderma sp. sclerodermataceae it was learnt from the local farmers that no artificial inoculation technique has been applied so far for mycorrhizal introduction in the agroforests. some of these mycorrhizal fungi have been artificially inoculated to shorea pinanga, s. stenoptera and hopea sangal (nuhamara et al. 1986), and developed successfully. it was also concluded that infected soil as source of inoculum performed better as compared to single mycorrhizal fungus. this suggests the need for application of infected soil to a neighboring site when a crop is to be experimented. however, on a long term basis, especially when drastic changes in agricultural practices can be foreseen, the use of pure culture will be the only advisable measure. shorea javanica is a special case because as a resin producer, the plant is expected to produce continuously. this requires optimum chemo-ecophysiological processes. to do this, both the above ground organs (e.g. leaves) and the under ground ones (roots), must function optimally. there should be a reciprocal balance of processes. it is in connection with this that the roots should be well fortified by typical mycorrhizal associations, to enable them to absorb and accumulate nitrogen, phosphorus, potassium and calcium more rapidly and for longer periods of time than non mycorrhizal ones. mycorrhizae, especially the ectomycorrhizae, appear to increase the tolerance of trees to drought, high soil temperatures, soil toxicity (organic and inorganic), and extreme low soil ph caused by high levels of sulfur or aluminium. ectomycorrhizae deter infection of feeder roots by 54 iii. mycorhizae in agroforestry: a case-study — nuhamara pathogens. hormone induced or the production of which is induced by fungal symbionts cause ectomycorrhizal roots to have a greater longevity (duration of physiological activity) than non mycorrhizal roots (marx 1973). sufficient water and other photosynthetic elements such as magnesium are essential for a proper functioning of the leaves. furthermore, according to the way carbon is fixed, plants belong to one of the c3, c4 or cam categories. figure 1. chart showing the complex ecophysiological processes in plant growth and development in relation to mycorrhiza, and possible co2 fixing ways. this implies that the different carbohydrate productions which in turn determine the different available simple carbohydrates might alter the different mycobionts. this proposed complex metabolic pattern is illustrated in fig. 1. as shown on the chart, the hormones, produced by or induced with the presence of mycorrhizae, activate the function of the rhizophere. regarding agroforestry, it is possible to manipulate the species composition and structure (michon 1983 and 55 biotropia vol. 1 no. 1, july-december 1987 torquebiau 1964) in such a way to facilitate the more stable eco-unit (oldeman 1983) which is also of good economical value for the farmers. the author suggests to construct agroforestry systems having a good combination of c3 plants like s. javanica trees and other fruit trees and c4 plants in combination with cam plants. it is believed that with such a mixed composition, a stable association can be obtained and productive compatibility achieved. another aspect of the possible manipulation of species in agroforestry concerns the relationships between host species diversity and the types of mycorrhizal association. the diagram of fig. 2 suggests that the zone where ecto and endo-mycorrhizae coexist is the best for mixed gardening or agroforestry, because the richer the mycobiont diversity, the higher the degree of productive compatibility. this practice is common in many tropical regions, e.g. java. oldeman (personal communication 1985) believed that such a model is stable and applicable to the tropical conditions. figure 2. theoretical curve showing the relationship between the host species diversity and the type of mycorrhizal association. the problem is how far it is possible to change or manipulate the symbiont composition. moving to the left of the figure would mean more endomycorrhizal plants and moving to the right a combination of ecto and endomycorrhizal plants. most of the annual herbaceous agricultural crops are endomycorrhizal, like paddy, 56 iii. mycorhizae in agroforestry: a case-study — nuhamara maize, soybean, etc, but some perennial woody agricultural crops such as coffee, cacao, oil palm are also endomycorrhizal plants. contrarily, most forest trees such as dipterocarps and pines are associated with ectomycorrhizae and there are also some important tropical forest trees such as teak and agathis which belong to endomycorrihizal plants. considering both the carbon metabolism and the mycorrhizal association of the plants, it must be possible to define the smallest agroforestry unit of good economical as well as ecological stability, and to propose it for development areas in different parts of the country. optimizing intercropping in agroforestry would mean the organizing of species composition, density and structure of both woody and herbaceous plants in order to facilitate their complementary productivity and at the same time minimizing their competitive relationships. in relation to resin production, it is suggested that further research be concentrated on tapping technique like its disturbance on the metabolic processes. the number and size of the scars affect the translocation of carbohydrates from the top down to the roots. this ultimately influences the availability of simple carbohydrates for the mycobionts. acknowledgments the author expresses his gratitude to dr. e.f. torquebiau, the french expert seconded to biotrop for his generous stimulation, inspiration as well as his invaluable criticisms on the author's ideas whithout which this paper would not be in this form. thanks are also due to dr. tri binarko suselo for his support and suggestions and to mr. koko iskandar for technical assistance. references bakshi, b.k. 1974. mycorrhiza and its role in forestry. forest research institute and colleges, dehra dun, india. 89 pp. marx, d.h. 1973. growth of ectomycorrhizal and non mycorrhizal short leaf pine seedlings in soil infested with phytopthora cinnamomi, phytopathology 63: 18-23. michon, g. 1983. village-forest-garden in west java. in: plant research and agroforestry. edited by: p.a. kuxley. international council for research in agroforestry, nairobi. 617 pp. nuhamara, s.t., s. hadi and s.s. tjitrosomo, 1986. the effect of different soil types and mycorrhizae on the growth of some dipterocarp seedlings. biotrop spec. publ. no. 26 (in press). oldeman, r.a.a. 1983. the design of ecologically sound agroforests. in: plant research and agroforestry. edited by: p.a. huxley, international council for research in agroforestry, nairobi. 617 pp. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 57 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 1. debi (endiandra).cdr biotropia vol. 19 no. 2, 2012: 59 63 endiandra kassamensis (lauraceae), a new species from new guinea debi arifiani , adi basukriadi & tatik chikmawati received 19 july 2012/accepted 29 november 2012 a new species of ( ) is described from new guinea. is described based on specimens collected over four decades ago. unlike most which grow in lowland forest, is found in high altitude forest. the species is characterized by the presence of staminodia with the absence of staminal glands.. , , staminal glands, staminodia, endemic, new guinea 1* 2 3 1 2 3 herbarium bogoriense, botani division, research center for biology-lipi jl. raya jakarta bogor km. 46, cibinong 16911, indonesia biology department, faculty of mathematics and natiural science, univesitas indonesia ui campus, depok 16424, indonesia biology department, faculty of mathematics and natiural science, bogor agricultural university bogor 16680, indonesia endiandra lauraceae endiandra kassamensis endiandra e. kassamensis endiandra lauraceae abstract introduction key words: endiandra lauraceae endiandra endiandra beilschmiedia cryptocarya potameia cryptocaryeae endiandra glauca endiandra endiandra is a medium-sized genus within the avocado family ( ) and consists of over 100 tree species. is generally known for its tree habit characterized by simple, spirally arranged and pinnately-veined leaves. according to van der werff and richter (1996), is grouped together with , and in the tribe based on the type of their inflorescences which is paniculate type ii in which the flowers of the ultimate cyme are not strictly oppposite. the flowers are bisexual with 3 stamens (rarely 2 or 6) having 2celled anthers; the ovary is superior, producing fruits in the form of drupe which are free on the receptacles. is the type species of the genus and was described from australia (brown 1810). australia houses 38 species of (hyland 1989) and rest of the species are distributed mostly in malesian regions with very few in east asia and pacific islands and extending north to southern china. new guinea is the largest island in malesian regions and a recent study on the species of revealed that * corresponding author : debyarifiani@yahoo.com 59 there are 46 species in that island with a high number of endemic species (arifiani 2012, unpubl.). the species of occurred generally in lowland forests (from 0 600 m above sea level) and the number of species decreases at higher altitude in montane forest (above 1000 m). delimiting species of requires both vegetative and floral characters evaluation. the characters evaluated include indument in all part surfaces, leaf, inflorescence, tepal, stamen, staminal glands, staminodia and fruit. detailed observation of the characters in several specimens suggested that the specimens of coode and dockrill 32655 and womersley and vandenberg 37195 represent an undescribed species. the description of was done using herbarium specimens available at herbarium bogoriense (bo) and loan specimens from singapore botanic gardens (sing). the study was carried out following methods of rifai (2011) in species enumeration. specimen characterization was done to collect morphological data by observing the characters of all specimens under the microscope (table 1). all measurements, the number and states of the characters were noted including their position, color, fragrance, texture, density and shapes. all measurements are for dried specimens or as otherwise stated. characterization method and botanical terms used endiandra endiandra endiandra kassamensis materials and methods 60 biotropia vol. 19 no. 2, 2012 plant parts characters habit height; width twig color; indument type, orientation and density terminal bud indument; shape; size leaf arrangement; texture leaf blade shape; size; apex; base; surface midrib texture lateral veins number; angle minor venation reticulation density petiole shape; indument; length inflorescence type; position; length; indument pedicel indument; length bract shape; size receptacle depth; indument tepal opening; shape; size; indument stamens number; shape; indument; size glands number; shape; indument; size staminodes number; shape; indument; size pistil shape; indument; length fruit shape; size table 1. characters observed in the taxonomic study (kostermans 1957, rohwer 1993) followed veldkamp (1987). characters observed were compared and correlated to each other in order to assign specimens to a discrete taxon. finally, description for each species was created, including information on distribution, habitat and ecology, and notes on the specific characters important for the taxon. similar to vegetatively but has more lateral veins and coarser reticulation. the flowers of bears no staminal glands but staminodes are present. type: (holo bo; iso sing), kassam pass, kainantu, eastern highlands district, png. tree up to 43 m high, 90 cm in diameter. solid, dark brown, glabrous. conical, big, with densely pubescent with short appressed hairs. alternate; petiole thin, canaliculate above, ca. 1 cm long, glabrous; blade coriaceous narrowly elliptic to elliptic, 11-14 x 3-6 cm, glabrous on both surfaces, apex acuminate, base cuneate; midrib flat to slightly impressed above, raised below, both surfaces glabrous; lateral veins diverging, 10-11 pairs, slightly raised, glabrous on both surfaces; minor venation coarsely reticulate, prominent. paniculate, bear many flowers, up to 12 cm long, terminal or axillary, with sparse short hairs; bracts caducous; pedicels slender, ca. 3 mm long, with dense curly hairs brown (fresh), erect, 2 mm in diameter; tepals thin, soft, subequal (inner ones smaller), narrowly ovate, outer ones results and discussions endiandra fulva e. kassamensis endiandra kassamensis womersley and vandenberg 37195 twigs terminal buds leaves inflorescences flowers , . 61 endiandra kassamensis et al.(lauraceae) a new species from papua new guinea – deby arifiani figure 1. arifiani. a. habit; b. intact flower; c. flower with 2 tepals removed; d. anther; e. staminode; f. pistil ( ). endiandra kassamensis womersley and vandenberg ngf 37195 1.2-1.5 x 1-1.2 mm, inner ones 1-1.2 x 0.8, with sparse long curly hairs outside, same inside especially basal part at anthers attachment; glands none; stamens 3; anthers somewhat triangular, 1 x 0.6 mm, glabrous; filament short, 0.1 mm long; locules small, roundish; staminodia 3, pentagonal, 0.4 x 0.3 mm, with curly hairs; receptacles deep, with curly hairs; ovary ovoid, 0.6-0.9 mm long, glabrous; style ca. 0.3 mm long; stigma inconspicuous. unknown distribution eastern highlands district (png). habitat & ecology rain forest, on hillside, subcanopy; alt. 1280-1372 m. specimens examined (3 sheets) coode & dockrill 32655 (bo); womersley & vandenberg 37195 (bo, sing). notes arifiani is different from other species of in new guinea because of the composition of its flowers. the species bears no glands but interestingly staminodia are present. vegetatively, is similar to teschner but bears more lateral veins and coarser reticulation. is endemic in new guinea and has a restricted distribution, i.e. in the eastern part of new guinea (papua new guinea) and was not found further west in west papua. futhermore the species grows only in a relatively high altitude forest. according to rohwer (1993), the diversity of lauraceae species are higher in lowland forests, which is in agreement with the diversity of in general except for several species of including that was only found in higher altitude forests, in the restricted area of kassam pass, kainantu subdistrict. this fact leads to be considered a rare species. detailed studies of endemic and rare species of are important for providing information for decision makers in allocating conservation efforts. is described for the first time in this study. it is a rare species and endemic to new guinea. the first author graciously thanks prof. mien a. rifai for valuable guidance and constructive suggestion during the research and to dr. sri s. tjitrosoedirdjo for valuable discussion and support during the research and preparation of the manuscript. we acknowledge the generosity of the curator of sing herbarium for the loan materials. we would like to thank the anonymous reviewer for the important comments and to mr. subari for the botanical line drawings. fruits . endiandra kassamensis endiandra e. kassamensis e. fulva endiandra kassamensis endiandra endiandra e. kassamensis e. kassamensis endiandra endiandra kassamensis conclusions acknowledgments 62 biotropia vol. 19 no. 2, 2012 63 references arifiani d. 2012. the biosystematic study of r.br. (lauraceae) in new guinea [dissertation]. depok, indonesia: universitas indonesia. 144 p. brown r. 1810. . typis richardi taylor et socii, london. hyland bpm. 1989. a revision of (excluding ). aust syst bot 2: 135-367. kostermans ajgh. 1957. . 4: 193-256. rifai ma. 2011. asas-asas sistematika biologi. herbarium bogoriense, pusat penelitian biologi-lipi, bogor. rohwer jg. 1993. . in: kubitzki k, rohwer jg, bittrich v. editors. the families and genera of vascular plants ii. berlin: springer verlag. van der werff h, richter hg. 1996. toward an improved classification of . ann mo bot gard 83: 40918. veldkamp jf. 1987. sequence of organs and terminology of characters. in: manual of herbarium taxonomy, theory and practice, de vogel e.f, editor. unesco. endiandra prodromus florae novae hollandiae et insulae van diemen lauraceae cassytha lauraceae reinwardtia lauraceae lauraceae endiandra kassamensis et al.(lauraceae) a new species from papua new guinea – deby arifiani doi: 10.11598/btb.2015.22.2.487 soil seed bank of an exotic sp. plantation acacia and an adjacent tropical heath forest in brunei darussalam adrian lee rahman suhaili , kushan u. tennakoon 1 2 and rahayu sukmaria sukri1* 1environmental and life sciences programme, faculty of science, universiti brunei darussalam, jalan tungku link, be 1410, brunei darussalam 2environmental and life sciences programme, faculty of science and institute for biodiversity & environmental research (iber), universiti brunei darussalam, jalan tungku link, be 1410, brunei darussalam received 28 april 2015/accepted 18 november 2015 abstract acacias are some of the most successful invasive plants in the tropics, causing significant negative impacts on the biodiversity and ecosystem services of invaded habitats. their successful invasiveness is partly attributed to the ability to accumulate large soil seed banks in the areas that they invade. seedling emergence and soil seed bank composition were compared under an plantation and an adjacent tropical heath (kerangas) forest in acacia mangium the andulau forest reserve, brunei darussalam. soil samples were collected from ten 20 x 20 m plots set up in three contrasting habitats: the plantation, the adjacent heath forest and the transition zone in between. soil samples acacia were subjected to smoke and heat treatments, following seedling emergence which was observed daily over a 12-week period. in a parallel investigation, variations in species richness, seed density and seed viability of the soil seed banks of the ten plots were investigated. seedling emergence was the highest in the plantation and the lowest in the heath forest plots, respectively. however, no significant differences among treatments and no significant treatment-habitat interactions were detected. species richness, seed density and seed viability in the plantation plots were significantly lower than those in the transition zone and intact heath forest plots. seeds were not recorded in the acacia mangium heath forest soil seed banks, but were detected in the plantation and transition zone plots. lower native plant species richness, seed density and viability in the plantation could imply higher regeneration potential for the a. mangium heath forest habitat if severe habitat destruction was to occur in this forest reserve. it is suggested that proper plantation management practices and close monitoring of soil seed banks are the best practices that could be adopted to minimize the gradual spread of invasive acacias into tropical heath forests of borneo. keywords: borneo, invasive alien plants, kerangas, seed extraction, seed viability, smoke treatment introduction invasive plants are a recognized as major threat towards biodiversity globally (davies & sheley 2007; simberloff 2009; corlett 2010). the genus acacia (fabaceae) is one of the most destructive invasive plants in the tropics, with 23 species listed as top invaders worldwide (richardson & rejmanek 2011). globally, the introduction of australian acacias began in the 1700s for aesthetical uses and production of woody pulp, fuel wood and timber (carruthers . 2011). the et al negative impacts of australian acacias are broad, often resulting in transformations to the biodiversity and services of invaded native ecosystems (le maitre . 2011). particularly in et al southeast asia, adverse impacts of acacia invasion, such as displacement of natives through competition, changes in ecosystem processes and allelopathy, have been reported (osunkoya . et al 2005; ismail & matali 2014; padmanaba & corlett 2014). the invasion successes of acacias are enhanced by their ability to accumulate large soil seed banks (richardson & kluge 2008). invasive acacias also alter the biotic resistance of resident * corresponding author : rahayu.sukri@ubd.edu.bn biotropia 2 140 150 vol. 22 no. , 2015: 140 mailto:rahayu.sukri@ubd.edu.bn seed bank communities, promoting their own spread and the secondary spread of other alien species (richardson & kluge 2008; gioria et al. 2012). invasive acacias reduce species richness, diversity and composition of soil seed banks of invaded ecosystems, thus changing the vertical structure of above-ground vegetation and community composition of the invaded ecosystems (gioria & osborne 2010). furthermore, -enriched soil seed banks act acacia as reservoirs that allow for the persistence of invasive behaviour (gioria . 2012). the success et al of acacias in the soil seed banks of invaded areas ar e o f t e n bo l st e r e d by an t h ro p og e ni c disturbances, such as forest fires, which alter environmental conditions of temperature, light intensity and humidity in favour of germination of these exotics (richardson & kluge 2008; le maitre 2011).et al. australian acacias ( willd., acacia mangium acacia cincinnata acacia auriculiformis f. muell. and benth.) were introduced to brunei darussalam in the 1990s to be used in timber plantations and as roadside plantings (osunkoya . 2005). in the et al twenty years following this initial introduction, a. mangium a. auriculiformis and are now regarded as invasive in brunei darussalam (osunkoya . et al 2005). their spread appears to be most intense along roadside embankments and in disturbed heath (kerangas) forests along fire-prone coastal areas (osunkoya . 2005), where trees et al acacia create almost monospecific habitats. in addition to invading these coastal heath forests, acacias also appear to invade further inland in brunei, particularly from roadsides and the original plantations into neighbouring natural habitats. tropical kerangas forests account for less than 1% of all forests in brunei darussalam and are rapidly under threat from development (wong & kamariah 1999; din . 2015; wong . 2015). et al et al plants in heath forests are well adapted to nutrient deficiencies caused by dry, nutrient-poor, acidic soils (proctor 1999; fujii 2014). kerangas soils in brunei darussalam are relatively low in nutrient levels, especially low nitrogen concentrations and low ph (moran . 2000; metali . 2015). due et al et al to its ability to fix atmospheric nitrogen, acacia effectively outcompetes native heath forest species in this nutrient-poor environment (osunkoya . 2005). as such, the invasions of et al acacias are a threat to these rare and vulnerable tropical ecosystems in borneo. a first step towards testing invasion severity is to determine whether the invasive species impact negatively upon native species in its introduced habitat (vilà . 2011). knowledge on soil seed et al bank dynamics can, therefore, help predict possible habitats susceptible to invasion according to specific attributes of invasive species (cordell 2002). the specific objective of this et al. preliminary study was to investigate the negative impacts of invasive acacias by exploring the soil seed bank composition and seedling emergence patterns of an plantation and a acacia neighbouring heath forest in the andulau forest reserve (andulau fr), brunei darussalam. the effects of different treatments with fire-related cues on seedling emergence and germination from the soil seed banks of the plantation and heath forest were also investigated. three hypotheses were proposed: 1. seed density and viability is the highest acacia in the soil seed bank of the plantation acacia and the lowest within the soil seed bank of the neighbouring heath forest; 2. in the seedling emergence study, seedling diversity of native species is the highest within soils from the heath forest and the lowest in the plantation soils;acacia 3. germination of seeds is higher in acacia response to fire-related cues (smoke and heat treatments) when compared to native heath forest species. materials and methods study sites and soil sampling a. mangiumthe study was conducted in an plantation and an adjacent heath forest in the belait district of brunei darussalam (4.4167° n, 114.5833° e). the plantation, covering a total area of 1 km , is located in an intact heath 2 (kerangas) forest within compartment 8 of the andulau forest reserve (fr). it was established by the brunei forestry department in the early 1990s as a commercial plantation for timber and harvested in 2010 for manufactured wood products (joffre ali ahmad, brunei forestry department, personal communications). since this harvest, the plantation has not been actively managed and saplings are now seen to a. mangium regenerate naturally. the adjacent heath forest is biotropia vol. 22 no. 2, 2015 141 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia also located within compartment 8 of the andulau fr and is separated from the acacia plantation by a 5 m firebreak. this firebreak comprised of layers of sand and gravel with ditches on either side of it, allowing drainage. a single transect line was established from the acacia plantation into the adjacent intact heath forest in a north to south direction. along this line, a total of ten 20 x 20 m plots were set up (fig. 1): (1) four plots were established in the heath forest (hf; plots 1–4); (2) two plots along the transition zone between the plantation and the adjoining heath forest (tz; plots 5–6) and (3) four plots in the abandoned plantation (p; plots acacia 7–10). plots within each habitat were located at 100 m from each other. within each plot, soils at five random points were sampled to a depth of 15 cm and bulked per plot for seedling emergence treatments. at three other random points in each plot, soils were separately sampled to 5 cm depth and bulked for the seed extraction study. seedling emergence for the seedling emergence experiment, soil samples were subjected to heat and smoke treatments. for the heat treatment, soil samples (n = 2 replicates per plot) were placed in an oven at 80 °c for 10 minutes (hanley & fenner 1998; read . 2000). to conduct smoke treatment, et al methods outlined by et al.dixon (1995) and read et al oil samples . (2000) were modified as follows: s (n = 2 replicates per plot) were placed in smoke a tent and exposed to smoke generated from the combustion of fresh leaf litter collected from the hf plots. soil samples were then allowed to saturate with smoke for 90 minutes at 40 °c. heator smoke-treated soil samples from each plot were separated into 500 g sub-samples and evenly spread out over sterilized potting mixture in 30 cm by 25 cm seedling trays (price . 2010). et al the potting mixture was first sterilized by autoclaving at 121 °c and 15 psi for 20 minutes (darbar & lakzian 2007). control trays containing only sterilized potting mixtures, but without any added soil samples were maintained for the duration of the experiment to monitor for contaminations during the observation period. all seedling trays (n = 60) from the three treatments were subsequently placed in a closed plant house at universiti brunei darussalam (ubd) and watered daily. mean of minimum and maximum temperatures recorded within the plant house were 26.5±0.4 °c and 31.6±0.3 °c, respectively, mean relative humidity of the plant house was 83.3±1.8% and mean of photosynthetically active radiation (par) was 449.1 µmol photons/m /s.2 seedling emergence, expressed as the number of germinating seedlings, was recorded for each tray over a period of two weeks. all emerging figure 1 schematic diagram illustrating the placement of the plots in relation to the plantation and the acacia mangium andulau forest reserve: four plots were established in the heath forest (hf; plots 1–4); two plots in the transition zone between the plantation and the adjoining heath forest (tz; plots 5–6); and four plots in the plantation acacia (p; plots 7–10). distances between plots in each habitat were 100 m. each plot was 20 by 20 m in size 100 m 10 30 m 8 9 7 450 m 100 m 100 m 30 m 5 3 1 6 4 2 acacic mangium plantation transition zone heath forest plantation edge key: line transect fire break (5 m width) 142 biotropia vol. 22 no. 2, 2015 seedlings were identified using leaf morphological characters (de vogel 1980; ng 1991). unidentified seedlings were transferred to separate pots to allow them to grow further for eventual identification using herbarium facilities. once seedling emergence had slowed or stopped, soils in the seedling trays were disturbed by stirring with a small spade to mix soil and further encourage the growth of remaining seeds (price . 2010). et al monitoring of seedling emergence in the treatment and control trays was completed after 12 weeks, at which point no seedling germination had been observed in any of the seedling trays for 2 consecutive weeks. seed density and viability to determine seed density in the seed bank, seed extraction methods described by ball and miller (1989) and price . (2010) were followed. et al subsamples of 100 g of soil were subjected to floatation, so that seeds could be extracted using a solution of 10 g sodium hexametaphosphate, 5 g sodium bicarbonate and 25 g magnesium sulphate diluted in 200 ml of water. this solution facilitated the density separation of seeds and organic matter from the soil mineral fraction. organic matter and seed fractions that remained after separation were washed with water through 150 µm and 75 µm sieves, respectively to remove remaining soil and debris. organic matter and seed fractions were oven-dried at 60 °c overnight and dried samples were placed in envelopes for storage until they were sorted and counted. seed sorting and counting was performed using a 10x magnification dissecting microscope (meiji techno co ltd, japan). seeds were a. mangium identified by cross checking the morphology with positively identified seeds collected from a fruiting a. mangium tree. seed density was quantified as the total number of seeds extracted from the soil sample. seed viability was determined using the forceps method (ball & miller 1989; price . 2010), et al whereby a pair of forceps was used to apply a gentle pressure onto the seed. if the seed resisted this pressure, it was considered as viable, however, if the seed did not resist this pressure then it was considered as non-viable. seed viability was quantified as the mean number of viable seeds identified from the soil sample. data analysis statistical analysis was done using r version 3.0.2 (r core team 2013). one-way anova was used to determine differences between plots in heath forest (hf), transition zone (tz) and plantation (p) for the following parameters: mean seedling emergence, mean species richness, mean seed density and mean viability of the seeds extracted. assumptions of heterogeneity of variances and normality were not violated. significant differences detected from one-way anova were further analysed using tukey's hsd test. results and discussion variation of seedling emergence in -acacia present and -absent habitatsacacia over the 12-week period, a total of 118 seedlings emerged. seedling emergence was observed in all plots, except for hf plots 1 and 4. overall, seedlings emerged at a much quicker rate for heat-treated soils compared to untreated and smoke-treated soils. the p plots had the highest mean seedling emergence (0.613), while the hf plots had the lowest mean seedling emergence (0.030) over the 12 week period (fig. 2). the oneway anova indicated that seedling emergence showed significant differences between habitats (f = 14.93, < 0.001), and was significantly the p highest in the p plots compared to the hf ( < p 0.001) and tz plots ( < 0.001). seedling p emergence did not differ significantly between hf and tz plots ( > 0.05; fig. 2)p . the significantly higher seedling emergence in the p plots compared to the tz and hf plots was possibly due to the higher proportion of a. mangium seeds, as well as seeds of pioneer species, such as grasses, that were recorded from the seed banks of the p plots. seeds of pioneers are better adapted for disturbance and harsh environmental conditions such as high light availability and relatively high temperatures (swaine & whitmore 1988; khurana & singh 2006), therefore, germinate faster in the open microhabitat conditions of the abandoned plantation. in acacia contrast, seedlings of non-pioneer trees which are more abundant in the heath forest sites, for 143 example , , agrostistachys longifolia madhuca curtisii syzygium caudatilimbum gymnostoma nobile and (maidin ., unpublished data), germinate slower et al as they are generally less tolerant to high irradiance and temperature (swaine & whitmore 1988; khurana & singh 2006). a total of 11 different seedlings species were recorded during the seedling emergence study, but the only species that could be positively identified was . the remaining 10 species were a. mangium monocotyledons ( ) and dicotyledons n = 4 species (n = 6 species). all of the monocotyledons recorded were identified as grasses. a. mangium seedlings were recorded emerging from soil samples collected in tz and p plots, but were not recorded from samples from hf plots. the p plots had the highest mean number of a. mangium seedling emergence (0.110) as compared to the tz plots (0.036). the mean number of a. mangium seedlings emerged were significantly higher in the p plots than the hf plots (f = 8.293, < 0.001), p but were not different between hf and tz plots or between tz and p plots (f = 8.293, > 0.05).p the significantly lower diversity of native seedlings recorded emerging from the soil seed banks of the p plots is consistent with findings on tree species diversity conducted by maidin . et al (unpublished data) using the same plots in our study, where the lowest tree diversity was recorded in the plantation, with most of the a. mangium trees comprising tropical pioneer species such as fagraea splendens macaranga conifera macaranga , and gigantea a. mangium, in addition to . seed bank composition is highly influenced by the diversity and composition of above-ground vegetation (dalling & denslow 1998), therefore, the lower tree diversity in the p plots appeared to have resulted in similarly lower diversity in their seed banks. additionally, the absence of late successional tropical species in the acacia plantations plots may also likely be due to the absence of specific environmental triggers, such as suitable temperature, light intensity and moisture levels, that are required for seed germination of such species in the p plots (baskin & baskin 2014). we had hypothesized that seeds would acacia be recorded in the hf plots, albeit in lower densities. this is because of the close proximity of the plantation to the hf plots acacia (separated from each other by less than 1 km), which had been expected to generate high propagule pressure (lockwood . 2005; et al colautti . 2006) that would facilitate et al acacia invasion into the intact heath forest. in contrast, results of this study revealed the absence of acacias in the soil seed banks of the hf plots. it was suggested that despite the potentially high figure 2 differences in the mean number (± se) of emerging seedlings among three different habitats: heath forest (hf), transition zone (tz) and plantation (p). different letters above the bars indicated a significant difference (at α = 0.05) in the mean number of seedlings among the three habitats compared 144 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia biotropia vol. 22 no. 2, 2015 propagule pressure, the spread of into a. mangium the hf plots may be affected by the lack of suitable dispersers. is known to be a. mangium bird-dispersed (gibson . 2011), which should et al allow for seed dispersal over long distances. it is possible that seeds from the plantation a. mangium were not adequately dispersed into the intact heath forests, perhaps because the birds that feed on seeds do not venture into intact forests. acacia however, it was difficult to ascertain this as further studies are needed to assess bird dispersal of seeds from the plantation into the a. mangium nearby heath forest. there was a lack of significant treatment effect on seedling emergence, although it had been expected that germination of seeds a. mangium would be especially stimulated by heat and/or smoke treatments (portlock . 1990; willis & et al read 2002; kulkarni . 2007). it was argued that et al our results likely indicated that the heat and smoke treatments employed in this study were insufficient to break dormancy and stimulate seed germination, and that further investigations using different heat and/or smoke treatments may result in a significant treatment effect. in this study, mean temperatures during the smoke treatment remained at ±40 °c and soil samples were exposed to smoke treatment for only 90 minutes. no reports are currently available on the optimum environmental manipulations required to enhance seed germination in the a. mangium tropics. however, auld (1986) reported that exposure to high temperatures (between 6080 °c) over any duration, or to extremely high temperatures (between 80-100 °c) for durations up to 1 hour, was needed to break seed dormancy in . these temperatures vary acacia suaveolens greatly between different species acacia depending on their respective degrees of heat tolerance (auld 1986; wahid . 2007). smoke et al and heat are complementary in stimulating seedling emergence from soil seed banks (read et al et al. 2000; zuloaga-aguilar . 2011), and studies have found that seeds typically require acacia smoke and/or heat as cues for germination (kulkarni . 2007; zuloaga-aguilar . 2011; et al et al rawson . 2013).et al a complicating factor in this study was inhibition of seedling emergence by fungal growth, which was observed in some of our heath forest soil samples. fungi are ubiquitous in soil and may increase seed mortality thus inhibiting seed germination (crist & friese 1993; wagner & mitschunas 2008). the presence of fungi in heat and smoke treated soils from the heath forest may indicate that these soils were subjected to very little temperature flux that could have otherwise eliminated soil fungal spores (neary . 1999; et al barcenas-moreno . 2009).et al soil seed bank composition a total of 31 different seed types were collected from the ten plots sampled, of which only seeds were successfully a. mangium identified. seeds were present in soil a. mangium samples from tz and p plots, but absent in the hf soil samples. hf plots registered the highest mean seed species richness, while p plots had the lowest (11.20 vs. 3.50, f = 41.81, < 0.001; fig. p 3). there was no significant difference (f = 41.81, p > 0.05) between tz and p plots in terms of species richness. comparable observations have been reported by wang (2009) where species richness in soil et al. seed banks of established exotic and a. mangium eucalyptus exserta plantations in south china were lower than those of the adjacent natural forest habitats. studies on -invaded areas of acacia saligna the south african fynbos (natural shrubland or heathland vegetation) have also revealed reduced native seed bank richness when compared to adjacent natural forest habitats (holmes & cowling 1997; gioria . 2014). low species et al richness in the soil seed bank of a plantation directly reflects the relatively low species richness in the standing vegetation of a plantation itself (wang . 2009). plantations typically comprise et al of monocultures with one dominant species being planted over a large area (evans & turnbull 2004), and this same pattern was evident in the andulau plantation in this study. a. mangium maidin (unpublished data) has clearly shown et al. that native heath tree species richness in the standing vegetation of the same a. mangium plantation plots were significantly lower than plots located in the heath forest. our observations, therefore, concurred with the hypothesis proposed by holmes and cowling (1997), where species richness of both the standing vegetation and seed banks tend to decrease with increasing duration of invasion by exotic species such as acacia. 145 figure 3 differences in mean species richness (± se) of seeds extracted from soil samples collected in plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different letters above the bars indicate a . significant difference (at α = 0.05) in the mean species richness between the two compared habitats figure 4 differences in seed density the mean number of seeds extracted from soil samples (± se) collected in , defined as plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different letters above . the bars indicate a significant difference (at α = 0.05) in the mean number of seeds extracted between the two compared habitats seed density was significantly the highest in soil samples from tz plots and the lowest in soil samples from p plots (83.50 vs. 6.83; f = 15.54, < 0.001; fig. 4). there was no significant p difference (f = 15.54, > 0.05) in seed density of p hf and tz plots. mean number of viable seeds was highest for hf plots, and lowest for p (46.0 vs. 2.5; f = 22.79, < 0.001; fig. 5). there was no p significant difference (f = 22.79, > 0.05) p between the hf and tz plots seed viability. exotic species are known to significantly reduce seed density in the soil seed banks of areas in which they establish and invade, and over time, further decrease in seed densities are typical (bossuyt & hermy 2003; gioria . 2014). thus, the et al conversion of parts of the andulau tropical heath forest to an exotic plantation for a. mangium commercial purposes have resulted in a subsequent decrease in native seed density in the soil seed bank of the plantation and its immediate surroundings. 146 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia the highest seed viability was detected within soils sampled from the heath forest and the transition zone, and the lowest was in the plantation plots. this may partly be due to the lower seed density from plantation plots, compared to the heath forest and plots in the transition zone. however, differences in seed viability in the soil seed banks among the different habitats may also indicate varying abilities of these habitats to recover after severe disturbance events (ashton et al et al. 1998; ghebrehiwot . 2012). the proportion of viable seeds present in the soil seed bank at the time of disturbance is important in determining initial floristic composition following a disturbance (hopkins & graham 1984). the appearance of early successional species after a disturbance event is related to the presence of viable seeds often long buried at the site (livingston & allesio 1968; luzuriaga . 2005). it is, therefore, suggested that et al if the study area was to experience severe disturbances, the heath forest would potentially be better equipped to recover through natural regeneration, than the plantation.a. mangium spread of acacia mangium a. mangium seeds were not observed in any of the plots within the heath forest habitat about 120 m away from the edge of the plantation. however, a. mangium seeds were found in one of the plots within the transition zone between these two contrasting habitats. our own observation within the study site in andulau fr was of extensive spread of into the open, disturbed a. mangium areas immediately surrounding the acacia plantation itself, thus indicating has a. mangium indeed escaped from the original plantation site. however, several factors may have allowed the adjacent intact heath forest to be protected from the spread. invasive alien plant species a. mangium may not be able to successfully establish in intact tropical forests due to light inhibition on the ground from the closed canopy and a thick litter layer with a low proportion of bare soil (pauchard & alaback 2004). canopy coverage in particular may be crucial in slowing down the spread of invasive seeds into the heath forest as a. mangium acacias are shade-intolerant plants (martin . et al 2008). low light availability, therefore, may limit the establishment of these shade-intolerant alien invasive species in a tropical rainforest, possibly conferring some resistance to intact forests from acacia et al invasions (fine 2002; osunkoya . 2005). t his c ur ren t stu dy was pr elim inar y investigation and exploratory that it only investigated seedling emergence potentials and seed bank compositions of an intact acacia plantation, an adjacent heath forest and the transition zone between these two contrasting habitats. however, the results of this study were an important first step towards understanding the potential of invasive acacias and their spread into the soil seed bank of an adjoining rare and vulnerable heath forest habitat in brunei. it is suggested that an important next step would be to conduct a comprehensive survey on soil seed bank composition, seed viability, seedling figure 5 differences in seed viability the mean number of viable seeds extracted (± se) from soil samples , defined as collected in plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different . letters above the bars indicate a significant difference (at α = 0.05) in the mean number of viable seeds between the two compared habitats biotropia vol. 22 no. 2, 2015 147 emergence patterns of all other natural habitats vulnerable to invasion in brunei acacia darussalam. a comparison of these factors and the composition of standing vegetation would provide a clear insight on the invasiveness of exotic acacias in brunei darussalam. conclusions comparisons of the soil seed banks between an plantation and the adjacent heath a. mangium forest have shown higher seed density, viability and species richness in the heath forest than in the a. mangium plantation. heath forests may have a higher chance of natural regeneration following future disturbance events due to their richer soil seed banks. clearing of intact forest for acacia plantations has a detrimental effect on seed bank diversity and composition, which can have negative knock-on effects on native plant regeneration. though is a known a. mangium invasive species, due to its shade intolerant nature, it appears to have some difficulty of spreading into intact tropical forests. it is, therefore, recommended close monitoring of escaping acacia seedlings from the intact parent plantations to mitigate further spread of acacias into natural habitats. acknowledgements the authors thank the forestry department, ministry of primary resources and tourism, brunei darussalam for granting permission and entry permit to work in the andulau forest reserve. we were grateful to haji ryni haji sofian, mr alex cheng and staff from the plantation section of the forestry department for advice and assistance with site selection. we thank two anonymous reviewers for comments that have improved the manuscript. funding for this study was provided by the universiti brunei darussalam research grant: ubd/pnc2/2/ rg/1(204). references ashton pms, harris pg, thadani r. 1998. soil seed bank dynamics in relation to topographic position of a mixed-deciduous forest in southern new england, usa. forest ecol manag 111: 15-22 . auld td. 1986. population dynamics of the shrub acacia suaveolens (sm.) willd.: fire and the transition to seedlings. aust j ecol 11:373-85. ball da, miller sd. 1989. a comparison of techniques for estimation of arable soil seed banks and their relationship to weed flora. weed res 29: 365 73. barcenas-moreno g, gomez-brandon m, rousk j, baath e. 2009. adaptation of soil microbial communities to temperature: 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ministry of industry and primary resources. wong km, kamariah as. 1999. forest and trees of brunei darussalam. bandar seri begawan (bn): universiti brunei darussalam. zuloaga-aguilar s, briones o, orozco-segovia a. 2011. seed germination of montane forest s p e c i e s i n response to ash, smoke and heat shock in mexico. acta oecol 37: 256-62. 150 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia http://www.r-project.org. biotropia book juni revisi 14 juli 09.indd 38 rapid detection of the africanized honey bee: a tool for indonesian animal quarantine rika raffiudin(1*, anifa bintar(1, m. chandra widjaja(2, ahmad farajallah(1, bambang purwantara(3 1department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia 2perhutani-west java and banten province, bandung, indonesia 3department of reproduction and obstetrics, faculty of veterinary medicine, bogor agricultural university, bogor, indonesia abstract molecular detection methods were used to determine if africanized honey bees (ahbs) are present in populations of imported apis mellifera in indonesia. the cytochrome b (cyt b gene) was amplified from mitochondrial dna and digested with the bglii restriction enzyme (cytb/bglii). two types of animal dna extraction kits were used and found suitable for rapid preparation of dna from a. mellifera by the animal quarantine facility. results showed that all 94 colony samples from beekeepers in java produced a 485 bp pcr product from the amplification of this gene. two dna fragments of 194 and 291 bp from all samples were produced after digestion with bglii. this cytb/bglii result together with the dna sequence of cyt b showed that all collected samples of a. mellifera were the non-ahb type. hence, this study did not detect ahb in indonesia. key words : apis mellifera, molecular detection methods, dna, cytochrome b, mitochondrial genetics introduction apis mellifera currently found in australia, america, and asia (included indonesia) are imported from europe, africa and the middle east (ruttner 1988). the most favorable for beekeepers is the a. mellifera ligustica subspecies due to its tame behaviour and high honey production. besides a. m. ligustica, an aggressive african honey bee subspecies, namely apis m. scutellata was imported to brazil in the mid-1950s. since the introduction of this african subspecies into brazil, descendent of 'africanized' honey bees (ahb) have spread throughout the neotropics and into temperate north america (schiff and biotropia vol. 16 no. 1, 2009: 38 44 *corresponding author: rika_r@cbn.net.id 39 sheppard 1993). it inbred with the european honey bee (ehb = a. m. ligustica or a. m. mellifera) became a killer ahb characterized as having a deadly sting (castro et al. 1994), a high wing beat frequency (spangler 1994), sensitivity to varrhoa parasitic mites (guerra et al. 2000), and rapid colony multiplication. schiff and sheppard (1993) have surveyed 422 feral honey bee colonies from non-africanized areas in the southern united states and it revealed that over 21% of them had mitochondrial dna (mtdna) derived from a european race established in north america in the 17th century, 77% of them had mtdna common in honey bees maintained by beekeepers and about 1% exhibited african mtdna. further analysis revealed that the african mtdna was derived from a north african subspecies imported to the us in the 19th century. the ahb can be distinguished from the non-ahb bees based on the mitochondrial cytochrome b (cyt b) gene digested with bglii (cyt b/bglii). the ahb cyt b does not have a restriction site for bglii, while the non-ahb has one bglii restriction site. therefore, the ahb type produces a single band of 495 bp, whereas the non-ahb pcr product produces a double band of 194 and 291 base pairs (bp) (crozier et al. 1991). this method has been used by australian bee keepers to detect the ahb imported to australia. in the united states, cyt b has also been reported to detect the ahb (pinto et al. 2003). however, currently the indonesian animal quarantine facility does not use a molecular detection method to recognize this ahb alien species. hence, the aim of this study was to establish a rapid molecular detection method for detecting ahb status in indonesia based on the cyt b/bglii. we used two dna extraction kits designed for animal tissue for rapid dna preparation from samples. this technique is appropriate for animal quarantine officers to make a rapid and accurate decision on the presence of the ahb in imported colonies in indonesia. materials and methods apis mellifera collection imported a. mellifera were collected from apiaries located in central java (pati, kudus, jepara) and in east java (jember, malang, kediri, pasuruan), indonesia. bees were anesthetized and preserved in absolute ethanol. dna extraction genomic dna was extracted from the thorax of single bee using phenol– chloroform extraction and ethanol precipitation. in addition, we tested the genomic dna mini kit from real biotech corporation (rbc) and the genclean column genomic dna isolation kit (generay biotech). for both kits, 20 mg of bee thorax was used for dna extraction, and single thorax in a 1.5 ml microtube was immersed in liquid nitrogen for 15 min prior to being crushed using a grinder. thereafter, the protocol according to the kit manufacturer was followed. a. mellifera dna amplifi cation. part of the cyt b region was amplified using primers of cytochrome b forward: 5’-tatgtactacctttgaggacaaatatc-3’ (11400); cytochrome b reverse 5’rapid detection of the africanized honey bee – rika raffi udin et al. 40 attacacctcctaatttattaaggaat-3’ (11859) (crozier et al. 1991). numbers in brackets indicate the position of a. mellifera cyt b in the complete mtdna (accession number l06178). cycle sequencing conditions protocol was 2 min. at 94 °c for initial denaturing, 35 cycles of 30 s at 94 °c, 30 s at 55°c and 1 min. at 72 °c for dna elongation, followed by 10 min. for the dna extension. dna sequencing followed the abi bigdye automated sequencing instruction from the supplier by using the same primers of as for dna amplication. dna restriction analysis apis mellifera cyt b pcr products were digested using bglii restriction enzyme. the digestion mixture contained the cyt b pcr product, dd h2o, buffer d, bglii restriction enzyme and were incubated at 37oc overnight. dna alignment we used clustalx (thompson et al. 1997) program to align the dna sequence generated in this study with the published ahb haplotypes provided in genbank database (http://www.ncbi.nlm.nih.gov; accession number ef016646 and ef016647). results and discussions apis mellifera cyt b amplifi cation and dna restriction analysis we collected samples from 94 colonies of imported a. mellifera from big and small apiaries in central and east java (data not shown). all samples from the 94 colonies produced the same size dna band approximately 500 bp ( figure 1). pcr products from 94 samples were digested with bglii and all produced two bands of approximately 200 and 300 bp (figure 2). figure 1. cyt b pcr products from imported a. mellifera in java, indonesia; m = 100 bp dna marker, 1-10 = a. mellifera colony sample number 1-10 from java, indonesia biotropia vol. 16 no. 1, 2009 41 figure 2 cyt b/bglii restriction fragments of imported a. mellifera in java, indonesia; m = 100 bp dna marker, 1-10 = a. mellifera colony sample number 1-10 from java. apis mellifera cyt b sequence data and alignment with ahb haplotypes since all cyt b pcr product of apis mellifera gave the same result (figure 1 and 2), we only sequenced one bee, i.e. a. mellifera from colony number 9 from pati, central java (am9pt). pcr product of am9pt cyt b sequence gave a total length of 485 bp (figure 3). the bglii sites (sequence agatct) digested the pcr product into two fragments of 194 and 291 bp (figure 3) . figure 3. restriction map of a. mellifera (am9pt) cyt b/bglii. resulted for two dna fragments of 194 and 291 bp these results were in agreement with the data of crozier (1991) from a. mellifera imported to australia and data from the united states (pinto et al. 2003) which have indicated that cyt b can be used to detect the ahb. a total of 451 colonies have been screened for the ahb characters in the usa. the united states department of agriculture, animal and plant health inspection service, plant protection and quarantine used trap lines and morphological identification techniques. based on morphology, the ahb and ehb could not be distinguished by beekeepers (montesinos 1995). hence, this accurate molecular technique can help solve the problem of detecting the ahb. africanized honey bees are one of the invasive alien species that are banned from entering all nations including indonesia. therefore, this effective system for warning about invasive alien species is needed, especially due to global trade. ahb and non-ahb cyt b alignment the dna sequence alignment showed that the am9pt cyt b sequenced in this study was exactly the same as that of genbank accession l06178; crozier & crozier 1993) denoted for a. mellifera ligustica (figure 4). hence, am9pt from this rapid detection of the africanized honey bee – rika raffi udin et al. 42 research was confirmed to be non-ahb bees. the primer amplified the partial cyt b gene started at nucleotide 396 of the complete cyt b gene sequence. the bglii site was located at nuclotide 694 based on whole sequence (figure 4). cytbl06178 attcttttaatatcaatagcagctgcatttataggatatgtactaccatgaggacaaata 420 am9pt ------------------------------------tatgtactaccatgaggacaaata 24 ef016646 ------------------------------------tatgtactaccatgaggacaaata 24 ef016647 ------------------------------------tatgtactaccatgaggacaaata 24 ************************ cytbl06178 tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 480 am9pt tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 84 ef016646 tcatattgaggtgcaacagtcattactaatcttttatcagcaattccttatattggtgat 84 ef016647 tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 84 ******************** *************************************** cytbl06178 acaattgtattatgaatttgaggtggattttcaattaataatgctacattaaatcgattt 540 am9pt acaattgtattatgaatttgaggtggattttcaattaataatgctacattaaatcgattt 144 ef016646 acaattgtattatgaatctgaggtgggttctcaattaataatgctaccttaaatcgattt 144 ef016647 acaattgtattatgaatctgaggtgggttctcaattaataatgctaccttaaatcgattt 144 ***************** ******** ** ***************** ************ cytbl06178 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 600 am9pt ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ef016646 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ef016647 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ************************************************************ cytbl06178 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 660 am9pt gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ef016646 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ef016647 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ************************************************************ cytbl06178 tcatttcatccatatttttcaattaaagatcttttaggattttatatcatcttatttatc 720 am9pt tcatttcatccatatttttcaattaaagatcttttaggattttatatcatcttatttatc 324 ef016646 tcatttcatccatatttttcaattaaagaccttttaggattttatattatcttatttatc 324 ef016647 tcatttcatccatatttttcaattaaagaccttttaggattttatattatcttatttatc 324 ***************************** ***************** ************ cytbl06178 tttatattcattaattttcaatttccatatcatttaggagatccagacaatttcaaaatt 780 am9pt tttatattcattaattttcaatttccatatcatttaggagatccagacaatttcaaaatt 384 ef016646 tttatattcattaattttcaatttccatatcatttaggagatccagataattttaaaatt 384 ef016647 tttatattcattaattttcaatttccatatcatttaggagatccagataattttaaaatt 384 *********************************************** ***** ****** cytbl06178 gcaaatccaataaatactccaactcatattaaacctgaatgatatttcctatttgcatat 840 am9pt gcaaatccaataaatactccaactcatattaaacctgaatgatatttcctatttgcatat 444 ef016646_1 gcaaatccaataaatactccaactcatattaaacctgaatgatattttctatttgcatat 444 ef016647_2 gcaaatccaataaatactccaactcatattaaacctgaatgatattttctatttgcatat 444 *********************************************** ************ cytbl06178 tcaattttacgagcaattcctaataaattaggaggtgtaatcggattagtaatatcaatt 900 am9pt tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ef016646 tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ef016647 tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ***************************************** 1 2 3 4 5 6 7 8 9 figure 4. a. mellifera am9pt from this study aligned with genbank cyt b sequences cytbl06178 (from a. m. ligustica acc num l06178), ef016646 (from ahb haplotype 1), and ef016647 (from ahb haplotype 2). * = nucleotide homology. underlined nucleotide = bglii sites. solid boxed nucleotides = nucleotide differences between ahb-1 and ahb-2. dashed boxed nucleotides = nucleotide change causing loss of bglii site in ahb type. numbers positioned above the dna alignment were all nucleotide differences between am9 pt, and ahb haplotype 1 and haplotype 2. biotropia vol. 16 no. 1, 2009 43 dna alignment between am9pt and ahb haplotype 1 and ahb haplotype 2 from genbank (accession numbers ef016647 and ef016647, respectively) showed nine nucleotide differences (figure 4, see the numbers above the nucleotide alignment). the difference between the ahb and the non-ahb at base number 295 was shown in figure 4, labelled mutation number 5. this is the position of the restriction site for bglii; it could be observed that the nucleotide “t” in cytbl06178 and am9pt were altered to be a “c” in both ahb haplotypes 1 and 2 (ef016646 and ef016647). this mutation removed the bglii restriction site, hence shows why a single band is seen in the ahb haplotypes. based on homology analysis of the two ahbs, these two haplotypes also differ at nucleotide number 65 (figure 4). the two ahb haplotypes obtained from genbank were submitted on february 1, 2007 by szalanski, a.l. and mckern, j.a from the department of entomology, university of arkansas, usa. based on the findings of this study, we suggest to carry out cooperation between the animal sections of the agency of agriculture quarantine (balai karantina pertanian) and the apiaries association in indonesia. the cooperation could commence by performing a socialization of honey bee biology. furthermore, the molecular detection for the ahb should be implemented at the animal quarantine in every province in indonesia, particularly at soekarno hatta and ngurah rai airports, so that imported bees could be monitored. conclusions molecular analysis of imported a. mellifera based on the cytochrome b gene from mitochondrial dna digested with bglii restriction enzyme was conducted 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1993. mitochondrial dna evidence for the 19th century introduction of african honey bees into the united-states. experientia, 49:530-532 spangler h. 1994. are the wingbeat frequencies of honey bees an indicator of population or behaviour. american bee journal, 134:53-55 tegelstrom h. 1986. mitochondrial dna in natural population: an improved routine for screening of genetic variation based on sensitive silver staining. electrophoresis, 7:226-229 th ompson j.d., gibson t.j., plewniak f., jeanmougin f. and d.g. higgins. 1997. th e clustal-x windows interface: fl exible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acid research, 25:4876-4882. biotropia vol. 16 no. 1, 2009 thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html 2. dyah perwitasari page 1 page 2 page 3 page 4 page 5 page 6 biotropia (2) 1988/1989: 32-37 a collection of ticks (ixodidae) from sulawesi utara, indonesia l.a. durden department of entomology, nhb 165, museum support center smithsonian institution, washington d.c. 20560, u.s.a. c.h.s. watts division of natural science south australian museum, north terrace, adelaide, south australia 5000, australia abstract ixodid ticks were collected from seven species of endemic murid rodents and from vegetation in sulawesi utara, indonesia. adult ticks belonging to the species, amblyomma babirussae, a. cyprium and dermacentor (indocentor) steini were taken from the vegetation. immature ticks assignable to the genera, haemaphysalis, amblyomma and ixodes infested the murids with parauromys dominator and maxomys musschenbroekii being the two most heavily tick-infested host species. the data are compared to other tick records from sulawesi. introduction ectoparasites were collected from murid rodents live-trapped in sulawesi utara (north sulawesi), indonesia, on august 1985. we report here on the ticks (ixodi-dae) collected during this project. eighty five murids belonging to seven species, all endemic to sulawesi, were examined for ticks. the comparatively small size of these host animals made infestation by adult ticks (which are identifiable to the species level) unlikely, so adult ticks were taken from vegetation when possible to supplement the collections. collection data for the 58 ticks recovered from the murids and from vegetation are given below, listed by collection locality. specimens are currently in the rocky mountain laboratory collections (now at the smithsonian institution, washington, washington b.c., u.s.a.) and accession numbers are also given. mammal specimens are either with collections of the south australian museum or the museum zoologicum bogoriense, indonesia; their original collection numbers are presented here. 1. gunung moajat (0°45'n 124°25'e). elevation, 1800 m. from vegetation. amblyomma cyprium neumann (1q) 32 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts ex. parauromys dominator (chsw no. 114) haemaphysalis sp. (1l*) 2. dumoga-bone nat. park (0°34'n 123°54'e). elevation, 200-300 m. on grass at forest edge dermacentor (indocentor) steini schulze (1°) ex. maxomys musschenbroekii (chsw no. 19) amblyomma sp. (4l, 2n*) haemaphysalis sp. (1l) ex. m. musschenbroekii (chsw no. 21) amblyomma sp. (3l) ex. rattus xanthurus (chsw no. 28) amblyomma sp. (in) amblyomma sp. (probably)(ln) ex. maxomys hellwaldii (chsw no. 42) haemaphysalis sp. (4n) (one specimen has three acaridid mites attached to its venter). ex. rattus hoffmanni (chsw no. 47) haemaphysalis sp. (1l, in) ex. p. dominator (chsw no. 48) amblyomma sp. (12l, in) ex. m. musschenbroekii (chsw no. 54) ixodes sp. (2l) ex. m. musschenbroekii (chsw no. 55) haemaphysalis sp. (7n) ex. r. hoffmanni (chsw no. 58) ixodes sp. (1l) ex. p. dominator (chsw no. 66) haemaphysalis sp. (in) ex. r. hoffmanni (chsw no. 80) ixodes sp. (in) 3. dumoga-bone nat. park. hog's back ridge (0°35'n 123°52'e) elevation, 490 m. ex. p. dominator haemaphysalis sp. (in) ex. r. hoffmanni (chsw no. 138) amblyomma sp. (4l, 4n 2 of these nymphs belong to one species and 2 to a second species) 4. gunung mogogonipa (0°27'n 123°57'e). elevation, 1008 m. on vegetation 33 biotropia no. 2, 1988/1989 amblyomma babirussae (19) dermacentor (i.) steini schulze (19) 5. by 'lake' (0°44'n 124°27'e). elevation, 1080-1200 m. ex. p. dominator (chsw no. 13) haemaphysalis sp. (in) *l = larvae, n = nymphs. the three species of adult ticks that were collected are fairly well known. amblyomma babirussae (figure 1) represents part of the endemic tick fauna of figure 1. amblyomma babirussae (adult female). sulawesi; adults are parasitic principally on larger artiodactyls and on endemic and feral pigs (suidae) (anastos 1950, keirans and robbins 1987) while immatures are parasitic on these same hosts and also on murids (keirans and robbins 1987, van peena et al. 1974). adults of a. babirussae are also known to attach to humans (keirans and robbins 1987). amblyomma cyprium (figure 2) has a wide distribution in the indo-pacific region (anastos 1950; kohls 1957; wilson 1969; marshall 1976; kemp and wilson 1979) with adults again parasitic largerly on wild and feral pigs, but also on humans and cattle, and immatures on a variety of smaller hosts, 34 figure 2. amblyomma cyprium (adult female). particularly murids (kohls 1957; kemp and wilson 1979). dermacentor (i.) steini (figure 3) has recently been redescribed by wassef and hoogstraal (1986) who document a wide distribution for this species in indo-malaysia (including the philippines and with establishment on papua new guinea); however, they give no records for sulawesi so the present collections extend the documented range for this tick. 35 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts figure 3. dermacentor (indocentor) steini (adult female). biotropia no. 2, 1988/1989 dermacentor (i.) steini adults again appear to be parasitic mainly on wild and feral pigs (wassef and hoogstraal 1986). unfortunately, adults of an undescribed species of tick probably assignable to the genus, amblyomma, collected by durden (1986) as a single nymphal specimen (accession no. hh112,315 in the hoogstraal tick collection is now housed at the smithsonian institution, washington d.c., usa) from a murid, m. musschen-broekii, in sulawesi utara, were not collected. this immature specimen is so atypical that hoogstraal (personal communication) could not be certain of its taxonomic status and hopefully, future ectoparasite collections from sulawesi will produce adults of this fascinating tick. analysis of the prevalence of tick infestation from the murid species examined shows that no ticks were taken from the 13 bunomys fratrorum or from the 11 congeneric b. chrysocomus rats processed. the other five murid species showed various levels of infestation. rattus hoffmanni (n = 10) showed a collective tick (three genera represented) prevalence of 40% and a mean intensity of 3.0 ticks per infested animal. the other murid species had the following comparable infestation figures: r. xanthurus (n = 7)(1 tick genus), 14.3%, 2.0; p. dominator (n = 16)(2 tick genera), 31.3%, 3.4; m. musschenbroekii (n = 17)(3 tick genera), 23.5%, 4.8; m. hellwaldii (n = 11)(1 tick genus), 9.1%, 4.0. the main difference between these data and those of durden (1986) is that, in this study, no dermacentor (indocentor) sp. ticks were collected from murids. in the earlier survey, b. fratrorum and especially m. musschenbroekii were quite heavily infested by ticks of this subgenus; this difference could reflect a seasonal influence. another difference, possibly attributable to phenology, is the presence of ixodes sp. immatures from murids trapped in the lowland forests (200-300 m elevation) of dumoga-bone national park in this collection; durden (1986) did not record this genus from murids from comparable elevations in this forest. the earlier collection (durden 1986) was made from january to march, 1985, during the latter part of the monsoon season (although heavy rains were not as concentrated into this season as is typical for much of southeast asia). acknowledgments deep gratitude is extended to dr. i.e. keirans who identified the ticks described here and to dr. g.g. musser for confirming host identities. appreciation is also extended to r.g. robbins for cataloguing ticks, to dr. n.a. wilson for sup 36 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts plying separates and to the late dr. h. hoogstraal for his personal communications and earlier tick identifications. this research was undertaken as part of project wallace organized by the royal entomological society of london and the indonesian institute of sciences (results of project wallace no. 30). references anastos, g., 1950. the scutate ticks, or ixodidae, of indonesia. entomol. am., 30: 1. durden, l.a., 1986. ectoparasites and other arthropod associates of tropical rain forest mammals in sulawesi utara, indonesia. nat. geogr. res., 2: 320. kemp, d.h. and wilson, n.a., 1979. the occurrence of amblyomma cyprium (acari; ixodidae) in australia, with additional records from the southwest pacific. pac. insects, 21: 224. kohls, g.m., 1957. insects of micronesia. acarina: ixodoidea. insects micronesia, 3: 85. marshall, a.g., 1976. host specificity amongst arthropods ectoparasitic upon mammals and birds in the new hebrides. ecol. entomol., 1: 189. van peenan, p.f.d., carney, w.p., sudomo, m. and sulianti saroso, j., 1974. parasites of mammals in gumbasa valley, central sulawesi, indonesia. trop. geogr. med., 26: 352. wassef, h.y. and hoogstraal, h., 1986. dermacentor (indocentor) steini (acari: ixodidae): identity of male and female. j. med. entomol., 23: 532. wilson, n.a., 1969. ticks (metastigmata: ixodidae) collected by the noona dan expedition to the philippine and bismarck archipelagos. entomol. med., 37: 285. 37 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf biotropia book final.indd 119 using streptomyces xylanase to produce xylooligosacharide from corncob anja meryandini1*, titi candra sunarti2, aprilia naomi1 and fery mutia2 1. department of biology, faculty of mathematics and natural sciences, bogor agriculture university, bogor, indonesia 2. department of agrotechno industry, faculty of agricultural technology, bogor agriculture university, bogor, indonesia abstract streptomyces 234p-16 and skk1-8 are xylanase-producing bacteria. corncob xylan were extracted using acidifi ed method. crude enzymes (produced by centrifuging the culture) were used to hydrolyze xylan from 2 varieties of corncob. crude extract activity was measured by using dns (dinitrosalisilic acid) method. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. skk1-8 xylanase can hydrolize corncob xylan (1% hawaii or 1.5% bisma xylan) within 4 hours and produce xylooligosacharide with polymerization degree of 4.76 and 6.37, respectively. key words: xylanase, xylooligosacharide, streptomyces. introduction besides rice, corn is the major carbohydrate sources in indonesia especially for feed and industrial raw materials. most of corn is used as raw material for food and nonfood industries. but these applications are limited to the corn kernel, and other parts of corn plant such as corncob was not utilized yet. about 30% of the corn is corncob; the rests are corn stover and kernels. data from badan pusat statistik (indonesian bureau of statistics) showed that maize production is increasing from 9.82 million ton in 2002 to 11.35 million ton in 2004. th is increase is also followed by the increase of corncob as an agricultural waste. corncob consists of protein, fat, nitrogen free extract and also hemicellulose and cellulose (johnson 1991). th e content of xylan (major part of hemicellulose) in corncob could go up to 40-g/100 g, the highest among all of the agricultural waste (yang et al. 2005). most xylan occurs as heteropolysaccharide, containing diff erent substituent groups in the backbone chain and in the side chain (beg et al. 2001). due to the corncob nutritional content, it can convert into a commercial product or be used as a medium to biotropia vol. 15 no. 2, 2008 : 119 128 *corresponding author: ameryandini@yahoo.com 120 cultivate microorganisms. in our previous research we have identifi ed xylanase-producing streptomyces (234p-16 and skk1-8). xylanase from 234p-26 has an optimum condition at ph 5 and 90 oc whereas from skk 1-8 the optimum condition is at ph 6 and 50 oc (meryandini 2005). in this research we will use this streptomyces xylanase to produce xylooligosaccharide from corncob xylan. materials and methods materials in this research we use 2 local varieties of corncobs (bisma and hawaii) and 2 acid-xylanases from indonesian streptomyces: streptomyces 234p-16 from padang and streptomyces skk1-8 from sukabumi. delignifi cation delignifi cation of corncobs methods had been investigated previously (widyani 2002). about 1000 g of corncob grits is immersed in 10 liters of 1% naocl solution for 5 hours at room temperature (28 oc). after 5 hours, the sample is decanted and rinsed by distilled water for several times and fi ltered, the solid part i.e. delignifi ed samples are then dried by oven drying at 50 oc for 48 hours. th e chemical composition of this sample will be examined as cellulose, hemicellulose, and lignin. xylan extraction & purifi cation th e method for separation of xylan using acidifi ed method from delignifi ed samples has been investigated by anggraini (2003). delignifi ed corncob grits are immersed in 15% naoh solution for 24 hours at room temperature (28 oc). th is step liberated the xylan into soluble fraction and neutralized by 6 n hcl solutions (ph 4.5-5.0). th e supernatant is centrifuged on 4000 rpm for 30 minutes to obtain residue such as xylan. purifi cation is conducted by re-dissolving the crude xylan into 4% naoh and then fi ltered. filtrate was acidifi ed using 6 n hcl (ph 4.5-5.0), and then centrifuged on 4000 rpm for 30 minutes. precipitates were dissolved in 95% ethanol and centrifuged, and then dehydrated using 50 o c drying oven. xylanase production th e streptomyces isolates are cultured on ym (yeastmalt) agar and then on oatspelt xylan agar medium (yeast extract 0.2%, sucrose 10%, k2hpo4 1.5%, mgso4 .7h2o 0.025%, nacl 0.23%, na2hpo4 .2h2o 5 %, oat spelt xylan 0.5%) for 4 days at room temperature. two cookbores of streptomyces are cultured in 100 ml liquid xylan media and incubated at room temperature using a shaking incubator with an agitation speed of 240 rpm. every day the culture should be centrifuged and the supernatans collected for enzyme assay. biotropia vol. 15 no. 2, 2008 121 xylanase production on several substrates two cookbores of streptomyces are cultured in 100 ml liquid xylan media (oatspelt and several concentration of 2 local corncob xylan) in a 500 ml erlenmeyer and incubated on a shaker for 10 to 12 days at room temperature. duration of xylan hydrolysis by crude xylanase streptomyces xylanase produced in 1% of corncob xylan was used to hydrolyze 1% of corncob xylan for 4 hours at optimum ph and temperature condition. degree of hydrolysis and degree of polymerization will be monitored every hour and the reducing sugars analyzed by dinitroasalicilycacid method, and total sugar by phenol-h2so4 method. assay of xylanase activity th e culture was centrifuged 5 minutes at 10.000 x g to obtain the xylanase crude extract. xylanase activity was measured with oat-spelt xylan as a substrate. enzyme solution (100 μl) was added to 1 ml substrate solution which contain 0.5% xylan and the mixture was incubated at optimum temperature for each enzyme (90 oc for 234p-26 and 50 oc for skk 1-8) for 30 minutes. crude extract activity was measured by using dns (dinitrosalisilic acid) method by miller (1959) with xylosa as the standard. th e reducing sugar of the references samples (substrate solution incubated without enzyme and diluted enzyme solution in buff er) were deduced from the values of the test samples. th e reducing-sugar was detected by spectrophotometer (λ = 540 nm). one unit xylanase activity was defi ned as the amount of enzyme which produces 1 μmol xylosa per minute. results and discussions xylan extraction th e chemical composition of corncob is described in table 1, and the delignifi cation eff ect of corncobs and recovery of cellulose fraction to the fi ber components are shown in table 2. table 1. chemical composition of corncobs constituent bisma variety hawaii variety ash (% db) 1.62 1.67 lipid (% db) 3.02 4.68 crude protein (%db) 2.41 4.82 crude fiber (% db) 38.07 40.65 carbohydrate (% db, by difference) 51.93 44.14 streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 122 table 2. composition of fibers before and after delignification. constituent before delignifi cation (%) after delignifi cation (%) bisma variety cellulose 65.96 44.36 hemicelulose 10.82 30.38 lignin 23.74 19.21 hawaii variety cellulose 60.04 41.88 hemicelulose 18.11 30.18 lignin 16.14 15.12 elimination of lignin should be conducted since lignin will reduce the eff ectiveness of the utilization of xylan as carbon source for microbial growth and will infl uence the production of enzyme (agustine 2005). post-delignifi cation, the product composition was analyzed and small amount of lignin liberated. th e initial lignin of bisma variety (23.74%) and hawaii variety (16.14%) were reduced to 19.27% (bisma variety) and 15.2% (hawaii variety), respectively. delignifi cation cannot eliminate lignin from lignocellulosic materials completely. cellulose microfi bril was integrated in hydrophobic matrix covered by lignin, and the lignin linked with cellulose and hemicellulose in covalent linkages (agustine 2005). according to fengel and wegener (1995), lignin, cellulose and hemicellulose cannot separate completely even by special separation and purifi cation. lignin and cellulose can be detected in purifi ed cellulose and lignin. delignifi cation was conducted using 1% naocl as strong oxidant (agustine 2005). anggraini (2003) and widyani (2002) stated that hypochlorite ion from naocl can cleave the carbon linkage on lignin structure, and delignifi cation caused the opening of the linkages between lignin and other polysaccharides and infl uenced the increasing use of xylan by bacteria. xylan extraction was conducted by submerging the delignifi ed materials into 15% naoh. anggraini (2003) and widyani (2002) concluded that hemicellulose can be dissolved in alkaline solution, such as 15% naoh can produce brighter and clear powder, relatively clean from impurities, and easily dissolved in water and produced high yield. acid solution 6 n hcl was added to neutralize up to ph 4.5-5.0 to precipitate again the xylan. th e yield of xylan recovery was 9.26% from bisma variety, and 9.94% from hawaii variety. based on research results of widyani (2002) and anggraini (2003) 7.6412.94 % and 7.31-11.45 %, respectively, of xylan can be produced from corncob using the same acidifi ed methods. production of xylanase th e daily production curve of xylanase streptomyces 234p-16 and skk 1-8 assayed at ph 7.2 and 37 oc is shown in figure 1. th e highest xylanase production was reached on day-10 with the activity of 0.625 unit/ml for skk1-8 and on day-5 with the activity of 0.27 u/ml for 234p-16. th e optimum time of xylanase production was then used as the standard harvest time for the next xylanase production. biotropia vol. 15 no. 2, 2008 123 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 4 5 6 7 8 9 10 11 12 days a ct iv ity ( u /m l) 234p-16 skk1-8 figure 1. production curve of streptomyces. 234p-16 and skk 1-8 xylanase assayed on 37 oc and ph 7.2. xylanase activity on several concentrations of corncob xylan 0 20 40 60 80 100 120 140 160 oat spelt hawai bisma substrate r el at iv e a ct iv ity (% ) 234p-16 skk1-8 figure 2. relative activity of streptomyces xylanases on 0.5% xylan from oat spelt, bisma dan hawaii corncob figure 2 shows diff erences in xylanase activity on several 0.5% substrates. xylanase from strain 234p-16 showed the highest activity on hawaii xylan, while xylanase from skk1-8 on oat spelt. xylanase 234p-16 showed the lowest activity on bisma xylan, whereas xylanase 45i-3 on hawaii xylan. hendarwin (2005) also reported activity diff erences in various substrates. xylanase activity in several concentrations of corn xylan and 0.5% oat spelt xylan streptomyces was inoculated in 0.5%, 1% and 1.5% bisma or hawaii xylan medium and compared with 0.5% oat spelt xylan. figure 3 shows the streptomyces 234p-16 xylanase activity on several concentrations of hawaii xylan (figure 3a) and several concentrations of bisma xylan (figure 3b ). xylanase from strain 234p-16 has the highest activity if cultivated in 1 % bisma xylan (0.57 u/ml) or in 1% hawaii xylan (0.947 u/ml) streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 124 a. activity of isolate 234p-16 xylanase 0 0,2 0,4 0,6 0,8 1 4 5 6 7 8 9 10 days a ct iv it iy ( u /m l) oat sp elt 0,5% hawai 0,5% hawai 1% hawai 1,5% b. activity of isolate 234p-16 xylanase 0 0,2 0,4 0,6 4 5 6 7 8 9 10 days a ct iv ity (u /m l) oat sp elt 0,5% bisma 0,5% bisma 1% bisma 1,5% figure 3. xylanase activities from streptomyces 234p-16 on (a) hawaii corncob ylan and (b) bisma corncob xylan compared with oat spelt xylan figure 4 shows the streptomyces skk1-8 xylanase activity on several concentrations of hawaii xylan (figure 4 a) and several concentrations of bisma xylan (figure 4b). xylanase from strain skk1-8 has the highest activity if cultivated in 1.5 % bisma xylan (49 u/ml) or in 1% hawaii xylan (5.75 u/ml). a. xylanase activity from isolate skk1-8 0 2 4 6 8 4 5 6 7 8 9 10 11 12 days a ct iv it y (u /m l) oatspelt 0,5% hawai 0,5% hawai 1% hawai 1,5% biotropia vol. 15 no. 2, 2008 125 b. xylanase activity from isolate skk1-8 0 10 20 30 40 50 60 4 5 6 7 8 9 10 11 12 days a ct iv it y (u /m l) oat spelt 0,5% bisma 0,5% bisma 1% bisma 1,5% figure 4. xylanase activities from streptomyces skk1-8 on (a) hawaii corncob xylan and (b) bisma corncob xylan compared with oat spelt xylan th e production of xylanase in 0.5% oat spelt xylan was compared with the production in several concentrations of corncob xylan. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan (figure 3 and 4 ). th e production of xylanase is usually induced in medium containing pure xylan or xylan-rich residues. several reports showed that xylanase can be induced with lignocellulose material such as wheat bran, rice straw, corncob and sugarcane bagase (beg et al. 2001). corncob xylan is a rich carbon source with several kinds of carbon content as according to fengel and wegener (1995) lignin, cellulose and hemicellulose cannot be separated perfectly although the usage of a special separation and purifi cation methods and this content gives an eff ect on xylanase production. all carbon sources will give an eff ect on enzym production as reported by ambarawati (2005) that medium containing mannan can induce xylanase from strain 45i-3 and vice versa. th e optimum concentration to induce xylanase was 1% for hawaii xylan and for bisma xylan. th is greater result on hawaii xylan could be due to the higher concentration of hemicellulose in hawaii xylan. hydrolysis of xylan sugar composition was monitored by the increasing of reducing sugar and the decreasing degree of polymerization and the result is shown in table 3. table 3 shows the decreasing of dp during hydrolysis, while the reducing sugar continued to increase. smaller number of dp shows that polysaccharide was depolymerized into short-chains compounds. li et al. (2000) also reported that each microorganism will liberate specifi c xylanase, and showed diff erent activity on the same substrate. high diversity of xylanase may be caused by many diff erent structures of xylan in the nature. isolates skk1-8, but not 234p-16, can be used for xos production using corncob xylan within 4 hours hydrolysis time. streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 126 chen et al. (1997) stated that xylo-oligosaccharide (xos) composed of 2-5 units xylose. vazque et al. (2000) reported that xos for food application should be 2-4 unit monomers, especially dp 2 xylobiose. table 3. reducing sugar, total sugar and the degree of polymerization isolate substrate time (hours) sugar production (mg/ml) total sugar (mg/ml) degree of polymerization 234p-16 1 % hawaii xylan 0 2.05 184.79 90.14 1 2.91 184.79 63.50 2 3.4 184.79 54.35 3 4.3 184.79 43.18 4 4.8 184.79 38.50 1% bisma xylan 0 1.53 151.47 99 1 2.06 151.47 73.53 2 2.39 151.47 63.38 3 3.13 151.47 48.39 4 3.39 151.47 44.68 skk1-8 1 % hawaii xylan 0 12.24 184.56 15.08 1 27.43 184.56 6.73 2 36.29 184.56 5.09 3 34.84 184.56 5.30 4 38.70 184.56 4.76 1.5% bisma xylan 0 1.36 182.61 134.27 1 2.45 182.61 74.53 2 26.84 182.61 6.80 3 24.72 182.61 7.39 4 28.65 182.61 6.37 conclusions xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. using 1.5% of bisma xylan, xylanase from skk1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. xylanase from strain 234p-16 is not suitable for producing xylooligosacharides. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. using 1.5% of bisma xylan, xylanase from skk1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. xylanase from strain 234p-16 is not suitable for producing xylooligosacharides. acknowledgments th is research was funded by dip pusat studi regional penelitian biologi tropika (seameo biotrop) with contract agreement no 044.1/psrp/sp-pen/iv/2006 on 6th april 2006 to anja meryandini. we thank dr. ir. yulin lestari for the isolate. biotropia vol. 15 no. 2, 2008 127 references adeola o. and m.r. bedford . 2004. exogenous dietary xylanase ameliorates viscosity-induced anti-nutritional eff ects in wheat-based diets for white peckin ducks (anas platyrinchos domesticus). british journal of nutrition 92:87-94. agustine w. 2005. penentuan kondisi optimum pertumbuhan dan produksi xilanase isolat aq1. undergraduate thesis (skripsi). department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia. anggraini f. 2003. kajian ekstraksi dan hidrolisis xilan dari tongkol jagung (zea mays l.). undergraduate thesis (skripsi). faculty of agricultural technology, bogor agricultural university, bogor, indonesia ali m.k., rudolph f.b. and g.n. bennett. 2004. th ermostable xylanases 10b from clostridium acetobotylicum atcc824. journal of industrial microbiology and biotechnology 31: 229-234. ambarawati d. 2005. karakterisasi mananase streptomyces sp galur 45i-3. undergraduate thesis. 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28 isolation and cloning of cdna of gene encoding for metallothionein type 2 from melastoma affine suharsono1,2, niken trisnaningrum1, lulut dwi sulistyaningsih1, utut widyastuti1,2 1research center for bioresources and biotechnology, and 2department of biology, bogor agricultural university indonesia abstract metallothionein is an important protein for detoxifying heavy metal ions. th is research was conducted to isolate and clone cdna of gene encoding for metallothionein type 2 from melastoma affi ne. total rna was isolated from young leaves. total cdna was synthesized from the total rna by reverse transcription. th e mamt2 cdna was successfully isolated by pcr technique. th e mamt2 cdna was inserted into pgem-t easy and the recombinant plasmid was successfully introduced into escherichia coli dh5α. dna sequencing analysis showed that this cdna is full length consisting of 246 pb encoding 81 amino acid residues. th is cdna is identical to mrna of atmt2 from arabidopsis thaliana. it does not contain any restriction sites found in the cloning sites of pgem-t easy. th e deduced protein of mamt2 contains 14 cysteine residues distributed in the cys-cys, cys-x-cys, and cys-x-x-cys motifs. key words: cdna, metallothionein, melastoma affine, cloning, cysteine introduction gene isolation has a very important role in the genetic improvement of crops. the genes can be isolated from genetic resources coming from microorganism, plants, or animals. melastoma malabathricum l. or m. affine d. don is an aluminum hyperaccumulator plant (watanabe et al. 2005) and used as an indicator for acid soil. since it is tolerant to acid and al stresses, it is very important to use this plant as genetic resources for tolerance genes to acid and al stresses. tolerant plants to al are very important to increase agricultural production in indonesia because indonesia has about 47.5 million hectares of pod soil yellow-red land which have low ph and high solubility of al (csar 1997). metallothionein (mt) is a protein having low molecular weight, around 4-8 kda (vallee 1991). it is constituted of 45-48 amino acid residues containing 12-17 conserved cysteine residues (kagi 1991). cobbett and goldsbrough (2002) biotropia vol. 16 no. 1, 2009: 28 37 corresponding author: sony@rcbio.org; sony-sh@ipb.ac.id, sony.suharsono@yahoo.com 29 have classified mt into four types based on distribution of cysteine residues in the c-terminal and n-terminal regions. type 1 of mt is consisted of two domains with metal binding motif cys-x-cys. type 2 is composed of two domains with metal binding motif in combination among cys-cys, cys-x-x-cys, and cys-x-cys. type 3 is also called as a phytochelatin (pc) and has a structure of (γ-glu-cys)n-gly, whereas type 4 contains rich in cysteine in repeated sequence. mt can bind heavy metal, and has an important role in the detoxifying of some heavy metal ions (clemens 2000, hall 2002), like zn (nigel et al. 1996). th e expression of bjmt2 in a. thaliana increased the seedling tolerance against co and cd (zhigang et al. 2006). th e expression of mt2 gene is induced by al stress in wheat (snowden et al. 1995), by h2o2 and metal ion in rice (zhou et al. 2005), by cu and co in a. thaliana (zhou and goldsbrough 1995), by drought stress in wild watermelon (akashi et al. 2004) but it is down regulated by small gtpase osrac i in rice (wong et al. 2004). mt2 gene had been isolated from soybean (kawashima et al. 1991), vicia faba (foley and singh 1994), rice (hsieh et al. 1995) and broccoli (yang et al. 2000). th is research has the objective to isolate and to clone the cdna of gene encoding for metallothionein type 2 from m. affi ne l. since the expression of mt2 is induced by al stress (snowden et al. 1995), we suppose that the over-expression of this gene can increase the plant tolerance to al. by this reason, this gene can be used to improve genetically the tolerance of important plant to al by over-expressing the mt2 gene. materials and methods materials young leaves of m. affine were used as plant material. pgem-t easy (promega) was used as cloning vector. escherichia coli dh5α was used as a host for recombinant plasmid. primers of actf (atggcagatgccgaggatat), and actr (cagttgtgcgaccacttgca) designed based on complete nucleotide sequence of a soybean actin gene (shah et al. 1982, genbank v00450) were used to amplify exon1-exon2 of β-actin as a control of cdna. primers of mf (tcgagaaaaatgtcttgctgtg), and m7r (cttcacttgcaggtgca agg) designed based on arabidopsis thaliana mt2a mrna (genbank nm_1117733) were used to isolate mt2 cdna of m. affine. total rna isolation young leaves (1 g) were ground with pestle in the mortar in the presence of 0.3 g sands and 10 ml warm extraction buffer (2% ctab, 2% pvp 25000, 100 mm trishcl ph8, 20 mm edta, 1.4 m nacl, 1% β-mercaptoethanol) previously heated at 65oc. this suspension was poured into 20 ml centrifuge tube, and incubated at 65oc for 10 minutes, and then added by 10 ml of chloroform:isoamylalcohol (24:1). after vortexing to mix this suspension, the tube was centrifuged at 42 000 xg (rotor sw11, sorvall ultra pro 80), 4°c for 10 mins. the upper liquid phase were collected and added by 0.25 volumes of 10 m licl. after incubation at -32oc for 2.5 hours, this suspension was centrifuged at 42 000 xg (rotor sw11, sorvall ultra pro 80), 4°c for isolation and cloning of cdna from melastoma affi ne suharsono et al. 30 10 mins. the pellet containing total rna was suspended by adding 500 ml te (10 mm tris-hcl ph 7.4, 10 mm edta). this total rna suspension was extracted by adding 1x volume of phenol ph 9, then vortexing and centrifuging at 14000 rpm (jouan br4i) at 20 oc for 10 mins. the total rna suspension was recovered from the upper phase, and then extracted by 1x volume of phenol:chloroform:isoamylalcohol (25:24:1), then centrifuged at 15 000 rpm, 20oc for 10 min. the total rna contained in upper phase was precipited by adding 0.25 volume of 10 m licl, and incubated at -32oc overnight. this total rna suspension was centrifuged at 15 000 rpm, 4oc, 10 min. total rna pellet was washed by 500 ml ethanol 70% and centrifuged at 15 000 rpm, 4oc, 10 min. after drying with vacuum dryer total rna was added by depc treated h2o to make a suspension. synthesis of total cdna total cdna was synthesized by mixing 5 μg total rna, 4 μl 5x buffer superscript iii reverse transcriptase (invitrogen), 20 pmol oligo(dt), 4 mm dntp, 10 mm dtt, 1 u enzyme superscripttmiii rtase dan depc treated h2o in the final volume of 20 μl. reverse transcription to synthesize total cdna was carried out at 52oc for 50 mins by using pcr machine (mj research tm 100). the purity of total cdna was verified by using pcr with specific primers of for cdna of exon1-exon2 of β-actin. the composition of pcr was 1 μl total cdna, 1x taq buffer, 40 mm mgcl2, 4 mm dntp mix, 20 pmol actf primer, 20 pmol actr primer, 4% dmso, 0.75 u taq dna polymerase (toyobo) and h2o in the final volume of 20 μl. the condition of pcr was pre-pcr 95°c, 5 mins, denaturizing at 94°c, 30 seconds, annealing at 57°c, 30 seconds, extension at 72°c, 1.5 min, and post-pcr at 72°c, 5 min. the pcr was conducted for 35 cycles. isolation of mamt2 cdna by pcr the composition of pcr was 1 μl total cdna, 2 μl 10x buffer taq, 4 mm dntp, 20 pmol mf primer, 20 pmol m7r primer, 4% dmso, 0.75 u taq dna polymerase (toyobo) and h2o in the final volume of 20 μl. the pcr was conducted for 35 cycles at 95°c, 5 min. for pre-pcr, 94°c, 30 seconds for denaturising, 60oc, 30 seconds for annealing, 72°c, 1.5 min. for extension and 72°c, 5 min. for postpcr. cloning mamt2 cdna into pgem-t easy mamt2 cdna of pcr product (3 ml) was mixed with 10 ng pgem-t easy (promega), 3 u t4 dna ligase (promega), 1x ligation buffer, and h2o in the final volume of 10 μl. the reaction was carried out at 4oc overnight. the ligation product was introduced into e. coli dh5α as described by suharsono (2002). selection of e. coli containing mamt2 e. coli containing mamt2 was selected based on the resistance to ampicillin and blue-white selection. the insert of mamt2 in the white ampicillin resistant colony was confirmed by pcr-colony. the colony was picked up by tooth-picker, then suspended in 5 ml h2o, and heated at 95 oc, 10 mins and cooled at 15oc for 5 mins. the suspension was added by 1.5 μl 10x buffer taq, 3 mm dntp, 15 pmol biotropia vol. 16 no. 1, 2009 31 mf primer, 15 pmol m7r primer, 4% dmso, 0.75 u enzim taq dna polymerase (toyobo). pcr was conducted in the same condition as for mamt2 cdna isolation. the insert of mamt2 cdna was also confirmed by plasmid dna isolation as described by suharsono (2002). the plasmid dna was digested by ecori at 37oc for 2 hours. analysis of cdna dna was sequenced by using automated dna sequencer abi prism 3100 version 3.7. local alignment analysis of cdna was carried out by using blast2 program (http://www.ebi.ac.uk/blast2) (mount 2001). restriction sites in the cdna of mamt2 was analyzed by neb cutter program (http://www.firstmarket.com/cutter/ cut2.html). results and discussions total rna isolation total rna of m. affine had been successfully isolated from young leaves and the efficiency of isolation was 118-213 μg rna/g leaves. the absorbent ratio of λ260/λ280 was 1.4-1.6. it showed that these total rnas were probably contaminated by remaining protein and/or phenol. the purity of total rna is high if the λ260/ λ280 absorbent ratio is around 1.8-2.0 (saunders and parker 1999). electrophoresis analysis in formaldehyde denaturized agarose gel in the mops buffer showed that the total rna contained two prominent bands. these two bands were corresponded to the integrated 28s and 18s ribosomal rna (figure 1). since the total rna contained integrated 28s and 18s rrna, the integrity of total rna was also high, and could be used as template for total cdna synthesis. the integrity of mrna is very important to isolate the full length of coding sequence of dna. to use the gene for genetic improvement by over-expression, the full length of gene is indispensible. figure 1. electrophoresis of total rna of m. affine isolated in different time from young leaves. 1=10 o’clock, 2=12 o’clock, and 3=14 o’clock. total cdna synthesis by using total cdna as template and specific primer for cdna of β-actin, amplification by pcr resulted to 450 bp band corresponding to cdna of exon1-exon2 of β-actin (figure 2). the result showed that the total cdna had been successfully synthesized and this total cdna was free from the contamination by genomic dna. if genomic dna contaminated the total cdna, the amplification of β-actin would result to two bands, 450 bp and 550 bp, corresponding to amplified cdna and isolation and cloning of cdna from melastoma affi ne suharsono et al. 32 genomic dna respectively. the 550 bp dna was resulted from the amplification of exon1-intron-exon2 of β-actin, and the size of intron beetwen exon1 and exon2 is around 100 bp (shah et al. 1982). this intron is spliced during the mrna synthesis, so cdna derived from mrna does not contain this 100 bp intron. figure 2. amplification of exon1-exon2 cdna of β-actin. 1= 100 bp dna marker, 2= exon1-exon2 cdna of β-actin cdna is very important for genetic engineering of eucaryotic organisms. this dna represents the coding region of gene. the size of cdna is equal or smaller than the corresponding dna of gene. since the size is smaller, the expression of gene by using cdna is more efficient than by using dna of gene. therefore, the cdna of gene is more preferable than dna although the isolation of gene by using cdna is more difficult than by using dna in eucaryotic organisms. isolation and cloning of mamt2 cdna pcr by using total cdna as template and mf and m7r as primers of resulted around 250 bp dna (figure 3). this result was the same as predicted because the size of mt2 in several plant species is around 250 bp as found in soybean (kawashima et al. 1991), wheat (snowden and gardner 1993), a. thaliana (zhou and goldsbrough 1994), and rice (zhou et al. 2006). this cdna was then inserted in pgem-t easy, and the recombinant plasmid dna was successfully introduced into e. coli dh5α shown by the white colonies grown in the selective media containing ampicillin, x-gal and iptg. pgem-t easy plasmid contains lacz in its cloning site (cs). only e. coli containing plasmid can survive in the media containing ampicillin and only e. coli containing recombinant plasmid can grow in white colony in the presence of x-gal and iptg in the media. the insertion of cdna in the lacz found in the cloning site caused the inactivation of lacz to be expressed. on the other hand, if there are no insertions in the lacz, this gene is expressed to produce β-galactosidase and this enzyme will convert uncolored x-gal to become blue color. as a result, the color of colonies which do not contain the recombinant plasmid is blue. biotropia vol. 16 no. 1, 2009 33 figure 3. cdna of mamt2 resulted by pcr to confirm the inserted cdna of mamt2, the white colony was picked up and used as template for pcr-colony. the pcr-colony resulted 250 bp dna (figure 4). the plasmid dna was successfully isolated from this white colony. the digestion of this plasmid dna with ecori resulted two fragments of dna, one is 250 bp and the other is 3 kb corresponding to the cdna of mamt2 and pgem-t easy cloning vector, respectively (figure 4). this result showed that the white colony contained mamt2 cdna found in the pgem-t easy. figure 4. analysis of cdna insert in pgemt-easy recombinant by pcr-colony (2) and ecori digestion (3). 1= 1 kb plus dna marker. analysis of cdna of mamt2 based on sequencing from sp6 primer, the recombinant pgem-t easy contained insert dna of 257 bp consisted of 246 bp open reading frame encoding for 81 amino acid residues (figure 5). alignment analysis by blastn showed that this cdna had similarity of 100% with mrna of atmt2a of a. thaliana (accession: nm_111773.3), 90% with mrna of bjmt2 of brassica juncea (y10850.1), 89% with mrna of brmt of b. rapa (d78498.1), 88% with mrna of bcmt of b. camprestris (l31940.1), and 88% with mrna of bomt2 of b. oleracea (af200712.1). this alignment analysis showed that the cdna isolated in this research is a full length of mamt2 of m. affine containing start and stop codons. isolation and cloning of cdna from melastoma affi ne suharsono et al. 34 figure 5. nucleotide and deduced amino acid sequences of mamt2. underline sequences indicate the forward and complementary reverse primers of used to isolate mamt2, bold letters indicate start and stop codons respectively, italic letters indicate the cys amino acids. restriction site analysis showed that mamt2 contains bbvi, bsabi, btsci, tsei, foki, apeki, cspci, hpych4iii, pshai, bsrfi, sgrai, acui, btgzi, mboii, sfani, cac8i and hpy188i sites (figure 6). these sites cannot be used to clone and engineer mamt2 because they can cut mamt2 in two or more fragments. fortunately, mamt2 does not contain any restriction site found in cs of pgem-t easy. it means that, all restriction sites found in cs can be used to isolate the mamt2 cloned into pgem-t easy. figure 6. restriction site map of mamt2. to perform genetic engineering, the information of the nucleotide sequence and the restriction sites of the gene are indispensable. the nucleotide sequence information can be used to modify the sequence by in vitro directed mutagenesis. the information of restriction sites of the gene is very important to decide the restriction enzymes which will be used to cut off the gene and then to fuse with other genes or expression elements as promoter and terminator. biotropia vol. 16 no. 1, 2009 35 deduced amino acid residues demonstrate that mamt2 contains 81 amino acid residues with 14 cysteine residues. this result is in accordance with the number of amino acid residues and cysteine residues of mt type 2 from several plants (kojima 1991). analysis of cysteine sequence motif showed that mamt2 has a motif cys-cys (3rd-4th residues), cys-x-cys (8-10, 14-16, 67-69, 73-75, 78-80) and cys-x-x-cys (20-23) (figure 5). this specific cys sequence demonstrates that mamt2 has the similar motif of cys distribution with mt type 2 of several plants (robinson et al. 1993; cobbett and goldsbrough 2002). the full length of mamt2 cdna isolated in this report is the first mt family genes isolated from m. affine as the indigenous plant in tropical rain forest like indonesia. since this plant is very tolerant to al, the mamt2 can be used to improve genetically the important plant for indonesia as soybean. the genetic improvement can be done by over-expressing mamt2. we are now targeting the over-expression of mamt2 in soybean to improve the resistance to al by using strong promoter. in the near future, we also would like to over-express this mamt2 cdna in jatropha curcas. conclusions the total rna of m. affine had been isolated and converted into total cdna. the full length of cdna of mamt2 gene had been isolated from this total cdna and cloned into pgem-t easy. this mamt2 cdna contains 246 bp and encodes 81 amino acids. mamt2 contains 14 cys amino acids distributing in cys-cys, cys-xcys, and cys-x-x-cys motifs. this full length of mamt2 cdna is the first cdna of mt family gene isolated from m. affine. it can be applied to improve genetically the al tolerance of plant. acknowledgments we would like to thank biotrop-seameo and directorate of research and community services, directorate general of higher education, ministry of national education, republic of indonesia for the financial support of this research by contract agreement no. 13.1/psrp/sp-pen/iv/2005 and spk 008/hikom/ dp2m/2008, respectively. references akashi k., n. nishimura, y. ishida, and a. yokota. 2004. potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon. biochemical and biophysical research communications, 323: 72-78. 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enzymology , 205: 613-626. kawashima i., y. inokuchi, m. chino, m. kimura, and n. shimizu. 1991. isolation of a gene for a metallothionein-like protein from soybean. plant cell physiology , 32: 913-916. kojima y., p.a. binz, and j.hr. kagi. 1999. nomenclature of metallothionein: proposal for a revision. in: metallothionein iv, c. klaassen. birkhauser-verlag. basel. mount d.w. 2001. bioinformatics: sequence and genome analysis. new york: cold spring harbor laboratory press. robinson n.j., a.m. tommey, c. kuske, and p.j. jackson. 1993. plant metallothionein. journal of biochemistry, 295: 1-10. nigel j. r., j.r. wilson, and j. s. turner. 1996. expression of the type 2 metallothionein-like gene mt2 from arabidopsis thaliana in zn2+-metallothionein-defi cient synechococcus pcc 7942: putative role for mt2 in zn2+ metabolism. plant molecular biology, 30: 1169-1179. saunders g.c., and h.p. parker. 1999. analytical molecular biology: quality and validation. teddington: lgc press. snowden k.c., 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osrac i in rice. plant physiology, 135: 1447-1456. zhigang a., l. cuijie, z. yuangang, d. yejie, a. wachter, r. gromes, and t. rausch. 2006 expression of bjmt2, a metallothionein 2 from brassica juncea, increases copper and cadmium tolerance in escherichia coli and arabidopsis thaliana, but inhibits root elongation in arabidopsis thaliana seedlings. journal of experimental botany, 57 (14): 3575-3582. yang c.y., y. lin, and j.f. shaw. 2000. cloning and characterization of a cdna encoding a mt2 type metallothionein from broccoli (brassica oleracea cv. green king) fl orest. plant physiology, 122: 1457. biotropia vol. 16 no. 1, 2009 37 zhou j., and p.b. goldsbrough. 1995. structure, organization and expression of the metallothionein gene family in arabidopsis. molecular and general genetics, 248: 318-328. zhou g., y.f. xu, and j.y. liu. 2005. characterization of a rice class ii metallothionein gene: tissue expression patterns and induction in response to abiotic factors. journal of plant physiology, 162: 686696. isolation and cloning of cdna from melastoma affi ne suharsono et al. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html doi: 10.11598/btb.2015.22.2.367 effect of substrate moisture and invasive grass competition on native fig ( ) seedling ficus fistulosa recruitment in limestone quarries annisa satyanti* center for plant conservation, bogor botanical garden, indonesian institute of sciences, bogor 16003, indonesia received 10 february 2014 /accepted 4 february 2015 abstract this study was conducted to determine the possibility of native fig ( ) for rehabilitating degraded ficus fistulosa habitats in limestone quarries. a greenhouse experiment was carried out to test the effect of different substrate moisture levels and competition between native fig ( ) and invasive grass on the ficus fistulosa pennisetum polystachyon native fig's growth and survival. was chosen as the studied species because it has high importance value ficus fistulosa index (ivi) compared to other fig species tested in ciampea limestone hill. the results showed that the substrate moisture levels did not affect the native fig biomass and the invasive grass biomass was not reduced by drought. the interaction of substrate moisture levels and competition from invasive grass reduced overall fig biomass, but not the leaf numbers and individual plant size. this study suggested that for quarry rehabilitation, invasive species management should be advocated along with soil treatment. keywords:ficus fistulosa competition , quarried limestone, rehabilitation, substrate moisture introduction limestone regions over the world along with their biodiversity are threatened mainly by limestone quarrying causing land degradation problems due to soil depletion and alteration of land topography. vegetation removal and lack of available soil on steep slopes induce a very high risk of erosion in limestone ecosystems (clemente . 2004). the common method of et al limestone quarrying increases drainage, physical and chemical erosion of the substrate which hinder natural germination and establishment of seedlings, thus delaying re-colonization (clemente et al. 2004). during the rainy season, water run-off is high, while the open sites are easy to dry up. plant recruitment in newly disturbed areas, like in an over-quarried limestone, is determined by water availability and soil moisture (soliveres 2012). the large number of abandoned quarries in many countries presents challenges for restoration of these degraded habitats (yuan . 2006). et al revegetation is among common strategies to restore the abandoned quarries (zhang chu & 2011). limestone quarries revegetation is challenging because not all plants are able to cope with relatively barren rock cliff and absence of topsoil or humus. substrate moisture is the most limiting environment variable influencing seedling emergence and survival, particularly in arid ecosystems (soliveres 2012), such as of these abandoned limestone quarries. in addition to water availability, competition against fast growing invader such as exotic grass can be critical during the seedling stage of the desired native plants. understanding factors that restrict the establishment and growth of native plant species can aid efforts for species conservation in their habitats and can expand options for species recovery, including restoration, translocation and ex-situ et al. conservation (maschinski 2004). native plant species is favoured for rehabilitating disturbed ecosystems (khater et al. 2009). is potential for limestone quarries ficus rehabilitation. in the abandoned limestone quarries in ciampea, bogor, west java, indonesia, * corresponding author : annisa.satyanti@lipi.go.id biotropia 2 95 101 vol. 22 no. , 2015: 95 mailto:annisa.satyanti@lipi.go.id 96 ficus is abundant in the remaining limestone forests (satyanti & kusuma 2010). previous study conducted by satyanti and kusuma (2010) ranked fourteen species ficus from ciampea limestone hill, i.e. , ficus annulata f. fistulosa f. grossularoides f. hirta f. montana f. , , , , pinnata f. sagittata f. septica , , and six other unidentified species based on their ficus importance value index (ivi). was ficus fistulosa found to have the highest ivi value in the limestone habitat in ciampea (satyanti & kusuma 2010) and hence, it is selected as the studied species in the current experiment. this study was conducted to determine the possibility of native fig ( ) for ficus fistulosa rehabilitating degraded habitats in limestone quarries. a greenhouse experiment was carried out to test the effect of different substrate moisture levels and competition between native fig ( ) and invasive grass ficus fistulosa pennisetum polystachyon .on the native fig's growth and survival materials and methods study site the limestone site in ciampea is a long coral reef raised to above 350 m above sea level, located at 106°41'00.0" e and 06°33'00.0" s. the formation of limestone is of the bojongmanik formation which is equal to a middle mioscene age (effendi . 1998). the forest used to have et al n u m e r o u s d i p t e r o c a r p u s h a s s e l t i i (dipterocarpaceae), stel echocar pus burahol (annonaceae) and a number of diospyros (ebenaceae), but no single species was dominant (van steenis 1931 in whitten & soeriaatmadja 1996). study species native fig reinw. ex. blume ( )ficus fistulosa common yellow stem fig (moraceae) is an evergreen tree and grows to about 10–15 cm in trunk diameter. it is native to malesia floristic region, including indonesia. it has a straight trunk with smooth bark ranging from light grey to yellowish in colour. the young twigs are hollow and easily breaks. leaves of this tree are alternately arranged, the young leaves are pale pink in colour. figs are pear shaped ±2.5 cm wide, borne on 2.5–5 cm long stalks and in cluster on woody knobs on the trunk and branches. figs ripened from yellow to green yellow. the flowers are found within the figs; with male and female flowers located at different trees. individual trees bears 4–7 crops a year. in java and borneo, fig is pollinated by fig wasps, and ceratosolen constrictus seed dispersal is mainly carried out by bats (genus dyacopterus ficus fistulosa). does not have aggressive roots like other strangler . hence, it is an ficus excellent tree to attract wildlife back to both native habitat and urban environment. it is also ethno-botanically important; in some parts of indonesia the young leaves are eaten as salads. a decoction of the leaves is given to women after childbirth and the latex has been used to treat headache (corner 1988). invasive grass schult )( ( )l.pennisetum polystachyon pennisetum polystachyon or widely known as mission grass is native to tropical africa. it is an annual or perennial plant; the culm is simple or branched; the branches are often flowering. spikelet length is 3–5 mm; false spike is 8–10 mm, excluding the bristle; the longest bristle is 15–55 cm long. when mature the spikelets break off at the control axis together with the bristles. it produces few tillers per plant. the distribution of this grass is throughout the tropics up to 1,500 m asl (above sea level). this grass requires high rainfall, but it is also grown in semi-arid regions. it tolerates both acid and alkaline soils. pennisetum polystachyon spreads readily by seeds which survive annual burning. seedling vigour is good, even in poor soil conditions (clayton . 2006).et al in indonesia it is considered invasive species (tjitrosoedirdjo 2005). seed germination of the native fig and the invasive grass fruits of the native fig and panicles of the invasive grass were collected from the limestone quarry in ciampea, bogor-west java. the limestone hill in this site is a long coral reef raised to above 350 m asl and has been quarried for more than thirty years. (dipterodipterocarpus hasseltii carpaceae), (annonaceae) stelechocarpus burahol and a number of (ebenaceae) species diospyros were used to be abundant in this limestone forest, but no single species was dominant (van steenis 1931 in whitten & soeriaatmadja 1996). the biotropia vol. 22 no. 2, 2015 e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – above mentioned species are now infrequently present in this habitat. ficus fistulosathe fruits of and panicles of the grass and limestone substrate were subsequently brought to the greenhouse of the bogor botanical garden, where fur ther experiment was conducted. the fruits of the native fig were then cut open and seeds were collected. seeds were then air dried for 1–2 hours prior to sowing. seeds were sown on sand in a tray. after 10–14 days seeds of the native fig were germinated and were kept to grow in the sowing tray before being transplanted to limestone substrate f or competition experiment. in parallel, panicles of the invasive grass were sown on sand in different sowing tray. after 3–4 weeks, seeds were germinated and grass seedlings were kept until leaf sheaths reached 5–10 cm length or about a week after sowing. fig and grass seedlings were subsequently transplanted to the treatment pots and watered until substrates were fully saturated and substrate moisture treatment was applied from the next watering top-up. competition experiment factors involved were substrate moisture and competition. substrate moisture treatments were 34 ml/week and 68 ml/week, inducing water stress (drought) for the low moisture treatment. this water volume was chosen based on personal observation that 68 ml/week was able to keep the substrate well saturated and the surface was adequately moist. each pot contained limestone substrate weighing 250 g. limestone substrates were collected from the quarried in ciampea. the substrate from these abandoned quarries was in the form of a mixture of coarse and fine gravel of limestone. two levels of competition were employed, i.e. with and without grass competition. in a competition pot, one seedling of native fig was planted with two seedlings of invasive grass. one-month-old native fig seedlings with 2–3 true leaves, ±2–3 cm tall were transplanted to limestone substrates in 9 cm (diameter) and 12 cm (depth) free draining pots for competition experiment. invasive grass seedlings were transplanted to competition pots at the same time. the experiment set–up was a factorial block design assigning each treatment and its combination within each block. for each treatment combination, six replicates were used and all were arranged in five blocks. thus, each block consisted of 24 pots, in which six pots of low soil moisture and no competition, six pots of low soil moisture and with competition, six pots of high soil moisture and no competition, and six pots of high soil moisture and with competition, leading to a total number of 120 pots. the competition experiment were maintained and observed for eight months. at the end of the experiment, measurements were made for native fig seedlings' height, leaf number and biomass. grass above and belowground biomass were also measured. data analysis in order to disentangle the effect of substrate moisture and competition on native fig seedling growth, a two-way anova was carried out. in addition, the interactions between substrate moisture and competition against invasive grass were also tested to determine whether or not those two factors affect the growth of native fig. for competition pot, invasive grass aboveand below-ground biomass were also tested against substrate moisture. subsequently, correlation between the native fig and invasive grass biomass was analysed. all calculations were performed using software spss ver. 15.0. results and discussion substrate moisture effect on native fig and invasive grass growth of native fig during the eight month obser vation was relatively slow period considering their nature as a woody tree growing in tropical climate. watering at 68 ml/pot/week significantly reduced native fig biomass compared to the 34 ml/pot/week but the effect of substrate moisture on native fig plant size and leaf number was on the other hand benign (fig. 1). grass biomass, however, was independent from the substrate moisture suggesting that invasive grass might be quite resistant to drought or water availability fluctuation (fig 2). . 97 biotropia vol. 22 no. 2, 2015 98 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 2 4 6 8 fig ure 1 p 38.537 173.251 native fig preferred lower water regime between competition treatments (f = 462.02, = 0.16 and f = 250.605, for aboveand below-ground, respectively). there is no effect of substrate moisture on fig seedling length and leaf number between competition treatments 34 ml/ week 68 ml/ week substrate moisture 0,0 0,2 0,4 0,6 34 ml/ week 68 ml/ week substrate moisture 0,0 0,2 0,4 0,6 fig ure aboveand below-ground grass biomass were not affected by the substrate moisture2 99 even though the pots were freely drained, the bulky and clayish nature of the limestone substrate may lead to biomass reduction of the native fig. , in general, tends to grow better in ficus aerated substrate and intolerant to submersion, even temporarily (parolin & wittmann 2010). there was no significant effect of substrate moisture to aboveand below-ground ficus biomass when grass was present. however, there was a significant reduction in biomass under higher moisture treatment in the absence of grass (fig. 3). effect of presence of invasive grass on native fig native fig aboveand below-ground biomass, leaf number and seedling length were reduced because of the presence of invasive grass (fig. 3 and 4). from the competition point of view, invasive grass contribution in suppressing native fig growth can either be from water and nutrients competition or light competition. moreover, at the end of the experiment seedlings height ficus were mostly exceeded by grass leaf sheaths. however, there was no correlation between native fig and invasive grass growth (table 1). it might be that other variables that were not measured had more influence. for instance, allelopathy from exotic grass had more influence on native fig biomass than merely moisture and neighbour growth. grass roots are known to have pennisetum allelopathic chemicals (zain 2013) and et al. adversely affects (tan . 2012), cyperus indica et al leptochloa chinensis hedyotis verticillata and (norhafizah . 2012). is, on the other et al ficus hand, known to have phytotoxic effect on grasses (siddiqui . 2009).et al figure 3 invasive g rass significantly reduced native fig aboveand below-ground biomass (f = 592.572, = 0.000 and 49.353 p f = 322.514, = 0.000, respectively). both aboveand below-ground biomass were very small at the presence 222,965 p of grass. there was a significant interaction effect between competition and substrate moisture to fig aboveground (f = 469.997; = 0.000) and below-ground biomass (f = 252.918; = 0.000). for both aboveand 39.144 174.851 p p below-ground biomass, there was no significant effect of substrate moisture when grass was present, while there was a significant reduction in biomass under higher moisture treatment in the absence of grass table 1 significance value for relationship between biomass biomass r2 p significance grass below-ground – fig below-ground biomass 0.010 0.436 ns grass below-ground – fig above-ground biomass 0.002 0.769 ns grass above-ground – fig above-ground biomass 0.001 0.770 ns grass above-ground – fig below-ground biomass 0.009 0.477 ns e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – 34 ml/ week 68 ml/ week substrate moisture 34 ml/ week 68 ml/ week substrate moisture 100 biotropia vol. 22 no. 2, 2015 interaction effect of substrate moisture and competition to fig growth and survival it was apparent that the interaction of substrate moisture and presence of competition from grass reduced biomass of fig above-ground and below-ground (fig. 3). there was no significant interaction effect on fig seedling size and leaf number even though it was evident that fig seedling leaf was reduced under the presence of grass (fig. 4). in general, native fig seedling length and number of leaf were not adversely affected by substrate moisture (fig.1), but rather by competition (f = 60.054; = 0.000 and 64.827 p f = 76.056; = 0.000, respectively; fig. 4). our 182.533 p current study showed that the effect of grass competition was larger than the differences in substrate moisture (fig. 1, 3 and 4). under greenhouse conditions, the native fig seeds germinated well on sand. the seedlings were able to grow on limestone substrates without addition of humus, but it was unknown whether seeds could germinate in novel soils under field conditions. until the end of the experiment, all fig seedlings survived regardless their small growth increment. it is not clear whether or not the seedlings will be able to cope further when the experiment is prolonged or when they are transplanted in the field. field trial showed that seedling establishment in limestone quarries varies dramatically with abiotic and biotic factors and it is often difficult to successfully reintroduce the seedling in the field (maschinski 2004).et al. as the native fig species showed very limited growth in this study, one perhaps would argue to use other fast growing species regardless the fact that they are non-indigenous. the use of native rhizobia-symbiosis forming plants can be used to enhance the success rate of limestone revegetation (jha . 1995). nevertheless, the et al use of native plants has many benefits in restoration schemes, such as their high adaptability to environmental stresses and low ecological risk (ballesteros 2012; kirmer et al. et al. 2012). in the field, it is quite challenging to find recognizable number of native fig recruitment in the quarried areas. besides inhospitable soil moisture and surface characteristics (soliveres et a l . 2 0 1 2 ) n a t u r a l e s t a b l i s h m e n t a n d reintroductions on unoccupied limestone field has probably been limited by poor natural dispersal. conclusions this study showed that the aboveand belowground native fig biomass was reduced mainly due to competition against invasive grass. the effect of grass was also pronounced to other fig traits, leaf number and seedling size. grass biomass, however, was not affected by substrate moisture. the effect of grass competition was larger than the differences in substrate moisture. f 4 igure competition against grass also significantly reduced the size of seedling and leaf numbers (f = 60.054; = 0.000 64.827 p and f = 76.056; = 0.000, respectively). there was no interaction effect between competition and substrate 182.533 p moisture to fig seedling size 34 ml/ week 68 ml/ week substrate moisture 34 ml/ week 68 ml/ week substrate moisture 101 study on the effect of allelopathy from invasive grass and neighbouring plants in a field experimental set-up as well as other potential native plants for limestone revegetation need to be carried out to develop better management of quarried limestone hill. acknowledgements the study was supported by grant no. 46.06.08 “an impact assessment of limestone quarries on flora diversity in ciampea, bogor” from the rufford foundation, uk. references ballesteros m, cañadas em, foronda a, fernándezondoño e, peñas j, lorite j. 2012. vegetation recovery of gypsum quarries: short-term sowing response to different soil treatments. app veg sci 15: 187-97. clayton wd, vorontsova ms, harman kt, williamson h. [internet]. 2006 onwards. grassbase – the online world grass flora. [cited 2013 april 15]; available from: http:// www.kew.org/data/grasses-db.html clemente as, werner c, máguas c, cabral ms, martinsloução ma, correia o. 2004. restoration of a limestone quarry: effect of soil amendments on the e s t a b l i s h m e n t o f n a t i v e m e d i t e r r a n e a n sclerophyllous shrubs. restoration ecol 12: 20–8. corner ejh. 1988. wayside trees of malaya third edition vol 1 – 2. kuala lumpur (my): malayan nature society. effendi ac, kusnama k, hermanto b. 1998. geological map of bogor quadrangle, java. bandung (id): geological research and development centre. jha pk, nair s, gopinathan mc, babu cr. 1995. suitability of rhizobia-inoculated wild legumes argyrolobium flaccidum astragalus graveolens indigofera gangetica , , and lespedeza stenocar pa in providing a vegetational cover in an unreclaimed limestone quarry. plant soil 177: 139–49. khater c, martin a, maillet j. 2009. spontaneous vegetation dynamics and restoration prospects for limestone quarries in lebanon. appl veg sci 6: 199-204. kirmer a, baasch a, sabine t. 2012. sowing of low and high diversity seed mixtures in ecological restoration of surface mined-land. appl veg sci 15: 198-207. maschinski j, baggs je, sacchi cf. 2004. seedling recruitment and survival of an endangered limestone endemic in its natural habitat and experimental reintroduction sites. am j bot 91 (5): 689–98. norhafizah mz, ismail bs, chuah ts. 2012. herbicidal activity of (napier grass). afr j pennisetum purpureum biotechnol 11(23): 6269–73. parolin p, wittmann f. 2010. struggle in the flood: tree responses to flooding stress in four tropical floodplain systems. aob plants 1-54. satyanti a, kusuma ywc. 2010. ecological study in two quarried limestone karsts hills in bogor west java: vegetation structure and floristic composition. biotropia 17 (2): 115–29. siddiqui s, yadav r, yadav k, wani fa, meghvansi mk, shar ma s, jabeen f. 2009. allelopathies potentialities of different concentration of aqueous leaf extracts of some arable tree on germination and radicle growth of var. c-235. glob j cicer arietinum mol sci 4 (2): 91-5. soliveres s, monerris j, cortina j. 2012. irrigation, organic fertilization and species successional stage modulate the response of woody seedlings to herbaceous competition in a semi_arid quarry restoration. appl veg sci 15(2) : 175-86. tan pw, chuah ts, ismail bs. 2012. allelopathic potential effect of on . in: pennisetum purpureum cyperus iria the 2 international conference on environmental nd and agriculture engineering. proceedings: 2012 june 29-june 30; jurong west (sg): iacsit press. p 109–13. tjitrosoedirdjo ss. 2005. inventory of the invasive alien plant species in indonesia. biotropia 25: 60–73. whitten aj , soeriaatmadja rs. 1996. the ecology of java and bali. singapore: periplus editions. yuan jg, fang w, fan l, chen y, wang d-q, yang z-y. 2006. soil formation and vegetation establishment on the cliff face of abandoned quarries in the early stages of natural colonization. restor ecol 14: 349–56. zain nm, yew oh, sahid i, seng ct. 2013. potential of napier grass ( ) extracts as a pennisetum purpureum natural herbicide. pak j bot 45: 2095–100. e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – http:// http://www.kew.org/data/grasses-db.html 5. samsuri (restoration) revi... biotropia vol. 21 no. 2, 2014: 111 124 restoration priority index development of degraded tropical forest landscape in batang toru watershed, north sumatera, indonesia samsuri , i nengah surati jaya , cecep kusmana and kukuh murtilaksono , received 15 april 2014/accepted 6 september 2014 tropical forest fragmentation has triggered forest degradation and decreased forest connectivity. further, degradation of tropical rain forest area reduces forest functions as global biological resources and affects livelihood of rural communities. to regain forest functions, a landscape restoration approach is needed. the research aimed to develop a restoration index model for degraded tropical forest landscapes in batang toru watershed. the proposed restoration index was constructed by four indices i.e. forest degradation, forest connectivity, forest fragmentation, and socio economic indices. regression analysis was conducted to develop models for four indices. discriminant analysis was conducted to obtain restoration index model. the forest landscape fragmentation index increased in the period of 1989-2013, while connectivity index tended to decrease during the same period. forest connectivity index had higher weight than other indices in calculating restoration index. this indicated that connectivity of batang toru forest landscape needed to be maintained and enhanced to ensure habitat quality and reduce loss of biodiversity. the research showed that sarula sub-watershed had high restoration index value, so it should be the first area to be restored. connectivity, fragmentation, fragstats, forest ecosystem, spatial 1* 2 3 4 1 department of forestry, faculty of agriculture sumatera utara university medan, indonesia department of forest management, faculty of forestry, bogor agricultural university bogor, indonesia department of silviculture, faculty of forestry, bogor agricultural university bogor, indonesia department of watershed management, faculty of agriculture, bogor agricultural university bogor, indonesia 2 3 4 abstract keywords: * corresponding author : gsamsuri@gmail.com doi: 10.11598/btb.2014.21.2.5 111 introduction recently, forest management is facing various problems arising either from natural disaster or from human activities such as forest fire, landslide, illegal logging, encroachment, shifting cultivation, and forest conversion. in some areas, the condition of degraded forest has been at a critical level; hence it might threaten the sustainability of forest ecosystem. forest degradation has also become a global issue because it gives significant influence on global climate change. according to itto/iucn (2005), the landscape of world's tropical forests remains 45%, and fragmented spatially in degraded condition. degradation of tropical rain forest reduces global biological resources, and causes poverty occurrence in communities in and around the degraded forest (lamb . 2005). during the period from 1970 to 1990, the degraded forest area in indonesia was estimated to be between 0.6 1.2 million ha per year. the forest degradation rate in the period of 1986 -1997 was about 1.7 million ha per year (fwi/gfw 2002); between 1994 and 2000 it was about 2.83 ha million per year (mof 2005), and in the period of 1970-1990 it was about 1.2 million per year. according to fao (2011), forest decrease in indonesia was at an estimated rate of 0.5% in the period of 2000-2010 and 1.7% in the period of 1990-2000 (fig. 1). this decrease is higher than the average of southeastern asia's which is at the level of 1% in the period of 1990-2000 and 0.4% in the period of 2000-2010 (fao 2011). et al figure 1. deforestation rate in several south east asia countries during 1990 2010 112 biotropia vol. 21 no. 2, 2014 in north sumatera, deforestation was estimated to be 6,508,525 ha (28% of total forest area) in the period of 1985-1997 (fwi/gfw 2002). batang toru forest as one of remaining tropical forest ecosystem in sumatera has deforestation rate of about 1.17% per year from 1994 to 2009. the area decreased from 160,000 ha to 150,000 ha in 2009. batang toru gets high pressure because of the openness of access resulting from forest encroachment and forest cover change. encroachment and forest cover change are the main drivers of forest degradation (mendoza . 2005; reddy 2013). degraded forest may cause flood, erosion and landslides (cotler & ortegalarrocea 2006). on the other hand, batang toru forest landscape is habitat for 67 species of mammals, 287 species of birds, 110 types of herpetofauna and 688 types of plants. batang toru is the habitat of the orang-utan ( ). the numbers of orang-utan are about 15% of total population of orang-utan in sumatera which is estimated to be about 6,600 individuals. besides providing important habitat for orang-utan, batang toru landscapes are also home for other rare animals such as , and antelopes. furthermore, batang toru forest landscape is also a habitat for unique flora of sumatera such as and several types of orchids (perbatakusuma & kaprawi 2011). the importance of the ecosystem functions of the batang toru as a regulator of hydrologic cycle and wildlife habitat should be maintained. therefore, forest and land degradation should be reduced by regaining landscape condition of batang toru forest through the restoration of forest landscape. forest restoration is done recently by activities to obtain a balancing function between forest conservation and rural community needed (reitbergen-mccraken 2007), regaining ecological integrity function and improving human welfare (mansourian . 1986) in deforested or degraded landscapes. such efforts can provide optimal results if well adapted to the characteristics of the ecosystem or the type of disturbances. recently, the ministry of forestry has a method to determine an index of land degradation based on biophysics condition. the ecosystem functions and socio economics aspects have not been considered as priority for area restoration. in this research, ecosystem functions and socio economics aspects are considered in determining the restoration index. to obtain the index, it needs a model design that expresses the index standard used in planning the activities of forest restoration. this research aimed to develop a restoration index model for degraded tropical forest landscapes in batang toru watershed (fig. 2a). this study was conducted in 3 sub-watersheds of batang toru watershed i.e. puli, sarula, and batang toru hilir sub-watersheds. the three study areas covered three districts i.e. tapanuli selatan, tapanuli utara and tapanuli tengah. this study used landsat images (batistella 2000, apan . 2002) to classify land cover types in several years (1989, 2001 and 2013). those images were landsat tm5 image of 1989, landsat etm7 image of 2001, and landsat oli image of 2013. this study required et al et al. pongo abelii tapirus indicus, panthera tigris sumatrae pardofelis marmorata, helarctos malayanus rafflesia gadutensis et al. et al et al. et al materials and methods 113 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. several thematic maps including watershed, river network, road network, and contour maps. the classification of land cover types was done according to classification scheme used by the ministry of forestry. field surveys were conducted to verify the land cover types and to collect vegetation data. field sample plots were determined by purposive sampling method and laid out by representation of part of watershed (upstream, middle stream and downstream), land system and land cover types. the equipments used in the field survey were gps, compass, haga hypsometer, diameter tape, and questionnaires. analysis of satellite images was conducted using erdas imagine 9.1. envi 4.5. arcgis 9.x was used in the spatial analysis of thematic maps (contour, road, and river maps). fragstats ver 3.3 (mcgarigal & marks 1995; mcgarigal 2002) was used to derive necessary landscape metrics. the research consisted of 5 stages to get the restoration index, as follows: landsat images were interpreted to get the normalized difference vegetation index (ndvi) and land cover types for year of 1989, 2000 and 2013 (jaya 2009). a field check was conducted to verify the classified land cover types. in addition, vegetation data were collected in several square sample plots of 2,500 m (fig. 2b). et al. 1. forest degradation index 2 figure 2. location of study site (a) and design of sample plot (b) 114 biotropia vol. 21 no. 2, 2014 trees in different growth stages were measured in four different size quadrants namely quadrant 1, 2, 3 and 4. an analysis of the vegetation data was conducted to get the degree of forest degradation (kreb 1989, magurran 1988). forest degradation was approached using ndvi value class. ndvi was predicted using vegetation parameters namely species diversity index (x ), basal area (x ), and stand density (x ). each range of ndvi was scored using likert scale (syarifi . 2007) (table 1), then it was used to model forest degradation index through regression analysis (puspaningsih 2011, rohyani 2012). the independent variables were distance to main road (x ), distance to main river (x ) and percent of slope (x ). forest land cover types were analyzed using the fragstats software. fragstats analysis was performed to derive landscape metrics. the range length of orang-utan was used as data input in fragstats analysis. flf degree was calculated based on landscape metrics (mcgarigal & marks 1995). landscape metrics used to determine the degree of flf were patch number per area (patch density), proximity and contiguity (fahrig 2003, mcgarigal 2002). each landscape metric was classified (table 1) and scored using likert scale (syarifi 2007) indicating the degree of flf. hence, it was used to develop flf index by regression analysis (puspaningsih 2011, rohyani 2012). the independent variables were value of contiguity between patch (x ), proximity value of each patch (x ), and the number of patch (x ) 1 2 3 4 5 6 7 8 9 . et al 2. forest landscape fragmentation (flf) index et al. no sub factor value score no sub factor value score 1 ndvi <0 0 – 0.25 0.25 – 0.50 0.50 – 0.75 >0.70 5 4 3 2 1 5 connectan <20 20 – 40 40 – 60 60 – 80 >80 1 2 3 4 5 2 number of patch per area (patch density) >325 285-325 245285 205 –245 < 205 5 4 3 2 1 6 radius of gyration <200 200-400 400-600 600 – 800 >800 1 2 3 4 5 3 proximity >3.270 2.551-3.270 1.234-2.551 555-1.233 <554 5 4 3 2 1 7 employment farmer 5 labour/official employee 3 un-employee 1 4 contiguity <0.2 0.2-0.4 0.4-0.6 0.6-0.8 >0.8 5 4 3 2 1 8 salary/income (million idr/month) <0. 6 6 – 3 3 -6 6– 8 >8 5 4 3 2 1 table 1 score of each sub factors 115 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. 3. forest landscape connectivity (flc) index et al. 4. socio economics index 5. developing restoration index et al. et al flc degree was calculated based on landscape metrics indicating connectivity i.e. radius of gyration and connectan of forest area (fahrig 2003; mcgarigal 2002). each metric was scored (table 1) to determine degree of flc, then it was used to model flc index by regression analysis (puspaningsih 2011; rohyani 2012). the independent variables were value of connectan among forest patch (x ) and value of radius of gyration of each forest patch (x ). socio economics perception of people living surrounding forest area was studied by interviewing them. interview was guided by semi-structured method using questionnaires. each answer was scored using likert scale (syarifi 2007) (table 1). regression analysis was used to model socio economics index (puspaningsih 2011; rohyani 2012). the independent variables were maximum score of income in millions rupiahs earned by people's group around the forest (x ), and score of occupation type group namely farmer group; company labor, government officer and informal sector labor group, and plantation estate owners group (x ). four map indices were overlaid and samples were taken from 225 unit (4 ha per unit). then, restoration index (z) was developed using four indices i.e. forest degradation index (y ), forest landscape fragmentation index (y ), forest landscape connectivity index (y ) and socio economics index (y ). discriminant analysis was perfomed to get a restoration index model (thatam 1998; sifriyani 2012). regression analysis resulted in this study showed that the model of forest degradation was y = 1 + 2.0771x + 0.0094x 1.4675x with determination coefficient of 90%. therefore, spatial and statistical analyses were also conducted and resulted the model of forest degradation index as y = 1.10571 0.00005x 0.00006x 0.00732x with determination coefficient of 63.19%. based on the model of forest degradation index, it was found out that higher stand density value and higher basal area proved that the forest condition were less degraded. vegetation density in three sub-watersheds was low and the estimates from different plots ranged from 12 to 93 individuals per ha, other sumateran lowland mountain natural forest could reach 120 tree per ha (whitten . 1987). the current stand density value indicated that forest cover decreased and it indicated degradation (lund 2009). morever, the nearness to road and river networks trigerred forest degradation because the road and river become people's access to go inside and outside the forest. generally, river and road networks are used to enter the forest. closer distance from river and road networks to the forest means the forest has more pressure than other forest locations. the road and river can be used to estimate forest degradation risk. 10 11 12 13 1 2 3 4 1 2 3 1 4 5 6 results and discussion forest degradation index 116 biotropia vol. 21 no. 2, 2014 forest in the study area was located on relatively high elevation and rough topography. in this condition, slope could cause difficulties in accessing and disturbing forest. human tends to choose land having easier accessibility to settle down, which leads to land conversion. the model of forest degradation was used to derive forest degradation map (fig. 3). forest degradation map divided forest landscape into degradation classes: lower, low, moderate, and high. degradation of forest landscape was located in area close to road and river networks. therefore, forest landscape close to the road should receive higher priority to be restored. figure 3. forest degradation index of batang toru watershed forest landscape fragmentation (flf) index orang-utan is endemic fauna in the forest region of batang toru needing proper habitat in batang toru's forest. one main goal of batang toru landscape restoration was to provide habitat for orang-utan and other biodiversity. the orang-utan habitat characteristics were used as input data in landscape metrics analysis. the day range length of orang-utan was at least 500 m per day. this radius was used as basic calculation for landscape metrics on every 500 m. this radius area was assumed as minimum habitat of orang-utan. furthermore, the day range length of orang-utan was not more than 1,000 m per day. this radius was used as an input for forest edge length of metrics landscape (sinaga 1992). 117 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. 2 7 8 9 regression model of flf index resulted from this study was y = 0.7819 0.576x 0.0000001x +0.0552x , with determination coefficient of 87.20%. based on landscape metrics value, it was known that flf degree increased from 1989 to 2013 which was indicated by the increasing of patch density (forman & godron 1986; forman 1995; fahrig 2003) per ha (fig. 4a) and by the increasing of patch proximity (fig. 4b). patch density in three sub-watersheds (puli, sarula and batang toru hilir) rose from about 0.10 in 1989 to 0.60 in 2013. the area of low flf index to medium flf index was bigger than that of high flf index. most flf index area was in the road distance of 1,000 m to 5,000 m where people could reach out and entered the forest in a day (jaya . 2007). flf index map showed that most forest landscape in batang toru watershed was in low flf index (<0.2), so it was categorized as low flf category (fig. 5a). low flf supported ecosystem function restoration because the material flow in the ecosystem still ran quite well due to low isolated forest patch which would not reduce organism mobility inside the forest. et al figure 4. forest patch density (a) and forest patch proximity (b) figure 5. forest fragmentation (a) and forest connectivity (b) of year 2013 in batang toru watershed 118 biotropia vol. 21 no. 2, 2014 forest landscape connectivity (flc) index forest landscape connectivity (flc) index in batang toru watershed decreased from 1989 to 2013. it was indicated by the decreasing of radius of gyration (fahrig 2003) from 400-700 range in 1989 to 50-200 range in 2013 (fig. 6a) and by the decreasing of connectan value from 3-9% range in 1989 to 0-2% range in 2013 (fig. 6b). regression analysis resulted from this study showed that the model for flc index was y =0.1144+0.0006x + 0.1954x with determination coefficient of 66.16%. most area of puli sub-watershed had medium connectivity index, while high connectivity index belonged to forest path in sarula sub-watershed. a small forest patch which was located in batang toru hilir sub-watershed had very low connectivity index because it was separated by other land cover types. non-forest cover area was located between small forest patches. larger non-forest cover area made small forest patches more separated from others. this will be a barrier for organism movement between forest patches. the derived map of flc index (fig. 5b) showed that puli sub-watershed had the highest flc index compared to sarula and batang toru hilir sub-watersheds. meanwhile, batang toru hilir and sarula sub-watersheds had low flc index. batang toru hilir sub-watershed was located in downstream of batang toru watershed where many lands clearing activities occurred, caused by long distance between forest patch remained. this condition was also occurred in puli sub-watershed located in upper stream of batang toru watershed. 3 10 11 figure 6. radius of gyration (a) and forest patch connectan (b) of each sub-watershed 119 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. flc map also showed that the most forest landscape of sarula sub-watershed had high connectivity index (0.5-0.6). the forest landscape with higher connectivity index was forest of sarula (0.6-0.8) located along main road through the sub-watershed. by restoring this forest, the forest landscape would be connected with the closest forest patches, so the connectivity between forest patches would be high and would improve the functional relationship (fry & tress 2007) in forest ecosystem. loss of natural connectivity of ecosystem is threat in distribution and survival of orangutan and other wildlife. as the habitat of wildlife, the flc of batang toru is important for diversity conservation. more serious attention is needed to improve the connectivity of forest landscape and habitat conservation, because landscape connectivity facilitates organism movement, genetical exchange, and other ecology of material flow (crooks & sanjayan 2006). habitat requirements for fauna and flora is the main key in biodiversity conservation, including stability and integrity of natural ecosystem (taylor . 1993; collinge 2000). therefore, it is very important to consider connectivity as a base in planning conservation and landscape change analysis. the model of socio economics index resulted from this study was y = 0.65450.043077x +0.004396x with determination coefficient of 24.78%. socio economics condition became a main key for the success of landscape restoration activities. people's perceptions on forest landscape restoration activities were different. the perceptions depended on people's level of income, education, main occupation types, age and duration of staying. the analysis showed that occupation types and level of income significantly affected people's participation possibility in forest landscape restoration. based on occupation types, employed people were highly depended on natural resources quality and therefore, they absolutely agreed and would participate in all restoration steps. farmer group was more supportive in landscape restoration compared to plantation workers or owners. the farmer group would participate in landscape restoration to have more income and land for cultivation. as well as farmers, low income people tended to participate in restoration activities to have more income (fig. 7a). people owning large estate area tended not to participate in landscape restoration. those people were worried their plantation would be disturbed, so it might reduce production of the plantation. people with high level of income disagreed and would not participate in forest landscape restoration. this condition should become consideration in determining location and determining partners in managing forest landscape restoration. forest landscape restoration programs should increase people's welfare and produce more woods from forest plantation (nawir . 2008). this study showed that the restoration index model was z = 0.133y + 0.024y + 0.939y + 0.079y with correlation coefficient of 55.7%. based on value of cannonical et al et al socio economics index restoration index 4 12 13 1 2 3 4 120 biotropia vol. 21 no. 2, 2014 discriminant coefficient, it was found out that socio economic index had less impact compared to the other four predictors, while the most influential model was connectivity index model. hit ratio value from discriminant analysis is 54.5%, which was sufficient for grouping into 3 different classes. based on test using alpha value of 0.05, the discriminant function result was accurate, because value was bigger than table value. hence, the restoration index model was used to create restoration index map (fig. 7b). the figure shows that high restoration index was located in sarula sub-watershed. it meant that when the restoration project was to be conducted, sarula sub-watershed should be the first area to be restored. based on matrix value of discriminant function, it was found out that the connectivity index had canonical coefficient value higher than other indices (selman 2006), therefore, it became the most important index followed by forest degradation index. connectivity index is important in some research because it is potential in mitigating habitat fragmentation impact (anderson & jenkins 2006). considering the landscape connectivity index would ensure the connectivity of wildlife habitat. socio economic index had less influence in the model indicating that people's participation possibility was relatively less important as consideration in restoration activity planning when the forest landscape was highly degraded. people's participation possibility should be encouraged to increase support in forest landscape restoration plan. press's q press's q x 2 figure 7. possibility participation map (a) and restoration index priority of forest landscape batang toru watershed (b) 121 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. et al et al increasing of ecosystem connectivity is one main objective of forest landscape management (saura . 2011), and also maintenance of stability and integrity of natural ecosystem (decout . 2010). forest landscape restoration is aimed to restore forest landscape as close as natural ecosystem. forest patch connectivity and forest degradation level become the most important indices, therefore, consideration to improve connectivity between separated patches and degradation levels becomes high priority. these indices will improve the current guidelines and methods for forest restoration. biophysical factor is the main consideration in planning site restoration (mof and jica, 2014). recently, land and forest biophysical characteristics are also used to determine site priority for land rehabilitation. ecologically, the connectivity index is one important factor in material flow and material cycle in forest ecosystem. in sarula sub-watershed, most forest degradation was in very high restoration index area. the index showed that sarula sub-watershed became the main priority for landscape restoration activities in batang toru watershed. restoring landscape of sarula sub-watershed will improve the whole condition of batang toru watershed because sarula sub-watershed is located in the upstream of batang toru watershed. batang toru forest landscape was heading for heavy degradation indicated by decreasing of species diversity level, increasing of medium and high ecosystem pressures, increasing of forest landscape fragmentation, declining of forest landscape connectivity. forest landscape restoration index was determined by forest landscsape connectivity index and forest degradation index. the forest connectivity index should be considered in planning site restoration to ensure wildlife habitat quality and biodiversity. the restoration index would enhance the current guidelines and methods for forest restoration. forest fragmentation was potential indicator to determine priority for site restoration. people's participation possibility gave less influence in landscape restoration index model. people needed to be encouraged to increase their involvement in forest landscape restoration activities in batang toru watershed. the map of forest landscape restoration index shows that sarula sub-watershed to be the first area restored before other sub watersheds. the restoration index needs other factors to fulfill all requirements such as land degradation index. therefore, further research is needed to design site restoration site in priority sub-watershed area. this research was part of phd thesis of the first author, funded by seameobiotrop dipa 2013. the authors extended deep appreciation to the ministry of education and culture, republic of indonesia for the scholarship and support to conclusions acknowledgements 122 biotropia vol. 21 no. 2, 2014 accomplish this paper. sincere appreciation was also extended to anonymous reviewers for corrections and comments. references anderson b, jenkins cn. 2006. new york, (us): columbia university press. apan aa, steven sr, mark sp. 2002. mapping and analysis of changes 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nazaruddin. 1987. . yogyakarta (id): gadjah mada university press. statistik kehutanan indonesia. pedoman dan tata cara restorasi di kawasan konservasi (hutan hujan tropis pegunungan dan hutan mooson tropis rehabilitasi hutan di indonesia, akan kemanakah arahnya setelah lebih dari tiga dasawarsa?. kajian spasial lahan kritis berbasis sistim informasi geografis untuk rehabilitasi kawasan koridor satwa liar dan harangan desa di kawasan hutan batang toru provinsi sumatera utara . phd thesis , the forest landscape restoration handbook (earthscan forestry library) phd thesis. . planning at the landscape scale pongo pygmaeus master thesis spatial decision support system multivariate data analysis the ecology of sumatera 124 biotropia vol. 21 no. 2, 2014 biotropia vol. 28 no. 1,2021: 21 28 doi: 10.1 1598/btb.2021.28.1.903 growth a n d development of oil palm shoots under different light qualities helena patricia manoh','*, yantil a n d nurita toruan-mathius' 'faculiy ofbiotechnology, atmajgya catholic universiiy ofindonesia, jakarta 12930, indonesia 'plant production and biotechnology divisian, sn/lart biotecbnology center, pt smart tbk., bogor 16810, indonesia received 22 september 2017/accepted 19 december 2019 abstract light quality is one important factor that affects the growth and development of in vitro plants. this study examines the influence of different light qualities on the in vitro growth and development of oil palm shoots which were cultured in murashige & skoog medium under white fluorescent lamp, white light-emitting diode (led), red led, blue led, combination of red and blue led, and in darkness. the results showed that the oil palm shoots grew and developed differently under different light qualities. root initiation and shoot elongation progressed well under red light, while chlorophyll and sugar content were better produced under white and blue light than under the red light. both white fluorescent lamp and the combination of red and blue led resulted in higher growth parameter compared to other light qualities. however, the results were not significantly different. keywords: chlorophyll, elaeisguineensis, hormone, in vitro, led light introduction in the production of oil palm (elaeis gzlineensis) clones, the use of tissue culture through somatic embryogenesis is widely applied. however, the efficiency of tissue culture in oil palm is very low (rohani et al. 2000; icushairi et al. 2010). hence, improvement on the efficiency of this technology on oil palm is important for its mass production. plant growth and development in tissue culture are regulated by various environmental factors, wherein light is one of the most important. undoubtedly, light is necessary for photosynthesis and photomorphogenesis, with certain specific wavelengths playing important roles in plant tissue culture efficiency (fujiwara & i<ozai 1995). the lighting system generally used for maintaining plant tissue culture is the tubular fluorescent lamps (tfl). however, tfl has a wide spectrum and contain unnecessary wavelengths for plant growth. the light-emitting diode (led), an alternative light source for plant tissue culture, has several advantages in comparison to other lighting systems. led has wavelength specificity, durabhty, longer operating lifetime, has the ability to select spectral composition, has a smaller size, and emits less heat (gupta & jatothu 2013). the disadvantage of led is the establishment cost. however, led can still be profitable in the long run, even though it may require a high capital investment, because the long-term operating costs of led are generally lower than other lighting systems. the led lighting system using various light qualities in plant tissue culture has been applied to a variety of species, such as cymbidizm (tanaka et al. 1998), strawberry (nhut e t al 2003), grape (poudel et al. 2008), antbam'zm (bucharto 2010), gosgpizlm birsatam (li et a/. 2010), onn'diam (mengxl et al. 201 i), brassica napas (li et al 2013), banana (vieira et al. 2015), 'corresponding author, email: biotechnology@sinarmasvanilla planfoolid (belle-bell0 et al. 201 6 ) , and agri.com sugarcane (ferreira et al 2017). the effect of biotropia vol. 28 no. 1,2021 light quality varied depending on the plant species yet little is known about led utilization with different light qualities in oil palm shoot. hence, the influence of various light qualities on in vitro growth and development of oil palm shoot was evaluated in this study. materials and methods plant materials and light quality treatments the i n v h o oil palm shoots used in t h s study were obtained through indirect somatic embryogenesis from young leaf explants following the wong e t al (1997) method. the different lights used were white tfl (phillips t5 tch086 ev 28w, white 6500 i<), white led (vanq vq-glt8020w-27, white 10000 i(), red led (vanq vq-glt8020w-27, wavelength: 660 nm), blue led (vanq vq-glt8020w-27, wavelength: 460 nrn) and red blue led (vanq vq-glt8020w-27, wavelength: 660:460 nm (3:l)). effect of light quality on shoot development the 4-6 cm shoots, which have more than two leaves were selected and transferred onto a modified murashige & skoog (1962) medium without plant growth regulator. all cultures were maintained in a culture room at a temperature of 28 + 2 "c, relative humidity 50 f 10°/o, and with 16/8 light/dark photoperiod under various light qualities. after two months of culture, the fresh weight, the number of new leaves, the shoot height, and its root formation were observed, and then whole shoot samples from each treatment were randomly collected for chlorophyll, sugar, and endogenous hormone analyses. the whole shoots were ground in liquid nitrogen and stored at -80 "c prior to analyses. chlorophyll content analysis for chlorophyll a and at 645 nm for chlorophyll b. the chlorophyll content was calculated according to lichtenthaler (1 987). sugar content analysis the sugar content was measured, according to the method described by dubois e t al. (1951). the 2 g sample was homogenized in 10 ml of distilled water and kept in water bath at 95 "c for 5 min. the sample was then stored overnight at 70 "c. subsequently, the sample was centrifuged at 5,000 rpm for 10 rnin and 2 ml of the supernatant was transferred into another tube then added with 0.02 ml of 80% phenol and 5 ml of sulfuric acid. the mixture was then incubated at 30 "c for 30 min. the optical density was measured using a spectrophotometer (biotek epoch microplate) at 490 nm. fructose and glucose were used as the standard. endogenous hormone content analysis the endogenous hormone content was measured, according to i<elen e t al (2004), with some modifications using ultra performance liquid chromatography (yuater uplc). auxin, gibberellic acid (ga), and abscisic acid (aba) were extracted from 5 g of sample and soaked overnight in 30 ml 80% methanol at 4 "c. the 5 g extracted sample was dissolved with 30 ml 0.1 m phosphate buffer (ph 8.5) and then partitioned with ethyl acetate two times. after removal of the ethyl acetate, the ph of the aqueous phase was adjusted to 2.5 with 1 n hc1. the sample was partitioned again with ethyl acetate 2 times and passed through anhydrous sodium sulphate. the ethyl acetate was evaporated in rotary evaporator at 50 "c. the dry residue containing hormones was dissolved in 2 ml of methanol and filtered into a uplc vial using a 0.2 pm millipore syringe filter. an injection volume of 1 pl was used for each analysis. the column used was water acquity uplc beh c18 1.7 pm (2.1 x 50 mm). the mobile phases constituted of acetonitrile: water (26:74) p h 4 with a flow rate of 0.2 ml/min and retention time of 3 minutes. the the chlorophyll content was extracted from signal of the compounds was monitored by a the sample using 10 ml of 100% acetone. the photodiode array (pda) detector at 208 nm. optical density was measured using a indole acetic acid (iaa), ga3, and aba (100 spectrophotometer (hitachi u-2900) at 662 nm ppm) were used as the standard compounds. growth and development of oil palm shoots under different light qualities patricia manoh e t al. for cytokinin isolation, 1 g of sample was extracted using 20 rnl 70% methanol for 4 hours at 4 "c. the extracted sample was filtered and injected into a uplc column as previously mentioned. the mobile phases constituted of water: methanol (2030) ph 2 with a flow rate of 0.35 ml/min and retention time of 0.6 minutes. the signal of the compounds was monitored by a pda detector at 210 nm. zeatin (100 ppm) was used as the standard compound. statistical analysis this experiment used randomized complete block design with three block and six treatments. data were analyzed statistically using analysis of variance (anova), and the differences among the treatment means were tested using duncan's multiple range test (dmrt) at p = 0.05. statistical analyses were conducted using sas version 9.1.3. results and discussion shoot growth and development the growth and development of oil palm shoots were influenced by the light quality (table 1). compared with other treatments, the no light (darkness) treatment gave the lowest growth. when under darkness, the number of new leaves and fresh weight were significantly lower than those under light treatments, while the results among different light treatments did not significantly differ from each other. however, the highest shoot height and fresh weight were observed in shoots growing under the combination of red and blue light-emitted diode (led). meanwhile, the highest number of new leaves was observed in shoots under white tubular fluorescent lamp (tfl) (table 1). white tfl is the most widely used light in plant tissue culture. however, whte tfl produces a wide range of wavelengths that contain unnecessary wavelengths for plant growth and development, whde led can emit light at specific wavelengths (gupta & jatothu 201 3). different wavelengths generally bring about different responses on plant growth and development. in this study, the red blue led gave the greatest growth compared to other led colors (table 1 & fig. 1). red and blue lights remarkably impacted plant growth, probably because these are the major energy sources for photosynthesis and photomor phogenesis. red and blue lights, either alone or in combination produce different responses in plant growth. red light induces shoot elongation (nhut e t al. 2003; cybularz-urban et al. 2007; poudel et al. 2008), while blue light induces chlorophyll and stomata formation (poudel et al. 2008; muneer et al. 2014). the combination of these two lights may have produced higher growth rate, due to the combined advantages of red and blue lights for inducing plant growth and development. the combination of red and blue lights also enhance the growth of other plant species, such as strawberry (nhut et al. 2003), cotton (l et al. 2010), ginseng (nhut etal. 2015), and vanilla (bello-bello et al. 201 6). table 1 effect of different light qualities on growth and development of oil palm shoots treatment number of new leaves fresh weight increase (mg) shoot height increase (cm) dark 0.51 f 0.03 white tfl 0.78 + 0.03 a white led 0.69 + 0.03 a red blue led 0.76 + 0.03 a blue led 0.76 f 0.04 a red led 0.70 + 0.03 a notes: values are mean + se for n = 330. different superscripts on the same column indicate significant differences at p = 0.05 by dmrt. biotropia vol. 28 no. 1,2021 figure 1 representative of oil palm shoots under different light qualities notes: d = dark w ?m = %bite tm;, w led = white led; rb led = combination red blue led; b led = blue led; r led = red led;, light quality and endogenous hormones light quality affects plant gzowth and development by regulating the synthesis of endogenous hormones: c y t o k h ~ ~ gibberellic acid (ga), auxin and abscisic acid (am). the endogenous hormone in the oil palm shoot showed varied responses to different light qualities (table 2). cytokinitl, ga and aba contents differed significantly from a c h other when exposed under the different hght quality treatments, indicating their different photo responses to certain wavelengths. the highest cytokinin content was observed under the white led and lowest in the darkness. gibberellic acid (ga] content was higher when the shoots were cultured under red, blue, or red and blue led when compared with white light. aba content was the highest under red blue led, and the lowest in dark vestment. auxin did not respond to any of the light quality treatments. althaugh the auxin content did not differ signifimdy among treatments, the highest was observed in red led treatment. interaction berween endogenous hormones and shoot growth synthesis of hormone in plant is regulated by plant photoreceptors, which include phpchromes, ~ t o & , ; o m e , and phototropin (de wit ~t .ti 20 1 6). the interaction between hght quality and endogenous hormone plays an important role in plant growth and development. ga and auxin are key players in plant elongation. the stem increment and ga content of norway spruce seedlings under red light were higher than under blue light, whereas u a content under red light were lower (ouyang t.t al', 2015). in contrast, ga eontent in petunia plants were higher under bhe light compared to red light (fukuda e t 8 2016). blue light enhances shoot elongation and red light inhibites shoot elongation in petunia plants. in this current study8 ga and auxin contents in oil palm shoots under led light were not significantly different, but ga content in shoots under blue led was higher than those under other lights. the. resulting differences may be attributed to plant species1 stage of plant growth, and other environmental factors. table 2 endogenous hormones cootent in oil palm shoots under different light qudties treatment c p k i n i n (mg/@ ga @/g) auxin @g/@ kg/$$ dark 0.48 f 0.04 3.39 f 0'33 3.51 f 0.68 8.13 f 5.63e white tfl 0.61 f 0.04 2.50 + 0.32 b 3.83 f 0.55 35.81 f 10,41 " wlhite led 0.70 f 0.03 2.24 -t 0,35 b 4.59 +. q.65 a 29.15 f 1034 * red blue led 0.64 f 0.02 ab 2.89 f 0.09 ah 4.37 1: 0.60 a 42.08 f 12.1 1 blue led 0.64 k 0.06 ab 3.06 f 9.26 3.99 f 0.78 a 19.77 f 9.70 bc red led 0.67 f 0.03 " 2.67 f 0.29 *b 4.79 + 0 9 3 a 30.40 f 8.51 * nates: values are mean f se f ~ r pr = 6. different letters in the same column indicate significant difference at p = 0.05 by d m growth and development of oil palm shoots under different light qualities patricia manoh e t al: shoot elongation seems to positively correlate with the interaction of endogenous hormones. in this study, shoot elongation under red led was higher than those under other lights (table 1) and this might be correlated with the higher auxin content under red led (table 2). ga and auxin are known to stimulate plant elongation through different mechanisms. auxin affects cell elongation by promoting the release of hydrogen ions from the plant cell and whch in turn reduces the stress on the cell wall. ga requires cell wall plasticity and cytoplasmic protein synthesis for cell elongation. to achieve this, cooperation with other hormones, such as auxin is required p a n g & irving 201 1). cytokinin has a different effect as it inhibits plant elongation by inhibiting auxin-promoted extension growth (cohen e t al. 1991). cytohnin and blue light induced inhibitory effects on hypocotyl growth of cucumber (cohen e t al. 1991). in this study, cytokinin content and shoot elongation under light treatments were not significantly different. t h s results showed that cytokinins might not be correlated with the elongation of oil palm shoots. auxin also plays a key role in root initiation. in this study, auxin content in the oil palm shoots increased under the red led (table 2) and it induced the root initiation in the shoots (fig. 2). the process of root formation is considerably complex and involves interaction with other hormones. auxin regulates root growth by modulating the effect of ga-induced destabilization of della protein growth repressor (fu & harberd 2003). in the presence of ga, della is degraded and thereby modulates other plant hormones, such as auxin. light quality and photosynthesis light quality also influences photosynthesis activity. in this study, the effect of light quality on photosynthesis was determined by the accumulation of photosynthetic pigments (chlorophyll a and b) and primary photo synthesis products (fructose and glucose). chlorophyll and sugar content of the oil palm shoots were significantly lugher under light treatment than those under dark treatment (figs. 3 & 4). light is an important factor that regulates chlorophyll biosynthesis. the shoots under white, red blue, and blue light accumulated higher chlorophyll, compared to those under the red or dark treatment (fig. 3). several studies showed that blue light is a good light for chlorophyll synthesis and that red light decreases chlorophyll content (tanaka e t al. 1998; li e t al. 2010). simdarly, in this study, the chlorophyll content in oil palm shoots decreased under red led, and increased under white and blue led (fig. 3). dark white white red blue blue red led tfl led led led figure 2 percentage of rooting in shoots under different light qualities notes: vertical bars indicate k se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. biotropia vol. 28 no. 1,2021 blue light is also important in chlorophyll synthesis. blue light stimulates chlorophyll synthesis by inducing the expression of genes encoding the enzyme for two early step of chlorophyll biosynthesis, glutamate-1 semialdehyde aminotransferase and 5 aminolevulinic acid dehydratase (matters & beale 1995). chlorophyll content were also correlated to cytolilnin content in oil palm shoots. chlorophyll content under light treatments was significantly higher than that in dark treatment (fig. 3) and t h s may be stimulated by high cytokinin content under light treatments (table 2). cytokinin affected chlorophyll biosynthesis by promoting the synthesis of 5-aminolevulinic acid, a precursor of chlorophyll synthesis, and enhancing activity of protochlorophyllide oxidoreductase, an enzyme in chlorophyll synthesis (cortleven & schmulling 201 5). sugar content in the oil palm shoots under different light treatments did not significantly &ffer (fig. 4). apparently, different light qualities did not affect sugar content because the in vitro shoots are mainly heterotrophic, where the shoots were grown in culture media with sugar. sugar is the main carbon source for heterotrophic growth (yasseen e t al 2013). the presence of sugar in culture media might reduce the need for sugar production and limits photosynthetic activity. mg/g chlorophyll a r chlorophyll b dark white white red blue blue red led tfl led led led figure 3 chlorophyll content in oil palm shoots under different light qualities notes: vertical bars indicate k se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. mg/g fructose f glucose dark white white red blue blue red led tfl led led led figure 4 sugar content in oil palm shoots under different light qualities notes: vertical bars indicate ? se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. growth and development of oil palm shoots under different light qualities patricia manoh et al. conclusions light quality influenced the growth and development of the oil palm shoots. the different wavelengths produced different effects on the root and shoot activities. root initiation and shoot elongation were well-produced under red light, while chlorophyll and sugar contents were better produced under the white and blue light. although the results were not significantly different, the combination of red and blue led resulted in higher growth than the other light treatments. besides the white light, the red and blue light combination provides good lighting for promoting the growth and development of oil palm shoots. hence, the red blue led may be used as an alternative for the whte tfl used in oil palm tissue culture. however, further study may be needed to analyze the ratio of red and blue led in order to optimize the protocols for oil palm tissue culture. acknowledgements this study was fully funded by the plant production and biotechnology division of pt smart tbk. the authors would like to express their great appreciation to the division head of plant production and biotechnology division for permission to publish this article. the authors would also like to thank all technicians in the tissue culture and biotechnology laboratory at smart biotechnology center, who provided their assistances in this study. references bello-bello jj, martinez-estrada e, caamal-velizquez jh, morales-ramos v. 2016. effect of led light quality on in vitro shoot proliferation and growth of vanilla (vanilla planifoolia andrews). afr j 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201 1. developing a model of plant vt, tung ht, vinh bt, luan tc. 2015. lighthormone interactions. plant signal behav 6(4):494 emitting diodes and their potential in callus 500. growth, plantlet development and saponin wong g, tan cc, soh ac. 1997. large scale propagation accumulation during somatic embryogenesis of of oil palm clones experiences todate. acta hort panax vietnamensir ha et grushv. biotechnol 447: 649-58. biotechnol equip 29(2):299-308. yaseen m. ahmad t. sablok g. standardi a. hafiz ia. ouyang f, mao jf, wang j, zhang s, li y. 2015. 2013. review: role of carbon sources for in transcriptome analysis reveals that red and blue vitro plant growth and development. mol biol rep light regulate growth and phytohormone 40:2837-49. 5. okky (the occurence) rev.cdr biotropia vol. 18 no. 2, 2011: 102 122 the occurrence of insects and fungi, and aflatoxin b contamination of stored sorghum in demak and wonogiri regencies, central java 1 okky setyawati dharmaputra , santi ambarwati , and ina retnowati recipient of biotrop research grant 2010/accepted 25 november 2011 the objectives of this study were to collect informations on the method of postharvest handling of sorghum and to investigate the moisture contents, insects infestation, fungal infection, and aflatoxin b contents of stored sorghum grains collected from various stages of the delivery chain in demak and wonogiri regencies, central java. in demak regency sorghum cultivation was monoculture, variety cultivated was upc-s1. in wonogiri regency sorghum cultivation was intercropping with secondary crop and cassava. sorghum varieties cultivated were kawali, numbu, zh30, mandau and hibrida hybrids. there was a difference between the method of postharvest handling of sorghum at farmer and collector levels in demak and wonogiri regencies. in general the method of postharvest handling of sorghum in demak regency was more appropriate and more advance compared to that in wonogiri regency. the moisture contents of sorghum at farmer as well as at collector level in demak regency (13.0%) and wonogiri regency (12.9%) were still lower that that of normal (safe) moisture content of sorghum. the number of insect species associated with sorghum in various distribution level in demak and wonogiri regencies was 10 and 17 species, respectively. the dominant insects species were and . the number of fungal species found in sorghum at various distribution level in demak and wonogiri regencies was 23 species, respectively. in general, the dominant fungal species were , and . in demak regency aflatoxin b contents of sorghum at farmer and collector levels were 22.50 and 15.45 ppb, respectively, while in wonogiri regency 2.27 and 10.28 ppb, respectively. insects, fungi, aflatoxin b , stored sorghum, demak and wonogiri regencies, central java 1,2 2 1 1 2 seameo biotrop, jl. raya tajur km. 6, po box 116, bogor16134, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum f. verticillioides abstract 1 1 1key words: 102 introduction a . t e : ● t m ● t . i s a foodstuff, sorghum ( (l) moench) is the fifth most important cereal after rice, wheat, maize and barley in the world. in many countries, sorghum is used as food and feedstuff, industrial raw materials such as for ethanol, beer, wine, syrup, glue, paint and modification of starch. compared to other foodcrops, sorghum can adapt to broad agroecology, resistant to dryness, high productivity, and more resistant to pests and diseases. apart from that, sorghum has nutritional content that is almost the same with milled rice as well as maize, consequently sorghum is good to be consumed either as alternative food or feedstuff. according to suarni (2001) protein contents in sorghum, milled rice and maize were 10.11, 9.28 and 11.02%, respectively; their lipid contents were 3.65, 1.88 and 5.42%, respectively; their carbohydrate contents were 80.42, 86.45 and 79.95%, respectively. although in indonesia the production of sorghum is still low, it is potential to be cultivated and developed, especially in marginal and dry areas (bp2aptp 2008). to anticipate food crisis caused by global warming, it is important to cultivate and to develop sorghum in indonesia. during storage grains could be infested by insects, microorganisms, mites and rats. insects are considered as the most significant cause of losses. among microorganisms, fungi are the most important cause of deterioration of stored grains. the role of insects on fungal infection cannot be disregarded. aside from injuring the grains, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce enough heat and moisture (especially during long-term storage) which stimulate fungal growth. fungal infection in grains can cause discolouration, decrease in physical quality and nutritional contents, and mycotoxin contamination (sauer . 1992). aflatoxins are the most dangerous mycotoxins, they can cause liver cancer in human and animal. there are four kinds of aflatoxins found in food and feedstuff, i.e. aflatoxins b , b , g and g . the most toxic of the four kinds of aflatoxins is aflatoxin b (afb1) he objectives of this study wer o obtain informations on postharvest handling methods of sorghu o investigate the degree of insect and fungal attacks, and afb1 contamination of stored sorghum collected from various stages of the delivery chain in demak and wonogiri regencies, central java. the moisture contents of sorghum were also determined, because moisture content is the important environmental factor for the development of insects and fungi n demak regency, surveys and sampling of sorghum were conducted in july and august 2010, while for wonogiri regency in april and july 2010. in demak regency (demak and mijen subdistricts), surveys and sampling of sorghum were conducted at farmer and collector levels and a traditional market in the city of demak. in ime and location of survey sorghum bicolor et al 1 2 1 2 1 materials and methods t s 103 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al biotropia vol. 18 no. 2, 2011 104 wonogiri regency (purwantoro, eromoko and pracimantoro subdistricts), surveys and sampling of sorghum were conducted at farmer level and collector level in pracimantoro subdistrict. in demak regency (demak and mijen subdistricts), surveys and sampling of sorghum were conducted in july and august 2010 at farmer and collector levels and a traditional market in the city of demak. in wonogiri regency (purwantoro, eromoko and pracimantoro subdistricts) surveys and sampling of sorghum were conducted during april and july 2010 at farmer level and at collector level in pracimantoro subdistrict nterviews were conducted during the surveys and sampling to collect information on postharvest handling of sorghum at farmer and collector levels. number of respondents from each level distribution chain in each regency could be different depending on the field conditions during the surveys. the questionnaires contained among others questions on postharvest handling carried out by farmers and collectors and problems encountered by them he number of sorghum samples collected from regency was 46 with the breakdown as follows : farmers (34), collectors (9) and retailers in traditional market (3). sorghum samples collected from regency was 38 consisting of samples from farmers (23) and collectors (15) (table 1). the samples were collected from places where the respondents stored sorghum at the time of the interview. the number of sorghum samples in each level distribution chain was determined based on the existence of sorghum at the time of interviews. as much as 2 kg of each sample was collected randomly from each respondent, it was then placed in a clean plastic bag. insects from each sample were separated using graded sieves, then they were preserved in vials containing ethanol 70%. . i . t i s s s nterviews using questionnaire ampling and method to obtain working sample demak wonogiri table 1. level of distribution chain, subdistrict origin of the sample, and the number of sorghum samples at various stages of the delivery chain in demak and wonogiri regenciess regency level of distribution chain subdistrict origin of the sample number of samples demak farmer demak and mijen 34 collector demak and mijen 9 retailer at traditional market demak 3 wonogiri farmer eromoko 13 pracimantoro 8 purwantoro 2 collector eromoko 1 pracimantoro 14 total 84 105 each sample was then divided three times using a sample box divider to obtain eight working samples for the determination of the percentage of sorghum grains infected by each fungal species, afb1 content, and a reserve sample oisture contents of sorghum grains were determined right after sample collections using moisture meter delmhorst model g-7. two replicates were used from each sample. insect species was identified using the publication of haines (1991) as the main reference. the degree of insect species infestation in each sample was determined based on its number per kg of sorghum. fungi were isolated using a plating method on dichloran 18% glycerol agar (dg18) (hocking and pitt 1980). two hundred sorghum grains were used from each sample. fungal species was identified using the publication of pitt and hocking (2009) as the main reference. in this study only afb1 content was determined, because it is the most dangerous aflatoxin compared to the other three kinds of aflatoxins (afb2, afg1 and afg2). afb1 content was determined according to thin layer chromatography method (aoac 2005). two replicates were used from each sample he results of interviews with farmers and collectors in demak regency are presented in tables 2 and 3. at level the method of sorghum cultivation was monoculture. the variety of sorghum cultivated was upc-s1. the range of land area for sorghum cultivation was 0.25-2 ha. sorghum was harvested three months after planting. the method of harvesting was by cutting its panicle using a sickle. the panicles of sorghum were sun-dried on tarpaulin or cement floor. in general sorghum grains were separated from panicles using a paddy thresher. most of sorghum was sold to collectors, although a small part was used as seeds to be replanted. sorghum was packed in polypropylene bag. in general storage duration of sorghum collected as samples was between 0-7 days. the condition of storage was dirty. no insects and fungi were found in sorghum samples, because the duration of storage was relatively short. the variety of sorghum bought by was upc-s1. sorghum was bought from farmers, collectors at village level, or from farmers and collectors at village/ subdistrict levels. if they were still not dry enough, further sun-drying was conducted on cement floor. the grains were separated from panicles using a paddy thresher and packed in polypropylene bags. sorghum samples have been stored for 0-7 days (66% of respondents) and 10-15 days (34% of respondents). the condition of storage (warehouse) was appropiate (78% of respondents) and not so appropriate (22% of respondents). sorghum was sold to big traders in wonosari (44% of . m . t d t interviews using questionnaires etermination of moisture content, insect infestation, fungal infection, and aflatoxin b conten farmer collector 1 results and discussion interviews using questionnaires in demak regency occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al 106 biotropia vol. 18 no. 2, 2011 table 2. results of interviews with on postharvest handling of sorghum in demak and wonogiri regencies farmers no. subject % respondent*) demak wonogiri 1. methods of sorghum cultivation: a. intercrop with secondary crop and cassava b. intercrop with secondary crop and cassava, and the use of fertilizer c. monoculture 0 0 100 57 43 0 2. variety of sorghum cultivated: a. kawali, numbu and zh 30 (white colour of grains) b. mandau and hibrida (red and reddish brown of grains) c. upc-s1 0 0 100 39 61 0 3. land area: a. 0.25 ha b. 0.25 ha c. 1.00 ha d. 1.50 ha e. 2.00 ha 29 44 15 3 9 48 13 39 0 0 4. harvesting time (days after planting): a. < 90 days b. 90 days 0 100 0 100 5. method of harvesting: a. by cutting the stalk of panicle using a sickle b. other method 100 0 100 0 6. method of drying(in the form of panicle): a. sun-drying on tarpaulin or cement floor b. hanging on racks c. inserted on wood located outside of the wall of the house made from zinc 100 0 0 83 13 4 7. method of threshing: a. manually b. beaten using a coconut leaf c. beaten using a of wood/bamboopiece d. beaten using a hammer e. beaten using a piece of stalk of tree f. using a paddy thresher 0 0 9 0 0 91 4 4 74 9 9 0 8. harvested sorghum were: a. sold to collector b. sold to collector and seed seller c. usedby farmers as cattle/chicken feed d. used by farmers as seeds and cattle feed e. b,d and consumed by farmers (by steaming) 0 100 0 0 0 30 57 5 4 4 9. method of storage: a. stored in plastic (polypropylene) bag b. spread out on tarpaulin 100 0 87 13 10. duration of storage of sorghum collected as sample: a. 0 -7 days b. 21 days c. 1 month d. 2 months e. 3 months 94 0 0 3 3 9 18 64 9 0 11. sanitation of storage: a. unappropriate (dirty) b. appropriate 100 0 100 0 107 respondents), big traders in other big cities (kudus, solo, yogyakarta, demak and bantul) (34% of respondents), and entrepreneur of oyster mushroom cultivation (22% of respondents) he results of interviews with farmers and collectors in wonogiri regency are presented in tables 2 and 3. in this regency, at level the method of sorghum cultivation was intercropping with cassava (57% of respondents); intercropping with secondary crop and cassava, and using fertilizer (43% of respondents). the sorghum varieties cultivated were kawali, numbu and zh 30 (zh-30 is a mutant line from batan), the colour of their grains were white (39% of respondents); while for mandau and hibrida varieties , the colour of their grains were red and brownishred, respectively (61% of respondents). the range of land area for sorghum cultivation was 0.25-1 ha. sorghum was harvested three months after planting, while the method of harvesting was by cutting its panicle using a sickle. in general, the panicles of sorghum were sun-dried on tarpaulin or cement floor. the grains were separated from panicles manually (4% of respondents) or beaten using a coconut leaf (4% of respondents), a stick of wood or bamboo (74% of respondents), a hammer (9% of respondents), and a piece of tree stalk (9% of respondents). sorghum was sold to collectors (30% of respondents), to collectors and for seeds to be replanted (57% of respondents), for feed (cattle or chicken) (5% of respondents), for seeds and cattle feed (4% of respondents); was sold to collectors, for seeds and consumption by steaming (4% of respondents). sorghum was packed in polypropylene bag (87% of respondents) and was spread out on tarpaulin (13% of respondents). the storage duration of collected sorghum for samples was 0-2 months. in general, the storage condition was dirty. during storage, insects were found (35% of respondents), while no fungi were found. the variety of sorghum bought by was kawali and numbu (53% of respondents), mandau hibrida varieties (47% of respondents). sorghum was bought from farmers (65% of respondents), from collectors at village level (12% of respondents), from farmers and collectors at village/subdistricts level (23% of respondents). panicles of sorghum were bought after sun-dried conducted by farmers (6% of respondents), after sun-dried by farmers in the form of grains (47% of respondents), after sun-dried by collector at village level in the form of panicle or grain . t interviews using questionnaires in wonogiri regency farmer collector table 2. continued no. subject % respondent*) demak wonogiri 12. insect pest was found in sorghum during storage: a. yes b. no 0 100 9 91 13. based on visual observation, fungi were found in sorghum during storage: a. yes b. no 0 100 0 100 *) number of respondent in demak regency : 34 number of respondent in wonogiri regency : 23 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al (47% of respondents). if sorghum bought from farmers were not yet properly dried, further sun-drying was carried out on cement floor. grains were separated from panicles using a paddy thresher and packed in polypropylene bag. the storage duration of sorghum samples was 0-7 days (59% of respondents), 1 month (29% of respondents), and 3 months (12% of respondents). in general, the condition of storage was not so appropriate. most of sorghum was sold to big traders in wonosari (71% of respondents). table 3. results of interviews with on post-harvest handling of sorghum in demak and wonogiri regencies collectors no. subject % respondent*) demak wonogiri 1. variety of sorghum bought: a. kawali and numbu (white colour of grains) b. mandau and hibrida (red and reddish brown of grains) c. upc-s1 0 0 100 53 47 0 2. sorghum was bought from: a. farmers b. from collectors at village level c. from farmers and collectors at village/subdistrict level 44 34 22 65 12 23 3. sorghum was bought: a. after being dried by farmer, in the form of panicle b. after being dried by farmer, in the form of grain c. after being dried by collector at village level, in the form of panicle or grain 0 100 0 6 47 47 4. method of drying of sorghum (not properly dried) bought from farmer : a. sun-drying on cement floor b. other method 100 0 100 0 5. method of threshing : a. using a paddy threshing b. other method 100 0 100 0 6. method of storage: a. stored in plastic (polypropylene) bag b. other method 100 0 100 0 7. duration of storage of sorghum collected as samples: a. 0 – 7 days b. 10 15 days c. 1 month d. 3 months 66 34 0 0 59 0 29 12 8. conditon of the storage(warehouse): a. not so appropriate b. appropriate 22 78 82 18 9. sorghum was sold to: a. big traders in wonosari b. big trader s in other big cities (kudus, solo, yogyakarta, demak, and bantul) c. entrepreneur of oyster mushroom cultivation 44 34 22 71 24 6 *) number of respondent in demak regency: 9 number of respondent in wonogiri regency: 15 biotropia vol. 18 no. 2, 2011 108 moisture content t . he range and mean of moisture contents of sorghum collected from farmers and collectors in demak and wonogiri regencies are presented in table 4. in demak and wonogiri regencies the mean moisture contents of sorghum collected from farmers were almost the same with those collected from collectors, i.e. 13.0 and 12.9%, respectively, because in general the storage period of sorghum at farmer level was relatively short. the moisture contents of sorghum collected from retailers at traditional market in demak regency were lower than those collected from farmers as well as collectors. the moisture contents of grains were always in equilibrium with the relative humidity of the storage. the moisture content will increase with the increase of the relative humidity, consequently fungal growth will be stimulated. in this study the mean moisture contents of sorghum at farmer and collector levels were still lower than the normal moisture content of sorghum. christensen . (1990) reported, that the normal moisture content of sorghum with superior and second qualities were 13 and 14%, respectively et al table 4. range and mean of moisture content of sorghum collected from farmers, collectors and retailers at traditional market in demak and wonogiri regencies regency level of distribution chain range (mean) of moisture content (% wet basis) demak farmer 10.7 – 14.2 (13.0) collector 12.5 – 13.8 (13.0) retailer at traditional market 12.0 – 12.6 (12.2) wonogiri farmer 11.9 – 13.8 (12.9) collector 11.9 – 14.2 (12.9) insect infestation insect infestation of sorghum in demak regency insect infestation of sorghum in wonogiri regency t . t he total number of insect species associated with sorghum at farmers, collectors and retailers at traditional market was 10 species (table 5). the insect species belong to order coleoptera (8 species), order hymenoptera (1 species) and order lepidoptera (1 species). most of the insect species were primary and secondary insect pest, and one species was a parasit insect. and were the dominant insects found (fig. 1). was only found in sorghum at retailer in traditional market and it was the dominant insect he total number of insect species associated with sorghum at farmer and collector levels was 17 species (table 6). the insects species belong to order coleoptera (10 species), order lepidoptera (3 species), order hymenoptera (2 species), order hemiptera (1 species) and order psocoptera (1 species). most of the species was primary and secondary insect pests, one species as a predator, and another one was a parasitic insect. s , and were the dominant insect species found (fig. 2). another insect found in some sorghum samples was , a parasite of egg sitophilus zeamais tribolium castaneum oryzaephilus surinamensis itophilus zeamais tribolium castaneum sitotroga cerealella anisoptermalus calandrae sitophilus zeamais . 109 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 fl cl l rl fl = farmer level; cl = collector level; rl = retailer level 1. 6. 2. sp. 7. 3. sp. 8. 4. 9. 5. 10. figure 1. percentage of sorghum samples infested by insects at farmer, collector and retailer levels in demak regency ahasverus advena sitophilus zeamais carpophilus tribolium castaneum cryptolestes typhaea stercorea oryzaephilus surinamensis anisoptermalus calandrae rhyzopertha dominica ephestia cautella fl = farmer level; cl = collector level 1. 7. 13. ants 2. 8. 14. 3. sp. 9. 15. 4. sp. 10. 16. 5. 11. 17. 6. 12. figure 2. percentage of sorghum samples infested by insects at farmer and collector levels in wonogiri regency ahasverus advena sitophilus zeamais araecerus fasciculatus thaneroclerus buqueti corcyra cephalonica carpohilus tribolium castaneum ephestia cautella cryptolestes typhaea stercorea sitotroga cerealella dinoderus minutes xylocoris flavipes liposcelis entomophilus rhyzopertha dominica anisoptermalus calandrae biotropia vol. 18 no. 2, 2011 110 t ab le 5 .s p ec ie s an d n u m b er o f in se ct s in so rg h u m at fa rm er s, co ll ec to rs an d re ta il er s in tr ad it io n al m ar k et in re g en cy d em ak n o in se ct s ta tu s n u m b er (% ) o f sa m p le s in fe st ed b y in se ct m ea n av er ag e o f n u m b er in se ct s / k g sa m p le f ar m er c o ll ec to r r et ai le r at tr ad it io n al m ar k et f ar m er c o ll ec to r r et ai le r at tr ad it io n al m ar k et l ar v a im ag o l ar v a im ag o l ar v a im ag o c o l e o p t e r a 1 a h as ve ru s ad ve n a (w al tl ) p es t 0 0 1 (3 3 .3 3 ) 0 0 0 0 0 1 .4 7 2 c ar po ph il u s sp . p es t 3 (8 .8 2 ) 2 (2 2 .2 2 ) 2 (6 6 .6 7 ) 0 -1 .8 3 (0 .6 1 ) 0 -0 .5 3 (0 .3 5 ) 0 0 .5 2 -1 .3 9 (0 .9 6 ) 0 0 .4 9 -3 .9 2 (2 .2 1 ) 3 c ry pt ol es te s sp . p es t 2 (5 .8 8 ) 2 (2 2 .2 2 ) 1 (3 3 .3 3 ) 0 8 .6 5 -3 6 .1 7 (2 2 .4 1 ) 0 .4 7 -0 .5 4 (0 .5 1 ) 0 0 .4 9 1 .4 7 4 o ry z ae ph il u s su ri n am en si s (l in n ae u s) p es t 0 0 2 (6 6 .6 7 ) 0 0 0 0 0 0 .9 8 (0 .9 8 ) 5 r hy z op er th a do m in ic a (f ab ri ch iu s) p es t 2 (5 .8 8 ) 0 0 0 2 .8 8 -7 .4 5 (5 .1 7 ) 0 0 0 0 6 s it op h il u s z ea m ai s m o ts ch u ls k y p es t 6 (1 7 .6 5 ) 1 (1 1 .1 1 ) 3 (1 0 0 .0 ) 0 0 .4 3 -3 8 9 .3 6 (1 1 1 .7 6 ) 0 3 .2 3 0 0 .4 9 -2 1 .0 8 (8 .5 0 ) 7 t ri bo li u m ca st an eu m (h er b st ) p es t 1 5 (4 4 .1 2 ) 6 (6 6 .6 7 ) 1 (3 3 .3 3 ) 0 -1 2 .6 4 (2 .4 0 ) 0 -2 .2 9 (0 .3 2 ) 0 -6 .3 6 (1 .6 9 ) 0 -7 .6 9 (1 .3 6 ) 0 0 .4 9 8 t yp h ae a st er co re a (l in n ae u s) p es t 2 (5 .8 8 ) 2 (2 2 .2 2 ) 0 0 0 .5 1 -0 .5 3 (0 .5 2 ) 0 0 .4 7 -1 .2 8 (0 .8 8 ) 0 0 h y m e n o p t e r a 9 a n is op te rm al u s ca la n dr ae (h o w ar d ) p ar as it e 2 (5 .8 8 ) 0 1 (3 3 .3 3 ) 0 5 2 .1 3 -5 5 .7 7 (5 3 .9 5 ) 0 0 0 0 .9 8 l e p id o p t e r a 1 0 e ph es ti a ca u te ll a (w al k er ) p es t 0 3 (3 3 .3 3 ) 1 (3 3 .3 3 ) 0 0 0 .4 5 -0 .8 5 (0 .5 9 ) 0 0 .4 9 0 111 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 6 . s p ec ie s an d n u m b er o f in se ct s in so rg h u m at fa rm er s an d co ll ec to rs in re g en cy w o n o g ir i n o in se ct s ta tu s n u m b er (% ) o f sa m p le s in fe st ed b y in se ct m ea n av er ag e o f n u m b er in se ct s / k g sa m p le f ar m er c o ll ec to r f ar m er c o ll ec to r l ar v a im ag o l ar v a im ag o c o l e o p t e r a 1 a h as ve ru s ad ve n a (w al tl ) p es t 0 1 (6 .6 7 ) 0 0 0 0 .5 6 2 a ra ec er u s fa sc ic u la tu s (d eg ee r) p es t 3 (1 3 .0 4 ) 5 (3 3 .3 3 ) 0 0 .4 2 -0 .8 1 (0 .6 4 ) 0 -1 0 .2 6 (2 .2 0 ) 0 .5 -8 .8 2 (3 .2 9 ) 3 c ar po ph il u s sp . p es t 2 (8 .7 0 ) 3 (2 0 .0 ) 0 0 .4 2 -0 .8 1 (0 .6 2 ) 0 0 .3 9 -0 .5 2 (0 .4 4 ) 4 c ry pt ol es te s sp . p es t 5 (2 1 .7 4 ) 2 (1 3 .3 3 ) 0 -1 2 .0 8 (2 .4 2 ) 0 -3 .1 4 (1 .7 8 ) 0 -0 .6 3 (0 .3 2 ) 0 -1 .0 1 (0 .5 1 ) 5 d in od er u s m in u tu s (f ab ri ci u s) p es t 1 (4 .3 5 ) 1 (6 .6 7 ) 0 0 .3 6 0 0 .6 3 6 r hy z op er th a do m in ic a (f ab ri ch iu s) p es t 3 (1 3 .0 4 ) 2 (1 3 .3 3 ) 0 0 .3 6 -2 .7 3 (1 .3 0 ) 0 0 .5 -1 .2 5 (0 .8 8 ) 7 s it op h il u s z ea m ai s m o ts ch u ls k y p es t 1 5 (6 5 .2 2 ) 1 1 (7 3 .3 3 ) 0 -2 5 .5 3 (3 .0 9 ) 0 -3 4 .6 8 (6 .0 2 ) 0 -3 .5 3 (0 .6 5 ) 0 -2 6 .5 0 (6 .5 8 ) 8 t h an er oc le ru s bu q u et i( l ef ev re ) p es t 0 1 (6 .6 7 ) 0 0 0 0 .5 2 9 t ri bo li u m ca st an eu m (h er b st ) p es t 1 4 (6 0 .8 7 ) 6 (4 0 .0 ) 0 -5 .1 2 (1 .3 2 ) 0 -2 .0 5 (0 .2 7 ) 0 -3 .7 5 (1 .1 8 ) 0 -1 .0 (0 .1 7 ) 1 0 t yp h ae a st er co re a (l in n ae u s) p es t 0 2 (1 3 .3 3 ) 0 0 0 0 .5 2 -0 .6 3 (0 .5 8 ) h e m ip t e r a 1 1 x yl oc or is fl a vi pe s (r eu te r) p re d at o r 1 (4 .3 5 ) 1 (6 .6 7 ) 0 0 .3 6 0 0 .5 0 h y m e n o p t e r a 1 2 a n is op te rm al u s ca la n dr ae (h o w ar d ) p ar as it e 4 (1 7 .3 9 ) 0 0 0 .6 8 -2 .4 2 (1 .6 5 ) 0 0 1 3 s em u t p es t 1 (4 .3 5 ) 0 0 3 0 .2 6 0 0 l e p id o p t e r a 1 4 c or cy ra ce ph al on ic a (s ta in to n ) p es t 1 (4 .3 5 ) 0 3 .1 5 0 0 0 1 5 e ph es ti a ca u te ll a (w al k er ) p es t 5 (2 1 .7 4 ) 2 (1 3 .3 3 ) 0 .4 2 -2 .2 2 (0 .9 8 ) 0 0 .5 -0 .7 4 (0 .6 2 ) 0 1 6 s it ot ro ga ce re al el la (o li v ie r) p es t 1 0 (4 3 .4 8 ) 4 (2 6 .6 7 ) 0 -1 7 .1 4 (2 .8 9 ) 0 -1 2 .3 8 (2 .6 1 ) 0 0 .6 3 -3 .0 (1 .3 1 ) p s o c o p t e r a 1 7 l ip os ce li s en to m op h il u s (e n d er le in ) p es t 1 (4 .3 5 ) 1 (6 .6 7 ) 0 0 .6 1 0 0 .3 9 biotropia vol. 18 no. 2, 2011 112 sitophilus zeamais sitotroga cerealella araecerus fasciculatus, thaneroclerus buqueti, dinoderus minutus, ephestia cautella typhaea stercorea carpophilus ahasverus advena sitophilus zeamais cryptolestes ferrugineus latheticca oryzae liposcelis bostrychophilus oryzaephilus surinamensis plodia interpunctella rhyzopertha dominica sitophilus zeamais tribolium castaneum cryptolestes ferrugineus r. dominica et al aspergillus candidus a. flavus a. niger a. tamarii a. wentii cladosporium cladosporioides colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens e. rubrum fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii cladosporium cladosporioides curvularia lunata c. pallescens fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae pestalotiopsis guepinii a. flavus a. niger a. tamarii a. wentii colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens, e. rubrum f. proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii and were the main insect pest in sorghum, while the other insect species were also the main insect pest of other foodstuff in storage. most collectors stored more than one kind of commodity, consequently some insect species found in other commodities were also found in sorghum. they were and . these insects were generally found in other commodities, such as milled rice, maize and mungbean. the existence of these insects was probably due to migration from one commodity to the other kinds of commodities. in general the number of insect species found in samples collected from collectors who stored sorghum for a longer period was relatively higher than collected from farmer. aside from sorghum, they also stored various agricultural commodities. other insects found in sorghum were sp. and . these insect species were not important, but the existence of these insects gave an indication, that the moisture content of sorghum was high, consequently sorghum could be easily infected by fungi. based on the result of inventory of insect species associated with stored sorghum at farmer and collector levels, was almost always found in all samples. as much as eight insect species was generally found in stored sorghum in taiwan, i.e. , , , , , , and . the dominant insects were and (peng 1998). according to mendesil . (2007) in ethiopia most farmers estimated the loss of stored sorghum caused by insect was up to 50%. insect infestation in sorghum was due to high temperature and unappropriate sanitation of the storage ercentage of sorghum grain infected by fungi in demak regency is presented in table 7. twenty three fungal species were isolated from sorghum collected from , i.e. , , , , , , sp., , , , , , , , , , , , , isolates 3, 7, 8 and 10. eight out of 23 fungal species belong to field fungi, i.e. , , , , , , , and . the existence of these fungi was probably due to optimum temperature or relative humidity for fungal growth before or after harvest. twenty fungal species were isolated from sorghum collected from , i.e. , , , , sp., , , , , , , , , , , , isolates 3, 8 and 10. field fungi were also still found in sorghum collected from collectors, because at farmer level sorghum was stored in a short period. it was also assumed, that the condition of storage was suitable for fungal growth. . p fungal infection fungal infection of sorghum in demak regency farmer collector 113 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 7 . f u n g al in fe ct io n in so rg h u m co ll ec te d fr o m fa rm er s, co ll ec to rs an d re ta il er s at tr ad it io n al m ar k et in re g en cy d em ak n o f u n g i n u m b er (% ) o f sa m p le s in fe ct ed b y fu n g i r an g e (m ea n ) p er ce n ta g e o f so rg h u m in fe ct ed b y fu n g i in in fe ct ed sa m p le s f ar m er c o ll ec to r r et a il er at tr ad it io n al m ar k et f ar m er c o ll ec to r r et ai le r at tr ad it io n al m ar k et 1 a sp er gi ll u s ca n di du s 2 (5 .8 8 ) 0 (0 .0 0 ) 1 (3 3 .3 3 ) 1 .0 -2 .5 (1 .7 5 ) 0 1 .5 0 2 a . fl a vu s 3 0 (8 8 .2 4 ) 9 (1 0 0 .0 0 ) 3 (1 0 0 .0 0 ) 0 .5 -1 0 0 .0 (7 1 .7 ) 2 .0 -9 9 .5 (7 4 .2 8 ) 1 3 .5 -8 7 .5 (6 1 .5 0 ) 3 a . n ig er 2 6 (7 6 .4 7 ) 7 (7 7 .7 8 ) 3 (1 0 0 .0 0 ) 0 .5 -5 2 .5 (6 .3 5 ) 1 .0 -4 3 .0 (1 4 .2 9 ) 0 .5 -3 .5 (2 .0 0 ) 4 a . ta m ar ii 1 7 (5 0 .0 0 ) 7 (7 7 .7 8 ) 1 (3 3 .3 3 ) 0 .5 -4 6 .0 (7 .2 1 ) 1 .0 -9 .0 (3 .5 0 ) 1 .5 5 a . w en ti i 7 (2 0 .5 9 ) 2 (2 2 .2 2 ) 0 (0 .0 0 ) 0 .5 -2 .5 (1 .3 6 ) 0 .5 (0 .5 ) 0 6 c la do sp or iu m cl ad os po ri oi de s 4 (1 1 .7 6 ) 0 (0 .0 0 ) 1 (3 3 .3 3 ) 0 .5 -1 .5 (0 .8 8 ) 0 1 1 .5 7 c ol le to tr ic h u m sp . 1 8 (5 2 .9 4 ) 8 (8 8 .8 9 ) 3 (1 0 0 .0 0 ) 0 .5 -2 3 .5 (6 .5 0 ) 0 .5 -1 3 .0 (4 .2 5 ) 3 .0 -1 0 .0 (6 .0 0 ) 8 c u rv u la ri a lu n at a 1 9 (5 5 .8 8 ) 8 (8 8 .8 9 ) 3 (1 0 0 .0 0 ) 0 .5 -2 2 .5 (7 .3 9 ) 4 .0 -1 6 .5 (1 0 .8 8 ) 1 .0 -7 .0 (4 .8 3 ) 9 c . pa ll es ce n s 2 9 (8 5 .2 9 ) 8 (8 8 .8 9 ) 3 (1 0 0 .0 0 ) 1 .0 -4 3 .5 (1 2 .0 5 ) 4 .5 -2 4 .0 (1 0 .8 1 ) 4 .5 -1 7 .0 (1 1 .1 7 ) 1 0 e n do m yc es fi bu li ge r 2 (5 .8 8 ) 1 (1 1 .1 1 ) 2 (6 6 .6 7 ) 3 .5 -8 0 .5 (4 2 .0 ) 5 .0 1 .0 -4 .0 (2 .5 0 ) 1 1 e u ro ti u m ch ev al ie ri 9 (2 6 .4 7 ) 1 (1 1 .1 1 ) 3 (1 0 0 .0 0 ) 0 .5 -1 2 .0 (2 .6 1 ) 1 .0 0 .5 -1 7 .5 (7 .0 0 ) 1 2 e . re pe n s 6 (1 7 .6 5 ) 1 (1 1 .1 1 ) 1 (3 3 .3 3 ) 0 .5 -2 .5 (1 .0 8 ) 0 .5 0 .5 1 3 e . ru br u m 5 (1 4 .7 1 ) 1 (1 1 .1 1 ) 2 (6 6 .6 7 ) 0 .5 -9 .0 (2 .6 0 ) 1 .5 0 .5 -1 .5 (1 .0 ) 1 4 f u sa ri u m pr ol if er at u m 1 (2 .9 4 ) 2 (2 2 .2 2 ) 0 (0 .0 0 ) 1 7 .5 0 .5 -1 .0 (0 .7 5 ) 0 1 5 f . se m it ec tu m 2 6 (7 6 .4 7 ) 9 (1 0 0 .0 0 ) 3 (1 0 0 .0 0 ) 1 .5 -6 7 .5 (1 9 .7 7 ) 1 .0 -2 9 .5 (1 2 .0 6 ) 2 .5 -8 .0 (3 .0 ) 1 6 f . ve rt ic il li oi de s 2 5 (7 3 .5 3 ) 8 (8 8 .8 9 ) 3 (1 0 0 .0 0 ) 0 .5 -9 .5 (3 .6 8 ) 1 .5 -2 2 .5 (6 .2 5 ) 1 .0 -4 .5 (2 .8 3 ) 1 7 l as io di pl od ia th eo br om ae 2 0 (5 8 .8 2 ) 7 (7 7 .7 8 ) 3 (1 0 0 .0 0 ) 0 .5 -7 .0 (1 .6 5 ) 0 .5 -4 .0 (1 .7 1 ) 0 .5 -1 .0 (0 .6 7 ) 1 8 p . ci tr in u m 2 9 (8 5 .2 9 ) 9 (1 0 0 .0 0 ) 3 (1 0 0 .0 0 ) 0 .5 -6 2 .5 (1 5 .7 2 ) 1 .0 -2 1 .5 (6 .8 9 ) 3 .5 -6 .0 (4 .5 ) 1 9 p es ta lo ti op si s gu e p in ii 1 6 (4 7 .0 6 ) 5 (5 5 .5 6 ) 2 (6 6 .6 7 ) 0 .5 -5 .5 (2 .0 6 ) 0 .5 -2 .5 (1 .4 0 ) 0 .5 -1 .0 (7 .5 0 ) 2 0 is o la te 3 9 (2 6 .4 7 ) 2 (2 2 .2 2 ) 0 (0 .0 0 ) 0 .5 -9 .5 (2 .3 9 ) 0 .5 -1 .0 (0 .7 5 ) 0 2 1 is o la te 7 1 (2 .9 4 ) 0 (0 .0 0 ) 1 (3 3 .3 3 ) 0 .5 0 2 .0 2 2 is o la te 8 1 (2 .9 4 ) 1 (1 1 .1 1 ) 1 (3 3 .3 3 ) 0 .5 1 .0 1 .0 2 3 is o la te 1 0 8 (2 3 .5 3 ) 1 (1 1 .1 1 ) 0 (0 .0 0 ) 2 .0 -8 .0 (4 .3 8 ) 1 2 .5 0 n u m b er o f sa m p le s at fa rm er le v el :3 4 n u m b er o f sa m p le s at co ll ec to r le v el :9 n u m b er o f sa m p le s at re ta il er in tr ad it io n al m ar k et : 3 biotropia vol. 18 no. 2, 2011 114 nineteen fungal species were isolated from sorghum collected from , i.e. , , , , sp., , , , , , , , , , , isolates 7 and 8. fungi which were often isolated from 34 sorghum samples collected from were (88.24% of samples), and (85.24%, respectively), and (76.47%, respectively) and (73.53%). caused highest percentage of infection (71.7%), with the range of 0.5 100.0%, while the lowest were isolates 7 and 8 (0.5%, respectively). fungi which were always isolated from 9 sorghum samples collected from were , and (100%, respectively), while fungi which were often isolated were sp., , and (88.89%, respectively), , and (77.78%, respectively). caused highest percentage of infection (74.28%), with the range of 2.0 99.5%, while the lowest were and (0.5%, respectively). fungi which were always isolated from three sorghum samples collected from were , , sp., , , , , , and (100%, respectively), while , , and (66.67%, respectively) were often isolated. caused highest percentage of infection (61.50%), with the range of 13.5 87.5%, while the lowest was (0.5%). in general, the dominant fungal species infecting sorghum grain collected from various stages of the delivery chain in demak regency were , and (fig. 3) retailer at traditional market farmer collector retailer at traditional market a. candidus a. flavus a. niger a. tamarii cladosporium cladosporioides, colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens, e. rubrum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii a. flavus curvularia pallescens penicillium citrinum a. niger f. semitectum f. verticillioides aspergillus flavus a. flavus f. semitectum penicillium citrinum colletotrichum curvularia lunata c. pallescens f. vercillioides a. niger a. tamarii lasiodiplodia theobromae aspergillus flavus a. wentii e. repens a. flavus a. niger colletotrichum curvularia lunata c. pallescens eurotium chevalieri f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum endomyces fibuliger eurotium rubrum pestalotiopsis guepinii aspergillus flavus e. repens a. flavus f. semitectum f. verticillioides . 88.24 100 100 76.47 100 100 73.53 88.89 100 0 20 40 60 80 100 aspergillus flavus fusarium semitectum f. verticillioides fl cl rl fl = farmer level; cl = collector level; rl = retailer level figure 3. percentage of sorghum samples infected by , and at farmer, collector and retail levels in demak regency aspergillus flavus, fusarium semitectum f. verticillioides 115 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 8 . f u n g al in fe ct io n in so rg h u m co ll ec te d fr o m fa rm er s an d co ll ec to rs in re g en cy w o n o g ir i n o f u n g i n u m b er (% ) sa m p le s in fe o f ct ed b y fu n g i r an g e (m ea n ) p er ce n ta g e o f so rg h u m in fe ct ed b y fu n g i in in fe ct ed sa m p le s f ar m er c o ll ec to r f ar m er c o ll ec to r 1 a sp er gi ll u s fl a vu s 1 8 (7 8 .2 6 ) 1 4 (9 3 .3 3 ) 0 .5 -9 9 .5 (1 7 .4 4 ) 0 .5 -8 6 .5 (2 3 .2 1 ) 2 a . n ig er 1 1 (4 7 .8 3 ) 1 4 (9 3 .3 3 ) 0 .5 -7 .0 (2 .9 1 ) 0 .5 -1 7 .5 (4 .7 9 ) 3 a . ta m ar ii 1 1 (4 7 .8 3 ) 9 (6 0 .0 0 ) 1 .0 -5 6 .5 (9 .5 5 ) 1 .0 -2 1 .5 (6 .7 2 ) 4 a . w en ti i 9 (3 9 .1 3 ) 1 4 (9 3 .3 3 ) 0 .5 -1 9 .0 (7 .1 7 ) 0 .5 -8 1 .5 (9 .9 6 ) 5 c la do sp or iu m cl ad os po ri oi de s 9 (3 9 .1 3 ) 0 (0 .0 0 ) 0 .5 -1 4 .5 (3 .0 0 ) 0 6 c ol le to tr ic h u m sp . 1 4 (6 0 .8 7 ) 2 (1 3 .3 3 ) 1 .0 -1 2 .0 (6 .4 3 ) 1 .0 -3 .5 (2 .2 5 ) 7 c u rv u la ri a lu n at a 1 0 (4 3 .4 8 ) 1 (6 .6 7 ) 0 .5 -1 3 .5 (7 .9 5 ) 5 .0 8 c . pa ll es ce n s 1 9 (8 2 .6 1 ) 9 (6 0 .0 0 ) 2 .0 -1 8 .0 (7 .2 6 ) 0 .5 -9 .5 (3 .4 4 ) 9 e n do m yc es fi bu li ge r 6 (2 6 .0 9 ) 1 (6 .6 7 ) 1 .0 -8 9 .5 (3 0 .6 7 ) 8 7 .0 1 0 e u ro ti u m ch ev al ie ri 7 (3 0 .4 3 ) 4 (2 6 .6 7 ) 0 .5 -9 .0 (3 .1 4 ) 2 .0 -3 .0 (2 .5 0 ) 1 1 e . re pe n s 6 (2 6 .0 9 ) 0 (0 .0 0 ) 0 .5 -1 .5 (0 .9 2 ) 0 1 2 e . ru br u m 8 (3 4 .7 8 ) 9 (6 0 .0 0 ) 0 .5 -6 0 .0 (1 3 .6 3 ) 0 .5 -1 8 .5 (4 .8 3 ) 1 3 f u sa ri u m pr ol if er at u m 1 8 (7 8 .2 6 ) 1 1 (7 3 .3 3 ) 0 .5 -4 6 .0 (1 5 .1 7 ) 2 .0 -4 5 .0 (1 5 .6 4 ) 1 4 f . se m it ec tu m 2 2 (9 5 .6 5 ) 1 4 (9 3 .3 3 ) 2 .0 -7 5 .5 (2 4 .4 8 ) 1 .0 -2 9 .0 (7 .1 8 ) 1 5 f . ve rt ic il li oi de s 2 2 (9 5 .6 5 ) 1 4 (9 3 .3 3 ) 1 .0 -7 0 .5 (2 9 .0 9 ) 1 .5 -9 5 .5 (5 1 .6 4 ) 1 6 l as io di pl od ia th eo br om ae 1 (4 .3 5 ) 2 (1 3 .3 3 ) 6 .5 1 (1 ) 1 7 p en ic il li u m sp .1 3 (1 3 .0 4 ) 8 (5 3 .3 3 ) 2 .0 -6 .5 (4 .5 0 ) 0 .5 -4 7 .0 (1 4 .6 9 ) 1 8 p en ic il li u m sp .2 4 (1 7 .3 9 ) 7 (4 6 .6 7 ) 1 .5 -3 0 .5 (1 1 .0 0 ) 9 .5 -4 9 .0 (2 8 .0 7 ) 1 9 p . ci tr in u m 2 3 (1 0 0 ) 1 2 (8 0 .0 0 ) 0 .5 -9 7 .0 (3 0 .2 4 ) 1 .5 -9 0 .5 (3 2 .3 3 ) 2 0 p . is la n di cu m 2 (8 .7 0 ) 2 (1 3 .3 3 ) 1 -2 .5 (1 .7 5 ) 2 .5 -3 .5 (3 .0 ) 2 1 p es ta lo ti op si s gu e p in ii 2 1 (9 1 .3 0 ) 1 2 (8 0 .0 0 ) 0 .5 -1 1 .1 (2 .8 6 ) 1 .0 -5 .5 (2 .8 ) 2 2 is o la te 3 1 5 (6 5 .2 2 ) 9 (6 0 .0 0 ) 0 .5 -1 7 .5 (4 .6 7 ) 0 .5 -1 7 .0 (4 .2 8 ) 2 3 is o la te 7 9 (3 9 .1 3 ) 0 (0 .0 0 ) 0 .5 -9 .5 (3 .3 8 ) 0 n u m b er o f sa m p le s at fa rm er le v el : 2 3 n u m b er o f sa m p le s at co ll ec to r le v el : 1 5 biotropia vol. 18 no. 2, 2011 116 fungal infection of sorghum in wonogiri regency percentage of sorghum infected by fungi in wonogiri regency is presented in table 8. twenty three fungal species were isolated from sorghum collected from , i.e. , , , , , sp., , , , , , , , , , , , , sp. 1, sp. 2, , isolates 3 and 7. eight out of 23 fungal species were belong to field fungi, i.e. , , , , , , , and . twenty fungal species were isolated from sorghum collected from i.e. , , , , sp., , , , , , , , , , , , sp. 1, sp. 2, , and isolate 3. field fungi were still found in sorghum collected from collectors, because the duration of storage was relatively short. it was also assumed, that the storage condition was suitable for fungal growth. (100% of samples) was always isolated from 23 sorghum samples collected from , while and (95.65%, respectively), (91.30%), (82.61%), and (78.26%, respectively) were often isolated. caused highest percentage of infection (30.67%), with the range of 1.0 85.5%, while the lowest was (0.92%), with the range of 0.50 1.50%. farmer collector, farmer aspergillus flavus a. niger a. tamarii a. wentii cladosporium cladosporioides colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens e. rubrum fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum p. islandicum penicillium penicillium pestalotiopsis guepinii cladosporium cladosporioides curvularia lunata c. pallescens fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae pestalotiopsis guepinii a. flavus a. niger a. tamarii a. wentii colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. rubrum f. proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum p. islandicum penicillium penicillium pestalotiopsis guepinii penicillium citrinum f. semitectum f. verticillioides pestalotiopsis guepinii curvularia pallescens a. flavus f. proliferatum endomyces fibuliger eurotium repens 78.26 93.33 95.6593.33 95.6593.33 0 10 20 30 40 50 60 70 80 90 100 aspergillus flavus fusarium semitectum f. verticillioides fl cl fl = farmer level; cl = collector level figure 4. percentage of sorghum samples infected by and at farmer and collector levels in wonogiri regency aspergillus flavus, fusarium semitectum f. verticillioides 117 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al table 9. aflatoxin b content in sorghum collected from farmers, collectors and retailers at traditional market in demak and wonogiri regencies 1 regency level of distribution chain number (%) of samples contaminated by afb1 range (mean) of afb1 content (ppb) in contaminated samples demak farmer 21 (61.76) 4.54-90.78 (22.50) collector 5 (55.56) 13.62-22.73 (15.45) retailer at traditional market 0 0 wonogiri farmer 1 (4.35) 2.27 collector 6 (40.0) 4.57-22.85 (10.28) fungi which were often isolated from 15 sorghum samples collected from were , , , and (93.33%, respectively), and (80%). caused highest percentage of infection (87.0%), while the lowest was (1.0%). and are field fungi, while are post-harvest (storage) fungi. in general, the dominant fungal species infecting sorghum grain collected from various stages of the delivery chain in wonogiri regency were also , and (fig. 4) ercentage of samples contaminated by afb1 in sorghum collected from farmers (61.76%) was higher than that of collected from collectors (55.56%). the mean of afb1 content of sorghum collected from farmes (22.50 ppb, with the range of 4.54 90.78 ppb) was higher than those of collected from collectors (15.45 ppb, with the range of 13.62 22.73 ppb) (table 9). this might be due to more toxigenic strains of available in sorghum collected from farmers compared to those collected from collectors. according to pitt and hocking (2009) aflatoxin production among others depend on the certain strains of and the existence of other fungal species which are antagonistic to aflatoxigenic . dharmaputra . (2001) reported that was able to inhibit growth, consequently aflatoxin production was also inhibited as much as 80% ercentage of samples contaminated by afb1 in sorghum collected from collectors (40%) was higher than collected from farmers (4.35%) (table 9). it was probably due to the duration of storage at collectors was longer than that of at farmers, or the existence of aflatoxigenic found in sorghum collected from farmers was lower that that of in sorghum collected from collectors. the main and range of afb1 content in sorghum samples at farmers was 2.27 and 2.27 ppb, respectively, while at collectors was 10.28 ppb (4.57 22.85 ppb) (table 9). da silva . (2004) reported that 59 strains were found from 10 fresh sorghum samples collected from farmers and 130 sorghum samples collected from collector a. flavus a. niger a. wentii f. semitectum f. verticillioides penicillium citrinum endomyces fibuliger lasiodiplodia theobromae fusarium semitectum f. verticillioides a. flavus, a. niger a. wentii a. flavus f. semitectum f. verticillioides a. flavus a. flavus a. flavus et al in vitro aspergillus niger a. flavus a. flavus et al a. flavus . p . p aflatoxin b content1 aflatoxin b content of sorghum in demak regency aflatoxin b content of sorghum in wonogiri regency 1 1 biotropia vol. 18 no. 2, 2011 118 collector in brazil. as much as 38 (64.4%) of 59 strains were able to produce afb +afb with the range of 12,00-3,282.50 ppb. aflatoxin production was affected among others by the kind of substrates, moisture content and relative humidity, water activity, damaged grains, concentration of oxygen and carbondioxide, temperature and the duration of storage, interaction among microorganisms and the existence of insects (diener & davis 1969). pitt and hocking (1996) stated that aflatoxin content exceeding 1 000 ppb toxic to animal and human. fao (2004) reported that in russia afb1 content in sorghum should not exceed 5 ppb, while in taiwan total aflatoxin content (afb1 + afb2 + afg1 + afg2) should not exceed 10 ppb. in indonesia there are no regulations concerning the maximum tolerable limit of aflatoxin content in sorghum. although in general sorghum at farmer and collector levels were not contaminated by aflatoxin, post-harvest handling in each level of distribution chain should be conducted appropriately to prevent aflatoxin contamination. the method of postharvest handling will affect fungal infection (including ) and aflatoxin contamination. in brazil the dominant fungi found in fresh harvested sorghum (10 samples) and stored sorghum (130 samples) were (57.1%), (42.7%), (25.1%), (21.4%) and 9 genera of other fungi with mycelia. , and were important fungi, because certain species of these fungi can produce toxin. the population range of these fungi were 1 x 10 36 x 10 , 1 x 10 295 x 10 and 1 x 10 20 x 10 /g, respectively. the most often fungi found was and . as much as 12.8% of the samples was contaminated by afb1. their range was 7 33 ppb (da silva . 2000). in nigeria nine pathogenic and saprophytic fungi were found in stored sorghum ( ) which were previously air-dried. pathogenic fungi found were , and , while most of saprophytic fungi belong to , especially , . and (ogundero 2007). was the important fungus of sorghum in argentine in 1991, 1992 and 1993. the dominant fungus was , while the most often fungi isolated were , , and (gonzales . 1997). the percentage of sorghum infected by , , , , , and , i.e. 93.75, 75.0, 62.75, 62.50, 62.50, 56.25 and 37.5%, respectively (hemanth . 2007). schroeder and boller (1973) reported that aflatoxin was found in sorghum samples collected in two of three periods in texas. in 1970, 6% of 114 sorghum samples were contaminated by aflatoxin. their range and mean of contents were 3 20 ppb and 10 ppb, respectively. in 1971, 16% of 25 sorghum samples were contaminated by aflatoxin. their range and mean of contents were 4 9 ppb and 6 ppb, respectively. in general, bandyopadhyay (2000) stated that some species of , , , , , , , and were pathogenic fungi in sorghum seeds. hemanth (2007) reported, that five ( , . , and ) fungal species often isolated were able to inhibit the germination of sorghum. aside from that, 1 2 a. flavus phoma aspergillus fusarium rhizopus fusarium aspergillus penicillium cfu aspergillus flavus fusarium moniliforme et al sorghum guineense cladosporium vignae macrophomina phaseolina helminthosporium turcicum aspergillus a. flavus a fumigatus a. niger fusarium fusarium moniliforme alternaria alternata phoma sorghina penicillium funiculosum aspergillus flavus et al alternaria alternata fusarium moniliforme colletotrichum graminicol;a acremonium strictum phoma sorghina macrophomina phaseolina pestalotia guepinii et al et al. aspergillus alternaria cladosporium diplodia fusarium curvularia phoma penicillium et al. fusarium semitectum, f. verticillioides, f. proliferatum c pallescens a. flavus 3 3 3 3 3 3 119 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al the diversity of fungi in sorghum collected from 10 regions in karnataka, india depended on the variety of sorghum, the structure and the location of the storage. according to schroeder and boller (1973) in 1969 and 1971 the percentage of sorghum samples collected in texas and infected by was 100%, respectively, while the percentage of samples contaminated by aflatoxin was 24 and 40%, respectively. reddy (2002) reported that in india 2, 2, 0 and 2 out of 29 sorg um samples were contaminated by aflatoxin with the range of 10 29 ppb, 30 49 ppb, 50 100 ppb, and >100 ppb, respectively here was a slight difference in the method of postharvest handling of sorghum at farmer and collector levels in demak and wonogiri regencies, especially the method of threshing sorghum grain. in demak and wonogiri regencies the farmers sun-dried sorghum in the form of panicles on tarpaulin or cement floor, while the collectors sundried the sorghum only on cement floor. in demak regency most of farmers threshed sorghum using a paddy thresher, while in wonogiri regency most of farmers threshed sorghum using a stick of wood. in these two regencies collectors threshed sorghum using a paddy thresher. in demak regency farmers and collectors stored sorghum grain in polypropylene bags. only in wonogiri regency most farmers and collectors stored sorghum in polypropylene bags. the moisture contents of sorghum at farmer and collector levels in demak and wonogiri regencies were still lower compared to normal moisture content of sorghum. various insect species associated with sorghum was found at various stages of the delivery chain in demak and wonogiri regencies. the dominant insects species were and . various field and postharvest storage fungi were isolated from sorghum at various stages of the delivery chain in demak and wonogiri regencies. the dominant fungal species found were , and . generally, afb1 content in sorghum at various stages of the delivery chain in demak and wonogiri regencies were low, i.e. ≤ 22.5 ppb he authors gratefully acknowledge the financial support of seameo biotrop through dipa 2010 by contract agreement no. 050.11/psrp/spkpnlt/iii/10. sincere thanks are also due to the head of the indonesian government's regional office of agriculture in demak and the indonesian government's regional office of agricultural crop and horticulture in wonogiri; to mrs. ratnaningsih, mr. edi suryadi, ms. nijma nurfadila, ms. amanda windyarani for their assistance, and the reviewers of this manuscript h . t . t . a. flavus et al. sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum 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(2013), one of the most important mechanisms responsible for suppressing pseudomonas sp. (a plant pathogen) is siderophoremediated competitions for iron. siderophores (from the greek “iron carriers”) are small ferric-ion-specific chelating agents produced by bacteria and fungi which grow in low iron condition causing them to scavenge iron from the environment and to make iron available to the microbial cell (neilands 1995; pal & gokarn 2010). siderophores are also known to bind molybdenum and lead (pal & gokarn 2010). sayyed et al. (2005) reported that siderophoreproducing pseudomonas sp. plays vital role in stimulating plant growth and in controlling several plant diseases. in pseudomonas fluorescens the pigment produced is a siderophore. pigment production is increased in the presence of sodium, potassium, lead, molybdenum, cadmium a n d a m m o n i u m s u l p h a t e ( ( n h ) s o ) 4 2 4 (bhattacharya 2010; sayyed et al. 2005). adding exogenous amino acid did not significantly increase the production of siderophore (hu & xu 86 biotropia vol. 24 no. 2, 2017 java province, indonesia. from each village, 1,500 g of rhizosphere soil samples (with tomato roots) were collected from five different plots (300 g per plot as subsample) and thoroughly mixed using trowel, until becoming a composite sample. from each composite sample, 10 g of rhizosphere soil was immersed in 90 ml 0.85% nacl. after a serial dilution, 0.1 ml of each dilution was inoculated on chrome azurol sulphate (cas) medium (gross 1990; louden et al. 2011). inoculation was repeated three times. plates containing the inoculation were incubated at room temperature (±28 c). numbers of o colony of siderophore-producing rhizobacteria were calculated at 24 48 hours after incubation, based on the production of orange zone around colony of the bacteria. successfully isolated bacteria were transferred to king's b agar medium and separated from each other to obtain pure culture. each isolate was preserved in nutrient broth (nb) with 20% glycerol and kept in -20 c environment. for daily maintenance, o isolates were preserved in nb and kept at room temperature (±28 c) o . hypersensitive reaction test hypersensitive reaction (hr) test was conducted to select the nonpathogenic bacteria as candidates of biocontrol agents. the test was 8 9 conducted by inoculating 10 – 10 cfu/ml of rhizobacteria on tobacco leaves. one milliliter of suspension was injected into tobacco leaf using 5 ml sterile syringe. successful injection was indicated by water soaked zone around the point of injection. inoculation of each isolate was conducted in duplo. inoculated leaves were o incubated at room temperature (±28 c) for 24 hours. leaves showing necrotic symptoms before 24 hours were counted as having positive reaction to the hypersensitive reaction and thus, be eliminated. antagonistic activities of siderophoreproducing rhizobacteria against in vitro ralstonia solanacearum bacteria isolates having negative reaction on the previous hypersensitive test were tested on their antagonistic activity toward r. solanacearum. both bacteria, i.e. r. solanacear um and rhizobacteria, were grown on a king's b agar 2011). sayyed et al. (2005) also reported that mn, hg and co showed inhibitory effect on siderophores growth and production. presence of potassium, magnesium and calcium had little inhibitory effect on siderophores production compared to controls (battacharya 2010). sayyed (2005) reported that production of et al. siderophores by bacteria was affected by growing media. in growing media such as nutrient broth (nb) and mackonkey's broth (mb), siderophores were not produced because both media are luxurious media having high content of fe. fefree succinic acid medium (sm) was found to provide maximum (92.25%) siderophores production in comparison to 88.00% in barbhayya rao broth (br), 79.00% in cassamino acid broth (caa) and 48.00% in enrichment medium (em) (sayyed 2010). a study on ph et al. effect to the production of siderophores by rhizobium sp. showed that the growth and production of siderophores started at ph 4.5, reaching maximum at neutral ph; at ph 10.0, there was no siderophores production (sridevi et al. 2008). study on genetic diversity of tobacco rhizosphere which produces siderophore demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium; some of them are , pseudomonas enterobacter serratia pantoea erwinia , , , and stenotrophomonas γ-proteobacteria which belong to (tian 2009).et al. this experiment was conducted to select and ch a r a c t e r i z e t h e s i d e r o p h o r e p r o d u c i n g rhizobacteria from tomato and to determine their potential as antagonistic agents for r. solanacearum. materials and methods isolation and quantification of siderophoreproducing rhizobacteria siderophore-producing rhizobacteria were isolated from rhizosphere samples taken from healthy tomato plants, collected from tomato field in cipanas sub-district (cianjur district) and lembang sub-district (west-bandung district). in cipanas sub-district, samples were collected from three villages, while in lembang sub-district samples were collected from two villages. both sub-districts are the center of tomato field in west 87 siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. plate. five hundred micro liter suspension of r. solanacearum (10 10 cfu/ml) was spread on the 8 9 surface of the king's b agar plate. after being air dried, three sterilized filter papers having 0.5 cm diameter were placed besides the agar plate with 2 cm distance from each other. filter paper in the center was inoculated with 50 µl of sterilized distilled water and designated as control. the two other filter papers were each inoculated with 50 µl suspension of one isolate of rhizobacteria. each treatment was repeated three times. inoculated plates were incubated at room temperature (±28 c). the antagonistic activity o was observed at 24 hours after incubation. antagonistic activity was indicated by the production of inhibition zone around the filter paper inoculated with siderophore-producing rhizobacteria. e f f e c t o f s i d e r o p h o r e p r o d u c i n g rhizobacteria on the viability of tomato seeds tomato seeds (arthaloka and ratna varieties) were dipped in 10 10 cfu/ml suspension of 7 8 siderophore-producing rhizobacteria for 16 hours before being planted on sterilized mixture of soil and compost (1 : 1 ratio). the soil mixture was put in 30 50 cm polyethylene pot tray having 128 holes. the experiment was arranged as completely randomized factorial design with 7 isolates of biocontrol agents and one control (without bacteria) as the first factor and two tomato varieties (arthaloka and ratna) as the second factor. thus, 16 treatments were applied in this experiment. each treatment was replicated three times. there were 48 units in total; each unit contained 15 seeds. one tomato seed was grown in one hole of polyethylene pot tray. the isolates of biocontrol agents used in this experiment were cp1c (code of one isolate of bacteria), cp2b, cp2d, cp3e, lb1a, lb1c and lb1l. seeds dipped in sterilized distilled water were used as control. layout of the treatments was shown in table 1. total of the emerging seedlings were calculated every day. seed viability (sv) was calculated using the following formula: total normal seedlings sv (%) = x 100% total seeds sown e f f e c t o f s i d e r o p h o r e p r o d u c i n g rhizobacteria on the height, fresh and dry weight of tomato plants an experiment to test the effect of siderophore-producing rhizobacteria toward the height, fresh and dry weight of tomato plants was conducted in a green house. tomato seeds of arthaloka and ratna varieties were dipped in suspension of siderophore-producing rhizobacteria (10 10 cfu/ml) for 16 hours 7 8 before being planted in 10 x 15 cm polybag filled with 2.5 – 3 kg of sterilized mixture of soil and compost (1 : 1 ratio). the experimental design applied was completely randomized factorial design having similar layout with the one presented in table 1. the only difference was that each unit contained 5 seeds. plant height was measured every 5 days starting from the day when the first leaf was fully opened. total area under height of plant growth cur ve (auhpgc) was calculated using table 1 layout of experiment to test the effect of siderophore-producing rhizobacteria toward the viability of tomato seeds tomato varieties isolate’s code cp1c cp2b cp2d cp3e lb1a lb1c lb1l control arthaloka repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds ratna repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 88 biotropia vol. 24 no. 2, 2017 modification of formula reported by van der plank (1963): where: y = increasing of plant height at the next i+1 observation y = increasing of plant height at the time of i observation t = the next observation (ii, iii, …, vi) i+1 t = time of observation (i, ii, …, v)i two months after planting, all plants (five plants) from each replication of each treatment were rooted and weighted using digital balance. the average of the five plants represented data of fresh weight for each replication. dry weight of plant was determined by drying the fresh plants in the oven at 100 c. the weight of the sample was o checked periodically until the weight of the sample was constant. characterization and identification of the siderophore-producing rhizobacteria seven isolates of the siderophore-producing rhizobacteria were characterized based on microscopic and colony appearances, as well as on physiological and biochemical properties, following the methods of klement . (1990) et al and schaad . (2001). two isolates of the et al siderophore-producing bacteria having potential as biocontrol agents were genetically identified by sequencing the 16s rdna gene. dna was extracted from log phase culture using phenolchlorofor m extraction procedure (sambrook & russel 2001). the 16s rdna gene was amplified using universal primer for prokaryotes which were the forward primer 27f (5'-agagtttgatcctggctcag-3') and reverse primer 1492r (5'ggttaccttacgactt-3'). total volume reaction for polymerase chain reaction (pcr) was 25 µl consisted of 1 µl of dna template; 12.5 µl of 1x ready mix pcr, 1.5 mm mgcl , 0.2 mm dntps and taq 2 polymerase 5 units/reaction; 9.5 µl of ddh o; 1 2 µl of 120 pmol primer 27f; and 1 µl of 120 pmol primer 1492r. pcr was performed under the following conditions: one cycle of preo denaturation at 95 c for 5 minutes, followed by 35 o cycles of denaturation at 95 c for 1 minute, o annealing at 55 c for 1 minute and extension at o 72 c for 2 minutes. the reaction was terminated o with a final extension at 72 c for 10 minutes. the pcr products were sent to the first base laboratory, malaysia for sequencing. blast searches were performed for sequences obtained to find the similarity with sequence data in genebank. data analysis data of the bacterial population was analyzed using t test of minitab program version 13.3. effects of the bacteria to the plant growth were statistically analyzed using anova for completely randomized factorial design with isolates of biocontrol agents as the first factor and tomato varieties as the second factor. treatment means were compared using the dmrt test at 5% level of significance. sas program version 9.1 was used for performing statistical analyses for completely randomized factorial design and for the dmrt test. results and discussion abundance and antagonistic activities of siderophore-producing bacteria based on the t test, the average of siderophoreproducing bacteria isolated from cipanas district 7 was 1.98 10 cfu/g, which was not significantly different with those from lembang district 7 having average of 5.3 x 10 cfu/g. colonies of the siderophore-producing bacteria on cas medium were shown in figure 1. siderophore production was indicated by the orange color around the colony of the bacteria. figure 1 shows that each colony produced different amount of siderophores, indicated by the diameter of orange area around each colony of bacteria. among the 60 isolates of siderophoreproducing rhizobacteria, 31 isolates positively showed hypersensitive reaction on tobacco, while 29 others showed negative reaction. bacteria were also tested further for their antagonistic activities against . based on the ralstonia solanacearum antagonistic test, 16 isolates positively produced inhibition zone (fig. 2) having diameter between 0.5 to 7.0 mm. among those 16 isolates, 9 isolates were hr positive which had to be eliminated from being candidate of biocontrol agents. the auhpgc n – 1 y y i i+ +1 2 t i +1 – tis( (( ( 89 other 7 isolates were hr negative, having respective diameter of the inhibition zone of cp1c (3.6 mm), cp2b (2.3 mm), cp2d (7.0 mm), cp3e (5.0 mm), lb1a (1.6 mm), lb1c (1.6 mm) and lb1l (0.5 mm). the widest diameter of inhibition zone was produced by isolate cp2d which was isolated from cipanas (table 2). microorganisms growing under aerobic conditions need iron for a variety of functions, including reduction of oxygen for atp synthesis, reduction of ribotide precursors of dna, for formation of heme and for other essential purposes. a level of at least one micromolar iron is needed for optimum growth (neilands 1995). figure 1 production of siderophores by tomato rhizobacteria on cas agar was indicated by yellow-orange color around colony of bacteria figure 2 production of inhibition zone (arrow sign) by the isolate of siderophore-producing rhizobacteria (note: the inset shows magnification of the inhibition zone) table 2 isolates of siderophore-producing rhizobacteria which produced inhibition zone against r. solanacearum on kings’s b agar isolate code1) diameter of inhibition zone (mm) isolate code diameter of inhibition zone (mm) isolate code diameter of inhibition zone (mm) cp1b2) cp1c cp2b cp2c cp2d cp2h 6.8 3.6 2.3 0.6 7.0 4.5 cp2l cp2s cp3e cp3m cp3t lb1a 1.6 2.6 5.0 2.2 4.0 1.6 lb1c lb1d lb1e lb1l 1.6 0.5 0.5 0.5 note: 1) cp = isolates from cipanas; lb = isolates from lembang 2) isolates written in bold were positively causing hypersensitive reaction (hr) siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. table 3 effect of siderophore-producing rhizobacteria on tomato seed viability of arthaloka and ratna varieties treatment seed viability (%)*) var. arthaloka control cp1c cp2b cp2d cp3e lb1a lb1c lb1l var. ratna control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 93.33 ab 86.63 abc 88.83 abc 84.40 abc 88.87 abc 75.50 abc 100.00 a 95.53 a 66.60 c 75.53 abc 79.97 abc 84.43 abc 75.53 abc 68.83 bc 66.63 c 84.30 abc note: *) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) 90 biotropia vol. 24 no. 2, 2017 table 4 effect of siderophore-producing rhizobacteria on the increase of tomato plant height of arthaloka and ratna varieties treatment plant height increase (cm)1) auhpgc3) (cm days) i 5 dap2) ii 10 dap iii 15 dap iv 20 dap v 25 dap vi 30 dap var. arthaloka control cp1c cp2b cp2d cp3e lb1a lb1c lb1l var. ratna 1.04 abcde 0.99 bcde 1.51 abc 1.69 ab 1.23 abcd 1.66 ab 1.71 ab 1.87 a 3.57 a 1.42 a 2.30 a 2.32 a 2.78 a 2.48 a 2.43 a 2.69 a 2.03 b 3.83 a 2.04 b 2.24 b 2.00 b 2.25 b 1.81 b 1.81 b 2.86 a 2.49 a 2.65 a 2.61 a 2.58 a 3.05 a 2.70 a 2.61 a 4.34 a 4.53 a 4.74 a 4.66 a 3.93 a 4.61 a 4.44 a 4.19 a 6.85 a 7.50 a 7.20 a 7.81 a 6.43 a 6.98 a 6.63 a 6.65 a 83.75 a 82.56 a 80.40 a 82.90 a 75.53 a 83.53 a 77.78 a 77.78 a control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 0.79 cde 1.09 abcde 0.29 e 0.48 de 0.87 bcde 0.91 bcde 0.53 de 0.40 de 1.99 a 0.99 a 1.51 a 1.91 a 1.23 a 1.78 a 1.45 a 2.00 a 1.89 b 3.73 a 1.89 b 2.03 b 1.87 b 1.46 b 2.0 b 1.81 b 1.97 a 2.11 a 1.31 a 1.85 a 1.86 a 1.57 a 1.44 a 1.35 a 3.19 a 3.98 a 2.85 a 2.97 a 2.89 a 2.29 a 2.57 a 2.27 a 5.09 a 6.47 a 3.81 a 4.37 a 4.23 a 3.67 a 3.77 a 3.55 a 59.90 a 72.92 a 48.05 a 56.00 a 52.00 a 46.97 a 48.17 a 47.09 a note: 1) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) 2) dap = days after planting 3) auhpgc = area under height of plant growth curve seven isolates of the bacteria were tested for their effects on seed viability, plant height and the fresh and dry weight of two varieties of tomato plants, i.e. arthaloka and ratna, as presented in table 3, 4, and 5, respectively. data in table 3, 4, and 5 show not only that the isolates of bacteria did not significantly increase the seed viability, plant height, fresh and dry weight of tomato compared to control, but also they did not have harmful effects to the tomato plants. the characteristics of colony morphology, physiology and biochemistry aspects of the seven isolates were presented in table 6 and 7, respectively. 91 table 6 morphological characteristic of colony of the seven isolates of siderophore-producing rhizobacteria on king's b agar isolate code1) colony characteristic diameter color elevation edge form cp1c cp2b cp2d cp3e lb1a lb1c lb1l ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 3 mm ± 1 mm white broken white broken white dark yellow greenish white pale white broken white convex domed convex convex convex umbonate convex wavy entire entire entire entire curly wavy circular circular circular circular circular irregular circular note: 1) cp = isolates from cipanas; lb = isolates from lembang table 7 physiological and biochemistry characteristics of siderophore-producing rhizobacteria having potential as antagonist for r. solanacearum isolate code1) fluorescence gram reaction phosphate solubilization resistance to 80 oc cp1c + + cp2b +++ + cp2d + + cp3e lb1a + + lb1c + + + lb1l + + note: 1) cp = isolates from cipanas; lb = isolates from lembang table 5 effect of siderophore-producing rhizobacteria on fresh and dry weight of tomato plants treatment1) fresh weight (g/plant)2) dry weight (g/plant) var. arthaloka control cp1c1) cp2b cp2d cp3e lb1a lb1c lb1l var. ratna control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 15.439 a 14.266 a 15.070 a 15.265 a 9.515 a 12.557 a 12.572 a 11.335 a 10.109 a 8.748 a 6.887 a 10.744 a 9.566 a 7.880 a 6.887 a 5.319 a 3.381 a 2.587 a 2.900 a 2.986 a 2.381 a 2.721 a 2.579 a 2.742 a 2.360 a 1.954 a 1.801 a 2.281 a 2.006 a 1.547 a 1.436 a 1.535 a note: 1) cp = isolates from cipanas; lb = isolates from lembang 2) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) based on the inhibition zone production, effect on seed viability, effect on plant height increase, and effect on fresh and dry weight of tomato product, two isolates of siderophoreproducing bacteria having the best effects were selected. the two isolates were cp1c and cp2d. the sequence of 16s rdna of those isolates were referred to the gene bank. using the blast program the isolate of cp1c was identified as brevundimonas sp., while the isolate cp2d was siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. 92 biotropia vol. 24 no. 2, 2017 · = enterobacter sp. cp2d = brevundimonas sp. cp1c figure 3 phylogenetic tree of isolates enterobacter sp. cp2d and brevundimonas sp. cp1c enterobacter sp. mth17 mth17 gi323218729 enterobacter sp. b49 b49 gi389827949 enterobacter ludwigii b-5 carrot gi444438257 enterobacter sp. acc2 acc2 gi399936203 enterobacter sp. m.d.e.na4-3 m.d.e.na4-3 gi326635001 enterobacter sp. e6-pcai-t2p21 e6-pcai-t2p21 gi358365187 enterobacter sp. enrichment culture clone gi355343574 cp2d brevundimonas sp. 13630g 13630g gi206581409 brevundimonas nasdae 13636e gi206581436 brevundimonas sp. mc8-1 mc8-1 gi300253164 cp1d brevundimonas vesicularis l17 gi375004753 blackwater bioreactor bacterium bw23 bw23 gi16589030 uncultured bacterium gi253770558 uncultured bacterium gi253770269 100 84 100 100 80 100 73 100 69 100 41 46 75 0.05 table 8 maximum score, e value and percentage of similarities of siderophore-producing bacteria isolate species homolog identity max score query cover e value accession number cp1c brevundimonas sp. 13630 g 16s ribosomal rna gene, partial sequence 95% 2021 92% 0.0 eu741063.1 cp2d enterobacter sp. enrichment culture clone dwsr 106 16s ribosomal rna gene, partial sequence 88% 998 69% 0.0 jn944751.1 identified as enterobacter sp. with percentage of similarity of 92% and 93%, respectively. maximum score, e value and the percentage of similarities of siderophore-producing bacteria were presented in table 8. phylogenetic tree of the related bacteria is presented in figure 3. tian et al. (2009) reported that enterobacter and pseudomonas were dominant in the rhizosphere of tobacco, with 44.5% and 24.7% total frequency, respectively. siderophores are produced by various bacteria and fungi, usually classified by the ligands used to chelate the ferric iron. the major groups of s i d e r o p h o r e s i n c l u d e t h e c a t e c h o l a t e s (phenolates), hydroxamates and carboxylates (e.g. derivatives of citric acid) (saharan & nehra 2011). rachid & ahmed (2005) reported that streptomycin and penicillin added to succinate medium acts differently on the siderophores production. streptomycin reduced siderophores production below 10 µm in different iron concentrations, while penicillin increased the production of siderophores in the presence of excess iron (above 100 µg/ml). the growth of p. fluorescens and siderophore production were inhibited by the occurrence of heavy metals (lead, mercury and cadmium), especially in iron-limited condition . 93 conclusions the average of siderophore-producing bacteria isolated from cipanas district (1.98 x 10 7 cfu/g) was not significantly different from those isolated from lembang district (5.3 x 10 cfu/g). 7 the highest diameter of inhibition zone to ralstonia solanacearum was 7.0 mm, produced by isolate cp2d. the selected bacteria producing the inhibition zone did not significantly affect seed viability, plant growth, fresh weight and dry weight of tomato compared to control. based on the characteristics of colony morphology, physiology, biochemistry and partial sequence of 16s rdna, two selected isolates, i.e. cp1c and cp2d were identified as sp. and sp., brevundimonas enterobacter respectively. acknowledgements this research was funded by dipa ipb with scheme of decentralized research program (program penelitian desentralisasi: hibah bersaing), contract no. 27/i3.24.4/spk/ pd/2010, on 5 march 2010. the authors also thank dr kikin hamzah mutaqin for the guidance in several molecular tests activities. references agrios gn. 2005. plant pathology. fifth edition. new york (us): academic press. 992 p. bhattacharya a. 2010. siderophore mediated metal uptake by pseudomonas fluorescens and its comparison to iron (iii) chelation. cey j sci (bio. sci.) 39(2):147-55. gross m. 1990. siderophores and fluorescent pigments. in: klement z, rudolph k, sands dc, editors. methods in phytobacteriology. budapest (hu): akadémiai kiadó. 568 p. hu qp, xu jg. 2011. a simple double-layered chrome azurol s agar (sd-casa) plate assay to optimize the production of siderophores by a potential biocontrol agent . afr j microbiol res bacillus 5(25):4321-7. jenifer mra, reena a, aysha os, valli s, nirmala p, vinothkumar p. 2013. isolation of siderophore producing bacteria from rhizosphere soil and their antagonistic activity against selected fungal plant pathogens. int j curr microbiol app sci 2(1):59-65. jeung y, kim j, kang y. 2007. genetic diversity and distribution of korean isolates of ralstonia solanacearum. plant dis 91(10):1277-87. klement z, rudolph k, sands dc. 1990. methods in phytobacteriology. budapest (hu): akadémiai kiadó. 568 p. louden bc, haarmann d, lynne am. 2011. use of blue agar cas assay for siderophore detection. j microbiol biol educ 12(1):51-3. neilands jb. 1995. siderophore: structure and function of microbial iron transport compounds. j biol chem 270(45):26723-6. pal rp, gokarn k. 2010. siderophores and pathogenicity of microorganisms. j biosci tech 1:127-34. rachid d, ahmed b. 2005. effect of iron and growth inhibitors on siderophores production by pseudomonas fluorescens. afr j biotechnol 4(7):6977 0 2 . av a i l a b l e o n l i n e a t h t t p : / / w w w. academicjournals. org/ajb. saharan bs, nehra v. 2011. plant growth-promoting rhizobacteria: a critical review. life sciences and medicine research, volume 2011:lsmr-21. http://astonjournals. com/lsmr [retrieved on 24 october 2011]. sambrook j, russell dw. 2001. molecular cloning. a laboratory manual. third edition. new york (us): cold spring harbor lab pr. p.6-62. sayyed rz, badgujar md, sonawane hm, mhaske mm, chincholkar sb. 2005. production of microbial iron chelators (siderophores) by fluorescent pseudomonads. indian j biotechnol 4:484-90. schaad nw, jones jb, chun w. 2001. laboratory guide for identification of plant pathogenic bacteria. third e d i t i o n . s t . pa u l ( u s ) : t h e a m e r i c a n phytopathological society. 373 p. sridevi m, kumar kg, mallaiah kv. 2008. production of catechol-type of siderophores by rhizobium sp. isolated from stem nodules of sesbania procumbens (roxb.) w and a. res j microbiol 3(4):282-7. tian f, ding y, zhu h, yao l, du b. 2009. genetic diversity of siderophore-producing bacteria of tobacco rhizosphere. braz j microbiol 40:276-84. wang jf, lin ch. 2005. integrated management of tomato bacterial wilt. the world vegetable center. h t t p : / / w w w. a v r d c . o r g / p d f / p r o d 5 management_bacterial_wilt.pdf. [retrieved on 26 september 2011]. yuliar, nion ya, toyota k. 2015. recent trends in control methods for bacterial wilt diseases caused by ralstonia solanacearum .. microbes environ. 30(1):1-11 siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia 1 (1) 1987: 26-40 studies on interference among trees in a plantation of altingia excelsa kan-ichi sakai*, andreas rumbino** national institute of genetics, mishina, japan and cenderawasih university, manokwari, irian jaya, indonesia respectively shinya iyama* and lilian u. gadrinab national institute of genetics, mishima, japan and tropical forest biology program, biotrop, bogor, indonesia respectively abstract a study was made with the use of a 50-year old altingia excelsa noronha plantation with 260 standing trees for separating components of density and intraspecific competition. a component of density effect causes overall decrease in growth while that of competition results in a contrasting effect in growth between any pair of neighboring trees. to detect this density effect, it is most appropriate to use an area of ca. 100 m 2 irrespective of the experimental area, e.g. circular or quadratic. competition effect cannot be detected when two individuals are apart more than two meters. an application of the density and competitive ability to tree breeding is briefly mentioned. introduction growth of trees standing in a monospecific population is under considerable influences of associate trees growing in the proximity. the first is the general effect of thickly planted trees and the second is the specific effect of competition between a given pair of neighboring trees. in some cases the so-called allelopathic effect goes on with them though in the present species no sign of its occurrence has been noticed. the density response and the inter-tree competition are very ubiquitous and consequently silviculturally very important, although the two have often been treated by plant ecologists and agronomists as phenomena of the same category. the senior writer of the present paper, however, is of the belief that the density effect is biologically different from the inter-tree competition. therefore, in the present study, the writers intend to investigate the density response and the intraspecific competition in a plantation planted more than fifty years ago with seedlings of a tropical tree species altingia excelsa noronha of the * temporarily seconded to biotrop. ** biotrop ten-month research training scholar on forest plant genetic resources, 1 septemb er 1980—30 june 1981. 26 interference among trees in a plantation of a/tingia excelsa — sakai et al. family hamamelidaceae. the species is locally called rasamala and is one of t he valuable timber species native to indonesia. it grows in natural stands in western java and is also planted for afforestation. its wood is heavy, hard and fine-grained and the color is red to blackish brown. it produces a yellow scented rasamala resin which is used in perfumery. it grows in an area of elevation of 500 to 1500 meters above sea level. the rasamala plantation is on a slow increase in the mountainous area of western java. materials and methods materials for the study have been obtained from an afforested plantation of altingia excelsa located in the mountainous region of bandung in the western part of java, indonesia. according to a brief document of the regional forestry office, saplings were initially planted at a distance of 3 x 1 meters in 1927. the first thinning was applied in 1932 (5 years after planting), while the second thinning in 1938 or 11 years after planting. detailed record, however, is not available at present. after 1939, the stand has been left without artificial thinning, though some trees died from the attack of insects or mammals such as forest pig, or due to some other reasons. data were taken in february 1981 when the number of trees still growing in the 60 x 90 meters rectangular plot was 268. measurement was taken on tree height, clear bole height and diameter at breast height (dbh) and exact location of trees. estimation of the effect of tree density on the growth of trees has been made in two ways: (1) effect of number of trees growing within a given radius on the growth of a tree standing in the center of the circle (circular plot) and (2) effect of number of trees within a quadrat or a circle on their average growth (quadrate plot or circular plot). the estimation is based on the assumption that the effect of density would be general for every tree standing in a neighborhood. for estimating the effect of tree density by measuring the central tree, various sizes of the circle from a radius of 2.5 to 7.5 meters have been taken, while the effect of tree number on their average growth has been measured in a quadrat of 10 x 10 meters or in circle of a radius of 5 to 6 meters. for an investigation of intraspecific competition, correlation of dbh between two nearest neighboring trees, on the one hand, and difference in dbh between them, on the other, have been measured for various inter-tree distances. this is on the assumption that the effect of competition on the growth of a pair of adjoining trees would be antagonistic, i.e. favoring one at the expense of another to yield negative correlation or to enlarge the difference between their dbh's. 27 biotropia vol. 1 no. 1, july-december 1987 results of the study trees of altingia excelsa in the present plantation have grown after 55 years of planting to an average height of 32.6 meters, though some trees exceeded 40 meters. the dbh has grown to 33.3 centimeters on the average, with the biggest tree with a value of more than 60 centimeters. the variations in the tree height and dbh are presented in tables 1 and 2, and graphically but with an increased number of class intervals in figures 1 and 2. figure 1. frequency histogram of tree height of an even -aged stand of altingia excelsa. the frequency histogram of the tree height is negatively skewed and leptokurtic (fig. 1), while that of dbh is positively skewed with an intuitive impression of some multimodality (fig. 2), though both of the tree height and the dbh were positively correlated with r = 0.6769** for 266 degrees of freedom. 28 i. growth of trees interference among trees in a plantation of altingia excelsa — sakai et al. figure 2. frequency histogram of diameter at breast height (dbh) of an even-aged stand of altingia excelsa. 29 biotropia vol. 1 no. 1, july-december 1987 ii. density effect at first, the density effect was measured by the dbh of a central tree being surrounded by various number of trees growing within a given area of a circular plot. the circular plots were constructed with various radii from 2.5 to 7.5 meters. if the tree density does exert some hindering effect on the growth of trees, the number of trees within a given circle should be negatively correlated with dbh of the central tree because the higher the density, the poorer is the growth of the central tree would be. the correlation coefficients between them are shown for various sizes of the circle (table 3). it is found that a smaller circle with a radius less than 4 meters is too small to include sufficient number of trees to yield apparent density effect. it looks that the most reliable size of the circle for detection of the density effect is a radius of 5 to 6.5 meters with the average of 4.5 to 7 trees growing within the plot. the distribution of dbh of a central tree, averaged for each class, against various number of trees within the radius of 5.5 meters, as an example, is shown in figure 3. figure 3. effect of number of trees coexisting within a radius of 5.5 meters on the dbh growth of a central tree. x: mean value. 30 interference mong trees in plantation of altingia excelsa sakai et al. 31 biotropia vol. 1 no. 1, july-december 1987 the second approach to the estimation of the density effect was made by measuring the average growth of all trees growing within a given circle. the size of the circular plot was confined to the radii ranging from 5 to 6.5 meters on the basis of the foregoing analysis. the correlation coefficients between the number of trees within a plot of a given radius and their average growth are shown in table 4. it shows that the most reliable size of a circle for estimating density response is with a radius of 5.5 to 6.0 meters. the 10 x 10 meter quadrat just corresponds to the area of a circle of a radius of 5.64 meters, giving an equivalent correlation of —0.351. the distribution of dbh in relation to the number of trees within a limited area with a corresponding regression line is depicted for a circle of 5.5 meter radius in figure 4 and 10 x 10 meter quadrat in figure 5. it is concluded from these studies that the response to density in the plantation of altingia excelsa can be properly detected by measuring mean dbh figure 4. effect of number of trees coexisting within a radius of 5.5 meters on their dbh growth. x: mean value. growth of trees in relation to their number growing within a radius of 5.5 to 6.0 meters or in quadrats of 10 x 10 meters, area of which being approximately between 95 and 110 m 2 or just 100 m 2 in the case of the quadrat. measurement of dbh of a single tree for detection of density effect is more or less inaccurate because of high fluctuation in measurements as found in figure 3 (compare with 32 interference among trees in a plantation of altingia excelsa — sakai et al. ** significant at the 0.01 level figure 4). granting that the coefficient of regression described in figure 4 is correct, we can mention that the response to tree density in altingia excelsa is -0.75 centimeters in dbh against an increase of one tree in the stand. figure 5. effect of number of trees coexisting in a 10 x 10 meter quadrat on their dbh growth. x: mean value. 33 biotropia vol. 1 no. 1, july-december 1987 iii. intraspecific competition as shown in figures 3 to 5, growth of dbh of trees decreases linearly as the number of trees in a limited area increases. competition between adjoining trees may interrupt the linear decrease in dbh due to the density effect. if competition occurs between two adjoining trees, the growth of the one is favored while that of another is handicapped on the basis of their inherent competitive ability. thus, a negative correlation results between them. table 5 shows that the correlation of dbh between two adjoining trees is negative if the inter-tree distance is less than 2 meters. the positive correlation found at the inter-tree distance larger than 2.01 meters indicates that inter-tree competition in altingia excelsa appears to come into play within a distance of two meters. in the right column of table 5 and also in table 6, the differences in dbh between two adjoining trees in relation to inter-tree distances are described. it is expected naturally that if competition occurs between two adjoining trees, the difference in dbh between them should be greater by the effect of competition than otherwise. it is found from table 5 that at a distance less than two meters, the difference in dbh is greater than others; while from table 6, one notes that at a distance of two meters or less, the distribution of various values of difference in dbh is not at random, but larger values than 11 are more often observed than theoretically expected. table 5. inter-tree competition in relation to inter-tree distance in altingia excelsa. inter-tree distance in meters number of pairs correlation of dbh between two adjoining trees difference in dbh between two adjoining trees in centimeters <1.75 11 — 0.4139† 14.71 ±7.96 1.75—2.00 16 —0.2276j† 12.63 ±7.62 2.01—2.25 14 + 0.6035* 9.65 ±7.93 2.26—2.50 28 + 0.1275† 12.25 ±8.08 2.51—3.00 30 + 0.4349* 9.94 ±7. 65 3.01—3.50 27 + 0.5858** 7.21 ±5.10 3.51—4.00 32 + 0.3703* 8. 35 ±7.65 4.01—4.50 30 + 0.0259† 11.12±7.38 4.51—5.00 25 + 0.6222** 6.84 ±5.27 5.01—5.50 15 + 0.1285† 7. 85 ±6.92 5.51—7.50 21 + 0.6155** 7. 56 ±5.78 † falling short of the significance level. *, ** significant at the 5 and 1% levels, respectively. 34 interference among trees in a plantation of altingia excelsa — sakai et al. figure 6 shows the distribution of mean differences in dbh between two adjoining trees at different inter-tree distances from 1.5 to 7.5 meters. the theoretical curve to fit the data can be obtained from the following formula : y = 20.574/ x ° 6 0 figure 6. mean differences in dbh against distances between two adjoining trees. the curve represents theoretical one calculated by y = 20.574/xo-60. 35 biotropia vol. 1 no. 1, july-december 1987 where y and x are dbh difference and inter-tree distance, respectively. the curve suggests that the competitive effect is apparent only when the inter-tree distance is small or less than 2 meters. discussion as early as some 30 years ago, koyama and kira (1956) investigated the frequency distribution of plant weight in experimental populations of several kinds of plants and found that the distribution of girth of trees or weight of annual plants generally showed a positive skewness or the l-shaped distribution though in the initial stage of their growth it took the form of a normal distribution. they concluded from a mathematical study that, assuming normal distribution in the initial plant weight and also in the growth rate, the distribution of plant weight in later stages had to become positively skewed or to show the l-type distribution without additional assumption of interaction among plants. they further found that the frequency distribution of plant height, on the contrary, showed a normal curve or in some cases negative skewness. ford (1975) investigated the frequency histograms of girth of trees or weight of annual plants in even-aged plant monocultures and concluded that positive skewness and platykurtosis showing bimodality were characteristic of their distribution. he also observed an even-spatial distribution of large plants in the population. he attributed such an occurrence of positive skewness with bimodality and the even-spatial distribution to the effect of intraspecific competition. in contrast to these observations, tanaka (1983) recently published a paper describing an apparently negative, instead of positive, skewness in the distribution histogram of dbh in 42-year-old cryptomeria japonica plantation. in altingia excelsa of the present investigation, the tree height showed highly significant negative skewness together with an apparent peakedness, whereas the dbh showed a significant positive skewness as depicted in figure 2. in addition, the histogram of dbh seems to be indicative of multimodality rather than bimodality. as described above, ford is of the opinion that bimodal distribution is an evidence of intraspecific competition, but we hold another view from ford in that multimodal distribution would be more general than bimodal one under the influence of competition. this is because the competitive ability of a plant is t h e joint product of environmental or non-heritable effect and t h e inherent genetic effect of the plant. the environmental effect involves, in addition to those generally regarded factors, such non-heritable effect as an acquired advantage due to earlier germination than others. that competitive ability of a plant is a genetic character and is mostly controlled by the so-called polygenes having so low a heritability as 0.3 or less has been repeatedly demonstrated (sakai 36 interference among trees in a plantation of altingia excelsa — sakai el al. 1955, oka 1960, akihama 1967, 1968). as a matter of fact, it is considered that the competitive ability of plants may involve a complex of capacities concerning exploitation of various kinds of resources with or without interference with their associates. in other words, plants in a seed-propagated population are expected to segregate for various morphological and physiological characters which could be positively or negatively responsible for competitive ability. taking such genetic complexity for granted and assuming random combination of genotypes for competitive ability in a pair of adjoining trees, multimodal distribution of tree girths will be more acceptable than bimodality. in the present study, density response has been measured by growth of dbh of a single tree growing in the center of a circular plot being surrounded by its associates, on the one hand, and by the mean dbh of all trees coexisting inside a circle, on the other. inter-tree competition, on the contrary, has been measured by either the correlation or the difference between dbh's of two neighboring trees. as briefly described in the opening paragraph of this paper, there has been a conflict of opinion among ecologists, agronomists and geneticists on the relation between density effect and inter-plant competition. agronomists and ecologists generally appear to take a view that competition is the response of plants to density induced shortages of common resources on the assumption that plants under such circumstances inevitably scramble for the short resources. for instance, kira, ogawa and sakazaki (1953) defined the effect of plant associates growing in a limited area on the growth of a plant as the "competition-density effect" without making a distinction between the two. not a few papers dealing with problems of inter-plant competition from a similar viewpoint have been published (see for example harper 1961). recently jacquard (1973) set forth his view that the density effect is one aspect of inter-plant competition and should be treated as of the same category. in this connection we can introduce a certain evidence from our experiment conducted before. sakai, mukaide and tomita (1963) investigated inter-tree competition in 13 plantations of cryptomeria japonica d. don., three of which were being planted with vegetatively propagated clonal strains and the remaining ten with seed-propagated ones. in this investigation, correlation of stem diameter between two adjoining trees was measured with an expectation that the correlation coefficient would theoretically be minus if competition occurred between them, because it should promote growth of one tree with higher competitive ability, and hinder that of another with lower ability. in practice, the effect of competition to make the inter-tree correlation minus may often counterpoise the plus effect of environments which is due to similarity in environmental conditions in neighborhood. the correlation coefficients between two adjoining trees in the clonal stands were found to be between 0.57 and 0.31 with a mean of 0.437 ± 0.130, while in seed-propagated stands, they were between 37 biotropia vol. 1 no. 1, july-december 1987 0.33 and -0.28 with a mean of 0.024 ± 0.195. thus, it has been concluded that in a clonal population, growth of individual trees may be suppressed by the effect of density, but little sign of inter-tree competition is observed. of interest in this connection is that kira, ogawa and sakazaki (1953) did find in their soya-bean experiment that in a population of a single cultivar, "the correlation coefficients between the weight of an individual and the mean weight of the nearest six plants were for the most part positive" going against their expectations. the values in the population were as high as 0.645 or 0.735. this result increased if inter-plant competition did not occur in the population of a soya-bean cultivar. two years later, some of them (hozumi, koyama & kira 1955) conducted a similar experiment with maize and found that the correlation between neighboring plants was negative. to the present writers, this appears quite reasonable because soya-bean is a highly self-fertilized plant and a cultivar is most likely a genetically homogeneous population, while maize is an allogamous plant and accordingly a single cultivar may without doubt be genetically heterogeneous. those who emphasize inter-plant scramble for common resources as the main cause of competition reason that the most widespread and intensive competition would occur in a group of plants with high genetic similarity, because the genetic resemblance among individuals will bring about the similarity in their requirements (sammeta & levin 1970). needless to say, the genetic resemblance among groups of plants is highest in a population of vegetatively propagated plants from a single parent (a clone) and a cultivar of self-fertilizing plants continuously propagated from a single plant in each generation. results of the experiments just mentioned above show that the reasonings are not correct, suggesting that the gene-to-gene bound competition must be an actual occurrence in plant populations. fasoulas and tsaftaris (1975) take the view that "density brings about competition" which suppresses the yield of plants either evenly (in a monogenotypic population) or unevenly, and defined the latter as "allo-competition" and the former as "isocompetition" instead of saying "no-competition". we have to add here the conclusion drawn from a spacing and mix-planting experiment of 12 barley cultivars conducted by sakai and lyama (1966). in the spacing experiment it was demonstrated that all cultivars equally showed a very poor growth at a very high density, but as the density decreased, they showed various patterns of growth promotion showing cultivar-specific density response. in the mix-planting experiment with the same cultivars, the twelve barley cultivars were found to be variable for competitive ability among them. by comparing both experiments, it was found that the density response and competitive ability were only partly correlated but not reaching the 0.05 level of statistical significance. 38 interference among trees in a plantation of altingia excelsa — sakai et al. thus, it was concluded that the response to plant density and competitive ability were different characters controlled by mostly different groups of genes. we finally state that the effect of plant density upon growth of plants is the general effect of decreasing growth for all plants concerned, though the amount of decrease may be more or less different due to genotypes. the effect of intraspecific competition is the specific effect of the interaction of genotypes for competitive ability in a given pair of plants to favor growth of one plant at the expense of another. so far not much attention seems to have been paid to the silvicultural significance of genetic variability of density response and intraspecific competition in forest trees, but the problem of density response and competition will be worthy of notice in future tree breeding, particularly in cross-fertilizing tree species with a rapid growth rate as found in the tropical forest trees. conclusion from the analytic study of density response, on the one hand, and competitive ability, on the other, in a plantation of more than 50-year-old altingia excelsa noronha trees, the following results have been obtained: (1) for estimation of density response in the species, measurement of average dbh of all trees growing in circular plots with radii of 5.5 or 6.0 meters or in 10 x 10 meter quadrate plots is effectual. in the present plantation it is expected that an increase of one tree would decrease growth of dbh of neighboring trees by 0.75 centimeters. (2) it has been found that inter-tree competition which favors, two adjoining trees, one tree at the expense of another has occurred in the plantation when two trees grow within a distance of two meters. (3) the writers hold the view that density response and competitive ability of trees are different characters each probably controlled by different groups of genes though the problem is seen differently among ecologists, agronomists and geneticists. (4) it is considered that density response as well as competitive ability will be important in future silviculture, particularly in cross-fertilized tree species wit h high growth rate. acknowledgments this study has been conducted as a part of the research activities of the tropical forest biology program of biotrop (seameo regional center for tropical biology), bogor, indonesia. the writers have to express their gratitude to 39 biotropia vol. 1 no. 1, july-december 1987 prof. dr. ishemat soerianegara, the former director of biotrop, and also to prof. dr. zoefri hamzah, the former tropical forest biology program manager of biotrop for their valuable advice and help in the study. literatures cited akihama, t. 1967. estimation of competitive ability and its relation to fitness in rice hybrid population. jap. j. breed. 17: 262-265. akihama, t. 1968. inheritance of the competitive ability and effects of its selection on agronomic characters. ibd. 18: 12-14. fasoulas, a. and a. tsaftaris. 1975. an integrated approach to plant breeding and field experimentation. dept. genet. &p1. br., aristotelian univ. of thessaloniki, greek, pub. no. 5: 5-37. ford, e.d. 1975. competition and stand structure in some even-aged plant monocultures. j. ecol. 63(1): 311-334. harper, j.l. 1961. approaches to the study of plant competition. symp. soc. exp. biol. 15: 1-39. hozumi, k., h. koyama and t. kira. 1955. intraspecific competition among higher plants. iv. a preliminary account on the interaction between adjacent individuals. j. inst. polytech., osaka city univ., d 6. p. 121-130. jacquard, p. 1973. glossary of terms and definition: fodder crops section, "competition and fodder crop breeding" (6th issue, april 1973, eucarpia). kira, t., h. ooawa and n. sakazaki. 1953. intraspecific competition among higher plants. i. ompetiton-yield-density interrelationship in regularly dispersed population. j. inst. polytech., osaka city univ., d 4. p. 1-16. koyama, h. and t. kira. 1956. intraspecific competition among higher plants. viii. frequency distribution of individual plant weight as affected by the interaction between plants. ibd., d. 7. p. 73-94. oka, h.i. 1960. variation in competitive abilities among rice varieties. jap. j. breed. 10: 61-68. sakai, k.i. 1955. competition in plants and its relation to selection. cold spring harbor symp. quant. biol. 20: 137-157. sakai, k.i. and s. iyama. 1966. studies on competition in plants and animals. xi. competitive ability and density response in barley. jap. j. breed. 16 (1): 1-9. sakai, k.i., h. mukaide and k. tomita. 1968. intraspecific competition in forest trees. silvae genetica 17 (1): 1-5. tanaka, k. 1983. changes in diameter and height distribution in a forest stand. j. jap. for. soc. 65: 473-476. 40 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 511 (sri wida potency).cdr potency of rhizosphere bacteria to promote rice growth under saline condition * sri widawati and i made sudiana research center for biology, indonesian institute of sciences csc-lipi, bogor 16911, indonesia received: 8 july 2015/accepted: 25 july 2016 abstract saline soil is a common problem in coastal paddy field, especially in indonesia. salinity affects rice growth and the activities of soil functional microbes, including functional bacteria, which play roles in plant growth. some of these microbes are associated with rice plants and are able to survive under saline condition. the presence of functional microbes is also important to improve soil quality. nitrogen and phosphate are essential soil nutrients and is available in soil due to the activities of nitrogen-fixing bacteria and free-living plant-associated bacteria. the objective of the present study was to obtain nitrogen-fixing, phosphate solubilizing and indole acetic acid (iaa)-producing bacteria that are able to survive and promote the growth of rice under saline conditions. from rice and peanut rhizosphere, caphosphate (ca-p) solubilizing and nitrogen-fixing bacteria were isolated separately using specific media. then, the cap solubilizing ability, phosphomonoesterase activity and iaa-producing ability were quantitatively examined. based on the abilities, 20 strains were selected and identified as burkholderia cepacia-complex, burkholderia anthina, burkholderia cenocepacia, bacillus cereus-complex (three strains), achromobacter spanius, azospirillum sp. (four strains), azotobacter sp. (three strains), rhizobium leguminosarum, rhizobium sp. (two strains), and pseudomonas sp. (three strains). the inoculation of several single strains or the mixture of the selected strains promoted the growth of rice under saline conditions. these inoculants could be potential as biofertilizer in saline paddy fields. keywords: indole acetic acid production, phosphate solubilization, plant growth promoting bacteria, nitrogen fixation, rhizosphere, rice introduction most of the fertile paddy fields in indonesia are located in coastal area and experiences soil salinization due to seawater intrusion (djufry et al. 2011). salinity affects not only the growth of rice (oryza sativa linn.), but also the activities of functional soil microbes, including bacteria, that play roles in mineralization of macro and microelements for plant growth (balser et al. 2006). some of these bacteria are associated with rice plants and are able to survive under saline condition. the activity of soil microbes is an important aspect of biogeochemical cycles of carbon, nitrogen, sulfur, phosphorus, etc. (banig et al. 2008). the presence of functional microbes is also important to improve the quality of soil (wijebandara et al. 2009). nitrogen and phosphate are essential nutrients and are available in soil due to the activities of nitrogen-fixing bacteria and free-living plant-associated bacteria (steenhoudt & vanderleyden 2000). several bacteria belonging to the genera rhizobium, azotobacter and azospirillum are able to fix nitrogen and solubilize phosphate (nosrati et al. 2014). some members of these genera also produce plant growth promoting hormone such as indole acetic acid (iaa), gibberellins and cytokinins (bhattacharyya & jha 2012). therefore, these genera are regarded as important components of biofertilizer (rao 1994; bhattacharjee & dey 2014). introduction of growth promoting bacteria can increase nitrogen availability for plants and enhance crop productivity. however, very little information is available for the effect of salinity on bacteria that have beneficial functions, such as nitrogen fixation, phosphate solubilization and the production of plant growth hormone (pliego et al. 2011; lugtenberg et al. 2013; nakbanpote et al. 2014). the purpose of this study is to obtain nitrogen-fixing, phosphate solubilizing and iaa producing bacteria that are able to survive and biotropia 3 2 6 116 123 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 511 * corresponding author: widadomon@yahoo.com 116 promote the growth of rice under saline conditions. materials and methods bacterial sources bacterial sources were obtained from collected rhizosphere of rice (oryza sativa) and peanut (arachis hypogaea) cultivated in the research field of cibinong science center, west java province, indonesia. the physical and chemical properties of this research field indicated that the soil is infertile soil (table 1). isolation of bacteria phosphate solubilizing bacteria were screened following the method of park et al. (2011). halo zone formation around colonies after 7 daycultivation at 30 °c on pikovskaya medium was used as an indicator of ca-phosphate (ca-p) solubilization (nguyen et al. 1992). nitrogenfixing bacteria were isolated targeting the genera rhizobium, azopirillum and azotobacter according to mubarik et al. (2011) and salamone et al. (2012) as well as aquilanti and clementi (2004), respectively. determination of ca-p solubilization ca-p solubilizing ability of the isolated strains was quantitatively determined according to chen et al. (2006) by measuring orthophosphate in the culture fluid after 7 days of cultivation. orthosphosphate determination was conducted according to vassileva et al. (2000). determination of phosphatase activity extracellular phosphomonoesterase (pmease) activity of the strains was determined following the method of tabatabai and bremner (1969) using p-nitrophenyl phosphate. the unit of the pmease activity was defined as µmol/hof pnitrophenol released in 1 ml of extracellular enzyme solution that was fractioned from 1.0 ml of the culture fluid after 7 days of cultivation. determination of iaa production the iaa production of the strains was investigated after 7 days of cultivation, following the methods of crozier et al. (1988) and gravel et al. (2007). selection and identification of bacteria based on the ca-p solubilization, pmease activity and iaa production abilities, a total of 20 strains were selected from the above strains for rice growth assays. identification of the 20 strains was performed following the method of otsuka et al. (2008) based on the 16s rrna g e n e s e q u e n c e w i t h 1 6 s 9 f ( 5 gagtttgatcctggctcag-3) and 16s1 5 1 0 r ( 5 g g c t ac c t t g t t ac g a 3 ) primers. rice growth assay at the stage of germination under saline condition one strain out of 10 taxonomic groups was selected and subjected to a root and shoot growth assay of rice at the stage of germination based on zaller (2007) and cerabolini et al. (2004). briefly, ten seeds of three rice cultivars, inpara-3, inpari-13 and inpara-6 were soaked in sterile water for 5 hours in their respective containers. these 10 rice seeds were then arranged based on their respective cultivars on top of filter paper which was put inside a petridish (20 cm in diameter). fifteen milliliter of 4.0 g/l nacl solution was poured onto the filter paper inside table 1 physical and chemical properties of soils in the research field parameter p (%) k (%) c (%) n (%) c/n ratio ca (%) exchangable mg (%) exchangable na (%) exchangable al dd (%) soil ph amount of bacteria population characteristic 0.173 0.045 1.303 0.36 3.61 11.41 0.57 0.30 0.04 5.8 10 4 -10 5 determined accoding to rowell (1994) very low very low low moderate very low high low low low acid infertile 117 promoting rice growth using rhizosphere – widawati and sudiana each petridish, on which 10 rice seeds were lined up, followed by 1.0 ml of bacterial inoculant 9 suspension containing 10 cells/ml. root and shoot lengths were measured at 7 days after germination. this experiment was set up using complete randomized design with three replications. rice growth assay at 45 days after planting under saline and non-saline conditions ten strains out of 20 isolates tested on germination test were then subjected to rice growth assay for 45 days under saline condition with 0.4% nacl. the number of cells for each 7 treatment was adjusted to about 3.2 x 10 . this value was selected based on the number of bacteria commonly found in paddy field soil. in a preliminary test (rice growth assay at the stage of germination), inpari-13 and inpara-6 could not grow well under the same saline condition. therefore, only inpara-3 was used in this assay. four seeds of inpara-3 were planted to experimental pots (0.5 gallon pots) containing sterile sands (1.5 kg) flooded with water(field capacity of sands = 24% or 360 ml ). treatments applied were: 1. saline condition (adding 360 ml of 0.4% nacl (6 g nacl) to the 0.5 gallon pots) and 2. non-saline condition (without 0.4% nacl). into each pot, 5 ml of bacterial inoculant suspension was added. the result of experiment is shown in table 5. after 7 days, the second inoculation with the same amount of bacterial suspension was conducted. the water level in pot was regulated by adding sterile water to compensate water decrease due to evaporation. the electrical conductivity (ec) value of the assay media under saline condition was kept at 7.5 ms/cm. at 45 days after planting the growth of rice was evaluated. this experiment was set as complete randomized design performed with three replications. results and discussion composition of the strains the selected 20 strains, originated from the rhizosphere of rice and peanut, belonged to the genera burkholderia, bacillus, achromobacter, pseudomonas, azospirillum, rhizobium and azotobacter (table 2). the selected strains were originated from non-saline soil. the reason for the selection was to compare the physiological characteristics of microbes isolated from saline and non-saline soil. the result of this study showed that the functional microbes for table 2 list of bacteria isolated from rice and peanut rhizosphere isolate code* phylum/class** taxon source (rhizosphere) csc p1 csc p2 csc p3 csc p4 csc p5 csc p6 csc p7 csc p8 csc p9 csc p10 csc p11 csc p12 csc n1 csc n2 csc n3 csc n7 csc n8 csc n9 csc n10 csc n11 proteobacteria/beta proteobacteria/beta firmicutes/bacilli proteobacteria/beta firmicutes/bacilli proteobacteria/gamma proteobacteria/alpha proteobacteria/alpha proteobacteria/alpha proteobacteria/gamma proteobacteria/gamma proteobacteria/beta proteobacteria/alpha proteobacteria/gamma proteobacteria/alpha proteobacteria/alpha firmicutes/bacilli proteobacteria/gamma proteobacteria/gammaproteobacteria/alpha rice rice rice rice rice rice rice rice rice rice rice rice peanut peanut peanut peanut peanut peanut peanut peanut notes: * = a strain with p in its code were isolated as ca-p solubilizing bacteria, and that with n were isolated as nitrogen fixing bacteria ** = alpha-, beta and gammadenote the classes alphaproteobacteria, betaproteobacteria and gammaproteobacteria, respectively burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius bacillus cereus-complex pseudomonas sp. azospirillum sp. azospirillum sp. rhizobium sp. azotobacter sp. azotobacter sp. burkholderia anthina rhizobium sp. pseudomonas sp. rhizobium leguminosarum azospirillum sp. bacillus cereus-complex azotobacter sp. pseudomonas sp. azospirillum sp. 118 biotropia vol. 23 no. 2, 2016 promoting rice growth were not different from that reported by susilowati et al. (2015). phosphate solubilizing ability of the strains ca-p solubilizing ability of the strains is shown in table 3. the difference in the strength of ca-p solubilizing ability was not related to the taxonomic property. all strains formed halo zone around colonies, and the area ratio of the halo zone to a colony was variable (data not shown) indicating the ability to solubilize ca-p differed among the strains. this was reflected in the ca-p solubilizing ability which was quantitatively determined (table 3). the highest ca-p solubilization ability was shown by pseudomonas sp. csc n2 and the lowest was shown by achromobacter spanius csc p4. the activity of pmease is shown in table 3. pseudomonas sp. cscn2 again showed the highest pmease activity, and achromobacter spanius csc p4 seemed to have no extracellular pmease activity. nitrogen-fixing ability of the strains all strains belonging to rhizobium, azotobacter and azospirillum genera were able to grow on nitrogen-limited media implying that these strains were able to fix nitrogen (chien et al. 1992). iaa production of the strains iaa production of the strains is shown in table 3. the amount of iaa produced varied depending on strains. the highest production was achieved by azospirillum sp. csc p8 and azospirillum sp. csc p7. the lowest iaa production was detected in achromobacter spanius csc p4. effect of bacterial inoculation on the rice during the 7-day germination assay with 0.4% nacl, the effect of bacterial inoculation varied depending on the strains (table 4). the best growth was obtained by the mixture of strains on inpara-3, with 7.46 cm and 6.5 cm in shoot and root length, respectively. medium level effect was observed in burkholderia cepacia-complex, bacillus cereus-complex, pseudomonas sp., azospirillum sp. and azotobacter sp. however, inoculation of burkholderia cenocepacia, achromobacter spanius, rhizobium sp., burkholderia anthina and rhizobium leguminosarum had no effect on shoot and root length. cultivars inpara-6 and inpari-13 could not grow without any inoculant (control) or with five single-strain-inoculants. in the 45-day growth assay (table 5), the growth of rice cultivar inpara-3 under saline burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius bacillus cereus-complex pseudomonas sp. azospirillum sp. azospirillum sp. rhizobium sp. azotobacter sp. azotobacter sp. burkholderia anthina rhizobium sp. pseudomonas sp. rhizobium leguminosarum azospirillum sp. bacillus cereus-complex azotobacter sp. pseudomonas sp. azospirillum sp. table 3 ca (po ) solubilization ability, pmease activity and iaa production of the strains3 4 2 isolate code taxon phosphate solubilization (mg/l)* pmease (unit)* iaa production (mg/l)* csc p1 csc p2 csc p3 csc p4 csc p5 csc p6 csc p7 csc p8 csc p9 csc p10 csc p11 csc p12 csc n1 csc n2 csc n3 csc n7 csc n8 csc n9 cscn10 cscn11 8.72 ± 0.89 1.06 ± 0.16 10.54 ± 0.16 0.30 ± 0.68 1.51 ± 0.11 11.26 ± 0.58 7.39 ± 0.42 6.68 ± 0.37 2.28 ± 0.63 5.71 ± 0.53 1.57 ± 0.95 0.47 ± 0.47 1.18 ± 0.05 11.39 ± 0.53 4.94 ± 0.32 2.00 ± 0.32 0.89 ± 0.95 0.83 ± 0.89 10.08 ± 0.26 1.86 ± 0.47 0.63 ± 0.71 0.13 ± 0.52 0.82 ± 0.85 0.01 ± 0.04 0.10 ± 0.86 0.75 ± 0.26 0.60 ± 0.86 0.68 ± 0.10 0.51 ± 0.70 1.27 ± 0.68 0.49 ± 0.67 0.10 ± 0.12 2.01 ± 0.34 2.22 ± 0.93 0.31 ± 0.27 0.47 ± 0.03 0.12 ± 0.88 0.45 ± 0.09 0.85 ± 0.89 0.14 ± 0.59 8.67 ± 0.92 2.63 ± 0.16 8.16 ± 0.90 1.94 ± 0.21 5.46 ± 0.58 8.27 ± 0.67 9.45 ± 0.06 9.56 ± 0.16 6.08 ± 0.42 8.75 ± 0.98 6.21 ± 0.32 2.13 ± 0.16 3.82 ± 0.89 8.16 ± 0.90 8.61 ± 0.10 7.61 ± 0.39 2.73 ± 0.68 8.39 ± 0.06 8.33 ± 0.84 8.09 ± 0.22 note: values represent mean±standard deviation (n = 3) 119 promoting rice growth using rhizosphere – widawati and sudiana table 4 the effect of bacterial inoculants on root and shoot length of rice (three cultivars) at the stage of seed germination isolate code taxon rice cultivar shoot length (cm)* root length (cm)* control csc p1 csc p2 csc p3 csc p4 csc p6 csc p8 csc n1 csc n9 csc n12 csc n3 mix (control: no inoculation) burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius pseudomonas sp. azospirillum sp. rhizobium sp. azotobacter sp. burkholderia anthina rhizobium leguminosarum mixture of strain inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 inpara -3 inpari -13 inpara -6 4.03 a dead dead 5.55 de 4.23 ab 4.37 ab 4.16 ab dead dead 5.54 de 4.51 abcd 4.59 abcd 4.09 a dead dead 5.58 de 4.55 abcd 4.79 abcd 6.10 e 4.80 abcd 4.94 abcd 4.30 ab dead dead 5.56 de 4.82 abcd 4.31 ab 4.03 a dead dead 4.34 dead dead 7.46 f 4.98 abcd 4.96 abcd 0.51 a dead dead 4.43 ghi 2,00 bcde 1.39 abc 1.47 abcd dead dead 4.24 ghi 2.99 defg 2.04 bcde 2.90 cdefg dead dead 4.70 hi 3.41 efgh 3.22 efgh 5.00 i 3.65 fghi 3.11 efgh 3.95 fghi dead dead 4.44 ghi 2.83 cdefg 2.39 bcdef 1.25 ab dead dead 3.62 dead dead 6.50 j 4.01 ghi 4.13 ghi note: values followed by the same letter in the same column are not significantly different based on duncan's multiple range test at 5% level condition was less than that under no saline condition. under saline condition, rice cultivar inpara-3 inoculated with the mixture of strains showed the best growth with 29 cm in plant height and 5.5 cm in root length. as a single isolate inoculation, pseudomonas sp. csc n6 showed the best effect. twenty strains with ca-p solubilizing, extracellular pmease producing and iaa producing abilities were successfully obtained, with an exception of a. spanius csc p4 that did not show clear pmease activity. these strains did not lean to a specific taxonomic lineage and composed of members of the phyla proteobacteria (the classes alphaproteobacteria, betaproteobacteria and gammaproteobacteria) and fermicutes. the fact that the strains were isolated as nitrogen-fixing bacteria including ca-p solubilizing members indicated that ca-p solubilizing ability was common among bacteria, at least among those living in the rhizosphere. it was also possible that p m e a s e a c t iv i t y wa s c o m m o n a m o n g 120 biotropia vol. 23 no. 2, 2016 table 5 the effect of bacterial inoculants on the growth of rice cultivar inpara-3, in sterile sand media under saline and non-saline conditions 45 days after planting isolate code inoculant salinity condition total dry biomass (g) plant height (cm) root length (cm) – csc p1 csc p2 csc p3 cscp4 csc p6 csc p8 csc n1 csc n9 cscn12 csc n3 – no bacteria burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius pseudomonas sp. azospirillum sp. rhizobium sp. azotobacter sp. burkholderia anthina rhizobium leguminosarum mixture of all the isolates non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline 0.02 a 0.01 a 0.09 cde 0.07 abcd 0.06 abcd 0.04 abc 0.09 cde 0.08 bcd 0.04 abc 0.02 a 0.13 e 0.09 cde 0.09 cde 0.07 abcd 0.07 abcd 0.04 abc 0.08 bcd 0.07 abcd 0.06 abc 0.02 a 0.05 abc 0.02 abc 0.14 e 0.12 de 14.25 ab 13.00 a 27.50 ghi 26.25 ghi 26.00 fgh 22.75 cdefg 27.30 ghi 26.00 fgh 25.50 efgh 17.00 abc 29.50 hi 26.88 ghi 27.50 ghi 26.25 ghi 24.50 defgh 20.75 cde 26.50 ghi 26.25 ghi 21.00 cdef 17.15 abc 23.00 defg 19.00 bcd 31.00 i 29.00 hi 1.50 ab 1.00 a 5.00 jk 4.00 hij 3.50 fgh 2.65 de 4.50 ij 3.75 ghi 3.50 fgh 3.00 ef 6.15 l 5.00 jk 4.25 ij 4.00 hij 3.25 fg 2.25 cd 4.50 ij 3.75 ghi 3.50 fgh 2.25 cd 3.25 fg 2.00 bc 6.50 l 5.50 k rhizosphere bacteria. interestingly, the strains with higher ca-p solubilizing ability generally showed higher pmease activity. iaa production was also reported as common among soil bacteria (hasan 2002; xin et al. 2009), which was supported by the present study. saline environment inhibits rice growth. this is because rice is a saline sensitive plant (ashraf & + + harris 2004); also because the uptake of ca , k 2 and inorganic n and p are disrupted under high na concentration (ashraf & harris 2004). in addition, the salinity also affected soil enzyme activities (siddikee et al. 2011), which could indirectly affect rice growth. the inoculation of the selected strains affected germination of rice under saline condition (table 4). the inoculation of mixture of the strains resulted in the best rice growth. as single strain, azospirillum sp. csc p8 and pseudomonas sp. csc p6 provided the best and the second best rice growth support, respectively. it was possible that the inoculants supported the growth of rice by supplying phosphate and iaa. azospirillum sp. is a potential nitrogen fixer and the mixture of the strains also includes nitrogen fixers. therefore, it was possible that nitrogen fixed by the inoculants might also promote rice growth. rice cultivars inpari-13 and inpara6 did not grow without the existence of inoculants. the present study showed that the inoculation of five strains and the mixture of strains enabled these cultivars to grow. this indicated that the inoculation not only promoted rice growth by supplying nutrient and iaa, but also enhanced rice tolerance towards salinity. it was interesting that some strains isolated from peanut rhizosphere could promote and support rice growth. among the rice cultivars tested in the present study, only inpara-3 grew in saline condition without inoculation of the strains. therefore, inpara-3 was then subjected to rice growth assay with 0.4% nacl. in this assay, the inoculation of the mixture of strains, pseudomonas sp. csc p6 and azospirillum sp. csc p8 provided notes: values followed by the same letter in the same column are not significantly different by duncan's multiple range test at 5% level. non-saline condition = 360 ml freshwater in 0.5 gallon pots. saline condition = 360 ml freshwater in 0.5 gallon pots was added with 0.4% nacl (6 g nacl). 121 promoting rice growth using rhizosphere – widawati and sudiana the best, the second best, and the third best rice growth support, respectively. these inoculants may be promising as biofertilizer to support rice growth in saline paddy fields. conclusions twenty strains of rhizosphere bacteria with ca-p solubilizing ability and iaa production were successfully obtained in this study. those bacteria mainly belonged to burkholderia cepacia-complex, burkholderia anthina, burkholderia cenocepacia, bacillus cereus-complex, achromobacter spanius, azospirillum sp., azotobacter sp., rhizobium leguminosarum, rhizobium sp. and pseudomonas sp. potential nitrogen fixing bacteria are azospirillum sp., azotobacter sp., rhizobium leguminosarum and rhizobium sp. most strains had pmease activity. some strains showed growthpromoting effect on rice under saline conditions and produced plant growth hormone. these strains could be candidates for biofertilizer for rice in saline paddy field. it is also important to consider using combination of inoculants and rice cultivars to obtain maximum result. acknowledgements this work was funded by jica-jst satrep 2011-2016. we thank dr shigeto otsuka from university of tokyo for research guidance. we are grateful to senlie oktavitana, anis mutirani and rinatu siwi for their assistance in laboratory works. references aquilanti lff, clementi f. 2004. comparison of different strategies for isolation and preliminary identification of azotobacter from soil samples. soil biol biochem 36(9):1475–83. ashraf m, harris pjc. 2004. potential biochemical indicators of salinity tolerance in plants. plant sci 166 (1):3–16. balser tc, mcmahon kd, bart d, bronson d, coyle dr, craig n, flores-mangual ml, forshay k, jones se, kent ae, shade al. 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112(2):191–9. 123 promoting rice growth using rhizosphere – widawati and sudiana page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 intraspecific variation on early growth of neolamarckia cadamba miq. in provenance-progeny tests in west java province, indonesia dede j. sudrajat , iskandar z. siregar , ulfah j. siregar ,1* 1 2 2,3 nurhasybi , irdika mansur nurul khumaida2,3 4 and 1forest tree seed technology research institute , , bogor 16143 indonesia 2department of silviculture ,, faculty of forestry, institut pertanian bogor bogor 16680, indonesia 3seameo-biotrop, bogor 16134, indonesia 4departmen of agronomy and ho ticulturt r e, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia received 21 october 2014/accepted 2 may 2016 abstract genetic parameters on early growth of , an indigenous species with potential as a source of neolamarckia cadamba wood timber, were estimated in open-pollinated provenance-progeny tests at two sites in west java province, indonesia. the experiment was conducted using randomized complete block design with 12 provenances, 105 families and 5 replications of 4-tree row plots. total height and root collar diameter were measured at the age of 18 months growth. significant differences among the provenances and families within provenances were observed for height and collar diameter at all sites, except for the collar diameter of among provenances at parungpanjang site. in general, garut (gsj) provenance performed better growth at the two sites than kualakencana (kkp) and nusa kambangan (nkj) provenances. the evaluation of component of variance at the two sites showed that the provenance effects (ranging from 0.5 to 1.7%) contributed more to total variance than family within provenance effects (ranging from 0.4 to 0.6%). genetic correlations between height and collar diameter were weak to moderate. heritability was low for all traits at limbangan, while at parungpanjang, the heritability was moderate. estimation of genetic gain for height and diameter by proportional selected family 0.30 was 0.13 and 0.18 for limbangan and 0.31 and 0.16 for parungpanjang. heritability measurement should be sustained to reach stable value. stable heritability combined with selection of family and selection within family will improve genetic gain. : keywords genetic correlation, genetic parameter, heritability, progeny, provenance, neolamarckia cadamba, selection introduction neolamarckia cadamba (roxb.) miq. commonly known as white jabon is a native fast growing tree species playing an important role in both commercial and traditional farming systems in several sites in indonesia (krisnawati 2011; et al. kallio . 2011). this species produces timber et al for pulp and light construction (soerianegara & lemmens 1993). various parts of the plant also have many bioactive substances, such as a n t i o x i d a n t , h y p o g l y c e m i c s u b s t a n c e , hypolipidemic substance, antibacterial substance, antimicrobial substance, etc. (acharyya et al. 2011; xu 2011; mishra & siddique 2011). et al. n. cadamba has a very extensive natural distribution extending from india, nepal, burma, sri lanka and malaysia across indonesia, philippines and papua new guinea (lamprecht 1989). in indonesia, is distributed white jabon almost across the entire country (soerianegara & lemmens 1993). evolutionary theory predicts that species with large population sizes, broad geographic ranges and varying climate condition will have large inter and intraspecific variation (rawat & bakshi 2011). population within plant species with wider geographic ranges has higher allozyme variation and the widely distributed species have more overall allozyme variation (hamrick & godt 1989). some studies showed that genetic variation of ha been n. cadamba detected among provenances for quantitative * corresponding author : djsudrajat@yahoo.com biotropia vol. 23 no. 1, 2016: 10 20 doi: 10.11598/btb.2016.2 . .3 1 439 10 mailto:djsudrajat@yahoo.com seed and seedling morpho-physiological parameters (sudrajat 2016), as well as among natural populations to aflp loci (sudrajat . et al 2015). given its wide ecological amplitude and a large amount of genetic variation, is n. cadamba a promising species for tree improvement program to increase its productivity and quality. tree improvement program of is n. cadamba not yet running well, so that large cultivation activities of this species is not yet supported by high quality seed. the main seed sources for all kinds of plantation activities are from unknown origin, which are collected from unimproved seed sources. however, the initiation of the tree improvement program of is very n. cadamba important as a basic for high qualified seed procurement. the success of tree improvement efforts depends upon many factors including genetic variation, heritability of desired parameters and potential gains derived (nebgen & lowe 1982; borralho 2001), so it is very important to use the genetic material from wide natural distribution of the species to establish a breeding population. in indonesia, establishment of breeding population with open pollinated progenies of plus trees is widely applied as the first step towards tree improvement for fast growing species (hashimoto 1996; soeseno 1988). it et al. might be the most reasonable and practical way to meet an immediate demand for genetically improved seed to be used in reforestation programs in indonesia. traditionally, forest genetic tests have been conducted sequentially with successive species, provenance and progeny trials. in practice, however, there is strong economic pressure to reduce the testing interval between these stages in a traditional tree improvement program. the use of combined provenance-progeny test has been advocated to reduce the testing interval between provenance and progeny stages (zheng 1994; sebbenn et al. et al. 2003; finkeldey 2005). it may be extended to combine provenance testing, progeny testing, seed production and conservation of ex situ n. cadamba in a single trial. the study investigated intraspecific variation for early growth parameters in 105 families from 12 provenances originated from seven n. cadamba indonesian islands. the objectives of this research were: i) to examine the distribution of genetic variation among and within provenances and families within provenances and ii) to determine the extent of genetic control for growth parameters in the form of provenanceprogeny test at two different locations in west java , indonesiaprovince . materials and methods open-pollinated seeds of 105 families of n. cadamba were collected from 12 natural populations distributed in sumatera, java, nusa kambangan, , celebes, kalimantan/borneo sumbawa and papua (table 1, fig. 1) from march to july 2012. the sampled trees were phenotypically average or above average with respect to stem diameter (diameter at breast height/dbh) and total height compared with surrounding trees in the population. the distance among mother trees within population was kept at a minimum of 100 m to minimize relation between seed lots. seeds were collected by climbing the tree samples. every seed lot was separated for each sample tree and information about location and number of samples was noted at every seed lot. the information was kept until the seed reached nursery stage to establish the provenance-progeny trials. seeds were sown in plastic pot filled with media consisted of sand, compost and charcoal (5:3:1, by volume). prior to sowing, the potting mixture was treated with 0.2% a fungicide to avoid any chances of fungus infection attacking newly germinated seedlings. pots were watersprayed with fine sprayer to keep them wet and were maintained in greenhouse. young four-leaf seedlings were transplanted on polythene bags (10 cm in diameter and 15 cm in height), containing media consisted of top soil, sand and compost in a ratio of 2:1:1 (volume) and were set up in nursery. seedlings were grown at 50% light intensity using shade net for 3 months. one month afterwards the seedlings were exposed at open area for hardening off. seedlings were irrigated based on the operational regime for the nursery. forty vigorous and healthy seedlings were selected from each family to be planted at two trial sites. the experiment was set up in two sites in west java , i.e. site i in limbangan, province garut (07°02'23” s, 108°00'43” e, 520 m altitude) and site ii in parungpanjang, bogor (06°20'42” s, 106°06'15” e, 52 m altitude). 11 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat 12 biotropia vol. 23 no. 1, 2016 average annual rainfall and temperature were 2,580 mm and at site i, respectively and 27 °c 2,440 mm and 28 °c at site ii, respectively. soil at site i and site ii had low level of n, p, k and corganic with ph of 5.1 and 4.2, respectively. site i was an open private land with slope ranged from 5 to 15%, having high incidence of domestic animal and was planted with irregular agricultural crops in several parts of area. site ii was an even area within state forest land covered with dense weed, that grew rapidly even after cleaning. the locations were selected based on several reasons. limbangan was selected to represent medium to high level of elevated land where many n. cadamba plantations were cultivated as dominant species in forest community areas, such as in garut district, west java province. parungpanjang was selected to represent the common site condition of forest plantation industries that are generally established in podsolic soil associated with low ph and low soil nutrients. four-month-old seedlings were planted at the fields in february 2013 (planting hole size 40 x 40 x 40 cm) administered with 3 kg manure per seedling as basic fertilizer. the spacing between planting hole was 3 x 3 m. the experiment was conducted in randomized complete block design (rcbd) with 5 replications of 4-tree row plots (fig. 2). within two months after planting, 100 g of npk fertilizer (15:15:15) was applied to each figure 1 geographic distribution of 12 populations was tested figure 2 design of planting test of at parungpanjang, bogor and limbangan, garutn. cadamba 13 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat plant. weed competition was kept to a minimum by manual weeding. all trees in each plot constituted the measuring unit in all replications. the first assessment was carried out at the age of 6 months and subsequently after 12 months. in this paper, results of 16 months growth (4 months in nursery + 12 months in field) planting have been described. the parameters assessed are height (m), collar diameter (cm) and survival percentage. data analysis the data were analyzed for each site, to determine the provenance and family effects using two-way analysis of variance (anova). before performing the analysis, data were examined for confor mity to nor mal distribution and homogeneity of the variance assumptions. anova for all parameters was conducted using the following statistical model (falconer & mackay 1996; sebbenn . 2003):et al y + p + f(p) ( ) + rp + ijkl i j k j ij = μ + r rf(p) ( ) + e ( )ik j l ijk an extra term (across site effect) was added to the model to investigate the significance of the difference of parameters across site. thus, the model used was: y = µ + s + p + f(p) ( ) + sp + sf(p) ijkl i j k j ij ik ( ) + e ( )j l ijk where : y = the measurement on the individual of ijkl l th the family from the provenance in k j th th the replicationi th μ = the overall mean r the effect of replication ( =1, 2, 3, ..,n)i = i i th s = the effect of site ( =1, 2)i i i th p = the effect of provenance ( =1, 2, 3, …, n)j j j th f(p) = the effect of family in provenance k( j) k j th th ( =1, 2, 3, …, n)k rp = the interaction effect between ij i th j replications and provenances th rf(p) = the interaction between ik( j) i th k j replications and family within th th provenance e = the residual.l(ijk) the sas proc glm was used to obtain the coefficients of the expected mean squares for the calculation of heritability. the components of variance were calculated using varcomp proc from sas statistical program (cary 1999). heritability was estimated from the variance components as described in falconer and mackay (1996). narrow sense individual ( ) and family h i2 heritability ( ) for each parameter were h f2 estimated using equations: where: = additive genetic variance = between-family-within-provenance variance component = phenotypic variance calculated as where: = variance due to interaction between r e p l i c a t i o n a n d f a m i l y w i t h i n provenance (experimental error) = variance among individual trees within family (sampling error) = family phenotypic variance, calculated as where: k k2 3 and = respectively, coefficients for and in the expected mean squares. phenotypic ( ) and genetic correlations rp xy( ) ( ) were estimated from the component of rg xy( ) variance and covariance (falconer 1981) substituted into the standard equation for the product moment correlation coefficient: where: and = components of variance of phenotypic and genotypic products of x parameter and = components of variance of phenotypic and genotypic products of y parameter and = components of covariance of phenotypic and genotypic products of x and y parameters, respectively. genetic gain (∆) was calculated by falconer's (1981) formula: ∆g = h s = h i σ2 2 p where: i = intensity of selection taken from zobel and talbert (1984) with proportion selected 0.70, 0.50 and 0.30 σp = phenotypic standard deviation. 14 biotropia vol. 23 no. 1, 2016 results and discussion growth rate and genetic variation significant differences were observed among provenances and among families within provenances for all parameters at limbangan and parungpanjang, except for collar diameter of among provenances at parungpanjang (table 2). at the limbangan, the kkp provenance attained a maximum height followed by gsj, pkc, pgc and nkj provenances. maximum collar diameter was recorded in gsj provenance followed by kkp, pgc, rps and nkj. field survival was recorded maximum in kkp provenance, followed by krs, pgs, rps and gsj. the kkp provenance had the highest height growth and presented approximately 44% faster than the slowest growth provenance. for root collar diameter, the gsj provenance was higher and presented about 59% higher growth than the least growth provenance. the range of variation at family level of irrespective provenances was large in respect to height and root collar diameter (table 3) as proven by values of coefficient of variation. over all kkp, gsj and pgc provenances were better than others. the range of family variation (irrespective provenance) at parungpanjang was high in all growth parameters. maximum height was recorded in gsj provenance, which was closely followed by apj, nkj, kkp and bhs provenances. maximum root collar diameter was attained by bhs provenance, however, it was at par with pkc, pgc and apj provenances. gsj provenance had the highest growth for height and presented about 17% higher growth than the least growth provenance. for root collar diameter, differences among provenances means were not statistically significant and the best provenance was only 10% larger than the least provenance. ten provenances showed field survival more than 70%, except apj and blb provenances. based on the values of cv and range of the family means, a wide range of variability was observed in respect of growth parameters ( ).table 3 the height and collar diameter growth of n. cadamba were better at limbangan than those at parungpanjang. the plant growth at parungpanjang was affected by heavy growth of weeds causing suppression and competition for nutrition and growth space. the factor might have led to slow growth of the plant at the site. on the other hand, survival percentage was higher at parungpanjang. survival percentage at limbangan was reduced by disturbance of domestic animal and irregular agricultural crops in several parts of the area covering the main plants in early growth of seedlings. in general, growth performance in both sites is better than the other progeny test of conducted in n. cadamba wonogiri, central java (setyadi . province et al 2013). differences in height and root collar diameter growth were greater among families than among provenances. at limbangan, the best growing family exceeded the least growing family by 243% and 234% for height and root collar diameter, table 2 mean square for height and collar diameter in provenance-progeny tests of at two sites in west java n. cadamba province, indonesia source of variation degrees of freedom mean square limbangan, garut parungpanjang, bogor height (m) collar diameter (cm) survival (%) height (m) collar diameter (cm) survival (%) blocks 4 61.281 ** 221.693 ** 9,310.667ns 8.604 ** 35.499 ** 25,694.552** provenance 11 3.858 ** 16.933** 3,949.994ns 0.833 * 1.6748 ns 2,902.045ns fam (prov) 93 2.165 ** 8.051** 2,018.897ns 0.864 ** 3.428** 2,167.913ns block*prov 44 1.859 ** 6.208* 1,860.280ns 1.196 ** 4.758** 2,799.698ns block*fam(prov) 359 1.973 ** 7.498 ** 2,644.036ns 1.053 ** 3.897** 2,841.399ns error 1,044 0.740 3.353 2,153.968 0.413 1.681 1,862.556 notes: ** = significant at <0.01 p * = significant at <0.05p ns = not significant 15 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat respectively. at parungpanjang, the best family exceeded the least family by 57% and 122% for height and root collar diameter, respectively (data are not shown). these results showed the potential of provenance-progeny test of for n. cadamba selection. analysis of variance across the sites revealed no significant provenance against site interaction and family within provenance against table 4 growth performance (mean ± standard deviation) of various provenances of across different sitesn. cadamba provenance height (m ) collar diameter (cm) survival (%) rps 2.15±0.94 4.42±2.16 70.0 krs 2.11±0.97 3.92±1.96 69.5 oks 2.25±1.19 4.19±2.10 64.8 gsj 2.50±1.22 4.77±2.27 66.9 nkj 2.35±1.16 4.35±2.16 60.7 apj 2.23±1.04 4.11±2.04 60.7 blb 1.97±0.77 3.63±1.82 58.2 ktb 2.15±0.71 4.06±2.19 67.0 pgc 2.30±1.10 4.49±2.23 70.1 pkc 2.33±1.10 4.36±2.08 63.2 bhs 2.31±1.05 4.33±2.10 61.9 kkp 2.50±0.86 4.59±1.83 61.3 f test: provenance 2.80 * 3.57 ** 1.43 ns provenance *site 2.49 ns 2.14 ns 0.70 ns family*site 1.55 ns 1.41 ns 0.84 ns notes: ** = significant at <0.01p * = significant at < 0.05p ns = not significant table 3 growth performance (mean ± standard deviation) of various provenances in provenance-progeny tests of n. cadamba provinceat two sites in west java , indonesia (number in parentheses are ranks) provenance limbangan, garut parungpanjang , bogor height (m) collar diameter (cm) survival (%) height (m) collar diameter (cm) survival (%) rps 2.34±1.20 (8) 4.78±2.77 (4) 62.5 (4) 1.97±0.58 (12) 4.06±1.59 (7) 77.5 (2) krs 2.14±1.12 (11) 3.92±2.26 (11) 63.9 (2) 2.08±0.82 (8) 3.93±1.66 (10) 75.0 (6) oks 2.46±1.48 (6) 4.45±2.53 (8) 58.6 (8) 2.04±0.78 (11) 3.94±1.54 (9) 70.9 (9) gsj 2.67±1.47 (2) 5.35±2.75 (1) 61.9 (5) 2.31±0.79 (1) 4.11±1.26 (5) 71.9 (8) nkj 2.47±1.34 (5) 4.68±2.59 (5) 50.7 (11) 2.26±0.99 (3) 4.09±1.73 (6) 70.7 (10) apj 2.17±1.10 (9) 4.08±2.31 (10) 53.2 (10) 2.28±0.98 (2) 4.13±1.78 (4) 68.2 (11) blb 2.17±1.16(10) 4.27±2.41 (9) 58.8 (7) 2.12±0.78 (6) 3.78±1.55 (12) 57.5 (12) ktb 1.89±1.30(12) 3.35±2.45 (12) 60.2 (6) 2.05±0.40 (10) 3.86±1.22 (11) 73.8 (7) pgc 2.57±1.30 (4) 4.87±2.61 (3) 63.7 (3) 2.06±0.81 (9) 4.14±1.74 (3) 76.4 (3) pkc 2.58±0.89 (3) 4.56±2.72 (6) 47.5 (12) 2.11±0.82 (7) 4.17±1.67 (2) 78.9 (1) bhs 2.42±0.75 (7) 4.49±2.07 (7) 53.8 (9) 2.21±0.92 (5) 4.19±1.78 (1) 76.3 (4) kkp 2.73±0.95 (1) 5.15±2.06 (2) 75.0 (1) 2.25±0.71 (4) 4.00±1.38 (8) 75.0 (5) family range 1.40-4.81 2.10-7.03 35-90 1.38-2.97 2.50-5.56 40-100 mean 2.42 4.49 59.1 2.14 4.08 72.7 cv (%) 35.50 40.76 30.06 31.76 notes: rps = rimbopanti nature reserve-sumatera, krs = kampar-riau-sumatera, oks = ogan komering ilir-sumatera, gsj = garut selatan-java, nkj = nusa kambangan, apj = alas purwo-java, blb = batulicin-kalimantan, ktb =kapuas-kalimantan, pgc = parangloe-sulawesi, pkc = pomalaa-sulawesi, bhs = batuhijau-sumbawa, kkp =kuala kencana-papua 16 biotropia vol. 23 no. 1, 2016 site interaction for all parameters (table 4). in across site, a significant difference was revealed by provenances for height and diameter parameters. among different provenances, the performance of the gsj and kkp provenances was found significantly superior than the others. the component of variance attributed to variation among provenances ranged from 0.5% for height at parungpanjang to 1.7% for root collar diameter at limbangan. on the other hand, component of variance attributed to variation among families within provenances were lower than component of variance among provenance, ranged from 0.4 to 0.6% at both sites (table 5). the higher values toward variation among provenance in relation to families within provenances suggested that provenances are isolated or gen flow is insufficient to overlap the effect of selection and/or genetic drift. larger genetic variation among provenances than genetic variation among families within provenances were also observed in provenanceprogeny test by zheng (1994) for et al. pinus caribaea in china, bali-uckas . (1999) for et al acer platanoides in sweden, sebbenn (2003) for et al. araucaria angustifolia et al in brazil, adinugraha . (2013) for tectona grandis in gunung kidul, and setiadi and fauzi (2015) for in araucaria cunninghamii bondowoso, indonesia. on the other hand, study based on aflp loci using 4 populations of n. cadamba showed that variation among population (27%) was much lower than variation within population (73%) (sudrajat . 2015). different et al trend was caused by different number and distribution of populationn. cadamba . genetic parameters phenotypic and genetic correlations between height and root collar diameter parameters at parungpanjang were higher than those at limbangan (table 6). this indicated that the possibility of selection in one parameter at parungpanjang is more efficient than those at limbangan. phenotypic correlations were higher than genetic correlations among height and root collar diameter parameters (table 5). high phenotypic correlation provided more support for combining these surveyed parameters as early selecting criteria. narrow sense individual ( ) and within h i2 families ( ) heritability at limbangan were poor h f2 (ranged from 0.031 to 0.055), while the table 5 components of variance and relative contribution (number in parentheses) of replications ( ), provenances ( ), σ σ2 2r p interaction between replications and provenances ( ), families within provenances ( ), interaction between σ σ2 2rp f(p) replications and families ( ) and individual within families ( ) of provenance-progeny tests at two sites in west σ σ2 2rf(p) e java , indonesiaprovince components of variance limbangan, garut parungpanjang, bogor height (m) collar diameter (cm) height (m) collar diameter (cm) σ2r 0.49330 (28.0%) 1.54578 (22.9%) 0.06746 (9.1%) 0.27601 (9.5%) σ2p 0.02681 (1.5%) 0.11501 (1.7%) 0.00377 (0.5%) 0.04134 (1.4%) σ2rp 0.00465 (0.3%) 0.04640 (0.7%) 0.01546 (2.1%) 0.07356 (2.5%) σ2f(p) 0.01045 (0.6%) 0.03875 (0.6%) 0.00359 (0.5%) 0.01216 (0.4%) σ2rf(p) 0.48827 (27.7%) 1.64063 (24.3%) 0.23901 (32.2%) 0.82821 (28.4%) σ2e 0.74002 (42.0%) 3.35359 (49.8%) 0.41369 (55.7%) 1.68163 (57.7%) table 6 genetic (upper diagonal) and phenotype (lower diagonal) correlation coefficients among traits in provenanceprogeny tests of two sites in west java , indonesian. cadamba at province limbangan, garut parungpanjang, bogor height collar diameter height collar diameter height 0.29 0.56 collar diameter 0.799** 0.885** note: ** = significant at <0.01p 17 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat heritabilities at parungpanjang site were moderate and ranged from 0.093 to 0.178 (table 7). values of heritability changed with sites with the same set of genotypes. lower value of heritability of the trial in limbangan could be due to high incidence of domestic animal and irregular agricultural crops covering several parts of area causing high environmental difference. setyadi et al. (2013) n. cadamba studied 75 half-sib families explored from java island and found higher family heritability of approximately 0.32 for height and 0.38 for stem diameter at breast height. poor values of heritability in this study could be due to plant age that was still young, indicating the genetic control of the parameters was still weak. for example at parungpanjang, family heritability at 6 months old plant (0.093 for height and 0.066 for root collar diameter) was lower than family heritability at 12 months old plant (0.150 for height and 0.178 for root collar diameter), indicating heritability was not yet stable. trend of increasing heritability with age was also reported in the studies of (osario eucalyptus grandis et al. 2001; gapare 2003), et al. pinus banksiana et (weng al. 2006) e. urophylla et al. and (kien 2009). the increased heritability with age for growth parameters could also be resulted from competitive effects occurred in later age in the stand (kien 2009). according to zobel and et al. talbert (1984), the heritability values often change with age when the environment changes and when the genetic control of the characteristic changes as the trees reach mature age. therefore, activities to improve site environment conditions will be very important to optimize growth and to reach stable heritability. heritability estimated should be interpreted carefully because of unequal number of families per provenances presented in the trials. in a multiprovenance-progeny test, the family variance is averaged across different provenances, with more information provided by the better-represented provenances (kien 2009). in all parameters at et al. both sites, family heritability was found to be higher than individual heritability. some studies in other species also revealed similar trend, such as in araucaria angustifolia et al.in brazil (sebbenn 2003) and in turkey (gulcu & celik 2009). pinus brutia this result indicated that higher genetic gain can be achieved with family selection. estimation of genetic gain is determined by heritability value, selection intensity and phenotypic standard deviation. in this research, simulation to predict the genetic gain used proportional selected family of 30%, 50% and 70% and the corresponding selection intensity were 1.15, 0.79 and 0.49 (zobel & talbert 1984). the higher value of genetic gain estimation for height and collar diameter by proportional selected family of 30% was 0.13 and 0.18 for limbangan and 0.31 and 0.16 for parungpanjang (table 8). predictions of genetic gain from family table 7 estimated heritabilities in provenance-progeny tests of two sites in west java , indonesian. cadamba at province notes : category of heritability values according to and cotterill dean (1990): y of poorheritabilit <0.1 = heritability of moderate0.1-0.3 = heritability of high>0.3 = table 8 estimates of genetic gain for height and diameter by within family of in provenance-progeny tests of n. cadamba proportion selected garut parungpanjang height diameter height diameter 0.30 0.13 0.18 0.31 0.16 0.50 0.05 0.09 0.09 0.07 0.70 0.03 0.05 0.05 0.04 18 biotropia vol. 23 no. 1, 2016 selection simulation revealed that height parameter showed higher values than collar diameter parameters. it was also revealed that genetic gain of height parameters at parungpanjang was higher than genetic gain at limbangan. johnson . (1955) and seghal . et al et al (1995) pointed out that heritability estimates along with genetic gain is more useful than heritability alone, because the heritability estimates indicated only the effectiveness of selection on genotype based on phenotypic performance, but fails to indicate the genetic progress. high heritability and genetic gain indicated the additive gen action on these parameters for their expression. conclusions levels of genetic variation in were n. cadamba found to be higher for all parameters among provenances than within provenances indicating possibility to use well performing provenances as seed sources for reforestation and regeneration practices. estimated family heritability was higher than narrow sense individual heritability, indicating the possibility of greater gains with selection among families rather than mass selection. however, heritability was low to moderate and not yet stable for all parameters at both sites, indicating that possibility of genetic gains with selection is limited and selection should be done until the heritability reached relatively high and stable values. a combination of family and within family selection would be effective in improving growth of this species. improving growth environment conditions with controlling the domestic animal and weed intensively should be applied immediately and continuously to optimize the plant growth, stabilize heritability and to optimize genetic gain. acknowledgements the authors wish to acknowledge seameobiotrop, pt. arutmin indonesia (batu licin, south kalimantan), pt. dasa intiga (kapuas, centr kalimantan), pt. newmont indonesia al ( b a tu h i j au , s u m bawa ) f o r p r ov i d i n g accommodations during genetic material exploration. the authors were grateful to the authorities of forest tree seed technology research institute, bogor, for financing the establishment of provenance-progeny trials at parungpanjang, bogor and limbangan, garut. references acharyya s, 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biotropla vol. 28 no. 1,2021: 38 45 doi: 10.11598/btb.2021.28.1.983 increasing risk of heavy metal contamination i n silvofishery ponds endah dwi hastutt, rini budihastuti and sri darmanti biology department, faczllo $science and mathematics, diponegaro universig, jalan prof: soedarto, s h tembalang, semarang 50275, indonesia received 18 january 201 8/accepted 17 april 2020 abstract this research aimed to determine the concentration of organic matter, pb and cd found in a silvofishery pond, to assess the toxicity level, to analyze the changes in their concentration within a year's period and to analyze the correlation between concentration and changes. the research was conducted by doing five field observation activities and laboratory analysis from may 2016 to july 2017. data were analyzed using anova and correlation tests. the concentration of pb and cd in the organic matter, was increasing in all five observations. throughout the research, the concentration level ranged at 1.60 3.30 mg/kg for organic matter, 3.130 8.230 mg/kg for pb, and 1.089 2.820 mg/kg for cd. in all observations, the toxicity level of cd concentration in the sediment exceeded the standards recommended by us epa (i 1.0 mg/kg) and anzecc & armcanz (5 1.5 mg/kg), while pb concentration was within the safe range (5 21 mg/kg and 5 50 mg/kg, respectively). moreover, the concentration and accumulation of pb and cd were strongly correlated, which indicated the possibility of having the same pollutant sources. therefore, a better management plan is recommended to avoid heavy metal accumulation in silvofishery ponds, a plan that would include the arrangement of mangrove plants in inlet canals and their periodic pruning to prevent the heavy metal from returning to the environment through the litter fall. keywords: aquatic toxicology, bioremediation, cadmium, heavy metal, sediment chemistry, silvofishery introduction silvofishery has supported the aquaculture activities through its emphasis on environ mental services (suwarto e t al 2015). the silvofishery system integrates mangrove plants into ponds to minimize environmental pollution caused by its effluent (hastuti & budihastuti 2016). the mangrove ecosystems has also improved the natural primary productivity because of nutrient cycling pudihastuti e t al 201 3). mangrove plays an important role in controlling the pollutants in the coastal ecosystem. its root ecosystem can trap the organic matter sediments which contain the heavy metal pollutants (yunus e t al. 201 1). thus, reducing the concentration of pollutants in the aquatic system. mangrove plants can also act as 'corresponding author, email: endah-pdil@yahoo.com bio-remediators that are capable of absorbing pollutants from the environment and accumulating these in its body parts (pakzadtoochaei 201 3). thus, mangroves have dual functions of controlling pollutant distribution, and remediating its growing environment. mangrove roots trap the polluted sediments and these sediments are then suspended around the tree. thus, as pollutants accumulate, their concentration below the mangrove stands increases (kathiresan e t al. 2014). however, by absorbing the pollutant, the mangroves would decrease its concentration in the sediments (selanno e t al. 2015). thus, the accumulation and diminishing rates of pollutants are affected by the balance between the deposition of pollutants and the absorption capacity of mangrove plants. heavy metal is one of the most dangerous types of pollutants that could be consumed and accumulated in plants and animals organs, increasing risk of heavy metal contamination in pond dwi hastuti e t al: resulting in toxicity (reis e t al. 2010; asati e t al. 2016). thus, food chain is the most important aspect in the accumulation of heavy metal toxicity (hajeb e t al. 2014). contamination of lower level organisms would lead to the accumulation of heavy metal in higher level organisms. obviously, the risk of heavy metal contamination may increase in a polluted environment. among the heavy metal contaminants, pb and cd are the most common aquatic pollutants produced from daily activities. sources of pb include volcanic explosions, forest fires, lead emissions from industry and transportation, metal processing, and manufacturing (zhang e t al. 2015). anthropogenic sources of pb pollution include paints and glazed budding materials (walraven e t al. 2016). the main sources of cd in the sediment are the industrial and mining wastes (yu e t al. 2010; donovan e t al. 2016). although still debatable, the application of p ferulizer was also considered as an additional source of cd in the agricultural soil (roberts 201 4). in order to secure environmental safety against pollutant contamination, some countries had developed environmental quality standards which differ in their applications. us epa applies a maximum value of 1 mg/kg for cadmium (cd) and 21 ppm for lead (pb), while anzecc & armcanz recommends 1.5 mg/kg for cd and 50mg/kg for pb (hubner e t al. 2009). in view of the environmental standards imposed by many agencies, the threat of heavy metal contamination is still very evident. moreover, heavy metal does not appear as single element but is mostly attached to organic matter in the environment. the accumulation of heavy metal is related to the accumulation of organic matter (yang e t al. 2010). hence, studies on the trend of organic matter and heavy metal accumulation are equally important. silvofishery has been considered as a "good management system" applied in conservative aquaculture (jonell & henriksson 2014). in most cases, however, the real application is different from the proposed model. for instance, plantations are set in the middle of ponds without any separating dikes w h c h are supposed to prevent direct contact between the pool and the mangrove. thus, the risk of pollutant contamination to the livestock cultivars might increase. silvofishery is mostly applied in degraded ponds (yunus e t al. 2015), making the heavy metal accumulation an additional risk to its already poor environmental quality. however, little concern has been directed towards organic matter and heavy metal accumulation in silvofishery. hence, this research was conducted to study the concentration of pb and cd in a silvofishery pond, and its toxicity level, to analyze the changes in their concentration level within a year's period, and to analyze the correlation between these concentrations and changes. materials a n d methods the research was conducted from may 2016 to july 2017 in a silvofishery pond in mangunharjo village, tugu district, semarang city, central java, indonesia. twenty-seven sampling points were selected and dstributed throughout the pond area (fig. 1). data were collected during the five sampling periods in may, july, and september 2016 and in may and july 2017. generally, the weather was bright with warm temperature during the sampling period. since the sampling was carried out during the dry season, the fresh water dominated the supply to the silvofishery pond. data collection was carried out to determine the concentration of organic matter, lead (pb) and cadmium (cd) in the sediment across a one-year period. samples obtained from the sedment accumulated below the mangrove stands were then analyzed in the laboratory. as much as 500 g sediment samples were taken from the surface to a depth of 30 cm using scoop and dpper. the sediment was readily washed. therefore, ekman grab could not be used for the sediment sampling. the water was filtered before the sediment was moved into the plastic bag. laboratory analysis was done at the wahana laboratorium, semarang. the organic matter was analyzed using the ashing method, whde total heavy metal content was analyzed using atomic absorption spectrophotometry (aas) method. biotropia vol. 28 no. 1,2021 inlet tunnel outlet tunnel ~ o o o o , o o o p o o o o o o ' 8 , o ~ o ' q o o o o p o ~ o o \ o o o o o p o o o o o \ /\\ -. \ / i \ ! ' / effluent \ \ / sampling points figure 1 schematic diagram of sampling points changes in organic matter and heavy metal concentration at each sampling point were also computed and analyzed. analysis of variance was performed on the mean values of organic matter and heavy metal taken from different sampling periods, as well as their changes over those periods. pearson correlation analysis was performed to determine the relation between periodic average of organic matter, pb and cd concentrations and their changes during the study period. results a n d discussion i pond decreased from the first to the second observation. among the sampling periods, the second period showed the most significant increase in the concentration of pb and cd (table 1). concentration of heavy metals indicated that pb was still within the safe range (table 1). the maximum pb concentration (8.090 mg/kg), was far below the critical value proposed by us epa (21 mg/kg) and anzecc & armcaz (50 mg/kg). thus, the silvofishery pond in mangunharjo vdlage was safe from pb contamination. however, the cd concentration has exceeded the recommended limit. the variations existed in the concentration of minimum concentration of cd in the first (1.089 organic matter, pb and cd in the silvofishery mg/kg) to third (1.460 mg/kg) observations pond over the five different sampling periods. exceeded the us epa limit of i 1 mg/kg. in data showed that there were increases in the fourth and fifth observations, the minimum average concentrations and decreases in range, concentration of cd even exceeded the limit set indicating a trend towards stabhation. the by anzecc & armcaz with the expected concentration of organic matter, pb and cd concentration of 5 1.5 mg/kg. table 1 concentration level of organic matter, pb and cd in the silvofishery pond sediments observation period no. parameters i i1 111 lv v 1. organic matter (%) a range 1.89-3.30 1.71-3.12 1.60-2.70 1.65-2.62 1.90-2.83 b average 2.23 + 0 . 2 8 ~ 2.21 f 0 . 3 1 ~ 2.27 + 0 . 2 7 ~ 2.29 + 0 . 2 5 ~ 2.38 f 0 . 2 0 ~ c averape c h a n ~ e -0.04~ 0.05x 0.02" 0.ogx " " 2. pb (mg/kg) a range 3.130-8.230 4.120-7.230 4.353-8.090 4.501-7.930 5.130-7.449 b average c average change " u 3. cd (mg/kg) a range 1.089'-2.4094 1.1 1 8'-2.4404 1.460'-2.8204 1.5109-2.7104 1.5834-2.7724 b average 1.8874 f 0.353p 1.8514 k 0.344 2.1104' k 0.339s 2.1204' k 0.3364 2.1714 f 0.2584 c average change -0.026x 0.259~ o.01ox 0.052" notes: e = exceeded the standard value of us epa, i$ = exceeded the standard value of us epa and anzecc & armcaz; pg 4, x, y = different letter across the same row indicates significant differences. increasing risk of heavy metal contamination in pond dwi hastuti e t al: the analysis of variance (anova) of organic matter did not show any significant difference among the mean values and concentration changes of the five sampling periods. t h s indicated that the management of organic matter concentrations and changes in the silvofishery pond was effective. however, the anova of pb and cd concentrations and changes showed that there was a significant difference among the different sampling periods. there were two groups of mean concentration: the first group which consisted of the first and second observations, and the second group which consisted of the third to fifth observations. this indicated that a significant increase in pb and cd accumulation happened in the second period. analysis of the concentration changes showed that a sipficant change occurred between the second period and the other periods. the sipficant increase in pb and cd concentration in the second period indicated the dominance of heavy metal accumulation in certain sampling periods. another important concern manifested by these findings is the increasing accumulation of pb and cd. in the first period, the concentration of both heavy metal elements was relatively low, but in the second to fourth periods it tended to increase. this indicated the increasing risk of heavy metal pollution in the silvofishery pond, which might trigger the contamination of the livestock cultivars. the concentration of cd alone was sufficient proof that the pond was contaminated. thus, a more effective pond management plan should be formulated in order to avoid the danger of continuous deposition of heavy metal pollution and subsequent contamination. heavy metal accumulation in the sediment is related to the sedimentation process. the sediment carries various substances including organic matter and heavy metal. thus, the accumulation of heavy metal might be interrelated with that of any other substances. this research also evaluated the correlation between heavy metal and organic matter (table 2). although the correlation coefficients were high, the organic matter concentration was not related to pb and cd concentration, as well as with concentration changes (table 2). however, the concentrations and changes of pb and cd correlated with each other. this indicated that the source of organic matter was not only sediment accumulation. low correlation level between concentration changes indcated the difference in the accumulation rate between the organic matter of pb and cd. the high and significant correlation between pb and cd concentrations and changes showed that both heavy metal elements came from the same source, i.e., the contaminated sediment. the silvofishery pond in mangunharjo village has accumulated organic matter and heavy metal elements, as indicated by the increasing concentration of these substances in the sediment. mangrove vegetations in the pond provide fine sedment trapping by slowing down the current; thus, resulting in an increase in the sedimentation rate (adame e t ul. 2010). an increase in heavy metal concentration in ponds is hghly related to the water sources. coastal ponds are supplied by both sea and river water. however, river streams are considered as the main source of pollutants in the coastal area since these are the channels of anthropogenic and industrial waste disposal (icumar e t ul. 2015). table 2 correlation among om, pb, and cd concentration and changes in a silvofishery pond correlation (sig.) average concentration o m p b cd average concentration change o m ---0.394 (0.606) 0.484 (0.516) p b ---0.995 (0.005)** cd --- notes: * = correlation is significant at the 0.05 level; ** = correlation is significant at the 0.01 level; some cells are left blank intentionally due to similar correlation items biotropia vol. 28 no. 1,2021 various inland activities produce heavy metal contaminated wastes which then enter the aquatic system (zhang e t al. 2010). when the freshwater flows to the estuaries, the heavy metal is dispersed throughout the surrounding ecosystem (ruzhong e t al. 2010), such as the ocean and ponds. most heavy metal pollutants are bound to fine sediments (zhang e t al. 2014). when the water enters vegetated ecosystem, the flow would slow down, increasing the chance of sedimentation (turgut e t al. 2015). thus, the accumulation of heavy metal occurs along with sediment deposition. the increasing heavy metal concentration in the silvofishery pond indicated that the water sources were polluted. the source could be from the freshwater stream or the seawater. the semarang rivers, as well as the city's coastal areas, have been known to be polluted due to high industrial activities (hastuti 2015). thus, there is a severe risk of pollutant contamination in the silvofishery ponds. moreover, the heavy metal concentration is expected to continually increase due to sediment trapping by mangrove roots in the ponds. although the mangroves are known as bioremediators of heavy metal pollutants (kdnnan e t al. 2016), their capacity is limited. the mangrove trees could accumulate heavy metal elements in their various parts but the elements would be returned to the environment through litter fall and the decomposition processes (martuti e t al. 2017). thus, the accumulation rate of heavy metal pollutants would depend on the supply rate from the sources and the uptake rate by the mangroves. the increasing trend of pollutants accumulation showed that the mangrove uptake rates could not match the pollutant supply rates. this requires the development of more effective management plans to prevent the increasing risk of heavy metal contamination and to reduce the toxicity level of the livestock cultivars. the extreme increase of organic matter and heavy metal concentration in the second period (from the 2nd to 31d observations) manifested the high input rate of sediment during that period. this might have occurred due to frequent rains that flooded the pond (gharbi e t al. 2016). and carried h g h amount of sediments from the river stream to the ponds. the ponds which generally occupy a single canal as water inlet and outlet would trap incoming water, thus increasing the chance of sediment deposition. cd concentration in the silvofishery pond indicated an unsafe environment. although heavy metal concentration in the sediment is not directly related to water quality, this may affect the availability of dissolved heavy metal in the water (hassaan e t al. 2016). us e p a recommends a maximum concentration of 1 mg/kg of cd in the sediment, wme anzecc & armcanz7s standard is hgher at 1.5 mg/kg (hubner e t al. 2009). however, in a l l observations, the average concentration of heavy metal exceeded both standards, indicating a high possibility of a polluted environment since the beginning. therefore, such increases in the concentration rates need thorough consideration in the management of silvofishery ponds. this research showed that pb and cd were accumulating in the silvofishery pond sediment. however, only cd exceeded the recommended value, while pb was still within the safe range. such condition increases the alarming possibihty of heavy metal contamination of cultivated fish (icumar e t al. 2011). any toxic elements in the aquatic system can be easily transported from one organism to another through the food chain. heavy metal pollutants would be absorbed by a low trophic level organism such as phytoplanktons, which are then consumed by zooplanktons and finally by fish (hassaan e t al! 2016). thus, the risk of contaminated fish consumed by humans increases as well. through the food chain phenomena, heavy metal elements will bioaccumulate in living organisms; plants, animals, and humans. the toxicity in a plant is usually shown by several symptoms, such as changes in leaf color or abnormal plant growth (nagajyoti e t al. 2010). unfortunately, toxicity in mangrove is usually unnoticeable as they have a high level of tolerance due to their bioaccumulative capability (icannan etal. 2016). thus, the effect of toxicity may expand to other aquatic animals including crabs, shells, and cultured fishes. some animals are also known to be tolerant to heavy metal toxicity, such as shells (shirneshan & bakhtiari 2012). however, the animals with low tolerance level may show toxicity symptoms, such as declining growth rate and damaged liver and kidney tissues (jayakumar e t al. 2016). increasing risk of heavy metal contamination in pond dwi hastuti e t al: the bioaccumulative capability of aquatic organisms increases the chance of heavy metal consumption and subsequent accumulation in the human body. consuming a small amount of contaminated fish would not affect the human body in a short period of time. however, the effects of heavy metal toxicity would be noticeable when the concentration is high enough. various symptoms caused by cd toxicity include kidney damage, osteoporosis, cardiovascular diseases, hypertension, diabetes, modification of several organs, infertility, and many others (bernhoft 2013). to avoid cd toxicity, humans can only take a maximum of 1 pg/kg per day (usepa 1998). the analysis shows that the concentration and accumulation of pb and cd correlated with each other indicating that a common factor had caused the accumulation of both elements. sedment input from contaminated sources was considered as one possible factor. thus, both heavy metal elements were deposited along with the sediments (zhang e t al. 2014). the insignificant correlation between the heavy metal and organic matter concentration and accumulation indicated that organic matter had further decomposed to nutrients. this research shows that even though silvofishery provides various beneficial environmental services, some negative impacts might arise after a considerable period of time. various heavy metal elements might accumulate over time, thereby resulting in their increasing concentration. after certain periods of time, the accumulated heavy metal may exceed the safe limit and cause environmental toxicity leading to environmental hazards, especially for aquaculture activities related to human livelihood. in order to maintain environmental quality by avoiding heavy metal toxicity in silvofishery ponds, a more effective management action plan is urgently needed. decreasing the heavy metal concentration in the pond sediments must be the main objective, especially in mangunharjo village. however, the process may take a long time, and controlling the heavy metal concentration in sediments is a gradual process and cannot be done instantly. an important management intervention to decrease the sediment input from the pond is by separating the mangrove plantation. dikes need to be constructed around it, mangroves should be planted in the inlet as a canal formation, thereby filtering the water input and sediment deposition before entering the pond. in so doing, the risk of heavy metal accumulation in the pond is decreased. another strategy to decrease heavy metal concentration in the ecosystem is by pruning the mangrove branches and leaves. thus, heavy metals absorbed and accumulated in the plant organs, particularly the leaves and branches, could be totally removed and prevented from reentering the ecosystem through litter falls. the pruning also stimulates the development of fresh branches and leaves, thereby providing more space for heavy metal absorption in the ecosystem. in this way, the concentration of heavy metal in the silvofishery pond would gradually decrease. the combination of both techniques may improve the effectiveness of heavy metal removal from the silvofishery pond ecosystem, thus, minimizing the risk of heavy metal toxicity. conclusion the silvofishery pond has accumulated organic matter rich with heavy metallic elements pb and cd, indicating an increased risk of environmental toxicity. the pond was contaminated with cd whose concentration has exceeded the recommended level by both us epa (i 1.0 mg/kg) and anzecc & armcanz (5 1.5 mg/kg). this excessive concentration might affect the aquatic organisms in the ecosystem. the increasing accumulation rate during the last periods of observation indicated an imbalance between pollutant supply and mangrove uptake rates. the concentration and accumulation rates of pb and cd show a h g h degree of correlation, indicating a possibility that both metallic elements come from the same sources. the recommendations for better management plans and actions include changing the pond setting by planting mangroves in the inlet canals, developing dikes to avoid direct rapid water flow from the mangroves to the pond, and pruning the mangrove leaves and branches to decrease heavy metal recycling. such pruning would also promote the development of fresh leaves and biotropia vol. 28 no. 1,2021 branches which can increase heavy metal absorption. acknowledgement this research was supported by the pnbp dipa university of diponegoro funding source batch 2017 no. 275-066/un7.5.1 /pg/2017. references adame mf, neil d, wright sf, lovelock ce. 2010. sedimentation within and among mangrove forests along a gradient of geomorphological settings. estuar coast shelf sci. 86(1):21-30. asati a, 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73:270-81. zhang h, cui b, xiao r, zhao h. 2010. heavy metals in water, soils and plants in riparian wetlands in the pearl river estuary, south china. procedia environ sci 2(5):1344-54. zhang r, wilson vl, hou a, meng g. 2015. source of lead pollution, its influence o n public health and the countermeasures. int j heal anim sci food saf 2(1):18-31. 705 ridesti (the growth strategies).cdr the analysis growth strategies of ten woody plant species for effective revegetation ridesti rindyastuti nd retno peni sancayaningsih 1* 2 a 1 , (lipi) ,purwodadi botanic garden indonesian institute of sciences , pasuruan 67163 indonesia laboratory of ecology and conservation, faculty of biology, gadjah mada, 2 department of biology, universitas yogyakarta 55281 indonesi, a received 05 october 2016/accepted 26 may 2017 abstract the growth strategies of plant species show the ecological role which is reflexed by their adaptation to environments and competitiveness. those are essential in the study of the revegetation effectiveness. however, the growth strategies of plant species in various types of habitats have not yet been fully investigated. the objective of this study was to investigate the growth strategies of ten woody plant species which were naturalized from mangrove to lowland habitats in relation to their effectiveness for revegetation program. the seedling's growth was recorded during 4 months in purwodadi botanic garden-lipi from october 2014 to february 2015. complete randomized design with plant species as a treatment using 3 replications was carried out to examine the plant's relative growth rates (rgrs), their components, leaf nitrogen productivity and growth strategies.the study showed that rgrs of ten woody plants species varied across species. based on the pearson correlations, the plant's net assimilation rates (nar) and two ecological traits related to the root trait i.e. nitrogen productivity and specific root length (srl) were strongly correlated with the rgrs. heritiera littoralis, diospyros discolor, antidesma bunius, schleichera oleosa, madhuca longifolia and syzygium cumini have high rgrs but low specific leaf area (sla). b. asiatica has relatively low rgrs and sla, while dracontomelon dao have high rgrs and sla. it showed that most of plant species studied, except d. dao achieve growth rates and competitiveness by developing strategies through forming fine roots to maximize its ecological function in nutrients uptake. most of woody plant species are adaptive to dry lowland habitat and only d. dao potentially occupy the ecosystem. furthermore, d. discolor and s. oleosa are highly recommended for revegetation in degraded tropical lowland areas. keywords: growth strategy, revegetation, rgr, root trait, woody plants introduction natural tropical forests are being destroyed at a rapid high rate, therefore the success of forest restoration continues to be a challenge. a restoration program could help reestablish a forest vegetation eventhough it is difficult to guarantee the successful recovery of its biodiversity (elliott et al. 2013). adequate knowledges on biology and ecology of plant species related to their adaptation to habitat conditions enhance their use for decission making in reforestation programs. especially in the early stage, plant species commonly develop the strategies for using limited resource in their environments, adapting to stresses, competing and associating with others species in order to preserve their existences in their distribution ranges. the growth strategies show the ecological role and contribution of plant species to an ecosystem and imply direct result of the evolutionary control by genetic (kolb et al. 1990)s . growth strategies can be identified by analysing and comparing the growth rates, leaf and root traits since these two organs are essential in photosynthesis and nutrient uptake (wright & westoby 2000). in a constant state and similar environment, plant species were reported to have interspecific differences in growth rate that indicate different results of ecological strategy of plant species (lambers et al. 1998; pugnaire & valladers 1999; laughlin et al. 2010). in plants, the survival depends on growth rate that can not be acurately measured only by observing plant size. * corresponding author: ride17@gmail.com biotropia 5 1 8 43 55 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.705 43 thus, relative growth rate (rgr) and its components were modeled to understand, compare and evaluate plant growth rate. rgr and its components such as nar, sla, lwr, and lar (described in table 2) tend to reveal more of the species traits than predict plant growth rate according to its habitat (lambers et al. 1998; pugnaire & valladers 1999). growth strategy is especially related to leaf and root trait reflected in sla (leaf area per which is unit leaf dry mass) and in srl (root length per unit root dry mass). sla as an aboveground seemed to be easier to investigate than srl. srl has been suggested as the belowground analogue to sla, and is considered as a morphological index of belowground species competitiveness (cornelissen . 2003). shipley (2002) reported et al that rgr of either woody and herbaceous plant s have strong and positive correlation with nar and have weak and negative correlation with sla. according to lambers . (1998) pugnaire et al and and valladers (1999), variation in rgr is determined mainly by sla. the opposite result is partly caused by differences of irradiance level used in the experiments. in general, the contribution of rgr's component is influenced by irradiance level. at low irradiance, sla becomes the most determinant factor of rgr , and nar is the main determinant of rgr at high irradiance. thus, plant species possess that different shade tolerance will show different s response in growth rate. in general, higher sla is s the main growth atribute support plant which s invasiveness in other type of habitat (shipley s 2002; lambers . 1998; pugnaire valladers et al & 1999), with an exception, the invasive woody species successful in mediterranean which are climates. this invasive woody species has a greater biomass allocation to roots than less-invasive species (grotkopp rejma´nek 2007).& the growth strategies of plant species adapted in tropic regions with various types of habitats and shade tolerances have not yet been fully investigated. on the other hand, plant interspecific variation in rgr and its components a r e i m p o r t a n t t o s t u d y t h e s p e c i e s competitiveness for particular purposes such as land restoration, carbon sequestration, species invasiveness etc. local plant species which possess high growth rates and are widely distributed in certain ecological range are highly recommended for restoration of degraded areas (cairns 1995). moreover, to grow the plant species from other types of habitats, the revegetation effectiveness and plant invasiveness should be managed by understanding plant ecological traits including growth rate, growth strategy related to sla, carbon gain and physiological performance of exotic species compared to native plants (pattinson . 1998; et al grotkopp & rejma´nek 2007). for this study, ten woody plant species were investigated because they originated from various types of habitats but were developed and planted for revegetation programs in tropical dry lowland areas. the effectiveness of these species for revegetation in dry lowland habitats should be investigated in terms of their growth strategies. this study could provide valuable insights on the variation among plant species and explain the ecological success and competitiveness of plants in certain habitats. thus, the objectives of this study were to: 1) determine the interspecific variation in rgrs and its components among plant species in similar irradiance level ; 2) find s out the ecological trait of ten woody plant species; and 3) identify the growth strategies of ten woody plant species and discuss their ecological trait related to their effectiveness for revegetation in dry low land habitat. materials and methods plant material the seedlings used in this study were from the plant collections of purwodadi botanic gardenlipi. all species used in this study are c3 woody plant species (rindyastuti hapsari 2017). the & botanic garden is located in pasuruan, east java, at 300 m asl. it has a semi-deciduous climate with monthly rainfall of 246.57 mm in the wet season and 38.2 mm in the dry season. the annual temperature ranges from 20.4 to 30.6 c. three o seedlings were used for each treatment. sixteenmonth old seedlings of ten local woody plant species used in this study are described in table 1 . 44 biotropia vol. 25 no. 1, 2018 at 16 months were harvested to obtain w and the 1 seedlings were harvested 4 months later at 20 months to obtain w . all seedlings were divided 2 into three parts: leaf, stem and root. plant materials were dried in an oven with temperature of 80 c to constant weight (crescente gratani o & 2013). rgr was calculated using the following equation ( :hoffmann & poorter 2002) rgr = (ln w -ln w )/(t -t )2 1 2 1 where: ln = natural logaritm 1t = time one 2 t = time two 1w = dry weight of plant in time 1 2w = dry weight of plant in time 2 the components of rgr observed in this study are listed and described in table 2. growth conditions, experimental design and rgrs measurement seedlings of ten woody plant species were grown in horticultural pots under controlled and favourable conditions in a glasshouse located at purwodadi botanic garden-lipi, pasuruan, east java which has a temperature range of 26.2-30.8°c in the dry season and 28.7-34.6°c in the rainy season. seedlings were watered ever y 2 days without fertilizer treatment. seedlings were grown in a mixture of silica sand and compost with a ratio of 1:1. the experiment was caried out in complete randomized design using 3 replications with variation of species as its treatment. all seedlings were grown until 16 months then were transferred into bigger polybags with new plant media to record maximum resource uptake for their growth. three seedlings for each replication 45 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih table 1 list of species, habitat, type of plant and shade tolerance of ten woody plant species observed in this study species habitat type shade tolerance barringtonia asiatica (lemmens et al. 1995) mangrove, tropical coast evergreen woody intermediate to tolerant dracontomelon dao (orwa et al. 2009) monsoon forest (deciduous and semi-deciduous) semi-deciduous, hardwood tolerant heritiera littoralis (lemmens et al. 1995) mangrove, tropical coast evergreen, hardwood tolerant diospyros discolor (verheij & coronel 1991; lemmens et al. 1995) dry lowland tropic evergreen hardwood tolerant calophyllum inophyllum (sosef et al. 1998; orwa et al. 2009) coast-lowland tropic evergreen hardwood intolerant antidesma bunius (sosef et al. 1998; orwa et al. 2009) highland tropic evergreen hardwood tolerant schleichera oleosa (kundu & schmidt 2011) dry low-highland mix deciduous forest deciduous hardwood tolerant syzygium cumini (verheij & coronel 1991; orwa et al. 2009) dry and moist tropicaldeciduous forest evergreen hardwood tolerant madhuca longifolia (sikarwar 2002) dry lowland tropics evergreen-semievergreen, hardwood tolerant adenanthera pavonina (orwa et al. 2009) evergreen-deciduous forest evergreen-deciduous, woody tolerant leaf area measurement leaf area were used as a component of parameters listed in table 2. leaf area was calculated by comparing the proportion of an area 2 of 16 cm paper weight with the weight of the entire paper using comparison formula as follows: where leaf area l2 = l1 = area of sample paper w2 = paper weight w1 = weight of sample paper. analysis of nitrogen productivity leaves of the ten woody plants were oven o dried at temperature of 80 c for 48 hours. five g of leaf sample were grinded and analyzed using the kjeldahl method (bremmer 1965). the analyses were performed at the soil chemistry laboratory, faculty of agriculture of brawijaya university in malang. nitrogen productivity was reflected by the value of lnp (rate of dry mass increase/unit leaf nitrogen/unit time) which was calculated using the formula as follow: seed mass seeds of the ten woody plant species were collected from the plant collection at purwodadi botanic garden-lipi, pasuruan, east java in march 2016. ten fresh seeds weight were measured for each woody plant species including the seed coat. the seed mass show plant strategies which were illustrated by l-h-s (leaf trait, height and seed mass) scheme in several growthforms (westoby 1998). this variable was used to determine plant strategies scheme of species studied, whether l-h-s scheme or others. data analyses the normality and randomity of data were tested using kolmogorov-smirnov and run test. the normal data were analyzed using variance analysis (anova) at confidence level of 95%. tukey's advance analysis was carried out for data grouping. the relationship among variables which have random data were analyzed using pearson correlation test at confidence level of 95% (p<0.05), while relationships between unrandom data were analyzed using spearman rank correlation test. results and discussion interspecific variation in rgr based on the normality test, the mean of rgr and its components of the ten woody plants were normal. based on the variance analyses, there was a significant difference in rgr (p=0.014) and sla, lar, swr, lwr and rwr with p values 0.0001, 0.0001, 0.0002, 0.0002, and 0.0001, 46 biotropia vol. 25 no. 1, 2018 table 2 list of abbreviations used in the text with definitions and units of measure abbreviations definitions units rgr relative growth r ate g g-1day-1 lar leaf area ratio (leaf area/plant dry mass) cm2 g-1 sla specific leaf area (leaf area/foliage dry mass) cm2 g-1 lwr leaf weight r atio (foliage mass/plant dry mass) g g -1 swr stem weight ratio (stem mass/plant dry mass) g g -1 rwr root weight ratio (root mass/plant dry mass) g g -1 nar net assimilation rate g cm -2 day-1 srl specific root length (root length/root dry mass) cm2g-1 lnp leaf nitrogen productivity (dry mass increase/unit leaf nitrogen/unit time) g g-1 day-1 leaf nitrogen productivity (lnp) = dry mass increase leaf nitrogen/time respectively (significant if p<0.05) among the ten woody plant species observed in this study. mean of rgr ranged from 4 x 10 g g day for -3 -1 -1 -3 -1 -1 cc. inophyllum and 10.7 x 10 g g day for s. umini (figure 1). mean of initial sla of the ten woody plant species ranged from 90.53 for h. littoralis to 298.56 for d. dao (figure 2). during the seedling growth, the mean of sla increased from 105.89 for h. littoralis to 321.58 for a. pavonina. the mean of initial lwr ranged from 0.094 for a. pavonina to 0.46 for d. discolor, while mean of lwr increased from 0.52 for s. cumini to 0.53 for d. discolor. the differences between species belonging to the lowest ratio and highest ratio of sla and lwr show the differences of succulence level and leaf dry matter content among species (wilson et al. 1999). the mean of initial swr ranged from 0.14 for d. discolor to 0.49 for s. cumini. the mean of swr increased from 0.29 for d. discolor to 0.61 for s. cumini in the end of observation. less mass was stored in the stem of d. discolor than in its other parts. mass allocations in the root was reflected by rwr (figure 3). mean of initial rwr ranged from 0.29 for h. littoralis to 0.54 for a. bunius. after 4 months of growth, the mean of rwr increased from 0.18 for d. discolor to 0.38 for b. asiatica. other than rwr, root trait was also reflected by srl (figure 3). srl ranged from 1.28 for b. asiatica to 7.2 for d. discolor in the initial growth and ranged from 1.52 to 10.86 at the end of the experiment. 47 figure 1 the mean of rgr and nar of ten woody plant species note: rgrs and nar were significantly different among species based on the variance analysis (p<0.05) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d . d ao h . l itt ro ra lis d . d isc olo r c. in op hy llu m a . b un iu s s. ol eo sa s. cu mi ni m . l on gif oli a a . p av on in a 48 biotropia vol. 25 no. 1, 2018 figure 2 the comparison between mean components of rgr related to leaf trait (lar, lwr and sla) of ten woody plant species (note: the component of rgr related to leaf trait were significantly different among species based on the variance analysis (p<0.05)) b. a sia tic a d . d ao h . l itt ro ra lis d . d isc olo r c. in op hy llu m a . b un iu s s. ol eo sa s. cu mi ni m . l on gif oli a a . p av on in a 49 figure 3 the mean of components of rgr related to stem and root traits represented by swr, srl and rwr of ten woody plant species (note: the components of rgr related to stem and root traits were significantly different among species based on the variance analysis (p<0.05) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d . d ao h . l itt ro ra lis d . d isc olo r c. in op hy llu m a . b un iu s s. ol eo sa s. cu mi ni m . l on gif oli a a . p av on in a 50 biotropia vol. 25 no. 1, 2018 correlation among components of rgr correlation tests were performed to reveal the relationships between plant rgr and its components. the mean of rgrs of the ten woody plant species was strongly correlated with nar (p value 0.0001) but not with other components (figure 1, table 3). the correlation explained the importance of nar in determining rgr of woody plant species. variations in other components did not explain the differences in -4 -2 -1 rgr. nar ranged from 1.2 x 10 g cm day for -3 -2 -1 c. inophyllum to 1.9 x 10 g cm day for s. oleosa. the nar values of two species which are known as fast growing species i.e. m. longifolia and a. pavonina were more than a fifteen-fold difference w i t h s l ow g r ow i n g p l a n t s ( f i g u r e 1 ) . interspecific variation in rgr and its components among species showed variation of ecological traits of plant species. the ecological traits can be shown by above ground trait supported by leaf trait or below ground trait supported by root traits which serves as a chosen strategy scheme of plant species in the habitat they occur (laughlin et al. 2010; grime 1979). in several previous studies, plant strategies were illustrated by l-h-s (leaf trait, height and seed mass) scheme. seed mass showed dependence with other growth component between different growth forms (westoby 1998). for example, seed mass dependence of woody c3 was different from those of graminoids (laughlin et al. 2010). in line with the previous study, the seed mass of ten woody plant species which included in woody c3 were not correlated with the height or leaf traits of plant species. therefore, it indicated that plant strategy across species within similar growth form were not supported by seed mass but by leaf or root trait. the plant size can not literally support the mass investment to plant structure because the value of rgr is mainly supported by dry matter content especially various lignin component (laughlin et al. 2010). the larger plant could show lower rgr maybe because of the high level of water content in the plant body. the larger plant sometimes consist of abundant water than its dry matter content, for example and . b. asiatica a. pavonina thus, the growth properties of plant species could not be observed only by the plant size thus, the study of growth rate and its components proves to be very important. the study result showed that the mean rgr of fast growing plants was more than two-fold difference from the slowest growing plant while nar was a five-fold difference from the slowest growing plant. rgr of woody plants showed different pattern from those of non-woody species. according to poorter and remkes (1990), in c3 non-woody species, rgrs of the fastest growing species was three-fold more than those of the slowest growing species, while nar of the fastest growing species was two-fold more than those of the slowest growing species. fast growing species obtain an ecological advantage of a high rgr related to high competitiveness and rapid occupation (poorter r mkes 1990). & e therefore, the difference in nar between the fast and slow growers seemed to be a trustworthy evidence of its contribution to differing rgr across plant species. a strong correlation between rgrs and nar of woody plant species showed how the high rgrs in fast growing species were achieved. theoritically, rgr is the product of lar and nar. however, lar did not seem to contribute to rgrs variation. while it is clear that two other growth components related to leaf formation (sla and lwr) did not show its association with growth rate, this could be concluded that there was other more important growth plant behavior in carbon gain than to invest more mass to the leaf. nar largely reflects the rate of photosynthetic carbon gain minus carbon released by respiration. loveys . (2002) et al reported that correlation between rgr and nar is more stable than the correlation between rgr and sla. the effect of sla to the variations in rgrs is more sensitive than nar because it showed greater response in slight changes of radiance level. moreover, sla is an important plant functional trait as species which have higher sla can obtain a more extensive foliage display that captures more light for biomass investment (crescente gratani 2013). & the mean of rgrs was correlated with srl and lnp but not with sla. nitrogen productivity showed an important role in root trait of plant growth. when rgrs only correlated with root length and nitrogen productivity, this means that the woody plants in this study could achieve more mass through root trait than leaf trait (wilson . 1999). it also explains how the et al 51 fast growth of species studied can be achieved and how different rgrs across species are formed. the fast growing species have high rgrs and tend to allocate more mass to the leaves than to the roots. therefore, the species studied have greater mass allocations to the roots and low rgr. hunt and cornelissen (1997) investigated that mean of rgrs of 59 temperate herbaceous species range from 0,1260 g g day for to -1 -1 brachypodium pinnatum 0,2741 gg day for . it indicated -1 -1 epilobium hirsutum that all woody plants studied were slower growing plant compared to the most herbaceous species. variation in seed mass seed mass was one of physiological plant trait that being included into l-h-s (leaf-heightseed) scheme proposed by westoby (1998). l-h-s scheme is a plant ecological strategy which defined by leaf trait, heigh and seed mass. seed mass data of ten woody plant were not normal and not random with a p value of normality test 0,01 (p>0.05), while the p value of a run test was p 0.0001 (p>0.05). thus, seed mass of ten woody plant species were analysed and grouped descriptically. seed mass of is the b. asiatica highest while those of is the lowest a. bunius (figure 4). seed mass can be grouped into big and small seed. clasifiedpoorter and rose (2005) the weight of small seeds is <0.1 g and large seed is >0.1 g. therefore, the seed of all species studied were clasified as big seeds. furthermore, there is no variation in seed mass across species studied that support interspesific variation in rgr. nitrogen productivity leaf nitrogen was reflected in leaf n concentration and lnp (leaf nitrogen productivity). leaf n probably correlated with above or below-ground traits of plant species (wright westoby 2000). the mean of n & concentration of ten woody plant ranges from 1.59 % for to 3.03 % for . h. littoralis a. pavonina mean of lnp value ranges from 0.035 gg day for -1 -1 -1 -1 c. inophyllum s. cumini to 0.24 g g day for . based on the variance analysis, both total n and lnp were significantly different among species. it showed that n productivity vary amongs species and probably contribute to variation in rgrs. based on the correlation test, lnp is correlated with rgrs and sla (p<0.05) (table 3). it showed that n productivity contribute to the variation in rgrs and leaf area. the mean of lnp of ten woody plant species are described in figure 5. the mean of rgr is strongly correlated with srl but is not correlated with sla while the mean of rgr is strongly correlated with lnp (table 3). it indicated that the growth of ten woody plants is achieved through the growth of root length and high leaf nitrogen productivity. the strong correlation between root length and rgrs showed an important role of root function in plant growth. two species which have high srl during the growth are and while d. discolor s. oleosa the species which has the lowest srl is b. asiatica. d. discolor s. oleosa and are considered native to dry lowland habitat while is a mangroveb. asiatica figure 4 seed mass of ten woody plants species (note: there was no variation in seed mass across species studied that support interspesific variation in rgr based on normality test (p>0.05) and run test (p>0.05)) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d . d ao h . l itt ro ra lis d . d isc olo r c. in op hy llu m a . b un iu s s. ol eo sa s. cu mi ni m . l on gif oli a a . p av on in a species that is likely to poorly survive in dry area. sla is strongly correlated with lnp. among the studied species, the highest sla was found in d. dao a. pavonina and . these two species are known as semi-deciduous species (table 1). leaf or root trait rgrs of ten woody species were correlated with srl but were not correlated with sla, while mean of rgrs was correlated with lnp. the strong correlation between rgrs with root length and leaf nitrogen productivity explained that most of woody plant in this study could achieve more mass through root trait than leaf trait (wilson . 1999). moreover, related to the et al growth properties of plant species, most species allocated greater mass to the leaves than to roots, but slow growing species with low rgr commonly partitioned greater proportion of new biomass to the roots rather than to the leaves (wright westoby 2000). therefore, most of & 52 biotropia vol. 25 no. 1, 2018 figure 5 the mean of lnp of ten woody plants species (note: lnp were significantly different among species based on the variance analysis (p<0.05)) table 3 pearson correlation coefficients between growth components (p<0.05) components p value correlation rgr and nar 0.0001 strongly correlated rgr and lar 0.64 not correlated rgr and sla 0.2 not correlated rgr and lwr 0.57 not correlated rgr and swr 0.88 not correlated rgr and rwr 0.48 not correlated rgr and srl 0.000 strongly correlated rgr and lnp 0.000 strongly correlated nar and lnp 0.056 not correlated sla and lnp 0.000 strongly correlated lwr and lnp 0.2 not correlated lar and lnp 0.1 not correlated swr and lnp 0.267 not correlated rwr and lnp 0.302 not correlated srl and lnp 0.58 not correlated seed mass and rgr 0.87 not correlated seed mass and lwr 0.67 not correlated seed mass and swr 0.97 not correlated seed mass and rwr 0.47 not correlated seed mass and srl 0.45 not correlated seed mass and height 0.28 not correlated b. a sia tic a d . d ao h . l itt ro ra lis d . d isc olo r c. in op hy llu m a . b un iu s s. ol eo sa s. cu mi ni m . l on gif oli a a . p av on in a species studied were clasified as slow growing species. the species which develop the root traits tend to be clasified as slow growing plant. based on the correlation among the rgr components, most of species studied were clasified as slow growing species which develop root trait in achieving mass for plant growth. among these species, d. discolor and are good examples for this growth s. oleosa strategies. in these two species, the highest rgr and srl were found, yet the sla was relatively low. m. longifolia a. pavonina and were known as fast growing woody plants. the rgr of m. longifolia was as high as those of other species while rgr of was low. has low rgr a. pavonina a. pavonina although it has the highest sla while m. longifolia has a high rgr yet low sla. because species which are confirmed as a fast growing species should show high rgr and sla, it is difficult to confirm that these two wellknown species are fast growing species. moreover, both species had among the largest increase in lar through time and largest decrease in rwr. a. pavoninathe which has high sla and high increase of lar yet large decrease of rwr may exhibit different ecological strategies from other species studied. this species tend to develop leave trait than root trait. in addition, in most of species studied, the increase of srl and the decrease of rwr were found. the formation of long root which is combined with the low roots dry mass could mean that plants in this study tended to form more fine roots to reach a wider range of water and nutrients. it showed a pattern of growth to form long root inspite of large foliage display to capture more light. the fine roots could clearly explain the growth strategies through root trait of the ten species studied. implication to land revegetation because of the massive biodiversity loss in recent years, forest restoration should be established in the framework of conservation to restore forest biodiversity and its ecological functions. cairns (1995) recommended native and local plant species to restore destroyed forest ecosystem. native and local species tend to have relatively slow growth and limited leaf expansion. on the other hand, fast growing plants which have wide ecological range distribution are highly recommended to restore degraded areas because of their ability for rapid establishment and coverage. however, non-native fast growing plants have greater potential to invade areas because of their ecological advantage of having a high rgr for high survival, competitiveness and rapid occupation (poorter remkes, 1990; & haggar . 1998; mahari giday 2014).et al & invaders are more likely to have higher rgrs, leaf area and tissue construction costs (daehler 2003), with an exception of woody plants thriving well in mediterranean climates which have greater allocation to roots than less invasive species (grotkopp rejma´nek 2007). further, higher & sla is strongly correlated to plant invasiveness (lambers et al. 1998; pugnaire valladers 1999). & most of the woody plant species in this study obtained their rgrs by developing root trait which showed that these species are less invasive and recommended for regenerating forest vegetation in dry lowland areas. dry tropical climate is a condition with relatively high temperatures and low water availability compared to other tropical climates such as rainforest areas. in dry tropical areas, plants are adapted to have mechanisms to reduce water loss through low transpiration and reach the water balance by absorbing water from the soil. root traits is a competitive advantage with respect to the dry climate with high air temperature and low water availability. adaptive roots have more fibers which can reach water in wider and deeper soil. because root function was correlated with the mean of rgrs, several species which have high rgrs yet relativelly low sla are considered to be well adapted to dry climate such as , , , h. littoralis d. discolor a. bunius s. oleosa m. longifolia, s. cumini, and . several species are known as native plants to dry lowland habitat. native species are preferred to be reintroduced in degraded areas to fix damaged soil by restoring soil fertility and enhancing microbe population which provides sufficient nutrients for plants (álvarez-sánchez 2009). based on this study, et al. native species are considered to have high rgrs which supported by root trait thus they are highly recommended for restoration programs in degraded areas. of the species studied, d. discolor and which are native to dry lowland s. oleosa, areas yet have high rgr and developed fine 53 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih roots, are highly recommended for revegetation in dry lowland habitat. b. asiatica is a native species to mangrove habitat which has relatively low rgr and sla, while which is adapted to monsoon forest d. dao has high rgr and sla this indicates that . b. asiatica exhibits relatively low competitiveness and occupation potential compared to other species. for being introduced to other types of habitat, non-native species are better of having low competition so that the species do not occupy the plant community in new habitat (bazzaz 1979). hence, can be more b. asiatica recommended for planting program in dry lowland habitat than . based on the d. dao d. daohigh rgr and sla, expands larger foliage to capture more sunlight. therefore, this species has the potential to occupy the ecosystem. other species which have high sla but low rgr, i.e. may develop a. pavonina, growth strategies through leaf trait but do not potentially invade the ecosystem. for further study, it is suggested to investigate whether d. dao release allelopathy, provide ground water and co-occur with other species and growthform to reveal the ecological suitability to new habitat in order to examine the effect of plant growth to soil and plant community. the ecosystem services of a species relating to root trait should be highlighted to maximize the advantage of restoration such as for hydrological services in degraded landscapes (bruijnzeel 2004; hall . 2011)et al . conclusions there are variations in rgrs across the ten woody plants species as determined by nar, nitrogen productivity and root trait. to achieve the growth rates and competitiveness, the woody plant species studied tend to develop strategies through forming fine roots to maximize its ecological function in nutrient uptake except d. dao. most of the woody plant species are adaptive to dry lowland habitat and only d. dao has the potential to occupy the ecosystem. d. discolor and s. oleosa are highly reccomended for revegetation programs in degraded tropical lowland areas. acknowledgements the authors would like to thank kemenristek dikti scholarship which support the whole project of ecophysiological study of woody plants and dr titut yulistyarini as co-promotor. we like to thank mr. riyadi and mr. roif marsono for their technical contributions during experiment in purwodadi botanic garden-lipi. we also like to thank ms. samantha tesoriero for writing consultation. references álvarez-sánchez j, sánchez-gallen i, guadarrama p. 2009. analyses of ecophysiological traits of tropical rain forest seedlings under arbuscular mycorrhization: implications in ecological restoration. in: varma a, kharkwal ac, editors. symbiotic fungi: principles and practice. berlin (de): springer-verlag berlin heidelberg. p. 293-305. bazzaz fa. 1979. the physiological ecology of plant succession. annu rev ecol syst 10:351-71. 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australian oody pecies. funct ecol 14(1):97-107.w s westoby m. 1998. a leaf-height-seed (lhs) plant ecology strategy scheme. plant soil 199:213 27. 55 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 biotropia (2) 1988/1989: 12-17 the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides s. tjitrosemito tropical agricultural pest biology program, biotrop, bogor, indonesia d. suwinarno department of agronomy, faculty of agriculture bogor agricultural university, bogor, indonesia abstract a field experiment was conducted to investigate the performance of soybean (c.v. americana) when established with zero tillage technique on imperata dominated area. four different techniques of alang-alang control i.e. imazapyr (20 kg ai/ha), glyphosate (25 kg ai/ha), glufosinate (3.0 kg ai/ha) and manual cultivation were arranged factorially with time of plantings i.e. 1,2 and 3 months after treatments. the alang-alang damages varied with herbicides and times, imazapyr (20 kg ai/ha) showed slow appearance of damage at 3 months after application it was only 69%, while that of glufosinate was already down to 48% due to regrowth. no phytotoxicity was recorded, but the yield was low. introduction soybean, glycine max l. merrill, is promising and a proven source of plant protein and edible oil, containing 39-45% (dry matter basis) high quality protein with excellent amino acid pattern and 20-23% (dry matter basis) edible oil with considerable unsaturated fatty acids (hittle 1974). in indonesia, the soybean production in 1973 was 541 000 tons with the domestic consumption of 505 000 tons. indonesia was exporting 36 000 tons, however, since 1975 indonesia has been importing an increasing amount of soybean reaching more than 500 000 tons in 1983, spending a considerable amount of precious devisa. actually, the indonesian production of soybean had been from 492 000 tons annually during pelita i (first five year development plan), to 568 000 tons annually during pelita ii (second five year development plan) and 622 000 tons during pelita iii (third five year development plan); however, this production could still not meet the domestic demand and a considerable amount of soybeans has to be imported (sihombing 1985). 12 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno various efforts have been tried to increase the production of soybean in indonesia; these are through intensification as well as extension program. areas available for further agricultural expansion are, unfortunately, those classified as marginal land (driessen & soepraptohardjo 1974); some of those areas are dominated by alang-alang (imperata cylindricd). this type of areas has been given a high priority for agricultural development (sastrosuwarno 1981); therefore, it is imperative to develop techniques applicable to areas dominated by this weed to utilize the area into an agricultural production system. manual and mechanical cultivation were reported to be successful, when done during the dry season (sembiring and supardjo 1981; mangunsong 1976), but the hazard of erosion can be of considerable magnitude (suwardjo 1986, coaster 1936), so manual and mechanical cultivation are considered less favorable and zero tillage may be a good alternative to those techniques. tjitrosemito et al. (1983), reported that glufosinate at 3 kg ai/ha was able to control alang-alang well; and the subsequent work in pots indicated that even glufosinate 1-3 kg ai/ha was enough to control alang-alang followed by establishment of soybean (tjitrosemito and wiroatmodjo 1983). however, the regrowth of alang-alang under treatments of glufosinate was usually quite high after 3 months. as pointed out by tjitrosemito (1985) there are several factors affecting the success of this technique among others, the appropriate planting time after herbicide application. ideally, crops should be planted immediately after spraying to take advantage of dying imperata with no germination of other weeds yet; but it may have to consider the residual activity of herbicide and avoid the possible phytotoxi-city of crops by that herbicide. this work reports on the effects of different planting time after spraying from 1-3 months on the growth performance of soybean (c.v. americana). materials and method the work was carried out from august 1985 to march 1986, at the biotrop experimental field, which had been grown with alang-alang for the last three consecutive years. the field was divided into 36 plots measuring 4 x 5 m 2 with pathways of 1 m wide in between plots. the pathway was cleared from alang-alang during the whole experimentation by slashing. the experimental design was split-plot. the main plot consisted of four different methods of alang-alang control, i.e. foliar application of herbicides imazapyr (20 kg ai/ha), glyphosate (2.5 kg ae/ha), glufosinate (3.0 kg ai/ha) and manual cultivation, while the subplot consisted of three different time of planting, i.e. 1, 2 and 3 months after spraying/treatment and replicated 3 times. 13 biotropia no. 2, 1988/1989 before planting, the standing dead alang-alang was flattened by rolling a drum over the plots. the soybean (c.v. americana) was planted at a distance of 40 x 20 cm by dibbling three grains in each hole. plastic strings were extended across the plot 40 cm apart and a very shallow furrow was made along the extended string to facilitate fertilizer application before dibbling. the fertilizer was applied as basal treatment as tsp, urea and k2so4 at 60 kg p2o5, 45 kg n, and 50 kg k2o/ha. to protect the soybeans from agromysa phaseoli, furadan 36 was applied and azodrin at 2 cc/1 was sprayed weekly for 4 weeks. the spraying was repeated whenever necessary. manual weeding was done 2 weeks after planting at all treatments by cutting weed at ground level. the percentage of damage to alang-alang at planting time was recorded and analyzed statistically. the growth performance of soybean was evaluated in terms of plant height, leaf number, leaf area, yield and weight of 100 grains of soybean. results and discussion 1. percentage of damage to alang-alang at planting time the percentage of damage to alang-alang is presented in table 1. table 1. the average percentage of damage to alang-alang at planting time. time after spraying (months) method of control 1 2 3 1. imazapyr (2.0 kg ai/ha) 18.3 30.0 69.3 2. glyphosate (2.5 ae/ha) 30.0 86.0 88.3 3. glufosinate (3.0 kg ai/ha) 99.3 84.3 48.3 4. manual 81.7 60.0 31.7 lsd (cxt) : 17 the average percentage of damage reflected the character of the herbicide used. imazapyr (2.0 kg ai/ha) produced a slow appearance of visual damage. one month after spraying, alang-alang exhibited only 18.3% damage increasing to 69.3%, two months later. glyphosate (2.5 ae/ha) caused a faster appearance of visual damage i.e. 30.0% at one month and 88.3%, 2 months later. these two herbicides are both systemic, affecting the synthesis of amino acids (shaner et al. 1985). glufosinate 14 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno showed a very rapid action in terms of visual symptoms which were almost complete one month after spraying. however, the affected alang-alang recovered soon and regrowth was quite extensive 3 months after spraying. it was not different from manual control. glufosinate is a strong inhibitor of glutamine synthesis in plants, inducing a rapid built-up of ammonia which reaches toxic level (kochler and lotzsch 1985). no phytotoxicity was observed in all treatments. this was contrary to the previous work on imazapyr where at 1.5 kg ai/ha it affected the growth of soybean. this may be due to possible leaching under field condition which did not occur in pots since they were not perforated and soybean exploited soil in pots extensively (less soil in pots). it is also possible that the variety americana is more tolerant to imazapyr than orba, the variety used in the previous work. further studies in this line are needed. time of planting after treatment did not affect the height of soybean plant as indicated by the absence of phytotoxicity. one month after spraying was sufficient to avoid the phytotoxic effect of glyphosate, glufosinate or imazapyr in terms of vegetative growth. contrary to the time of planting after treatment, alangalang control affected the growth of soybean considerably. at two weeks old, soybean under manual cultivation grew shorter (10.1 cm) compared to those with herbicide treatment (table 2). at 8 weeks, the growth of so.ybean under glufosinate (3.0 kg ai/ha) slowed down, but it did not differ from those under manual cultivation, while those under glyphosate (2.5 kg ai/ha) grew tallest (53.4 cm). table 2. the average plant height (cm) at 2, 4 and 8 weeks after planting. time after planting (weeks) method of control 2 4 8 1 . imazapyr (2.0 kg ai/ha) 11.9 b 19.6 b 51. 3 bc 2 . glyphosate (2.5 kg ae/ha) 12.0 b 20.2 b 53.4 c 3 . glufosinate (3.0 kg ai/ha) 12.3 b 19.2 b 48.1 ab 4 . manual 10.1 a 17.2 a 44.7 a nb: numbers in a column followed by the same letter do not differ significantly at p≤0.05. 2. the development of leaves the soybean performance in terms of leaf number and area is presented in table 3. 15 biotropia no. 2, 1988/1989 table 3. the average leaf number/plant and leaf area recorded 8 weeks after planting. leaf method of control number leaf area index 1 . imazapyr (2.0 kg ai/ha) 10.9 b 1.9b 2 . glyphosate (2.5 kg ae/ha) 9.8 ab 1.7 ab 3 . glufosinate (3.0 ai/ha) 8.6 a 1.5 a 4 . manual cultivation 8.9 a 1.4 a nb: numbers in a column followed by the same letter do not differ significantly at p≤0.05 the vegetative growth in terms of plant height, leaf number and leaf area indicated an excellent growth exhibited by soybean when alang-alang was controlled by imazapyr (2.00 kg ai/ha). the growth of soybean under glufosinate was reasonable and was as good as those under manual cultivation. 3. yield of soybean the yield of soybean is presented in table 4. table 4. the yield of soybean (kg/ha) under various techniques. method of control kg/ha 1. imazapyr (2.0 kg ai/ha) 750 2. glyphosate (2.5 kg ae/ha) 750 3. glufosinate (3.0 kg ai/ha) 690 4. manual cultivation 680 the yield of soybean gave a very different picture from its vegetative growth. the yield of manual cultivation was as high as those under glyphosate treatment. soybean grown with zero tillage when alang-alang was controlled with imazapyr was lower than manual control of glyphosate. it was rather surprising that the vigorous growth of leaves did not produce a high yield as was expected. plant population and calculated leaf area index did not support the contention that this low yield was due to overcrowding. it may be worth to mention that this experiment was carried out during the very wet season, it probably affected the availability of par, thereby reducing the yield. 16 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno references coster, c. 1938. bovengrondse afstroming en erosie op java. tectona 31: 613-729. driessen, p.m. and soepraptohardjo. 1974. soil for agricultural expansion in indonesia. bull. no. 1. soil research institute. hittle, c.n. 1974. soybeans around the world. soybean production protection and utilization. intsoy 6: 6-17. hymowitz. 1970. on the domestication of soybeans. econ. bot. 24(4): 408-421. ismail, inu g., w.s. ardjasa and s. effendi. 1981. prospek penggunaan herbisida roundup dalam pengelolaan lahan alang-alang untuk tanaman pangan. pres. higi ke 6: 9-14. koecher, h. and k. loetzsch. 1985. uptake, translocation and mode of action of the herbicide glufosinate-ammonium in warm climate weed species. proc. xth apwss conf.: 193-198. mangunsong, m., d. siregar and h. harahap. 1975. pemberantasan alang-alang dalam skala besar pada tanah podsolik merah kuning. symp. pencegahan dan pemuliaan tanah kritis dalam rangka pengembangan wilayah. 20 hal. morse, j. 1950. history of soybean production. in soybeans and soybean products, vol. 1, k.s. markly, ed. interscience publishers, inc. ny. 3-59. sastrosuwarno, s. 1980. padang alang-alang sebagai prioritas pembukaan daerah transmigrasi. pros. p.p. padang alang-alang, biotrop, bogor, 7 hal. sembiring, e.n. and s. supardjo. 1981. studi penyiapan lahan alang-alang secara khemis dan mekanis untuk pemukiman transmigrasi. proc. vlth indonesian weed science society conf. l-88a. shaner, d.l., p.l. anderson, m.a. stidham, m. muhitch, m.l. reider, p.a. robson and t.r. peoples. 1985. the mode of action of ac 252, 925. proc. xth apwss: 185-192. sihombing, d.a. 1985. prospek dan kendala pengembangan kedelai di indonesian, dalam kedelai, puslitbangtan, bogor. suwardjo, h. and n. sinukaban. 1986. masalah erosi dan kesuburan tanah di lahan kering podsolik merah kuning di indonesia. pros. lokakarya usaha tani konservasi. 17 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf biotropia 1 (1) 1987: 58-66 iv. storage and germination tests on shorea javanica seeds m.i.j. umboh tropical forest biology program, biotrop, bogor, indonesia keywords: germination tests, seed treatment, shorea javanica abstract seeds of shorea javanica k & v (dipterocarpaceae), were subjected to different treatments of temperature (27 ± 2°c, 20 ± 2°c and < 10°c), coating (ash and paraffin) and relative humidity (20, 66 and 86%) and their germination capability as well as moisture content assessed after 3, 7, 14 and 30 days of storage. the germination percentage of the seeds stored 30 days in a cloth bag under different conditions of temperature showed highly significant differences (a = 0.01). no significant differences were found on the effect of coating, duration of storage as well as on the interaction between these two factors. the moisture content of the seeds between 13 and 14% gave a germination percentage above 50% after 30 days. introduction like in other dipterocarpaceae, the production of seeds in shorea javanica is irregular (ashton 1960, torquebiau 1984). knowledge of seed storage technology for preserving planting stocks from one fruiting season until the next is therefore a very important prerequisite for making genetic and breeding studies of this timber species. this work is a preliminary study on the effect of storage temperature, coating, relative humidity and moisture content on the germination capacity of shorea javanica. materials and methods seeds were collected in august and september 1985 from the forest plantations near krui, lampung (sumatra). immediately after collection from the trees, seeds with complete structure (i.e. with wings) were put in a cloth bag for transport to bogor. two experiments were conducted : experiment 1 : three days after harvest, 3600 seeds were divided into 3 groups: 2 groups coated with ash or paraffin wax and uncoated control. the seeds were then stored under three different temperature conditions: room temperature (27 ± 2°c), air conditioned room (20 ± 2°c) and inside refrigerator ( < 10°c) for 3, 7, 14 and 30 days. there were 50 seeds per treatment and each treatment was in replicate. 58 iv. storage and germination test on shorea javanica seeds — umboh experiment 2 : 1800 seeds were stored in eight dessicators (200 seeds per dessicator) four of which were kept at room temperature (27 ± 2°c) and the others at air conditioned room temperature (20 ± 2°c). three of the four dessicators of each series were filled with saturated solutions of potassium acetate (kc2h3o2), sodium nitra te (nanco2) and potassium chlorite (kcl) to keep relative humidities (rh) at around 20%, 66%, and 86%, respectively (suchslands 1980). one dessicator of each series was without chemical and had approximately 67% rh at room temperature or less than 60% rh in air conditioned room. in each dessicator, the seeds were stored for 3, 7, 14 and 30 days and fifty seeds were taken at the end of each period. the germination tests were done in two sand-filled seed beds which measured 5 x 1.2 m. these seed beds were covered with plastic sheets to keep moisture at ca. 90% rh. the emergence of the shoot on the surface of sand was indicated by a small stick. the data on germination percentage of the first experiment were subjected to analysis of variance for the three factors (i.e. temperature, coating and duration of storage) with completely randomized block, whereas the data on t he second experiment were presented as germination percentage for each experiment. determination of seed moisture content in relation wi t h t he germination capacity was also made. for this purpose, five seeds were added for each treatment and they were dissected and weighed before and after they were placed in an oven at 110°c for 20 hours. the formula of moisture content was on a wet weight basis : fresh weight of seed — dry weight of seed % moisture content = x 100 fresh weight of seed results experiment 1. the development of the seedling until the appearance of cotyledons and a small part of the apex needed one to two weeks after sowing. results are shown in table 1. table 1. percent germination of s. javanica seeds at different temperatures and with different duration of storage and coating (means of two replications). 59 biotropia vol. 1 no. 1, july-december 1987 effect of temperature the analysis of variance of the data showed that there were highly significant differences in the germination percentage of seeds according to the different temperature conditions (table 2). the comparison of the germination percentages of seeds stored at different temperatures was performed by tukey's identification and is shown in table 2a. there was no significant difference of germination percentage between seeds placed at 27 _+ 2°c (52.42%) and 20 ± 2°c (54.42%) but percent germination of table 2. analysis of variance on the effect of temperature, coating and duration of storage and the interaction of these factors on the percent germination of s. javanica seeds. source of variation degree of freedom sum square mean square f value blocks 1 41953.3872 41953.3872 temperature 2 35643.1078 17821.5341 39.2678 ** coating 2 172.4444 86.2222 0.1900 ns tc 4 48.2222 12.2556 0.0266 ns duration 3 876.6111 292.2037 0.6438 ns td 6 306.2222 51.0370 0.1125 ns cd 6 104.8889 17.4815 0.0385 ns tcd 12 463.7778 38.6481 0.0385 ns residual 35 15884.6178 453.8462 notes: ** = highly significant at level α = 0.01 ns = not significant at level α = 0.05 tc : interaction between temperature and coating td : interaction between temperature and duration cd : interaction between coating and duration tcd : interaction among temperature, coating and duration table 2a. tukey's identification of means of germination percentage (mean of all data of table 1 for each temperature). temperature 2 7 ± 2 ° c 20±2°c 10°c mean of germination (%) 52.42 54.42 6.25 ns ns: non-significant at level a = 0.05 * significant difference at level a = 0.05 60 iv. storage and germination test on shorea javanica seeds — umboh seeds placed at both temperatures compared with those at < 10°c (6.25%) was significantly different. effect of presence and absence of coating as shown in table 2, the effect of coating the seeds, and of the duration of storage as well as the interaction among factors of temperature, coating and duration of storage gave germination percentages which were not significantly different. the seeds coated with ash germinated better than those coated with paraffin which had a lower germination percentage than untreated seeds (table 2b). however, statistical test showed that the differences were not significant. table 2b. percent germination according to different coatings (means of all data of table 1, for each coating). control ash paraffin germination % means 38.25 39.25 35.58 effect of the duration of storage the means of germination percentages decreased for all storage duration of treatments 3 to 30 days (table 2c) but the differences were not statistically significant. table 2c. percent germination according to the duration of storage (means of all data of table 1, for each duration of storage). duration (days) 3 7 14 30 germination % means 42.78 38.00 35.11 33.89 experiment 2: effect of relative humidity the germination percentage of seeds stored at 27 ± 2°c with 20% rh (r20) and 66% rh (r66) went down to almost zero after 14 and 30 days of storage respectively (fig. 1). however, the seeds stored without chemicals i.e. rh aproximately 67% (rc) had a germination capacity slightly above 50% after 30 days of storage. 61 biotropia vol. 1 no. 1, july-december 1987 62 iv. storage and germination test on shorea javanica seeds umboh 63 biotropia vol. 1 no. 1, july-december 1987 this difference may be due to a negative effect on germination capability of sodium nitrite (nano2) used to maintain the rh at 66%. however, this effect still remains to be investigated. at 20 +. 2°c, the germination percentage of seeds stored at 66% rh (r66), 86% rh (r86) and 60% rh (without chemicals), were still above 80% after 30 days (fig. 2). at 20% rh (r20), the germination percentage was almost zero after 14 days. influence of temperature and relative humidity on the moisture content of the seeds : table 3 shows the values of moisture content of seeds during experiment 2. at 27 ± 2°c and 86% rh, the moisture content of the seeds was stable from 7 to 30 days, and it showed a slow decrease under the other rh conditions. at 20 ± 2°c, the moisture content of the seeds was stable up to 30 days under 60, 66 and 86% rh, while it was very low from day 7 on at 20% rh. table 3. moisture content (%) of seeds, after various durations of storage and under different temperatures and relative humidities. temperature relative humidity (%) 27 ±2°c 20 ±2°c 67 20 66 86 60 20 66 86 days 7 14.83 12.71 15.24 15.50 14.40 11.36 15.14 15.31 14 14.48 10.58 14.34 15.06 14.51 10.27 14.87 14.96 30 13.21 10.58 12.21 14.86 13.04 7.88 13.17 14.44 the value of moisture content of the seeds, when related to the germination percentages under different relative humidities, indicate that the best germination rate was achieved by those seeds with a moisture content between 13 and 14% after 30 days. results of the second experiment indicate that s. javanica seeds are preferably kept at 20 ±. 2°c (air conditioned room) under 60 to 66% rh or at 27 ± 2°c (room temperature) under 67% rh. discussion it is generally agreed that viability of seeds may be defined as the capability of t h e seeds to germinate under favorable conditions. the conditions which influence the germinating capacity of seeds are: temperature, moisture content, rela tive humidity, physiology of seed and some genetic factors. 64 iv. storage and germination test on shorea javanica seeds — umboh effect of temperature, coating and duration of storage there are interrelationships among temperature, moisture content and storage time on the life of seeds (justice & bass 1979). the germination capability of s. javanica seeds was influenced by temperature. for example, the percent germination of seeds stored at 27 ± 2°c or at 20 ± 2°c and those stored at < 10°c varied significantly (at level a = 0.01). the germination percentage of seeds at < 10°c was almost zero after 30 days of storage, or 33 days after collection from the trees. however, different coatings and storage up to 30 days had no effect on the percent germination. it is therefore considered better to store s. javanica seeds in a cloth bag at air conditioned room temperature without coating (table 1). such physiological characteristics allow one to consider shorea javanica seeds as recalcitrant seeds (chin & robert 1980). effect of temperature, relative humidity and moisture content the higher the relative humidity, the higher the moisture content of the seeds (nuhamura 1958 cited by justice & bass 1979). it is also known that, as temperature is increased when seeds are held at a constant relative humidity, the seed moisture content decreases. in the present experiment with s. javanica, temperature had a more important effect on seed germination than relative humidity. at 20°c, all seeds under 60, 66 and 86% rh still had very good germination capability (above 80%) after 30 days of storage. when the relative humidity was decreased to 20%, the moisture content decreased and it influenced negatively the germination capacity at both 27 or 20°c. this is similar with the observations of tompsett (1985) on the decrease of germinability of s. robusta seeds when their moisture content was decreased. due to the limitation of seed production at the beginning of the experiment in august and september 1985, it is recommended that further research on the effect of moisture content of the seeds on germination should be conducted. also, it is important to determine the composition of s. javanica seeds because there may be a relation between the lipid contents of a seed and its moisture content. this is based on the observation of barton (1941, cited by justice & bass 1979) i.e. seeds containing high percentage of carbohydrates, proteins, or both like rice, other grains and soybean, can have moisture contents of about 13-15% at 25°c and 75% rh whereas peanuts, which are rich in oil, would have a moisture content of approximately 9-11% under the same conditions. conclusion there were up to 30 days no significant differences in the percent germination of seeds stored at 27 ±. 2°c (52.42%) and at 20 ± 2°c (54.42%) but 65 biotropia vol. 1 no. 1, july-december 1987 the germination of seeds stored at both temperatures compared with those at 10 °c (6.25%) was significantly lower. presence or absence of paraffin or ash seed coating had no effect on the percent germination of see ds. the percent germination of seeds decreased for all treatments with an increase of the duration of storage from 3 to 30 days. the seeds stored without chemicals at a relative humidity (rh) of about 67% (rc) still had a germination capacity above 50% after 30 days of storage. at 20 ±_ 2°c (room air-conditioned), the percent germination of seeds stored at 66% rh (r66), 86% rh (r86) and 60% rh (without chemicals), were still above 80% after 30 days. in all experiments, at 20% rh, the percent germination was reduced to zero after 30 days. at room temperature (27 ±. 2°q with 86% rh, the moisture content of seeds was stable from 7 to 30 days (±_ 15%), but it showed a slow decrease under other rh conditions. at room air-conditioned (20 +. 2°c), the moisture content of seeds under 60, 66 and 86% rh were about 14% up to 30 days but it was very low at 20% rh. acknowledgments i wish to thank the forest administrator of lampung utara (krui) especially mr. tajudin nur and his staff for their help in providing materials for this study. thanks are also due to mr. iwan setiawan for his technical assistance, mrs. lilian gadrinab for her kind help with english correction and dr. torquebiau for his comments. references ashton, p.s. 1980. the biological and ecological basis for the utilization of dipterocarps. bio-indonesia, 7: 43-54. chin, h.f. & e.h. roberts. 1980. recalcitrant crop seeds. tropical press son, kuala lumpur, malaysia. justice, o.c. & l.n. bass. 1979. principles and practices of seed storage. william cloves & sons limited, london, 275 pp. mahdi, a. 1982. the viability and storage characteristics of shorea stenoptera burck and citrus sinensis o. seeds. thesis presented for msc degree in birmingham university. suchsland, o. 1980. determination of sorption isotherms in minidessicator. wood science, vol. 12: n°4. tompsett, p.b. 1985. the influence of moisture content and storage temperature on the viability of shorea almon, s. robusta, and s. roxburghii seeds. can. j. for. res. vol. 15. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry system, 2: 103-127. 66 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf 65.pdf 66.pdf fungal infection and aflatoxin contamination in stored nutmeg ( ) kernelsmyristica fragrans at various stages of delivery chain in north sulawesi province okky setyawati dharmaputra , santi ambarwati , ina retnowati1,2* 1 1 and nijma nurfadila1 1seameo biotrop, jalan raya tajur km 6, bogor 16134, indonesia 2 16680, adepartment of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor indonesi received 22 january 2015/accepted 21 may 2015 abstract fragrant nutmeg ( ) is an important commodity that has been used in food and pharmaceutical myristica fragrans industries, hence its quality should be monitored. the objectives of the research were obtain information on the (a) to postharvest handling of nutmeg investigate the occurrence of fungi (includ ) and aflatoxin kernels; (b) to ing a. flavus contamination in stored nutmeg oisture content and percentage of damaged kernels and (c) mto measure nutmeg kernels. methods used in this study included survey, interviews and sample collection in the delivery chain. this study was ions conducted in april and may 2013, in three reg (north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud) and two cities (bitung and manado). the total number of nutmeg samples collected from kernels different point of the delivery chain was 76. it consisted of samples collected from farmers (25 samples), collectors s (22) and exporters (29). the results showed that , the moisture content of nutmeg kernels collected from north sulawesi province was not higher than the maximum limit of moisture content determined by indonesian national standard or sni (10%). nutmeg kernels collected from farmers and collectors had a high percentage of damaged kernels. and were the dominant fungi infecting nutmeg kernels collected from aspergillus niger endomyces fibuliger farmers and collectors, while was the dominant fungus found infecting nutmeg kernels stored by eurotium repens the exporters. aflatoxin b and total aflatoxin contents nutmeg kernels samples collected from farmer and exporter1 s s of were based on statistical analysis using non-parametric analysis, the effect of delivery chain did not give relatively high. any significant difference on moisture content, percentage of damaged kernels, total fungal pulation and s nutmeg po total aflatoxin content. our study suggested that methods the postharvest handling of nutmeg kernels conducted by farmers, collectors and exporters in north sulawesi province (north minahasa, sitaro and sangihe talaud regions), bitung and manado cities should be improved to minimize aflatoxin b contamination1 and total aflatoxin . keywords: aflatoxin, delivery chain fungi, , north sulawesi rovince, p , nutmeg kernelsmyristica fragrans introduction nutmeg is native to the moluccas islands of indonesia, but nutmeg is also grown in nowadays penang island in malaysia, in the caribbean (particularly grenada), in the southern state of kerala in india and in zanzibar islands. fragrant nutmeg ( ) is an important myristica fragrans c o m m o d i t y w i d e l y u s e d i n f o o d a n d pharmaceutical industries, therefore , it is important monitor its quality (direktorat to jenderal perkebunan 2012). based on statistical data from the directorate general of estate crops in indonesia, area s planted with nutmeg were 75,062 ha . the in 2008 distribution areas covered 19 provinces. the largest plant area was in north ation of nutmeg moluccas (33%), followed by n aanggroe ceh d (23%), north sulawesi arussalam or nad (18%), moluccas (12%), west java (5%) and the rest (9%) in other provinces. indonesia contributes 75% (8,943 tonnes) of nutmeg production in the world (revitalisasi perkebunan pala siau, sulawesi utara 2010). north sulawesi province is one of the most important nutmeg producing in . nutmeg from provinces indonesia* corresponding author : okky@biotrop.org biotropia vol. 22 no. 2, 2015: 129 139 129 doi: 10.11598/btb.2015.22.458 mailto:okky@biotrop.org biotropia vol. 22 no. 2, 2015 130 this is exported to t netherlands, italy, province he japan and vietnam. during and storage s, postharvest period nutmeg kernels can be infested by insects and c o l o n i z e d by m i c r o o r g a n i s m s. a m o ng microorganisms, fungi important are the most cause of stored foodstuff deterioration. fungal infection in foodstuff can cause discolouration and mycotoxin contamination, as well as decrease in physical quality and nutritional content (sauer et al. 1992). aflatoxins are toxins produced by certain fungi, such as aspergillus flavus a. and parasiticus are considered dangerous . aflatoxins due to associat with various diseases their ion in human and animals, such as aflatoxicoses and liver cancer. there are four naturally occurring aflatoxins in many stored commodities, i.e. aflatoxins b , b , g and g . the most common 1 2 1 2 and toxic aflatoxin is aflatoxin b (1 (afb ) basappa 1 2009). the european union has determined maximum tolerable limits (mtl) of b and af 1 total aflatoxin in nutmeg kernels to be 5 and 10 ppb, respectively (fao 2004). since nutmeg is a m o n g e x p o r t i m p o r t a n t a g r i c u l t u r a l commodit for indonesia, it is important to ies conform to nutmeg kernels importation rules the from countries. the first step in the importing postharvest handling is to monitor and improve the current procedure of nutmeg storage. kernels in relation to storage monitoring, several objectives in this study were set as follow : to (a) s obtain information on postharvest nutmeg handling methods to (b) ; investigate fungal population, fungal diversity (including aspergillus flavus of stored ) and aflatoxin contamination nutmeg kernels collected from different points of the delivery chain in north sulawesi province and (c) to determine and percentage moisture content of damaged nutmeg kernels in the postharvest chain. materials and methods time and ocation of urveyl s s of nutmeg kernels weresurvey and sampling conducted in april and may different 2013 at point of delivery chain s , i.e. at farmer, collector and exporter levels in five locations in north sulawesi province. the five locations chosen for the survey based on recommendation from the agricultural service of north sulawesi province were north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud regions; bitung and manado cities (table 1). interview using uestionnairesq interviews during the survey were conducted to collect information on postharvest nutmeg handling at different point the delivery chain s in (farmer collector ). the , and exporter levels que sti onnai re s on c ont aine d que sti ons postharvest handling carried out by procedures farmers, collectors , and exporters as well as problems encountered by them umber of . the n respondents from each level of delivery point was different depending on the conditions in the field during the survey. sampling ethodm s the samples were collected from the places where the respondents obtained the nutmeg kernels. the number of samples nutmeg kernels at each level determined of delivery point was proportionally, based on the number of farmers, collectors and . exporters as much as shelled kernels 500 g of nutmeg and , in-shell kernels were1 000 g of nutmeg collected randomly from each respondent ach . e sample packed in a was clean plastic (polyethylene) and then was double packed in bag hermetic bags to minimize any changes to the nutmeg kernels samples due to long distance transportation between the location of sampling and laboratory in bogor were, where the samples analyzed. method for btaining orking ampleso w s the nutmeg shelled manually to in-shell s were obtain the skernel . each sample was mixed homogeneously, then was into four parts. divided one part was used to determine the percentage of damaged nutmeg kernels and as a reserve sample, while the other three parts were ground using a mill powder tech model rt 04 the ground . nutmeg samples were then divided into eight parts to deter mine moisture content, fungal population, dominant fungal species infecting kernels aflatoxin ontent.and c fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans deter m inatio n of moi stur e content, percentage of damaged kernels, fungal p a n copulation and flatoxi ontent moisture content of (based on wet nutmeg basis) w determinedas using the distillation method (sni 1993). weretwo replicates used for each sample. the percentage of damaged kernels was calculated from the weight of damaged the kernels ed by the and divid the weight of working sample damaged kernelfrom which the s were taken damaged kernels included cracked, . broken, shriveled and mouldy kernels. werefungi isolated using serial dilution the method followed by pour plate method with the dichloran 18% glycerol agar (dg18) (hocking & & pitt 1980 pitt hocking 2009). each fungal ; species identified according to pitt and were hocking (2009) as the main reference. aflatoxin b (afb ) and total aflatoxin1 1 contents were determined using hplc with post-column derivatization (vicam 2007). data collection and analysis data collection on nutmeg postharvest handling methods at farmer, collector and exporter levels were carried out by conducting inter views with far mers, collectors and exporters. data of moisture content (mc), percentage of damaged kernels, fungal total population and total aflatoxin content were collected from farmers (25 samples), collectors (22 samples) and exporters (29 samples) at north sulawesi province. total samples at farmer and collector levels were collected from north minahasa, sitaro and sangihe talaud regions. meanwhile, total samples at exporter level were collected from bitung and manado cities. data of mc, percentage of damaged nutmeg kernels, total fungal population and total aflatoxin content were analyzed with nonparametric one way kruskal-wallis test. 131 tabl 1 , samplese level of delivery chain sub-district origin of the sample and the number of nutmeg kernels at various stages of delivery chain in north sulawesi province region/ city level of delivery chain sub-district origin of the sample number of samples north minahasa farmer kauditan 16 collector kauditan 17 exporter siau tagulandang biaro (sitaro) farmer east siau 1 west siau 1 collector east siau 3 exporter sangihe talaud farmer kendahe 2 central tabukan 1 tamako 1 east tahuna 1 west tahuna 1 mangawito 1 collector kendahe 2 exporter bitung farmer collector exporter halmahera island 1 lembeh island 1 kauditan 1 some locations 1 bitung 2 manado farmer collector exporter siau 12 tahuna 1 manado 10 total number of samples 76 results and discussion results of with ar mer s, inter views f c eollectors and xporters the total number of respondents was 26 (14 farmers, 8 collectors and 4 exporters). interviews related to nutmeg postharvest handling at farmer and collector levels were conducted only in north minahasa, sitaro and sangihe talaud reg , ions while interviews at exporter level were conducted in bitung and manado cities. method of collecting nutmeg at farmer level, farmers picked generally nutmeg fruits from the trees . they also (±50%) collected nutmeg fruits which had fallen on the ground . armers collected nutmeg (±50%) f also fruits that had fallen on the ground, because this method was easier to be conducted compared to harvesting nutmeg fruits directly from the trees. price of nutmegs picked directly from the trees was similar to price of nutmegs that had fallen on the ground. ,therefore farmers did not pay attention to the method of proper nutmeg postharvest handling. at collector level, a total of 57% of collectors bought nutmegs from farmers in three conditions, i.e. wet, semi-dry and dry conditions; for both shelled and in-shell .nutmeg s if a collector bough nutme st gin-shell , then a wooden stick was used for conducting the shelling process. as much as 12.5 and 87.5% of farmers sold nutmeg s in semi-dry and dry conditions respectively. in , general farmers sold nutmeg in dry condition, , s because price higher than sold in the was those semi-dry or wet conditions. at exporter level, up to 75% of exporters bought shelled nutmegs from collectors, while 25% of exporters bought a mixture of shelled and in-shell . nutmegs most of exporters bought shelled s the weight of shelled nutmeg , because nutmegs was that of lighter than in-shell nutmegs, which could the costdecrease transportation . method of drying and storing n s utmeg at the farmer level, 87.5% of farmers dried shelled and in-shell nutmegs using sun-drying method by spreading the nutmegs on tarpaulin placed on the ground. the dried nutmegs were subsequently stored in plastic bags. after 1 kg of nutmegs were collected and shelled, they were sold to collectors. in general, farmers did not do any attempts to sort dried nutmegs based on physical quality, because they wanted to sell dry nutmeg as soon as possibles . at the collector level, drying of nutmegs was conducted using two methods, i.e. sun-drying and smoke drying methods. in general, collectors used smoke drying method, especially during rainy season. the smoke drying method used coconut shells as fuel. up to 57% of collectors dried both shelled and in-shell nutmegs using sundrying method on tarpaulin placed on the ground. a total of 43% of collectors smoke-dried nutmegs on wire and wooden racks, followed by sun-drying method. for better result, collectors dried the nutmeg kernels using wire and wooden racks at elevated position ±1 m from the floor, to avoid contamination by animal faeces or dirt. in the night or when it rained, the semi-dried and wet nutmeg kernels were packed in the same plastic bags. as much as 71% of collectors sold nutmeg kernels to exporters, while 29% of nutmeg kernels were sold to large traders, without being sorted. before being sold, the nutmeg kernels were stored for about a month. during storage, 71% of collectors did not monitor nutmeg kernels for aflatoxin contamination, due to lack of aflatoxin detection equipment, although they knew the impact of aflatoxin on human and animal health. at the exporter level, 75% of exporters grouped the nutmeg kernels manually based on qualities. the exporters usually stored the sorted nu tm e g ker n e ls i n gu nn y a nd p la sti c (polypropylene) bags. those were exporters aware of potential aflatoxin contamination and the impact on human and animal health. they monitored the nutmeg kernels for aflatoxin contamination using a long wave ultraviolet lamp, in which aflatoxin contaminated nutmeg kernels would produce blue green yellow fluorescence and be removed prior to export. source and umber of amplesn s the total number of samples nutmeg kernels collected from different points in the delivery chain was 76 consisted of nutmeg kernels . this samples from farmers (25 samples), collectors (22 samples) and exporters (29 samples) (table 1). 132 biotropia vol. 22 no. 2, 2015 moisture content the moisture content (mc) of foodstuff at the beginning of storage is one of the important factors influencing the quality of foodstuff during storage. a high initial mc value at the beginning of storage provides an opportunity for growth and development of spoilage and mycotoxigenic fungi. according to indonesian national standard or sni, maximum moisture content value for nutmeg kernels and their processed products should be 10% (sni 01-0006-1993). the range and mean mc values of nutmeg kernels collected from farmers, collectors and exporters in north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud regions; bitung and manado cities are presented in table 2. the highest and the lowest mc values of nutmeg kernels collected from farmers in north minahasa region were 18.00 and 8.00%, respectively. for collectors, those mc values were 15.50 and 7.50%, respectively. the mean mc value of nutmeg kernels collected from farmers was similar to that collected from collectors, i.e. 10.88 and 11.07%, respectively. the mean mc value of nutmeg kernels collected from farmers and collectors were higher than the maximum limit determined by . the high mc value of sni nutmeg kernels collected from farmers was probably due to the short period of drying after the harvesting process, low intensity of sun light during the sun-drying process, limited storage space and short duration of storage. the highest and lowest mc values of nutmeg kernels from farmers in sitaro region were 10.50 and 10.00%, respectively, while the highest and lowest mc values of nutmeg kernels from collectors were 11.50 and 6.50%, respectively. the mc values of nutmeg kernels from farmers in sitaro region exceeded the maximum limit determined by . sni in sangihe talaud region the range and mean of mc values obtained from the farmers' and collectors' samples were lower than the maximum limit determined by . the highest and sni lowest mc values of nutmeg kernels from farmers were 9.00 and 6.98%, respectively; while the highest and lowest mc values of nutmeg kernels from collectors were 8.00 and 7.99%, respectively. the range of mc values of nutmeg kernels collected from exporters in manado (7.00 11.50%) was wider than that collected from bitung (7.50 10.00). the mean mc value of nutmeg kernels from exporters in bitung (8.75%) and manado (9.48%) cities were lower than the maximum limit determined by . sni 133 tab 2 , undamaged and damaged kernelsle range and mean of moisture content of nutmeg collected from farmers, collectors and exporters in north sulawesi province region / city level of delivery chain range (mean) of moisture content (% wet basis) range (mean) of undamaged kernels (%) range (mean) of damaged kernels (%) north minahasa farmer 8.00 – 18.00 (10.88) 0 – 88 (22.78) 12.00 –100 (77.22) collector 7.50 – 15.50 (11.07) 0 – 81.42 (21.20) 18.58 – 100 (76.12) exporter siau tagulandang biaro (sitaro) farmer 10.00 – 10.50 (10.25) 52.91 – 61.88 (57.40) 38.13 – 47.09 (42.61) collector 6.50 – 11.50 (8.83) 0 – 47.85 (15.95) 52.15 – 100 (84.05) exporter sangihe talaud farmer 6.98 – 9.00 (7.85) 0 – 61.54 (36.71) 38.46 – 100 (63.29) collector 7.99 – 8.00 (8.00) 0 – 58.78 (29.39) 41.22 – 100 (70.61) exporter bitung farmer collector exporter 7.50 – 10.00 (8.75) 0 – 75.31 (35.25) 24.69 – 100 (64.75) manado farmer collector exporter 7.00 – 11.50 (9.48) 0 – 71.33 (26.42) 28.67 – 100 (73.58) fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans the mean mc value of nutmeg kernels collected from farmers and collectors in north minahasa region were higher than that collected from other delivery chains in north sulawesi province. overall, the highest mean mc value was recorded from nutmeg kernels collected from collectors in north minahasa region, followed by those collected from farmers in sitaro region, from exporters in manado city, from collectors in sitaro region, from exporters in bitung city and from farmers and collectors in sangihe talaud region. nutmeg kernels are very hygroscopic, therefore, storage at semi-dried condition caused the kernels to absorb moisture which led to initiation of mould growth, spoilage and mycotoxin contamination. based on non-parametric statistical analysis, d significantly influenceelivery chain did not moisture content (mc) value kernelss of nutmeg ( ).table 3 percentage of u damaged ndamaged and kernels damage to nutmeg kernels may contribute to infection by mycotoxigenic fungi such as a. flavus and increase the chances for af latoxin contamination during the postharvest storage. the r nge of damaged nutmeg kernels percentage a in north minahasa region from farmers and collectors were 12.00 – 100 and 18.6 – 100%, respectively. in sitaro region, the range of damaged nutmeg kernels from farmers and collectors were 38.1 – 47.1% and 52.2 – 100%, respectively. means of damaged nutmeg kernels percentage from farmers and collectors were 42.6 and 84.1%, respectively. in sangihe talaud region, the mean of damaged nutmeg kernels percentage collected from farmers (63.29%) was lower than that collected from collectors (70.6%). in bitung city the percentage of damaged nutmeg kerne s collected from exporters was l 64.8%. in manado city the percentage was 73.6% (table ). means of damaged nutmeg kernels 2 percentage collected from farmers and collectors in north sulawesi p were higher than rovince those collected from exporters. this might be due to improper sorting regimes used by farmers and collectors. in addition, many farmers and collectors still manually shelled the nutmegs using wooden stick, which can increase the percentage of damaged nutmeg kernels. based on statistical analysis using nonparametric analysis, elivery chain did not d significantly influence the percentage of damaged nutmeg table 3kernels ( ). fungal population diversity and dominance the highest diversity of fungal species was found in nutmegs collected from farmers and collectors in north minahasa region, 13 and i.e. 12 species, while the lowest number respectively, of fungal species were observed in samples obtained from farmers in sitaro region (7 species) and those obtained from collectors in sangihe talaud region (2 species; tables 4, 5, 6). aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in north minahasa region. fungal population isolated from samples from farmers was more contaminated than those from collectors (table 5). the dominant fungi found in nutmeg samples from farmers was (81%) penicillium citrinum followed by (69%) and a. niger eurotium repens (63%). three dominant fungi found in nutmegs collected from collectors were endomyces fibuliger (76%), (76%) and (76%) which a. niger p. citrinum may have been caused by high moisture content. 134 table 3 the effect of nutmeg delivery chain on moisture content (mc), percentage of damaged nutmeg kernels, fungal total population and total aflatoxin level of delivery chain mc (%) percentage of damaged kernels (%) fungal total population (cfu/g wet basis) total aflatoxin content (ppb) farmer 9.98 ± 2.62 a 70.55±29.66 a 3.9x105±1.7x106 a 141.10±392.11 a collector 10.49 ± 2.52 a 76.70±32.39 a 1.3x106±3.2x106 a 2.15±4.54 a exporter 9.33 ± 1.13 a 71.75±30.27 a 9.9x103±1.1x104 a 50.63±213.43 a note: means in the same group followed by the same letter in a column are not significantly different at 5% level biotropia vol. 22 no. 2, 2015 no was isolated from nutmeg samples a. flavus obtained from collector in the sitaro region. at farmer level, a. niger, a. penicillioides, eurotium repens p. citrinum and were isolated from all samples, dominated by only a a. penicillioides. small number of nutmeg samples were colonized by in the sangihe talaud region. no a. flavus nutmeg samples from collectors appeared to contain . all samples of nutmegs a. flavus obtained from farmers and collectors in all sampling sites contained a. niger. the total fungal population and diversity of samples from exporters were lower in bitung city than in manado city (table 7). in bitung city, a. flavus was only isolated from 2% of samples. the dominant fungi were xerophilic spoilage fungi such as (all samples) followed by e. repens e. chevalieri (83%). in manado city, much higher number of samples contained (39%). a. flavus again, the dominant fungal population isolated were (87%), followed by (70%) e. repens a. niger and (65%).a. penicillioides aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in north minahasa region. fungal population isolated from samples from farmers was more contaminated than those from collectors (table 5). the dominant fungi found in nutmeg samples from farmers was (81%) penicillium citrinum followed by (69%) and a. niger eurotium repens (63%). three dominant fungi found in nutmegs collected from collectors were endomyces fibuliger (76%), (76%) and (76%) which a. niger p. citrinum may have been caused by high moisture content. no was isolated from nutmeg samples a. flavus obtained from collector in the sitaro region. at farmer level, a. niger, a. penicillioides, eurotium repens p. citrinum and were isolated from all 135 table 4 fungal populations and diversity in nutmeg samples obtained from farmers collectors in north minahasa and region no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus flavus 9 (56) 9 (53) 0.5 x 10 – 8.8 x 103 (1.5 x 103) 0.7 x 10 – 5.2 x 103 (7.8 x 102) 2. a. niger 11 (69) 13 (76) 0.2 x 10 – 2.2 x 104 (4.3 x 103) 0.1 x 102 – 8.8 x 103 (2 x 103) 3. a. ochraceus 1 (6) 0.7 x 10 (0.7 x 10) 4. a. penicillioides 2 (13) 3 (18) 2.2 x 102 – 1.3 x 103 (7.8 x 102) 4.5 x 10 – 2.7 x 102 (1.7 x 102) 5. a. tamarii 8 (50) 4 (24) 0.8 x 10 – 1.2 x 103 (3.3 x 102) 1.5 x 102 – 7.3 x 102 (3.7 x 102) 6. a. sydowii 2 (13) 1 (6) 1.2 x 10 – 1.8 x 103 (9.2 x 102) 8.2 x 102 (8.2 x 102) 7. a. wentii 1 (6) 1.2 x 102 (1.2 x 102) 8. endomyces fibuliger 7 (44) 13 (76) 5.2 x 10 – 8.4 x 106 (1.3 x 106) 1.5 x 10 – 1.1 x 107 (2.2 x 106) 9. eurotium chevalieri 5 (31) 3 (18) 1.5 x 10 – 0.4 x 10 4 (1.2 x 103) 3.3 x 10 – 7.3 x 10 3 (2.6 x 103) 10. e. repens 10 (63) 9 (53) 0.5 x 10 – 6.8 x 103 (1.7 x 10 3) 1.3 x 10 – 3.2 x 10 3 (9.0 x 102) 11. e. rubrum 7 (41) 0.2 x 10 – 3.5 x 10 3 (9.4 x 10 2) 12. penicillium citrinum 13 (81) 13 (76) 0.3 x 10 – 0.6 x 10 5 (6.3 x 103) 1.3 x 10 – 0.4 x 10 4 (7.8 x 102) 13. rhizopus sp. 2 (13) 2.5 x 10 2 – 7.2 x 10 2 (4.8 x 10 2) 14. syncephalastrum racemosum 1 (6) 0.2 x 10 3 (0.2 x 10 3) 15. trichoderma sp. 2 (13) 0.1 x 102 – 1 x 103 (5.1 x 102) notes: number of samples collected from farmers: 16 number of samples collected from collectors: 17 fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans samples, dominated by only a a. penicillioides. small number of nutmeg samples were colonized by in the sangihe talaud region. no a. flavus nutmeg samples from collectors appeared to contain . all samples of nutmegs a. flavus obtained from farmers and collectors in all sampling sites contained a. niger. the total fungal population and diversity of samples from exporters were lower in bitung city than in manado city (table 7). in bitung city, a. flavus was only isolated from 2% of samples. the dominant fungi were xerophilic spoilage fungi such as (all samples) followed by e. repens e. chevalieri (83%). in manado city, much higher number of samples contained (39%). a. flavus again, the dominant fungal population isolated were (87%), followed by (70%) e. repens a. niger and (65%).a. penicillioides nutmegs imported from india, sri lanka, indonesia and brazil were infected by , a. niger a. flavus rhizopus stolonifer and . the dominant fungi in these samples were (mandel 2005). a. flavus the water availability of semi-dried and damaged nutmeg kernels provided environmental conditions which are conducive to xerophilic and xerotolerant fungi, including mycotoxigenic species. the boundary conditions for growth and my c o t ox i n p r o d u c t i o n su g g e s t e d t h a t aflatoxigenic and ochratoxigenic fungi may be able to th ve under storage conditions (lacey & ri magan 1991; sanchis & magan 2004; magan & aldred 2007). 136 tabl and siau tagulandang e 5 fungal population and diversity in nutmeg samples obtained from farmers collectors in biaro (sitaro) region no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus niger 2 (100) 2 (67) 0.2 x 10 – 2.5 x 10 (1.3 x 10) 5.5 x 102 – 4.2 x 104 (2.1 x 104) 2. a. penicillioides 2 (100) 1 (33) 1.8 x 10 – 2.0 x 103 (0.1 x 104) 3.7 x 102 (3.7 x 102) 3. endomyces fibuliger 1 (50) 0.5 x 10 (0.5 x 10) 4. eurotium chevalieri 1 (50) 1 (33) 6.7 x 10 (6.7 x 10) 2.8 x 10 (2.8 x 10) 5. e. repens 2 (100) 1 (33) 0.2 x 10 – 3.3 x 102 (1.7 x 102) 1.8 x 10 (1.8 x 10) 6. e. rubrum 1 (50) 1 (33) 1.7 x 10 (1.7 x 10) 0.3 x 10 (0.3 x 10) 7. penicillium citrinum 2 (100) 0.2 x 10 – 0.8 x 10 (0.5 x 10) notes: number of samples collected from farmers: 2 number of samples collected from collectors: 3 tab and sangihe talaud ionle 6 fungal population and diversity in nutmeg samples obtained from farmers collectors in reg no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus flavus 1 (14) 0.2 x 10 (0.2 x 10) 2. a. niger 7 (100) 2 (100) 0.7 x 10 – 2.4 x 105 (5.1 x 104) 4.8 x 10 – 0.1 x 105 (5.4 x 103) 3. a. penicillioides 3 (43) 0.1 x 102 – 3.7 x 102 (1.3 x 102) 4. endomyces fibuliger 1 (14) 1.5 x 105 (1.5 x 105) 5. eurotium repens 4 (57) 0.5 x 10 – 8.3 x 102 (2.2 x 102) 6. e. rubrum 3 (43) 0.3 x 10 – 0.5 x 103 (1.7 x 102) 7. fusarium solani 1 (14) 5.2 x 104 (5.2 x 104) 8. penicillium citrinum 3 (43) 1 (50) 0.3 x 102 – 1.8 x 104 (6.8 x 103) 4.3 x 102 (4.3 x 102) notes: number of samples collected from farmers: 7 number of samples collected from collectors: 2 biotropia vol. 22 no. 2, 2015 based on statistical analysis using nonparametric analysis, elivery chain did not d significantly influence the total fungal population ( ).table 3 aflatoxin t a contentb and otal flatoxin s1 the range of afb and total aflatoxin content 1 in nutmeg samples from farmers in north sulawesi province were 0.40 – 1,632.19 ppb and 0.58 – 1,831.48 ppb, respectively. survey conducted at farmer level provided information a that postharvest handling was not conducted properly, i.e. mixed nutmeg picked the farmers s from the tree th fell on the ground s with ose ; consequently afb and total t the aflatoxin conten1 in these samples (table 8)were high . according to horn (2003) soil serves as a reservoir for and that a. flavus a. parasiticus produce aflatoxins in agricultural commodities. aflatoxigenic fungi reside in soil as conidia, sclerotia and hyphae, while act as primary inocula for directly infect peanuts (and possibly nutmeg ing fruit whic fell on the ground). rh ange of afb 1 and total aflatoxin in nutmeg samples content obtained from collectors in north sulawesi province were 0.11 – 14.59 ppb and 0.11 – 16.65 ppb, respectively. sun-drying method was faster and more effective than smoke-drying method. however, if the weather is extreme, sun-drying method is not recommended, because it could reduce the quality of atsiri oil in nutmeg kernels. therefore, many collectors used smoke-drying method, because the temperature can be controlled. smoke-drying the weakness using method the of long drying duration, includes need which may allow colonization of spoilage and mycotoxigenic fungi. based on statistical analysis using nonparametric analysis elivery chain did not , d 137 table 7 fungal population and diversity in nutmeg samples collected from exporters in citbitung and manado ies no. fungi bitung city manado city number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) number (%) samples infected by fungi range (mean) of fungal population in nutmeg 1. aspergillus flavus 2 (33) 0.5 x 10 – 7.7 x 102 (3.9 x 102) 9 (39) 0.1 x 102 – 5.3 x 102 (1.3 x 102) 2. a. niger 3 (50) 0.3 x 10 – 1.7 x 102 (7.1 x 10) 16 (70) 0.7 x 10 – 0.4 x 105 (3.1 x 103) 3. a. penicillioides 2 (33) 0.5 x 102 – 3.5 x 103 (1.8 x 103) 15 (65) 1.3 x 102 – 2.9 x 104 (6.6 x 103) 4. a. sydowii 1 (17) 0.5 x 10 (0.5 x 10) 5. a. tamarii 1 (17) 0.2 x 103 (0.2 x 103) 1 (4) 2.2 x 102 (2.2 x 102) 6. a. versicolor 2 (9) 4.5 x 10 2 – 1.2 x 10 3 (8.1 x 102) 7. endomyces fibuliger 2 (33) 1.7 x 10 2 – 6.7 x 10 2 (4.2 x 102) 4 (17) 2.8 x 10 2 – 5.3 x 10 3 (1.7 x 103) 8. eurotium chevalieri 5 (83) 0.2 x 10 – 2.5 x 10 3 (5.4 x 10 2) 5 (22) 0.5 x 10 2 – 3.7 x 10 3 (1.1 x 103) 9. eurotium repens 6 (100) 0.1 x 10 2 – 8.8 x 10 2 (3.6 x 10 2) 20 (87) 2.5 x 10 2 – 1.8 x 10 4 (0.5 x 104) 10. e. rubrum 1 (17) 6.8 x 10 2 (6.8 x 10 2) 15 (65) 2.8 x 10 – 0.2 x 10 4 (5.2 x 10 2) 11. paecilomyces variotii 1 (4) 4.5 x 102 (4.5 x 102) 12. penicillium citrinum 4 (67) 0.5 x 10 – 9.3 x 102 (2.7 x 102) 11 (48) 1.8 x 10 – 1.3 x 103 (2.4 x 102) note : in bitung city 6s number of samples collected from exporters : in manado city 23 number of samples collected from exporters : fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans (cfu/g wet basis) significantly influence total aflatoxin content of nutmeg ( ).table 3 several exporters in north sulawesi province possess facilities for drying, shelling, sorting and storage facilities. thus, aflatoxin contamination could be minimized. in this research, we also collected a sample of sorted nutmeg kernels visually assessed using the long wave ultraviolet lamp. this potentially contaminated sample from an exporter contained a total aflatoxin content of 0.18 – 1,112.58 ppb. tabata (1993) reported et al. that aflatoxin was found in 3,054 foodstuff and their product samples, especially nutmeg samples. the highest aflatoxin contamination was found in nutmeg (80%), while afb was also found in 1 pistachio nuts (1,382 ppb). takahashi (1993) reported that in 1986 until 1991, as much as 29 (43%) of 67 nutmeg samples collected from japan, were contaminated by aflatoxin. according to okano (2012) the distribution of et al. aflatoxigenic fungi in 25 imported indonesian nutmeg samples were contaminated with aflatoxins b or b and g. the incidence of aflatoxigenic fungi in the samples contaminated with high levels of aflatoxin was significantly higher than that in the samples with low levels of the toxins (r = 0.752). the toxin production of isolates from the samples in cultures of yeast extract sucrose broth was examined by means of tlc and hplc analyses. the ability of isolates to produce aflatoxins did not correlate with the contamination levels of aflatoxin in the samples. overall, the postharvest handling procedures need to be standardized to minimize aflatoxin contamination. conclusions generally, moisture content (mc) of nutmeg samples collected from north sulawesi province was below the maximum recommended limit of indonesian national standard or sni. nutmeg samples collected from farmers and collectors generally had a higher percentage of damaged kernels. and were the a niger e. fibuligerspergillus 138 tabl b e 8 aflatoxin and total aflatoxin contents in nutmeg collected from farmers, collectors and exporters in north 1 sulawesi province region/city level of delivery chain number of samples number (%) samples contaminated by aflatoxin range (mean) of afb1 content in contaminated samples (ppb) range (mean) of total aflatoxin content in contaminated samples (ppb) north minahasa farmer 16 6 (37.50) 0.40 – 762.24 (128.52) 0.58 – 910.48 (153.28) collector 17 5 (29.41) 0.19 – 14.59 (4.70) 0.19 – 16.65 (5.67) exporter siau tagulandang biaro (sitaro) farmer 2 2 (100) 1.03 – 1.44 (1.23) 1.44 – 1.55 (1.49) collector 3 3 (100) 0.11 – 0.69 (0.40) 0.11 – 1.34 (0.58) exporter sangihe talaud farmer 7 7 (100) 1.65 – 1 632.19 (335.92) 1.65 – 1831.48 (371.99) collector 2 2 (100) 3.28 – 13.94 (8.61) 3.28 – 13.94 (8.61) exporter bitung farmer collector exporter 6 3 (50) 1.40 – 799.25 (267.74) 1.40 – 1112.58 (372.18) manado farmer collector exporter 23 15 (65.22) 0.10 – 266.72 (18.37) 0.18 – 334.49 (20.69) biotropia vol. 22 no. 2, 2015 dominant fungi in nutmeg kernels from farmers and collectors, while was the dominant e. repens species in samples obtained from nutmeg exporters in north sulawesi province. aflatoxin b and total aflatoxin contents in nutmeg samples 1 collected from farmers and exporters were relatively high. although based on statistical analysis using non-parametric analysis, elivery d chain did not significantly influence moisture content ( ) nutmeg mc , percentage of damaged kernels, total fungal population and total aflatoxin content of nutmeg, the method of nutmeg postharvest handling especially at farmer and , c o l l e c t o r l e ve l s s h o u l d b e c o n d u c t e d a p p r o p r i a t e l y t o m i n i m i z e a f l a t o x i n contamination. acknowledgements the authors would like to acknowledge seameo biotrop for providing financial support through dipa 2013. thanks due to were the indonesian government's office of plantation crop of north sulawesi province in manado and to cv multi rempah sulawesi for their information and cooperation during the survey; to mrs ratnaningsih, mr edi suryadi and mr. iswadi for their technical support. references basappa sc. 2009. . aflatoxins; formation, analysis and control new delhi (in): narosa publishing house. direktorat jenderal perkebunan. 2012. peningkatan produksi, produktivitas dan mutu tanaman rempah dan penyegar. pedoman teknis perluasan tanaman pala tahun 2012. jakarta. 20 hal. fao. 2004. worldwide regulations for mycotoxins in food and feed in 2003. fao food and nutrition paper 81. rome (it): food and agriculture organization of the united nations. 165 p. hocking ad, pitt ji. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low moisture foods. appl env microbial 39: 488-92.. horn bw. 2003. ecology and population biology of aflatoxigenic fungi in soil. toxicol rev 22 j toxin (2 & 3): 351-79. lacey j, magan n. 1991. fungi in cereal grains: their occurrence and water and temperature relationship. in: chelkowski j, editor. cereal grain, mycotoxins, fungi and quality in drying and storage. amsterdam (nl): elsevier. p 77-118. magan aldred n, d. 2007. post-harvest control strategies: minimizing mycotoxins in the food chain. jint of food microbiology. 119 (1-2): 131-9. mandel qa. 2005. fungal contamination of some imported spices. mycopathologia 59 : 291-8. okano k, tomita t, ohzu y, takai m, ose a, kotsuka a, ikeda n, sakata j, kumeda y, nakamura n, ichinoe m. 2012. aflatoxins b and g contamination and aflatoxigenic fungi in nutmeg. shokuhin eiseigaku zasshi 53 (5): 211-6. pitt ji, hocking ad. 2009. new yorkfungi and food spoilage. (us): springer. revitalisasi perkebunan pala siau, sulawesi utara. 2010. warta penelitian dan pengembangan pertanian 32 (1): 4-6. sauer db, meronuck ra, christensen cm. 1992. microflora. in: sauer db, editor . storage of cereal grains and their product 4 editionth (us). . minnesota : american association of cereal chemist. p 313 – 40. sanchis v, magan n. 2004. environmental conditions affecting mycotoxins. in: magan, n, olsen m, editors. mycotoxins in food: detection and control. florida (us): crc press. p 496. standar nasional indonesia. biji pala1993, . sni 01-00061993. jakarta : badan standardisasi nasional. (id) tabata s, kamimura h, ibe a, hashimoto h, iida m, tamu r a y, ni s h i m a t. 1 9 9 3 . af l at ox i n contamination in foods and foodstuffs in tokyo: 1986-1990. j aoac int 76 (1): 32-5.ern. takahashi t. 1993. aflatoxin contamination in nutmeg: analysis of interfering tlc spots. j food sci 58: . 197-8. vicam. 2007. aflatest instruction manual for hplc. watertown : vicam (us) . 139 fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans sri-13 mei 2017-556 (iyan predicting).cdr predicting the impact of climate change on the distribution of f. muell.flindersia pimenteliana in indonesian papua and papua new guinea iyan robiansyah * center for plant conservation bogor botanic garden, indonesian institute of sciences, bogor 16003, indonesia received 12 december 2015/accepted 12 august 2016 abstract population of flindersia pimenteliana (maple silkwood) in indonesian papua and papua new guinea is severely fragmented and experiencing a continuing decline due to habitat destruction and illegal logging. this species is very susceptible to environmental changes and at greater risk of extinction due to its small and fragmented geographic ranges and low abundance. using maximum entropy (maxent) method, the present study predicted the impact of climate change on the distribution of the species across its native distribution area. elevation and 19 bioclimatic variables commonly used in species distribution modeling were used as predictors. the prediction model of the 2 current potential distribution identified a total area of 156,214 km in indonesian papua and papua new guinea (18% of total land area) as suitable habitat for f. pimenteliana. elevation and precipitation of the wettest, coldest and warmest quarters contributed most to the model. based on the average of hadgem2-es and miroc-esm models, potential distribution projections under rcp8.5 scenario suggested a habitat gain of 16% for 2050 and 8% for 2070 in the species distribution. whereas under rcp4.5, an average habitat gain of 7% was predicted for both 2050 and 2070. the newly suitable habitats were predicted to be found mainly in southern and western highland of papua new guinea. protection of these areas from habitat destruction and land use change is needed to assist f. pimenteliana find the most suitable climate for its survival. keywords: climate change, distribution models, flindersia pimenteliana, maxent, prediction introduction climate is one of the most important factors influencing plant distribution. at population scale, seed germination, growth and survival of plants are strongly influenced by temperature and precipitation (walck et al. 2011). thus, the rapidly changing climate for the coming decades will undoubtedly alter local environment where plants grow and consequently change their abundance and distribution (ipcc 2013). plants have only two alternatives in dealing with future climate change in order to survive i.e. either adapting in situ or dispersing to find the most suitable climate for their survival. failing to find the most suitable environment will result in the extinction of the plants. in this context, understanding and predicting the response of plant species and possible distribution alternatives under climate change condition are important to develop proactive strategies to reduce the impact of climate change on plant species diversity. species distribution models (sdms) is an important tool in ecology and biodiversity conservation, both for understanding the factors that affect species distribution and for predicting the response of species to climate change (peterson et al. 2011; franklin 2013; guisan et al. 2013; guillera-arroita et al. 2015). these models c o r r e l a t e s p e c i e s o c c u r r e n c e d a t a a n d environmental variables to estimate species distribution using statisticalor machine-learning procedures (phillips et al. 2006; roberts & hamann 2012). among available models, maximum entropy (maxent) (phillips et al. 2006) is one of the best sdms for analyzing the presence-only data in terms of ability to distinguish between areas where a species is biotropia 4 1 7 59 70 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 556 * corresponding author: iyan.robiansyah@lipi.go.id 59 present, versus those where it is absent (elith et al. 2006). with more than 1,000 applications since 2006, maxent is also one of the most popular sdms, mainly due to the user-friendliness of the software and its high predictive accuracy compared to other sdms (merow et al. 2013). flindersia pimenteliana f. muell. (rutaceae) (maple silkwood) is a tree native to queensland (australia), indonesian papua and papua new guinea (australian tropical rainforest plants, http://keys.trin.org.au). the species is usually found in well developed rain forests and in various sites, but reaches its best development in upland and montane rain forests. evaluation on population status of in indonesian f. pimenteliana papua and papua new guinea revealed that the tree is categorized as according to endangered the international union for conservation of nature (iucn) redlist category and criteria (eddowes 1998). illegal logging is identified as major threat for this species, leading to severe population fragmentation and decline in the population size. such species is very sensitive to environmental changes and at greater risk of extinction. in this study, maxent was used as a tool to predict the impact of climate change on the distribution of across its native f. pimenteliana environment. this study was aimed to: (1) predict current potential distribution of in f. pimenteliana indonesian papua and papua new guinea; (2) identify the environmental factors associated with habitat distribution of and (3) f. pimenteliana estimate the impact of climate change to the future potential distribution of the tree. the results of this study may serve as a basis in developing long term adaptation strategies for assisting in dealing with climate f. pimenteliana change. materials and methods target species and occurrence data flindersia pimenteliana f. muell. (synonym: f. chrysantha merr. & l.m. perry and f. mazlini f.m. bailey) (rutaceae) is a tree having height up to 40 m. this plant species can be a canopy or subcanopy tree. its habitat ranges from near sea level to 1,300 m asl. the tree has a straight, cylindrical trunk which can be unbranched for up to 20 m and its diameter can reach 100 cm (conn & damas 2006). f. pimenteliana is heavily exploited from the wild due to its good quality timber, which is suitable for wood craft, furniture, moldings and interior construction purposes (purnawati 2013). there had been several efforts to plant this tree in order to minimize timber harvesting from the wild. however, due to the trunk and canopy large sizes, it is economically impossible to grow this tree in plantation until reaching the same good timber quality as it is from the wild (australian tropical rainforest plants, http://keys.trin.org.au). based on population assessment in indonesian papua and papua new guinea, the iucn classifies f. pimenteliana as endangered species, without considering population of this species in queensland (australia). occurrence data of f. pimenteliana were obtained from the global biodiversity information facility (gbif) (www.gbif.org), an open access data portal that provides rich information about the known presence of organisms. a total of 74 records with geographical coordinate were initially obtained from gbif. this low numbers of occurrences were mainly due to lack of occurrence information from indonesian papua. the data were then filtered for duplicate and autocorrelated occurrence points using spatially rarefy occurrence data tool in sdmtoolbox (brown 2014). the tool removes spatial cluster of localities according to climate heterogeneity. for this purpose, the tool was set as having two classes, natural breaks classification type and 10 km minimum distance. the resulting data were composed of 23 unique distribution records which were then used for building the model. this filtering method can maximize the number of spatially independent localities and can enhance model performance by removing over-fit of the models towards environmental bias (veloz 2009; hijmans 2012; boria et al. 2014). creation of bias file maxent typically selects background points from a large area and compares them with the present data to differentiate between the suitable and unsuitable environmental conditions. van der wal et al. (2009) showed that model 60 biotropia vol. 24 no. 1, 2017 this purpose, pairwise pearson correlation coefficient of current bioclimatic data was calculated using sdmtoolbox and r ≤ ±0.9 was used as a cut-off threshold to determine the exclusion of highly correlated variables (table 1). the resulting variable set was composed of 9 predictors (table 2). variables commonly used in ecological studies and best represented the original input of climate data (bio1, bio2, and bio12) as well as those with the least correlation to others were retained in the model. together with elevation data, these variables were considered to be the representatives of predictor candidates and were assessed through maxent's jackknife test. as a consequence, variables that contributed < 1.0% were eliminated and the final explanatory variables obtained were then used to build maxent models for (table 2).f. pimenteliana future climate projections to p r e d i c t f u t u r e d i s t r i b u t i o n o f f. pimenteliana, downscaled and calibrated global circulation model (gcm) of hadgem2-es (hadley centre global environment model, version 2-earth system) and miroc-esm (model for interdisciplinary research on climate-earth system models) for 2050 (average for 2041 2060) and 2070 (average for 2061 2080) were used (hijmans 2005; et al. http://www.worldclim.org). these data were among the most recent gcm climate projections that were used in the fifth assessment intergovernmental panel on climate change (ipcc) report. each of these climate models had 30 arc-seconds resolution and projected two representative concentration pathway (rcp) emission scenarios, namely rcp4.5 and rcp8.5. while the rcp4.5 represented low emission scenario, the rcp8.5 was a scenario of high greenhouse gas emissions and represented the worst case scenario of climate model simulation in the fifth assessment ipcc report (see riahi (2011) for more et al. details of the scenarios). final models for future habitat prediction were obtained by averaging results from hadgem2-es and miroc-esm future climate models. performance was lower when background points were selected from a large area. this happened because the background points selected, which were very distant from all presence points, were more likely to show environmental conditions that were very different from those for the presence data (anderson & raza 2010; barbetmassin et al. 2012). thus, the larger the study area, the higher proportion of less informative background points is included in the model. to overcome this problem, a bias file was created in the present study to restrict background point selection and hence, increase the model performance. using buffered minimum-convex polygon tool in sdmtoolbox, the bias file was built with maximum radial distance of 10 km from the occurrence points. selection of environmental variables the elevation and current bioclimatic data set was obtained from the shuttle radar topography mission (srtm) global elevation data (http:// srtm.csi.cgiar.org/) and worldclim 1.4 database (hijmans et al. 2005; http:// www.worldclim.org), respectively. the current bioclimatic data (nix 1986) consisted of 19 variables and were derived from monthly rainfall, while the temperature data were obtained from weather stations across the globe within the period of 1950 2000. these data showed annual trends, seasonality and extreme environmental factors; and were frequently used in predicting species distribution. hence, a total of 20 environmental variables were initially considered for model building. all the environmental layers used in the model had 30 arc-seconds or ≈1 km resolution. these layers were clipped to the indonesian papua and papua new guinea political boundaries and then converted to ascii raster files using esri arcmap 10.1. due to the limitation of data availability, the present study did not include other important variables influencing the distribution of plant species, such as soil characteristics, distance from water bodies and groundwater table. to reduce model overfitting and minimize high co-linearity, highly correlated bioclimatic variables were removed from the model. for 61 impact of climate change on flindersia pimenteliana – robiansyah 62 biotropia vol. 24 no. 1, 2017 b io 1 b io 2 b io 3 b io 4 b io 5 b io 6 b io 7 b io 8 b io 9 b io 1 0 b io 1 1 b io 1 2 b io 1 3 b io 1 4 b io 1 5 b io 1 6 b io 1 7 b io 1 8 b io 2 -0 .6 1 b io 3 -0 .1 7 0 .3 1 b io 4 -0 .1 1 0 .0 1 -0 .9 0 b io 5 0 .9 8 -0 .4 9 -0 .2 5 b io 6 0 .9 9 -0 .7 0 -0 .1 5 -0 .1 5 0 .9 5 b io 7 -0 .5 4 0 .8 9 -0 .1 6 0 .4 5 -0 .3 8 -0 .6 5 b io 8 0 .9 9 -0 .5 8 -0 .2 0 -0 .0 8 0 .9 8 0 .9 8 -0 .5 0 b io 9 0 .9 9 -0 .6 3 -0 .1 2 -0 .1 6 0 .9 6 0 .9 9 -0 .5 9 0 .9 7 b io 1 0 1. 0 0 -0 .6 1 -0 .2 3 -0 .0 5 0 .9 9 0 .9 8 -0 .5 1 0 .9 9 0 .9 8 b io 1 1 1. 0 0 -0 .6 0 -0 .0 9 -0 .2 0 0 .9 7 0 .9 9 -0 .5 7 0 .9 9 0 .9 9 0 .9 9 b io 1 2 -0 .0 7 0 .0 8 0 .1 3 -0 .0 4 -0 .0 6 -0 .0 5 0 .0 2 -0 .1 2 0 .0 1 -0 .0 7 -0 .0 7 b io 1 3 0 .0 7 0 .0 4 0 .0 0 0 .0 2 0 .1 0 0 .0 7 0 .0 4 0 .0 3 0 .1 2 0 .0 7 0 .0 7 0 .8 7 b io 1 4 -0 .1 6 0 .0 5 0 .2 5 -0 .1 3 -0 .1 7 -0 .1 2 -0 .0 8 -0 .2 0 0 .0 7 -0 .1 7 -0 .1 4 0 .9 0 0 .6 3 b io 1 5 0 .2 3 -0 .0 4 -0 .4 1 0 .2 6 0 .2 7 0 .1 7 0 .1 6 0 .2 6 0 .1 4 0 .2 5 0 .2 0 -0 .5 9 0 .2 0 0 .8 3 b io 1 6 0 .0 5 0 .0 4 -0 .0 3 0 .0 6 0 .0 8 0 .0 4 0 .0 6 0 .0 1 0 .0 9 0 .0 5 0 .0 4 0 .8 8 0 .9 9 0 .6 3 0 .1 9 b io 1 7 -0 .1 3 0 .0 3 0 .2 2 -0 .1 1 -0 .1 5 -0 .0 9 -0 .0 7 -0 .1 8 0 .0 4 -0 .1 4 -0 .1 2 0 .9 3 0 .6 6 0 .9 9 0 .8 1 0 .6 6 b io 1 8 -0 .2 4 0 .3 4 0 .1 4 0 .0 1 -0 .1 9 -0 .2 5 0 .2 8 -0 .2 3 0 .2 3 -0 .2 4 -0 .2 3 0 .7 6 0 .7 0 0 .6 3 0 .3 4 0 .7 1 0 .6 4 b io 1 9 -0 .0 5 -0 .1 0 0 .0 9 -0 .0 3 -0 .0 7 0 .0 0 -0 .1 5 -0 .1 2 0 .0 6 -0 .0 5 -0 .0 4 0 .8 9 0 .7 3 0 .8 7 0 .6 5 0 .7 3 0 .8 9 0 .4 5 t ab le 1 p ai rw is e p ea rs o n c o rr el at io n c o ef fi ci en t o f 1 9 b io cl im at ic v ar ia b le s u se d f o r p re d ic ti n g p o te n ti al d is tr ib u ti o n o f f lin de rs ia p im en te lia na n o te s: v al u es ≥ ± 0 .9 a re s h o w n i n b o ld m ea n in g o f e ac h v ar ia b le c an b e re fe rr ed t o t h e w o rl d c li m w eb si te ( h tt p :/ / w w w .w o rl d cl im .o rg ) 63 t ab le 2 p er ce n ta g e co n tr ib u ti o n o f e n v ir o n m en ta l v ar ia b le s to t h e m ax en t m o d el s fo r f lin de rs ia p im en te lia na p o te n ti al d is tr ib u ti o n . v ar ia b le s ex cl u d ed f ro m t h e fi n al m o d el a re a ls o li st ed a n d t h u s h av e p er ce n t co n tr ib u ti o n o f 0 . a ls o s h o w n f o r ea ch v ar ia b le a re m ea n s an d s ta n d ar d d ev ia ti o n s va lu es f o r cu rr en t an d f u tu re c li m at e (2 0 5 0 a n d 2 0 7 0 ) u n d er r ep re se n ta ti ve c o n ce n tr at io n p at h w ay (r c p ) 4 .5 a n d r c p 8 .5 s ce n ar io s v ar ia b le s p er ce n t co n tr ib u ti o n c u rr en t 2 0 5 0 2 0 7 0 r c p 4 .5 r c p 8 .5 r c p 4 .5 r c p 8 .5 p re ci p it at io n in t h e w et te st m o n th (b io 1 3 ) 3 7 .6 3 5 2 .5 ± 8 5 .5 3 7 0 .1 ± 6 9 .3 3 7 2 .2 ± 9 6 .0 3 7 4 .9 ± 9 8 .2 3 7 8 .8 ± 1 0 2 .5 e le va ti o n 2 8 .8 5 5 0 .6 ± 7 9 1 .4 5 5 0 .6 ± 7 9 1 .4 5 5 0 .6 ± 7 9 1 .4 5 5 0 .6 ± 7 9 1 .4 5 5 0 .6 ± 7 9 1 .4 p re ci p it at io n in t h e co ld es t q u ar te r (b io 1 9 ) 1 4 .3 6 4 2 .4 ± 3 3 7 .1 6 2 4 .4 ± 3 4 8 .9 6 3 8 .6 ± 3 5 6 .1 6 5 7 .4 ± 3 5 .3 6 5 2 .8 ± 3 7 2 .9 p re ci p it at io n in t h e w ar m es t q u ar te r (b io 1 8 ) 9 .9 8 3 6 .4 ± 1 8 6 .9 7 9 4 .4 ± 1 9 3 .5 7 6 5 .3 ± 1 9 4 .0 7 9 9 .8 ± 2 0 1 .9 7 6 6 .8 ± 2 2 6 .3 is o th er m al it y (b io 3 ) 7 .2 8 3 .2 ± 5 .7 8 2 .8 ± 6 .0 8 2 .6 ± 5 .5 8 2 .0 ± 6 .2 8 1 .9 ± 6 .2 m ea n d iu rn al r an g e in t em p er at u re (b io 2 ) 2 .2 8 6 .4 ± 1 2 .9 8 6 .7 ± 1 3 .9 9 0 .6 ± 1 5 .3 8 5 .7 ± 1 3 .2 8 7 .3 ± 1 5 .0 a n n u al m ea n t em p er at u re ( b io 1 ) 0 2 4 0 .8 ± 3 8 .9 2 5 7 .6 ± 3 8 .9 2 6 1 .1 ± 3 8 .8 2 6 2 .7 ± 3 8 .6 2 7 2 .6 ± 3 8 .7 t em p er at u re a n n u al r an g e (b io 7 ) 0 1 0 3 .4 ± 1 4 .6 1 0 4 .4 ± 1 6 .5 1 0 9 .5 ± 1 9 .3 1 0 4 .5 ± 1 2 .3 1 1 0 .3 ± 2 1 .3 a n n u al p re ci p it at io n ( b io 1 2 ) 0 3 0 6 9 .2 ± 8 6 2 .5 3 0 5 8 .7 ± 8 9 4 .6 2 7 3 4 .4 ± 7 9 0 .6 3 3 5 0 .6 ± 9 9 0 .4 3 0 5 4 .6 ± 9 8 0 .7 p re ci p it at io n s ea so n al it y (c o ef fi ci en t o f v ar ia ti o n ) (b io 1 5 ) 0 2 5 .4 ± 1 4 .3 2 9 .3 ± 1 5 .9 3 1 .2 ± 1 5 .4 2 8 .9 ± 1 6 .2 3 1 .3 ± 1 6 .7 impact of climate change on flindersia pimenteliana – robiansyah the potential range of loss and gain of f. pimenteliana was calculated for the year of 2050 and 2070. to determine whether the species assemblages would be constant or change in the future compared to the current potential habitats, the turnover rate ( ) was t calculated using the following formula (hu et al. 2010): where: t = species turnover rate g = species gain l = species loss sr = current species potential distribution t value ranges from 0 to 100, where value of 100 indicates that the species assemblages are different, while value of 0 indicates that the species assemblages are similar with previous conditions (trisurat 2011).et al. model development and evaluation t h e m a x i m u m e n t r o p y m e t h o d , a s implemented in maxent (version 3.3.3k), was used to model the potential distribution range of f. pimenteliana. to validate and calibrate the model, the maxent modeling was run through spatially jackknife tool of sdmtoolbox. model validation was done using geographically structured threefold cross-validation method. furthermore, the tool tested different combinations of five model feature class types (1=linear; 2=linear and quadratic; 3= hinge; 4=linear, quadratic, and hinge; and 5=linear, quadratic, hinge, product, and threshold) to optimize the model performance. each of these model parameter classes was run in five replicates. finally, the best model was automatically selected by evaluating the omission rate, area under the curve (auc) and model feature class complexity of each model. to measure the importance of environmental variables, the procedure was used spatially jackknife which created response curves. the best model selected was used to run the final model using all of the occurrence points. the model was also projected to the future climates of 2050 and 2070 using rcp4.5 and rcp8.5 emission scenarios. maximum training sensitivity plus specificity logistic threshold was used to convert the continuous suitability index into suitable and unsuitable areas for f. pimenteliana. the predicted suitable areas were then clipped by 25 m resolution palsar-2/palsar forest cover of 2015 obtained from the japan aerospace exploration agency (www.eorc.jaxa.jp). results and discussion model performance and environmental variable responses although there are some drawbacks attributed to maxent, such as overfitting, model complexity dependency and independent evaluation data requirement (radosavljevic & anderson 2014), its user-friendliness has attracted many application in sdms. furthermore, maxent has been shown to perform better than other models for analyzing presence-only data. the model also has the best predictive power even with very low sample size (wisz et al. 2008). in the present study, using 23 occurrence data, the maxent model developed for predicting the potential distribution of f. pimenteliana was significantly better than random expectations. the average test auc for the replicate runs was 0.710 (sd = 0.043). peterson et al. (2011) argued that the auc value of 0.7 0.9 indicated moderate performance of the model. the present study was the first attempt on building sdms to predict potential current and future distribution of f. pimenteliana in indonesian papua and papua new guinea. after removing highly correlated bioclimatic variables and those with < 1% contribution, there were 6 variables identified as being important in creating model fit for (table 2). f. pimenteliana precipitation-related variables (bio13, bio18 and bio19) had the most influence to f. pimenteliana distribution. these variables together contributed 61.8 % to the model. on the other hand, temperature-related variables (bio3 and bio2) together contributed only 9.4% to the model. furthermore, elevation was also important variable for distribution with 28.8% f. pimenteliana contribution. the significant role of precipitation on tree species distribution in tropical forests was well documented (baltzer 2008; brenes-et al. arguedas 2009; baltzer & davies 2012). soil et al. water availability affected by precipitation is a direct determinant of tropical trees distributions in both local and regional scales (engelbrecht et al. 2007; toledo 2012)et al. . 64 biotropia vol. 24 no. 1, 2017 t = 100 × g+ l sr + g response curves of the top four of highly contributed variables for f. pimenteliana were shown in figure 1. habitats with elevation between 400 – 1,500 m had high potential suitability for the species. furthermore, habitats with precipitation of the wettest month between 160 300 mm (bio13) had high suitability for sustaining f. pimenteliana. in addition, the species preferred habitat having precipitation of the coldest quarter (bio19) and the warmest quarter (bio18) between 100 400 mm and 360 760 mm, respectively. habitat suitability of f. pimenteliana decreased with the increasing value of the precipitation-related variables. this pattern is understandable as, according to schuur (2003), increased precipitation in humid tropical forests is known to have negative effect on plant growth and net primary productivity. the author argued that high rainfall in humid ecosystem may reduce plant growth and productivity by decreasing radiation inputs, increasing nutrient leaching or reducing soil oxygen availability. prediction of current distribution using maximum training sensitivity plus specificity logistic threshold (0.396), model prediction of the current distribution identified a 2 total area of 156,214 km (18% of total land area) as suitable habitat for f. pimenteliana. liu et al. (2013, 2016) suggested that for presence-only data, maximum training sensitivity plus specificity logistic threshold can be used confidently for threshold selection as it produced higher sensitivity compared to other methods. most of the suitable habitats were obser ved in mountainous regions and southern coastal areas of the main island where the precipitation intensity was relatively low (fig. 2). in indonesia, potentially suitable habitats for f. pimenteliana were mostly located along mountain chains of indonesian papua, extending from the mountain ranges in sorong regency to the jayawijaya mountains in pegunungan bintang regency. there were also relatively separated suitable 65 figure 1 response curves of the top four of highly contributed variables for maxent model of flindersia pimenteliana. the red center line represents the mean values derived from the cross-validation runs, while the blue curve delineates the standard deviation. variable definitions: bio13, elevation (m), bio19 and bio18 represent precipitation (mm) of the wettest month as well as the coldest and warmest quarters, respectively impact of climate change on flindersia pimenteliana – robiansyah l o g is ti c o u tp u t (p ro b a b il it y o f p re se n c e ) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 100 200 300 400 500 600 700 800 bio13 elevation 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 bio19 bio18 400 600 800 1000 1200 1400 1600 18002000150010005000 regions for such as in waigeo island f. pimenteliana in raja ampat regency, fakfak mountains in fakfak regency, kumawa mountains in kaimana regency and coastal areas in the southern region of merauke regency. for papua new guinea, most of the potentially suitable habitats were observed to be along mountain regions in eastern highlands and owen stanley range. furthermore, suitable habitats were also detected in southwestern corner of the western province, coastal areas of east sepik province, east part of new britain island and new ireland island. very little is known about the habitat distribution of f. pimenteliana in indonesia as study examining this species is very scarce. recent study conducted by purnawati et al. (2012) and purnawati (2013) reported that the species was observed in teluk cendrawasih national park, teluk wondama regency, which was also included as a suitable habitat in the present model prediction. further surveys in other predicted distribution areas of the plant are needed to validate the prediction of the present study. for papua new guinea, some of the predicted areas agree with areas mentioned in the specimen records of f. pimenteliana held by the royal botanic garden of sydney (http://plantnet. rbgsyd.nsw.gov.au). these areas included many localities in morobe province (more than 53 records), eilogo in central province, bewani in west sepik province, mount obree and toma village in northern province, mount kilkerran and rabaraba in milne bay province and goroko and aiyura in eastern highlands province. further surveys are required in other predicted areas to validate the model prediction, especially in western province where specimen record of f. pimenteliana is absent. in addition, new surveys in previously known areas are also considered necessary as most of the records mentioned above are very old (some records are dated back to 1923). protected areas are one of the most important tools in plant conservation. in the present study, most of the predicted suitable habitats of f. pimenteliana were located outside the existing protected areas (fig. 3). only 24,393.9 km 2 (15.6%) of these habitats were currently covered by terrestrial protected areas in indonesian papua and papua new guinea. as can be seen in figure 3, protected areas with high coverage of predicted suitable habitats for are mostly f. pimenteliana found in indonesia, including tambrauw mountain nature reserve, lorenzt national park, jayawijaya nature reserve, enarotali nature reserve and kumawa mountain nature reserve. to protect and conserve , expansion f. pimenteliana of existing protected areas or establishing the new one is necessary to cover the predicted suitable areas as much as possible. this action is especially needed in papua new guinea where only small percentage of the predicted suitable habitats is covered by the existing protected areas. although the present study was able to predict suitable habitats of with high f. pimenteliana success rate, care should be taken when implementing the results in the field-based conservation programs. since maxent estimates the fundamental niche of a species, the predicted distribution presented in this study might be 66 biotropia vol. 24 no. 1, 2017 figure 2 predicted potential distribution of flindersia pimenteliana for current (2016) and future climate conditions (2050 and 2070) under rcp4.5 and rcp8.5. future habitat predictions were obtained by averaging results from hadgem2-es and miroc-esm future climate models overestimated (pearson 2007). the model does not consider other factors influencing the distribution of plant species such as dispersal process, anthropogenic influences, biotic interactions or geographic barriers (pearson 2007; soberón 2007). prediction of future distribution future distribution of f. pimenteliana predicted by hadgem2-es and miroc-esm model generally had low agreement in papua new guinea compared to that in indonesian papua. standard deviation between the two models was 67 figure 3 current (2016) potential distribution of flindersia pimenteliana and existing protected areas in papua and papua new guinea according to world database on protected areas (wdpa, http://www.protectedplanet.net/) figure 4 standard deviation of predicted probabilities of flindersia pimenteliana occurrence from hadgem2-es and miroc-esm future climate models. black dots are occurrence data used in modeling impact of climate change on flindersia pimenteliana – robiansyah higher in papua new guinea, especially in regions where no sample data were recorded (fig. 4). based on the average value of the models, the suitable habitat for f. pimenteliana was predicted to decline by 3% for rcp4.5 and 2% for rcp8.5 in the year 2050. the suitable habitat gains for the two rcps were 7% and 16%, respectively. hence, the percentage turnover in 2050 was estimated to be 41% and 54% for rcp4.5 and rcp8.5, respectively (table 3). for 2070, the suitable habitat for the species was predicted to decline by 4% for both rcp4.5 and rcp8.5, whereas the habitat gains were 7% and 8%, respectively. therefore, the percentage turnover in 2070 was estimated to be 44% for rcp4.5 and 48% for rcp8.5 (table 3). under rcp4.5, the species was predicted to gain suitable habitats in southern and western highland of papua new guinea in 2050. this gain was relatively stable and still could be observed in 2070. higher suitable habitat gain was observed under rcp8.5 in 2050. this gain, however, was not stable and greatly decreased in 2070 (fig. 2). in contrast, the suitable habitat along jayawijaya mountains, coastal area of merauke regency and east sepik province, and the most east of owen stanley range in papua new guinea were predicted to disappear in 2050. these habitat losses were similar for both rcps and relatively stable until 2070 (fig. 2). since the species had turnover rate of more than 30%, major shift in distribution was predicted to occur in the future for both rcps. while some of predicted suitable habitats in s o u t h e r n c o a s t a l r e g i o n s d i s a p p e a r e d , mountainous areas located in the middle of papua new guinea were predicted to become suitable habitats for f. pimenteliana in 2050 and 2070. changes in timing and seasonality of rainfall in the future may be responsible for this rang e shift. australian bureau of meteorology and csiro (2011) predicted rainfall pattern change in papua new guinea with more than 15% increase in annual and seasonal rainfall by 2090. this range shift of geographic distribution towards higher elevation is also observed in many terrestrial organisms and is commonly linked to increased growth and dispersal success due to warmer climate (chen et al. 2011). although the temperature was predicted to increase at 0.11 °c per decade over papua new guinea (australian bureau of meteorology and csiro 2011) and in the range of 0.2 0.3 °c per decade over indonesian papua (boer & faqih 2004), the present study, however, was unable to detect this correlation as all temperature-related variables had little effect to the model. conclusions it is likely that f. pimenteliana will benefit from climate change, as environmentally suitable ranges for this species are projected to increase by 2050 and 2070. the present study predicted major range shift of geographic distribution for the species towards higher elevation. the newly suitable habitats were predicted to be found mainly in southern and western highland of papua new guinea. these are the areas where the protection of the species might be the most feasible and cost-effective in the future. thus, protection of these areas from habitat destruction and land use changes is needed to make sure that f. pimenteliana can adapt to the climate change. further field surveys are required to validate the prediction of the present study. furthermore, it is recommended to obtain additional occurrence data and include other important predictors (e.g. edaphic factors) to minimize uncertainty in climate projections. 68 biotropia vol. 24 no. 1, 2017 table 3 predicted gain, loss and total suitable habitats, and turnover rate of flindersia pimenteliana for future climate conditions (2050 and 2070) under representative concentration pathway (rcp) 4.5 and rcp 8.5 2050 2070 rcp4.5 rcp8.5 rcp4.5 rcp8.5 suitable (km2) 195,058 275,956 188,578 190,397 gain (km2) 64,516 140,551 64,818 71,940 loss (km2) 25,671 20,810 32,454 37,757 turnover (%) 41 54 44 48 references anderson rp, raza a. 2010. the effect of the extent of the study region on gis models of species geographic distributions and estimates of niche evolution: preliminary tests with montane rodents (genus nephelomys) in venezuela. 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10 page 11 page 12 biotropia vol. 28 no. 1,2021: 1 10 doi: 10.1 1598/btb.2021.28.1.623 reproductive biology of freshwater clam pokea (ba tissa violacea var. celebensis, v o n marten 1897) (bivalvia: corbiculidae) i n pohara river, kendari, southeast sulawesi province, indonesia bahtiarl, la anadi1, wa nurgayahl, m hamzah* and udhi e. hernawanz 'faczllty offideries and marine science, huluoleo university, jalan hea mokodompit no. 1 maluka andzlonohzl, kendari 93232, indonesia 2reseurch centerfor oceanogr@h_y, indonesidn institnte ofsciences, jalun pan' putih i , ancol timur, jakarta 14430, indonesia received 15 february 2016/accepted 23 december 2019 abstract the freshwater clam, locally known as pokea, (butissa violaceu var celebensis, von martens 1897; bivalvia: corbulidae) is a popular and widely consumed food in icendari, southeast sulawesi. despite its popularity, basic information required for conservation management, such as reproductive biology, is lacking. hence, this study aims to examine the reproductive biology of the clam obtained from pohara river, icendari, southeast sulawesi province, indonesia. pokea samples were collected monthly from february 2012 to january 2013. its reproductive biology, including sex ratio, stage of gonadal maturity, gonadosomatic index (gsi), fecundity, and size of the first mature gonad from each sample were recorded. data were analyzed using chi-square test and linear regression in the package sigma plot v.6.o. pokea population in pohara river was male-biased. the population spawns throughout the year and the peak spawning season was in august-september. mature gonads were found at small shell size (indicating early sexual maturity). this gonadal development in pokea that might have been influenced by food availability, is a very relevant baseline information for the conservation of pokea population in the pohara river. keywords: clams, freshwater, gonadal maturity, spawning introduction the freshwater clam, locally known as pokea, ( b a t k uiolacea var celebensis, von martens 1897, bivalvia: corbulidae) is a popular and widely consumed food in icendari, southeast sulawesi (fig. 1). geograplucally, the genus batissa is widely distributed in the western and southern pacific (from malaysia, philippines, papua new guinea, western australia to fiji) (dudgeon & morton 1989). b. uiolacea was known to be distributed in southeast asia and northern australia (sastrapradja 1977). in indonesia, this species occurs in some of the big islands, incluchng sumatra (putri 2005), java *corresponding author, email: udhiehernawan@gmail.com (sastrapradja 1977), papua (djajasasmita 1977) and sulawesi (i<usnoto 1953). in southeast sulawesi, the clams are found in some of the big rivers in the southeast peninsular, such as pohara river, lasolo river, roraya river and laeya ever (bahtiar 2005). ecologically, this species occupies the substrate surface or lives beneath the substrate in the river estuary (sastrapradja 1977; djajasasmita 1977; bahtiar 2007a). pokea can be found in a variety of sediment types, from gravel to clay (bahtiar 2007b; bahtiar et al. 2008). this clam species lives in small and big colonies (bahtiar 2007a). these clams were believed to have different reproductive characteristics from other species of freshwater bivalves. this is because individuals of the clams are unisexual, while other species are mostly hermaphrodite. biotropia vol. 28 no. 1,2021 figure 1 the freshwater clam pokea ( b a t h a violacea var cehbensis) notes: a: width; b: length. very likely due to a combination of uncontrolled harvesting and sand mining in its habitat, the pokea populations in pohara river have been declining. this is manifested by the declining trend of both the number and size of the sampled pokea. in addition, localized extinction has been reported in the mining areas. this situation calls for some form of conservation management intervention of this species (bahtiar e t al. 2012). despite its high demand for consumption, coupled with its declining populations, some basic information required for effective conservation management, such as reproductive biology, is lacking (bahtiar 2012). accurate information on the reproductive biology of this species is critically relevant for several conservation practices, particularly in determining the harvesting season and minimum clam size for sustainable harvesting (cantillaneza e t al. 2005). hence, this research aims to examine the reproductive biology of the pokea clam in pohara river, kendari, southeast sulawesi. material and methods sample and data collection 122023'556" e), from february 2012 to january 2013 (fig. 2). during the first week of each month, a total of 120 individual clams were collected, then separated based on the gender. male individuals have milky white colored gonad, while female ones have brown colored gonad. the recorded parameters for reproductive biology, included sex ratio, fecundity, stage of gonadal maturity, gonado somatic index, and size of the first mature gonad from each samples. gonadal development was divided into 5 levels: inactive (stage i), early development (stage 11), final development (stage 111), mature (stage iv), spawning/post-spawning (stage v). this gonadal development was examined based on the gonad morphology and histology (bahtiar 2012). the gonado somatic index (gsi) was measured based on illanes e t al. (1985) and wolff (1987) methods. fecundity was assessed based on gravirnetric method, using egg weight, total gonad weight, and body weight (eiffendi 2002). size of the first mature gonad was screened from all class sizes (arocha & barios 2009). the phytoplankton abundance was also recorded at the sampling sites. all laboratory works were conducted at the faculty of pokea samples were collected monthly from fisheries, haluoleo university, icendari, the estuary of pohara river (03"58'551m s and southeast sulawesi province, indonesia. reproductive biology of freshmter dam pokea in p o h m river bahtiar d al. '4 0 160 3201(m c v 1 s e a 0 345mwm d mars di-t i i n e g u l i . . i west uyrroeiol(met r o r i e a . 0 66 1m km tmm! ,a&=s 'ns%w€ 1-re figure 2 map of the sampling sites at pohara river, kendari, southeast sulawesi province data analysis the significant difference of the sex ratio was determined using the chi-square (x? test where: (mzighani 2005), using the following formula: = fecundity number where: " (oi ei)' x2 = c i=l o i n = number of eggs from sampled gonads g = total gonad weight g = weight of the sampled gonads oi = the number of males and females the relationship between fecundity and the observed at i shell width was analyzed using regression ei = the expected number of males and analysis (steel & torrie 1981). the probability females at i of mature gonad was calculated using nonlinear regression functions with a logarithmic curve gonado somatic index (gsi) was calculated (arocha & barios 2009), using the following using the formula described by sastry (1979): formula: bg gsi = ~ 1 0 0 % bt where: gsi = gonado somatic index bg = gonad weight (g) bt = body weight includmg gonad (g) pokea's fecundity was calculated based on the gravimetric method (effendi 2002), using the following formula: where: y = probabihty of mature gonad (yo) e = exponential function a = intercept b = slope x = width at i (cm) biotropia vol. 28 no. 1,2021 the relationshp between the gsi and gonadal development represents the phytoplankton abundance was analyzed using reproductive processes that occur until the the pearson correlation. all statistical analyses spawning event. the processes were divided were done using sigma plot v.6.o. into five stages, from inactive phase to the spawning phase. the parallel gonadal stages of both male and female pokea observed in this results a n d discussion study confirmed the studes of bahtiar (2012). sex ratio the number of male individual clams was higher than that of the female individuals at each sampling month, at an average male/female ratio of 65/35 (table 1). the sex ratio was significantly male biased, indicated by the chi square test value = 0.0003). this male-biased sex ratio conforms with previous studies by bahtiar (2005) and bahtiar (2012). however, sex ratio in bivalve population is not always male biased but varies with different species. for example, the clam gam' elongata population exhbited a slightly female-biased sex ratio (nabuap & campos 2006). the male-biased sex ratio might be the consequence of the fact that (i) pokea reproduces by external ferohzation, and (ii) males of pokea are generally smaller in size than females. in external fertilization, the sperms are subjected to greater mortality in the aquatic environment. therefore, to increase the probability of a successful fertilization, the number of male individuals should be higher than that of female indviduals (bahtiar 2012). table 1 sex ratio of the freshwater clam poliea furthermore, the peak periods of spawning events were also similar. partial spawning was also observed based on the changes in the egg dameter. egg diameters at gonadal stage iv consisted of two size groups, with median values of 21.60 pm and 159.16 pm (bahtiar 2012). at the post-spawning stage or), three size groups were reported, with median value of 22.80 pm, 172.06 pm and 262.82 pm (fig. 4). t h ~ s condition implied that the eggs reached maturity at different periods, indicating that the pokea spawns throughout the year. therefore, pokea can be classified as a partial/multiple spawner, although it has a peak period of spawning. partial/multiple spawner is common in bivalves; for example the clam v e n w veracosa spawns all year long, however the spawning event peaks in august. some populations spawn in december firado etal. 2003), or the clam donax tmnc~lzls shows two peaks of spawning periods (march and august) with gametogenesis cycle starts in late november and finishes by the end of august (gaspar e t al. 1999). month male (%) female (o/o) march 2012 april 201 2 may 2012 june 2012 july 2012 august 2012 september 2012 october 2012 november 2012 december 2012 january 201 3 f e b m a ~ 201 3 total 65.0591 34.9409 reproductive biology of freshwater clam pokea in pohara river bahtiar e t al. male loo 1 c, c e a 20 0 0 ? a g m u female -0 0 40 20 " i o 2012 20 13 m a r apr may jun jul a u g sep oct nov dec jan feb figure 3 stages of gonadal development in male and female pokea notes: stage i (inactive); early stage i1 (development); stage i11 (final development); stage iv (mature); and stage v (spawning/post-spawning). i gonad stage 1 v gonad stage v gonadal stage egg diameter ( l m ) egg diameter (pm) figure 4 bhattacharya analysis on the distribution of egg diameter on gonadal stage iv and v (modified from bahtiar 2012) gonado somatic index (gsi) from 1.46 to 15.69, and peaked in july. after the both male and female pokea showed similar peak event in july, the gs1 value temporal patterns of gsi. averaged gsi ranged declined in august-september 2012 (fig. 5). biotropia vol. 28 no. 1,2021 female male 0 i i i i i i i i i i i i i 2012 20 13 mar apr may jun jul aug sep oct nov dec jan feb observation month figure 5 mean gsi of male and female h k e a in each observation month gsi indicates annual reproductive cycle and it links with the stages of gonadal maturity. therefore, a concordance of temporal pattern existed between gsi and gonadal stages, in whch gsi peaked in july, followed by the peak of spawning event in august-september. bahtiar (2012) also reported a similar pattern. reproductive cycles in bivalves varies with different species. for example, corbicula australis spawns at the end of september and october (bryne 2000), while corbicula puminea spawns twice a year in early may or june and late september (mouthon 2001). this intraspecific variation in the reproductive cycle is probably related with different geographical locations. however, this is not conclusive as evidences or studies on the reproductive biology of pokea are still lacking. gonadal maturity is determined based on egg diameter changes in each month, as egg size represents reproductive growth in the gonad (haggertyet al 2005). when the gsi values were compared, changes in egg diameter showed a similar temporal pattern. this is because the gsi value is determined by egg size (bahtiar 2012) (fig. 6). a similar pattern was also observed in the species megalonaias nervosa showing the number and size of oosit increased at the end of june (mature gonad) unul the end of september (spawning), then decreased in early october (inactive phase) (haggerty et al 2005). fecundity the relationship between fecundity (i?) with the median class of shell width (l) was determined from a simple regression equation, f = 36815"l-81706 with a determination coefficient (r) = 0.97. an individual with a minimal shell width of 2.97 cm contained a total of 4652 eggs, while another one with a width of 5.93 cm contained 130,465 eggs (fig. 7). numbers of eggs foundin this study were relatively smaller compared with the findings of bahtiar (2012) ranging from 4,950 to 1,007,384. the small number of eggs observed in this study might be a consequence of the environmental disturbances resulted from the sand mining activities. the dsturbances might have triggered the early maturity of the clams resulting in the decreased number of eggs. compared with other species, the egg number recorded in t h s study was much lower. for example, the unionid mussels (unionidae) contained 200,000 to 17,000,000 eggs (vaughn 2005) and the macker mussel actinonaias hgamentina contained 80,616 to 1,561,224 eggs (moles & layzer 2008). biotropia vol. 28 no. 1,2021 shell width (cm) figure 7 linear relationship between fecundity and shell width in pokea o male 4 female 0 2 4 6 8 shell width (cm) figure 8 size of the first mature gonad in male and female pokea relationship between the reproductive the bivalve reproductive performance. the parameters and environmental factors important role of food availability in triggering simple h e plots showed a concordance between the reproductive performance of pokea (gsi) and environmental conditions (phytoplankton abundance). this pattern was further examined in a simple linear regression showing that the relationship has a coefficient of determination, r2 = 69.05% (fig. 9). as pokea feeds on phytoplankton, the increase of phytoplankton abundance in july might be related with gonadal maturity in pokea, as it reached a spawning peak in august-september. hence, environmental condition has influenced sexual maturity has been well documented, particularly in bivalve farming. the increased gsi in a m e l t e n iradians was stimulated by an external factor that increased availability of food in terms of organic matter (gosling 2002). the increase of gsi in gam' elongata occurred following the increased of food supply, again in terms of organic matter (nabuab & campos 2006). other environmental factors, such as ph, temperature, organic matter, and seston, might also be important pahtiar 2012). reproductive biology of freshwater clam pokea in pohara river bahtiar et al. f + phytoplankton abundance i apr may jun jul aug sep oct nov dec jan feb observation month figure 9 relationship between gsi and phytoplankton abundance conclusion (tetraptzlw albidus) from the western central atlantic. aust j mar freshwater res 43:393-402. the pokea (batissa violacea var celebensis) bahtiar. 2005. icajian populasi pokea (batissa violacea var. population in pohara &ver was male-biased. celebensis, von martens, 1897), 1897 di sungai the population spawned throughout the year pohara, icendari, sulawesi tenggara. vhesis]. bogor (id): institut pertanian bogor. and the peak spawning season was in august september. mature gonad was found at small bahtiar. 2007a. iconservasi populasi pokea (batissa violacea var. celebensis von martens, 1897), di sungai pohara, shell size (indicating early sexual maturity). food icendari, sulawesi tenggara. laporan hibah availability might have influenced the gonadal bersaing. jakarta (id): dp2m-dikti. development in pokea. t h s is a very relevant baseline information for the conservation management of pokea population in the pohara river. acknowledgment the authors would like to thank all the field assistants during the sampling period and the dean of the faculty of fisheries and marine sciences, haluoleo university, i<endari, southeast sulawesi, indonesia for the support and suggestions for the completion of this study. references arocha f, barios a. 2009. sex ratio, spawning seasonality, sexual maturity, and fecundity of white marlin bahtiar. 2007b. preferensi habitat dan lingkungan perairan pokea (batissa violacea celebensis martens, 1897) di sungai pohara, icendari, sulawesi tenggara. aqua hayati 5(2):81-7. bahtiar, yulianda f, setyobudiandi i. 2008. icajian aspek pertumbuhan populasi pokea (batissa violacea var. celebensis, von martens 1897) di sungai pohara, icendari, sulawesi tenggara. jurnal ilmu-ilmu perairan dan perikanan indonesia 15(1):1-5. bahtiar. 2012. studi bioekologi dan dinamika populasi pokea (batirsd violacea var. celebensis, von martens 1897) yang tereksploitasi sebagai dasar pengelolaan di sungai pohara, icendari, sulawesi tenggara. pissertation]. bogor (id): insitut pertanian bogor. bone 0, marshal nb. 1982. biology of fishes. first edition. new york (us): blackkie and sons ltd. 205 p. cantillaneza m, avendan-oa m, thouzeaub g, le pennec m. 2005. reproductive cycle of a p p e c t e n pzlrpzlratzls (bivalvia: pectinidae) in la rinconada biotropia vol. 28 no. 1,2021 marine reserve (antofagasta, chile): response to environmental effects of el nin-o and la nin-a. aquaculture 246:181-95. djajasasrnita m. 1977. an anotated list of the species of the genus corbicda from indonesia (mollusca: corbiculidae). bulletin zoologisch museum. amsterdam (nl): universiteit van amsterdam. dudgeon d, morton b. 1989. the population dynamics and sexual strategy of anodanta woodiana (bivalvia: unionidae) in plover cove reservoir, hongkong. j zoo1 lond 201:161-83. gosling e. 2002. bivalve molluscs: biology, ecology and culture. new york (us): blackwell publishing. gaspar mb, ferreira r, monteiro cc. 1999. growth and reproductive cycle of donax tmnmiz/s l. (mollusca: bivalvia) of faro, southern portugal. fish res 41 :309-16. haggerty tm, garner jt, rogers rl. 2005. reproductive phenology in megalonaias nervosa (bivalvia: unionidae) in wheeler reservoir, tennessee river, alabama, usa. j hydrobiologia 539:131-6. heino m, kaitala v. 1996. optimal resource allocation between growth and reproduction in clams: why does indeterminate growth exist? funct ecol 10:245-51. illanes je, akaboshi s, uribe e. 1985. efectos de la temperature en la reproduccio'n del ostio'n del norte appectenpcr7puratms en la bah'a de tongoy durante el feno'meno el nin-o. investig pesq (chile) 32: 167-173. korniushin av, glaubrecht m. 2005. anatomy and reproduction of viviparous pisidzcrm (parapisidium) reafiuiatm icuiper, 1966: implications for the phylogeny of sphaeriidae (mollusca: bivalvia: heterodonta). org divers evol 6:185-95. kusnoto. 1953. kebun raya indonesia (botanic gardens of indonesia). bogor. jurnal treubia 22: 53-7. mzighani s. 2005. fecundity and population structure of cockles, anadara antiqcrata l. 1758 (bivalvia: arcidae) from a sandy/muddy beach near dar es salaam, tanzania. western indian ocean j. mar. sci. 4(1):77-84. moles kr, layzer jb. 2008. reproductive ecology of actinonaias igawentina (bivalvia-unionidae) in a regulated river. j n am benthol soc 27(1):212-22. mouthon j. 2001. life cycle and population dynamics of the asian clam corbictrla pcrminea (bivalvia: corbiculidae) in the saone river at lyon (france). j hydrobiologia 452:109-19. nabuap f, campos an. 2006. some aspects of the reproduction in the elongate sunset clam, garz elongata (lamarck, 181 8) from banate bay area, west central philippines. j science diliman 18(2):34-46. sastrapradja. 1977. sumber protein hewani. bogor (id): lembaga biologi nasional-lipi. sastry an. 1979. pelecypoda (excluding ostreidae). v. mollusks: pelecypoda and lesser classes. new york (us): academic. press. sousa r, antunes c, guilherrnino l. 2008. ecology of the invasive asian clam corbicda p.minea (miiller, 1774) in aquatic ecosystems: an overview. ann limnol-int j lirn 44(2):85-94. steel rgd, torrie jh. 1980. prinsip dan prosedur statistika: suatu pendekatan biometric (diter jemahkan oleh sumantri). jakarta (id): gramedia. tirado c, salas c, mirquez i. 2003. reproduction of venas vemcosa l., 1758 (bivalvia: veneridae) in the littoral of mhlaga (southern spain). fish res 63:437-45. vaughn cc. 2005. freshwater mussel population in southeastern oklahoma: population trends and ecosystem services. stillwater (us): oklahoma water resources research institute. wolff m. 1987. population dynamics of the peruvian scallop appecten pcrrpztratz/s during the el nino phenomenon of 1983. can j fish aquat sci 44(10):1684-91. biotropia (2) 1988/1989: 25-31 removal and leaching of nutrients by salvin1a molesta mitchel and eichhornia crassipes (mart.) solms y.c. wee and h.h. yeoh department of botany, national university of singapore, singapore 0511, republic of singapore abstract profuse growth of eichhornia crassipes and salvinia molesta in singapore reservoirs required their regular manual removal as their prolonged presence can lead to deterioration in the quality of the potable water. clearing of the reservoir catchments, together with regular removal of the weeds and dumping them away from the catchments, should, in the long term, reduce their presence in the reservoirs. laboratory experiments showing the removal of chloride, sulphate, phosphorus and nitrate from the growing medium and the release of chloride, phosphorus and nitrate by rotting plants should convince the administrators of the benefit of proper management of the problem. introduction eichhornia crassipes (water hyacinth) and salvinia molesta (water spangle) are two of the most troublesome water weeds of southeast asia. the former is bulky and grows profusely in eutrophic waters, doubling its number every eight to ten days (wolverton and mcdonald 1976). the latter, a water fern of oligotrophic waters, is similarly profuse in growth, with a doubling time under favorable growth conditions of four to ten days (mitchell and tur 1975). both plants were introduced into the country: water hyacinth as an ornamental in 1893 and water spangle as a teaching specimen around the 1950s (wee 1986, wee and corlett 1986). water hyacinth existed in rural fish ponds where it was grown as a feed for pigs. it became a problem when the kranji reservoir was constructed in 1970 by the barraging of the kranji river. as the catchment of this reservoir was agricultural areas, pollutants from pig farms and run-off fertilizers from fruit and vegetable farms provided enough nutrients to trigger a population explosion five years later. problems of water spangle was seen in the seletar reservoir. its catchment of secondary forests ensured less pollution but, agricultural pollutants still found their way into the water, resulting in proliferation of the plants around 1978. removal of these plants has traditionally been by mechanical means but use o f herbicides like 2,4-d and paraquat has proven to be more efficient (penfound and earle 1948, widyanto 1976). as both bodies of water were reservoirs, use of herbicides was not considered. the use of beetles to control water spangle, as reported in austra lia (room 1984) has yet to be tried in this region. thus, at the 25 biotropia no. 2, 1988/1989 two reservoirs, mechanical removal of the weeds was regularly undertaken, as the presence of large quantities of the weeds besides being aesthetically objectionable could cause deterioration of the quality of the potable water. this work investigated the removal and leaching of nutrients by these plants under laboratory conditions, as under proper supervision, these plants could be used to our advantage, to remove the pollutants from the water. materials and methods water spangle and water hyacinth were obtained from the garden of the department of botany, national university of singapore. they were grown in rain water contained in concrete tanks. for experiments, 35 x 29 x 12 cm plastic trays were used to contain the plants. uniform sized plants were first washed in tap water a few times, then rinsed twice with distilled water. five to ten water spangle plants (total weight 250 g) and five water hyacinth plants (total weight 350 g) were placed in each basin containing 5 1 hoagland's complete nutrient solution (hewitt 1952). nine replicates per plant material were set up. the basins were left in the greenhouse partially exposed to sunlight. loss of water through evaporation was made up by the addition of distilled water up to the original level. samples of water were removed at regular intervals and analyzed for sulphate, chloride, nitrate and phosphorus using standard chemical methods (anonymous 1973). the nutrient uptake by the plants was then calculated, based on the amount of nutrient left in the medium. a parallel series of experiments were conducted using tap water (43.6 ppm chloride, 656 ppm sulphate, 3 ppm phosphorus) instead of the complete solution. the fresh weight of water hyacinth used here was 612 g while that of water spangle was 555 g. leaching experiments were done by leaving 100 g fresh weight each of water hyacinth and water spangle in basins to rot. at regular intervals the rotting plants were washed with 300 ml distilled water and the water analyzed for sulphate, c hloride, nitrate and phosphorus. results and discussion figure 1 shows the removal of chloride, sulphate, phosphorus and nitrate from the complete media by water spangle and water hyacinth on a per gram fresh weight basis with time. chloride removal was rapid, 0.54 mg and 0.29 mg were removed 26 removal and leaching of nutrients – y.c. wee & h.h. yeoh figure 1. removal of nutrients (mg g fr wt -1 ) by water hyacinth (•) and water spangle (o) from hoagland's complete solution with time. 27 biotropia no. 2, 1988/1989 by water spangle and water hyacinth, respectively, by the third day. the former removed 0.57 mg of the chloride after 16 days, after which there was a net decrease in chloride removal, perhaps due to the leaching of the anion into the growing medium from decaying leaves. water hyacinth however, showed a constant and gradual increase in removal of the anion with time, resulting in 0.57 mg removal after 40 days. sulphate was also efficiently removed; within three days water spangle removed 7.88 mg and water hyacinth 4.43 mg of the sulphate present. phosphorus was slowly removed from the nutrient solution by both plants. water spangle took 33 days to remove some 0.53 mg of the phosphorus while water hyacinth removed about 0.43 mg. nitrate removal showed a rather erratic course. it was rapidly removed during the first 12 days by both plants, after which there was a net decrease in nitrate removal, attributed to leaching of the nutrient from the plants before a further increase. the data showed that the anions, chloride, sulphate and nitrate were rapidly removed while phosphorus uptake by these plants was generally slower. water spangle and water hyacinth grown in tap water also showed uptake of chloride and sulphate (figure 2), but the profiles were different from those of plants grown in figure 2. removal of nutrients (mg g fr wt -1 ) by water hyacinth (•) and water spangle (o) from tap water with time. 28 removal and leaching of nutrients-y.c. wee & h.h. yeoh the complete medium. it took water spangle and water hyacinth 22 days to remove 0.19 mg and 0.23 mg of the chloride respectively after which removal was very gradual. sulphate removal, however, was slightly more efficient. by 16 days, 5.22 mg and 5.07 mg were removed by water hyacinth and water spangle respectively. there were no further removals of sulphate after this period. no data were available for phosphorus and nitrate as there was only a trace of the former and no trace of the latter at all in the tap water. rotting plants were also observed to release chloride, phosphorus and nitrate into the water (figure 3). these were found to increase with time over a period of figure 3. nutrients leached into distilled water (mg g fr wt -1 ) during decay of water hyacinth (•) and water spangle (o). 29 biotropia no. 2, 1988/1989 18 days, after which the plants were totally disintegrated. sulphate released by both plants was negligible. the general profiles for both water spangle and water hyacinth were much the same. the use of aquatic plants to bring eutrophic waters into proper nutrient balance is well known (steward 1970). so far, most have been with water hyacinth. their abilities to remove phosphorus from sewage effluent (ornes and sutton 1975) and nitrogen and phosphorus from eutrophic waters (duningan, phelan and shamsuddin 1975) have also been reported. wolverton and mcdonald (1976) showed that in an experimental lagoon enriched with sewage effluent, these plants can reduce the pollutant level by 75-80%. the plants can also absorb toxic heavy metals like gold, silver, cobalt, strontium, cadmium, nickel, lead and mercury (wolverton and mcdonald 1976). the excessive presence of nitrogen and phosphorus is the main cause of eutrophication and the consequent prolific growth of aquatic weeds (sheffield 1967). thus removal of these primary nutrients can significantly reduce weed growth. a single water hyacinth plant has been shown capable of absorbing over 3 mg of phosphorus per day (rogers and davis 1972). however, haller and sutton (1973) showed that the maximum accumulation of 9.07 mg phosphorus per g dry plant weight was seen after four weeks when the phosphorus content of the growing medium was around 40 ppm. results from the experiments show that the presence of water spangle and water hyacinth in the two reservoirs can be put to advantage if they are allowed to proliferate, regularly harvested and the harvested plants dumped. the ability of these plants to remove nutrients as well as pollutants rapidly from the water means that with every harvesting, the water gets a little cleaner. in time, the purity of the water would be such that it would not be able to support the excessive growth of these weeds. however, low nutrient status does not necessarily mean that growth will be eradicated as it has been shown that water spangle can survive for long periods under conditions of severe limiting nutrient status (gaudet 1973). the above practice will need to go hand in hand with the cleaning of the reservoir's catchments, which, since 1977, has been in progress as a result of resettlement of pig farms elsewhere (wee and corlett 1986). however, the past practice of dumping the harvested plants along the edge of the reservoirs just to save cost of transportation was counter-productive in that, nutrient leached from the rotting plants gets back into the water to support growth of a new crop of plants. data collected in the above experiments should go a long way in convincing administrators the advantages of investing in transportation costs in the long term. 30 removal and leaching of nutrients-y.c. wee & h.h. yeoh acknowledgments this project was funded by a grant from the singapore institute of biology. references anonymous, 1973. chemical methods of water testing. bdh chemicals ltd., poole, england. 47 pp. 2nd ed. dunioan, e.p., r.a. phelan, and z.h. shamsuddin, 1975. use of waterhyacinths to remove nitrogen and phosphorus from eutrophic waters. hyacinth control journal 13: 59-61. gaudet, jj. 1973. growth of a floating aquatic weed, salvinia, under standard conditions. hydro-biologia, 41: 77-106. haller, w.t. and d.l. sutton. 1973. effect of ph and high phosphorus concentrations on growth of water hyacinth. hyacinth control journal 11: 50-61. hewitt, e.j. 1952. sand and water culture methods used in the study of plant nutrition. commonwealth bureau of horticultural plantation crops, tech. comm. no. 22. mitchell, d.s. and n.m. tur. 1975. the rate of growth of salvinia molesta (s. auriculata auct.) in laboratory and natural conditions. journal of applied ecology 12: 213-225. ornes, w.h. and d.l. sutton. 1975. removal of phosphorus from static sewage effluent by water-hyacinth. hyacinth control journal 13: 56-58. penfound, w.m. t. and t.t. earle. 1948. the biology of the water hyacinth. ecological monograph 18: 448-472. rogers, h.h. and d.e. davis. 1972. nutrient removal by waterhyacinth. weed science 20: 423-427. room, p.m. 1984. biological control of floating weeds in australia. paper presented at sympos. in weed science, april 1984, bogor, indonesia. 2 pp. sheffield, c.w. 1967. water hyacinth for nutrient removal. hyacinth control journal 6: 27-30. steward, k.k. 1970. nutrient removal potentials of various aquatic plants. hyacinth control journal 8: 34-35. wee, y.c. 1986. aquatic weed problem in singapore's freshwater reservoir. in: biotrop special publication no. 24, symposium weed science (eds. j.v. pancho, s.s. sastroutomo and s. tjitrosemito) 25-29. wee, y.c. and r. corlett. 1986. the city and the forest: plant life in urban singapore. singapore university press. widyanto, l.s. 1976. studies on the growth and control of waterhyacinth (eichhomia crassipes (mart.) solms). proceedings of the 5th asian-pacific weed science society conference, tokyo. 429-434. wolverton, b. and r.c. mcdonald. 1976. don't waste waterweeds. new scientist 71: 318-320. 31 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 685 siti mariam (light-color).cdr light-color-induced changes in fatty acid biosynthesis in chlorella sp. strain ks-ma2 in early stationary growth phase 1 2 3 4 siti-mariam osman , tse seng chuah , saw hong loh , thye san cha and aziz ahmad 1* 1 school of fundamental sciences, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 2 school of science and food technology, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 3 school of marine and environmental sciences, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 4 institute of marine biotechnology, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysi a received 08 august 2016/accepted 24 june 2017 abstract optimization of light supply remains a critical issue in microalgae biotechnology. the impacts of light color on fatty acid production and biosynthesis in microalgae are poorly understood. the aim of this study was to determine the effect of light color on growth and fatty acid content in chlorella strain ks-ma2. cells were cultured on f/2 medium and incubated under blue, green, red or white light. the cells' growth, fatty acid composition and the expression levels of the ketoacyl synthase 1 (kas-1), omega-6 desaturase (ω-6 fad) and omega-3 desaturase (ω-3 fad) genes were measured at the early stationary growth phase. results of this study indicated that light color affected cell density and fatty acid profile produced by chlorella sp. strain ks-ma2. cells cultured under blue, red and white light had higher cell density than those cultured under green light. palmitic acid (38.62 ± 3.29% of biomass dry weight) and linolenic acid (7.96 ± 0.88% of biomass dry weight) were highly accumulated under white light. stearic acid was dominant under blue light (11.11 ± 0.14% of biomass dry weight), whereas oleic acid was dominant under red light (30.50 ± 0.14% of biomass dry weight). linoleic acid was highly produced under green and blue light (28.63 ± 1.36% and 26.00 ± 0.81 % of biomass dry weight, respectively). kas-1 and ω-6 fad were highly expressed under blue light, whereas ω-3 fad was highly expressed under green light. the production of particular fatty acids of interest from chlorella could be achieved by shifting color of light used during the incubation of the cell cultures. blue-light is the most suitable light color for producing biomass and stearic acid by chlorella strain ks-ma2. keywords: light spectrum, linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid introduction demand for microalgae as food, animal feed supplements, feedstock and biofuel is increasing tremendously (wacker et al. 2016). the success of algae-based industrial products depends on high biomass productivity and quality with low production cost (abu-ghosh et al. 2016). biomass productivity is closely interrelated with the amount of light energy received, which is processed and stored in chemical compounds (chen et al. 2015; liu et al. 2012). sunlight is the most cost-effective energy source for microalgae production, but it has drawbacks, including changes in weather, day and night cycles, and seasonal changes, which affect light intensity and spectrum (abu-ghosh et al. 2016). optimization of light supply remains a critical issue in microalgae biotechnology. varying light intensity might reduce the photosynthetic rate, hindering microalgae growth due to shortage of energy (ge et al. 2013). light intensity also contributes to t e m p e r a t u r e c h a n g e s a n d a f f e c t s t h e accumulation of polyunsaturated fatty acids on * corresponding author: aaziz@umt.edu.my biotropia 5 1 8 33 42 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.685 33 microalgae (wacker et al. 2016; piepho et al. 2011). cell division is an energy-consuming process and microalgae tend to accumulate sufficient energy before proliferation (zachleder et al. 2016) the compounds of energy storage, such as lipids and starch in particular, tend to accumulate in the algal cells under stress conditions, when cell growth halts (chen et al. 2015). in addition, light intensity can affect microalgae physiological characteristics, including pigment and chemical composition, growth rate, ion transport (kwon et al. 2013) and lipid classes (wacker et al. 2016; liu et al. 2012). sun et al. (2014) showed that the total lipid content of neochloris oleoabundans increases when the light intensity is increased from 50 to 200 µmol/m /s, 2 but decreases with light intensity higher than 200 µmol/m /s. the rate of photosynthetic activity 2 declines during limited light, but linearly increases with increasing light availability (choi et al. 2013). generally, light intensity refers to the number of photons (energy) per unit area and unit time. the energy of each photon is inversely proportional to the wavelength of the associated electromagnetic wave i.e. the shorter the wavelength, the more energetic the photon. for example, the energy of a photon of blue light ( = λ 450 nm) is 2.76 ev, while the energy of a photon of red light ( = 700 nm) is 1.8 ev. white light λ contains a spectrum of visible light with different wavelengths. microalgae usually absorb photons in the range of 400 to 700 nm for photosynthesis; the wavelengths absorbed are species-specific (kim . 2014). therefore, light color has been et al shown to have a significant impact on microalgae productivity. for example, de mooij . (2016) et al showed that the highest productivity of chlamydomonas reinhardtii is obtained under yellow light, whereas the lowest is obtained under blue and red light. the lowest biomass of spirulina platensis culture is obtained with blue led, and the highest is obtained with pink and red led (markou 2014). on the other hand, ge et al. (2013) found that red light enhances biomass production of . however, the chlorella vulgaris impacts of light color on fatty acid production and expression of fatty acid biosynthesis genes, particularly in microalgae, are poorly understood. previous research has focused on the photoautotrophic production of microalgae oil using white light as energy source. however, no explicit data are available concerning the differences in cell proliferation and energy accumulation with different wavelengths of light. the objectives of this study were to determine the effects of different-colored light on growth, fatty acid composition, total oil content and expression of genes for fatty acid biosynthesis proteins ketoacyl-acp synthase-i ( ), kas-1 omega-6 desaturase ( ) and omega-3 ω-6 fad desaturase ( ) in strain ks-ma2. ω-3 fad chlorella kas is required for the addition of malonyl-coa in the elongation of butyryl-acp from a 4to 14carbon chain (kainou 2006). fad enzymes et al. ( fad and fad) are involved in the ω-6 ω-3 insertion of double bonds at the and ω-6 ω-3 positions of pre-formed fatty acid chains in reactions requiring oxygen, nadh and cytochrome and reducing equivalent (zhang b5 et al. . 2016) materials and methods a stock culture of mangrove-isolated chlorella strain ks-ma2 was obtained from the microalgae stock culture collection of the universiti malaysia terengganu, malaysia and used in this study. the chlorella cells were maintained on f/2 medium (guillard & ryther 1962) agar plates, which were prepared using natural seawater with salinity of 30 mg/l. medium was solidified with 10 g/l of bacteriological agar and sterilized by autoclaving at 121 °c for 20 minutes. a single colony was streaked onto a sterilized agar plate and subsequently incubated in a growth chamber at 25 o with 55 c under 24 hour white-light illumination 70 µmol photons/m/s light intensity with constant aeration. this culture was considered the chlorella stock culture and was re-streaked onto a newly prepared agar plate every two months to maintain the cells in the institute of marine biotechnology, universiti malaysia terengganu. light color treatments 6 chlorellaa total of 5 × 10 cells/ml of strain ks-ma2 was aseptically transferred into 2.5 l freshly prepared f/2 medium. the salinity level was adjusted to 30 mg/l. the cultures were incubated at 25 ± 1 c, with illumination for 24 o hous per day at 55 70 µmol photons/m/s light intensity and constant aeration. the light colors used during incubation were white (control; λ = 400 750 nm), red (λ = 650 nm), blue (λ = 475 nm) 34 biotropia vol. 25 no. 1, 2018 35 and 350 ml/minute, respectively. the temperatures were programmed as follows: an o initial temperature of 35 c for 0.5 minute; o o increased to 195 c at a rate of 25 c/minute; o subsequently increased to 205 c at a rate of 3 o c/minute; and finally increased to 320 o o c/minute at a rate of 8 c/minute and held for 6.64 minutes. the fatty acid components were identified by comparing their retention time and fragmentation pattern with established standards. the supelco 37 component fame reference standard mix (sigma-aldrich) was used to identify and quantify the percentage of cisand trans-fatty acid isomers in the samples. examination of gene expression levels by real-time pcr total rna was isolated with gf-1 total rna extraction kit (vivantis) according to the manufacturer's instructions and treated with dnase i (fermentas) to remove contaminating dna. dna-free rna was confirmed by pcr amplification of the 18s rdna gene using the rna as templates. subsequently, 1 μg of the rna was reverse transcribed with iscript reverse transcription supermix (bio-rad) in accordance with the manufacturer's instructions. the cdna generated was directly used for quantitative realtime pcr. the real-time pcr was performed in a myiq single color real-time pcr detection system (bio-rad) using sybr green real-time pcr master mix (bio-rad) according to the manufacturer's instructions. the pcr mixture consisted of cdna (50 ng, 1 l), 0.4 μ μm final concentration of each forward and reverse primer (table 1), 10 l 2× iq sybr green supermix μ and nuclease-free water to the final volume of 20 μl. after heating at 95 °c for 15 seconds, the real or green (λ = 510 nm) provided by a fluorescent lamp source covered with colored cellophane. cell densities were measured every two days until a constant reading was obtained, indicating the cells had attained early stationary phase, and the cells were later harvested. three replications were prepared for each treatment. at harvest, cell numbers, biomass as dry weight, and total oil and fatty acid content were determined. the cell numbers and absorbance at 600 nm were measured on a 10-fold serial dilution of culture using a haemocytometer (neubauer) and biophotometer (bio-rad), respectively, in triplicate. cell density was calculated based on plotted calibration curve at 600 nm. quantification of oil and fatty acid contents oil extraction and esterification of fatty acids to methyl esters (fames) was carried out according to the modified methods of cha et al. (2011). oil was extracted from a dried sample of chlorella cells. a 500 mg of sample was vortexed in 10 ml of hcl (concentrate) for 3 minutes and subsequently boiled using double-boiling technique for 30 minutes. after cooling to room temperature, the oil were extracted with 25 ml hexane for 1 minute and repeated twice with 15 ml hexane. extracts containing fames were combined, hexane residue was removed using vaporization with a rotavapor r-210/215 o (buchi). the extracted oil was incubated at 80 c until a constant weight was achieved. fames were analyzed using a gas chromatograph (gc-6890, agilent) equipped with a flame ionization detector fitted with a capillary column (db-225ms, supelco). the injector temperature was set at 250 o c, and the flow rate of carrier gas (he) was 2.4 ml/minute. the flow rate for h gas and air were 2 primers name primers sequence (5' 3") size of amplicon (bp) 18s f1 5'-cct gcg gct taa ttt gac tca aca cg -3' 172 18s r2 5'-tag cag gct gag gtc acg ttc g -3' kas1 f5 5'-cca tga ttg gtc att gct tgg gag c -3' 151 kas1 r5 5'-gct ctt gct tca tgt ttg gga cca c-3' o6d f3 5'-ctt cac cca cga agg cac agg c -3' 129 o6d r3 5'-cct gca cac tgc tgg gaa cg -3' o3d f2 5'-cat gtt gag aac gac gag tcc tgg tat c -3' 162 o3d r2 5'-gtc aaa gtg gga gcc agt ctt gc -3' 35 fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. table 1 forward and reverse primers for quantitative real-rime pcr b a b time pcr amplification was programmed for 40 cycles of 95 °c for 35 seconds, 64.2 °c for 35 seconds and 72 c for 30 seconds. specificity of all o pcr amplifications was verified by a melting curve at the completion of each run from 55 to 95 °c at 0.5 °c increment. the gene expression data were analysed using the 2 method (schmittgen −δδct & livak 2008; livak & schmittgen 2001). the data obtained were represented as the fold change in the target gene in the treatments relative to the control and normalized to the expression level of the 18s rrna reference gene. statistical analysis differences in total oil and fatty acid content among the light-color treatments of the chlorella strain were statistically analysed by one-way anova using spss version 16.0. significant differences among means were identified by tukey's test at p = 0.05. (www.ibm.com/software/ analytic/spss). results and discussion growth of chlorella results of this study showed that light color did not affect the cell growth of chlorella strain ks-ma2. nevertheless, light color changed fatty acid composition. increased cell proliferation led to the increase of cell densities and biomass obtained. cell densities varied among light treatments (fig. 1). cells cultivated under blue light reached early stationary phase after 16 days of culture. cells cultivated under green, white and red light reached early stationary phase after 18, 20 and 22 days of culture, respectively (fig. 1a). comparatively, cells incubated under red light took longer to attain stationary phase compared to those under other light colors. cell densities in red-, whiteand blue-light treatments did not differ significantly (p > 0.05). cell numbers recorded were fluctuated from 2.08 10 cells/ml 8 to 1.72 10 cells/ml. biomass dry weight 8 36 biotropia vol. 25 no. 1, 2018 figure 1 effect of light colors on: (a) cell density and biomass at the early stationary phase and (b) growth of chlorella strain ks-ma2 on f/2 medium (note: bars represent cell densities and lines represent biomass dry weight (mean ± sd; n=3); values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) obtained also did not significantly differ (p > 0.05; fig. 1b). cell proliferation is dependent on the available energy in forming atp or nadph. the available energy is derived from photosynthetic activity (zachleder et al. 2016). in addition, photosynthetic efficiency is associated with photon energy, which is inversely proportional to light wavelength (kim et al. 2014), i.e. the shorter the wavelength, the more energetic the photon (choi et al. 2013). thus, blue light is more efficient for photosynthesis and lipid biosynthesis (markou 2014). however, blue light appeared to cause photo-inhibition, inducing cell damage and reduced the time to attain early stationary phase. blue light has been reported to up-regulate the lhcx1 protein and zeaxanthin epoxidase, a proteins involved in photoprotection in phaeodactylum tricornutum (costa et al. 2013). on the other hand, red light has longer wavelength than blue light and thus, might prevent photoinhibition (george et al. 2014; xu et al. 2013). therefore, the highest cell density and biomass were obtained in the red-light treatment (fig. 1b). similar findings have been reported for scenedesmus sp. (kim et al. 2013) and c. vulgaris (xu et al. 2013; yan et al. 2013). however, different algal classes have different light requirements for growth and photosynthesis (kwon et al. 2013; cheirsilp & torpee 2012). total oil content and fatty acid composition total oil content (fig. 2) and fatty acid composition (fig. 3 & 4) varied among the light color treatments (p < 0.05). cells under blue-light and white light treatments contained the highest percentage of total oil, up to 22.6% of total dry weight (fig. 2). this was also 3.4and 3.7-fold higher than that of cells under redand greenlight treatments, respectively. saturated fatty acid accumulation did not differ significantly ( > 0.05) p in the treatments of white-, blueand green-light (fig. 3a), but it was higher than the red-light. meanwhile, monounsaturated fatty acids were highly accumulated under the red-light treatment (30.9%) (fig. 3b). this accumulation was 1.19-, 1.44and 1.47-fold higher than under white, blue and green light, respectively. in contrast, cells cultured under green light produced higher proportions of polyunsaturated fatty acids (pufas: 34.5%) compared to those under white (25.7%) or red (30.3 %) light (fig. 3c). however, this value did not significantly differ ( > 0.05) p from the blue-light treatment (32.2%). figure 4 shows the relative amount of the five major fatty acids i.e. palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, in chlorella strain ks-ma2 cultured under different light colors. palmitic acid (38.6%) and linolenic acid (7.9%) were highly accumulated ( < 0.05) p under white-light conditions (fig. 4a & 4e) compared to the other treatments. meanwhile, the accumulation of stearic acid (11.1%) was the highest under blue-light treatment, 2.3-fold higher than that under white light (fig. 4b). conversely, oleic acid (30.5%) was highly accumulated under red light (fig. 4c), and linoleic acid (28.6%) was highly accumulated under green light (fig. 4d). results of this study are in line with previous reports stating that different light colors change the total oil accumulated (markou 2014; liu et al. 2012; marchetti et al. 2012) and fatty acid profile (wacker et al. 2016; piepho et al. 2011). light with higher 37 fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. figure 2 effect of light color on total oil (mean ± sd) (note: values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) 38 biotropia vol. 25 no. 1, 2018 energy, such as blue or white light, might increase the atp and nadph available for triacylglycerol synthesis (fig. 2). addition of carbon in the elongation of fatty acid chains requires nadph, derived from photosynthesis (chen et al. 2015). moreover, blue light is used for enzyme activation and regulation of gene transcription (das et al. 2011). high transcription of kas-1 under bluelight treatment (fig. 5a) might be due to the increased photosynthetic activity. a b c figure 3 effect of light color on: (a) saturated fatty acid; (b) monounsaturated fatty acid; and (c) pufa content at the early stationary phase of chlorella strain ks-ma2 cultured on f/2 medium (mean ± sd; n=3) (note: values followed by the same letter were not significantly different by tukey's test with p = 0.05 significance level) 39 therefore, more carbon flux generated from photosynthesis is channelled to lipid biosynthesis (liu 2012), involving the elongation of fatty et al. acid chains; thus, resulting in higher accumulation of unsaturated fatty acids (fig. 3a), the palmitic acid (fig. 4a) and stearic acid (fig. 4b) under high energy light. in addition, higher total oil production is important for light-stress adaptation (choi 2013). the lipid layers et al. function as a light filter to reduce irradiation on the cell components, to prevent photo oxidative damage and to reduce water loss (liu 2012)et al. . fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. a b e c d figure 4 effect of light color on: (a) palmitic acid; (b) stearic acid; (c) oleic acid; (d) linoleic acid; and (e) linolenic acid content at early stationary phase of chlorella strain ks-ma2 cultured on f/2 medium (mean ± sd; n=3) p(note: values followed by the same letter were not significantly different according to tukey's test with = 0.05 significance level) 40 biotropia vol. 25 no. 1, 2018 expression levels of kas-1, -6 fad and -3 ω ω fad results of this study showed that kas-1 (fig. 5a) and ω-6 fad (fig. 5b) were highly transcribed under blue light. it was 5.3and 2.2-fold of the reference gene, respectively. the ω-3 fad was more highly transcribed under green light (2.8fold of the reference gene; fig. 5c). this was 3.8-, 1.8and 2.3-fold higher than that under white light, respectively. these genes exhibited the lowest transcription levels under red-light treatment. fatty acid desaturation is important for microalgae tolerance toward strong light (choi et al. 2013; solovchenko et al. 2008). pufas are a b c figure 5 effect of light color on mean fold change in relative expression of: (a) kas-1; (b) ω-6 fad; and (c) ω-3 fad of chlorella strain ks-ma2 (mean ± sd; n=3) (note: values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) 41 necessary for the maintenance of photosynthetic membrane function. pufas also play an important role in acclimation to low light conditions (solovchenko 2008). high pufa et al. content obtained in blueand green-light was mainly contributed by linoleic acid (fig 4d). the -α linolenic (fig. 4e) produced in white-light was the least i.e. 8 % of biomass dry weight. blue light also induced the expression of acyl-lipid desaturase (kis 1998), which might be et al. responsible in higher accumulation of -linolenic α (fig. 4e) and transcription levels of (fig. ω-6 fad 5b). on the other hand, higher transcription levels of under green-light (fig. 5c) did not ω-3 fad contribute to the accumulation of -linolenic acid. α the increase of and transcription kas-1 ω-6 fad levels might be associated with photo-protection against higher light intensity (yoshioka 2012). et al. nonetheless, knowledge on the post translation of these genes is remained unknown. this study showed that there was inconsistent requirement of light in production among microalgae pufas species (wacker et al. 2016). this species-specific l i g h t a c c l i m a t i o n s t r a t e g y r e f l e c t e d t o photosynthetic factors and differences in light spectra. conclusions light color played a major role in the proliferation of chlorella strain ks-ma2 and its fatty acids profile. mass production of chlorella biomass could be obtained under blue-light conditions, whereas total oil was better obtained under whiteor red-light conditions. higher production of specific fatty acids could be achieved using different light color during the cultivation of cells i.e. white light for palmitic acid and linolenic acid, blue light for stearic acid and red light for oleic acid. linoleic acid accumulation was not influenced by light color. the transcription level of kas-1 and ω-6 fad were highly activated by blue-light, while the transcription level of ω-3 fad was highly activated by green-light. acknowledgements this study was funded by science fund project under the ministry of agriculture of malaysia (grant number sf-05-01-12-sf0007). references abu-ghosh s, fixler d, dubinsky z, iluz d. 2016 flashing light in microalgae biotechnology. bioresour technol 203:357-63. cha ts, chen jw, goh eg, aziz a, loh sh. 2011. differential regulation of fatty acid biosynthesis in two species in response to nitrate chlorella treatments and the potential of binary blending microalgae oils for biodiesel application. bioresour technol 102:10633-40. cheirsilp b, torpee s. 2012. enhanced growth and lipid production of microalgae under mixotrophic culture 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24:721-32. yoshioka m, yago t, yoshie-stark y, arakawa h, morinaga t. 2012. effect of high frequency of intermittent light on the growth and fatty acid profile of isochrysis galbana aquaculture 338-341:111–7.. zachleder v, bisova k, vitova m. 2016. the cell cycle of microalgae. in: borowitzka, bearddall j, raven j, e d i t o r s. t h e p hy s i o l o g y o f m i c r o a l a g e : development in applied phycology volume 6. cham (zg): springer international publishing. available from: https://doi.org/10.1007/978-3319-24945-2_1 zhang y, wang h, zhang j, hu y, zhang l, wu x, liang ... b. 2016. the cytochrome b5 reductase hpo-19 is required for biosynthesis of polyunsaturated fatty acids in caenorhabditis elegans. biochim biophys acta 1861(4):310-9. doi: 10.1016/j.bbalip.2016. 01.009 42 biotropia vol. 25 no. 1, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia book juni revisi 14 juli 09.indd 45 biotropia vol. 16 no. 1, 2009: 45 54 corresponding author: cginting2001@yahoo.com physic nut (jatropha curcas l.) diseases in lampung province cipta ginting and tri maryono department of plant protection, university of lampung bandar lampung 34145, indonesia abstract intensifi ed cultivation of physic nut (jatropha curcas l.) could raise the importance of plant diseases. th e objectives of this research were to diagnose diseases occurring on physic nut in lampung province and to determine their intensity. field observation was conducted in four districts: south lampung, tanggamus, bandarlampung, and tulang bawang. disease intensity, whether expressed as disease incidence or severity, was recorded from plant samples determined by making diagonal lines across the fi eld on which fi ve observation spots were made. on each spot, fi ve plant samples were observed. specimens were also collected and placed individually in plastic bags for laboratory observation. th e diseases found on physic nut in lampung province were cercospora leaf spot, alternaria leaf spot, fusarium wilt, and bacterial wilt. in addition, leaf malformation fi rst thought to be viral disease was commonly found in many locations. further mechanical transmission failed to produce similar symptom on tested plants and higher population of mites were found on malformed leaves than that in normal leaves. based on the disease distribution and intensity, the most likely threatening disease in physic nut cultivation is bacterial wilt. fusarium also caused wilt, but it was only found in one subdistrict with low incidence. key words: alternaria, cercospora, fusarium wilt, bacterial wilt, jatropha curcas diseases introduction recently, eff orts to develop and utilize alternative energy sources have been increasing due to the need to meet energy demand and to reduce the dependence on fossil energy sources. utilizing plants as alternative sources is a priority because of being renewable energy resources. physic nut (jatropha curcas l.) is one of the prospective plants. it can grow on soil with medium and low fertility, its oil is inedible and of high quality for fuel (sudradjat 2006; syah 2006). physic or also known as “jarak pagar” in indonesia is used to be grown less intensive such as for fence. in the last few years, however, physic nut has been cultivated intensively in a monoculture pattern. for example, in katibang subdistrict, south lampung district, alone the acreage of physic nut was 192 ha (anon. 2007a). furthermore, the head of mining and energy department of lampung province stated that the potential area for physic nut planting in lampung province is about 5000 ha (anon. 2007b). banuwa (2007) also reported that lampung province has a good prospect to grow jatropha. 46 biotropia vol. 16 no. 1, 2009 change of planting pattern could increase the importance of some plant diseases. intensifi cation of a plant generally tends to increase the threat of pests and diseases (fry 1982; agrios 2005). plants that are grown less intensive usually do not suff er severely from pests and pathogen attack. however, when they are cultivated intensively such as in intensive monoculture pattern, plants are of high risk to be attacked severely by pests and diseases, especially when they are not properly managed. accordingly, the change in cultivation pattern of physic nut into intensive monoculture pattern should be followed by raising awareness about its pests and diseases. information about physic nut diseases in literature is very limited. lozano (2007) listed pathogens infecting the plant without mentioning their geographical distribution, including phytophthora spp., pythium spp., and fusarium spp., heminthosporium tetramera, pestalotiopsis paraguarensis, p. versicolor, and cercospora sp. it has also been reported that several pathogens infect physic nut in indonesia: ralstonia solanacearum, rhizoctonia bataticola (republic of indonesia department of agriculture 2006a), phytophthora nicotianae (republic of indonesia department of agriculture 2006b), alternaria sp. and cercospora sp., fusarium sp., botrytis and xanthomonas campestris (hambali et al., 2006; prihandana and hendroko 2006). latha et al. (2009) reported the occurrence of root and collar rot diseases of physic nut in india. to our knowledge, there is no published information on diseases of physic nut in lampung province so far. th e objectives of this research were to diagnose diseases occurring on physic nut in lampung province and to determine the intensity of the disease in the fi eld. materials and methods observation in the fi eld th e disease diagnosis started with fi eld observation of physic nut diseases. field observation was conducted in four districts, considered to be the most important areas of physic nut cultivation in lampung, and in each district two subdistricts were visited. th e districts and subdistricts were south lampung (natar and ketibung subdistricts), tanggamus (gunung alip and talang padang), bandarlampung (rajabasa and tanjung seneng), and tulang bawang (tanjung raya and simpang pematang). due to wide geographical distribution of physic nut fi elds, some fi elds were only visited once or twice. observation was focused on symptoms of diseases as they occur in the fi eld and factors that might aff ect the diseases such as plant age, plant spacing (row width), plant growth, plant canopy, and topography. to determine the intensity of diseases, plant samples were selected with the following procedure. two diagonal lines were made across the fi eld on which fi ve observation spots were made. on each of fi ve spots (x) fi ve plants were observed. for systemic diseases, the disease intensity was expressed as disease incidence determined by using the formula of di = n/n x 100%, where di = disease incidence, n = number of plants showing symptoms, and n = the total number of plants observed. 47 jatropha curcas diseases – cipta ginting et al. for local diseases, the disease intensity was expressed as diseases severity determined by the formula of where ds = disease severity, v = disease score of sample ith, n = number of plant samples with a particular score, v = the highest score, and n = total number of samples. disease score range from 0 (no symptom) to 4 (more than 50% of leaf surface showed symptoms). regarding the kinds of the diseases, in addition to the plant samples, any plants showing symptoms were observed. at fi rst the distribution of diseased plants was determined whether it was evenly distributed in the fi eld or it was in clustered (made gradient). th en, for each diseased plant it was determined whether the symptoms were local or systemic. specimens, whole or part of plant samples showing symptoms, were collected and placed individually in plastic bags. th e specimens were placed in a cooler during transportation to the laboratory for further examination. observation in the laboratory each of the specimens was observed in the laboratory soon upon arrival or the next day. if the suspected pathogen causing particular symptoms was a fungus, the specimen was incubated at relative humidity of 100% by placing sterile cotton dampened with sterile water in plastic bags or trays covered with plastic. pathogenic fungi usually form spores in 2 – 3 days at room temperature. th e spores and other structures were observed with a compound microscope. some other specimens were used to isolate infecting fungi. for this purposes, the media used was potato dextrose agar (pda) supplemented with lactic acid 1.4 ml per liter media. small pieces (1 – 2 mm) of plant tissue between the healthy and the infected tissue were taken and then placed on the media in petri dishes. fungi growing in the media was transferred into new media and identifi ed to genus. identifi cation was based on barnett and hunter (2001). diseased plants suspected to be infected by virus was diagnosed from the symptoms. to verify whether or not the causal agent was a virus, the suspected agent was transmitted mechanically. further observation was also conducted to determine whether causal agent other than virus might cause the symptoms. on the malformed leaves, mites were found. to determine whether or not the severity of leaf malformation was correlated to the population of mites, the leaves were divided into three groups, i.e.: leaves with severe symptoms, leaves with mild symptoms, and those without symptoms. from each category, the number of mites was determined. if the symptom was suspected to be caused by pathogenic bacteria, diagnosis was based on internal and external symptoms. in addition, eff ort was made to observe bacterial ooze. to do this, a few steam cuts were placed on test tubes containing tap water. about 30 minutes later, bacterial ooze was streaming out of the tissue. 48 biotropia vol. 16 no. 1, 2009 results and discussions locations and cultivation manners table 1 summarizes the locations and cultivation manners of physic nut fi elds in four districts. it shows that the cultivation system including plant age, row width, and planting pattern was similar in the subdistricts of every district. however, the cultivation system greatly varied among districts. th e area used for physic nut cultivation is generally fl at except that of ketibung, south lampung. physic nut was cultivated in a monoculture system in all fi elds except in tulang bawang district where it was grown in mixed culture with rubber and oil palm plants. physic nut generally grew well except in both fi elds of tulang bawang district and tanjung seneng where it grew poorly. in bandarlampung, only two physic nut fi elds were found and those were in rajabasa and tanjung seneng subdistricts. in addition to these two fi elds, there were a few locations where seedlings are produced for sale. in tulang bawang district, three fi elds were reported to be planted with physic nut. however, when we visited the locations it appeared that one fi eld was grown with another jathropa species locally known as “jarak kepyar”. th erefore, only two fi elds about 0.5 ha, each was observed which are located in tanjungraya and simpang pematang subdistrits. table 1. locations and cultivation manners of physic nut in the field observed in four districts in lampung province locations cultivation manners plant age (months) row width (m) planting pattern land condition south lampung district: natar subdistrict ketibung subdisrict 8 9 2 x 2 monoculture fl at 2 x 2 monoculture aslant tanggamus district: gunung alip subdistrict talang padang subdisrict 6 1 x 1 monoculture fl at 6 1 x 1 monoculture fl at bandarlampung district: rajabasa subdistrict tanjung seneng subdisrict 6 1 x 1 monoculture fl at 12 1 x 1.25 monoculture fl at tulang bawang district: tanjung raya subdistrict 2 1 x 1.25 mixed culture with rubber plants fl at simpang pematang subdisrict 2 1 x 1.25 mixed culture with oil palm plants fl at physic nut diseases th e disease and disorder of physic nut found in lampung were cercospora leaf spot, bacterial wilt, alternaria leaf spot, fusarium wilt, and leaf malformation (table 2). cercospora leaf spot was found in all fi elds except in tanjung seneng, bandarlampung. th e diagnosis of cercospora leaf spot was conducted by fi eld observation followed by 49 jatropha curcas diseases – cipta ginting et al. laboratory examination. th e form of leaf spot was irregular. th e leaf spot was whitish brown in the center with brown at the edge. th e spot was a necrotic tissue that was encircled by chlorotic tissue seen as yellowish zone (figure 1). in the laboratory, fungal reproductive structure taken from the leaf spot was identifi ed as cercospora based on the identifi cation key used (barnett and hunter 2001). cercospora is reported to cause such leaf spot on physic nut and the species is reported to be c. ricinella (hambali et al. 2006) and c. jatrophae (prihandana and hendroko 2006). based on this observation and reference, it was concluded that the disease was cercospora leaf spot caused by cercospora sp. figure 1. symptom of cercospora leaf spot. the symptom was described in the teks. alternaria leaf spot was found only in tanjung seneng. diagnosis of alternaria leaf spot was also conducted in the fi eld and confi rmed in the laboratory. th e symptoms of this disease are similar to that of cercospora leaf spot except that all spot areas are brown in color without whitish area (figure 2). in addition, after 2-day incubation at 100% relative humidity at room temperature, conidia were found in the spot area. th e conidia consisted of several dark cells. according to the identifi cation key (barnett and hunter 2001), the conidia were formed by alternaria. alternaria ricini was reported to be the causal agent of leaf spot of physic nut (hambali et al. 2006). based on the observation in the fi eld and in the laboratory by hambali et al. (2006), it was concluded that the disease was alternaria leaf spot caused by alternaria sp. figure 2. symptom of alternaria leaf spot (arrow). 50 biotropia vol. 16 no. 1, 2009 another disease of physic nut was wilt caused by a fungus, fusarium. th e disease was only found in tanjung raya subdistrict, tulang bawang district. th e symptoms of the disease were the aff ected plant became wilt and the leaves remained attached to the plants. isolation was done on pda containing lactic acid (1.4 ml/l) media and the resulted culture was whitish in color. observation under a microscope revealed that there were two kinds of conidia, i.e.: macroconidia and microconidia. according to barnett and hunter 2001, the fungus is fusarium. hambali et al. (2006) reported that f. oxysporum could cause wilt symptoms in physic nut in the seedbeds as well as in the fi eld. bacterial wilt was found at least in one subdistrict of all districts in lampung province (table 2). th e main symptoms were that the aff ected plants became wilted and the leaves remained attached to the plants. th e base stem showed necrosis. internal observation revealed that the internal tissues showed brownish color in the middle parts (figure 3). when the stem was submerged in tap water, bacterial ooze streamed out of the plant tissue. th e bacterial ooze looked like white strand but when the water was shaking the bacterial ooze soon broke and the suspension became clouded. based on the external and internal symptoms as well as the bacterial ooze, it was assumed that the disease was bacterial wilt caused by pseudomonas (ralstonia). in addition to the diseases, a disorder called leaf malformation was observed on the leaves, mostly on the young ones. leaf malformation occurred in all fi elds except the fi elds in tulang bawang district. it was thought to be caused by a plant virus because several plants showed similar symptoms when attacked by certain plant viruses (agrios 2005; semangun 2000). some farmers also believe that the problem was caused by a virus. however, mechanical transmission failed to give positive results that the inoculated plants did not show any symptom after more than 4 weeks after inoculation. since no symptoms occur in the tested plants indicating that the agent could not be transmitted mechanically, further observation was conducted in two fi elds which were natar and rajabasa subdistricts. during 5-week observation in both fi elds, some plants showed reduced symptoms and the incidence of leaf malformation was decreased (table 2). furthermore, it was observed that mites were found to be associated with the disordered plants. when the population of mite was determined on leaves with severe, mild, and no symptoms, it was consistently found that mite population on leaves with symptoms was higher than that on leaves without symptoms (table 3). table 2. locations, diseases, and disease intensity of physic nut locations (date of first observation) diseases disease intensity (%) at 1 – 5 observations1) 1 2 3 4 5 south lampung district: natar subdistrict (6 juni 2007) cercospora leaf spot2) bacterial wilt3) leaf malformation3) 22 7 98 17 4 84 13 2 58 6 4) 44 6 33 ketibung subdisrict (8 oktober 2007) cercospora leaf spot leaf malformation 10 66 9 56 tanggamus district: sinar banten subdistrict (3 september 2007) cercospora leaf spot bacterial wilt leaf malformation 20 12 80 51 table 2. continued jatropha curcas diseases – cipta ginting et al. locations (date of first observation) diseases disease intensity (%) at 1 – 5 observations1) 1 2 3 4 5 talang padang subdisrict (3 september 2007) cercospora leaf spot bacterial wilt leaf malformation 21 32 40 bandarlampung district: rajabasa subdistrict (3 juni 2007) cercospora lea1f spot bacterial wilt leaf malformation 22 12 84 22 12 84 17 12 52 25 16 40 25 20 36 tanjung seneng subdisrict (7 agustus 2007) alternaria leaf spot2) leaf malformation 4 96 5 88 tulang bawang district: tanjung raya subdistrict (19 juni 2007) cercospora leaf spot fusarium wilt3) 5 8 4 6 3 5 simpang pematang subdisrict (1 oktober 2007) cercospora leaf spot bacterial wilt 3 13 2 11 1 9 note: 1)obervation was conducted 1 – 5 times. in any fi eld observed more than once, the observation interval was 2 week. 2)cercospora leaf spot and alternaria leaf spot were expressed as disease disease severity. th e formulas used to determine disease severity is stated in materials and method. 3)bacterial wilt, fusarium wilt, and leaf malformation were expressed as disease incidence. th e formulas used to determine disease intensity are stated in materials and method. 4) means no data. figure 3. part of wilted physic nut and longitudinal section of stem revealed brownish internal tissues showed. 52 biotropia vol. 16 no. 1, 2009 table 3. population of mites on leaves with or without symptom at two subdistricts observation total number of mites per 25 leaves spots1) severe symptom mild symptom no symptom natar subdistricts 1 25 16 7 2 34 30 5 3 25 30 8 4 15 18 4 5 19 25 6 average 23.6 23.8 6.0 rajabasa subdistricts 1 24 16 5 2 33 30 7 3 25 23 7 4 19 15 6 5 18 11 4 average 23.8 19.0 5.8 1)on each observation spot, 25 leaves of each category were observed. intensity of physic nut diseases disease intensity was determined by two methods, disease severity and disease incidence, according to the kind of diseases. disease severity was used for diseases local in nature such as leaf spot and disease incidence was used for systemic in nature such as wilt diseases and leaf malformation. table 2 shows that disease severity of cercospora leaf spot on physic nut was low at tulang bawang district where the plants were 2 months old. in the other three districts, where physic nut was 6 – 12 months old, the disease severity was relatively higher than that in tulang bawang. based on the severity of cercospora leaf spot and its distribution almost all fi elds were contaminated by the disease. more attention should be paid to the diseases of physic nut cultivation. alternaria leaf spot was only found in tanjung seneng subdistrict and its severity was 4 – 5% (table 2). based on the results, it could be concluded that alternaria leaf spot is unlikely to be the major disease of physic nut. fusarium wilt was found only in tanjung raya subdistrict, tulang bawang district. its incidence was 5 – 8% (table 2). based on the incidence its occurrence was relatively low, so the fusarium wilt could be regarded as a minor disease of physic nut. since it causes wilt that is very destructive, the disease including factors aff ecting disease development and transmission should be monitored. at locations where disease intensity was measured more than once with 2 week interval, disease intensity was either remained constant or tended to decrease overtime except alternatia leaf spot. it should be noted that, the observation was made started on may 2007 when the relative humidity was relatively high, and then it was decreasing during the period of observation. based on the data, wilting diseases (fusarium wilt and bacterial wilt) also tended to decrease. th e decrease was partly due to destructive 53 jatropha curcas diseases – cipta ginting et al. observation that some wilted plants were taken and brought to the laboratory for further observation. th ese uprooted plants were not counted at subsequent fi eld observation. th e most likely to be controlled in physic nut cultivation is bacterial wilt which is suspected to be caused by pseudomonas (ralstonia) solanacearum. th e disease was found in all districts visited. its incidence was 2 – 7% in natar subdistrict, 9 – 13% in simpang pematang subdistrict, and 12% and 32% in gunung alit and talang padang subdistricts, respectively (table 2). in addition, it causes wilt so that the disease potentially devastates the plants. according to the facts gathered from this study, it is interesting to note the recovery phenomenon shown by a plant. th e plant wilted as a result of attack by pseudomonas solanacerum. however, few months later, the plant recovered. one tends to assume that the recovery phenomena found in bacterial case might be due to the dry condition. th is phenomenon needs to be studied further to determine the factor involved. information obtained from this study is relevant to formulate disease control strategy. th e observation was conducted during the dry season in lampung province so that relative humidity was low. relative humidity is an important environmental factor aff ecting disease incidence and development and low relative humidity reduce the incidence and development of plant diseases (agrios 2005; fry 1982). similar observation should be conducted during the rainy season with high relative humidity. conclusions diseases found on physic nut in lampung province were cercospora leaf spot, alternaria leaf spot, fusarium wilt, and bacteial wilt. in addition, leaf malformation was also found in many locations. th e symptom was fi rst thought to be the result of a pathogenic virus attack. however, the symptom is not mechanically transmitted and the severity of diseases seemed to be correlated with the occurrence and population of mites. th e disease severity of cercospora leaf spot and alternaria leaf spot was relatively low to moderate. disease incidences of bacterial and fusarium wilts were also considered moderate. based on the distribution and intensity of the diseases, the most likely threatening disease in physic nut cultivation is bacterial wilt. fusarium also caused wilt, but it was only found in one subdistrict with low incidence. acknowledgments th e authors would like to thank the southeast asian regional centre for tropical biology (seameo biotrop) bogor for the fi nancial support through dipa 2007 and to didik purwanto and tedy achmad rijaya for technical assistance in the fi elds and in the laboratory. 54 biotropia vol. 16 no. 1, 2009 references agrios, g.n. 2005. plant pathology. 5th ed. elsevier academic press, burlington, m.a., as. anon.. 2007a. lamsel – petani budidayakan jarak. lampung post, april 16, 2007. anon.. 2007b. bahan bakar nabati: potensi lampung 724,354 hektar. lampung post, january 24, 2007. banuwa, i.s. 2007. prospek pengembangan tanaman jarak pagar (jatropha curcas l.) di provinsi lampung. paper presented at seminar held in bandar lampung, may 29, 2009. 14 pp. barnett, h.l. and hunter, b.b. 2001. illustrated genera of imperfect fungi. 5th ed. macmillan pub. co., new york. fry, w.e. 1982. principles of plant disease management. academic press, new york. hambali, e. suryani, a., dadang, hariyadi, hanafi e, h., reksowardojo, i.k., rivai, m., ihsanur, m., suryadarma, p., tjitrosemito, s., soerawidjaja, t.h., prawitasari, t., prakoso, t., and w. purnama. 2006. jarak pagar: tanaman penghasil biodiesel. penebar swadaya, depok. latha, p., v. prakasam, a. kamalakannan, c. gopalakrishnan, t. raguchander, m. paramathma and r. samiyappan. 2009. first report of lasiodiplodia theobromae (pat.) griff on & maubl causing root and collar rot diseases of physic nut (jatropha curcas l.) in india. australasian plant disease notes 4: 19 – 20. lozano, j. a. d. 2007. jatropha. www.gvedinternational.org/fi le_117/jatropha/ pdf.eng.pdf. accesed on february 18, 2008. prihandana, r. and r. hendroko. 2006. petunjuk budi daya jarak pagar. agromedia pustaka, jakarta. republic of indonesia , department of agriculture. 2006a. penelitian dan pengembangan tanaman jarak pagar (jatropha curcas l.) sebagai bahan pembuatan enerji-bio di indonesia perlu mengikuti peta jalur yang rasional. info tek jarak pagar (jatropha curcas l.) vol. 1 no. 1. in www.deptan.go.id/berita/ update27juli06/infotek/jp/no.201/ 2006.pdf. accessed on january 17, 2006. republic of indonesia department of agriculture. 2006b. peluncuran perdana benih unggul jarak pagar (jatropha curcas l.). info tek jarak pagar (jatropha curcas l.) vol. 1 no. 7, 2006 in www.deptan. go.id/berita/update27juli06/infotek/jp/no.207/2006.pdf accessed on january 17, 2006. semangun, h. 2000. penyakit-penyakit tanaman perkebunan di indonesia. revised ed. gadjah mada university press, yogyakarta. sudradjat, h.r. 2006. memproduksi biodiesel jarak pagar, solusi hasilkan biodiesel bekualitas tinggi. penebar swadaya, depok. syah, a. n. a. 2006. biodiesel jarak pagar, bahan alternatif yang ramah lingkungan. agromedika pustaka, jakarta. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html 65 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo white oyster mushroom (pleurotus florida) mutant with altered antioxidant contents ira djajanegara1* and harsoyo2 1center of bioindustry bppt indonesia, laptiab puspitek serpong, tangerang 15314, banten, indonesia 2batan (indonesian atomic agency), patir-batan jakarta, indonesia abstract radiation using gamma ray (60co) at 0.75 kgray with dose velocity of 1.149 kgray/ hour on white oyster mushroom (pleurotus florida) mycelia yielded several mutants. based on isozyme analysis using two enzyme markers such as esterase (est) and acid phosphatase (acp) showed that 3 putative mutants (po-3, po-4 and po-5) among 5 mutants are positive. even though the isozyme patterns indicated that those 3 putative mutants are positively mutated, only po-5 showed higher productivity compared to control (po-k) which is reflected by significantly higher number of fruit bodies, higher fresh weight and dry weight yield of three successive flush periods. it was assumed that the mutation which occurred in po-3 and po-4 may affect other trait(s) of the white oyster mushroom. antioxidant analysis of those mutants indicate that mutant po-4 has significantly higher antioxidant content compared to control (po-k) and the two other mutants (po-3 & po-5). this finding leads to the possible application of white oyster mushroom as a natural antioxidant source. key words : mutant, white oyster mushroom, gamma ray, production, isozyme introduction one way to introduce genetic variability is through mutation using chemical agents or ionizing radiation. mutation may induce one or more change in characteristic of the mutated organisms. the characteristic changes may occur at the gene level which will be passed to the next generation (carlile & watkinson 1994). mutation is applied to edible mushrooms to obtain better quality and productivity. the desired mutant characteristic can be economically beneficial for example resistant to pathogen, higher yield, etc. there are two types of mutation based on the site in nucleus namely point mutation and chromosomal mutation. mutation can alter the coding region or noncoding region. mutation at coding region of a gene will cause the cell to experience silent, missense, frameshift and nonsense mutations. mutation altering the non-coding *corresponding author : idjajanegara@yahoo.com biotropia vol. 15 no. 1, 2008 : 65 73 66 biotropia vol. 15 no. 1, 2008 region will cause different protein production or have no effect on mrna maturation. other types of mutation are null and leaky mutations. these two mutations occurred at the active site of a protein (griffiths et al. 2000) gamma radiation is an ionizing radiation often applied to eukaryotic organisms. according to esser (1971), gamma radiation is an effective ionizing radiation due to its ability to penetrate cell walls of mushroom mycelia. gamma rays has higher energy which makes this type of radiation better penetrates into the target cells. several sources of gamma rays are cobalt-60, cesium-137 and technetium-99 (busby 2003). on eukaryotic cells, gamma radiation is able to break dna molecules and change the purine and pirimidine bases of the target dna (kaiser 2001). in several microbiology studies, mutation that produced higher metabolites are mostly obtained by radioisotope mutagenesis (slater 2000). ionizing radiation has been a common practice for extending storage life in mushrooms especially champignon. radiation at 1.5 – 3 kgy on champignon fruit bodies will increase storage life from 8 weeks to 12 weeks (maha & pangerteni 1989). ionizing radiation has also been used to generate mutants in several edible mushrooms. elliot (1982) used ultra violet (uv ) radiation to produce an agaricus bisporus mutant strain that is resistant to fungicide. in this case, there is a possibility to produce mutant of white oyster mushroom (pleurotus florida) that has higher productivity through ionizing radiation treatment. white oyster mushroom (pleurotus florida) is an edible mushroom that gained popularity lately due to its nutritional values and ease of cultivation. this mushroom contains higher amount of protein, lipid, phosporous, iron, thiamine and riboflavin compared to other edible mushrooms. in addition, this mushroom also contains 18 essential amino acids such as isoleucine, lysine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, valine, arginine, histidine, alanine, aspartic acid, glutamic acid, glycine, proline and serine (djarijah & djarijah 2001). according to bobek et al. (1989), oyster mushroom also rich in antioxidant makes this type of edible mushroom as an excellent source of natural antioxidant. the demand for natural antioxidant sources by food industries increased in the last decade due to the latest findings that indicated the harmful effect of the commonly used synthetic antioxidants such as bha (butyl hydroxy anisole) and bht (butyl hydroxy toluene). bht and bha are correlated to the damage of liver (namiki & osawa 1981). however, the antioxidant content of natural resources is very low. therefore, it will be beneficial to obtain a white oyster mushroom strain that produces higher amount of antioxidant to fulfill the food industry demand. one step to overcome this problem is to apply radiation in the form of gamma rays (60co) to create mutants with commercial characteristics such as higher antioxidant content and many others. this experiment is intended to apply gamma radiation to white oyster mushroom to obtain genetic variability which may lead to commercially superior white oyster mushroom strains. 67 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo materials and methods the mutation material used in this experiment was white oyster mushroom (pleurotus florida) local strain called tp. media used for growing the mycelia were pda (potato dextrose agar) and pdb (potato dextrose broth). all the mycelia were grown in pda and pdb media at room temperature (± 28oc). differences in mycelia diameter were observed on mycelia grown in pda media while mycelia samples for isozyme analysis were grown in pdb media. mutant and control were grown on substrates composed of saw dust (76.3%), rice husks (18.5%), chalk (2.96%) and gypsum (1.27%) as sugested by chang & miles (1989) for productivity observation. chemicals used in this experiment were chloramphenicol as an antibiotic which was added to media while ethanol 70% and 96% were used for sterilization. chemicals used for isozyme analysis were liquid n, potato starch, ascorbic acid, cysteine, tritonx-100, pvp-40, na2hpo4.2h2o, histidine monohydrate, citric acid monohydrate, tris hydoxylmethyl aminomethane, sodium phosphate, 1-naphtyl acetate, 2-naphtyl acetate, fast blue rr salt, sodium acetate, cacl2, h2o2 3%, 3-amino-9-ethylcarbasol, bromophenol blue, nad, malic acid, nbt, pms, tris-hcl, na-1-naphtyl phosphate acid, fast garnet gbg salt and aquadest. approximately 0.5 x 0.5 cm2 mycelia grown on pda media were transferred to the fresh pda media for radiation and some were reserved for stock. the same amount of mycelia (with and without radiation) were transferred to the pdb media for isozyme analysis. all the transfer and multiplication activity were done in the laminar air flow cabinet according to djarijah & djarijah (2001). radiation procedure was done according to esser (1971) with a radiation dose of 0.75 kgray using radiation velocity at 1.149 kgy/hour. radiation was applied to mycelia grown on pda media at exponential phase which is approximately 10 days after inoculation (djajanegara et al. 2004). growth curve of the white oyster mushroom (pleurotus florida ) used in this experiment was based on previous experiment by djajanegara et. al. (2004). isozyme analysis using acid esterase and acid phosphatase were done according to wendel & weeden (1989). antioxidant analysis was carried out by dpph method (yamaguchi et al. 1998) using bha (butyl hydroxy anisole) as a standard. results and discussion gamma rays radiation was done on mycelia culture occupying approximately a quarter of the agar plate (10 days after inoculation). at 12 days after inoculation, the mycelia of white oyster mushroom were in the exponential growth period (djajanegara et al. 2004). just like any other edible mushroom, the white oyster mushroom has an extensive growth pattern as long as substrate is available. mushroom cells are composed of vesicles excreting many types of ezymes and polymers to support the growth at the tip of hyphae. hyphae cells at the tip of mycelium have high metabolism rate. division on 68 biotropia vol. 15 no. 1, 2008 those cells are extensive to produce new nuclei for the newly formed cell compartments (alexopoulus et al.1996). radiation application on those cells during that stage will be expected to produce maximum effect due to maximum radio-sensitivity of the highly metabolic rate of the undifferentiated cells (prassad 1999). there are a number of publications on plant improvement using mutation. this is in contrast with strain improvement in cultivated mushroom especially the oyster mushroom which cultivation has been recently introduced. in order to verify the mutation, isozyme analysis was conducted. isozyme which is also known as multiple molecular forms of enzymes is defined as enzymes that share a common substrate but differ in electrophoretic mobility (markert & moller 1959). they are revealed when tissues extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains this type of genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (prakash et al. 1969). the data in isozyme analysis show number and relative mobilities of various enzyme products when combined with appropriate genetic analysis become transformed into single or multilocus genotypes for each analyzed individual. notes : 1) control (po-k), 2) po-1, 3) po-2, 4) po-3, 5) po-4, 6) po-5 manifestations of mutation range from changes in intensity or relative intensity of bands to appearance or disappearance of bands (tyson et al.1985). in this experiment, enzyme specific stains used are acid phosphatase (acp) and esterase (est) (figures 1 & 2). according to de charisey (1985), subcellular localization of acp is various in the cell with 2 – 4 numbers of isozymes. on the other hand, subcellular localization of est is at the cytosol with 2 – 10 numbers of isozymes (wehling & schmidt-stohn 1984). isozyme analysis using those two enzyme markers will cover any mutation which occurs in the cell. + 1 2 3 4 5 6 figure 1. starch gel of pleurotus sp. assayed for acid phosphatase activity. 69 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo notes : 1) control (po-k), 2) po-1, 3) po-2, 4) po-3, 5) po-4, 6) po-5 isozyme analysis using acid phosphatase (acp) as enzyme marker in this experiment showed 2 4 isozymes (charisey 1985). in this experiment, we observed 3 bands in the control of wildtype white oyster mushroom using acid phosphatase (acp) as enzyme marker which is in agreement with the reference. those 3 bands constantly appeared in the zymogram of the wild-type white oyster mushroom sample as repeated 4 times (data not shown). there was no difference in the isozyme pattern of putative mutants po-1 and po-2 compared to control (po-k). however, there was 1 additional band present in putative mutant po-3 and po-4 compared to control , while in the putative mutant po-5 the top band that was present in control was not observed. based on this analysis the mutation occurred in po-3, po-4 and po-5. in order to be certain with the mutants, another isozyme analysis using different enzyme marker was done (fig. 2). isozyme analysis using esterase (est) specific stain was chosen to verify the gamma ray mutation. zymogram pattern of enzyme esterase (est) revealed 2 – 3 bands which is in agreement with the observation reported by wehling & scmidt-stohn (1984). there was no difference between putative mutant po-1 and control (po-k). however, there was a different isozyme pattern between control and putative mutants po-3 & po-4 in which the relative migration of the top band was shifted. in the putative mutant po-2 and po-5, additional band was observed when the pattern was compared with control (po-k). interestingly, isozyme analysis using acid phosphatase enzyme marker on the putative mutant po-2 did not show any mutation event but when the same sample was analyzed using esterase enzyme marker mutation event was observed. differences of the enzyme marker systems such as sub-cellular localization may contribute to this observation. zymogram pattern of esterase (est) revealed that mutation occurred in po-2, po-3, po-4 and po-5. figure 2. starch gel of pleurotus sp. assayed for esterase activity. 70 biotropia vol. 15 no. 1, 2008 however, based on 2 zymogram patterns of esterase (est) and acid phosphatase (acp), it could be concluded that the mutants are po-3, po-4 and po-5. the manifestation of the mutation was investigated further by growth and morphological observations. there are no morphological differences between the mutants (po-3, po-4 and po-5) and control (po-k) in term of the shape and color of the fruit bodies. data on productivity were reflected by amount of fruit bodies/bag log, diameter of fruit bodies, fresh weight (g) harvest/1 kg media (1 bag log) and fresh weight (g) of harvest/1 kg media (1 bag log). all the bag logs were treated equally and opened for aeration at the same time. harvests were conducted at 3 successive flushing periods. based on productivity observation, only mutant po-5 showed significantly higher productivity compared to control (po-k) (table 1). biological efficiency ration (ber) of mutant po-5 which is reflected by fresh weight of harvest/weight of media was higher than 10% for each harvest. the ber observed for po-5 mutant was 21% (10 weeks post-inoculation), 14% (12 weeks post-inoculation) and 10.5% (14 weeks post-inoculation), while for the wild type were 10% (10 weeks post-inoculation), 8% (12 weeks post-inoculation) and 6.5% (14 weeks post-inoculation, respectively. according to aryantha & rachmat (1999), by reaching biological ratio efficiency at least 10%, the wood mushroom cultivation will be categorized as economically feasible. the mutant po-5 also showed faster flushing period (4 days after aeration) compared to control (2 weeks after aeration). in this case, mutant po-5 is a very useful strain to increase production. table 1. growth performance of the white oyster mutants (po-3, po-4 & po-5) compared to control (po-k) types of white oyster mushroom number of fruit bodies/bag log diameter of fruit bodies (cm) fresh weight harvest (g)/ 1 kg media (1 bag log) dry weight harvest (g) / 1 kg media (1 bag log) 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi control (po-k) 28a 26a 12a 7.5a 6.8a 7a 100a 80a 65a 13a 10.4a 8.5a mutant po-3 27a 26a 11a 8a 7a 7a 98a 78a 65a 12.7a 10.1a 8.5a mutant po-4 28a 27a 12a 7.1a 6.5a 6.8a 97a 77a 67a 12.6a 10a 8.7a mutant po-5 50b 42b 28b 9ab 9.6ab 9.5ab 210b 140b 105b 27.3b 18.2b 13.7b notes : wpi = weeks postinoculation to the bag log, a & ab = growth performance was not significantly different between genotypes based on t-test (p>0.05), b = significantly different (p<0.05) 71 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo further analysis on the mutants (po-3, po-4 and po-5) showed that mutant po-4 has significantly higher antioxidant content in the fruit body compared to control (po-k) and other mutants (po-3 and po-5). antioxidant activity of the mutant po-4 is equal to antioxidant activity of 47.627 mg/g bha (table 2). higher antioxidant activity was always correlated with higher antioxidant content (namiki & osawa 1981). as a comparison, the antioxidant content of soymilk is equivalent to 29.5mg bha/g while fermented soybean is equivalent to 72.8 mg bha/g (shahidi 1997). table 2. antioxidant content of the white oyster mutants (po-3, po-4 & po-5) compared to control (po-k) types of white oyster mushroom antioxidant content (equivalent to mg/g bha) white oyster mushroom control (po-k) 17.184a white oyster mushroom mutant po-3 16.945a white oyster mushroom mutant po-5 16.772a white oyster mushroom mutant po-4 47.627b notes: a & ab = antioxidant content was not significantly different between genotypes based on t-test (p>0.05) b = significantly different (p<0.05) several diseases are caused by strong oxidants such as cardiovascular disease, cancer and many others which can be inhibited by the use of antioxidants. most of the damaging actions of oxidants are from reactive oxygen species (ros) such as free radicals. free radicals can be produced by dust, pollution, or as a side product of metabolism. antioxidant is a chemical that is able to react with oxidant to inhibit the oxidation of bio-molecules (langseth 1995). in addition, antioxidant is also required in food industry as a substance that is able to prevent lipid oxidation which leads to decay of food. lipid oxidation is the first step for other changes in food that will have impact on nutrition value, food safety, color, flavor and texture of food (shahidi 1997). in this case, the mutant po-4 has a potential to be used commercially in the food industry as a natural antioxidant source. conclusions mutation using gamma rays (60co) on white oyster mushroom (pleurotus florida) yielded 4 putative mutants which are po-2, po-3, po-4 and po-5. isozyme analysis using acid phosphatase (acp) and esterase (est) as marker enzymes confirmed that mutation occurred at putative mutants po-3, po-4 and po-5. there were no differences in the morphology of the mycelia and fruit bodies between those mutants and control. observation on mutants po-3, po-4 and po-5 showed that the productivity of po-5 was significantly higher compared to control and 72 biotropia vol. 15 no. 1, 2008 the two other mutants (po-3 and po-4). antioxidant analysis showed that mutant po-4 has a significantly higher antioxidant content compared to control (po-k) and the two other mutants (po-3 and po-5). this leads to the possible application of this particular white oyster mushroom (pleurotus florida) mutant as a source for natural antioxidant which is beneficial for medical and food industry. references alexopoulus, c. j., c. w. mims & m. blackwell. 1996. introductory mycology. 4 th ed. john wiley & sons, inc., new york. aryantha, i. n. p. & b. rachmat. 1999. dasar-dasar usaha budidaya jamur. bio agro lestari publ., bandung. bobek et. al. 1991. effect of mushroom pleurotus ostreatus and isolated fungal polysaccharide on serum and liver lipids in syrian hamsters with yperlipidemia. nutrition 7:105-108. bueche, f. & d. l. wallach. 1994. technical physics. 4 th ed. john wley & sons, inc., new york. busby, b. 2003. radiation & radioactivity. http://www.physics.isu.edu/radinf.htm. accessed on 6 april 2003. carlile, m. j. & s. c. watkinson. 1994. the fungi. academic press, london. chang, s. t. & p. g. miles. 1989. edible jamurs and their cultivation. crc press, florida. djajanegara, i., lestari, r., harsoyo & p. wahyudi. 2004. penelitian pendahuluan mutasi dengan sinar gamma (co60) untuk meningkatkan kandungan metabolit sekunder dan analisis isozyme pada 3 varietas jaur tiram (pleurotus sp.). jurnal ilmu kefarmasian indonesia 2(1):10 17 djarijah, n. m. & a. b. djarijah. 2001. budidaya jamur tiram. penerbit kanisius, yogyakarta. de charesey, h., m. t. barreneche, m. jusuf. c. ouin and j. pernes. 1985. inheritance of some marker genes in setaria italica. theor. appl. genet. 71:57-60. elliot, t. j. 1982. genetic and breeding of cultivated jamurs in tropical jamur (ed.) s. t. chang & t. h. quimio. the chinese university press, hongkong. 35-67. elliot, t. j. & f. a. langton. 1981. strain improvement in the cultivated mushroom agaricus bisporus. euphytica 30:175-182. esser, k. 1971. application & importance of fungal genetics for industrial research in : radiation & radioisotopes for industrial microorganisms. iaea, viena : 83 – 91. griffiths, a. j. f., j. h. miller, d. t. suzuki, r. c. lewontin & w. m. gelbart. 2000. an introduction to genetic analysis. w. h. freeman, new york. hartana, a. pelatihan singkat teknik analisis dengan metode dan peralatan mutakhir di bidang hayati dan kimia. pusat studi ilmu hayati lembaga penelitian ipb, bogor. holme, d. j. & h. peck. 1994. analytical chemistry. 2 nd ed. longman scientific & technical, burnt mill. kaiser, g. e. 2001. mutation. http://student.ccbc.cc.md.us/biotutorial/protsyn/mutate.html accessed on 5 june 2003. langseth, l. 1995. oxidants, antioxidants and disease prevention. ilsi europe, belgium. larraya, l. m., g. perez, e. ritter, a. g. pisabarro & l. ramirez. 2000. genetic linkage map of the edible basidiomycete pleurotus ostreatus. appl. and environ. microbiol. 66(12): 5290 – 5300. maha, m. & d. s. pangerteni. 1989. pengawetan jamur merang (volvariella volvaceae) dengan kombinasi pemanasan dan iradiasi in risalah simposium iv aplikasi isotop dan radiasi, jakarta, 13-15 des. 1207-1215. markert, c. l., & f. moller. 1959. multiple forms of enzymes: tissue, ontogenic and species specific patterns. proc. natl. acad. sci. usa 45:753-763. moss, g. p. 1961. iubmb enzyme nomenclature: e. c. 1.1.1.37. http://www.chem.qmul.ac uk/iubmb/ short com 805 azimah haji (assessment).cdr assessment of seedling abundance, survival and growth of two dipterocarp species in peat swamp forests of brunei darussalam hazimah haji mohd din, nor basirah bakiri, rahayu sukmaria sukri and faizah metali* environmental and life sciences programme, faculty of science, universiti brunei darussalam, gadong be1410, brunei darussalam received 31 january 2017 / accepted 21 march 2018 abstract dryobalanops rappa becc. and shorea albida sym. are bornean endemics of high conservation value and increasingly threatened by anthropogenic disturbances. in-situ study of seedling abundance and growth performance of these dipterocarp species was conducted in two selected peat swamp forests of brunei darussalam, following a mast fruiting event in march–may 2014. within six 6 x 6 m plots at each forest site, d. rappa seedlings at the anduki peat swamp forest and s. albida seedlings at the badas peat swamp forest were measured for abundance at the initial census in september 2014, as well as survival and relative growth rates (rgr) after a period of 5 months, with the final census in february 2015. we found significantly higher seedling abundance for d. rappa (1885 ± 208) than s. albida (160 ± 71). significantly higher percentage survival was recorded for d. rappa seedlings (90.8 ± 2.2%) in comparison to s. albida -1 -1 seedlings (81.7 ± 2.2%). s. albida seedlings (0.24 ± 0.02 mm mm month ) showed significantly greater rgr in stem -1 -1 diameter than d. rappa seedlings (0.18 ± 0.02 mm mm month ), however, there were no significant differences in the rgrs based on seedling height, leaf number and biomass between d. rappa and s. albida seedlings. in terms of seedling abundance and percentage survival, d. rappa seedlings appeared to be more successful in regeneration and may potentially be used for rehabilitation of degraded tropical peat swamps and other forest types. our results suggested that greater conservation efforts of peat swamps must be made to protect the bornean endemic plant species, in particular s. albida. keywords: borneo, dipterocarpaceae, dryobalanops rappa, relative growth rates, shorea albida introduction peat swamp forest is a unique ecosystem that represents the second major forest type in brunei darussalam, accounting for 15.6% (or 90,884 ha) of brunei's forest (wong et al. 2015; forestry department 2016). it is mainly located in the belait district, interconnecting with the peat swamps of the baram basin in sarawak. bornean peat swamp forests are ombrogenous and have six 'phasic communities', each consisting of distinct plant communities (anderson 1963). over the years, there has been a significant reduction in peat swamps in brunei darussalam mainly due to infrastructure development and forest fires (pg harun 2015; wong 2016). peat swamps house unique flora, some of which are endemic and increasingly threatened in their natural ranges due to anthropogenic disturbances such as legal or illegal logging, forest fires and land-use changes (yule 2010; posa et al. 2011). most tree families in lowland dipterocarps are found in peat swamps, but many peat swamp species are restricted and physiologically adapted to this extreme forest environment (ng & ibrahim 2001). in brunei darussalam, extensive stands of dryobalanops rappa becc. or 'kapur paya' exist on shallow peat overlying sand in lumut and anduki, while pure stands of shorea albida sym. occur in the 'padang alan' and 'alan bunga' peat swamp forests of badas, both in the belait district (anderson 1964; ashton et al. 2003; wong et al. 2015). the anduki peat swamp forests experienced massive forest fires during the * corresponding author: faizah.metali@ubd.edu.bn biotropia 5 2 8 148 154 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.805 148 1997–1998 el niño drought (wooster et al. 2012), resulting in fragmented stands of intact d. rappa existing within an otherwise developed area. low regeneration rates have been observed in natural d. rappa populations (wong & kamariah 1999), and this species is increasingly threatened from illegal logging (phillips 1998). the badas peat swamps consist of gregarious stands of s. albida that are decreasing in population density and have been showing few signs of natural regeneration for the past 30 years (kobayashi 1998). the iucn red list classifies s. albida as endangered due to rapid population loss within its natural forest ranges, coupled with a regeneration status that is reported to be virtually non-existent (ashton 1998). furthermore, s. albida is endemic to north and west borneo (ashton 1998) and so the remaining stands in the badas peat swamps in brunei are likely to be one of the few remaining intact stands of s. albida in borneo and globally (forestry department 2014). both d. rappa and s. albida are important timber tree species that rely on irregular mass flowering and mast fruiting episodes as a means of their natural regeneration (appanah & turnbull 1998). although several studies have been conducted on the survival and growth of dipterocarpaceae seedlings in bornean forests (turner 1990; delissio et al. 2002; nakagawa et al. 2005; shimamura et al. 2006; takeuchi & nakashizuka 2007; daisuke et al. 2013), only a few have focussed specifically on peat swamp species (ibrahim 1996; gavin & peart 1997; saito et al. 2005; jans et al. 2012), and no study has been conducted on s. albida seedlings to date. following a small mass flowering and a mast fruiting event in the badas and anduki peat swamps in march–may 2014, the abundance, survival and growth of d. rappa and s. albida seedlings at the understorey of these two peat swamp forests were assessed in br unei darussalam. our study provides preliminary findings on two native dipterocarp species that can potentially be utilised in efforts to naturally regenerate and rehabilitate disturbed peat swamp forests. materials and methods the study was conducted in the anduki and badas peat swamps located in the belait district, brunei darussalam, north-west borneo, at distances of ca. 14 km from each other by road. a dominant dipterocarp species (dipterocarpaceae) in each peat swamp site was selected. the anduki peat swamp (4°37'39.00"n, 114°22'1.14"e) is located in the coastal areas of belait district within the anduki forest reserve and is dominated by dryobalanops rappa becc. the badas peat swamp (4°34'9.12"n, 114°24'40.08"e) is located further inland, south of seria and is mainly populated by shorea albida sym. a total of six plots (6 m x 6 m or 2 36 m each) plots were set up: three plots of shorea albida at badas and three plots of dryobalanops rappa at anduki. plots were located ca. 50 m away from each other and each plot was set up with a mother tree of the respective species located at the centre of the plot. abundance of d. rappa seedlings in anduki and s. albida seedlings in badas were quantified per plot at the initial census in september 2014, ca. 3–5 months after the mass flowering and mast fruiting events. within each plot, 40 seedlings of either d. rappa (at anduki) or s. albida (at badas) were randomly chosen and tagged in september 2014. the seedling censuses were conducted twice, in september 2014 and after 5 months, in february 2015. the tagged seedlings were measured for percentage survival, stem height (measured from the point of measurement painted white on soil surface to the top leaf bud), stem diameter and number of leaves (counted fully expanded leaves, excluding heavily eaten leaves i.e. approximately 70% of lamina eaten and dead leaves) at both censuses. a total of 10 seedlings of each species were randomly chosen from outside each of the three study plots per census and harvested to provide estimates of initial and final dry biomass. at both harvests, seedlings were washed with distilled water, oven-dried at 60 c for 48 h and weighed. o the relative growth rate (rgr) based on stem height (rgrh), stem diameter (rgrd), number of leaves (rgrl) and biomass (rgrb) per 149 seedling abundance, survival and growth of two dipterocarp species – mohd din et al. recorded higher survival and rgr for d. rappa seedlings planted in lowland mixed dipterocarp forest (sukri 2010) and degraded heath forest (w. h. tuah, unpubl. data). natural disturbance within the understorey may influence the survival of tree seedlings (appanah & turnbull 1998) and can be an explanation for the low percentage survival of s. albida. yamada (1997) reported that badas peat swamp has higher incidences of fallen trees and presence of buttress roots on its forest floor compared to anduki peat swamp. during the rainy season between december 2014 – january 2015 in badas, s. albida seedlings may not survive when hit by fallen trees and branches, thus decreasing its survival percentage. through the mast fruiting period, the s. albida seeds may fall on the false forest floor in badas, which is formed from litter accumulation on buttress roots. this may lead to s. albida seedlings mortality as the false floor may fall to the peat when disturbed. seedling predation and microenvironment also influence seedling survival, and it has been recorded elsewhere in borneo that late fruiting of shorea species increased the possibility of seed losses to predation (curran & webb 2000). anduki peat swamp has different physiological environment compared to badas peat swamp forest in terms of its natural disturbance. fragmentation of the anduki forest due to rapid forest fires may cause its forest floor to develop shallow litter and peat overlying sandy soil (yamada 1997). the anduki forest reserve is dependent on waterlogged conditions to maintain the peat bog (yussof 2015) and d. rappa seedlings are well-adapted to waterlogged peat (yamada & suzuki 2004). survival and growth of s. albida seedlings have been found to be negatively affected by low light intensity (kobayashi 1998), water shortage during dry spells (kobayashi 1998) and excessive water during rainy period (dixon et al. 2013), which may be factors that explain their low survival rates in badas. d. rappa is a light-demanding species (ashton et al. s. albida 2003), while seedlings have been recorded as shade-intolerant (kobayashi 1998; pers. obs.). regardless of the light environment, light-demanders tend to possess larger specific leaf area and higher photosynthetic capacity, which result in greater carbon gain, and potentially higher survival, compared to shadetolerant species (ghazoul & sheil 2010). light has seedling over the five-month census period were calculated following hunt (1982): rgr = (log we 2 – log w ) / (t – t ), w and we 1 2 1 2 1 where are stem height, stem diameter, total number of leaves or biomass and . to compute rgrb, t – t is 5 months2 1 seedlings were paired by ranked values of seedling biomass at the initial and final harvests. b e t we e n s p e c i e s d i f f e r e n c e s s e e d l i n g abundance, percentage seedling survival and rgr values were determined using t-tests in r 2.15.2 (r core team 2014). all data were first explored to confir m the nor mality of residuals and homogeneity of variances, and where necessary, data was log -transformed, with the exception of 10 percentage sur vival, which was arcsinetransformed. results and discussion using the initial census survey, d. rappa seedlings at the anduki peat swamp forest showed 2 significantly higher mean abundance per 36 m plots compared to s. albida seedlings at the badas peat swamp (d. rappa: 1885 ± 208 vs. s. albida: 160 ± 71, p< 0.01). this is a probable reflection of the higher density of adult d. rappa trees in anduki, compared to the more sparsely populated adult s. albida trees in badas (pers. obs.). additionally, the higher d. rappa seedling abundance may be due to higher abundance of flowering mother trees, as almost all mature d. rappa trees flowered at anduki whilst not all mature s. albida trees flowered at badas during the same mass flowering event in 2014 (pers. obs.). other studies have similarly documented a positive relationship between the number of mother trees and seedling density, with the highest seedling densities found in areas with high adult abundance (itoh et al. 1997; webb & peart 1999; backlund 2013). mean percentage survival of tagged d. rappa seedlings over the period of 5 months was significantly greater than s. albida seedlings (90.8 ± 2.2% vs. 81.7 ± 2.2%, p< 0.05). all relative growth rates (rgrs) data of both species showed similar growth rates (fig. 1) that there were no significant differences in all relative growth rates (rgr) except for rgr in stem diameter (rgrd), in which d. rappa seedlings had significantly lower rgrd than those of s. albida seedlings (0.18 ± 0.02 vs. 0.24 ± 0.02 mm mm month ; p< 0.01; -1 -1 fig. 1b). other studies in brunei darussalam have biotropia vol. 25 no. 2, 2018 150 been recognised to significantly influence survival and growth of dipterocarp seedlings (scholes et al. 1996; whitmore & brown 1996). for example, d. rappa s. albidaand saplings were planted in the open in ongoing reforestation trial plots of degraded waterlogged heath forests ( kerapah forests) in brunei darussalam, and have performed successfully in terms of its survival and growth rates over a period of more than a year (w. h. tuah, unpubl. data). it was found that s. albida seedlings had significantly higher rgrd compared to d. rappa seedlings. this suggests that in the shaded understorey of peat swamp forests, s. albida seedlings allocated more resources to stem diameter growth than height growth. tropical tree species that preferentially invest resources into stem diameter growth and wood density typically exhibit traits that defend against herbivory and pathogens, as well as for the figure 1 differences in relative growth rates (rgr) for seedlings in anduki and seedlings in dryobalanops rappa shorea albida badas: (a) rgrh (stem height; cm cm month ), (b) rgrd (stem diameter; mm mm month ), (c) rgrl -1 -1 -1 -1 (number of leaves; leaf number month ) and (d) rgrb (biomass; g g month ). rgr data were determined for -1 -1 -1 the 40 tagged and seedlings, except for rgrb which was determined for the 10 seedlings d. rappa s. albida harvested outside each plot at each census. the asterisk sign within each rgr denotes significant differences of means between species (marked with red dots) using t-tests (*, < 0.05; **, <0.01; ***, < 0.001). ns=non-p p p significant. 151 seedling abundance, survival and growth of two dipterocarp species – mohd din et al. development of their root system (kitajima 1994), all of which decrease investment into height growth. the badas peat swamp forest has a flat, densely crowded canopy (yamada 1997), which restricts light entry to the understorey and may further inhibit height growth of s. albida seedlings. these shade-intolerant s. albida seedlings (kobayashi 1998) require ample light to grow in height. higher growth rates of s. albida in the open areas than in the closed canopy forest was recorded in the ongoing rehabilitation trial plots in the kerapah forests of brunei darussalam (w. h. tuah, unpubl. data). both the dryobalanops rappa peat swamp forest in anduki as well as the shorea albida peat swamp forest in badas have been identified as critical habitats that are of high conservation value (forestry department 2014). peat swamps are highly vulnerable to fires, particularly during the dry season and when the water table have been lowered (yule 2010). changes in drainage and hydrological conditions significantly increase repeated fire events (posa et al. 2011). repeated fires in anduki and badas, especially during drier periods and drought events, coupled with slow seedling growth rates, will undoubtedly result in further losses of both d. rappa and s. albida populations. in the case of s. albida, this loss will be further exacerbated by the low number of survived seedlings and mother trees flowering at the same time (pers. obs.). conclusion this study recorded significant findings on dryobalanops rappa seedlings in anduki and shorea albida seedlings in badas. d. rappa seedlings were more abundant than s. albida seedlings, and also showed higher percentage seedling survival. s. albida seedlings, however, appeared to invest more in stem diameter growth, as their relative growth rate in terms of stem diameter was significantly higher compared to d. rappa seedlings. we suggest that d. rappa seedlings may be suitable as a species that can be used to rehabilitate degraded peat swamp forests, but detailed, longer term studies are needed to further support this conclusion. finally, given that peat swamps forests are fast disappearing due to natural and anthropogenic disturbances, their protection and conservation efforts should be prioritised to protect the bornean endemic plant species, in particular s. albida. acknowledgements the authors thank the forestry department, ministry of primary resources and tourism (mprt), brunei darussalam for granting permission to conduct research in the anduki and badas peat swamps (no. [84]/jph/und/17 pt.1), and the brunei national herbarium, mprt for providing information on the mass flowering and mast fruiting episodes. we thank the technical staff of environmental and life sciences, universiti brunei darussalam (ubd) for their assistance with field equipment. funding for this project was provided by ubd for nbb. references anderson jar. 1963. the flora of the peat swamp forests of sarawak and brunei, including a catalogue of all recorded species of flowering plants, ferns and fern allies. singapore (sg): gard. bull. singapore. p. 131228. anderson jar.1964. the structure and development of peat swamps of sarawak and brunei. j trop geo 18:7-16. appanah s, turnbull jw. 1998. a review of dipterocarps: taxanomy, ecology and silviculture. bogor (id): cifor. ashton p [internet]. 1998. , the iucn red list shorea albida of threatened species 1998. e.t33099a9751699; [ c i t e d 2 0 1 6 d e c 8 ] . av a i l a b l e f r o m : http://dx.doi.org/10.2305/iucn.uk.1998.rlts. t33099a9751699.en ashton ps, kamariah as, said im. 2003. field guide to the forest trees of brunei darussalam and the northwest borneo hotspot. bandar seri begawan (bn): university of brunei darussalam in association with brunei forestry department, brunei shell petroleum and sultan haji hassanal bolkiah foundation. backlund s. 2013. the effects of mother trees and site conditions on the distribution of natural regeneration establishment in a bornean rainforest disturbed by logging and fire [thesis]. retrieved from swedish university of agricultural sciences. curran lm, webb co. 2000. experimental tests of the spatiotemporal scale of seed predation in mast fruiting dipterocarpaceae. ecol monogr ‐ 70(1):129-48. biotropia vol. 25 no. 2, 2018 152 daisuke h, tanaka k, jawa kj, ikuo n, katsutoshi s. 2013. rehabilitation of degraded tropical rainforest using dipterocarp trees in sarawak, malaysia. int j forestry res: 683017. doi: 10.1155/2013/683017 delissio lj, primack rb, hall p, lee hs. 2002. a decade of canopy-tree seedling survival and growth in two bornean rain forests: persistence and recovery from suppression. j trop ecol 18(5):645–58. dixon c, fyson gf, pasiecznik n, praciak a, rushforth k, sassen m, … van heist m. 2013. the cabi encyclopedia of forest trees. oxfordshire (uk): cabi publishing. th forestry department. 2014. the 5 national report to the convention on biological diversity. bandar seri begawan (bn): the forestry department, ministry of industry and primary resources. forestry department [internet]. 2016. forest types: peat swamp. bandar seri begawan, bn: forestry department; 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[cited 2016 apr 11]. available from: http://bt.com.bn/ wong km, kamariah as. 1999. forests and trees of brunei darussalam. bandar seri begawan (bn): universiti 153 seedling abundance, survival and growth of two dipterocarp species – mohd din et al. brunei darussalam, forestry dept., ministry of industry and primary resources and brunei shell petroleum company sendirian berhad. wong km, ahmad ja, low yw, kalat maa. 2015. rainforest plants and flowers of brunei darussalam. bandar seri begawan (bn): forestry department, ministry of industry and primary resources. wooster mj, perry glw, zoumas a. 2012. fire, drought and el niño relationships on borneo (southeast asia) in the pre-modis era (1980–2000). biogeosciences 9(1):317-40. yamada i. 1997. tropical rainforests of southeast asia. honolulu (us): university of hawaii press. yamada t, suzuki e. 2004. ecological role of vegetative sprouting in the regeneration of dryobalanops rappa, an emergent species in a bornean tropical wetland forest. j trop ecol 20(4):377-84. yule cm. 2010. loss of biodiversity and ecosystem functioning in indo-malayan peat swamp forests. biodivers conserv 19(2):393-409. yussof m [internet]. 2015. hob initiative to battle global warming. borneo bulletin; [cited 2015 oct 3]. available from: http://borneobulletin.com.bn biotropia vol. 25 no. 2, 2018 154 page 1 page 2 page 3 page 4 page 5 page 6 page 7 biotropia book final.indd 135 taxometrics classification (hierarchical and ordination) of aquatic and semiaquatic mosses: a preliminary model to bryodiversity management m.j. loo¹*, t. a. delvalls casillas¹ and l. martin diaz¹ ² ¹unesco/unitwin/wicop..department of physical chemistry, faculty of marine and environmental sciences, universidad de cádiz – rio san pedro campus, 11510 puerto real, cádiz, spain. ¹²instituto de ciencias marinas de andalucía. csic. rio san pedro campus, 11510 puerto real, cádiz, spain. abstract bryodiversity is naturally serving the ecosystems sustainably. it serves the environments by preventing natural disaster (fl ooding), maintaining the quality of the water body and fi lter or treats the pollutants naturally. effi cient bryodiversity management is needed for environmental cost cutting and have a cost-eff ective management strategy. to achieve this, cluster and principal component analyses (pca) were manipulated to produce the linkage distance between the otus and identify the important groups of characters, respectively. in return, it becomes a guideline for bryofl ora and environmental managements. in this study, 23 otus and 156 characters were analyzed. th e output from the reliability and item analysis showed that the data set is highly reliable (cronbach’s alpha = 0.9627). from the cluster analysis, it showed that 5 clustered groups (manageable units) could be derived from the produced phenogram. th is is based on the nearest neighbour amalgation rule and euclidean distances. as for the principal component analysis, three factors were derived and explained 75.1064% of the variation with 56.0485%(pc1), 11.7346%(pc2) and 7.3233%(pc3), respectively. th e ordination showed that 5 manageable units were derived from pc1 and 3 manageable units for pc2 and pc3, respectively. in conclusion, conservation should precede any biodiversity management plans. key words: aquatic mosses, semi-aquatic mosses, cluster analysis, principal component analysis (pca), classifi cation *corresponding author: loominjet@googlemail.com biotropia vol. 15 no. 2, 2008 : 135 154 136 introduction bryodiversity management is a new discipline in management science. bryodiversity refers to the richness of bryophytes (mosses, liverworts and hornworts). management signifi es planning conservational strategy, organizing conservational plans, implementing organized conservational approach and controlling or sustaining the on-going of the implemented plan with the aim to conserve the nature (raffi eld and bingham 1994). based on stuessy (1990), biosystematics is crucial in understanding the biodiversity of a particular ecosystem. in this context the focus is on the richness of aquatic and semiaquatic mosses. without knowing the richness, no conservation plan will be implemented and thus, fl oral extinction is highly potential. in this study, aquatic and semi-aquatic mosses were studied phenetically to fi nd out the rarity and commonness among the studied populations. th is is very crucial in conservation where rare species should be urgently conserved and less threatened spesies should be sustained too. cost-eff ective is the success key in any management activities (raffi eld and bingham 1994). phenetic analysis (cluster analysis and principal component analysis) will statistically group species with the most similar characters together (scotland and carine 2000; komosinki et al. 2001; and aguilar et al. 2004) and forms few manageable units. instead of over-consuming time and costs for few related or familiar species and neglecting other populations, managing clustered group will be the solution in the successful bryodiversity management. th is new approach aims for conservation and at the same time continues serving the needs of the ecosystem. in term of costs, no artifi cial fl ood mitigator and barrier, no water quality tester and no waste water contamination might be required if aquatic and semi-aquatic mosses are present in the natural habitats (ando and matsuo 1984; frahm 1996; welch 1948; conrad 1935; whitehouse and mcallister 1954; ando 1957; grout 1912; coupal and lalancette 1976). materials and methods moss material and characterization twenty-three species or operational taxonomic units were selected and 156 characters with diff erent level of character states (table 1) were chosen for numerical classifi cation. further phenetic methodology referred to stuessy (1990), stotler and stotler (2000), frahm (2003), smith (1978), holmes (1998), tsai et al. (2002); and yamagishi et al. (2005). th e main sources for analyses were morphological and anatomical data: vegetative (gametophyte) and reproductive (sporophyte) components. both taxonomic sources were measured quantitatively and qualitatively. table 1. characters and character states for the taxometric analyses (1)plant size(0-small,1-big/large,2-others); (2)plant habitat and submergence (0-semi-aquatic,1aquatic,2-not submerged,3-sometimes partly submerged,4-occasionally submerged,5-others); (3)plant colour(0-greenish to blackish and rarely whitish,1-others); (4)ephemerality of plant(0-no,1-yes,2others); (5)plant growth form(0-acrocarpous,1-pleurocarpous,2-others); (6)plant covered by glaucous biotropia vol. 15 no. 2, 2008 137 or bluish(0-no,1-yes,2-others); (7)plant: prostate to erect(0-no,1-yes,2-others); (8)plant branching form(0-simple to pinnately branched,1-others); (9)rarity of plant(0-rare to common,1-others); (10)plant: terete or julaceous form(0-no,1-yes,2-others); (11)plant: means of asexual reproduction(0without,1-with,2-without or with,3-others); (12)plant: coarseness(0-not coarse,1-coarse,2-others); (13)plant with fl attened shoots(0-no,1-yes,2-others); (14)autoicous (autoecious)(0-without archegonia and antheridia in separate infl uorescences,1-with archegonia and antheridia in separate infl uorescences,2-others); (15)plant with innovative branches beneath infl orescences(0-no,1-rare,2often,3-others); (16)plant: more than 5mm(0-no,1-yes,2-others); (17)rhizoids(0-obvious/with rhizodal tubers,1-not obvious,2-others; (18)leaves unbordered by row of cells(0-no,1-yes,2-others); (19)leaf bordered by(0-elongate cells,1-smooth cells,2-others); (20)leaf sheathing(0-rarely,1-often,2others); (21)leaf costa ending below the apex to excurrent(0-no,1-yes,2-others); (22)leaf without hair-points(0-no,1-yes,2-others); (23)leaf hair-points(0-hyaline,1-others); (24)leaves all of one kind(0-no,1-yes,2-others); (25)leaves direction(0-homomallous,1-others); (26)leaves arrangement(0attached all around the stem,1-attached in two rows on opposite sides of the stem (distichous),2others); (27)leaf lamina(0-conspicious,1-others); (28)leaf lamina unistratose(0-no,1-yes,2-others); (29)leaf layer(0-unistratose,1-multistratose,2-unistratose to multistratose,3-others); (30)leaves inconspiciously ranked(0-no,1-yes,2-others); (31)leaves apex(0-ovate to spatulate,1-others); (32)leaves tip(0-acuminate to acute (awned),1-others); (33)leaves(0-various,1-undulate,2-straight or straight when dry,3-plicate or deeply plicate,4-plicate or not,5-concave,6-others); (34)leaf (dorsal view)(0-keeled or fl at,1-others); (35)leaves position(0-at extreme apex,1-others); (36)leaf alteration(0little altered when dry,1-others); (37)adaxial surface of the leaf costa(0-without lamellae or fi laments,1with lamellae or fi laments,2-broadly channeled or fl at,3-others); (38)leaf without cancellinae(0-no,1yes,2-others); (39)spreading leaves(0-without,1-with,2-with and wide,3-others); (40)diff erentiation of branch and stem leaves(0-strongly,1-weakly/scarcely,2-others); (41)apical cells of branch leaves(0about 1/2 length of those at midleaf,1-scarcely shorter than those at midleaf,2-others); (42)leaf bases(0-without cancellinae,1-with cancellinae,2-others); (43)leaf base with appear split(0-no,1yes,2-others); (44)sheathing base of leaves(0-without,1-rarely,2-with,3-others); (45)concave leaf bases(0-not concave,1-not concave and with a narrow insertion,2-with,3-others); (46)leaf longer than 1mm(0-no,1-yes,2-others); (47)leaf cross-section(0-recurved only on one side,1-plane to recurved,2recurved to revolute,3-revolute,4-others); (48)leaf bases never or gradually expanded(0-no,1-yes,2others); (49)propagula in leaf apices(0-without,1-with,2-others); (50)leaf apices at extreme apex(0margins entire or papillose-crenulate,1-others); (51)leaf apices(0-acuminate to bluntly acute,1cuspidate,2-cuspidate to piliferous,3-without piliferous or aristate (awn),4-with piliferous or aristate (awn),5-others); (52)channeled leaf apices(0-no,1-yes,2-others); (53)leaf cell diametry(0isodiametric,1-more or less isodiametric,2-others); (54)leaf cell surface(0-fl at, smooth and papillose,1smooth, bulging or prorulose,2-smooth,3-papillose or prorulose,4-smooth and papillose,5-fl at,6papillose (uni to pluri) or prorulose,7-rarely with minute cuticular roughenings,8-others); (55)leaf cell type(0-one type,1-others); (56)leaf cell colour(0-green,1-others); (57)size of leaf cells(0-shorts,1longs,2-others); (58)relative size of leaf cells(zero.1(-2):1,one.above about 3:1 or longer,two.4:1 or less,three.1-4(-5),four.more than 10:1,fi ve.(3-)4:1 or longer,six.others); (59)relative size of upper leaf cells(zero. more than 5:1,one. 2-6:1,two.others); (60)leaf cell papillose(0-no,1-pluripapillose,2-closely set, simple to branched papillae/simple to branched papillae,3-unipapollose to pluripapillose,4-with papillae stellate from a stipitate base to c-shaped,5-others); (61)leaf cells in obvious rows(0-no,1yes,2-others); (62)leaf cell shape(0-long-hexagonal,1-short-rectangular to linear,2-conic, clavate or branched and rarely c-shaped,3-rounded to quadrate,4-linear,5-merely rounded and not stellate,6taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 1. continued 138 long, linear and hexagonal,7-rectangular,8-rectangular to long-hexagonal,9-others) ; (63)upper leaf cells(0-smooth or with low and indistinct papillae,1-smooth,2-fi rm-walled and short-oblong to rhombic,3prorulose,4-densely pluripapillose with c-shaped papillae,5-others); (64)basal leaf cells(0usually without thickened transverse walls,1-others); (65)papillose over the lumina or prorulose in leaf cells(0-no,1-yes,2-only papillose over the lumina,3-others);(66)mid leaf cells of stem leaf(0-40–120 μm long,1-others);(67)branch and stem leaves scarcely diff erentiated(0-no,1-yes,2-others);(68)leaf margin(0-single teeth or entire,1-entire,2-toothed,3-abruptly serrate at the shoulder,4-entire and denticulate,5-serrate margins whose teeth are often refl exed,6-entire or papillose-crenulate,7-with paired teeth,8-others);(69)numbers of leaf margin(0-single,1-double,2-two to multilayered,3others);(70)near midleaf or below recurved to revolute(0-no,1-yes,2-others);(71)upper leaf margins plane to revolute (with cells undiff erentiated or paler than median cells)(0-no,1-yes,2others);(72)stomates(0-absent,1-present,2-others);(73)leaf costa(0-without a costa or costa short and double, double or single with 2-3 lateral spurs,1-single,2-distinct throughout,3-others);(74)adaxial surface of leaf along costa(0-broadly channelled or fl at,1-others);(75)single leaf costa to at least midleaf(0-yes,1-others);(76)apical of leaf costa(0-subpercurrent,1-bluntly excurrent,2-excurrent to ending in the cusp,3-others);(77)transverse section of leaf costa(0-2 stereid bands,1-single and dorsal stereid band,2-diff erentiated stereid bands,3-others);(78)both dorsal and ventral stereid bands present(0-no,1-yes,2-others);(79)cells of abaxial surface of costa(0-oblong and elongate,1-quadrate to short-oblong,2-others);(80)dorsal part of leaf costa(0-smooth or toothed at back (ridged),1-not ridged,2-others);(81)size of leaf costa(0-narrow or much narrower,1-wide or broad,2others);(82)lamellae or fi laments on the adaxial surface of the costa(0-without,1-with,2others);(83)ventral costal epidermis(0-absent,1-present,2-others);(84)cells of adaxial (upper) surface of costa similar to or smaller than laminal cells in transverse section(0-no,1-yes,2-others);(85)costa more than 100μm wide at base(0-no,1-yes,2-others);(86)costa ending in the leaf apex(0-no,1-yes,2others);(87)costa ending below the apex to excurrent(0-no,1-yes,2-others);(88)costa ending in a spine(0-no,1-yes,2-others);(89)costa occupying less than 1/4 the leaf base(0-no,1-yes,2others);(90)axilliary hairs(0-hyaline,1-brown,2-others);(91)basal cells of axilliary hairs(0-slender,1others);(92)alar cells(0-scarcely diff erentiated,1-infl ated in well marked groups,2-not at all infl ated,3alar group not extending more than 20 – 40 % up leaf,4-others);(93)stem paraphyllia(0-no,1-no or lacking,2-abundant and fi lamentous,3-others);(94)stem paraphyllia foliose(0-no,1-yes,2-others); (95)foliate stem(0-sometimes complanate,1-foliate throughout and without rhizome-like connections between erect stems,2-symmetrical,3-sometimes fl attened,4-others);(96)stem form(0-erect,1occasionally branched beneath infl urouscences,2-others);(97)stem branching(0-not branched,1mostly prostrate with lateral branches,2-prostrate with erect branches bearing terminal sporophytes (cladocarpous),3-branching various (e.g. complanate-foliate, fl attened-foliate, prostrate with lateral branches and ranches curved downwards),4-others);(98)stem ranked leaves(0-without,1-with,2others);(99)stem: central strand(0-absent,1-present,2-others);(100)stem: hyalodermis(0-absent,1present,2-others);(101)stem abundance(0-sparse to abundant,1-others);(102)stem sclerodermis(0clearly diff erentiated,1-not or weakly developed,2-others); (103)stem size(0-up to 2.7mm long,1others); (104)stem epidermal cells(0-small,1-big/large,2-others); (105)stems round in transverse section(0-no,1-yes,2-others); (106)sporophytes(0-various types,1-terminal,2-not clustered,3-lateral,4others);(107)size of capsule(0-small,1-big/large,2-others);(108)capsule projection(0-long-exserted,1exserted,2-others); (109)capsule symmetry(0-symmetric,1-asymmetric,2-others); (110)capsule(0operculate or cleistocarpous,1-others); (111)capsule: valvate(0-never,1-always,2-others); (112)capsule growth form(0-erect or straight,1-inclined to pendulous,2-horizontal to pendulous,3-never furrowed biotropia vol. 15 no. 2, 2008 table 1. continued 139 or strumose,4-globose and rugulose to furrowed when dry,5-horizontal or pendulous,6others);(113)neck of capsule(0-short and inconspicious/inconspicuous,1-others);(114)surface of capsule(0-smooth,1-smooth or furrowed,2-others);(115)capsule narrower than urn(0-no,1-yes,2others);(116)position of capsule(0-distinctly terminal/terminal,1-others);(117)shape of capsule(0cylindric to oval,1-others);(118)capsule longer than 1mm(0-no,1-yes,2-others);(119)capsule apex and unlobed at base(0-no,1-yes,2-others);(120)calyptrae(0-cucullate,1-others);(121)calyptrae: plicate(0-no,1-yes,2-others);(122)covering of calyptrae(0-covering only operculum,1-covering only operculum and capsule apex,2-others);(123)calyptrae unlobed at base(0-no,1-yes,2-others);(124)size of calyptrae(0-small,1-large/big,2-others);(125)peristome(0-absent,1-present,2-present with papillose,3-others);(126)number of peristome(0-single,1-single or absent,2-single or double,3double,4-others);(127)peristome teeths(0-16,1-with teeth united in a high or rarely low,2others);(128)peristome state(0-not refl exed,1-spirally twisted above,2-with tubular basal membrane ,3-others);(129)development of peristome(0-weakly developed,1-better developed,2-strongly developed,3-others);(130)basal membrane of endostome(0-keeled,1-others);(131)segments of endostome(0-keeled and perforate,1-others);(132)endostome with cilia(0-no,1-yes,2others);(133)exostome(0-without or free of,1-with,2-others);(134)operculum(0-conic to apiculate,1others);(135)projection of seta(0-exserted,1-others);(136)size of seta(0-longer than 2mm,1-others);(1 37)perichaetial position(0-terminal,1-usually low and simple to bifi d,2-others);(138)perichaetia with papillae(0-no,1-yes,2-absent to large,3-others);(139)perichaetial surface(0-not scablike,1-scablike,2-ot hers);(140)perichaetial leaves (bract)(0-slightly or not diff erentiated,1-not diff erentiated,2-less diff erentiated,3-others);(141)shape of bract(0-never long awned,1-others);(142)propagula cup(0absent,1-present,2-others);(143)propagula(0-absent,1-present,2-with or without,3-sometimes present,4-never on leaf apices but sometimes elsewhere on leaves or in axils,5-others);(144)axillary propagula(0-without,1-with,2-others);(145)cells without nodulose-waxy walls(0-no,1-yes,2others);(146)cell characteristics(0-smooth, lax, thin-walled and hexagonal to rhombic,1others);(147)cells of old plants without colour change to bluish-green(0-no,1-yes,2-others);(148)cell walls not thickened on abaxial side(0-no,1-yes,2-others);(149)angular cells(0-not opaque,1others);(150)end walls of basal cells not thickened(0-no,1-yes,2-others);(151)hyaline basal cells (if present)(0-extending equal to costa,1-others);(152)laminal cells surface view(0-obscure,1-welldefi ned,2-others);(153)basal laminal cells(0-diff erentiated, hyaline and elongated,1-little diff erentiated, green and short-rectangular,2-others);(154)tomentum (if present)(0-restricted to extreme base of stems,1-others);(155)gemmae(0-without,1-with,2-others);(156)nerve(0-(60-) 65 – 115μm wide near base,1-others). data analysis two analyses: cluster analysis (depicting similarities among otus) (madeira et al. 1999; ferguson et al. 2000; sharma et al. 2004) and principal component analysis (pca) (non-hierarchical relationships among otus) (mcnulty 2004) were chosen and performed on the matrix data (table 2-11). for cluster analysis, single linkage amalgation rule and euclidean distances measure were manipulated for classifi cation. statistica 6.0 (by statsoft, inc. 2001) was utilized in this taxometric study (mazak and groves, 2006). further numerical taxonomic methodology followed luna et al. (2000); romero et al. (2000); and kim et al. (2003). taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 1. continued 140 data validation reliability and item analysis was performed to measure the overall representation of the data analyzed and degree of bias. th is test was run with statistica 6.0 (by statsoft, inc, 2001). results and discussions th e output from the reliability and item analysis showed that the data set is highly reliable (cronbach’s alpha = 0.9627). th is means that more than 96% of the data analyzed were true score variability and refl ecting the real situation. th is value is higher than the standardized alpha (0.9612). th e character states (156 characters examined) for 23 otus (table 2–11) were analyzed and produced 22 nodes for classifi cation. table 2. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses operational taxonomic unit (otu) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 barbula bolleana (müll. hal.) broth. 1 0 0 0 0 0 1 0 0 2 3 2 2 2 3 2 1 1 bryum caespiticium hedw. 1 2 1 0 0 0 1 0 0 0 2 1 0 2 3 2 1 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 3 1 0 0 0 1 0 0 0 2 1 0 2 3 2 1 1 cratoneuron fi licinum (hedw.) spruce 2 0 1 2 2 2 1 0 1 2 3 2 2 2 3 2 2 2 didymodon tophaceus (brid.) lisa 1 0 0 0 0 0 1 0 0 2 3 2 2 2 3 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 1 0 0 0 0 0 1 0 0 2 2 2 2 2 3 2 1 0 eurhynchium speciosum (brid.) jur. 1 0 0 0 1 2 0 1 0 2 3 2 2 2 3 2 2 0 eurhynchium hians (hedw.) sande lac. var. hians 1 0 0 0 1 2 0 1 0 2 3 2 2 2 3 2 2 0 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 0 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 fissidens grandifrons brid. 2 4 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 fontinalis antipyretica hedw. 2 1 1 2 1 2 2 1 1 2 3 2 2 2 3 2 2 2 fontinalis duriaei schimp. 2 4 1 2 1 2 2 1 1 2 3 2 2 2 3 2 2 2 gymnostomum calcareum nees & hornsch. 1 0 0 2 0 0 1 0 0 2 2 2 2 2 3 2 1 0 hygroamblystegium tenax (hedw.) jenn. 1 1 0 0 1 2 1 0 0 2 3 1 2 2 3 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 2 0 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 leptodictyum humile (p. beauv.) ochyra 2 0 0 0 1 2 1 0 0 2 3 2 1 1 3 2 2 0 leptodictyum riparium (hedw.) warnst. 2 0 0 0 1 2 1 0 0 2 3 2 1 1 3 2 2 0 palustriella commutata (hedw.) ochyra 2 0 0 2 2 2 1 0 1 2 3 2 2 2 3 2 2 2 philonotis fontana (hedw.) brid. 1 4 0 0 0 0 1 0 0 2 3 2 2 2 2 2 1 2 platyhypnidium riparioides (hedw.) dixon 1 1 0 0 1 2 1 0 0 2 3 2 2 2 3 2 2 0 pohlia melanodon (brid.) a.j. shaw 1 0 0 0 0 0 1 0 0 0 3 1 2 2 3 1 1 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 1 0 0 0 0 0 1 0 0 0 3 1 2 2 3 1 1 1 tortula marginata (bruch & schimp.) spruce 1 0 0 0 0 0 1 0 0 2 2 2 2 2 3 2 0 0 biotropia vol. 15 no. 2, 2008 141 table 3. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 barbula bolleana (müll. hal.) broth. 2 2 2 1 1 1 1 0 0 2 2 1 1 1 0 1 0 1 bryum caespiticium hedw. 0 2 1 1 1 1 2 0 0 2 3 2 0 0 0 1 1 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 2 1 1 1 1 2 0 0 2 3 2 0 0 0 1 1 1 cratoneuron fi licinum (hedw.) spruce 2 2 2 2 1 2 1 0 1 2 3 2 1 1 5 1 1 1 didymodon tophaceus (brid.) lisa 2 2 2 0 1 1 1 0 0 2 1 1 1 1 0 1 0 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 2 1 1 1 1 0 0 2 0 2 1 1 0 1 1 1 eurhynchium speciosum (brid.) jur. 0 2 2 1 1 1 2 0 0 2 3 2 1 1 3 1 1 1 eurhynchium hians (hedw.) sande lac. var. hians 0 2 2 1 1 1 2 0 0 2 3 2 1 1 4 1 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 2 2 2 1 2 1 1 1 2 3 2 1 1 6 1 1 1 fissidens grandifrons brid. 2 2 2 2 1 2 1 1 1 2 3 2 1 1 6 1 1 1 fontinalis antipyretica hedw. 2 2 2 2 1 2 1 0 1 1 3 2 1 1 6 0 1 1 fontinalis duriaei schimp. 2 2 2 2 1 2 1 0 1 1 3 2 1 1 6 0 1 1 gymnostomum calcareum nees & hornsch. 2 2 2 1 1 1 1 0 0 2 0 2 1 1 0 1 0 1 hygroamblystegium tenax (hedw.) jenn. 0 2 2 1 1 1 1 0 0 2 3 2 1 1 0 1 0 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 2 2 1 2 1 0 1 2 3 2 1 1 6 1 0 1 leptodictyum humile (p. beauv.) ochyra 2 2 1 1 2 1 1 0 0 2 3 2 1 1 4 1 1 1 leptodictyum riparium (hedw.) warnst. 2 2 1 1 2 1 1 0 0 2 3 2 1 1 4 1 1 1 palustriella commutata (hedw.) ochyra 2 2 2 2 1 2 1 0 1 2 3 2 1 1 3 1 1 1 philonotis fontana (hedw.) brid. 2 0 2 0 1 1 1 0 0 2 0 1 1 1 2 1 1 1 platyhypnidium riparioides (hedw.) dixon 2 2 1 2 1 1 1 0 0 2 3 2 1 1 4 1 1 1 pohlia melanodon (brid.) a.j. shaw 2 2 2 1 1 1 1 0 0 2 3 1 1 1 6 1 1 0 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 2 1 1 1 1 0 0 2 3 1 1 1 6 1 1 0 tortula marginata (bruch & schimp.) spruce 1 2 2 0 0 1 1 0 0 2 0 2 1 1 6 1 1 1 table 4. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 barbula bolleana (müll. hal.) broth. 0 2 3 2 2 1 2 0 3 2 1 1 0 0 5 2 0 0 bryum caespiticium hedw. 1 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 0 cratoneuron fi licinum (hedw.) spruce 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 8 didymodon tophaceus (brid.) lisa 2 1 3 2 2 2 2 0 3 2 1 1 0 0 5 2 2 0 eucladium verticillatum (brid.) bruch & schimp. 3 1 3 2 2 2 2 0 3 2 1 2 2 1 5 2 2 6 eurhynchium speciosum (brid.) jur. 3 2 1 0 0 2 2 0 0 2 1 2 2 1 0 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 3 2 1 0 0 2 2 0 0 2 1 2 2 1 0 2 2 2 taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 142 operational taxonomic unit (otu) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 3 2 3 2 2 2 1 3 3 2 4 2 2 1 5 2 2 8 fissidens grandifrons brid. 3 2 3 2 2 2 1 3 3 2 4 2 2 1 5 2 2 8 fontinalis antipyretica hedw. 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 fontinalis duriaei schimp. 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 gymnostomum calcareum nees & hornsch. 2 1 3 2 2 2 2 0 3 1 4 0 0 1 5 2 2 6 hygroamblystegium tenax (hedw.) jenn. 0 2 2 2 2 2 2 0 3 2 1 2 2 1 0 2 2 8 hymenostylium recurvirostrum (hedw.) dixon 3 2 3 2 2 2 2 3 3 2 0 2 2 1 5 2 2 7 leptodictyum humile (p. beauv.) ochyra 1 2 2 1 1 2 2 0 1 2 1 2 2 1 0 0 2 2 leptodictyum riparium (hedw.) warnst. 1 2 2 1 1 2 2 0 1 2 1 2 2 1 0 0 2 2 palustriella commutata (hedw.) ochyra 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 philonotis fontana (hedw.) brid. 0 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 3 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 2 2 2 0 3 2 1 2 2 1 5 2 2 2 pohlia melanodon (brid.) a.j. shaw 0 2 3 2 2 2 2 3 3 1 1 0 2 1 5 2 2 4 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 2 3 2 2 2 2 3 3 1 1 0 2 1 5 2 2 4 tortula marginata (bruch & schimp.) spruce 0 1 3 2 2 0 2 1 3 2 4 0 0 1 2 2 1 0 table 5. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 barbula bolleana (müll. hal.) broth. 0 0 0 0 1 2 3 5 1 2 1 2 0 3 2 2 2 3 bryum caespiticium hedw. 0 0 2 1 2 5 0 0 0 1 3 1 2 1 0 2 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 0 2 1 2 5 0 0 0 1 3 1 2 1 0 2 2 1 cratoneuron fi licinum (hedw.) spruce 0 0 2 6 2 5 2 9 5 2 3 2 2 8 3 2 2 2 didymodon tophaceus (brid.) lisa 0 0 0 3 2 2 2 2 5 0 2 1 2 0 3 2 2 2 eucladium verticillatum (brid.) bruch & schimp. 0 0 2 6 2 3 2 3 5 1 1 1 2 0 3 2 2 2 eurhynchium speciosum (brid.) jur. 0 0 2 4 0 2 4 5 1 1 3 1 2 5 3 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 0 0 2 4 0 2 4 5 1 1 3 1 2 5 3 2 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 fissidens grandifrons brid. 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 fontinalis antipyretica hedw. 0 0 2 6 2 5 2 9 1 1 3 1 2 1 3 2 2 2 fontinalis duriaei schimp. 0 0 2 6 2 5 2 9 1 1 3 1 2 1 3 2 2 2 gymnostomum calcareum nees & hornsch. 0 0 2 3 2 2 2 5 5 0 1 1 2 0 3 2 2 2 hygroamblystegium tenax (hedw.) jenn. 0 0 2 6 0 5 2 9 2 1 2 1 2 5 3 2 2 2 hymenostylium recurvirostrum (hedw.) dixon 0 0 2 6 2 1 2 9 5 1 3 1 2 6 3 1 2 2 leptodictyum humile (p. beauv.) ochyra 0 0 1 4 0 5 2 6 5 1 2 0 1 4 2 2 2 2 leptodictyum riparium (hedw.) warnst. 0 0 1 4 0 5 2 6 5 1 2 0 1 4 2 2 2 2 table 4. continued biotropia vol. 15 no. 2, 2008 143 operational taxonomic unit (otu) 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 palustriella commutata (hedw.) ochyra 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 philonotis fontana (hedw.) brid. 0 0 2 6 2 5 2 7 3 1 3 1 2 7 3 2 2 2 platyhypnidium riparioides (hedw.) dixon 0 0 2 4 0 5 2 4 5 1 3 1 2 5 2 2 2 2 pohlia melanodon (brid.) a.j. shaw 0 0 1 5 2 5 0 8 5 1 3 1 2 0 3 2 2 2 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 0 1 5 2 5 0 8 5 1 3 1 2 0 3 2 2 2 tortula marginata (bruch & schimp.) spruce 0 0 2 6 2 4 2 3 4 0 1 1 2 8 0 2 1 2 table 6. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 barbula bolleana (müll. hal.) broth. 1 1 0 0 2 0 0 0 1 2 2 3 3 3 3 3 3 0 bryum caespiticium hedw. 3 1 1 3 3 2 2 0 0 1 2 2 2 2 1 2 2 2 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 3 1 1 3 3 2 2 0 0 1 2 2 2 2 1 2 2 2 cratoneuron fi licinum (hedw.) spruce 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 didymodon tophaceus (brid.) lisa 1 0 1 3 2 2 1 0 0 0 1 2 2 2 2 2 2 1 eucladium verticillatum (brid.) bruch & schimp. 3 1 0 3 2 1 2 0 0 2 2 2 2 2 2 2 2 2 eurhynchium speciosum (brid.) jur. 3 1 1 3 3 2 2 2 0 2 2 2 2 2 2 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 3 1 1 3 3 2 2 2 0 2 2 2 2 2 2 2 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fissidens grandifrons brid. 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fontinalis antipyretica hedw. 0 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fontinalis duriaei schimp. 0 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 gymnostomum calcareum nees & hornsch. 1 0 0 3 1 2 2 1 0 0 1 2 2 2 2 2 2 2 hygroamblystegium tenax (hedw.) jenn. 4 2 0 1 3 2 2 2 1 0 2 2 1 1 2 2 2 3 hymenostylium recurvirostrum (hedw.) dixon 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 leptodictyum humile (p. beauv.) ochyra 2 1 0 3 3 2 2 2 0 1 2 2 2 2 1 2 2 2 leptodictyum riparium (hedw.) warnst. 2 1 0 3 3 2 2 2 0 1 2 2 2 2 1 2 2 2 palustriella commutata (hedw.) ochyra 3 1 0 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 philonotis fontana (hedw.) brid. 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 2 2 platyhypnidium riparioides (hedw.) dixon 3 1 0 3 3 2 2 2 0 2 2 2 2 2 1 1 2 2 pohlia melanodon (brid.) a.j. shaw 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 1 2 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 1 2 tortula marginata (bruch & schimp.) spruce 1 1 0 2 1 2 2 0 0 2 2 1 2 2 2 2 2 2 table 5. continued taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 144 table 7. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 barbula bolleana (müll. hal.) broth. 1 0 1 2 0 1 0 0 1 0 1 2 1 2 2 bryum caespiticium hedw. 1 0 1 2 1 0 0 2 2 1 1 2 1 2 2 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 0 1 2 1 0 0 2 2 1 1 2 1 2 2 cratoneuron fi licinum (hedw.) spruce 2 1 1 1 2 2 4 2 2 2 0 2 1 2 2 didymodon tophaceus (brid.) lisa 0 0 1 2 0 0 0 0 1 2 1 0 1 2 2 eucladium verticillatum (brid.) bruch & schimp. 1 0 1 2 4 0 0 2 2 2 1 2 1 2 2 eurhynchium speciosum (brid.) jur. 1 4 1 2 4 2 1 2 2 2 1 2 1 2 2 eurhynchium hians (hedw.) sande lac. var. hians 1 4 1 2 4 2 1 2 2 2 1 2 1 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 1 4 3 2 4 2 4 2 2 2 1 2 1 2 2 fissidens grandifrons brid. 1 4 3 2 4 2 4 2 2 2 1 2 1 2 2 fontinalis antipyretica hedw. 1 4 3 2 3 2 4 2 2 2 1 2 1 2 2 fontinalis duriaei schimp. 1 4 3 2 3 2 4 2 2 2 1 2 1 2 2 gymnostomum calcareum nees & hornsch. 1 0 1 2 3 1 0 0 1 0 1 1 0 0 2 hygroamblystegium tenax (hedw.) jenn. 1 4 1 2 3 2 1 2 2 2 1 2 1 2 2 hymenostylium recurvirostrum (hedw.) dixon 1 4 3 2 4 2 4 0 1 2 1 2 1 2 2 leptodictyum humile (p. beauv.) ochyra 1 3 1 2 4 2 2 2 2 2 1 2 1 2 2 leptodictyum riparium (hedw.) warnst. 1 3 1 2 4 2 3 2 2 2 1 2 1 2 2 palustriella commutata (hedw.) ochyra 1 1 2 2 2 2 4 2 2 2 1 2 1 2 2 philonotis fontana (hedw.) brid. 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 3 2 1 2 2 2 1 2 1 2 2 pohlia melanodon (brid.) a.j. shaw 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 tortula marginata (bruch & schimp.) spruce 1 0 1 2 3 0 0 2 2 2 1 2 1 2 2 table 8. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 barbula bolleana (müll. hal.) broth. 0 0 0 0 0 0 0 0 0 1 1 1 2 2 0 bryum caespiticium hedw. 0 0 1 1 0 0 1 1 2 1 0 0 1 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 0 1 1 0 0 1 1 2 1 0 0 1 2 1 cratoneuron fi licinum (hedw.) spruce 0 2 2 2 0 0 6 1 1 2 1 1 2 2 1 didymodon tophaceus (brid.) lisa 0 0 0 0 0 0 0 0 1 1 1 2 1 0 0 eucladium verticillatum (brid.) bruch & schimp. 0 2 2 2 1 2 6 1 2 2 0 1 2 2 1 eurhynchium speciosum (brid.) jur. 3 2 2 2 0 2 6 1 2 1 1 1 2 2 1 eurhynchium hians (hedw.) sande lac. var. hians 3 2 2 2 0 2 6 1 2 1 1 1 2 2 1 biotropia vol. 15 no. 2, 2008 145 operational taxonomic unit (otu) 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fissidens grandifrons brid. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fontinalis antipyretica hedw. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fontinalis duriaei schimp. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 gymnostomum calcareum nees & hornsch. 0 0 1 0 0 0 0 1 2 1 1 0 2 2 0 hygroamblystegium tenax (hedw.) jenn. 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 3 2 2 2 1 2 6 1 2 2 1 1 2 2 1 leptodictyum humile (p. beauv.) ochyra 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 leptodictyum riparium (hedw.) warnst. 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 palustriella commutata (hedw.) ochyra 0 2 2 2 0 0 6 1 2 2 1 1 2 2 1 philonotis fontana (hedw.) brid. 1 0 1 0 0 0 4 1 2 1 1 1 2 2 1 platyhypnidium riparioides (hedw.) dixon 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 pohlia melanodon (brid.) a.j. shaw 4 0 1 0 0 0 0 0 2 1 0 1 2 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 4 0 1 0 0 0 2 0 2 1 0 1 2 2 1 tortula marginata (bruch & schimp.) spruce 1 0 1 0 0 0 6 1 1 1 1 0 2 1 0 table 9. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 barbula bolleana (müll. hal.) broth. 0 2 2 2 1 0 0 3 3 1 1 2 2 1 0 bryum caespiticium hedw. 2 2 2 2 1 4 2 3 1 0 0 1 0 2 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 2 2 2 2 1 4 2 3 1 0 0 1 0 2 0 cratoneuron fi licinum (hedw.) spruce 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 didymodon tophaceus (brid.) lisa 0 2 2 0 1 2 2 3 1 1 1 2 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 2 2 3 4 2 3 3 1 1 2 2 2 1 eurhynchium speciosum (brid.) jur. 2 2 2 2 3 4 2 3 2 1 1 2 2 1 1 eurhynchium hians (hedw.) sande lac. var. hians 2 2 2 2 3 4 2 3 2 1 1 2 2 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fissidens grandifrons brid. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fontinalis antipyretica hedw. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fontinalis duriaei schimp. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 gymnostomum calcareum nees & hornsch. 2 1 1 2 1 1 2 3 3 1 1 2 2 1 1 hygroamblystegium tenax (hedw.) jenn. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 2 2 0 4 2 3 3 1 1 2 2 1 1 leptodictyum humile (p. beauv.) ochyra 2 2 2 2 3 2 2 0 3 1 1 2 2 0 1 leptodictyum riparium (hedw.) warnst. 2 2 2 2 3 2 2 0 3 1 1 2 2 0 1 taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 8. continued 146 operational taxonomic unit (otu) 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 palustriella commutata (hedw.) ochyra 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 philonotis fontana (hedw.) brid. 2 2 2 2 3 4 2 3 1 1 1 2 2 1 1 platyhypnidium riparioides (hedw.) dixon 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 pohlia melanodon (brid.) a.j. shaw 2 2 2 2 3 3 2 3 3 1 1 2 2 1 0 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 2 2 3 3 2 3 3 1 1 2 2 1 0 tortula marginata (bruch & schimp.) spruce 2 0 2 0 1 4 1 2 3 1 1 2 2 1 1 table 10. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 136 137 138 139 140 141 142 143 144 145 146 barbula bolleana (müll. hal.) broth. 0 0 1 0 0 0 0 5 2 1 1 bryum caespiticium hedw. 0 2 3 2 3 1 2 2 0 1 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 2 3 2 3 1 2 2 0 1 0 cratoneuron fi licinum (hedw.) spruce 1 2 3 2 3 1 2 5 2 2 1 didymodon tophaceus (brid.) lisa 1 0 2 0 0 0 2 0 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 1 2 3 2 1 1 2 2 1 1 1 eurhynchium speciosum (brid.) jur. 1 2 3 2 3 1 2 5 2 1 1 eurhynchium hians (hedw.) sande lac. var. hians 1 2 3 2 3 1 2 5 2 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.nann. 1 2 3 2 3 1 2 5 2 2 1 fissidens grandifrons brid. 1 2 3 2 3 1 2 5 2 2 1 fontinalis antipyretica hedw. 1 2 3 2 3 1 2 3 2 2 1 fontinalis duriaei schimp. 1 2 3 2 3 1 2 3 2 2 1 gymnostomum calcareum nees & hornsch. 1 0 2 2 2 1 2 2 2 1 1 hygroamblystegium tenax (hedw.) jenn. 1 2 3 2 3 1 2 5 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 1 2 3 2 3 1 2 5 2 2 1 leptodictyum humile (p. beauv.) ochyra 1 2 3 2 3 1 2 0 2 1 1 leptodictyum riparium (hedw.) warnst. 1 2 3 2 3 1 2 0 2 1 1 palustriella commutata (hedw.) ochyra 1 2 3 2 3 1 2 5 2 2 1 philonotis fontana (hedw.) brid. 1 2 3 2 1 1 2 2 2 1 1 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 3 1 2 5 2 1 1 pohlia melanodon (brid.) a.j. shaw 0 2 3 2 3 1 2 2 1 1 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 2 3 2 3 1 2 2 1 1 1 tortula marginata (bruch & schimp.) spruce 1 2 3 2 1 1 2 2 2 1 1 biotropia vol. 15 no. 2, 2008 table 9. continued 147 table 11. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 147 148 149 150 151 152 153 154 155 156 barbula bolleana (müll. hal.) broth. 2 2 1 2 0 0 0 1 2 1 bryum caespiticium hedw. 1 1 1 2 0 2 2 1 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 1 1 2 0 2 2 1 2 1 cratoneuron fi licinum (hedw.) spruce 2 2 1 2 1 2 2 1 2 1 didymodon tophaceus (brid.) lisa 2 2 1 2 0 1 1 1 2 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 1 2 0 2 2 1 2 1 eurhynchium speciosum (brid.) jur. 2 2 1 2 1 2 2 0 2 1 eurhynchium hians (hedw.) sande lac. var. hians 2 2 1 2 1 2 2 0 2 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.nann. 2 2 1 2 1 2 2 1 2 1 fissidens grandifrons brid. 2 2 1 2 1 2 2 1 2 1 fontinalis antipyretica hedw. 2 2 1 2 1 2 2 1 2 1 fontinalis duriaei schimp. 2 2 1 2 1 2 2 1 2 1 gymnostomum calcareum nees & hornsch. 2 2 1 2 0 2 2 1 2 1 hygroamblystegium tenax (hedw.) jenn. 1 2 2 1 2 2 2 1 2 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 1 2 1 2 2 1 2 1 leptodictyum humile (p. beauv.) ochyra 2 2 0 2 1 2 2 0 0 0 leptodictyum riparium (hedw.) warnst. 2 2 0 2 1 2 2 0 0 0 palustriella commutata (hedw.) ochyra 2 2 1 2 1 2 2 1 2 1 philonotis fontana (hedw.) brid. 2 2 1 2 0 2 2 1 2 1 platyhypnidium riparioides (hedw.) dixon 2 2 1 2 1 2 2 0 2 1 pohlia melanodon (brid.) a.j. shaw 2 2 1 2 0 2 2 1 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 1 2 0 2 2 1 2 1 tortula marginata (bruch & schimp.) spruce 2 2 1 1 0 2 2 1 2 1 th e output of the analysis is presented in phenogram (figure 1). in the fi rst, second and third node, the linkage distance value is 1.0000 for bryum caespiticium and bryum pseudotriquetrum, eurhynchium speciosum and eurhynchium hians var. hians; and, leptodictyum humile and leptodictyum riparium, respectively. th e distance linkage between pohlia melanodon and pohlia wahlenbergii is 2.0000 (node 4). as for node 5, the value is 3.0000 between fontinalis antipyretica and fontinalis duriaei.two diff erent genera of cratoneuron fi licinum and palustriella commutata showed the value of 3.7417 in node 6. fissidens crassipes subsp. warnstorfi i and fissidens grandifrons in the seventh node are distantly valued 4.0000. at the eighth node (6.4807), cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i and fissidens grandifrons were linkaged. next, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons and hymenostylium recurvirostrum were clustered under node 9 with linkage distance value of 8.0000. at the tenth node, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica and fontinalis duriaei were distantly measured with value of 9.6954. taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 148 with distance linkage value of 10.2956, three species of leptodictyum humile, leptodictyum riparium and platyhypnidium riparioides were linkaged under node 11. next, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium and platyhypnidium riparioides were analyzed to have distance linkage value of 10.3923 (twelfth node). under node 13, there are eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana (11.3137). cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica, fontinalis duriaei, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana are under one cluster group (node 14 and distance linkage equals to 11.5326). under node 15, didymodon tophaceus and gymnostomum calcareum were found with 11.7898 value. for the sixteenth node, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica, fontinalis duriaei, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides, philonotis fontana and hygroamblystegium tenax were having distance value of 11.8743. with linkage distance value of 12.0831, didymodon tophaceus, gymnostomum calcareum and eucladium verticillatum were grouped under node 17. node 18 showed the combination between node 16 and 17 with 12.4097. distance linkage value of 12.4499 grouped all otus under node 18 and node 4 to become node 19. as for node 20, the combination is between node 19 and tortula marginata (12.5300). barbula bolleana and node 20 (12.8452) formed node 21. th e distance linkage value for all otus is 12.8564. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 linkage distance bryum pseudotriquetrum bryum caespiticium tortula marginata pohlia wahlenbergii pohlia melanodon eucladium verticillatum gymnostomum calcareum didymodon tophaceus hygroamblystegium tenax philonotis fontana platyhypnidium riparioides leptodictyum riparium leptodictyum humile eurhynchium hians var. hians eurhynchium speciosum fontinalis duriaei fontinalis antipyretica hymenostylium recurvirostrum fissidens grandifrons fissidens crassipes subsp. warnstorfii palustriella commutata cratoneuron filicinum barbula bolleana figure 1. dendogram showing the single linkage (nearest neighbours) clustering relationship based on euclidean distance among the 23 species of mosses biotropia vol. 15 no. 2, 2008 149 according to the principal component and classifi cation analysis, the quality of representation value is 100% or most reliable. th e fi rst three components explained 75.1064% of the variation with 56.0485%(pc1), 11.7346%(pc2) and 7.3233%(pc3) respectively (table 12). components with eigenvalues lower than 1 were eliminated and not signifi cant statistically. for pc1, the main variables are from the vegetative parts (plant and leaf ) and major morphometric characters are numbered 3, 8, 9, 26, 27, 33 (negative loading), 51 (negative loading), 54 (negative loading), 55, 56, 58 (negative loading), 60 (negative loading), 63 (negative loading), 68 (negative loading) and 143 (negative loading). less important on the reproductive part (capsule): 110, 112 (negative loading) and 126 (negative loading). pc2 showed the major variable is alar cells of leaf (92). th e focal part is on the vegetative component of the bryophyte. factor loading scores for pc3 were less correlated to the variables (characters) as compared to pc1 and pc2 . any factor score lower than 5.0000 is considered insignifi cant and eliminated from the factor loading tables. table 12. taxometric variables for the first three principal components component eigenvalue % total variance cumulative % 1 12.89116 56.04853 56.0485 2 2.69895 11.73459 67.7831 3 1.68436 7.32332 75.1064 figure 2. shows that mosses are skewed obviously to the negative side for pc1. pc1 grouped species obviously into three groups (i, j, o, d, r, n, k, l, p, q, h and g; s, v, u and f; and e, b and c) and 4 identical individual species (t, w, m and a). as for pc2 (figure 2), 2 groups were segregated into the positive (i, j, o, d, r, n, k, l, p, q, h and g) and negative (s, v, u, f, m, a, e, b and c) sides; and 2 individual species near to the intermediary part (t and w). taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. a b c d e f g h i j k l m n o p q r s t u v w -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 factor 1 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 f actor 2 figure 2. scatter diagram between pc1 and pc2 from principal component analysis of 23 species of aquatic and semi-aquatic mosses using 156 characters 150 in figure 3, pc3 grouped otus into one positive group of h, g, b, c, p, q, t, n and w; negative group of m, e, a, k, l, r, i,d, j, k, l, o, v and u; one species under intermediary line (f ); and one species near to intermediary line (s). pc1 (figure 3) segregated the individuals inconspicuously. basically, individual species grouped under intermediary area is sharing characters from positive and negative sides. figure 3. scatter diagram between pc1 and pc3 from principal component analysis of 23 species of aquatic and semi-aquatic mosses using 156 characters for cluster analysis, the algorithm chosen was hierarchical (aggromerative) (tipirdamaz et al. 2006). th is is because the main objective of this study is to group otus from smaller clusters into a larger groups (polythetic) (stuessy 1990). th e end result is to divide clustered groups for effi cient aquatic and semiaquatic bryodiversity management. th e amalgation rule for analysis was single linkage (nearest neighbour) as the purpose to study the species relationship among otus and the measurement for distance between species was based on euclidean distance. euclidean distance is the most common and easy to interpret (statistica 6.0, 2001). in this analysis, numbers of variables, cases and subcases analyzed were massive. indirectly, biases and standard deviations were minimized. as the result, the reliability of the output is more than 96%. on the other hand, clustering bryodiversity into few manageable units are very crucial and cost-eff ective. th e relationship between a cluster of mosses refl ects the generic, familial or higher taxonomic similarity. genetically, they are sharing a closer gene pool (genotypes) and morphologically, the phenotypes are signifi cant characters for identifi cation and serving the ecosystem. mosses are natural bioindicator for water quality, soil erosion controller and fi ltering the wastewater naturally (ando and matsuo 1984; biotropia vol. 15 no. 2, 2008 a b c d e f g h i j k l m n o p q r s t u v w -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 factor 1 -0.50 -0.25 0.00 0.25 0.50 f actor 3 151 frahm 1996; welch 1948; conrad 1935; whitehouse and mcallister 1954; ando 1957; grout 1912; coupal and lalancette 1976). th us, this approach can help bryodiversity managers to conserve the aquatic and semi-aquatic mosses in a collective way. in a simple manner, we effi ciently manage all the clusters of mosses equally. equality helps in balancing the habitat (ecosystem) for the benefi ts of human beings. ironically, wrong management strategy can be bias to certain species, the other species will be neglected and the ecosystem will not be served naturally. from the dendogram (figure 1), there are 22 nodes. th us, management strategy can be based on the nodes in the phenogram. for node 10, one management cluster can be formed from few subclusters which consists of cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica and fontinalis duriaei. subclusters of node 13 (eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana), node 18 (didymodon tophaceus, gymnostomum calcareum, hygroamblystegium tenax and eucladium verticillatum), node 20 (tortula marginata, pohlia wahlenbergii and pohlia melanodon) and node 22 (bryum caespiticium, bryum pseudotriquetrum and barbula bolleana). in short, fi ve management units of bryodiversity are proposed for management. under principal component analysis (pca), management of clustered groups are strengthened (tipirdamaz et al. 2006). based on statistica 6.0 (2001), pca is reducing the numbers of variables and transform important variables into numbers of principal component. th is is benefi cial in management, where precise group of identifi able characters (in this context) are known for management and conservation. furthermore, pca is a very cost-eff ective tool for biodiversity management. in cluster analysis, bryodiversity management is based on hierachical clustered manageable unit and pca is based on group of related characters that forms one factor. in this case, we have three principal components. in the fi rst principal component (figure 2), we have three distinct groups and four independent otus (can form two minor groups). th us, 5 managable units can be formed. all otus were skewed to the negative side. statistically, it signifi es all the otus were likely characterized diff erently from the common character states. th is is very similar to the numbers of manageable units derived from cluster analysis, but with distinct species combination. as for the second component, 3 managable units were formed. it consists of a positive group that agrees with most of the common character states, a negative group that complies likely to the opposite character states and two intermediary individuals (skewed a bit to the negative side) where sharing both common and uncommon (more) character states. in figure 3, three manageable units were observed: one positive-skewed group, one negative-skewed group and one individual in the intermediary line. comparing both taxometric classifi cations, cluster analysis is useful in hierachically linked otus for relationship-based management approach. all characters have the same weight and will be used totally. as for pca, it groups otus on the scatter plot that refers to the group of important characters (principal component). th us, only critical characters are used for effi cient management. relatively, both approaches to bryodiversity management are highly appreciated. only through cluster analysis, the linkage distance will be known and important for future populational references. th is means that populations are taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 152 evolving and further revisions will further change the taxonomic structure. for instance, pca does not show this feature. in a nutshell, both approaches are supplementing each ones. both combinations will help in solving managerial dilemma and problems. conclusion bryodiversity in the mediterrean area of spain is relatively lower (23 species of aquatic and semi-aquatic mosses) as compared to the 46 species recorded in the tropical region of sabah, malaysia.bryofl ora conservation cum management have to be taken place. th is is critical as the bryophytes are naturally serving the ecosystems continually and sustainably. th e studied mosses were phenetically related and could be divided into 5 cluster groups through cluster analysis. th e clustered group can be managed as a manageable unit. th e rationale is that no single population will be overmanaged or neglected; and equal conservational plan to be implemented among the phenetically related units. th e manipulation of principal component analysis (pca) in this study reduced the least important characters used for management. th e output produced three components with each group contain numbers of vital characters within. th is is costeff ective for bryodiversity managers. th is analysis allowed managers to identify, manage and conserve populations based on the components. from this study, output from cluster analysis will be an alternative to the results produced from the pca. nevertheless, both outputs are highly reliable and ready to be used for management. in a nutshell, it is more meaningful to conserve natural environmental regulator rather than creating man-made 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63-146. yamagishi m., s. matsumoto s., a. nakatsuka and h. itamura . 2005. identifi cation of persimmon (dyopyros kaki) cultivars and phenetic relationships between diopyros species by more eff ective rapd analysis. scientia horticulturae 105: 283-290. biotropia vol. 15 no. 2, 2008 37 application method of antimicrobial substances – a. dikin et al. *corresponding author : antario_dikin@yahoo.com biotropia vol. 15 no. 1, 2008 : 37 49 application method of antimicrobial substances for the control of schizoph yllum commune fr. causing brown germ and seed rot of oil palm antario dikin1*, kamaruzaman sijam2 and idris abu seman3 1agricultural quarantine agency of indonesia, jakarta, indonesia 2department of plant protection, faculty of agriculture, universiti putra malaysia, selangor d.e, malaysia 3plant pathology and weed science group, biological research division malaysian palm oil board, selangor, d.e. malaysia abstract biological seed treatment promotes to save the environment from toxic chemicals in the agricultural practices. schizophyllum commune is one of the important seedborne pathogenic fungi causing brown germ and seed rot of oil palm which required effective and efficient treatment based on environmental friendly approaches. anti-microbial substances are extracted from antagonistic bacteria of b. multivorans and m. testaceum after mass production in the liquid media. application method of anti-microbial substances for the control of schizophylllum commune was done by seed dipping for 30 minutes and vacuum infiltration at 400 mm hg. vac. for 2 min. in supernatant of anti-microbial substances diluted in sterilized distilled water with concentration ratio of 1:4. application method using anti-microbial substances from antagonistic bacteria inhibited the growth of pathogenic fungus, enhanced seed germination, and without causing any abnormal growth of oil palm seedlings. key words : anti-microbial substances, seed treatment schizophyllum commune fr. introduction seed treatment as part of integrated pest management practically minimizes the infection of any plant pathogens associated with seeds. the application of seed treatment can be used as preventive and curative method against plant pathogens. pre-treatment is normally applied to protect the seeds from soil-borne pathogens and to control seedborne pathogens. schizophyllum commune fr. causes brown germ and seed rot of oil 38 biotropia vol. 15 no. 1, 2008 palm. heavy infection of the pathogen decreased seed germination of oil palm about 60% (dikin et al. 2003). fifty two antagonistic bacterial isolates from oil palm plantation in peninsular malaysia were potential agents for suppressing the growth of s. commune. of the 52 isolates which produced anti-microbial substances burkholderia cepacia rb47, b. multivoras ru50 and microbacterium testaceum ru7 inhibited the growth of s. commune (dikin et al. 2006). formulation of active compounds in the bio-pesticide such as pyrrolnitrin from pseudomonas cepacia was developed by micro-encapsulation using gluten and casein. the micro-encapsulations protected active agents which are sensitive to direct light exposure and extreme temperature (yu and lee 1997). bacillus subtillis is an antagonistic bacteria of s. commune formulated as biopesticide in dry powder form. dry powder as the carrier of bacterial spores in the slurry is to protect seeds from the infection of rhizoctonia solani, fusarium spp., alternaria spp., and aspergillus spp. (fravel et al. 1998; desai et al. 2002). potential supernatant of anti-microbial substances from b. multivorans ru50 and m. testaceum ru7 suppressed the growth of s. commune in vitro. meanwhile, in vivo application for the control of brown germ and seed rot diseases of oil palm requires further studies. the objectives of the study were to determine the anti-microbial substances from b. multivorans ru50 and m. testaceum ru7 to control brown germ and seed rot diseases and to evaluate their potential preventive measures of oil palm seeds. materials and methods antimicrobial substance one loop-full of b. multivorans ru50 and m. testaceum ru7 isolates were inoculated to 1 liter of sterilized liquid media (10 g of neo peptone, 10 g of lactose and 10 g of peptone, 10 g of maltose, respectively, for each species of bacteria). liquid media containing the cell of each bacterial isolate were fermented in electric rotator (new brunswick scientific g-25kc) at 125 rpm for 5 days, and then added with 50 ml of absolute methanol. supernatant from fermented liquid media was separated using centrifugation and evaporated using buchi rotavopor r-200 at 45oc to reach 100 ml (dikin et al. 2006). effect of anti-microbial substances on non-germinating oil palm seeds inoculated with s. commune twenty five pre-heated oil palm seeds were artificially inoculated with s. commune by placing the seeds onto s. commune cultural plates, and then incubated in polythene bags for 7 days at room temperature for mycelial colonization. inoculated seeds were 39 application method of antimicrobial substances – a. dikin et al. treated with anti-microbial substances from mixing supernatant of b. multivorans ru50 and m. testaceum ru7. the ratios for treatment concentration of seed dipping were 1 : 2 (r2), 1:4 (r4), 1 : 6 (r6), and as control dipped into distilled water (sc-dip) for 30 minutes. other treatments of inoculated seeds were infiltrated in the anti-microbial substances of various concentrations namely 1:2 (v2), 1:4 (v4), 1:6 (v6) and as control the seeds were infiltrated into distilled water at 400 mm hg vac. for 2 min. the preheated seeds non inoculated with s. commune were used as negative control. treated non germinating seeds and the control were air dried in laminar chamber, and then incubated in polythene bags at 26±2oc. observation of seed germination was carried out at 14, 21, 28 days interval. four replicates were made for each treatment including control (dikin et al. 2003). effect of anti-microbial substances on the fungal colonization ten pre-heated oil palm seeds were treated with the supernatant of anti-microbial substances by dipping, vacuum infiltration, and seed coating. as control, the seeds were not treated with anti-microbial substances. as comparison, the seeds were dipped in a synthetic fungicide, 0.2 % of mancozeb. dipping treatment was conducted by soaking oil palm seeds in diluted anti-microbial substances 1:4 (r4) for 30 minutes. vacuum treatment was conducted by soaking and infiltration the seeds in diluted anti-microbial substances with ratio of 1:4 (v4) at 400 mm hg vac. for 2 min. seed coating was conducted by coating oil palm seeds with freeze-drying of anti-microbial substances in talc. the rate of concentration between talc and anti-microbial supernatant suspension was 1:1. seeds treated with anti-microbial supernatant were mixed with the talc powder in a polythene bag and shaken until the powder covered the whole seeds. the seeds were air dried, and then placed on the culture plate of 7day-old s. commune. plates were incubated at room temperature in polythene bags. the colonized seeds were recorded for each treatment. five replicates were made for each treatment including control. comparison of various pre-treatments of artificial inoculation of oil palm seeds on seed germination twenty five pre-heated oil palm seeds were treated with supernatant of antimicrobial substances by dipping (r4), vacuum (v4), seed coating by freeze dry solidification of anti-microbial substances in talc with concentration ratio of 1:1, dipping oil palm seeds in the suspension of peptone maltose, neo peptone lactose for 30 minutes. as negative control. as comparison oil palm seeds were dipped in the synthetic fungicide of 0.2% mancozeb. for dipping treatment seeds were shaken in diluted antimicrobial substances with concentration of 1 part of supernatant of anti-microbial substances diluted with 4 parts of distilled water (r4) for 30 minutes. for vacuum treatment the seeds were soaked and vacuumed in diluted anti-microbial substances with concentration of 1 part of supernatant of anti-microbial substances diluted with 4 parts of distilled water (v4) at 400 mm hg. vac. for 2 min. treated seeds were placed 40 biotropia vol. 15 no. 1, 2008 on cultural plates of 7–day-old s. commune and then incubated for 7 days in polythene bags at room temperature. then the seeds were collected and placed in a polythene bag for percentage of germination. seeds were incubated for 28 days at room temperature and the germination recorded. three replicates were made for each treatment. effect of pre-treated artificial inoculation of oil palm seeds on the growth of seedling a number of 10 germinated oil palm seeds from each treatment including control i.e. the protective treatment including seed dipping in diluted anti-microbial substances with ratio of 1:4 (r4) in distilled water, vacuum seed in diluted anti-microbial substances with ratio of 1:4 (v4), seed coating by freeze-dry solidification of antimicrobial substances in talc with ratio of 1:1, seed dipping in mancozeb, inoculated seeds treated with liquid solution of neopeptone, peptone, maltose, lactose as negative control and healthy seeds as positive control was sown in plastic pots (29 x 39 x 10 cm). seedling growth was maintained by watering and measuring the height of seedlings every week and the dry weight of seedlings were recorded at 12 weeks. three replicates were made for each treatment (dikin et al. 2003). results and discussion the mixed supernatant of antimicrobial substances from b. multivorans and m. testaceum were solidified in talc and kaolin as carrier by dry air heating and freeze -drying. both solidified anti-microbial substances in talc and kaolin affected the diametric clear zone of s. commune. dissolved freeze-dried anti-microbial substances in talc with distilled water 1:1 showed diametric clear zone highest formation of 21 mm, followed by freeze dried kaolin, air heating dried talc, and air heating dried kaolin with the diametric clear zone of s. commune i.e. 17.3 mm, 15.3 mm and 14.6 mm respectively. the formulation of anti-microbial substances in freeze-dried talc suppressed the growth of s. commune at dissolved ratio, 1:25, followed by freeze-dried kaolin. both freeze-dried talc and kaolin showed diametric clear zone of 14 mm and 13.6 mm, respectively. meanwhile, the air heating of dried talc and kaolin dissolved in distilled water at ratio 1:10 suppressed the growth of s. commune (table 1.). 41 application method of antimicrobial substances – a. dikin et al. table 1: clear zone diameter of s. commune against diluted anti-microbial substances in powder form formulated antimicrobial substances diluted ratio (anti-microbial substance in powder form : sterilized water)a average 1:1 1:5 1:10 1:15 1:20 1:25 1:30 freeze dry of kaolin 17.3ab 17ab 16bb 15.3cb 14.6db 13.6eb 0fa 13.4 freeze dry of talc 21aa 20ba 16.3ca 15.6da 15ea 14fa 0ga 14.5 dry air heating of kaolin 14.6ad 14.3ac 14.3ac 0bc 0bc 0bc 0ba 6.1 dry air heating of talc 15.3ac 13.3bd 12.6cd 0dc 0dc 0dc 0da 5.8 average 17 16.1 14.8 7.7 7.4 6.9 0 note: a average of three replications. means followed by different small letters within row (lsd0.05 =0.3 mm) and different capital letters within column (lsd0.05= 0.2 mm) are significantly different. among the two carriers with two drying techniques, freeze-dried talc was the best formulation of anti-microbial substances of b. multivorans and m. testaceum. dry air heating , freeze-dried talc and kaolin had different rigidity in grinding to obtain small size particles. it was easier to grind the solidified anti-microbial substances by freeze-drying technique than dry air heating. effect of supernatant of antimicrobial substances on non-germinating oil palm seeds inoculated with s. commune liquid form of supernatant of anti-microbial substances from b. multivorans and m. testaceum significantly suppressed the growth of s. commune in the oil palm seeds (p=0.05). the potential anti-microbial substances reduced the oil palm seed infection and enhanced the seed germination of oil palm. seed treatment of oil palm by dipping and vacuum showed that both treatments affected s. commune infection in the seed. seed germination of healthy oil palm seeds at 28 days after incubation was 95%. seed germination of artificial inoculated seeds dipped and vacuumed in distilled water was 17 and 56%, respectively. seed treatment with supernatant of anti-microbial substances significantly recovered seed germination. dipping (r4) and vacuuming (v4) treatment with supernatant of anti-microbial substances reached germination of 94 and 93%, respectively, and was not significantly different compared to healthy seeds i.e. 92% (table 2). seed treatment of oil palm with supernatant of anti-microbial substances suppressed fungal growth. mycelia colonized the whole seed and penetrated up to the kernel through germ pores. germ pores of seeds were blocked by compact mycelia and caused a failure in germination. the inhibition of mycelial growth of s. commune on 42 biotropia vol. 15 no. 1, 2008 the whole seeds using anti-microbial substances i.e. dipping and vacuum treatment induced seed germination. at the early stage, the germination plugs of the treated seeds release from germ pores; radicle and plumule gradually came out with soft tissue and white in colour. table 2. percentage of seed germination of oil palm seeds after incubation treatment*) days of incubation 14 21 28 control without sc 92a 92a 95a sc vac 53d 56e 56f sc dip 16f 16f 17g r2 22e 60d 78e r4 70c 82c 94ab r6 72c 82c 86d v2 72c 88b 90c v4 89ab 92a 93ab v6 86b 92a 92bc notes: average of four replications. means within column with the same small letter are not significantly different at p=0.05 according to least significant different test *) r2 = dipping treatment with one part of supernatant anti-microbial substances and added two parts of distilled water r4 = dipping treatment with one part of supernatant anti-microbial substances and added four parts of distilled water r6 = dipping treatment with one part of supernatant anti-microbial substances and added six parts of distilled water v2 = vacuum treatment with one part of supernatant anti-microbial substances and added two parts of distilled water v4 = vacuum treatment with one part of supernatant anti-microbial substances and added four parts of distilled water v6 = vacuum treatment with one part of supernatant anti-microbial substances and added 6 parts of distilled water control without sc = healthy seeds (control) sc vac = vacuum treatment of artificial inoculated seeds in distilled water scdip = dipping treatment of artificial inoculated seeds in distilled water comparison of various pre-treatments of artificial inoculation of oil palm seeds on seed germination pre-treatment of seed using anti-microbial substances was able to protect the fungal growth and was reflected by the seed germination recovery. from the different pre-treatments in the application of anti-microbial substances as preventive measures, 43 application method of antimicrobial substances – a. dikin et al. 28-day treatment after incubation showed that dipping and vacuum significantly increased the percentage of seed germination. both dipping and vacuum pre-treatments increased the germination rate, compared to seeds without artificial inoculation. both dipping and vacuum pre-treatments were also significantly different with the application of synthetic fungicide, mancozeb (p = 0.05). seed coating with freeze dry anti-microbial substances in talc was not significantly different with non-inoculated seeds and significantly different with artificial inoculated seeds treated with distilled water or liquid medium (neo peptone, lactose, peptone, maltose) as negative control (table 3.). table 3: percentage of seed germination of pre-treated seeds before artificial inoculation with s. commune*) pre-treatment days of incubation 14 21 28 vacuum 37b 72a 84a dipping 31c 72a 84a seed coating 25d 51b 80b mancozeb 26cd 51b 81b without s. commune (positive control) 73a 76a 81b inoculated s. commune + water (negative control) 11e 12c 33c inoculated s. commune + neopeptone lactose peptone maltose (negative control) 13e 13c 25d note : average of four replications. means followed by different small letters within column are significantly different (lsd p=0.05) supernatant of anti-microbial substance as seed protector against infection of s. commune supernatant of anti-microbial substances from b. multivorans and m. testaceum protected oil palm seeds from infection of s. commune (figure 1.). the anti-microbial substances in the liquid form and solidified anti-microbial substances in talc significantly inhibited colonization of s. commune on all oil palm seeds. vacuum pre-treatment of oil palm seeds significantly reduced the colonization of fungus up to 88%, followed by seed coating with solidified anti-microbial substances in talc which reduced the colonization of fungus by 66% and dipping pre-treatment reduced the colonization of fungus by 16%. the synthetic fungicide, mancozeb at 2 g/l in distilled water did not protect the seeds from the colonization of s. commune (table 4.). 44 biotropia vol. 15 no. 1, 2008 a c b d table 4: percentage of pre-treated oil palm seeds colonized by s. commune (%) pre-treatment seed coloniza-tion (%)* dipping in anti-microbial substances with concentration 1:4 84b vacuum in anti-microbial substance with dose 1:4 12d seed coating with solidified anti-microbial substances in talc 34c mancozeb 100a without anti-microbial substances (control ) 100a note : average of five replications. means followed by different small letters within column are significantly different (lsd p =0.05). d b a figure 1: a. oil palm seeds after vacuum pre-treatment with anti-microbial substances b. seed coating with freeze-dry solidified anti-microbial substances in talc c. dipping pre-treatment with anti-microbial substances d. seeds without anti-microbial substances as control 45 application method of antimicrobial substances – a. dikin et al. a b effect of pre-treated artificial inoculation of oil palm seeds on the growth of seedlings seed treatment of artificially inoculated seeds with anti-microbial substances induced seed germination. schizophyllum commune with its compact mycelia blocked the germ pores and was found to be the main cause of germination loss. when the radicle and plumule already emerged from seed, and the seed infection of s. commune took place, germinated seeds were still able to continue its growth. the anti-microbial substances showed impacts on suppressing the fungal growth during germination, and did not inhibit seedling growth. seedling height from inoculated seeds and then treated with dipping in distilled water showed growth inhibition (figure 2). seedling height at 12 weeks old showed that all treatments of vacuumed and dipped in anti-microbial substances and non inoculation of s. commune were not significantly different. mean dry weight of 12-week-old seedling treated with v2, v4, v6, r2, r4, r6, water dipping (c-wt) and water vacuum (c-cv ) were significantly different. vacuum treatment (v2, v4) enhanced dry weight of seedling followed by others (table 5). figure 2. oil palm seedlings from artificially inoculated seeds with s. commune a. without treatment with anti-microbial substances b. treated artificially with anti-microbial substances 46 biotropia vol. 15 no. 1, 2008 table 5: height of oil palm seedling from the treated germinating seeds (cm) seedling age (week) treatment* c-wt no-sc v2 v4 v6 r2 r4 r6 c-vc 6 13.7d 15.7ab 15.3ab 15.4ab 15.1bc 15.4ab 16.3a 14.9bc 14.1dc 7 15.3c 17.6b 18.4ab 18.5ab 17.8ab 17.9ab 19.3a 18.5ab 17.1b 8 18.3c 19.1bc 19.1bc 21a 19.6abc 20abc 21.2a 19.9abc 20.1ab 9 19.7c 20.1bc 21.7a 21.9a 21abc 21.3ab 21.7a 20.5abc 20.9abc 10 19.7b 21.6a 22.8a 22.7a 22.6a 22.1a 22.7a 21.7a 21.4ab 11 21.4d 23.8ab 24.5a 23.6ab 23.8ab 23.7ab 23.2bc 23.3bc 22.5c 12 22.5b 24.6a 25.8a 26a 25.6a 25.1a 25.8a 25.7a 24.9a dry weight (mg) 1242b 1250ab 1425a 1431a 1265ab 1281ab 1345ab 1337ab 1368ab note : average of thirty replications. means followed by different letters within the same row are significantly different (lsd p=0.05). formulation of supernatant of anti-microbial substances from b. multivorans and m. testaceum were solidified in the carrier of talc and kaolin which affected the mic of ant-imicrobial substances against s. commune. the carrier of talc and kaolin as aggregate ingredients has the function to release and dissolve anti-microbial substances easily and faster in water without reduction of active compounds to suppress the growth of s. commune. freeze-dry solidification of supernatant of anti-microbial substances was not easy to keep it in dry powder form. the freeze-dried powder of supernatant of anti-microbial substances is hygroscopic and trapped water from the air. after 24 hours in open air, the freeze-dried powder of anti-microbial substances becomes liquid. talc and kaolin as the carriers of anti-microbial substances with concentration ratio of 1:1 formulated the anti-microbial substances to the powder form and act as bio-pesticide. the concentration ratio of anti-microbial substances in talc or kaolin was 1:1 and avoided water trapped by the formulated bio-pesticide from the air. dry technique of freeze-drying and dry air heating in the solidification of supernatant of anti-microbial substances in talc and kaolin had different values of minimum inhibitory concentration (mic). dry air heating of talc and kaolin at 60oc for 48 hours reduced the potential of anti-microbial substances to inhibit s. commune. actually, the stability of ant-microbial substances from b. multivorans and m. testaceum against the temperature in the powder form was up to 60oc within 2 hours. other limiting factors in dry air heating of solidified anti-microbial substances in talc and kaolin were hardening, difficult to grind and not easy to dissolve in water compared with freeze dry solidification of anti-microbial substances in talc and kaolin. lower 47 application method of antimicrobial substances – a. dikin et al. value of mic anti-microbial substances in talc and kaolin powder compared to the liquid form was due to reduction half of the active compounds and also the content of active compounds, such as phenazine, pyrrolnitrin and phenylpyrrol which was too small based on hplc analysis. synthetic fungicide such as thiram with a dose of 2 g/l is used for seed treatment of oil palm (turner and gillbanks 2003). although, the ratio of concentration of this synthetic fungicide was relatively high, the potential of suppressing pathogenic fungus was still effective. the pre-treatment of oil palm seeds using formulated anti-microbial substances in liquid form was effective to control s. commune. schizophyllum commune attacked oil palm seeds through direct contact between infected seeds and healthy seeds, and then the mycelia colonized the whole seed. based on the dry weight of 12-week-old seedlings, seed treatment of inoculated seeds with antimicrobial substances did not inhibit seedling growth. the liquid formulation protected the attack of s. commune by the colonizing oil palm seeds. vacuum pre-treatment was more effective than dipping pre-treatment in the application of liquid formulation as protective measure. vacuum pre-treatment quantitatively enhanced the absorption of anti-microbial substances by the oil palm seeds. mean while, dipping pre-treatment showed that the anti-microbial substances covered the whole seeds, dry faster in the open air and less absorption of anti-microbial substances. the difference in absorption quantity of anti-microbial substances on both vacuum and dipping pre-treatment in the seeds is equal to different applications of the concentration of anti-microbial substances. effective dose in the application of liquid formulation was one part supernatant of anti-microbial substances from b. multivorans and m. testaceum dissolved into four parts of distilled water. however, for the application of freeze-dry solidified antimicrobial substances in talc it was equal to one gram dissolved in two ml of distilled water. this dose of powder formulation was able to suppress the fungal growth, but the application of seed pre-treatment caused less germinating seeds, due to the formulated talc in the wet powder to cover the whole seeds which strongly blocked the germ pores for the absorption of the required oxygen in respiration activity before and after germination. coating pre-heated seeds with solidified freeze dry anti-microbial substances in talc apparently protected the oil palm seeds from contaminant fungi for long period in store. coating pre-heated seeds with synthetic fungicide is commonly used to protect oil palm seeds from contaminant fungi in the intercontinental trade. slurry pre-treated seeds were washed with water to remove the fungicide on seed surface before germination process (dikin et al. 1995). pre-treatment of seeds using supernatant anti-microbial substances from b. multivorans and m. testaceum controlled artificially inoculated seeds with s. commune. the recovery of seed germination of oil palm was due to treatment of supernatant antimicrobial substances. the effect of application of anti-microbial substances, as curative or preventive measure on seed germination and seedling growth showed no significant 48 biotropia vol. 15 no. 1, 2008 differences with healthy seeds. based on the results, the application of anti-microbial substances for the control of s. commune confirmed that the supernatant anti-microbial substances from b. multivorans and m. testaceum contain at least 3 active compounds of pyrrolnitrin, phenylpyrrol and phenazine which did not cause negative effect on seedling growth. these active compounds were reported to inhibit fungal growth (cartwright et al. 1995). conclusions supernatant of anti-microbial substances from b. multivorans and m. testaceum have been formulated in liquid form and freeze-dried talc form. both formulations had different functions for the control of s. commune as preventive and curative measures. formulated anti-microbial substances in liquid form and freeze dry solidification in talc had different values of minimum inhibitory concentration. application of the supernatant anti-microbial substances in liquid form for preventive and curative measures was conducted by seed dipping for 30 minutes and vacuum 400 mm hg vacuum for 2 minutes with ratio concentration of 1 part supernatant of anti-microbial substances diluted in 4 parts of distilled water (1:4). formulation of freeze-dry solidification of anti-microbial substances in talc was applied for seed dressing. formulation of anti-microbial substances in powder form is a preventive measure against fungal infection particularly s. commune. finally, seed pre-treatment using anti-microbial substances enhanced seed germination, and did not inhibit seedling growth of oil palm. acknowledgement part of the ph.d. thesis of the author is financially supported by irpa project, malaysian government (vote no. 54400). references cartwright, d.k., w.s. chilton and d.m. benson. 1995. pyrrolnitrin and phenazine production by pseudomonas cepacia, strain 5.5b, a biocontrol agent of rhizoctonia solani. appl. microbiol. and biotechnol., 43: 211-216. desai, s., m.s. reddy and j.w. kloepper. 2002. comprehensive testing of biocontrol agents. in biological control of crop diseases, ed. s.s. gnanamacikam. 17: 387-420. new york: marcel dekker, inc. 49 application method of antimicrobial substances – a. dikin et al. dikin, a., hermawan and z. zubir. 1995. temuan schizophyllum commune pada benih kelapa sawit asal costa rica.. prosiding kongres nasional xiii dan seminar ilmiah perhimpunan fitopatologi indonesia. mataram, indonesia. p 303-305 dikin, a., kamaruzaman sijam, zainal abidin mior ahmad and idris b abu seman. 2003. biological control of seedborne pathogen of oil palm, schizophyllum commune fr. with antagonistic bacteria. int. j. agric. and biol., 5(4): 507-512. dikin, a., kamaruzaman sijam, jugah kadir, idris b. abu seman. 2006. effect of different carbon sources and peptones on the production of antimicrobial substances from bacteria against schizophyllum commune fr. international journal of agriculture and biology. vol. 7. no. 3. 1560-8530/2005/07-3385-388. http://www.ijab.org fravel, d.r., w.j.connick jr and j. a. lewis. 1998. formulation of microorganisms to control plant disease. in formulation of microbial biopesticides, ed. h.d. burges. kluwer academic publishers, london 5:187-202. turner, p.d. and r.a. gillbanks. 2003. oil cultivation and management. the incorporated society of planters. kuala lumpur. malaysia. p.34. yu, j.j. and w.c. lee. 1997. microencapsulation of pyrrolnitrin from pseudomonas cepacia using gluten and casein. j. fermentation and bioengin., 84 (5): 444-448. fainmarinat inabuy_manuscript for biotropia 1 isolation and characterization of partial cdna of sucrose synthase putative gene in palmyra palm (borassus flabellifer) fainmarinat inabuy, adi pancoro research center for biotechnology, institute technology bandung, jl. ganesha no.10 bandung, indonesia40132. email: fimaluvbio@yahoo.com abstract intensification of biofuel resources is urgently needed considering decreased availability of world’s fossil fuel. palmyra palm (borassus flabellifer) is highly potential to be developed as bioethanol source regarding the high sucrose content in its nira. it was observed that nira produced in dry season is sweeter than that in rainy season, which presumed to be influenced by a difference in expression level of sucrose-related genes during the two seasons. study of sucrose synthase (sus) gene of palmyra are therefore required prior to study of the gene expression. palmyra sus gene sequence is currently unavailable in genbank, thereby pair of primers was designed from highly conserved region of sus proteins among monocots. a 1866 bp partial cdna fragment of sus putative gene has been succesfully isolated from rna of the young leaves of b. flabellifer. blastn and blastp aligments showed that either bfsus cdna or bfsus polypeptide has high similarity with sus cdna and proteins from diverse plant species with the highest similarity shown by tulipa gesneriana. the phylogenetic tree showed that sus protein sequences of monocot species were distinctively grouped and splitted from those of dicot species. the bfsus was clustered in monocot group, although not specifically grouped with particular monocot species. nevertheless, b. flabellifer showed nearest genetic distance with tulipa gesneriana and oncidium cv.’goldiana’. characterization of bfsus polypeptide using geneious 4.6.2 indicated the presence of sucrose synthase (sus) and glycosyl transferase (gt) domains, four putative udp-glucose binding pockets within the gt domain, and a calciumdependent ser/thr protein kinase binding site within the sus domain. these domains and motifs are highly conserved in sus proteins across plant species, confirming that the cdna fragment obtained in this study is very likely cdna encodes sucrose synthase in b. flabellifer. keywords: borassus flabellifer, nira, sucrose, sucrose synthase, sus domain, gt domain, palmyra palm. introduction palmyra (borassus flabellifer) is a palmae species with high potency to be developed as a source of bioethanol. the plant is typically grown in dry areas in strictly seasonal tropical or subtropical climate. the palmyra palm is known to be very adaptive in dry areas with only 500-900 mm annual rainfall (flach and rumawas, 1996). the main product of palmyra is the sweet liquid produced from its inflorescence, called nira, or locally known as tuak (fox, 1977). the nira of palmyra palm contains 17-20% dry matters comprises of sucrose, amino acids, proteins, vitamins, and other essential minerals. sucrose (c6h12o6) that constitutes 13-18% per liter nira is the principal material in the production of bioethanol through the process of fermentation. it was observed in east nusa tenggara, particularly in timor and rote islands, that nira tapping is more 2 preferable in dry season rather than in rainy season. beside the safety reason, it is probably because palmyra nira in dry season is sweeter than in rainy season. it becomes interesting to study whether this phenomena is solely caused by high level of water contained in the nira, by a higher rate of photosynthesis occurred in dry season, or it has something related with sucrose genes expression level. a preliminary study on sucrose related gene expressed in palmyra palm is therefore needed to answer the question. sucrose is the most important plant disaccharide; it is the principle form by which photosynthetic product is transported throughout plant tissues from the source photosynthetic tissue to the sink non-photosynthetic tissues (bush, 1999). this disaccharide consists of one molecule of glucose and one molecule of fructose that is bounded by a glycosidic bond (lodish et al., 1999). many enzymes involved in metabolism of sucrose. the closest related enzymes are sucrose-synthase (sus: ec 2.4.1.13), sucrose phosphate synthase (sps: ec 2.4.1.14), sucrose-6-phosphatephosphatase (sppase: ec 3.1.3.24), and invertase (ec 3.2.1.26). the reversible and irreversible reactions of sucrose hydrolysis are catalyzed, respectively, by sus and invertase. the biosynthesis of sucrose is catalyzed by the sequential action of sps and sppase (winter and huber, 2000). sucrose can be synthesized from hexose monophosphates by sus or sps. in the case of sus, the in vivo sucrose concentrations are always much higher than fructose or udpglucose, resulting in reaction that is essentially always towards the direction of sucrose cleavage. invertase, on the other hand, only catalyzes the cleavage of sucrose into glucose and fructose. all enzymes catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules by forming glycosidic bonds are classified in the glucosyltranferase (gt) family of enzyme. sucrose synthase (ec 2.4.1.13) is a key enzyme involved in sucrose metabolism, included in the gt4 family regards to its role in transferring the glucose from udpglucose to fructose molecule to form sucrose or, in reverse, hydrolyze the sucrose into glucose and fructose. sus enzyme plays significant role in food storage of many plants, either in the form of starch or sugar (baud et al., 2004). sus was shown to control the mobilization of sucrose into various pathways that important for the metabolic, structural, and storage functions of the plant cell. phloem-loading, a process by which sucrose is transported from photosynthetic to nonphotosynthetic tissues, is facilitated by sus. inhibition of sus-encoding genes had shown significant reduction in sucrose import capacity of floem tissues, thereby causing less sucrose content in tomato fruit (d’aoust et al., 1999). conversion of sucrose to udp-glucose that is catalyzed by sus provides substrate for cell wall biosynthesis and starch synthesis in plant storage organs (sun et al., 1992; zrenner et al., 1995; dejardin et al., 1997; hendrik, 1990). sus activity also associated with development of nodules in legume plants and in regulation of apical meristem function (craig et al., 1999). studies on sus-encoding genes in many plant species revealed that in vitro environmental conditions, such as light, temperature, and water availability have a significant influence on the expression of sus-encoding genes. palmyra (b. flabellifer) which has higher sugar content in dry season than in wet season, probably reflects a likewise relation. sus genes have been isolated from various starch and sugar-storing plants, such as citrus (citsusa and citsus2) (komatsu et al., 2002), sugarcane (kumar et al., 2007), rice (wang et al., 1992), maize (sus1 and sh1 gene) (mccarty et al., 1986), wheat (marana et al., 1988), cotton (ruan et al., 2003), tomato (sun et al., 1992), and potato (zrenner et al., 1995).the enzyme is found in all plant tissues but is highly expressed particularly in sink tissues (baud et al., 2004). according to those studies, the full length sus gene has in average 4970 bp length, while its cdna length is only about half of it, ranging from 2400 to 2500 bp. this research was aimed to study the characteristic of sus gene in b. flabellifer. a comprehensive knowledge of the sus gene characteristics is an initial requirement to study its expression level, and further to conduct manipulation or control of those genes, which enables efforts to enhance the productivity of b. flabellifer as a potential source of sugar and bioethanol. 3 results and discussion rna isolation from borassus flabellifer total rna of b. flabellifer was isolated from the young leaves of 35-40 years old-plants. young leaves was chosen as it was reported that sus genes are commonly expressed in newly developed tissues that require supply of either sucrose or sucrose derived-compounds (kumar et al., 2007). the results of five methods tested for rna isolation from b. flabellifer is summarized in table 1. the modified method of apt et al. (1995) resulted in good quality of rna although the purity and quantity were rather low. the rna isolation was best accomplished by using trizol ® reagent (invitrogen). the reagent, a mono-phasic solution composed of phenol and guanidine isothiocyanate, is an improvement to the single-step rna isolation method developed by chomczynski and sacchi (1987). this method successfully generated two distinct bands of 18s and 28s ribosomal rna that were clearly occurred in 1% gel electrophoresis (figure 1), which indicated a good rna quality. the rna yield was relatively high, 1600 µg/ml, with low protein contamination as shown by the ratio 1.62. we noted that the success in obtaining high rna yield from borassus leaves is greatly determined by the finest powder that could be recovered from the leaves tissue. reverse transcriptionpcr a total cdna from the young leaves of b. flabellifer were obtained by cdna synthesis. they then used as template for the next pcr reaction. the annealing of sus-spesific primers to the target cdna were best achieved in temperature 52ºc. as well, 2 mm of mg2+ and 1.5 µg/50 µl of template cdna were found to be the most suitable concentration for the pcr mix. total 30 cycles of amplification using the gene specific primers, ssfw and ssrv, has successfully produced a single fragment of 1866 bp that was detected in 1% agarose gel (figure 2). according to the expected product size, this 1866 bp fragment was predicted as the target fragment of the sus gene. cloning of sus putative gene and transformation of e. coli prior to sequencing reaction, the sus putative fragment is purified from the agarose gel and then cloned into a plasmid vector, pgem®-t easy (promega), which then delivered to e. coli strain dh5α. e. coli cells that had been transformed were able to grow in the ampicilincontaining medium. transformed cells containing sus gene fragment were selected by bluewhite screening method. transformation of e. coli strain dh5α with pgem®-t containing sus fragment resulted in blue and white colonies growth on the ampicillin contained-lb media. plasmid isolation and confirmation of gene insertion plasmid isolation from the e. coli white colony and subsequent restriction cut using particular restriction enzymes are purposed to confirm the presence of the sus gene fragment within the plasmid of e. coli. when cut with ecori, the plasmid produced two unexpected fragments, ± 1300 bp and ± 500 bp, instead of 1866 bp (figure 3a). it was presumed that the sus fragment of b. flabellifer has an ecori restriction site within its sequence, thereby produced shorter fragment than expected. it was found later that the sequence of sus gene of b. flabellifer does have the recognition site for ecori. the plasmid was then cut with another endonuclease enzyme, noti. as expected, cutting with noti generated a single 1866 bp fragment (figure 3b). this result indicated that the sus gene fragment had been successfully inserted into the pgem®t easy plasmid. pcr screening with sp6 and t7 primers was further conducted to prove the presence of sus fragment within the multiple cloning sequence (mcs) region in the plasmid. those two primers 4 flank the mcs region in the pgem®-t easy plasmid. as expected, a ±1995 bp fragment was occurred (figure 3c). this length corresponds to the length of the sus fragment, ±1866 bp, plus 129 bp lengths of the sp6 and t7 mcs region. sequencing and characterization of sus putative gene and polypeptide two steps of sequencing were required since sus putative fragment of b. flabellifer, further stated as bfsus, is relatively long for a single reading by the sequencer system. the sp6 and t7 primers were used in the former sequencing, followed by specific internal primers to read the gap sequence within the fragment. the later primers were designed from the read of the former sequence. using geneious 4.6.2 program, an overlapped reading occurred from those two steps of sequencing, thus finally assembly the whole sequence of the bfsus putative gene fragment. the assembled bfsus fragment was trimmed from pgem®t vector region by using vecscreen program (www.ncbi.nlm.nih.gov), followed by determination of ssfw and ssrv primers annealing region. the final construction of bfsus partial gene, which total length is 1866 bp, was successfully obtained (figure 4). the sequence of bfsus putative gene was first analyzed by blastn program (altschul et al., 1997) to figure out the similarity level of bfsus putative gene with other sus gene sequences recorded in the genbank. the percentage of identical sites and query coverage indicated that bfsus putative gene shares quite high similarity, 70.8% – 80.6%, with sus genes from various monocots. thus, confirming that the isolated 1866 bp bfsus fragment is most likely a sucrose synthase gene. bfsus sequence shows the highest similarity with sus genes of tulipa gesneriana (80.6%), followed by x. mokara (78.8%), potamogeton distinctus (78.7%), oncidium sp. cv goldiana (78.6%), and bambusa oldhamii (78.5%). translation of bfsus cdna sequence resulted in bfsus polypeptide sequence consists of 622 amino acids (figure 5). the blastp program showed that bfsus polypeptide sequence is highly similar with sus protein sequences of many plant species, ranged from 78.3 % to 87.5%, confirming that the bfsus gene expresses sus protein. the highest similarity, 87.5%, are showed by tulipa gesneriana, a liliaceae plant widely known as tulip, and oncidium cv ‘goldiana’, a genus that contains about 330 species of orchids from the orchidaceae family. oryza sativa (86.8%), bambusa oldhamii (86.5%), zea mays (86.2%), and other monocots also showed high similarity. in order to elucidate relationship between bfsus putative protein and sus proteins from other species, and between monocot and dicot species, a phylogenetic tree is generated using neighborjoining method (figure 6). the following things can be interpreted from the phylogenetic tree. first, the similarity of sus polypeptide sequence in certain taxonomic group tends to be higher than another group. the similarity of sus among monocot species, for instance, is distinctively higher than those of dicot species. this also showed by species coming from the same genus, for instance, between solanum tuberosum and solanum lycopersicum. second, b. flabellifer is shown to be clustered with the monocot group, although not specifically grouped with particular monocot species. it is however shown that borassus flabellifer has the nearest genetic distance with tulipa gesneriana. although these two species live in distinctively different climate regions, presumably they both evolve similar mechanism to adapt to osmotic stress in their surroundings, probably by accumulating sucrose. in water stress conditions, plants are able to keep their osmotic gradient lower than their surrounding by accumulating more solutes, including sucrose, in their tissues, thereby preventing loss of water. multiple sequence alignment of sus polypeptides indicated two main domains that typically occurred in all plant species. those are a sucrose synthase domain located upstream toward the n-terminal and a glucosyltransferase domain that is located downstream towards the c-terminal 5 of the protein. these domains differs sus from other glycosyltransferase enzymes. using pfam database, it was shown that those two domains are occurred in the bfsus polypeptide albeit in partial length. the moiety of sucrose synthase and glucosyltransferase domain is found toward the n and c-terminal region, respectively, of the bfsus polypeptide. the overlapped region between these domains is spanned from asn283 to ser559. despite found to be highly conserved among plant species, the structural and functional sites and motifs within sucrose synthase domain are still unrevealed yet. instead, four putative functional motifs have been reported for the second domain, the glycosyl transferase. it is known that structure of a protein determine its function. therefore, function of a protein could be deduced by comparing protein sequences and structures with homolog proteins of known function. similar motifs between two proteins generally will have same function, especially when they are homolog (horton et al., 2006). functional motifs within the bfsus polypeptide were elucidated by finding functional conserved motifs within other enzymes of gt family which had been previously annotated. buschiazzo et al. (2004) had successfully isolated and crystallized another gt family enzyme from agrobacterium tumefaciens, the glycogen synthase (gs). gs catalyses the synthesis of the α-1,4-glucose backbone in the reaction of glycogen biosynthesis. this enzyme possesses the gt domain, a domain also found in sus enzyme, with some annotated motifs within it. according to homolog motifs in gs (buschiazzo et al., 2004), four functional motifs predicted as urasile diphosphate (udp)-binding pockets were detected in the bfsus polypeptide. the first putative udp binding pocket found in bfsus polypeptide is gly169, a residue located in a glycine rich motif, 166-dtggq-170 (figure 7) (huber and huber, 1996; buschiazzo et al., 2004). the glycine (g) residue is predicted to directly contact with phosphate group of udp molecule that binds to sus protein. buschiazzo et al. (2004) discovered that, in the close conformation of the gs protein, the glycine residues in the kxggl motif come into contact with the phosphate groups of udp. this role had been reported also in the gs of escherichia coli (furukawa et al., 1993). they reported that only the glycine residues but not the basic side chain appear to be essential for gs enzymatic activity. the dtggq motif was found to be conserved in sucrose phosphate synthase (sps), another member of gt family, which functioned as fructose-6-p binding site (huber and huber, 1996). the three subsequent udp-putative binding pockets in bfsus polypeptide are 444-mar-446 residues, n520, and t546 (figure 8 and 9). it was observed that the mar motif within the bfsus polypeptide is located at the c-terminal end of a β-strand (figure 8), a pattern that is also occurred in isr motif of gs polypeptide, albeit in different number of β-strand. this probably reflects their similar role. it was observed that the guanidinium group of r299, in gs, interacts with the phosphate group of adp via a hydrogen bonding (buschiazzo et al., 2004). the mar residues of bfsus polypeptide thereby suggested as binding site for phosphate group of udp via a hydrogen interaction. the asn520 (n520) motif of bfsus polypeptide is predicted to bind with the adenine ring of the udp molecule. according to gs, the carbonyl group of protein backbone in asn520 may be interacted with atom n6 of the adenine ring of udp molecule via a weak hydrogen interaction. the last putative udp binding pocket, the t546 was suggested to bind ribose sugar of udp molecule. the side chain of t546 may interact via hydrogen bond with o2 atom of the ribose sugar. the last conserved region of sus protein that is also found in bfsus polypeptide is the ser170 putative phosphorylation site. this essential site was found within a typical serine residue containingmotif, ‘rhlss’, which lay between arg167 and ser171. in the partial bfsus polypeptide this motif is located between arg30 and ser34 (30-rhlss-34). this motif was firstly detected in zea mays sus protein, spanned from arg159 to ser163 (hardin et al., 2003). this motif was also found in oryza sativa, saccharum officinarum (r159 to s163), bambusa 6 oldhamii, triticum aestivum (r167 to s171), and tulipa gesneriana (r161 to s165). hardin et al (2003) reported that sus protein of zea mays is phosphorylated by calcium-dependent protein kinases (cdpks) at the ser170 residue, within the rhlss motif, in addition to ser15 (asano et al., 2002). ser15 was reported as a major phosphorylation site that affects cleavage activity and membrane association, whereas ser170 is a minor phosphorylation site that may trigger enzyme degradation via the ubiquitin/26s proteasome (qiu et al., 2007). since phoshorylation of ser170 is important for enzyme degradation but not directly involved in the catalytic activity of sus, this site is suggested as one of the allosteric sites of the enzyme. those all results confirm that the isolated 1866 bp of bfsus gene fragment is most likely the sucrose synthaseencoding gene in b. flabellifer that encodes sus protein. further expression study however is required to prove that the bfsus gene actually expresses sucrose synthase protein in b. flabellifer. the sequence of partial bfsus has been submitted to the genbank with accession number gq265926. materials and methods plant material young leaves were picked from the apical shoot of 35-40 years age-plant of palmyra palm (borassus flabellifer) grown in lasiana shore area in kupang, east nusa tenggara. leaves samples were cleaned, wrapped, and immediately stored in cold condition before being taken to bandung. the whole processes of sampling were done with rnase-free standard work procedure. leaves samples were then frozen in liquid nitrogen and were stored in -800c refrigerator before rna isolation. all the laboratory works took place in the laboratory of genetics and molecular biology, in sith-itb, bandung. rna isolation total rna of b. flabellifer was extracted from the fresh young leaves that were previously stored in -80ºc. all chemicals were diluted in rnase-free water and prepared using rnase-free equipments. all equipments were formerly treated with dietylpyrocarbonate (depc) before use. grinding equipment, such as mortar, pestel, and scissors were chilled before use, either by storing in -80ºc or by soaking in liquid nitrogen. five protocols have been tested to optimize rna isolation method from young leaves of b. flabellifer. those are method of seki et al. (2002), method of khemvong and suvachittanont (2005), modified-method of apt et al. (1995), trizol-modified (i) method, and trizol modified (ii) method (invitrogen). quality of rna is determined by absorbance ratio in 260 nm and 280 nm uv light in the range of 1.80 – 2.00 which indicated low contamination of protein. appearance of two typical ribosomal rna bands, 18s and 28s, in electrophoresis gel also indicated good quality of rna. the quantity of rna was determined by rna yield (µg) per total solution volume (ml). primer design sequence of b. flabellifer sus gene is currently unavailable in the genbank, thereby pair of primer was designed from highly conserved region of sus proteins among monocot plant species. sucrose synthase protein sequences of oryza sativa, zea mays, saccharum officinarum, and other monocots, were accessed from ncbi genbank database (www.ncbi.nlm.nih.gov). multiple sequence alignment of sus proteins was conducted using clustal x software. the most conserved regions in sus proteins among those species were chosen for primer sequence 7 determination. primer sequences were deduced from those regions by choosing a particular group of species whose cdna sequence is highly conserved. this was accomplished by bioedit and codehop programs. nucleotide number 412 to 432 of sus gene of zea mays was chosen as forward primer; while reverse primer was taken from nucleotide number 2277 to 2252. primer physical characteristics were measured by sigma-genosys primer calculator. specificity of both primer pairs were tested by nucleotide blast program (altschul et al., 1997). the chosen primer sequences showed eligible characteristics, and more importantly, exhibited high specificity for sus gene (table 2). reverse transcription-pcr isolation of sucrose synthase (sus) gene from b. flabellifer was accomplished by two step reverse-transcription polymerase chain reaction (rt-pcr) method. in the first step, total cdna is synthesized from the total rna by using superscript ii cdna synthesis kit (invitrogen). in the second step, pcr amplification using sus gene-specific primers was carried out to specifically amplify fragment of sus gene from the previously isolated cdna. total cdna synthesis total cdna synthesis of b. flabellifer was conducted using superscript ii cdna synthesis kit (invitrogen). composition of each component in cdna synthesis reaction is described in table 3. all solutions are diluted in depc-treated deionized water. all steps were carried out according to rnase-free standard work procedure to avoid or minimize degradation of the rna. a master mix solution was prepared by mixing buffer rt with mgcl2, dithiothreitol (dtt), and rnase out tm recombinant rnase. separately, rna sample was mixed with oligo dt primer and dntps prior to five minutes incubation in 65ºc and then one minute in ice. the master mix was then added to the rna sample mixture and then diluted with depc-treated water to 20 µl final volume. this final mixture was incubated for two minutes in 420c for primer annealing. superscripttm iireverse transcriptase was subsequently added to the mixture, gently homogenized, and then incubated for 50 minutes in 42ºc for complete reaction of cdna synthesis. the reaction was terminated by 15 minutes incubation in 70ºc. to eliminate any remaining rna strands in the cdna solution, rnaseh were added to the solution and then incubated in 370c for 20 minutes. total cdna of b. flabellifer young leaves was obtained at the end of this step. pcr amplification for isolation of sus gene a standard pcr amplification technique was conducted following the cdna synthesis to specifically isolate the sus gene. optimization of pcr profile, especially annealing temperature, was carried out to find the best condition that favor specific amplification of the sus gene fragment. a range of annealing temperature, from 45ºc to 55ºc, was tested. concentration of cdna template, ranging from 0.5 to 2 µg /50 µl, and mg2+, ranging from 1 to 2.5 mm, were tested as well to obtain best fragment of the target gene. the pcr reaction was carried out using a long pcr enzyme mix product (fermentas). composition of the pcr mixture is described in table 4. gene cloning and transformation of e. coli the product of rt-pcr was observed by electrophoresis using 1% agarose gel. detected sus fragment was purified from the agarose gel and then cloned to the pgem-t easy® vector by employing t4 ligase enzyme. to amplify the sus fragment, the recombinant plasmids were subsequently delivered into bacterial cell of escherichia coli strain dh5α using heat-shock 8 treatment. the transformed cells of e. coli that contain recombinant plasmid were selected through screening of blue-white colony. the transformed cells were cultured for 16 hours in luria-bertany (lb) media containing 0.1 ng/µl ampicillin. the plasmids dna of the transformed cells were then extracted by alkaline lysis method (sambrook et al., 1989). insertion of sus fragment into plasmid was confirmed by cutting the plasmid with two endonuclease restriction enzymes, ecori and noti. sequencing and characterization of bfsus gene and polypeptide sus putative fragment that have been cloned to pgem-t easy plasmid were further analyzed by dideoxy chain-termination method to read the nucleotide sequence of the isolated fragment. minimum 150 ng/µl of plasmid dna is required to a single sequencing reaction. since the 1866 bp gene fragment is relatively long for a single accurate reading by the sequencer system, two steps of sequencing are needed to accomplish the total fragment reading. the first sequencing employed t7 and sp6 universal primers, which followed by second sequencing using gene internal–primers. gene-internal primers were designed from the first sequencing result. sequencing result from those four primer directions were finally assembled using geneious 4.6.2 program. all sequencing process was conducted by macrogen inc. in south korea. characterization of bfsus putative gene comprises nucleotide and polypeptide sequence analysis. the nucleotide sequence of the putative bfsus was aligned with sus cdna sequences deposited in genbank (www.ncbi.nlm.nih.gov) using blastn program in order to find their similarity. the bfsus cdna sequence was then translated into bfsus polypeptide using geneious 4.6.2, followed by searching in the genbank using blastp program. the highly conserved regions, including essential domains and motifs, were searched within bfsus putative polypeptide, either by employing geneious 4.6.2 program or by comparing the polypeptide with other related proteins that have been previously annotated. other proteins from the same family are preferable since it is expected to share similar properties with the sus. to reveal genetic relationship between borassus flabellifer and other plant species based on their sus polypeptide sequence, a neighborjoining (nj) phylogenetic tree was generated using geneious 4.6.2 tree builder. conclusion a partial cdna fragment of sucrose synthase (sus) putative gene has been successfully isolated from the young leaves of b. flabellifer, henceforth named as bfsus. the sequence has been submitted to the genbank with accession number gq265926. the sequence of bfsus cdna, and the corresponding bfsus polypeptide, is highly similar with sus cdna and sus protein from various plant species recorded in genbank, with highest similarity shown by tulipa gesneriana and oncidium sp. the bfsus putative polypeptide performs all typical characteristics of sus protein, including most conserved domains and motifs. presence of sucrose synthase (sus) and glycosyl transferase (gt) domains, four putative udp-glucose binding pockets within the gt domain, and a calcium-dependent ser/thr protein kinase binding site (ser170) within the sus domain, confirms that the bfsus cdna fragment obtained in this study is most likely encodes sucrose synthase in b. flabellifer. this also assuring that bfsus gene is expressed in the young leaves of b. flabellifer. 9 table 1. summary of five tested methods for rna isolation from young leaves of b. flabellifer. trizolmodified ii found to be the best method for rna isolation from the young leaves of b. flabellifer *) rna yield = (absorbance in 260 nm) x (42.5 µg/ml) x (dilution factor) figure 1. comparison of rna quality obtained by the five tested methods. two bands of ribosomal rna, the 18s and 28s rrna, that were occurred in electrophoresis gel indicated that total rna of b. flabellifer has been successfully obtained. trizol-modified ii method is the best method for rna isolation from the young leaves of b. flabellifer. figure 2. a single cdna fragment of 1866 bp, generated by reverse transcription-pcr, predicted as the target fragment of the sus gene of b. flabellifer. m e t h o d ratio of uv absorbance (a260/a280) rna quality in gel electrophoresis rna yield (µg/ml) *) total time (hour) trizol-modified i (invitrogen) 1.23 – 1.24 average 114.75 5 seki et al., 2002 1.44 – 1.47 poor 93.50 8 khemvong &suvachittanont (2005) 1.67 – 2.00 poor 44.625 12 apt et al. (1995)-modified 1.45 – 1.47 good 148.75 18 trizol-modified ii (invitrogen) 1.61 – 1.62 good 1600 4 28s rrna 18s rrna 1 2 3 5 4 2000 bp 1500 bp 1866 bp 10 figure 3. (a) the recombinant plasmid cut with ecori generated two unexpected ± 1300 and ± 500 bp fragments indicating gene internal cut by this enzyme; (b) plasmid cut with noti generated a single expected fragment of length ±1866 bp, indicating that sus gene of b. flabellifer had been successfully inserted in the mcs region of the plasmid; (c) pcr screening using sp6 and t7 primers resulted in ±1995 bp fragment confirmed the presence of sus fragment within the mcs region of the pgem®-t easy plasmid. table 2. pair of forward and reverse primers used to isolate sucrose synthase gene from total rna of borassus flabellifer young leaves. primer code primer sequence start primer length product size (bp) gc (%) tm (ºc) sec. struc ture blast output (σ species) susfw cttgagctggactttgagcca 412 21 52.38 62.92 weak 10 susrv cttccaggtgtacttctcctcgat ac 2277 26 1866 50.00 62.72 weak 8 table 3. chemical composition in cdna synthesis no. component final concentration 1 total rna of b. flabellifer 2 µg/20 µl 2 dntps 0.5 mm 3 oligo (dt)12-18 primer 0.025 µg/µl 4 buffer rt 1x 5 mgcl2 5 mm 6 dithiothreitol (dtt) 0.01 m 7 rnase outtm recombinant rnase 1 u/20 µl 8 superscripttm iireverse transcriptase 1u/20 µl 9 depc-treated deionized water until 20 µl 10 e. coli rnaseh 1 µl/20 µl table 4. composition of pcr mixture for sus cdna isolation no. component final concentration 1 10x long pcr buffer 1x 2 2 mm dntp mix 0.2 mm 3 25 mm mgcl2 2 mm 4 primer (forward and reverse) 0.2 µm 5 total cdna 0.03 µg/µl 6 long pcr enzyme mix 1.25 unit/ 50 µl 7 nuclease free water to 25 µl ± 3000 bp ± 1300 bp ± 500 bp a ± 1866 bp ± 3000 bp b ± 1995 bp c 11 12 13 figure 4. sequence of partial cdna of borassus flabellifer sus putative gene. the yellow arrow indicates forward primer, ssfw, and the green arrow indicates reverse primer, ssrv. the red arrow indicates internal primer p1, and the blue arrow indicates internal primer p2. the red box indicates restriction site of ecori endonuclease restriction enzyme. the brown box indicates putative phosphorylation site (30-rhlss34). the blue boxes indicate four putative udp binding pockets (166-dtggq-170; 444mar-446; n520; and t546) figure 5. the bfsus putative polypeptide sequence with total length 622 amino acids was generated by translating the sequence of bfsus cdna, using geneious 4.6.2. 14 figure 6. a phylogenetic tree based on sucrose synthase polypeptide sequence of various monocot and dicot species. similarity of sus polypeptide among monocot species is distinctively higher than those of dicot species. borassus flabellifer is clustered with the monocot group, although not specifically grouped with particular monocot species. b. flabellifer showed the nearest genetic distance with tulipa gesneriana. 15 figure 7. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 16 figure 8. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 17 figure 9. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 18 acknowledgement we thank the southeast asian organization of tropical biology (seameo-biotrop) for funding this research. our work has been supported by a research grant form seameo-biotrop in the period of 2008-2009. our gratitude to the school of life sciences and technology for the facilities we were able to use in the laboratory of genetics and molecular biology. thanks to mikael tupa for his favor in obtaining the borassus flabellifer leaves in lasiana coastal area. we also thank dr. suminar achmadi and the biotropia reviewers for their careful reading, comments, and corrections of the manuscript. references altschul, stephen f., thomas l. madden, alejandro a. schäffer, jinghui zhang, zheng zhang, webb miller, and david j. lipman. 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(2002). monitoring the expression profiles of 7000 arabidopsis genes under drought, cold and high-salinity stresses using a full-length cdna microarray. plant journal 31: 279292. biotropial(l) 1987: 1-25 studies on the breeding structure of tree species in the tropical rain forest. i: family clumps and intrapopulation differentiation kan-ichi sakai *, toru endo *, shinya iyama* national institute of genetics, mishima, japan yasusada miyazaki*, shigesuke hayashi*, yoshiya shimamoto * kyushu university (tukuoka), kagoshima university (kagoshima) and hokkaido university (sapporo), japan respectively lilian u. gadrinab & ulfah juniarti tropical forest biology program, biotrop, bogor, indonesia abstract breeding structures of two tropical rain forest tree species, altingia excelsa in java and agathis borneensis in kalimantan were investigated. assuming that similarity in the assortment pattern of the isoperoxidase bands tells genetic relationship between trees, on the one hand, and that inbreeding increases smaller values of the disagreement counts, on the other, it has been concluded that inbreeding occurs considerably in altingia excelsa and to some extent in agathis borneensis. finding that trees showing very low disagreement counts are located close to each other, they were grouped as an assumptive family. it was found that different families were quite dissimilar with respect to isoperoxide constitution and in several leaf characters as well. the distance between two trees at which they can mate is estimated to be 16 to 18 meters or 16.5 meters and the area one family occupies is 200 to 250 m ,̂ assuming that a family clump can be a breeding unit in altingia excelsa, within which trees mate at random. some families were distributed mixed with each other within the mating distance, but they were found still genetically differentiated from each other. this reproductive isolation among families is interpreted to be due to genetic differences between families in flowering time. in agathis borneensis, there was no indication of family clump formation. related trees may have been widely scattered in the forest, and the inbreeding of the species may be due to self-fertilization of individual trees and not to outcrossing between relatives. introduction one of the characterisitic features of a tropical rain forest is its high species diversity. it stimulates our interest to inquire into two relevant problems. one is how trees could propagate in such a forest with an extremely low population density for each species, or what the breeding system of tropical tree species is. * temporarily seconded to biotrop. 1 biotropia vol. 1 no. 1, july-december 1987 another problem is how speciation takes place in such a tropical rain forest of high diversity of species. in the meantime, we recollect two hypotheses already advanced concerning the speciation problem: one is t he so-called genetic drift hypothesis by fedorov (1966), and another is the selection hypothesis by ashton (1969). fedorov assumes that a very small number of conspecific trees with asynchronous flowering among them should naturally induce each individual tree to propagate by self-fertilization which after many generations results in speciation by genetic drift. ashton, on the contrary, assumes that notwithstanding more or less difficulty in outbreeding, it would still occur frequently enough to allow gene exchange throughout populations in a continuous habitat causing speciation as a result of allopatric differentiation between populations in response to differential selection pressures. this paper describes results of an investigation into the breeding structure of two species, altingia excelsa noronha (hamamelidaceae) in java and agathis borneensis warb. (araucariaceae) in kalimantan,with an aim to find a way to pierce an opening to the problem of speciation. materials and methods two natural forests were investigated. the stand in which altingia excelsa was investigated was located on the outskirts of the village of ciwidey near bandung in western java. all trees with diameter at breast height (dbh) exceeding 6 cm, growing in quadrat of 50 x 50 m in the stand were measured for their growth, and their position in the quadrat was mapped on a section-paper. in all, 148 trees of various species were counted, among which 38 were trees of aliingia excelsa. twigs with mature leaves were collected from each of these 38 trees for laboratory research. the mature leaves from each tree were divided into two parts, one for measurement of several leaf characters while the remaining part for the electrophoretic analysis of the isoperoxidases. preliminary studies showed that the zymogram patterns for acid phosphatase and esterase were rather simple and thus only peroxidase was used in this study. agathis borneensis was sampled from a quadrat of the same size plotted in a natural forest of international timber corporation indonesia in kalimantan. there were about 230 trees with dbh exceeding 6 cm of various species in the quadrat, of which 21 were agathis borneensis. growth measurement and mapping of individual trees were made and their leaves were collected for the study of leaf characters and eletrophoretic analysis. leaf characters measured in both species were leaf length, leaf width, leaf size (length x width), number of veins, and length and thickness of petioles. 2 breeding structure of tree species in the tropical rain forest — sakai et al. methods applied for the electrophoretic analysis were as follows: enzyme extractant m4 containing 10% triton x-100, 0.5 m nacl tris and 0.2 m ascorbic acid, was adjusted with acetic acid to ph 7.5. one hundred mg of finely cut leaf pieces were crushed in a mixture of 0.5 ml of the extractant, 100 mg of quartz sand and 50 mg of polyvinyl-polypyrrollidone (endo 1981). this system proved to be generally suitable for the extraction of peroxidase in altingia as well as in agathis. starch gel electrophoresis was subjected to the system modified by brewer (1970). the gel buffer contains 0.42 g of histidine hc1 and 0.072 g of naoh per 0.5 1 mixed with 60 to 66 g of hydrolyzed potato starch, with the final ph of the solution around 6.1. the tray buffer was wi t h a ph of about 6.0 and it contained 117.6g(or 0.6 m) trisodium citrate and 9.4 g of citric acid per liter. the gel mold of toyo model ha-1 devised by endo (1968) was used. electrophoretic run was done under constant 250 voltage per 20 cm for 4.5 hours in a refrigerator with an ice cooling box. reaction mixture of peroxidase stain contains 4 mm eugenol, 4 mm 3-amino-9-ethylcarbazole in 10% acetone solution at final concentration, and 0.03% hydrogen peroxide, and 0.02m tris-acetic acid buffer, ph 4.0 (endo 1978). the figure 1. schematic explanation for measuring the disagreement count between x and y. 3 biotropia vol. 1 no. 1, july-december 1987 isoperoxidase zymograms of 38 altingia excelsa and 21 agathis borneensis trees are schematically drawn in appendices 1 and 2. on t he assumption that the pattern of the isoperoxidase bands of a tree would be nothing but the reflection of its genetic make-up, genetic similarity was measured among trees with the aid of the disagreement count which gives the total number of missing mate to each band between a given pair or trees (sakai & miyazaki 1972). in practice, there may occur variation in activity of bands, but it was neglected for the present study. figure 1 explains the method of calculation of the disagreement counts between two zymograms. detection of family structure of a tree species in a natural stand with the aid of the disagreement counts was made on the following premises: (1) it was assumed that the plural isoperoxidase bands appearing in the population would be distributed at random among individual trees if mating occurs at random, while inbreeding, either self-fertilization or mating between relatives, would tend to produce the isoperoxidase combination of the parental type. (2) the disagreement counts obtained between trees of the same family were thus lower in value than those obtained between trees not related with each other. it should be borne in mind, however, that converses are not always true; that is, trees with low disagreement count cannot immediately be regarded as sib-members of a family. (3) propagated sib-trees would be apt to form a clump around their mother-tree. (4) trees growjng in a clump would have a higher chance of mating with each other than with trees of a separate group, particularly in the tropical rain forest with low population density. (5) trees belonging to a single family would be more alike in their biochemical as well as vegetative characters, and it may naturally enlarge character variation among families in contrast to its curtailment within families. results and discussion i. altingia excelsa (a) formation of family clumps individual variation of dbh of 38 trees is presented in table 1. the table shows that trees exceeding 61 cm in dbh were five among 38, or 13%. in order to determine if the actual distribution of disagreement counts occurs according to the random assortment of the isoperoxidase bands in individual trees, we calculate the theoretical expectation of the latter (appendix 3). 4 breeding structure of tree species in the tropical rain forest — sakai et al. the comparison between theoretical expectations and observed disagreement of bands by the method described is given in table 2. data presented in table 2 show an apparently excess occurrence of observations in the lower classes of 0, 1 and 2, suggesting that the fertilization in altingia excelsa is not panmictic, but a fair amount of inbreeding could be occurring. 5 biotropia vol. 1 no. 1, july-december 1987 in figure 2 we find that actual observations exceeded the theoretical expectations in such lower classes as 0, 1 and 2, and also in those of higher classes exceeding 8. that disagreement counts of several smallest and largest values appeared more frequently than theoretical expectation suggests that inbreeding on the stand tended to construct two contrary groups of trees, one with very similar isoperoxidase patterns and another with highly dissimilar ones. inbreeding in a natural forest can occur in two ways: self-fertilization of individual trees and crossing among relatives. for the latter to occur, trees belonging to the same family growing together in a clump would be more advantageous. are consanguineous trees of the same family really growing in a clump? in order to answer this question, the relationship between inter-tree distance and disagreement counts was investigated (table 3). table 3. relationship between disagreement counts and inter-tree distances in altingia excelsa. inter-tree distance (m) disagreement count total x 2 p 0.1 2.3 4.5 6< 0-10 expectation 6.7 21.6 30.5 33.3 observation 15 25 25 27 92 13.17 <0.01 11-20 expectation 123 39.8 56.3 61.6 observation 11 29 70 60 170 6.43 >0.05 21-30 expectation 14.3 46.4 65.5 71.7 observation 15 42 58 83 198 3.05 >0.20 31< expectation 17.7 57.2 80.8 88.4 observation 10 69 80 85 244 5.90 >0.10 table 3 shows that the difference between expected and observed value was statistically significant only for the smallest inter-tree distance of 0-10 m. it was found that the difference was in the conspicuous excess of observations in the 0 and 1 classes of the disagreement counts, that is, trees growing nearby or so close to each other at a distance of less than 10 m were likely to bear a striking resemblance in the pattern of the isoperoxidase zymograms, giving the impression that genetically, closely related trees grow together in small groups. in other words, tree pairs showing low disagreement count as 0 to 2 and growing nearby in natural forest may be considered as members of a family. thus five assumptive families: a, b, c, d and e were depicted in figure 3. broken lines connecting individual trees with arabic numerals 0, 1 or 2 in the figure are presumed ties between parent and child trees, the numerals indicating the corresponding disagreement counts. until trees in question are proven to be related members of genetically separate 6 breeding structure of tree species in the tropical rain forest — sakai et al. figure 3. family clumps inaltingia excelsa. arabic numerals represent disagreement counts, while those in parentheses disagreement counts between trees of presumed relation. 7 biotropia vol. 1 no. 1, july-december 1987 families, the "assumptive families" designated above will be called merely as tree "groups" for the time being. of the five groups, two, i.e. b and e are worthy of note because both have the largest groups, each with 7 trees. the largest trees of both groups were connected with small disagreement count 1 or 2, indicating that the two groups are more or less related or maybe descendants of a common progenitor. of more interest is that both groups appeared to show a within-group segregation in width of leaves (table 5). although we are not yet in a position to speak definitely, it is supposed that b and e groups might be both heterozygous for a gene probably with major effect on leaf width. (b) differentiation in the constitution of isoperoxidase components the occurrence of various isoperoxidase bands in trees of five groups was examined (table 4). table 4. interpopulation differentation in distribution of isoperoxide bands *) the maximum number of isoperoxidase bands per tree in altingia excelsa was 16. it was found that trees of each group were characterized by specific isoperoxidase bands. for instance, c group is very peculiar in not having such bands as a and / which are rather ubiquitous in altingia excelsa. instead, it possessed e, g, and k bands which are more or less uncommon to the other groups. table 4 shows that those five groups were very variable in their biochemical characteristics demonstrating an intrapopulation genetic differentiation. (c) intrapopulation differentiation in vegetative characters now we should further inquire into differentiation among groups in some leaf characters. six leaf characters were investigated and their variation is given in a form of frequency distribution in table 5. it was noted that in almost all characters, variation among these groups was apparent. b group had the biggest size in many leaf characters, e the next, while c and d groups were the smallest, leaving a intermediate. the analysis of variance of the leaf characters is shown in table 6. 8 breeding structure of tree species in the tropical rain forest — sakai et al. table 5. variation of six leaf characters in five family groups of altingia excelsa 9 biotropia vol. 1 no. 1, july-december 1987 significant at the 5%* and 1%** levels, respectively. ns ) non-significant. from table 6 we find that vein number was not significantly variable among groups, but measurements in leaves and petioles were all significantly variable. from the facts described above, one can conclude that groups a to e defined up to now are clumps of trees of single families or family clumps, each of which is being genetically differentiated from others. for genetic differentiation among clusters within a population to occur, it is necessary that propagation is by mating within the same, but not between different families. if one assumes that trees of a single family form a breeding group within which mating occurs at random, then what would be the area occupied by a group in a natural stand? of the five families depicted in figure 3, families a and c are not included in the following discussion because of too small number of trees in them. one notices in figure 3 that in each of the three remaining families, one tree always stood relatively far from the others of the same family. thus, the inter-tree * inter-tree distance. 10 breeding structure of tree species in the tropical rain forest — sakai et al. distance was measured in two ways: one for all trees, and another for all trees but one standing apart. the results are presented in table 7 and figure 4. figure 4. distribution of inter-tree distances in three families of altingia excelsa. black circles are those between the farthest tree and the remaining ones in each family. the distribution of inter-tree distances in three families is shown in figure 4, wherein distances involving the farthest tree are depicted in black circles. excluding the black circles, one may state that the mean distance of the mean plus one standard deviation as the family distance for e = 10.79 + 5.71 = 16.5 m (table 7). this distance is expected to include 84% of cases in a theoretical normal distribution and could be taken as the mating distance in altingia excelsa, if we assume 16 to 18 meters as the mating distance, then the area as a circle of a family clump in the species is roughly estimated to be π r 2 = 200 m 2 to 250 m2 with r = 8 to 9 meters. if 16 to 18 meters is the mating distance, then why do b and c families look genetically isolated from each other in spite of growing mixed together in the forest (figure 3)? we have already concluded that the two families b and c are very different biochemically as well as morphologically. table 8 shows the distribution of the disagreement counts obtained between trees within and between b and c families, based on which we have distinguished b from c. it indicates that both families are very dissimilar in the assortment pattern of the isoperoxidase bands. 11 biotropia vol. 1 no. 1, july-december 1987 in order to find whether trees of both families could be regarded as growing mixed together in an area of the mating distance, the inter-tree distances have been measured among six trees excluding the farthest one of the b family and three of t he c. the data are presented in table 9. table 9. inter-tree distances of six trees* in family b and distances between three trees of family c and six of family b. inter-tree distance (m) 0.1 2.6 5.1 7.6 10.1 12.6 15.1 17.6 total mean sd 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 within b 2 3 3 4 1 2 0 0 15 7.09 3.71 between 0 0 3 3 3 4 4 1 18 12.25 3.98 b and c * one farthest tree was discarded. the inter-tree distance between the two families are from 5 to 20 meters, but 13 of 18 or 72% of the inter-tree distances was less than 15 meters, and 17 of 18 or 94% was less than 17.5 meters, which are for the present understood as within the range of the mating distance for altingia excelsa. why are trees of both families isolated sexually in spite of growing within the range of 16 to 18 meters? at present we guess that the two families may not flower at the same time inducing sexual isolation between them. details of the argument is given in the discussion. ii. agathis borneensis a comparison was made between theoretical distribution of disagreement counts and the actual observations (table 10). from table 10 one notes that the number of actual observations appeared to exceed the expectations of the disagreement counts in the respective classes of 0 and 1, and that of 6 or more although the excess is not enough to reach the level of statistical significance. in figure 5, we find that the general situation of the curve is 12 breeding structure of tree species in the tropical rain forest — sakai et al. figure 5. comparison between observed and expected frequencies of disagreement counts in agathi.s borneensis. table 10. comparison between theoretical expectations and actual observations of disagreement counts in agathis borneensis similar with that of altingia, and it is considered that inbreeding occurs to some extent in agathis borneensis, too. the next step is to examine if the occurrence of low disagreement counts was related to small inter-tree distances (table 11). disagreement counts in agathis borneensis looked to be randomly distributed and quite independent of inter-tree distances. thus, one can conclude that in agathis borneensis parental and child trees do not form the clusters of a single family group in a natural stand. occurrence of inbreeding in the species may have been due to self-fertilization of individual trees. 13 biotropia vol. 1 no. 1, july-december 1987 table 11. distribution of disagreement counts in relation to inter-tree distances in agathis borneensis. discussion plants growing in a population naturally involve related and nonrelated individuals more or less intermingled with each other. in fact, there is an indication that a plant population can be divided into these two groups. for instance, levin (1977) gave a brief description in his paper dealing with the distribution of genetic variation in the annual herb phlox drummondii hook that crossing between plants growing within 2 meter distance gave about three times more degenerative embryos than those from plants growing farther than 15 meters. price and waser (1979) in their crossing experiment with plants of delphinium nelsonii in wild populations collected pollen from plants growing at different distances of 0 (self), 1, 10, 100 and 1000 meters. it was found that the cross between plants growing at an intermediate outcrossing distance of 10 meters gave the best results in seed set and survival rate of seedlings, while cross with plants growing nearby seemed to show inbreeding depression. coles and fowler (1976) found in picea glauca that seeds obtained from crossing between trees growing within a radius of 100 meters showed some inbreeding effect in seed set, germination and seedling growth. how do parental and child trees distribute in a forest? hubbell (1979) found in a tropical dry forest that there were three groups of tree species with respect to the distribution pattern of the adult and juvenile densities around given adult trees. of the 30 species examined, 15 belonging to the first group showed the highest mean juvenile density in the 0 to 5 meters annulus, closest to the adult. the ten 14 breeding structure of tree species in the tropical rain forest — sakai el al. species of the second group showed horizontal density curves, while the remaining 5 species constituting the third group showed the maximal juvenile density in the area between 5 and 15 meters from the adult. there are few papers describing that seedlings are not likely to grow in t he neighborhood of their mother trees. for instance, janzen (1970, 1971) described the effect of herbivores in preventing growth of saplings around their mother trees in tropical forests. webb, tracy and haydock (1968) found an autoallelopathic effect of subtropical rain forest trees that produced a substance toxic to seedlings of the same species. watkinson (1978) observed that in an annual grass species vulpia fasciculata (gramineae) the seed drops first around the mother plant (first phase), but later they migrate or are buried in the soils (second phase). the present study aims at detection of family clumps, if any, in two tree species, altingia excelsa noronha and agathis borneensis \varb. both are the tropical rain forest tree species having not a few conspecific trees in the same stand. the number of trees of altingia in the 50 x 50 m quadrat in the ciwidey natural forest that was investigated was 38 among a total 140 trees. this seems to be not exceptional for the species, because federov (1966) wrote in his paper that altingia excelsa was one of those rare species which could attain a relatively large population density in the tropical rain forests in east java. another species, agathis borneensis also had a relatively large population density, i.e. 21 among 230 trees in the 50 x 50 m quadrat in kalimantan. for detection of familial structure in these two species, the results of isoperoxidase analysis have been used. two of the present authors, sakai and miyazaki (1972) published a paper reporting the analysis of family groups in the natural forests of thujopsis delabrata sileb. et zucc. by means of the so called disagreement counts which measures the degree of dissimilarity in the assortment pattern of isoenzyme bands between individual trees. based on the accepted views that the behavior of isoenzymes is monopolitically controlled by genes, and that isoenzymes have nothing to do with adaptability and are thus free from natural selection, it is interpreted that the degree of dissimilarity in the assortment pattern of isoenzymes could tell the genetic dissimilarity. as a matter of fact, schwartz and armitage (1983) have hypothesized in the study of wild marmots that related individuals should have a higher average electrophoretic genetic similarity than unrelated individuals, although their case is a little different from ours. this disagreement counts of isoenzymes described above have thenceforth been employed for detecting familial structure in fagus crenata blume by hashizume and sugimoto (1980), cryptomeria japonica d. don by hashizume and sugimoto (1982), chamaecyparis obtusa endl. by hashizume, watanabe and ookita (1983) and abies sachalinensis mast, by matsuura (1983). 15 biotropia vol. 1 no. 1, july-december 1987 all these studies depend on a few premises, among which is that random mating between trees of a population would give rise to random assortment of individual bands of the isoenzymes in individual trees, while inbreeding would induce resemblance in the combination pattern of t heir bands. it was found in the present study that in altingia, the disagreement counts of such a low value as 0, 1 or 2, i.e. trees with very high similarity, were more significant than the theoretical expectation calculated on the basis of random assortment of isoenzyme bands. in other words, trees similar in the assortment pattern of the isoperoxidases were more than randomly expected, indicating that propagation by inbreeding has most probably taken place in the stand. further analysis of the distribution of the disagreement counts in relation to the inter-tree distances has shown that the lowest counts were found mostly in the distances of 0 to 10 meters between trees. this means that trees with similar assortment patterns of isoperoxidase bands are located very close to each other, or that related trees form a small subgroup or clump in the stand. in this connection, it is interesting to remember the work of hashizume and sugimoto (1980) on fagus crenata blume. they found that the disagreement counts measured between mother-trees and their respective offspring were mostly so small as 0 and 1. thus presumptive families have been depicted for altingia excelsa in figure 3, where five groups, a, b, c, d and e were considered. in some papers concerning formation of small groups of trees in a forest, roe (1967) reported that in picea engelmannii, the seed dispersal occurs in the vicinity around the mother trees. fedorov (1966) wrote that in the tropical rain forest, "not only rare species, but dominant species too, usually form small populations". ashton (1969) wrote that "contagious distribution of individuals is in fact general among rain-forest trees". according to ashton, the clumping is related to the means of dispersal on the one hand, and to the chance of saplings of a single species to take their place in a gap, on the other hand. what role would these small groups of trees in a forest play with respect to t he reproduction and propagation of trees? sarvas (1967) divided pollen dispersal of tree species into 4 levels: (1) wit hi n individual trees, (2) between trees within a subpopulation, (3) between sub-populations within a population and (4) between populations. he investigated the problem in wind-pollinating pinus silvestris by the pollen catch technique and found that 50% of pollen affecting fertilization came from outside the population. contrary to sarvas, however, langner (1953) made an experimental study on the fertilization problem in picea abies (l.) karst. he made use of the "aurea" mutant and found that pollination occurred mostly between immediately neighboring trees in the stand. 16 breeding structure of tree species in the tropical rain forest — sakai et al. ashton (1969) described that in the tropical forest, group or clump is likely to be the principal breeding group in most tree species although outcrossing with other groups also occurs. chan (1980) observed that in shorea leprosula, trees in a cluster produced more fruits than isolated ones. appanah (1980) described that in the tropical rain forest, inter-tree movements of pollinators occurs mainly between neighboring trees in a clump, suggesting short distance pollen transfer. if reproduction occurs within a clump, or the effective breeding group is very small and with little exchange of genes between clumps, genetic differentiation should be great among them (wright 1946). in a perennial herb liatris cylindracea, schaal (1975) and schaal and levin (1978) found striking variation in gene frequencies of isoenzymes and in plant growth characters among subpopulations. levin (1977) investigated distribution of genetic variation in wild populations of phlox drummondii hook., a complex annual species consisting of six subspecies. the isoenzyme studies showed that most genetic variation was found within populations, less between populations within subspecies and least among subspecies. on t he contrary, schaal and smith (1980) investigated isoenzymes in populations of a leguminous allogamous species desmodium nudicaule and found that no genetic differentiation within the population or no genetic substructuring was found in the species. in tree species, curies and ledig (1982) found in 11 populations of pinus rigida that 97% of genetic diversity was among individual trees within populations and only 3% was between them. a similar result was obtained by hiebert and hamrick (1983) in pinus longaeva bailey. linhart, mitton et al. (1981) investigated genetic variation at seven isoenzyme loci in six clusters of trees in a population of pinus ponderosa laws. they found that the tree clusters differed significantly from each other in the isoenzyme loci as well as in several gross morphology. in pinus monticola dougl., rehfeldt (1979) grew 8 full-sib families from each of 12 populations to investigate variation in several growth characters. he found significant variation among families within populations, but litt le or no differentiation among populations. in pseudotsuga menziesii (mirb.) franco, yeh and o'malley (1980) investigated 21 isoenzyme loci in 11 populations. they found that almost all genetic variation was found within populations and very little among them. in the tropical rain forest, gan, robertson et al. (1977) have found spatial heterogeneity in several isoenzymes as well as in some leaf characters in a population of shorea leprosula and xerospermum intermedium. they attributed this heterogeneity to the short-range pollen flow and fruit dispersal causing spatial isolation between subpopulations. in aitingia excelsa, it was found that each of the five presumptive families or groups was apparently different from others in several respects. as seen in table 4, every group possesses its specific bands which other 17 b1otropia vol. 1 no. 1, july-december 1987 groups do not. the groups were also variable in several leaf characters. these facts tell us that the five groups were genetically differentiated, suggesting that each group had been genetically separated from others. in other words, reproduction may have occurred mainly within each group without exchanging genes with others. thus, the five groups are admitted to be separate as breeding groups, and because of very low values of disagreement counts within each group, each of them is certainly considered to be a single family. assuming that trees within a single family clump could mate freely, we are able to estimate the mating distance in the species as 16 to 18 meters or 16.5 meters. for estimating the area occupied by the family clump as a breeding group, it would be a circle of 200 or 250 m 2 with a radius of 8 or 9 meters. a panmictic unit of breeding or a group of plants in which their gametes may come together has been defined by wright (1946) as a neighborhood in a plant population. schaal and levin (1978) estimated the area of the neighborhood of a perennial herb, liatris cylindracea to be 33 m 2 on the basis of flying distance of insects and the distance of seed dispersal. the family clump in the present paper may correspond to the neighborhood in some respects, because the two concepts equally emphasize an occurrence of random mating among members of a group. a remarkable difference between these two is breeding in the family clump, mating occurs among related individuals, while in the neighborhood it occurs among all individuals. attention should be paid to the two families b and c (figure 3). granting that 16 to 18 meters are the mating distance of aitingia excelsa, a question arises as to why trees of both families could be so much differentiated from each other in electrophoretic as well as in morphological characters, in spite of their mixed distribution in the forest. in nature, there may be various kinds of reproductive barriers. an extreme case may be found among provenances from a wide region. for instance, dogra (1981) found in pinus wallichiana a.b. jacks native to himalayas that flowering time was different from provenance to provenance, particularly between low and high elevation. the mountain ridge could also be a very effective barrier. in the present case of altingia, however, this geographical barrier was not present in the population. flowering time of a plant is of course dependent on environmental conditions, on the one hand, and genetic constitution of the plant, on the other. for the genetic control on flowering of plants, stern and roche (1974) have given forcible discussions in their book, "genetics of forest ecosystems". stam (1983) took a view of natural selection of environments on within-population differentiation of flowering time in plants. 18 breeding structure of tree species in the tropical rain forest — sakai et al. it is often stated that the lack of seasonality in the tropical forest leads to irregularity and lack of coincidence in flowering among not only related species, but also among individual trees of the same species (fedorov 1966, ashton 1969, frankie 1975, chan 1980). this maybe due to shortage in the tropics of environmental effect like day/night length or vernalizing temperature which serves as an incentive to flower induction. thus, in the tropical rain forest where climatic control is absent, genetic make-up or genotype should be responsible for the initiation of flowering in each tree. here we can recollect that trees of the same family are likely to have many of the same genes in common. so, it is to be expected that they will show synchronized flowering so far as they belong to the same family, flowering time of which, however, may be different from trees of the other families. the two families b and c would have been sexually isolated from each other though their trees grow mixedly distributed within the same area of the mating distance. thus different families in altingia excelsa would be subjected to the effect of genetic drift. (additional investigations are needed). as described previously, two opposite views have been proposed for reproductive system of trees in the tropical rain forest. one is that of fedorov (1966) who thinks that in the tropical rain forest, tree species reproduces predominantly by inbreeding, probably by self-fertilization due to scarcity of conspecific individuals combined with asynchronous flowering among them, leading to genetic drift and finally to speciation. another view is that of asthon (1969) who looks that the mode of speciation in the tropical rain forest is essentially not different from other terrestrial plant ecosystems, the speciation being expected to occur as a result of outcrossing and gene recombination combined with natural selection. data obtained from the present study on altingia excelsa seem to favor fedorov although we take a view of intra-family propagation instead of fedorov's self-fertilization hypothesis. in agathis borneensis, inbreeding occurs to some extent but there is no sign of clustering of a group of trees or formation of family clumps as detected in altingia excelsa. it is assumed that related trees are separated widely in the forest without forming a small group per family. effect of the inbreeding observed may be attributed to self-fertilization of individual trees. conclusion from the present study it has been found that a population of altingia excelsa involved several breeding groups, each of which consisted of related trees of a single family. families were very variable biochemically as well as 19 biotropia vol. 1 no. 1, july-december 1987 morphologically, suggesting that they were genetically differentiated subgroups within a population. the mating distance, that is, the distance within which trees can mate at random has been estimated to be 16 to 18 meters in altingia excelsa. if trees of different families were mixedly growing within the mating distance, they still maintained their peculiarities suggesting the presence of inter-familial sexual isolation due perhaps to genetic difference in flowering time among families. in agathis borneensis, formation of no family clump was noticed, though there was an indication of occurrence of inbreeding to some extent. inbreeding in this species may probably be due to self-fertilization in individual trees. literature cited appanah, s. 1980. pollination in malaysian primary forests. tropical ecology and development. p. 177-182. ashton, p.s. 1969. speciation among tropical forest trees: some deductions in the light of recentevidence. biol. j. linn. soc. 1. p. 155-196. brewer, g.j. 1970. "introduction to isozyme techniques". academic press (new york). 186 p. chan, h.t. 1980. reproduction biology of some malaysian dipterocarps. tropical ecology and development, p. 169-175. coles, j.f. &d.p. fowler. 1976. inbreeding in neighboring trees in two white spruce population. silv. gen. 21 (1): 29-34. dogra, p.d. 1981. variability in biology of flowering in blue pine provenances of northwestern himalayas in relation to reproductive barriers and gene flow. proc. symp. on flowering physiology. xvii iufro world cong. p. 8-15. endo, t. 1968. zymography (in japanese). sci. rep. toyo kagaku sangyo 26: 1-5. endo, t. 1978. a new method for peroxidase isozyme stain. ann. rep. nat. inst. gen. 28: 41. endo, t. 1981. an extraction system for leaf zymographic analysis in woody plants. ibid. 31: 41. fedorov, an.a. 1966. the structure of the tropical rain forest and speciation in the humid tropics. j. ecol. 54: 1-11. frankie, g.w. 1975. tropical forest phenology and pollinator plant coevolution. "coevolution of animals and plants", ed. by l.e. gilbert and p.h. raven. univ. texas press, p. 192-209. gan, y.y., f.w. robertson, p.s. ashton, e. soepadmo & d.w. lee. 1977. genetic variation in wild population of rain forest trees. nature 269 (5626): 323-325. curies, r.p. & f.t. ledig. 1982. genetic diversity and population structure in pitch pine (pinus rigida) mill.). evolution 36 (2): 387-402. hashizume, h. & s. sugimoto. 1980. a study of the breeding system in natural forests of buna (fagus crenata blume) using the peroxidase isozyme technique. hardwood research 1: 59-71. hashizume, h. & s. sugimoto. 1982. genetic and thremmatologic studies on the natural forest of sugi (cryptomeria japonica d. don) in the chugoku mountain district. (i) the reproductive system of naturally grown sugi. bull. fac. agr. tottori univ. 34: 73-81. hashizume, h., y. watanabe & m. ookita. 1983. genetic studies on natural forests of hinoki (chamaecyparis obtusa endl.) in the chugoku mountain district. (i) studies of genetic 20 breeding structure of tree species in the tropical rain forest — sakai et al. variability and reproductive system in naturally grown hinoki using the peroxidase isozyme technique. ibid. 35: 24-33. hiebert, r.d. & j.l. hamrick. 1983. patterns and levels of genetic variation in great basin bristle cone pine, pinus longaeva. evolution 37 (2): 302-310. hubbell, s.p. 1979. tree dispersion, abundance, and diversity in a tropical dry forest. science 203 (4387): 299-1307. janzen, d.h. 1970. herbivores and the number of tree species in tropical forests. amer. nat. 104 (940): 501-528. janzen, d.h. 1971. escape of juvenile dioclea megacarpa (leguminosae) vines from predators in a deciduous tropical forest. amer. nat. 105 (942): 97-112. langner, w. 1933. eine mendelspatung bei aurea-formen von picea abies (l.) karst. als mittel zur klarung der befruchtungs-verhaltnisse im walde. z. f. forstgenetic 2 (3): 49-51. levin, d.a. 1977. the organization of genetic variability in phlox drummondii. evolution 31 (3): 477-494. linhart, y.b., j.b. mitton, l.b. sturgeon &m.l. davis. 1981. genetic variation in space and time in a population of ponderosa pine. heredity 46 (3): 407-426. matsuura, t. 1983. family analysis in natural forest of abies sachalinensis. "ecological-genetic studies in natural forests and their practical applications" hoppo ringyo-kai (sapporo). p. 265-281. price, m.v. & n.m. waser. 1979. pollen dispersal and optimal outcrossing in delphinium nelsonii. nature 277: 94-297. rehfeldt, g.e. 1979. ecotypic differentiation in population of pinus monticola in north idaho – myth or reality? amer. nat. 114 (5): 627-636. roe, a.l. 1967. seed dispersal in a bumper spruce seed year. u.s. for. serv. res. paper 1nt-39. p. 1-10. sakai, k.i. & y. miyazaki. 1972. genetic studies in natural populations of forest trees. ii. family analysis. silv. gen. 21 (3): 149-154. sarvas, r. 1967. pollen dispersal within and between subpopulations: role of isolation and migration in microevolution of forest tree species. proc. xiv cong. 1ufro, vol. 3: 332-345. schaal, b.a. 1975. population structure and local differentiation in liatris cylindracea. amer. nat. 109(969): 511-528. schaal, b.a. & d.a. levin. 1978. morphological differentiation and neighborhood size in liatris cylindracea. amer. j. bot. 65 (9): 923-938. schaal, b.a. & w.g. smith. 1980. the apportionment of genetic variation within and among populations of esmodium nudiflorum. evolution 34 (2): 214-221. schwartz, o.a. & k.b. armitage. 1983. problems in the use of genetic similarity to show relatedness! evolution 37 (2): 417-420. stam, p. 1983. the evolution of reproductive isolation in closely adjacent plant populations through differential flowering time. heredity 50 (2): 105-118. stern, k. & l. roche. 1974. "genetics of forest ecosystems". springer-verlag (berlin). 330 p. watkinson, a.r. 1978. the demography of a sand dune annual: vulpiafasciculata. iii. the dispersal of seeds. j. ecol. 66 (2): 483-498. 21 biotropia vol. 1 no. 1, july-december 1987 webb, l.j., j.g. tracy & k.p. haydock. 1968. a factor toxic to seedlings of the same species associated with living root of the nongregarious subtropical rain forest tree grevillea robusta. j. appl. ecol. 5 (1): 13-25. wright, s. 1946. isolation by distance under diverse systems of mating. genetics 31: 39-59. yeh, f.cn.-h. & d. o'malley. 1980. enzyme variations in natural populations of douglas f ir , pseudotsuga menziesii (mirb.) franco, from british columbia. i. genetic variation patterns in coastal populations. silv. gen. 29 (3-4): 83-92. 22 breeding structure of tree species in the tropical rain forest — sakai et al. appendix 1 23 b1otropia vol. 1 no. 1, july-december 1987 appendix 2. 24 breeding structure of tree species in the tropical rain forest — sakai et al. appendix 3 calculations of the theoretical expectations of disagreement counts according to the random assortment of isoperoxidase bands in individual trees let the frequency of occurrence of i-th band in the population be designated as xj. the probability that the disagreement count will be either zero or one for the band is given in t he following: probabiliy that the ppulation will give zero or one disagreement count for the i-th isoperoxidase band the probability of getting zero disagreement count for the i-th band is given as (xj + (1 — xj) 2 ), and that of getting one is given as 2xi(l — xi), since plus meeting with plus or minus meeting w it h minus ( + /+ or — / — ) gives zero, while minus meeting with plus or the inverse ( + / — or — / + ) gives disagreement count of value one. let the total number of isoperoxidase bands which are assumed to be genetically independent with each other be p, then the probability of getting zero disagreement count in the population will be ( x 1 2 + (1— x 1 2 ) x . . . . . . . . . . x (x 2 i + ( 1— xi) 2 ) x . . . . . . . . . . x (x 2 p + (1-x 2 p) = p π i = 1 ( x 2 i + (1-x j )2) the probability that the disagreement count gets the value of one in the population w it h p bands will be given by: p p σ π i = 1 [2xi (-xj) j = 1 (x2j + (1-xj)2)] the probability that the disagreement count may take the value larger th an 2 can be calculated in t h e similar way. 25 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf biotropia (2) 1988/1989: 18-24 the distribution and potential problems of mimosa pigra l. in indonesia sri s. tjitrosoedirdjo tropical agricultural pest biology program, biotrop, bogor, indonesia abstract mimosa pigra l. (mimosa asperata l.) of the family mimosaceae is an introduced species from south america which is locally naturalized in indonesia. the present known distribution sites are still restricted to java, sumatra, and kalimantan. it is found in almost all provinces of java and many heavily infested areas have been noted in jakarta and west java. the introduction of the plant to other islands has not been reported yet. the separation of the islands by sea is preventing the further spread of the plant in indonesia. control is basically occasional and on an individual basis. there is no sustained effort yet to control the plant. the urgency of controlling and restricting its spread cannot be over emphasized. introduction mimosa pigra l. is an introduced species in indonesia from south america which has been locally naturalized for a long time (backer and van den brink 1963). soerjani (1979) included this species as one of the ten serious aquatic weeds in indonesia. the plant is very difficult to control because its spiny and woody character makes it very difficult to penetrate when growing very densely. recently, there is a large number of areas in jakarta and its vicinity, as well as some areas in west java infested with m. pigra. in thailand, m. pigra was introduced from indonesia in 1947. it was first planted in amphus mae taeng where it was used as a ground cover to improve soil quality and to help prevent bank erosion. within 30 years, it has infested large areas of northern thailand (lamar robert 1982). among the institutes working on m. pigra in indonesia are: the central research institute for plantation which conducted the study on its biology and ecology (sumaryono and soedarsan 1983); the biological research and development center, indonesian institute of sciences, which conducted studies on its ecology, biological control and taxonomic aspects (uji 1988, partomihardjo 1988, and kahono 1988); and biotrop which studied its potential problems at bening and saradan reservoir, east java (anonymous 1983). research on the biology, distribution, ecological role in the environment and control methods in indonesia should be intensified and the problem must be dealt with before it expands to larger areas and become more serious. 18 the distribution and potential problems of mimosa pigra l. -sri s. tjitrosoedirdjo in this paper, the present distribution of m. pigra at the known sites and the potential problems in the future are discussed and evaluated. distribution of mimosa pigra in indonesia the distribution of m. pigra in indonesia was obtained from the examination of herbarium specimens at the herbarium bogoriense, bogor, indonesia and information from several other sources (soedarsan 1980, sumaryono and soedarsan 1983). mimosa pigra was first introduced to indonesia by the bogor botanical garden from mexico (thysmann and binnendijk 1866). the earliest record of its presence was in 1844 from bogor, west java (hasskarl 1844). the oldest existing herbarium specimens were found in 1902 from ciliwung riverside in bogor identified as mimosa asperata l.; in the same year, a specimen was also found in jakarta. the present distribution of m. pigra in indonesia is still restricted to java, sumatra and kalimantan (figure 1). most of the specimens were found in java, i.e. from all the provinces, jakarta, west java, central java and east java. in sumatra it was reported from solok, west sumatra (soedarsan 1980) and sibolangit, north sumatra in 1917. the only specimen from kalimantan was found in samarinda in 1979. it seems that in kalimantan m. pigra was newly introduced. there is no report yet from other islands. according to the recent information in indonesia there are two types of the habits of m. pigra i.e. erect and prostrate stenis. field observations showed different morphological characteristics of the two types of m. pigra. the prostrate stem type is only strictly distributed around jakarta, bogor, tangerang and bekasi at an altitude of 5-260 m a.s.l. the erect stem type is widely distributed all over java (partomihardjo 1988, uji 1988). backer and van den brink (1963) indicated that in indonesia it can be found at an altitude of 1-700 m above sea level. in different places m. pigra is called by different names such as klampis air, putri main hitam (sumaryono and soedarsan 1983) andputri malu raksasa. in west java it is known as jerujut, gehgeran and cucuk buset, rondo kaget (sunda), pis kucing (java). 19 biotropia no. 2, 1988/1989 20 the distribution and potential problems of mimosapigra l.-sri s. tjitrosoedirdjo potential problems in the future there are some factors assisting the dispersal and rapid colonization of m. pigra (miller 1982). among them are the intrinsic characteristics of the plant, like the ability of whole seed pods on their segments to float on water for long periods, and the hairy nature of pods allowing segments to stick to hair or clothing, which enable them to spread easily. this is combined with dispersal both by natural uncontrollable forces and by man as well as animals. in a river, the spread of m. pigra downstream from its original location has been largely through uncontrollable water movement. its spread to isolated sites away from the river is mainly by human activity through removal of sand from the river for use in construction of roads or buildings. the ability of m. pigra to rapidly colonize areas is due to its inherent ability to flower and seed all year round under favorable conditions, the production of large quantities of seeds, their long term viability and tolerance to both flood and drought. wanichanantakul and chinawong (1979) reported that in thailand, an average plant is seeded twelve times per year. they also found that prolonged seed dormancy was due to the hard seed coat. in indonesia, (anonymous 1983) the rate of growth of m. pigra at bening and saradan reservoirs, east java, was observed. the rate of growth was 11.54 g dry weight/day -1 or 4.42% day -1 . although it is less than the rate of growth of the floating aquatic weeds, it is relatively high for a woody perennial plant. the high rate of growth is also supported by the high production of viable seeds. the number of mature seeds found in 5 cm depth of soil in a plot 25 x 25 cm 2 was 200. the laboratory experiments showed that the seed germination rate was very high, i.e. approximately 93.5% under light and 89.5% under dark conditions (anonymous 1983). sumaryono and soedarsan, 1983 reported that in bogor, the stem diameter reached up to 4.6 cm after 8 months. the first flowers appeared 4 months after seed germination. they also reported that after cutting, regeneration is very rapid. it grew better in a soil water of 43-67% and the plant could survive in flooded land although its growth was retarded. in java, m. pigra has spread faster than on other islands. colonization by m. pigra is basically through movement of seeds by water. mostly, the plants were found in wet places-such as riverside, irrigation canal or at the shore of the lake (table 1). introduction to isolated places not reached by water can also be noticed. it is also found along the road, railway or in waste lands. 21 biotropia no. 2, 1988/1989 table 1. distribution of m. pigra in indonesia based on the herbarium specimens at the herbarium bogoriense", other sources (soedarsan 1980 2) ), anon, 1983 3 ), and own observation 4) . 22 the distribution and potential problems of mimosa pigra l. sri s. tjitrosoedirdjo increasing development of housing, industrial estates, highways, airports, bridges and roads in jakarta use large amount of sand. this sand comes from the rivers or places already contaminated by m. pigra seeds. heavy infestation can be found in waste lands close to housing and industrial estates, along the jagorawi highway, and along the way to soekarno-hatta airport. at ciapus bogor, there is a big sand quarry where the product is distributed mostly to jakarta and west java. m. pigra can be found easily in the areas surrounding the quarry. this condition contributes to the further spread of m. pigra elsewhere. in central java, m. pigra can be found at the roadside between yogyakarta and magelang. there are some rivers such as krasak, putih and blongkeng, between these two cities. the rivers are rich in sand from merapi mountain, which is collected and distributed to many places in central java. if the sand from these rivers is contaminated with m. pigra seeds, it can be spread further within a short time. anonymous (1983) reported that in east java, bening and saradan reservoirs are infested with m. pigra. the plants are mostly found at the lake shore. at saradan reservoir m. pigra is a dominant species. at bening reservoir where the fluctuation of the water is very high (approximately 13 m), m. pigra appears to be particularly well adapted to grow in a seasonally flooded habitat. during the dry season, m. pigra grows well and biomass production is very high. during this time at bening reservoir, the flooded area is 570 ha and there are 370 ha which can be potentially colonized by m. pigra. after flooding during the wet season, m. pigra will decompose and the nutrients released would enrich the water. its decay will cause an increase in the water's carbon dioxide content, chemical oxygen demand, ammonia and nitrate, and nitrogen, and reduce its ph and dissolved oxygen concentration. the plants recover by germination of viable seeds. anonymous (1983) suggested that a control program should be carried out during the dry season before flowering, to prevent the production of seeds. in sumatra, soedarsan (1980) reported that m. pigra was found close to the river. it reached 4 m in height. if there is no control treatment m. pigra will spread further. with the construction of the trans sumatra highway from aceh to java and crossing the sunda channel, there is a possibility that m. pigra will be spread from java to sumatra through the movement of man and vehicles. 23 biotropia no. 2, 1988/1989 summary although m. pigra has been introduced in indonesia for a long time, the existing known places are still restricted to java, sumatra and kalimantan. the introduction of the plant to other islands has not been reported yet. the separation of the islands by the sea is preventing the further spread of the plant in indonesia. in java, m. pigra is widely distributed, it is found in almost all provinces. the high rate of growth, the ability of producing large amounts of seed, the great regenerative power and also the resistance to flood and drought support the rapid colonization of m. pigra. in jakarta and west java, many places have been infested heavily by m. pigra. there is no sustained effort yet to control the plant, only occasionally by individual initiative. the urgency of controlling and restricting its spread cannot be over emphasized. references anonymous. 1983. penyelidikan tanaman air dan perikanan waduk pada proyek irigasi widas (studies on the reservoir's aquatic weeds and fishery at the widas irrigation). internal report biotrop, bogor, indonesia. backer, c.a. and r.c.b. van der brink. 1963. flora of java. vol. i. n.v.p. noordhof-groningen the netherlands. lamar robert, g. 1982. economic returns to investment in control of m. pigra in thailand. ippc document no. 42. a-82. mpc agricultural economics report no. 15. hasskarl, j.k. 1844. catalogus plantarum in horto botanico bogoriense cultarum alter. (ter lands-drukkerij : batavia). miller, i.l. 1982. the distribution and threat of m. pigra in australia proc. international symposium on m. pigra management chiang mai, thailand. partomihardjo, t. 1988. studies on the biology of mimosa pigra for supporting its control in indonesia. proceeding wssi conference vol. ii. soedarsan, a. 1980. potensi klampis air (m. pigra l.). sebagai gulma di masa depan (the potential of m. pigra as a weed in the future). menara perkebunan 48(6): 179-180. soerjani, m. 1979. recent trends in aquatic weed management in indonesia proc. 7th asian pac. weed sci. conf. sydney, australia. supp. vol. sumaryono and soedarsan, a. 1983. beberapa aspek pertumbuhan klampis air (some aspect of the growth of m. pigra). menara perkebunan 51 (6): 160-164 . thysmann, j.e. and s. binnendijk. 1866. catalogus plantarum quae in horto botanico bogoriense colunter-ter lands drukkerij: batavia. uji, t. 1988. mimosa species in java. proceeding indonesian weed science society conf. vol. i. wanichanantakul, p. and s. chinawono. 1979. some aspects on the biology on m. pigra in northern thailand. proc. 7th asian pac. weed sci. soc. conf., sydney, australia. 24 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf biotropia 1 (1) 1987: 46-52 ii. soils from cultivated stands of shorea javanica m.j. sheehy skhffington* tropical forest biology program, biotrop, bogor, indonesia keywords: soils, shorea javanica, sumatra abstract soils from shorea javanica plantations in different parts of lampung province, south sumatra were sampled to a depth of 50 cm and described. they varied from quite deep loamy alkaline soils near krui, to sticky acid clays behind ngaras. the cation content of most samples was quite high, but organic nutrients were low, suggesting volcanic origin of the soils. preliminary observations of performance in situ of 5. javanica would suggest a requirement for deep loamy, fairly alkaline soils with a moderately high exchangeable cation content for optimal yield and performance. introduction an important aspect in considering sites of s. javanica plantations is whether they can support a sufficiently productive tree crop. resin is not always abundantly provided at all existing sites and this may be a function of environmental factors, such as soil, local climate or topography. this study is proposed to deal with soil characteristics suitable for s. javanica cultivation. two approaches can be taken in the determination of the effect of soils on s. javanica performance. the first entails a survey of existing s. javanica stands, sampling the soils and gathering information on the health and resin productivity of the trees, and the second involves controlled growth experiments of seedlings on a range of soils, to estimate growth performance and thus select sites with soils yielding optimal production. this report covers the first aspect, namely analysis of soils collected at various s. javanica sites in the lampung province of sumatra. sites 1) some time was spent in the region of krui where resin production is good and villagers derive a reasonable income from its harvest. soils have already been analysed from a site ca. 5 km from krui (torquebiau 1984), but as plantations are extensive within a radius of 30 km of the town, a second sample site was chosen at palnam, 19 km from krui on the liwa road. about 3-400 m south of this road an outcrop of pink reef limestone was visible in a plantation and soil samples were taken from there. * present address: department of botany, university college, go/way, ireland. 46 ii. soils from cultivated stands of shorea javanica — skeffington 2) to the south of krui, along the coast 'road' at way biha, a plantation was found in very sandy soil about 2-300 m from the beach. the trees, most of them ca. 20-30 years old were healthy and straight, ca. 20-30 m high, but resin production is poor, the bark thick, ca. 2-3 cm, and flower and fruit production occasional. 3) further south, at ngaras, s. javanica plantations extend up the hills at t h e back of t h e village. near the village and halfway up the hill (site ngaras i) soils were brown and fairly deep. resin production is good on these lower slopes. above these, at the top of the hill (site ngaras ii), soils were redder and more acidic. resin production here is not so good. further inland on higher slopes, soils were brown and less acidic, yet resin production was no better than at ngaras ii, indicating that caution should be used when correlating resin production and soil properties. 4) two other sites were examined in lampung, one at wana, near metro where a small stand of fairly old damar trees yielded soils of good crumb structure. the other, near bakauheni was even smaller and probably disused for a longer time. in each site, soils were sampled three times and subsampled at 5 cm and 30 cm depth. methods for laboratory analyses were standard methods currently used in biotrop tropical forest biology program laboratory. results field soil descriptions these are summarized in table 1. the soils at palnam indicate a fairly rapid litter decomposition and the resulting humus grades into the clayey substratum giving a fairly uniform brown color. at ngaras, however, soils are only brown on the lower slopes (ngaras i) and these overlay a stickier yellow-brown clay. on the upper slopes they were yellowish to yellow/orange throughout and more blocky in structure, with a thin litter/humus layer above. the soil at way biha was very sandy and poorly developed below 10 cm, whereas at wana and bakauheni, soils had a good surface humic crumb structure, but beneath was reddish clay and a harsh smell of metal oxides (signs of temporary waterlogging?) was detectable. laboratory analyses the range of texture (fig. 1 and table 2) relating to particle size is quite striking but if one considers only the soils where resin production is good (i.e. at palnam and ngaras i), these are loams with a fairly even proportion of each particle size class. also among the loams are the samples from 5 cm depth at wana and bakauheni. however at 30 cm, these contain very little sand and have a high proportion of clay. the ngaras ii soils are also low in sand content, whereas the coastal soils at way biha comprise little else. the ph also shows a wide range, the most acid soils being those at ngaras, especially ngaras ii. the others are not far from neutral (ph 7). this relatively 47 biotropia vol. 1 no. 1, july-december 1987 table 1. summary of profile descriptions 48 ii. soils from cultivated stands of shorea javanica skeffington 49 biotropia vol. 1 no. 1, july-december 1987 figure 1. soil texture triangle, showing position of soils listed on table 2. high ph is borne out by a high cation exchange capacity (c.e.c) and base saturation, which are comparatively low at ngaras. analysis of individual cations (table 2) shows that calcium occupies most of the exchangeable sites in soils where the c.e.c. is high. the other three, especially k and mg are relatively high, but vary little between sites. of notable exception again are the samples from 5 cm depth at wana and bakauheni which both have a very high % base saturation and also high k and mg concentrations. the limestone bedrock must contribute to the high ph and calcium content of the palnam soils. on the other hand, the organic constituents, total carbon and nitrogen, are very low for all soils, being lowest again at the ngaras sites, which in this case differ little. the c/n ratio is also low but this is indicative that n is being mineralized, suggesting rapid decomposition, 50 s s a percent s and ii. soils from cultivated stands of shorea javanica — skeffington and thus, low concentrations of these elements are not indicative of their probable rapid turnover. inorganic phosphate is relatively low, being highest at way biha and palnam. accumulation of marine shells and detritus may be a high source of inorganic p at the coastal site of way biha. comparison with other soils because of high rainfall, tropical soils generally rapidly become acidic and poor in bases due to fast weathering and leaching (sanchez 1976). only relatively recent alluvial or volcanic soils tend to be fertile and to a lesser extent those overlying exchangeable base-rich rocks such as limestone. there is evidence that the krui soils contain volcanic ash (torquebiau 1964) and it is likely that those at ngaras may also have received ash in the past. in terms of c.e.c. and exchangeable cations all soils compare favorably with fertile andosols developed on volcanic tuffs near bogor in java (hardjosoesastro et al. 1983) where mg, k and especially ca are in fact lower than in these soils. other soils from lampung have similar chemical properties (adiningsih et al. 1983) including low carbon and nitrogen percentages, suggesting that the range of soil types presented here is relatively common in the province. in general, the soils of sumatra (and to a greater extent of java), are more fertile than those of islands with little or no recent volcanic activity or large alluvial deposits. islands such as borneo have older, more weathered soils which are more acidic with a lower cation content and exchange capacity (proctor et al. 1983). thus soils where s. javanica should grow relatively well would not be very acidic (ph > 4.5), contain a moderate to high amount of cations, but not necessarily with high carbon, nitrogen or phosphate concentrations. for a good resin yield it may be important to select fairly deep base-rich soils which also have a good mix of sand, clay and silt resulting in a loamy texture. prospects for future research the second part of the proposal outlined above, i.e. growth experiments, should follow directly from this preliminary investigation. for proper and vigorous growth of 5. javanica seedlings, they would require to be grown in fertile base-rich soils of a good loamy texture. however, some estimate of their tolerance to less fertile soils can be derived from their experimental growth in a range of soils of different ph and fertility. podzolic soils would have a ph similar to the more acid soils analyzed here and can be collected locally (e.g. at jasinga). however, these may have moderately high concentrations of exchangeable cations (adiningsih et al. 1983) and all soils thus used should be analyzed as described above to assess their comparability to the soils in the field. 51 biotropia vol. 1 no. 1, july-december 1987 performance of seedlings in poor soil conditions will probably be compounded by effects of over-shading or exposure to too much light (bravo 1960). a two-way experiment to test the combined effects of range of soils (possibly adding fertilizer to some, sundralingam 1983), and light conditions, would resolve optimal conditions for seedling growth as well as demonstrating which soil types are not conducive to healthy growth in either seedlings or adult trees. examples of more fertile soils which could be used locally are the andosols at sukamantri described in hardjosoesastroe et al. (1983). at cisarua are some brown regosols, also volcanically-derived and fertile, which may prove to be the more suitable of the fertile soils. measurement of growth performance could include size, leaf number and co2 uptake and subsequent harvest for biomass and nutrient content measurement. acknowledgments thanks are due to dr. emmanuel torquebiau for his help and advice as leader of the expedition. also thanks to mr. ahmad zainuddin for field assistance and mr. agus nasir who carried out the laboratory analysis. references adiningsih, j.s. and m. sudjadi, 1983. the effect of submergence and fertilization on some characteristics of red yellow podsolic soils from central lampung (in indonesian). pemberitaan penelitian tanah dan pupuk no. 2. pp. 1-8. bravo, p.r. 1980. germination and initial growth of shorea stenoptera and s. comoressa as affected by shading and different potting media. b1otrop report on training course in biological aspects of silviculture, sept. 1979—july 1980, bogor, indonesia. hardjosoes astro, r., h. suyanto and a.m. satari. 1983. andosol from the sukamantri area of bogor district (in indonesian). pemberitaan penelitian tanah dan pupuk no. 2. pp. 18-29. marali, m., o.k. husein and bachri, 1980. hubungan sifat tanah podsolik dengan pertumbuhan tanaman tengkawang di haurbentes, jasinga, bogor, presiding no. 1, penelitian tanah, pp. 37-44. proctor, j., j.m. anderson, s.c.l. fooden and h.w. vallack, 1983. ecological studies in four contrasting lowland rain forest in gunung mulu national park, sarawak. i forest environment and floristics. journal of ecology, 71. pp. 237-260. sanchez, p.a. 1976. properties and management of soil in the tropics. wiley, chichester. sundralingam, p. 1983. responses of seedlings of dryobalanops aromatica and d. oblongifolia to commercial fertilizers. malaysia forester 46 (1) 86-92. torquebiau, e. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2. pp. 103-127. 52 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf biotropia 1 (1) 1987: 67-74 v. preliminary study on isozymes of shorea javanica u. juniarti and m. i. j. umboh tropical forest biology program, biotrop, bogor, indonesia introduction the detection of genetic variability in natural or man-made populations/ plantations is useful in both basic and applied biology. in addition to the various facets of studies on shorea javanica already initiated by torquebiau (1984) and alongside with his recommendations on focus for future research, a study on the genetic aspects of the species should be given important considerations. as the trees are tapped for resin, an important forest product, the genetic basis of the production as well as the range of variation in amount of resin production among t he trees must be known. coupled with this is a thorough investigation on the differences in pest resistance/susceptability among the trees and their genetic basis. while the assumption (torquebiau 1984) that trees in natural forest areas are-rarely attacked by diseases because of mycorrhizal fungi is interesting, its confirmation is necessary. if this is true, problems would arise when plants are introduced into a new plantation site as experienced by the forest research institute (ardikoesuma 1954). thus, we need to look for pest resistant plants i.e. those that can remain healthy even in the absence of mycorrhizae. the above studies on possible genetic variation could give vital information for development of forest plantations of the species and for breeding and tree improvement strategies. by knowing the extent of genetic variation in natural population or in plantations one could be guided to maintain or increase the genetic base in these areas. biochemical characters such as isozyme banding patterns have been useful in several areas of plant biology, population genetics, evolution and breeding. isozymes are detected by starch gel electrophoresis and when their genetic control is established, they could be genetic markers in analyzing variation in morphological or physiological characters. the present study is an attempt to detect the isozymes in leaves, seeds and cotyledons of shorea javanica by gel electrophoresis. materials and methods mature leaves from six mother trees and seeds from one tree were collected from krui, lampung (sumatra) in september 1985. the leaves were kept inside a plastic ice cooling box immediately after harvest and stored in an incubator at 0°c until used for electrophoresis. fifty mg of the leaf blade, seed or cotyledons of 67 biotropia vol. 1 no. 1, july-december 1987 germinating seedlings were excised and ground in a mortar containing 20 mg of quartz sand, 20 mg of polyvinylpolyrrolidone (pvpp) and ca. 0.6 ml of an extractant containing triton-x-100, tris (0.5m), ascorbic acid (0.5m) adjusted to ph 7.0 by acetic acid. crude extract was absorbed into a 5 mm x 9 mm toyo no. 50 filter paper and the wicks were inserted into a 10% starch gel mediated with either 30 mm borate buffer (ph 8.0) or 5 mm histidine buffer (ph 6.6) at the position of 10 cm from the anodal end of the gel. the gel was prepared following smithies (1955) with a toyo starch gel moulder. electrophoresis was conducted with the use of a toyo electrophoretic apparatus containing either 0.3 m borate buffer (ph 8.0) or 0.6 m sodium citrate (ph 6.2) at a constant voltage of 300 v for borate system and at 250 v for sodium citrate system. running time was 3.5—4 hours in a toyo model is-2200 incubator at 0°c. the gel surface was covered with plastic wrap and chilled by ice throughout the run. each gel was sliced horizontally with a sharp blade and gel slices were assayed for the following enzymes: alcohol dehydrogenase (after schwartz and endo 1966), malic acid dehydrogenase (after shaw and prasad 1970), peroxidase (after endo 1978) and superoxide dismutase (after beauchamp and fridovich 1971). the zymograms were recorded by drawing. results and discussion 1. alcohol dehydrogenase (adh) a. leaves fig. 1 shows 3 anodally-moving bandmorphs. one tree showed bandmorph 1 consisting of 3 bands a l, a2, and a3. another tree showed bandmorphs ii consisting of 2 bands a2 and a3. four trees showed a single band, a2. fig. 1. adh bandmorph in leaves. 68 v. preliminary study on isozymes of shorea javanica — jun ia rt i & umboh b. seeds fig. 2 shows 5 anodally-moving bandmorphs. out of 18 seeds, 5 showed bandmorph 1 consisting of 3 bands al , a3 and a4; four showed bandmorph ii consisting of bands a3 and a4; one showed bandmorph iii consisting of band a4; four showed bandmorph iv consisting of bands a2, a4, a5 and a6 and four showed bandmorph v consisting of bands a l , a2, a4 and a5. fig. 2. adh bandmorphs in seeds. c. cotyledons of germinating seeds only one adh bandmorph was found, i.e. an anodally-moving al band. 2. malate dehydrogenase (mdh) a. leaves fig. 3 shows 2 anodally-moving bandmorphs. four trees showed bandmorph i with band al , a3, a4, and a5 while two trees showed bandmorph ii consisting of bands al , a2, a3 and a4. fig. 3. mdh bandmorphs in leaves. 69 biotrop1a vol. 1 no. 1, july-december 1987 b. seeds fig. 4 shows 3 anodally-moving bandmorphs. twelve seeds showed bandmorph i consisting of bands a l , a2, a4 and a5; four seeds showed bandmorph ii consisting of bands a l, a2, a3, a4 and a5; two showed bandmorph iii consisting of bands al, a2 and a5. fig. 4. mdh bandmorphs in seeds. c. cotyledons of germinating seeds variation in bandmorph was also found in cotyledons. three mdh bandmorphs (fig. 5) were found; all have 3 anodally-moving bands, but migration rates differ. bandmorph i consists of bands a3, a5, a7; bandmorph ii consists of bands a2, a4, a6 and bandmorph iii consists of bands al, a3 and a5. fig. 5. mdh bandmorphs in cotyledons of germinating seeds. 3. peroxidase (pox) a. leaves three bandmorphs were detected in the leaves (fig. 6). these three have 4 similar cathodally-moving bands cl, c2, c3 and c4. bandmorph i consists of 6 anodally-moving-bands a l, a2, a3, a4, a5 and a6. bandmorph ii consists of 5 anodally-moving bands a l, a2, a4, a5 and a6. bandmorph iii consists of 4 anodally-moving bands al, a2, a4 and a6. 70 v. preliminary study on isozymes of shorea javanica — ju n ia rt i & umboh fig. 6. pox bandmorphs in leaves. b. seeds eight different bandmorphs were detected in the seeds (fig. 7), each one consisting of anodally and cathodally-moving bands. the range in total number of bands detected is 4-12. fig. 7. pox bandmorphs in seeds. 71 b1otropia vol. 1 no. 1, july-december 1987 c. cotyledons of germinating seeds eight pox bandmorphs were found; each one consisting both of anodally-moving and cathodally-moving bands (fig. 8). fig. 8. pox bandmorphs in cotyledons of germinating seeds. 4. super oxide dismutase (sod) a. leaves fig. 9 shows 2 sod bandmorphs i with anodally-migrating al and a2 bands and bandmorph ii with anodally-migrating al and a3 bands. four trees showed bandmoprh i and two trees showed bandmorph ii. fig. 9. sod bandmorphs in leaves. 72 v. preliminary study on isozymes of shorea javanica — juniarti & umboh b. seeds fig. 10 shows 4 bandmorphs. eight seeds showed 4 anodally-migrating bands al, a2, a3 and a4; two seeds showed 1 anodally-moving band al; four seeds showed 4 anodally-moving bands a2, a3, a4 and a5 bands and 4 seeds showed one anodally-moving a2 band. fig. 10. sod bandmorphs in seeds. following the method of starch gel electrophoresis, isozymes of 4 enzymes were detected as bands. the variation found in each of the enzymes assayed in leaves as well as among the seeds seems to be an indication of the diversity of this species in the plantations. however, considering the very limited sampling done from the plantation, the extent of this diversity could not be ascertained. knowing the number of loci involved and the genetics of the observed differences will be prerequisites in understanding the genetic diversity. the mother tree of the seeds that were analyzed were inadvertently not recorded for its isozyme bandmorphs thus the variation in bandmorphs that was exhibited by the seeds could not be analyzed in relation with the bandmorphs of the mother tree. otherwise, an insight into the breeding system of the tree could have been obtained. analysis of the isozymes in cotyledons of germinating seeds could possibly show differential gene activity at different developmental stages. suggestions for future research a sufficient number of trees must be sampled for isozyme detection from leaf extracts. to explain the genetic basis of the locus and its variation will require evidence from crosses between known bandmorphs. since this is difficult to do with this large dipterocarp tree, a progeny test could suffice. this would involve collection of seeds per mother tree as basis of known bandmorph or genotype for the 6 enzymes and raising seedlings. analysis of the enzyme pattern in the seedling leaves will have to be done. 73 biotropia vol. 1 no. 1, july-december 1987 sampling in a few plantation sites will be needed for the comparison of the genetic diversity among these sites. the analysis of the isozyme variation will provide data for quantifying the extent of the genetic variation in these sites. a possible correlation could be searched between presence/absence of certain isozyme bands or whole genotypes and soil/habitat characteristics. it would be interesting to compare the frequency of certain isozyme alleles or whole genotypes among the sites. this may give a clue to the adaptive significance of some isozyme as found in some crop plants. when linkages are found between specific isozyme alleles and genes for economically important characters, e.g. growth, yield, pest resistance, susceptibility for mycorrhizal association etc., these isozyme markers could be used for selection of planting materials to establish plantations. note the research described in this paper was originally carried out by mrs. u. juniarti and dr. m.i.j. umboh; the paper was written by mrs. l.u. gadrinab, whose assistance is gratefully acknowledged (editor). references ardikoesoemo, r.i. 1954. tanaman shorea javanica di djawa. rimba indonesia 3-4 (1954), hal. 141-151. beauchamp, c. and i. fridovich. 1971. superoxide dismutase: improved assays and assay applicable to acrylamide gels. anal. biochem. 44: 276. endo, t., b.b. shahi and c. pai. 1971. genetic convergence of the specific acid phosphatase zymograms in oryza saliva. jpn. j. genet. 46: 147. ______, 1978. a new method for peroxidase isozyme stain. ann. rep. nat. inst. genet. no. 28: 41. gadrinab, l. 1984. a biosystematic study on section pachycarpae in dipterocarpaceae: shorea macrophylla ashton and shorea stenoptera burck. biotrop internal report. gan, y. and f. robertson. 1981. isozyme variation in some rain forest trees. biotropica 13 (1): 20-28. ihara, m. 1985. a report on forest genetic studies. biotrop internal report. schwartz, d. and t. endo. 1966. the genetic control of alcohol dehydrogenase in maize-simple and compound loci. genetic 53: 709. shaw, c.r. and e. prasad. 1970. starch gel electrophoresis of enzymes. a compilation of recipes. biochem. genet. 4: 297. smithies, o. 1955. zone electrophoresis in starch gels. biochem. j. 61: 629. torquebiau, e. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 74 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf 796 siti meliah (isolation) revisi.cdr isolation, characterization and molecular identification of myxobacteria from two outermost islands of indonesia 1* 2 siti meliah and puspita lisdiyanti 1 (lipi) science center, bogor research center for biology, indonesian institute of sciences , cibinong 16911, indonesia 2 (lipi) science center,research center for biotechnology, indonesian institute of sciences , cibinong bogor 16911, indonesia received 30 december 2016 / accepted 03 november 2017 abstract myxobacteria are gram negative bacteria commonly found in soil, tree bark, and decay wood. these bacteria have unique social behaviors by forming fruiting bodies, moving by gliding motility and preying on other microorganisms. the research was conducted to isolate, characterize, and identify indigenous myxobacteria from sumba and papua islands of indonesia as a preliminary step to utilize their potential in the pharmaceutical industry. myxobacteria were isolated using filter paper and baiting with escherichia coli to obtain cellulolytic and bacteriolytic myxobacteria, respectively. characterization of myxobacteria was performed with gram staining, observation on pigmentation, morphology of vegetative cells, fruiting bodies, and myxospores. molecular identification was conducted based on 16s rrna gene sequence analysis. a total of 10 myxobacterial strains were successfully isolated and purified. all isolates obtained were gram negative, rod shaped with yellow or orange pigmentation. fruiting bodies observed contained spherical myxospores. molecular identification of these bacterial strains showed that they belong to myxobacteria from suborder cystobacterineae, namely myxococcus fulvus, myxococcus stipitatus, and melittangium lichenicola. to our knowledge, this is the first record of their occurrence in indonesia. keywords: characterization, identification, isolation, myxobacteria introduction in 1809, a german botanist heinrich friederich link reported his observation on myxobacterial fruiting bodies of polyangium vitellinum and described them as “gasteromycete”, member of fungi basidiomycota (link 1809). two more species, stigmatella aurantiaca and chondromyces crocatus, were described 48 years later (berkeley 1857). they were classified as hyphomycetes, a fungi imperfecti. they continued to be mistaken for fungi until a united state botanist roland thaxter introduced them as myxobacteria in 1892 (thaxter 1892). it took about 20 years before thaxter's work became widely accepted. until 2006, there were approximately 50 species of recognized myxobacteria which were grouped into 3 suborders, 6 families, and 17 genera (shimkets et al. 2006). myxobacteria are gram negative bacteria predominantly found in terrestrial habitats, such as soil, decaying plant materials, and bark of living or dead trees. however, some reports revealed that myxobacteria can also be isolated from marine samples (zhang et al. 2005; brinkhoff et al. 2012). among the reported myxobacteria collected, those from marine samples were novel, including haliangium ochraceum, h. tepidum (fudou et al. 2002), enhygromyxa salina (iizuka et al. 2003a), plesiocystis pacifica (iizuka et al. 2003b) and pseudenhygromyxa salsuginis (iizuka et al. 2013). myxobacteria in general are characterized by their ability to form fruiting bodies and their gliding motility on solid surface. the fruiting bodies are formed after exhaustion of the food supply and contain dry resistant myxospores. colonies can spread into an unoccupied area. this spreading * corresponding author: siti.meliah@lipi.go.id biotropia 5 2 8 121 129 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.796 121 movement is called swarming behavior. the shape, size, color, or arrangement of vegetative cells, swarms, fruiting bodies, and myxospores are important in determining genus of myxobacteria. in contrast to most bacteria, myxobacteria are also capable of lysing living cells. for this reason, they are called predators. based on the specialization of myxobacteria in degrading biomacromolecules, they are divided into bacteriolytic myxobacteria that lyse whole cells of other microorganisms and cellulolytic myxobacteria that efficiently decompose cellulose instead of living cells (singh 1947; hou et al. 2006). myxobacteria are known for their enormous potential in producing secondary metabolites with various biological activities. for certain reasons, their utilization as potential secondary metabolite producers is limited. they are relatively difficult to isolate and purify. in addition, only some of their species are easily grown in liquid culture during fermentation. thus, they are often overlooked by industrial sectors. however, due to the emerging of multiple drugs resistance in pathogenic microorganisms, the need to find new alternatives of potential bioactive compounds from natural resources, including myxobacteria is inevitable. myxosporin, myxovalargin, and myxothiazol are some of the secondary metabolites produced by myxobacteria with antimicrobial activity. these compounds are extracted from typical bacteriolytic myxobacteria, myxococcus fulvus (dawid 2000). the genus myxococcus is also reported to produce myxovirescin (williams & mcgill 1990) and myxalamides (gerth et al. 1983; konovalova et al. 2010). thuggacin antibiotics active against mycobacterium tuberculosis; the causative agent of tuberculosis, were reported to be chondromyces crocatus (buntin et al. 2010). another secondary metabolite, epothilone that acts upon cancer cells was extracted from cellulolytic myxobacteria, sorangium cellulosum (gerth et al. 1996). the myxobacteria were isolated from various samples and places, such as seawater and sediment samples from shandong province in china (li et al. 2002); marine sediment from santa barbara in us, texel in netherlands, and bokum in germany (schäberle et al. 2010); and also soil samples from yunnan, qinghai, hebei and yuenan in china (zhang et al. 2003); india (singh & singh 1971); and kiritimati island, republic of kiribati (mohr et al. 2016). there is no sufficient record of this bacterial group that has been isolated in indonesia despite their medical importance, except a report on a comparative analysis of predation of myxococcus xanthus isolated from sulawesi in 2010 (morgan et al. 2010). however, studies on diversity and composition of prokaryotic communities in sumatera using culture independent methods revealed that fungi-like myxobacteria (sorangium and haliangium) were detected in the soils samples and were slightly more abundant in the managed soils than in rainforest soils (schneider et al. 2015). sumba and papua islands are some of the outermost islands of indonesia. the geography and ecology of these islands are different from each other. however, like most indonesian areas, they are assumed to be a biodiversity rich habitat, particularly for microbes. therefore, this research is conducted to isolate, characterize, and identify indigenous myxobacteria in order to record their occurrence in indonesia and is a preliminary step to exploit their potential as a natural producer of anti-infective. materials and methods materials and sampling methods materials used in this research are listed in table 1. these samples were collected from two different locations of indonesia in april 2016. soils were sampled using composite method. soil and limestone samples were air dried overnight to reduce the growth of untargeted microorganisms. in total, the number of samples used in this study was 37. isolation of cellulolytic and bacteriolytic myxobacteria the isolation of cellulolytic and bacteriolytic myxobacteria was conducted using methods described by reichenbach and dworkin (1992) w i t h s o m e m o d i f i c a t i o n s . c e l l u l o l y t i c myxobacteria were isolated by placing a drop of soil sample, limestone sample, and a fragment of decay wood onto a piece of whatman no. 1 filter 2 paper sized 1 cm on stan 21 agar media (a mix of a solution: 1 g k hpo , 0.02 g yeast extract, 10 g 2 4 agar in 700 ml distilled water; and b solution: 1 g kno , 1 g mgso .7h o, 1 g cacl .2h o, 0.2 g 3 4 2 2 2 fecl , 0.1 g mnso .7h o in 300 ml distilled 3 4 2 122 biotropia vol. 25 no. 2, 2018 cyanocobalamin, supplemented with 25 µg/ml cycloheximide using sterilized syringe needle. the transferring process was conducted several times until pure cultures of myxobacteria were obtained. pure cultures were stored in 10% gycerol stock solution supplemented with 0.1% cacl .2h o at -80 c. o 2 2 phenotypic characterization the isolates obtained were morphologically observed with the help of dissecting (olympus sz) and binocular (olympus bx43) microscopes. several characters were observed including fruiting bodies that emerge days after incubation, vegetative cells, and also the pattern and the color of swarm colonies. the fruiting bodies were crushed to examine the myxospores. each of the pure isolates was subjected to gram staining using crystal violet, iodine, and safranin reagents. molecular identification based on 16s rrna gene analysis molecular identification was conducted based on 16s rrna gene analysis. genomic dna was extracted using a set of processes started by rinsing bacterial cells with 500 µl te buffer ph 8.0. after centrifugation at 13,000 rpm for 5 minutes, pellet obtained was resuspended with 50 µl te buffer ph 8.0 and 300 µl extraction buffer consist of tris-hcl, edta, sodium dodecyl sulfate, and nacl. this suspension was homogenized using vortex mixer for 5 minutes. an amount of 150 µl 3m sodium acetate was added to the suspension and incubated for 10 minutes at room temperature. after incubation, the suspension was centrifuged at 13,000 rpm for water). these solutions were autoclaved separately. solution a and b were combined and t h e n s u p p l e m e n t e d w i t h 2 5 µ g / m l cycloheximide. this medium was designated as st21cx. the samples in st21cx media were o incubated at 30 c for 2-4 weeks. bacteriolytic myxobacteria were isolated using baiting technique with escherichia coli. e. coli cells were cultured on luria broth (lb) medium (10 g/l tryptone, 10 g/l nacl, 5 g/l yeast extract suspended in 1l distilled water) for 24 hours. e. coli suspension was then centrifuged for 10 minutes at 10,000 rpm. the bacterial pellet was resuspended with cycloheximide solution 25 µg/ml enough to make a thick slurry of bacterial cells. e. coli cells were then cross striked on a water agar (wcx) medium (1 g/l cacl .2h o, 15 g/l 2 2 a g a r , s u p p l e m e n t e d w i t h 2 5 µ g / m l cycloheximide). in the center of the cross, a peasized amount of soil and limestone sample was inoculated. the samples in wcx media were then o incubated at 30 c for 2-4 weeks. purification of myxobacteria myxobacteria obtained from both methods were transferred using sterilized syringe needle to a fresh wcx medium cross striked with autoclaved e. coli to purify them. dissecting microscope was used to recognize the myxobacteria so that this direct purification technique can be done. the bacteria were then o incubated at 30 c for 1-3 weeks. the fruiting bodies or swarm cells that produce clear zones around the dead were e. coli transferred to a modified vy/2cx medium consisting of 5 g/l baker's yeast fermipan, 1 g/l c a c l . 2 h o, 1 5 g / l a g a r, 0 . 5 µ g / m l 2 2 table 1 sampling locations and materials used to isolate myxobacteria sampling location altitude (masl) ph t ( oc) samples number of samples wanggameti national park , sumba, east nusa tenggara e 120o 15.360’ s 10o 04.696’ – e 120o 16.703’ s 10o 03.496’ 983-1164 7 23-36 soil 13 limestone 3 decay wood 10 tambraw, west papua e 132o 15’ 05.2” s 00o 45’ 0.99” – e 132o 44’ 05.8” s 00o 52’ 0.94” 443-891 6.5 26.5-32 soil 7 decay wood 4 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti 123 another 5 minutes. the supernatant obtained was transferred to a new microtube and gently mixed with isopropanol in the same volume. this mixture was centrifuged for 10 minutes at 13,000 rpm. the pellet obtained was suspended with 70% ethanol and centrifuged for 1 minute at 13,000 rpm. the dna or pellet was air dried and resuspended with 50 µl te buffer ph 8.0. the quality and quantity of genomic dna was examined by biospec-nano micro-volume uvvis spectrophotometer (shimadzu). universal eubacterial primers 27f (5'agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') were used to amplify 16s rrna gene (lane 1991). gene amplification was performed under the following o conditions: pre denaturation at 94 c for 2 minutes, subsequently followed by 35 cycles of o denaturing at 94 c for 15 seconds, annealing at 55 o o c for 30 seconds, elongation at 72 c for 1 o minute, and final extension at 72 c for 10 minutes in mastercycler gradient (eppendorf). pcr products were checked on 1% agarose gel stained with ethidium bromide solution and observed under uv transilluminator. these dna fragments were sequenced by macrogen inc. (south korea) using 27f and 1492r primers in abi 3730xl dna analyzer. the sequences of the isolates obtained were analyzed using bioedit program (hall 1999). the identification of phylogenetic neighbors was initially carried out by the blastn (altschul et al. 1997) program against the database containing type strains with validly published prokaryotic names and representatives of uncultured phylotypes (kim et al. 2012). the top thirty sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using global alignment algorithm, which was implemented at the eztaxon server (http://www.ezbiocloud.net/eztaxon). the 16s rrna gene sequences in this study were submitted to genbank ncbi under these following accession numbers; mg561397 (smdw06.2), mg561394 (smcv05.1), mg561395 (smcv05.2), mg561396 (smcv05.3), mg561388 (ps3.1), mg561389 (ps3.2), mg561390 (ps3.3), mg561398 (sms05.1), mg561399 (sms05.2) and mg561400 (sms05.3). all the identified isolates were also deposited in indonesian culture collection (inacc) using the number smdw06.2 (inacc b1220), smcv05.1 (inacc b1221), smcv05.2 (inacc b1222), smcv05.3 (inacc b1223), ps3.1 (inacc b1224), ps3.2 (inacc b1225), ps3.3 (inacc b1226), sms05.1 (inacc b1227), sms05.2 (inacc b1228) and sms05.3 (inacc b1229). phylogenetic analysis multiple alignment of all the dna sequences were performed by muscle program (edgar 2004). type strains and their sequences were collected from a list of prokaryotic names with standing in nomenclature (lpsn) website ( w w w. b a c t e r i o . n e t ) a n d g e n b a n k (www.ncbi.nlm.niv.gov). kimura 2-parameter model was selected to calculate the distance matrices between sequences (kimura 1980). phylogenetic tree was constructed using neighbor-joining method (saitou & nei 1987) with 1,000 replicates of bootstrap. all these programs are implemented in mega version 6 (tamura et al. 2013). desulfovibrio desulfuricans acc. no. m34113 which belongs to deltaproteobacteria, was used as out-group in constructing the phylogenetic tree. . results and discussion taxonomically, the fruiting gliding myxobacteria belong to the phylum of proteobacteria, subphylum delta-proteobacteria, order myxococalles, and consist of 3 suborders (cystobacterineae, nannocystineae, sorangiineae). they are characterized by rod and yellow, orange, or red pigmented cells. in this study, yellow and orange pigmented swarms and fruiting bodies emerged on whatman no. 1 filter paper on st21cx and on streaked e. coli on wcx agar media, after 2-3 weeks of incubation. visible clear zones also appeared around the streaked e. coli on wcx media after 5-7 days of incubation. it is known that bacteriolytic myxobacteria consume other living bacteria or yeasts as their nutrient source. hence, these clear zones were an indication of predatory activity of bacteriolytic cells. the predatory activity of myxobacteria on other bacteria and yeasts is supported by the production of extracellular lytic enzymes and antibiotics (xiao et al. 2011). this mechanism also plays an important role in competing with their natural opponents in a habitat. 124 biotropia vol. 25 no. 2, 2018 under a dissecting microscope, swarms and fr uiting bodies grown on samples were transferred to a new wcx media with dead e. coli as a nutrient source and subsequently transferred to vy/2cx agar using a sterile syringe needle. this purification method was able to produce a more uniform colony appearance (fig. 1). from a total of 37 samples using st21cx and wcx medium as isolation media, only 10 myxobacterial isolates were successfully recovered and purified. compared to common bacteria, purification of myxobacteria is relatively tricky. they easily carry contaminates, such as fungi, other bacteria, and soil amoeba, because they produce slime to help them move on a solid medium. a number of purification techniques have been developed to increase proportion of pure cultures. improved methods, such as purification with crystal violet and second baiting technique subjected to fruiting body can improve the proportion of pure culture obtained up to 42.5% and 69.7%, respectively (zhang et al. 2003). all the 10 bacterial isolates were gram negative bacteria. they were rod shaped with slight differences in size. morphologically, the isolates obtained vary in color, shape and swarming pattern. these isolates showed conspicuous characteristic of myxobacteria by producing fruiting bodies. round or spherical myxospores were observed under the microscope from crushed fruiting bodies. these characteristics were identical to morphology of myxobacteria described in bergey's manual (reichenbach 2005). based on their morphology, these isolates were grouped into three morphological groups (table 2). morphological identification based on vegetative cells, swarms, fruiting bodies, and myxospores is still valid for some myxobacteria genera. however, these morphological characters are not stable and may change or be lost under artificial growth condition despite expressed by their genotype. hence, identification and classification of myxobacteria remains challenging for most of the genera and species (garcia et al. 2010). this is also partly due to the fact that some recently isolated myxobacteria strains do not usually have typical myxobacterial morphological characteristics. therefore, molecular identification is needed to confirm the identity of the isolates. analysis on 16s rrna gene sequences of 10 isolated bacteria revealed that these isolates were member of myxobacteria group. they belong to the genera and with more myxococcus melittangium than 99% similarities (table 3). and myxococcus melittangium genera were often found in soil samples throughout the world, from tropical rain f o r e s t s, c e n t r a l e u r o p e a n f o r e s t s, t o mediterranean regions and so far, to our a b c figure 1 morphological appearance of purified myxobacterium collected from sumba sms05.1 (a), smdw06.2 (b), and papua ps3.1 (c) on vy/2cx agar medium after incubation for 5-10 days. fruiting bodies appeared as yellow/orange cells aggregate. table 2 isolates grouping based on morphological appearance morphological characteristics of colony myxospores total isolates orange, centrifugal pattern, orange fruiting body spherical 3 yellow swarms, orange fruiting body spherical 4 orange swarms, short stalked orange fruiting body spherical 3 125 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti knowledge this is the first record of the three species occurrence in indonesia. based on mor phological obser vation supported by molecular identification, diversity of myxobacteria obtained from this work was not numerous. some isolates might not form typical fruiting bodies at the time of observation, hence overlooked during transferring process. isolation techniques, incubation time, and media used to isolate myxobacteria still unable to culture various species occur in a habitat in this study. every method would be a compromise, as different myxobacteria species would grow well on one medium, others would grow less well and show little tendency to form fruiting bodies (dawid 2000). hence, the variation on isolation technique, purification technique, and enrichment media are expected to increase the probability to obtain more diverse myxobacteria. phylogenetic analysis of the 10 myxobacteria sequences showed that all the isolates are placed in suborder cystobacterineae (fig. 2). taxonomically, genus myxococcus along with aggregicoccus, corallococcus and pyxidicoccus are classified into the family myxococcaceae in the suborder cystobacterineae. on the other hand, the genus melittangium along with angiococcus, archangium, cystobacter, hyalangium, stigmatella and newly described genus vitiosangium (awal et al. 2 0 1 7 ) a r e c l a s s i f i e d i n t o t h e f a m i l y cystobacteraceae also in the suborder cystobacterineae. table 3 molecular identification of isolated myxobacteria 126 biotropia vol. 25 no. 2, 2018 figure 2 phylogenetic tree constructed on the basis of 16s rrna gene sequences using neighbor-joining method. isolates from sumba (sm) and papua (ps) islands were placed in the suborder cystobacterineae among myxobacteria species. bar means 1 substitution per 200 nucleotides. numerals at branch points indicate the bootstrap value as percentages derived from 1000 replications. only values greater than 60% are shown. 127 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti conclusion a total of 10 myxobacterial isolates was successfully isolated from soil, limestone and decay wood samples collected from two outermost islands of indonesia, sumba and papua. they were isolated using filter paper and baiting with e. coli methods. characterization of sumba and papua isolates revealed that they were gram negative and rod shaped. their colonies were yellow and orange in pigmentation, and produce fruiting bodies. molecular identification showed that they are members of myxobacterial species myxococcus fulvus, myxococcus stipitatus and melittangium lichenicola. to our knowledge, this is the first record of the occurrence of the three species in indonesia. all the isolates were deposited in indonesian culture collection (inacc) under the name myxococcus fulvus inacc b1220, myxococcus fulvus inacc b1221, myxococcus fulvus inacc b1222, myxococcus fulvus inacc b1223, myxococcus stipitatus inacc b1224, myxococcus stipitatus inacc b1225, myxococcus stipitatus inacc b1226, melittangium lichenicola inacc b1227, melittangium lichenicola inacc b1228 and melittangium lichenicola inacc b1229. acknowledgements this research was financially supported by dipa 2016 lipi. we wish to thank maman rahmansyah and everyone involved in ekspedisi widya nusantara (e-win) and nkri expedition during the samples collection. we also thank tri ratna sulistiyani, 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introduction the trees of the dipterocarpaceae family fill a prominent place in the southeast asian tropical forests as most of the species are used as timber trees or source of secondary products (e.g. resin "damar"). their economic value is therefore of high interest to the local population and other countries (symington 1974, ashton 1982). up to the present, the exploitation of these forests consists of extracting timber from natural stands or at a lesser extent, in traditional management of agroforestry systems (torquebiau 1984, michon 1985). the possible fungal infections of these trees have been taken into account as debilitating factor of production. during researches devoted to the dipterccarp pathology, fungal attacks on seeds, seedlings and saplings have been observed frequently. they seem to be able to induce important damages in young stands and nurseries. fungi and their damages were observed in several places in west java and sumatra (figure 1). most of them belong to the imperfect genera fusarium and cylindrocarpon (tuberculariaceae). * this study was conducted as part of a doctoral dissertation research under the supervision of prof. g. durrieu of laboratory of botany and forestry, university of p. sabatier, through the tropical forest biology programme of seameo-biotrop. 25 biotropia no. 3, 1989/1990 figure 1. location of study sites materials and methods the pathogens were isolated from diseased organs (roots, collar, stem, twigs, leaves) and cultivated on potato-dextrose-agar (pda) in petri dishes, at room temperature (26 32°c). inoculation experiments were performed on seedlings to confirm the virulence of the isolated strains. the species identification were based on recent literature (booth 1971, barnett & hunter 1972, renard et al. 1972, nirem-berg 1976) and was checked by dr. m.f. roquebert of museum d' histoire naturelle laboratoire de cryptogamie, paris. results and discussion among the pathogens isolated on dipterocarps were different species of the genera fusarium (i.e. f. sacchari var. sacchari, f. moniliforme and f. oxysporum) and cylindrocarpon (i.e. c. destructans). among the associated fungi which can 26 notes on some fusarium and cylindrocarpon — c. elouard be considered as secondary parasites were pestalotia, cylindrocladium, nigrospora and marasmius. fusarium sacchari (bull.) w, gams. var. sacchari 1. hosts vatica pauciflora (korth.) bl. this species was used to prevent fermentation of palm wine. the parasite was isolated from a sapling of about 70 cm height. shorea seminis (de vriese) sloot. the fruits are used in borneo as source of illipe butter (ashton 1982). the hosts were about 2 years old and measured 20 to 30 cm in height. shorea leprosula miq. this dipterocarp tree is one of the main sources of timber classified as light red meranti (symington 1974). hosts were saplings of about one year old and 10-25 cm'in height. both of these hosts came from the experimental plantation of cikarawang of bogor agricultural university, darmaga, bogor, west java (figure la). 2. symptoms of the disease observed in the field (figures 2 & 3) defoliation and weathering of saplings of vatica pauciflora. on shorea seminis, beige-coloured spots delimited by a brown line, at first at the apex of leaves and later on covering the whole leaf surface, leading to its fall; at the base of the twig, necrosis could be observed; the bark of both collars and roots were necrosed and became dark; some of the plants were dry from root to apex. some shorea leprosula were dry and dead, with darkish bark, cambium of collars and roots. necrosis could be observed at the base of twigs. alive plants have cracked collar's bark and darkish roots; leaves have brown spots, beginning from the extremity and later on covering the whole surface. 3. description of the pathogen (figure 4) this fusarium was isolated from the cambium of the branch of v. pauciflora and from leaves, twigs, collars and roots of s. seminis. the pigmentation of the medium was brown to vinaceous. the mycelium was cream-coloured, pink to violet, aerial, cottony to fibrous. the growth rate was 6.8 cm after 4 days (figure 5). microconidia were initially formed by simple phialides, and further more by branched conidiophores ended by polyphialides. the older the culture, the more 27 biotropia no. 3, 1989/1990 figure 2. spots on leaves of shorea seminis caused by fusarium sacchari var. sacchari figure 3. vatica pauciflora inoculated with fusarium sacchari var. sacchari 28 notes on some fusarium and cylindrocarpon c. elouard figure 4. fusarium sacchari var. sacchari figure 5. growth rate of the fungi 29 biotropia no. 3, 1989/1990 complex is the organization of conidiophores. microconidia were oval to clavate, one-celled, and measured 3 10 x 1-3 um. more often, there was no macroconidia in culture, or only few in number. macroconidia were formed by simple phialides, generally straight, 3 to 6 septate, and measured 6-55 x 1.7-2.4 um. no mycelial and conidial chlamydospores were observed. 4. associated fungi two marasmius species were associated with f. sacchari var. sacchari: m. equicrinis: it developed rhizomorphs like long black hairs, on which there were small carphophores (2-8 mm of diameter) on twigs, branches and leaves of hosts. marasmius sp. developed white himanthium on twigs, branches and leaves. fusarium monitiforme scheld. sensu wollenw. et reinking = f. verticilloides (sacc.) niremberg comb. nov. 1. hosts shorea javanica k. & v. this shorea species has been exploited in agroforestry systems for about 150 years by local population in the province of lampung, south sumatra (figure ic). trees produce a resin ("damar") which is exported to europe and america for the production of varnish, linoleum and paint. the trees were tapped and the resin was collected every month (torquebiau 1984). the parasite was collected on saplings of s. javanica of about 1 m to 1 . 5 m height and one to two years old. 2. symptoms of the disease observed in the field defoliation and weathering of the plant; the bark of twigs and roots were black and the cambium was dry. 3. description of pathogens (figure 6) this fusarium was isolated from twigs and roots. the pigmentation of the medium was brown to violet. the mycelium was cream-coloured, pink to violet, violet-grey; at first, appearing like a small cushion and later on becoming aerial, cottony to fibrous. the growth rate after 4 days was 2.2 cm (figure 5). microconidiophores were simple, lateral to the hyphae and measured 27 — 54 x 1 5 um. microconidia were in chains and measured 5 17 x 2 4 cm; they were 30 a. microconidiophores and microconidia b. macroconidia figure 6. fusarium moniliforme one-celled, clavate to oval, sometimes with the basal cell lightly flattened. macroconidiophores were lateral branches of hyphae. macroconidia were curved, with a flat basal cell and sometimes a sharp apical cell. they were 3-8 septate when mature and measured 40-72 x 4-5 um and seldom produced in culture. no mycelial and conidial chlamydospores were observed. 4. associated fungi two parasites were found associated with fusarium moniliforme: cylindro cladium sp. from collar and pestalotia sp. from leaves with symptoms of beige spots. these two fungi were secondary parasites. fusarium oxysporum schlecht. 1. hosts shorea pinanga scheff. the fruits of s. pinanga were used as illipe-nuts. the pathogen was collected 31 notes on some fusarium and cylindrocarpon-c. elouard biotropia no. 3, 1989/1990 on saplings of about one to three years old, in population under an adult tree of s. pinanga at the arboretum of haurbentes, jasinga, west java (figure 1b). shorea javanica k. & v. this species could also be a host for f. oxysporum. it was isolated from saplings of 60 cm height at krui (figure 1c). 2. symptoms observed in the field roots, barks of collars and twigs of s. pinanga became darkish; some of the saplings were dry and dead, while others had root rot. no symptoms were observed on s. javanica. 3. description of the parasite (figure 7) this fusarium was isolated from twigs, collar and roots of saplings of s. figure 7. fusarium oxysporum 32 notes on some fusarium and cylindrocarpon — c. elouard pinanga and from twig of 5. javanica. the pigmentation of the medium was brown to violet. the mycelium was cream-coloured, violet, like a small cushion at the beginning, aerial and cottony when older. the growth rate after 4 days was 5.2 cm (figure 3). microconidiophores were simple phialides, lateral to hyphae, measuring 7-9 x 1-2 um, or branched conidiophores, until 30 um long. microconidia were variable in form, oval to ellipsoid and sometimes allantoid; they were one-celled and measured 4.7-14.4 x 1.5-4.7 um. macroconidiophores were lateral to hyphae, measuring 10—12x1 — 1.5 um. macroconidia were 3 to 5 septate, most frequently 3 septate. they were straight to lightly curved generally sharped at both ends. some macroconidia had a pedi-form basal cell and a hooked and curved apical cell. they measured 16 44( 80) x 24.5 um. chlamydospores were formed by mycelium or conidia. the mycelial chlamy-dospores were simple, of 7.7 8.9 x 6.5 8.3 um, double, of 10.6 x 6.5 um, both smooth and rough walled. fusarium sp.l 1. hosts shorea pinanga scheff. this fusarium sp. was collected on saplings of about 2 years old and 40 cm to 1 m in height in the nursery of biotrop, bogor; seeds were previously collected in the arboretum of haurbentes (figures la and b). 2. symptoms observed in the field red-brown spots developed on leaves with a grey-coloured powder on the upper surface; spots appeared at the apex of leaves and were delimited by a red line; more often, young leaves were necrosed first and then spots came into sight. development of spots on leaves led to necrosis and death of cells, and later on leaves fell. on twigs, cankers appeared and bark became darkish. on collars and roots, the pathogen caused dry rot, and collar's bark became darkish. the propagation was not aerial but by the vascular system. healthy leaves were wrapped with pieces of cellophane to avoid possible aerial contamination. spots appeared on these wrapped leaves as well as on the unwrapped ones. the proga-gation then seems to be internal, by vessels. 33 biotropia no. 3, 1989/1990 3. description of the pathogen (figure 8) this fusarium was isolated from leaves, twigs, collars and roots of s. pinanga. no pigmentation of the medium was observed. the mycelium was cream-coloured to violet, like a small cushion. the growth rate was 5 cm after 4 days (figure 5). microconidia were initially formed by long conidiophores of 45 62 x 1.2-1.8 um. microconidia were oval, cylindrical, allantoid, one-celled, and measured 1.8-17 x 1.2-3.7 um. macroconidia were formed by simple phialides or simple conidiophores, 7 -59 x 1.2-1.5 um. straight to curved, fusiform, they were 2 to 6 septate, and measured 14-41 x 1.8-5.4 um. chlamydospores were simple, 8.9x6.5 um, double, 12.4 x 7.1 um intercalary in mycelium or at the apex of hyphae. figure 8. fusarium sp.1 34 note on some fusarium and cylindrocarpon — c. elouard 4. associated fungi a secondary parasite, cylindrocladium sp., was cultured from collar with this fusarium sp. fusarium sp.2 1. host shorea javanica k. & v. this other fusarium sp. was collected on saplings of 25 cm height, at krui (figure 1c). 2. symptoms observed in the field defoliation and weathering of the plant was noted with bark darkish in appearance. 3. description of the pathogen (figure 9) it was isolated from roots and twigs. the pigmentation of the medium was pink, violet to vinaceous. the mycelium was violet, vinaceous, cottony and aerial. the growth rate after 4 days was 4.2 cm (figure 5). microconidiophores were simple phialides or short conidiophores, 21.8-22.4 x 1.6 — 3.2 um. microconidia were oval, allantoid to oblong. one-celled, they measured 2.6-16.7 x 0.9-3.7 um. macroconidia were straight to lightly curved, 2 to 7 septate, measured 10-57.6 x 2-3.5 um, and were produced by short conidiophores. chlamydospores were intercalary or terminal to hyphae, simple, of 8.3 x 7.7-9.4 um, double, of 10-14.4 x 6.9 um. 4. associated fungi a secondary parasite, cylindrocladium sp., was cultured from roots. cylindrocarpon destructans (zinssm.) scholten, anamorph of nectrica radicicola 1. hosts shorea pinanga scheff. and hopea mengerawan miq. h. mengerawan was a commercial timber tree of second class value. seeds of 5. pinanga were collected in the botanical garden (kebun raya) of bogor, west java. 35 a. microconidiophore and microconidia b. macroconidia c. chlamydospores (1) smooth wall, (2) rough wall figure 9. fusarium sp.2 saplings of h. mengerawan were 2 years old and were collected at the experimental plantation of benakat, near palembang in south sumatra (figure id). 2. symptoms observed in the field (figures 10 & 11) necrosis and weathering of the seeds of 5. pinanga were observed. the saplings of h. mengerawan had brown spots on leaves delimited by dark lines. the spots first appeared at the apex of the leaf but they covered little by little the entire surface causing the leaf to fall. thus, photosynthesis activity decreased or stopped leading to the weathering of the host. 3. description of the parasite (figure 12) c. destructans was isolated from cotyledons of s. pinanga and was responsible for damping-off. it was also cultured from roots and twigs of sapling of h. mengerawan. the pigmentation of the medium was brown to vinaceous. the mycelium was cream-coloured, violet to violet-grey, layer on medium in small cushion or powdery. the growth rate after 4 days was more than 4 cm (figure 3). 36 biotropia no. 3, 1989/1990 notes on some fusarium and cylindrocarpon -c. elouard figure 10. spots on leaves of hopea mengerawan caused by cylindrocarpon destructans. figure 11. hopea mengerawan inoculated with cylindrocarpon destructans. 37 biotropia no. 3, 1989/1990 figure 12. cylindrocarpon destructans microconidiophores were simple phialides, lateral to hyphae, 8 17 x 1.5 um, or branched conidiophores, 29 83 x 1.7 2.7 um. microconidia were oval, allantoid to oblong, with sometimes a sharp end. one-celled, they measured 5.4-21.2 x 1.8-4.8 um. macroconidiophores were phialides in groups, 13-17x2.4-3 um, or simple conidiophores lateral to hyphae, 27 x 2 um. macroconidia were thick-set straight to lightly curved, with sometimes curved ends. most of them were 3 septate and measured 18.5-33.3 x 3.5-5.2 um. chlamydospores were both conidial and mycelial; abundant, intercalary or terminal to hyphae; simple, 7-11 x 5 9 um, double 12-16 x 6.9 um, triple 21 x 7.7 um, smooth and rough walled. 4. associated fungi cylindrocladium sp. was associated with c. destructans on seeds of s. pinanga and on saplings of h. mengerawan. it was isolated from roots of h. mengerawan and can be considered as a secondary parasite. on the other hand, cylindrocladium sp. was responsible for damping-off of s. pinanga. thus, cylindrocladium sp. seems to be easily associated with c. destructans. 38 notes on some fusarium and cylindrocarpon-c. elouard conclusion these few observations showed that fusarium and cylindrocarpon were not infrequent as disease agents of young dipterocarps and their attacks very often end in death of the infected plants. if the prevalence of the fungi in natural stands is probably of less importance because of the dispersion of saplings, it may not be the same in nurseries and planted stands. the fungal attacks may become harmful on account of the heavy density of susceptible seedlings and the disease could develop at an epidemic proportion. thus, it is necessary to have better knowledge of the biology and the ecology of these pathogens, to help prevent their damages in artificial forest stands. acknowledgments this workisapartof aresearch programme supported by agrant from the french "ministere des affaires etrangeres". i want to express all my thanks to the director, prof. dr. h. sitti soetarmi tjitrosomo, the deputy director, dr. r. umaly and to the staff of biotrop, particularly to dr. okky s. dharmaputra, for the help they provided me, and to dr. m.f. roquebert, laboratoire de cryptogamie, museum d'histoire naturelle, paris, for her invaluable assistance in the identification of fusarium and cylindrocarpon specimens. references ashton, p.s. 1982. dipterocarpaceae. flora malesiana, series i, spermatophyta, flowering plants, vol. 9, part 2. p. 552. barnett, h.l. and b.b. hunter 1972. illustrated genera of imperfect fungi. burgers publishing company, third edition, 241 p. booth, c. 1971. the genus fusarium. commonwealth mycological institute, ferry lane, kew, surrey, england. 237 p. michon, g. 1985. de i'homme de la foret au paysan de 1'arbre; agroforesteries indonsiennes. these de doctorat, universit des sciences et techniques du languedoc, montpellier. 273 p. nirenberg, h. 1976. untersuchungen liber die morphologishe und biologishe differenzierung in der fusarium section liseola. mitteilungen aus der biologishen bunderanstalt fur land und forstwirt-schaft. berlin. dahlem. 117 p. 39 biotropia no. 3, 1989/1990 renard, j.l., j.p. gascon et a. bachy. dec. 1972. recherches sur la fusariose du palmier a huile-research on vascular wilt disease of the oil palm. oleagineux, 27° annee, no. 12, pp. 581 591. symington, c.f. 1974. foresters' manual of dipterocarps. malayan forest record no. 16. university malaya, kuala lumpur, malaysia. 114 text-fig., 114 planches, 244 p. torquebiau, e. 1984. man-made dipterocarp forest in sumatra agroforestry systems 2(2), pp. 103-128. 40 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 724 siti sofiah (flora diversity) revisi.cdr flora diversity, composition and ecology in besiq bermai tropical forest of damai district, east kalimantan * siti sofiah , destario metusala, trimanto and siti nurfadilah purwodadi botanic garden indonesian institute of sciences pasuruan , , (lipi), 67163 indonesia received 17 november 2016 / accepted 16 november 2017 abstract besiq bermai forest is part of kalimantan forests known for vast plant diversity. the present study aimed to investigate flora diversity, composition, and ecology in besiq bermai forest to support the management of biodiversity and forest conservation. thirteen plots were established with different sizes of plots (100 m x 20 m plots for trees; 40 m x 5 m plots for saplings; and 5 m x 5 m plots for understory). data recorded included plant species name and individual number of each plant species. data analysed were shannon-wiener diversity index, relative density, relative frequency, relative dominance and important value index. the principal component analysis (pca) was performed to determine relationship between edaphic components and flora occurrence. the results showed that there were 93 species of trees (belonging to 48 genera and 22 families), 112 species of saplings (belonging to 62 genera and 43 families), and 48 species of understory (belonging to 28 genera and 20 families). shannon-wiener diversity index (h') were 6.05, 6.25 and 3.26 for tree, saplings and understory, respectively. the most common family for tree and saplings in the forest ecosystem in this area was dipterocarpaceae (shorea spp). species of tree with the highest importance value index were dillenia excelsa, syzygium sp. and shorea parvifolia. the highest importance value index for species of saplings were macaranga triloba and shorea parvifolia; and for species of understory were phrynium jagorianum. ecological (edaphic) factors affecting the occurence and establishment of flora in bermai forest were total n and c/n ratio. the present study has implication for the management of biodiversity and forest conservation. keywords: composition, ecology, flora diversity, kalimantan, tropical forest introduction tropical forest is one of the hotspots-regions of biodiversity, with high plant diversity, complex ecosystems, and greatest variety of flora and fauna (richards 1952; whitmore 1988; de bruyn et al. 2014). borneo is recognised as one of the biodiversity hotspots regions, along with 24 other hotspot regions in the world (marchese 2015). east kalimantan, located in the eastern part of borneo, possess thousands of potential plants in the flora diversity of indonesia. tropical forest along indonesia-malaysia (indo-malay region) is known for its largest tropical forest ecosystem in the world (gaither & rocha 2013). inventory to investigate plant diversity and floristic composition in natural forests is important to the level of adaptation to the environment and their ecological significance (reddy . 2011), and is absolutely essential et al understanding the forest ecosystem dynamics (reddy . 2008). it plays an important role in the et al management of biodiversity as essential bioresources and in the management of conservation of species and forest ecosystem (malouin . 2015; lukác . 2013; meng et al z et al et al et al. 2011; nurfadilah 2015; you . 2016; huang . 2016). plant diversity and floristic et al composition in several tropical forests addressed for the management of biodiversity and ecosystem conservation have been widely studied accross the world, in costa rica (gilman . et al 2016), in china (meng . 2011), in ghana et al (addo-fordjour . 2009), in ethiopia (kebede et al et al et al. 2016), in tanzania (giliba . 2011), in bangladesh (feroz 2016).et al. despite its great diversity, tropical forests have experienced the highest diversity loss. tropical * corresponding author: sofie2291@yahoo.com biotropia 5 2 8 85 94 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.724 85 forests in indonesia, especially in kalimantan, is amongst experiencing higher diversity loss those (bruhl & eltz 2010; margono 2014; hansen et al. et al. 2009). approximately 57 % of low land rain forests in kalimantan conservation area (hutan lindung) has disappeared 1885-2001 between ( et al. ; stibig et al. 2007).gaveau 2013 exploitation of tropical forests, especially in kalimantan, has occured for a long period (gaveau 2016). et al. generally, the loss of forests in kalimantan has been caused by the land use change into agricultural areas (busch 201 ), & ferretti-gallon 7 plantation of oil palms, and mining. restoration should be conducted for the recovery of forest ecosystems. restoration is vital for recovery of tropical deforestation and degraded land, and for conservation of biodiversity (ashton 2014; et al. kettle 2012). in the management of restoration, biodiversity and forest conservation, analysis of the flora diversity and composition, and combined understanding of ecolog y and environmental factors that influence the diversity in forests is important. the present study aimed to investigate the flora diversity, composition and ecology in besiq bermai forest damai district of in east kalimantan. the study site was coal mining area and targeted for reforestation restoration after mining (post-mining). the results of the present study on the flora diversity, composition, and ecology of forests is important for the rehabilitation of coal mining program areas. 86 biotropia vol. 25 no. 2, 2018 figure 1 a map of study site area in besiq bermai forest, east kalimantan, indonesia materials and methods the study was conducted in low land rain tropical forests of village besiq bermai of damai 0 district in east kalimantan, gps coordinate 00 0 51'.02.6”s, 115 26'30.7”e (fig. 1). this forest area was a primary forest that started undergoing degradation and becoming a secondary forest. in the present study, nested plots were established, with stratified random sampling method. thirteen plots were made with different sizes of plots, out of the total forest area of about 2000 ha; 100 m x 20 m plots for trees, 40 m x 5 m plots for saplings, and 5 m x 5m plots for understory. category of trees and saplings were determined by the size of diameter at breast height (dbh) of woody plants; tree (dbh > 30 cm) and saplings (dbh between 5 cm 30 cm). understory are groundcover plants growing on the forest floor which are typically herbs. all plant species within the plots were recorded. to investigate the floristic composition, some parameters including shannon-wiener diversity index, important value index, density (d), relative density (rd), frequency (f), relative frequency (rf), dominance (c) and relative dominance (rc) for each species, were analysed. number of individuals of a taxon x 100rd = total number of individuals number of plots containing a taxon x 100rf = total frequencies of all taxa and the establishment of flora in their habitats, the principal component analysis (pca) method with minitab 16 software was used. results and discussion flora diversity the study showed that the flora diversity in besiq bermai forest, east kalimantan was relatively high. an index of plant diversity of greater than 3 (>3) is regarded as high-value area (khan et al. 2012). plant diversity index indicating the degree of diversity of plants at the study site in the study was high, with the shannon-wiener diversity index (h') for trees, shrubs and understory in this area being 6.05, 6.25 and 3.26, respectively (fig. 2). comparatively, the diversity index at the study site was higher than that of other parts of kalimantan or borneo, such as in lubuk kakap, west kalimantan with h' for tree being 3.54 (budiharta 2010) and in a lowland dipterocarp forest of north borneo with h' for tree was less than 3.5 (godoong et al. 2014). the difference in the degree of diversity at the study site which is located in east kalimantan and other parts of kalimantan/borneo might be due to the difference in regions within borneo. guhardja et al. (2012) reported that the diversity varied within kalimantan/borneo, with one of the highest diversity found in east kalimantan. furthermore, kurniawan and parikesit (2008) reported that plant diversity and composition in an area depends on some factors including altitude, humidity, nutrient availability, illumination, topography and soil types. basal area of a taxon x 100rc = total basal area of taxa h'= -∑ pi ln pi ; pi= ni/ ni = number of individual from species-i n = total number of individual to analyse edaphic factors that influence the growth and the establishment of flora at the study site, soil samples were collected. soils were collected in each plot for 3 replicates. the collection of soils for soil physical and chemical profiles were collected from the depth of 0 cm 30 cm, and 30 cm 60 cm and 3 replicates for each plot. the soil physicochemical properties were assessed in the soil science laboratory of agriculture research institute of mulawarman university, samarinda and were analysed through the drying stage temperature of 105°c. soil physical factors analysed included soil texture (sand, silt and clay), while chemistry factor s included soil acidity (ph), organic c, c/n ratio, organic matter (om) and n-total. soil texture analysis was conducted by separating sand, silt and clay particles by quantitative method through mechanical analysis process. this process consists of spreading aggregate soil into single grains, followed by sedimentation. soil acidity (ph) was measured in soil and water mixture extracts with a ratio of 1: 5, c content was analysed by walkley and black (1934) method, while total n was determined by kjeldahl (188 ) method. to 3 investigate ecological factors focussing on edaphic variables that influenced the occurence 87 flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. figure 2 shannon – wiener diversity index (h') of tree, sapling and herb in forest of besiq bermai trees 6.05 7 6 5 4 3 2 1 0 6.247 3.264 saplings habitus s h a n n o n -w ie n er d iv er si ty i n d ex ( h ’) herbs floristic composition of trees this study showed that, there were 93 species of trees from 48 genera and 22 families in besiq bermai forest. some of the most important species for tree included dillenia excelsa, syzygium sp. and (fig. 3). , shorea parvifolia dillenia excelsa was the most important species in terms of basal area based on rc, however, it had the lowest number of plants. sp. was the second syzygium most important species in terms of species frequency, which was often found in plots at the study site. ranked number shorea parvifolia three in ten most important species, it had the largest number of plants (fig. 3). genus of shorea dominated the study site with nine species, and three of the species were in the list of ten most important species in terms of their abundance. the present study also demonstrated that the most important plant family for trees was dipterocarpaceae, followed by lauraceae and myrtaceae (fig. 4). dipterocarpaceae also had the largest number of species (17 species) including the genera shorea, hopea and dipterocarpus; followed by lauraceae with 12 species (i.e genera of eusideroxylon, litsea and actinodaphne), burseraceae with seven species (i.e. genera of canarium, dacryodes and santiria), leguminosae with seven species (i.e. genera of sindora, koompassia and dialium), and myrtaceae with six species in a single genus of syzygium. 88 figure 4 ten most important plant families in the category of trees in besiq bermai forest. family important value is the sum of important value index of all species contained in a single family. biotropia vol. 25 no. 2, 2018 figure 3 ten most important tree species in besiq bermai forest (note: rd = relative density; rf = relative frequency; rc = relative dominance; ivi=important value index) the results of this study was similar with other studies that showed that the most important family in other forests within borneo/kalimantan was dipterocarpaceae. kartawinata (2008) et al. reported that a family with the highest important value index in the lowland dipterocarp forest at wanariset saboja, east kalimantan, was dipterocarpaceae. arbainsyah (2014) also et al. reported that the most abundant seedling of the species in the primary forest in labanan east kalimantan was dipterocarpaceae. budiharta (2010) also showed that dipterocarpaceae dominated the biodiversity protection area of lubuk kakap, west kalimantan. saner (2012) et al. also reported that tropical lowland dipterocarp rain forests in sabah, malaysian borneo, was also dominated by dipterocarp species. the dominance of dipterocarpaceae in the forests might be in part due to its mast fruiting, the synchronous production of fruits in multi-years cycle at intervals of several years. this irregular fruiting may enhance pollination efficiency, seed survival, and seedling establishment (visser et al . 2011; rodriguez et al. 2013; oshima et al 2015). . the predator satiation hypothesis stated that starvation of seed predators in time of no production of fruits decreases seed predation and therefore, increase tree regeneration (visser et al . 2011). floristic omposition of saplingsc revealed the present study also the floristic composition of saplings. the most important species for saplings was in terms macaranga triloba of basal area, followed by . shorea parvifolia syzygium . xylopia malayana sp and (fig. 5). the pattern of the most important species for trees 89 figure 5 ten most important species for sapling in besiq bermai forest (note: rd=relative density; rf=relative frequency; rc=relative dominance; ivi=important value index) figure 6 ten most important plant families in besiq bermai forest in the category of saplings. family important value is the sum of important value index of all species contained in a single family. flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. 90 and saplings in study was different. budiharta this (2010) also showed variation of pattern of the flora composition between tree and saplings in west kalimantan that might be due to the difference in mast flowering and fruiting frequencies between species which influence the survival of seedlings. the most important family for saplings was dipterocarpaceae, followed by euphorbiaceae and annonaceae (fig. 6). dipterocarpaceae also had the highest number of species (10 species) including the genera shorea and hopea; followed by euphorbiaceae with 9 species (i.e genera of xylopia and monocarpia). floristic omposition of understoryc the results of the study also showed the flora composition for understory. the most important species for understory was calathea sp., followed by pandanus sp., and alpinia sp. (fig. 7). analysis of compositional diversity of understory flora in the study showed that the understory flora was dominated by calathea sp. that belongs to the plant family marantaceae (fig. 7). the dominance of calathea in the shaded forest canopy with low light intensity in the forest floor in this study may be related to its low tolerance of light intensity and its capacity to grow in shaded areas with low figure 7 ten most important species for understory (note: rd=relative density; rf=relative frequency; ivi=important value index) biotropia vol. 25 no. 2, 2018 figure 8 ten most important families for understory bl ec hn um o ri en ta le 91 light intensity. matlaga (2008) reported that clonal offspring of calathea grew and survived well in shaded understory. the most important family for understory in besiq bermai forest was marantaceae, followed by zingiberaceae and pandanaceae (fig. 8). in terms of the number of species, marantaceae and zingiberaceae were high with six (6) species from the genera calathea, phrynium and stachyphrynium. zingiberaceae at the study site consisted of 6 species from the genera zingiber, amomum and alpinia. ecology of flora in besiq bermai forest the establishment of flora in their habitats showed the adaptation of the flora to their environment. the diversity and distribution of many plant species are influenced by the ecological factors including edaphic factors (heineman et al. 2015; vleminckx et al. 2015). through (pca),principal component analysis environmental (soil) factors consisting of five soil variables (ph, organic c, total n, organic matter, and c/n ratio) and environment variables (temperature, humidity, light intensity and elevation) could be classified into two principal components. the two components principal explain 68.8% of all edaphic factors measured. s the first component explained 35.4% of all edaphic factors measured, while the second component only explained 33.4%. this showed that the first component provided more information than the second component in describing and identifying environmental conditions in besiq bermai forest in which many plant species occured. c/n ratio of soils was the most influential variable towards the factors in the first component (pc1), while total n was the most influential variable towards the factors in the second component (pc2) (table 1). the model of the habitat of flora in besiq bermai forest with its edaphic factors can be formulated based on table 1. pc1 = 0.27 ph + 0.35 oc + 0.17 tn+ 0.36 om + 0.37 c/n r; pc2 = -0.35 ph + 0.26 oc+0.46 tn + 0.27 om -0.10 c/n r. each soil variable in pc 1 and in pc2 was not strongly correlated (table 1). there are many soil factors (variables) that influence the diversity and distribution of plant species, such as organic matter, exchangable cation, soil types and other soil factors. these soil factors form the characteristics of habitats and can be the determinants of the distribution and diversity of flora in their habitats (peña-claros et al. 2012; vleminckx et al. 2015; zhang et al. 2016). some studies have shown that species diversity and composition in some ecosystems were related to the site characteristic including edaphic factors (gautam et al. 2016; khan et al. 2017). gautam et al. (2016) showed that the most important factors influencing tree diversity in the north indian moist deciduous forests were exchangeable base cations and phosphorous. furthermore, peñaclaros et al. (2012) showed that in the dry forest in bolivia, correlation between soil characteristics and plant diversity was more related to ca, organic matter, n, mg and na. in this study, the first factor that mostly influenced the diversity and establishment of flora in besiq bermai forest was total n in the soil. nitrogen is an essential element for the growth and development of plants. the availability of n in the soil affect the growth, development, and establishment of flora in besiq bermai forest. table 1 eigen value of soil elements in besiq bermai forest pc1 (component factors 1) pc2 (component factors 2) eigen value 5.11 2.92 proportion 0.3 0.17 cumulatif 0.3 0.47 variabels: ph (ph) 0.27 -0.35 organic c (oc) 0.35 0.26 total n (tn) 0.17 0.46 organic matter (om) 0.36 0.27 c/n ratio (c/n r) 0.37 -0.10 flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. 92 the second factor that influenced the occurence and establishment of flora in besiq bermai forest was c/n ratio. c/n ratio shows the rate of decomposition of organic matter and mineralisation of nutrient elements in soils (tisdale 1985). ross et al. (2011) showed the relationship between the establishment of plant species and c/n ratio indicating that c/n ratio influenced the diversity of plant species in their h a b i t a t . t h e c / n r a t i o i n d i c a t e s t h e decomposition of organic matter and in relation to the nutrient provision in soils plays key roles in the function of forest ecosystem for the establishment of the flora. implications for conservation the present study has shown the flora diversity, compositon, and ecology in besiq bermai forest. the selected study site was a coal min in g a rea . w ith th e o b lig a tio n a n d responsibility to rehabilitate and restore sites after the coal is mined, the results of the present study is required for the reconstruction of the sites and reverting the flora diversity and composition, and ecology of the forest. the present study contributes to support the management of biodiversity and forest conservation after coal mining. the study is also required for rehabilitation program of degraded post mining lands. the soil chemistry that most influences the existence of species of the shorea family is c/n ratio and total nitrogen. the c/n ratio can explain the speed of overhauling organic matter in the form of chemical-bound nutrient decomposition and mineralization in the form of complex compounds. when planting at post-mining areas, it's advisable to use additional organic materials, such as compost, manure, or the addition of calcium, depending on the deficiency in organic matter and acidity level of the soil. there are several species included in the iucn red list, with critically endangered category, namely , and shorea leprosula dipterocarpus cornutus hopea ferruginea. the dominance of the various shorea plants that are included in the iucn redlist, as well as other dominant local species should be given priority as the species used in the restoration of post-mining areas due to their ecological suitability with forest ecosystems in the study sites. conclusion the diversity of plants in besiq bermai forest was relatively high, with shannon-wiener diversity index (h') 6.05, 6.25 and 3.26 for tree, s a p l i n g a n d u n d e r s t o r y, r e s p e c t i v e l y. dipterocarpaceae was the most commmon plant family for tree and saplings, while marantaceae was the dominant family for understory. edaphic factors that had the highest influence on the occurence and establishment of flora in besiq bermai forest were total n and c/n ratio. the present study has an implication to support management of biodiversity and forest conservation. acknowledgements we would like to thank the team of exploration for the assistance in the field. this research was supported by research program on plant diversity and habitat in coal mining exploration areas of pt. bharinto ekatama and pt. indo tambangraya megah tbk branch, west kutai, east kalimantan. references addo-fordjour p, obeng s, anning ak, addo mg. 2009. floristic composition, structure and natural regeneration in a moist semi-deciduous forest following anthropogenic disturbances and plant invasion. int j biodivers conserv 1(2):021-037. arbainsyah, de longh hh, kustiawan w, de snoo gr. 2014. structure, composition and diversity of plant communities in fsc-certified, selectively logged forests of different ages compared to primary rain forest. biodivers conserv 23(10):2445-72. ashton ms, gunatilleke cvs, gunatilleke iaun, singhakumara bmp, gamage s, shibayama t, tomimura c. 2014. restoration of rain forest beneath pine plantations: a relay floristic model with special application to tropical south asia. forest ecol manag 329:351–9. bruhl ca, eltz t. 2010. fuelling the biodiversity crisis: species loss of ground-dwelling forest ants in oil palm plantations in sabah, malaysia (borneo). biodivers conserv 19(2) 519-29.: budiharta s. 2010. floristic composition at biodiversity protection area in lubuk kakap, district of ketapang, west k alimantan biodiversitas . 11(3):151-6. biotropia vol. 25 no. 2, 2018 93 busch j, ferretti-gallon k. 2017. what drives deforestation and what stops it? 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h, jin h , khaldi a , kwak m , lee t , khaine i , y h j y k woo s 2016. plant diversity in different y. bioclimatic zones in tunisia. j asia pac biodivers 9 :56-62.(1) zhang c, li x, chen l, xie g, liu c, pei s. 2016. effects of topographical and edaphic factors on tree community structure and diversity of subtropical mountain forests in the lower lancang river basin. forests 7 222(10): . 94 biotropia vol. 25 no. 2, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia vol. 28 no. 1,2021: 29 37 doi: 10.11598/btb.2021.28.1.907 dominance, association and distribution pattern of tree species i n burnt forest i n east kalimantan subekti rahayu', agus priyono iozrton02, sambas basuni2 and agus hii(mat2 ' worldagraforesbty, jalan c@r, situ gede, sindang barang, bogor 16680, indonesia 'forest resources conseruntion and ecotourism department, faculg $foresty, institut pertanian bogor, kavzpzrs ipb dramaga, bogor 16680, indonesia received 4 october 2017/accepted 17 february 2020 abstract repeated forest fues remarkably impact species composition. pioneer species colonize the burnt forest and widely develop up to 30 years after a fire but late-succession species regenerate gradually or even disappear owing to direct impact of fires or other ecological consequences related to fires. hence, forest restoration through assisted natural regeneration needs some information about the state of post-fire species composition. t o better evaluate tree species composition after repeated fires, the species dominance as an indicator of species composition was used in this research, with additional information o n the species association and distribution patterns. a 1.8-hectare plot, divided into 180 subplots with size of 10 x 10 m, was established in a secondary forest in samboja research forest, east icalimantan. the sample plot was burnt in 1982/1983 and 1997/1998. all trees above 10 cm dbh were measured and leaf specimens were collected for species identification at the herbarium bogoriense, cibinong, west java. for comparison, the 1981 data from the indonesian institute of science were used. analysis of the importance value index used the species dominance data. a 2 x 2 matrix based o n the presence-absence of species for each subplot was used to analyse the association index among species. variance and average value ratio of certain species present in each subplot were expressed in a dispersion index. a chi-square was used to test the significance between the association and dispersion index. thirteen years after a second fire, pioneer species of macarangagigantea were most dominant, followed by vernonia arborea, a sub climax species. this indicated that the forest was in an early succession process. pholidocarpw mqadtm was consistently dominant before and after the fire. a total of 38 pairs of species were significantly positively associated and 4801 pairs negatively associated. about 60% of species association, both negatively and positively, were among the 'native species' (species that existed before the fire events) and 'non-native species' (new comer species that regenerated after the fire) in the plot sample. a non-native species, vernonia arborea, associated negatively with the non-native species tabernaemontana sphaerocarpa, and native species, oncospem homdum, palaquizlm das_yph_vllzm and endiandra mbescens. the distribution pattern of four native species, artocarpzls anisoph_vlhs, cananga odorata, croton laev~olius and macarangagigantea, changed after repeated fires, from uniform to clumped. keywords: association, distribution pattern, dominance, east icalimantan, forest fire introduction samboja research forest is a remnant, lowland, mixed, dipterocarp forest ecosystem in east icalimantan, indonesia, in whch the commercial and high-quality timber species grow, such as meranti (shorea sp.) and ulin (ezlsideroydon ~agem) (delmy 2001). fifty-five (55) species of dipterocarpaceae were preiously *corresponding author, email: s.rahayu@cgiar.org growing in the area (i<artawinata et al. 2008). however, the forest was disturbed by major fires during the dry seasons of 1982/1983 and 1997/1998, leading to a decline in its tree species richness, from 254 to 148 species in 1.65 hectares (simbolon 2005). fires dramatically increased the canopy openness and light penetration owing to drastic declines in aboveground biomass immediately after the fires (slik et al. 2008). the high light environment was associated with an abundance of light biotropia vol. 28 no. 1,2021 demanding, light wood, large-leaved, small-seed spatial distribution of species and inter-species and long-seed dormancy species which are association are fundamental information for typically characteristics of early succession tree understanding species structures and coexistence species (slik e t al. 2010; swaine & whitrnore in communities &i e t al. 2014). however, inter 1988). specific association (between species) and spatial dominant pioneer species mostly occurred in distribution pattern of species in secondary the first decade of forest succession and then growth after fire disturbances have been rarely followed by climax species commonly studied. characterized-by the shade-tblerant species ( ~ l i k e t al. 2008). species interactions during the succession process are of central importance in the ecology of a species because a number of biotic and abiotic factors influence the species interaction. in addition, inter-specific association plays a role in controlling species' diversity and composition (ludwig & reynolds 1988; condit e t a/. 2002). positive association will support the forest regeneration process because species will develop together, but negative association may lead to forest regeneration failure owing to the objectives of this research therefore, were (1) to analyze species' dominance affected by repeated fires; (2) to analyze associations between species and spatial distribution patterns after repeated fires; and (3) to provide recommendations for future restoration planning based on current conditions in ecosystem succession. materials a n d methods competition among species (wright 2002). however, species have natural mechanisms to study area avoid competition both from other individual trees and from other species through developing spatial distribution patterns (janzen 1970). restoration of degraded forest, instead of focusing only on preservation and protection of intact systems, has emerged as a conservation effort in the past few decades (dobson e t a/. 1997). traditional restoration efforts focusing on re-establishing abiotic conditions and relying on succession of biotic communities that remained resilient in degraded forest are faced with many challenges such as changes in landscape connectivity, loss of native species, shift in species dominance, trophic interaction and/or exotic invasion, and the effects of changes in the biogeochemical processes. identifying the constraints is a critical step toward providing recommendations on addressing these challenges (sudng e t dl. 2004). a shift in species dominance due to repeated fires was the constraint faced in samboja research forest (simbolon 2005; slik e t al. 2008). shifting dominance as a result of fire disturbances may affect the recovery interactions between existing species and future species composition. understanding the condition of dominant species, species association between species and spatial distribution patterns is important in forest-restoration planning. the a permanent 1.8 ha. plot was established amidst the 3,504 hectares of samboja research forest, icutai icertanegara and penajam paser utara district, east iealimantan, indonesia with location coordinates of 0'59'23"-0'59'27" s and 1 16'57'31"-1 16'57'51" e (fig. 1). the study site was a remnant lowland mixed dipterocarp forest that was affected by repeated fires in 1982/1983 and 1997/1998. methods the 1.8 hectare (120 x 150 m), divided into 180 subplots with size of 10 x 10 m, was established in the secondary forest of samboja research forest in 2011. the plot was affected by major fires in 1982/1983 and 1997/1998. trees above 10 cm diameter at breast height (dbh) were recorded from the sampling units of 10 x 10 m, with their dbh recorded and positions geo-mapped. leaf specimens were collected for species identification at the herbarium bogoriense, bogor, west java, indonesia. the collected data were then compared with those published by the indonesian institute of sciences pis) in 1981 (icartawinata e t al. 2008) and in 2003 (simbolon 2005). dominance, associakion and &stxibudon pattern of tree species in bum fo~est in east w a n t a n rahayu @ s/. figure mamboja research forest, east kalimantan, indonesia source: balitek ksda samboja khdtk sarnboja data analysis the association between species was the importance value index (ivl) of each analyzed by measuring the degree of association based solely on the presence or absence species was computed from the relative of species in the sampling units of 10 x frequency, relative density and relative dominance of the species (curtis 1959). higher 10 m subplots established in the sample plot m value indicates more dominant species (ludwig & reynolds 1988). presence and absence of single species in the sampling unit compared to others. was presented in binary data of 2 x 2 contingency (table 1). table 1 association index between two species species b presence absence species a presence a absence b where: a = number of subplots where both species, a and b, are present. b = number of subplots where species a is present, but species b is absent. c = number of subplots where species b is present, but species a is absent. d = number of subplots where both species, a and b, are absent. n = number of subplots. biotropia vol. 28 no. 1,2021 expected value of a species associated with other species was calculated using: (a + 6) (a + c) e (a) = n and association index (ai) is a e(a). chi-square test was used to test the significance of the association, using yates correction factor: degree of freedom for association index is r statistic software was used in this data analysis. there were two types of association: 1. positive, if a > e(a), that is, the pair of species occurred together more often than expected if independent. 2. negative, if a < e(a), that is, the pair of species occurred together less often than expected if independent. index of dispersion od) used to express the species distribution pattern based on the variation to mean ratio (ludwig & reynolds 1988), was computed as: where: o2 is the variance and p is the mean the three types of dispersion index are namely; (1) random, if o2 = p, (2) clumped, if o2 > p and (3) uniform, if o2 < p. chi-square statistics used to test significantly different id, was computed as: degree of freedom (db) is n-1. results and discussion species dominance the five most dominant species observed in the sample plots of the 10.5 hectare undisturbed ulin and dipterocarp forest in samboja research forest in 1981 were sborea laevis, pbolidocarpw mdjadun, dio@yros borneensis, ezlsideroylon zpvagem' and scapium macropopdurn (icartawinata et al. 2008). during the 1981 observation, three most dominant species were found in the smaller plot of 1.8 ha of undisturbed pbolidocarpas majadam) dio@yros borneensis and eztsideroylon pagem'. further analysis was focused on the smaller sample plots in the 1.8 ha. five years after the second fire of 1997/1998, three most dominant species based on stem basal area were found in the smaller plots, namely; pbolidocarpas myadan) easideroylon pagem' and dipterocarpas cornatas (simbolon 2005). these three species had individual trees that survived the repeated fires. the surviving individual trees were recognised from tree mapping analysis through comparing coordinate position and stem diameter of 1981 and 2011 data. large surviving trees having > 40 cm diameter of easideroylon pvagem' and dipterocarpas cornatas) as well as the palm species, pbolidocarpas mdjadan, having 20 30 cm diameter contributed both high basal area and ivi. other species that started to establish during the period had contributed to small ivi. the burnt area was dominated by trees having < 5 cm diameter and 10 15 cm tree diameter (sirnbolon 2005). thirteen years after the second fire (2011 observation), the three most dominant species dramatically changed from the climax species of ezasideroylon pagem' and dipterocarpas cornata~ to pioneer species of macaranga gigdntea and sub climax species of vernonia arborea. pbolidocarpus mdjadan was consistently in the third place fable 1). the ten most dominant species were mostly pioneer, except ezlsideroylon ?wagem' and dipterocarpw cornatas. this clearly indicated that 13 years after a fire disturbance, pioneer species colonized the open area. however, surviving species continued to grow among the pioneer species. the surviving individuals of pbolidocarpas mdjadan provided h g h levels of seed for the regeneration process. this endemic palm in icalimantan, mostly growing in swamp forest in the study area, was unaffected by the fires (simbolon 2005). moreover, mortality of this palm was relatively low, reaching 10% in the burnt forest, probably due to the physiological characteristics of its stem vascular structure (van nieuwstadt & shiel2005). dominance, association and distribution pattern of tree species in burn forest in east icalimantan rahayu e t al. table 1 species with the 10 highest m at 1 3 years after the second fire (2011 observation) no. family species local name importance value index 1 euphorbiaceae macaranga gigantea merkubung 35.29 2 asteraceae vernonia arborea merambung 30.54 3 arecaceae pholidocarpus majadum liran 12.98 4 moraceae artocarpus anisoph_ylhs mentawa 12.27 5 rutaceae melicope glabra sampang/terutup 12.19 6 euphorbiaceae croton laevfoli,vs lasa-lasa/belanti 7.46 7 lauraceae eusideroylon .ywagen' ulin 7.20 8 verbenaceae peronema canescens sungkai 6.71 9 annonaceae cananga odorata icenanga 5.73 10 dipterocarpaceae dipterocarpw cornutus keruing gajah 5.62 easideroqlon pagem' is another surviving species that had a slight decrease in m from 9.3 before to 7.2 after the fire. e. ?wagem' has the capacity to re-sprout from damaged trees after fire (delmy 2001), even from stumps and roots (van nieuwstadt & shiel 2005). this species is categorized as heavy wood, ranging 0.88 1.19 g/cm3, on average 1.04 g/cm3 wartawijaya e t al. 1992). heavy wood species (> 0.8 g/cm3) have the capacity to survive fire (van nieuwstadt & sheil 2005; brando e t a l 2012). high density wood produces some extractive material, such as cellulose, hemicelluloses, and lignin, and other properties, that reduce the fire distribution index and ignition time (brando e t al. 2012). before fire, macaranga gigdntea was a minor species in the permanent plot of samboja research forest (icartawinata e t a/. 2008). after repeated fires, the number of trees increased rapidly from 35 in 2003 (simbolon 2005) to 167 in 2011. three years after the fire, macaranga gigantea was the most dominant species in the twice-burned forest, (slik e t al. 2008) and was consistently dominant at 5, 7 and 13 years. this species was regularly found as one of the most dominant species after 10 20 years in regenerating forest (silk e t al. 2008). however, in samboja research forest, m . ggigdntea was the most dominant species three years after the fire. the level of disturbance after the fire might have affected the early establishment of macaranga gigdntea. macaranga is an early succession genus that prefers a frequently disturbed habitat that is characteristic of a highly-disturbed forest (slik e t al. 2003). m. gigdntea traits of being a pioneer and light demanding species are suitable for growth in burnt forest where high exposure to sunlight occurs. its typically orthodox seeds are able to lie dormant in the soil and germinate as soon as the forest canopy opens up (suita & nurhasybi 2009; susanto e t a l 201 6). vemonia arborea, a sub-climax species (desitarani e t al. 2015), was not found in the sample plot before the fire events but developed rapidly and became the second dominant species in 201 1. however, t h s species was found in an undisturbed forest located at 1 2 krn from the sample plot (jgisnawati e t al. 201 1, atmoko e t a l 2015). the small seeds of v: arborea can be dispersed by wind. moreover, this species was a co-dominant in the once-burned forest of sungai wein but not in the twice-burned samboja (slik e t al. 2008b). increasing a13+ after fires probably affected v. arborea colonization in burnt areas. a study in samboja lestari, east icalimantan documented that al+ content in the soil significantly increased four years after a fire and i/. arborea was associated with high a13+ content (j'asir e t al. 2010). high a13+ content in the soil becomes a limiting factor of root growth (mossor-pietraszewska 2001) for common species, but not for v. arborea whch is adapted to high a13+. in samboja, the v. arborea population developed widely after 2003 (five years after the second fire). the occurrence of pioneer and s u b c h a x species as the most dominant, together with establishing climax species in the burned forest has indicated that the succession process was in a competition phase (clement 191 6). in order to address restoration management goals, the assisted natural regeneration and species enrichment activities can be applied in the forest succession phase (elhot e t al. 2013; lamb & gilmour 2006). assisted natural regeneration should focus on species priorities and enrichment, particularly for sub-climax and climax species, to improve the forest function and therefore, conserve biodiversity. biotropia vol. 28 no. 1,2021 species association negative association occurred more frequently thirteen years after the second fire, an analysis of the 191 established species in the sample plots of secondary forest showed that 18,336 pairs of species pairing with themselves, consisted of 1,220 (7%) pairs with positive association, 17,115 (93%) pairs with negative association and a pair of species as neutral. chi square test found 38 pairs with significantly positive association and 4,801 pairs with negative association. t h s positive association rarely occurred but negative association was common, particularly, among 'non-native' species (icuebbing & nunez 2015). non-native and native species in this research refers to the existence of species before and after the fires. non-native species were found in the plot only after the fires but native species were found before and after the fires. however, positive association is important in establishing management priorities (icuebbing & nunez 2015). i t is critical in community structure or habitat modification, in which one individual or species alters the condition of the local environment, often making a stressful habitat more hospitable to other individuals or species (stachowicz 2001). furthermore, positive association between species in mutual interaction will enhance each other's survival probabilities (ludwig & reynolds 1988). positive interactions between different species are of particular interest because of their potential to 'cascade' throughout the com munity, with a major effect on the ecosystem structure and function (stachowicz 2001). some 53% positive association and 60% negative association in the sample plot occurred between species that existed before the fire ('native') and newcomer species that regenerated after the fire ('non-native'). in this research, the between 'native7 and 'non-native' rathe; than among 'non-native'. the more frequent occurring association among the 'non-native' species indicated that 'non-native' species indirectly provide environmental support (flory & bauer 201 4). improving performance of the 'non-native' species after fires is associated with natural resources availability or changes in disturbance regime (daehler 2003). however, light competition as an indirect impact of fire disturbance was an important factor affecting interaction among individual trees or species (icunstler e t al. 2012). vernonia arborea, a 'non-native' species, was positively associated with one 'native7 species anthocephalm chinensis but negatively associated with other 'native7 species, oncospem homa'um, palaquium d a q p b l h m , e didndm mbescens and 'non-native7 species, tabernaemontana sphaerocapa (table 2). a. chinensis, a surviving individual tree provided a suitable environment for sub-climax species of v. arborea seedlings. anthocephal,w chinensis grows in the swampy area of the plot unaffected by fires (simbolon 2005). v. arborea generally grew in the upper elevations of the sample plot. only a few individual trees grew on the swamp area close to anthocephalzls chinensis. as a sub-climax species, v. arborea probably took advantage of a. chinensis' canopy for shade during early regeneration. negative association between i/. arborea with tabernaemontana sphaerocapa, oncospem hom'dum, palaquium daqpbllzm and endiandra mbescens was probably because of different habitat requirements. tabernaemontana sphaerocarpa, oncospem bom'dum, palaquium daqpkyllum and endidndm mbescens grew on lower elevations, in the swampy part of the sample plot. table 2 ten species' pairs with highest yates chi-square value pair of species yates x2 critical association a ea df correction value type knema laterigia neonauclea cabcina 2 0.07 15 32.24 25.00 + calopbylhm soulattri di0.pyro.r pilosanthera 2 0.08 8 25.59 15.51 + anthocephalus chinensis vernonia arborea 4 2.92 12 24.31 21.03 + knema cinerea pobalthia mmphii 2 0.09 3 23.80 7.82 + pobalthia m p h i i q p g i m brachyrachis 2 0.09 3 23.80 7.82 + tabernaemontana sphaerocarpa vernonia arborea 0 0.83 2 1861.84 5.99 tabernaemontana hauilandii vitex pinnata 0 0.01 2 129.56 3.84 oncosperma homdum vernonia arborea 0 0.83 2 127.65 3.84 palaquium dasyphyllzm vernonia arborea 2 2.50 2 91.47 25.00 endiandra mbescens vernonia arborea 0 0.83 2 68.78 3.84 notes: a= number of subplots where both species, a and b, are present; ea= expected value of a species associated with other species. dominance, association and distribution pattern of tree species in burn forest in east icalimantan rahayu et al. v. arborea generally grew in the upper elevations of the sample plot. only a few individual trees grew on the swamp area close to anthocephazas chinensis. as a sub-climax species, v. arborea probably took advantage of a . chinensis' canopy for shade during early regeneration. negative association between v. arborea with tabernaemontana sphaerocarpa, oncosperma hom'dam, paiaqaiam dagpbiiam and endiandm rabescens was probably because of chfferent habitat requirements. tabernaemontana .phaerocarpa, oncospema homzmzd~m, pazaqaiam dagtp&lzam and endidndm mbescens grew on lower elevations, in the swampy part of the sample plot. species distribution pattern species with significantly distributed clumped (table 3). the changing distribution pattern of artocarpzls anisopbizus, cananga odo~ata, croton zaevfolizls and macaranga gigdntea from uniform to clumped, after repeated fires, was due to various factors, such as tree survival in the cooler site and their stem size (davis et al. 2005). the survivor trees in the cooler site functioned as seed sources when most trees were killed, while their small stem size tended to clump. the nature of its fruits and seeds had also affected their distribution pattern after the fire. artocarpas anisop&zlzs has a compound fruit. it contains many 1.7 x 1.0 cm seeds that are difficult to chsperse without a dispersal agent, such as a big mammal. the swampy area was suitable for cananga odorata and croton zaevfohas. swampy in the 1981 observation, the species distribution in the 1.8-hectare sample plot of area depletion and droughts after the fires unchsturbed forest was a balance between became a limiting factor for c. odorata and c. uniform (48%), where the distance between iaevfohas growing in the sample plot. macaranga neighbouring inchviduals is maximised due to gigdntea, a minor species and distributed competition of resources, and random (52%) uniforml~, changed dramatically to dominant where the spacing between individual is and clumped. after biomass burning, open areas unpredictable, usually occurring in the habitat were associated with pioneer species, such as m. with consistent environmental condition. gigdfftea, due to hgh light as an repeated fires affected the species' distribution pattern. analysis of 191 species in the 1.8 hectare secondary growth forest indicated that 69 species (369'0) were distributed randomly, 28 species (14'/0) were clumped and 95 species (50%) were uniformly distributed. clumped dispersion of species was found in the secondary growth forest after repeated fires. further statistical testing using the chi-square found six source during the colonization process. clumped pattern of peronema canescens might affect the planting of this species in a forest rehabilitation program. during the forest rehabilitation activities in samboja researh forest in 1990s p. canescens was one of the species planted in the area for fire breaks and boundary area signs to community land. then, it has spread out after the repeated forest fire. table 3 dispersion index of six species in the 1.8-hectare secondary forest after repeated fires in samboja research forest species name dispersion variance average index d f critical value adocarpus anisap~llz.r 1.97 0.37 5.28 348.74 66 85.98 cananga odorata 0.21 0.1 3 1.66 36.61 22 32.67 croton laevfolizls 0.31 0.1 8 1.77 54.86 31 44.98 macaranga gigantea 1.37 0.93 1.48 245.95 166 197.06 peronema canescens 0.33 0.1 8 1.80 57.48 32 46.19 vernonia arborea 1.29 0.75 1.73 231.32 134 162.01 biotropia vol. 28 no. 1,2021 conclusion the dominance of macaranga gigantea indicated a high level of forest disturbance in that part of samboja research forest affected by two fire events. co-dominance of vemonia arborea indicated the changing environmental conditions, such as increasing a13+, whch was limiting to the species growth. occurrence of pioneer, sub-climax and climax species indicated that the forest succession was in a competition phase. assisted natural regeneration and species enrichment would be possible restoration activities for biodiversity conservation. improving non-native species performance in the twice-burned area might occur due to changing natural resources availabihty and &sturbance regime after a fire. lastly, the changing environmental conditions, species characteristics and human activities affected the distribution pattern of species. references atrnoko t, yassir i, sitepu bs, mukhlisi, widuri sa, muslim t, mediawati i, ma'mf a. 2015. keanekaragaman hayati hutan rintis wartono icadri: hutan tropis i g m a n t a n di iwdtic samboja. balikpapan (id): balai penelitian teknologi konservasi sumber daya alam. 104p. brando pm, nepstad dc, balch ji<, bolker b, christman mc, coe m, putz fe. 2012. fire-induced tree mortality in a neotropical forest: the roles of bark traits, tree size, wood density and fire behavior. global change biology 18:630-41. delmy a. 2001. fire resistant of tree species in bukit soeharto education forest, east kalimantan, indonesia. in: icobayasi e t al., editors. rehabilitation of tropical forest ecosystems. bogor (id): center for international forestry research. desitarani, wiriadinana h, miyakawa h, rachman i, rugayah, sulistyono, partomihardjo t. 2015. buku panduan lapangan jenis-jenis tumbuhan restorasi. jakarta (id): kementerian lingkungan hidup dan icehutanan-jica-lembaga ilmu pengetahuan indonesia. dobson ap, bradshaw ad, baker ajm. 1997. hopes for the future: restoration ecology and conservation biology. sciences 277:515-20. elliot sd, blakesly d , hardwick k. 2013. restoring tropical forests: a practical guide. richmond (uic): royal botanic gardens ice, 344p. flory sl, bauer j. 2014. experimental evidence for indirect facilitation among invasive plants. j ecol 102:12-8. janzen d h . 1970. herbivores and the number of tree species in tropical forests. american naturalist 104(940):501-28. kartawinata i<. 2010. dua abad memgungkap kekayaan flora dan ekosistem indonesia. presented in sanvono prawirohardjo memorial lecture x, indonesia institute of science, jakarta. 23 august 2010. bogor (id): lipi. igrtawinata i<, purwaningsih, partomihardjo t, yusuf r, abdulhadi r, riswan s. 2008. floristic and a structure of low land dipterocarp forest at wanariset samboja, east kalimantan, indonesia. reinwartia 12(4) :301-23. igisnawati h, wahjono d, imanuddin r. 2011. changes in the species composition, stand structures and aboveground biomass of a lowland dipterocarps forest of samboja, east kalimantan. j for res clement fe. 1916. plant succession: an analysis of the 8(1):1-16. washingt0n dc (us): kuebbing se, nunez ma. 2015. negative, neutral, and carnegie institution of washington. positive interactions among nonnative plants: condit r, pitman n , leigh e g jr, chave j, terborgh j , patterns, processes, and management implications. foster rb. 2002. beta-diversity in tropical forest glob chang biol21:926-34. trees. science 295:666-9. kunstler g, lavergne s, courbaud b, thuiller w, curtis jt. 1959. the vegetation of wisconsin: an ordination of plant communities. madison (us): university of wisconsin press. davis ma, curran c, tietmeyer a, miller a. 2005. dynamic tree aggregation pattern in a species-poor temperate woodland disturbed by fire. j veg sci 16:167-74. daehler cc. 2003. performance comparison of co occuring native and alien invasive plants: implication for conservation and restoration. annu rev 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departement of forestry. simbolon h. 2005. dinamika hutan dipterocarp campuran wanariset semboja, kalimantan timur setelah tiga kali kebakaran tahun 1980-2003. biodiversitas 6(2):133-8. slik jwf, icebler pja, van welzen pc. 2003. macaranga and malotm species (euphorbiaceae) as indicator for disturbance in the mixed lowland dipterocarp forest of east igdimantan (indonesia). ecol indic 2:311-24. slik jwf, bernard c, van beek m, breman fc, eichhorn kao. 2008. tree diversity, composition, forest structure and aboveground biomass dynamic after single and repeated f i e in a bornean rain forest. oecologia 158(3):579-88. slik jwf, breman fc, bernard c, van beek m, cannon ch, eichhorn kao, sidiyasa i<. 2010. fire as a selective force in a bornean tropical evenvet forest. oecologia 164: 841-9. stachowicz jj. 2001. mutualism, facilitation, and the structure of ecological communities. bioscience 51 (3):235-46. suding isn, gross kl, houseman gr. 2004. alternative states and positive feedbacks in 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ecosyst environ 137:172-82. biotropia book final.indd 110 effect of phytase supplementation in soybean meal based diet on nutrient digestibility and growth performance of green catfish (hemibagrus nemurus) yulisman, dedi jusadi* and ing mokoginta department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university, kampus ipb darmaga, bogor, indonesia abstract th is experiment was conducted to evaluate the eff ect of phytase supplemented to the diet on phosphorus (p) digestibility and growth performance of the green catfi sh hemibagrus nemurus. five kinds of experimental diets were used in this experiment, namely diets a, b, c, d and e. diet a, as a control, was supplemented with inorganic p, without phytase supplementation. diet b, c, d and e were supplemented with 0, 20, 40 and 60 mg phytase/100 g soybean meal (sbm), respectively, without inorganic p supplementation. fifteen fi sh with initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. fish were fed on the diets for 60 days. results indicated that p digestibility increased from 64.5% to 87.0% as phytase supplement increased from 0 mg in diet b to 60 mg phytase/100 g sbm in diet e. p digestibility in diet e was higher than that in diet a (77.6%). th e daily growth rate and feed conversion ratio followed similar trend. p, ca and zn concentration in the whole body and bone of fi sh fed diet e were higher than the fi sh fed diet b, c and d, but were insignifi cant compared to the fi sh fed diet a. nitrogen (n) and p loading by fi sh fed diet e were, respectively, 76% and 20% lower than those in fi sh fed the control diet. it is concluded that the inclusion of 60 mg phytase/100 g sbm in the diet of the green catfi sh could replace the utilization of inorganic p increase the digestibility of the diet thereby resulting in increased growth rate and reduced excretion of p and n into the waters. key words: hemibagrus nemurus, phytase, soybean meal, phosphorus. introduction th e expansion of global aquaculture production increases the demand for aquaculture feeds. for carnivore species as green catfi sh hemibagrus nemurus, protein can form up to 60% of the diet and fi shmeal is the main source of dietary protein. fishmeal is one of the most expensive and demanded ingredients and has become the main and most critical ingredient in aquafeed production. th e increasing cost and demand of fi shmeal has encouraged feed manufacturers to search for cheaper alternative protein source as plant protein. soybean meal (sbm) is a common plant protein source used as a substitute for *corresponding author: dedijusadi@yahoo.com biotropia vol. 15 no. 2, 2008 : 110 118 111 fi shmeal in the fi sh diet. it could replace 75% of fi shmeal as a protein source in the diet of the green catfi sh (pebriyadi 2004). a major obstacle in using sbm is their high phytic acid content. phytic acid is a substance that contains unavailable phosphorus (p) to monogastric animals, including fi sh, because these animals lack phytase, which can hydrolyze phytic acid (baruah et al. 2004). by using phytase supplement to the diet, the p digestibility can be improved. li et al. (2004) reported that 250 units of phytase per kg diet could eff ectively replace di-calcium phosphate supplement in the diet of channel catfi sh without aff ecting growth, feed effi ciency or bone phosphorus deposit. masumoto et al. (2001) showed that 0.2 g of phytase supplement to the diet containing 67% sbm increased the growth rate and feed effi ciency of the japanese fl ounder paralicthys olivaceus. th erefore, using phytase supplement in the fi sh diet could increase the bioavailability of phosphorus of sbm. hence, replacing the inorganic p of the diet, resulting in lower fecal p loss. th is experiment was conducted to evaluate the eff ect of phytase supplement to the diet on p digestibility and growth performance of the green catfi sh hemibagrus nemurus. materials and methods experimental diets five experimental diets were used in this experiment, namely diets a, b, c, d and e. diet a, as a control, was supplemented with inorganic p (nah2po4.h2o, kh2po4, ca2(po4)3) only, without phytase supplement. diet b, c, d and e were supplemented with 0, 20, 40 or 60 mg phytase (natuphos® 5000)/100 g sbm, respectively, without inorganic p supplement (table 1). th e proximate composition of experimental diets is shown in table 2. table 1. ingredients composition of experimental diet (g/kg diet) ingredients treatments a b c d e fish meal1 130 130 130 130 130 soybean meal1 640 640 640 640 640 fish oil 40 40 40 40 40 soybean oil 44 44 44 44 44 tapioca1 57 57 47,87 47,74 47,62 vitamin mix 15 15 15 15 15 choline chloride 5 5 5 5 5 l-methionine 5 5 5 5 5 taurin 6 6 6 6 6 mineral mix2 58 0 0 0 0 p-free mineral mix3 0 58 58 58 58 phytase 0 0 0.13 0.26 0.38 citric acid 0 0 9 9 9 p content (%): total p 1.46 0.55 0.55 0.56 0.58 water-soluble p 0.59 0.19 0.45 0.48 0.53 phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 112 ingredients treatments a b c d e water-soluble p/total p ratio 40.29 34.87 82.79 86.06 91.13 1 crude protein (dry weight) concentration of: fish meal 70.39%, sbm 42.75%, tapioca meal 0.91%. 2 the mineral mix had the following composition (g/kg dry diet): nacl 0.5; mgso4.7h2o 7.5; nah2po4h2o 12.5; kh2po4 16; ca2(po4)3 14.49; fe-citric 1.25; filler 3.60 and trace element mix (0.5 g) had the following composition: znso4.7h2o 17.65; mnso4 8.1; cuso4.5h2o 1.55; cocl.6h2o 0.05; kio3 0.15; and filler 30.5 (takeuchi 1988). 3 p-free mineral mix had the following composition (g/kg dry diet): nacl 0.5; mgso4.7h2o 7.5; kcl 17.53; fe-citric 1.25; cacl2.2h2o 13.34; filler 30.5 and trace element mix (0.5 g) had the following composition: znso4.7h2o 17.365; mnso4 8.1; cuso4.5h2o 1.55; cocl.6h2o 0.05; kio3 0.15; and filler 30.5. table 2. proximate composition of experimental diets (% wet weight) proximate composition treatments a b c d e crude protein 31.19 29.49 29.30 30.11 29.19 crude lipid 10.58 10.62 10.18 10.30 9.92 ash 11.09 8.74 8.92 9.12 8.79 water 14.01 18.98 20.38 17.04 20.23 fiber 4.20 3.21 2.62 2.92 2.24 nfe1 28.93 28.96 28.60 30.51 30.87 total energy2 (kcal/100g) 392.73 383.71 377.03 390.53 387.38 energy/protein (kcal/g protein) 12.59 13.01 12.87 12.97 13.27 1 nitrogen free extract. 2 total energy (ge) was calculated based on equivalent values of: protein 5.6 kcal/g, lipid 9.4 kcal/g, and nfe 4.1 kcal/g (takeuchi, 1988). for the pretreatment of sbm with phytase, 640 g of sbm was mixed with 2240 ml water, and then phytase was added to the sbm-water mixed at 20, 40, 60 mg/100g, respectively, in the diets c, d and e. th e ph was adjusted to 5.5 with citric acid. th e mixtures were incubated at 37 °c for 2 h (matsumoto et al. 2001). th en, the mixtures were mixed with other ingredients and formed into granules. th e pellets were stored at -20 °c until use. feeding trial green catfi sh juveniles were obtained from the main center for freshwater aquaculture development, sukabumi, west java, indonesia. upon arrival at the laboratory of fish nutrition, of the bogor agricultural university, the fi sh were acclimated to experimental conditions for 10 days and fi sh readily adjusted to the experimental diets. th ereafter, fi fteen juveniles with an initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. each aquarium was part of a closed recirculation system. during table 1. continued biotropia vol. 15 no. 2, 2008 113 rearing period, each aquarium was supplied with continuous aeration and water was allowed to fl ow through at the rate of 200-300 ml/min. every day, impurities in the water of each aquarium were removed and 50% of the water was renewed to maintain water quality. from each aquarium, water was fl own to the physical and biological fi lters, then in the conditioning tank. th ereafter, the water was drained back to each aquarium by using pump. dissolved oxygen contents were 4.18-5.13 mg/l, water temperatures 28 to 30 ºc, and ph 6.30 to 6.35. each experimental diet was fed to the fi sh in three aquaria. th e diets were randomly assigned to groups of fi sh in the aquarium. th e fi sh were fed on the diets to satiation three times a day at 08.00, 13.00 and 18.00 hrs for 60 days. at the end of the feeding trial, the fi sh of each aquarium were bulk weighed from each aquarium. growth of the fi sh (as measured by the percentage of daily growth rate), feed conversion ratio (fcr), protein retention (pr) were calculated as described previously (huisman 1976; steff ens 1989; takeuchi 1988). after the fi nal weighing, fi ve fi sh were randomly sampled from each aquarium for body proximate and mineral compositions analysis, and three fi sh were randomly sampled from each aquarium for analyses of bone mineral (p, ca and zn) composition. th e proximate composition and mineral analyses were carried out according to the methods described by takeuchi (1988). digestibility trial th e eff ect of phytase supplementation to the diet on the p digestibility was determined by the indirect method with 0.5% of chromic oxide (cr2o3) as an inert reference substance. th e green catfi sh with the same size as that for the feeding trial were used in this digestibility trial. twelve fi sh were stocked into each 60-l aquarium. triplicate groups of fi sh were fed on each experimental diet to satiation three times a day for 7 days prior to collection of feces. feces were collected after 2 h of feeding by siphoning into a plastic sieve. after each collection, the samples from each aquarium were pooled, frozen at -20 ºc and stored for subsequent analyses. feces collections were conducted for 21 days. th ereafter, the samples of feces were analyzed for chromium (cr), p and protein content according to the methods described by takeuchi (1988). apparent digestibility of total nutrient, protein, and p were calculated as follows: % digestibility = 1 – (% cr in feed/ % cr in feces) x (% ingredient in feces/ % ingredient in feed) x 100 p and nitrogen (n) losses through feces were calculated as follows: p in feces (g) = undigestible p (%) x p consumption during culture period n in feces (g) = (undigestible n (%) x n consumption during culture period)/6.25 statistical methods th e data were statistically analyzed for diff erences among the means by the oneway analysis of variance. th e duncan’s test was used to compare treatment means using the statistical software spss 12 for windows. diff erences were considered signifi cant at p<0.05. phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 114 results and discussion results indicated that p and protein digestibility increased from 64.5% to 87.0%, and 87.1% to 90.5%, respectively, as phytase supplement increased from 0 mg in diet b to 60 mg phytase/100 g sbm in diet e. p digestibility of diet e was higher than that of diet a (77.6%) (table 3). p digestibility was correlated with the water-soluble p. th e water-soluble p content in control diet was 0.59 %. as the phytase supplement increased, water-soluble p increases from 0.19 % in diet b to 0.53 % in diet e (table 1). it could be observed that the lowest p loss through feces was found for diet e, and the lowest nitrogen (n) loss through feces for diet d. diet e gave the second lowest n loss through feces. level of n and p loading by fi sh fed diet e were 76% and 20%, respectively, lower than those in the fi sh fed control diet (a). table 3. phosphorus (p) and protein digestibility of experimental diets parameters experiment a b c d e p digestibility (%) 77.6 64.5 70.4 73.3 87.0 p consumption (g) 10.4+0.1d 3.2+0.1a 3.2+0.1a 3..4+0.1b 4.0+0.0c p digested (g) 8.1+0.1e 2.0+0.0a 2.3+0.0b 2.5+0.1c 3.5+0.1d p output via feces (g) 2.3+0.0e 1.1+0.0d 1.0+0.0c 0.9+0.0b 0.5+0.0a protein digestibilty (%) 89.1 87.1 85.1 89.7 90.5 protein consumption (g) 257.4+2.8d 207.6+3.5a 215.6+3.5b 218.2+4.1b 251.8+1.0c nitrogen (n) digested (g) 36.7+0.4c 28.9+0.5a 29.4+0.5a 31.3+0.6b 36.5+0.2c n output via feces (g) 4.5+0.1d 4.3+0.1c 5.1+0.1e 3.6+0.1a 3.8+0.0b *) values within the same row with different superscript letters are significantly different, p<0.05. concentrations of p, ca and zn in the whole body and bone of the fi sh fed diet e were higher than those for the groups of fi sh fed on the diets supplemented with 0-40 mg phytase/100 g sbm, but were not signifi cantly diff erent from those of the fi sh fed diet a (table 4). table 4. p, ca and zn content in green catfish (% dry weight) fed experimental diets parameters experiment a b c d e whole body p 3.18 + 0.28b 2.08 + 0.27a 2.34 + 0.39a 2.97 + 0.19b 3.20 + 0.12b ca 3.26 + 0.06c 1.92 + 0.30a 2.12 + 0.07a 2.90 + 0.20b 3.30 + 0.09c zn 0.012 + 0.001b 0.009 + 0.001a 0.012 + 0.000b 0.012 + 0.001b 0.013 + 0.002b biotropia vol. 15 no. 2, 2008 115 parameters experiment a b c d e bone p 21.41+ 1.12c 13.44 + 0.26 a 16.65 + 0.77b 16.99 + 1.24b 21.46 + 0.43c ca 33.33 + 0.11d 21.44 + 1.15a 24.42 + 0.76b 26.04 + 1.15c 33.60 + 1.66d zn 0.032 + 0.002b 0.021 + 0.002a 0.023 + 0.002a 0.024 + 0.003a 0.032 + 0.001b *) values within the same row with different superscript letters are significantly different, p<0.05. th e mean body weight gain of the fi sh fed diet e was higher than that of the fi sh fed with the other diets. th e daily growth rate and feed conversion ration (fcr) followed similar trend, while the fi sh fed diet b had the lowest daily growth rate and fcr (table 5). th e protein retention had the similar trend with the protein digestibility, which increased from 36.8% from 0 mg phytase/100 g sbm in the diet b to 44.4% from 60 mg phytase/100 g sbm in the diet e. table 5. initial body weight (wo), final body weight (wt), protein retention (pr), daily growth rate (dgr), feed consumption (fc), feed conversion ratio (fcr) and survival rate (sr) of green catfish parameters treatments a b c d e wo (g) 7.0+0.01 7.0+0.02 6.9+0.12 6.8+0.09 6.8+0.05 wt (g) 43.8+0.8 31.9+1.1 35.2+ 0.9 40.2+0.4 47.7+0.4 dgr (%) 3.1+0.0d 2.6+0.1a 2.8+0.1b 3.0+0.0c 3.3+0.0e pr (%) 39.5+0.2b 36.8+0.4a 40.1+1.4b 41.6+1.4b 44.4+1.9c fc (g) 709.7+7.6e 570.4+9.7a 585.9+9.5b 601.1+11.2c 688.1+2.7d fcr 1.3+0.0 c 1.5+0.1e 1.4+0.0 d 1,2+0.0 b 1.1+0.0 a sr (%) 100+0.0 100 + 0.0 100+0.0 100+0.0 100+0.0 *) values within a row with different superscript letters are significantly different, p<0.05. increasing levels of phytase level from 0 mg to 60 mg/100g of sbm resulted in increasing levels of water-soluble p, thereby increasing water-soluble p/total p ratios in the diets from 34.87% to 91.13%. diet e produced the highest p and protein digestibility, followed by diet a (control diet) (table 3). it means that phytase released p from the insositol ring of phytate to be water-soluble p (baruah et al. 2004). th e water-soluble p was digested in the intestine of fi sh, and improved the absorption and utilization of p. phytase also released some protein and amino acids from the phytic acid, hence the absorption and utilization of protein also increased. th e same results were found for other fi sh species, such as the atlantic salmon salmo salar (sajjadi and carter 2004), striped bass morone saxatilis (papatryphon and shoares 2001), rainbow trout onchorhynchus mykiss (cheng and hardy 2004), korean rockfi sh sebastes schlegeli (yoo et al. 2005), and tilapia oreochromis niloticus (liebert and portz 2005). on the other hand, the fi sh fed on the diet b had the lowest p digestibility. it appears that the concentration of phytase in diet b could not enough release all p from phytic acid in the sbm. table 4. continued phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 116 table 4 shows that not only p availability in the diet could be improved by phytase supplementation, but also the other minerals as ca and zn which could be available to the fi sh. accumulation of ca, p and zn in the body and bone of fi sh also increased as the phytase levels of the diets increased from 0 to 60 mg/100 g sbm. other experiments also showed that phytase supplemented diet increased ca, p and mn contents of the african catfi sh clarias gariepinus (nwanna et al. 2005), striped bass morone saxatilis (hughes and soares 1998), and atlantic salmon salmo salar (sajjadi and carter 2004). it is known that phytic acid is the major p storage compound in plant seeds, including sbm. phytic acid is also a strong chelator of important minerals such as ca, mg, k, fe, cu, zn and forms poorly soluble complexes. apart from minerals, phytic acid also forms complexes with protein and amino acids. th e digestibility of these complexes by fi sh are very limited due to the lack of intestinal phytase (pointillart et al.1987). th e supplementation of phytase in this experiment could release those minerals and protein from phytic acid, resulted in increasing their (minerals and protein) digestibility for fi sh (table 1 & 3), hence the absorption and utilization of minerals and protein also increased (table 4 & 5). on the other hand, minerals also play a role in many processes of protein, lipid and carbohydrate metabolisms. increasing the availability of some minerals in the diet by phytase supplementation also appears to improve the protein synthesis, which can be seen from the protein retention data (table5). th e highest protein retention was found in the groups of fi sh fed on diet e, while the fi sh fed on diet b had the lowest protein retention. th e increased protein retention also improved the protein deposit in the body, hence increasing the daily growth rate (table 5). in this experiment, the fi sh fed on diet e had the highest protein retention and daily growth rate. th is indicates that phytase could increase the nutrient (protein) digestibility leading to higher protein retention, resulting in the increase of the growth rate and reduce feed conversion ratio. th e same conclusions were also indicated by other authors that the phytase supplementation to sbm improved the weight and growth rate of juvenile korean rockfi sh sebates schlegeli (yoo et al. 2005), tilapia oreochromis niloticus (liebert and ports 2005; furuya et al. 2001), rainbow trout oncorhynchus mykiss (vandenberg et al. 2003), and pangasius pangasius (debnath et al. 2005). nwanna et al. (2005) also found that supplementation of phytase to a diet containing sbm gave better feed conversion than that for diet without phytase supplementation for the african catfi sh clarias gariepinus, and the striped bass morone saxatilis (hughes and soares 1998). as earlier explained some of the diet consumed by fi sh was undigested, and will be excreted through feces. increasing phytase concentration in the diet from 0 mg to 60 mg/100g sbm reduced the levels of p and n excretion through feces. th is indicated that phytase was able to release nutrient (protein) and minerals of phytate, resulting in the improvement of fi sh growth and also reduced p and n excretion through feces. hughes and soares (1998) concluded that phytase in a diet containing high level of plant protein could reduce the p excretion of striped bass morone saxatilis, seabass dicentrarchus labrax (teles et al. 1998), japanese fl ounder paralichthys olivaceus (masumoto et al. 2001), rainbow trout oncorhynchus mykiss (vielma et al. 2002), atlantic salmon salmo salar (sajjadi and carter 2004), tilapia oreochromis niloticus (furuya et al. 2001; liebert and portz 2005), and the african catfi sh clarias gariepinus (van weerd et al. 1999; nwanna et al. 2005), thereby reducing the loading of p waste into the environment. biotropia vol. 15 no. 2, 2008 117 conclusions th e supplement of 60 mg phytase/100 g sbm in the diet improved p digestibility and the growth of the green catfi sh hemibagrus nemurus, hence reducing the loading of p and n waste to the environment. references baruah k., n.p. sahu, a.k. pal, and d. debnath . 2004. dietary phytase: an ideal approach for a cost eff ective and low-polluting aquafeed. naga, worldfi sh center quarterly 27 (3) 3 & 4 jul-dec 2004. p. 15-19. cheng zj, and r.w. hardy . 2004. eff ect of microbial phytase supplementation in corn distiller’s dried grain with solubles on nutrient digestibility and growth performance of rainbow trout, oncorhynchus mykiss. journal of applied aquaculture. 15 (3/4): 83-100. debnath d., a.k. pal, n.p. sahu, k.k. jain, s. yengkokpam and s.c. mukherjee. 2005. eff ect of dietary microbial phytase supplementation on growth and nutrient digestibility of pangasius pangasius fi ngerlings. aquaculture research (36) 2: 180 – 187. furuya w.m., g.s. gonclaves, v.r.b. furuya , c. and hayashi. 2001. phytase as feeding for nile tilapia (oreochromis niloticus). performance and digestibility. rev. bras. zootec. 30: 924 – 929. hughes k.p. and j.h. soares jr. 1998. effi cacy of phytase on phosphorus utilization in practical diets fed to striped bass morone saxatilis. aquaculture nutrition, 4: 133 – 140. huisman e.a. 1976. food conversion effi ciencies at maintenance and production levels for carp (cyprinus carpio linn) and rainbow trout (salmo gairdneri ric.). aquaculture, 9 (2): 259-273. liebert f. and l. portz . 2005. nutrient utilization of nile tilapia (oreochromis niloticus) fed plant based low phosphorus diets supplemented with graded levels of diff erent sources of microbial phytase. aquaculture, 248: 111 – 119. li m.h., b.b. manning and e.h. robinson. 2004. summary of phytase studies for channel catfi sh. mississippi agricultural and forestry experiment station (23) 23 no. 13: 1-5. masumoto t., b. tamura and s. shimeno. 2001. eff ects of phytase on bioavailabilty of phosphorus in soybean meal-based diets for japanese fl ounder (paralichthys olivaceus). fisheries science, 67:1075-1080. nwanna l.c., o.a. fagbenro and a.o. adeyo. 2005. eff ect of diff erent treatments of dietary soybean meal and phytase on the growth and mineral deposition in african catfi sh clarias gariepinus. journal of animal and veterinary advances. 4: 980 – 987. papatryphon e., and j.h. soares jr. 2001. th e eff ect of phytase on apparent digestibility of four practical plant feedstuff s fed to striped bass, morone saxatilis. journal aquaculture nutrition. 161-167. pebriyadi, b. 2004. supplementation of methionine and tryptophan in the high soybean meal diets of juvenile green catfi sh (mystus nemurus). th esis (in indonesian). graduate school, bogor agricultural university. 53 p. pointillart a., a. fourdin and n. fontaine. 1987. importance of cereal phytase activity for phytate phosphorus utilization by growing pigs fed diets containing triticale or corn. journal of nutrition 29: 907912. sajjadi m. and c.g. carter. 2004. dietary phytase supplementation and the utilization of phosphorus by atlantic salmon (salmo salar l.) fed a canola-meal-based diet. aquaculture 240: 417 – 431. takeuchi t. 1988. laboratory work-chemical evaluation of dietary nutrients. p. 179-233, in watanabe (ed) fish nutrition and mariculture. kanagawa international fisheries training. japan international cooperation agency (jica), japan. phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 118 teles a.o., j.p. pereira, a. gouveia and e. gomes. 1998. utilization of diets supplemented with microbial phytase by seabass (dicentrarchus labrax) juvenils. aquaculture living resources, 11 (4): 255259. vandenberg g.w., v. dallaire, s.l. scott and j. de la noue . 2003. encapsulation of microbial phytase : eff ects on phosphorus bioavailability in rainbow trout (oncorhynchus mykiss). aquaculture nutritioncontributed papers. http://www.aquacultureassociation.ca/ac03/abstracts/nutrition.htm van weerd j.h., k.h.a. khalaf, f.j. aartsen and p.a.t. tjissen. 1999. balance trials with african catfi sh clarias gariepinus fed phytase-treated soybean meal based diets. aquaculture nutrition, 5:135142. vielma j., k. ruohonen and m. peisker . 2002. dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, oncorhynchus mykiss. aquaculture. 204: 145 – 156. watanabe t. 1988. fish nutrition and mariculture. jica textbook. th e general aquaculture course. department of aquatic biosciences. tokyo university of fisheries. 233 p. yoo g.y., x. wang, s. choi , k. han, j.c. kang and s.c. bai sc. 2005. dietary microbial phytase increased the phosphorus digestibility in juvenile korean rockfi sh sebastes schlegeli fed diets containing soybean meal. aquaculture 243. 315 – 322. biotropia vol. 15 no. 2, 2008 biotropia no. 3, 1989/1990: 41-49 antagonistic effect of four fungal isolates to ganoderma boninense, the causal agent of basal stem rot of oil palm okky setyawati dharmaputra and h.s. soetarmi tjitrosomo seameo-biotrop, p.o. box 17, bogor, indonesia abdul latief abadi faculty of agriculture, brawijaya university, malang, indonesia abstract four fungal isolates from soils obtained from three sites of the oil palm plantations in north sumatra were found antagonistic to ganoderma boninense, the causal agent of basal stem rot of oil palm. penicillium citrinum inhibited the growth of the pathogen and formed a zone of inhibition on the agar media. trichoderma harzianum bio 1 as well as bio 2 and t. viride not only repressed the growth of the pathogen but also caused lysis of the hyphae, and the colony was totally overgrown by the antagonists. introduction ganoderma boninense pat. is an important basal stem rot pathogen of oil palm (elaeis guineensis jacq.) in north sumatra. some control methods have been applied to overcome the disease but none seemed to be satisfactory (parnata 1974; suyoto & djamin 1981; sipayung & purba 1986). the use of antagonistic soil microorganisms to control the pathogen has not been investigated so far. taking into consideration the statement asserted by cook & baker (1983) that certain species of penicillium and trichoderma have been reported to be antagonistic to plant pathogenic fungi, we at biotrop were curious to find out whether some antagonists could be isolated from soil samples taken from the oil palm plantations in north sumatra and investigate their antagonistic properties against ganoderma boninense. this paper describes the results of an investigation on the effect of four fungal isolates on the growth of the pathogen in vitro. materials and methods isolation of fungi from soil soil samples were taken randomly from three locations (adolina, gunung bayu, and tinjowan) of the oil palm plantations in north sumatra. the dilution 41 biotropia no. 3, 1989/1990 plate method (johnson & curl 1972) was used to isolate the fungi at a concentration of 10 -2 ml. into each petri dish, 1 ml of the soil suspension was transferred aseptically and added to it was 10 – 12 ml of melted selective medium containing chloramphenicol and rose bengal. the dishes were then incubated for 5 days at room temperature. isolates of g. boninense isolates of g. boninense used in this experiment were ga, gb and gd. they were isolated from basidiocarps obtained from the basal stem of oil palm in north sumatra. the name of the isolates were based on the colour and morphology of the basidiocarp. antagonism between g. boninense isolate gb and the fungal isolates the fungal isolates obtained were tested for their antagonistic property against the pathogen using the direct opposition method as recommended by dennis & webster (1971). the promising antagonistic molds were then put aside for further studies. the direct opposition method was prepared by placing the pathogen (4 mm in diameter) on the pda medium and 2 days later at a distance of 3 cm from the pathogen the antagonist was inoculated on the same dish (figure 1). each treatment was in three replications and incubated at room temperature. observations were made on the growth of the pathogen and the presence of an inhibition zone that might develop between the two colonies (figure 2). antagonism between g. boninense isolates ga, gb and gd and the four promising isolates of fungi in this study the four isolates of fungi that were found antagonistic to the pathogen were further tested individually for their effect on the pathogen when placed together using the direct opposition method. the inoculation method of the pathogen and the antagonist is the same as previously mentioned. observations were made 3 days after the inoculation of the antagonists on the inhibition of mycelial growth of the pathogen by the antagonist using the formula of fokkema (1973): r1 r2 i = x 100% r1 where i = percentage of inhibition r1 = radius of the pathogen away from the antagonist r2 = radius of the pathogen towards the antagonist 42 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi figure 1. antagonistic test between g. boninense and the fungal isolate. a, inoculum of g. boninense; b, inoculum of the fungal isolate tested. figure 2. colony measurement of g. boninense (a) to calculate the percentage of inhibition by the antagonist (b). radius of the pathogen (r, and r2) and the zone of inhibition (d) were also noted. 43 biotropia no. 3, 1989/1990 for p. citrinum, the distance (d) of the zone of inhibition was also measured. hyphal interaction in the zone of confrontation between the colonies was observed by direct examination under the microscope with 10 x 200 magnification 2 days after the inoculation of the antagonist (aia). interactions observed between the pathogen and antagonist were scored according to the categories presented in table 1. table 1. score of interaction between ganoderma boninense and trichoderma. score criteria/pathogen hyphae lysed 1 ±25% 2 ±50% 3 ±90% the scores were added by 1 if pathogen hyphae were smaller than the normal ones the scores were reduced if ±10% of the antagonist by 1/2 hyphae were also lysed the scores were reduced by 1 if ± 25% of the antagonist hyphae were also lysed results and discussion the fungi isolated from the soil samples are presented in table 2: 4 isolates of penicillium, 2 isolates of aspergillus, 3 isolates of trichoderma, and 1 unidentified isolate, totaling 10 isolates obtained by the use of dilution plate method. table 2. the fungi isolated from adolina, gunung bayu and tinjowan, north sumatra. 44 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi cook and baker (1983) stated that penicillium and trichoderma have long been recognized as antagonists to plant pathogenic fungi. all of the ten isolates obtained were further tested on the possibility of inhibiting the growth of the pathogen (isolate gb) in vitro using the direct opposition method as recommended by dennis & webster (1971). of the 10 isolates, only four showed promising result, that is penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2, and trichoderma sp. 3 (figure 3). figure 3. antagonism between ganoderma boninense isolate gb and penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2 and trichoderma sp. 3. further identification of the four isolates showed that penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2, and trichoderma sp. 3 were respectively p. citrinum, t. harzianum bio-1, t. harzianum bio-2 and t. viride. antagonism between p. citrinum and g. boninense isolates ga, gb, and gd a zone of inhibition (d) was observed when p. citrinum was paired with the pathogen. the growth of the pathogen towards the antagonist was inhibited since the 2 nd day aia and it stopped to grow on the 6 th day aia. the distance (d) of the zone of inhibition on the 3 rd day aia were 11.25 mm, 12.88 mm and 10.88 mm for isolates ga, gb and gd, respectively. it was assumed that an antibiotic diffused into the medium. 45 biotropia no. 3, 1989/1990 statistical analysis using f indicated that i on the 3 rd day aia was not significantly different among the three isolates of the pathogen: 29.95% for isolate ga, 25.28% for isolate gb and 19.08% for isolate gd, respectively. direct examination under the microscope showed that the hyphae of the pathogen were not lysed, but they were abnormal, i.e. they had more septa and their cells were shorter than the normal ones. antagonism between trichoderma isolates and g. boninense isolates ga, gb and gd having examined all the dishes where the pathogen colonies and the antagonists were grown, it was noted that there were no inhibition zones. for all treatments, it was observed that mycelial contact between the two colonies on the same dish started on the 2 nd day aia. t. harzianum bio 2 stopped the growth of the pathogen on the 3 rd day aia, whereas t. harzianum bio1 and t. viride on the 4 th day aia. the percentage of inhibition of the pathogen mycelial growth by the antagonist is presented in table 3. in general, the three isolates of trichoderma inhibited the growth of the pathogen in vitro. the lowest percentage occurred when isolate gb was placed against t. harzianum isolate bio-2, while the highest was noted when isolate gd was inoculated against t. harzianum bio-2. it is interesting to report here that t. harzianum isolate bio 1 was able to inhibit at statistically the same degree, the growth of all isolates ga, gb, and gd, whereas the inhibition table 3. average percentage inhibition of mycelial growth of ganoderma boninense isolate ga, gb and gd by trichoderma harzianum (isolate bio 1 and bio 2) and t. viride. combination of treatment average of inhibition*) (pathogen vs. antagonist) (%) ganoderma boninense vs. trichoderma harzianum 28.96 a isolate gb isolate bio 2 g. boninense isolate gd vs. t. viride 37.71 b g. boninense isolate gb vs. t. viride 38.57 b g. boninense isolate ga vs. t. harzianum isolate bio1 39.53 be g. boninense isolate ga vs. t. harzianum isolate bio-2 39.62 be g. boninense isolate gd vs. t. harzianum isolate bio1 40.29 be g. boninense isolate gb vs. t. harzianum isolate bio1 41.57 be g. boninense isolate ga vs. t. viride 41.84 be g. boninense isolate gd vs. t. harzianum isolate bio-2 45.18 c *) means within columns followed by the same letter are not significantly different. lsd 0.05 = 6.14. 46 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi of t. harzianum bio 2 and t. viride was determined by the type of isolate of the pathogen. direct examination under the microscope showed that the pathogen hyphae underwent lysis; the percentage of which was determined by the type of isolate of the antagonist (table 4). in any case, the hyphae of the antagonist were also lysed in certain treatment, e.g. t. harzianum bio1 vs. g. boninense isolates ga and table 4. microscopic examination of hyphal interaction between ganoderma boninense isolates gb and gd with trichoderma harzianum (isolates bio -1 and bio 2) and t. viride. 47 biotropia no. 3, 1989/1990 gd, t. harzianum bio-2 vs. g. boninense isolates ga and gd, t. viride vs. g. boninense isolates ga, gb and gd. in general t. harzianum bio 1 had the same effectivity score against the three pathogen isolates. the ef fectivity score of t. harzianum bio 2 and t. viride was determined by the type of isolate of the pathogen. if we look at either the percentage of inhibition of mycelial growth of the pathogen by the antagonist or the capability of the antagonist to cause hyphal lyses of the pathogen, t. harzianum bio1 was the most potential antagonist. chet et al. (1979) found that t. harzianum could control damping-off disease on bean, peanuts and egg plants caused by sclerotium rolfsii and rhizoctonia solani. sivan and chet (1986) also found that t. harzianum could control fusarium spp. in cotton, wheat and muskmelon. conclusion p. citrinum, t. harzianum (isolate bio1 and bio-2), and t. viride isolated from oil palm plantation in north sumatra were antagonistic to g. boninense. t. harzianum isolate bio 1 was the most potential antagonist. p. citrinum probably produces an antibiotic substance that diffuses into the medium, while tricho-derma causes lyses of the pathogen's hyphae. acknowledgments the authors gratefully acknowledge the funds given by marihat research center, pematang siantar, north sumatra. thanks are also due to dr. r.a. samson of central bureau voor schimmelcultures (cbs), baarn, the netherlands, and dr. m.a. rifai of the research centre for development in biology, indonesian institute of science, bogor, indonesia, in having confirmed the identification of the fungal isolates. references chet, i., y. hadar, y. elad, j. katan and y. henis. 1979. biological control of soil-borne plant pathogens by trichoderma harzianum. in schippers, b. and w. gamsa (eds.). soil-borne plant pathogens. academic press, london: 585-591. cook, r.j. and k.f. baker. 1983. the nature and practice of biological control of plant pathogens. the american phytopathological society, st. paul, minnesota. 48 antagonistic effect of four fungal isolates -o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi dennis, c. and j. webster. 1971. antagonistic properties of species groups of trichoderma. iii. hyphal interaction. trans. brit. mycol. soc. 57: 363 -369. fokkema, n.j. 1973. the role of saprophytic fungi in antagonism against drechslera sorokiniana (helminthosporium sativurri) on agar plates and on rye leaves with pollen. physiological plant pathology 3: 195-205. parnata, y. 1974. control of basal stem rot of oil palm using urea in accelerating stem and stump decay. bulletin balai penelitian perkebunan medan 5(3): 89 -94. sipayung, a. and r.y. purba. 1986. penelitian dan usaha penanggulangan penyakit busuk pangkal batang (ganodermd) di perkebunan kelapa sawit (research and control of basal stem rot (ganoderma sp.) in oil palm plantation). special edition. pt perkebunan vi-vii, marihat research center, marihat ulu, pematang siantar. sivan, a. and i. chet. 1986. biological control of fusarium spp. in cotton, wheat and muskmelon by trichoderma harzianum. j. phytopathology 116: 39-47. suyoto, s. and a. djamin. 1981. sistem pemberantasan penyakit busuk pangkal batang (ganodermd) di perkebunan kelapa sawit ptp vi. (system of controlling basal stem rot (ganodermd) in oil palm plantation ptp vi). paper presented at 6 th indonesian phytopathology society congress, bukittinggi. 49 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 4. dwi andreas santosa biotropia vol. 17 no. 2, 2010: 98 104 lipid producing microalgae from several ecosystems in west and central java, indonesia 1,3*) 2) dwi andreas santosa , and sulastri , 1) department of soil science and land resources, faculty of agriculture, bogor agricultural university, bogor, indonesia 2) indonesian center for biodiversity and biotechnology (icbb), bogor, indonesia 3) seameo-biotrop, bogor, indonesia received as dipa 2008 / accepted 17 july 2010 abstract this study is aimed to get lipid producing microalgae as feedstock for biofuel production. the microalgae were isolated from 355 collected water samples which represented many distinct ecosystems such as paddy fields, rivers, agricultural dams, ponds, swampy areas and unique ecosystem of volcano and mud-volcano craters in westand central java, indonesia. a total of 267 strains of microalgae were isolated from the samples of which 221 strains of them have capability to produce lipid. there were four promising strains that produce lipid between 14.7 45.7 percent dry weight in optimal condition that were identified as chlamydomonas sp. ko-7267 and pk-7195, chlorella sp. ks-7300 and desmodesmus sp. bk-7291. keywords: microalgae, lipid, biofuel, indonesian ecosystems introduction global oil production is rapidly approaching its peak, even if natural gas liquids and expensive, destructive, and risky deepwater and polar oil are included. based on several scenarios, the peak oil will happen sometime between 2010 2030 (robert 2005). peak oil means that half of oil reserves has been exploited and used. peak production of oil in indonesia has occurred in the 1990 (full report workbook 2004). in 2004, indonesia (formerly opec member) became a net importer. the lack of stability of future energy supplies has motivated the development of alternative energy sources in order to eliminate the possibility of a future energy shortage. furthermore, one of the most environmental problems today is global warming, caused primarily due to the heavy use of fossil fuel. in the world, large amount of co 2 * corresponding author : dsantosa@indo.net.id 98 are released into the atmosphere. photosynthetic microalgae are potential candidate for substitution of the fossil energy. some strains can produce lipid which can be converted into biodiesel, while others produce petroleum like hydrocarbon. the cultivation of algae is also important to reduce the effect of climate change through utilizing excessive amount of co , since cultivated these organisms are capable of 2 fixing co to produce energy and chemical compounds upon exposure to sunlight. 2 microalgae are photosynthetic renewable resources with high lipid content, have faster growth rate than plant cells and are capable to grow in saline waters which are unsuitable for agriculture. the lipid content of microalgae on a dry cellular weight basis varies between 20 and 40%. lipid content as high as 85% has been reported for certain microalgal strains. botryococcus braunii, is a unique microalgal strain, having a long hydrocarbon chain of between 30 and 40% (dry weight basis) which produces almost similar compounds as crude oil. both physical and chemical processes are applicable in the production of liquid fuels from algal strain of high lipid content. these processes include direct lipid extraction in the production of diesel oils substitutes, transesterification in the formation of ester fuels, and hydrogenation in the production of hydrocarbons (borowitzka 1988). microalgae contain lipids and fatty acids as membrane components, storage products, metabolites and sources of energy. algal fatty acids and oils have a range of potential applications. the characteristics of algal oils are similar to those of fish and vegetable oils, and can thus be considered as potential substitutes for the products of fossil oil (as a biodiesel) (miyamoto 1997). cultivation of microalgae in a large scale can be considered as potential substitutes for the petroleum-based diesel used in transportation. the study conducted by nerl (national renewable energy laboratory, us) found that 7.5 billion gallon of biodiesel from microalgae could be produced on 200,000 ha of desert land per year. this value equals to 214 million barrels or equivalent to 65% of total annual petroleum production in indonesia. this value could never be reached by cultivating other energy producing plants. indonesian microalgae as sources of biofuel production are almost unexplored. very few related reports and publications could be found in indonesia. the exploration of indonesian microalgae as a source of sustainable energy is therefore very promising. materials and methods samples collection samples were collected from rivers, lakes, paddy fields, ponds, dams, craters and saline waters in the south area of west and central java, and also some areas in the northern coast of coastal area of central java (fig. 1). those areas represent a large variety in soils, ph, temperature and salinity. a number of 355 samples were collected from 36 different sites (table 1). 99 lipid producing microalgae from several ecosystem dwi andreas santosa et al. figure 1. the sampling sites isolation and screening of microalgae the soil and fresh water samples were cultured in 616 medium (atcc) and mj medium (modified jorgensen's media for diatoms). the samples from the saline habitats were cultured in modified noro medium (takagi et al. 2005) containing (per litre): nacl 29.2g; kno 1g; mgcl h o 1.5g; mgso .7h o 0.5g; kcl 0.2g; cacl 0.2g; 3 2. 2 4 2 2 k hpo 0.045g; tris(hydroxymethyl)aminomethane 2.45g; edta.2na 1.8g; 2 4 znso47h2o 0.087 mg;h bo 0.61 mg; cocl .6h o 0.015mg; cuso .5h o 0.06 mg; 3 3 2 2 4 2 mncl 0.23mg; (nh ) mo o .4h o 0.38mg; fe(iii)edta 3.64 mg. the ph was 2 4 6 7 24 2 adjusted to 8.0 with 1n hcl. other culture media were used in order to obtain suitable media for their growth. the algae were subjected to purification by serial dilution followed by plating. the individual colonies were isolated and inoculated into liquid o medium and incubated at 27±2 c under 1.2±0.5 klux light intensity with 12:12 hrs photoperiods. the purity of culture was ensured by repeated plating and by regular observation under microscope. the isolates were characterized with respect to lipid and/or oil production capabilities. the lipid and oil producing microalgae were determined by using nile red (nr) staining (qin 2005). a stock solution of nr was prepared by adding 2.5 mg of nr to 100ml of acetone. the solution was kept in an amber-coloured bottle and stored in the dark at room temperature. the microalgae cell was stained by placing 5 ml of culture in a petridish, then added with 200µl of nr stock solution to the dish. staining of lipids and oil was completed within 30 minutes. no destaining or rinsing was required. after staining, a drop of stained cell culture was transferred to a depression slide and cover slip added. cell culture was examined at 1000x magnification using a phase contrast microscope and epiflourescence microscope. when viewed under epiflourescent illumination, neutral lipid and oil of microalgae stained with nr will show a bright yellow/orange color (carman1991; qin 2005). 100 biotropia vol. 17 no. 2, 2010 the growth of microalgal culture was determined daily based on the appearance of green or brown color on the medium. the cell density was measured with a spectrophotometer using 680 nm wavelengths (lee et al. 1998). the terminal optical density (od) will mark at 0.2, at this point the alga biomass is equivalent to 0.37g of dry weight per liter (qin 2005). the isolates were screened based on the period of time to reach od 0.2. the total lipid production of the selected strains was measured by gravimetry methods. results and discussions sampling sample collection was conducted in west and central java from april 10 until april 14, 2008. samples were taken from diverse ecosystems i.e. sampling in mountain ecosystem in west java (gunung gede, gunung tangkuban perahu), volcanic lakes (crater of tangkuban perahu, pre-historic crater of bledug kuwu), rivers, lakes, ponds, and rice fields in west and central java, as well as swampy areas in northern part of central java. sampling has also been conducted in two agricultural dams i.e. waduk sempor, kebumen and waduk kedung ombo, purwodadi. a number of 355 water samples have been collected from those areas. the samples and sampling location are shown in table 1. table 1. samples and sampling locations code of samples sampling locations characteristic cp7041-cp7050 ciputri, pacet, cianjur, west java water from paddy fields and ponds sk7051-sk7055 sukamantri, karangtengah, west java water from paddy fields cb7056-cb7060 ciburung, padalarang, west java water from dam tp7061-tp7080 tangkuban perahu mountain, west java water from crater, hot-sulfuric water jl7081-jl7085 jaya giri, lembang, west java water from fishponds pt7086-pt7090 highway kopo, west java water from paddy fields cg7091-cg7095 limbangan street, garut, west java water from paddy fields ct7095-ct7100 ciawi, tasik, west java water from paddy fields be7101-be7110 bojongsari, cijenjing, ciamis, west java water from fishponds mh7111-mh7120 kota banjar, west java water from river pd7121-pd7130 panulisan, dayeuh harja, cilacap, central java water from paddy fields cr7131-cr7140 ciraja, kt pucung, cilacap, central java water from paddy fields jl7141-jl7145 jatilawang, banyumas, central java water from paddy fields kj7146-kj7155 kr.anyar, jatilawang, banyumas, central java water from paddy fields tb7156-tb7165 purwodadi, tambak, banyumas, central java water from paddy fields smp7166-smp7190 waduk sempor, kebumen, central java water from agricultural dam pk7191-pk7195 pekunden, kota winangun, kebumen, central java water from paddy fields pr7196-pr7200 purworejo, diy water from paddy fields tpk7201-tpk7205 taji, prambanan, diy water from paddy fields mc7210-mc7225 metese, ceper, klaten, diy water from paddy fields 101 lipid producing microalgae from several ecosystem dwi andreas santosa et al. code of samples sampling locations characteristic pw7226-pw7235 pakis, wadung getas, delanggu, klaten, diy water from paddy fields sg7236-sg7245 siwali, gondangrejo, central java water from paddy fields ls7246-ls7255 ngadul, sumbu lawang, sragen, central java water from paddy fields ko7256-ko7275 waduk kedung ombo, purwodadi, central java water from agricultural dam mt7276-mt7280 mayahan, tawangharjo, purwodadi, central java water from paddy fields bk7281-bk7295 bledug kuwu, grobogan, central java water from crater ks7296-ks7300 kuwu, grobogan, central java water from paddy fields tb7301-tb7310 tambaksari, blora, central java water from paddy fields ksr7311-ksr7315 kemadu, sulang, rembang, central java water from paddy fields bkr7316-bkr7340 banyudono, kaliori, rembang, central java water from fishponds, salt producing ponds kpr7341-kpr7350 karangpandan, rembang, central java water from fishponds gr7531-gr7360 growong, juwana, pati, central java water from paddy fields tn7361-tn7370 teban, njekulo, kudus, central java water from paddy fields tkj7371-tkj7380 tanjang, karangjati, central java water from paddy fields yw7386-yw7390 jogoloyo, wono salam, demak water from paddy fields kd7391-kd7395 pangan, kandanghaur, indramayu water from paddy fields table 1. continued isolation and screening of algae microalgae were isolated from samples collected from rivers, lakes, brackish and freshwater ponds, paddy fields, wetland soils in west and central java. the method and suitable medium for the isolation of microalgae were developed during this research. the major source of microalgae isolation (155 samples) came from paddy fields. a total of 355 samples have been used for algae isolation and 267 isolates of microalgae were obtained. some samples produced colony of algae quite fast and the medium became green after 2-3 weeks, while other samples grew slowly or did not grow. two medium have been used i.e. modified jorgensen's medium and atcc 617 medium. there were no differences between the two medium in supporting the growth of algae. the algal growth depended on the origin sample. the isolates obtained from this research were deposited at laboratory of soil biotechnology of ipb and the duplicates were deposited at icbb-culture collection of micoorganisms. distribution of microalgae based on the origin samples it was shown that origin samples play important role on the success of algae isolation. samples can be grouped into 5 : 1) paddy field, 2) river, 3) ponds, 4) agricultural dam, and 5) crater. most samples were collected from the paddy fields. the percentage of samples from paddy fields producing colony of algae is shown in table 2. 102 biotropia vol. 17 no. 2, 2010 table 2. the percentage and distribution of samples from paddy field that produced algal growth sampling site % sampling site % sampling site % west java central java cianjur 70 cilacap 85 purwodadi 80 karangtengah 60 banyumas 65 grobogan 80 bandung 80 kebumen 45 blora 90 garut 80 purworejo 70 rembang 80 tasik 83 prambanan 80 pati 63 indramayu 60 klaten 70 kudus 100 karanganyar 100 karangjati 70 sragen 80 demak 90 the geographical locations of sampling showed no significant difference in algal growth. there were also no differences between samples collected from west java and central java. samples collected from four sites produced microalgae in the range of 90 100 percent i.e. karanganyar, blora, kudus and demak. microalgae are abundant and can be found not only in paddy fields but also in all of this project studied ecosystems, both man-made ecosystems such as fish ponds and agricultural dams as well as natural ecosystems. interestingly, microalgae can also be isolated from harsh environment such as tangkuban perahu crater with very low ph (1-2) as well as bledug kuwu, a mud volcano crater that contains high concentration of salt. more than 60 percent of the samples, except samples collected from padalarang dam, produced microalgae after incubated in an appropriate media (table 3). table 3. percent growth of microalgae from different ecosystems sampling sites % sampling sites % volcano crater, tangkuban perahu 68 salt producing ponds, rembang 87 mud-volcano crater, bledug kuwu 79 river, banjar 67 dam, padalarang 20 fish pond, lembang 80 dam, sempor 64 fish pond, ciamis 60 dam, kedung ombo 78 fish pond, rembang 90 lipid producing microalgae around 267 strains of algae could be isolated from the samples. screening on lipid production of microalgae cell showed that 79 percent of the strains (212 isolates) have capability to produce lipid. those strains are then selected for further study in order to obtain the best strain which has fast growth and high lipid production to be developed further as a source of biodiesel. screening had been conducted on those strains and four promising isolates were selected i.e. pk-7195 produced 25.5 percent lipid, bk7291 produced 14.7 percent lipid, ko-7267 produced 35.7 percent lipid, and ks7300 produced 45.7 percent lipid under optimum condition. pk-7195 was isolated from a paddy field sample of pekunden, kota winangun, kebumen, central java; bk-7291 was isolated from a sample of mud-volcano crater of bledug kuwu, grobogan, central java; ko-7267 was from kedung ombo dam, purwodadi, central java and ks-7300 from sample of paddy field in kuwu, grobogan, central 103 lipid producing microalgae from several ecosystem dwi andreas santosa et al. java. based on the morphological characters, isolate ko-7267 and pk-7195 are identified as chlamydomonas sp., isolates ks-7300 identified as chlorella sp., while bk7291 is desmodesmus sp. acknowledgment authors would like to thank seameo-biotrop for funding this research. thanks are also due to the dean of faculty of agriculture, bogor agricultural university for his valuable advices and help in contractual terms. asih karyati, juleha, salmah shahab, and hartatik are acknowledged for the laboratory works. references borowitzka m.a. 1988. in” micro-algal biotechnology”. eds. borowitzka, m. a. and borowitzka l. j., p. 257287. cambridge university press, cambridge. carman k.r., thistle d., ertman s.c. and m. foy. 1991. nile red as a probe for lipid-storage products in benthic copepods. marine ecology progress series, 74: 307-311. full report workbook. 2004. statistical review of world energy. full report workbook. lee s.j., yoon b.d. and oh, h.-m. 1998. rapid methods for determination of lipid from the green botryococcus braunii. biotechnology techniques, 12:553-556. ministry of state for population and environment. 1992. indonesian country study on biological diversity. cooperation between the united nations environment programme, the republic of indonesia, and the kingdom of norway. miyamoto k. 1997. renewable biological system for alternative sustainable energy production. fao agricultural services bulletin-128. takagi m., karseno and t. yoshida. 2005. effect of salt concentration on intracellular accumulation of lipids and triacyglyceride in marine microalgae dumaliella cell. journal of bioscience and bioengineering. 101(3) : 223-226. qin j. 2005. bio-hydrocarbon from algae: impact of temperature, light and salinity on algae growth. a report for the rural industries research and development corporation. australian government. robert p. 2005. the end of oil. on the age of a perilous new world. a mariner book, boston. 104 biotropia vol. 17 no. 2, 2010 page 1 page 2 page 3 page 4 page 5 page 6 page 7 742 alimuddin (resistance) revisi.cdr resistance against aeromonas hydrophila infection and growth of (f2)second generation african catfish selected [clarias gariepinus] using molecular markers alimuddin , fadhila maharani putri , dinamella wahjuningrum , 1* 1 1 2 2 1 dian hardiantho , ade sunarma and sri nuryati 1 department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor, bogor 16680, indonesia 2 a main center for freshwater aquaculture, sukabumi 43114, indonesi received 15 december 2016 / accepted 01 august 2017 abstract aeromonas hydrophila is a pathogenic bacteria that causes mass mortality in catfish. in previous studies, specific pathogen resistant (spr), a. hydrophila-resistant african catfish first generation (f1) has been cultivated by marker assisted selection using the major histocompatibility complex (mhc) 1 as a molecular marker. in this study, growth performance, inheritance of the mhc dna marker in the second generation (f2) of catfish and disease resistance against a. hydrophila infection were observed. the f2 progenies were produced by crossing f1 fish between themselves. nursery was performed in 80-l glass aquaria, 4 replications for each cross, at the same initial density, for 2 months of rearing. the results showed that daily growth rate of f2 progenies from the spr broods was significantly higher than those from broods without the marker. results of the pcr analysis showed that average number of f2 progenies from spr broods carrying the mhc marker was about 91% higher than that of control. after the fish 6 reached about 12 cm body length, they were challenged by intramuscularly injecting of 0.1 ml a. hydrophila (ld : 10 50 -1 cfu ml ) for 7 days. results of challenge test showed that survival of f2 offspring from the crosses of spr broods (77.2%) was about two times higher than those from brood without mhc marker (38.3%). differential leukocyte count supported the high resistance of f2 progenies from f1 broods having mhc i marker against a. hydrophila infection. in conclusion, african catfish farming carrying mhc marker potentially have higher productivity and reduces fish lose due to infection by a. hydrophila. keywords: aeromonas hydrophila, african catfish, disease resistance, growth, mhc i introduction african catfish (clarias gariepinus) is one of indonesia's leading freshwater aquaculture species (mmaf 2016). efforts to increase african catfish production is continuously done through aquaculture intensification. however, farmers often suffer loss from low survival rate, whether during nursery or growing phase. low survival is usually caused by aeromonas hydrophila bacterial infection which can result in 90% mortality in catfish (zhang et al. 2016). genetically specific pathogen resistant african catfish cultivation can be an alternative in preventing loss caused by the infection of this disease. cultivating disease resistant catfish can be done through conventional selection, however, this method takes relatively long time; for example, dropsy-resistant kasnodar common carp (cyprinus car pio) development took 9 generations (kirpichnikov 1999). the alternative effort is molecular marker assisted selection. this method has been successfully utilized to develop majalaya variety of common carp resistant against khv infection (alimuddin et al. 2011), and khv resistant common carp variety has passed variety evaluation test and spread to farmers in 2015. in our previous research through challenge test, differences of endurance against a. hydrophila infection was found in african catfish (azis et al. 2015a). pcr analysis with specific primer for * corresponding author: alimuddin@apps.ipb.ac.id biotropia 5 2 8 95 102 vol. 2 no. , 201 : 10.11598/btb.2018.25.2.742 95 major histocompatibility complex i (mhc i) gene showed that there are differences between fishes that lived or died post a. hydrophila bacterial infection (azis et al. 2015a). mhc's role in the immune system is to represent antigen. with this result, we suggested that mhc i can be used as a molecular marker of a. hydrophila infection resistant african catfish. afterward, challenge test with a. hydrophila against the first generation (f1) of crossing between fishes exhibiting molecular marker showed that the resistance can be inherited. the survival rate is about 2.2 times higher than f1 of fish with no molecular marker. mhc i molecular marker can be inherited by f1 generation with 62.5-83.4% percentage, while for control its only 25.0% (azis et al. 2015b). in this research, second generation (f2) of african catfish was produced by crossing f1 fishes exhibiting mhc i markers. the purpose of this research s to evaluate marker inheritance, wa resistance a. hydrophila against and growth during nursery. materials and methods identification of f1 broods carrying mhc marker identification of fish having the mhc i marker was conducted by following azis et al. (2015a) method. a total of five pairs matured broods were randomly taken from the pond. their genomic dna was extracted from fin tail tissue using dna isolation kit (puregene, minneapolis, usa) by following manufacturer instruction. dna was diluted with 50ml sterile distilled water (sdw). purity and dna content was measured using spectrophotometer (gene quant) at 260 nm and 280 nm wavelength. pcr amplification was performed at 25 ml final reaction volume. the solution of reaction consisted of 2.50 l 10x pcr buffer, 2 l dntp μ μ mix, 2 l of forward and reverse clmhah-01 μ primers, 0.25 l dna polymerase (kapa μ taq biosystems), 1 l genomic dna and 17.25 l μ μ sdw. pre-denaturation of pcr process was performed at 94°c for 3min, 35 cycles of amplification with denaturation at 95°c for 30 sec, annealing at 68°c for 20 sec, and extension at 72°c for 30 sec, and a final extension at 72°c for 5 min. pcr products were separated by electrophoresis 1.5% (w/v) agarose gel, at 70 on volts for 90 min. dna was visualized using red gel (biotum inc. california, us) and uv transilluminator. pr oduction and maintenance of f2 generation three pairs of f1 african catfish broodstock carrying mhc i marker and sexually mature were chosen. ovulation and spermiation were induced by injecting ovaprim (syndel laboratories ltd) -1 with 0.2 mlkg dosage. artificial fertilization was done by mixing eggs with sperm in a plastic container. f2 progeny was also developed by mating catfish broodstocks that did not exhibit mhc i marker as a control. eggs were incubated in 80 liter aquarium. african catfish were reared for 2.0 months, at the same initial density. fishes were given tubificid as live food from 3 days until 30 days after hatching (dah), ad libitum. in 28-32 dah, in addition to tubificid, fishes were also fed with pf-1000 commercial feed until 60 dah. afterward the fishes were fed hi-provit commercial feed until end of research. commercial feed was given three times daily, at satiation. to keep good water quality, aquarium water replacement was conducted for 80% of total amount every two days. water quality was measured before and after water replacement. oxygen and ammonia concentration was measured by titration method (apha 2005), whereas temperature and ph were directly measured by using thermometer and phmeter. analysis of marker inheritance and growth performance f2 progenies having the mhc i marker identification was carried out by pcr method as described above. mhc i marker identification was conducted on 20 dah f2 fishes. a total of 30 individual f2 progenies were randomly taken from each cross to determine mhc i marker inheritance. growth performance test on f2 progenies was conducted for two months. a total of 30 fishes (body weight 0.11±0.03 g) were taken from each cross which carried and did not carried mhc i marker. three replications were taken from each cross. weighing were conducted every two weeks. 96 biotropia vol. 25 no. 2, 2018 survival we . . of a, b and c cross re 76 7±7 6%, 80 0± 5 0% and 75 0±5 0%, respectively. . . . . meanwhile, negative control fish from every cross injected with pbs all survived. this showed that fish mortality was a result of a. hydrophila bacterial infection. our previous research has produced f1 afican catfish through mhc i molecular marker based selection (azis et al. 2015b). in this research, f2 african catfish were challenged against aeromonas hydrophila infection. f2 african catfish resistant was consistent with the f1 fish, in which the offsprings of broodstock exhibiting mhc i marker have higher resistance compared to fishes whose broodstocks did not (table 1). the high survival of f2 fish post a. hydrophila challenge was consistent with the percentage of individuals carrying the marker, which was higher compared to fishes whose parent did not carry mhc i marker. the number of f2 fish from marker exhibiting parent cross was approximately 2 times higher compared to those whose parent did not exhibit mhc i marker (table 1). the inheritance of marker in f2 fishes (77.0-97.0%) was relatively similar to what was reported in f1 fish by azis et al. (2015b), which is 62.6-83.4%. this suggests that the mhc i marker was passed on to f2 offsprings. similar pattern has also been found in the inheritance of cyca-dab1*05 mhc ii marker in indonesian common carp resistant to pathogen infection; 70.0% and 83.3% in the f1 and f2 generation, respectively (decree of marine and fishery minister no.24/kepmenkp/2015). mhc consists of many genes and is polymorphic (rakus 2008). the oligonucleotide primer used in this study generates three dna bands of pcr amplification product (azis et al. 2015b). these three dna pcr products are suspected to be different genes of the mhc i group (azis et al. 2015a). the selection performed on african catfish has not yet utilized one of those genes, thus the inheritance of the mhc i gene marker did not follow mendelian segregation pattern. in addition, f2 fish from parents without mhc i marker were also found to be positive for the dna marker. the number of f2 fish suffering from ulceration was fewer than in fishes whose parent did not exhibit mhc i marker. the same occurrence was found in f1 fish (azis et al. 2015b). dna analysis by pcr revealed that 76% the number of fish was counted at the end of the research to determine survival rate. c h a l l e n g e t e s t a g a i n s t a e r o m o n a s hydrophila aeromonas hydrophila isolate was obtained from fish health laboratory, department of aquaculture, ipb. a total of 20 fishes (total body length 11-12 cm) of each cross were challenged by intramuscularly injecting 0.1 ml of a. hydrophila 6 -1 (ld : 10 cfu ml ; this was obtained from our 50 preliminary study). negative control fishes were injected with 0.1 ml phosphate buffer saline. the fish were kept in 80-l volume glass aquaria and each cross had three aquariums as replications. observations during challenge test treatment involved erythrocyte count, leucocyte count and differentiation, hematocrit, clinical signs, and mortality. blood samples of nine fishes from each cross were taken before and after challenge. the number of erythrocytes and leukocytes were analyzed by using the method of blaxhall and daisley (1973), and hematocrit by anderson and siwicki (1995). data analysis growth rate, survivability and blood count were analyzed with anova with the help of microsoft excel 2011 and spss version 21.0 program with 95% confidence interval. data that were significantly different was analyzed further by duncan multiple range (dmr) test. mhc i marker inheritance data on f2 generation, and quality of water rearing were analyzed by descriptive statistics. results and discussion mhc i marker inheritance and post challenge survivability utilizing method introduced by azis et al. (2015b), mhc i marker inheritance percentage from marker exhibiting broods obtained values ranging between 77.0-97.0%, whereas broods tock without marker (control) had 46.6% (table 1). challenge test result showed that of f2 survival african cat from mhc i marker exhibiting fish broods s higher (p<0.05) compared to tocks wa control without marker (cross d: 38 3±7 6%). the . . growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. 97 of dead fish from parents not exhibiting mhc i marker (n=25) did not carry mhc i marker (data not shown). this showed a strong correlation between the presence of mhc i marker with fish survival against a. hydrophila infection. blood count and clinical symptoms blood count analysis result is presented in table 2. generally, erythrocyte count, leucocyte count and hematocrit from fishes of all cross before challenge were similar. erythrocyte count of african catfish before challenge ranged between 2.13-2.83 (×10 cell mm ), leucocyte 6 -3 count was about 2.93-4.35 (×10 cell mm ) and 4 -3 hematocrit around 35.80-39.40%. after the challenge, leucocyte count between challenged crosses was similar, and each lower (p<0.05) compared to control that were not challenged by a. hydrophila or only injected by pbs (ka, kb, kc, and kd). meanwhile, hematocrit level of all cross challenged by bacteria suffered decrease compared with pbs injected control (table 2). leucocyte counts of fishes injected by pbs (ka, kb, kc and kd) were lower compared to a, b, c and d cross that were challenged (table 2). test obefore the challenge , bl od count analysis showed that f2 african catfish were hea thy. it l was shown by the er throcyte count, leucocyte y count, and hematocrit which s within the range wa of healthy catfish (table ). the er throcyte 2 y count range of healthy catfish is 2.0-3.0 (×10 6 cellmm ) (hastuti & subandiyono 2015), total -3 leucocyte count ranged 1.83-4.22 (x10 cell/mm ) 4 3 (triyaningsih 2014) and hematocrit value et al. between 30.8-45.5% (yanto 2015). mmune et al. i response against bacterial of f2 fish a. hydrophila infection can be inferred from complete blood count parameter. after challenge, er throcyte y count decreased to 1.50-1.82 (×10 cellmm ), and 6 -3 hematocrit value to 20.2-25.0% (table ). ulcer in 2 infected fish caused blood vessel to burst and decrease er throcyte count. ul eration s the y c wa result of bacterial hemolysin toxin on fish's body surface (del coral . 1990). in contrast, as et al shown in table , after the challenge, leucocyte 2 count increased to 6 11-7 04 (×10 cellmm ). 4 -3 . . this increase is the fish's response to defending table 2 erythrocyte count, leucocyte count, and hematocrit of second generation african catfish from parents exhibiting mhc i marker before and after aeromonas hydrophila injection treat ment erythrocyte (x106 cell/mm3) leucocyte (x104 sel/mm3) hematocrit (%) prechallenge postchallenge prechallenge postchallenge prechallenge postchallenge a 2.38 ± 0.09ab 1.58 ± 0.11a 4.35 ± 0.09a 6.97 ± 0.37 a 35.80 ± 1.02a 23.20 ± 3.22a b 2.42 ± 0.43ab 1.82 ± 0.79a 3.66 ± 0.38 a 6.27 ± 0.87 a 37.20 ± 1.95ab 25.00 ± 0.69 a c 2.13 ± 0.25 a 1.50 ± 0.33 a 3.87 ± 0.25 a 6.11 ± 0.84 a 36.60 ± 0.65 ab 22.50 ± 3.21 a d 2.64 ± 0.44 ab 1.74 ± 0.11 a 3.95 ± 0.96 a 7.04 ± 0.72 a 37.60 ± 2.17 ab 20.20 ± 4.27 a ka 2.63 ± 0.38 ab 3.09 ± 0.17 b 4.07 ± 0.64 a 3.27 ± 1.06 b 37.70 ± 1.14 ab 33.30 ± 3.89 b kb 2.61 ± 0.33 ab 3.27 ± 0.21 b 3.75 ± 0.26 a 3.05 ± 0.33 b 37.70 ± 2.29 ab 38.80 ± 1.89 b kc 2.58 ± 0.30 ab 3.32 ± 0.11 b 3.32 ± 0.11 a 3.26 ± 0.44 b 38.90 ± 0.93 b 40.60 ± 2.82 b kd 2.83 ± 0.16 b 2.93 ± 0.13 b 2.93 ± 0.13 a 2.98 ± 0.04 b 39.40 ± 0.55 b 38.80 ± 2.65 b note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. ka, kb, 3 3 kc, and kd respectively are fish from a, b, c, and d crossing injected by phosphate buffer saline (pbs). different superscript letter in the same coloumn showed a significant difference (p<0.05) biotropia vol. 25 no. 2, 2018 98 table 1 percentage of mhc i marker inheritance and survival of african catfish second generation post aeromonas hydrophila bacterial infection parameter crosses a b c d fish carrying the marker (%) 93.0 77.0 97.0 46.7 survival rate (%) 76.7 ± 7.6 b 80.0 ± 5.0 b 75.0 ± 5.0 b 38.3 ± 7.6 a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. different 3 3 superscript letter in the same row showed a significant difference (p<0.05). against pathogen infection (li . 2013; hastuti et al & subandiyono 2015). leucocyte differentiation was only observed post-infection (table 3). lymphocyte count of fishes challenged by a. hydrophila bacteria was lower compared to those injected by pbs (p<0.05). lymphocyte count of d cross fish (62.00± 2.52%) challenged by a. hydrophila was lower (p<0.05) compared to a cross (72.00 ± 2.08%), b cross (75.00 ± 6.00%), and c cross (79.00 ± 1.53%). neutrophyl count of fish injected with pbs (4.00-7.00 %) was lower compared to those challenged by a. hydrophila (p<0.05). between challenged crosses, d cross fishes (26.00 ± 2.52%) had higher neutrophyl count compared to a cross (18.00 ± 4.51%), b (16.00 ± 3.79%), and c cross (12.00 ± 3.00%). monocyte count was also higher in fishes challenged by a. hydrophila bacteria (p<0.05), and monocyte count between all crosses were similar (p>0.05). significantly different leucocyte profile between f2 fish from broods exhibiting tock marker and those from broods that did not are leucocyte differentiation, especially lymphocyte and neutrophyl (table ). the decrease of 3 lymphocyte in fish whose parents exhibiting mhc i marker post bacterial infection is lower compared to those whose parent did not exhibit mhc i marker. the lymphocyte decreased in count after challenge in f2 fishes whose parent exhibit mhc marker s lower compared to wa those whose parent did not exhibit mhc i marker. in contrast, the increase in neutrophyl count of their progenitor did not exhibit mhc i marker was higher compared to those whose parent exhibit mhc i marker. the difference in lymphocyte and neutrophyl count indicated the potential of using leucocyte differential parameter to identify fishes resistant to pathogenic bacterial infection. in addition, difference in lymphocyte and neutrophyl count showed their role in a. hydrophyla bacteria phagocytosis that resulted in f2 fish exhibiting mhc i marker showing higher (hastuti survival & subandiyono 2015). lymphocyte count in healthy catfish ranges between 80.0-90.0%, monocyte less than 15.13%, and neutrophyl about 6.0-8.0% (andayani et al. 2014). the lymphocyte count of fish challenged by bacteria (62.0-79.0%) s less compared to wa unchallenged fish (88.0-93.0%). this is in line with what was reported by ( ), martins et al. 2008 that lymphocyte percentage decreased when an individual is suffering from disease . infection neutrophyl of fish that were not challenged (4.07.0%) s lower compared to fishes that were wa challenged (12.0-16.0%). neutrophyl has an active role in first phase of inflammation. neutrophyl would be produced around the first 6 hour after inflammation (kuby 1997). thus, neutrophyl count would increase if inflammation caused by aeromonas hydrophila bacterial infection occurs. f2 fish percentage with inflamation was a bit lower compared to fish whose parent did not exhibit mhc i marker. this support the lower neutrophyl count compared to fish whose parent did not exhibit mhc i marker. monocyte count of fishes that were challenged was higher (9.014.0%) compared to monocyte count of fish injected by pbs (2.0-5.0%). the increase in monocyte count showed non-specific immune table 3 lymphocyte, neutrophil, and monocyte percentage of second generation african catfish from parents exhibiting mhc i marker challenged by aeromonas hydrophila bacteria treatment leucocyte differentiation (%) lymphocyte neutrophyl monocyte a 72.00 ± 2.08b 18.00 ± 4.51c 10.00 ± 3.46cd b 75.00 ± 6.00 bc 16.00 ± 3.79 bc 9.00 ± 2.89 bc c 79.00 ± 1.53 c 12.00 ± 3.00 b 9.00 ± 3.79 bcd d 62.00 ± 2.52 a 26.00 ± 2.52 d 14.00 ± 3.61 d ka 88.00 ± 3.61 d 7.00 ± 1.53 a 5.00 ± 3.06 a kb 93.00 ± 1.53 d 4.00 ± 0.58 a 4.00 ± 1.15 ab kc 91.00 ± 2.08 d 5.00 ± 0.33 a 2.00 ± 1.00 a kd 91.00 ± 0.58d 6.00 ± 0.01a 3.00 ± 0.58a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. ka, kb, 3 3 kc, and kd respectively are fish from a, b, c, and d crossing injected by phosphate buffer saline (pbs). different superscript letter in the same column showed a significant difference (p<0.05). 99 growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. response from catfish's body against h a. ydrophila pathogen. clinical symptoms observation was conducted 24-96 hours post challenge. observed clinical symptoms were ulceration on skin and dropsy (edema) by the under belly of african catfish. from visual observation, about 40% of f2 offsprings of broods exhibiting mhc i marker suffered ulceration, however all control fish suffered ulceration. other studies support the assertion that f2 fish from parents carrying the mhc i marker has a higher resistance than that of from parents without the marker. clinical symptoms shown by f2 catfish were similar with those reported by previous research. clinical symptoms can be observed in 12-96 hours post infection. clinical symptoms commonly observed in 24 hours post infection were swelling skin and inflammation around injection area of a. hydrophila. afterward, fish would show dropsy, followed by ulceration (wulandari et al. 2014). the behavior of fishes during challenged test were lowering appetite and irregular swimming pattern. fishes tend to hang floating in the surface or passively at the bottom of the aquaria. most of the fishes died with damaged fin and exuded mucus. according to rey et al. (2009), fish infected by aeromonas hydrophila will show dropsy or liquidfilled swelling belly, damaged meat or ulcer indicated by skin injury, and loss of blood. infected fish will also show clinical symptoms such as irregular swimming or fish damage. weight gain and survival rate during nursery f2 fish body weight gain and survival rate during nursery are presented in table 4. the initialbody weight of fishes reared for growth rate test was 0.11±0.03 g. the results showed that daily growth rate (dgr) from two mhc i marker exhibiting f2 cross (cross b: 7.87% day and c: -1 8.32% day ) was higher (p<0.05) compared to -1 control which did not exhibit the marker effect (d cross: 7.15% day ). however, dgr of fish cross -1 a was the same as cross d (p>0.05). meanwhile, survival rate of all treatment fish was the same (p>0.05). the water quality during nursery was 24.7-30.30c temperature, 7.40-7.60 mg l -1 dissolved oxygen, 0.02-0.95 mgl ammonia and -1 6.4-7.70 ph. growth and determine aquaculture survival productivity level. in this research, f2 generation of african catfish was nursed in aquar with ia relatively good water quality. in that condition, f2 african catfish from parents with mhc i marker (crosses b and c) had higher weight gain, while the was the same with african catfish survival with those that did not exhibit the marker (table 4). thus, cultivation of african catfish exhibiting mhc i marker has the potential in having higher productivity. however, weight gain vary among crosses, necessary to choose parent therfore, its that produces higher growth. in addition, it is most likely that no link between weight gain and p r e s e n c e o f m h c m a r ke r i n p a r e n t . furthermore, a. hydrophilaif bacterial infection exist, farmers that rear african catfish exhibiting mhc i marker has greater probability in getting higher harvest compared to those with fish without mhc i marker. the statement is supported by the result of this research, through a. hydrophila survival challenge with 2 higher compared to f2 fish whose parent did not exhibit the mhc i marker. this result is relatively consistent with what was reported by azis . et al (2015b) in f1 generation of african catfish. this result also showed that mhc i marker expressing african catfish's is stable. in resistance table 4 daily growth rate (dgr) and survival rate of second generation african catfish that exhibited mhc i marker and that did not exhibit mhc i marker, in relation to their endurance against aeromonas hydrophila bacterial infection while kept in 80-l volume aquaria for 2.0 months parameter crosses a b c d dgr (%/day ) 7 .53 ± 0.57 ab 7 .87 ± 0.28 bc 8 .32 ± 0.60 c 7 .15 ± 0.18 a survival rate (%) 90 .00 ± 3.33 a 98 .30 ± 3.33 a 95 .80 ± 3.85 a 96 .70 ± 3.30 a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. different 3 3 superscript letter in the same row showed a significant difference (p<0.05). biotropia vol. 25 no. 2, 2018 100 accordance to varieties from breeding in indonesia, at least generation required to test f3 is the performance consistency of mhc i marker exhibiting african catfish before it could be released in fact khv resistant for public use. the common carp strain developed through mhc ii marker based selection (alimuddin . 2011) has et al pass releas as a new fish strain at third generation in 2015. conclusion second generation african catfish from parents exhibiting mhc i marker is known to have higher growth rate and resistant against aeromonas hydrophila bacterial infection compared to fish whose parents did not exhibit mhc i marker. mhc i marker is inherited by f2 generation, similar percentage to what is found in f1 generation. further study is needed to observe whether the oligonucleotide primers anneals to another gene that having the same nucleotide at 3', and the pcr products are the same gene as that found in f2 fish from parents carrying the mhc i marker. acknowledgements this research was partially supported by psu research grant no.: 627/it3.11/ pn/2016 from ministry of research and higher education, indonesia. references alimuddin, mubinun, santika a, carman o, faizal i, sumantadinata k. 2011. identification of the majalaya common carp strain resistance to khv infection using cyca-dab1*05 allele as a marker. indonesian aquaculture journal 6(2):157-63. american public heatlh association (apha) [internet]. 2005. standard methods for the examination of water and wastewater. [cited 2016 aug 28]. available from: http://www.mwa.co.th/download/file_ upload/smww_1000-3000.pdf andayani, marsoedi s, sanoesi e, wilujeng ae, suprastiani h. 2014. profil hematologis beberapa spesies ikan air tawar budidaya. [hematology profile of freshwater fish species]. national conference – green technology 3. p. 363-5. anderson dp, siwicki ak. 1995. basic hematology and serology for fish health programs. manila (ph): fish health section, asian fisheries society. azis, alimuddin, sukenda, zairin mjr. 2015a. identifikasi k a n d i d a t m a r k a m h c i p a d a i k a n l e l e (clarias sp.) tahan infeksi aeromonas hydrophila. [identification of mhc i marker candidate in catfish (clarias sp.) resistance to aeromonas hydrophila infection]. jurnal riset akuakultur 10(2):261-9. azis, alimuddin, sukenda, zairin mjr. 2015b. mhc i molecular marker inheritance and first generation catfish (clarias sp.) resistance against aeromonas hydrophila infection. pak j biotechnol 12(2): 131-7. blaxhall pc, daisley kw. 1973. routine hematological methods for use with fish blood. j fish biol 5(6):771-81. del coral f, shotts eb, brown j. 1990. adherence haemagglutination and cell surface characteristics of motile aeromonads virulent for fish. j fish dis 13(4):255–68. hastuti s, subandiyono. 2015. catfish (clarias gariepinus burch) well-being which maintained with biofloc technology. ijfst 10(2):74-9. kirpichnikov vs. 1999. genetics and breeding of common carp. paris (fr): inra editions. kuby j. 1997. immunology. california (us): w.h freeman and company. li c, wang r, su b, luo y, terhune j, beck b, peatman e. 2013. evasion of mucosal defenses during aeromonas hydrophila infection of channel catfish (ictalurus punctatus) skin. dev comp immunol 39(4):447-55. martins ml, mouriño jlp, amaral gv, vieira fn, dotta g, jatobá amb, … pereira-jr g. 2008. h a e m a t o l o g i c a l c h a n g e s i n n i l e t i l a p i a experimentally infected with enterococcus sp. braz j biol 68(3):657-61. ministry of marine affairs and fisheries republic of indonesia (mmaf) [internet]. 2016. mmaf developed environmental friendly method in catfish farming with biofloc. [cited 2016 aug 28]. available from: http://www.djpb.kkp.go.id/arsip/c/451/ kkp-kembangkan-teknologi-budidaya-ikan-leler a m a h l i n g k u n g a n m e t o d e b i o f o k u n t u k keberlanjutan/?category_id= rakus lk. 2008. major histocompatibility (mh) polymorphism of common carp link with disease resistance [thesis]. retreived from wageningen university. rey a, verjan n, ferguson hw, iregul c. 2009. pathogenesis of aeromonas hydrophilla strain kj99 infection and its extracelullar products in two species of fish. vet rec 164(16):493-9. doi: 10.1136/vr.164.16.493 101 growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. triyaningsih, sarjito, prayitno sb. 2014. pathogenicity of aeromonas hydrophila isolated from catfish (clarias gariepinus) derived from boyolali. jamtech 3(2):117. wulandari a, prayitno sb, sarjito. 2014. pathogenicity of k14 isolate which isolated from catfish (clarias gariepinus) derived from demak. jamtech 3(2):1439. yanto h, hasan sunarto. 2015. studi hematologi untuk h, diagnosa penyakit ikan secara dini di sentra produksi budidaya ikan air tawar sungai kapuas kota pontianak. [early fish disease diagnose through haematological analysis in main center for freshwater aquaculture, kapuas river, pontianak]. jurnal akuatika 6(1) 11-20.: zhang d-h, shoemaker experimental d, xu c. 2016. induction of motile in channel aeromonas septicemia catfish ( ) by waterborne challenge ictalurus punctatus w aeromonas hydrophila. ith virulent aquaculture reports 3 18 23. : biotropia vol. 25 no. 2, 2018 102 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 808 endah retnaningrum (production) revisi.cdr production and characterization of biosurfactants produced by pseudomonas aeruginosa b031 isolated from a hydrocarbon phytoremediation field 1* 2 endah retnaningrum and wahyu wilopo 1 faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2 department of geological engineering, faculty of engineering, universitas gadjah mada, yogyakarta 55281, indonesia received 31 january 2017 / accepted 29 january 2018 abstract the biosurfactants are used by several industrial sectors such as petroleum, agriculture, food production, chemistry, cosmetics, and pharmaceuticals. because of their hydrophobic and hydrophilic moieties, they have potency to reduce surface tension, interfacial tension between water-hydrocarbon systems, and low micelle concentration. their characteristics strongly depend on the producer strain as well as on the medium composition, such as carbon and nitrogen sources. this study was conducted to investigate the influence of different sources of carbon (n-hexadecane, glycerol and glucose) and nitrogen (urea, nh cl and nano ) for the production of biosurfactants by a new strain of 4 3 pseudomonas aeruginosa b031 isolated from a rhizosphere of paraserianthes falcataria l. nielsen, a hardwood plant species at a phytoremediation field. the biosurfactant characteristics of the strain were evaluated, particularly its surface-active properties and potential to remove hydrocarbon. glycerol was found to be the optimum carbon source, with rhamnose concentration, emulsification index, and critical micelle concentration (cmc) of 718 mg/l, 37%, and 35 mn/m, respectively. sodium nitrate (nano ) was observed as the optimum nitrogen source, with rhamnose 3 concentration, emulsification index, and cmc of 290 mg/l, 30%, and 24 mn/m, respectively. these biosurfactants efficiently reduced surface tension of culture broth from 42 mn/m to 31 mn/m for the glycerol treatment and from 37 mn/m to 24 mn/m for the sodium nitrate treatment. the crude biosurfactants from the glycerol and sodium nitrate treatments also removed 87.5% and 84%, respectively, of crude oil from sand. these rates were higher than those of the chemical surfactants (sds and triton x-100). these findings indicate that the biosurfactants produced by the strain from both glycerol and nano treatments can efficiently decrease the interfacial tension of culture broth 3 dilution and have a high emulsion index, thus hold promise in hydrocarbon bioremediation application. keywords: bioremediation, biosurfactant, glycerol, nano , optimum3 introduction biosurfactants are amphiphatic compounds produced by a wide variety of microorganisms that either adhere to cell surface or are excreted extracellular in the growth medium (al-bahry . et al 2013; geetha . 2018). they contain et al hydrophobic and hydrophilic moieties that reduce surface tension, interfacial tension between waterhydrocarbon systems, and low micelle concentration (zdziennicka & jańczuk 2018). as such, biosurfactants are used by several industrial sectors such as petroleum, agriculture, food p r o d u c t i o n , ch e m i s t r y, c o s m e t i c s, a n d pharmaceuticals (lai . 2009; pacwa-et al plociniczak . 2011). they could be used also to et al remove hydrocarbon contamination (mnif & ghribi 2015; ma . 2018).et al biosurfactants are composed of lipopeptides, glycolipids, phospholipids, fatty acids, neutral lipids, and polymeric biosurfactants (moya et al. 2015). often produced during the stationary phase of bacterial growth, they exhibit considerable substrate specificity (calvo et al. 2009). their characteristics strongly depend on the producer strain as well as on the medium * corresponding author: endahr@ugm.ac.id biotropia 5 2 8 130 139 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.808 130 composition, such as carbon and nitrogen sources (janek et al. 2010; liu et al. 2015). microbial strains belonging to species bacillus sp. (barakat . 2017), (rufino et al candida lipolytica et al rhodococcus et al. 2011), sp. (shavandi . 2011), pseudomas et al et alsp. (thavasi . 2011; sakthipriya . 2015) and (burgos-diaz sphingobacterium detergens et al. 2013) have been reported to produce biosurfactants. of these, the rhamnolipids biosurfactants produced by sp. is of pseudomonas interest in that it could remove hydrocarbon contamination (deepika . 2016; ma . 2016; et al et al ma . 2018). due to economic considerations in et al the industry, biosurfactants are commonly applied as whole-cells culture or crude biosurfactant. as reported by patowary . (2018), purification et al process of biosurfactants consumed almost 60% of the total production costs. a phytoremediation field in balikpapan, east kalimantan, indonesia, uses paraserianthes falcataria l. nielsen, a hardwood plant species, for hydrocarbon removal. previous studies show that plants for phytoremediation could remove hydrocarbon pollutants through mechanisms such as biodegradation, phytovolatilization, accumulation, and transformation (kong . et al 2016; guo . 2017). when soil hydrocarbon et al concentration in the phytoremediation field was measured in may 2014, it was found that the plants removed 50% of the hydrocarbon pollution within 3 months. in comparison, other hardwood plants, such as and , tectona grandis gmelina arborea are reported to remove only 10% and 15%, respectively (agbogidi . 2007; yenn . 2014). et al et al the differences in the hydrocarbon removal ability are influenced by the plant species, which create unique habitats for the soil microbial population (cristaldi . 2017). that is, the soil et al microbial population is strongly influenced by the plant species used for phytoremediation. the process of removing hydrocarbon in a phytoremediation field results from the complex interaction between roots, rhizosphere and soil microorganisms (khan . 2013; fatima . et al et al 2018). these association induce the particular physiology and biochemistry of plant roots, resulting in the process of hydrocarbons removal. as such, phytoremediation processes can make nutrients available, such as carbon and nitrogen (hou . 2015). glucose, a carbon source, is et al produced during photosynthesis, and is exuded into the soil by plant roots (khan . 2013). on et al other hand, the degradation process of hydrocarbon contamination in the soil environment induces n-hexadecane and glycerol, which are carbon sources also (varjani 2017). during the phytoremediation process, adding n fertilizer (e.g., urea, nh cl, nano ) enhances 4 3 hydrocarbon degradation because nitrogen becomes available to both plant and soil microorganism. a strain of pseudomonas aeruginosa b031, which was isolated from the rhizosphere of paraserianthes falcataria l. nielsen, probably has unique characters, as shown by thavasi et al. (2011) and sakthipriya et al. (2015), especially in terms of biosurfactant production. in this regard, it is i m p o r t a n t t o i n ve s t i g a t e t h e m e d i u m compositions involving variations in carbon sources (n-hexadecane, glycerol, and glucose) and nitrogen sources (urea, nh cl and nano ) for 4 3 the production of biosurfactants by pseudomonas aeruginosa b031. the characteristics of the strain were evaluated, particularly its surface-active properties and potential to remove hydrocarbons. materials and methods biosurfactant-producing bacterial strain a biosurfactant-producing bacterial strain belonging to pseudomonas aeruginosa b031 was isolated from a hydrocarbon phytoremediation field in balikpapan, east kalimantan, indonesia. the strain was isolated and screened using methods described by hassanshahian and emitazi (2008). it was identified following the methods described in bergey's manual systematic of bacteriology (brenner et al. 2005). this bacterial isolate was then sub-cultured on luria bertani medium agar tubes as a stock culture before use as inoculum. inoculum propagation p. aeruginosathe strain stock culture of b031 was inoculated into a plate containing luria bertani and incubated at 37ºc. after 24 h, one loop of culture was inoculated to 50 ml of stone mineral salt solution (smss) medium in a 250 ml erlenmeyer flask. the culture was then incubated at 37ºc for 18 h and agitated at 200 rpm in a shaking incubator (thorsen . 2013). at the et al end of the incubation period, a culture sample 131 biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo prepared using different concentrations of rhamnose (vinogradov et al. 2016). emulsification index (e24) the emulsification index of the culture samples was determined using the method described by panjiar et al. (2015). two ml of hexadecane and 2 ml of the cell free supernatant were mixed in a test tube and homogenized in a vortex at 3,500 rpm high speed for 2 min. the emulsification stability was measured after 24 h. the emulsification index was calculated as follows: e24 = x 100 height of emulsion formed (cm) total height of solution (cm) surface tension measurement the surface tension of the cell-free supernatant was measured in a k6 tensiometer (shimadzu), using the du nouy ring method. the values reported are the mean of three measurements (joshi & shekhawat 2014). critical micelle concentration (cmc) cell-free culture media of different carbon and nitrogen sources were diluted into various extents with sterile medium to analyze for surface tension (1/128, 1/64, 1/32, 1/16, 1/8, ¼, ½, and 1). the dilutions at which a surfactant begins to aggregate and had no further significant reduction in surface tension were assessed as cmcs. these measurements were estimated graphically by plotting the surface tension values versus the culture broth dilution values (cheng 2013). hydrocarbon removal ability the potential of the crude biosurfactant to remove hydrocarbons from contaminated sand was evaluated using the methodology described by aparna et al. (2012). sandy soil samples were collected from the parangtritis beach, indonesia; the samples were air dried and then sieved using a 2-mm mesh. furthermore, samples were mixed with tap water up to the moisture content 19 % (v/w). soil samples (20 g each) were polluted by 2 g of crude oil (pertamina, balongan, cilacap, indonesia), then transferred to 250-ml erlenmeyer flasks. the flask was added with 40 ml of the cell-free fermented broth and then was taken and adjusted with sterile smss medium in order to obtain a cell suspension of 0.7 od ( o p t i c a l d e n s i t y ) , d e t e r m i n e d b y a spectrophotometer. the number of bacterial cells corresponded to an inoculum of 10 cfu/ml. 7 the smss medium used for these experiments consisted of the following (g l ): 0.05% kh po , -1 2 4 0.1% k hpo , 0.05%, mgso .7h o, 0.01% kcl, 2 4 4 2 and 0.001% feso .7h o; the ph was adjusted to 4 2 7.0 by 1.0 m hcl (ph 7.2). optimization of medium and cultivation condition the influence of carbon and nitrogen sources on the biosurfactant yield and its properties was studied in the smss medium supplemented with 2% (v/v) of their sources. the carbon sources were n-hexadecane, glycerol, and glucose. the nitrogen sources were urea, nh cl and nano . 4 3 a 2% cell suspension of 0.7 od at 600 nm was inoculated into a 500-ml flask containing 100 ml smss medium. the culture was then incubated at 37ºc under agitation of 200 rpm. after 96 hours of incubation, culture samples were collected and further evaluated for bacterial biomass and biosurfactant characteristics. biomass and biosurfactant characteristics the culture samples collected previously were centrifuged at 36,000 g, temperature of 4ºc for 1 h. the pellet cells were dried overnight at 105ºc and then weighed to measure the pseudomonas aeruginosa b031 biomass. the crude biosurfactants in the cell-free culture medium obtained were c h a r a c t e r i z e d i n t e r m s o f r h a m n o s e concentration and surface-active properties, including emulsification index (e24), surface tension, and critical micelle concentration (cmc). rhamnose concentration rhamnose concentration in the cell-free culture medium was measured using the phenolsulphuric method (seedevi et al. 2018). one ml of the cell-free culture broth was mixed with 0.5 ml of 80% phenol and 2.5 ml of concentrated sulphuric acid. after the mixture was incubated for 10 min at room temperature, the absorbance w a s m e a s u r e d a t 4 9 0 n m u s i n g a spectrophotometer. the rhamnose concentration was then calculated using a standard curve 132 biotropia vol. 25 no. 2, 2018 incubated at 37ºc under agitation of 200 rpm in a shaking incubator. after 18 h incubation, the s a m p l e w a s a d d e d w i t h 1 2 0 m l o f dichloromethane as the extracting solvent, then centrifuged at 10,000×g for 15 min. the amount of residual hydrocarbon in the sample was determined by gravimetric analysis (villalobos et al. 2008). for comparison, the hydrocarbon removal ability of sds, triton x-100 and distilled water (control) was also analyzed under the same conditions. statistical analysis all statistical analyses were performed using stat view for windows (sas institute, cary, nc, usa) based on p<0.05. the effects of the treatments and their differences were assessed using analysis of variance (anova) and duncan multiple rang e test (dmrt) method, respectively. results and discussion bacterial strain identification table 1 lists the morphological, cultural, and biochemical characteristics of the bacterial strain selected for this study. this isolate developed pale green pigmentation on asparagine medium and released a sweet grape-like odor. its colony was small, rough, and convex. the isolate was gram negative and showed motility under microscope investigation. the biochemical analysis showed that it had abilities on denitrification, gelatin liquefaction, and starch hydrolysis. it showed positive results in the oxidase and catalase tests. it could use glucose, fructose, and mannitol as carbon source to ferment, resulting in acid production. in addition, the isolate could grow in a temperature range of 30°c to 42°c and in a ph range of 5-9. according to bergey's manual of systematic bacteriology, along with the results of the analyses, the isolate was identified as pseudomonas aeruginosa et al . (brenner . 2005) effect of carbon on strain biomass and biosurfactant properties table 2 presented the bacterial biomass and characteristics of the biosurfactant produced by pseudomonas aeruginosa b031 using different carbon sources, such as glycerol, glucose and ncharacteristic observation* morphology colony color pale green colony type small, rough, convex cell shape rods gram staining negative motility + biochemistry denitrification + gelatin liquefaction + starch hydrolysis + oxidase test + catalase test + carbon utilization glucose + fructose + mannitol + culture temperature 30˚c + 37˚c + 42˚c + ph 5 + 7 + 8 + 9 + table 1 morphological, biochemical and cultural characteristics of the selected strain b031 * _ note: + indicates growth of strain, indicates no growth of strain table 2 effect of carbon source on cell growth, rhamnolipid concentration, emulsion index, surface tension and final ph during biosurfactant production by pseudomonas aeruginosa b031 carbon source biomass (g/l)* ph* rhamnose (mg/l)* emulsion index (%) surface tension (mn/m)* n-hexadecane 0.3±0.01b 5.1±0.03a 449±19c 20±0.10.3b 37±0.3c glycerol 0.5±0.02 a 7.0±0.02 c 718±15 d 37±0.2 a 31±0.2 b glucose 0.1±0.01 c 3.4±0.01 b 180±11 a 10±0.01 c 44±0.1 a control 0±0d 7.2±0.01d 0±0b 0±0d 63±0d * note: data in this table are presented as mean ± standard deviation. their values with different superscripts in the same column are significantly different (p < 0.05). 133 biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo hexadecane. as observed, the strain was able to use all the evaluated carbon sources to grow and produce biosurfactants. all the carbon sources were found to have significantly influenced bacterial biomass, ph, rhamnose concentration, emulsion index and surface tension. among the tested carbon sources, glycerol was found to be the optimum for biosurfactant production of the strain. its yield was higher than those reported by previous researchers who also used pseudomonas sp and obtained rhamnolipids (amani et al. 2013; sodagari et al. 2018). the highest cell biomass and rhamnose concentration among the observations were 0.5 g/l and 718 mg/l, respectively. these results were expected since glycerol as a carbon source is taken up more easily than the others. on other hand, the use of nhexadecane, which is a very complex and heterogeneous carbon source, resulted in the lowest biomass and biosurfactant yield. moreover, the biosurfactant yield was lower than the results obtained by varjani and upasani (2016) who reported that the pseudomonas aeruginosa ncim 5514 strain produced 1.450 mg/l of rhamnolipid when glycerol was added at 3% w/v as carbon and energy source. further, the biosurfactant produced by the strain using glycerol had the highest significant reduction in surface tension and formed a stable emulsion on cultivation media compared with nhexadecane and glucose (p<0.05). the surface tension and emulsion index values were 31 mn/m and 37 %, respectively. the use of glycerol also stabilized the ph condition, whereas the use of glucose and n-hexadecane resulted in a decreased ph. this lower ph value was probably due to the production of secondary metabolic acid (müller et al. 2012). critical micelle concentration (cmc), which indicates the efficiency of a surfactant, is another important characteristic to consider. to measure the cmcs of the biosurfactant under study, culture broths of three treatments (n-hexadecane, glycerol, glucose) were diluted to various extents with a sterile medium and analyzed for surface tensions (fig. 1). the point of inflection of the curve, which was obtained by measuring the surface tensions of the various dilutions, corresponds to the surfactant concentration equal to cmc. analysis of the cmcs values of the glycerol, glucose, and n–hexadecane treatments were 35 mn/m, 44 mn/m, and 41 mn/m, respectively (p<0.05). the lower cmc value of glycerol treatment indicates that glycerol is the most efficient carbon source among the three sources tested (kłosowska-chomiczewska . 2017). et al monteiro . (2018) reported that glycerol could et al be obtained abundantly and cheaply as a byproduct of biodiesel from animal fats and vegetable oil production. glycerol could be obtained also from trans-esterification of vegetable oils and fossil sources (petroleum, natural gas, coal). therefore, glycerol is a promising and abundant substrate for biosurfactant production. figure 1 changes in surface tension in culture broths of pseudomonas aeruginosa b031 at various dilutions using three carbon sources. arrows correspond to the dilution values that are equal to cmcs. vertical bars indicate ± standard deviation of means (n=3). 134 biotropia vol. 25 no. 2, 2018 effect of nitrogen sources on strain biomass and biosurfactant properties sodium nitrate was found to be significantly more effective than urea and ammonium chloride (p<0.05) (table 3). the data obtained imply that the use of sodium nitrate as nitrogen sources is better for supporting the growth of the strain and stabilizing the ph of the cultivation medium. these conditions induced a significantly higher surface tension reduction and the formation of a stable emulsion on the cultivation media using sodium nitrate treatment compared with the urea and ammonium chloride treatments (p<0.05). the results were in accordance with previous studies (ma et al. 2016). according to kryachko et al. (2016), the bacteria cell uses nitrates, ammonia, and amino a c i d s a s n i t r o g e n s o u r c e s t o p r o d u c e biosurfactant. during these processes, the no 3 have to be reduced to no and then to nh . the 2 3 nh can be assimilated either by glutamate 3 dehydrogenase to produce glutamate or, with glutamine, by glutamine synthetase to produce glutamine. glutamine and α-ketoglutarate are then transformed to glutamine by l-glutamine 2oxoglutarate aminotransferase. therefore, in comparison with nh , the assimilation of no as 3 3 a nitrogen source is slower, simulating a nitrogenlimiting condition that is favorable to biosurfactant production (rizzo et al. 2017). the evaluation of the cmcs of the culture broths of three treatments (sodium nitrate, urea, and ammonium chloride) shows significant differences among the treatments (p<0.05) (fig. 2). the cmc values of the treatments were 24 mn/m for sodium nitrate, 35 mn/m for urea, and 43 mn/m for ammonium chloride. the reduction in surface tension and cmc of the biosurfactants is used as primary criterion for s e l e c t i o n o f b i o s u r f a c t a n t p r o d u c i n g microorganisms (thavasi 2011). this research demonstrated that the crude biosurfactant produced by the strain using glycerol and sodium nitrate as substrates decreased surface tension of the cultivation media by more than10 mn/m, thus they can be considered good surfactants (vijayakumar & saravanan 2015). as presented in fig. 1 and 2, surface tension decreased from 42 mn/m to a minimum value of 31 mn/m for the glycerol treatment and from 37 mn/m to 24 table 3 effect of nitrogen source on cell growth, rhamnolipid concentration, emulsion index, surface tension and final ph during biosurfactant production by pseudomonas aeruginosa b031 nitrogen source biomass (g/l)* ph* rhamnose (mg/l)* emulsion index (%)* surface tension (mn/m)* urea 1.8±0.01a 6.8±0.01a 220±19c 28±0.06a 35±0.04a nh4cl 1.0±0.01b 4.8±0.02b 90±15d 17±0.2b 42±0.5b nano3 2.5±0.02c 7.3±0.01c 290±11a 30±0.1c 24±0.1c control 0±0d 7.2±0.01c 0±0b 0±0d 63±0d figure 2 changes in the surface tension of culture broths of pseudomonas aruginosa b031 at various dilutions using three nitrogen sources. arrows correspond to the dilution values that are equal to cmcs. vertical bars indicate ± standard deviation of means (n=3). 135 note: *data in this table are presented as mean + standard deviation. their values with different superscripts in the same column are significantly different (p< 0.05). biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo mn/m for the sodium nitrate treatment. these capacities are comparable to the outcomes of similar previous research wherein surface tension was reduced to a minimal value of 27-28 mn/m (petrikov . 2013). other studies had been et al carried out also on biosurfactants by . , p fluorescens for which the minimal value of surface tension was 27.5 mn/m (i̇kizler . 2017). therefore, et al this study showed that both glycerol and sodium nitrate are efficient and promising substrates for biosurfactant production from pseudomonas aruginosa b031. application of biosurfactants in hydrocarbon removal an increasing number of sites have been polluted by hydrocarbons become a seriously effect to ecosystem and human health critical. for removing these pollutions, environmentally and low-cost technology was highly required. because oil has low water solubility, which increase its sorption by soil particles, researchers have been exploring the use of biosurfactants to accelerate hydrocarbon removal from the contaminated sites (arslan . 2017).et al the use of crude biosurfactant to remove hydrocarbon contaminants is common in bioremediation technologies. therefore, this study investigated the application of the biosurfactant from b031 in its crude p. aeruginosa form, without prior costly extraction or purification steps. the experiment used cell-free supernatant containing the crude biosurfactant and chemical surfactants (sds and triton x-100) to verify the former's capacity to remove crude oil from sand samples. the biosurfactant produced b031, p. aeruginosa sds, triton x-100, and distilled water (control) showed significantly different (p<0.05) capabilities in removing crude oil from contaminated sand (table 4). the crude biosurfactants from both glycerol and sodium nitrate treatments yielded higher values (87.5% and 84%, respectively), but no significant difference (p<0.05) was observed between them. the removals obtained by sds and triton x-100 as synthetic surfactants at the same concentration were lower (73% and 71.6%, respectively), which that of distilled water (control) was only 30.2%. the results imply that the cell-free supernatant containing the crude biosurfactant was practically as effective as the isolated biosurfactant in removing crude oil, indicating the possible use of the unpurified biosurfactant to minimize production costs. the ability of biosurfactants to remove crude oil can be explained as follows: biosurfactants promote the transport of hydrophobic contaminants toward an aqueous phase through some particular interactions, resulting in emulsification and micellization, thus leading to their removal (costa . 2010). the results of et al the experiments indicate that these biosurfactants are more effective in oil recovery than the chemical surfactants (sds or triton x-100). these results were in accordance with the results of lai . (2009) who observed that et al biosurfactants such as rhamnolipids and surfactin are more efficient in removing hydrocarbon than tween-800 and triton x-100. therefore, these biosurfactants indicate good prospects for applications, especially for bioremediation. conclusion the biosurfactant produced by pseudomonas aeruginosa b031 was found to produce the highest biomass and the best surface-activities and emulsification properties when glycerol and sodium nitrate were used as carbon and nitrogen sources, respectively. the crude biosurfactants table 4 comparison of the hydrocarbon removal ability of the crude bacterial biosurfactant, sds, triton x-100 and distilled water (control) sample crude oil removal (%) crude bacterial biosurfactant from glycerol treatment 87.5±7 d crude bacterial biosurfactant from nano3 treatment 84.± a 6 d sds 73.0±3 b triton x-100 71.6±2c distilled water (control) 30.2±2a note: * data in this table are presented as mean ± standard deviation. their values with different superscripts in the same column are significantly different (p < 0.05). 136 biotropia vol. 25 no. 2, 2018 produced by the strain were able to efficiently decrease the interfacial tension of culture broth dilution. these biosurfactants were capable of efficiently removing crude oil from sand samples, performing better than chemical surfactants, thus hold promise for hydrocarbon bioremediation application. acknowledgements we acknowledge financial support from boptn 2015 research grant of faculty of biology, universitas gadjah mada, yogyakarta, indonesia (contract number: ugm/bi/2460/ um/03/02). authors also thank ms sofianingtyas for her help in preparing the strain. references agbogidi mo, dolor ed, okechukwu em. 2007. evaluation of (linn.) and (roxb.) tectona grandis gmelina arborea for phytoremediation in crude oil contaminated soils. agric conspec sci 72(2):149-52. al-bahry sn, al-wahaibi ym, elshafie ae, al-bemani as, joshi sj, al-makhmari hs, al-sulaimani hs. 2013. 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84:275-82. 139 biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 840 dewi yuniati (growth performance) revisi.cdr growth performance and enzyme activit in ies catfish fed water [pangasianodon hypophthalmus] with hyacinth-based diet * dewi yuniati , nur bambang priyo utomo, mia setiawati and alimuddin department of aquaculture, faculty of fisheries and marine science, ,institut pertanian bogor bogor 16680, indonesia received 26 april 2017 / accepted 14 august 2017 abstract an alternative subtitution of pollard as an imported feed ingredient is a neccesity and one of the potential ingredients is water hyacinth (eichhornia crassipes). this study was conducted to evaluate growth performance and enzyme activity in catfish (pangasianodon hypophthalmus). diet with five different levels of water hyacinth subtitute of pollard (0%, 25%, 50%, 75%, and 100%) were fed to catfish for 60 days. seventy (70) catfish fry with average initial body weight of 2.45±0.15 g were maintained in 100cm x 80cm x 60cm aquaria. fish fed at satiation level three times daily at 8 am, 12 pm, and 4 pm. with dietary of 25% water hyacinth, growth performance and protease activity similar to 0% treatment. feed intake, protein digestibility, feed efficiency, protein efficiency ratio, and protease and amylase enzyme activities decreased (p<0.05) in those fed with more than 25% water hyacinth. catfish fed 25% water hyacinth showed significantly (p<0.05) higher daily growth rate, feed efficiency and protein digestibility than those with other treatments. based on the growth performance and enzyme activity, we can conclude that the optimum dietary level of water hyacinth subtitute pollard for fry catfish is 25%. keywords: catfish, growth performance, pollard, water hyacinth introduction feed is the main component to meet the energy needs of the fish. has a high pollard digestibility and bioavailability as carbohydrate source in fish feed (suprayudi et al. 2010). munir et al. 2015) ollard contain ( report that p s 53.40% nfe, lipid14.78% protein, 7.77% , 9.78% crude fibre, and 4.34% ash owever , h an alternative subtitution of pollard as an imported feed ingredient is a neccesity. research on the use of ingredients of fish feed has been done ulva ; lactuca havea brasiliensis lemna, singgle cell protein , , ( et al.mahasu 2016; inara 2011; bag 2011; utomo et al ). the research among others, focused . 2007 on the efforts of using local ingredients and creating efficiency of feed cost. one of the potential ingredients to substitute pollard in fish feed is water hyacinth (eichhornia crassipes). water hyacinth has lower cost than pollard, water hyacinth cost rp 2000/kg while pollard cost . . s with base on therp 4800/kg and change time dollar rate. water hyacinth is a south american native plant that has spread to more than 50 countries, including indonesia. water hyacinth is one of the aquatic plants that grow in abundance in rivers, rice fields, and lakes or dams. its characteristics include rapid growth and the ability to compete with other aquatic plants (shu et al. 2014). water hyacinth can cover the surface of water bodies, thereby reducing the amount of sunlight and air penetrating into the water. in addition, the plant can cause shallowing and increase evaporation due to evapotranspiration. in a dense population, water hyacinth can impede water flow and block irrigation channels (sotolu & sule 2011). therefore, the presence of water hyacinth in water bodies is more often regarded as harmful weeds that can negatively impact the aquatic ecosystem (tellez et al. 2008).* corresponding author: dewiyun25@gmail.com biotropia 5 2 8 140 147 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.840 140 in previous study, water hyacinth has been used as fish feed in tilapia (khalil et al. 2015; bag et al. 2011; hontiveros et al. 2015; muchtaromah et al. 2012), cyprinus carpio (mohapatra 2015), clarias gariepinus (sotolu & sule 2011), and labeo rohita (saha & ray 2011). the use of water hyacinth flour as a substitute of fish meal for clarias gariepinus feed for 26.72% and 31.63% showed a lower digestibility rate (sotolu & sule 2011). however, according to sotolu and sule (2011), it is still possible to use the plant for fish feed. hence, water hyacinth was chosen to be the raw material of fish feed in this research. furthermore, chemical analysis of water hyacinth shows that the plant contains nutrients similar to that of pollard, 30.81% nfe, 12.40% protein, 4.72% lipid with digestibility of water hyacinth leaves reaching up to76.4% (chang et al. 2013; hontiveros & serrano 2015). the utilization of nutrients in fish feed is closely linked to enzyme activity in fish. the use of different feeds in fish can affect their enzyme activities. marzuqi (2015) revealed that milkfish given different contents of carbohydrates experience different amylase enzyme activities in line with the increasing contents of carbohydrates in the feed. according to tibin et al. (2012) the use of water hyacinth indicated a decrease in feed utilization in tilapia fish. there is still gap in knowledge about the use of water hyacinth as a source of plant material in catfish feed. thus, the present research was carried out to evaluate the use of water hyacinth flour in fish feed as a source of carbohydrate in the growth performance and enzyme activity of catfish pangasianodon hypophthalmus, an important farmed fish in southeast asia. materials and methods water hyacinth flour the water hyacinth flour made from leaves and stems used in this research was taken from the sukabumi centre for freshwater aquaculture. the water hyacinth was first cleaned and then 0 dried in oven at 60 c for 24 hours. the dried water hyacinth was then grinded into flour. before being used as the ingredient for the feed, a test of heavy metal content was conducted to determine the presence of lead (pb), cadmium (cd) and mercury (hg). the heavy metal content analysis was done using the atomic absorption spectrophotometry method (apha 2012). the test results showed that no heavy metal contents of lead, cadmium, and mercury were detected in the water hyacinth flour. experimental diets diet containing the five different levels of water hyacinth (0%, 25%, 50%, 75% 100%) and as pollard substitute. the premix used were commercial product called lagantor from kalbe. 1 note: nitrogen free extract 2 gross energy, 1 g protein = 5.6 kcal, 1 g lipid = 9.4 kcal, 1 g nfe = 4.1 kcal (takeuchi 1988). -1 table 1 composition and nutrients contained of the experimental diets (% kg dry matter) 141 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. webster and lim (2002); protein and lipid retention were calculated based on takeuchi (1988). after the final weighing, three pieces of fish were randomly taken from each aquaria for a proximate analysis. water content analysis was done by drying the sample in an oven at the temperature of 105 110 c for 6 hours; analysis 0 – of crude fibre was carried out with dissolving method using strong acids and bases and heating; protein analysis with kjeldahl method, lipid analysis of dried samples with soxhlet method, lipid analysis of wet samples with folch method, and ash content analysis by heating sample in a furnace at 600 c (takeuchi 1988). 0 enzyme activity analysis the enzyme activities analysed included those of amylase, protease, and cellulase. the enzyme activities were observed at the end of the experiment. amylase enzyme activity was measured by the method of worthington (1993). cellulase enzyme activity was measured by the method of kader and omar (1998). protease enzyme activity was measured using bradford's method (bradford 1976). digestibility test the effect of water hyacinth used in feed digestibility was measured using the indirect method by adding chromium oxide cr o as an 2 3 indicator in the feed. digestibility test was conducted after growth performance tests using the same fish. the fish were fed three times a day at satiation at 8 am, 12pm, and 4 pm. faecal wastes were first to be collected on the fourth day after giving the feed containing cr o . the faeces were 2 3 collected 30 minutes after feeding. the collected faeces from each aquaria were stored in a freezer. the collection of the faeces was done for 25 days. once collected, the faeces were dried in the oven 0 at 110 c for 4-6 hours. the faeces sample was analysed for its cr o and protein contents. the 2 3 total digestibility value and protein digestibility were calculated using formula of takeuchi (1988). the ingredients used were weighed according to their composition and mixed . in a mixing machine after getting evenly mixed, the feed was moulded and heated in the oven at 60 c for 4 hours. the 0 composition of the feed and the nutrients contained in it are presented in table 1. fish rearing the catfish pangasianodon hypopthalmus used originated from cibanteng, bogor, indonesia. the fish were maintained in the aquatic laboratory of the southeast asian regional centre for tropical biolog y (seameo biotrop). they were reared in 100cm x 80cm x 60cm aquaria. the aquaria were cleaned and disinfected using 30 ppm chlorine before use. each of the aquaria was filled with 200l of water. fish acclimatization was done in two weeks before the rearing period. during the acclimatization, the fish were fed with 50% treatment feed at satiation level. after acclimatization, the fish were weighed, and the average weight was 2.45±0.15 g. the fish were then placed randomly into the 15 aquaria with a stocking density of 70 fishes/aquaria. the rearing of the fish was done for 60 days. feeding was done at satiation for three times daily at 8 am, 12 pm, and 4 pm. the growth was measured by sampling every fifteen days. every three days 70% of the water was replaced to maintain the quality of the water during the rearing. each aquaria was equipped with aeration 0 and heater regulated at 28 c. the temperature of the water was measured daily, while the water quality was measured three times during the farming process, with parameters including dissolved oxygen, ph, and ammonia. the water quality during farming was at the optimum range for catfish, with a temperature ranging from 0 -1 28–29 c, dissolved oxygen of 5.6–6.3 mg l , ph -1 7.6–7.8 and ammonia 0.0005–0.02 mg l . at the end of the rearing period, the fish biomass in each of the aquaria was measured. the parameters of survival rate, the amount of feed intake, feed efficiency, and daily growth rate were calculated based on halver and hardy (2002); protein efficiency ratio was calculated based on 142 biotropia vol. 25 no. 2, 2018 statistical analysis the design employed in this research was the completely randomized design with five treatments and three replications. data were analysed using spss software ver. 22.0. to find the effects of treatments, data were tested with anova and followed by duncan test with a 95% confidence level. the difference between treatments was found with a significance value of p<0.05. results and discussion growth performance the results showed that the use of water hyacinth of more than 25% in diet caused a decrease in feed intake, daily growth rate, protein digestibility, protein efficiency ratio, and daily growth rate. lipid retention and protein retention in the 75% and 100% treatments were lower compared to those in other treatments, where in the 100% treatment, the retention rates were 7.85% and 19.87%, respectively (table 2). the use of water hyacinth in fish feed at certain levels gave different results in the feed intake (fi) of the catfish. in this research, the use of more than 25% water hyacinth in feed caused a decreased in fi. the decreases of fi in the 50%, 75% and 100% treatments compared to 0% treatment were 11.83%, 38.89%, and 45.88% respectively. in tilapia, the use of water hyacinth in feed did not affect feed consumption, indicating that tilapia could accept feed containing water hyacinth better than pangasianodon hypopthalmus (hontiveros et al. 2015). the difference in the feed intake levels in each treatment was assumed to be caused by a difference in the feed palatability. palatability is a response to certain feed, which is influenced, among others, by feed characteristics. in addition, the acceptance of fish to raw material is different for each species (setiawati et al. 2014). venero et al. (2008) mentioned that the use of plant materials can reduce feed acceptability and feed palatability. palatability is linked to attractability that will influence responses in feed searching, intake, and ingestion or acceptability (inara 2011). the decreased rate of fi was followed by a decreased in digestibility rate. digestibility is the process of breaking down feed into a simpler form that is readily absorbed by intestinal walls and into the blood vessel system. there was no difference in the total digestibility of feed in all treatments (p>0.05). this is in contrast to protein digestibility that experienced a decrease in line with the increasing amount of water hyacinth in feed (p<0.05). diet contain 0% and 25% water ing hyacinth showed similiar result 82%, whilst in of tabel 2 final biomass (bt), feed intake (fi), total digestibility (td), protein digestibility (pd), protein retention (pr), lipid retention (lr), protein efficiency ratio (per), daily growth rate (dgr), feed efficiency (fe), survival rate (sr) of catfish (pangasianodon hypophthalmus) fed with feed containing water hyacinth note: *) the values in the same rows with different superscript letters indicate significant differences (p<0.05). 143 td water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. -1dgr (%day ) the 50%, 75%, and 100% treatments, it decreased to 79.92%, 74.57%, and 75.96% respectively. the factors affecting digestibility are, among others, the treatments before and after feed making, material sources, particle size, fish size, and nonprotein components in the feed (usman 2002). the decrease protein digestibility value 50-in of 100% treatment in this study were caused by the increasing amount of crude fibre that can disturb feed utilization and growth. mohapatra (2015) reports that the use of water hyacinth significantly increase the crude fibre and affect the feed s s utilization of cyprinus. in the study the current , crude fibre content of 0–100% treatments were at the range of 3.47–13.06%. the higher the fibre content in the feed, the lower digestibility of protein. this result is the in accordance with report of suprayudi et al. (2010). the end products of the consumed and digested feed are nutrients that can be used and then stored in the fish body. the levels of protein and lipid retentions in the 0–50% treatments were the same, and they declined in the 75% and 100% treatments. the different protein and lipid retention levels were caused by differences in the amount of feed intake and feed digestibility value. carbohydrate, protein, and lipid are the three components that contribute the highest amount of energy for the fish. if low amounts of nutrients are absorbed, their potential to be stored in the fish will also be low, because the nutrients will be first used for the fish activities. this also applies to growth: if a small amount of energy is received by the fish, then the energy for its growth will be even much less. the use of certain levels of water hyacinth in feed gave positive effect on the growth of (mohapatra 2015). in this research, the cyprinus use of more than 25% water hyacinth negatively impacted the growth of the catfish. compared to 0% treatment the percentages of daily growth in , the 50%, 75% and 100% treatments decreased. the decreased of daily growth in the 50%, 75%, and 100% treatments were 27.1% day , 55.6% day -1 1 -1 , and 69.9% day , respectively. the lowering growth rate in the fish can be attributed to the unfulfilled needs of nutrients in the fish and the inability of the fish to use the energy and materials in their feed (usman 2002). feed intake in the 0% and 25% treatments were better than that in the other treatments, hence the amount of the ingested feed was greater in the 0% and 25% treatments. the difference presumably caused more energy to be received in 0% and 25% treatments, resulting in better growth. in this research, the crude fibre content in the feed increased along with the increasing proportion of water hyacinth flour in the feed. the crude fibre derived from vegetable materials is in general difficult to be digested by fish. in addition, one of the components of cell walls in plants is cellulose that is intractable (inara 2011). the better growth in the 0% and 25% treatments reflects feed efficiency value. this value indicates that the antinutritional substances and the high fibre content in the 0% and 25% treatments did not disturb the use of feed by the catfish. the survival rate ranged from 97.14–98.57%, indicating that water hyacinth did have any harmful effects on the fish. enzymes ctivitiesa similar protease enzyme activities were found in the 0% and 25% treatments, thus have different result compared to the 50%-100% treatments (fig. 1). similar cellulase enzyme activities were found in all treatments (fig. 2). different amylase enzyme activities were observed in all treatment (fig. 3). enzymes have an important role in digestion process. the protease enzyme activities in the 0% and 25% treatments were greater than those in the 50–100% treatments. the declining protease enzyme activities in the 50–100% treatments were assumed to be caused by the anti-nutritional substances in the feed. water hyacinth contains anti-nutritional substances such as tannin and phytic acid of 0.98% and 0.42%, respectively. anti-nutritional substances such as tannin can affect growth and enzyme profiles of fish. tannin impedes protease enzyme activity and therefore can reduce protein digestibility, while phytic acid can reduce protein and mineral bioavailability (saha & ray 2011). in addition, the declining enzyme activities can also be caused by the amount of the substrates present feed in the intake. in this study, fish fed diet containing 50–100% water hyacinth showed lower feed intake than those fed 0% and 25% water with hyacinth, which was directly proportional to the amount of substrates for the enzyme in their digestive systems. the higher feed intake and protein digestibility in the 0% and 25% treatments compared to those in other treatments had an impact on the high value of protein 144 biotropia vol. 25 no. 2, 2018 figure 1 protease enzyme activities in catfish fed with feed containing different levels of water hyacinth figure 2 cellulase enzyme activities in catfish fed with feed containing different levels of water hyacinth figure 3 amylase enzyme activities in catfish fed with feed containing different levels of water hyacinth 145 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. efficiency ratio in these treatments, namely 2.30 and 2.37, respectively. the cellulase enzyme activities observed were not different in all treatments. this is attributed to the low amount of cellulase enzyme in the fish that worked maximally in all treatments, although there were differences in the amounts of feed intake and water hyacinth in each feed. the amylase enzyme activities were found to be different in all treatments. the amylase enzyme activities in the 50–100% treatments were lower than those in other treatments. amylase enzymes break down starch in feed. the amylase enzyme activites decreased with increasing level of water hyacinth assumed to be caused by the low carbohydrate content as the level of water hyacinth increased. conclusion the pangasianodon hypopthalmus fed diet containing 25% water hyacinth subtitute pollard showed similar growth performance and protease activity with 0% treatment. the use of water hyacinth of more than 25% showed lower growth performance than other treatments. water hyacinth flour can be used for up to 25% or 6.25% in the diet of pangasianodon hypopthalmus. acknowledgements we would like to express our gratitude to the southeast asian regional centre for tropical biolog y (seameo biotrop) for the opportunity to conduct our research in its aquatic laboratory, and our gratitude also goes to the centre for freshwater aquaculture, sukabumi, for providing water hyacinth flour for the study. references american public health association (apha). 2012. standard method for examination of water and wastewater, 22th edition. 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[single cell protein as substitution for fish meal in juvenile common carp (cyprinus carpio) diets]. jurnal perikanan 9(2):188-93. venero ja, davis da, lin c. 2008. use of plant protein sources in crustacean diets. new york (us): the howort press. p. 163–203. webster cd, lim ce. 2002. nutrient requirements and feeding of finfish for aquaculture. new york (us): cabi publishing. p. 4-5. worthington v. 1993. worthington enzyme manual. new jersey (us): worthington biochemical corporation. 147 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia (2) 1988/1989: 1-7 fruit production of a six-year old shorea stenoptera plantation at haurbentes, bogor, indonesia eizi suzuki department of biology, college of liberal arts, kagoshima university, kagoshima 890, japan lilian u. gadrinab tropical forest biology program, biotrop, bogor, indonesia abstract a six-year old plantation of shorea stenoptera at haurbentes, bogor flowered for the first time on october, 1987. in plots of 270 m 2 , 12 of the 32 trees had opened flowers. the average heights of flowering and non-flowering trees were 400 cm and 270 cm, respectively. the flowers and fruits were counted four times from october 28, 1987 until february 17, 1988. in october, a total of 24313 flowers existed and 1.9% of them became mature fruits on february, 1988. the fruit production was 308 kg/ha and 133 kg/ha in fresh and dry weights, respectively. introduction shorea stenoptera burck produces large and oil-rich fruits called illipe nuts. many shorea plantations are found in west kalimantan where fruits are collected for oil. there are many studies about the phenology of dipterocarpaceae (burgess 1972, cockburn 1975), mast flowering (ng 1977, yap 1987) and pollination (appanah 1981, appanah and chan 1981). there are, however, no quantitative studies on the change of the number of flowers that mature to fruits in dipterocarpaceae. there are only a few records on the age at sexual maturity of dipterocarpaceae (foxworthy 1932, ng 1966 and 1977) but none exists for s. stenoptera. thus, when a six-year old plantation of s. stenoptera in bogor began to flower, it was deemed interesting to trace the number of flowers that mature to fruits. study site and methods shorea stenoptera and other dipterocarp species have been planted in an experimental forest at haurbentes, bogor (6°32'-33's, and 108°26'e, and 200 m above sea level) which was established in 1940 by the center for forestry research and development. the total of monthly average precipitation from 1957 to 1965 was 4196 mm. 1 biotropia no. 2, 1988/1989 plots p-l to p-3 and p-4 were made in a 6-year old and 18-year old plantation of shorea, respectively. s. stenoptera seedlings had been planted every 3 m x 3 m. the plot sizes were 10 m x 10 m, 12 m x 15 m, 12 m x 15 m, and 20 m x 25 m, for p-l to p-4, respectively. p-l was a few meters distant from p-3 and about 20 m from p-2. p-2 was on a 16° slope with a north aspect while the others were on gentle slopes of less than 10°. the tree height (h) and trunk diameter at the 1.3 m height (dbh) were measured. it was noted whether the faces were in flowering or not. in p-l and p-2 all the panicles were tagged with numbered tapes and a stapler. the length of the panicles was measured and the number of flower buds, flowers, young fruits, flower scars was counted. after the flower fell, two bracteoles and the scar of the peduncle remain at the point of the panicle where the peduncle was attached. later the bracteoles also fell. a few trees were in blossom earlier than others and lost many small branches of panicles. the flower number of the former trees was therefore underestimated. on 8 december, 1987, 8 january, and 17 february, 1988 the fruits were counted again. one tree had a few new panicles in december and january. they were counted as in the above process. on 17 february, 337 fruits were collected randomly, their length, diameters and weight were measured. the calices were separated from 57 fruits and the fruits were weighed. the calices and fruits were cut into small pieces and oven dried at 105°c for 16 hours, and weighed. results and discussion the first trees of shorea stenoptera in haurbentes were grown from seeds which came from pontianak, west kalimantan about 40 years ago (masano et al. 1987). table 1 shows the amount of illipe nuts, group of shorea species, called teng-kawang in indonesia, s. stenoptera, s. pinanga scheff., and s. seminis sloot, harvested in west kalimantan from 1983 to 1987. the annual crop fluctuated greatly. tengkawang species have a characteristic of mast flowering as many other shorea species. in 1987, s. stenoptera began to flower in april in west kalimantan table 1. annual crop of illipe nuts harvested from plantations and natural forests in west kalimantan from data of the department of forestry of west kalimantan at pontianak. april 1983 march 1984 fresh weight 10 640 500 kg 1984 1985 0 1985 1986 396 900 1986 1987 1 982 980 1987 1988 7 822 480 2 fruit production of a six-year old shorea stenoptera-e\7\ suzuki & lilian u. gadrinab while at haurbentes, it was in september. the flowering season in different localities is not the same. twenty three species of dipterocarpaceae are planted at haurbentes, and most of them were flowering in september or october of 1987. in mt palung, west kalimantan, 22 species of dipterocarpaceae flowered in april of 1987 (simbolon at herbarium bogoriense, personal communication). the tree sizes and basal area (ba, summed area of stem cross sections at 1.3 m height) of 5. stenoptera in p-l to p-3 are shown in table 2. the trees were rarely shaded by neighboring trees and the areas were manually weeded. the flowering trees were significantly bigger in dbh and taller than the nonflowering ones. they began to flower after 6 years from germination. the ages at first flowering of 65 species of dipterocarpaceae in the arboretum at kepong, near kuala lumpur were from 17 to 45 years (ng 1966 and 1977), dipterocarpus baudii flowered at 6.5 years old (foxworthy 1932). 5. stenoptera is one of the earliest flowering species in dipterocarpaceae. in p-4 which is an 18 year-old-plantation all trees flowered. table 2. tree size and area of s. stenoptera in p-l, p-2 and p-3 (450 m 2 in total), and p-4 (500 m 2 ). p-l, p-2, and p-3 (6 yr old) flowering tree non-flowering all trees p-4 (18 yr old) no. 20 25 45 20 mean dbh (cm) 4.36* 2.59* 3.37 20.02 standard deviation 1.47 1.10 1.54 7.99 mean height (cm) 400* 270* 328 1568 standard deviation 105 90 116 428 basal area (m 2 /ha) 0.74 0.34 1.08 14.5 * the difference is significant at the level of p = 0.01. shorea stenoptera is a small tree, but is a remarkably variable species (ashton 1982). gadrinab (1984) has found two genetic types, big and small types. the big type becomes 40 m or more in height while the small type is usually shorter than 20 m. they can be distinguished even when still young and small because branches of the big type are usually thick and extended upward while those of the small type are slender and hanging down. the trees in p-l to p-3 seemed to be of the small type. a few trees in p-4 are of the big type. the changes in number from flowers to mature fruits of 12 flowering trees in p-l and p-2 are shown in table 3. the flowering season of shorea stenoptera at haurbentes started in september, and was nearing its end at the time of study which was october 28-30. s. pinanga bore fruits but no more flowers at that time. 3 biotropia no. 2, 1988/1989 eighty four percent of all flowers (24 312) had already fallen by october 30. thirteen percent of all the flowers were lost from november 1 to december 8, 1987. two trees which had only 74 flowers lost all flowers by december 8. from december 9 to february 17, 1988, 1.3% of all the flowers were lost. the mature fruits in february were 1.9% of all flowers in october, and there were only 1.7 fruits/m 2 . one tree which had 47 fruits on january 8, 1987 lost all of them by february 17. the fruits of many trees were nearly mature on january 8. hence, the real fruit set ratio (fruit no./flower no.) was from 1.9% to 2.6%. the mean fruit set ratios of 187 species of self-incompatible and 129 self-compatible hermaphroditic plants were 22.1% and 72.5%, respectively (sutherland and delph 1984). they, however did not include dipterocarpaceae. this ratio in 5. stenoptera was very low. many flowers fell soon after their opening. they might not have been pollinated. table 3. change of flower and fruit number of s. stenoptera in p-l and p-2 (270 m 2 ) and fruit weight. no. % later flower oct. 28-30 no. of panicle 337 1987 bud 779 3.2 flower & fruits 3 181 13.1 flower scar 20 352 83.7 total 24312 100 dec. 8 young fruits 777 3.2 200 jan. 8'88 almost mature fruits 643 2.6 2 141 feb. 17 mature fruits 458 1.9 0 0 feb. 17 mean length of fruits 42 mm mean diameter of fruits 28 mm fresh fruit weight without calyx 18.16g dry fruit weight without calyx 7.84g fruit production (fresh) 308 kg/ha (dry) 133 kg/ha one tree continued to produce new panicles and flower until january 1988. the flowers in december and january were, however, 1.4% of all flowers in october. they did not contribute to fruit production in february. some trees outside the plots also continued to make a few flowers in december and january. in west kalimantan, we found a tree bearing a few flowers in november. there seemed to be some variation in the flowering period among individuals. ng (1977) has reported that s. stenoptera flowered frequently for very extended periods in kepong. 4 fruit production of a six-year old shorea stenoptera-eizi suzuki & lilian u. gadrinab the fruits without calyx were 42 mm and 28 mm in mean length and diameter respectively, and 18.16 g in fresh weight. there were significant variations among some trees in the mean length and diameters of fruits from each tree (figure 1). figure 1. relationships between mean length and diameter of fruits from each tree (solid circle) and all trees (open circle). cross lines show the confidence limits of the means (p = 0.05). the shape and length of five wings of calyx were also different among trees. the wing length varied from one third to 2 from that of fruit length. as a result, the wings have no role in wind dispersal. the productivity of fruits was 133 kg/ha in dry weight. the relationships between tree sizes and number of flowers in october are shown in figure 2. there was a rough correlation between them. the tree with the least flowers was shaded by a big tree outside the plot. the trees in p-l tended to make more fruits than trees in p-2 of the same size. only the relationships between height of the tree in p-2 and number of flowers was significantly correlated at 5% level as shown by a straight line in fig. 1 (correlation coefficient = 0.750). 5 biotropia no. 2, 1988/1989 figure 2. relationships between flower number of a tree and tree height (left figure), and dbh (right figure). the flower number is the sum of buds, flowers, young fruits, and scars in october. circle, trees in p-l. triangle, in p-2. conclusion the age at first flowering of s. stenoptera trees which are being grown at the experimental forest at haurbentes was 6 years old. two months after the start of flowering, 84% of flowers counted (24 312 flowers from 12 trees in a 270 m 2 plot) had already fallen. then after another month, 97.1% of all the flowers had fallen and by the end of another month, 98.4% of the total flowers were lost. mature fruits at this time were 1.9% of all flowers found in the first months of flowering. the productivity of fruits was 133 kg/ha in dry weight. acknowledgments the authors gratefully acknowledge the invaluable support by the japan international cooperation agency (jica) and seameo regional center for tropical biology (biotrop), and the forestry center for research and development in bogor for the permission of research in the experimental forest. thanks are also due to the field workers. 6 fruit production of a six-year old shorea stenoptera eizi suzuki & lilian u. gadrinab references appanah, s. 1981. pollination in malaysian primary forests. malayan forester, 44: 37-42. appanah, s. and h.t. chan. 1981. thrips: the pollinators of some dipterocarps. malayan forester, 44: 234-252. ashton, p.s. 1982. dipterocarpaceae. flora malaysiana, series 1, vol. 9: 237-552. martinus nijhoff pub., the hauge. burgess, p.p. 1972. studies on the regeneration of the hill forests of the malay peninsula. the phenology of dipterocarps. malayan forester, 35: 103-123. cockburn, p.p. 1975. phenology of dipterocarps in sabah. malayan forester, 38: 160-170. foxworthy, f. w. 1932. dipterocarpaceae of the malay peninsula. malayan forest records 10. gadrinab, l.u. 1984. a biosystematic study on section pachycarpae in dipterocarpaceae: shorea macrophylla ashton and shorea stenoptera burck. biotrop. masano, h. alrasyid, and z. hamzah. 1987. planting trials of dipterocarp species outside their natural distributional range in the haurbentes experimental forest, west java. proceed. third round table conference on dipterocarps (ed. a. j.g.h. kostermans), 19-37. unesco, jakarta. no. f.s.p. 1966. age at first flowering of dipterocarps. malayan forester, 29: 290-295. no. f.s.p. 1977. gregarious flowering of dipterocarps in kepong, 1976. malayan forester, 40: 126-137. sutherland, s. and l.f. delph. 1984. on the importance of male fitness in plants: patterns of fruit-set. ecol., 65: 1093-1104. yap, s.k. 1987. gregarious flowering of dipterocarps: observations based on fixed tree populations in selangor and negri sembilan, malay peninsula. proceed. third round table conference on dipterocarps (ed. a.j.g.h. kostermans), 305-317. unesco, jakarta. 7 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf biotropia no. 3, 1989/1990: 1 -24 assessment of spot satellite data for tropical vegetation inventory and monitoring in sumatra y. laumonier and u..r. djailany syafii tropical forest biology programme, seameo-biotrop bogor, indonesia abstract following a previous vegetation mapping in sumatra island (indonesia), an assessment of spot satellite capability to handle specific problems related to vegetation identification and monitoring from remote sensing data has been undertaken. results of visual interpretation and multispectral analysis have shown the usefulness of spot data for the appraisal of tropical vegetation at medium scale. this was particularly striking for the swampy vegetation types including mangroves and for the secondary vegetation, for which significant improvements have been brought by multispectral classifications. a 20 m ground resolution is neither sufficient to provide information on primary forest patterns, nor to identify properly logged over areas. never theless, several degrees of depletion of the forest and all the serial stages have been identified, which is a considerable progress compared with previous remote sensing means. spot is a very good alternative to medium scale aerial photographs for the production of medium scale (1 : 100 000 to 1 : 250 000) vegetation and land-use maps. background and justification since 1980 biotrop, the seamed regional centre for tropical biology, has conducted not only a mapping inventory of land-use units and natural vegetation types in sumatra which involves remote sensing analysis, but also very detailed field studies on various plant formations and their dynamics, floristics, structure and ecology. results have been synthesized in the form of ecological vegetation maps (laumonier 1983, laumonier et al. 1986, 1987) and related surveys (laumonier 1981, blasco et al. 1983). the mapping was mostly done using conventional means, i.e. interpretation of a 1/100 000 scale panchromatic aerial photograph cover (1974-78) which appeared to be of rather poor quality. moreover, the landsat mss imageries have proved insufficient in discriminating many peculiar vegetation or land-use types. this necessitated an unusually detailed field work to supplement the lack of information obtained from space, but it gave researchers of the team a fairly good knowledge of the area. the second phase of the biotrop study implemented in 1988 is concerned with specific areas selected according to management problems spotted during the small-scale mapping phase. such development schemes would require an elabora 1 biotropia no. 3, 1989/1990 various shrubby secondary vegetation types identical from space, but floristically entirely different often mixed with cultivation and usually classified as "mosaics". a. visual interpretation and manual classification the visual interpretation of black and white prints allows to differentiate 19 landcover and vegetation units (table 4 and figure 3). the standard fcc gives less information compared with the black and white interpretation of the 3 bands. improved false colour composites (xs bi vgi and xs3 bi vgi) allow a better discrimination of secondary types, burnt or newly planted areas. b. digital classification for the scene of pasirmayang, a multispectral classification has been performed for two windows, one in pasirmayang area (512 x 512 pixels) corresponding mostly to natural vegetation cover, the other in sitiung area (512 x 512 pixels) consisting of cultivation and settlements. xs1, xs2 and xs3 means and standard deviations plotted on bi-dimensional diagrams provide a satisfactory discrimination of vegetation and land cover units (figure 4). in pasirmayang window, 10 classes corresponding to 6 vegetation types and 4 other land-use units were recognized (figure 4, plate 2), whereas in sitiung 5 vegetation types and 3 land-use units could be distinguished (figure 4, plate 3). table 4. classification of vegetation in muarabungo's lowland area by visual interpretation 1. lowland primary forest 2. hill forest 3. logged-over or depleted forest 4. tall secondary forest mixed with rubber trees 5. secondary growth forest mixed with remnant primary forest trees 6. secondary growth forest mixed with rubber 7. shrubby, thicket secondary vegetation 8. young secondary growth forest mixed with shrubs and herbaceous vegetation 9. newly planted rubber mixed with young secondary growth forest 10. grassland 11. riparian forest 12. fresh water swamp forest 13. swampy secondary shrubby vegetation and grassland 14. paddy field 15. newly opened, recently burnt area 16. mosaic of secondary growth and newly planted food crops (paddy, cassava, banana & fruit trees) 17. fruit trees, coconut and habitat (homestated mixed gardens) 18. rubber tree estate 19. bare soil 10 biotropia no. 3, 1989/1990 tion of larger scale vegetation and land-use maps and more precise knowledge and understanding of the land-use patterns identified on remote sensing documents. the very high ground resolution obtained with spot offers new perspectives in the mapping of those patterns. a first evaluation of such data was initiated during the peps program (programme devaluation preliminaire de spot) organized by the french space agency (laumonier et al. 1987). its aim was to assess the capabilities of spot satellite to tackle some of the most striking problems met during the first phase of the project in sumatra, specially with the identification and classification of vegetations and land-use units. the present study is a follow up of this investigation backed up by related researches (barkey 1987, djailany 1987, gastellu-etchegorry 1988) concerned with an evaluation of visual versus digital interpretations and classifications, a comparison of spot data with conventional aerial photographs and landsat mss data already analyzed in the area (deshayes 1981, ducros-gambart & gastellu-etchegorry 1984, ducros-gambart et al. 1984) and an assessment of panchromatic (p) versus multi-spectral (xs) mode. it is also supported by the ground sampling already completed by the biotrop team for the characterization of complex heterogeneous success-ional stages, primary forest patterns and land-use units in sumatra (laumonier 1981, torquebiau 1986, djailany 1987). materials and methods four scenes have been requested both in p and xs modes at the processing level 1b (table 1) corresponding to specific problem areas in central and south sumatra (figure 1). the muarabungo area (western part of jambi province) has been chosen as an example of a forested area with problems of local agricultural practices and modern plantation schemes. baturaja (south sumatra) is the test site for another type of land use where the original vegetation has almost disappeared and has been replaced by complex secondary types. lastly, the delta of musi-banyuasin was selected as a good example of management problems in swampy areas. the completion of data acquisition during the requested period (1986) occurred over a region where the cloud cover was always very high at the equator. the spot lateral viewing capability could be responsible for the relative ease in their acquisition. the materials used in the study included computer compatible tapes (cct), negative films at 1/400 000 and 1/200 000 scales for each xs band, standard false colour composites (2 scenes only) at 1/80 000 or 1/100 000 scales, and negative films at 1/200 000 in panchromatic mode. 2 biotropia no. 3, 1989/1990 figure 3. manual interpretation of vegetation types and land-use units on the black and white spot imageries, scale 1/40 000, 1986 (legend see table 4). 11 biotropia no. 3, 1989/1990 figure 4. xs3/xs2 diagram of the 15 spectral classes selected in pasir mayang scene plate 2. multispectral classification of window pasir mayang (512 x 512 pixels) 12 assessment of spot satellite data —y. laumonier & u.k. djailany-syafii legend: sitiung 1. logged-over or depleted forest 2. tall secondary forest mixed with rubber 4. rubber tree estate 11. recently burnt area 12. burnt area long time ago 13. open dry field and shrubby vegetation 14. gardens mixed with food-crops 15. habitation plate 3. multispectral classification of window sitiung (512 x 512 pixels) the multispectral analysis has brought additional information for some nonvegetated types (5,7,11,12,15; table 5) which are more or less homogeneous or difficult to separate properly on the black and white prints. the too long period of time separating data acquisition from field check and the dynamic nature of the land use unit types have hampered the proper evaluation of the results of the classification. some areas classified as "dry open fields" in july 1986 appeared to be rainfed paddy areas in october 1987. all burnt areas (shifting cultivation) face similar problems in that, the spectral response varies a lot between nearby windows (types 11 and 12 in sitiung and in pasirmayang for instance). once again, the problem of having the same spectral value for entirely different object on two different windows and viceversa hampered the classification in figure 4. the confusion between classes 8 and 14 or 9 and 13 is obvious. the haze occurring in the sitiung area may be responsible for the slightly different spectral values compared with the same object in pasirmayang. a general classification cannot be applied to these two windows. moreover, the very simple classification method used may be responsible for some difficulties in separating the objects. separation of spectral value using the box classification method faces the problem of a statistical distribution represented by the standard deviation of the centre point of the box. furthermore, 13 biotropia no. 3, 1989/1990 table 5. spectral characteristics of the 15 classes, defined on a 2 x (512 x 512 pixels) of pasirmayang scene unknown spectral values are always classified into smaller box and this results in unsolved problems in decision boundaries delineation in a bi-dimensional space. in the muarabungo scene, three windows have been chosen (sei pemunyin 1 and 2, rimbo bujang), each of 512 x 512 pixels. the first two corresponds mainly to tropical rain forest and its degradation types, the third (rimbo bujang) to agricultural landcover types found in transmigration areas. for this scene, the box classification of xs1, xs2 and xs3 means and standard deviations gathered from the three windows gives 18 classes, 13 representing vegetation types, and 5 corresponding to open areas, roads and rivers (figure 5 and table 6). results are very similar to those of pasirmayang. implementation of that classification into each window needs some adjustments to avoid misclassification. plate 4, table 7 and plate 5, table 8, are the results of such an implementation. the same problems of some pixel being not classified as in pasirmayang scene has occurred. compared with the classification by visual interpretation of the black and white prints, certain classes (11, 12, 13; table 4) are very difficult to identify properly in the digital classification process. 14 assessment of spot satellite data-y. laumonier & u.k. djailany-syafii figure 5. xs3/xs2 diagram of the 18 spectral classes selected in muarabungo scene. table 6. spectral characteristics of the 18 classes, defined on a 3 x (512 x 512 pixels) of muarabungo scene 15 biotropia no. 3, 1989/1990 plate 4. digital classification of window rimbo bujang, muarabungo table 7. digital classification of rimbo bujang, muarabungo class vegetation types pixels surface (ha) percentage (%) colour 1 unclassed 5953 238.12 2.3 black 2 logged-over or depleted forest 16283 651.32 6.2 red 3 tall secondary forest 9648 385.92 3.7 whitish mixed with rubber trees (bright) red 5 secondary growth forest 24661 986.44 9.4 pale mixed with rubber and shrubs pink 7 secondary growth forest 5940 237.6 2.3 green mixed with remnant primary forest trees 8 mosaic of food crops and 20400 816 7.8 yellowish sparse fruit trees green 9 rubber tree estate 31970 1278.8 12.2 violet 10 newly opened, recently 62956 2518.24 24 brown burnt area 11 fruit trees, coconut and 26999 1079.96 10.3 dark habitat (homestated mixed purple gardens) 15 shrubs and grassland 8315 332.6 3.2 yellow alang-alang 16 newly planted rubber 37104 1484.16 14.2 whitish mixed with young secondary grey growth forest 17 herbaceous and slightly 11915 476.6 4.5 dark open area yellow 16 plate 5. digital classification of window sei pemunyin, muarabungo table 8. digital classification of window sei pemunyin, muarabungo class vegetation types pixels surface (ha) percentage (%) colour 0 unclassed 723 28.92 0.3 black 1 lowland primary forest 21623 864.92 8.3 red 2 logged-over or depleted 62353 2494.12 23.8 pale forest pink 3 tall secondary forest 49591 1983.64 18.9 bright mixed with rubber trees red 4 young secondary forest 11824 472.96 4.5 whitish (bright) red 7 secondary growth forest 79407 3176.28 30.3 green mixed with remnant primary forest trees 10 newly opened, recently 7042 281.68 2.7 brown burnt estate 15 shrubs and grassland 11840 473.6 4.5 yellow alang-alang 16 newly planted rubber 7191 287.64 2.7 whitish mixed with young secondary grey growth forest 17 herbaceous and slightly 2885 115.4 1.1 dark open area yellow 18 river 7665 306.6 2.9 blue assessment of spot satellite data-y. laumonier & u.r. djailany-syafii 17 biotropia no. 3, 1989/1990 3. sungai sembilang — banyuasin delta sungai sembilang banyuasin delta has undergone drastic changes since it has been chosen as a pilot area for transmigration resettlement. in spite of the numerous development aid studies conducted there, it still needs an urgent and accurate monitoring. mangroves used to flourish in the area and the remaining ones are likely to suffer from high population pressure in the future if not managed properly now. besides the mangrove belt, the lowland plain is consisting of extensive peat swamps which are also very difficult to manage for agricultural purposes. vegetation types which are often difficult to identify and segregate properly are: the mangrove forest and the adjacent fresh water swamp forest just behind it (this usually leads to an over-estimation of the actual mangrove area). the fresh water swamp forest on alluvium or shallow peat and the peat swamp forest itself on deeper peat soils, altogether with various peat swamp forest types. the swampy grasslands and the paddy fields occurring within swampy areas. the various secondary shrubby swamp vegetation types and the low natural "pole" swamp forest which look identical when seen from the air. important to investigate also is the proper identification of such secondary vegetation types like the "gelam" (melaleuca cajuputi) formations. a. interpretation and manual classification the visual interpretation gives very interesting results as all the types are perfectly distinct (12 land cover types), from mangrove swamps, fresh water swamp forest and various peat swamp forest types. b. digital classification the multispectral classification performed with spot gives 9 classes among which 6 are vegetation types (plates 6a & 6b). several types or communities are visible within the mangrove itself, and the distinction between mangroves and fresh water swamp forest is easy. possible applications 1. well-drained lowlands a) primary lowland forest appraisal of the mosaic patterns was studied in panchromatic mode at 1/50 000 scale and with multispectral data. it was impossible in any case to recognize any pattern even if some degrees of natural disturbance could be pointed 18 assessment of sport satellite data – y . laumonier & u. r. djailany-syafii 19 p la te 6 a . d ig it a l cl a ss if ic a ti o n o f w in d o w s u n g a i s e m b il a n g b a n y u a si n d e lt a ( a ft e r b a rk e y 1 9 8 7 ) p la te 6 b . t h e m a ti c m a p o f w in d o w s u n g a i s e m b il a n g -b a n y u a si n d e lt a ( a ft e r b a rk e y 1 9 8 7 ) biotropia no. 3, 1989/1990 out. riparian forest types, even along small rivers, have been located with accuracy. b) the logging activities have been easily spotted, but indirectly through the visualization of the logging roads. in fact, it was impossible to differentiate the undisturbed forest from the logged-over one in the surroundings. this is a major concern for an evaluation of the intensity of the depletion of forest resources in the region. however, when the human impact on the forest increases, some degrees of depletion appear. this is a real progress compared with landsat mss documents. c) for the monitoring of serial stages, spot capabilities appeared to be most striking. the complete vegetation series from newly clear-cut, burnt, newly planted to young regrowth stages mixed with rubber cultivation can be pointed out. in addition, several secondary forest types mixed with rubber trees, as well as various shrubby secondary vegetation types or low secondary forest types, usually classified as "mosaics" have been discriminated. these results constitute a very noteworthy input for agricultural development of the area. 2. swamps a) it has been sometimes difficult in the past to separate the mangrove forest from the adjacent fresh water swamp forest. this usually led to an over estimation of the actual mangrove area. the ecotone of those two formations is very clear on our spot document (especially on black and white prints). this is due to the perfect delineation of the back-mangrove belt dominated by the palm onco-sperma tigillariutn which is indirectly responsible for the perfect delineation of the mangrove area. in other places, back mangrove hills produced by mud lobsters and invaded by the fern acrostichum aureum give also a very clear white border to the inland limit of the formation. moreover, at least two if not three mangrove communities can be differentiated. b) one of the major challenges for the agricultural development of such an area is the distinction between the fresh water swamp forest on alluvium or shallow peat and the peat swamp forest itself on deeper peat soils. too deep peat areas are very difficult to manage for agricultural purposes. we were able to separate properly on the image the fresh water swamp forest from other types. it was even possible to differentiate several areas according to the thickness of the peat itself by locating easily the "fresh water swamp", "mixed peat swamp", and "peat swamp" forest types. 20 assessment of spot satellite data —y. laumonier & u.r. djailany-syafii c) natural shrubby or very low forest types, sometimes known as "padang" vegetation do occur in the area and are generally confused with secondary types. the spectral analysis has allowed to separate them perfectly. discussion and conclusion it was possible during the present study to compare the usefulness of spot xs and landsat mss data for forestry applications using research findings from previous landsat mss analysis in baturaja martapura area (ducros gambart & gastellu etchegorry 1984; gastellu etchegorry et al. 1985). some important remarks are stressed below: the number of spectral classes that can be discriminated with spot xs, i.e. 32, is much larger than the number of spectral classes that can be obtained with landsat mss, i.e. 8. this is due not only to the finer spatial resolution but also to better spectral characteristics of spot data. in order to derive a reliable and precise forest mapping (1:150 000) with landsat mss data, it was necessary to define complex algorithms (i.e. multi-textural analysis), whereas far better results are directly obtained with simple photointerpretation of 1:50 000 spot imagery. the fine resolution of spot data allows to map many earth features such as roads and rivers, which are particularly useful for an accurate spatial registration of the spot derived documents and provide opportunities for obtaining at the same time cartographic products. numerous ground control points which could not be recognized in the landsat image can easily be located in the spot image. the only advantage of landsat imagery was its larger synoptic overview. panchromatic documents were only available for two scenes and only in the form of negative films at a scale of 1/200 000 since no cct were available, the existing black and white prints were not very useful for this work as the level of information for vegetation is quite low when compared with xs data. the high resolution of spot has proved to be of high value in field checking for exact location of the image's features (e.g. small football field in a little village). however, this emphasizes the real necessity for a very fast delivery of data for proper use of remote sensing techniques in tropical environment where the character of most of the land-use and vegetation patterns is so dynamic. regarding the identification and classification of the humid tropical vegetation in sumatra for environmental management purposes, spot has brought several advantages and encouraging application prospects: 21 biotropia no. 3, 1989/1990 — forestry management several forest types, especially within mangrove and swamp forest areas and also in the uplands, have been discriminated with spot data. even if some of the needs cannot be perfectly fulfilled, e.g. proper distinction between primary and logged-over forest, the fact that degrees in the forest depletion can be pointed out will be of great interest to the forester. considerable improvement in the identification of serial stages will also facilitate the monitoring of reforestation projects. — land-use management the improvement in the identification of some land uses is also striking. such possibilities as the distinction between newly clear cut fields, burnt areas, and newly planted food crops, will allow easy monitoring of such areas where the shifting cultivation is an important feature of the landscape. — mangrove and swamps management the perfect delineation of the mangrove is one of the most beneficial results of spot. many different figures exist concerning the exact mangrove surface in the region. possibilities of studying on satellite documents what could be the mangrove communities themselves ("rhizophora type" or "sonneratia type") was another interesting aspect which should be investigated cautiously. for agricultural management of the peat area, spot provides an excellent tool for an indirect evaluation of the thickness of the peat through identification of specific vegetation types. many of the identification problems mentioned above could probably be solved by photogrammetry using aerial photographs at scale of 1:5000 to 1:10 000. however, the cost of aerial cover in such remote places prohibits in most cases their use before implementation of development project. moreover, there were often difficulties in the past for any extrapolation between the general data provided by landsat mss, and the detailed features of large scale aerial photographs. only simple digital image processing has been applied in this study. it gives nevertheless interesting results and the improvement of multispectral classifications of high resolution data is challenging for site quality assessment through vegetation appraisal. increased revisiting capabilities and lateral viewing, together with a better accuracy in the localization of samples offer additional opportunities in the data acquisition and processing. the dynamic character of most of the land-use and vegetation patterns implies that ground verification has to be carried out as soon as possible after data recording. on one hand, the use and interpretation of classes along the "brightness axis" was interesting and must be worked out in the future for the classification of spectral classes with sparse or non-vegetation cover. on the other hand, the 22 assessment of spot satellite d a t a -y . laumonier & u. r. djailany-syafii "vegetation axis" gives information about the vegetation cover and biomass. the synthesized information that is displayed by figure 2 is undoubtedly very useful for surveying forested areas, and more especially for estimating the density of the vegetation cover of land cover classes. it can provide preliminary assessment about the vegetation cover of spectral classes that were not identified or checked in the field. finally, the validity of classes that were supposedly identified either in the field or with aerial photographs can easily be tested. for example, classes corresponding to forested areas should not be located on the "brightness axis". this study suggests that spot is certainly a very good alternative to medium altitude aerial photograph coverage (1/100 000, 1/50 000) and emphasizes its value for tropical vegetation mapping at a medium scale (1/100 000 to 1/250 000) since comparisons of the cost of spot data versus other remote sensing tools (hadjar 1987, antikidis et al. 1988) give also a striking advantage to spot. acknowledgments this study was conducted as a part of the research project supported by the peps program (programme d' evaluation preliminaire de spot) of the french space agency, in cooperation with iciv, toulouse and seameo-biotrop, bogor. the authors would like to thank dr. j.p. gastellu-etchegorry and dr. r.a. barkey for their contributions in the digital treatments of the martapura and sungai sembilang regions. references antikdis, j.p., a. hudson and j.p. gastellu-etchegorry 1988. a spot/landsat receiving station in indonesia s.c.o.t. report. 57pp. barkey, r.a. 1987. etudes des mangroves de l' indo-malaisie et application des techniques de la tele-detection a sulawesi. these doctorat de l'ups, iciv toulouse, france. biotrop-bakosurtanal 1988. pemetaan vegetasi daerah baturaja, propinsi sumatra selatan. skala 1:250 000. laporan kerjasama seameo-3iotrop dengan bakosurtanal. blasco, f., y. laumonier and purnadjaja 1983. tropical vegetation mapping: sumatra. biotrop bull, in tropical biology. no. 22. deshayes, m. 1981. traitement numerique des donnees landsat. application a la cartographic auto matique de la vegetation tropicale. these docteur-ingenieur, ups-iciv, toulouse. djailany, u.r. 1987. formations vegetales naturelles de sumatra (indonesia): structure et caractristi-ques radiometriques. these doctorat, de l'upt, iciv, toulouse, france. 23 biotropia no. 3, 1989/1990 ducros-gambart, d. and j.p. gastellu-etchegorry, 1984. automatic analysis of bi-temporal land-sat data: application to the study of the evolution of vegetation-covered areas in a tropical region. igarss symp., ref. esa sp-215: 187-192. ducros-gambart, d., t. l.e toan and m. deshayes 1984. utilisation des donnees landsat pour la cartographic des formations ve'getales tropicales dans le sud de sumatra. l'espace ge'ographique, n°3, 205-214, 290. gastellu-etchegorry, j.p. 1988. remote sensing with spot; an assessment of spot capability in indonesia. gadjah mada university press. yogyakarta. hadjar, n. 1987. forest observation by satellite. paper presented at the acrs, jakarta, a 16/1 13. laumonier, y. 1981. classification of southern sumatra forest types. biotrop unpublished report. laumonier, y. 1983. international map of the vegetation, scale 1:1 000 000; sheet southern sumatra. biotrop, bogor, iciv, toulouse. laumonier, y., purnadjaja and setiabudi 1986-87. international map of the vegetation, scale 1:1 000 000; sheet central sumatra (1986), sheet northern sumatra (1987). biotrop, bogor; iciv, toulouse. laumonier, y. u.r. djailany, j.p. gastellu-etchegorry and r. barkey 1987. assessment of spot satellite based system in tropical vegetation identification, classification and monitoring in sumatra (indonesia). spot first result in flight conference, paris nov. 87. cnes. p ? purnadjaja 1980. evolution de la vegetation 19691976 dans la region de palembang, sumatra, me'moire dea, upsi c i v , toulouse. richardson, a.j. and c.l. wiegand 1977. distinguishing vegetation from soil background information. photogram. eng. and remote sens. 43(2): 207 — 216. rouse, j.w., r.h. haas, j.a. schelland d.w. deering 1973. monitoring vegetation systems in the great plains with erts. 3rd erts symp., nasa sp -351, 1:309-317. torquebiau, e.f. 1986. mosaic patterns in dipterocarp rain forest in indonesia and their implications for practical forestry. j. of trop. ecol. 2:301 325. tucker, c.j. 1977. spectral estimation of grass canopy variables. remote sens, of environment, 6: 11-26. 24 assesment of spot satellite data – y. laumonier & u.r. djailany-syafii figure 1. location of study areas table 1. requested products and acquisition scored 3 biotropia no. 3, 1989/1990 both visual and digital analysis techniques have been used. vegetation sketch maps and land cover classifications were prepared by manual interpretation of black and white xs or standard false colour composite prints at scales varying from 1/50 000 to 1/100 000. for mangroves and swampy areas, other false colour combinations have been used such as xs2 – bi vgi or xs1 pci pc2. a supervised multispectral classification was performed simultaneously using a pericolor 1000 system in france and an ibm at/xt system in indonesia, by simple visualization of each band using gray scales or colour table and simple transformations, such as band ratios, vegetation indices and principal component analysis. the procedure is similar to the information on techniques extracting developed for soil and leaf area indices (richardson & wiegand 1977; tucker 1977; rouse et al. 1973). means, standard deviation, range of radiance values as well as coefficient of correlation for the 3 bands were computed for all samples, together with covariance matrix of all classes to assess the value of the classification. to facilitate the visualization of the field site data, xs1, xs2 and xs3 means and standard deviations were plotted on bi-dimensional diagrams. all classes have been checked in the field (one year after data acquisition by the satellite) and eventually redefined. results 1. baturaja martapura the original vegetation is much degraded with typical huge areas of grasslands dominated by imperata cylindrica, various stages of shrub savannas and pseudoclimactic forest type dominated by schima wallichii. some very degraded remnants of the original forest types and a land-use composed of irrigated or rainfed paddy fields, orchards, rubber plantations, "damar" plantations and reforestation areas are other important features of the landscape. some small depressions are common along the streams. a rather large one is found north east of the scene, around lake (lebak) datuk. the water level in these depressions fluctuates considerably and during the dry season they are mostly dry. , a. visual interpretation the classification produced is given in table 2. these results were used to produce an ecological vegetation map at a scale of 1/250 000 at the request of the indonesian national coordination agency for survey and mapping, the bakosurtanal. a total of 23 types were identified and used later as legend of the published map (biotrop bakosurtanal 1988). 4 assessment of spot satellite data y. laumonier & u.r. djailany-syafii table 2. classification of the vegetation in martapura region by visual interpretation 1. very depleted lowland forest (high secondary forest with remnant primary forest tree species) 2. high secondary forest (often mixed with rubber) 3. mosaic of high secondary forest and food crop cultivation 4. high secondary forest dominated by schima wallichii 5. shrub, secondary regrowth 7. grassland 8. swampy tall secondary vegetation dominated by melaleuca sp. 9. swampy shrubby vegetation 10. swampy grassland dominated by cyperaceae 11. rubber estate 12. coffee gardens, often overtopped by fruit trees, mixed with food crop fields 13. pepper gardens 14. orchards, fruit trees 15. newly opened field for rubber and oil palm estate 16. dry fields, food crops, mixed with secondary regrowth 17. village, habitat and fruit trees 18. rice field b. digital classification the analysis of visual displays of spot data provided the selection of thirty two spot spectral classes. computer screen displays of enhanced spot data and spot photographic products at a 1/100 000 scale of bands xs1, xs2 and xs3 allowed the identification of most of them. however, some classes (20 to 31) could be distinguished only during digital processing stages (density slicing, image enhancement, etc.). they were not detected with conventional photo-interpretation, and the question of their real existence also arises since, unfortunately, most of these classes could not be identified properly. however, qualitative values of the density of their biomass were tentatively assessed with the aid of techniques developed for extracting information on soil and leaf area indices (rouse et al. 1973; richardson & wiegand 1977; tucker 1977). they can be classified as "sparse vegetation" (29, 30, 31), "rather sparse vegetation" (26, 27, 28), "vegetation" (22, 23, 24, 25) and "green vegetation" (20, 21). the 32 classes and their characteristics are presented below (gastellu-etche-gorry 1988): they are grouped per land cover type and according to their relative position on the "vegetation axis". forest (1) primary forest: tall trees up to 45 55 m. compared to other classes, it is characterized by the smallest xs1 and xs2 radiometric values; i.e. highest absorption by vegetation. 5 biotropia no. 3, 1989/1990 (2) very depleted forest: result of former exploitation of primary forest followed by local depletion. (3) remnant primary forest and secondary growth: external features (roads, etc.) reveal some degradation. radiometries in bands xsi and xs2 are slightly larger than those of (1). (4) remnant primary forest and secondary growth. the proportion of primary forest is less important than that of (3). (5) swamp vegetation: small xs3 radiometric values: xsi and xs2 radio-metric values are larger than those of dry land forests. a "spotted" textural pattern may reveal the presence of the swampy areas. secondary forest (6) old tall secondary forest: compared to (7), the trees and texture are similar but the vegetation index (i.e. leaf area index and/or biomass density) is larger. (7) tall secondary forest: taller and older than (8); texture may be similar to that of (10). (8) low secondary forest: compared to primary forest, the xsi and xs2 radiometric values are larger and the xs3 radiometric values are similar. (9) low secondary forest: successional stage before (8), younger and smaller. rubber plantation (10) rubber plantation: homogeneous aspect on xsi and xs2 imageries. it is identified thanks to the presence of roads that usually display a geometric pattern. (11) young rubber plantation: successional stage after (12). (12) newly planted rubber trees: xsi radiometric values are between those of rubber plantation and secondary forest growth, whereas its xs3 radiometric values are smaller than those of rubber plantation, primary forest and secondary forest. settlements and mixed gardens (13) fruit tree gardens: close to settlements; should be verified in the field. (14) gardens and sparse vegetation cover (alang-alang grasses). shrubs, herbaceous vegetation and bare soil (15) herbaceous vegetation: vegetational stage after (17), it is more densely vegetated (larger vegetation index). 6 assessment of spot satellite d a t a -y. laumonier & u . k . djailany-syafii (16) shrubs and thicket vegetation. (17) sparse vegetation cover: bare soil and more or less sparse herbaceous vegetation. (18) recently opened area: vegetational stage before (17). (19) bare soil. non-identified land cover units (assessed through their respective vegetation index; leaf area! index/biomass): (20), (21) green vegetation (22), (23), (24), (25) vegetation (26), (27), (28) rather sparse vegetation (29), (30), (31) sparse vegetation. water (32) moreover, figure 2 shows that some spectral classes which s ha re s i mi l a r spectral characteristics cannot be reliably discriminated by spectral analyses only figure 2. xs3/xs2 diagram of the 32 spectral classes selected in martapura scene. 7 biotrop1a no. 3, 1989/1990 i.e. classes (26) and (28), (21) and (11), (8) and (22), etc. in this case, knowledge of the field (if any) and the spatial and textural information which can be extracted from simple photo interpretation of spot xs1, xs2 and xs3 imageries must also be inputed (gastellu etchegorry 1988). in this study, the identification of these classes obviously required additional field checks which were conducted in june 1988. plate 1 and table 3 give an example of the reformulation of the 32 classes for one "window of the martapura scene", after field work. only 13 classes (11 vegetation types) were actually identified for that particular area, which means that several spectral values correspond actually to one vegetation types. classes 5,8,22,23,26 and 28 for instance, should be regrouped into class 7 e.g. grasslands, at least as far as identification of the types is concerned. table 3. digital classification of window batu marta, martapura vegetation types pixels surface (ha) percentage (%) colour 0 unclassed and home 14926 597.04 4.8 black stated mixed gardens (settlement) 3 secondary forest 40617 1624.68 13.2 whitish mixed with rubber bright red 4 low secondary forest 59524 2380.96 19.4 bright and schima wallichii green 6 shrub, secondary 4235 169.4 1.4 whitish regrowth (newly grey felled) 7 grassland 52676 2107.04 17.1 yellow 8 rubber tree estate 93328 3733.12 30.4 red 9 swampy shrubby 10737 429.48 3.5 orange vegetation 11 dry field, food crop, 12398 495.92 4 dark mixed with secondary green regrowth 12 longtime burnt and 6900 276 2.3 dark slightly covered by grey regrowth 13 recently burnt for 7828 313.12 2.5 brown estate area 14 swampy area 2388 95.52 0.8 violet 17 river 811 32.44 0.3 blue 18 sand sediment in the 782 31.28 0.3 bright river blue 8 plate 1. digital classification of window batu marta, martapura 2. muarabungo pasirmayang a very peculiar land-use feature is found there. local people have grown rubber trees since 1910. after about two years of rice cropping, the field is planted with rubber trees and it gradually becomes a secondary forest mixed with rubber which is rarely cleaned but still used for tapping. this leads to various stages of secondary forests mixed with rubber which are impossible to differentiate from pure serial stages on remote sensing documents. the area still harbours pieces of quite undisturbed forest and large tracks of logged over forests. the landscape is also characterized by large transmigration areas. in that area, remote sensing encountered problems in identifying and classifying the following vegetation and land cover types: the mosaic nature of the primary lowland forest: the tropical forest is highly dynamic and grows in patches, a mosaic of higher and lower forest parts which have their origin in the natural fall of trees or part of the tree crowns. the locally depleted forest (local exploitation) and the really undisturbed ones (only industrial logging is recognizable but only indirectly through the visualization of the logging roads). low secondary forest types mixed with rubber tree cultivation (managed by local farmers) and pure serial stages without human interferences. 9 assessment of spot satellite data —y. laumonier & u.r. djailany-syafii 1.pdf 10.pdf 2.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf