RESPONSE LONKIDA ( L.) OF TOWARDS Nauclea rientaliso MYCORRHIZ AL INOCULUM IN WATERLOGGED CONDITION FAISAL DANU TUHETERU , CECEP KUSMANA , IRDIKA MANSUR1, 2* 3 3, and ISKANDAR4 1Tropical Silviculture Major, Post-graduate chool of S Institut Pertanian Bogor, Bogor 16680, Indonesia 2Department of Forestry, Faculty of Forestry and Environmental Science, Halu Oleo University , Indonesia 3Department Silvi ultur Institut Pertanian Bogor, Bogor 16680, Indonesiaof c e, Faculty of Forestry, 4Department of Soil Sciences and Land Resources, Faculty of Agriculture, Institut Pertanian Bogor, Bogor 16680, Indonesia Received 1 August 2014/Accepted 6 October 2014 ABSTRACT W . aterlogged condition is one of environmental conditions not favorable for plants and mycorrhizal fungi The tolerant are more adaptive to ed condition Arbuscular Mycorrhizal species waterlogg . The documented effects of Fungi ( ) esides increasing the tolerance of plants, AMF inoculation on wetland plant performance are inconsistent. B AMF waterlogged . was carried out also causes plants depression in condition This study to examine the morpho- anatomical adaptation, survival, growth, biomass and nutrient uptake of onkida ( L.) from different l Nauclea orientalis habitats inoculated with Arbuscular Mycorrhizal Fungi (AMF) in waterlogged condition for 90 days. Experiment procedures include : seed germination, preparation of inoculums and inoculation of and waterlogg d AMF ed treatment. ling adventitious rootsThe results showed that lonkida seed s formed lenticels (100%), (41%) and a under waterlogg Seedling dry landerenchym ed condition. s from habitats, both mycorrhizal and non-mycorrhizal, had lower height growth rate than those from other habitat types. The same results happened relative growth rates for for shoot (RGRs), root (RGRr) and total (RGRt) and the ratio of root shoots. Treatments of temporal dry weight swamp habitat without ed condition and with ed condition and treatment in savannah with waterlogg waterlogg waterlogg plant dry weight root shoot and ed condition had RGRt greater than those in other treatments. In general, ( , total between swamp with ) and total N in roots and shoots were greater in the interaction temporal habitat non- mycorrhizal in ed condition. waterlogg However, lonkida seedling from dry land habitat required AMF under waterlogged to improve biomass and N a cumulation in roots. , waterloggcondition c Independently ed condition increased the pool of average height gain (18%), diameter (46%), leaf area (40%), leaf length (17%) and leaf width (21%). Nonetheless, ed condition treatment decreased the number of leaves (9%) colonization waterlogg and AMF (71%) The results of this study indicate that seed s from temporal had good growth and high biomass in . d ling swamp condition with or without ed condition waterlogg . : Keywords Arbuscular Mycorrhizal Fungi (AMF), morpho-anatomical adaptations, ., lonkida, LNauclea orientalis swamp ed condition, waterlogg INTRODUCTION Waterlogged is one of environmental conditions not favorable for plants and mycorrhizal fungi (Helgason Filters 2009). & Waterlogged condition causes anaerobic condition indicated by hypoxia and anoxia (Elzenga van Veen 2010). These conditions can & affect growth, development and adaptability of plant (Kreuzwieser Gessler 2010; species & Parolin Wittman 2010) as well as species & composition of forest stands (Kozlowski 1984, 1997). Effects of waterlogged condition is highly varied depending on genetic and age of the plants, time and duration of inundation (Kozlowski 1984) as well as depth of inundation (Iwanag a & Yamamoto 2008). Plant can tolerate waterlogged condition through morpho-anatomical and physio logica l adaptat ion mechani sms. Physiological adaptation can be done by accumulating reserves in the root in the form of carbohydrates and alcohol fermentation as an * Corresponding author : faisaldanu_28@yahoo.com BIOTROPIA Vol. 22 No. 1, 2015: 61 - 71 DOI: 10.11598/btb.2015.22.1.416 61 mailto:faisaldanu_28@yahoo.com BIOTROPIA Vol. 22 No. 1, 2015 62 alternative (Kreuzwieser Gessler 2010; Parolin & & Wittman 2010), while morpho-anatomical adaptation is done by forming lenticels, adventi ous root and erenchyma (Folzer . ti s a et al 2006; Parolin Wittman 2010)& . Plant tolerance to ed condition varies waterlogg and is determined by the (Kozlowski species 1984). The tolerant are more adaptive to species waterlogg ed condition (Kogawara 2006). et al. However, differences in habitat of the species also largely determine the success of the plant to survive in inundation condition (Ferreira . et al 2009; Nielsen . 2010). Several studies have et al reported that growing naturally in the species inundated area are more tolerant to waterlogged condition, such as (Keeley 1980), Nyssa sylvatica female (Nielsen 2010) and Populus angostifolia et al. Himatanthus sucuba et al. (Ferreira 2009). Information on tropical tolerance and species adaptation to inundation still needs to be studied (Parolin 2009). In Indonesia, research on tolerant species toward different types of inundated habitat is still very limited. Lonkida ( L.) is a tropical Nauclea orientalis multipurpose tree species that grows naturally in a variety of habitat types including wetlands (Petty & Douglas 2010; Kartikasari 2012) and has et al. natural distribution in Indonesia (Whitmore et al. 1997; Keβler 2002). This species has potential et al. uses for agroforestry, phytoremediation and rehabilitation of degraded land, including wetlands (Marghescu 2001; Amihan-Vega & Mendoza 2005; Mawaddah 2012) and medicinal plant (Lim 2013). A preliminary study of lonkida response to ed condition greenhouse waterlogg in scale had been conducted and it was confirmed that this species was tolerant to waterlogged condition (Kurniawati 2011). However, studies related to growth response, adaptation, and plant nutrient uptake ability of lonkida from various habitats under waterlogged condition had not been reported. This was also reported to be colonized species by Arbuscular Mycorrhizal Fungi ( AMF) Glomus mossea in India (Sudha Ammani 2010) and & showed good growth response to the application of AMF in waterlogged condition (Kurniawati 2011). Tolerance to waterlogged condition could be enhanced through inoculation. AMF Association of inundated plants with is part AMF of strategy to improve nutrient uptake and oxygen circulation (Elzenga & van Veen 2010). In waterlogged condition, could AMF association improve growth (Osundina 1998; Fougnies et al. 2007), biomass and nutrient uptake, especially phosphorus (Muok Ishii 2006; Fougnies & et al. 2007), the growth of root systems (Qiang-Sheng et al. 2013) and the structure of plant communities (Bauer 2003). However, colonization, et al. abundance and benefits of in edAMF waterlogg condition can vary among plant species (Bauer et al. 2003) due to of used (Secilia species AMF & Bagyaraj, 1994; Sah 2006), availability of et al. phosphorus (Stevens 2002; Garcia 2008) et al. et al. and distance from the rhizospher (Keeley AMF 1980). Biomass of mycorrhizal Nyssa sylvatica seedlings from inundated seed sources were higher than that from dry areas (Keeley 1980). The purpose of this study was to examine the morpho-anatomical adaptation, survival, growth, biomass production and nutrient uptake of AMF- inoculated lonkida originated from different habitat types in waterlogged condition. MATERIALS AND METHODS Experiment Procedures Seed germination Lonkida seeds were collected from mother trees grown in different habitat types (swamps, temporary , temporary swamps savannah swamps and dry land areas) in C andKendari ity, Konawe South Konawe District (Fig. 1), Southeast Sulawesi rovinceP . Lonkida seeds were germinated in plastic boxes having dimension of 20x20x5 cm containing sterile soil media (±1 kg) and were grown until reaching seedling stage at age of 150 days before being used in this experiment. There were 144 seedlings ready to be used in this experiment. Inoculum preparation and AMF inoculation AMF inoculum used was mycofer IPB containing 4 (four) species of AMF, namely Glomus etunicatum, Glomus manihotis, Acaulospora tuberculata Gigaspora margaritaand . The number of spores per 5 g of inoculum was 57-125 spores. Before inoculation, polybags having size of AMF 15x20 cm were filled with sterile soil media (1.5 kg soil media/bag). There were 72 lonkida seedlings having age of 150 days inoculated with AMF inoculum and subsequently were called mycorrhizal seedlings. The other 72 lonkida Response o lonkida ( L.) mycorrhiz i condition Faisal – et al. f owards al noculum n aterloggedNauclea orientalis t i w seedlings were not inoculated with AMF inoculum and served as control treatment; subsequently were called non-mycorrhizal seedlings. Chemical properties of the sterile soil media were pH 5.2, organic C (Walkey Black) & 0.27%, total N (Kjeldahl) 0.06%, C/N ratio 5, available P (Bray I/I ) 2.5 ppm, exchangeable Ca, I Mg, K, Na, and CEC were 10.32 cmol/kg, 1.49 cmol/kg, 0.66 cmol/kg, 1.76 cmol/kg and 19.79 cmol/kg (Extract Buffer 1.0 N NH OAc pH 7.0), 4 respectively. Waterlogged treatment Mycorrhizal and non-mycorrhizal seedlings at age of 150 days after planting were then treated with ed condition. Each seed was waterlogg ling placed in a polybag having size of 26.5x23 cm containing sterile soil media. A total of 72 seedlings inside polybags were immersed in water, while the other 72 seedlings inside polybags were not immersed, but watered every day as control (Garcia . 2008). Water level was monitored et al every 3 days. Experimental design This study used factorial in a completely randomized design (CRD) consisting of 3 factors: 1. Treatment A= habitat origin of lonkida consisted of: A =dry land;0 A = ; 1 swamp A =temporal ;2 swamp A = temporal savannah.3 swamp 2. Treatment B = AMF inoculum treatment consisted of: B = uninoculated non-mycorrhizal);0 ( B = inoculated with AMF inoculum 1 (mycorrhizal). 3. Treatment C = ed treatment waterlogg consisted of: C = (non-waterlogged);0 control C =waterlogged.1 Each treatment was replicated 3 times and each replication consisted of test plants. The 3 experiment was conducted for 90 days. At the end of experiment, the seedlings' age was 240 days old. Data Collection Growth Measurements were conducted at the end of the experiment (at the end the 90-day experiment) for seedling height, seedling diameter, number of leaves per seedling and plant . survival Measurement of leaf area was conducted using green leaf area meter GA-5 model. Relative growth rates (RGR) for total (RGTt), shoots (RGTs) and roots (RGTr) for control dry mass and waterlogged condition were then calculated using the formula of (200 ):Mendoza 5et al. 63 Figure 1. Distribution map of lonkida tree sampling RGRi = (ln Wt - ln W )/(t -t )f f oto where: RGR= relative growth of plants; rate i= total dry weight, shoots and roots; Wt = dry weight shoots and roots f plant (total, ) at the end of the (seedlings' experiment age was 240 days old); Wt = dry weight o plant at the beginning of experiment period (seedlings' age was 150 days old); t -t = the difference between the and the f o initial total period growth (experiment of period was 90 days). Dry weight and in plant tissuenutrients At the end of 90 days, the shoots and roots were harvested. Fresh shoots and roots were ovendried at 70 C for 48 hours (Garcia . 2008). o et al Determination of P was carried out using HNO -3 HClO method, and N by Kjeldahl (Balai 4 Penelitian Tanah 2009). For N and P, two expressions of nutrient uptake efficiency was calculated con entration per unit of dry weight i.e. c and total (a cumulation) uptake per unit of dry c weight. Morpho-anatomical adaptation Observation and counting of the number of lenticels and were done every adventitious roots week - . using non destructive samples The data of lenticel and adventitious roots obtained at s were the end of . The proportion data of experiment lenticels number and adventitious roots the were ratio between the number of sample forming s lenticel and adv ootss entitious r . Aerenchyma observations for the lenticles and adventitious roots were made by following the method of freezing microtome . Roots of 3 (Longstreth 1879) cm long (diameter <3 mm) were immersed in 70% alcohol solution ( ours The roots were then 24 h ). cut using scissors into root pieces of 0.5 cm long. The root pieces were then put in Yamato Mc- 802A Electro Freezer for 2-3 minutes at -15 C. o The roots were then cut using Yamato RV-240 microtome with thickness of 20 µm. Before the roots were placed on object glasses, several drops of 20% glycerol solution were applied on the object glasses, while the roots were stained with safranin. Aerenchyma observation was made using Axio Imager microscope A1m/Axiocam MRc5 x magnification . (200 ) Susceptibility (SI) Index For all measured plants variables, and soil S Index (SI) was calculated using the usceptibility method of Hiler (1972):et al. SI = 1 - (waterlogged plant/ )non-waterlogged plant SI value will be positive if the waterlogged condition decreases the variables measured. On the other hand, SI value will be negative if waterlogged condition increases the variables measured. AMF spore density and colonization Thirty grams of soil samples were collected from soil medium in polybags used as mychorrizal lonkida medium. Spores were extracted from soil using wet sieving and decanting method (Gerdemann & Nicolson 1963) followed by centrifugation; supernatant acquired was added with 50% sugar solution (Walker 1982). AMF et al. spores extracted were observed and counted under a dissecting microscope with 35x magnification. Root colonization was observed by using root staining technique of Brundrett et al. (1996). Fresh roots cleaned in 10% KOH for were 2 days, then were immersed in H O for 10-20 2 2 minutes and were rinsed thoroughly with water. The roots were then soaked in HCl 0.2% for 20 minutes and then were stained with trypan Blue solution . . Ten root samples (1 cm long) (0 05%) from each plant were observed under a microscope with 200x magnification. oot R colonization was calculated using the formula of Brundrett (1996):et al. % = root colonization Number of fields-of-view containing mycorrhizae Total observed fields-of-view Data Analysis Data were analyzed using analysis of variance (ANOVA). Abnormal data were normalized using logarithmic transformation. The data were further analyzed using Duncan's multiple range test (DMRT) for means comparison. Data analyses were computed using SAS 9.1.3 portable program. BIOTROPIA Vol. 22 No. 1, 2015 64 x 100% 65 Table . Measurement of the observed variables for lonkida seedlings grown in ed condition and in non-1 waterlogg waterlogged condition Variables Measurement for lonkida seedlings grown in waterlogged condition (mean±SE) Measurement for lonkida seedlings grown in non-waterlogged condition (mean±SE) SI Effect of waterlogged condition Plant growth variables Survival 100±0.00 a 100±0.00 a ns ns Height (cm) 8.13±2.45 a 6.18±1.177 b -0.32 + Diameter (mm) 1.81±0.31 a 1.24±0.25 b -0.46 + Total of leaves (blade) 13.1±0.838 b 14.3±1.183 a 0.09 - Leaf area (cm2) 9.8±3.045 a 7.0±0.659 b -0.40 + Leaf length (cm) 8.2±0.878 a 7.0±0.363 b -0.17 + Leaf width (cm) 2.9±0.431 a 2.4±0.132 b -0.21 + RGRs (per day) 0.0084±0.0032 a 0.0060±0.0015 b -0.42 + RGRr ( per day) 0.0075±0.0035 a 0.0069±0.0020 a ns ns Shoot/root ratio 1.89±0.25 a 1.77±0.32 a ns ns Adaptation to waterlogged condition Total adventious root 1.16±0.552 a 0.0±0.00 b - + Adventious root (%) 48.6±17.005 a 0.0±0.00 b - + Total lenticels 30.9±2.91 a 1.3±0.35 b -22.7 + Lenticels (%) 100±0.00 a 81±16.95 b -0.23 + AMF symbiosis AMF colonization (%) 11.5±2.64 b 29.1±6.25 b 0.61 - Nutrient uptake N in root (%) 1.091±0.13 a 1.185±0.10 a ns ns N in shoot (%) 0.73±0.059 b 0.83±0.061 a 0.12 - P in root (%) 0.208±0.14 a 0.203±0.016 a ns ns Total P in root (mg) 0.255±0.05 a 0.199±0.033 b -0.28 + P in shoot (%) 0.124±0.02 a 0.108±0.013 a ns ns Total P in shoot (mg) 0.276±0.11 a 0.178±0.029 b -0.55 + Le Ad A B C Notes: 1) Figures followed by the same letters in different columns are not significantly different according to DMRT at 95 % confidence level 2) differentns=not significantly 3) Susceptibility index=1-(waterlogged plant/non-waterlogged plant) SI/ Figure . lonkida magnification aerenchyma2 Cross-section of root tissue (x200 ) showing the presence of in (A) non- waterlogged (B waterlogged (C) formation adventitious roots condition; ) condition; of lenticels (Le) and (Ad) Response o lonkida ( L.) mycorrhiz i condition Faisal – et al. f owards al noculum n aterloggedNauclea orientalis t i w RESULTS AND DISCUSSION Mor o Anatom Adaptationph - ical Seed of lonkida have morpho-anatomical lings adaptation mechanisms to survive in ed waterlogg condition. The results showed that lonkida seedlings d , lenticels produce adventitious roots and erenchyma (Fig. and Table ). Lenticels a 2 1 began to form within 3 to 5 days after being in the waterlogged condition. All seed under lings waterlogged condition formed 100% lenticels (Table 1). Lonkida seedlings grown in RESULTS AND DISCUSSION Mor o Anatom Adaptationph - ical Seed of lonkida have morpho-anatomical lings adaptation mechanisms to survive in ed waterlogg condition. The results showed that lonkida seedlings d , lenticels produce adventitious roots and erenchyma (Fig. and Table ). Lenticels a 2 1 began to form within 3 to 5 days after being in the waterlogged condition. All seed under lings waterlogged condition formed 100% lenticels (Table 1). Lonkida seedlings grown in w ed aterlogg condition had an increase in lenticels and adventitious roots compared with lonkida seedlings grown in non- ed conditionwaterlogg (Table 1). Aerenchyma was formed in the root cortex of lonkida in both waterlogged and non- waterlogged conditions. Aerenchyma proportion in root of lonkida grown in waterlogged tissue condition was greater than that of lonkida grown in non- ed condition (Fig. )waterlogg 2 . Seed s of lonkida adapt to waterlogged ling ed condition by modifying the morphology and anatomy of the body through lenticels, adventitious root and aerenchyma formations. In the treatment where lonkida seedlings were grown in non-waterlogged condition and swamp habitat, the lenticels percentage was below 100 %. The same finding was found in aerenchyma in root tissue. A formation were absent in dventitious root treatment where lonkida seedlings were grown in non-waterlogged condition. The adaptation mechanism improves the oxygen uptake ability and oxygen transfer to plant tissues in waterlogged condition (Yin 2012). Thus, et al. morphological and anatomical adaptations could reduce hypoxic condition and contribute to the restoration and maintenance of aerobic respiration of seedlings in waterlogged condition (Ashraf 2012). Lenticels were crucial in the diffusion of oxygen and anaerobic metabolism resulting products (ethanol, CO and CH ) and 2 4 played a role in plant water homeostasis (Ashraf 2012). The of plants tolerant to adventitious root waterlogged condition was formed to play the role as the main root replacement to maintain water and mineral supplies when the main roots were not functioning normally, to maintain aerobic respiration of seedlings and to initiate stomata opening (Kozlowski 1997; Ashraf 2012). Network of aerenchyma contributed to the life and growth of plants in a long term waterlogged condition (Ashraf 2012). Adaptation mechanisms mentioned above were found both in plants tolerant or intolerant to water log ged condit ion. Lent ice ls and adventitious root woody plant, were found in such as Calophyllum brasilienses Camb (de Oliveira & Joly 2010), (Medina . 2009), Erythrina speciosa et al Larix laricina Populus (Islam & Macdonald 2004), deltoids et al Salix gracilistylaBéjaoui ( . 2012), (Nakai et al Salix martiana. 2010) and (Parolin 2009). Aerenchyma formation in root also occurs in Erythrina speciosa et al. Melaleuca (Medina 2009), cajuputi et al. Quercus petraea (Tanaka 2011) and (Folzer 2006). In addition to the above et al. mechanisms, the roots of lonkida seedlings also appeared on the surface of the media and out through the holed polybags. These facts indicated that the roots could grow well in waterlogged condition and could absorb oxygen and nutrients for the plants. The formation of leaves and roots in ed condition was thought to support waterlogg growth through the activity of oxygen uptake, increased water and nutrient contents and increased photosynthesis activity (Kozlowski 1997). Increased seedling growth in ed waterlogg condition indicated that lonkida seed s had ling high efficiency in the improvement of biomass per each nutrient absorbed and maintained aerobic respiration (Kozlowsk 1997; Tanaka .i et al 2011 ). Plant Growth He t of s ling igh lonkida eed s in temporal swamp and savannah habitats without mycorrhiza showed significant difference from other treatments, except for treatment in swamp habitats either with or without mycorrhiz ( . a Fig 3a waterlogged). Lonkida seedlings grew better in condition those in non-water-compared with logged condition, with h igh growth differences e t of 32%, diameter differences of 46%, leaf area differences of 40%, leaf length differences of 17% and leaf width differences of 21% between the two treatments (Table ). Nonetheless, 1 waterlogged condition reduced the number of leaves 9%. Temporal habitat by swamp on waterlogged condition had the highest RGRt value (0.0125 g/day) and the lowest RGRt value occurred in the dry land ed under waterlogg condition (0.0047 g/day) . . Treatment (Fig 3b) of BIOTROPIA Vol. 22 No. 1, 2015 66 waterlogg ed condition did not significantly affect RGRr, but RGRs increased by 42% . (Table 1) Non-mycorrhizal lonkida seedlings had higher RGRs, RGRr and RGRt compared with mycorrhizal lonkida seedlings The (Table 3). results of this study indicated that edwaterlogg condition did not inhibit vertical growth (root and shoot) and horizontal growth (diameter) of plant. Lonkida seed s originated from ling dry land habitats have lower growth rate, RGRs, height RGRr , RGRt and root shoot ratio compared with lonkida seedlings originated from other habitat types, for both mycorrhiz and non-mycorrhiza al l treatments. The results of this study indicated that lonkida originated habitat seedlings from dry land were not suitable to be planted in ged waterlog condition. Several studies have reported the same results, such as study on the female P uop lus angustifolia that grew naturally in inundated area was more tolerant to waterlogged condition within 15 weeks compared to male P. angustifolia male which originated from the dry land (Nielsen et al. et al 2010). Ferreira . (2009) reported that Himatanthus sucuba originated from non-flooded areas (terra-firme) was intolerant to waterlogged condition compared with that originated from inundated area (Varzea) in Amazonian flood plain. However, shoot root ratio of seedlings originated from dry habitats were smaller than those from other habitats. The low was shoot root ratio assumed as being a protective mechanism from dehydration through transpiration (van Splunder et al. 1996). Another strategy that might occur was that seed s from dry habitats had more C ling land translocation to the roots than to the shoots (Martinez 2012).et al. In contrast to the dry habitats, lonkida seedlings originated from temporal swamp habitat in waterlogged and non-waterlogged conditions and lonkida seedlings originated from savannah habitat waterlogg in ed condition had greater RGRt compared with RGRt in other treatments. The average height growth of lonkida seed s ling from temporal swamp and savannah habitats were quite high, indicating that lonkida seedlings from both habitats were tolerant to waterlogged condition; which also indicated that the physiological adaptation mechanism of the seedlings was not disrupted and morpho- anatomic change was formed well in the al waterlogged condition. In addition, lonkida seed s in the temporal grew well in dry ling swamp condition and in ed condition. Parolin waterlogg (2009) explained that the differences in the type of tolerance to waterlogged condition were strongly associated with ecotype differences among habitats due to natural selection. The results of this experiment indicated that lonkida is a species having relatively broad ecological distribution, compared to most species from inundated area, which generally had relatively small ecological amplitude (Parolin 2009). Plant Biomass Table 2 shows significant difference on shoot dry weight was shown for mychorrizal lonkida seedlings originated from dry land in waterlogged condition, non-mychorrizal lonkida seedlings originated from swamp in waterlogged condition, non-mycorrhizal lonkida seedlings originated from savannah in waterlogged condition and mycorrhizal lonkida seedlings originated from savannah in non-waterlogged condition. The same table also shows that root dry weight of mycorrhizal lonkida seedlings originated from dry land in waterlogged condition was significantly different from all other treatments. Significant difference on total dry weight was shown for mycorrhizal lonkida seedlings originated from dry land in waterlogged condition, non-mycorrhizal lonkida seedlings originated from savannah in waterlogged condition and mycorrhizal lonkida seedlings originated from savannah in non- waterlogged condition (Table 2). hoot- ratio S root of non-mycorrhizal lonkida seedlings was higher (1.95) than that of mycorrhizal lonkida seedlings (1.65) . (Table 3) The results of this study showed that lonkida seed s had various adaptation strategies to ling overcome environmental stress. Seedlings from dry habitats required mycorrhizae fungi in waterlogged condition. Some studies also indicated that AMF improved plant tolerance in waterlogged condition (Osundina 1998; Neto et al. et al. The role of AMF 2006; Fougnies 2007). in waterlogg condition ed can be seen from increase of Stevens 2002; nutrient uptake especially P ( et al. Muok and Ishii 2006; ; et al.Fougnies 2007 Garcia et al. ) enhanc growth ( 2008 in ing Secilia & 67 Response o lonkida ( L.) mycorrhiz i condition Faisal – et al. f owards al noculum n aterloggedNauclea orientalis t i w BIOTROPIA Vol. 22 No. 1, 2015 68 Bagyaraj 1994 Osundina 1998 Fougnies ; ; et al. 2007 Secilia & ) and plant biomass (Keeley 1980; Bagyaraj 1994). Nutrient Uptake The highest total N in root occurred in mycorrhizal lonkida seedlings originated from dry land habitat in ed condition. The highest waterlogg total N in shoot occurred in non-mycorrhizal lonkida seedlings originated from temporal swamp in waterlogged condition The (Table 2). results of this study revealed that was AMF needed by seedlings of habitat in dry land waterlogged condition. N uptake by in AMF waterlogg ed condition had also been reported in Aster tripolium et al. (Neto 2006). In contrast to the dry habitats, seed s of habitat ling temporal swamp did not require mycorrhizae i waterlogged n condition. Non-mycorrhizal lonkinda seed lings from temporal swamp in waterlogged condition had higher N accumulation in leaves and roots. Total P in root of lonkida seedlings grown in waterlogged condition was higher than that of lonkida seedlings grown in non-waterlogged condition (Table 1) which was in line with research on plants of Mill. (Mendoza . Lotus Glaber et al 2005; Garcia . 2008).et al Arbuscular Mycorrhizal Fungi (AMF) Colonization and Spore Density A fungi (AMF)rbuscular mycorrhizal colonization in waterlogged condition was 61% lower than that in non-waterlogged condition (Table ). AMF structures found in the roots of 1 lonkida were internal hyphae, external hyphae, hyphal coils, vesicles and arbuscular. The internal structure of the hyphae commonly AMF was good without waterlogg . The ed condition numbers of vesicles per 1 cm roots were more in lonkida seedlings grown in condition waterlogged (Table ). inhibit 2 Waterlogged condition ed colonization and spore density of AMF. AMF colonization believed to decrease the density was of spores in waterlogged condition due to anaerobic environment (Escudero & Mendoza 2005; Mendoza 2005; Garcia 2008; et al. et al. Stevens 2011; 2013). The low et al. et al.Wu number of spores in this study attributed to the fact that the species of AMF used were not originated from areas with waterlogged condition (Qiang-Sheng 2013), or oxygen -deficient et al. condition (Kozlowski 1997), limited or had supply of C from the host (Neto 2006). Thus, et al. the AMF was not able to sporulate and had limited external hyphae resulting from waterlogg condition (Secilia & Bagyaraj 1994; ed Sah 2006). Nevertheless, other studies et al. showed higher colonization of AMF in waterlogg condition (Sah 2006; Neto ed et al. et al. 2006; Faougnies 2007). In addition to the et al. species of AMF, plant species (Bauer 2003), et al. availability of P (Stevens 2002; Garcia et al. et al. 2008) distance from the AMF rhizospher (Keeley , 1980) as well as depth and duration of waterlogg also contribute to the ed condition d AMF symbiosis (Graham . 2013).et al Arbuscular mycorrhizal structure found were the internal and external hyphae, vesicles, arbuscular, coil and spores. Arbuscules found and the numbers of vesicles per cm root were more in waterlogged condition in non waterlogged-than condition. Arbuscule is a structure of AMF in the root that acts as a terminal for the transfer of nutrients from the host to the AMF and the transfer of carbon to fungi (Smith Read 2008) & . Vesicles are storage structures and can support the growth of the internal hyphae (Smith Read & 2008). n , vesicle I waterlogged condition structures found in large quantities. The were results of this study indicate that the AMF save d d a lot of energy in the vesicles needed to adapt to the (Garcia 2008). waterlogged condition et al. This fact ha also been reported in the roots of d Lotus tenuis et al. Citrus junos (Mendoza 2005) and (Qiang-Sheng 2013). ow et al. L number of arbuscules the root of lonkida also show that in ed the transfer of C from plants to fungi was low and inhibit sporulation processed CONCLUSIONS lings hadLonkida seed high survival rate and adapt to waterlogg by modifying ed ed condition the morpho-anatom through formation of ical lenticels, and aerenchym . adventitious roots a Lonkida seed s inundated habitat tend ling from ed to have higher growth and biomass. In waterlogged condition, AMF treatment increased biomass and N accumulation in roots of lonkida seedlings from dry land habitat. originated A fungi (AMF)rbuscu la r mycor rh iza l colonization in waterlogged condition was lower 69 Response o lonkida ( L.) mycorrhiz i condition Faisal – et al. f owards al noculum n aterloggedNauclea orientalis t i w than that in non-waterlogged condition. Based on these results, lonkida has the potential to be used for rehabilitating degraded mining lands. REFERENCES Amihan-Vega B, Mendoza JD. 2005. Benefits rom ree f t g t d u e rrowing In he egraded plands: mpirical ealities f t :rom Tabango, Leyte, he Philippines. In Harrison S, Herbohn J, Suh J, Mangaoang E Vanclay J editors., , AC IAR Sm a l l ho l der Fores t r y Pro j ec t - Redevelopment of a Timber Industry Following Extensive Land Clearing : from TProceedings he E P W . nd-of- roject orkshop Ormoc city, The Philipines , 19-21 August 2004. p 93-106. Ashraf MA. 2012. Waterlogging stress in plants : a review. Afr J Agric Res. 7(13): 1976-81. Balai Penelitian Tanah. 2009. Petunjuk Teknis Analisis Kimia Tanah, Tanaman, Air dan Pupuk . Bogor (ID) : Balai Besar Litbang Sumber Daya Lahan Pertanian, Balai Penelit ian dan Pengembangan Pertanian Departemen Pertanian. Bauer CR, Kellogg CH, Bridgham SD, Lamberti GA. 2003. Mycorrhizal olonization across hydrologic c gradients in restored and reference freshwater wetlands. Wetlands 23(4): 961-68. Béjaoui Z, Albouchi A, Lamhamedi MS, Abassi M, El Aouni MH. 2012. Adaptation and morpho-physiology of three Marsh. x L. Clones after Populus deltoides P. Nigra preconditioning to prolonged waterlogging. Agroforest Syst 86:433-42. Brundrett M, N Bougher, B Deu, T Grove, Majalaczuk . 1996. Working with Mycorrhizas in Forestry and Agriculture ( ) :. Canberra AU Australian Centre for International Agriculture Research. de Oliveira VC, Joly CA. 2010. Flooding tolerance of Calophyllum brasili ense Camb. (Clusiaceae): morphological, physiological and growth responses. Trees 24:185-93. Elzenga JTM, van Veen H Waterlogging and lant . 2010. p n u Mancuso S, Shabala S, editor. utrient ptake In : . Waterlogging Signalling and Tolerance in Plants New . York Springer (US): . p 23-36. Escudero V, Mendoza R. 2005. Seasonal variation of arbuscular mycorrhizal fungi in temperategrasslands along a wide hydrologic gradient. Mycorrhiza 15: 291-99. Ferreira C da Silva, Piedade MTF, Tine MA , Rossatto DR, S Parolin P, Buckeridge MS. 2009. The role of carbohydrates in seed germination and seedling establishment of , an Amazonian Himatanthus sucuuba tree with populations adapted to flooded and non- flooded condition. Ann Bot 104: 1111 9. -1 . Folzer H, D JF, Capelli N, Rieffel D, B PM. 2006. at adot Response of sessile oak seedlings ( ) to Quercus petraea flooding: an integrated study. Tree Physiol 26 759. : - 66. Fougnies L, Renciot S, Muller F, Plenchette C, Prin Y, de Faria SM, Bouvet JM, Nd Sylla S, Dreyfus B, Bâ AM. 2007. Arbuscular mycorrhizal colonization and nodulation improve flooding tolerance in Pterocarpus officinalis Jacq. eedlings. Mycorrhiza 17: 159 66s - . Garcia I, Mendoza R, Pomar MC. 2008. Deficit and excess of soil water impact on plant growth of Lotus tenuis by affecti g nutrient uptake and arbuscular n mycorrhizal symbiosis. Plant Soil 304: 117 31 - . Gerdemann JW Nicolson TH. 1963. Spores of mycorrhizal , endogone species extracted from soil by wet sieving and decanting. Trans. Brit. Mycol. Soc. 46: 235-44 . Graham LL, Turjaman M, Page SE. 2013. Shorea balangeran and (syn. ) as tropical peat Dyera polyphylla Dyera lowii swamp forest restoration transplant species: effects of mycorrhizae and level of disturbance. Wetlands Ecol Manage 21(5):307-21. Helgason T, Fitter AH. 2009. Natural selection and the evolutionary ecology of the arbuscular mycorrhizal fungi ( ) [DARWIN REVIEW]. Phylum Glomeromycota J Exp Bot 60(9): 2465 80 - . Hiler EA, van Bavel CHM, Hossain MM, Jordan WR. 1972. Sensitivity of southern peas to plant water deficit at three growth stages. Agron. J. 64: 60-4. Islam MA, Macdonald SE. 2004. Ecophysiological adaptations of black spruce ( ) and Picea mariana tamarack ( ) seedlings to flooding. Trees Larix laricina 18: 35-42. Iwanaga F, Yamamoto F. 2008. Effects of flooding depth on growth, morphology and photosynthesis in Alnus japonica species. New For 35:1 14. - . Kartikasari SN, Marshal AJ, Beehler BM Ekologi . 2012. Papua Seri Ekologi Indonesia Jilid VI: . Jakarta(ID): Yayasan Pustaka Obor Indonesia dan Conservation International. Keeley JE. 1980. Endomycorrhizae influence groth of blackgum seedlings in flooded soils. Am J Bot 67(1): 6-9. Keßler PJA, Bos MM, Daza SEC , Kop A, Willemse LPM, S Pitopang R, Gradstein SR. 2002. Checklist of oody w p , Suplementlants of Sulawesi, Indonesia. Blumea 14. National Herbarium NederelandLeiden (DE): and U p 160.niversiteit Leiden branch. Kogawara S, Yamanoshita T, Norisada M, Masumori M, Koj im a K . 2 00 6 . P hoto syn the s i s an d photoassimilate transport during root hypoxia in Melaleuca cajuputi, a flood-tolerant species, and in Eucalyptuscamaldulensis, a moderately flood-tolerant species. Tree Physiol 26 1413 23. : - . Kozlowski TT Responses of oody lants to . 1984. w p f . : Kozlowksi TT, editor. looding In Flooding and Plant BIOTROPIA Vol. 22 No. 1, 2015 70 Growth San Diego Academic Press. . (US): p 129-59. Kozlowski TT. 1997. Responses of oody plants to w flooding and salinity. Tree Physiol Monograph No. 1:1-29. Kreuzwieser J, Gessler A. 2010. Global climate change and tree nutrition: influence of water availability [Invited Review: part of an invited issue on tree nutrition]. Tree Physiol 30 1221 34: - . Kurniawati P Pengaruh pemberian inokulum . 2011. m pikoriza dan emupukan NPK terhadap p lertumbuhan semai ongkida ( L.) Nauclea orientalis pada kondisi tergenang dan tidak tergenang . Skripsi. Bogor (ID): .Institut Pertanian Bogor Lim TK. 2013. Edible Medicinal and Non-Medicinal Plants: Volume 5, Fruits. New York (US) : Springer. Longstreth MMD. 1879. The use of the freezing microtome. Boston Med Surg J 100:632-36. Marghescu T. 2001. Restoration of degraded forest land in Thailand: the case of Khao Kho. Unasylva 207 (52) 52-6.: Martínez-Alcántara B, Jover S, Quiñones A, Forner-Giner MÁ, Rodrígues-Gamir J, Legaz F, Primo-Millo E, Iglesias DJ. 2012. Flooding affects uptake and distribution of carbon and nitrogen in citrus seedlings J Plant Physiol 169:1150-57.. Mawaddah M Pertumbuhan kayu putih (Melaleuca . 2012. leucadendron Nauclea orientalis Linn.) dan Longkida ( Linn.) pada kondisi tergenang air asam tambang. Skripsi. Institut Pertanian BogorBogor (ID): . Medina CL, Sanches MC, Tucci MLS, Sousa CAF, Cuzzuol GRF, Joly CA. 2009. (Leguminosae-Erythrina speciosa Papilionoideae) under soil water saturation: morphophysiological and growth responses. Ann Bot. 104: 671-80. Mendoza R, Escudero V Garcia I. 2005. Plant growth, , nutrient acquisition and mycorrhizal symbioses of a waterlogging tolerant legume ( Mill.) in a Lotus glaber saline-sodic soil. Pl Soil 275: 305-15ant . Muok BO, Ishii T. 2006. Effect of arbuscular mycorrhizal fungi on tree growth and nutrient uptake of Sclerocarya birrea under water stress, salt stress and flooding. J. Jpn. Soc. Hortic. Sci. 75:26 31- . Nakai A, Yurugi Y, Kisanuki H. 2010. Stress responses in Salix gracilistyla cuttings subjected to repetitive alternate flooding and drought. Trees 24:1087-95. Neto D, Carvalho LM, Cruz C, Martins-Loução MA. 2006. How do mycorrhizas affect C and N relationships in flooded plants?. Plant Soil 279:51-63.Aster tripolium Nielsen JL, Stewart BR, Pearce DW, Letts MG, J H. iskoot 2010. Streamside trees: responses of male, female and hybrid cottonwoods to flooding. Tree Physiol 30 1479 88: - . Osundina MA. 1998. Nodulation and growth of mycorrhizal J.R. and G. First in Casuarina equisetifolia response to flooding. Biol Fertil Soils 26:95 9- . Parolin P. 2009. Submerged in darkness: adaptations to prolonged submergence by woody species of the Amazonian floodplains . Annals of Botany 103:359- 76. Parolin P, F Wittmann. 2010. Struggle in the flood: tree responses to flooding stress in four tropical floodplain systems. AoB Plants 2010: plq003 doi: , 10.1093/aobpla/plq003. Petty AM, Douglas MM. 2010. Scale relationships and linkages between woody vegetation communities along a large tropical floodplain river, north Australia. J Tropical Ecol 26:79 92. - Qiang-Sheng Wu, Ying-Ning Zou, Yong-Ming Huang. 2012. The arbuscular mycorrhizal fungus Diversispora spurca ameliorates effects of waterlogging on growth, root system architecture and antioxidant enzyme activities of citrus seedlings. Fungal Ecology 6 (1): 37-43. Sah S, S Reed, K Jayachandran, C Dunn, JB Fisher. 2006. The effect of repeated short-term flooding on mycorrhizal survival in snap bean roots. HortScience 41:598 602- . Secilia J, Bagyaraj DJ. 1994. Selection of efficient vesicular- arbuscular mycorrhizal fungi for wetland rice - a preliminary screen. Mycorrhiza 4: 265-68. Smith SE, Read DJ . . 2008. Mycorrhizal ymbiosis Third dS . E ition New York (US): .Academic Press Stevens KJ, Spender SW, Peterson RL. 2002. Phosphorus, arbuscular mycorrhizal fungi and performance of the wetland plant L. under Lythrum salicaria inundated condition. Mycorrhiza 12: 277 83 - . Stevens KJ, Christopher BW, Joel AJ. 2011. Effects of arbuscular mycorrhizal fungi on seedling growth and development of two wetland plants, Bidens frondosa Eclipta prostrata L., and (L.) L., grown under three levels of water availability . 21:279-Mycorrhiza 288. Sudha K, Ammani K. 2010. Arbuscular mycorrhizal fungi in medicinal plants in Thrissur district, Kerala. Mycorrhiza News 21(4): 13-8. Tanaka K, Masumori M, Yamanoshita T, Tange T. 2011. Morphological and anatomical changes of Melaleuca cajuputi under submergence. Trees 25: 695-704. Van Splunder I, Voesenek LACJ, Coups H, De Vries XJA, Blom CWPM. 1996. Morphological responses of seedlings of four species of Salicaceae to drought. Can. J. Bot 74: 1988-95. Walker C, Mize CW Menabb HS Jr. 1982. Population of , Endogonaceous fungi at two location in Central Iowa. 60:2518-29.Can. J. Bot. 71 Response o lonkida ( L.) mycorrhiz i condition Faisal – et al. f owards al noculum n aterloggedNauclea orientalis t i w