Biodiversitas vol. 14, no. 2, October 2013

ISSN: 1412-033X E-ISSN: 2085-4722

Journal of Biological Diversity Volume

14 – Number 2 – October 2013

ISSN/E-ISSN: 1412-033X (printed edition) 2085-4722 (electronic)

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Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 55-60

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140201

Genetic diversity among fourteen different Fusarium species using RAPD marker SHITAL R. BONDE♥, ANIKET K. GADE, MAHENDRA K. RAI Department of Biotechnology, S.G.B. Amravati University, Amravati-444602, Maharashtra, India. Tel: +91-721-2662208-9; Ext 267, Fax: +91-7212662135, 2660949, email: shitalbonde@gmail.com Manuscript received: 4 February 2013. Revision accepted: 22 May 2013.

ABSTRACT Bonde SR, Gade AK, Rai MK. 2012. Genetic diversity among fourteen different Fusarium species using RAPD marker. Biodiversitas 14: 55-60. We report genetic diversity of total fourteen different Fusarium species by RAPD-PCR analysis using 25 random primers. The genus Fusarium is food borne pathogen responsible for T-2 toxin production which affects human and animal health. In the present study, total 14-different species of Fusarium were analyzed on the basis of genetic diversity using RAPD method. A dendrogram was developed by UPGMA method. RAPD analysis was carried out by using 25 different universal primers each of them consisted of 10 bases. Genetic similarity coefficients between pair wise varied from 0.00 to 0.9 based on an unweighted paired group method of arithmetic average (UPGMA) cluster analysis. RAPD-PCR technique can be used as an important tool for the genetic differentiation Fusarium species. Key words: Fusarium, genetic diversity, RAPD, UPGMA.

INTRODUCTION The word ‘mycotoxin’ is used for the toxic chemical products produced by fungi that readily colonize crops in the field or after harvest (Richard et al. 2007; Turner et al. 2009). Mycotoxins are secondary metabolites produced by certain filamentous fungi, which can be produced in food and food-products as a result of fungal growth. They cause a toxic response, termed as mycotoxicosis, when ingested by higher vertebrates and other animals (Menaka et al. 2011). T-2 toxin is a Type A chemical class of nonmacrocyclic trichothecenes. The principle fungus responsible for the production of T-2 toxinis is Fusarium sporotrichioides (CAST 2003). T-2 toxin is produced by various species of Fusarium, which are widespread on a variety of plants and in soil throughout the cold temperate regions (Omurtag et al. 2001). T-2 toxin is generally found in various cereal crops such as wheat, corn, barley, rye, oats and processed grains (malt, beer and bread) (SCF 2001). Symptoms of T-2 toxin include nausea, emesis, dizziness, chills, abdominal pain, diarrhea, dermal necrosis, irreversible damage to the bone marrow, reduction in white blood cells (aleukia), inhibition of protein synthesis, and is toxic for the hematological and lymphatic systems (Omurtag et al. 2001).T-2-contaminated products can cause severe effects in humans/animals which can result in death (Moss and Long 2002). T-2 toxin also alters the level of dopamine, tryptophan, serotonine and serotonine metabolites in the brain of rodents and pigs (Pestka and Smolinski 2005). T-2 toxin is quite known for inhibition of DNA, RNA and protein synthesis, mitochondrial function as well as other subcellular processes, and to cause death of

eukaryotic cells (Gyongyossy-Issa et al. 1985). The genus Fusarium is commonly associated with many economically important crop diseases, however, distribution and diversity of this species is very important (Latiffah et al. 2007). Wang et al. (Wang et al. 1993) reported human toxicosis caused by mouldy rice contaminated with Fusarium and T2 toxin. Mycotoxin ingestion by humans, which occurs mainly through plant-based foods and the residues and metabolites present in animal-derived foods can lead to deterioration of liver or kidney function The mycotoxigenic fungi involved with the human food chain belong mainly to three genera Aspergillus, Fusarium and Penicillium. While Fusarium species are destructive plant pathogens producing mycotoxins before, or immediately alters harvesting. The use of molecular markers based on the polymerase chain reaction for species identification and as diagnostic tool has become very popular during the last decade (Sabir 2006). RAPD assays have been used extensively to define fungal populations at species, intraspecific, race and strain levels (Miller 1996; Ingle et al. 2009) and RAPD-PCR is technique for detecting genetic variability (Edwards et al. 2002; Sabir 2006). Different molecular markers are available for the differentiation of fungal taxa (Steinkellner et al. 2008). Mostly used Random amplified polymorphic DNA (RAPD) (Gupta et al. 2009) and amplified fragment length polymorphisms (AFLP) (Niessen 2007) are specific PCR-based molecular markers. These markers demonstrated remarkable genetic variation (Skaria et al. 2011). RAPD technique has been used since a long time for phylogenetic studies (Niessen 2007; Gupta et al. 2009).

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B I O D I V E R S IT A S 14 (2): 55-60, October 2013

There are several reports on differentiation of Fusarium species using RAPD markers. Gupta et al. (2009) reported the genetic polymorphism among six isolates of F. solani causing wilt disease in guava, isolated from different places in India. Ingle and Rai (2011) reported genetic diversity of F. semitectum, Fusarium associated with mango malformation were identified and analysed for their genetic diversity among Fusarium isolates (Smith et al. 2001; Arif et al. 2011). Similarly, a genetic variation in F. oxysporum f. sp. fragariae causing wilt disease in strawberry was characterized by Nagarajan et al. (2004). RAPD markers are easy, rapid for an evaluation of genetic variation (Niessen 2007). The study of RAPD analysis has been used widely in phylogenetic analysis of bacteria, fungi and plants (Singh et al. 2011). The aim of the present study was to estimate the genetic diversity of 14-different secreting Fusarium species studied by RAPD-PCR.

MATERIALS AND METHODS Fungal species Different Fusarium were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India (Table 1). Table 1. Fourteen different Fusarium cultures were used for RAPD analysis Cultures

Fusarium species

MTCC-3325 MTCC-3731 MTCC-350 MTCC-7375 MTCC-349 MTCC-3730 MTCC-636 MTCC-156 MTCC-1755 MTCC-6580 MTCC-2086 MTCC-1983 DBT-18 DBT-21

F. avenaceum F. equiseti F. solani F. sporotrichioides F. culmorum F. tricinctum F. lateritium F. moniliforme F. oxysporum F. nivale F. poae F. acuminatum F. graminearum F. semitectum

DNA Isolation Different Fusarium species were grown on Potato Dextrose Agar (PDA) at 25 ± 20C for 3 days. The mycelia grown were harvested and total DNA was extracted using fungal genomic DNA isolation kit from Chromous Biotech Pvt. Ltd, Bangalore, India according to manufacturer’s instructions. RAPD analysis Twenty five fungal primers from Random Fungal Primer Kit (RFu ‘D’) Genie Pvt. Ltd, Bangalore, India, were evaluated for PCR amplification of 14 Fusarium species. In the preliminary experiments, 12 out of the 25 primers tested produced distinct and reproducible band profile, and polymorphisms produced by ten primers. Four of 12 primers were used for comparative analysis of the

forteen Fusarium species. The primers, including Rfu-9 (5’-CCTGGGTGCA-3’), Rfu-10 (5’-CCTGGGTGAC-3’), Rfu-23 (5’-CCGGCCATAC-3’) and Rfu-25 (5’CCGGCTGGAA-3’) (Table 2). Table 2. Primer and their sequences tested in RAPD analysis. Sequences 5’-3’

Primer

CCTGGGCCAG RFu 1 CCTGGGCGAG RFu 2** CCTGGGCTGG RFu 3 CCTGGGCTAT RFu 4** CCTGGGCTTG RFu 5 CCTGGGCTAC RFu 6 CCTGGGCTTA RFu 7** CCTGGGTCGA RFu 8 CCTGGGTGCA RFu 9* CCTGGGTGAC RFu 10* CCTGGCTTAC RFu 11 CCTGGGTTAC RFu 12** CGGGGGATGG RFu 13 CTCCCTGACC RFu 14 GAGCACCTGT RFu 15** GAGCACGTCA RFu 16 GAGCACGGCA RFu 17 GAGCACGGAG RFu 18** GAGCTCGCAT RFu 19 GAGGGCATGT RFu 20 CCGGCCCCAA RFu 21 CCGGCCTTAA RFu 22** CCGGCCATAC RFu 23* CCGGCCTTCC RFu 24** CCGGCTGGAA RFu 25* Note: *) Primers used in this study; **) Primers produced distinct and reproducible band, but not used in this study.

Preliminary amplifications determined the optimal concentration of the component in the PCR reaction mixture and amplification conditions. Amplifications were performed in a total volume of 25 µL containing 12.5 µL PCR master mix (2X) (Fermentas Life Sciences, Canada) 5 µL of template DNA (20 ng), 1.5 µL MgCl2 (25 mM), 0.3 µL Taq DNA polymerase (Genexy, 5U/µL), 1 µL each primer and 4.7 µL nuclease free distilled water (supplied with Fermentas PCR master mix). PCR was carried out on gradient PCR machine (PalmCycler from Corbett Research, Australia). The program included an initial denaturation at 940C for 2 min, 35 cycles with denaturation at 940C for 30 sec, annealing 40 0C for 1 min, extension at 720C for 2 min and final extension at 720C for 5 min with holding temperature at 40C for 10 min. All experiments were repeated for three times. PCR products were electrophorezed on 1.5% agarose by using 1X TAE buffer (Fermentas Life Sciences, Canada), stained with ethidium bromide, visualized in a UV-transilluminator and the gel were photographed using Gel Doc (AlphaImager, Gel documentation system, USA), system. Data analyses Statistical analyses of all 14-different Fusarium were carried out using software PAST PAleontological STatistics (Version 2.07). While, Unweighted Pair Group

BONDE et al. – Genetic diversity of Fusarium

Method with Arithmetic Mean Analysis (UPGMA) was used to construct phylogenetic dendrogram. This method is one of the oldest techniques to be used in phylogenetic analysis, is an offshoot of the linkage methods that were popular in numerical taxonomy studies. Its simplicity and ease of interpretation has made its survival in phylogenetic studies. It works on the assumption that the rates of evolution in all lineages are same and gives output clustering in increasing order of distance (Sahoo et al. 2010).

RESULTS AND DISCUSSION RAPD analysis Genomic DNA isolated from 14 different species was subjected to RAPD-PCR analysis with 25 random decamer primers of Fungal RAPD Primer (RFu ‘D’) kit (Table 2). In the preliminary experiments, 12 out of the 25 primers tested produced distinct and reproducible band profile, and polymorphisms produced by ten primers. Four of 12

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primers were used for comparative analysis of the fourteen Fusarium species. The primers, including RFu 9, RFu 10, RFu 23 and 25 generated polymorphic bands in all 14 different species (Figure 1). All the amplified fragments were ranged 1 kb to 3.0 kb. RAPD assays of all 14 species with four above mentioned primers yielded 180 bands which were found to be polymorphic. Above data showed that RAPD is a convenient method for distinguishing the different species of Fusarium and also reveal a significant genetic variation among these species. There was other most studied Fusarium species viz. F. oxysporum also showed the genetic variation (Ingle and Rai 2011). Assigbetse et al. (1994) differentiated races of F. oxysporum f. sp. vasinfectum on cotton by using RAPD as molecular tool and Bonde et al. (2013) studied genetic variation of F. equiseti isolated from fruits and vegetables. In another study carried out by Edel et al. (2001) it was observed that the isolates of F. oxysporum isolated from soil samples in France showed genetic diversity. While, Nagarajan and

A

B

C

D

Figure 1. RAPD patterns on 1.5% agarose gel of amplified fragments generated from different Fusarium sp. with primers RFu-9 (B) RFu-10 (C) RFu-23 (D) RFu-25. Lane M, DNA marker (1 kb), lane 1. F. avenaceum, lane 2. F. equiseti, lane 3. F. solani, lane 4. F. sporotrichioides, lane 5. F. culmorum, lane 6. F. tricinctum, lane 7. F. lateritium, lane 8. F. moniliforme, lane 9. F.oxysporum, lane 10. F. nivale, lane 11. F. poae, lane 12. F. acuminatum, lane 13. F. graminearum, lane 14. F. semitectum

B I O D I V E R S IT A S 14 (2): 55-60, October 2013

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group (2004) studied the genetic variation in F. oxysporum f. sp. fragariae population causing wilt in strawberry using RAPD and RFLP analysis. The work carried out by Leslie et al. (2006) supports the findings of present study. They observed inter-and intra specific genetic variation in different Fusarium species. RAPD-PCR technique as suitable method was used to rapid identification and differentiation of Fusarium species (Pujo et al. 1997; ElFadly et al. 2008). Gupta et al. (2009) reported the genetic polymorphism and diversity in isolates of F. solani isolated from wilt disease of Guava in India. Ingle et al. (2009) suggested RAPD marker is important, reliable tool for genetic variation among ten phytopathogenic isolates of F. semitectum from India.

UPGMA RAPD markers along with appropriate statistical procedures are suitable for genetic variation analyses at both intra and inter-population levels (Leon et al. 2011). Fusarium species secreting T-2 toxin were analyzed with several UPGMA dendrograms with bootstrap analysis. In addition, bootstrap values of UPGMA dendogram obtained with the utilization of RAPDs were slightly higher. Genetic relationship calculated in the form of similarity coefficient from dendrogram showed high level of genetic similarity among all different Fusarium, which ranges from 0 to 0.9. Clustering was performed by UPGMA method. UPGMA analysis of the RAPD data separated the Fusarium species in two clusters (Figure 2).

F. equiseti

F. solani

F. sporotrichioides

F. culmorum

F. tricinctum

F. lateritium

F. moniliforme

F. oxysporum

F. nivale

F. poae

F. acuminatum

F. graminearum

F. semitectum

F. avenaceum F. equiseti F. solani F. sporotrichioides F. culmorum F. tricinctum F. lateritium F. moniliforme F. oxysporum F. nivale F. poae F. acuminatum F. graminearum F. semitectum

F. avenaceum

Table 3. Distance matrix

0 1.00 1.10 1.00 1.11 1.10 1.00 1.10 1.00 1.11 1.00 1.01 1.09 1.11

0 1.11 1.01 1.00 1.10 1.00 9.89 1.01 1.00 1.00 1.00 8.91 1.00

0 1.00 1.10 9.89 8.90 1.11 1.00 1.10 1.11 1.10 1.10 1.10

0 1.00 9.99 9.00 1.01 1.11 1.00 1.01 1.00 1.00 1.00

0 1.10 1.00 1.10 1.00 9.99 1.00 9.99 1.10 1.00

0 9.91 1.10 9.99 1.10 1.10 1.10 1.10 1.10

0 1.00 9.00 1.00 1.00 1.00 1.00 1.00

0 1.01 1.10 9.90 1.10 9.90 1.10

0 1.00 1.01 1.00 1.00 1.00

0 1.00 9.99 1.10 9.00

0 1.00 8.91 1.00

0 1.10 9.99

0 1.10

0

F. tricinctum F. semitectum F. lateritium F. acuminatum F. nivale F. culmorum F. moniliforme F. poae F. graminearum F. avenaceum F. equiseti F. solani F. sporotrichioides F. oxysporum

Figure 2. Phylogenetic analysis using UPGMA method

BONDE et al. – Genetic diversity of Fusarium

UPGMA dendrogram showed the F. sporotrichioides and F. oxysporum in one clade and other species in another clade. In upper clade F. equiseti and F. solani showed greater similarity than other F. tricinctum, F. semitectum, F. lateritium, F. acuminatum, F. nivale, F. culmorum, F. moniliforme, F. poae, F. graminearum and F. avenaceum. UPGMA analysis thus carried out in the present study showed the genetic variation in these 14 different Fusarium species. A distance matrix on simple matching coefficients was calculated from the data based on the RAPD of all 14 Fusarium species. The matrix was used to construct a dendrogram using distance tool with UPGMA method of PHYLIP for establishing to analyze the level of relatedness among the ten isolates. The dendrogram obtained from the data showed that hierarchical clustering separated the isolates into three groups according to their similarity coefficients. The similarity coefficients among the all isolates ranged from 0 to 0.9. Distance matrix of different 14 Fusarium species was obtained (Table 3). UPGMA is a simple agglomerative or hierarchical clustering method used in bioinformatics for the phylogenetic analysis. The results obtained in the present study are noteworthy and showed the similarity with the observations of Ingle and Rai 2009, Bonde et al. (2012), Gupta et al. (2009) and Nagarajan et al. (2004). In their studies on isolates of F. semitectum, F. equiseti, F. solani and F. oxysporum respectively, they used data generated from RAPD banding pattern for the UPGMA analysis and found that there was genetic variations in different isolates of same Fusarium (Abd-Elsalam et al. 2003). Statistical analysis were carried out of all 14 Fusarium species using PAST software in which diversity graph (Figure 3) which computes a number of similarity or distance measures between all pairs of rows.

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which is the basis for the calculation of p 0.002596 in which three statistical tests for normal distribution of one or several samples of univariate data, given in columns. Diversity indices statistics apply to association data, where numbers of individuals are tabulated in rows (taxa) and possibly several columns (associations). The available statistics are as follows, for each association: Number of taxa (S), total number of individuals (n), Dominance = 1-Simpson index. Ranges from 0 (all taxa are equally present) to 1 (one taxon dominates the community completely), Simpson index 1-D. Measures 'evenness' of the community from 0 to 1. Diversity indexes, taking into account the number of individuals as well as number of taxa. Varies from 0 for communities with only a single taxon to high values for communities with many taxa, each with few individuals. Buzas and Gibson's evenness, Brillouin’s index, Menhinick's richness index, Margalef's richness index, Equitability. Shannon diversity divided by the logarithm of number of taxa. This measures the evenness with which individuals are divided among the taxa present. Fisher's alpha-a diversity index, defined implicitly by the formula S=a*ln (1+n/a) where S is number of taxa, n is number of individuals and a is the Fisher's alpha. Berger-Parker dominance is simply the number of individuals in the dominant taxon relative to n. The data below were generated by a random number generator with uniform distribution and tests such as Shapiro-Wilk test, Jarque-Bera test and Chi-square tests (Table 5) were studied to check the univariate normal distribution of data. Table 4. Diversity indices 0 Taxa_S Individuals Dominance_ Shannon_H Simpson Evenness_e^H/S Menhinick Margalef Equitability_J Fisher_alpha Berger-Parker

59 14 2.4212309315201E58 D 0.383 1.152 1-D 0.617 0.2261 8.997E-29 0.0967 0.4365 0.1024 0.4544

Table 5. Shapiro-Wilk test, Jarque-Bera test and Chi-square tests as it is other statistical analysis

Figure 3. Diversity profile of 14 different Fusarium species

The diversity indices are applied in statistics of association data, where numbers of individuals are tabulated in rows (taxa) and possibly several columns (associations) and test for normal distribution (Table 4) asymptotically normal distribution with mean 0 and variance 1 under the null hypothesis of zero correlation,

Statistical parameter

Values

Min Max Sum Mean Std. error Variance Stand. dev Median Geom. mean

1E30 1.10011E58 2.42123E58 1.72945E57 1.00174E57 1.40487E115 3.74815E57 1.11111E55 4.01722E55

B I O D I V E R S IT A S 14 (2): 55-60, October 2013

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Our results suggest existence of significant genetic variation among these Fusarium species secreting mycotoxin, on the basis of RAPD analysis. Fusarium species secrets mycotoxin and to reduce mycotoxin contamination in food and feed by these species as well as to search for remedy for infected food and feed the present study of genetic diversity of Fusarium species will be useful.

CONCLUSION RAPD marker was found to be powerful tool to analyze the genetic variation among the Fusarium species. These Fusarium species are responsible for producing mycotoxin, which is hazardous to animals and human beings. The results of the present study provide evidence that RAPD technique can be used for identification and differentiation of different Fusarium species. Study of mycotoxin secreting Fusarium is necessary to avoid T-2 toxin contamination in food and feed. We suggest that RAPD marker may be used as one of reliable alternative for the determination of genetic variation among the different Fusarium species.

ACKNOWLEDGEMENTS The authors are grateful to Defense Research and Development Organization (DRDO), New Delhi for providing financial assistance for the present research.

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Gupta VK, Misra AK, Gaur R, Pandey R, Chauhan UK. 2009. Studies of genetic polymorphism in the isolates of Fusarium solani. Austr J Crop Sci 3:101-106. Gyongyossy-Issa MI, Khanna V, Khachatourians GC. 1985. Characterization of hemolysis induced by T-2 toxin. Biochim Biophys Acta 838: 252-256. Ingle AP, Karwa A, Rai MK, Gherbawy Y. 2009. Fusarium: Molecular detection, mycotoxins and biocontrol. In: Gherbawy Y, Mach R, Rai M (eds.). Curent Advance in Molecular Mycology. Science Publishers Inc., Enfield, New Hampshire. Ingle AP, Rai MK. 2011. Genetic Diversity among Indian phytopathogenic isolates of Fusarium semitectum Berkeley and Ravenel. Adv Biosci Biotech 2: 142-148. Latiffah Z, Zariman M, Baharuddin S. 2007. Diversity of Fusarium species in cultivated soils in Penang. Malay J Microb 3: 27-30. Leon T, Raj S, Britto JD, Benjamin P, Kumar JR. 2011. RAPD-PCR fingerprint analysis of Cassia angustifolia Vahl. in Tirunelveli District of Tamil Nadu. Int J BioTechnol 2: 17-20. Leslie JF, Summerell BA. 2006. The Fusarium laboratory manual, 3rd ed. Blackwell publishing professional, Ames IA, USA. Menaka M, Phanni M, Malathy NS . 2011. Isolation and identification of microcystin and mycotoxin from food borne fngus and to study about its antibacterial activity. EJLS 1:5-13. Miller S. 1996. Detecting Propagules of plant pathogenic fungi. Adv Bot Res 23: 73-102. Moss MO, Long MT. 2002. Fate of patulin in the presence of yeast Saccharomyces cerevisiae. Food Addit Contam 19: 387-399. Nagarajan G, Nam MH, Song JY, Yoo SJ, Kim HG. 2004. Genetic variation in Fusarium oxysporum f. sp. fragariae populations based on RAPD and rDNA RFLP analyses. Pl Pathol J 20: 264-270. Niessen L. 2007. PCR based diagnosis and quantification of mycotoxin producing fungi. Int J Food Microb 119:38-46. Omurtag GZ, Yazıcıoglu Z. 2001. Occurrence of T-2 toxin in processed cereals and pulses in Turkey determined by HPLC and TLC. Food Addit Contam 18: 844-849. Pestka JJ, Smolinski AT. 2005. Deoxynivalenol: Toxicology and potential effects on humans. J Environ Sci Health B 8: 39-69. Pujo I, Guarro J, Gene J, Sala JP. 1997. In vitro antifungal susceptibility of clinical and environmental Fusarium spp. strains. J Antimicrob Chemoth 39: 163-167. Richard JL. 2007. Some major mycotoxins and their mycotoxicoses-an overview. Int J Food Microbiol 119 (2) 3-10. Sabir SM. 2006. Genotypic identification for some Fusarium sambucinum strains isolated from Wheat in Upper Egypt. World J Agri Sci 2 (1) 610. Sahoo L, Das BK, Parhi K, Mukherjee SC. 2010.DNA fingerprinting of Flavobacterium columnare using RAPD-PCR. Ind J Microbiol 50: S10-S16. SCF [Scientific Committee on Food]. 2001. Opinion on Fusarium Toxins. Part 5: T-2toxinandHT-2toxin. www.europa.eu.int/comm/food/fs/sc/scf/out88en.pdf._3. Singh M, Chaudhuri I, Mandal SK, Chaudhuri RK. 2011. Development of RAPD Markers linked to Fusarium Wilt Resistance Gene in Castor Bean (Ricinus communis L). Genet Eng Biotech J (GEBJ-28): 1-8. Skaria R, Sen S, Muneer PM. 2011. Analysis of genetic variability in rice varieties (Oryza sativa L) of Kerala using RAPD markers. Genet Eng Biotech J (GEBJ-24): 1-9. Smith SN, Decay JE, Hsui HW, Jen LH, Hsieh WH, Lee HL. 2001. Soilborne populations of Fusarium oxysporum f. sp. vasinfectum, cotton wilt fungus in California fields. Mycologia 93:737-743. Steinkellner S, Mammerler R, Vierheilig H. 2008. Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains. Eur J Plant Path 122: 395401. Turner NW, Subrahmanyam S, Piletsky SA. 2009. Analytical methods for determination of mycotoxins: a review. Anal Chim Acta 632 : 168180. Wang ZG, Feng JN, Tonq Z. 1993. Human toxicosis caused by moldy rice contaminated with Fusarium and T-2 toxin. Biomed Environ Sci 6: 65-70.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 61-66

ISSN: 1412-033X EISSN: 2085-4722 DOI: 10.13057/biodiv/d140202

Fungal species isolated from Quercus castaneifolia in Hyrcanian Forests, North of Iran MOHAMMAD REZA KAVOSI1, FERIDON FARIDI1, GOODARZ HAJIZADEH2,♼ 1

Department of Forest Science, Faculty of Forest Ecology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran. 2 Department of Forestry, Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Mazandaran, Iran. Tel./fax. +98 151 3822715, ď‚Šemail: goodarzhajizadeh@gmail.com Manuscript received: 6 May 2013, Revision accepted: 17 July 2013.

ABSTRACT Kavosi MR, Feridon F, Hajizadeh G. 2013. Fungal species isolated from Quercus castaneifolia in Hyrcanian Forests, North of Iran. Biodiversitas 14: 61-66. In order to isolate and identify of fungi associated with Quercus castaneifolia seed, sampling carried out in Shast-Kalate, Ghorogh, Loveh and Golestan forest. Collected seeds sterilized and then separated sections including: outer section of seed (crust) and inner seed section (endosperm). Each section of seed tissue is cultured on potato dextrose agar media. After sub-culture and providing of the fungi pure cultures, various species isolated and identified by spores characteristics, their size and color, including: Aspergillus flavus, A. niger, Curvularia affinis, Trichoderma harzianum, Trichothecium roseum, Eurotium rubrum, E. amstelodami, Penicillium implicatum, P. fellutanum, Diplodia sp. Nigrospora gossypi, Alternaria alternata , Fusarium oxysporum and Beltrania santapaui. The most frequency of fungus in Shast-Kalate forest was P. implicatum by 74% of frequency within seed section, the most frequency in Ghorogh and Loveh Forest was P. fellutanum with 63 and 66% of frequency within seed section respectively and the most frequency in Golestan forest was B. santapaui by 51% of frequency outer seed section. The result showed diversity of the fungi on the outer seed section is higher than within seed section. The results also showed during several isolation a saprophyte fungus always could be finding on the acorn seeds. This is the comprehensive report on fungi associated with Quercus castaneifolia seed in Hyrcanian forest, North of Iran. Key words: crust, endosperm, fungal, Hyrcanian forest, Quercus castaneifolia, seed

INTRODUCTION Forest tree seeds continuously are affected by physical and physiological disturbance which most of these diseases caused by fungi. Health and growth ability sapling considerably depend on seed quality (Mittal and Mathur 1998). Most of fungi associate with seeds of forest trees are molds which expand on surface of seed and sometime they are inner pollution factor (Huss 1956). Effect of mold on seeds is that they seem health apparently but they originally have spoiled on basis of vitality considerable (Shea 1957). Recently known that all seeds contain microscopic fungi spores whether on surface of seed or inside of seed (Singh and Mathur 1993). Urosevic (1961) was specified that some of fungi spores have germinated and after growing and mycelium penetrating, it influences in to the cotyledon which through have nourished from germs. Fungus associate with seed can cause weakness of seed germination directly and indirectly and can dispose these seeds to earthborn pathogen fungi attack (Gibson 1957). Healthy seed in forest for natural regeneration is important issues which future life forest depends on it. Disease and damaged seeds even under suitable environmental condition cannot have desired regeneration for forest survival or cannot cause specific species. The trees appeared from damaged and diseased seeds have slight growth and seeds produced by these trees will be had low

vitality (Rai and Mamatha 2005). The purpose of this study was identification of inner and outer fungi of Chestnut-leaved oak (Quercus castaneifolia) seed and specifying their frequency in Golestan province forests.

MATERIALS AND METHODS Sampling site In this research four forest regions in Golestan province, north of Iran including (i) Shast-Kalate Research and Education Forest, (ii) Ghorogh Forest Park, (iii) Loveh Research Forest, and (iv) Golestan National Park were selected (Figure 1). In each region, four trees were chosen randomly, and 25 seeds of each tree were selected randomly. Collected seeds have been settled in new and sterile bags and after recording region specification and collection date, they transferred to laboratory and settled in a place with suitable temperature and ventilation. In finally, four samples of 25 kinds of seeds of each region were collected. Isolation and purification seed fungi For fungus isolation, seeds were divided less segments and also inner (crust) and outer (endosperm) portion. Separation of inner portion from outer portion of seed

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Figure 1. Location of the study site inside the Hyrcanian forests of Golestan Province, North of Iran, including: A. Shastkalate Research and Education Forest, B. Ghorogh Forest Park, C. Loveh Research Forest, and D. Golestan National Park.

due to comparing mentioned two portion fungi was conducted. After seed was divided less segment, its surface was disinfected with 0.5% sodium hypochlorite and ethanol during 1-2 minutes and was washed three times with sterile Distilled water and settled in to sterile filter paper for desiccating. Then segments of inner and outer portion of seed separately and with four repetitions on nutrition medium of potato-dextrose-agar (PDA) extract contain lactic acid and preserved in incubator in 25±1°C. After three days, grown fungi were subculture on medium and hereby fungi become sterilization.

This identification was on basis of various criteria such as presence or absence of septum; shape and size of ascus; ascospore; conidia and phialid, kind of ascospore; number of ascospore in each ascus; number of conidia laid on conidiophore or phialid; being one or more cellular of ascospore and conidia; in some species, presence or absence of metulae; diameter growth of colonies; colonies color and made.

Identification of fungi Identification of fungi genus after their growing on the seed segments was used valid reference of Barnett and Hunter (1998) and Ellis (1976) and their classification on basis of Eriksson (2006) and Alexopoulos et al. (1996). For identification of species was used various medium and valid reference. Further species were identified and purified on PDA medium and 25°C in absolute darkness. For some of species like Fusarium which do not know spore Carnation leaf-piece Agar (CLA) medium and optical period LD 12:12 in 25°C according to Nelson et al. (1983) and Saremi (1998) method were used. For Alternaria, for denoting spore number in spore chain, LD 16:8 optical period in 20-23°C and Agar-Water (AW) medium in addition to PDA were used according to Dingra and Sinclair (1995) method and on basis of Ellis (1971, 1976) cognition key. About Trichoderma, LD 12:12 optical period and 25°C according to Dingra and Sinclair (1995) method and, Kubicek and Harman (1998) cognition key were used. Pitt (1997, 2000) cognition keys was applied for identification of Eurotium, Aspergillus, Penicillium fungi. Litvinov (1967) and Ellis (1976) description the identification of Trichothecium, Curvularia, Beltrania and Nigrospora fungi and Barnett and Hunter (1998) for identification Diplodia fungus.

Species specification Results of this study showed that all seeds polluted with one or more species of separated fungi which most of them were imperfect fungi or Ascomycetes. After sterilization and specification of thallus and colonies, 12 species including: Nigrospora gossypii, Aspergillus flavus, A. niger, Trichoderma harzianum, Alternaria alternate, Trichothecium roseum, Fusarium oxysporum, Beltrania santapaui, Penicillium implicatum, Eurotium rubrum, Curvularia affinis and Diplodia sp. become isolation and identification on Quercus castaneifolia seeds that frequency and description of specification of each one in detail is following in Table 1. All identification fungi on Q. castaneifolia seeds were reported from Hyrcanian Forests, North of Iran for the first time.

RESULTS AND DISCUSSION

Alternaria alternata (Fr.) Keissl. Colonies usually was approximately olivaceous to black and sometimes grey with pubescent appearance on PDA medium and 25°C. Colonies diameter growth after three days was 3-3.5 cm (Figure 2A). Conidiophores were partly small with 7-10×43-50 μm dimensions, simple and branched, approximately brown and even surface. Conidia were formed on WA medium in 6 to 17 fold chains (Figure 2C) and ovoid to obclavate or pear form and contain

KAVOSI et al. – Fungal species isolated from Quercus castanifolia Table 1. Fungi frequency percent of inner and outer portion of oak seed in four regions in Golestan province

ShastFungi kalate I O Alternaria alternata 0 0 Diplodia sp. 0 23 Aspergillus flavus 27 46 Aspergillus niger 11 16 Curvularia affinis 39 0 Eurotium rubrum 0 35 Nigrospora gossypii 0 0 Penicillium implicatum 74 31 Beltrania santapaui 0 0 Fusarium oxysporum 0 0 Trichothecium roseum 0 41 Trichoderma harzianum 0 7 Note: I = inner, O = outer

Regions Ghorogh Loveh park I O I O 20 30 13 0 0 0 0 0 0 8 0 0 16 14 0 0 0 0 0 0 0 34 0 0 0 17 0 15 39 26 39 18 0 0 0 56 0 0 0 35 13 5 0 0 0 0 0 0

Golestan park I O 16 0 0 0 5 3 0 0 0 0 0 0 0 0 11 6 0 51 0 19 0 0 0 0

surface covered by tiny tubers. Conidia have 2 to 7 transverse walls and 2 to 4 vertical walls and 12-34×6.512.5 μm dimensions and the end of the conidium nearest the conidiophore was round while it tapers towards the apex with 2.5-4 μm width (Figure 2B). Diplodia sp. Colonies was specified with whitish yellow on PDA medium and 25°C (Figure 3A). Pycnidia were black, individual, spherical and stomatous (Figure 3B). Conidiophores were tiny and simple and conidia were dark, bicellular, 5-7 μm, elliptical or ovoid and in some of them there was curve (Figure 3C). Aspergillus flavus Link Colonies on PDA medium and 25°C was olive to lime green with a cream reverse. Colonies has fast diameter growth, after three days it was about 5.2 cm and has woolly to cottony texture which contain small granular (Figure 4A). Hyphae have light and septum. Conidia were settled on vesicle radially or perpendicular. Conidiophores were coarse and colourless and up to 800 μm length and 15-20 μm width. Vesicles were spherical to semi spherical (20-40 μm) and phialids (3-4×8-12 μm) covered approximately all surface of vesicle. Conidia were 3-6 μm, even, tiny and spherical to semi spherical (Figure 4B). Aspergillus niger Tiegh. nom. cons. Colonies at first was white but due to producing conidia become black and the reverse side seemed light yellow or pail on PDA medium and 25°C which during the growth, they produced radial gaps on medium (Figure 5A). Colonies diameter growth after three days was 4.3 cm. Hyphae had transparent and septum that conidia were settled on vesicle radially. This species produced metulae. Conidiophores were long (400 to 3000 μm), even and transparent that were dark in tip and end to bubble or cell of spherical vesicle (30-75 μm). Metulaes and phialids

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cover all surface of vesicle. Conidia were brown to black, uneven and with tuber, spherical and 4-5 μm (Figure 5B). Curvularia affinis Boedijn. Colonies was black to dark greenish black on PDA medium and 25°C which in white margins, colonies texture was cotton and its diameter growth after three days was 4.4 cm (Figure 6A). Conidiophores mostly were simple and had spores that formed with two sympodial geniculate. Conidia were 2 to 4 cells, 23-33×8-14 μm, fusiform and curve so that the central cell was typically darker and enlarged compared to the end cells in the conidium and the swelling of the central cell usually gave the conidium a curved appearance (Figure 6B). Eurotium rubrum Jos. König etal Bainier & Sartory Colonies on PDA medium and 25°C was reddish orange that in white margin, colonies texture was cotton and its diameter growth after three days was 3.5 cm (Figure 7A). Cleistothecia were occurred spherical to nearly elliptical form and with yellow colour (Figure 7B). Ascuses were almost egg form to elliptical, 12-13 μm and they had a tiny and unstable wall. Ascospores were unicellular, oblate (like a flattened sphere) and have equatorial ridges, thus resembling pulleys, 4.4-5×6.2-6.8 μm, elliptical, yellow with even margin and with eight fold form in to the Ascus (Figure 7C). Nigrospora gossypii Jacz. Colonies on PDA medium and 25°C was dark grey with small and large while point in its background which were basically cotton form that stick fungus mycelium to top of the container. It’s colour was black grey and approximately dark-blue behind of container and colonies diameter growth after 3 days was 7-7.5 cm (Figure 8A). Conidiophores were simple, transparent and were settled vertically on mycelium which their length was 10-12.5 μm. Conidia were black, unicellular and semi spherical and partiy elliptical form with even surface and flat section that their size was 11-15 μm. This fungus also had middle chlamydospores (Figure 8B). Penicillium implicatum Biourge Colonies on PDA medium and 25°C at first was cotton white that finally will become powdery blue-green (Figure 9A). Colonies diameter growth after 3 days was 2.6 cm. Conidiophores were out of growth mycelium individually and ended to phialids. Conidiophore height was 25-50 μm and phialids length was 8-10 μm. Conidia were spherical, dark green, 2-3.5 μm, unicellular, and were formed from chains which youngest conidia settled in base of chain (Figure 9B). Beltrania santapaui Pirozynski & Patil Colonies on PDA medium and 25°C was grayish dark brown which behind of container was light grey and colonies growth on PDA medium after three days was 3.5 cm (Figure 10A). Conidiophores were simple and had septum which at the end, they were branched and conidia were formed on each of these branches (Figure 10B). Conidiophore length was 87.5 μm and conidia were seen

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Figure 1. Alternaria alternata (a: colony on PDA medium, b: conidia, c: fold chains on WA medium). Bar = 300 µm Figure 3. Diplodia sp. (a: colony on PDA medium, b: pycnidia, c: conidia). Bar = 300 µm Figure 4. Aspergillus flavus (a: colony on PDA medium, b: conidiophore and conidia). Bar = 300 µm Figure 5. Aspergillus niger (a: colony on PDA medium, b: conidiophore and conidia). Bar = 20 µm Figure 6. Curvularia affinis (a: colony on PDA medium, b: conidiophore and conidia). Bar = 20 µm Figure 7. Eurotium rubrum (a: colony on PDA medium, b: cleistothecia, bar = 150 µm, c: ascus and ascospore). Bar = 30 µm Figure 8. Nigrospora gossypii (a: colony on PDA medium, b: conidiophore and, b1 : conidia). Bar = 20 µm Figure 9. Penicillium implicatum (a: colony on PDA medium, b: conidiophore and conidia). Bar = 20 µm Figure 10. Beltrania santapaui (a: colony on PDA medium, b: conidiophore and conidia, bar = 45 µm, c: conidia). Bar = 30 µm Figure 11. Fusarium oxysporum (a: colony on PDA medium, b: monophialide, bar = 75 µm, c: macroconidia and microconidia). Bar = 30 µm Figure 12. Trichothecium roseum (a: colony on PDA medium, b: conidiophore and conidia). Bar = 20 µm Figure 13. Trichoderma harzianum (a: colony on PDA medium, b: conidiophore and conidia). Bar = 20 µm

bicellular and elliptical form with one appendage that was dark brawn. Conidia size without appendage was equal to 5-7.5×15-21 μm and its length was 2.5-3.5 μm (Figure 10C). Fusarium oxysporum Schltdl. Colonies diameter growth was measured 3.1-3.8 cm on PDA medium and 25°C, its color at first was light pinkish white and finally become violet that its center was lighter and its margin was dark violet. Mycelium was cotton and scatter that were condensed by growth completion. Behind of container in the margin was dark violet and in the center was opaque orange (Figure 11A). Middle chlamydospores were formed frequently on mycelium. Macroconidia on abundant sporodochia that were sickle from and partly longitude, most of them had three tiny septum and their length was 3-5×24-30 μm (Figure 11C). Macroconidia and also Microconidia on short and individual phialides were formed which microconidia were false-heads on these phialides (Figure 11B). Microconidia were most of time egg form or longitude elliptical unicellular or kidney form (Figure 11C). Trichothecium roseum (Pers.) Link Colonies approximately grow up rapidly. Its diameter growth after three days on PDA medium and 25°C was 2.8 cm and was whitish light pink and partly powder form (Figure 12A). Until first conidia produce, conidiophores were not separation from growth section hyphae. They were vertical, without branch and most of time they had septum nearby base of conidiophore. Two conidia were formed alternatively and with overlap in tip of conidiophore. Conidia were bicellular, elliptical or pear form with joint place to curve, transparent, even to partly coarse and were 11-16×7-10 μm (Figure 12B).

Trichoderma harzianum Rifai Colonies on PDA medium had rapid growth which at first was cotton white but after 2 days was approximately light green (Figure 13A). hyphae had septum were branched and 2.5-5.5 μm diameter. Chlamydospores at the end or in the middle of hyphae were elliptical to fusiform with even wall and 8.5-10×5-7.5 μm diameter. Conidiophores were branched that through end of conidiophore, length of these branches were smaller. Phialids were short, bar form, on the base they were narrower than middle area and on top of its conic, its dimensions were 5-7.5×2.5-3.5 μm. Conidia individually collected at the end of Phialids and were egg form to spherical with even wall and 2.5-3.4 μm dimensions (Figure 13B). Discussion In this study, specified that Penicillium fungus rather than identified fungi have more frequency which its species had most frequency in all range site. This genus along with fungi such as Fusarium and Trichoderma caused for discolor of seeds (El-Gali 2003). Fungi grow both on seed crust and on seed cotyledon but a variety of fungi of seed crust are more than cotyledon. In inward section, Penicillium had further frequency in all regions whilst Trichoderma become isolation on seed crust. This is corresponded to Winston (1956) and El-Gali (2003) studies which isolated Penicillium, Fusarium and Trichoderma fungi on Red Oak (Quercus rubra) seeds. Dorsey et al. (1962) separated Penicillium on seeds of Q. velutina and Q. rubra. Aspergillus and Penicillium are genera that have generality in color change of cotyledon and even seed crust and finally causal lesion and crack on seed crust (Swiecki et al. 1991). In Swiecki studies,

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Fusarium oxysporum and Trichothecium sp. obtain on seed and seedling of Q. macrocarpa in northern California which similar to our study. In this study, isolated Fusarium that was isolated on Q. alba and Q. macrocarpa seeds by Vozzo (1984). Agbaba and Gradecki (2005) isolated Ciboria batschiana, Phomopsis quercella, Fusarium sp. Ophiostoma sp. Penicillium sp. Trichothecium roseum, and Trichoderma viride from Q. pubescens seeds which Fusarium, Penicillium, Trichoderma genus and Trichothecium roseum species is corresponded to our study. Tiberi et al. (2002) isolated Fusarium solani, Fusarium eumartii, Verticillium dahliae, Diplodia mutila and Phomopsis quercina from oaks seed of Italy which Diplodia and Fusarium is observed in our study. Gallego et al. (1999) isolated Fusarium oxysporum seen in our study from Q. ilex seed for testing of being pathogen the fungi. Santos et al. (2005) and Merouani et al. (2001) separated many fungi on Q. suber seed which among them can be referred to Penicillium implicatum, Trichoderma harzianum, Trichothecium roseum, Fusarium oxysporum, Diplodia mutila, Aspergillus niger, Aspergillus flavus and Alternaria alternata that whole of these species were seen in our study. Also some studies conducted on fungi along with major forest trees that fungus similar our study was including Alternaria, Fusarium, Aspergillus, Penicillium, Trichoderma, and Beltrania (Vladimir et al. 2005; Swapna and Nagaveni 2008).

CONCLUSION The results show that all acorn seeds collected were infected with one or more species of fungi have been isolated which are often classified to Ascomycetes fungi. Since the length of oak seed dormancy and physiological process is very long period. This could be due to opportunistic fungi such as contact with the surface of the seed coat and the seed easily reach and thereby is prevented from germinating. This is the comprehensive report on fungi associated with Quercus castaneifolia seed in Hyrcanian forest, North of Iran.

REFERENCES Agbaba SN, Gradecki M. 2005. Health condition of common oak acorn (Quercus pubescens) and protection measures in Croatia. 5th ISTASHC Seed Health Symposium. 10-13 May 2005, Angers France. 4243. Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory Mycology. 4th ed. John Wiley and Sons, New York. Barnett HL, Hunter BB. 1998. Illustrated Genera of Imperfect Fungi. 4th ed. ASP Press, St. Paul, Minnesota, USA. Dorsey CK, Tryon EH, Carvell KL. 1962. Insect damage to acorns in West Virginia and control studies using granular systematic insectidies. Econ Entomol 55: 885-888. El-Gali ZI. 2003. Histopathological and biochemical studies on bean seeds infected by some seed-borne fungi. [PhD. Dissertation]. Department of Agricultural Botany. Alexandria University. Egypt.

Ellis MB. 1971. Dematiaceous Hyphomycetes. C.A.B International Mycological Institute, Kew, UK. Ellis MB. 1976. More dematiaceous Hyphomycetes. C.A.B International Mycological Institute, Kew, UK. Eriksson OE. 2006. Outline of Ascomycota. Myconet. www.field museum. org/myconet/printed_v12_a. asp: 1-82. Gallego FJ, de Algaba AP, Fernandez-Escobar R. 1999. Etiology of oak decline in Spain. Eur J For Path 29: 17-27. Gibson IAS. 1957. Saprophytic fungi as destroyers of germinating pine seeds. E Afr Agric For J 22: 203-206. Huss E. 1956. Research into damage to tree seeds by dewinging. Skogsforskinings-Institute, Stockholm. Kubicek CP, Harman GE. 1998: Trichoderma and Gliocladium. Vol. 1. Basic Biology, Taxonomy and Genetics. Taylor & Francis, London. Litvinov AM. 1967. Identify Microscopic Soil-born Fungus. Leningrad Science Publisher, Leningrad. Merouani H, Branco C, Almeida MH, Pereira JS. 2001. Effect of acorn storage duration and parental tree on emergence and physiological status Cork oak (Quercus suber L.) seedlings. Ann For Sci 58: 534554. Mittal RK, Mathur SB. 1998. Seed Pathology. Indian Council of Agricultural Research, New Delhi, India, and Danish Government Institute of Seed Pathology, Denmark. Nelson PE, Toussoun TA, Marasas WFO. 1983. Fusarium species: An illustrated manual for identification. Penn State University. University Park, Pennsylvania. Pitt JI, Hocking AD. 1997. Fungi and food spoilage. 2th ed. Blackie Academic & Professional, Chapman & Hall, London. Pitt JI. 2000. A Laboratory Guide to Common Penicillium Species. 3th ed. N.S.W. Food Science Australia, North Ryde. Rai VR, Mamatha T. 2005. Seedling diseases of some important forest tree species and their management. In: Diseases and Insects in Forest Nurseries. Proceedings of the 5th Meeting of IUFRO Working Party S7.03.04, May 6-8 2003, at Peechi, Kerala, India. Santos MN, Braganca MH, Casimiro PP. 2005. Cork oak associated microorganisms throughout cork manufacture process. EFN 13 (1): 75-93. Saremi H. 1998. Ecology and Taxonomy of Fusarium Species. Ferdowsi University of Mashhad, Mashhad. Shea KR. 1957. Problem analysis: Molds of forest tree seed. Weyerhaeuser Timber Company, Forestry Research Centre, [Place of publication unknown]. Singh P, Mathur SB. 1993. Disease problems of forest tree seeds: diagnosis and management. 309-324. In Proc. IUFRO Symp. On Tree Seed Problems, with special reference to Africa. Project Group P. 2.04.00-Seed Problems, Ougadougou, Burkina Faso, 23-28 Nov. Swapna PK, Nagaveni HC. 2008. Seed health problems and their impact on seedling production. National Seminar on Medicinal plants and herbal products. S.V University, Tirupati, A.P. on 7-9th March 2008. Swiecki TJ, Bernhardt EA, Arnnold RA. 1991. Insect and disease impacts on blue oak acorns and seedlings. Pages 149-155 in Standiford RB, technical coordinator. Proceedings of the symposium on oak woodlands and hardwood rangeland management; October 31November 2, 1990; Davis, California. General Technical Report PSW-GTR-126. USDA Forest Service, Pacific Southwest Research Station, Berkeley, California, USA. Tiberi R, Alessandro RA, Marianelli L, Peverieri S, Roversi PF. 2002. Insects and Fungi Involved in Oak Decline in Italy. IOBC/wprs Bulletin. Urosevic B. 1961. The influence of saprophytic and semi-parasitic fungi on the germination of Norway spruce and Scots pine seeds. Proc Int Seed Test Assoc 26 (3): 537-556. Vladimir L, Zlatan R, Bozica J. 2005. Mycoses of forest seed in object for production and warehouse. Bull Fac For Univ Banja Luka 4: 15-30. Vozzo JA. 1984. Insects and fungi associated with acorns of Quercus sp. Department of Agriculture, Forest Service, and Southeastern Forest Experiment Station. No. 6: 40-43. Washington DM. 2003. Fungi associated with northern red oak (Quercus rubra) acorns. [M.Sc. Thesis]. West Virginia University. Morgantown, WV. Winston PW. 1956. The acorn microsphere, with special reference to arthropods. Ecology 37: 120-132.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 67-72

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140203

Observations on arbuscular mycorrhiza associated with important edible tuberous plants grown in wet evergreen forest in Assam, India RAJESH KUMAR1,♼, ASHWANI TAPWAL2, SHAILESH PANDEY1, RAJA RISHI1, DEVAPOD BORAH1 1

Rain Forest Research Institute, P.O. 136, Jorhat 785001, Assam, India. Tel.: +91-0376-2305106, ♼e-mail: rajeshicfre@gmail.com 2 Forest Research Institute, Dehradun 248006, Uttrakhand, India Manuscript received: 9 May 2013. Revision accepted: 16 July 2013.

ABSTRACT Kumar R, Tapwal A, Pandey S, Rishi R, Borah D. 2013. Observations on arbuscular mycorrhiza associated with important edible tuberous plants grown in wet evergreen forest in Assam, India. Biodiversitas 14: 67-72. Non-timber forest products constitute an important source of livelihood for rural households from forest fringe communities across the world. Utilization of wild edible tuber plants is an integral component of their culture. Mycorrhizal associations influence the establishment and production of tuber plants under field conditions. The aim of present study is to explore the diversity and arbuscular mycorrhizal (AMF) colonization of wild edible tuber plants grown in wet evergreen forest of Assam, India. A survey was conducted in 2009-10 in Sunaikuchi, Khulahat, and Bura Mayong reserved forest of Morigaon district of Assam to determine the AMF spore population in rhizosphere soils and root colonization of 14 tuberous edible plants belonging to five families. The results revealed AMF colonization of all selected species in all seasons. The percent colonization and spore count was less in summer, moderate in winter and highest in rainy season. Seventeen species of arbuscular mycorrhizal fungi were recorded in four genera viz. Acaulospora (7 species), Glomus (5 species), Sclerocystis (3 species) and Gigaspora (2 species). Key words: AMF, root colonization, wild edible tuber

INTRODUCTION Wild edible plants refer to species that are neither cultivated nor domesticated, but available from their natural habitat and used as source of food (Beluhan and Ranogajec 2010). They are collected by forest fringe communities for their requirement of food and livelihoods. Earlier works have reported the wild edible plants as a potential source of nutrition and many of them have higher nutrition than conventionally eaten crops (Grivetti and Ogle 2000). Arbuscular mycorrhizal fungi (AMF) colonize the roots of higher plant as obligate symbionts, where the host generally benefited through increased nutrient uptake, improved growth and better survival (Linderman 1994; Akhtar and Siddiqui 2007; Smith and Read, 2008). Soil is characterized by the presence of a diverse population of microorganisms of which mycorrhizal fungi constitutes one of important component. Arbuscular mycorrhizal (AM) fungi are the most common types among all mycorrhizae and represent a major group of soil microbial community (Linderman 1992). Arbuscular Mycorrhiza is a widespread mutualistic symbiosis between land plants and fungi belonging to the phylum Glomeromycota. Their occurrence as root symbionts has been reported from exceptionally wide range of plants (Sharma et al. 2007). The AMF association may also increase the tolerance of host plant against biotic (Hol and Cook 2005; Akhtar and Siddiqui 2007) and abiotic stresses, including salinity and drought (Cartmill et al. 2007). In modern years, AM fungi gained

considerable importance in horticulture, agriculture, afforestation and land reclamation (Javot et al. 2007) because of their potentially to improve growth and yield of the plants by increasing the nutrient uptake (Jensen 1984). AM fungal association found in all organs of plants which are concerned with the absorption of substances from the soil (Srivastava et al. 1996). The occurrence of AM fungi association with the portions other than roots was reviewed by Nazim (1990). Presence of AM association has been reported in tubers of Pueraria tuberosa (Willd.) DC (Rodrigues 1996), Colocasia esculenta (L.) Scott (Bhat and Kaveriappa 1997), garlic bulbs (Kunwar et al. 1999) and tubers of Gloriosa superba L. (Khade and Rodrigues 2003). AMF colonization varies with season and its effects also influence the establishment of plants under field condition (Giovannetti and Nicolson 1983). Information on AM association with tuberous plants is scanty. Therefore, the present study is aimed to determine the AMF spore population in rhizosphere soils and its colonization for wild edible tuberous plants during different seasons in Sunaikuchi, Khulahat and Bura Mayong reserve forest of Morigaon district in Assam, India.

MATERIALS AND METHODS Study area The Sunaikuchi, Khulahat, and Bura Mayong Reserved Forests are situated in Morigaon district of Assam, India

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between 26.15° to 26.5° Northern latitude and 92° to 95.5° Eastern longitude (Figure 1). These three Reserved Forest, (RF) of Morigaon district formed under Assam Forest Regulation Act, 1891. The area receives annual rainfall is about 1530.9 mm and the annual average maximum temperature is 30.4°C and the minimum is 19.8°C. Fringe area of the RF is inhabited by a few ethnic groups such as Karbis, Bodos, Kukis, Dimasas, Hmars, Garos, Rengma Nagas and Tiwas. These communities are dependent on forest for habitat and other needs for well-being; the forest contributes livelihoods to many households as well. Target species The root and rhizosphere soil samples of 14 wild edible tuberous plants belonging to five families were collected

viz; Ipomoea batatas (L.) Lam., Pueraria thomsonii Benth., Pueraria tuberosa (Wild.) D.C, Vigna vexillata (L.) Rich., Alocasia odora (Roxb.) C. L. Koch, Alocasia cucullata Schott., Colocasia esculenta (L.) Schott., Sagittaria sagittifolia L., Amorphophallus campanulatus Roxb., Dioscorea pentaphylla L., Dioscorea puber (Bl.), Dioscorea alata L., Dioscorea esculenta Burk., Dioscorea batatas Decene (Figure 2), belonging to four families Fabaceae, Araceae, Araceae and Dioscoreaceae respectively) and studied. AMF spore isolation, enumeration and identification A total of 150 soil samples were collected from the rhizosphere of 14 plants species having tuber from a depth of 5-30 cm during mid-May, late July, and early September

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Figure 1. Study sites at Sunaikuchi, Khulahat and Bura Mayong Reserved Forests (●) in Morigaon district of Assam, India

KUMAR et al. – Arbuscular mycorrhiza in wet evergreen forest in Assam

A

B

C

E

F

H

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G

I

L

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Figure 2. A. Ipomoea batatas, B. Amorphophallus campanulatus, C. Alocasia cucullata, D. Alocasia odora, E. Colocasia esculenta, F. Sagittaria sagittifolia, G. Dioscorea alata, H. Dioscorea esculenta, I. Dioscorea batatas, J. Dioscorea pentaphylla, K. Pueraria thomsoni, L. Pueraria tuberosa, M. Vigna vexillata (photos from many sources).

in 2009-10. The samples (about 500 g for each) were airdried for 2 weeks and stored in sealed plastic bags at 4°C. AMF were isolated by a wet sieving and decanting technique (Gerdemann and Nicholson 1963; An et al. 1990; Singh and Tiwari 2001). Fifty grams of soil was suspended in 250 ml of water, stirred with a magnetic stirrer for 10

min and sieved. Spores and debris were collected on 150, 100, 70 and 40 μm sieves under tap water, filtered through Whatman filter paper and placed in a 90 mm Petri-dish for examination under a binocular stereomicroscope (Olympus BX 50F4, Japan). Each type of AMF spore was sequentially mounted in water, lactophenol, Poly vinyl

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alcohol and Melzer’s reagent (Morton 1988; Morton and Benny 1990) for identification. The spores were identified up to the species level with the help of a VAM fungi identification manual (Schenck and Perez 1990). The identification was based on spore color, size, surface ornamentation and wall structure with reference to the descriptions and pictures provided by the International Collection of Vesicular and Arbuscular Mycorrhizal Fungi (http://invam.caf.wvu.edu) and originally published species descriptions. Analysis of AMF and DSE colonization Roots were washed thoroughly in tap water and cut into approximately 1cm long segments. The roots were cleared in 10% (w/v) KOH by heating at 90°C for 1 to 2 h, depending on the degree of lignifications of the roots, then washed and stained with stamp pad ink (Das and Kayang 2008). The stained root samples were mounted on slides and examined for AM colonization under a light microscope. The colonization of root length with arbuscules, vesicles, hyphae and dark septate endophytes per sample were quantified by the magnified intersections method (McGonigle et al. 1990). Percent root colonization was determined using the following formula: % Root colonization = No. of positive segments x 100 No. of segments observed

RESULTS AND DISCUSSION Five-hundred and eighteen arbuscular mycorrhizal fungal spore samples were wet-sieved from the 150 soil samples. Seventeen species of arbuscular mycorrhizal fungi were identified. The morphological characters of some identified arbuscular mycorrhizal fungi are illustrated in (Table 1). All the fourteen plant species studied exhibited AM fungal association. AMF colonization in roots and the spore population in the rhizosphere soil samples of all

fourteen plant species having tubers showed wide range of variation under different seasons (Table 1). The level of AM fungal association depends on root morphology, metabolism and rate of plant growth (Warmer et al. 1980). Percent root colonization and mycorrhizal spore counts steadily increased in rainy season. Earlier reports also revealed higher percent root colonization during rainy season (Raghupathy and Mahadevan 1993; Kumar et al. 2013). The maximum infection (73%) was recorded in Sagittaria sagittifolia whereas minimum infection (45%) in Amorphophallus campanulatus were observed during rainy season in 2009-10. However, the maximum percent colonization was (53%) in Vigna vexillata in winter and (51%) in summer only. In the present study, the percent root colonization recorded higher in rainy season than in winter and summer. Least activity of AM fungi in other seasons may be due to reduced translocation of carbohydrates towards the roots. The spore population was also least in summer and gradually increased in July. The spore population varied from 15-61 spores, 13-41 spores and 7-27 spores during rainy, winter and summer seasons respectively (Table 2). Khade and Rodrigues (2007) also observed maximum number of spore density while studying the occurrence of AM fungi in plants with underground storage organs. The identified species of arbuscular mycorrhizal fungi belonged to the genera of Acaulospora (7 species), Glomus (5 species), Sclerocystis (3 species) and Gigaspora (2 species).The occurrence frequency of the five genera was 42.62%, 36.67%, 12.92%, and 7.71%, respectively (Table 2). The results indicated that Acaulospora and Glomus were the dominant genera, and A. denticulata, A. spinosa, A. tuberculata, G. clarum, G. constrictum and G. monosporum and S. clavispora were the dominant species (Table-3). It is also observed that Acaulospora and Glomus species usually produce more spores than Gigaspora and Sclerocystis species in the same environment. This may be due to their smaller spore size and require a short time to produce spores (Hepper 1984; Bever et al. 1996).

Table 1. Important wild edible plants with tubers in Sunaikuchi, Khulahat, and Bura Mayong Reserved Forest, Assam in 2009-2010 and seasonal variation of arbuscular mycorrhizal association in wild edible tuberous plants Name of plants Alocasia cucullata Schott. Alocasia odora (Roxb.) C.L. Koch Amorphophallus campanulatus Roxb. Colocasia esculenta (L) Schott. Sagittaria sagittifolia L. Ipomoea batatas (L.) Lam. Dioscorea alata L. Dioscorea batatas Decene. Dioscorea esculenta Burk. Dioscorea pentaphylla L. Dioscorea puber BL Pueraria thomsonii Benth. Pueraria tuberosa (Wild.) D.C Vigna vexillata (L.) Rich

Family

Local name

Araceae Araceae Araceae Araceae Araceae Convolvulaceae Dioscoreaceae Dioscoreaceae Dioscoreaceae Dioscoreaceae Dioscoreaceae Fabaceae Fabaceae Fabaceae

Panchamukhi Kachu Baibing Pani kachu Kachu Ole kachu Ranga alu, Mitha alu Kath alu Gosh alu Mua alu Paspatia alu Jangali alu Mayong (Mis), Pani alu Urahi alu Bonoria urahi

% root colonization No. of spores/ 50g of soil Rainy Winter Summer Rainy Winter Summer 58 33 40 21 19 16 66 35 16 44 31 21 45 32 41 21 18 14 66 42 34 26 19 18 73 45 37 38 25 13 53 41 22 44 31 14 62 41 32 26 24 16 66 38 31 39 25 19 58 39 28 15 13 7 51 40 32 22 18 13 67 38 35 24 23 9 42 35 24 39 32 18 68 44 32 58 33 21 72 53 51 61 41 27

KUMAR et al. – Arbuscular mycorrhiza in wet evergreen forest in Assam

CONCLUSION The study revealed that the plants with tubers growing in the tropical wet ever green forest of Sunaikuchi, Khulahat, and Bura Mayong Reserved Forest of Assam, India are colonized by arbuscular mycorrhizal fungi. It is also apparent that rainy season may considered as the best season for the propagation of plants by the application of AMF as bioinoculants even for the plants of rare and threatened species. Our results also revealed that uneven spatial distribution (clumped distribution) of arbuscular mycorrhizal fungal spores and the complex below ground structure of tropical wet ever green forests are major factors that affect the spore density.

ACKNOWLEDGEMENTS The authors are thankful to the Indian Council of Forestry Research and Education (ICFRE) for funding the research project No: RFRI13/2008-09/FP.

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Table 2. Identified arbuscular mycorrhizal fungi and their occurrence frequencies

Arbuscular mycorrhizal fungi

Absolute occurrence

Acaulospora Acaulospora bireticulata Rothw. & Trappe Acaulospora denticulata Sieverding & Toro Acaulospora foveata Trappe & Janos Acaulospora mellea Spain & Schenck Acaulospora scrobiculata Trappe Acaulospora spinosa Walker & Trappe Acaulospora tuberculata Janos & Trappe Glomus Glomus claroideum Schenck & Smith Glomus clarum Nicol. & Schenck Glomus constrictum Trappe Glomus fasciculatum (Thaxter) Gerd. & Trappe Glomus monosporum Gerd. & Trappe Sclerocystis Sclerocystis clavispora (Trappe) Almeida & Schenck Sclerocystis coremioides Berk. & Broome 8 1.52 Sclerocystis sinuosa (Gerd. & Bakshi) Almeida Gigaspora Gigaspora gigantea (Nicol. & Gerd. ) Gerd. &Trappe Gigaspora margarita W.N. Becker & I.R. Hall Total AMF = 17Species

221 16 67 18.4 19.4 13.2 53.3 33.7 190 20 83 33 17 37 67 33 19 15 40 19 21 518

REFERENCES Akhtar MS, and Siddiqui ZA. 2007. Biocontrol of a chickpea root-rot disease complex with Glomus intraradices, Pseudomonas putida and Paenibacillus polymyxa. Aust Plant Pathol 36: 175-180. An ZQ, Hendrix JW, Hershman DE, and Henson GT. 1990. Evaluation of the most probable number (MPN) and wet-sieving methods for determining soil-borne populations of endogonaceous mycorrhizal fungi. Mycologia 82: 516-581. Beluhan S, and Ranogajec A. 2010. Chemical composition and nonvolatile components of Crotial wild edible mushrooms. Food Chemistry 124: 1076-1082. Bever JD, Morton JB, Antonovics J, Schultz PA. 1996. Host-dependent sporulation and species diversity of arbuscular mycorrhizal fungi in a mown grassland. J Ecol 84: 71-82. Bhat RP, Kaveriappa KM. 1997.Occurrence of vesicular Arbuscular mycorrizal fungi in the tubers of Colocasia esculenta (L.) Schott., Mycorrhiza News 912-13. Cartmill AD, Alarcon A, Valdez-Aguilar LA. 2007. Arbuscular mycorrhizal fungi enhance tolerance of Rosa multiflora cv. Burr to bicarbonate in irrigation water. J Plant Nutr 30: 1517-1540. Das P, Kayang H. 2008. Stamp pad ink, an effective stain for observing arbuscular mycorrhizal structure in roots. World J Agric Sci 4: 58-60 Gerdemann JW, Nicolson TH. 1963. Spores of mycorrhizal Endogone extracted from soil by wet sieving and decanting. Trans Br Mycol Soc 46: 235-244. Giovannetti M, Nicolson TH. 1983. Vesicular-arbuscular mycorrhizas in Italian sand dunes. Trans Br Mycol Soc 80: 552-557. Grivetti LE, Ogle BM. 2000. Value of traditional foods in meeting macroand micronutrient needs: the wild plant connection. Nutr Res Rev 13: 31-46. Hepper CM. 1984. Isolation and culture of VA mycorrhizal (VAM) fungi. In: Powell CL, Bagyaraj DJ (eds). VA Mycorrhizae. CRC Press, Florida.

Relative occurrence/ frequency (%) 42.62 3.08 12.93 3.55 3.74 2.54 10.28 6.50 36.67 3.86 16.02 6.37 3.28 7.14 12.92 6.37 3.66 2.89 7.71 3.66 4.05 100

Hol GW, Cook R. 2005. An overview of arbuscular mycorrhizal funginematode interactions. Basic Appl Ecol 6: 489-503. Javot H, Pumplin N, Harrison MJ. 2007. Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles. Plant Cell Environ 30 (3): 310-322. Jensen A.1984. Responses of barley, pea and maize to inoculation with different vesicular Arbuscular Mycorrhizal fungi in irradiated soils. Pl Soil 78: 315-323. Khade SW, Rodrigues BF. 2003. Incidence of Arbuscular Mycorrhizal colonization in tubers of Gloriosa superba L., Mycorrhiza News 15: 14-16. Khade SW, Rodrigues BF. 2007. Incidence of arbuscular mycorrhizal (AM) fungi in some angiosperms with underground storage organs from Western Ghat region of Goa. Trop Ecol 48 (1): 115-118. Kumar R, Tapwal A, Jaime A Teixeira da Silva, JA, Pandey S, Borah DP. 2013. Biodiversity of arbuscular mycorrhizal fungi associated with mixed natural forest of Jeypore, Assam. Bioremed Biodiv Bioavail 7 (1): 91-93. Kunwar IK, Reddy PJM, Manoharachary C. 1999. Occurrence and distribution of AMF associated with garlic rhizosphere soil. Mycorrhiza News 11: 4-6. Linderman RG. 1992. Vesicular-arbiscular mycorrhizae and soil microbial interactions, In: Bethlenfalvay GJ, Linderman RG (eds). Mycorrhizae in sustainable agriculture. Soil Science Society of America, Madison, WI. Linderman RG. 1994. Role of VAM fungi in biocontrol. In: Pfleger FL, Linderman RG (eds). Mycorrhizae and plant health. The American Phytopathological Society, St. Paul, Minnesota. McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. 1990. A new method which gives an objective measure of colonization of roots by vesicular arbuscular mycorrhizal fungi. New Phytol 115: 495-501. Morton JB. 1988. Taxonomy of VA mycorrhizal fungi: classification, nomenclature, and identification. Mycotaxon 37: 267-324. Morton JB, Benny GL. 1990. Revised classification of arbuscular mycorrhizal fungi (Zygomycetes), a new order Glomales, two new suborders Glomineae and Gigasporinae and two new families Acaulosporaceae and Gigasporaceae with an emendation of Glomaceae. Mycotaxon 37: 471-479.

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Nazim G. 1990.Vesicular Arbuscular Mycorrhiza in portions other than roots. In: Jalali BL, Chand H (eds). Current Trends in Mycorrhizal Research. Sankat Mochan Art Press, Hisar, India. Raghupathy S. Mahadevan A.1993. Distribution of vesicular Arbuscular mycorrhizae in plants and rhizosphere soils of the tropical plains, Tamilnadu, India. Mycorrhiza 3: 123- 136. Rodrigues BF. 1996. Occurrence of VAM fungi in the tubers of Pueraria tuberosa (Willd.) DC. Mycorrhiza News 8-9. Schenck NC, Perez Y. 1990. Manual for the Identification of VA Mycorrhizal Fungi (2nd End), International Culture Collection of VA Mycorrhizal Fungi (INVAM), University of Florida, Gainesville, FL.

Sharma S, Aggarwal A, Kaushish S. 2007. Biodiversity of endomycorrhizal fungi associated with some medicinally important plants of Himachal Pradesh. J Indian Bot Soc 86: 14-17. Singh SS, Tiwari SC. 2001. Modified wet-sieving and decanting technique for enhanced recovery of spores of vesicular-arbuscular mycorrhizal (VAM) fungi in forest soils. Mycorrhiza News 12: 12-13. Smith SE, Read DJ. 2008. Mycorrhizal Symbiosis. 3rd ed. Academic Press, London. Srivastava D, Kapoor R, Srivastava SK, Mukerji KG. 1996. Vesicular arbuscular mycorrhiza-an overview In: Mukerji KG (ed). Concepts in Mycorrhizal Research. Kluwer, Netherlands. Warner A, Mosse B. 1980. Independent spread of Vesicular Arbuscular Mycorrhizal fungi in soil. Trans Br Mycol Soc 74: 407-410.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 73-78

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140204

Diversity and frequency of macrofungi associated with wet ever green tropical forest in Assam, India ASHWANI TAPWAL1,♥, RAJESH KUMAR2, SHAILESH PANDEY2 1

Forest Pathology Division, Forest Research Institute, P.O. New Forest, Dehradun 248006, Uttrakhand, India. Tel.: +91-0135-222-4259, ♥ email: ashwanitapwal@gmail.com 2 Rain Forest Research Institute, P.O. 136, Jorhat 785001, Assam, India. Manuscript received: 17 May 2013. Revision accepted: 18 July 2013.

ABSTRACT Tapwal A, Kumar R, Pandey S. 2013. Diversity and frequency of macrofungi associated with wet ever green tropical forest in Assam, India. Biodiversitas 14: 73-78. A study was conducted in Jeypore Reserve Forest located in Assam, India to investigate the diversity of macrofungi associated with different tree species. The diversity of broad leaves trees and high humidity during monsoon period favours ideal growth of diverse group of macrofungal fruiting bodies. Thirty macrofungal species representing 26 genera belonging to 17 families were collected from six different sites in the study area. Out of these maximum six genera assignable to family Polyporaceae, five genera to Russulaceae, three genera to Agaricaceae, two genera to Ganodermataceae and Cantharellaceae each and rest of the families were represented by single genus only. The ecological preference of the species reveled that maximum (17) species were saprophyte, living on dead substrates or decaying wood debris, ten species were found associated with roots of higher trees, while three species were found parasitic. Overall 20 species were found edible including some species having medicinal utilization. The present study revealed that maximum frequency of occurrence was exhibited by Trametes versicolor and Schizophyllum commune (83.33%), followed by Microporus xanthopus, Pycnoporus sanguineus (66.67%) and Coprinus disseminates (50%). The rest of the species exhibited the frequency distribution ranging between 16.67-33.33%. The maximum density was recorded for Schizophyllum commune (126.67%) followed by Trametes versicolor (120%) and Xylaria polymorpha (93.33%) . The density of rest of the species were ranged between 3.33- 6.67%. The key objective of the present study was to generate a database on macrofungal diversity of Jeypore Reserve Forest along with their ecological preferences and utilization, which is not earlier documented. Key words: Jeypore Reserve Forest, macrofungi, mycorrhiza

INTRODUCTION The Jeypore Reserve Forest is an important wet ever green tropical forest patch of eastern Assam which constitutes a part of the Eastern Himalaya biodiversity hotspot region. This reserve forest is relatively less disturbed by humans beings in comparison to other protected areas of the state (Saikia and Devi 2011). The major tree species of the area are Dipterocarpus retusus, Shorea assamica, Baccaurea ramiflora, Begonia roxburghii, Gmelina arborea, Litsea salicifolia, Mesua ferrea, Syzygium cumini, Terminalia myriocarpa, Vatica lanceaefolia etc in addition to diverse population of herbs and shrubs. Plantations provide a habitat for diverse macro fungal communities, which vary markedly in composition from site to site. Fungi are some of the most important organisms in the world, because of their vital role in ecosystem function, influence on humus and human-related activities (Mueller and Bill, 2004). Mushrooms are cosmopolitan heterotrophic organisms that are quite specific in their nutritional and ecological requirements. They can grow in soil or degrading plant residues as saprophytes, wood decaying and many live in symbiotic association with the roots of higher plant species. They play important role in nutrient recycling; growth and

establishment of seedlings in forest floor. While some fungal species forms parasitic association with trees and cause considerable damage. The peak season for the formation of fruit body of macrofungi is different for each ecological climate (Arora 1991). Defining the exact number of fungi on the earth has always been a point of discussion and several studies have been focused on enumerating the world’s fungal diversity (Crous 2006). Current studies have estimated about 1.5 million species of fungi on globe (Hawksworth 2004). One-third of which exists in India and of this only 50% are characterized till date (Manoharachary et al. 2005). More than 27,000 fungal species are recorded from India, which is the largest biotic community after insects (Sarbhoy et al. 1996). Despite the great bio-geographic significance of the Jeypore Reserve Forest, it remains poorly documented in terms of macrofungal diversity. This broad leaf forest presumably possesses great diversity not only in plant species but also in macrofungi. Some selected pockets of this forest have been surveyed for the diversity of macrofungi. The macrofungi observed in the study area are either edible, medicinal, saprophyte or wood rotting fungi. These fungal species vary in their abundance and phenology of fruiting. Most of the macrofungal species producing hypogeous/ epigeal sporocarp are thought to be

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ectomycorrhizal (Trappe 1962; Lakhanpal 1997; Beig et al. 2008). The importance of macrofungi has been well established since ancient times. Many Asian countries use traditionally wild edible mushrooms as delicious and nutritional foods and medicine. Wild edible mushrooms are appreciated not only for texture and flavor but also for their chemical and nutritional characteristics (Manzi et al. 1999; Sanmee et al. 2003). Mushrooms provide minerals, vitamins and proteins with high nutritional value as do the best local legumes (Buyck 1994). Mushrooms are also reported as therapeutic foods, useful in preventing diseases such as hypertension, hypercholesterolemia and cancer (Bobek and Galbavy 1999; Bobek et al. 1991). These functional characteristics are mainly due to the presence of

dietary fiber and in particular chitin and beta glucans (Manzi et al. 2001). Studies have also shown antitumor, antiviral, antithrombotic and immunomodulating effects of mushrooms (Mau et al. 2002). The aim of present investigation was to generate base-line information on prevailing macrofungi of Jeypore Reserve Forest with their ecological relationship and utilization. MATERIALS AND METHODS Study site The Jeypore Reserve Forest is located in Dibrugarh District of Assam, India (Figure 1) lies between 27°06′27°16' N and 95°21’-95°29’E longitude at an elevation of 1100-2600 m. The climate of the study site is humid

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Figure 1. Study sites at Jeypore Reserved Forests (○) in Dibrugarh district of Assam, India

TAPWAL et al. – Diversity of macrofungi in a Jeypore reserve forest

tropical characterized by high rainfall and high humidity (up to 90%). The annual mean precipitation in the last three years ranged from 3600 to 5500 mm of which 82% is received during the monsoon season from May to August and 17% during dry periods from September to March. The mean ambient temperature is 27°C. Sample collection and diversity analysis Periodic surveys were made to the study area for the collection of macrofungi during rainy season (June to September) and winter (October to December) in 20102011. Six sites in Jeypore Reserve Forest (JRF) have been surveyed in winter and rainy season for the collection of macrofungi. The collected samples were wrapped in wax paper and brought to the laboratory for identification. The macroscopic characters like shape, size, color, texture, attachment of stipe, smell, spore print, habit and habitat has documented during the survey and collection work. The taxonomy has been worked on the basis of macro and microscopic characteristic following available literatures (Zoberi 1973; Alexopoulos et al. 1996; Purakasthya 1985). The soft textured specimens were preserved in 2% formaldehyde and leathery textured were preserved in 4% formaldehyde. The utilization of different mushroom species for food and as medicine has been documented from the available literature. The frequency and density of different species has been determined by the following formulas: No. of site in which the sp. is present Freq. of fungal sp. (%) = ----------------------------------------- x 100 Total no. of sites Total no. of individual of a particular species Density = ------------------------------------------------------- x 100 Total no. of species

RESULTS AND DISCUSSIONS Species diversity of macrofungi is related to their particular habitats. The factors like geographic location, elevation, temperature, humidity, light and surrounding flora greatly influence the growth and development of macrofungi. Thirty macrofungal species representing 26 genera belonging to 17 families were collected from the study area (Figure 1). Maximum six genera assignable to family Polyporaceae, five genera to Russulaceae, three genera to Agaricaceae, two genera to Ganodermataceae and Cantharellaceae each and rest of the families were represented by single genus only. The diversity analysis revealed that maximum frequency occurrence was exhibited by Trametes versicolor and Schizophyllum commune (83.33%), followed by Microporus xanthopus (66.67%), Pycnoporus sanguineus (66.67%) and Coprinus disseminates (50%). The frequency distribution of rest of the species was ranged between 16.67-33.33%. Almost in a similar trend maximum density was recorded for Schizophyllum commune (126.67%) followed by Trametes

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versicolor (120.00%), Xylaria polymorpha (93.33%) and the rest were ranged between 3.33- 76.67% (Table 1).

Figure 1. Macrofungal distribution in families, ecological relationship and utilization

Twenty species were found edible, out of which some have medicinal properties. Mushrooms are delicious food due their high quality protein, vitamins and minerals. Fresh mushrooms contain about 90% moisture and 10% dry matter. Dry mushrooms contain about 90% dry matter and 10% moisture (Chang and Buswell 1996). For local populations, mushrooms are usually considered as substitutes for animal protein, and are known as meat for the poor (Buyck 1994). Most common edible macrofungi found in JRF are the species of Agaricus, Lactarius, Lycoperdon, Russula, Scleroderma, Cantharellus, Pleurotus, Lentinus, Schizophyllum etc. In India, mushrooms are a non wood forest produce and popular as food among the ethnic people of North east India. Some of the edible species like Termitomyces eurrhizus, Lentinus conatus, Schizophyllum commune, Tricholoma giganteum and Pleurotus are sold in the markets of Kohima district of Nagaland by the local people (Tanti et al. 2011). In addition to these Kumar et al. (2013) described 15 edible fungi along with their macronutrient content collected from different forest areas of Nagaland. The ecological preference of the species revealed that maximum number of (17) species were saprophyte and 10 species were found associated with higher trees. The mycorrhizal fungi basically serves as an extension of the plant root system, exploring soil far beyond the roots and transporting water and nutrients to the roots. The fungus grows from the colonized roots into the surrounding soil. Mycelial colonization of the soil varies among ectomycorrhizal fungi; some may only grow a few centimeters into the soil and others can grow several meters from the ectomycorrhiza. Some fungi produce dense, hyphal mats that strongly bind the soil and organic matter (Molina 1994). Mycorrhizae increase the survival, growth and development of associated plants by performing essential physiological processes i.e. increased absorption surface, selective ion absorption and accumulation

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(Jorgenson and Shoulders 1967; Marks and Kozlowski 1973), and help seedlings to resist infection by certain feeder root pathogens (Marx 1971). A single tree can host different species of mycorrhizal fungi and one fungus can be associated with different trees at the same time. Such type of multiple association forms an underground network

of hyphae to connect fungi, trees and shrubs in the forest to share water and nutrients. In the present study, ten species were found associated with roots of higher trees forming mycorrhizal association while three species were found parasitic. High population of mycorrhizal and saprophytic species indicated the good health of the forest.

Table 1. List of macrofungi recorded in Jeypore reserve forest with uses and ecological relationship Fungi Agaricus arvensis Lycoperdon pyriforme Coprinus disseminatus Amanita pantherina Auricularia auricula-judae Boletus badius Cantharellus lateritius Craterellus sp. Clavaria sp. Ganoderma lucidum Ganoderma applanatum Ramaria sp. Laccaria bicolor Phellinus gilvus Marasmius androsaceus Pleurotus sp. Panus fulvus Earliella scabrosa Lentinus sp. Microporus xanthopus Pycnoporus sanguineus Trametes versicolor Lactarius hygrophoroides Russula amoena R. delica R. pectinata R. nobilis Schizophyllum commune Scleroderma sp. Xylaria polymorpha

Family Agaricaceae Agaricaceae Agaricaceae Amanitaceae Auriculaceae Boletaceae Cantharellaceae Cantharellaceae Clavariaceae Ganodermataceae Ganodermataceae Gomphaceae Hydnangiaceae Hymenochaetaceae Marasmiaceae Pleurotaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Polyporaceae Russulaceae Russulaceae Russulaceae Russulaceae Russulaceae Schizophyllaceae Sclerodermataceae Xylariaceae

Ecological relationship Saprophyte Mycorrhizal Saprophyte Mycorrhizal Dead wood Mycorrhizal Saprophyte Saprophyte, dead wood Saprophyte, dead & decaying wood Parasitic Parasitic Saprophyte, dead wood Mycorrhizal Parasitic Saprophyte, plant debris Dead wood Dead and decaying wood Dead wood Dead wood stumps Dead wood Saprophyte, Dead wood Wood decaying Mycorrhizal Mycorrhizal Mycorrhizal Mycorrhizal Mycorrhizal Dead wood Mycorrhizal Dead wood

Utilization Edible Edible Non edible Non edible Edible, Medicinal Non edible Edible Edible Non edible Medicinal Medicinal Edible Non edible Non edible Non edible Edible Edible Non edible Edible, medicinal Medicinal Non edible Medicinal Edible Edible Edible Edible Edible Edible, medicinal Edible Non edible

Frequency of occurrence 33.33 16.67 50.00 16.67 33.33 33.33 33.33 33.33 16.67 16.67 16.67 16.67 33.33 16.67 16.67 16.67 16.67 33.33 33.33 66.67 66.67 83.33 16.67 33.33 16.67 33.33 16.67 83.33 16.67 16.67

Density 10.00 13.33 66.67 23.33 43.33 13.33 20.00 20.00 6.67 6.67 10.00 3.33 16.67 6.67 10.00 56.67 3.33 16.67 6.67 76.67 20.00 120.00 10.00 36.67 20.00 13.33 3.33 126.67 13.33 93.33

Table 2. List of mushroom species having medicinal uses Mushroom species

Utilization

Reference

Ganoderma lucidum

Promotes health and longevity, lowers the risk of cancer and heart disease and boosts the immune system. Antioxidant, hypoglycemic and antihypertension To stop a child from breast feeding To stop a child from bed wetting Anti-candida, anti-tumor and anti-viral properties, antitumor, anticancer and immunomodulating activities anti-diabetic, antitumor, antihypertensive, anti-inflammatory, immunomodulatory and antibacterial agents immunomodulatory and anti-cancer effects Biodegrading textile dyes and lignosulphonates arthritis, gout, styptic, sore throats, ulcers, tooth aches, fevers, hemorrhages and antibacterial Antiinflammatory, antitumor, antioxidant, antihepatotoxicity Tendon relaxation, pain alleviation and antihypertension Protect from cancer, environmental allergies, fungal infection, frequent flu and colds, bronchial inflammation, heart disease, hyperlipidemia, hypertension, infectious disease, diabetes, hepatitis and regulating urinary inconsistancies

Wachtel-Galor et al. (2004) Oyetayo (2011) Chang and Lee (2004) Chang and Lee (2004) Wasser (2002); Kidd 2000 Gurusamy and Arthe (2012) Ramberg et al. (2010). Trovaslet et al. (2007); Eugenio et al. (2008) Kim et al. (2011) Zhang et al. (2009) Bisen et al. (2010)

Ganoderma applanatum Microporus xanthopus Xylaria polymorpha Schizophyllum commune Auricularia auricula-judae Trametes versicolor Pycnoporus sanguineus Phellinus gilvus Marasmius androsaceus Lentinus sp.

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A

B

C

D

E

F

G

H

I

Figure 2. A. Ganoderma lucidum, B. Ganoderma applanatum, C. Pycnoporus sanguineus, D. Auricularia auricula-judae, E. Schizophyllum commune, F. Microporus xanthopus, G. Trametes versicolor, H. Marasmius androsaceus, I. Xylaria polymorpha

Only three species viz. Ganoderma applanatum, G. lucidum and Phellinus gilvus recorded in JRF were parasitic in nature. The pathogenic fungi directly kill or weaken the forest plants and decline the forest health and productivity. But fungal diseases also have positive influences on ecosystem productivity and biodiversity (Trappe and Luoma 1992). For example, the trees killed by diseases open the forest for the growth of light demanding plants. Standing dead trees also provide habitat for cavitynesting birds and mammals. In boarder sense it is important to realize that pathogens below the threshold population are a natural component of the forest ecosystem and contribute to landscape diversity (Molina 1994). Although the species of Ganoderma and Phellinus were recorded in some tree species but their population was very less. Beside their pathogenic nature, they are being used for the manufacture of various drugs by pharmaceutical

companies. G. lucidum is well known to promote health and longevity, lowers the risk of cancer and heart disease and boosts the immune system (Wachtel-Galor et al. 2004) while the G. applanatum have antioxidant, hypoglycemic and antihypertension activity (Oyetayo, 2011). P. gilvus has been reported to have antiinflammatory, antitumor, antioxidant, antihepatotoxicity potential (Kim et al. 2011). Other medicinal mushrooms recorded in JRF includes Microporus xanthopus, Pycnoporus sanguineus, Xylaria polymorpha, Schizophyllum commune, Auricularia auricula-judae, Trametes versicolor, Marasmius androsaceus and Lentinus sp. (Table 2; Figure 2). Mushrooms in North eastern India sold in traditional markets or commercially exploited for food or medicines (Tanti et al. 2011). Gogoi and Sarma (2012) documented 12 macrofungal species from Dhemaji district of Assam with their ethnomycological utilization. Kumar et al.

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(2013) described four medicinal mushrooms Nagaland along with their nutrient contents.

from

CONCLUSION The mushrooms grown in the wild plays an important role to maintain the forest health besides their medicinal importance and nutritional value. Therefore, it becomes quite necessary to explore, document and conserve this natural wealth. The present study provides a database on macrofungal diversity of Jeypore Reserve Forest, Assam, India along with their ecological preferences and utilization, which was not documented earlier.

ACKNOWLEDGEMENTS The authors are gratefully acknowledged to Indian Council of Forestry Research and Education (ICFRE) for funding the research project: No-RFRI-39/2010-11/FP.

REFERENCES Alexopoulos CJ, Mims CW, Blackwell. 1996. Introductory Mycology. John Wiley and Sons Inc., New York. Arora D. 1991. All that the Rain Promises and More. A hip pocket guide to Western Mushroom. Ten Speed Press, New York. Beig MA, Dar GH, Ganai NA. 2008. Some Hitherto unrecorded macrofungi from India. J Mycol Pl Pathol 38:158-160. Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GBKS. 2010. Lentinus edodes: A Macrofungus with Pharmacological Activities. Curr Med Chem 17: 2419-2430. Bobek P, Galbavy S. 1999. Hypocholesterolemic and antiatherogenic effect of oyster mushroom (Pleurotus ostreatus) in Rabbit. Nahrung 43: 339. Bobek P, Ginter E, Jurcovicova M, Kunlak L. 1991. Cholesterol lowering effect of mushroom Pleurotus ostreatus in hereditary hypercholesterolomic rats. Ann Nutr Metab 35: 191-195. Buyck B. 1994. UBWOBA: Les Champignons Comestibles de l'Ouest du Burundi. AGCD, Rue du Trône, 4-1050, Bruxelles. Publ. Agricole #34 [French]. Chang ST, Buswell JA. 1996. Mushroom nutraceuticals. World J Microbiol Biotechnol 12: 473-476. Chang YS, Lee SS. 2004. Utilisation of macrofungi species in Malaysia. Fungal Diversity 15: 15-22. Crous PW. 2006. How many species of fungi are there in tip of Africa. Stud Mycol 55:13 Eugenio ME, Carbajo JM, Terrón MC, González AE, Villar JC. 2008. Bioremediation of lignosulphonates by lignin-degrading basidiomycetous fungi. Biores Technol 99 (11): 4929-4934. Gogoi Y, Sarma TC. 2012. An ethnomycological survey in some areas of Dhemaji district (Assam). Proceedings of International Conference on Anthropogenic Impact on Environment & Conservation Strategy. Ranchi, November 2-4, 2012. Gurusamy R, Arthe R. 2012. Effect of medicinal mushroom, Auricularia auriculajudae, polysaccharides against EAC cell lines. Res J Biotech 7 (2): 14-17. Hawksworth DL. 2004. Fungal diversity and its implifications for genetic resource collections. Stud Mycol 50: 19. Jorgenson JR, Shoulders E. 1967. Mycorrhizal root development vital to survival of slash pine nursery stock US Dep. Agric. For. Serv., Tree Plant Notes 18: 7- 11. Kidd PM. 2000. The use of mushroom glucans and proteoglycans in cancer treatment. Alter Med Rev 5 (1): 4-27.

Kim SH, Lim JH, Moon C, Park SH, Kim SH, Shin DH, Park SC, Kim CJ. 2011. Antiinflammatory and antioxidant effects of Aqueous extracts from Phellinus gilvus in Rats. J Health Sci 57 (2): 171-176. Kumar R, Tapwal A, Pandey S, Borah RK, Borah D, Borgohain J. 2013. Macro-fungal diversity and nutrient content of some edible mushrooms of Nagaland, India. Nusantara Biosci 5 (1): 1-7. Lakhanpal TN. 1997. Diversity of mushroom mycoflora in the NorthWest Himalaya. In: Sati SC, Saxena J, Dubey RC (eds) Recent researches in ecology, environment and pollution. Today and Tomorrow’s Printers and Publishers, New Delhi. Manoharachary C, Sridhar K, Singh R, Adholeya, Suryanarayanan TS, Rawat S, Johri BN. 2005. Fungal Biodiversity: Distribution, Conservation and Prospecting of Fungi from India. Curr Sci 89 (1): 58-71. Manzi P, Gambelli L, Marconi S, Vivanti V, Pizzoferrato L. 1999. Nutrients in edible mushrooms: An interspecies comparative study. Food Chem 65: 477-482. Manzi P, Aguzzi A, Pizzoferrato L. 2001. Nutritional value of mushrooms widely consumed in Italy. Food Chem 73: 321. Marks GC, Kozolowski TT. 1973. Ectomycorrhizae: Their ecology and Physiology. Academic Press, New York. Marx DH. 1971. Ectomycorrhizae as biological deterents to pathogenic root infections. In: Hacskaylo E. (ed). Mycorrhizae US Govt. Printing Office, Washington. Mau LL, Lim HC, Chen CC. 2002. Antioxidant properties of several medicinal mushrooms. J Agric Food Chem 50: 6072. Molina R. 1994. The role of mycorrhizal symbioses in the health of giant redwoods and other forest ecosystems. USDA Forest Service Gen. Tech. Rep. PSW-151 Mueller GM, Bills GF. 2004. Introduction. In: Mueller GM, Bills GF. Foster MS (eds). Biodiversity of Fungi Inventory and Monitoring Method. Elsevier Academic Press, San Diego. Oyetayo OV. 2011. Medicinal uses of mushrooms in Nigeria: towards full and sustainable exploitation. Afr J Tradit Compl Altern Med 8 (3): 267-274. Purakasthya RP, Chandra A. 1985. Manual of Indian Edible Mushrooms. Today and Tomorrow’s Publication, New Delhi. Ramberg JE, Nelson ED, Sinnott RA. 2010. Immunomodulatory dietary polysaccharides: A systematic review of the literature. Nutrition J 9: 1-22. Saikia PK, Devi OS. 2011. A checklist of avian fauna at Jeypore Reserve Forest, eastern Assam, India with special reference to globally threatened and endemic species in the Eastern Himalayan biodiversity hotspot. J Threat Taxa 3 (4): 1711-1718. Sanmee R, Dell B, Lumyong P, Izumori K, Lumyong S. 2003. Nutritive value of popular wild edible mushrooms from northern Thailand. Food Chem 84: 527-532. Sarbhoy AK, Agarwal DK, Varshney JL. 1996. Fungi of India 1982-1992. CBS Publi. & Distributors, New Delhi. Tanti B, Gurung L, Sarma GC. 2011. Wild edible fungal resource used by the ethnic tribes of Nagaland, India. Indian J Trad Know 10 (3):512515. Trappe JM. 1962. Fungus associates of ectotrophic mycorrhizae. Bot rev 28: 538-606. Trappe JM., Louma D. 1992. The ties that bind: fungi in ecosystems. In: Carroll GC, Wicklow DT (eds). The fungal community, its organization and role in the ecosystem. Marcel Dekker, New York. Trovaslet M, Enaud E, Guiavarc'h Y, Corbisier AM, Vanhulle S. 2007. Potential of a Pycnoporus sanguineus laccase in bioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye. Enz Microb Technol 41 (3): 368-376. Wachtel-Galor S, Tomlinson B, Benzie IFF. 2004. Ganoderma lucidum (‘Lingzhi’), a Chinese medicinal mushroom: biomarker responses in a controlled human supplementation study. Br J Nutr 91: 263-269. Wasser SP. 2002. Review of medicinal mushrooms advances: good news from good allies. Herbal Gram 56: 28-33. Zhang L, Yang M, Song Y, Sun Z, Peng Y, Qu K, Zhu H. 2009. Antihypertensive effect of 3,3,5,5-tetramethyl-4-piperidone, a new compound extracted from Marasmius androsaceus. J Ethnopharmacol 123: 34-39. Zoberi MH. 1973. Some edible mushrooms from Nigeria. Nigerian Field 38: 81-90.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 79-88

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140205

Fungal diversity associated with bamboo litter from Bambusetum of Rain Forest Research Institute, Northeast India RAJESH KUMAR1,♼, ASHWANI TAPWAL2, SHAILESH PANDEY1, RAJA RISHI1 1

Rain Forest Research Institute, P.O. 136, Jorhat 785001, Assam, India. Tel.: +91-0376-2305106, ♼email: rajeshicfre@gmail.com 2 Forest Research Institute, Dehradun 248006, Uttrakhand, India Manuscript received: 22 May 2013. Revision accepted: 29 July 2013.

ABSTRACT Kumar R, Tapwal A, Pandey S, Rishi R. 2013. Fungal diversity associated with bamboo litter from Bambusetum of Rain Forest Research Institute, Northeast India. Biodiversitas 14: 79-88. Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocelluloses matrix, which other organisms are unable to digest. Diversity of bamboo leaf litter fungi from fallen leaves and undergoing active decomposition leaves in different season and different depth was carried out in 2009-10. Twenty four samples were collected from Bambusetum of Rain Forest Research Institute (RFRI), Northeast India. The moist chamber, direct isolation and dilution plate methods were used to assess the diversity of fungal species. Fungi were cultivated on 3% malt extract agar and half strength potato dextrose agar. The litter was divided into freshly fallen senescent leaves (grade 1) and leaves already undergoing active decomposition (grade 2). Moist chamber incubation of the litter revealed 45 fungal taxa belonging to 22 genera. fungal taxa were found on grade I and 39 fungal taxa found on grade II litter. Although 24 fungal taxa were common to both grades, Differences were observed in percentage occurrence of fugal species between the two grades of litter. Periodic surveys were carried out to collect macrofungi. Young and matured carpophores of 16 macro fungi species were collected in different seasons. Out of these macrofungi, 3 species belongs to family Entolomataceae and Agaricacea, two species belongs to Tricholomataceae and Geoglossaceae one species belongs to each family Dacrymycetaceae, Pluteaceae, Coprinaceae, Marasmiaceae Lycoperdaceae and Phallaceae. The bamboo leaf-litter was selected for the present syudy because of the dominance and great economic value of bamboo vegetation in North-east India. Key words: carpophores, decomposition, leaf litter, RFRI

INTRODUCTION Fungi are one of the most important organisms in the world, because of their vital role in ecosystem functions and human-related activities (Mueller and Bill 2004). Fungi play a significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, natural cycling and decomposing the dead organic matter present in soil and litter. (Molina et al. 1993; Keizer 1998; Pilz 2001; Cowan 2001; Chang and Miles 2004, Hunt 1999; Gates 2005). The peak mushrooms and macrofungi season for each region vary with ecological climate (Arora 1991). The number of existing fungi worldwide has been estimated to 1.5 million species (Hawksworth 2004). One-thirds of the fungal diversity of the globe exists in India and of this, only 50% are characterized yet (Manoharachary et al 2005). The number of fungi recorded in India exceeds 27,000 species, the largest biotic community after insects (Sarbhoy et al. 1996). Macrofungal biodiversity also play an important role in balancing ecological services. Fungi are one of the key functional components of forest ecosystems (Brown et al. 2006). They are omnipresent but drawing less attention than animal and plants. They are highly diverse in nature (Piepenbring 2007). Having a stable and estimate of taxonomic diversity for fungi is also necessary to enable fungi to be included in considerations of biodiversity

conservation, land-use planning and management (Mueller and Schmit 2007). Decomposition on the forest floor is a very complex phenomenon and is achieved by different groups of microorganisms. The major component of the top soil consists of different parts of plant materials. These are immediately colonized by diverse groups of microorganisms as they fall on the soil surface and soon after the processes of decomposition starts. Litter decomposition is also an important link in nutrient cycling of the forest (Grigal and McColl 1977). During the last few years various workers have developed interest to understand the nature of fungi both in forest and cultivated fields. The study on diversity of leaf litter fungi from various host plants were reported earlier (Bills and Polishook 1994; Saravanan 2004; Tokumasu et al. 1997). Some fungi were found to be common on leaf litter in previous studies, while many new fungal taxa have been described from decaying leaves and dead wood (Hughes 1989). A total of 26 genera, 31 species of Hyphomycetes, 8 species of Coelomycetes and 5 species of Ascomycetes were reported in Thiland. Two leaf litter fungi, Myrothecium verrucaria and Ciliochorella sp. were found to supress the growth of Alternaria alternata, Colletotrichum capsici, Curvularia lunata and Fusarium oxysporum under in vitro conditions (Manoch et al. 2006). In addition, morphological study of 42 genera 48 species leaf litter fungi was reported using light microscope (Manoch et al. 2006). Six new species of dematiaceous hyphomycetes from dead wood

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and bark in New Zealand were also illustrated and described (Hughes 1989). Alternaria, Aspergillus, Cladosporium, Penicillum and Trichoderma were reported as dominant fungi on decomposing bamboo litter. Deka and Mishra (1982) and Schmit et al. (1999) reported 30 species from bamboo litters. (Osono and Takeda 2002) observed the ability of 79 fungal isolates on litter decomposition of deciduous forest in cool temperate in Japan, and reported 6 species of Basidiomycetes causing 15.10 to 57.67% of weight losses, 14 species of Xylaria and Geniculosporium causing weight losses upto 14.4%. Some ectomycorrhizal fungi associated with Sal forest are Astraeus hygrometricus, Boletus fallax, Calvatia elata, Colletotrichum dematium, Corticium rolfsii, Mycena roseus, Periconia minutissima, Russula emetica, Scleroderma bovista, S. geaster, S. verrucosum and Scopulariopsis alba were documented by (Soni et al. 2011). Keeping the above facts in mind, the present study was focused on the isolation and identification of fungi associated with decomposition of litter of bamboo in different seasons and in different depths from Bambusetum of RFRI, Jorhat, Northeast India. Bamboo leaf-litter was selected for the present study because of the dominance of bamboo vegetation and its great economic value in North-east India.

present, it houses 39 species of bamboo (green gold) under 13 generic heads. Out of these special attention has been given on the exotic, endangered, rare and ornamentals that were collected from different regions of the Indian subcontinent (Figure 1).

MATERIALS AND METHODS

Moist chamber incubation technique Twenty five leaves of each grade of leaf litter were randomly selected and incubated in sterile moist chambers at 25±2°C. Petri plates (20 cm diam.) were sterilized (Keyworth 1951) and used as moist chambers with sterilized filter paper and periodically moistened with sterile distilled water. Leaves were incubated for 48 hours and then examined under a binocular stereomicroscope for the fungal fructifications. All fungi found sporulating were isolated, examined and identified to species level. Isolation frequency and percentage occurrence were used to explain the colonization efficiency of the microfungi on the leaf litter (Table 1, Figure 2). Isolation frequency denotes the number of samplings in which a particular fungus was recorded as against the total number of samplings (24).

Study area The study was conducted in 2009-2010 at Bambusetum of Rain Forest Research Institute (RFRI), which is situated in the Northeastern part of India having longitude of 95°17´ E and latitude 26°46´ N and at an altitude of 107 m above the sea level. The climate of the region is semi arid. It is warm and moist from May to September. December and January are usually the colder months. The area receives an average mean annual rainfall of 2029 mm, average temperature 26ºC in summer and minimum temperature is 10ºC in the month of January. The soil is lateritic sandy loam of pH 4.5-5.0. Bambusetum was established in the year 2002, occupying an area of about 1 hectare. At

Figure 1. Bambusetum of RFRI, Jorhat, Assam, India

Study on litter decomposing fungi The fungi were isolated from leaf litter on culture media, then purified and identified as per methods briefly described below. Direct observation Twenty four samplings were made during the period of study. Litter samples were collected at random from the study site and brought to the laboratory in sterile polythene bags. The litter was sorted into two grades representing the two stages of decomposition. These were ‘grade 1’ representing freshly fallen and senescent leaves and ‘grade 2’ representing leaves in an advanced stages of decomposition, usually thin, fragmentary and tightly compressed. Leaf litter samples were cut into 5x5 mm2 small pieces with a sterile parallel razor at random from the base, middle and apex. These pieces were cleaned, stained ,observed under stereo-microscope and fungal colonization were recorded (Shipton and Browns 1962).

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Figure 2. Micro fungal colony of Mucor, Aspergillus, Penicillum, Rhizopus, Cunninughumella and Trichoderma, from leaf litter culture of Bambusitum of RFRI, Jorhat, Assam, India.

Based on this, the fungi were categorized into 5 groups; most common(81-100%);common(61-80%); frequent(4160%); occasional (21-40%) and rare(1-20%). Percentage occurrence was used to denote the number of leaves on which a particular fungus was present as against the total number of leaves (25) examined per grade by moist chamber incubation. Leaf litter washing technique In addition to the moist chamber incubation, a second technique of washing fresh leaves removed from the plant and leaf litter was performed (Subramanian and Vittal 1979). Fifteen fresh leaves and fifteen litter leaves were randomly selected from each grade of litter. From each leaf, five 1 cm2 pieces were cut with a pair of sterile scissors. The samples were washed in 100 mL of sterile water in a 250 mL Erlenmeyer flask for 30 minutes on a shaker. From this initial suspension, serial dilutions were prepared. One mL of the required dilution (1/1000) was pipetted into each of six replicate plates. Potato dextrose agar (potato 200 g, dextrose 20 g, Agar 20 g, distilled water 1 L) with streptomycin sulfate (300μg/mL) was cooled to 45°C and poured into each Petri dish. The plates were incubated at room temperature in glass chambers under aseptic conditions for 4 days and examined for fungal growth. All fungal colonies were recorded and the fungi were sub-cultured and identified.

classified as most common (8-100%); common (61-80%); frequent (41-60%); occasional (21-40%); rare (1-20%). Collection of macro fungal and diversity analysis The periodic surveys were made for the collection of macrofungi during rainy season (June to September) and winter (October to December) in 2009-2010. The collected samples were wrapped in wax paper and brought to the laboratory for identification and proximate analysis. The taxonomy has been worked on the basis of macro and microscopic characteristic following available literatures (Zoberi 1973; Alexopolous et al. 1996; Purakasthya 1985). The soft textured specimens were preserved in 2% formaldehyde and leathery textured were preserved in 4% formaldehyde and kept in museum of Forest Protection Division, Rain Forest Research Institute, Jorhat, Assam by assigning identification number. The frequency and density of different species has been determined by the following formulas: No. of site in which the sp. is present Freq. of fungal sp. (%) =----------------------=-------------------x 100 Total no. of sites Total no. of individual of a particular species Density =-------------------------------------------------------x 100 Total no. of species RESULTS AND DISCUSSION

Identification of fungi Fungi were identified on the basis of their growth characteristics, morphological characteristics and ontogeny with the help of manuals, monographs and taxonomic papers of various authors (Gilman 1957; Grove 1967; Subramanian 1971; Ainsworth et al. 1972; Barnett and Hunter 1972; Ellis 1971, 1976; Sutton 1980; and von Arx 1981). Identification was based on morphological study examined under stereo, light, microscopes (Olympus BX 50 F4, Japan and Axio Scope A, Carl Zeiss). Frequency of occurrence and percentage contribution were calculated as per the procedures described by (Saksena 1955). Where frequency of occurrence refers to the number of samplings in which a fungus was recorded out of the total number of samplings made during the period of study. This was converted to a percentage and on this basis the fungi were

The rapid bamboo leaf litter decomposition can be attributed mainly due to the soft cuticle, low lignin content, high moisture content and suitable temperature. Many workers have reported that changes in the relative proportions of chemical constituents of litter may influence the rate of decomposition (Frankland 1966; Van Cleve 1974). In grade 1 litter, 29 species belonging to 22 genera were isolated. Thirty nine species belonging to 17 genera were isolated from grade 2 litter (Table 1). Significant variation in microbial quantity was recorded in different seasons of the year. Our study revealed that the highest micobial population in all the sampeling sites was recorded in the month of September and second highest number of fungal propagules.was recorded in the month of March and April. The lowest microbial population in all the sampling sites

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Table 1. Average percentage occurrence and isolation frequency of species isolated from two grades of bamboo litter Species Acropkialophora nainiana Edward Alternaria brassicae (Berk) Sacc. Arthrinium phaeospermum (Corda) M.B. Ellis Aspergillus flavus Johann Heinrich Friedrich Link Aspergillus fumigates Fresenius Aspergillus nidulans G Winter Aspergillus niger van Tieghem Aspergillus tamari Kita. Aspergillus terreus Thom Aspergillus wentii Wehmer Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker, Chaetomium bostrychoides Zopf and. C. crispatum Chaetomium globosum Kunze ex Fr. Cladosporium berbarum (Pers.) Link Cladosporium cladospoides Link Cladosporium cladosporioides Link Cladosporium oxysporum (Schlecht.) Snyder & Hansen Curvularia eragrostidis (Henn.) J.A. Mey. Fusarium concolor Reinking Fusarium equiseti (Corda) Sacc. Fusarium solani (Mart.) Sacc. Fusarium solenoid Sacc. Humicola grisea (Traaen) Mason Myrothecium verrucaria (Alb. & Schwein.) Ditmar Nigrospora sphaerica (Sacc.) E.W. Mason Penicillium funiculasum, Thom, Penicillium nigricans Thom Penicillium ulaiense Thom, Penicillium vermiculatum P. A. Dang. Periconia digitata (Cooke) Sacc., Pestalotiopsis theae (Sawada) Steyaert, Pestalotiopsis versicolor (Speg.) Steyaert Tetraploa aristata Scheuer. Trichoderma harzianum Rifai Trichoderma koningii Oudem. Trichoderma virens Miller, Gidden and Foster Trichoderma viride Pers Volutella concentric Penz. & Sacc. Choanephora cucurbitarum (Berk. & Ravenel) Thaxt., Cunninghamella echinulata (Thaxt.) Thaxt. ex Blakeslee Cunninnghumella elegans (Lendner) Lunn & Shipton Mucor circinelloides Tiegh. Mucor mucedo de Bary & Woron. Rhizopus nodosus (Namysl.) Hagem, Rhizopus stolonifer (Ehrenb. & Fr.) Vuill.

Phyllum Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Zygomycota Zygomycota Zygomycota Zygomycota Zygomycota Zygomycota Zygomycota

Average % occurrence Grade 1 Grade 2 1.65 0.45 3.32 2.07 0.66 3.17 4.08 3.62 3.57 4.69 4.39 4.14 4.44 5.24 3.32 4.55 3.18 2.30 1.71 2.21 1.65 0.66 2.72 1.93 0.80 1.24 1.71 2.76 1.92 5.66 1.71 7.04 2.97 6.07 2.77 1.71 4.14 1.24 1.93 4.69 1.96 2.31 2.21 2.41 1.79 2.90 1.10 3.17 1.10 0.66 4.23 4.44 3.88 7.59 3.57 1.24 1.20 2.07 1.31 1.96 4.55 2.21 4.69 3.72 3.88 4.55 3.32 3.52

Isolation frequency Grade 1 Grade 2 R R C R R O MC C C O MC O MC O C O C R O F R R F R R R O R R F R F F F O O R R O R F F F R O O O R R MC MC C F C R O R O O O F O C C O C C

Table 2 Frequency of occurrence and density of macrofungi associated with bamboo leaf litter Species name Agaricus augustus Fr. Cystoderma carcharias (Pers.) Fayod Termitomyces albuminosus (Berk.) R.Heim Coprinus plicatilis (Fr.) Fr. Dacryopinax spathularia (Schwein.) G.W.Martin Entoloma cetratum (Fr.) M.M. Moser Entoloma lividoalbum (Kühner & Romagn.) Kubicka Entoloma rhodopolium (Fr.) P. Kumm Geoglossum defforme (Fr.) Durand Geoglossum fallax Durand Morganella pyriformis (Schaeff.) Kreisel & D. Krüger Marasmius siccus (Schwein.) Fr. Dictyophora indusiata (Vent) Desv. Volvariella murinella (Quél.) M.M. Moser Clitocybe nuda (Fries) Bigelow & Smith Clitocybe phyllophila (Fr.) Kummer

Family Agaricacea Agaricacea Agaricaceae Coprinaceae Dacrymycetaceae Entolomataceae Entolomataceae Entolomataceae Geoglossaceae Geoglossaceae Lycoperdaceae Marasmiaceae Phallaceae Pluteaceae Tricholomataceae Tricholomataceae

Frequency of occurrence (%) 25.0 41.6 8.30 33.3 41.0 58.3 66.6 33.0 25.0 41.6 25.0 16.6 8.30 33.3 8.30 8.30

Density 18.75 37.5 6.25 12.25 31.25 56.25 62.50 25.0 18.75 56.25 12.25 37.5 6.25 25.0 6.25 12.25

ID number RFRI/000336 RFRI/000343 RFRI/000330 RFRI/000299 RFRI/000339 RFRI/000337 RFRI/000335 RFRI/000340 RFRI/000295 RFRI/000296 RFRI/000334 RFRI/000294 RFRI/000329 RFRI/000338 RFRI/000292 RFRI/000302

KUMAR et al. – Fungal diversity from Bambusetum of RFRI, India

was recorded either in May or June. It was observed that 70-85 % of the total population was shared by Ascomycota, 1-10% by Zygomycota and other by the macrofungi. The major groups of fungi in order of their dominance were the genera Aspergillus, Penicillium, Fusarium, Trichoderma and Cladosporium. In total, 7 species of Asperqillus and 4 species of each Penicillium, Fusarium, Trichoderma and Cladosporium were recorded. Among them A. terreus, A. tamari and A. wentii occasionally occurred in grade 1 litter isolation plates. Most common members of the group were A. niger, A. tamari and A. flavus in grade 2 litter isolation plates. Similarly, Trichoderma viride frequently present in litter1 and T. harzianum and T. koningii were the most common in litter 2. A. terreus, A. tamarii and A. niger. A. fumigatus were isolated in greater numbers during summer months, whereas, A. tamarii and A. nidulans in winter months. Although, A. niger and A. flavus were recorded regularly throughout the year but they were more prominent during June to October after the monsoon break. The second dominant group was the genus Penicillium which shared 10-15 % of the Deuteromycetes population. It was isolated in good numbers during winter months extending from November to March. Frequently isolated species were P. funiculasum, P. nigricans P. vermiculatum and Penicillium ulaiense. The genus Fusarium were quite frequent in rainy and winter months which comprised about 5 % of the population. Winter months were also favourable for Cladosporium but in summer it was recorded infrequently. Second dominant class was the Phycomycetes which shared 15-20 % of the total population. Rainy season was highly congenial for their occurrence. Frequently listed members were Choanephora cucurbitarum, Cunninghamella echinulata and Cunninghamella elegans. Mucor mucedo, M. circinelloides, Rhizopus nodosus and R. stolonifer are the common occurrence fungi and the rarely noted ones were Acropkialophora nainiana, Cladosporium cladosporioides, Tetraploa aristata, Curvularia eragrostidis, Bipolaris maydis and Arthrinium phaeospermum (Fugure 3 and 4). The fungal community composition was found to be distinct at each stage of succession (Promputtha et al. 2002). The method used for assessing the phylloplane mycota of green as well as litter leaves in the present study was also used by several earlier workers (Dickinson 1965, 1967; Hering 1965; Hogg and Hudson 1966; Tokumasu 1980; Shirouzu et al. 2009). The reason for using these techniques was to establish if any fungi that were missed by the direct observation would be found. Environmental variables exert great influence on their occurrence in different seasons. Therefore, some members were predominantly isolated in one season rather than other seasons. But certain fungi which consistently occurred throughout the year perhaps did not suffer much from such extremes as the soil environment is physically better buffered than subaerial environment to support them (Garrett 1955). The occurrence and distribution of microfungi studied in different seasons in bamboo leaf litter of RFRI were mostly governed by the temperature and moisture contents of soils. The abundance of fungi in different soils depends on the organic and nitrogen contents together with the other nutrient factors. The surface layer

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always exhibits maximum population, isolates and species numbers which gradually decline with depth increased. The periodic surveys were made for the collection of macrofungi, young and matured carpophores of 16 macro fungi species were also collected in different seasons. (Table 2.) The description of the collected specimens recorded as follows: Entoloma rhodopolium (Fr.) P. Kumm (Figure 1A, 2A). The cap is 5-12 cm; convex, sometimes with a slight central bump, becoming broadly convex, broadly bellshaped, or nearly flat; sticky when fresh; tan to yellowbrown or grayish brown, fading and drying out to grayish or almost whitish; the margin lined, at least by maturity. The gills are attached to the stem; close or nearly distant; white at first, becoming pink with maturity. The stem is 410 cm long; 6-12 mm thick; more or less equal; fairly dry; smooth, or very finely hairy at the apex; white; becoming hollow. The spore print is pink. The spores measure 6.5-11 x 7-9 µm, angular and inamyloid. Cystidia absent. Clamp connections present. It is inedible. Dacryopinax spathularia (Schwein.) G.W.Martin (Figure 3B,4B). The fruit bodies of Dacryopinax spathularia are spatula-shaped, usually 1-1.5 cm (0.4-0.6 in) tall and between 0.5-3 mm wide. The color is orange when fresh, but it darkens to orangish-red when dry. The spore print is white. Spores are ellipsoid, smooth-surfaced, translucent, and measure 7-10 by 3-4 μm. It has fourspored basidia that are 25-35 by 3-5 μm. It is edible. Cystoderma carcharias (Pers.) Fayod (Figure 3C, 4C). The cap is 2-5 cm across, sometimes white but usually shaded with pinkish or, more rarely, pale lilac, convex, flat, often umbonate, covered with minute granules, with appendiculate margin or cap edge. The gills are white, crowded, adnate. The stipe is 3-6 x 0.4-0.8 cm long, cap colored below ring and covered with small, pointed warts, white higher up, slightly enlarged at base and slightly narrower at top. Ring of the same color, smooth on interior, like the lower part of the stipe externally. The flesh is whitish or ochreous, strong fetid smell and unpleasant flavor. The spores measure 4-5x3-4 μm, white, elliptical, smooth, microns, and amyloid. It is edible. Volvariella murinella (Quél.) M.M. Moser (Figure 3D, 4D). The cap is 3.5 cm across oval becoming convex to broadly convex to nearly flat; whitish, sometimes very slightly darker over the center; the margin lined; slightly sticky when fresh but soon dry. The gills are free from the stem; whitish becoming pink to salmon; close or almost distant. The stem is 1-5 cm long; 1-3 mm thick; more or less equal; dry; white; smooth; without a ring; the base encased in a thick, white to grayish, sack-like volva which may be buried. The spore print is Salmon pink. The spores measure 5.5-8 x 4-6 µm, elliptical and smooth. Clamp connections absent. Entoloma cetratum (Fr.) M.M. Moser (Figure 3E, 4E). The cap is 2-5cm across, domed to bell-shaped with a nipple, transparently striate, yellowish-brown darker when wet. The stem is 4-8x2.5mm long, same colour as the cap. The gills are whitish at first then ochraceous-pink. The spores measure 11-12.5x6.5-7.5 µm. The spore print is pink. It is inedible.

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Figure 3. A. Entoloma rhodopolium, B. Dacryopinax spathularia, C. Cystoderma carcharias, D. Volvariella murinella, E. Entoloma cetratum, F. Agaricus augustus, G. Entoloma lividoalbum, H. Morganella pyriformis, I. Termitomyces albuminosus, J. Dictyophora indusiata, K. Clitocybe phyllophila, L. Geoglossum defforme, M. Geoglossum fallax, N. Coprinus plicatilis, O. Marasmius siccus, P. Clitocybe nuda

Agaricus augustus Fr. (Figure 3F, 4F). The cap shape is hemispherical in button stage, and then expands, becoming convex and finally flat, with a diameter of up to 22 cm. The cap cuticle is dry, and densely covered with concentrically arranged, brown-color scales on a white to yellow background. The gills are crowded and pallid at first, and turn pink then dark brown with maturity. The gills are free from the stem. The stem is clavate up to 20 cm tall, and 4 cm thick. In mature specimens, the partial veil is torn

and left behind as a pendulous ring adorning the stem. Above the ring, the stem is white to yellow and smooth. Below, it is covered with numerous small scales. Its flesh is thick, white and sometimes has a narrow central hollow. The stem base extends deeply into the substrate. The spores measure 7-10 by 4.5-6.5 Îźm, ellipsoid and smooth. The basidia are 4-spored. It is edible. Entoloma lividoalbum (KĂźhner & Romagn.) Kubicka (Figure 3G, 4G). The cap is 5-9 cm across; convex

KUMAR et al. – Fungal diversity from Bambusetum of RFRI, India

becoming broadly convex or broadly bell-shaped; dry to greasy; smooth; yellow-brown, fading with age. The gills are attached to the stem; nearly distant; at first white, becoming pink with maturity. The stem is 7-20 cm long; 12.5 cm thick; more or less equal; dry; smooth but finely lined longitudinally; white, often discoloring and bruising brownish near the base. The flesh is thin; fragile; white. The spore print is pink. The spores measure 7-12 x 5-12 µm; mostly 5-and 6-sided; angular; inamyloid. Cystidia is

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absent. Clamp connections present. It is inedible. Morganella pyriformis (Schaeff.) Kreisel & D. Krüger (Figure 3H, 4H). The fruiting body is pear shaped, 1.5-5 cm wide; 2.5-5 cm high; dry; often covered with tiny white spines when young and fresh, but the spines usually disappearing by maturity; typically with a pinched-off stem base; by maturity developing a central perforation through which spores are liberated by rain drops and wind currents; whitish to yellowish brown; with a white, fleshy interior at

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Figure 4. A. Entoloma rhodopolium, B. Dacryopinax spathularia, C. Cystoderma carcharias, D. Volvariella murinella, E. Entoloma cetratum, F. Agaricus augustus, G. Entoloma lividoalbum, H. Morganella pyriformis, I.Termitomyces albuminosus, J. Dictyophora indusiata, K. Clitocybe phyllophila, L. Geoglossum defforme, M. Geoglossum fallax, N. Coprinus plicatilis, O. Marasmius siccus, P. Clitocybe nuda

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Figure 5. A. Micro fungal spores and colony of A. Cunninghamella elegans, B. Alternaria brassicae, C. Fusarium equiseti, D. Penicillum funiculasum, E. Rhizopus stolonifer, F. Pestalotiopsis theae, G. & H. Trichoderma harzianum, I. Trichoderma virens

first; later with yellowish to olive granular flesh and eventually filled with brownish spore dust. The spores measure 3.5-4.5 µm; round; smooth; without a pedicel. Capillitial threads measure 3-6 µm wide. Termitomyces albuminosus (Berk.) R.Heim (Figure 3I, 4I). The cap is 5-11 cm, flat, acutely umbonate, pale brown to brown, glabrous, cracked, striate. The gills are free, crowded of several lengths, white to pale brown. The stem is central, 7-16 × 1.2-1.5 cm long, solid, white, glabrous, base enlarged with black brown rhizomorphs. The spores measure 6-10 × 4-5 µm, elliptical, hyaline, smooth, Cystidia broadly clavate, hyphae with clamps. It is edible. Dictyophora indusiata (Vent) Desv. (Figure 3J, 4J). Egg 5 cm in diameter, globose, ovoidal, white or grayish. Carpophore 15-20 x 2.5-3.5 cm, fusiform or cylindrical, barbed toward the top, white, porous, hollow, head ogival for a short time, then bell-shaped, yellowish under the gleba, white if stripped, with rugose surface, reticulate with apex perforated and delimited by a raised and distnict collar. Veil white, hanging almost to the ground, with wide polygonal chains formed by elliptical strands. Gleba olive-

green, mucilaginous, not very fetid. The spores measure 3.5-4.5 x1.5-2 µm colorless, elliptical, and smooth. It is reportedly eaten at the egg stage but not recommended. Clitocybe phyllophila (Fr.) Kummer (Figure 3K, 4K. The cap is 3-10cm broad, funnel-shaped with a wavy margin. The stem is 20-60 x 5-13mm, swollen at the base, whitish or light tan, hairy. The gills are decurrent, crowded, moderately broad; whitish to flesh-colored. The spores measure 3.5-4.5 x 3-3.5µm, white to cream, ovoid to ellipsoid and smooth. It is inedible. Geoglossum defforme (Fr.) Durand (Figure 3L, 4L). The fruit body is 4-12 cm high, club-shaped, compressed; black, smooth and sticky. The spores measure 5-7 x 90125µm, asci up to 245 x 270µm, mostly 15 septate, Light to dark brown, smooth, club-shaped to cylindrical, packed with eight spores. The spore print is black. It is inedible. Geoglossum fallax Durand (Figure 3M, 4M). It grows scattered or in small groups, occurring on soil in well drained areas. The sporocarp measures up to 3-7 cm high, club-shaped, upper part 0.1-0.3mm broad the length of the fruitbody, flattened and dark brown to black. The stem is

KUMAR et al. – Fungal diversity from Bambusetum of RFRI, India

0.06-0.3 cm wide, slender, dark brown to black, viscid, bald and minutely downy. The ascospores measure (40)6078(90) x 4.6-6.7 um, straight or somewhat curved, dark brown; asci mostly 8-spored. paraphyses colorless to brown. The spore print is brown. It is inedible. Coprinus plicatilis (Fr.) Fr. (Figure 3N, 4N). The cap is 10-30 mm, bell shaped, grooved from the margin, yellow to light brown, gray in the groves. The stem is 30-90 mm long and 2.5 mm thick, fragile, hollow and white. The gills are white at first, becoming gray, free from the stem. The spore print is black. The spores measure 9-15 x 7-11 µm, ellipsoid to almond shaped, large, and have an eccentric pore. It is Inedible. Marasmius siccus (schwein.) Fr. (Figure 3O, 4O).. The cap up to 0.4-3cm across, bell-shaped with deep wide radial pleats; rust-orange to rust-brown, minutely velvety. The stem is 2.4-6.5 cm long, 1 mm thick, equal, yellowish above, brown toward the base; smooth basal. The spore print is white. The spores measure 14-20 x 3-4.5 µm, spindle-to club-shaped, smooth, often curved. It is inedible. Clitocybe nuda (Fries) Bigelow & Smith (Figure 3P, 4P). The cap is 3-20 cm; convex to nearly flat, surface smooth, dull purple, flesh-colored, tan. The stem is 2.5-9 cm long, 1-2 cm In diameter, pale purple colored like the gills, base covered with buff mycelium. The gills are attached to the stem, crowded, lilac, pinkish-buff. The spore print is pinkish. The spores measure 4.5-7 x 4.5-5 µm; ellipsoid and smooth. It is edible.

CONCLUSION It is clear that in different grades of litter shifts in activity of the various species of the mycota occurred. As assessment of such activity is based on percentage occurrence of these fungi in different grades of litter, computed on the basis of sporulating colonies on the litter, and not on dilution plate counts, the data so obtained may be considered sufficiently reliable. It is obvious that the fungi colonizing the phylloplane or litter must be already present in that area. The phylloplane serves as a settling area for propagules of numerous fungi, several of which are components of the air spora. The host leaf allows the development of only a few species and inhibits others. Those fungi which are able to establish on living leaves are foliicolous. These can, in turn, be classified in to: (i) those whose activity is confined to living leaves and (ii) those that continue to be active after colonizing a living leaf even after it is shed. The true litter fungi are perhaps those that colonize the leaves after they are shed and show activity for varying periods.

ACKNOWLEDGEMENTS The authors are gratefully acknowledged to Indian Council of Forestry Research and Education (ICFRE) for funding the research project: No-RFRI-39/2010-11/FP.

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REFERENCES Ainsworth GC, Sparrow FK, Sussan AS. 1972. The Fungi, an Advanced Treatise. Vol. IV A. Academic Press, New York. Alexopoulos CJ, Mims CW, Blackwell. 1996. Introductory Mycology. John Wiley and Sons Inc., New York. Arora D. 1991. All that the Rain Promises and More. A Hip Pocket Guide to Western Mushroom. Ten Speed Press, Berkeley. Barnett HL, Hunter B. 1972. Illustrated Genera of Imperfect Fungi. 3rd ed. Burgess Pub. Co., Minneapolis. Bills GF, Polishook JD. 1994. Abundance and diversity of microfungi in leaf litter of a lowland rain forest in Costa Rica. Mycologia 86: 187198. Brown NS, Bhagwat S, Watkinson. 2006. Macrofungal diversity in fragmented and disturbed forests of the Western Ghats of India. J Appl Ecol 43 (1): 11-17. Change ST, Miles PG. 2004. Mushrooms-Cultivation, National Value, Medicinal Effect and Environmental Impact. 2nd ed. CRC Press, New York. Cowan A. 2001. Fungi-Life Support for Ecosystems. Essential ARB4, West Wickham, Kent, UK. Deka HK, Mishra RR. 1982. Decomposition of bamboo (Dendrocalamus hamiltoni Mess) leaf litter in relation to age of jhum fallows in northeast India. Pl Soil 68:151-159. Dickinson CH, Pugh GJF. 1974. Biology of Plant Litter Decomposition, Vol. I & II. (eds CH Dickinson, GJF Pugh). Academic Press, London. Dickinson CH. 1967. Fungal colonization of Pisum leaves. Canadian J Bot 45: 915-927. Ellis MB. 1971. Dematiaceous Hyphomycetes. CMI, Ferry Lane, Kew, Surrey, England. Ellis MB. 1976. More Dematiaceous Hyphomycetes. CMI Ferry Lane, Kew, Surrey, England. Frankland JC. 1966. Succession of fungi on decaying petioles of Pteridium aquilinum. J Ecol 54: 41-63. Garrett S D. 1955. Microbial ecology of the soil. Trans Br Mycol Soc 38: 1-9. Gates GM, Ratkowsky DA, Grove SJ. 2005. A Comparision of macrofungi in young Silvicultural regeneration and mature forest at the Warra LTER siet in the southern forests of Tasmania. Tasforests 16: 127. Gilman JC. 1957. A Manual of Soil Fungi. Rev 2nd ed. Oxford and IBH Publishing Co. Calcutta, India. Grigal DF, McColl JG. 1977. Litter decomposition following forest fire in northeastern Minnesota. J Appl Ecol 14: 531-538. Grove WB. 1967. British stem and leaf fungi (Coelomycetes). Vol. 2. Cambridge University Press, London. Hawksworth DL. 2004. Fungal diversity and its implifications for genetic resource collections. Stud Mycol 50: 19. Hering TF. 1965. Succession of fungi in the litter of a Lake District Oakwood. Trans Br Mycol Soc 48: 391-408. Hogg BM, Hudson HJ. 1966. Microfungi on leaves of Fagus sylvatica. I. The microfungal succession. Trans Br Mycol Soc 49: 185-192. Hudson HJ. 1968.The ecology of fungi on plant remains above the soil. New Phytol 67: 837-874. Hughes SJ. 1989. New Zealand fungi 33. Some new species and new record of dematiaceous hyphomycetes. New Zealand J Bot 27: 449459. Hunt GA. 1999. Assessing macrofungi of special concern for conservation in forested ecosystems. In: Proc Biology and Management of Species and Habitats at Risk. Vol 2, 15-19 Feb 1999, University College of the Cariboo, Kamloops. Keizer GJ. 1998. The Complete Encyclopedia of Mushrooms. Rebo Publishers, Netherland. Keyworth PK. 1951. A petridish moist chamber. Trans Br Mycol Soc 34: 291-292 Manoch L, Kokaew J, Jeamjitt O, Dethoup T. 2006. Leaf litter fungi and studies on antagonistic effect against plant pathogenic fungi in vitro. In: The Proceedings of 44th Kasetsart University Annua Conference, Subject: Plants. Kasetsart University, Bangkok. 30 January-2 February 2006. Manoharachary C, Sridhar K, Singh R, Adholeya, Suryanarayanan TS, Rawat S, Johri BN. 2005. Fungal biodiversity: Distribution, conservation and prospecting of fungi from India. Curr Sci 89: 58-71. Molina R, O’Dell T, Luoma D, Amaranthus M, Castellano and Russell K. 1993. Biology, Ecology and Social aspects of Wild Edible

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Shirouzu T, Hirose D, Fukasawa Y, Tokumasu S. 2009. Fungal succession associated with the decay of leaves of an evergreen oak, Quercus myrsinaefolia. Fungal Divers 34: 87-109. Soni KK, Pyasi A, Verma RK. 2011. Litter decomposing fungi in sal (Shorea robusta) forests of central India. Nusantara Biosci 3: 136144. Subramanian CV, Vittal BPR. 1979. Studies on litter fungi II. Fungal colonization of Atlantia monophylla Corr. leaves and litter. Nova Hedwigia 63: 361-369. Subramanian CV. 1971. Hyphomycetes. Indian Council of Agricultural Research, New Delhi. Sutton BC. 1980. The Coelomycetes, fungi imperfectii with pycnidia, ascervulii and stromata. CMI, Kew, England. Tokumasu S. 1980. Observations on the fungal flora in pine leaf litter. In: Kenkyu K (ed). Biseibutsu no Seitai (Ecology of Microorganism). Vol. 7. Gakkai Shuppan Center, Japan. Tokumasu S, Tubaki K, Manoch L. 1997. Microfungal communities on decaying pine needles in Thailand. In: Janardhanan KK, Natarajan KR, Hawksworth DL. (eds.). Tropical Mycology. Science Publishers Inc, Enfield, New Hampshire. Van Cleve K. 1974. Organic matter quality in relation to decomposition. In: Holding AJ, Heal OW, MacLean SF Jr, Flanagan PW (eds). Soil Organisms and Decomposition in Tundra. Tundra Biome Steering Committee, Stockholm, Sweden. Venkobachar C. 1995. Screening of tropical wood-rotting mushroom for copper biosorption. Appl Env Microbiol 61 (9): 3507. Von Arx JA. 1981. The genera of fungi sporulating in pure culture. 3rd ed.. J Cramer, Vaduz, Germany. Zoberi MH. 1973. Some edible mushrooms from Nigeria. Nigerian Field 38: 81-90.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 89-94

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140206

The influence of gap size on plant species diversity and composition in beech (Fagus orientalis) forests, Ramsar, Mazandaran Province, North of Iran HASSAN POURBABAEI1,♥, HAMIDREZA HADDADI-MOGHADDAM1, MARZIEH BEGYOM-FAGHIR2, TOOBA ABEDI3 1

Department of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, Iran. P.O. Box 1144, Tel.: +98-182-3220895, Fax.: +98-1823223600, ♥email: H_pourbabaei@guilan.ac.ir 2 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran 3 Environmental Research Institute of Academic Center for Education, Culture and Research (ACECR), Rasht, Iran

Manuscript received: 28 March. 2013. Revision accepted: 18 July 2013.

ABSTRACT Pourbabaei H, Haddadi-Moghaddam H, Begyom-Faghir M, Abedi T. 2013. The influence of gap size on plant species diversity and composition in beech (Fagus orientalis) forests, Ramsar, Mazandaran Province, North of Iran. Biodiversitas 14: 89-94.This study was conducted to investigate the influence of gap size on plant species diversity and composition in beech (Fagus orientalis Lipsky.) forests, Ramsar, Mazandaran province. Fifteen gaps in small, medium, and large sizes were randomly selected. Abundance of tree saplings, shrubs and herbaceous species were counted on 4 m2 micro-plots within the gaps. Diversity indices including Shannon-Wiener, Simpson, Mc Arthur's N1, Hill's N2, species richness and Smith-Wilson’s evenness index were computed. The results revealed that there was significant difference among three gap categories in terms of diversity. The highest diversity values of tree and herbaceous species were obtained in the large gaps, while the highest diversity value of shrub species was in the medium gaps. Species composition of small gaps (28 species: 7 trees and 21 herbaceous), medium gaps (37 species: 7 trees, 5 shrubs and 25 herbaceous) and large gaps (40 species: 7 trees, 4 shrubs and 29 herbaceous) were recognized. Therefore, based on the results of this study, it is recommended that in order to maintain plant diversity and composition up to 400 m2 gap size cloud be used in this forests. Key words: Fagus orientalis, gap size, plant diversity

INTRODUCTION The oriental beech (Fagus orientalis Lipsky.) is a deciduous tree species (Salehi et al. 2011), distributed from Macedonia, Bulgaria, northwest Turkey (Asia Minor), Azerbaijan, Caucasus to Iran (Rechinger 1963-2010; Komarov 1934-1963). Iranian beech forests are dominant in the Montane and submontane zones of central and western Caspian forests (Mobayen and Tregubov 1970; Asli and Nedialkov 1973). These forests occupy approximately 18% of the forested area (Bayramzadeh et al. 2012) and comprise the most productive and important commercial forests in the Caspian zone (Salehi et al. 2011). However, these forests are subjected to constant changes (Sampson and DeCoster 1998), a variety of natural and anthropogenic perturbations (Odum and Barrett 2004; Thompson 2010; Alongi 2007). Several researches were carried out on forest structural changes, silvicultural system and especially gap silviculture system (Tuomela et al. 1996; Albanesi et al. 2005; Boudreau and Lawes 2005; Renato and De Lima 2005). A planned program of silvicultural treatments (British Colombia 2003) ensures the conservation and maintenance of biological diversity and richness for sustainable forestry

(Torras and Saura 2008; Schumann et al. 2004; Battles and Fahey 2000; Simila et al. 2006). Whenever one or several number of trees fall in the forest, certain physical space is created this is called gap (Denslow 1987; Runkle 1991). Based on Gray and Spies (1996) gaps are two types: I) Natural gaps formed by falling single tree or a small group of trees, produced by windfall or broken trunk and II) Artificial gaps created by man as a result of single or group cutting of trees. Several investigators reported the effect of gaps on maintaining and enhancing biological diversity (Poulson and Platt 1989; Coates 2002; Gray and Spies 1996; Albanesi et al. 2005), their importance to the species dynamics of forests types (White and Pikett 1985; Platt and Strong 1989) and their impact on soil (Haghverdi et al. 2012). Several investigations about gap’s characteristics especially gap size (Sagheb-Talebi 1995; Mousavi et al. 2003), shape, dynamics (e.g. McCarthy 2001; Fujita et al. 2003; Zeibig et al. 2005; Kenderes et al. 2008) and its relation to plant diversity and species richness (Gray and Spies 1996; Goleij 2006; Scheller and Mladenof 2002; Heywood and Watson 1995) have been carried out in different forests of the temperate regions.

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Many outstanding studies have been done in the Caspian forests. Tabari et al.(2003, 2007), Tabari (2008) Amanzadeh et al. (2009), Esmailzadeh et al. (2011), Fallahchai et al. (2011), Parhizkar (2011a, b) and Sefidi et al. (2011). However, there is no information about effect of selective cutting method gap size on plant species diversity in Ramsar’s beech forests. So, the main objectives of the present study were to investigate plant species diversity and compositions among different gap categories created by this method of forest management and identify the best gap size which can help achieving sustainable diversity in these forests.

large) (Table1). Then, 5 gaps were randomly selected from each category (totally, 15 gaps) (Berg and Van Lear 2004). 2 m×2 m sampling plots were systematically taken along two diameters of each gap in with 1m interval (Figure 2). Gaps areas were calculated using ellipse method based on the following equation (Runkle 1991; Renato and De Lima 2005). S = R1 R2π /4 S = ellipse area, R1 = Large diameter, R2 = Small diameter

MATERIALS AND METHODS The study area is components No. 13 and 14 of district No. 5 locate in watershed No. 30 of Ramsar’s Safaroud forest management plan, Mazandaran, Iran (Figure 1). This area has approximately 140 hectare. Altitude range from 1000 to 1200 m asl. and the slop is 25% to 50%. General aspect is northwest. This area locate at 50° 35΄12˝ E and 36° 55΄ 8˝N. Safaroud forest has moderate to cold temperate climate according to the Emberger formula. The mean annual temperature is 15.8 °C (the hottest month is June (24°C) and the coldest is January (7°C)). Mean annual rainfall is 1366 mm. The parent material of the region is limestone, with moderate to good permeability. Soil type is washed brown with Argillic horizon containing loam-clay and coarse and polygonal structure. This region has moderate to deep soil depth (60-70 cm) and mull humus (OFRW 2007). The components were identified by forest surveying. Gaps derived from logging which are located at north west aspect, with approximately similar slope were identified and divided into three size categories (small, medium and

Figure 2. Position of sampling plots in the gaps

The number of individuals of tree saplings and shrubs were counted and coverage percent of herbaceous species were estimated using Domin’s criteria in each sampling plot (Mueller-Dombois and Ellenberg 1974). Then, number of species within each gaps were measured and the Simpson (1-D), Shannon-Wiener (H'), Mc Arthur's N1 and Hill's N2 indices, species richness and Smith- Wilson’s evenness index (Evar) were calculated in different vegetation layers using ecological methodology software (Krebs 1999). The Kolmogrov-Smirnov test was used to study the normality of diversity; richness and evenness data in different gaps, then ANOVA and Tukey’s tests were performed using SPSS software.

Iran, Islamic Republic of

Study site in Ramsar’s Safaroud forest Scale 1: 250.000

Ramsar of district, Mazandaran Province

Figure 1. Location of study area in the Ramsar’s Safaroud forest, Mazandaran Province, Iran

POURBABAEI et al. –Effect of gap size in Fagus orientalis

RESULTS AND DISCUSSION

Table 2. List of plant species in the gaps

Totally 8 trees, 5 shrubs and 30 herbaceous species were identified in the studied area. The species composition of three different gaps categories were as follows: Small gaps include 28 plant species (including 7 trees and 21 herbaceous species), medium gaps include 37 species (including 7 trees, 5 shrubs, and 25 herbaceous species) and large gaps include 40 species (including 7 trees, 4 shrubs, and 29 herbaceous species) (Table 2). Tree saplings had maximum diversity in large and minimum diversity in small gaps, respectively. Tukey’s test revealed that there were no significant differences among diversity values of sampling layer in different gaps (P>0.05) (Table 3). The diversity values of shrub species were significant differences among different gaps (P<0.05) and had maximum amount in the medium gaps (this values were not calculated in small gaps because no shrubs were observed) (Table 4). Herbaceous layer indicated maximum diversity value in large gaps and minimum diversity value in small gaps. Tukey’s test showed that there were significant differences in herbaceous species diversity values among the gaps (Table 5). The highest species richness was observed in herbaceous layer and the lowest was found in shrub layer. The large gap indicated the highest mean species richness (Table 6). Maximum evenness value was obtained in tree saplings and shrub layers, and minimum was found in herbaceous layer in the medium gaps. There were significant differences between large and medium gaps in shrub layer and also between three gaps categories in herbaceous species layer (Table 7). The Jaccard’s index indicated that there were maximum similarity between woody species in large and medium gaps, and minimum similarity was between medium and small gaps. In the herbaceous layer, maximum similarity was obtained between medium and large gaps and, minimum similarity was between small and large gaps (Table 8).

Scientific name

Gaps Small Medium Large

Number of identified gaps 13 11 6

Gaps Small Medium Large

Family

Tree layer Acer cappadocicum Gled. Acer insigne Boiss. Alnus subcordata C. A. Mey. Carpinus betulus L. Fagus orientalis Lipsky. Fraxinusexcelsior L. Tilia begonifolia Stev. Ulmus glabra Huds.

Aceraceae Aceraceae Betulaceae Betulaceae Fagaceae Oleaceae Tiliaceae Ulmaceae

+ + + + + + +

+ + + + + + +

+ + + + + + +

Shrub layer Crataegus microphylla (Wild) Jac. Ilex spinigera Loes. Mespilus germanica L. Prunus divaricata Ledeb. Ruscus hyrcanus Juz.

Rosaceae Aquifoliaceae Rosaceae Rosaceae Asparaginaceae

-

+ + + + +

+ + + +

+ + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Herbaceous layer Acalypha australis L. Euphorbiaceae Atropa belladonna L. Solanaceae Carex oreophila L. Cyperaceae Ceterach officinarum DC. Aspleniaceae Epipactis atrorubens Hoffm. Orchidaceae Equisetum sp. Equisetaceae Euphorbia heliscopiaL. Euphorbiaceae Galium rotundifolium L. Rubiaceae Geranium robertianum L. Geraniaceae Hypericum fursei N. Robson. Hypericaceae Melissa officinalis L. Lamiaceae Mentha pulegium L. Lamiaceae Mercurialis annua L. Guphabaceae Nepeta involucrata (Bunge)Bornm. Lamiaceae Oplismenus undulatifolius (Ard.) P. Beauv. Poaceae Periploca graeca L. Asclepiadaceae Phlomis ghilanensis C. Koch. Lamiaceae Phyllitis scolopendrium L.(Newm.) Aspleniaceae Potentilla reptans L. Rosaceae Primula heterochroma Stapf. Primulaceae Pteridium aquilinum (L.) Kuhn. Hypolepidaceae Rubus hyrcanus Juz. Rosaceae Sambucus ebulus L. Caprifoliaceae Sanicula europaea L. Apiaceae Scopolia carniolaca L. Solanaceae Scutellaria velenovskyi L. Lamiaceae Urtica dioicia L. Urticaceae Veronica sp. Scrophulariaceae Viola alba Bess. Violaceae Xanthium strumarium L. Asteraceae Note: +: presence, - : absence

Table 1. Characteristics of gaps derived from selection logging Area (m2) 100-200 200-300 300-400

91

Number of selected gaps 5 5 5

Table 3. Diversity measures and their standard errors of tree species saplings in the gaps Diversity indices 1-D N2 H' N1

Small 0.71±0.002 3.52±0.029 1.94±0.011 3.88±0.031

Gap size Medium 0.70±0.003 3.48±0.026 1.97±0.012 3.91±0.028

Large 0.72±0.001 3.58±0.012 2.05±0.002 4.13±0.007

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Table 4. Diversity measures and their standard errors of shrubs species in the gaps Diversity indices 1-D N2 H' N1

Gap size Medium 0.43±0.001* 1.81±0.003* 0.97±0.001* 1.89±0.002*

Small -

Large 0.18±0.001* 0.72±0.003* 0.17±0.001* 0.76±0.002*

Table 5. Diversity measures and their standard errors of herbaceous species layer in the gaps Diversity indices 1-D N2 H' N1

Small 0.89±0.001 9.68±0.072 3.64±0.010 10.89±0.068

Gap size Medium 0.87±0.001 7.80±0.046 3.33±0.005 10.06±0.038

Large 0.91±0.001* 11.72±1.022* 3.76±0.017* 13.83±1.042*

Table 6. Richness values of different vegetation layers in the gaps Vegetation layers Tree Shrub Herbaceous Mean

Small 7 0 21 9.33

Gap size Medium 7 5 25 12.33

Large 7 4 27 12.67

Mean 7 3 24.33

Table 7. Evenness measures of different vegetation layers in the gaps Vegetation layers Tree Shrub Herbaceous

Small 0.60±0.004 0.73±0.003*

Gap size Medium 0.59±0.005 0.73±0.001* 0.37±0.001*

Large 0.54±0.006 0.55±0.003* 0.61±0.002*

Table 8. Jaccard’s similarity index of woody and herbaceous species among the gaps Gap sizes Small-Medium Medium-Large Small-Large

Vegetation Layers Woody Herbaceous 0.46 0.76 0.76 0.79 0.63 0.65

Based on our results, abundance of tree saplings varied in different gaps. The most variation was observed between small gaps and other categories, While the difference between medium and large gaps were not significant. Maximum abundance of tree saplings was found in the medium gaps. The total species abundance (especially Fagus orientalis) severely declined with increasing in gap size. Large gaps are exposed to direct sunlight which caused the establishment of invasive herbaceous and shrub species (as competing elements) and increasing soil dryness. Therefore, it will prevent the establishment of

beech regeneration (Takeh et al. 2004; Peltier et al. 1997; Mousavi 2001). Several researches have reported that some herbaceous species (e.g. Rubus sp., Petris sp.) influenced the survival of beech saplings by providing canopy (Taheri 2000; Espahbodi and Tabari 2004). But some others claimed that beech saplings were not able to compete with Rubus sp. or other herbaceous species (Savill 1991; Harmer 1995). Ersali (1999) reported that presence of competing herbaceous species increases water consumption, and on the other hand reduces the establishment of tree seedlings. However, based on Helliwell (1982), the beech saplings were more successful than the light-demanding plants (e.g. oak and maple species) in competing with herbaceous species. Diversity The important impact of cutting in species diversity has been reported in several researches (e.g. Heywood and Watson 1995; Nagaike et al. 1999; Okland et al. 2003). Our results revealed that the high diversity of tree saplings was in large gaps which it is consistent with previous researches of Yamamoto (1989), and Hall et al. (2003). However, the diversity differences among three categories of gaps were not significant. Shrub species diversity was significantly high in medium gaps and declined in small (with the light shortage) and large gaps (with increasing in light and herbaceous competition). This result is in agreement with previous studies of De Granper and Bergeron (1997), Pourbabaei and Ranjavar (2008). The herbaceous species diversity was increased in large gaps (300-400 m2). Vast cutting area and more light penetration favored the growth of light-demanding species, increased species richness and cover percentage. This result supports the previous surveys by Scheller and Mladenoff (2002), Schumann et al. (2004), Nelson and Halpern (2005), Pourbabaei and Ranjavar (2008). Our findings indicated that the richness of tree species (included 7 species) were similar in three gaps categories, While shrub species richness varied among different gaps size, and it was minimum in small gaps due to lack of light. The herbaceous species richness was different among different gaps. Species composition The current result showed that species composition varied in three categories of gaps and supported the previous result of Boudreau and Lawes (2005). However, the medium and large gaps composed of more similar woody and herbaceous species. Many factors including environmental and physical evidences such as soil moisture, texture and fertility (Hutchinson et al. 2007), light variations (Rozenbergar et al. 2007) and selection methods especially single selection (Malcolm and Ray 2000) influence the composition and abundance of plant species. Species composition changes with increasing in gap size (Coates 2002) and it is often from pioneers in the early successional stage, towards climax species in later

POURBABAEI et al. –Effect of gap size in Fagus orientalis

successional phases (Mc Evoy 2004). In open canopy, the heliophyte pioneers species will grow very fast and this will cause establishment of shade tolerant species in understory. But very large open canopy the heliophyte pioneers species grow rapidly and change the species composition (Moore and Vankat 1986; Deal 1997; Leniere and Houle 2006). The significant role of gap size in providing rich species composition, diversity and creating desired successional communities were reported earlier (Whitmore 1989; Mc Carthy 2001; Goleij 2006; Liu et al. 2011).

CONCLUSION The gap size has significant effects on plant species diversity which it has major role in forest stability and sustainable production. Based on the results of present study, gap sizes up to 400 m2 improve the species diversity and this is recommended for forest harvesting. So, different gap size should be prepared in broad-leaved forest management strategies, which create multiple storey, heterogeneity, species diversity, mixed composition and regeneration to provide ecological stability of these forests.

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B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 95-100

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140207

Population dynamics of cuscus in tourist island of Ahe, District of Nabire, Papua ANTON SILAS SINERY1,♼, CHANDRADEWANA BOER2, WARTIKA ROSA FARIDA3 1

Faculty of Forestry, State University of Papua, Jl. Gunung Salju, Amban-Manokwari 98314, West Papua, Indonesia. Tel & Fax.: +62-986-211364, ♼ email: anton_sineri@yahoo.com 2 Faculty of Forestry, Mulawarman University, Samarinda 75119, East Kalimantan, Indonesia 3 Zoology Division, Reserach Center for Biology, Indonesian Institute of Sciences, Cibinong, Bogor 16911, West Java, Indonesia Manuscript received: 25 April 2013. Revision accepted: 14 July 2013.

ABSTRACT Sinery AS, Boer C, Farida WR.2013. Population dynamics of cuscus in tourist island of Ahe, District of Nabire, Papua. Biodiversitas 14: 95-100. Cuscus is a pouched herbivorous mammal of the family Phalangeridae which is arboreal and nocturnal.. The animals are protected by law because, in addition to having a low reproduction and limited distribution area, they face a very high level of hunting. Hunting in the wild by people is done not only in production forest areas but also in forest conservation areas such as recreational forest of Table Mountain, Arfak Mountains Nature Reserve, and other places. Directly or indirectly, the hunting affects the quality of the ecosystem in these areas, especially the cuscus population. Better management efforts are required in these areas to ensure the survival of many organisms in it, especially the cuscus. This study aimed to determine the cuscus population in Ahe Island, and the method applied was descriptive method using direct observation. The study was conducted in one month. The results demonstrate that cuscus in Ahe Island consisted of common spotted cuscus (Spilocuscus maculatus) and eastern cuscus (Phalanger orientalis). The number of individuals of S. maculatus was 24, consisting of 14 females and 10 males, whereas P. orientalis consisted of 2 individuals and both were males. The number of adult cuscus individuals was 16, while adolescents and children, were respectively 8 and 2. At least 10 plant species were identified as a source of feed for cuscus in Ahe Island recreation area. Plant parts consumed by cuscus were fruit and young leaves, but based on level of need, most of the cuscus consumed fresh fruit because of its sweet taste and high water content that helps the digestive process. Key words: Ahe Island, cuscus populations, feed resources, Papua, plant species

INTRODUCTION Cuscus, a pouched mammal (marsupials), is a herbivore which is arboreal and nocturnal. Menzies (1991), Flannery (1994), and Petocz (1994) mention that the distribution areas of cuscus include the islands of Indonesia (Papua, Sulawesi, Maluku and Timor Islands), Papua New Guinea (PNG), New Britain, Solomon Islands, Cape York, and Queensland Australia. In New Guinea (PNG and Papua) there are 11 species of the genus Spilocuscus (spotted cuscus) and genus Phalanger (unspotted cuscus). In Papua, there are 7 species of cuscus, namely common spotted cuscus (Spilocuscus maculates), spotted black cuscus (S. rufoniger), Waigeo cuscus (S. papuensis), cuscus Timor (Phalanger orientalis), ground cuscus (P. gymnotis), hair silk cuscus (P. vestitus) and hill forest cuscus (P. permixtio) (Menzies 1991; Petocz 1994; Aplin and Helgen 2008; Saragih et al. 2010). All seven species of cuscus in Papua are protected by the decree of the Minister of Agriculture No. 247/KPTS/UM/4/1979 and Government Regulation No. 7 Year 1999 on the Preservation of Plants and Animals. Globally, cuscuses are listed in the Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Although there have been government rules issued to protect the cuscus, its implementation is still considered less effective and needs

to be improved (Sinery 2002; Sinery 2010). Cuscus utilization for consumption and for other purposes in Papua nowadays is increasing. In addition, the cuscus fur is also used to make various ornaments like bags, hats, and for decoration in the customary fashion. Such utilization can affect the cuscus population (Ariantiningsih 2000). The consumption of cuscus meat by local people shows an increasing trend. It can be seen from the number of hunting results which reaches 2-5 heads every hunting activity done at least once every month. Although it is generally done in areas with a high density of cuscus populations, hunting is still a serious threat to the existence of these animals. The condition is influenced by various factors, including lack of public awareness about the legal status of cuscus as protected animals according to both the national laws and local customs. This has implications for the pattern of utilization, which in turn affects the existence of wildlife such as cuscus. People in the islands of Numfor, Biak, Arui, Moor, Auki, Yoop, Napan, and Yapen tend to use cuscus for consumption, and so do the residents of mainland Papua in areas such as Arfak Mountains, Meja Mountain, Jayapura, Sarmi, Sorong, and a few other areas. Ahe Island with an area of approximately 2.5 ha is one of the smallest islands in the island-chain of Mambor around Cenderawasih National Park. As one of the isolated

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areas which are not inhabited, Ahe Island has good lowland forest formation dominated by Ficus sp., Intsia sp., Syzygium sp., Linociera macrophylla, Morinda citrifolia, and Glochidion sp., and the coastal vegetation dominated by Terminalia sp. and Barringtonia sp. Forests in the region spreads from the middle of the island to the shore surrounded by white sand beaches. The species of wildlife found on the island are green lizard (Mabouya multifasciata), Lizard (Varanus sp.), mambruk bird (Goura sp.), maleo bird (Megapodius freycinet), and cuscus (Spilocuscus and Phalanger). Other potential tourist attraction is the remains of Allied Forces aircraft relics in World War II largely been transferred to the mainland. The expanse of water adorned with coral reefs and a wealth of other biotas add to the beauty of this island. At this time, Ahe Island is managed by society with coordination of the Agent of Tourism of Nabire District and the Government of Papua Province. Legally, the management of the tourist area of Ahe Island is done by CV Ahe (a private business) based on the decree of the Governor of Papua Province in 2007, and the operation began in 2009. Since its establishment, the management has successfully developed a variety of this island’s potential with the main goal of improving the potential of tourism, education, and research through the provision of various facilities, such as accommodation, lighting, and facilities of recreation. To add value to the potential of this island, the management has introduced four species of wildlife: maleo bird, mambruk bird, lizards, and cuscus. In 2007, a total of 7 species cuscus were introduced in Ahe Island, consisting of the species that are distinguished based on plumage characters, namely eastern cuscus (Phalanger orientalis) and common spotted cuscus (Spilocuscus maculatus). The current population is estimated to have increased, which can be seen from the number of juveniles. This condition is a positive thing in terms of the protection and conservation of cuscus. However it is necessary to consider the possibility that an increase in population will affect the carrying capacity of the island's cuscus habitat. Taking into account the very small size of the forest, it is necessary to carry out wellplanned management to control cuscus populations and develop their habitats in this island. For this purpose it is necessary to study the cuscus population and its habitat conditions in Ahe Island. This study aimed to determine the condition of cuscus population and habitat’s carrying capacity based on the availability of cuscus feed. The results are expected to be sources of information and consideration for all parties in the wildlife management efforts, both in situ and ex situ, particularly for C.V. Ahe (a private business) in managing cuscus in Ahe Island in the future.

The method used in this research was descriptive method based on observations. Taking into account the location of the study area of 2.5 ha and the solitary nature of the cuscus, data collection was done using census method by monitoring cuscus populations. To facilitate the process of data collection, the study site was divided by several transects or observation lines. Results of preliminary observations indicated that the distribution of cuscus in the research area was uniform so the Ahe Island’s beach was used as a baseline. The transects were made parallel to the shoreline or cutting the contour lines.. Furthermore, the baseline was divided into 5 transects perpendicular to north-south baseline. All transects were set proportionally, and the distance between transects was 50m. The length of the transects were 100m, 335m, 320m, 150m, and 120m, so that the total length of all transects was 1.025m while transect width was adjusted with minimal visibility (40m or 20m either side of the transect). According to Sinery (2010), the effective width of observation transect for dense forest types such as forest types in Arfak Mountains is 50m (25m either side of the transect) and we should use a narrower measure which is more effective in the observation of the population (Sinery 2009). Monitoring of cuscus was performed simultaneously by 5 groups of 2 people (1 identifying and recording and 1 measuring the distance from objects to transect). Cuscus population monitoring was not accompanied by the capture (sampling), but if possible, limited capturing was done. Identification was done for each species using Flannery (1994, 1995). Data collected consisted of (i) primary data, i.e. data from field observations, and (ii) secondary data, i.e. data obtained from the relevant agencies. Primary data consisted of: species, cuscus descriptions, cuscus populations, type of feed and the general condition of cuscus habitat. Secondary data included data on climate and the general state of research locations obtained from the relevant authorities. The data of cuscus morphological were analyzed using the tabulation and were used to identify the species of cuscus. The estimation of cuscus population density as the result of observation was carried out using the equation from Lewis (1994) as follows. n (2n - 1) A N = -----------------2L Σ r N = population density, n = number of individuals encountered, A = area of region (plot observations), L = length of line/transect, Σr = distance from the point where cuscus found to the line of transect

MATERIALS AND METHODS The research was conducted on the island of Ahe, Mambor, Nabire District, Papua Province, Indonesia (Figure 1) and lasted for 1 month, i.e. in November 2012.

Furthermore, the result analysis of population density was tabulated according to the structure and species composition. Structure and species composition included stratification by type of cuscus species, sex, and age.

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DISTRICT OF NABIRE

Figure 1. Study site in Ahe island, Mambor islands, Nabire District, Papua Province

RESULTS AND DISCUSSIONS Composition of cuscus species Monitoring results indicated that number of cuscus in the tourist region of Ahe Island was large enough, ie 26 individuals consisting of two species of cuscus, namely: Spilocuscus maculatus (common spotted cuscus) and Phalanger orientalis (eastern cuscus). Detailed description of the number of individuals, sex, and age class of cuscus by species is shown in the Table 1. Table 1 above shows that of the 26 individuals cuscus encountered, 24 individuals were Spilocuscus maculatus (common spotted cuscus) with a population density of 9.6/ha while 2 others were Phalanger orientalis (eastern cuscus) with a population density of 0.8/ha. In quality, evenness individual cuscus by species in this area was low because the percentage of Spilocuscus maculatus was much higher (92.3%) than that that of Phalanger orientalis (7.7%). Differences in the number of individuals of both species were affected by the low number of individuals introduced, especially Phalanger orientalis, in the early management of this area. The results showed the highest average density was found in transect 2 with an average density of 3.56 individuals per square meter, followed by transect 3 with

an average density of 3.24 individuals per square meter, and transects 4 with an average density of 2.4 individuals per square meter.. There were 10 species of vegetation as sources of feed for cuscus in Ahe Island, namely Ficus benjamina, Ficus microstoma, Ficus prolixa (paka), Ficus pisocarpa, Ficus infectoria, Merremia peltata, Pongamia pinnata, Intsia bijuga, Syzygium sp, and Cocos nucifera. In general, the active time of cuscus in Ahe Island, which is the period cuscus starting out of the nest/hideout to return to rest or hide, was from 18:00 to 05:00 EIT (Eastern Indonesian Time). Cuscus was usually found in the conditions after raining and under the moonlight with an average air temperature of 23 Âş C and the average humidity of 82%, and in a region with an elevation of 2-12 m asl. In Ahe Island Spilocuscus maculatus had higher gender equity than Phalanger orientalis. This species of cuscus at least had 7-10 pairs with the number of reproductive couples of approximately 7 pairs. It is quite good in terms of the survival of species and individuals, as the more reproductive couples there are the more likely mating occur, which in turn will produce offspring. However, this should not necessarily be a major factor in the forecast of cuscus species existence, due to the polygamy nature of cuscus that can change partners.

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Table 1. Individuals density of cuscus by type

Species of cuscus Spilocuscus maculatus Phalanger orientalis Total

Number of individual (ni) 24 2 26

Sex Male

Female

10 2 12

14 14

Adult (>8) 14 2 16

Class age (months) Adolescent Child (3–8) (< 3) 8 2 8 2

Population density (individuals/ha) 9.6 0.8 10.4

Table 2. Population dynamics of cuscus in Ahe Island Species of cuscus Spilocuscus maculatus Phalanger orientalis Total

First introduction (2007) 5 2 26

Last introduction (2008) 9 9

Monitoring result (2012) 22 2 24

Table 3. List of plants as feed sources for cuscus in Ahe Island Scientific name

Local name

Parts consumed

Ficus benjamina L. Ficus microstoma Wall. Ficus pisocarpa Bl. Ficus prolixa G. Forst. Ficus inferctoria Roxb. Merremia peltata (L) Merr. Pongamia pinata (L) Pierre Intsia bijuga (Colebr.) Kuntze Syzygium sp. Cocos nucifera L.

Beringin daun lebar Beringin pantai Beringin daun halus Makuku buah halus Makuku buah halus di daun Tali Wuraram Kayu besi pantai Kayu besi hutan Jambu pantai merah Kelapa

Fruit Shoots fruit Fruit Fruit Fruit Shoots Shoots Shoots Fruit Fruit (young)

Data showed that Spilocuscus maculatus had equitable distribution of age classes, and dominance by adult age class was followed by adolescents age class and children age class, while Phalanger orientalis consisted of two individuals both at adult age class. Based on this condition it can be expected that ecologically Spilocuscus maculatus has a better survival chance in the future than Phalanger orientalis. It is based on the existence of male and female adults who will play a role in the regeneration of the species, and age class adolescents as candidates for adults age class, and then age class of children who will be the next adolescents age class. Population dynamics To find out the adaptation process of cuscus to the conditions of Ahe Island as its new habitat, the population dynamics of cuscus was carried out from the tabulation. Table 2 indicates that the dynamics of cuscus population is not too big in the tourist area of Ahe Island. Such changes are progressive or increasing, particularly in Spilocuscus maculatus. An increasing number of individuals of this type can be seen from the existence of new individuals in children class age which showed the birth rate (birthrate). In contrast, no increase occurs in Phalanger orientalis individuals because it does not have the type of female individual as discussed previously.

Quality of density Many Moderate Little Little Little Little Little Little Little Little

Naturally, cuscus has a fairly low rate of reproduction, namely one child in each reproductive period with an average frequency of reproduction of once a year. According to Sinery (2002, 2010), the average number of offspring generated in each time of reproductive period is one. Petocz (1994) mentions that cuscus has a low rate of reproduction, so it is estimated that its population in the wild is quite low. When connected to the existing number of reproductive couples of cuscus (7 pairs), then cuscus in the region, particularly Spilocuscus maculatus, is quite productive, ie 7-8 children in the 3-year period (20072012). This suggests that this species of cuscus can adapt to the habitat conditions in Ahe Island although it has not yet reached the level of normal reproduction rate. The conditions are certainly influenced by many factors, both internal factors and external factors. Internal factors are factors derived from these animals which include hormones and genes. Both factors can not be predicted quantitatively and affect cuscus in relation to its reproduction, but in general each cuscus has the ability to reproduce more than once in a year with the number of offspring can reach four heads. The number of offspring is greatly affected by reproductive condition of the parent, the availability of food, and other conditions. An adult female cuscus generally produces more than one offspring and can even reach four offspring with a pregnancy period of 20 to

SINERY et al. – Population dynamics of cuscus in Ahe Island, Nabire

42 days. Not all offspring can be raised by the parent. Usually a female can raise only one offspring until it is able to feed itself. External factors or contributing factors are the physical and biotic factors which directly influence the reproduction of cuscus such as vegetation (food, shelter, and activity), the availability of space (home range and territory area) and human activity. According to Alikodra (1990), habitat is an area consisting of both physical and biological components that are used as a place to live as well as breeding ground for wildlife. In general, the conditions of Ahe Island such as landscape, weather conditions, and vegetation are not varied, so it is expected to affect the cuscus breeding in the island. The measurement results showed that the elevation of the island ranges from 1 to 12 m asl. with an average air temperature of 27 ºC and an average relative humidity of 82%. The weather factors do not significantly affect the cuscus while the topography was considered giving quite an effect on the distribution of vegetation that directly affect the variation of cuscus’ feed types. These types of feed that are generally the vegetations of coastal forests and lowland forests are listed in Table 3. This table shows that cuscus lives on the type of leafy forest vegetation such as Pometia sp., Myristica sp., Ficus sp., Intsia sp., and liana species commonly encountered in primary forest and secondary forest. Habitat components consist of the physical and biotic components, forming a system that controls wildlife. Physical factors include water, climate, soil, and topography, whereas biological factors include vegetation and other wildlife. Feed, water, shelter, human activities, nature events and other wildlife greatly affect the existence of wildlife (Alikodra, 1989). Cuscus is a nocturnal mammal that is active (foraging, mating and playing) at night. In general, the active time cuscus in Ahe Island, starting from the cuscus out of the nest to return to the nest to rest or hide, is from 18:00 to 05:00 EIT (Eastern Indonesian Time). Cuscus is usually found in the conditions after raining and when the moon shines brightly with average air temperature of 23º C and average humidity of 82%. On conditions after the rain, cuscus does its foraging by utilizing part of the new vegetation growth/shoots and other activities. In addition, when moon shines brightly, cuscus uses moonlight to look for sources of feed and to find and determine partner. Cuscus is active at night and rest during the day in the grove of trees, holes in the ground, or in a rock crevice. Sometimes this animal rests (sleeps), bends over and hugs branches or tree trunks which are not dense or open (Flannery 1994). The results showed that cuscus is generally found in locations with an altitude of 2-12 m asl. Ahe Island conditions are in accordance with the opinion of Flannery (1994) that the cuscus spread in the area with altitude of 0 to 2,900 m asl., especially in wooded areas. According to Warmetan (2004), trees such as Intsia sp., Lithocarpus sp., Ficus sp., Pterocarpus indica and Macaranga sp. are used by cuscus as nesting places (sleeping places). The species of feed consumed by cuscus in Ahe Island include forest vegetation and plantation crops

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such as Ficus benjamina, Ficus microstoma wall, Ficus pisocarpa BI, Ficus prolixa, Ficus infectoria Roxb, Merremia peltata, Pongamia pinnata, Intsia bijuga, Syzygium sp, Cocos nucifera. Parts of the plant widely consumed by cuscus are fruit (mature) and young leaves (shoots or buds). As stated by Kocu (2006), parts of the feed consumed by cuscus are flowers, young fruit, ripe fruit, young shoots and young leaves. The comparison between the parts of plants that are consumed shows that cuscus eat more ripe fruit, because the ripe fruit physiologically has a sweet flavor with a high water content so it is easy to digest.

CONCLUSIONS There were 26 individual cuscuses in Ahe Island consisting of 24 common spotted cuscuses (Spilocuscus maculatus) and 2 timor cuscuses (Phalanger orientalis), 12 males and 14 females. As many as, 14 individuals were adult, 8 adolescent, and 2 juvenile. There was a progressive population dynamics in cuscus of Ahe Island, although it is limited only to the common spotted cuscus (Spilocuscus maculatus) because of the balance of reproductive couples, while the Phalanger orientalis was not experiencing dynamics. There were 10 species of trees as sources of feed for cuscus in Ahe Island including forest vegetation and plantation crops such as Ficus benjamina, Ficus microstoma, Ficus pisocarpa, Ficus paka, Ficus infectoria, Merremia peltata, Pongamia pinnata, Intsia bijuga, Syzygium sp., and Cocos nucifera. In general, the active time of cuscus in Ahe Island was from 18:00 to 05:00 EIT (Eastern Indonesian Time), the period since these animals began to move until he returned to rest or hide. Cuscus was usually found in the conditions after raining and when the moon shines brightly, with an average air temperature of 23ºC and an average humidity of 82%, and with altitude of 2-12 m asl.

REFERENCES Alikodra HS. 1989. Management of Wildlife Vol. I. Bogor Agricultural University, Bogor. [Indonesian] Aplin K, Helgen K. 2008. Spilocuscus wilsoni. In: IUCN 2008. IUCN Red List of Threatened Species. www.iucn.org Ariantiningsih F. 2000. Hunting Systems and Public Attitudes Towards Efforts Elk in Rumberpon Island Manokwari Regency. [Hon. Thesis] Department of Forestry, Faculty of Agriculture, University of Cenderawasih, Manokwari. [Indonesia]. Flannery T. 1994. Possums of the World. A Monograph of the Phalangeroidea. Geo Production Pty Ltd, Australia. Flannery TF. 1995. Mammals of New Guinea. 2nd ed., Comstock/Cornell, USA. Kocu Y. 2006. Exploration of Cuscus Species at Kokas Village South Sorong Regency. [Hon. Thesis]. Faculty of Forestry, University of Papua, Manokwari. [Indonesia] Lewis MA. 1994. Spatial coupling of plant and herbivore dynamics: the contribution of herbivore dispersal to transient and persistent “waves” of damage. Theor Pop Biol 45: 277-312. Menzies JI. 1991. A Handbook of New Guinea Marsupials and Monotermes. Kristen Pres, Madang, PNG. Petocz RG. 1994. Terrestrial Mamalia of Irian Jaya. PT Gramedia Pustaka Utama, Jakarta. [Indonesian]

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Saragih EW, Sadsoeitoeboen MJ, Pattiselanno F. 2010. The diet of spotted cuscus (Spilocuscus maculatus) in natural and captivity habitat. Nusantara Biosci 2: 78-83. [Indonesian] Sinery A. 2002. Exploration of Cuscus at Numfor Island, Biak Numfor Regency. [Hon. Thesis]. Faculty of Forestry, University of Papua, Manokwari. [Indonesian]. Sinery A. 2009. Utilization of Cucsus as Animal Protein in Papua. Cahaya Papua, Manokwari. [Indonesian]

Sinery A. 2010. Population of cucsus at Arfak Nature Reserve, Manokwari Regency, West Papua. Agrifor 9 (2): 79-88 [Indonesian] Warmetan H. 2004. Exploration of Cuscus Species at Central Yapen Nature Reserve and its Surroundings, South Yapen Sub district, Yapen Waropen Regency. [Hon. Thesis]. Faculty of Forestry, University of Papua, Manokwari. [Indonesian]

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 101-105

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140208

Evolution of oviposition behavior in gypsy moth (Lymantria dispar) in Hyrcanian forests, North of Iran GOODARZ HAJIZADEH1,♥, MOHAMMAD REZA KAVOSI2, HAMID JALILVAND1 1

Department of Forestry, Faculty of Natural Resources, Sari Agricultural Sciences & Natural Resources University, P.O.Box:#578, Sari, Mazandaran Province, IR-Iran. Tel./Fax. +98 151 3822715, ♥email: goodarzhajizadeh@gmail.com 2 Department of Forest Ecology, Faculty of Forest Sciences, Gorgan University of Agricultural Sciences & Natural Resources, Gorgan, Golestan Province, IR-Iran. Manuscript received: 26 June 2013. Revision accepted: 24 July 2013.

ABSTRACT Hajizadeh G, Kavosi MR, Jalilvand H. 2013. Evolution of oviposition behavior in gypsy moth (Lymantria dispar) in Hyrcanian forests, North of Iran. Biodiversitas 14: 101-105. Oviposition behavior has been introduced at the center of many of the major debates on the ecology and evolution of interactions between insects and plants. The objective of this research was to determine the number of egg masses gypsy moth in relation to diameter at breast height (dbh), egg placement, orientation and host tree species. Sampling was carried out in Daland national park, Gorgan province. By global position system (GPS) device using polygons with width of 20 m and determined azimuth, defoliated trees were recorded. Data and means were compared using Duncan's multiple range tests. Results showed that the diameter at breast height was not significantly affected by the number of egg masses. The effect of oviposition place on number of egg masses gypsy moth were significant (P<0.01). The highest number of egg masses (2.148 egg masses/tree) was observed at trunk of defoliated trees; also, minimum (1.65 egg masses/tree) occurred in branches of defoliated trees. The effects of oviposition orient were significant (P<0.05). The means comparison showed that the maximum rates of egg masses was occurred in the south geographical position (2.04 egg masses/tree), the least of defoliation was related to the north direction (1.57 egg masses/tree). The primary host tree species was Persian iron wood (Parrotia persica). In finally, the selectivity of oviposition females may often provide the initial basis for divergence of insect populations on to different plant species, and it may drive the evolution of some plant defenses. Key words: behavior, egg masses, gypsy moth, Lymantria dispar, oviposition

INTRODUCTION The gypsy moth Lymantria dispar L. (Lepidoptera: Lymantriidae) is a major pest of forests and shade trees in the north-eastern United States (Thorpe et al. 2007). Subsequent to its introduction from Europe in approximately 1868, it has defoliated more than 34 million ha and more than five million ha have been treated with insecticides to suppress populations (Gypsy Moth Digest 2005). Defoliation stresses and kills trees; and indirect effects of defoliation can reverberate throughout forested ecosystems. Social impacts are also substantial. Recreational use of parks grounds is sharply curtailed during outbreaks; and the substantial nuisance created by large number of wandering larvae and frass raining from trees exacerbates its pest status in urban areas (Herms 2003). The female moth does not fly, even though she has large wings. Egg masses or clumps are usually found near empty pupal cases of females. Eggs are placed in dark sheltered areas, bark crevices, under loose bark, and the undersides of limbs, rocks, stumps, leaf litter, vehicles, and outdoor household equipment (Leonard 1981). The gypsy moth is a highly polyphagous folivore which will feed on over 300 species of woody plants (Leonard 1981). Among its favored foods are oaks and aspens.

Newly hatched gypsy moth larvae are carried to hosts by wind dispersal in the spring, landing on plants and then either remaining to feed or redispersing (Capinera and Barbosa 1976; Lance and Barbosa 1981). Gypsy moth is one of the most important pests in Hyrcanian forests, north of Iran. It was observed for the first time in 1937 in Guilan region, Hyrcanian forest zone. The largest outbreaks of gypsy moth occurred in Talesh forest in Guilan forests in 1975 (Kavosi 2008). It is speared in Hyrcanian, Arasbaran and Zagros forests (oak forests) during this time. It was recognized that gypsy moth is distributing in thorough Hyrcanian forests and the most importantly, its focus are, Daland park (Golestan province), Zare and Noor parks (Mazandaran province) and Rezvanshahr and Masal forests (Guilan province) (Hajizadeh 2010). The activity of this pest in central parts and the south western forests of IR-Iran has been admitted outside these regions. The defoliated rate in Hyrcanian zone is further more than the other zones and thousands of hectares of forests in this zone are getting extinct (rate of defoliated in Guilan region has reached to the fields and houses) (Hajizadeh and Kavosi 2011). Hajizadeh et al. (2012) studied the effects of oviposition height and host tree species on some L. dispar’s biological parameters of gypsy moth in Hyrcanian forests. Samples were taken on five oviposition heights (0.5, 1, 1.5,

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2, 2.5 m) on trunk of four common host tree species including, Zelkova carpinifolia, P. persica, Quercus castaneifolia and Carpinus carpinifolia. Results showed that the oviposition heights significantly affected pest biological parameters (egg clutch size, egg hatching percent, larval body length and mortality percent of first instars), but the effects of host tree species and interacting effects were not significant. The highest survival percent, egg clutch size and body length was observed at oviposition height of 0.5 m on the P. persica species, and the most egg mortality of first instars was recorded at oviposition height of 2.5 m on the Q. castaneifolia species. Lechowicz and Jobin (1983) studied the effects of estimating the susceptibility of tree species to attack by the gypsy moth. Numbers of gypsy moth larvae feeding on each of 922 randomly sampled trees in a Quercu-AcerFraxinetum forest in the southwestern Quebec, Canada were counted in 1979 and in 1980 to quantify the larval feeding preferences as observed in the field for eighteen deciduous and one coniferous tree species at the northern range limit of the gypsy moth. Both the diameter height (dbh) and the estimated foliage biomass of the sampled trees were used to calculate the relative proportions of foliage represented by each of the nineteen tree species in the forest canopy. The objective of this research was to determine the effects of diameter at breast height (dbh), egg placement, orientation and host tree species on number of egg masses gypsy moth, Lymantria dispar (L.) in Hyrcanian forests at the north of Iran.

MATERIALS AND METHODS The experiment was conducted in Daland park, which is part of the larger Golestan forest in Hyrcanian zone, IRIran (latitude 36°2′S-36°4′S, longitude 36°3′E-41°5′E) (Figure 1). This area is approximately, 3750 m long and 2900 m wide and has a total area of 608 ha. The study region has an average temperature of 16.5°C, a total annual

rainfall of 660 mm and an altitudinal range of 75-119 m above sea level. The park consists almost entirely of P. persica, Q. castaneifolia, Z. carpinifolia and C. betulus with a few small areas of other species (Populus alba, Ficus carica, Morus alba, Cupressus S.V. horizentalis, Pinus eladerica, Thuja orientalis and Acer insigne). The study site was recently infested by the gypsy moth. It was considered to be part of the eastern leading edge of the generally infested area (Anon 2005). To coordinate the egg masses gypsy moth, to zigzag between the trees were moving. By global position system (GPS) device with a width of 20 m and azimuth polygon specific coordination of defoliated trees was recorded (Figures 2 and 3). Data and means were compared using Duncan’s multiple range tests.

RESULTS AND DISCUSSION Results showed that the diameter at breast height (dbh) of host tree species has no significant effect on the number of egg masses gypsy moth (Table 1). Maximum of egg masses were observed at 80-90 cm dbh (2.37 egg masses/tree) (Figure 4). In geographical direction of the trunk of host tree species, there was no significant difference (P<0.01) (Table 2). The compare of means showed that the maximum rates of egg masses in defoliated trees occurred in the south position (2.04 egg masses/tree), the least of defoliation was related to the north (1.57 egg masses/tree) (Figure 5). The effects of oviposition place were significant (P<0.01). The highest number of egg masses (2.148 egg masses/tree) was observed at the trunk of defoliated trees (Table 3). As expected, tree species had significant effect (ɤ = 0.05) on egg masses of gypsy moth (Table 4). The maximum of egg masses of defoliated trees was observed on Persian iron wood, Parrotia persica (average 1.92 egg masses per defoliated tree). Minimum (average 1.15 egg masses per defoliated tree) occurred in the trunk of Cupressus Sempervirences var horizontalis (Figure 6).

Daland Park

Islamic Republic of Iran

Figure 2. Location of the study site inside Daland park, the part of Hyrcanian forests, Golestan, North of Iran.

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Figure 2. Defoliation and tree mortality associated with gypsy moth outbreaks in Guilan province forests of Hyrcanian Forest (Hajizadeh and Kavosi 2011).

A

B

C

D

A

B

C

D

Figure 3. Life stages of gypsy moth, Lymantria dispar; A. egg, B. larva, C. pupa, D. imago

Table 1. Analysis of variance of gypsy moth egg masses in diameter at breast height of defoliated trees. df

MS

F

Sig

Between Groups 11 1.364 0.532 0.882ns Within Groups 581 2.564 Total 592 Note: Asterisks (nsP > 0.05) indicate not significant differences between the treatments. Table 2. Analysis of variance of gypsy moth egg masses, as influenced by oviposition orients.

df

MS

F

Sig

Between Groups 3 6.757 2.684 0.046* Within Groups 589 2.517 Total 592 Note: Asterisks (*P < 0.05) indicate significant differences between the treatments. Table 3. Comparision of oviposite place in gypsy moth, Lymantria dispar

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104 Test Value Egg nests

95% Mean confidence t df Sig SE difference interval of the difference 3.643 348.069 0.00 0.528 0.145 -0.814 0.243

Table 4. Analysis of variance of gypsy moth egg masses as influenced by tree species

Average of egg masses

df MS F Sig Between Groups 4 8.247 3.308 0.011* Within Groups 588 2.493 Total 592 Note: Asterisks (*P < 0.05) indicate significant differences between the treatments.

dbh (cm)

Average of egg masses

Figure 4. Average of egg masses gypsy moth in classified diameter at breast height (dbh) of host tree species in Daland Park, Golestan State region.

Oviposition orients

Average of egg masses

Figure 5. Average of egg masses gypsy moth in geographical direction of trunk in defoliated trees.

Host tree species

Figure 6. Mean egg masses of gypsy moth in 2009 for insects that fed on the various host tree species. PP = Parrotia persica; ZC = Zelkova carpinifolia; CB = Carpinus betulus; QC = Quercus castanifolia: and CS = Cupressus sempervirens. Same letters indicate mean values that are not significantly different.

Discussion The gypsy moth, Lymantria dispar L., is one of the most important pests of forest trees throughout the world, including Hyrcanian forests of the northern of IR-Iran. Larval herbivory can result in leaf area reductions, leaves abscission, and eventually, yield quality and quantity losses. The average of egg masses gypsy moth, as an index, which indicates the status invasion. Recognition of oviposition place and diameter at breast height (dbh) of host trees is a way to study the population dynamic and sampling programs to monitoring gypsy moth. Criteria such as the defoliation, reducing the diameter of the trunk and killing the host tree species to determine the economic damage of gypsy moth, are used (Barbosa 1978). The relationship between infestation and diameter at breast height of host tree species varies depending on the forest types. However, the infestation rate in the mixed forest types of trees with a low canopy is less (Smitley et al. 1993). In this study, the highest infestation rate was observed in the diameter of 80-90 cm, this result was in consistent with other researchers (Roden et al. 1992; Smitley et al. 1993; Nesslage et al. 2007). Kurt et al. (1999) studied the effect of silviculture treatments in the management of gypsy moth, they concluded destruction and persistency of forest trees areas of activity provide the pest. Construction of facilities in fringes of forest areas and degraded forests into agricultural lands and orchards in the areas of the forest canopy is open. Opening the forest canopy, high temperatures, low humidity and light on the forest environment are followed. The better conditions for growth and development of gypsy moth in forest areas make available (Ghent and Onken 2004). The highest infestation rate in south direction of the trunk defoliated trees was observed, which was consistent with findings of other researchers. Gypsy moth, in Hyrcanian forests, north of Iran, the second half of June to August according to altitude and weather conditions, at night on leaves, the skin split tree trunks, rocks and even man-made forest in the oval-shaped mass oviposition on them with a bunch of hair and fluff coats. So after leaving the pupal skin, usually in the same location will start oviposition. Then, all part of the summer and autumn and winter as eggs in diapauses State spends the life cycle gypsy moth, eggs categories that are easy to biopsy. High population densities in the gypsy moth, the eggs on the trunks of host trees are found in most categories. However, at low population densities, a large percentage of egg categories, under the rocks and trees along streams are observed. Categories of eggs of this pest, the outbreak had a small organ, each are containing 75 to 100 eggs. But the growing population and a static number of eggs in very few categories of rebellion, but their larger size, each containing 700 to 1000 eggs. In this study, the highest rate of egg masses gypsy moth on the trunks of host trees was the lowest of the branches of trees, which is consistent with findings of other researchers (Barbosa and Capinera 1974; Elkinton and Liebhold 1990).

HAJIZADEH et al. – Evolution of oviposition behavior in Lymantria dispar

CONCLUSION The gypsy moth, Lymantria dispar L., is one of the most important pests of forest trees throughout the world. Larval herbivory can result in leaf area reductions, leaves abscission, and eventually, yield quality and quantity losses. In fact, in this study, we found significant differences in defoliation levels among tree species. We found that the primary host tree species of gypsy moth in Iran was Persian ironweed, Parrotia persica. In finally, identification of suitable host trees and high spawning of gypsy moth a appropriate way to run a program of sampling and population dynamics of the pest smoothly.

REFERENCES Anon. 2005. Revision plan of national park Daland. Forest, Range and Watershed Management Organization Press. Gorgan. Barbosa P, Capinera JL. 1974. The influence of food on developmental characteristics of the gypsy moth. Can J Zool 55: 1427-1429 Barbosa P. 1978. Distribution of egg masses and endemic larvae of gypsy moth population among various tree species. Can J Zool 56: 28-37. Capinera JL, Barbosa P. 1976. Dispersal of first-instar gypsy moth larvae in relation to population quality. Oecologia 26: 53-64. Elkinton JS, Liebhold AM. 1990. Population dynamics of gypsy moth in North America. Ann Rev Entomol 35: 571-596. Ghent JH, Onken AH. 2004. Trip report on assistance to Mongolian Ministry for Nature and Environment for the control of forest defoliators, FAO Project TCP/MON/2902, Asheville, NC. Gypsy Moth Digest. 2005. http://www.na.fs.fed.us/fhp/gm/. Hajizadeh G, Kavosi MR, Afshari A, Shataee S. 2012. Effects of oviposition height and host tree species on some biological

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parameters of gypsy moth, Lymantria dispar L. J For Wood Sci 19 (1): 149-162. [Persian] Hajizadeh G, Kavosi MR. 2011. Primary Host Tree Species of the gypsy Moth Lymantria dispar (Lepidoptera: Lymantriidae) in Hyrcanian Forests. J Agri Sci Tech B 1: 342-346. Hajizadeh G. 2010. The comparison of integrated and pheromone control on the intensity and spatial distribution of Lymantria dispar L., [M.Sc. Thesis], Gorgan of Agricultural Sciences and Natural Resources, University Press. [Persian] Herms DA. 2003. Assessing management options for gypsy moth. Pesticide Outlook 14: 14-18. Kavosi MR. 2008. Study of distribution gypsy moth, Lymantria dispar L. in the North forests. The First Symposium of Climate Change and Dendrochronology, Sari University, Mazandaran. Kurt W, Gottschalk R, Mark J. 1999. Managing forest for gypsy moth silviculture treatments in reducing foliation and mortality, 12th Central Hardwood Forest Conference of Natural Resources, Univ. Missouri, Columbia. Lance D, Barbosa P. 1981. Host tree influences on the dispersal of first instar gypsy moths, Lyrnantria dispar L. J Ecol Entomol 6:411-416. Lechowicz MJ, Jobin L. 1983. Estimating the susceptibility of tree species to attack by the gypsy moth. J Ecol Entomol 8: 171-183. Leonard DE. 1981. Bioecology of the Gypsy Moth. In: Doane CC, McManus ML (eds.). The Gypsy Moth: Research toward Integrated Pest Management. USDA Technical Bulletin, Washington DC. Nesslage GM, Maurer BA, Gage SH. 2007. Gypsy moth response to landscape structure differs from neutral model predictions: implications for invasion monitoring. J Biol Invas 9: 585-595. Roden DB, Miller JR, Simmons GA. 1992. Visual stimuli influencing orientation by larval gypsy moth, Lymantria dispar L. J Can Entomol 122: 304-617. Smitley DR, Rao RP, Roden DB. 1993. Role of tree trunks foliage type, and canopy size in host selection by Lymantria dispar (Lepidoptera: Lymantriidae). J Environ Entomol 22: 134-140. Thorpe KW, Hickman AD, Tcheslavskaia KS, Leonard DS, Roberts A. 2007. Comparison of methods for deploying female gypsy moths to evaluate mating disruption treatments. Agric For Entomol 9: 31-37.

B I O D I V E R S IT A S Volume 14, Number 2, October 2013 Pages: 106-111

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d140209

Ethnobotanical study and nutrient content of local vegetables consumed in Central Kalimantan, Indonesia HASTIN E.N.C. CHOTIMAH1,♥, SUSI KRESNATITA1, YULA MIRANDA2 1

Department of Agronomy, Faculty of Agriculture, Palangkaraya University. Jl. Yos. Sudarso, Palangkaraya 73112, Central Kalimantan, Indonesia. Tel. +62-536-3326196, ♥email: hastinwindarto@yahoo.com 2 Department of Mathematics and Natural Sciences Education, Faculty of Teacher Training Education, Palangkaraya University, Central Kalimantan, Indonesia Manuscript received: 4 May 2013. Revision accepted: 15 May 2013.

Abstract. Chotimah HENC, Kresnatita S, Miranda Y. 2013. Ethnobotanical study and nutrient content of local vegetables consumed in Central Kalimantan, Indonesia. Biodiversitas 14: 106-111. People in Central Kalimantan consume vegetables collected from the wild or traditionally cultivated. Documentation effort of them is very important because the diversity of local vegetables is threatened with extinction due to the conversion of peat land and forest fires. This study aimed to determine the diversity of local vegetables in Central Kalimantan, its use as a vegetable and nutrient content of some vegetables. The method used was the exploration and interviews. Exploration was carried out in three districts, namely Palangkaraya, Pulang Pisau, and Seruyan. Sampling of plants was done randomly and selectively. Data analysis was performed descriptively. The results showed that we recorded 42 plant species belonging to 30 families. There were many vegetables processing: stir-fry, make into clear soup, a light coconut milk soup, acidic soup, or just consumed as fresh vegetables. Based on the nutritional value, Helminthostachys zeylanica (L.) Hook had a potential to be developed as vegetables or medicinal plant. It had the highest protein, carbohydrate and minerals, namely P, Fe, Na and K among the vegetables analyzed. Key words: ethnobotany, indigenous vegetables, nutritional value, Central Kalimantan

INTRODUCTION Conserving the world’s biodiversity is very important to support sustainable living. Kalimantan island is endowed with agro-biodiversity like local vegetables which have high nutritional value, health benefits, income-generation potential, and agronomic advantages that can be exploited. Major constraints that hinder optimal production and utilization of the local vegetables include neglect by stakeholders, lack of quality seed, lack of technical production and utilization packages, and poor marketing channels. Consequently, their potential has not been fully exploited. In this study, the term ‘local vegetables’ is used to refer to both native and introduced vegetables. Native vegetables are edible plants indigenous to an area, while introduced vegetables are those that have been introduced into a particular area. Introduced vegetables have adapted to local condition after their introduction with the result that they are considered as local or even thought as native (Laker 2007; Dweba and Mearns 2011). It is reported that in Central Kalimantan more than 200 plants are used as local vegetables. Some of them are believed to have properties to maintain a healthy body from disease. In African communities, African indigenous vegetables have been reported to have high nutritional value, where consumption of 100 g of the vegetables provides over 100% of the daily requirement of vitamins and minerals and 40% of proteins (Onyango 2003). Some local vegetables that are currently found and consumed a lot by people in Central Kalimantan are

Stenochlaena palustris, Ceratopteris thalictroides, Calamus sp., Cnesmone javanica, Nauclea sp. and others (Irawan et al. 2006). Meanwhile research on the utilization of plant fruits and wild vegetables by the Dayak Kenyah of East Kalimantan showed that many species of fruit bearing plants are cultivated by the tribe, but it is not the case with vegetables. The reason is that many wild plants can be utilized for the vegetable, making it less necessary to cultivate. Leaves, shoots and roots of various wild plants can be eaten as a vegetable. Buds and shoots of Cyperus bancanus, shoot of Imperata cylindrica are consumed as fresh vegetables. Young leaves and stems of Cyathea contaminans as well as Diplazium ferns, Nephrolepis bisserata, and Stenochlaena are boiled or pan-fried vegetables and sometimes traditionally cooked in bamboo tubes. Likewise, other species of Zingiberaceae such as Alpinia sp., Kaempferia sp., Nicolaia speciosa are source of vegetables and the preferred flavoring. The tip of the harvested rattan trunk is usually processed by fire until withered, then the tough skin and thorn are peeled. The inside is then used as a vegetable. Likewise, young rattan trunk of Eugeissona utilis, Oncosperma and Pinanga are vegetables usually cooked along with fish (Hendra 2002). In Central Kalimantan, documentation effort is very important because the diversity of local vegetables are threatened with extinction due to land conversion for plantations and transmigration areas. The condition was further exacerbated by the presence of peat forest fires which almost always occur every dry season. This research was intended to conserve local vegetables in Central

CHOTIMAH et al. – Indigenous vegetables consumed in Central Kalimantan

Kalimantan by conducting an initial survey to collect basic information on their nutritional content. The abundance and nutrient information of them are very important for the establishment of baseline information for creating food consumption guidelines for local communities, applying cultivation technology to support the food security, and for determinating the phytochemical and pharmaceutical potential.

MATERIALS AND METHODS The objectives of this study were to determine the availability of local vegetables in Central Kalimantan, Indonesia and to assess the current and possible future utilization as a food source. The method used was the exploration and interviews. Exploration carried out in three districts namely Palangkaraya, Seruyan, and Pulang Pisau in Central Kalimantan in the middle of Indonesia

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(Figure 1). The Dayaks tribe are natives who inhabit the island of Kalimantan. Literally 'dayak' means the rural community and is a collective term for a variety of ethnic groups, which differ in language, art forms, and many elements of culture and social organization (MacKinnon et al. 2000). They have consumed and taken advantage of local vegetables for generations. Some of the vegetables are not specifically cultivated or grow wild in the forest without human intervention. They can survive in poor soils; require less inputs and resources, chemical fertilizers and pesticide. Vegetables were sampled randomly and selectively. The sampling included the vegetative parts (shoots, stems and leaves) and the generative (flower, fruit and seeds) as well as other parts such as bulbs and others. Exploration was also done with the interview method. Target informants for the interview per district were ten traditional vegetable traders in market t and three key informants. The key informants were community leaders and local people

A

B C

Figure 1. Locations of study in Districts of (A) Palangkaraya, (B) Pulang Pisau, and (C) Seruyan, in Central Kalimantan Province, Indonesia

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who make use of existing local plants around to meet the daily need. Data collected included: name of plant species (local name and scientific name), the parts of plant consumed, method of cooking, natural habitat, the seasonal abundance, and the economic value. Identification was done using the key determination of the book Flora of Java (Backer and Bakhuizen van den Brink 1963; 1965; 1968). Data analysis was performed descriptively. Moisture and ash contents were analyzed by gravimetric methods. Fat was determined by hydrolysis soxhlet methods. Crude protein was estimated by the micro Kjeldahl method. Total protein was calculated by multiplying the evaluated nitrogen by 6.25. Phosphor content was determined by spectrophotometry, meanwhile Ca, Fe, Na and K nutrient by AAS (AOAC 1990). Analysis of vitamin C was determined by spectrophotometry.

RESULTS AND DISCUSSION Abundance of local vegetables From the observation and exploration in traditional markets and in the field 42 species belonging to 30 families of local vegetables have been identified. A list of species and plant parts used are presented in Table 1. There were some vegetables found in the market but not at the site of exploration, and vice versa. Vegetables Ardisia sp. and Lepisanthes alata were not found in traditional markets, but were found at the site of exploration in the District of Seruyan. Ardisia sp. now very rare, while L. alata was found in the vicinity of the riverside. The others found in the market were Ceratopteris thalictroides and Stenochlaena palustris, Curcuma domestica, Helminthostachys zeylanica and various species of mushrooms.

Table 1. List of local species consumed as vegetables in Central Kalimantan Vernacular Name

Latin Name

Family

Part being used

Bakung Pisang Uwei Enyoh Undus Segau Kulat bitak Kanas Genjer Mantela Kujang Tantimun batu Tantimun Baluh bahenda Kanjat Paria Uwi turus Lampinak Jawau Kulat siaw Bawang suna Jagung belanda Uru mahamen Kalamenyu Teken parei Katu Kulat enyak Kulat baputi Kulat danum Humba betung Sarai Kalakai Bajei Taya Kenyem Kulat kritip Rimbang asem Terung tanteloh Sanggau Kedondong Henda Potok

Crinum asiaticum L. Musa paradisiaca L. Calamus sp. L. Cocos nucifera L. Elaeis guineensis Jacq Lactuca virosa L. Auricularia sp. (Bull.) J.Schrot. Ananas comosus Merr Limnocharis flava (L.) Buchenau Carica papaya L. Colocasia esculentum Schott Cucumis sativus L. Cucumis sativus L. Cucurbita moschata Duch Gymnopetalum cochinense Kurz Momordica charantia L. Dioscorea aculeata Roxb. Cnesmone javanica Blume Manihot esculenta Crantz Hygrocybe conica (Schaeff.: Fries) Kumm Allium schoenoprasum L. Abelmoschus esculentus (L.) Moench Mimosa pudica L. Ardisia sp. Sw. Helminthostachys zeylanica (L.) Hook Sauropus androgynus (L.) Merr Oudemansiella sp. Speg. Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm. Pleurotus sp. (Fr.) P. Kumm. Dendrocalamus asper (Schult. & Schult. f.) Backer Cymbopogon citratus (DC.) Stapf Stenochlaena palustris (Burm.) Bedd Ceratopteris thalictroides (L.) Brongn Nauclea sp. L. Lepisanthes alata (Blume) Leenh Schizophyllum commune Fries Solanum ferox L. Solanum mammosum L. Solanum torvum Sw. Spondias pinnata (L. f.) Kurz Curcuma domestica Val. Alpinia sp. Roxb.

Amaryllidaceae Araceae Arecaceae Arecaceae Arecaceae Asteraceae Auriculariaceae Bromeliaceae Butomaceae Caricaceae Colocasiaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Cucurbitaceae Dioscoreaceae Euphorbiaceae Euphorbiaceae Hygrophoraceae Liliaceae Malvaceae Mimosaceae/Fabaceae Myrsinaceae Ophioglossaceae Phyllanthaceae Physalacriaceae Pleurotaceae Pleurotaceae Poaceae Poaceae Polypodiaceae Pteridaceae Rubiaceae Sapindaceae Schizophyllaceae Solanaceae Solanaceae Solanaceae Anacardiaceae Zingiberaceae Zingiberaceae

Bulb Flower, fruit Young shoot Young shoot Young shoot Leave Fruit body Young fruit Shoot, young leave, flower Flower, fruit, young leaves Runner Fruit Young leave Flower, fruit, young leave Young fruit Young leave Bulb Young leave Young leave Fruit body Bulb, leave Fruit Young leave Young leave Young leave Young leave Fruit body Fruit body Fruit body Young shoot Inner shoot Young leave Young leave Young leave Fruit Fruit body Fruit Fruit Fruit Young leave Flower Young shoot

CHOTIMAH et al. – Indigenous vegetables consumed in Central Kalimantan

The various species of mushroom were P. ostreatus, Oudemansiella sp., A. auricula, H. conica, and S. commune. The Pleurotus sp. is a kind of oyster mushrooms, having different texture of the fruit flesh. The mushroom of Auricularia sp. or better known as jelly ear mushroom has pale brown color, while Hygrocybe conica has the red color. The mushrooms are commonly found on decomposed tree trunks. The mushrooms are sold by local people in the marketplace and on the sides of one road that connects the district with other districts. The abundance of a variety of mushroom is strongly influenced by the season. They are usually abundant during the rainy season. Edible mushroom exploration by Nion et al. (2010) reported that the wild Pleurotus sp. and S. commune were abundant in the months from May to July, while Oudemansiella sp. which usually grows on the decaying trunks of rubber trees was found only in the month of May and Auricularia sp. only in November. The most widely sold vegetables in the market were S. palustris and C. thalictroides. These vegetables are commonly found on the roadside, agricultural area, in the former area of open land and land burned. Most of the local vegetables are grown wild without cultivation. Rattans (Calamus sp.), for example, are widely spread, and climb the stems of large trees. There are various types of rattan namely bajungan, uwei irit, rua and lepu. The differences are found in stem size and color (white, pink and green). The part plant consumed is young shoot which has bitter taste. Other vegetables that grow wild are C. asiaticum, L. flava and M. pudica that grow wild in peat swamps. This abundance result is similar to that of Irawan et al. (2006). Species of wild plants which have been cultivated lately are G. cochinense, A. esculentus, H. zeylanica, A. schoenoprasum, Alpinia sp., S. torvum, S. ferox and S. torvum. The fruit vegetable S. ferox is a type of Solanum which was originally considered a weed plants, but it is now cultivated by a resident in Berengbengkel Palangkaraya. The round fruit shape of S. ferox is larger than that of S. torvum. The fruit is sour and can be consumed either when it is raw (green) or ripe (yellow). According to the local residents, another vegetable which has been cultivated by local residents is H. zeylanica. The vegetable is sometimes found under a rubber tree stands, meanwhile Chiu and Chang, (1992) state that H. zeylanica is rare plant in lightly shaded region and it is the only species of the genus Helminthostachys. The rhizome of the plant contains antioxidant flavonoids (Huang et al. 2003) and is widely used in Chinese herbal medicine as an antipyretic and antiphlogistic agent (Chiu and Chang 1992). Another wild plant which has been cultivated is A. esculentus. This plant is native to Africa and is now grown in many areas such as Asia, Middle East and Southern States of the USA (Calisir et al. 2005; Adelakun et al. 2009; Sengkhamparn et al. 2010), but is little known in Indonesia. Vegetable consumed only in Central Kalimantan is taro C. esculentum runner. It is a vegetative part (stolon) of taro plant, horizontally growing on top of the ground usually more than 30 cm long. A single clump of taro plant can have 4-5 pieces of runner. The more fertile and friable the

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soil, the more runner comes out, but not all of taro can be consumed due to the itchy-inducing substance. Ethnobotany For the Dayaks people of Central Kalimantan, S. palustris is a favorite food. In addition to the distinctive and delicious taste S. palustris is also believed to be the drug of youth. It can be stir-fried, boiled, and made into clear soup or just consumed as fresh vegetable. According to Irawan et al. (2006), S. palustris, C. thalictroides and runner of C. esculentum can be a good source of iron and folic acid. The vegetables may be given to women during the childbearing and post delivery periods. For most people of Indonesia, rattan (Calamus sp.) is known as an industrial raw material, mainly for handicrafts and furniture but not so with the Dayaks people in Central Kalimantan. They actually take advantage of young rattan stems commonly called singkah. Rattan is usually cooked with fish, S. ferox, and taro runner. This tasty dish is also quite bitter, so it has the distinctive taste of local cuisine. Meanwhile, Alpinia sp. and M. pudica have a slightly sour taste. Sour taste is believed to reduce the fishy smell of fish when Alpinia sp. cooked and mixed with fish. Likewise, young taya (Nauclea sp.) leaves are usually cooked with pork with a slightly bitter and sour distinctive taste. G. cochinense (Irawan et al. 2006) has an ability to absorb bitterness and is frequently used as sweetener. S. torvum (pea eggplant) is cooked with mashed cassava leaves or used by boiling. H. zeylanica is a seasonal plant and the population is not too much. Utilized part of this plant is the young leaves. The vegetable may be stir-fried, made into clear soup, a light coconut milk soup and acidic soup. It is also used by the Dayaks people as a substitute for the flavor in dishes by adding a few pieces of leaves into the dishes. According to local people the vegetable also has medicinal properties. Ethnomedical investigation by Sarker et al. (2012) reported that H. zeylanica roots were crushed and added to three finger widths of water and taken thrice on an empty stomach to treated severe fever, red color of urine and pain in the urinary bladder. G. cochinense has been reported to be used for treating various types of ailments including diabetes and malaria (Syiem and Lyngdoh 2009). Some communities in other regions eat taro on the leaves and tubers, but in Central Kalimantan, runner that grows above the ground around the parent plant is also used as a vegetable. The method of cooking is to peel the thin outer skin and then to cut the length of Âą 4-5 cm, wash, boil in advance to get rid of itchy-inducing substance. Meanwhile A. esculentus commonly named okra is processed to be clear soup or just consumed as fresh vegetables. Adelakun et al. (2009) reported that nutritionally, the richest part of the okra plant was the dried seed. Previously, Odelaye et al. (2003) noted that okra seed could serve as alternate rich sources of oil and protein to both the temperate regions and the tropics. Okra seed oil is also rich in unsaturated fatty acids such as linoleic acid, which is an essential fatty acid in human nutrition.

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Nutrient content Table 2. Proximate analysis of some indigenous vegetables of Central Kalimantan Table 2 shows the proximate analysis results of some local Moisture Ash Fat Protein Carbohydrate Vegetables vegetables in Central Kalimantan. In g 100 g-1 general, vegetables have a moisture Crinum asiaticum 91.92 1.03 0.31 0.80 5.94 content ranging from 83.91% to Abelmoschus esculentus 91.44 0.86 0.28 1.94 5.48 91.44%, while the ash content was 0.62% to 1.23%. Table 2 also reveals Allium schoenoprasum 88.40 0.62 0.03 1.64 9.31 that H. zeylanica leaves had the Elaeis guineensis 90.62 1.26 0.30 1.37 6.45 highest content of protein (4.50 g Helminthostachys zeylanica 83.91 1.23 0.26 4.50 10.10 100g-1) followed by A. esculentus -1 Solanum ferox 88.35 0.86 0.25 1.54 9.00 fruit (1.94 g 100g ) and A. -1 schoenoprasum bulb (1.64 g 100g ). The protein content of A. esculentus is smaller than protein content when Table 3. Mineral and vitamin C content of some indigenous vegetables of Central it is made into flour. Because the Kalimantan roasting is reported to improve flavor and color, the seeds of mature A. Phosporus Calcium Iron* Sodium Potassium Vit C esculentus are reported to be roasted, Vegetables mg 1000 g-1 g 100 g-1 ground and used as a coffee substitute in Turkey (Calisir et al. Abelmoschus esculentus 70.25 802.04 nd 92.46 2851.57 1.47 2005). The range means obtained for Allium schoenoprasum 85.25 368.69 nd 517.75 2056.56 1.66 roasted seeds protein contents were Crinum asiaticum 11.35 1226.57 nd 539.11 1819.36 1.41 42.14-38.10% (Adelakun et al. Elaeis guineensis 49.80 935.81 nd 171.08 3436.24 1.38 2009). A. esculentus seeds are also reported richer in phenolic compound Helminthostachys zeylanica 97.50 1058.02 136.72 678.33 3980.92 27.19 mainly composed by oligomeric Solanum ferox 28.50 268.38 nd 207.25 2340.73 4.31 catechins and flavonol derivative Note : * limit detection value 0.2 ppm; nd = not detected. (Arapitsas 2008) The content of carbohydrates in the form of vegetable starch, cellulose and sugar for H. zeylanica leaves, A. schoenoprasum bulbs and S. ferox fruit were during the childbearing and post delivery periods (Irawan 10.10 g 100g-1, 9.31 g 100g-1 and 9.00 g 100g-1, whereas fat et al. 2006). Vegetables account for a small part of our daily caloric content were 0.26 g 100g-1, 0.03 g 100g-1, and 0.25 g 100g1 intake: however their benefits to health surpass their caloric , respectively. From the seeds of S. ferox, a yellow colored oil has been obtained in 27% yield. The fatty and found to contribution. The contributory factors are due to the be palmitic 12.15%, stearic 9.96% and linoleic acid 38.06% presence of vitamins and provitamins (Ismail et al. 2004). Many vegetables also contain high phenolics that provide a (Garg and Gupta 2006). Mineral analysis of some vegetables observed revealed source of dietary anti-oxidants (Kaur and Kapoor 2002). that (Table 3) phosphorus ranged from 11.35 to 97.50 mg The results of analysis of vitamin C (Table 3) also showed had the 1000 g-1, calcium 268.38 to 1226.57 mg 1000 g-1, sodium that among vegetables analyzed, H. zeylanica -1 -1 highest vitamin C content (27.19 g 100 g ), followed by A. 92.46 to 678.33 mg 1000 g , potassium 1819.36 to 3980.92 -1 -1 schoenoprasum 1.66 g 100 g , C. asiaticum 1.41 g 100 g-1, mg 1000 g whereas iron content only detected in H. -1 -1 -1 zeylanica by 136.72 mg 1000 g . Nutrient-rich foods are E. guineensis-1 1.38 g 100 g , S. ferox 1.43 g 100 g and the vitamin vital for proper growth both in adults and children. If we 1.47 g 100 g for A. esculentus. Compared with -1 C content in tomatoes (17.8-19 mg 100 g ) and tapioca take into account the recommended dietary allowance -1 -1 leaves (77.2-1100 mg 100 g ) (Tee et al. 1988), vitamin C (RDA) for mineral : phosphorus 700 mg day , calcium -1 -1 -1 in some local vegetables studied are still higher. 1000 mg day , iron 8 mg day , sodium 1500 mg day and potassium 4700 mg day-1 for adults (Institute of Medicine Food and Nutrition Board, National Academies 2005) some local vegetables can provide 1.7-14%, 26.9-122.6 %, 6.1345.2%, 38.7-84.68% of phosphorus, calcium, sodium and potassium, respectively. Meanwhile, H. zeylanica is a good source of iron. Many of the Dayaks traditional vegetables are good sources of iron and have great potential to overcome nutritional anemia among the Indonesian people, especially women. Leaves of S. palustris, taro runner and leaves of C. thalictroides can become good sources of iron and folic acid. The vegetables may be given to women

CONCLUSION The exploration conducted in three districts found 42 species of local vegetables consumed by the local people of Central Kalimantan. They consume the vegetables by boiling, steaming and eating them fresh. Some vegetables are also believed to have properties to maintain a healthy body from disease. Some vegetables also have potential as sources of nutrients for humans.

CHOTIMAH et al. – Indigenous vegetables consumed in Central Kalimantan

ACKNOWLEDGMENTS We would like to thank to Direktorat Jenderal Pendidikan Tinggi Kementerian Pendidikan Kebudayaan Indonesia for granting financial support under Fundamental Grant No. 0541/023-04.1.01/00/2011, and to Cenarung as native Dayak person for his ethnobotanical knowledge of vegetables.

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Authors Index Abdelreheem A Abedi T AlKhanjari SS Ammar MSA Arafat A Arisoesilaningsih E Begyom-Faghir M Belal A Boer C Bonde SR Borah D Borgohain J Bramandito A Budiharta S Chotimah HENC El-Gammal F El-Haddad K El-Mesiry G El-Nagerabi SAF Elshafie AE Fallah A Farag W Farida WR Faridi F Gade AK Haddadi-Moghaddam H

43 89 10 43 17 37 89 43 95 55 67 79 17 37 106 43 43 43 10 10 25 43 95 61 55 89

Hajizadeh G Hojjati SM Hosseini SM Jalilvand H Kavosi MR Kooch Y Kresnatita S Kumar R Madduppa HH Miranda Y Nassar M Orabi A Pandey S Pourbabaei H Rai MK Rishi R Setyawan AD Shaaban A Siahaan AB Sinery AS Soejono Subhan B Sugiyarto Sutarno Tapwal A Wiryono

61, 101 25 25 101 61, 101 25 106 67, 73, 79 17 106 43 43 67, 73 89 55 67 1 43 31 95 37 17 1 1 67, 73, 79 31

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Subject Index Ahe Island Al-Jabal Al-Akhdar AMF anthropogenic aquaculture artificial reefs behavior biodiversity bulk density carpophores Central Kalimantan coal mined coral transplantation crust cuscus populations decomposition degraded areas egg masses endophytic fungi endosperm ethnobotany Fagus orientalis feed resources fish-habitat association fungal Fusarium gap size genetic diversity gypsy moth Hyrcanian forest indigenous Java Jeypore Reserve Forest leaf litter

95, 96, 97, 98, 99 10, 11, 12 67, 68, 69, 70, 71 17, 19, 26, 89 43 43, 45, 47, 48, 49, 50, 51 101 2, 10, 11, 12, 13, 14, 15, 17, 18, 23, 41, 43, 44, 51, 73, 77, 79, 106 25, 27, 28, 29 79, 83, 86 106, 107, 108, 109, 110 31, 32, 33, 34, 35 43, 44, 48, 49, 51 61, 65 95, 96, 98 25, 79, 80, 81 37, 41 101, 102, 103, 104 10, 11, 12, 13, 14, 15 61 106, 109 25, 26, 29, 89, 91, 92 95 17 10, 11, 12, 13, 14, 55, 56, 57, 61, 67, 70, 71, 73, 75, 76, 77, 78, 79, 80, 81, 83, 86 10, 12, 13, 14, 55, 56, 57, 58, 59, 60, 62, 63, 65, 66, 79, 82, 83, 86 89, 90, 91, 92, 93 44, 55, 56, 57, 60 101, 102, 103, 104, 105 25, 26, 27, 29, 61, 62, 66, 101, 102, 103, 104 10, 34, 106, 107, 108, 110 1, 2, 4, 5, 6, 7, 8, 17, 18, 38 73, 74, 75, 76, 78 25, 79, 80, 81, 82, 83, 101

Lymantria dispar macrofungi mariculture moisture Mount Lawu multivariate analysis mycorrhiza nursery nutritional value old trees Oman oviposition Papua plant diversity plant species Quercus castaneifolia RAPD rehabilitation RFRI root colonization seed soil texture species diversity species-specificity spring taxonomy tissue tree diversity understory UPGMA vegetables wild edible tuber Z. hajanensis Ziziphus spina-christi

101, 102, 103, 104, 105 73, 74, 75, 76, 79, 81, 82, 83 43, 44, 49 25, 27, 28, 29, 75, 81, 83, 92, 108, 110 1, 2, 3, 4, 5, 7 17, 19, 22 31, 67, 70, 71, 73, 74, 75, 76, 79, 80 43, 47, 48, 49, 51 74, 78, 106 25, 27, 28 10, 11, 12, 13, 14 101, 102, 103, 104 95, 96 1, 41, 89 11, 13, 14, 33, 70, 73, 89, 90, 91, 92, 93, 95, 101, 106, 108 61, 62, 66, 102 55, 56, 57, 58, 59, 60 37, 39, 40, 41, 43, 44, 45, 49 79, 80, 81, 82, 83 67, 70 39, 40, 61, 62, 63, 65, 66, 106, 107, 109, 110 25, 26, 27, 29 1, 2, 4, 5, 13, 17, 19, 21, 22, 25, 26, 33, 33, 37, 40, 75, 89, 90, 91, 92, 93 10, 11, 14 6, 7, 14, 37, 38, 39, 40, 41, 101 1, 57, 75, 81 10, 12, 13, 14, 43, 48, 51, 61 37 31, 33, 34, 35 55, 57, 58, 59 57, 106, 107, 108, 109, 110 67, 68, 70 10, 11, 12, 13, 14 10, 11, 12, 13, 14

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List of Peer Reviewers Ahmad Dwi Setyawan

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Surakarta 57126, Central Java, Indonesia

Amir Hamidy

Zoology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong Bogor 16911, West Java, Indonesia

Dharmendra K. Gupta

Departamento de Bioquimica, Biologia Cellular y Molicular de Plantas, Estacion Experimental Del Zaidin, CSIC, Apartado 419, Granada 18008, Spain

Fahmi

Research Centre for Oceanography, Indonesian Institute of Sciences (LIPI), Jakarta 14430, Indonesia

Hassan Pourbabaei

Department of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, P.O. Box 1144, Iran.

Irvan Sidik

Zoology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong Bogor 16911, West Java, Indonesia

Kiomars Sefidi

Department of Range and Watershed Management, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran

Leena Hamberg

Finnish Forest Research Institute, P.O. Box 18 (Jokiniemenkuja 1), FI-01301, Vantaa, Finland

Mahendra K. Rai

Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati 444602, Maharashtra, India

María de los Ángeles La Torre-Cuadros

Departamento de Manejo Forestal, Facultad de Ciencias Forestales, Universidad Nacional Agraria La Molina, Peru

Mirza Dikari Kusrini

Department of Forest Resources Conservation and Ecotourism, Faculty of Forestry, Bogor Agricultural University, Darmaga Campus, West Java, Indonesia

Mousa Najafiniya

Jiroft and Kahnooj Center for Agricultural Research, P.O. Box 78615-115, Jiroft, Iran

Nataša Radić

Celica, Biomedical Center, Technology Park, Ljubljana, Slovenia.

Rajesh Kumar

Rain Forest Research Institute, P.O. Box 136, Jorhat 785001, Assam, India.

Rui Jorge Miranda Rocha

Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal

Showket Ahmed Pala

Department of Botany, Faculty of Biological Sciences, University of Kashmir, Hazratbal, Srinagar 190006, Jammu and Kashmir, India.

Suman Sankar

Birbal Sahni Institute of Palaeobotany (BSIP), 53 University Road, Lucknow 226007, Uttar Pradesh, India.

Sutarno

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Surakarta 57126, Central Java, Indonesia

Sutomo

Bali Botanic Garden, Indonesian Institute of Sciences, Candikuning, Baturiti, Tabanan 82191, Bali, Indonesia.

Tytti Sarjala

Finnish Forest Research Institute, Kaironiementie 15, FI-39700 Parkano, Findland

Wiryono

Department of Forestry, Faculty of Agriculture, University of Bengkulu. Bengkulu 38371A, Bengkulu, Indonesia.

Xiao-Long Yang

College of Pharmaceutical Science, Hebei University, Baoding 071002, P.R. China

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Table of Contents Vol. 14, No. 1, Pp. 1-53, April 2013

SPECIES DIVERSTY Species diversity of Selaginella in Mount Lawu, Java, Indonesia AHMAD DWI SETYAWAN, SUTARNO, SUGIYARTO ECOSYSTEM DIVERSTY Endophytic fungi associated with Ziziphus species from mountainous area of Oman and new records SAIFELDIN A.F. EL-NAGERABI, ABDULQADIR E. ELSHAFIE, SULEIMAN S. ALKHANJARI Dynamics of fish diversity across an environmental gradient in the Seribu Islands reefs off Jakarta HAWIS H. MADDUPPA, BEGINER SUBHAN, DONDY ARAFAT, ADITYA BRAMANDITO Variability of soil physical indicators imposed by beech and hornbeam individual trees in a local scale YAHYA KOOCH, SEYED MOHSEN HOSSEINI, SEYED MOHAMMAD HOJJATI, ASGHAR FALLAH Species composition of understory vegetation in coal mined land in Central Bengkulu, Indonesia WIRYONO, ARIF BUHA SIAHAAN Proposing local trees diversity for rehabilitation of degraded lowland areas surrounding springs SOEJONO, SUGENG BUDIHARTA, ENDANG ARISOESILANINGSIH REVIEW Review: Current trends in coral transplantation – an approach to preserve biodiversity MOHAMMED S.A. AMMAR, FAHMY EL-GAMMAL, MOHAMMED NASSAR, AISHA BELAL, WAHID FARAG, GAMAL EL-MESIRY, KHALED EL-HADDAD, ABDELNABY ORABI, ALI ABDELREHEEM, AMGAD SHAABAN

1-9

10-16 17-24 25-30 31-36 37-42

43-53

Vol. 14, No. 2, Pp. 55-111, October 2013 GENETIC DIVERSTY Genetic diversity among fourteen different Fusarium species using RAPD marker SHITAL R. BONDE, ANIKET K. GADE, MAHENDRA K. RAI ECOSYSTEM DIVERSTY Fungal species isolated from Quercus castaneifolia in Hyrcanian Forests, North of Iran MOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH Observations on arbuscular mycorrhiza associated with important edible tuberous plants grown in wet evergreen forest in Assam, India RAJESH KUMAR, ASHWANI TAPWAL, SHAILESH PANDEY, RAJA RISHI, DEVAPOD BORAH Diversity and frequency of macrofungi associated with wet ever green tropical forest in Assam, India ASHWANI TAPWAL, RAJESH KUMAR, SHAILESH PANDEY Fungal diversity associated with bamboo litter from Bambusetum of Rain Forest Research Institute, Northeast India RAJESH KUMAR, ASHWANI TAPWAL, JAYASREE BORGOHAIN Effect of gap size of selective cutting method on plant species diversity and composition in beech (Fagus orientalis) forests, Ramsar, Mazandaran Province, North of Iran HASSAN POURBABAEI, HAMIDREZA HADDADI-MOGHADDAM, MARZIEH BEGYOM-FAGHIR, TOOBA ABEDI Cuscus population dynamics in tourist island of Ahe, District of Nabire, Papua ANTON SILAS SINERY, CHANDRADEWANA BOER, WARTIKA ROSA FARIDA

55-60

61-66 67-72 73-78 79-88 89-94

95-100

A-5 Evolution of oviposition behavior in gypsy moth (Lymantria dispar) in Hyrcanian forests, North of Iran GOODARZ HAJIZADEH, MOHAMMAD REZA KAVOSI, HAMID JALILVAND ETHNOBIOLOGY Ethnobotanical study and nutrient content of indigenous vegetables consumed in Central Kalimantan, Indonesia HASTIN E.N.C. CHOTIMAH, SUSI KRESNATITA, YULA MIRANDA

101-105

106-111

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ISSN: 1412-033X E-ISSN: 2085-4722

GENETIC DIVERSTY Genetic diversity among fourteen different Fusarium species using RAPD marker SHITAL R. BONDE, ANIKET K. GADE, MAHENDRA K. RAI

55-60

ECOSYSTEM DIVERSTY Fungal species isolated from Quercus castaneifolia in Hyrcanian Forests, North of Iran 61-66 MOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH Observations on arbuscular mycorrhiza associated with important edible tuberous plants 67-72 grown in wet evergreen forest in Assam, India RAJESH KUMAR, ASHWANI TAPWAL, SHAILESH PANDEY, RAJA RISHI, DEVAPOD BORAH Diversity and frequency of macrofungi associated with wet ever green tropical forest in 73-78 Assam, India ASHWANI TAPWAL, RAJESH KUMAR, SHAILESH PANDEY Fungal diversity associated with bamboo litter from Bambusetum of Rain Forest Research 79-88 Institute, Northeast India RAJESH KUMAR, ASHWANI TAPWAL, JAYASREE BORGOHAIN Effect of gap size of selective cutting method on plant species diversity and composition in 89-94 beech (Fagus orientalis) forests, Ramsar, Mazandaran Province, North of Iran HASSAN POURBABAEI, HAMIDREZA HADDADI-MOGHADDAM, MARZIEH BEGYOM-FAGHIR, TOOBA ABEDI Population dynamics of cuscus in tourist island of Ahe, District of Nabire, Papua 95-100 ANTON SILAS SINERY, CHANDRADEWANA BOER, WARTIKA ROSA FARIDA Evolution of oviposition behavior in gypsy moth (Lymantria dispar) in Hyrcanian forests, 101-105 North of Iran GOODARZ HAJIZADEH, MOHAMMAD REZA KAVOSI, HAMID JALILVAND ETHNOBIOLOGY Ethnobotanical study and nutrient content of local vegetables consumed in Central Kalimantan, Indonesia HASTIN E.N.C. CHOTIMAH, SUSI KRESNATITA, YULA MIRANDA

106-111

Front cover: Microbial colonies (PHOTO: RAJESH KUMAR)

Published semiannually

PRINTED IN INDONESIA ISSN: 1412-033X

E-ISSN: 2085-4722