BIOTROPLA Vol. 28 No. 3,2021: 221 - 230 DOI: 10.1 1598/btb.2021.28.3.1339 DIVERSITY OF ENDOPHYTIC FUNGI ASSOCIATED WITH FRUITS A N D LEAVES OF TAMARIND (Tamarindus indica L.) BASED O N ITS RIBOSOMAL D N A SEQUENCES NURUL ASYIQIN MOHD ZAINI', NURUL HUWAIDAH MD NIZAM, DAYANG FATIN ZAFIRA AWG ZAINAL ABIDIN, NOR IZANIS AZNI MOHD NAZRI AND NUR AIN IZZATI MOHD ZAINUDIN* Department $Biology, Factllo $Science, Universiti Putra Mala_ysia, 43400 Serdang, Selangor, Malysia Received 18 February 2020 /Accepted 2 May 2020 ABSTRACT Plant-associated microbes are among essential natural resources that abundantly exist in a natural environment, such as endophyuc fungi. Studies o n endophytic fungi in medicinal plants have allowed the discovery of numerous fungi species and their hidden potentials. Therefore, this study focused o n the isolation and identification of endophyuc fungi from several plant parts of tamarind (T. indica), such as leaves and fruits. A total of 69 fungal cultures were successfully isolated and identified into 31 distinct species from 15 genera based on morphological characteristics and internal transcribed spacer (ITS) sequence analysis using a Maximum Likelihood method. A high diversity of endophytic fungi associated with T . indica were observed by Shannon- Wiener index H' (3.083). There were six different species obtained from the genus Colletotrichzlm (C. aenigma, C. brevisporum, C. cobbittiense, C. fmcticola, C. gloeosporiaides and C. siamense), and Diaporthe (D. arecae, D. ceratoxamiae, D. phaseolorzrm, D. pseudomangzjirae, D. pseudooctlii and D. pseudophoenicicala), four species of Aspergilzlzls (A. aczlleatzrs, A. carbonarizls, A.flautls and A. tzlbingensis), two species of Czlrvzrlank/Cochliobolzrs (C. geniculatzrs and C. lunata) and lV&rospora (N. lacticolonia and N. oryxae), two species of Lasiod$lodia (L. psetldotheobromae and L theobmmae) and Penicillm (P. m@ii and P. verruczllos~m). Other fungal species that were also identified are Botyosphaeria mamane, Fusaritlm solani, Tmncospora tephropora, Ph_yllostictafallopiae, Sarcostroma bisettllatnm, Tiichodema asperelhm and Xylarid j2ejeensi.r. Keywords: Endophyuc fungi, internal transcribed spacer (ITS), phylogenetic tree, tamarind INTRODUCTION Endophytic fungi are microorganisms inhabiting plant tissues in a part of their life without showing any harm toward the host plants. The species of endophytic fungi are expected in over a million species, which arisen from the natural surroundings W s h r a e t al. 2018). They are widely dstributed, which have been found in many plant species that can grow in natural environments such as terrestrial plant communities (Nisa e t al. 2015). Endophytic fungi such as Aspel.gllus, Colletotm'cbum, Fusam'um, Penicillm and Tm'cbodema may colonize several parts of plants, includmg fruits and leaves *Corresponding author, email: ainizzati@upm.edu.my (Hanada e t al. 2010). There are many research studes reported the abundance of fungi associated with plants, however, there is a lack of study in the endophytic fungi associated with T. indica. Bourou e t al (2010) reported, three genera of arbuscular mycorrhzal fungi (Acaulospora, Glomzls and Scutellospora) were associated with T. indim. Tamarind tree has been reported to be infected by some wood decay fungi such as Daldinid concentm'ca, Scbixopbylh commune, Flavodon jlavus, @ex bydnoides, and Pbellinus fastuosus (Nnagadesi & Arya 2015). In previous reports, Aspep!lIus n&eq Rhixopus stolonifeq Ulocladium cban'drum, Penicillium cb ysogenum, P. citm'num and Pbomopsis liquidambaris were associated with infected-tamarind fruit BIOTROPIA Vol. 28 No. 3,2021 (Danggomen e t al. 2013; Peter & Patrick 2017). Isolation, Purification and Preservation of Penin'Ilium cblysgenum and P. n'tm'nam are Microfungi confirmed pathogens and caused spoilage in All plant samples were washed in running tap fruits (Peter & Patrick 2017). Recently, water for 30 rnin to remove any debris or soil A.pergiIIus nniger has been proven as a pathogen before being processed. The leaves were cut into that causes black pod of tamarind (Meena e t al. segments of 5 x 5 mm. Then, the surface of the 2018). leaves and fruits was surface sterilized by Due to the regarbg e n d O ~ h ~ c following the described by Ravindran fungal diversity associated with L indica is et al (2012) by immersing in 700,0 ethanol lacking, this study will provide important (5 sec), 4% sodium hypochlorite (NaOCI) information regarding the diversity of fungal (90 set., with sterile distilled water e n d o p h ~ e s associated with L ifldica. This study (30 sec) and blotted dry with sterile filter paper. was aimed to determine the ~ulturable All of the segments were placed (3 segments endophytic fungal diversity associated with each plate) on potato dextrose agar (PDA) L indicd using molecular phylogenetic analysis of supplemented with streptomycin (0.05 g/rnl) ITS rDNA sequences. and neomycin (0.01 g/L) using sterilized forceps. The culture plate was incubated at room temperature (27 f 2OC) for 5 to 7 days or MATERIALS AND METHODS until there was an appearance of mycelium or colony from the sample fragments. Plant Samples The fungal mycelia grown from the parts of the sample were streaked on 4% water agar Collection of leaves and fruits samples of F A ) for purification. The WA plate was T. was cOm~leted in 2018 and 2019 at incubated for another 24 hours. Then, the single Jalan Asam Jaws, Universiti tip of hyphae was cut and transferred onto a Serdang Selangor located at 3"00709.0"N n, pDA plate and incubated at 2 7 & 2 0 ~ for 101"42'34.gnE (Fig. 1). The fruits and leaves seven days. The pure isolated fungi were samples were collected using fruit picker from preliminarily identified by examining their 20 L indicd trees with 2 m apart. All samples morphological characteristics. All isolates were were further placed in paper bags, properly maintained and preserved at -20 "C using a labeled, and brought to the Mycology modified filter paper method for working and Laboratory, Department of Biology for fungal stock cultures with slight modifications (Fang e t isolation. al. 2000). Figure 1 Samples of fruits (A) and leaves (B) of T. indica were collected in Persiaran Asam Jawa, Universiti Putra Malaysia Endophytic fungi from tamarinds - Mohd Zaini eta/ DNA Extraction, PCR Amplification and Sequencing All isolates were cultured on PDA and incubated for 5 days. DNA of the isolates was extracted using UltraCleanB Microbial DNA Isolation IGt (MO BIO, Carlsbad, CA, USA) according to manufacturer's instruction. Amplification of the ITS regions was conducted using Polymerase Chain Reaction (PCR) machine (Hercuvan Lab Systems, California, USA) involved primers ITS1 (5'- TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (Xkte e t al. 1990). The PCR master mix was prepared from 4 pL of 5xPCR buffer, 2 pL of 2 mM dNTP, 2 pL of 25 mM MgClz, 1 pL of 10 mM for each primer, 0.1 pL of Taq DNA polymerase with concentration 5 U pL, 6.9 pL of nuclease- free water and 3 pL of DNA in a total volume of 20 pL. The PCR protocol with initial denaturation step was done for 30 sec at 95 OC, followed by 35 cycles of denaturation (95 OC for 10 sec), annealing (59 OC for 15 sec) and extension (72 OC for 30 sec), and was completed by final extension step at 72 OC for 5 min. Then, the PCR product was prepared for gel electrophoresis or stored at -20 OC. The PCR products were gel-electrophoresed using 1.5% agarose gel. The mixture of 2.5 yL of 6x loading dye (blue/orange) and 2.5 yL of 100 bp DNA marker were used as a ladder. The DNA and ladder were pipetted with 5 yl in volume into the holes using a micropipette and electrophoresed. The amplicon size was visualized under a UV trans-illuminator. The PCR products were purified using a QIAquick gel extraction kit (QIAGEN, USA), following the manufacturer's instructions. The purified PCR products were sequenced by using an Applied Biosystem 3730x1 DNA Analyzer (MyTACG Bioscience Company, MY). Phylogenetic Analysis Evolutionary analyses of ITS sequences were conducted in Molecular Evolutionary Genetics Analysis @EGA) 6.0 software to obtain alignment sequences (Tamura e t al. 2013). Homologous sequences were obtained from The GenBank database NCBI (http://blast. ncbi.nlm.nih.gov/) using BLASTN search (https://blast.ncbi.nlm.nih.gov/Blas t.cgi? PAGE-TYPE=BlastSearch) of the ITS sequences. The phylogenetic analysis was conducted using the Maximum Likelihood method based on the Tamura-Nei model with 1000 bootstrap test (Tamura & Nei 1993) in MEGA version 6.0. Saccharomyces cerevisde CBS 1171 (AB018043) was used as an outgroup (Fig. 3). The GenBank accession number of new sequences were listed in Table 1. Species Diversity The species diversity was calculated by using the Shannon-Weiner Index (Spellerberg 2008) as formula below: where: H' = Value of Shannon Wiener's diversity index pi = Proportion of species s = Number of species in community 1 = Number of indviduals in species RESULTS AND DISCUSSION A total of 69 isolates of fungi were obtained from 20 fruit and leaf samples of T. indica, and were identified based on their morphological characteristics (Fig. 2) and ITS sequence analysis (Table 1 and Fig. 2). Thirty-two species belong to 15 genera were found in the present study including Aspergillas (4 species), Botyospbaerid (a single species), Colletotm'chmn (6 species), Cochliobolzls/ Czawalarid (2 species), Diaporthe (6 species), Fzuam'am (a single species), Lasiod$lodia (2 species), Nigrospoa (2 species), Penin'IIium (2 species), Tmncospora (a single species), Pbyllostictd (a single species), Sarcostroma (a single species), Tm'cboderma (a single species), and Xylarid (a single species) (Table 1). BIOTROPIA Vol. 28 No. 3,2021 E L I 1;igut-e 2 1:ungal morphological retrieved in the culture media isolation procedure. F,ndoph\-tic fungi were isolated From fruits and leaves o f T ilzdi~u. I u n g i a-ere cultivated i n PDA m e d i u m a t 27 O C foi- 7 da1.s Based o n phylogenetic analysis of ITS Basidiomycota). Clade A was divided into 2 sub- sequences of the 69 endophytic fungi isolated clades; Clade A1 represents isolates of from tamarind fruits and leaves, two major A.pe?gi/h.r, Bot!yoshaerid, Co//etot~icbzm, I>iapof-t/3e, clades ( A and B) were generated (Fig. 3). The Fi~samUm, Nigrospa, ,.I'arcostroma, Tncboderma, first clade (Clade A) comprises isolates of fungi P~e/?ici//iunz, Ph~~lhsticta, and X y l a t a , whereas Clade under Phylum Ascomycota and Clade B A2 represents isolates of Lasiod$lodia and contains Tru~zcospora tephropora B3 148 (Phylum Cu~-~j~t/aria/ Cocbliobolus. Endophyuc fungi from tamarinds - M o h d Zaini e t al. Table 1 ITS sequences GenBank accession number o f deposited fungal isolates from fruits and leaves of T. indicd No. Isolates S~ecies Plant oart GenBank accession number Aspeqi/lus aculeatus A . carbonarizls A . Javus A. tubingensis A. tubingensis Boflyosphaeria mamane Colletotrichnm aenigma C. brevispom C. cobbittiense C.ficticola C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gheosporioides C. siamense C. siamense C. siamense C. siamense C. sidmense C. siamense C. siamense C. siamense Cumlaria lunata C. lnnata Cochliobolus geniculatns C. lunata Diqorfhe arecae D. ceratozamiae D. phaseolorzlm D. phaseolom D. phaseolom D. phaseolomm D. phaseolomm D. phaseolorcrm D. psendomang$rae D. pseudooculi D. psendoocnli D. psendophoenicicola D. psendophoenicicala Fusarium solani k o d i p l o d i a psendo fheobmae L theobromae L theobromae Nigrospora lacticolonia N. lacticolonia N . lactiolonia N . olyzae N. olyzae N . oryxae N. ovyxae Penicillinm roIfsii P. ro&ii P. rofsii P. rofsii P. ro&si; P. uemculosum Ph_yllostictaf.ll@iae Sarcostmma bisetnlatum Trichodemza asperellurn T . asperellurn T . asperellurn Fruit Fmit Fmit Fruit Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Fruit Fruit Fruit Fruit Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Fruit Leaf Fruit Leaf Leaf Leaf Leaf Leaf Fruit Leaf Leaf Leaf Leaf Fruit Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Fmit Fruit Fruit Leaf Leaf Fruit Leaf Leaf Fruit Fmit Fmit Leaf Leaf BIOTROPIA Vol. 28 No. 3,2021 Figure 3 Phylogenetic tree generated from the Maximum Likelihood method based on the ITS sequences of 69 fungal endophytes sequences associated with T. indim. The tree generated using Tamura-Nei model with 1000 Bootstrap replications. All Bootstrap scores with less than 50% are not shown in the tree Mt043776 C gloeosporrodes 83154 MT043780 C glaeosponades 83158 MT043796 C glaeosponades 83186 MT043799 C gloeosporiodes 83169 The Shannon index (H' = 3.083) indicated most diverse fungal genera isolated from that the tamarind fungal community possesses a tamarind leaves was Colletot?ichzlm and Diaporthe vast diversity of endophyuc fungi (Table 2). The (Fig. 3, Table 1). 58 - MT043784 C glaeosponardedes 83162 MT043786 C cobbiffiense 83170 MT043776 C glaeosponardedes 83154 MT043781 C glaeosporiardes 83154 - MT043792 C glaeosporiaides 83182 MK204297 C. siamense 82885 MK204295 C siamense 82892 MT043774 C g1aeospo"oides 83152 MT043801 C glaeosporioides 83191 MK204289 C froct,cola 82961 MK204314 C. aengma 82881 93 - MK204292 C. siamense 82907 MK204294 C. siamense 82921 ~ l i a x 69 MT043769 C. brev,sparum 83145 MK204285 F salani82964 MK204288 T asperellum 82963 MT043803 N aryrae 83193 MT043779 N lact,calonra 83157 MT043782 N lact,calonra 83160 MK204313 N a w e 82889 MT043788 S biswfulatum 83172 MT043785 X feejeensis 83163 5 3 MT043765 D phaseolomm 83141 A l i A 1 76 -1g MT043763 D phaseolomm 83161 MT043777 D phaseolomm 83155 MT043770 D phaseolomm 83147 MT043800 D. phaseolomm 83190 MK204303 D. phaseolomm 82940 MK204305 D pseudomangtferae 82928 MT043798 D pseudooculr 83168 MT043772 D ceatozamiae 83150 MT043773 D pseodophoenicicola 83151 MT043790 D pseudoculr 83180 MT043793 D pseodophoenicicola 83183 71 MK204301 D. arecae 82952 MK204300 P venuculasum 82958 89 - A l i b 99 MK204304 A aculeatus 82931 - 52 MK204306 P mlfw, 82925 MK204308 P mlfsir 82919 MK204310 P mlfw, 82899 MK204309 P mlfw, 82894 MT043804 P falloprae 83194 A l i i 9 3 MT043767 B mamane 83143 MT043789 L theobmmae 83179 MT043794 L pseudotheobmmae 83184 MK204311 A tubrngensrs 82916 MK204302 A carbonanus 82948 - - A 2 MK204299 A Lvus 82959 B MK204312 C lunsta 82902 MT043766 C lunata 83144 MT043771 T tephmpora 83148 NR 111007 Sacchammyces cerevrsrae Endophyuc fungi from tamarinds - Mohd Zaini e t al. Table 2 Endophyuc fungal percentage and Shannon-Wiener Index obtained from culture media isolation using fruits and leaves of T. indicd No. Species Number of isolate C. breuisporm C. cobbittiease C. fmcticola C. &eosporioides C. siamense C. lunata Cochliobolm geniculatu~ Diaporthe arecae Fusarium solani Lasiodiplodia theobromae L pseudotheobromae Nigrospora lactz'colonia N . olyxae Penicillum ro@ii Tmncospora tephropora Ph_yllostictafallqbiae Sarcostroma bisetulatum Tiicboderma asperelhi Xylaria feejeensis Percentage (O/O) 1.45 1.45 1.45 2.90 1.45 1.45 1.45 1.45 1.45 14.49 11.59 4.35 1.45 1.45 1.45 8.70 1.45 2.90 2.90 1.45 2.90 1.45 4.34 5.79 7.25 1.45 1.45 1.45 1.45 4.34 1.45 Shannon-Wiener Index (H') Total 69 100 3.083 In this study, the most abundant fungal (26 isolates) species obtained from T. indicd leaves was from genus Colletotm'chum where 10 isolates were identified as C. gloeosporioides with 14.49% (H' = 0.280). Endophyuc C. fructicola and C. siamense have been recovered from healthy Cymbopogon citratgs (Manamgoda e t al. 201 3). Weir et al. (2012) stated that C. siamense is geographically diverse with a varied host range and is a common saprobe or endophyte. Colletotm'chum species can be found abundantly forming its association with temperate plants and they are widely distributed in the tropical and subtropical areas (Cannon e t al. 2012), but no report on associations with T. indim. A study by Boddy (2016) also reported that Colletotm'chum species could be existed w i h n plant tissues without causing any harm while it is in an inactive state. These studies showed that members of Colletotm'chum exhibit a multiple life styles. Six isolates of endophytic Diaporthe phaseolomm have been isolates from healthy fruits and leaves of T. indica. Diapon'he spp. are known to be existed symbiotically alongside plants as saprobic, endophytic or phytopathogenic (Udayanga e t aL 201 1; Tan e t al. 201 3; Gomzhina & Gannibal 201 8). According to Gonzalez and Tello (2011), endophytic Diapon'he species are commonly isolated from several hosts in the temperate and tropical region. Research on Diapon'he species by Gomes e t al. (2013) collected several species of Diaporthe from Vaccinium growing regions in Europe i n c l u h g D. phaseolomm and D . arecae. Diaporthe pseudomangiferae has been reported cause inflorescence rot, rachis, canker, and flower abortion of mango (Serrato-Diaz e t al. 2014). BIOTROPIA Vol. 28 No. 3,2021 Endophytic C. lanata and Cochliobolas Pbyllostica species have been known to form genicalata; (telemorph of C. genicalata) have been isolated from leaves of T. indim. Two distinct species from genus Lasidioplodid that were isolated from the leaves of tamarind were Lasidioplodia theobromae and Lsidioplodia pseadotheobromae with a simdarity percentage of 99% and 97% respectively. Similar to Colletotn'cham species, Carvalaria/Cochliobolas and Ldsiodiplodia are well-known plant pathogens and can also be endophytes. In t h s study, Aspe@ills tabengensis was found associated with the T. indicd leaves. This species was found to form an association with many plant species such as the mangrove plant, Sonora desert plant (Nadumane et al. 201 6), and strawberry (Palmer et al. 2019). Previously, other species of Aspe@llas which is Aspe~illas niger was isolated from diseased-fruits of T. indica and caused black pod (Meena et al. 2018). Two species of Penicillm, P. ro@ii and P. vemcdosam have been isolated from healthy fruits and leaves of T. indim. Penicillizlm spp. are common pathogens and caused spoilage in fruits (Peter & Patrick 2017). The assemblage of endophytic fungi in healthy tissue of T. indica may indicate that some of the fun@ are possible latent pathogens and some may saprophytic. The other genus dominated the T. indica leaves was Nigrospora sp. Wang et al. (2017) claimed that Nigrospora sp. is a common in forming symbiosis with plants as pathogens, endophytes or saprophytes. Nigroqora sphaevica (synonym of N. oy~ae) was found inhabiting numerous hosts such as the Zea, Andropogon and Cymbopogon as reported by Wang et al. (2017). Supaphon and Preedanon (2019) also claimed, the species was isolated from Hehntbas annas as an endophyte. Botyo~haerid mamane was only one isolate obtained from this genus. According to Phdlips et al. (2013), this species that belonged to the Botryosphaeriaceae is existed diversely in nature as pathogenic, endophytic or saprobic with more preferable to woody plants. A study by Li et al. (201 8), also recorded the discovery of species of Botryosphaeriaceae from plantation trees including Canninghamina lanceolata, Dimocaqas longan, Melastoma sanguineam and Phoenix hanceana, whch were growing adjacent to Eaca4pta-r. their association with plants widely and can be either pathogens or endophytes. In this study, one isolate of Phyllostictd fallopide with a 100% percentage of similarity with the established sequence in the GenBank database. The morphology of the isolate characterized as P. fallpiae also fit the description of this species by Zhang et al. (2013). One isolate was identified as Xylarid feejeensis which was isolated from healthy leaves samples with 98.90% similarity to the GenBank sequences. According to Chen et al. (201 3) xylariaceous fungi are dominantly associated with the Dendrobiam species of class Orchdaceae. This f i n h g had supported the existence of Xyb& sp. as an endophyte. Trancospora tephropora (synonym of Perenniporid tephropora) was the only basidiomycete found associated with healthy T. indicd leaves with similarity percentage of 99.84% from the sequence from GenBank database. CONCLUSION ' I h s study revealed that various endophytic fungi were isolated from the fruits and leaves of tamarind. The 31 species that have been successfully identified were A. acaleatas, A . carbonakas, A . flavas, A . tabingensis, B. mamane, C. aenigma, C. brevispomm, C. cobbittiense, C. fmcticola, C. gloeo~;pokoides, C. sidmense, C. genicalatas, C. lanata, D. arecae, D. ceratoxamiae, D. phaseoloram, D. pseadomangiferae, D. pseadoocali, D. pseadophoenicicoh, F. solani, L. psezldotheobromae, L. theobromae, N. lacticolonia, N. ogqae, P. ro@ii, P. vemcalosam, T. tephropora , P. fallopiae, S. bisetalatzlm, T. asperelltlm and X.feejeensis. 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