Endophytic Colletotrichum from tropical grasses ...

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Institute of Excellence in Fungal Research, Mae Fah Luang. University, Chiang Rai ..... spored C. axonopodi Crouch, B.B. Clarke, J.F. White & B.I.. Hillman, C.
Fungal Diversity DOI 10.1007/s13225-013-0256-3

Endophytic Colletotrichum from tropical grasses with a new species C. endophytica Dimuthu S. Manamgoda & Dhanushka Udayanga & Lei Cai & Ekachai Chukeatirote & Kevin D. Hyde

Received: 16 April 2013 / Accepted: 3 July 2013 # Mushroom Research Foundation 2013

Abstract Endophytic fungi are a diverse and important group of microorganisms. We investigated the occurrence of Colletotrichum species as endophytes in two common tropical grass species, Pennisetum purpureum (dwarf napier) and Cymbopogon citratus (lemon grass) in Thailand. Combined phylogenetic analysis of ITS, partial sequences of actin (ACT), calmodulin (CAL) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene regions and morphology were used to characterize the species. This is the first report of an association as endophytes of Colletotrichum fructicola, C. tropicale and C. siamense with Pennisetum purpureum, and C. fructicola and C. siamense with Cymbopogon citratus. Colletotrichum endophytica sp. nov. associated with Pennisetum purpureum, is introduced based on multi-locus phylogenetic analysis with descriptions and illustrations. The potential hyperdiversity of the endophytic Colletotrichum species associated with tropical grasses is discussed with an emphasis on future research. Keywords Phylogeny . Poaceae . Taxonomy . Pennisetum purpureum . Cymbopogon citratus

Introduction Colletotrichum is an important pathogenic genus causing anthracnose of various plant hosts including grasses and D. S. Manamgoda : D. Udayanga : L. Cai (*) State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No 3 1st West Beichen Road, Chaoyang District, Beijing 100101, People’s Republic of China e-mail: [email protected] D. S. Manamgoda : D. Udayanga : E. Chukeatirote : K. D. Hyde Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand D. S. Manamgoda : D. Udayanga : E. Chukeatirote : K. D. Hyde School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

cereals worldwide (Crouch et al. 2009a, b; Prihastuti et al. 2010; Hyde et al. 2009a, b). Colletotrichum species have been recorded as endophytes in almost all major groups of angiosperms (Rivera-Orduña et al. 2011; Hofstetter et al. 2012; Tadych et al. 2012), conifers (Damm et al. 2012; Cannon et al. 2012), ferns (McKenzie et al. 2009) and lichens (Petrini et al. 1990). The genus has been recorded in association with more than 42 genera in the family Poaceae (Crouch and Beirn 2009). Most graminicolous Colletotrichum species generally produce falcate conidia (Sutton 1980; Shenoy et al. 2007; Crouch et al. 2009a). Some exceptional cases of Colletotrichum species producing elliptic conidia have been reported as pathogens on Sorghum sp. and rye grass (Lolium perenne L.) (Neill 1940; Mathur et al. 2002). Molecular data are essential for the accurate identification of the species in Colletotrichum and the current number of accepted species supported with molecular data is likely to rise with future studies (Hyde et al. 2009a; Cai et al. 2011a, b; Weir et al. 2012; Cannon et al. 2012). Colletotrichum graminicola (Ces.) G.W. Wilson and C. sublineola Henn. ex Sacc. & Trotter are important graminicolous species responsible for leaf anthracnose disease of Zea maize L. (Sutton 1980) and Colletotrichum graminicola has been used as a model organism for studying genetics and pathogenicity (Crouch and Beirn 2009). Several endophytic species of Colletotrichum have been isolated from a single host in recent studies. For example, 17 different Colletotrichum species, including seven new species have been reported from Bletilla ochracea Schltr. (Chinese Butterfly Hardy ground orchid) (Tao et al. 2013). In another study, 39 Colletotrichum isolates of C. gloeosporioides (Penz.) Penz. & Sacc. sensu lato, C. boninense Moriwaki, and C. simmondsii R.G. Shivas & Y.P. Tan have been isolated from Schinus terebinthifolius Raddi (Brazilian pepper tree) (Lima et al. 2012). There have been several studies on grass endophytes (Sánchez Márquez et al. 2007, 2010; Ghimire et al. 2011), but the diversity of Colletotrichum species found as endophytes is still poorly understood. Many endophytic

Fungal Diversity

Colletotrichum species are identified into broad species complexes such as C. gloeosporioides and C. boninense. Endophytic taxa within these complexes are rarely identified to actual phylogenetic species (Guo et al. 2008; Pimentel et al. 2006). Although there have been a number of studies on endophytic Colletotrichum species from temperate grasses (Crouch et al. 2009b; Su et al. 2010; Mouhamadou et al. 2011), the endophytic taxa on tropical grasses are poorly studied. The objective of this study is to determine the species of endophytic Colletotrichum associated with the tropical grasses Pennisetum purpureum Schumach. and Cympogon citratus (DC.) Stapf. in northern Thailand. Multi-gene phylogenetic analysis is used to determine species relationships with a description of a new species from tropical grasses. A discussion is provided with an account of the potential hyperdiversity of Colletotrichum species occurring in tropical grass hosts with emphasis on future research.

Materials and methods Collection and Isolation Healthy grass leaves and sheaths of Pennisetum purpureum and Cymbopogon citratus were collected from Chiang Kong teak forest, and several other locations in Muang District, Chiang Rai, Thailand. Fungal endophytes were isolated according to the method described by Unterseher et al. (2012). After 3–5 days, the leading edges of mycelium growing from the sides of sterilized grass pieces were sub cultured onto PDA. The Colletotrichum strains were primarily identified from the other fungal species based on cultural characteristic on PDA, morphology of the spores and phylogenetic evidence of ITS sequence data. DNA extraction, PCR amplification and Sequencing DNA was extracted from the fresh mycelium, using the protocol as described in Manamgoda et al. (2011). Amplification of ITS, GAPDH, ACT and CAL gene regions was carried out using primers ITS4/ITS5 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT512F/ACT783R (Carbone and Kohn 1999) and CL1/CL2A (O’Donnell et al. 2000) respectively. PCR conditions were followed as described by Prihastuti et al. (2009). PCR purification and sequencing were carried out by the SinoGenoMax Company, Beijing, China. Phylogenetic analysis Sequences were assembled by with Sequencher 4.9 for Windows (Gene Codes Corp., Ann Arbor, Michigan) aligned

initially with Clustal X (Thompson et al. 1997) and optimized by the online sequence alignment tool MAFFT version 7 (Katoh et al. 2002; Katoh and Toh 2008). Additional reference nucleotide sequences were downloaded from GenBank (Table 1). Single and combined gene analyses of ITS, GAPDH, CAL and ACT were carried out. Parsimony trees were inferred by PAUP v4.0b10 (Swofford 2002) using a heuristic search option with 1,000 random sequence additions. All gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1,000 replicates. Descriptive tree statistics for parsimony (Tree Length [TL], Consistency Index [CI], Retention Index [RI], Related Consistency Index [RC] and Homoplasy Index [HI]) were calculated. Model of evolution for all data sets were estimated by MrModeltest 2.3 (Nylander 2004; Nylander et al. 2008). Posterior probabilities (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.1 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Six simultaneous Markov chains were run for 1×106 generations and trees were sampled every 100 generation resulting in 20,000 total trees (in two simultaneous analyses). The first 2,000 trees, representing the burn-in phase of the analyses were discarded and the remaining trees in each analysis were used to calculate posterior probabilities in the majority rule consensus tree (Cai et al. 2006; Liu et al. 2012). ML genetrees were estimated using the software RAxML 7.2.8 Black Box (Stamatakis 2006; Stamatakis et al. 2008) in the CIPRES Science Gateway platform (Miller et al. 2010). For the concatenated dataset all free modal parameters estimated by RAxML with ML estimate of 25 per site rate categories. The concatenated dataset was partitioned by loci in RAxML platform. The RAxML software accommodated the GTR model of nucleotide substitution with the additional options of modeling rate heterogeneity (Γ) and proportion invariable sites (I). Trees were figured in Treeview (Page 1996) and MEGA 5 (Tamura et al. 2011).

Results Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (1 isolate), Colletotrichum siamense (9 isolates), Colletotrichum tropicale E.I. Rojas, S.A. Rehner (1 isolate), and an unidentified Colletotrichum species (2 isolates) were isolated as endophytes from Pennisetum purpureum. A further three isolates were identified as a new species, C. endophytica, which is introduced in this paper. Three endophytic Colletotrichum isolates from Cymbopogon citratus were identified as C. siamense (3 isolates) and C. fructicola (1 isolate). According to this study the most common endophytic species on tropical grasses was

Fungal Diversity Table 1 Sources of isolates and GenBank accession numbers used in the study Species

Code

Host

Location

GenBank accession numbers

Ref.

ITS

GAPDH

ACT

CAL

MFLUCC090233* Coffea arabica

Thailand

FJ972612

FJ972576

FJ907424

FJ917506

Prihastuti et al. 2009

MFLUCC090232

Coffea arabica

Thailand

FJ972605

FJ972571

FJ903188

FJ917501

Prihastuti et al. 2009

MFLUCC090234

Coffea arabica

Thailand

FJ972615

FJ972573

FJ907421

FJ917503

Prihastuti et al. 2009

C. clidemiae

ICMP 18658*

Clidemia hirta

USA

JX010265

JX009989

JX009537

JX009645

Weir et al. 2012

C. cliviae

CBS 125375*

Clivia miniata

China

GQ485607 GQ856756 GQ856777 GQ849464 Yang et al. 2009

C. cordylinicola

MFLUCC 090551* Cordyline fruticosa Thailand

C. asianum

JX010226

JX009975

HM470235 HM470238 Weir et al. 2012 Phoulivong et al. 2010

C. endophytica

C. fructicola

C. gloeosporioides

C. horii C. kahawae

C. musae

LC0324*

P. purpureum

Thailand

KC633854 KC832854 KF306258 KC810018 This study

LC1216

P. purpureum

Thailand

KC633853 KC832853 KC692467 KC810017 This study

LC0327

P. purpureum

Thailand

KC633855 KC832846 KC692468 KC810016 This study

MFLUCC 100676 Wild fruit

Thailand

KF242123

KF242181

KF157827

KF254846

Udayanga et al. 2013

MFLUCC090228* Coffea arabica

Thailand

FJ972603

FJ972578

FJ907426

FJ917508

Prihastuti et al. 2009

MFLUCC090226

Coffea arabica

Thailand

FJ972602

FJ972579

FJ907427

FJ917509

Prihastuti et al. 2009

LC0542

C. citratus

Thailand

KC633852 KC832841 KC692466 KC810019 This study

LC0540

P. purpureum

Thailand

KC633859 KC832842 KC669628 KC835391 This study

IMI 356878*

Citrus sinensis

Italy

JX010152

CORCG 5

Orchid

Thailand

HM034809 HM034807 HM034801 HM034803 Phoulivong et al. 2010

CORCG4

Orchid

Thailand

HM034808 HM034806 HM034800 HM034802 Phoulivong et al. 2010

JX010056

JX009531

JX009731

Weir et al. 2012

ICMP10492*

Persimmon sp.

China

GQ329690 GQ329682 GU133374 GU133376 Xie et al. 2010

TSG002

Persimmon sp.

China

AY791890 GQ329680 GU133379 GU133381 Xie et al. 2010

IMI 319418*

Coffea arabica

Africa

JX010231

JX010012

JX009452

JX009642

Weir et al. 2012

IMI363578

Coffea arabica

Africa

FJ972607

FJ972607

FJ907433

FJ917515

Prihastuti et al. 2009

CBS 116870*

Musa

USA

HQ596292 HQ596299 HQ596284 -

MFLUCC100976

Musa

Thailand

HQ596293 HQ596300 HQ596285 HQ596296 Su et al. 2011

Su et al. 2011

MFLUCC100977

Musa

Thailand

HQ596294 HQ596301 HQ596286 HQ596297 Su et al. 2011

MFLUCC100978

Musa

Thailand

HQ596295 HQ596302 HQ596287 HQ596298 Su et al. 2011

C. psidii

CBS 145.29*

Psidium sp.

Italy

JX010219

JX009967

JX009515

JX009743

Weir et al. 2012

C. queenslandicum

ICMP 1778*

Carica papaya

Australia

JX010276

JX009934

JX009447

JX009691

Weir et al. 2012

C. salsolae

ICMP 19051*

Salsola tragus

Hungary

JX010242

JX009916

JX009562

JX009696

Weir et al. 2012

C. siamense

MFLUCC090231

Coffea arabica

Thailand

FJ972614

FJ972574

FJ907422

FJ917504

Prihastuti et al. 2009

MFLUCC090230* Coffea arabica

Thailand

FJ972613

FJ972575

FJ907423

FJ917505

Prihastuti et al. 2009

LC0548

P. purpureum

Thailand

KC633856 KC832849 KC669625

LC1212

P. purpureum

Thailand

KC633857 KC832848 KC669626 KC692471 This study

LC0532

C.citratus

Thailand

KC633858 KC832852 KC669627 KC692472 This study

LC1232

P. purpureum

Thailand

KC633860 KC832845 KC676709 KC692473 This study

This study

LC0326

P. purpureum

Thailand

KC633861 KC832856 KC676710 KC692474 This study

LC0347

C. citratus

Thailand

KC633862 KC832851 KC676711

LC0329

P. purpureum

Thailand

KC633863 KC832843 KC676712 KC692475 This study

This study

LC1235

P. purpureum

Thailand

KC633865 KC832850 KC676714 KC692477 This study

LC0323

P. purpureum

Thailand

KC633866 KC832855 KC676715 KC692478 This study

Fungal Diversity Table 1 (continued) Species

Code

Host

Location

GenBank accession numbers ITS

C. siamense ( syn. C. hymenocallidis C. siamense ( syn. C. jasminisambac)

GAPDH

ACT

Ref. CAL

LC1234

P. purpureum

Thailand

KC633867 KC832844

LC1225

P. purpureum

Thailand

KC633869 KC832847 KC676716

LC0328

P. purpureum

Thailand

KC633868 KC832858

CBS 125378*

Hymenocallis sp.

China

GQ485600 GQ856757 GQ856775 GQ849463 Yang et al. 2009

MFLUCC 100277* Jasminum sp.

Thailand

HM131511 HM131497 HM131507 HM131492 Wikee et al. 2011

LC 0922

Jasminum sp.

Thailand

HM131513 HM131499 HM131508 HM131494 Wikee et al. 2011

Australia

FJ972601

FJ972580

FJ907428

FJ917510

Prihastuti et al. 2009

*

KC692479 This study This study KC692480 This study

C. simmondsii

BRIP 28519

Carica papaya

C. theobromicola

CBS 124945*

Theobroma cacao

Panama

JX010294

JX010006

JX009444

JX009591

Weir et al. 2012

C. ti

ICMP 4832*

Cordyline sp.

JX010269

JX009952

JX009520

JX009649

Weir et al. 2012

C. tropicale

CBS 124949*

Theobroma cacao

New Zealand Panama

JX010264

JX010007

JX009489

JX009719

Weir et al. 2012

LC0537

P. purpureum

Thailand

KC633864 KC832857 KC676713 KC692476 This study

C. viniferum

gg11

Vitis vinifera

China

JN412807

JN412799

JN412790

C. xanthorrhoeae

BRIP 45094*

Australia

JX010261

JX009927

JX009478

LC0551

Xanthorrhoea preissii P. purpureum

Thailand

KC633850 KC835389 KC633871 KC692470 This study

LC1238

P. purpureum

Thailand

KC633851 KC835390 KC633870 KC692469 This study

Colletotrichum sp.

Peng et al. 2013 JX009653

Weir et al. 2012

Endophytic cultures isolated from grasses and sequences generated in this study are shown in bold, * ex-type cultures

C. siamense sensu lato. Two isolates, LC 1238 and LC 0551 from P. purpureum are genetically closely related to C. cliviae Yan L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai, C. orchidearum Allesch. and C. destructivum O’Gara according to the NCBI’s BLAST search of ACT, GAPDH and CAL genes. Type sequences for C. destructivum are not available for accurate comparison in the phylogenetic analysis, therefore these isolates were not unequivocally identified in this study. The combined ACT, CAL, GAPDH and ITS alignment included 54 sequences with C. simmondsii as the outgroup taxon. The parsimony analysis revealed that from the 1,579 characters, 951 characters were constant, 414 characters were parsimony informative, while 214 variable characters are parsimony-uninformative. MP, BI and ML trees generated were identical. One of the most parsimonious trees generated from parsimony analysis of combined dataset is presented in Fig. 1, with Bayesian posterior probabilities and rapid bootstrapping estimations of RAxML, indicated on the branches of MP tree (Fig. 1) (TL=1,071, CI= 0.799, RI=, 0.870, RC=0.695, HI=0.201). Taxonomy Colletotrichum endophytica Manamgoda, Udayanga, L. Cai & K.D. Hyde, sp. nov. (Figure 2) MycoBank: MB 803923

Etymology: named after its original habitat as a grass endophyte Colonies on PDA attaining 75 mm diam. in 6 days at 25 °C, growth rate per day 6.5 ±0.3 mm (n=6), white to grey, reverse dark grey to black at the centre, aerial mycelium, dense and raised, with orange conidial masses. On culture on PDA or slide cultures on PDA, Sexual state: not produced Asexual state: Conidiophores 22–24 (−27) μm (x̄=23 SD=1.2 n=26) long, cylindrical or clavate, the base of the conidiophore is wide up to 5 μm, hyaline, unbranched and arranged in clusters. Conidia 13–19 (−21)×(3.6–) 4.5–5.5 μm (x̄=16.2 SD=2.7 n=40; x̄= 4.83 SD=0.45 n=40), unicellular, hyaline, cylindrical sometimes slightly ovoid, with rounded ends. Appressoria formed in slide culture on PDA measured 8–12 (−15)×(4–8) μm (x̄=10.4 SD =2.4 n=30; x̄=5.7 SD=1.6 n=30), formed from branched mycelia, terminal, brown to dark brown, variable in shape, irregular unlobed or slightly lobed, rarely obviously lobed, appressoria complex formation observed. Known host: Pennisetum purpureum, unknown wild fruit in northern Thailand Known distribution: Northern Thailand Material examined: THAILAND, Chiang Rai Province, endophytic on healthy leaves of Pennisetum purpureum, 5 May 2010, Dimuthu S. Manamgoda GEN005B (MFLU13-0004, dried culture, holotype; ex-type living culture=MFLUCC 130418 = LC0324); ibid., 4 April 2010, Dimuthu S.

Fungal Diversity

LC 1235 Pennisetum LC 0323 Pennisetum

100/91/NS

LC 1232 Pennisetum LC 0326 Pennisetum LC 1234 Pennisetum LC0 328 Pennisetum LC 0922 Jasminum MFLUCC 100277 Jasminum (C.jasmini-sambac) MFLUCC 090231 Coffea LC 0548 Pennisetum LC 1225 Pennisetum

C. siamense s.l

MFLUCC 090230 Coffea LC 1212 Cymbopogon

94/70/NS

LC 0532 Cymbopogon LC 0329 Pennisetum 83/NS/NS

LC 0347 Cymbopogon CBS 125378 Hymenocallis (C.hymenocallidis) LC 0537 Pennisetum CBS 124949 Theobroma

98/58/92

C. tropicale

MFLUCC 090226 Coffea MFLUCC 090228 Coffea

96/92/96

C. fructicola

LC 0540 Pennisetum

100/NS/99

LC 0542 Cymbopogon

99/88/92

gg11 Vitis 97/NS/NS

C. viniferum

CBS 116870 Musa 100/99/100 MFLUCC

100978 Musa

MFLUCC 100977 Musa

C. musae

MFLUCC 100976 Musa

97/NS/NS

ICMP 19051 Salsola ICMP 1778 Carica

C. salsolae C.queenslandicum

MFLUCC 090234 Coffea NS/82/88

MFLUCC 090233 Coffea

C. asianum

MFLUCC 090232 Coffea

100/100/99

78/75/86

100/84/93

LC 0324 Pennisetum

100/100/85

LC 1216 Pennisetum

C. endophytica

LC 0327 Pennisetum MFLUCC 100676 wild fruit 100/99/92 100/100/99

70/71/NS

CORCG5 Orchid IMI 356878 Citrus

C. gloeosporioides

CORCG4 Orchid 100/100/100

ICMP 10492 Diospyros TSG002 Diospyros

70/NS/91

BRIP 45094 Xanthorrhoea 100/100/100 97/76/78

IMI 363578 Coffea IMI 319418 Coffea CBS 145.29 Psidium

100/100/NS 99/98/91

MFLUCC 090551 Cordyline ICMP 4832 Cordyline ICMP 18658 Clidemia

100/100/NS 100/79/86

CBS 124945 Theobroma

100/100/100 100/100/100

LC 1238 Pennisetum

C. xanthorrhoeae C. kahawae C. psidii C. cordylinicola C. ti C. clidemiae C. theobromicola Colletotrichum sp.

LC 0551 Pennisetum CBS 125375 Clivia

BRIP 285519 Carica

C. horii

C. cliviae C. simmondsii

10

Fig. 1 Phylogram generated from maximum parsimony analysis based on combined ITS, GAPDH, ACT and CAL gene sequences, showing the phylogenetic relationships of graminicolous endophytic Colletotrichum species (shown in green). Bayesian posterior

probabilities of more than 95 %, parsimony bootstrap of more than 75 %, and RAxML rapid bootstrapping estimations of more than 75 % are indicated above the branches. NS=not significantly supported

Manamgoda GEN005F (MFLU13-0003, dried culture; living culture=MFLUCC 130417=LC1216); ibid., 8 April 2010, Dimuthu S.Manamgoda, GEN002B (MFLU13-0005, dried

culture; living culture=MFLUCC 130419=LC0327); ibid., on an unknown wild fruit, 27 February 2010, D. Udayanga DNCL0075 (Living culture=MFLUCC 10–0676)

Fungal Diversity Fig. 2 Morphology of Colletotrichum endophytica (holotype MFLU 130418) a, b) culture grown on PDA c) orange coloured spore masses produced in culture d, e) Appressoria produced on slide cultures of PDA f) Conidiophores and conidia g) Conidia. Scale bars: c=50 mm d, = 10 μm e, f, g=5 μm

Notes: Endophytic Colletotrichum species from tropical grasses are poorly known. Strains of C. endophytica clustered in a distinct clade with high support in the multi-gene phylogenetic tree (Fig. 1), which is also the case in the single gene tree from GAPDH, CAL and ACT. On the other hand, ITS sequence data does not distinguish C. endophytica as a distinct species (data not shown). ITS data does not provide adequate species resolution in the C. gloeosporioides species complex. Colletotrichum clidemiae B.S. Weir & P.R. Johnst., C. tropicale, C. ti Weir & P.R. Johnst., and C. fructicola and some strains of C. siamense sensu lato have identical ITS sequences (Weir et al. 2012). Our analyses showed that C. endophytica is distinguished by genealogical concordance, and therefore is introduced as a novel species. Most Colletotrichum species found as endophytes in the tropics are also known to occur as pathogens and saprobes on wide range of hosts. This may be the same for C. endophytica. The species sits between the musae and kahawae clades, in the multi-gene phylogeny of the C. gloeosporioides species complex.

Discussion Colletotrichum species are pathogens and ubiquitous asymptomatic foliar endophytes in a vast range of hosts (Cannon and Simmons 2002; Osono 2008). It has been suggested that endophytic fungi may be latent saprobes (Promputtha et al. 2010; Purahong and Hyde 2011), latent pathogens (Brown et al. 1998; Photita et al. 2005) and/or mutualists (Saikkonen et al. 2010). However the interaction between most plants

and endophytes are still undetermined (Hyde and Soytong 2008; Rojas et al. 2010). Some Colletotrichum endophytes are potential bio-control agents and sources of important secondary metabolites (Aly et al. 2011; Debbab et al. 2011, 2012; Lima et al. 2012) and may be responsible for human disease due to aerospores (Sutton 1992; Vázquez de Aldana et al. 2013). Four species Colletotrichum endophytica, Colletotrichum fructicola, C. siamense sensu lato, Colletotrichum tropicale and two unidentified strains of Colletotrichum were recorded as endophytes from tropical grasses in this study. Most of the strains are C. siamense sensu lato which is known to have a wide host range (Weir et al. 2012). Colletotrichum fructicola was originally described from Coffea arabica (Prihastuti et al. 2009), but this species has also been recorded from Crinum asiaticum (Amaryllidaceae) Fragaria×ananassa, Malus domesticta, Pyrus pyrifolia (Rosaceae), Limonium sp. (Plumbaginaceae), Dioscorea alata (Dioscoreaceae), Arachis (Fabaceae) and Theobroma cocao (Malvaceae) (Hyde et al. 2009a; Yang et al. 2009; Weir et al. 2012). Colletotrichum tropicale Rojas, Rhener & Samuels was recorded as an endophyte in tropical regions associated with Theobroma cacao L. (Malvaceae), Trichilia tuberculata (Meliaceae), Viola surinamensis (Myristicaceae), Cordia aliodora (Boraginaceae), Annona muricata (Annonaceae) and Litchi chinensis (Sapindaceae) (Rojas et al. 2010; Weir et al. 2012). Colletotrichum endophytica was also found as a saprobe on an unknown wild fruit (Udayanga et al. 2013). The tropical Colletotrichum grass endophytes isolated in this study did not form a separate phylogenetic clade indicating that the species are host generalists and do not appear

Fungal Diversity

to cause disease of grass species. A recent study focusing on the relationship between airborne spores and allergen exposure found that air borne spores of fungal genera such as Cladosporium and Fusarium are common grass endophytes (Vázquez de Aldana et al. 2013). Endophytic Colletotrichum species commonly found on grasses may also become pathogens or saprobes and be responsible for airborne allergies in humans. The endophytic isolates from cacao in Panama were thought to comprise part of the background endophytic community in the local forest ecosystems (Rojas et al. 2010). The occurrence of multiple species from the two tropical grass species in this study shows the potential hyperdiversity of endophytic Colletotrichum. Further investigations are required to clarify the ecological relationships of the pathogenic and endophytic Colletotrichum species on crops, and tropical wild grasses. The presence of Colletotrichum species as endophytes adds an extra dimension to the understanding of host-specificity in the genus (Rojas et al. 2010; Cannon et al. 2012). Common temperate grass associated Colletotrichum species are falcatespored C. axonopodi Crouch, B.B. Clarke, J.F. White & B.I. Hillman, C. caudatum (Peck ex Sacc.) Peck, C. cereale Manns, C. falcatum Went, C. graminicola, C. hanaui Crouch, B.B. Clarke, J.F. White & B.I. Hillman, C. jacksonii Crouch, B.B. Clarke, J.F. White & B.I. Hillman, C. nicholsonii Crouch, B.B. Clarke, J.F. White & B.I. Hillman, C. paspali Crouch, B.B. Clarke, J.F. White & B.I. Hillman, C. sublineolum Henn. ex Sacc. & Trotter and C. eremochloae J.A. Crouch & TomasoPeterson (Crouch et al. 2009b; Crouch and Tomaso-Peterson 2012). There are a few Colletotrichum species reported from grasses as endophytes. For example, C. cereale, C. graminicola and C. phyllachoroides have previously been reported from the cool season grasses, Panicum virgatum and Suaeda fruticosa respectively (Fisher and Petrini 1987; Crouch et al. 2009a; b; Ghimire et al. 2011). An unidentified Colletotrichum species has been isolated from Dactylis glomerata and Holcus lanatus (Sánchez Márquez et al. 2007; 2010). In this study, C. endophytica, C. fructicola, C. siamense, and C. tropicale were isolated from Cymbopogon citratus and Pennisetum purpureum in Thailand. This is the first report of latter four species associated with tropical grasses. Future studies are needed on endophytic Colletotrichum species on a wide range of grass hosts and locations in the tropics to assess the occurrence and host range of species in grasses and to reveal their ecology and biology related to host distribution and life modes. In this study combined ACT, CAL, GAPDH, and ITS analysis enabled us to identify most of the isolates to species level, with the exception of two isolates. A large number of type sequences are available in GenBank for taxa in the C. gloeosporioides species complex, and therefore we could resolve most species accurately and with confidence using combined ACT, CAL, GAPDH, and ITS gene sequence data.

According to Weir et al. (2012), ITS, GAPDH, CAL and ACT is unable to distinguish C. aeschymones B.S. Weir & P.R. Johnst. and C. tropicale from all other species. Recent studies have shown that apn2/matIGS (intergenic spacer bridging the DNA lyase and mating type locus) provides greater resolution in the C. gloeosporioides species complex. Combined analysis of ITS, apn2, tub2 and ApMat gene-markers were reported to give a better resolution of cryptic C. gloeosporioides species (Doyle et al. 2013; Sharma et al. 2013). Colletotrichum siamense sensu lato also appears to be a species complex and was the most common grass endophyte isolated in this study. Combined analysis of the apn2/matIGS gene has revealed several different lineages within Colletrichum siamense sensu lato, including C. hymenocallidis, C. jasmini-sambac, C. siamense sensu stricto, C. melanocaulon and three undescribed clades which are probably new species (Doyle et al. 2013; Sharma et al. 2013; Udayanga et al. 2013). Generally, in future studies of Colletotrichum endophytes, we expect that most of species can be accurately identified using available molecular markers and the type sequence data available in public databases. Acknowledgments Dimuthu S. Manamgoda thanks Mushroom Research Foundation, Thailand and the State Key Laboratory of Mycology, Chinese Academy of Science, Beijing, China for Postgraduate Scholarships. K.D. Hyde thanks the National Research Council of Thailand, Colletotrichum grant number 54201020003 and King Abdulaziz City of Science and Technology, Riyadh, Saudi Arabia, project No. 10-Bio-965-02 to study Colletotrichum. L. Cai acknowledges grants NSFC 31070020 and CASKSCX2-YW-Z-1026. Rungtiwa Phookamsak and Fang Liu are thanked for laboratory assistance. We are very grateful to Dr. Roger G. Shivas, who gave valuable suggestions to this work during his academic visit to China (funded by NSFC 31110103906).

References Aly A, Debbab A, Proksch P (2011) Fifty years of drug discovery from fungi. Fungal Divers 50:3–19. doi:10.1007/s13225-011-0116-y Brown KB, Hyde KD, Guest DI (1998) Preliminary studies on endophytic fungal communities of Musa acuminata species complex in Hong Kong and Australia. Fungal Divers 1:27–51 Cai L, Jeewon R, Hyde KD (2006) Phylogenetic investigations of Sordariaceae based on gene sequences and morphology. Mycol Res 110:137–150. doi:10.1016/j.mycres.2005.09.014 Cai L, Giraud T, Zhang N, Begerow D, Cai G, Shivas RG (2011a) The evolution of species concepts and species recognition criteria in plant pathogenic fungi. Fungal Divers 50:121–133. doi:10.1007/ s13225-011-0127-8 Cai L, Udayanga D, Manamgoda DS, Maharachchikumbura SSN, Liu XZ, Hyde KD (2011b) The need to carry out re-inventory of tropical plant pathogens. Trop Plant Pathol 36:205–213. doi:10. 1590/S1982-56762011000400001 Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553– 556

Fungal Diversity Cannon PF, Simmons CM (2002) Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana. Mycologia 94:210–220 Cannon P, Damm U, Johnston P, Weir B (2012) Colletotrichum-current status and future directions. Stud Mycol 73(1):181–213. doi:10. 3114/sim0014 Crouch JA, Beirn LA (2009) Anthracnose of cereals and grasses. Fungal Divers 39:19–44 Crouch JA, Beirn LA, Cortese LM, Bonos SA, Clarke BB (2009a) Anthracnose disease of switchgrass caused by the novel fungal species Colletotrichum navitas. Mycol Res 113:1411–1421. doi:10. 1016/j.mycres.2009.09.010 Crouch JA, Clarke BB, White JF Jr, Hillman BI (2009b) Systematic analysis of the falcate-spored graminicolous Colletotrichum and a description of six new species from warm-season grasses. Mycologia 101:717–732. doi:10.3852/08-230 Crouch JA, Tomaso-Peterson M (2012) Anthracnose disease of centipedegrass turf caused by Colletotrichum eremochloa, a new fungal species closely related to Colletotrichum sublineola. Mycologia 104:1085–1096. doi:10.3852/11-317 Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012) The Colletotrichum acutatum species complex. Stud Mycol 73:37–113 Debbab A, Aly AH, Proksch P (2011) Bioactive secondary metabolites from terrestrial endophytes and associated marine derived fungi. Fungal Divers 49:1–12. doi:10.1007/s13225-011-0114-0 Debbab A, Aly AH, Proksch P (2012) Endophytes and associated marine derived fungi—ecological and chemical perspectives. Fungal Divers 57:45–83. doi:10.1007/s13225-012-0191-8 Doyle VP, Oudemans PV, Rehner SA, Litt A (2013) Habitat and Host Indicate Lineage Identity in Colletotrichum gloeosporioides s.l. from Wild and Agricultural Landscapes in North America. PLoS One 8:e62394 Fisher PJ, Petrini O (1987) Location of fungal endophytes in tissues of Suaeda fruticosa: a preliminary study. T Brit Mycol Soc 89:246–249 Ghimire SR, Charlton ND, Bell JD, Krishnamurthy YL, Craven KD (2011) Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tallgrass prairie of northern Oklahoma. Fungal Divers 47:19–27. doi:10. 1007/s13225-010-0085-6 Guo B, Wang Y, Sun X, Tang K (2008) Bioactive Natural Products from Endophytes: A Review. Appl Biochem Microbiol 44:136–142. doi:10.1134/S0003683808020026 Hofstetter V, Buyck B, Croll D, Viret O, Couloux A, Gindro K (2012) What if esca disease of grapevine were not a fungal disease? Fungal Divers 54:51–67. doi:10.1007/s13225-012-0171-z Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754–755 Hyde KD, Cai L, Cannon PF, Crouch JA, Crous PW, Damm U, Goodwin PH, Chen H, Johnston PR, Jones EBG, Liu ZY, McKenzie EHC, Moriwaki J, Noireung P, Pennycook SR, Pfenning LH, Prihastuti H, Sato T, Shivas RG, Tan YP, Taylor PWJ, Weir BS, Yang YL, Zhang JZ (2009a) Colletotrichum – names in current use. Fungal Divers 39:147–182 Hyde KD, Cai L, McKenzie EHC, Yang YL, Zhang JZ, Prihastuti H (2009b) Colletotrichum: a catalogue of confusion. Fungal Divers 39:1–17 Hyde KD, Soytong K (2008) The fungal endophyte dilemma. Fungal Divers 33:163–173 Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066. doi:10.1093/nar/ gkf436 Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9:286–298. doi:10. 1093/bib/bbn013 Lima JS, Figueiredo JG et al. (2012) Genetic Diversity of Colletotrichum spp. an Endophytic Fungi in a Medicinal Plant, Brazilian Pepper

Tree. International Scholarly Research Network ISRN Microbiology 2012: Article ID 215716: 7 doi:10.5402/2012/215716 Liu JK, Phookamsak R et al (2012) Towards a natural classification of Botryosphaeriales. Towards a natural classification of Botryosphaeriales. Fungal Divers 57:149–210. doi:10.1007/ s13225-012-0207-4 Mathur K, Thakur RP, Neya A, Marley PS, Casela CR (2002) Sorghum anthracnose—Problem and management strategies. In: Leslie J (ed) Sorghum and Millets Diseases. Iowa State Press, Ames, pp 211–220 Mckenzie SJ, Peres NA, Barquero MP, Arauz LF, Timmer LW (2009) Host range and genetic relatedness of Colletotrichum acutatum isolates from fruit crops and leatherleaf fern in Florida. Phytopathology 99:620–631. doi:10.1094/PHYTO-99-5-0620 Manamgoda DS, Cai L, Bahkali AH, Chukeatirote E, Hyde KD (2011) Cochliobolus: an overview and current status of species. Fungal Divers 51:3–42. doi:10.1007/s13225-011-0139-4 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop 2010 (GCE): 1–8 Mouhamadou B, Molitor C, Baptist F, Sage L, Clément J, Lavorel S, Monier A, Geremia RA (2011) Differences in fungal communities associated to Festuca paniculata roots in subalpine grasslands. Fungal Divers 47:55–63 Neill JC (1940) The endophyte of Rye-grass (Lolium perenne). New Zeal J Sci Tech 21:280A–291A Nylander JAA (2004) MrModeltest 2.0. Program distributed by the author. Evolutionary Biology Centre, Uppsala University Nylander JAA, Wilgenbusch JC, Warren DL, Swofford DL (2008) AWTY (are we there yet?): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics 24:581–583. doi:10.1093/bioinformatics/btm388 O’Donnell K, Nirenberg HI, Aoki T, Cigelnik E (2000) A Multigene phylogeny of the Gibberella fujikuroi species complex: Detection of additional phylogenetically distinct species. Mycoscience 41:61–78 Osono T (2008) Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations. Mycologia 100:387–391. doi:10.3852/07-110R1 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358 Peng LJ, Sun T, Yang YL, Cai L, Hyde KD, Bahkali AH, Liu ZY (2013) Colletotrichum species on grape in Guizhou and Yunnan provinces, China. Mycoscience 54:29–41. doi:10.1016/j.myc.2012.07.006 Petrini O, Hake U, Deryfuss MM (1990) An analysis of fungal communities identified from fructicose lichens. Mycologia 82:444– 451 Photita W, Taylor PWJ, Ford R, Lumyong P, McKenzie HC et al (2005) Morphological and molecular characterization of Colletotrichum species fromherbaceous plants in Thailand. Fungal Divers 18:117–133 Phoulivong S, Cai L, Chen H, McKenzie EHC, Abdelsalam K et al (2010) Colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal Divers 44:33–43. doi:10.1007/s13225010-0046-0 Pimentel IC, Glienke-Blanco C et al. (2006) Identification and colonization of endophytic fungi from soybean (Glycine max (L.) Merril) under different environmental conditions. Braz. arch. biol.technol. [online] 49: 705–711. ISSN 1516–8913 Prihastuti H, Cai L, Chen H, McKenzie EHC, Hyde KD (2009) Characterization of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Divers 39:89–109 Prihastuti H, Cai L, Crouch J, Phoulivong S, Moslem M, McKenzie E, Hyde KD (2010) Neotypification of Colletotrichum falcatum, the causative agent of red-rot disease in sugarcane. Sydowia 62:283– 293

Fungal Diversity Promputtha I, Hyde KD et al (2010) Can leaf degrading enzymes provide evidences that endophytic fungi becoming saprobes? Fungal Divers 41:89–99. doi:10.1007/s13225-010-0024-6 Purahong W, Hyde KD (2011) Effects of fungal endophytes on grass and non-grass litter decomposition rates. Fungal Divers 47:1–7. doi:10.1007/s13225-010-0083-8 Rannala B, Yang Z (1996) Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. J Mol Evol 43:304–311 Rivera-Orduña FN, Suarez-Sanchez RA, Flores-Bustamante ZR, Gracida-Rodriguez JN, Flores-Cotera LB (2011) Diversity of endophytic fungi of Taxus globosa (Mexican yew). Fungal Divers 47:65–74. doi:10.1007/s13225-010-0045-1 Rojas EI, Rehner SA, Samuels GJ, Van Bael SA, Herre EA et al (2010) Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish pathogen and endophyte clades. Mycologia 102:318–1338. doi:10.3852/09-244 Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572– 1574. doi:10.1093/bioinformatics/btg180 Saikkonen K, Saari S, Helander M (2010) Defensive mutualism between plants and endophytic fungi? Fungal Divers 41:101–113. doi:10.1007/s13225-010-0023-7 Sánchez Márquez S, Bills G, Zabalgogeazcoa I (2007) The endophytic mycobiota of the grass Dactylis glomerata. Fungal Divers 27:171–195 Sánchez Márquez S, Bills GF, Dominguez Acuna L, Zabalgogeazcoa I (2010) Endophytic mycobiota of leaves and roots of the grass Holcus lanatus. Fungal Divers 41:115–123. doi:10.1007/s13225009-0015-7 Sharma G, Kumar N, Weir BS, Hyde KD, Shenoy BD (2013) Apmat gene can resolve Colletotrichum species: a case study with Mangifera indica. Fungal Divers (In press) Shenoy BD, Jeewon R, Lam WH, Bhat DJ, Than PP et al (2007) Morpho-molecular characterisation and epitypification of Colletotrichum capsici (Glomerellaceae, Sordariomycetes), the causative agent of anthracnose in chilli. Fungal Divers 27:197–211 Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690. doi:10.1093/bioinformatics/btl446 Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 57:758–771 Su YY, Guo LD, Hyde KD (2010) Response of endophytic fungi of Stipa grandis to experimental plant function group removal in Inner Mongolia steppe, China. Fungal Divers 43:93–101. doi:10. 1007/s13225-010-0040-6 Su YY, Noireung P, Liu F, Hyde KD, Moslem MA et al (2011) Epitypification of Colletotrichum musae, the causative agent of banana anthracnose. Mycoscience 52:376–382. doi:10.1007/ s10267-011-0120-9 Sutton BC (1980) The Coelomycetes. Commonwealth Mycological Institute, Kew Sutton BC (1992) The genus Glomerella and its anamorph Colletotrichum. In: Bailey JA, Jeger MJ (eds) Colletotrichum Biology, Pathology and Control. CAB International, Wallingford, pp 1–26

Swofford DL (2002) PAUP 4.0b10: phylogenetic analysis using parsimony. Sinauer Associates, Sunderland Tao G, Liu ZY, Liu F, Gao YH, Cai L (2013) Endophytic Colletotrichum species from Bletilla ochracea (Orchidaceae) in Guizhou, China. Fungal divers (In press) Tadych M, Bergen M, Johnson Cicalese J, Polashock J, Vorsa N (2012) Endophytic and pathogenic fungi of developing cranberry ovaries from flower to mature fruit: diversity and succession. Fungal Divers 54:101–116. doi:10.1007/s13225-012-0160-2 Tamura K, Peterson P, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28:2731–2739. doi:10.1093/ molbev/msr121 Templeton MD, Rikkerink EHA, Solon SL, Crowhurst RN (1992) Cloning and molecular characterization of the glyceraldehyde-3phosphate dehydrogenaseencoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene 122:225–230 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876. doi:10.1093/nar/25.24.4876 Udayanga D., Manamgoda DS, Liu XZ, Chukeatirote E, Hyde KD (2013) What are the common anthracnose pathogens of tropical fruits?, Fungal Divers FUDI-D-13-00082 (accepted) Unterseher M, Petzold A, Schnittler M (2012) Xerotolerant foliar endophytic fungi of Populus euphratica from the Tarim River basin, Central China are conspecific to endophytic ITS phylotypes of Populus tremula from temperate Europe. Fungal Divers 54:133–142. doi:10.1007/s13225-012-0167-8 Vázquez de Aldana BR, Bills G, Zabalgogeazcoa I (2013) Are endophytes an important link between airborne spores and allergen exposure? Fungal Divers. doi:10.1007/s13225-013-0223-z, Online March 2013 Weir B, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115–180. doi:10.3114/sim0011 Wikee S, Cai L, Pairin N, McKenzie EH, Su YY, Chukeatirote E, Hyde KD (2011) Colletotrichum species from Jasmine (Jasminum sambac). Fungal Divers 46:171–182 White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: A Guide to Methods and Applications. Academic, New York, pp 315–322 Xie L, Zhang JZ, Cai L, Hyde KD (2010) Biology of Colletotrichum horii, the causal agent of persimmon anthracnose. Mycology 1:242–253 Yang YL, Liu ZY, Cai L, Hyde KD, Yu ZN, McKenzie EHC (2009) Colletotrichum anthracnose of Amaryllidaceae. Fungal Divers 39:123–146 Zhaxybayeva O, Gogarten JP (2002) Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses. BMC Genomics 3(1):4. doi:10.1186/ 1471-2164-3-4