What are the common anthracnose pathogens of tropical fruits? Dhanushka Udayanga, Dimuthu S. Manamgoda, Xingzhong Liu, Ekachai Chukeatirote & Kevin D. Hyde Fungal Diversity An International Journal of Mycology ISSN 1560-2745 Fungal Diversity DOI 10.1007/s13225-013-0257-2 1 23 Your article is protected by copyright and all rights are held exclusively by Mushroom Research Foundation. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Fungal Diversity DOI 10.1007/s13225-013-0257-2 What are the common anthracnose pathogens of tropical fruits? Dhanushka Udayanga & Dimuthu S. Manamgoda & Xingzhong Liu & Ekachai Chukeatirote & Kevin D. Hyde Received: 16 April 2013 / Accepted: 7 July 2013 # Mushroom Research Foundation 2013 Abstract Species of Colletotrichum are associated with anthracnose of a wide range of host plants including cultivated and wild tropical fruits. The genetic and ecological diversity of species associated with wild fruits are poorly explored, as compared to those associated with pre and postharvest diseases of cultivated fruits. In the present study, isolates of Colletotrichum were obtained from commercially available cultivated fruits, wild fruits (from native trees in natural habitats) and a few herbaceous hosts collected in northern Thailand. These isolates were initially characterized based on analysis of complete sequences of nuclear ribosomal internal transcribed spacer (ITS), into the genetically defined species complexes of Colletotrichum gloeosporioides, C. acutatum, C. boninense and C. truncatum. The isolates were primarily identified in the C. gloeosporioides species complex, based on a strongly supported clade within the ITS gene tree and were further characterized using multi-gene phylogenetic analyses and morphology. Phylogenetic analyses of ITS, partial sequences of actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and βtubulin (TUB2) genetic markers were performed individually and in combination. Colletotrichum gloeosporioides sensu stricto was identified from lime (Citrus aurantifolia) and rose D. Udayanga : D. S. Manamgoda : X. Liu (*) 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: liuxz@im.ac.cn D. Udayanga : D. S. Manamgoda : E. Chukeatirote : K. D. Hyde Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand D. Udayanga : D. S. Manamgoda : E. Chukeatirote : K. D. Hyde School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand apple (Syzygium samarangense). Colletotrichum fructicola was isolated from dragon fruit (Hylocerous undatus) and jujube (Ziziphus sp.). Colletotrichum endophytica was found only from an unknown wild fruit. We observed a considerable genetic and host diversity of species occurring on tropical fruits within the clade previously known as Colletotrichum siamense sensu lato. The clade consists of isolates identified as pre and postharvest pathogens on a wide range of fruits, including coffee (Coffea arabica), custard apple (Annona reticulata), Cerbera sp., figs (Ficus racemosa) mango (Mangifera indica), neem (Azadirachta indica) and papaya (Carica papaya) and was the dominant group of species among most wild fruits studied. With the exception of one isolate from banana, which grouped in the C. siamense clade, all the other isolates were identified as Colletotrichum musae. A new species, Colletotrichum syzygicola, associated with Syzygium samarangense in Thailand, is introduced with descriptions and illustrations. This study highlights the need to re-assess the evolutionary relationships of Colletotrichum species occurring on cultivated and wild fruits with emphasis on their ecology and cryptic diversification including sampling at regional and global scales. Keywords Colletotrichum gloeosporioides . Multi-gene phylogeny . Postharvest diseases . Quarantine . Systematics . Species complex . Tropical Asia . Wild fruits Introduction Colletotrichum Corda, is an important plant pathogenic genus causing anthracnose of a wide range of fruits, vegetables, cereals, grasses and ornamental plants in tropical and temperate regions (Mills et al. 1992; Johnston and Jones 1997; Freeman et al. 2001; Chung et al. 2006; Yang et al. 2009; Rojas et al. 2010). Fruit production is mostly affected in both high-value crops and wild fruits in natural habitats. However, Author's personal copy Fungal Diversity Colletotrichum species associated with wild fruits are poorly known (Rampersad 2011; Cannon et al. 2012). Colletotrichum spp. were voted as the eighth most important plant pathogens in the world in a recent survey among fungal pathologists, for its perceived scientific and economic importance (Dean et al. 2012). The fruits infected by Colletotrichum generally have small, water soaked, sunken, circular spots that may increase in size with age and the centre of an older spot becomes blackish and develops gelatinous pink or orange spore masses (Waller et al. 2002; Agrios 2005; Nelson 2008). Taxonomic and phylogenetic studies on the genus Colletotrichum are in an expansive phase following the epitypification of C. gloeosporioides (Penz.) Penz. & Sacc. (Cannon et al. 2008), the list of current names in use (Hyde et al. 2009b), and the subsequent recommendation for a polyphasic approach to the taxonomy of the genus (Cai et al. 2009). Recent studies have focused on phylogenetic reassessments of species complexes (Damm et al 2012a, b; Weir et al. 2012) and have determined that what were previously thought to be a single species, comprise multiple distinct lineages. For example the boninense clade (Colletotrichum boninense species complex) now comprises about 18 species (Damm et al. 2012a), while the acutatum clade (C. acutatum species complex) now comprises 31 species (Damm et al. 2012b) and the gloeosporioides clade (C. gloeosporioides species complex) comprises more than 22 species (Weir et al. 2012). The species numbers in the major clades are likely to rise, unravelling the cryptic taxa based on multigene phylogenetic analyses and incorporating a large number of isolates in worldwide collections (Cannon et al. 2012). In addition to the major species complexes in Colletotrichum, several intermediate clades have been studied. These intermediate clades, for example C. orbiculare, C. spaethianum, C. destructivum species complexes comprise comparatively less cryptic species (Crouch et al. 2006, 2009a, b; Choi et al. 2011; Noireung et al. 2012; Liu et al. 2013). Epitypification of Colletotrichum gloeosporioides by Cannon et al. (2008) and subsequent use of multi-gene phylogeny have resulted in this taxon being revealed as a species complex. Colletotrichum gloeosporioides was originally described from Citrus in Italy, thus the chosen epitype culture derived from a necrotic spot on leaves of Citrus sinensis from same country (Cannon et al. 2008). Colletotrichum gloeosporioides was previously thought to be a cosmopolitan species infecting a wide range of plant hosts including tropical fruits (Alahakoon and Brown 1994; Freeman et al. 2000; Abang et al. 2002). Phoulivong et al. (2010a) tested this hypothesis by molecular and morphological characterization of Colletotrichum strains from anthracnose symptoms on tropical fruits in Laos and Thailand. Colletotrichum gloeosporioides sensu stricto was not found from any of the fruit examined in their study, however many strains from various common fruits were not assigned to any known taxa based on the five genes employed. Recent molecular studies have resulted in the discovery of new species or accurate identification of known taxa as common causal agents of tropical fruit diseases. This includes C. fructicola Prihastuti, L. Cai & K.D. Hyde, C. gloeosporioides, C. kahawae Waller & Bridge, C. musae (Berk. & M.A. Curtis) Arx, C. theobromicola Delacroix, C. siamense Prihastuti, L. Cai & K.D. Hyde, C. tropicale Rojas, S.A. Rehner & G.J. Samuels, and several distinct species within the C. acutatum and C. boninense species complexes (Prihastuti et al. 2009; Rojas et al. 2010; Maharaj and Rampersad 2012). Many species of Colletotrichum have wide host ranges while relatively host specific exceptions are C. musae (Berk. & M.A. Curtis) Arx, and C. horii B. Weir & P.R. Johnst. associated with Musa spp. and Diospyros sp. respectively. Studies on Colletotrichum in tropical Asia have shown that there is a remarkable species diversity found on wide range of hosts. The species of the genus also causes considerable pre and postharvest fungal diseases on fruits, vegetables, ornamentals, other crops and forest trees in India, Indonesia, Laos, Sri Lanka, Thailand, Tropical China and Vietnam ( Wijesundera et al. 1989; Alahakoon and Brown 1994; Galván et al. 1997; Dinh et al. 2003; Gunawardhana et al. 2009; Phoulivong et al. 2010a; Gupta et al. 2010; Mukherjee et al. 2011). Tropical Asian countries produce, export and consume a diverse array of edible fruits, and therefore accurate identification of postharvest pathogens of fruits has a significant impact on agriculture, bio security and quarantine (Than et al. 2008a; Adikaram et al. 2010; Ko Ko et al. 2011; Phoulivong et al. 2012; Sharma et al. 2013). In this study, we focus on the Colletotrichum species associated with commercially available, cultivated fruits and wild fruits (from native trees in natural habitats) in Thailand. The aim of the study was to re-assess the Colletotrichum species commonly associated with anthracnose of tropical fruits, which is was dominated by the C. gloeosporioides complex. Phylogenetic relationships were initially inferred by comparison of ITS sequence data with ex-type sequences. The multi-gene phylogenetic analyses were then applied to infer phylogenetic relationships and boundaries of taxa in the C. gloeosporioides species complex. Colletotrichum siamense sensu lato was identified as the dominant, most ecologically and genetically diversified clade among the isolates assessed. A new species, C. syzygicola associated with anthracnose of Syzygium samarangense, is introduced as a novel species with a full description and illustrations. Materials and methods Fungal isolates and morphological studies A total of 55 Colletotrichum strains (Table 1) from randomly collected fruits including banana (Musa sp.), Cerbera sp., Taxon identification Isolate/Culture collection Host GenBank accession numbers ACT TUB2 Reference CAL GS GAPDH ITS ICMP18608 Persea americana JX009443 JX010389 JX009683 JX010078 JX010044 JX010244 Weir et al. 2012 ICMP17673 JX009483 JX010392 JX009721 JX010081 JX009930 JX010176 Weir et al. 2012 C. alienum ICMP12071 Aeschynomene virginica Malus domestica JX009572 JX010411 JX009654 JX010101 JX010028 JX010251 Weir et al. 2012 C. aotearoa ICMP 18537 Coprosma sp. JX009564 JX010420 JX009611 JX010113 JX010005 JX010205 Weir et al. 2012 C. asianum BMLI3/ MFLUCC 09-0232 Coffea sp. FJ 903188 FJ 907434 FJ 917501 FJ 972586 FJ 972571 FJ 972605 Prihastuti et al. 2009 BPDI4/ MFLUCC 09-0233 Coffea sp. FJ 907424 FJ 907439 FJ 917506 FJ 972595 FJ 972576 FJ 972612 Prihastuti et al. 2009 BML-I14/ MFLUCC 09-0234 Coffea sp. FJ 907421 FJ 907436 FJ 917503 FJ 972598 FJ 972573 FJ 972615 Prihastuti et al. 2009 C. clidemiae ICMP18658 Clidemia hirta JX009537 JX010438 JX009645 JX010129 JX009989 JX010265 Weir et al. 2012 C. cordylinicola LC955/ BCC 38864 Codyline fruticosa HM470233 HM47028 HM470236 HM470242 HM470239 HM470245 Phoulivong et al. 2010a, b LC856/ BCC 38872 Codyline fruticosa HM470234 HM47029 HM470237 HM470243 HM470240 HM470246 Phoulivong et al. 2010a, b DNCL075/ MFLUCC 10-0676 Unknown wild fruit KF157827 KF254857 KF254846 KF242154 KF242181 KF242123 This study MFLUCC 13-0418 Pennisetum sp. KC692467 – KC810018 – KC832854 KC633854 Manamgoda et al. 2013 DNCL013/ MFLUCC 10-0616 Ziziphus sp. KF157822 KF254898 KF254874 KF242146 KF242184 KF242115 This study C. endophytica C. fructicola C. gloeosporioides C. horri C. hymenocallidis C. jasmini-sambac C. kahawae subsp. ciggaro DNCL055/ MFLUCC 10-0656 Hylocereus undatus KF157823 KF254898 KF254875 KF242147 KF242185 KF242116 This study BPDI16/ MFLUCC 09-0228 Coffea sp. FJ 907426 FJ 907441 FJ 917508 FJ 972593 FJ 972578 FJ 972603 Prihastuti et al. 2009 ICMP18646 JX009581 JX010409 JX009674 JX010099 JX010032 JX010173 Weir et al. 2012 BPDI18/ MFLUCC 09-0226 Tetragastris panamensis Coffea sp FJ 907427 FJ 907442 FJ 917509 FJ 972592 FJ 972579 FJ 972602 Prihastuti et al. 2009 BPDI12/ MFLUCC 09-0227 Coffea sp. FJ 907425 FJ 907440 FJ 917507 FJ 972594 FJ 972577 FJ 972611 Prihastuti et al. 2009 CBS93587 Citrus sinensis FJ 907430 FJ 907445 FJ 917512 FJ 972589 FJ 972582 FJ 972609 Prihastuti et al. 2009 CORCG4 An unknown Orchid HM034800 HM03480 HM034802 – HM034806 HM034808 Phoulivong et al. 2010a, b DNCL053/ MFLUCC 10-0654 Citrus aurantifolia KF157807 KF254856 KF254852 KF242131 KF242162 KF242100 This study DNCL052/ MFLUCC 10-0653 Citrus aurantifolia KF157806 KF254855 KF254851 KF242130 KF242161 KF242099 This study DNCL022/ MFLUCC 10-0625 Syzygium samarangensis KF157804 KF254853 KF254853 KF242128 KF242159 KF242097 This study DNCL027/ MFLUCC 10-0629 Citrus aurantifolia KF157805 KF254854 KF254854 KF242129 KF242159 KF242098 This study ICMP12938 Citrus sinensis JX009560 - JX009732 - JX009935 JX010147 Weir et al. 2012 Weir et al. 2012 ICMP10492 Diospyros kaki JX009438 JX010450 JX009604 JX010137 GQ329681 GQ329690 TSG002 Diospyros kaki GU133379 GU13330 GU133381 GU133382 GQ329680 AY791890 Xie et al. 2010 ICMP18642 Hymenocallis americana GQ856775 JX010410 JX009709 JX010100 JX010019 JX010278 Weir et al. 2012 ICMP19118 Jasminum sambac HM131507 JX010415 JX009713 JX010105 HM131497 HM131511 Weir et al. 2012 ICMP18575 Capsicum annuum JX009455 – JX009717 – JX010059 JX010256 Weir et al. 2012 ICMP18618 Capsicum annuum JX009512 – JX009718 – JX009945 JX010257 Weir et al. 2012 ICMP18539 Olea europaea JX009523 JX010434 JX009635 JX010132 JX009966 JX010230 Weir et al. 2012 Author's personal copy C. aenigma C. aescynomenes Fungal Diversity Table 1 Isolates and sequences used in this study Table 1 (continued) Taxon identification Isolate/Culture collection Host GenBank accession numbers Reference ACT TUB2 CAL GS GAPDH ITS Hypericum perforatum JX009450 JX010432 JX009636 JX010120 JX010042 JX010238 Weir et al. 2012 ICMP19122 Vaccinium sp. JX009536 JX010433 JX009744 JX010134 JX009950 JX010228 Weir et al. 2012 IMI363578 Coffea sp. GU133374 GU13335 GU133376 GU133377 GQ329682 AY787483 Prihastuti et al. 2009 IMI319418 Coffea arabica JX009452 JX010444 JX009642 JX010130 JX010012 JX010231 Weir et al. 2012 ICMP17915 Coffea arabica JX009474 JX010435 JX009638 JX010125 JX010040 JX010234 Weir et al. 2012 CBS116870 Musa sp. HQ596284 HQ59620 JX009742 HQ596288 HQ596299 HQ596292 Su et al. 2011 DNCL045/ MFLUCC 10-0646 Musa sp. KF157828 KF254902 KF254843 KF242151 KF242178 KF242120 This study DNCL039/ MFLUCC 10-0640 Musa sp. KF157829 KF254903 KF254844 KF242152 KF242179 KF242121 This study DNCL067/ MFLUCC 10-0668 Musa sp. KF157830 KF254904 KF254845 KF242153 KF242180 KF242122 This study BTL32 Musa sp. HQ596285 HQ59621 HQ596296 HQ596289 HQ596300 HQ596293 Su et al. 2011 BTL25 Musa sp. HQ596286 HQ59622 HQ596297 HQ596290 HQ596301 HQ596294 Su et al. 2011 BTL31 Musa sp. HQ596287 HQ59623 HQ596298 HQ596291 HQ596302 HQ596295 Su et al. 2011 C. nupharicola ICMP18187 Nuphar lutea JX009437 JX010398 JX009663 JX010088 JX009972 JX010187 Weir et al. 2012 C. psidii CBS145.29 Psidium sp. JX009515 JX010443 JX009743 JX010133 JX009967 JX010219 Weir et al. 2012 C. queenslandicum ICMP1778 Carica papaya JX009447 JX010414 JX009691 JX010104 JX009934 JX010276 Weir et al. 2012 C. salsolae ICMP19051 Salsola tragus JX009562 JX010403 JX009696 JX010093 JX009916 JX010242 Weir et al. 2012 C. siamense BPDI12/MFLUCC09-230 Coffea sp. FJ 907423 FJ 907438 FJ 917505 FJ 972596 FJ 972575 FJ 972613 Prihastuti et al. 2009 BML-I15/MFLUCC09-231 Coffea sp. FJ 907422 FJ 907437 FJ 917504 FJ 972597 FJ 972574 FJ 972614 Prihastuti et al. 2009 DNCL043/ MFLUCC 10-0644 Ficus sp. KF157816 KF254891 KF254870 KF242140 KF242171 KF242109 This study COF005/ MFLUCC 10-0681 Coffea arabica KF157808 KF254883 KF254862 KF242132 KF242163 KF242101 This study DNCL050/ MFLUCC 10-0651 Azdirachta indica KF157826 KF254901 KF254878 KF242150 KF242177 KF242119 This study C. musae C. syzygicola C. ti C. tropicale DNCL072/ MFLUCC 10-0673 Mangifera indica KF157815 KF254890 KF254869 KF242139 KF242170 KF242108 This study DNCL018/ MFLUCC 10-0621 Citrus aurantifolia KF157800 KF254879 KF254858 KF242124 KF242155 KF242093 This study DNCL021/ MFLUCC 10-0624 Syzygium samarangense KF157801 KF254880 KF254859 KF242125 KF242156 KF242094 This study DNCL028/ MFLUCC 10-0630 Syzygium samarangense KF157802 KF254881 KF254860 KF242126 KF242157 KF242095 This study DNCL051/ MFLUCC 10-0652 Syzygium samarangense KF157803 KF254882 KF254861 KF242127 KF242158 KF242096 This study ICMP4832 Cordyline sp. JX009520 JX010442 JX009649 JX010123 JX009952 JX010269 Weir et al. 2012 Weir et al. 2012 Theobroma cacao JX009489 JX010407 JX009719 JX010097 JX010007 JX010264 Annona muricata JX009570 – JX009720 – JX010014 JX010277 Weir et al. 2012 C. simmodsii BRIP28519 Carica papaya FJ 907428 FJ 907443 FJ 917510 FJ 972591 FJ 972580 FJ 972601 Prihastuti et al. 2009 CBS294.67 Carica papaya FJ 907429 FJ 907444 FJ 917511 FJ 972590 FJ 972581 FJ 972610 Prihastuti et al. 2009 Colletotrichum spp. indet. DNCL054/ MFLUCC 10-0655 Carica papaya KF157817 KF254892 KF254871 KF242141 KF242172 KF242110 This study DNCL056/ MFLUCC 10-0657 Carica papaya KF157818 KF254893 KF254872 KF242142 KF242173 KF242111 This study DNCL068/ MFLUCC 10-669 Musa sp. KF157819 KF254894 KF254873 KF242143 KF242174 KF242112 This study Fungal Diversity CBS129949 ICMP18651 Author's personal copy ICMP17922 = Glomerella cingulata var. migrans =Glomerella rufomaculans var. vaccinii Vaccinium C. kahawae subsp. kahawae Author's personal copy CBS Centraalbureau voor Schimmelcultures (The Netherlands), ICMP International Collection of Microorganisms from Plants, MFLUCC Mae Fah Luang University Culture Collection (Thailand), DNCL, COF Isolate codes of Dhanushka Udayanga housed in MFLUCC, BRIP Queensland Plant Pathology Herbarium and culture collection (Australia), IMI CABI Genetic Resource Collection (UK), BCC BIOTEC Culture Collection (Thailand), BML, LC, BTL, BPD, CORCG isolate codes in Prihastuti et al. 2009; Su et al. 2011 Sequences generated in this study are in bold This study This study KF242114 KF242113 KF242182 KF242183 KF242145 KF242144 KF254847 KF254848 KF254896 KF254895 KF157820 Coffea arabica Coffea arabica COF003/ MFLUCC 10-0679 COF004/ MFLUCC 10-0680 KF157820 This study This study KF242107 KF242104 KF242166 KF242169 KF242138 KF242135 KF254865 KF254868 KF254889 KF254886 KF157811 Ficus racemosa Cerbera sp. DNCL076/ MFLUCC 10-677 DNCL034/ MFLUCC 10-0635 KF157814 This study This study KF242105 KF242106 KF242168 KF242167 KF242136 KF242137 KF254867 KF254866 KF254887 KF157812 KF157813 Cerbera sp. Cerbera sp. DNCL035a/ MFLUCC 10-0636a DNCL035b/ MFLUCC 10-0636b KF254888 This study This study KF242102 KF242103 KF242165 KF242164 KF242133 KF242134 KF254864 KF254863 KF157810 KF157809 Annona reticulata Annona reticulata DNCL059a/ MFLUCC 10-0660a KF254885 This study DNCL059b/ MFLUCC 10-0660b KF254884 KF242118 KF242117 KF242175 KF242176 KF242149 KF242148 KF254876 KF254877 KF254900 KF254899 KF157824 KF157825 Aeschynanthus radicans Aeschynanthus radicans DNCL073a/ MFLUCC 10-0674 DNCL073b/ MFLUCC 10-0674b ACT TUB2 CAL GS GAPDH ITS Reference GenBank accession numbers Host Isolate/Culture collection Taxon identification Table 1 (continued) This study Fungal Diversity custard apple (Annona reticulata), coffee (Coffea arabica), and pods of Delonix sp., dragon fruit/pitaya (Hylocereus undatus), fig (Ficus racemosa), jujube (Ziziphus sp.), lime (Citrus aurantifolia, mango (Mangifera indica), neem (Azadirachta indica), papaya (Carica papaya), rose apple (Syzygium samarangense), strawberry (Fragaria sp.), solitary fishtail palm (Caryota urens) and several unidentified wild fruits were obtained for this study (Fig. 1). Wild fruits (e.g. Azadirachta indica, Cerbera sp., Delonix sp., Ficus racemosa) with visible anthracnose symptoms were collected from forest trees and common cultivated fruits (e.g. Annona reticulata, Coffea arabica, Hylocereus undatus, Musa sp., Ziziphus sp. were collected from home gardens, orchards and local markets in northern Thailand (in Chiang Mai and Chiang Rai Provinces). Isolates from coffee berries were endophytes from healthy fruits collected from an orchard in Chiang Mai Province. Single spore isolates were obtained from symptomatic tissues with visible conidiomata, using the protocol outlined by Udayanga et al. (2012). Endophytic isolates were obtained from the sterile pericarp of coffee berries as described in Photita et al. (2005) and Than et al. (2008a). Additional specimens were collected from some common weeds and several unidentified wild herbaceous hosts. Cultures were initially maintained on potato dextrose agar (PDA), and all the cultures were deposited in MFLUCC (Mae Fah Luang University Culture Collection, Thailand). For observation of colony characters and growth rates, a 4 mm plug was aseptically cut from the actively growing edge of 5 day old cultures on PDA and transferred to new PDA plates in triplicate. The inoculated plates were then incubated at 25 °C for 7 days, in the dark. Three cultures of each isolate were observed for colony characters and growth rates. Colony diameter was recorded at 24 h intervals for 7 days for estimating fungal growth rates. After 7 days, colony size and colour (Rayner 1970) of the conidial masses and zonation were recorded and conidial size and shape from 50 arbitrary conidia were measured under the microscope (Nikon ECLIPSE 80i). Morphology of appressoria, setae, conidiophores and conidia were obtained using a slide culture technique on PCA medium (potato carrot agar) as described in Cai et al. (2009). The morphology of the isolates was recorded and used in species identification where appropriate and for the description of the new species. DNA extraction, PCR amplification and sequencing Isolates were grown on PDA and incubated for 5 days at 25 °C in the dark. DNA was extracted using the protocol as outlined by Udayanga et al. (2012) using the actively growing mycelia from the edge of cultures. The ITS region was sequenced using the forward/reverse primer pair ITS5/ITS4 (White et al. 1990), following the PCR protocols outlined by Author's personal copy Fungal Diversity Fig 1 Anthracnose symptoms of tropical fruits caused by species in the Colletotrichum gloeosporioides complex in northern Thailand a Azadirachta indica (C. siamense), b Annona retciulata (Colletotrichum sp. indet. 4), c Ficus racemosa (Colletotrichum sp. indet. 5), d Cerbera sp. (Colletotrichum sp. indet. 5), e Caryota urens (C. siamense), f Hylocerus undatus (C. fructicola ) g Mangifera indica (C. siamense), h Musa sp. (C. musae) i Syzygium samarangense (C. syzygicola) j Citrus sp. (C. gloeosporioides sensu stricto) Udayanga et al. (2012). A selected set of strains in the C. gloeosporioides species complex predetermined based on ITS sequence analysis (gloeosporioides clade in Fig. 2) were used for the amplication and sequencing of multiple gene regions. Partial actin (ACT): ACT512F/ACT783R (Carbone and Kohn 1999), β-tubulin (TUB2): Bt2a/Bt2b (Glass and Donaldson 1995), calmodulin (CAL): CL1/CL2A (O’Donnell et al. 2000), glutamine synthetase (GS): GSF1/GSR1 (Stephenson et al. 1997) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GDF1/GDR1 (Templeton et al. 1992) were amplified following the thermal cycles outlined by Prihastuti et al. (2009). All PCR products were visualized on 1 % agarose gel in the presence of Goldview (Geneshun Biotech, China) with D2000 DNA ladder (Realtimes Biotech, Beijing, China). PCR products were then purified and sequenced using the same forward and reverse primers at SinoGenomax Company, Beijing, China. Sequence alignment, phylogenetic analyses and species recognition Raw sequences were assembled with Sequencher 4.9 for Windows (Gene Codes Corp., Ann Arbor, Michigan). The assembled consensus sequences were initially aligned with ClustalW, and optimized by online sequence alignment editor MAFFTv.7 in default settings (mafft.cbrc.jp/alignment/server/) and manually where necessary (Katoh and Standley 2013). Extype and authentic sequences were obtained from several contemporary phylogenetic studies (Phoulivong et al. 2010a, b; Su et al. 2011; Cannon et al. 2012; Weir et al. 2012). Evolutionary Author's personal copy Fungal Diversity models for phylogenetic analyses were selected independently for each locus using MrModeltest 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC) implemented in both PAUP V4.0b10 and MrBayes v.3.1.2. Phylogenetic reconstructions of concatenated and individual gene-trees were performed using Parsimony (MP), Bayesian (BI) Markov Chain Monte Carlo and Maximum Likelihood (ML) criteria. PAUPv4.0b10 (Swofford 2002) was used to conduct the parsimony analysis to obtain the phylogenetic trees. Trees were inferred using the heuristic search option with 1,000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Bayesian analyses (BI) were performed using MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001 and Ronquist and Huelsenbeck 2003). Six Markov chains were run for 1,000,000 and the 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 each of the analyses were discarded and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. ML gene-trees 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). Phylogenetic trees and data files were viewed in MEGA v. 5 (Tamura et al. 2011), Treeview (Page 1996) and Fig Tree v1.2.2 (Rambaut and Drummond 2008). ITS sequence analysis was initially used to place isolates in different clades of Colletotrichum (Fig. 2). Additional alignments were analyzed individually and in combination to infer placement of selected isolates in the C. gloeosporioides species complex (Fig. 3). The isolates were identified to species based on multi-gene phylogeny. All the sequences used in multi-gene analyses were deposited in GenBank (Table 1) and the additional ITS alignments and trees in TreeBASE (Study ID = 14511: www.Treebase.org). Results Analysis of ITS sequence data and phylogenetic placement of isolates in the clades within the genus The ITS sequence alignment comprised 91 sequences including the outgroup taxon and 509 characters were included in the analysis. Parsimony analysis revealed that 353 characters were constant, 69 characters were parsimony informative, while 87 variable characters were parsimony uninformative. The analysis of the alignment yielded 463 equally parsimonious trees and the first tree is presented here (Fig. 2) (TL=211, CI=0.844, RI=0.961, RC=0.811, HI=0.156). MP/BI and RAxML trees were identical. This tree provides a preliminary guide for identification of isolates used in this study. The ITS analysis demonstrated that the 55 isolates of Colletotrichum from fruits in Thailand belonged to the acutatum, boninense, gloeosporioides, and truncatum species complexes in the genus. Forty-one isolates (75 % of total isolates) clustered in the gloeosporioides clade and were isolated from the fruits of Annona reticulata, Azadirachta indica, Carica papaya, Cerbera sp., Citrus aurantifolia, Coffea sp., Ficus racemosa, Hylocerus undatus, Mangifera indica, Musa sp., Syzygium samarangense, Ziziphus sp. and several unidentified wild fruits. The isolates that clustered in the acutatum clade were from Fragaria sp., Mangifera sp., Musa sp. and Ziziphus sp. and may represent different phylogenetic species known in the clade. The two isolates in the boninense clade were from coffee berries, and one isolate from a Delonix pod clustered in the truncatum clade. The other isolates in truncatum clade were from a stem of Amaranthus sp., and leaf spot of Piper sp. (wild pepper). These results illustrate the diversity of Colletotrichum species associated with tropical fruits in northern Thailand with the dominated group belonging to the C. gloeosporioides species complex. Multi-gene phylogeny and species recognition The combined aligned data matrix contains 77 sequences including the outgroup and 3,275 characters (including gaps), 148 characters were excluded in the parsimony analysis. The parsimony analysis revealed that 1961 characters were constant, 987 characters were parsimony informative, while 184 variable characters were parsimony uninformative. The parsimony analysis of the alignment yielded 48 equally parsimonious trees and the first tree is presented here (Fig. 3) which enables identification of the isolates to species level (TL=1903, CI=0.762, RI=0.898, RC=0.684, HI=0.238). MP/BI and RAxML trees were identical. In combined analysis of six loci of C. gloeosporioides complex, the 33 isolates grouped into seven subclades including C. endophytica Manamgoda, D. Udayanga, K.D. Hyde, C. fructicola, C. gloeosporioides sensu stricto, C. musae, C. siamense sensu lato, C. syzygicola and an undetermined phylogenetic species closely related to C. fructicola represented by two nonsporulating endophytes from coffee berries collected in Thailand (Colletotrichum sp. indet. 6). Most of the isolates from wild fruits clustered in the C. siamense sensu lato clade, and one isolate was C. endophytica (see Manamgoda et al. 2013 for descriptions and illustrations). The other isolates from cultivated fruits were C. fructicola, C. gloeosporioides, C. musae and several distinct lineages within C. siamense clade. Author's personal copy Fungal Diversity MFU09-0627 Mangifera Thailand MFU09-0632 Carica Thailand DNCL059 Annona Thailand DNCL066 Mirabilis Thailand COF005 Coffea Thailand DNCL043 Ficus Thailand MFLUCC09-0230 Coffea Thailand (C. siamense) MFLUCC09-0231 Coffea Thailand COF004 Coffea Thailand DNCL055 Hylocereus Thailand DNCL013 Ziziphus Thailand COF003 Coffea Thailand DNCL072 Mangifera Thailand MFU09-0638 Dimocarpus Thailand MFU09-0630 Carica Thailand CBS953.97 Citrus Italy (C. gloeosporioides) DNCL053 Citrus Thailand CBS112999 Citrus Italy NS/NS/75 DNCL027 Citrus Thailand DNCL052 Citrus Thailand DNCL022 Syzygium Thailand MFU09-0631 Carica Thailand 88/100/96 MFU09-0626 Mangifera Thailand MFLUCC09-0233 Coffea Thailand (C. asianum) DNCL050 Azadirachta Thailand DNCL070 Mangifera Thailand DNCL049 Azadirachta Thailand MFU09-0635 Syzygium Thailand MFU09-0637 Syzygium Thailand DNCL035b Cerbera Thailand DNCL076 Ficus Thailand DNCL073 Aeschynanthus Thailand DNCL023 Caryota Thailand gloeosporioides DNCL034 Cerbera Thailand DNCL035 Cerbera Thailand DNCL064 Shlegera Thailand MFU09-0622 Ziziphus Thailand MFU09-0623 Ziziphus Thailand DNCL039 Musa Thailand DNCL048 Musa Thailand DNCL067 Musa Thailand DNCL047 Musa Thailand 87/100/80 DNCL038 Musa Thailand DNCL036 Musa Thailand DNCL046 Musa Thailand DNCL037 Musa Thailand DNCL029 Musa Thailand DNCL045 Musa Thailand 100/95/100 ICMP10492 Diospyros Japan (C. horii) TSG002 Diospyros China MFU09-0634 Syzygium Thailand MFU09-0629 Mangifera Thailand ICMP18642 Hymenocallis China (C. hymenocallidis) DNCL054 Cariaca Thailand DNCL075 Wild fruit Thailand DNCL056 Carica Thailand DNCL068 Aeschynanthus Thailand MFU09-0639 Ziziphus Thailand MFU09-0633 Carica Thailand 71/100/84 LC0886 Syzygium Thailand 80/100/81 MFU090-636 Syzygium Thailand BCC38864 Cordyline Thailand (C. cordylinicola) BCC38872 Cordyline Thailand DNCL051 Syzygium Thailand 86/100/78 DNCL028 Syzygium Thailand DNCL021 Syzygium Thailand DNCL018 CitrusThailand IMI319418 Coffea Kenya (C. kahawae) IMI363578 Coffea Kenya DNCL044 Fragaria Thailand MFU09-0628 Mangifera Thailand MFU09-0624 Ziziphus Thailand DNCL019 Fragaria Thailand DNCL024 Musa Thailand DNCL041 Fragaria Thailand acutatum DNCL040 Fragaria Thailand DNCL004 Fragaria Thaialnd DNCL001 Ziziphus Thailand BRIP28519 Carica Australia (C. simmondsii) 100/100/97 CBS29467 Carica Auatralia IMI117620 Carica Australia (C. acutatum) IMI117619 Carica Australia (C. acutatum) COF01 Coffea Thailand COF09 Coffea Thailand boninense 100/100/100 MAFF305972 Crinum Japan (C. boninense) DNCL058b Amaranthus Thailand CBS151.35 Phaseolus USA (C. truncatum) 98/100/97 DNCL058c Amaranthus Thailand truncatum DNCL061 Piper Thailand DNCL058 Amaranthus Thailand 99/100/94 DNCL065 Delonix Thailand AB369259 Pyrus Japan Fusarium oxysporum Author's personal copy Fungal Diversity Phylogram inferred from parsimony analysis of ITS sequences of isolates of Colletotrichum obtained in this study to show their phylogenetic placement in species complexes of C. gloeosporioides (gloeospirioides), C. acutatum (acutatun), C. boninense (boninense) and C. truncatum (truncatum). Strains from which new sequences were generated are in original codes and green (DNCL or COF) annotated with host and location, other sequences were obtained from Phoulivong et al. (2010a, b), Su et al. (2011) and Cannon et al. (2012). MP/ Bayesian posterior probabilities/ RaxML bootstrap values ≥70 % are displayed above or below each branch. Ex-type and ex-epitype isolates are in bold. GenBank accessions are given for downloaded sequences. The tree is rooted with Fusarium oxysporum (AB369259) ƒFig. 2 Colletotrichum siamense sensu lato was recognized based on the strong bootstrap support (99 %) in the combined phylogenetic tree. Isolates from Azadirachta (DNCL050), Coffea (COF005), Ficus (DNCL043) and Mangifera (DNCL072) clustered with the ex- type isolate of C. siamense (MFLUCC 09-030) which are recognized to be the same species. The other isolates did not cluster with C. siamense, C. jasminesambac or C. hymenocalidis were assigned to the distinct lineages of Colletotrichum sp. indet. 1-5. We restrain from defining new species within the C. siamense clade until the phylogeny is revisited with all recently described cryptic taxa. Taxonomy Isolates associated with fruit anthracnose of Syzygium samarangense (Myrtaceae) and one isolate associated with a fruit of Citrus aurantifolia (Rutaceae) clustered in a unique sub-clade in Colletotrichum gloeosporioides species complex in multi-gene phylogeny inferred by six gene combined alignment (Fig. 3). The individual gene genealogies were compared with each other and with the combined analysis. The new species, C. syzygicola (Fig. 4) described here, is closely related to C. cordylinicola Phoulivong, L. Cai & K.D. Hyde, originally described from Cordyline fruticosa (an ornamental plant belongs to Asparagaceae) in Thailand (Phoulivong et al. 2010b). Colletotrichum syzygicola Udayanga, Manamgoda, K. D. Hyde, sp. nov. MycoBank: MB803938 Etymology: primarily associated with fruit anthracnose of Syzygium samarangense Colonies on PDA attaining 72 mm diam., in 7 days at 25 °C, growth rate 5.5±0.5 mm/day (n=6), white to grey, reverse grey at the centre, aerial mycelium, dense and raised, with orange conidial masses, sclerotia present. Setae absent in culture on PDA or slide cultures in PCA. Conidiophores (10.5–) 12–16 (–21)×4–5 μm (mean ± SD=14±2×4.5±0.5 μm, n=50), cylindrical or clavate, wide at the base, hyaline, unbranched, occurring in densely arranged clusters. Conidiogenous cells enteroblastic, hyaline, cylindrical to clavate. Conidia (12–) 13–15 (–18.1)×5.5–6.5 μm (mean ± SD=14.7±0.9×5.9± 0.5 μm), unicellular, hyaline, ovoid to cylindrical or clavate, with rounded apices. Appressoria formed in slide culture (18–) 19–22 (–24) μm×7–8 (mean ± SD=20.1±1.5× 7.6±0.5 μm, n=50), formed from branched mycelia, terminal, brown to dark brown, variable in shape, irregular or knobbed. Sexual state not observed in culture. Known Hosts: On fruits of Syzygium samarangense and Citrus aurantifolia associated with anthracnose symptoms Known distribution: Northern Thailand Holotype: THAILAND: Chiang Rai Province, Nang Lae, Fah-Thai market, on fruits of Syzygium samarangense, 18 April 2010, Dhanushka Udayanga (MFLU12-2476, dried sporulating culture on PDA; ex –holotype culture DNCL021= MFLUCC 10-0624) ; Isotype ibid. (MFLU 12-2477: dried culture; ex-isotype culture DNCL028=MFLUCC 100630). Additional material examined: THAILAND: Chiang Rai Province, Nang Lae, Fah-Thai market, on fruits of Citrus aurantifolia, 27 February 2010, Phongeun Sysouphanthong (DNCL018, living culture MFLUCC10-0621); ibid., Chiang-Kong city market, on fruits of Syzygium samarangense, 10 April 2010, Dhanushka Udayanga (DNCL051, living culture MFLUCC 10-0652). Notes: Isolates of Colletotrichum syzygicola primarily from Syzygium samarangense formed a divergent lineage from species of the kahawae clade (i.e. C. aotoreae B. Weir & P.R. Johnst., C. clidemia B. Weir & P.R. Johnst, C. cordylinicola, C.psidii Curzi, C. kahawae, C. kahawae subsp. ciggaro B. Weir & P.R. Johnst., C. ti B. Weir & P.R. Johnst. and Glomerella cingulata f. sp. camelliae ) in the combined phylogenetic analysis (Weir et al. 2012),. Isolates from Syzygium sp. in Phoulivong et al. (2010a, b, 2012) were named as C. cordylinicola, however they stated that these isolates represent a different pathotype infecting tropical fruits, whereas C. cordylinicola originally isolated from Cordyline does not infect fruits. The isolate BCC38864 from Syzygium sp. which forms a distinct lineage (Phoulivong et al. 2010b) was regarded as C. cordylinicola in their multi-gene phylogenetic tree, could be due to insufficient sampling to recognize a unequivocally distinct lineage. To our knowledge, there are no any distinct morphological or phylogenetic species originally described within C. gloeosporioides complex from Syzygium sp., which is the common host of C. syzygicola. The other recently defined species associated with ericaceous hosts in north America (Colletotrichum fructivorum V. Doyle, P.V. Oudem. & S.A. Rehner, Colletotrichum temperatum V. Doyle, P.V. Oudem. & S.A. Rehner and epitype of Colletotrichum rhexiae Ellis & Everh.), which are closely related to C. kahawae were regarded as distinct from C. syzygicola by comparison of available single genes and relative phylogenetic relationships within the genus. Author's personal copy Fungal Diversity MFLUCC09-0230 Coffea Thailand (C. siamense) MFLUCC09-0231Coffea Thailand DNCL043 Ficus Thailand COF005 Coffea Thailand DNCL050 Azadirachta Thailand DNCL072 Mangifera Thailand ICMP18575 Capsicum Thailand 90/100/99 ICMP18618 Capsicum Thailand ICMP19118 Jasminum Vietnam (C. jasmini-sambac) C. siamense s.l. DNCL054 Carica Thailand (sp. indet. 1) 100/90/100 DNCL056 Carica Thailand DNCL068 Musa Thailand (sp. indet. 2) 100/97/100 DNCL073 Aeschynanthus Thailand (sp. indet. 3) DNCL073b Aeschynanthus Thailand DNC059b Annona Thailand (sp. indet. 4) 100/100/100 DNCL059 Annona Thailand DNCL035 Cerbera Thailand (sp. indet. 5) DNCL035b Cerbera Thailand 98/100/96 98/100/99 DNCL076 Ficus Thailand DNCL034 Cerbera Thailand ICMP18642 Hymenocallis China (C. hymenocallidis) CBS124949 Theobroma Panama 99/100/100 C. tropicale ICMP18651 Annona Panama ICMP17673 Aeschynomene USA C. aeschynomenes 98/100/99 DNCL013 Ziziphus Thailand DNCL055 Hylocereus Thailand C. fructicola MFLUCC09-0228 Coffea Thailand 90/94/88 ICMP18646 Tetragastris Panama MFLUCC09-0226 Coffea Thailand MFLUCC09-0227 Coffea Thailand 73/100/92 100/100/100 COF004 Coffea Thailand (sp. indet. 6) Colletotrichum sp. COF003 Coffea Thailand C. alienum ICMP12071 Malus NZ ICMP18187 Nuphar USA C. nuphricola 97/100/100 DNCL045 Musa Thailand DNCL039 Musa Thailand DNCL067 Musa Thailand NS/71/71 C. musae BTL32 Musa Thailand 100/100/100 BTL25 Musa Thailand BTL31 Musa Thailand CBS116870 Musa USA C. aenigma ICMP18608 Persea Israel ICMP19051 Salsola Hungary C. salsolae ICMP1778 Carica Australia C. queenslandicum MFLUCC09-0232 Coffea Thailand MFLUCC09-0233 Coffea Thailand C. asianum 100/100/100 ICMP18580 Coffea Thailand DNCL075 wild fruit Thailand C. endophytica 97/100/98 MFLUCC13 -0418 Pennisetum Thailand ICMP12938 Citrus NZ CORCG4 Orchid Thailand DNCL053 Citrus Thailand 93/99/92 DNCL052 Citrus Thailand C. gloeosporioides DNCL022 Syzygium Thailand DNCL027 Citrus Thailand 100/100/100 CBS95397 Citrus Italy 94/100/99 IMI363578 Coffea Kenya IMI319418 Coffea Kenya ICMP17915 Coffea Angola C. kahawae 100/83/100 NS/78/100 ICMP18539 Olea Australia 90/100/95 ICMP19122 Vaccinium USA ICMP17922 Hypericum Germany 88/100/91 Glomerella cingulata “f. sp.camelliae” ICMP10643 Camellia UK ICMP18658 Clidemia USA C. clidemiae 84/100/97 DNCL018 Citrus Thailand 100/100/100 DNCL021 Syzygium Thailand C. syzygicola 100/100/100 98/100/97 DNCL028 Syzygium Thailand DNCL051 Syzygium Thailand 91/100/85 BCC38864 Cordyline Thailand C. cordylinicola 100/100/100 BCC38872 Cordyline Thailand 100/92/95 C. psidii CBS145.29 Psidium Italy ICMP18532 Vitex NZ C. aotearoa ICMP4832 Cordyline NZ C. ti ICMP10492 Diospyros Japan C. horii 100/100/100 TSG002 Diospyros China BRIP28519 Carica Australia C. simmondsii CBS294.67 Carica Australia 97/100/100 Author's personal copy Fungal Diversity Phylogram inferred from the parsimony analysis of combined sequences of selected strains in the Colletotrichum gloeosporioides species complex. Strains originally isolated in this study are given in green, other sequences were obtained from Phoulivong et al. (2010a, b), Su et al. (2011) and Weir et al. (2012). Coloured boxes within C. siamaense clade indicate the distinct lineages observed in combined analysis. Each colour assigned to the cryptic taxa recognized as C. siamense, C. jasmine-sambac, C. hyenocallidis and the isolates not identified to species level (sp. indet. 1-5). MP/ Bayesian posterior probabilities/ RaxML bootstrap values/ ≥70 % are displayed above or below each branch. Ex-type and ex-epitype cultures are in bold. The tree is rooted with Colletotrichum simmondsii (BRIP 28519, CBS 294.67) ƒFig. 3 Discussion Molecular phylogenetic studies have revolutionized the understanding of species diversity and their ecological and evolutionary significance in Colletotrichum (Hyde et al. 2009a; Damm et al. 2009, 2010; O’Connell et al. 2004, 2012; Silva et al. 2012b). Within the C. gloeosporioides species complex, 90 % of the phylogenetically distinct taxa (e.g. C. aenigma, C. alienum, C. asianum, C. fructicola, C. fructivorum, C. gloeosporioides sensu stricto, C. musae, C. kahawae, C. psidii, and C. queenslandicum, C. siamense, C. syzygicola, C.tropicale, C. temperatum and C. viniferum) are originally described from fruits causing anthracnose or similar symptoms. In this study, we recognized that the dominant anthracnose pathogens in tropical Asia comprise several species in C. gloeosporioides complex, although C. gloeosporioides sensu stricto has a comparatively limited range of hosts. We identified C. gloeosporioides only from Citrus aurantifolia and Syzygium in northern Thailand, while Peng et al. (2012) and Huang et al. (2013) found this species to be the most common on Citrus leaves and fruits in China. Colletotrichum tropicale is a common species associated with Annona and Theobroma in Panama, and C. theobromicola with Annona and Theobroma cacao in Panama and Mexico are not found from the fruits obtained from Thailand in this study. Colletotrichum endophytica, a common endophyte in tropical grasses (Manamgoda et al. 2013), was also found from an unidentified wild fruit in this study. Colletotrichum syzygicola is a common pathogen infecting Syzygium fruits and less frequently on Citrus obtained from local markets in Thailand. Colletotrichum fructicola (from coffee and dragon fruit), C. musae (from banana), C. siamense sensu lato (a wide range of hosts) were also dominant species on tropical fruits sampled. In the ITS analysis (Fig. 2) the isolates in acutatum clade were commonly associated with Fragaria (strawberry) and Ziziphus (jujube). Many species within the C. acutatum species complex have also been reported as common causative agents of the fruit diseases of tropical and temperate hosts in different studies (Than et al. 2008a, b; Shivas and Tan 2009). A multi-gene phylogenetic analysis is required for the accurate identification of species within C. acutatum complex from Fragaria since C. fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan, C. godetiae Neerg, C. nymphaeae (Pass.) Aa, C. salicis (Fuckel) Damm, P.F. Cannon & Crous and C. simmondsii R.G. Shivas & Y.P. Tan (Damm et al. 2012a) have been reported from this host. Weir et al. (2012) indicate the occurrence of C. fructicola, C. theobromicola (syn. C. fragariae A.N. Brooks) and C. siamense associated with Fragria in United States and Cananda. The species within the Colletotrichum boninense and C. truncatum species complexes were less frequently found in this study but can also be considered as important species associated with tropical fruits. Colletotrichum siamense was originally described as a pathogen associated with anthracnose of coffee berries in northern Thailand (Prihastuti et al. 2009). In the present study, we identified C. siamense sensu stricto as an endophyte in pericarp of healthy coffee berries and as a pathogenic species causing anthracnose on fruits of Ficus racemosa, Azadirachta india and Mangifera indica. The conventional six gene analysis of C. gloeosporioides complex results a strong monophyly of a broad phylogenetic group of C. siamense (Weir et al. 2012), however it is less reliable to resolve the species relationships and boundaries within the clade. Considerable genetic diversity was observed in the clade recognized as C. siamense sensu lato from sampling within a limited geographic region including six collection sites in northern Thailand (Fig. 3). The distinct lineages strictly correlated with host association in case of isolates fruits of Annona, Cerbera and leaves of Aeschynanthus. The close inspection of six gene combine alignment and single gene analyses reveal that the C. hymenocallidis, C. jasmini- sambac and C. siamense can be considered as recently diverged species with minimum phylogenetic distinction to establish their strong monophyly. However, the single gene phylogenetic analyses of ITS, GPDH and GS (trees not shown), support the distinction of the above three taxa from the other groups (sp. indet. 1-5) not assigned to a known species. Therefore, we recognize C. siamense as a species complex which urgently needs phylogenetic reappraisal. Doyle et al. (2013) and Sharma et al. (2013) also showed C. siamense to be a species complex based on APN2 (DNA lyase) and APN2/MAT-IGS (intergenic spacer between 3′ end of the DNA lyase and mating type locus MAT1-2) genetic markers. Colletotrichum siamense sensu lato was also reported as the most common endophyte associated with two tropical grass species in Thailand (Manamgoda et al. 2013). In our analysis we observed that the GS gene in the C. siamense sensu lato clade has a higher variability as compared to that of other Colletotrichum species. This was also noted by Silva et al. (2012a) and considered as discordance of gene-trees within C. siamense which Author's personal copy Fungal Diversity Fig. 4 Morphology of Colletotrichum syzygicola based on the ex-type culture DNCL021 a Infected fruits of S. samarangense (orange colour spore masses in the middle of the lesions) b Culture on PDA (2 weeks old) c Conidiomata with spore masses on PDA d, e Appressoria f, g Conidiophores h Conidia Scale bars: c=1000 μm d, e=20 μm, f, g, h=15 μm they recognized as a common issue in resolving evolutionary relationships in the C. gloeosporioides complex. Although we observed several distinct lineages within in C. siamense species complex, novel species were not introduced in this study until it is revisited in a future comprehensive assessment. Therefore, this study lays the groundwork to refine the understanding of the extent of cryptic diversification within the C. siamense clade in future. It has been common desirable practice to link existing species names of Colletotrichum with new collections via epitypification (Hyde et al. 2009b; Weir et al. 2012; Rossman 2013). The general unwritten rules of epitypification require that an epitype should be collected from the same host and host organ, and same country or region as the protologue and have the same characteristics (Zhang and Hyde 2008). In genera such as Colletotrichum, species have wide host ranges and large numbers of names exist in the literature. Previous authors have linked newly resolved species with old scientific names and have designated epitypes for these earlier names. For example, Colletotrichum musae from Musa sp., C. Author's personal copy Fungal Diversity rhexiae Ellis and Everh. from Rhexia virginica are recently epitypified based on fresh collections (Su et al. 2011; Doyle et al. 2013). Some authors have introduced novel species only when the taxonomic entity cannot be reliably linked to earlier names (Crouch and Tamaso-Peterson 2012; Lima et al. 2013; Peng et al. 2013). However, the description of novel taxa should be well supported by adequate molecular datasets and state- of- the-art analysis with the inclusion of frequent and abundant fresh collections (Hyde et al. 2013; Sharma et al. 2013). Tropical Asia is an economically and physiographically rich region with a unique, hyperdiverse flora and fungi (Hyde 2003; Hawksworth 2004; Mueller and Schmit 2007; Karunarathna et al. 2012). Post-harvest management of fruit and vegetables in most developing countries in the Asian region is, however, far from satisfactory (Report of International Centre for Science and High Technology, United Nations Industrial Development Organization, 2012). Major constraints include inefficient handling and transportation and loss due to fungal and bacterial diseases due to lack of implementation of postharvest technologies (Adikaram 1986; Aidoo 1993; Choudhury 2006). Cultivated fruits in Southeast Asia suffer from great yield losses due to factors including fungal disease encouraged by favourable environmental factors (Li 1970; Yaacob and Subhadrabandhu 1995; Rolle 2006). Phytosanitary standards maintained by some developed countries limit the exports of fruits from developing countries which often impact on agricultural trade (Romberg and Roberts 2008). The species associated with the diseases of commercial fruits are critical to re- regulating movement of pathogens and establish effective disease control measures. Therefore, it is important to integrate the dynamic changes taking place in classification and nomenclature of pathogenic fungi with the broad applications in biosecurity, quarantine and disease control (Rossman and Palm-Hernández 2008; Cai et al. 2011; Zhang et al. 2013). Incorporation of molecular data derived from the isolates in regional and global sampling of Colletotrichum species, improves the knowledge of diversity, population structure, extent of host and geographic distribution. Acknowledgments Dhanushka Udayanga thanks the State Key Lab of Systematic Mycology, the Chinese Academy of Sciences, Beijing for a visiting postgraduate scholarship (2010-2011). This project is supported by the Chinese Academy of Sciences, Beijing (NFSC Y2JJ011002). Kevin D. Hyde thanks the National Research Council of Thailand for the award of grant No. 54201020003 and a grant from the National Plan of Science and Technology, King Abdulaziz City of Science and Technology, Riyadh, Saudi Arabia, project No. 10-Bio-965-02 to study Colletotrichum. Sawonee Wikee, Samantha Karunarathna and Phongeun Sysouphanthong (MFLU, Thailand) are thanked for providing specimens. Cai Lei (Chinese Academy of Sciences, Beijing) is thanked for suggestions to improve the manuscript. References Abang MM, Winter S, Green KR, Hoffmann P, Mignouna HD et al (2002) Molecular identification of Colletotrichum gloeosporioides causing yam anthracnose in Nigeria. Plant Pathol 51:63–71 Adikaram NKB (1986) A survey of postharvest losses in some fruits and vegetables and the fungi associated with them. Ceylon J Sci (Bio Sci) 19:1–10 Adikaram NKB, Karunanayake C, Abayasekara C (2010) Role of preformed antifungal compounds in the resistance of fruit to postharvest pathogens. In: Gullino ML, Prusky D (eds) Postharvest pathology. ISBN 978-1-4020-8931-2, Springer, pp 1–11 Agrios GN (2005) Plant pathology, 5th edn. Academic, New York Aidoo KE (1993) Postharvest storage and preservation of tropical crops. Int Biodeterior Biodegrad 32:161–173 Alahakoon PW, Brown AE (1994) Host-range of Colletotrichum gloeosporioides on tropical fruit crops in Sri Lanka. Int J Pest Manage 40:23–26 Cai L, Hyde KD, Taylor PWJ, Weir BS, Waller J et al (2009) A polyphasic approach for studying Colletotrichum. Fungal Divers 39:183–204 Cai L, Udayanga D, Manamgoda DS, Maharachchikumbura SSN, Liu XZ, Hyde KD (2011) The need to carry out re-inventory of tropical plant pathogens. Trop Plant Pathol 36:205–213 Cannon PF, Buddie AG, Bridge PD (2008) The typification of Colletotrichum gloeosporioides. Mycotaxon 104:189–204 Cannon PF, Damm U, Johnston PR, Weir BS (2012) Colletotrichum – current status and future directions. Stud Mycol 73:181–213 Carbone I, Kohn L (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556 Choi KJ, Kim WG, Kim HG, Choi HW, Lee YK, et al (2011) Morphology, molecular phylogeny and pathogenicity of Colletotrichum panacicola causing anthracnose of Korean ginseng. Plant Pathology J 27:1–7 Choudhury ML (2006) Recent developments in reducing postharvest losses in the Asia-Pacifi c region. In: Rolle RS (ed) Reports of the APO seminar on reduction of postharvest losses of fruit and vegetables. Tokyo, Japan, pp 15–22 Chung W-H, Ishii H, Nishimura K, Fukaya M, Yano K et al (2006) Fungicide sensitivity and phylogenetic relationship of anthracnose fungi isolated from various fruit crops in Japan. Plant Dis 90:506– 512 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 104:108–1096 Crouch JA, Clarke BB, Hillman BI (2006) Unraveling evolutionary relationships among the divergent lineages of Colletotrichum causing anthracnose disease in turfgrass and maize. Phytopathology 96:46–60 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 Crouch JA, Clarke BB, Hillman BI (2009b) What is the value of ITS sequence data in Colletotrichum systematics and species diagnosis? A case study using the falcate-spored graminicolous Colletotrichum group. Mycologia 101:648–656 Damm U, Woudenberg JHC, Cannon PF, Crous PW (2009) Colletotrichum species with curved conidia from herbaceous hosts. Fungal Divers 39:45–87 Damm U, Barroncelli R, Cai L, Kubo Y, O’Connell R, Weir B, Yoshino K, Cannon PF (2010) Colletotrichum: species, ecology and interactions. IMA Fungus 1:161–165 Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012a) The Colletotrichum acutatum species complex. Stud Mycol 73:37–113 Author's personal copy Fungal Diversity Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir B et al (2012b) The Colletotrichum boninense species complex. Stud Mycol 73:1–36 Dean R, Van Kan JAL, Pretorius ZA, Hammond-Kosack KE, Di Pietro A et al (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430 Dinh SQ, Chongwungse J, Pongam P, Sangchote S (2003) Fruit infection by Colletotrichum gloeosporioides and anthracnose resistance of some mango cultivars in Thailand. Australas Plant Pathol 32:533–538 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(5):e62394 Freeman S, Minz D, Jurkevitch E, Maymon M, Shabi E (2000) Molecular analyses of Colletotrichum species from almond and other fruits. Phytopathology 90:608–614 Freeman S, Minz D, Maymon M, Zveibil A (2001) Genetic diversity within Colletotrichum acutatum sensu Simmonds. Phytopathology 91:586–592 Galván GA, Wietsma WA, Putrasemedja S, Permadi AH, Kik C (1997) Screening for resistance to anthracnose (Colletotrichum gloeosporioides Penz.) in Allium cepa and its wild relatives. Euphytica 95:173–178 Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330 Gunawardhana PLT, Seneviratne AMWK, Adikaram NKB, Yakandawala DMD (2009) A phonetic analysis of Colletotrichum gloeosporioides (Penz.) Penz & Sacc. Cey J Sci (Bio Sci) 38:57–66 Gupta VK, Pandey A, Kumar P, Pandey BK, Gaur RK, Bajpai V, Sharma N, Sharma S (2010) Genetic characterization of mango anthracnose pathogen Colletotrichum gloeosporioides Penz. by random amplified polymorphic DNA analysis. Afr J Biotechnol 9:4009–4013 Hawksworth DL (2004) Fungal diversity and its implications for genetic resource collections. Stud Mycol 50:9–18 Huang F, Chen GQ, Hou X, Fu YS, Cai L, Hyde KD, Li HY (2013) Colletotrichum species associated with cultivated Citrus in China. Fungal Divers (in press). doi:10.1007/s13225-013-0232-y Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755 Hyde KD (2003) Mycology in the future in the Asia-Pacific region. Fungal Divers 13:59–68 Hyde KD, Cai L, McKenzie EHC, Yang YL, Zhang JZ et al (2009a) Colletotrichum: a catalogue of confusion. Fungal Divers 39:1–17 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 (2009b) Colletotrichum—names in current use. Fungal Divers 39:147–182 Hyde KD, Udayanga D, Manamgoda DS, Tedersoo L, Larsson E, Abarenkov K, Bertrand YJK, Oxelman B, Hartmann M, Kauserud H, Ryberg M, Kristiansson E, Nilsson RH (2013) Incorporating molecular data in fungal systematics: a guide for aspiring researchers. Curr Res Environ Appl Mycol 3:1–32 Johnston PR, Jones D (1997) Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences. Mycologia 89:420–430 Karunarathna SC, Udayanga D, Maharachchikumbura SN, Pilkington M, Manamgoda DS, Wijayawardene DNN, Ariyawansa HA, Bandara AR, Chukeatirote E, McKenzie EHC, Hyde KD (2012) Current status of knowledge of Sri Lankan mycota. Curr Res Environ Appl Mycol 2:18–29 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. doi:10.1093/molbev/mst010 Ko Ko TW, Stephenson SL, Bahkali AH, Hyde KD (2011) From morphology to molecular biology: can we use sequence data to identify fungal endophytes? Fungal Divers 50:113–120 Li HL (1970) The origin of cultivated plants in Southeast Asia. Econ Bot 24:3–19 Lima NB, Batista A, Vinicius M, Morais MA Jr, Barbosa AGM, Michereff S, Jand Hyde KD, Câmara MPS (2013) Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil. Fungal Divers (in press). doi:10.1007/s13225-013-0237-6 Liu F, Cai L, Crous PW, Damm U (2013) Circumscription of the anthracnose pathogens Colletotrichum lindemuthianum and C. nigram. Mycologia 12:315 Maharaj A, Rampersad SN (2012) Genetic differentiation of Colletotrichum gloeosporioides and C. truncatum associated with anthracnose disease of papaya (Carica papaya L.) and bell pepper (Capsium annuum L.) based on ITS PCR-RFLP fingerprinting. Mol Biotechnol 50:237–249 Manamgoda DS, Udayanga D, Cai L, Chukeatirote E, Hyde KD (2013) Endophtic Colletotrichum associated with tropical grasses with a new species C. endophytica. Fungal Divers (accepted): FUDI-S-13-00098 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA pp 1–8 Mills PR, Hodson A, Brown AE (1992) Molecular differentiation of Colletotrichum gloeosporioides isolates infecting tropical fruits. In: Bailey JA, Jeger MJ (eds) Colletotrichum: biology, pathology and control. CABI, Wallingford, pp 269–288 Mueller GM, Schmit JP (2007) Fungal biodiversity:what do we know? What we can predict? Biodivers Conserv 16:1–5 Mukherjee A, Khandker S, Islam MR, Sonia B, Shahid J (2011) Efficacy of some plant extracts on the mycelial growth of Colletotrichum gloeosporioides. Bangladesh Agric Univ 9:43–47 Nelson SC (2008) Mango anthracnose (Colletotrichum gloeosporioides). Plant Dis 48. College of Tropical Agriculture and Human Resources (CTAHR), University of Hawai at Mänoa, Honolulu, Hawai. www. ctahr.hawaii.edu/oc/freepubs/pdf/pd-48.pdf Noireung P, Phoulivong S, Liu F, Cai L, Mckenzie EHC, Chukeatirote E, Jones EBG, Bahkali AH, Hyde KD (2012) Novel species of Colletotrichum revealed by morphology and molecular analysis. Cryptogam Mycol 33:347–362 Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Center, Uppsala University O’Connell R, Herbert C, Sreenivasaprasad S, Khatib M, EsquerréDugayé M-T et al (2004) A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions. Mol Plant Microbe Interact 17:272–282 O’Connell RJ, Thon MR, Hacquard S, Amyotte SG, Kleemann J et al (2012) Life-style transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nat Genet 44:1060–1065 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 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358 Peng L, Yang Y, Hyde KD, Bahkali AH, Liu Z (2012) Colletotrichum species on Citrus leaves in Guizhou and Yunnan Provinces, China. Cryptogam Mycol 33:267–283 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 Photita W, Taylor PWJ, Ford R, Lumyong P, McKenzie HC et al (2005) Morphological and molecular characterization of Colletotrichum species from herbaceous plants in Thailand. Fungal Divers 18:117–133 Author's personal copy Fungal Diversity Phoulivong S, Cai L, Chen H, McKenzie EHC, Abdelsalam K et al (2010a) Colletotrichum gloeosporioides is not a common pathogen on tropical fruits. Fungal Divers 44:33–43 Phoulivong S, Cai L, Parinn N, Chen H, Abd-Elsalam K et al (2010b) A new species of Colletotrichum from Cordyline fruticosa and Eugenia javanica causing anthracnose disease. Mycotaxon 114:247–257 Phoulivong S, McKenzie EHC, Hyde KD (2012) Cross infection of Colletotrichum species; a case study with tropical fruits. Curr Res Environ Appl Mycol 2:99–111 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 Rambaut A, Drummond A (2008) FigTree: tree figure drawing tool, version 1.2. 2. Institute of Evolutionary Biology, University of Edinburgh Rampersad SN (2011) Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease of papaya in Trinidad. Plant Dis 95:1244–1254 Rayner RW (1970) A mycological colour chart. - Commonw. Mycol. Inst., Kew 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 Rolle RS (2006) Improving postharvest management and marketing in the Asia-Pacifi c region: issues and challenges trends in the fruit and vegetable sector. In: Rolle RS (ed) Reports of the APO seminar on reduction of postharvest losses of fruit and vegetables. Tokyo, Japan, pp. 23–31 Romberg M, Roberts D (2008) Phytosanitary regulations shape fruit and vegetable trade patterns. Amber Waves 6:36–37 Ronquist F, Huelsenbeck J (2003) Mrbayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574 Rossman AY (2013) Colletotrichum: Complex speciesor species complexes? [Review of the book Colletotrichum: Complex species or species complexes? 2012. U.Damm, P.F. Cannon, P.W. Crous (Eds.). CBS Studies in Mycology No.73. CBS Fungal Biodiversity Centre]. Inoculum 64:21 Rossman AY, Palm-Hernández ME (2008) Systematics of plant pathogenic fungi: why kit matters. Plant Dis 92:1376–1386 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) Shivas RG, Tan YP (2009) A taxonomic re-assessment of Colletotrichum acutatum, introducing C. fioriniae comb. et stat. nov. and C. simmondsii sp. nov. Fungal Divers 39:111–122 Silva DN, Talhinas P, Várzea V, Cai L, Paulo OS, Batista D (2012a) Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts. Mycologia 104:396–409 Silva DN, Talhinhas P, Cai L, Manuel L, Gichuru EK, Loureiro A, Várzea V, Paulo OS, Batista D (2012b) Host-jump drives rapid and recent ecological speciation the emergent fungal pathogen Colletotrichum kahawae. Mol Ecol 21:2655–2670 Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690 Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 57:758–771 Stephenson SA, Green JR, Manners JM, Maclean DJ (1997) Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis. Curr Genet 31:447–454 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 Swofford DL (2002) PAUP 4.0b10: phylogenetic analysis using parsimony. Sinauer Associates, Sunderland Tamura K, Peterson D, 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 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 Than PP, Jeewon R, Hyde KD, Pongsupasamit S, Mongkolporn O, Taylor PWJ (2008a) Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand. Plant Pathol 57:562–572 Than PP, Shivas RG, Jeewon R, Pongsupasamit S, Marney TS et al (2008b) Epitypification and phylogeny of Colletotrichum acutatum J.H. Simmonds. Fungal Divers 28:97–108 Udayanga D, Liu XZ, Crous PW, McKenzie EHC, Chukeatirote E, Hyde KD (2012) A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis). Fungal Divers 56:157–171 Waller JM, Lenné JM, Waller SJ (2002) Plant pathologists’s pocketbook. CABI, Wallingford Weir B, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115–180 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 press Inc. Harcourt Brace Javanovich publishers, New York. pp 315–322 Wijesundera RLC, Bailey JA, Byrde RJW, Fielding AH (1989) Cell wall degrading enzymes of Colletotrichum lindemuthianum: their role in the development of bean anthracnose. Physiol Mol Plant Pathol 34:403–413 Xie L, Zhang JZ, Cai L, Hyde KD (2010) Biology of Colletotrichum horii, the causal agent of persimmon anthracnose. Mycology 1:242–253 Yaacob O, Subhadrabandhu S (1995) The production of economic fruits in south east Asia pp. 443 Yang YL, Liu ZY, Cai L, Hyde KD, Yu ZN, McKenzie EHC (2009) Colletotrichum anthracnose of Amaryllidaceae. Fungal Divers 39:123–146 Zhang Y, Hyde KD (2008) Epitypification: Should we epitypify? J Zhejiang Univ-Sc B 9:842–846 Zhang N, Rossman AY, Seifert K, Bennett JW, Cai G, Cai L, Hillman B, Hyde KD, Luo J, Manamgoda D, Meyer W, Molnar T, Schoch C, Tadych M, White JF Jr (2013) Impacts of the International Code of Nomenclature for algae, fungi and plants (Melbourne Code) on the scientific names of plant pathogenic fungi. Online. APSnet Feature. American Phytopathological Society, St. Paul