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Persoonia : Molecular Phylogeny and Evolution of Fungi logoLink to Persoonia : Molecular Phylogeny and Evolution of Fungi
. 2016 Aug 2;38:20–37. doi: 10.3767/003158517X692788

Colletotrichum species causing anthracnose disease of chili in China

Y-Z Diao 1,2, C Zhang 1,✉, F Liu 2, W-Z Wang 1, L Liu 1, L Cai 2, X-L Liu 1
PMCID: PMC5645185  PMID: 29151625

Abstract

Anthracnose caused by Colletotrichum species is a serious disease of more than 30 plant genera. Several Colletotrichum species have been reported to infect chili in different countries. Although China is the largest chili-producing country, little is known about the species that have been infecting chili locally. Therefore, we collected samples of diseased chili from 29 provinces of China, from which 1285 strains were isolated. The morphological characters of all strains were observed and compared, and multi-locus phylogenetic analyses (ITS, ACT, CAL, CHS-1, GAPDH, TUB2, and HIS3) were performed on selected representative strains. Fifteen Colletotrichum species were identified, with C. fioriniae, C. fructicola, C. gloeosporioides, C. scovillei, and C. truncatum being prevalent. Three new species, C. conoides, C. grossum, and C. liaoningense, were recognised and described in this paper. Colletotrichum aenigma, C. cliviae, C. endophytica, C. hymenocallidis, C. incanum, C. karstii, and C. viniferum were reported for the first time from chili. Pathogenicity of all species isolated from chili was confirmed, except for C. endophytica. The current study improves the understanding of species causing anthracnose on chili and provides useful information for the effective control of the disease in China.

Keywords: DNA phylogeny, multi-gene analysis, plant pathogen, systematics

INTRODUCTION

Chili (Capsicum spp.) is an important vegetable crop worldwide. China maintains the largest planted area of chili, producing more than 28 M tons per year for domestic consumption and export (Li et al. 2009). One of the most destructive diseases restricting chili production is anthracnose, caused by Colletotrichum spp. (Bailey & Jeger 1992, Poonpolgul & Kumphai 2007, Than et al. 2008), resulting in up to 40 % yield loss in China (Lin et al. 2004).

Colletotrichum species can infect more than 30 plant genera (Perfect et al. 1999, Dean et al. 2012, Farr & Rossman 2016). More than 10 Colletotrichum species have been reported from chili, with different distributions among countries (Than et al. 2008, Liao et al. 2012, Kanto et al. 2014, Sharma et al. 2014, Diao et al. 2015). For example, anthracnose on chili is caused by C. coccodes, C. fructicola, C. siamense, and C. truncatum in India (Sharma & Shenoy 2014); by C. acutatum, C. coccodes, and C. gloeosporioides in the USA; by C. acutatum, C. dematium, C. gloeosporioides, and C. truncatum in Australia; by C. acutatum, C. coccodes, C. dematium, C. gloeosporioides, and C. panacicola in Korea (Than et al. 2008); and by C. acutatum, C. gloeosporioides, C. truncatum, and C. coccodes in China (Shin et al. 1999, Liao et al. 2012). Most of these reports, however, were based on morphology and ITS sequences or a combination of ITS and TUB2 sequences, which have been shown to be insufficient in distinguishing closely related taxa in several species complexes. In addition, these records were mostly based on a small sampling from restricted areas, and, thus, may underestimate the species diversity.

The current study aimed to investigate the Colletotrichum species causing anthracnose on chili in China, by employing large-scale sampling and isolation, and via morphological characterisation and multi-locus phylogeny of the obtained strains.

MATERIALS AND METHODS

Sample collection and isolation

From 2008 to 2014, fruits and leaves of chili (Capsicum spp.) with anthracnose symptoms were collected from 50 locations in 29 provinces of China (Fig. 1). In each location, a hierarchical sampling method was used as previously described (Kohli et al. 1995). Five fields were chosen at each sampling location, and 25 chili fruits and also leaves in some cases were collected from each field along a diagonal transect. Colletotrichum species were isolated as described by Cai et al. (2009). All isolates were grown at 28 °C for further study. Type specimens of new species from this study were deposited in the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), and ex-type living cultures were deposited in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China.

Fig. 1.

Fig. 1

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Map showing locations in China where chili was sampled for Colletotrichum species. Each coloured circle represents one species by preliminary identification, and the size of the circle indicates the number of isolates collected from that location.

Morphological characterisation

Mycelial plugs (5 mm) were transferred from the edge of actively growing cultures to fresh potato dextrose agar (PDA, 1.5 %, Difco) plates. Cultures were kept at 28 °C with a 12/12 h fluorescent light/dark cycle. The morphological characters for all isolates, including colony and conidial characteristics, were observed. Microscopic characters were examined with the Carl Zeiss Imager A2 microscope after 1 mo of cultivation. Among the 1 285 obtained isolates (Table 1), 121 representative isolates were selected for further multilocus phylogenetic analyses based on geographical location, morphology (colony shape and colour and characteristics of aerial mycelia and conidia), and ITS sequences. Different morphological types were selected from each location, and the number of representative isolates selected depended on the number of isolates with different morphologies. Furthermore, if the number of isolates with high morphological and ITS sequence similarities was less than 10 in one location, then one isolate was randomly selected as a representative. If the number was more than 10, on the other hand, then an additional isolate (one from each of the 10 isolates) was selected for multi-locus phylogenetic analyses. The length and width of 30 conidia for each isolate were measured in lactic acid, and mean values calculated. The formation of appressoria was induced as described by Cai et al. (2009).

Table 1.

A list of all Colletotrichum isolates collected from chili in China based on preliminary identification.

Species Location Host tissue Year Number of isolates Longitude Latitude
C. aenigma Yangliuqing, Tianjin Fruit 2012 1 39.4 117.01
C. cliviae Xingcheng, Liaoning Fruit 2012 1 40.63 120.74
C.conoides Nanjing, Jiangsu Fruit 2010 3 32.06 118.79
C. endophytica Mile, Yunnan Fruit 2011 1 24.41 103.41
C. fioriniae Fengxiang, Shanxi Fruit 2011 17 34.55 107.4
Changchun Fruit 2011 47 43.81 125.32
Xining, Qinghai Fruit 2011 42 36.61 101.78
Sanya, Hainan Fruit 2012 2 18.25 109.51
Guiyang, Guizhou Fruit 2012 3 26.64 106.63
Xingcheng, Liaoning Fruit 2012 29 40.63 120.74
Yinchuan, Ningxia Fruit 2012 4 38.48 106.23
Guyuan, Ningxia Fruit 2012 8 36.01 106.24
Fengxian, Shanghai Fruit 2012 16 30.91 121.47
Harbin, Heilongjiang Fruit 2012 9 45.8 126.53
C. fructicola Fuzhou, Fujian Fruit 2011 2 26.07 119.29
Guilin, Guangxi Fruit 2011 10 25.27 110.29
Hengyang, Hunan Fruit 2012 4 29.03 111.69
Changsha, Hunan Fruit 2012 20 28.23 112.94
Laiyang, Shandong Fruit 2011 32 36.99 120.74
Wucheng, Shandong Fruit 2012 15 37.16 116.08
Zhangzhou, Fujian Fruit 2008 5 24.51 117.64
Quanzhou, Fujian Fruit 2009 23 24.87 118.67
Fengxiang, Shanxi Fruit 2011 5 34.55 107.4
Xinxiang, Henan Fruit 2011 5 35.3 113.93
Yichun, Jiangxi Fruit 2011 6 27.81 114.41
Jianyang, Sichuan Fruit 2011 8 30.41 104.55
Mile, Yunnan Fruit 2011 10 24.41 103.41
Yangliuqing, Tianjin Fruit 2012 4 39.4 117.01
Wuhan, Hubei Fruit 2012 4 30.28 114.29
Xingcheng, Liaoning Fruit 2012 4 40.63 120.74
Shizhu, Chongqing Fruit 2013 4 30.6 108.29
C. gloeosporioides Guilin, Guangxi Fruit 2011 1 25.27 110.29
Qingyuan, Guangdong Fruit 2013 21 23.28 112.48
Qingyuan, Guangdong Fruit 2014 1 23.28 112.48
Laiyang, Shandong Fruit 2011 64 36.99 120.74
Wucheng, Shandong Fruit 2011 30 37.16 116.08
Fengxiang, Shanxi Fruit 2011 10 34.55 107.4
Xinxiang, Henan Fruit 2011 10 35.3 113.93
Taizhou, Zhejiang Fruit 2011 8 28.65 121.42
Changsha, Hunan Fruit 2012 20 28.23 112.94
Mile, Yunnan Fruit 2011 5 24.41 103.41
Jianyang, Sichuan Fruit 2011 4 30.41 104.55
Guyuan, Ningxia Fruit 2012 2 36.01 106.24
Wuqing, Tianjin Fruit 2012 1 39.38 117.04
Xingcheng, Liaoning Fruit 2012 4 40.63 120.74
C. grossum Haikou, Hainan Fruit 2011 3 20.04 110.19
C. hymenocallidis Changsha, Hunan Fruit 2012 35 28.23 112.94
C. incanum Helingeer, Inner Mongolia Fruit 2012 6 40.37 111.82
C. karstii Sanya, Hainan Fruit 2012 3 18.25 109.51
Jianyang, Sichuan Fruit 2011 5 30.41 104.55
Mile, Yunnan Fruit 2011 1 24.41 103.41
C. liaoningense Xingcheng, Liaoning Fruit 2012 11 40.63 120.74
Shapingba, Chongqing Fruit 2012 1 29.54 106.46
C. scoville Yanqing, Beijing Fruit 2011 4 40.45 115.97
Changping, Beijing Fruit 2013 8 40.22 116.23
Fuzhou, Fujian Fruit 2011 68 26.07 119.29
Gangu, Gansu Fruit 2012 21 34.73 105.33
Jida, Jilin Fruit 2013 33 43.88 125.31
Changzhi, Shanxi Fruit 2011 84 36.19 113.11
Shuozhou, Shanxi Fruit 2012 14 39.33 112.43
Sanya, Hainan Fruit 2012 1 18.25 109.51
Zhijiang, Hunan Fruit 2011 1 27.44 109.68
Zhanjiang, Guangdong Fruit 2011 18 21.27 110.35
Qingyuan, Guangdong Fruit 2012 20 23.28 112.48
C. truncatum Qingyuan, Guangdong Fruit 2013 80 23.28 112.48
Qingyuan, Guangdong Fruit 2014 10 23.28 112.48
Maoming, Guangdong Fruit 2013 13 21.55 110.88
Yichun, Jiangxi Leave 2011 20 27.81 114.41
Shizhu, Chongqing Fruit 2013 23 30.6 108.29
Wuhan, Hubei Fruit 2013 25 30.28 114.29
Fengxiang, Shanxi Fruit 2011 12 34.55 107.4
Wucheng, Shandong Fruit 2011 125 37.16 116.08
Laiyang, Shandong Fruit 2011 10 36.99 120.74
Yangliuqing, Tianjin Fruit 2012 11 39.4 117.01
Langfang, Hebei Fruit 2011 20 39.52 116.61
Chengde, Hebei Fruit 2013 5 40.95 117.96
Daxing, Beijing Fruit 2011 9 39.73 116.34
Shunyi, Beijing Fruit 2011 10 40.13 116.65
Xingcheng, Liaoning Fruit 2012 16 40.63 120.74
Changchun, Jilin Fruit 2012 7 43.71 125.54
Chengdu, Sichuan Fruit 2011 3 30.57 104.07
Hefei, Anhui Fruit 2011 2 31.82 117.23
Fuzhou, Fujian Fruit 2011 2 26.07 119.29
Luoyang, Henan Fruit 2011 1 34.62 112.45
Xinxiang, Henan Fruit 2011 2 35.3 113.93
Changsha, Hunan Fruit 2012 1 28.23 112.94
Hengyang, Hunan Fruit 2012 1 26.89 112.57
Changde, Hunan Fruit 2012 3 29.03 111.69
Mile, Yunnan Fruit 2011 4 24.41 103.41
Zhanjiang, Guangdong Fruit 2011 5 21.27 110.36
Xining, Qinghai Fruit 2011 1 36.61 101.78
Shuozhou, Shanxi Fruit 2012 1 39.33 112.43
C. viniferum Mile, Yunnan Fruit 2011 1 24.41 103.41
Total 1285
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DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted from 121 representative isolates as previously described (Murray & Thompson 1980, Diao et al. 2015). The following loci were amplified with the indicated primers: the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) with primers ITS4/ITS5 (White et al. 1990); partial sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primers GDF1/GDR1 (Templeton et al. 1992); actin gene (ACT) with primers ACT512F/ACT783R (Carbone & Kohn 1999); beta-tubulin (TUB2) with primers T1/Bt2b (Glass & Donaldson 1995, O’Donnell & Cigelnik 1997); calmodulin (CAL) with primers CL1/CL2A (O’Donnell et al. 2000); chitin synthase 1 (CHS-1) with primers CHS-79F/CHS-345R (Carbone & Kohn 1999); and histone3 (HIS3) with primers CYLH3F/CYLH3R (Crous et al. 2004b). PCR reactions were performed as described by Damm et al. (2009). DNA sequencing was conducted by Sunbiotech, Beijing, China with a 3730 DNA Analyzer (Applied Biosystems, USA). The sequences obtained from forward and reverse primers were used to obtain consensus sequences with DNAMAN v. 6.0 (Lynnon Biosoft, USA). Sequences were aligned using MAFFT v. 6 (Katoh & Toh 2010).

Phylogenetic analysis

All sequences of the 121 representative isolates were blasted in GenBank. Sequences with high similarities were selected and included in the analyses (Table 2a, 2b, 2c). Concatenated analyses of ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2 were conducted for the C. acutatum species complex and Colletotrichum species with curved conidia, while ITS, GAPDH, CAL, ACT, CHS-1, and TUB2 were concatenated for the analysis of the C. gloeosporioides species complex and other species. Maximum parsimony (MP) analyses based on the combined datasets were conducted using PAUP v. 4.0b10 (Swofford 2002). Phylogenetic trees were generated using the heuristic search option with TBR branch swapping and 1 000 random sequence additions. Maxtrees were unlimited, with branches of zero length collapsed, and all multiple parsimonious trees were saved. Clade stability was assessed using a bootstrap analysis with 1 000 replicates. Afterward, tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated. Bayesian inference (BI) was used to reconstruct the phylogenetic tree using MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003). Best-fit models of nucleotide substitution were selected using MrModelTest v. 2.3 (Nylander 2004). Two analyses of four MCMC chains were run from random trees for 1 000 000 generations, and trees were sampled every 100 generations resulting in 20 000 total trees. The first 25 % of the trees were discarded as the burn-in phase of each analysis, and the remaining trees were used to calculate posterior probabilities (Cai et al. 2006, Liu et al. 2012, 2013). An additional Maximum likelihood (ML) analysis was implemented in the C. gloeosporioides species complex using the CIPRES Science Gateway v. 3.3 (www.phylo.org), and the RAxML-HPC BlackBox was selected with default parameters. Sequences derived in this study were deposited in GenBank (Table 2), and the concatenated alignments were deposited in TreeBASE (http://treebase.org/treebase-web/home.html; study S17061), and the descriptions and nomenclature in MycoBank (Crous et al. 2004a).

Table 2a.

Strains used for the phylogenetic analysis of the Colletotrichum gloeosporioides species complex and other species with details about host, location, and GenBank accession numbers.

Species Isolate Host Location GenBank accessions
ITS GAPDH CAL ACT CHS-1 TUB2
C. aenigma ICMP 18686 Pyrus pyrifolia Japan JX010243 JX009913 JX009684 JX009519 JX009789 JX010390
ICMP 18608* Persea americana Israel JX010244 JX010044 JX009683 JX009443 JX009774 JX010389
C. aeschynomenes ICMP 17673* Aeschynomene virginica USA JX010176 JX009930 JX009721 JX009483 JX009799 JX010392
C. alatae CBS 304.67* Dioscorea alata India JX010190 JX009990 JX009738 JX009471 JX009837 JX010383
ICMP 18122 Dioscorea alata Nigeria JX010191 JX010011 JX009739 JX009470 JX009846 JX010449
C. alienum ICMP 12071* Malus domestica New Zealand JX010251 JX010028 JX009654 JX009572 JX009882 JX010411
ICMP 18621 Persea americana New Zealand JX010246 JX009959 JX009657 JX009552 JX009755 JX010386
C. aotearoa ICMP 18537* Coprosma sp. New Zealand JX010205 JX010005 JX009611 JX009564 JX009853 JX010420
C. asianum ICMP 18580* Coffea arabica Thailand FJ972612 JX010053 FJ917506 JX009584 JX009867 JX010406
ICMP 18696 Mangifera indica Australia JX010192 JX009915 JX009723 JX009576 JX009753 JX010384
C. boninense MAFF 305972* Crinum asiaticum var. sinicum Japan JX010292 JX009905 JQ005674 JX009583 JX009827 JQ005588
CBS 128547 Camellia sp. New Zealand JQ005159 JQ005246 JQ005680 JQ005507 JQ005333 JQ005593
C. brevisporum BCC 38876* Neoregalia sp. Thailand JN050238 JN050238 JN050222 JN050216 KF687760 JN050244
MFLUCC100182 Pandanus pygmaeus Thailand JN050239 JN050228 JN050217 JN050245
C. clidemiae ICMP 18706 Clidemia hirta USA JX010274 JX009909 JX009639 JX009476 JX009777 JX010439
C. cliviae CBS 125375* Clivia miniata China JX519223 JX546611 JX519240 JX519232 JX519249
CSSS1 Clivia miniata China GU109479 GU085867 GU085861 GU085869
CSSS2 Clivia miniata China GU109480 GU085868 GU085862 GU085870
CAUOS6 Capsicum annuum China KP890123 KP890100 KP890131 KP890115
C. conoides CAUG17* Capsicum annuum China KP890168 KP890162 KP890150 KP890144 KP890156 KP890174
CAUG33 Capsicum annuum China KP890169 KP890163 KP890151 KP890145 KP890157 KP890175
CAUG34 Capsicum annuum China KP890170 KP890164 KP890152 KP890146 KP890158 KP890176
C. cordylinicola ICMP 18579 Cordyline fruticosa Thailand JX010226 JX009975 HM470238 HM470235 JX009864 JX010440
C. dracaenophilum CBS 118199* Dracaena sp. China JX519222 JX546707 JX519238 JX519230 JX519247
C. endophytica CAUG28 Capsicum annuum China KP145441 KP145413 KP145357 KP145329 KP145385 KP145469
LC0324* Pennisetu purpureum Thailand KC633854 KC832854 KC810018 KF306258
MFLUCC 100676 Pennisetu purpureum Thailand KF242123 KF242181 KF254846 KF157827
C. fructicola CAUG1 Capsicum sp. China KP145416 KP145388 KP145332 KP145304 KP145360 KP145444
CAUG5 Capsicum sp. China KP145420 KP145392 KP145336 KP145308 KP145364 KP145448
CAUG6 Capsicum sp. China KP145421 KP145393 KP145337 KP145309 KP145365 KP145449
CAUG8 Capsicum sp. China KP145422 KP145394 KP145338 KP145310 KP145366 KP145450
CAUG10 Capsicum sp. China KP145424 KP145396 KP145340 KP145312 KP145368 KP145452
CAUG11 Capsicum sp. China KP145425 KP145397 KP145341 KP145313 KP145369 KP145453
CAUG16 Capsicum sp. China KP145430 KP145402 KP145346 KP145318 KP145374 KP145458
CAUG18 Capsicum sp. China KP145431 KP145403 KP145347 KP145319 KP145375 KP145459
CBS 125395 Theobroma cacao Panama JX010172 JX009992 JX009666 JX009543 JX009873 JX010408
ICMP 17789 Malus domestica USA JX010178 JX009914 JX009665 JX009451 JX009809
ICMP 12568 Persea americana Australia JX010166 JX009946 JX009680 JX009529 JX009762
ICMP 18581* Coffea arabica Thailand JX010165 JX010033 FJ917508 FJ907426 JX009866 JX010405
ICMP 18613 Limonium sinuatum Israel JX010167 JX009998 JX009675 JX009491 JX009772 JX010388
ICMP 18727 Fragaria ananassa USA JX010179 JX010035 JX009682 JX009565 JX009812 JX010394
CBS 238.49* Ficus edulis Germany JX010181 JX009923 JX009671 JX009495 JX009839 JX010400
CBS 125397* Tetragastris panamensis Panama JX010173 JX010032 JX009674 JX009581 JX009874 JX010409
C. gloeosporiodes CAUG2 Capsicum sp. China KP145417 KP145389 KP145333 KP145305 KP145361 KP145445
CAUG3 Capsicum sp. China KP145418 KP145390 KP145334 KP145306 KP145362 KP145446
CAUG12 Capsicum sp. China KP145426 KP145398 KP145342 KP145314 KP145370 KP145454
CAUG13 Capsicum sp. China KP145427 KP145399 KP145343 KP145315 KP145371 KP145455
CAUG14 Capsicum sp. China KP145428 KP145400 KP145344 KP145316 KP145372 KP145456
CAUG15 Capsicum sp. China KP145429 KP145401 KP145345 KP145317 KP145373 KP145457
CAUG19 Capsicum sp. China KP145432 KP145404 KP145348 KP145320 KP145376 KP145460
CAUG20 Capsicum sp. China KP145433 KP145405 KP145349 KP145321 KP145377 KP145461
CAUG22 Capsicum sp. China KP145435 KP145407 KP145351 KP145323 KP145379 KP145463
CAUG23 Capsicum sp. China KP145436 KP145408 KP145352 KP145324 KP145380 KP145464
CAUG24 Capsicum sp. China KP145437 KP145409 KP145353 KP145325 KP145381 KP145465
CAUG25 Capsicum sp. China KP145438 KP145410 KP145354 KP145326 KP145382 KP145466
CAUG26 Capsicum sp. China KP145439 KP145411 KP145355 KP145327 KP145383 KP145467
CAUG29 Capsicum sp. China KP145442 KP145414 KP145358 KP145330 KP145386 KP145470
IMI 356878* Citrus sinensis Italy JX010152 JX010056 JX009731 JX009531 JX009818 JX010445
CORCG4 Vanda sp. Thailand HM034808 HM034806 HM034802 HM034800
CORCG5 Vanda sp. Thailand HM034809 HM034807 HM034803 HM034801 HM034805 HM034811
C. grevillea CBS 132879* Grevillea sp. Italy KC297078 KC297010 KC296963 KC296941 KC296987 KC297102
C. grossum CAUG7* Capsicum sp. China KP890165 KP890159 KP890147 KP890141 KP890153 KP890171
CAU31 Capsicum sp. China KP890166 KP890160 KP890148 KP890142 KP890154 KP890172
CAUG32 Capsicum sp. China KP890167 KP890161 KP890149 KP890143 KP890155 KP890173
C. hebeiense JZB330024 Vitis vinifera cv. Cabernet Sauvignon China KF156873 KF377505 KF377542
JZB330028* Vitis vinifera cv. Cabernet Sauvignon China KF156863 KF377495 KF377532 KF289008 KF288975
C. hymenocallidis CAUG9 Capsicum sp. China KP145423 KP145395 KP145339 KP145311 KP145367 KP145451
ICMP 18642* Hymenocallis americana China JX010278 JX010019 JX009709 GQ856775 GQ856730 JX010410
C. kahawae subsp. ciggaro ICMP 12952 Persea americana New Zealand JX010214 JX009971 JX009648 JX009431 JX009757 JX010426
ICMP 18539* Olea europaea Australia JX010230 JX009966 JX009635 JX009523 JX009800 JX010434
C. karstii CGMCC 3.14194* Vanda sp. China HM585409 HM585391 HM581995 HM585428
CAUOS1 Capsicum sp. China KP890103 KP890134 KP890118 KP890096 KP890126 KP890110
CAUOS7 Capsicum sp. China KP890108 KP890139 KP890124 KP890101 KP890132 KP890116
CAUOS8 Capsicum sp. China KP890109 KP890140 KP890125 KP890102 KP890133 KP890117
C. liaoningense CAUOS2* Capsicum sp. China KP890104 KP890135 KP890119 KP890097 KP890127 KP890111
CAUOS3 Capsicum sp. China KP890105 KP890136 KP890120 KP890098 KP890128 KP890112
CAUOS4 Capsicum sp. China KP890106 KP890137 KP890121 KP890099 KP890129 KP890113
CAUOS5 Capsicum sp. China KP890107 KP890138 KP890122 KP890130 KP890114
C. musae ICMP 19119* Musa sp. USA JX010146 JX010050 JX009742 JX009433 JX009896 HQ596280
IMI 52264 Musa sapientum Kenya JX010142 JX010015 JX009689 JX009432 JX009815 JX010395
C. nupharicola ICMP 17938 Nuphar lutea subsp. polysepala USA JX010189 JX009936 JX009661 JX009486 JX009834 JX010397
ICMP 17940 Nymphaea ordorata USA JX010188 JX010031 JX009662 JX009582 JX009836 JX010399
ICMP 18187* Nuphar lutea subsp. polysepala USA JX010187 JX009972 JX009663 JX009437 JX009835 JX010398
C. psidii ICMP 19120* Psidium sp. Italy JX010219 JX009967 JX009743 JX009515 JX009901 JX010443
C. queenslandicum ICMP 1778* Carica papaya Australia JX010276 JX009934 JX009691 JX009447 JX009899 JX010414
C. salsolae ICMP 19051 Salsola tragus Hungary JX010242 JX009916 JX009696 JX009562 JX009863 JX010403
C. siamense CBS 130420 Jasminum sambac Vietnam HM131511 HM131497 JX009713 HM131507 JX009895 JX010415
C. thailandicum BCC 38879* Hibiscus rosasinensis Thailand JN050242 JN050231 JN050220 JN050248
MFLUCC10092 Alocasia sp. Thailand JN050243 JN050232 JN050221 JN050249
C. theobromicola CBS 124945* Theobroma cacao Panama JX010294 JX010006 JX009591 JX009444 JX009869 JX010447
ICMP 17814 Fragaria vesca USA JX010288 JX010003 JX009589 JX009448 JX009819 JX010379
ICMP 17957 Stylosanthes viscosa Australia JX010289 JX009962 JX009597 JX009575 JX009821 JX010380
C. ti ICMP 4832* Cordyline sp. New Zealand JX010269 JX009952 JX009649 JX009520 JX009898 JX010442
ICMP 5285 Cordyline australis New Zealand JX010267 JX009910 JX009650 JX009553 JX009897 JX010441
C. tropicale CBS 124949* Theobroma cacao Panama JX010264 JX010007 JX009719 JX009489 JX009870 JX010407
ICMP 18672 Litchi chinensis Japan JX010275 JX010020 JX009722 JX009480 JX009826 JX010396
C. tropicicola BCC 38877* Citrus maxima Thailand JN050240 JN050229 JN050218 JN050246
MFLUCC100167 Paphiopedilum bellatolum Thailand JN050241 JN050230 JN050219 JN050247
C. viniferum CAUG27 Capsicum sp. China KP145440 KP145412 KP145356 KP145328 KP145384 KP145468
GZAAS 5.08601* Vitis vinifera, cv. ‘Shuijing’ China JN412804 JN412798 JQ309639 JN412795 JN412813
GZSSS 5.08608 Vitis vinifera, cv. ‘Hongti’ China JN412802 JN412800 JQ412782 JN412793 JN412811
C. xanthorrhoeae ICMP 17903* Xanthorrhoea preissii Australia JX010261 JX009927 JX009653 JX009478 JX009823 JX010448
C. yunnanense CBS 132135* Buxus sp. China JX546804 JX519248 JX546706 JX519239 JX519231
Glomella cingulata ICMP 10643 Camellia williamsii UK JX010224 JX009908 JX009630 JX009540 JX009891 JX010436
‘f. sp. camelliae’
Monilochaetes infuscans CBS 869.96* Ipomoea batatas South Africa JQ005780 JX546612 JQ005843 JQ005801 JQ005864
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* = Ex-type culture. Strains studied in this paper are in bold font.

Table 2b.

Strains used for the phylogenetic analysis of the Colletotrichum acutatum species complex with details about host, location, and GenBank accession numbers.

Species Isolate Host Location GenBank accessions
ITS GAPDH CHS-1 HIS3 ACT TUB2
C. acutatum CBS 112996* Carica papaya Australia JQ005776 JQ948677 JQ005797 JQ005818 JQ005839 JQ005860
CBS 144.29 Capsicum annuum Sri Lanka JQ948401 JQ948732 JQ949062 JQ949392 JQ949722 JQ950052
C. chrysanthemi CBS 126518 Carthamus sp. Netherlands JQ948271 JQ948601 JQ948932 JQ949262 JQ949592 JQ949922
IMI 364540 Chrysanthemum coronarium China JQ948272 JQ948602 JQ948933 JQ949263 JQ949593 JQ949923
C. fioriniae CAUA18 Capsicum annuum China KP145016 KP145096 KP145048 KP145064 KP145032 KP145080
CAUA20 Capsicum annuum China KP145226 KP145162 KP145290 KP145194 KP145130 KP145258
CAUA24 Capsicum annuum China KP145017 KP145097 KP145049 KP145065 KP145033 KP145081
CAUA25 Capsicum annuum China KP145018 KP145098 KP145050 KP145066 KP145034 KP145082
CAUA26 Capsicum annuum China KP145019 KP145099 KP145051 KP145067 KP145035 KP145083
CAUA27 Capsicum annuum China KP145020 KP145100 KP145052 KP145068 KP145036 KP145084
CAUA28 Capsicum annuum China KP145021 KP145101 KP145053 KP145069 KP145037 KP145085
CAUA29 Capsicum annuum China KP145022 KP145102 KP145054 KP145070 KP145038 KP145086
CAUA30 Capsicum annuum China KP145023 KP145103 KP145055 KP145071 KP145039 KP145087
CAUA31 Capsicum annuum China KP145024 KP145104 KP145056 KP145072 KP145040 KP145088
CAUA32 Capsicum annuum China KP145025 KP145105 KP145057 KP145073 KP145041 KP145089
CAUA37 Capsicum annuum China KP145026 KP145106 KP145058 KP145074 KP145042 KP145090
CAUA38 Capsicum annuum China KP145027 KP145107 KP145059 KP145075 KP145043 KP145091
CAUA39 Capsicum annuum China KP145028 KP145108 KP145060 KP145076 KP145044 KP145092
CAUA40 Capsicum annuum China KP145029 KP145109 KP145061 KP145077 KP145045 KP145093
CAUA41 Capsicum annuum China KP145030 KP145110 KP145062 KP145078 KP145046 KP145094
CAUA48 Capsicum annuum China KP145031 KP145111 KP145063 KP145079 KP145047 KP145095
CBS 125396 Malus domestica USA JQ948299 JQ948629 JQ948960 JQ949290 JQ949620 JQ949950
CBS 127537 Vaccinium sp. USA JQ948317 JQ948647 JQ948978 JQ949308 JQ949638 JQ949968
CBS 128517* Fiorinia sp. USA JQ948292 JQ948622 JQ948953 JQ949283 JQ949613 JQ949943
CBS 129916 Vaccinium sp. USA JQ948317 JQ948647 JQ948978 JQ949308 JQ949638 JQ949968
CBS 293.67 Persea sp. Australia JQ948310 JQ948640 JQ948971 JQ949301 JQ949631 JQ949961
CBS 127601 Mangifera sp. Australia JQ948311 JQ948641 JQ948972 JQ949302 JQ949632 JQ949962
CBS 129947 Vitis sp. Portugal JQ948343 JQ948673 JQ949004 JQ949334 JQ949664 JQ949994
CBS 200.35 Rubus sp. USA JQ948293 JQ948623 JQ948954 JQ949284 JQ949614 JQ949944
C. gloeosporioides IMI 356878* Citrus sinensi Italy JX010152 JX010056 JX009818 JX009531 JX010445
C. godetiae CBS 133.44* Godetia sp. Denmark JQ948402 JQ948733 JQ949063 JQ949393 JQ949723 JQ950053
C. guajavae IMI 350839* Psidium sp. India JQ948270 JQ948600 JQ948931 JQ949261 JQ949591 JQ949921
C. laticiphilum CBS 112989* Hevea sp. India JQ948289 JQ948619 JQ948950 JQ949280 JQ949610 JQ949940
C. nymphaeae CBS 515.78* Nymphaea sp. Netherlands JQ948197 JQ948527 JQ948858 JQ949188 JQ949518 JQ949848
C. orchidophilum CBS 632.80* Dendrobium sp. USA JQ948151 JQ948481 JQ948812 JQ949142 JQ949472 JQ949802
C. salicis CBS 607.94* Salix sp. Netherlands JQ948460 JQ948791 JQ949121 JQ949451 JQ949781 JQ950111
C. scovillei CAUA1 Capsicum annuum China KP145208 KP145144 KP145272 KP145176 KP145112 KP145240
CAUA2 Capsicum annuum China KP145209 KP145145 KP145273 KP145177 KP145113 KP145241
CAUA3 Capsicum annuum China KP145210 KP145146 KP145274 KP145178 KP145114 KP145242
CAUA4 Capsicum annuum China KP145211 KP145147 KP145275 KP145179 KP145115 KP145243
CAUA5 Capsicum annuum China KP145212 KP145148 KP145276 KP145180 KP145116 KP145244
CAUA6 Capsicum annuum China KP145213 KP145149 KP145277 KP145181 KP145117 KP145245
CAUA7 Capsicum annuum China KP145214 KP145150 KP145278 KP145182 KP145118 KP145246
CAUA8 Capsicum annuum China KP145215 KP145151 KP145279 KP145183 KP145119 KP145247
CAUA9 Capsicum annuum China KP145216 KP145152 KP145280 KP145184 KP145120 KP145248
CAUA10 Capsicum annuum China KP145217 KP145153 KP145281 KP145185 KP145121 KP145249
CAUA11 Capsicum annuum China KP145218 KP145154 KP145282 KP145186 KP145122 KP145250
CAUA12 Capsicum annuum China KP145219 KP145155 KP145283 KP145187 KP145123 KP145251
CAUA13 Capsicum annuum China KP145220 KP145156 KP145284 KP145188 KP145124 KP145252
CAUA14 Capsicum annuum China KP145221 KP145157 KP145285 KP145189 KP145125 KP145253
CAUA15 Capsicum annuum China KP145222 KP145158 KP145286 KP145190 KP145126 KP145254
CAUA16 Capsicum annuum China KP145223 KP145159 KP145287 KP145191 KP145127 KP145255
CAUA17 Capsicum annuum China KP145224 KP145160 KP145288 KP145192 KP145128 KP145256
CAUA19 Capsicum annuum China KP145225 KP145161 KP145289 KP145193 KP145129 KP145257
CAUA21 Capsicum annuum China KP145227 KP145163 KP145291 KP145195 KP145131 KP145259
CAUA22 Capsicum annuum China KP145228 KP145164 KP145292 KP145196 KP145132 KP145260
CAUA23 Capsicum annuum China KP145229 KP145165 KP145293 KP145197 KP145133 KP145261
CAUA33 Capsicum annuum China KP145230 KP145166 KP145294 KP145198 KP145134 KP145262
CAUA34 Capsicum annuum China KP145231 KP145167 KP145295 KP145199 KP145135 KP145263
CAUA35 Capsicum annuum China KP145232 KP145168 KP145296 KP145200 KP145136 KP145264
CAUA36 Capsicum sp. China KP145233 KP145169 KP145297 KP145201 KP145137 KP145265
CAUA42 Capsicum sp. China KP145234 KP145170 KP145298 KP145202 KP145138 KP145266
CAUA43 Capsicum sp. China KP145235 KP145171 KP145299 KP145203 KP145139 KP145267
CAUA44 Capsicum sp. China KP145236 KP145172 KP145300 KP145204 KP145140 KP145268
CAUA45 Capsicum sp. China KP145237 KP145173 KP145301 KP145205 KP145141 KP145269
CAUA46 Capsicum sp. China KP145238 KP145174 KP145302 KP145206 KP145142 KP145270
CAUA47 Capsicum sp. China KP145239 KP145175 KP145303 KP145207 KP145143 KP145271
CBS 120708 Capsicum sp. Thailand JQ948269 JQ948599 JQ948930 JQ949260 JQ949590 JQ949920
CBS 126529* Capsicum sp. Indonesia JQ948267 JQ948597 JQ948928 JQ949258 JQ949588 JQ949918
CBS 126530 Capsicum sp. Indonesia JQ948268 JQ948598 JQ948929 JQ949259 JQ949589 JQ949919
C. simmondsii CBS 122122* Carica sp. Australia JQ948276 JQ948606 JQ948937 JQ949267 JQ949597 JQ949927
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* = Ex-type culture. Strains studied in this paper are in bold font.

Table 2c.

Strains used for the phylogenetic analysis of Colletotrichum species with curved conidia with details on host, location, and GenBank accession numbers.

Species Isolate Host Location GenBank accession numbers
ITS ACT TUB2 CHS-1 GAPDH HIS3
C. anthrisci CBS 125335 Anthriscus sylvestris Netherlands GU227846 GU227944 GU228140 GU228336 GU228238 GU228042
C. chlorophyti IMI 103806* Chlorophytum sp. India GU227894 GU227992 GU228188 GU228384 GU228286 GU228090
CBS 142.79 Stylosanthes hamata Australia GU227895 GU227993 GU228189 GU228385 GU228287 GU228091
C. circinans CBS 111.21 Allium cepa USA GU227854 GU227952 GU228148 GU228344 GU228246 GU228050
CBS 221.81* Allium cepa Serbia GU227855 GU227953 GU228149 GU228345 GU228247 GU228051
C. dematium CBS 125.25* Eryngium campestre France GU227819 GU227917 GU228113 GU228309 GU228211 GU228015
CBS 125340 Apiaceae Czech Republic GU227820 GU227918 GU228114 GU228310 GU228212 GU228016
C. fructi CBS 346.37* Malus sylvestris USA GU227844 GU227942 GU228138 GU228334 GU228236 GU228040
C. incanum ATCC 64682* Glycine max USA KC110789 KC110825 KC110816 KC110807 KC110798
IL6A Glycine max USA KC110787 KC110823 KC110814 KC110805 KC110796
IL9A Glycine max USA KC110788 KC110824 KC110815 KC110806 KC110797
CAUCT34 Capsicum sp. China KP145641 KP145505 KP145675 KP145539 KP145573 KP145607
C. lilii CBS 109214 Lilium sp. Japan GU227810 GU227908 GU228104 GU228300 GU228202 GU228006
C. lindemuthianum CBS 151.28 Lilium sp. Netherlands GU227800 GU227898 GU228094 GU228290 GU228192 GU227996
C. lineola CBS 125337* Apiaceae sp. Czech Republic GU227829 GU227927 GU228123 GU228319 GU228221 GU228025
CBS 125339 Apiaceae sp. Czech Republic GU227830 GU227928 GU228124 GU228320 GU228222 GU228026
C. liriopes CBS 119444* Lirope muscari Mexico GU227804 GU227902 GU228098 GU228294 GU228196 GU228000
CBS 122747 Lirope muscari Mexico GU227805 GU227903 GU228099 GU228295 GU228197 GU228001
C. phaseolorum 1 CBS 157.36 Phaseolus radiatus var. aureus Japan GU227896 GU227994 GU228190 GU228386 GU228288 GU228092
C. phaseolorum 2 CBS 158.36 Vigna sinensis Japan GU227897 GU227995 GU228191 GU228387 GU228289 GU228093
C. rusci CBS 119206* Ruscus sp. Italy GU227818 GU227916 GU228112 GU228308 GU228210 GU228014
C. spaethianum CBS 167.49* Hosta sieboldiana Germany GU227807 GU227905 GU228101 GU228297 GU228199 GU228003
CBS 100063 Lilium sp. South Korea GU227808 GU227906 GU228102 GU228298 GU228200 GU228004
CBS 101631 Hemerocallis sp. New Zealand GU227809 GU227907 GU228103 GU228299 GU228201 GU228005
C. spinaciae CBS 128.57 Spinacia oleracea Netherlands GU227847 GU227945 GU228141 GU228337 GU228239 GU228043
IMI 104607 Spinacia sp. Italy GU227850 GU227948 GU228144 GU228340 GU228242 GU228046
C. tofieldiae CBS 168.49 Lupinus polyphyllus Germany GU227802 GU227900 GU228096 GU228292 GU228194 GU227998
CBS 495.85 Tofieldia calyculata Switzerland GU227801 GU227899 GU228095 GU228291 GU228193 GU227997
C. trichellum CBS 118198 Hedera sp. Guatemala GU227813 GU227911 GU228107 GU228303 GU228205 GU228009
CBS 217.64 Hedera helix UK GU227812 GU227910 GU228106 GU228302 GU228204 GU228008
C. truncatum CBS 182.52 Glycine max USA GU227866 GU227964 GU228160 GU228356 GU228258 GU228062
CBS195.32 Glycine max USA GU227865 GU227963 GU228159 GU228355 GU228257 GU228061
CBS 345.70 Glycine max Denmark GU227867 GU227965 GU228161 GU228357 GU228259 GU228063
CBS 151.35* Phaseolus lunatus USA GU227862 GU227960 GU228156 GU228352 GU228254 GU228058
CAUCT1 Capsicum annuum China KP145608 KP145472 KP145642 KP145506 KP145540 KP145574
CAUCT2 Capsicum annuum China KP145609 KP145473 KP145643 KP145507 KP145541 KP145575
CAUCT3 Capsicum annuum China KP145610 KP145474 KP145644 KP145508 KP145542 KP145576
CAUCT4 Capsicum annuum China KP145611 KP145475 KP145645 KP145509 KP145543 KP145577
CAUCT5 Capsicum annuum China KP145612 KP145476 KP145646 KP145510 KP145544 KP145578
CAUCT6 Capsicum annuum China KP145613 KP145477 KP145647 KP145511 KP145545 KP145579
CAUCT7 Capsicum sp. China KP145614 KP145478 KP145648 KP145512 KP145546 KP145580
CAUCT8 Capsicum sp. China KP145615 KP145479 KP145649 KP145513 KP145547 KP145581
CAUCT9 Capsicum sp. China KP145616 KP145480 KP145650 KP145514 KP145548 KP145582
CAUCT10 Capsicum sp. China KP145617 KP145481 KP145651 KP145515 KP145549 KP145583
CAUCT11 Capsicum sp. China KP145618 KP145482 KP145652 KP145516 KP145550 KP145584
CAUCT12 Capsicum sp. China KP145619 KP145483 KP145653 KP145517 KP145551 KP145585
CAUCT13 Capsicum sp. China KP145620 KP145484 KP145654 KP145518 KP145552 KP145586
CAUCT14 Capsicum sp. China KP145621 KP145485 KP145655 KP145519 KP145553 KP145587
CAUCT15 Capsicum sp. China KP145622 KP145486 KP145656 KP145520 KP145554 KP145588
CAUCT16 Capsicum sp. China KP145623 KP145487 KP145657 KP145521 KP145555 KP145589
CAUCT17 Capsicum sp. China KP145624 KP145488 KP145658 KP145522 KP145556 KP145590
CAUCT18 Capsicum sp. China KP145625 KP145489 KP145659 KP145523 KP145557 KP145591
CAUCT19 Capsicum sp. China KP145626 KP145490 KP145660 KP145524 KP145558 KP145592
CAUCT20 Capsicum sp. China KP145627 KP145491 KP145661 KP145525 KP145559 KP145593
CAUCT21 Capsicum sp. China KP145628 KP145492 KP145662 KP145526 KP145560 KP145594
CAUCT22 Capsicum sp. China KP145629 KP145493 KP145663 KP145527 KP145561 KP145595
CAUCT23 Capsicum sp. China KP145630 KP145494 KP145664 KP145528 KP145562 KP145596
CAUCT24 Capsicum sp. China KP145631 KP145495 KP145665 KP145529 KP145563 KP145597
CAUCT25 Capsicum sp. China KP145632 KP145496 KP145666 KP145530 KP145564 KP145598
CAUCT26 Capsicum sp. China KP145633 KP145497 KP145667 KP145531 KP145565 KP145599
CAUCT27 Capsicum sp. China KP145634 KP145498 KP145668 KP145532 KP145566 KP145600
CAUCT28 Capsicum sp. China KP145635 KP145499 KP145669 KP145533 KP145567 KP145601
CAUCT29 Capsicum sp. China KP145636 KP145500 KP145670 KP145534 KP145568 KP145602
CAUCT30 Capsicum sp. China KP145637 KP145501 KP145671 KP145535 KP145569 KP145603
CAUCT31 Capsicum sp. China KP145638 KP145502 KP145672 KP145536 KP145570 KP145604
CAUCT32 Capsicum sp. China KP145639 KP145503 KP145673 KP145537 KP145571 KP145605
CAUCT33 Capsicum sp. China KP145640 KP145504 KP145674 KP145538 KP145572 KP145606
CAUCT33 Capsicum sp. China KP145640 KP145504 KP145674 KP145538 KP145572 KP145606
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* = Ex-type culture. Strains studied in this paper are in bold

Genealogical concordance phylogenetic species recognition analysis

New species and their most closely related neighbours were analysed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model with a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014). The PHI tests were performed in SplitsTree4 (Huson 1998, Huson & Bryant 2006) to determine the recombination level within phylogenetically closely related species using a six-locus concatenated dataset (ACT, CAL, CHS, GAPDH, ITS, and TUB2) for C. conoides, C. grossum, and C. liaoningense and their respective related species. A pairwise homoplasy index below a 0.05 threshold (Φw < 0.05) indicated the presence of significant recombination in the dataset. The relationship between closely related species was visualised by constructing a split graph.

Pathogenicity assay

Seven chili cultivars, Capsicum annuum cv. Chaotianjiao, Denglongjiao, Sanyingjiao, Zidantou, C. frutescens cv. Shuangla, Xiaomila and C. chinense cv. Huangdijiao, were inoculated with representative strains of 15 Colletotrichum species respectively (Table 3, 4). Pathogenicity tests were conducted on chili following the methods described by Montri et al. (2009) and Mongkolporn et al. (2010). Healthy, ripe red and green chili fruits were surface sterilised in 1 % NaClO for 5 min separately, washed twice with sterile-distilled water, and air dried on sterile filter paper. Each fruit was inoculated with 1 μL of a conidial suspension (1 × 106 conidial/mL), which was injected onto the non-wounded fruit surface using a microsyringe (Shanghai, China). Control fruits were treated with 1 μL of distilled water. Each isolate was inoculated to five replicate fruits. The inoculated fruits were incubated in a moist chamber at 28 °C and were examined for symptoms daily for 9 d. The virulence and pathotypes were evaluated as described by Montri et al. (2009). The experiment was conducted twice.

Table 3.

Anthracnose severity scores on a 0–9 scale and pathotypes of 15 Colletotrichum species isolates at the ripe red fruit stage of seven chili cultivars.

Isolate Capsicum annuum
 C. frutescens
 C. chinense
 Mean Pathotype
Chaotianjiao Denglongjiao Sanyingjiao Zidantou Shuanla Xiaomila Huangdijiao
C. aenigma CAUG26 5 5 7 5 5 7 5 6 PC1-R
C. cliviae CAUOS5 7 7 5 7 7 5 0 5 PC2-R
C. conoides CAUG17 7 7 7 7 7 5 5 6 PC1-R
C. endophytica CAUG28 0 0 0 0 0 0 0 0 PC3-R
C. fioriniae CAUT34 7 7 9 9 9 9 7 8 PC1-R
C. fructicola CAUG1 7 7 9 9 9 9 7 8 PC1-R
C. gloeosporioides CAUG2 7 7 9 9 7 7 5 7 PC1-R
C. grossum CAUG7 7 7 7 7 5 5 7 6 PC1-R
C. hymenocallidis CAUG9 5 7 9 9 7 7 0 6 PC2-R
C. incanum CAUT34 5 7 7 5 7 5 5 6 PC1-R
C. karstii CAUOS1 7 5 7 7 9 9 7 7 PC1-R
C. liaoningense CAUOS2 9 7 9 9 5 9 5 8 PC1-R
C. scovillei CAUA1 7 9 9 9 9 7 9 8 PC1-R
C. truncatum CAUT1 7 7 9 7 9 9 7 8 PC1-R
C. viniferum CAUG27 5 7 9 9 9 9 5 8 PC1-R
Mean 6 6 7 7 7 7 5 6 –
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Table 4.

Anthracnose severity scores on a 0–9 scale and pathotypes of 15 Colletotrichum species isolates at the mature green fruit stage of seven chili cultivars.

Isolate Capsicum annuum
 C. frutescens
 C. chinense
 Mean Pathotype
Chaotianjiao Denglongjiao Sanyingjiao Zidantou Shuanla Xiaomila Huangdijiao
C. aenigma CAUG26 5 5 5 5 0 0 0 3 PC1-G
C. cliviae CAUOS5 7 9 5 7 7 5 7 7 PC2-G
C. conoides CAUG17 7 5 0 5 7 5 0 4 PC3-G
C.endophyticaCAUG28 0 0 0 0 0 0 0 0 PC4-G
C. fioriniae CAUT34 9 9 7 7 9 9 7 8 PC2-G
C. fructicola CAUG1 7 9 9 5 0 7 7 6 PC5-G
C.gloeosporioidesCAUG2 5 9 7 5 0 7 7 6 PC5-G
C. grossum CAUG7 3 3 0 5 5 5 0 3 PC3-G
C.hymenocallidis CAUG9 5 5 5 5 7 7 7 6 PC2-G
C. incanum CAUT34 5 5 5 3 7 5 5 5 PC2-G
C. karstii CAUOS1 5 7 7 7 0 0 0 4 PC1-G
C.liaoningense CAUOS2 5 5 9 7 5 3 7 6 PC2-G
C. scovillei CAUA1 7 7 7 7 9 9 7 7 PC2-G
C. truncatum CAUT1 7 5 5 7 7 7 5 6 PC2-G
C. viniferum CAUG27 5 5 5 7 9 9 7 7 PC2-G
Mean 5 6 5 5 5 5 4 5 –
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RESULTS

Disease survey and strain isolation

Symptoms of anthracnose were circular or angular sunken lesions on chili fruits and irregularly shaped brown spots with dark brown edges on leaves. A total of 1 285 isolates of Colletotrichum spp. were obtained from 29 provinces in China (Fig. 1, Table 1). Attempts were made to collect samples from multiple locations in Tibet and Xinjiang provinces for several years but failed to locate symptomatic plants. All strains were isolated from fruits except those from Jiangxi province, where serious damage was found on chili leaves rather than on fruits.

Group assessment

Based on megablast searches in GenBank using ITS sequences and the colony morphologies on PDA, all strains were assigned to four groups, i.e., those that produce cylindrical conidia with round ends were assigned to the C. gloeosporioides species complex; those that produce acute ends or ± cylindrical conidia with only one acute end were assigned to the C. acutatum species complex; those that produce dark setae and curved conidia were assigned to the Colletotrichum species with curved conidia; and the remaining strains were assigned to a fourth group. Among the 121 isolates, 31 belonged to the C. gloeosporioides complex; 48 belonged to the C. acutatum complex; 34 belonged to the Colletotrichum species with curved conidia, and eight belonged to the fourth group (Damm et al. 2012a, b, 2013, 2014, Weir et al. 2012, Crouch 2014).

Multi-locus phylogenetic analyses

The 121 representative isolates from chili were subjected to multi-locus phylogenetic analyses (Table 2a, 2b, 2c). The trees generated from the Bayesian and RaxML analyses were essentially similar to that from the MP analysis (Fig. 2) and are therefore not shown. In Fig. 2, the 31 isolates in the C. gloeosporioides complex clustered in eight clades, eight with C. fructicola, 13 with C. gloeosporioides, and four with C. aenigma, C. endophytica, C. hymenocallidis, and C. viniferum, respectively. In addition, two distinct lineages, which clustered distantly from any known species in the complex, were recognised as new species and herein described as C. conoides and C. grossum (Fig. 2). In Fig. 3, the isolates of the C. acutatum complex clustered in two clades, 31 with C. scovillei and 17 with C. fioriniae. In the Colletotrichum species with curved conidia, 33 isolates clustered with C. truncatum, and one clustered with C. incanum (Fig. 4). The remaining isolates were assigned to C. cliviae and C. karstii. A new lineage belonging to the fourth group, distinct from all known species, is herein described as a new species, C. liaoningense (Fig. 6).

Fig. 2.

Fig. 2

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Maximum parsimony tree of isolates in the Colletotrichum gloeosporioides species complex obtained from a heuristic search of combined ACT, CAL, CHS-1, GAPDH, ITS, and TUB2 gene sequences. Colletotrichum boninense was used as the outgroup. Bootstrap support values ≥ 50 %, Bayesian posterior probability values ≥ 0.95 and RAxML bootstrap support values (ML ≥ 50 %) are shown at the nodes. Tree length = 1665, CI = 0.672, RI = 0.889, RC = 0.597, HI = 0.328. Ex-type strains are emphasised in bold.

Fig. 3.

Fig. 3

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Maximum parsimony tree of isolates in the Colletotrichum acutatum species complex obtained from a heuristic search of combined ACT, CHS-1, GAPDH, HIS3, ITS, and TUB2 gene sequences. Colletotrichum gloeosporioides was used as the outgroup. Bootstrap support values ≥ 50 % and Bayesian posterior probability values ≥ 0.95 are shown at the nodes. Tree length = 943, CI = 0.757, RI = 0.912, RC = 0.691, HI = 0.243. Ex-type strains are emphasised in bold.

Fig. 4.

Fig. 4

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Maximum parsimony tree of Colletotrichum species with curved conidia obtained from a heuristic search of combined ACT, CHS-1, GAPDH, HIS3, ITS, and TUB2 gene sequences. Colletotrichum lindemuthianum was used as the outgroup. Bootstrap support values ≥ 50 % and Bayesian posterior probability values ≥ 0.95 are shown at the nodes. Tree length = 2853, CI = 0.467, RI = 0.859, RC = 0.401, HI = 0.533. Ex-type strains are emphasised in bold.

Fig. 6.

Fig. 6

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Maximum parsimony tree of isolates of Colletotrichum species in the fourth group obtained from a heuristic search of combined ACT, CAL, CHS-1, GAPDH, ITS, and TUB2 gene sequences. Monilochaetes infuscans was used as the outgroup. Bootstrap support values ≥ 50 % and Bayesian posterior probability values ≥ 0.95 are shown at the nodes. Tree length = 2913, CI = 0.717, RI = 0.870, RC = 0.624, HI = 0.283. Ex-type strains are emphasised in bold.

Pathogenicity

All tested isolates except that of C. endophytica were pathogenic to most of the detached ripe red chili fruits (Table 3). All Capsicum annuum and Ca. frutescens cultivars were susceptible to all tested Colletotrichum species (except C. endophytica), with disease scores from 5 to 9. Capsicum chinense was susceptible to most of the Colletotrichum species, except C. cliviae CAUOS5, C. endophytica CAUG28, and C. hymenocallidis CAUG9. Capsicum annuum and Ca. frutescens were the most susceptible, with average scores of 7. Three pathotypes (PC1-R, PC2-R, and PC3-R) were identified (Table 3) based on three differential reactions of tested strains with Capsicum chinense cv. Huangdijiao, Ca. annuum and Ca. frutescens. Host reactions of the mature green fruit were similar to those of the ripe fruit (Table 4). Similar to the ripe fruit, all three cultivars of the mature green fruit of Capsicum annuum, Zidantou, Denglongjiao, and Sanyingjiao were susceptible to all tested Colletotrichum species, except C. endophytica, with average scores from 5 to 6. Most of the Capsicum frutescens cultivars and Ca. chinense cv. Huangdijiao at the green fruit stage were susceptible to most isolates, except C. aenigma CAUG26, C. conoides CAUG17, C. gloeosporioides CAUG2, C. grossum CAUG7, C. fructicola CAUG1 and C. karstii CAUOS1. Five pathotypes were identified based on the differential reactions with Xiaomila, Shuangla, and Huangdijiao (Table 4). All of the pathogenic isolates formed sunken, brown to dark lesions on the fruits. No symptoms developed on the negative controls.

Prevalence of Colletotrichum species

To determine the prevalence of the Colletotrichum species associated with chili in China, the sample locations and the number of isolates were assessed for each species. Isolates with highly similar morphology and ITS sequences to those of the ex-type of C. truncatum appear to be most common (N = 422), representing 33 % of all isolates, and presenting in 56 % of all sampling locations (Fig. 5). All 34 isolates chosen from this group for multi-locus phylogenetic analysis were confirmed to be C. truncatum (Fig. 4). It therefore appears that C. truncatum is the most prevalent species of Colletotrichum on chili in China. The next most prevalent species included C. scovillei, C. gloeosporioides, C. fioriniae, and C. fructicola, which accounted for 21, 14, 14, and 13 % of all the isolates, respectively. The remaining species were detected in less than 3 % of the sampling locations.

Fig. 5.

Fig. 5

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Prevalence of Colletotrichum species on chili in China based on preliminary identifications. a. The percentage of isolates represented by the indicated Colletotrichum species on chili; b. number of sampling locations where the seven most prevalent species were isolated.

Taxonomy

Based on the morphology and the multi-locus phylogeny, the 121 isolates were assigned to 15 species. Seven species (C.aenigma, C. cliviae, C. endophytica, C. hymenocallidis, C. incanum, C. karstii, and C. viniferum) were reported from chili for the first time. Three other species (C. fioriniae, C. fructicola, and C. scovillei) were reported for the first time in China, and a further three species newly described.

Colletotrichum conoides Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, sp. nov. — MycoBank MB812003; Fig. 7

Fig. 7.

Fig. 7

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Colletotrichum conoides (CAUG17). a–b. Colonies on PDA above and below; c. conidiophores; d–e, g–h: appressoria; f. conidia. — Scale bars: c–h = 10 μm.

Etymology. Referring to the host variety (Capsicum annuum var. conoides) from which the fungus was first collected.

Colonies on PDA attaining 53–55 mm diam in 4 d at 28 °C; aerial mycelia greyish white; reverse light grey to medium grey with white margin. Chlamydospores not observed. Vegetative hyphae hyaline, smooth-walled, septate, branched. Conidiomata and setae not observed. Conidiophores formed directly on aerial mycelium, hyaline, aseptate. Conidiogenous cells hyaline, cylindrical to clavate, 22–30 × 3.5–5 μm, opening 2.5–3 μm. Conidia hyaline, aseptate, smooth-walled, cylindrical to clavate, both ends obtusely rounded, contents granular and mostly equally distributed, 13–17.5 × 5–6.5 μm (av. = 15.9 × 5.9 μm), L/W ratio = 2.7. Appressoria single or in small groups, medium to dark brown, aseptate, mostly ellipsoidal to irregular in outline, and crenate or deeply lobed at margin, 4–11.5 × 6–10.5 μm (av. = 8.35 × 7.1 μm), L/W ratio = 1.2. Sexual morph not observed after 8 wk.

Specimen examined. CHINA, Jiangsu Province, Nanjing City, on fruits of Capsicum annuum var. conoides, Sept. 2010, Y.Z. Diao (holotype HMAS 246481, ex-type living culture CGMCC 3.17615 = CAUG17 = LC6226); ibid., NJ26, living culture CAUG33; ibid., NJ27, living culture CAUG34.

Notes — Colletotrichum conoides is phylogenetically most closely related to C. hebeiense (Fig. 2). Sequence data from ITS and CHS-1 could not separate the two species, but they can be distinguished by GAPDH (12 bp), ACT (4 bp), or TUB (3 bp). The two species also differ in the following characteristics: the granules are uniformly distributed in the conidia of C. conoides but mostly present at the polar ends in the conidia of C. hebeiense; most appressoria of C. conoides are ovoid ellipsoidal with crenate or deeply lobed margin, while those of C. hebeiense are clavate to subglobose; conidia of C. conoides are slightly larger than those of C. hebeiense (13–17.5 × 5–6.5 μm vs 11.6–15.3 × 4.47–6.88 μm). In addition, C. conoides was described from Capsicum annuum var. conoides, while C. hebeiense was described from Vitis vinifera (Yan et al. 2015). A PHI test revealed no significant recombination event between C. conoides and C. hebeiense (Fig. 8).

Fig. 8.

Fig. 8

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The results of the pairwise homoplasy index (PHI) test of closely related species using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.

Colletotrichum grossum Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, sp. nov. — MycoBank MB812006; Fig. 9

Fig. 9.

Fig. 9

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Colletotrichum grossum (CAUG7). a–b. Colonies on PDA above and below; c–e: conidiophores; f. conidia; g–k: appressoria. — Scale bars: c–f, j–k = 10 μm (j applied to g–j).

Etymology. Referring to the host variety (Capsicum annuum var. grossum) from which the fungus was first collected.

Colonies on PDA attaining 49–52 mm diam in 4 d at 28 °C; aerial mycelia white, reverse light grey with white margin. Chlamydospores not observed. Vegetative hyphae hyaline, smooth-walled, septate, branched. Conidiomata and setae not observed. Conidiophores formed directly on aerial mycelium, hyaline, aseptate. Conidiogenous cells hyaline, cylindrical to clavate, 22–32 × 3–3.5 μm, opening 2–2.5 μm. Conidia hyaline, aseptate, smooth-walled, cylindrical to clavate, both ends rounded or one end acute, contents granular and mostly present at the polar ends, 14.5–20.5 × 5–7.5 μm (av = 16.8 × 6.3 μm), L/W ratio = 2.7. Appressoria single, medium brown, aseptate, mostly ovoid or ellipsoidal to irregular in outline, and crenate in margin. 5.5–11.5 × 4–10.5 μm (av = 8.65 × 6.1 μm), L/W ratio = 1.4. Sexual morph not observed after 8 wk.

Specimen examined. CHINA, Hainan Province, Haikou city, on chili fruits (Capsicum annuum var. grossum), Oct. 2010, Y.Z Diao (holotype HMAS 246480, ex-type living culture CGMCC3.17614 = CAUG7 = LC6227); ibid., HN2, living culture CAUG31; ibid., HN3, living culture CAUG32.

Notes — Colletotrichum grossum is phylogenetically most closely related to C. theobromicola (Fig. 2). The sequence data of ITS and CAL do not separate the two species, but they can be distinguished by GAPDH (3 bp), ACT (5 bp), and TUB (8 bp). In morphology, C. grossum differs from C. theobromicola by having wider conidia (14.5–20.5 × 5–7.5 μm vs 14.5–18.7 × 4.5–5.5 μm) and colonies that are flat white rather than black as in C. theobromicola (Rojas et al. 2010). A PHI test revealed no significant recombination event between C. grossum and C. theobromicola (Fig. 8).

Colletotrichum liaoningense Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, sp. nov. — MycoBank MB812007; Fig. 10

Fig. 10.

Fig. 10

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Colletotrichum liaoningense (CAUOS2). a–b. Colonies on PDA above and below; c–e: conidiophores; f. conidia; g–k: appressoria. — Scale bars: d–k = 10 μm (d applies to c–d).

Etymology. Referring to the province in China where the fungus was first collected.

Colonies on PDA attaining 48–51 mm diam in 4 d at 28 °C; aerial mycelia light grey, reverse medium to dark brown with white margin. Chlamydospores not observed. Vegetative hyphae hyaline, smooth-walled, septate, branched. Conidiomata acervular. Setae medium grey, smooth-walled to verruculose, 3–6-septate, 46–68 μm long, base cylindrical, conical, or slightly inflated, 4.5–6.5 μm diam at the widest part, tip rounded. Conidiophores formed directly on aerial mycelium, hyaline, aseptate. Conidiogenous cells hyaline, cylindrical to clavate, 27–30 × 3.5–4.5 μm, opening 2–4 μm. Conidia cylindrical to clavate, both ends rounded or one end acute, contents granular and mostly present at the polar ends, hyaline, aseptate, smooth-walled, 14–18.5 μm × 5–7.5 μm (av. = 16.3 × 6.1 μm), L/W ratio = 2.7. Appressoria single, medium to dark brown, aseptate, mostly ellipsoidal to irregular in outline, and crenate at margin, 3.5–5 × 2.5–4.5 μm (av. = 4.1 × 2.9 μm), L/W ratio = 1.4. Sexual morph not observed after 8 wk.

Specimen examined. CHINA, Xingcheng city, Liaoning Province on chili fruits (Capsicum annuum var. conoides), Oct. 2012, Y.Z. Diao (holotype HMAS 246479, ex-type living culture CGMCC3.17616 = CAUOS2 = LC6228); ibid., LN3, living culture CAUOS3; ibid., LN4, living culture CAUOS4; ibid., LN6, living culture CAUOS6.

Notes — Colletotrichum liaoningense is phylogenetically most closely related to C. brevisporum (Fig. 6). The sequence data from ITS and ACT could not separate the two species; however, they can be distinguished from each other via GAPDH (10 bp) or TUB (12 bp). The granules are equally distributed in the conidia of C. liaoningense but mostly present at the polar ends in conidia of C. brevisporum. The appressoria of C. liaoningense are smaller than those of C. brevisporum (3.5–5 × 2.5–4.5 μm vs 10–13 × 8–11 μm) (Noireung et al. 2012). A PHI test revealed no significant recombination event between C. liaoningense and C. brevisporum (Fig. 8).

DISCUSSION

Colletotrichum truncatum, the most frequently isolated species in this study, has been reported from more than 460 plant species (Farr & Rossman 2016). This taxon has also been shown to cause serious damage to chili production in Australia, China, India, Thailand, and other countries (Poonpolgul & Kumphai 2007, Than et al. 2008, Sharma et al. 2014, Diao et al. 2015). In China, C. truncatum has been reported from tomato, dragon fruit, pumpkin, and other crops (Chai et al. 2014, Cheng et al. 2014, Diao et al. 2014, Guo et al. 2014). Geographic populations of C. truncatum in China exhibit significant genetic differentiation and recombination abilities, which can probably be attributed to the prevalence of this species (Diao et al. 2015).

Colletotrichum gloeosporioides has been reported to infect chili in Australia, China, India, Korea, Thailand, the USA, and other countries (Shin et al. 1999, Kim et al. 2008, Than et al. 2008). However, a recent study revealed this taxon to be a species complex comprising many morphologically similar taxa (Weir et al. 2012). Therefore, this new classification system necessitates a re-investigation of species in the C. gloeosporioides species complex on chili, as species in this complex exhibit biological and physiological differences. In the current study, C. gloeosporioides s.str. and C. fructicola were revealed to be most prevalent in this complex, representing 47 % and 42 % of the isolates, respectively (Fig. 2). Colletotrichum fructicola was originally isolated from coffee berries (Prihastuti et al. 2009), and has since been found on a wide range of host plants (Weir et al. 2012). However, this is the first report of C. fructicola infecting chili. In previous studies, C. gloeosporioides s.str. was shown to be an uncommon pathogen on chili and other fruits in the tropics (Phoulivong et al. 2010). Additionally, we failed to isolate C. gloeosporioides s.str. from chili in the tropical regions of China, e.g. Hainan, south of Guangdong, and Yunnan provinces (Table 1), which suggested a significant effect of climate on the distribution of these pathogens. Pathogenicity of all obtained species from chili in this study was confirmed by inoculation tests, except for that of C. endophytica. Colletotrichum endophytica, which was originally reported as an endophytic fungus in tropical grasses (Manamgoda et al. 2013), did not show pathogenicity to any chili cultivars in our test, further underlining the possible endophytic nature of this species.

Colletotrichum acutatum is a commonly reported species, and causes anthracnose on numerous plants worldwide (Damm et al. 2012a). It was originally described from Carica papaya, Capsicum frutescens, and Delphinium ajacis in Australia (Simmonds 1965), but has subsequently been reported to infect chili in almost all pepper-growing countries, such as Australia, China, India, Korea, New Zealand, Thailand, and the USA (Than et al. 2008). Like C. gloeosporioides, C. acutatum has also been shown to represent a species complex (Damm et al. 2012a). Interestingly, C. acutatum s.str. was not found on chili in China (Fig. 3). Only C. scovillei and C. fioriniae were identified from this complex (Fig. 3).

No Colletotrichum species were detected on chili in Tibet and Xinjiang, despite the fact that several field trips have been made to these provinces, and attempts have been made for to isolate these fungi. The failure to detect Colletotrichum species from these regions might be explained by the high latitude, small growing area, dry climate, and high day/night variation in temperature. Colletotrichum fructicola and C. truncatum were isolated from leaves in the Jiangxi province, and were also found from fruits in other sampling regions. In previous studies, these two species were primarily isolated from fruits from various plants (Poonpolgul & Kumphai 2007, Than et al. 2008, Alaniz et al. 2015, Diao et al. 2015).

In summary, the current study represents the hitherto most intensive investigation of Colletotrichum species on chili in China, which revealed 15 species, with the dominant species being C. fioriniae, C. fructicola, C. gloeosporioides, C. scovillei, and C. truncatum. The information provided here could prove useful for the control of anthracnose on chili, as well as for the screening of new chili cultivars against anthracnose.

Acknowledgments

This work was supported by the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201303023), and also partially supported by the National High Technology Research and Development Program of China (2012CB111401).

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