Colletotrichum species causing anthracnose disease of chili in China.

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.

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). One of the most destructive diseases restricting chili production is anthracnose, caused by Colletotrichum spp. (Bailey & Jeger 1992, Poonpolgul & Kumphai 2007, Than et al), resulting in up to 40 % yield loss in China (Lin et al).

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, Liao et al, Kanto et al, Sharma et al, Diao et al). 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); and by C. acutatum, C. gloeosporioides, C. truncatum, and C. coccodes in China (Shin et al, Liao et al). 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). 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. 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

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.

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

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); 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); chitin synthase 1 (CHS-1) with primers CHS-79F/CHS-345R (Carbone & Kohn 1999); and histone3 (HIS3) with primers CYLH3F/CYLH3R (Crous et al). PCR reactions were performed as described by Damm et al. 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, Liu et al, 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

* = 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

* = 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

* = 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. 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	–

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	–

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, b, 2013, 2014, Weir et al, 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

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

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

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

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

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.

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

Fig. 7

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). A PHI test revealed no significant recombination event between C. conoides and C. hebeiense (Fig. 8).

Fig. 8

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.

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

Fig. 9

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).

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

Fig. 10

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). 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, Sharma et al, 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.