Unravelling Colletotrichum species associated with Camellia: employing ApMat and GS loci to resolve species in the C. gloeosporioides complex

Abstract

We investigated the phylogenetic diversity of 144 Colletotrichum isolates associated with symptomatic and asymptomatic tissues of Camellia sinensis and other Camellia spp. from seven provinces in China (Fujian, Guizhou, Henan, Jiangxi, Sichuan, Yunnan, Zhejiang), and seven isolates obtained from other countries, including Indonesia, UK, and the USA. Based on multi-locus (ACT, ApMat, CAL, GAPDH, GS, ITS, TUB2) phylogenetic analyses and phenotypic characters, 11 species were distinguished, including nine well-characterised species (C. alienum, C. boninense, C. camelliae, C. cliviae, C. fioriniae, C. fructicola, C. gloeosporioides, C. karstii, C. sia-mense), and two novel species (C. henanense and C. jiangxiense). Of these, C. camelliae proved to be the most dominant and probably host specific taxon occurring on Camellia. An epitype is also designated for the latter species in this study. Colletotrichum jiangxiense is shown to be phylogenetically closely related to the coffee berry pathogen C. kahawae subsp. kahawae. Pathogenicity tests and the pairwise homoplasy index test suggest that C. jiangxiense and C. kahawae subsp. kahawae are two independent species. This study represents the first report of C. alienum and C. cliviae occurring on Camellia sinensis. In addition, our study demonstrated that the combined use of the loci ApMat and GS in a phylogenetic analysis is able to resolve all currently accepted species in the C. gloeosporioides species complex.

INTRODUCTION

Camellia, a genus of flowering plants in the family Theaceae, is cultivated in eastern and southern Asia, from the Himalayas east to Japan and Indonesia. Many species of Camellia (Ca.) are of major commercial importance. For example, leaves of Ca. sinensis are processed to produce tea, a popular beverage, while Ca. japonica, Ca. oleifera, and Ca. sasanqua and their hybrids are cultivated as ornamentals. Camellia production is affected by a large number of diseases, of which anthracnose, caused by species of the genus Colletotrichum, is one of the most important (Copes & Thomson 2008, Farr & Rossman 2014, Guo et al. 2014). Several Colletotrichum species have been reported from Camellia, e.g. C. boninense (Damm et al. 2012b), C. camelliae (Thompson & Johnston 1953, Tai 1979, Alfieri et al. 1984), C. carveri (Cash 1952), C. coccodes (Thaung 2008), C. gloeosporioides (Alfieri et al. 1984, Shivas 1989, Lu et al. 2000, Chen 2003, Guo et al. 2014), C. pseudomajus (Liu et al. 2014), C. queenslandicum (Simmonds 1966; syn. C. gloeosporioides var. minor, Weir et al. 2012), and Glomerella major (Tunstall 1934).

The genus Colletotrichum was also considered as one of the dominant endophytic genera in Camellia plants (Lu et al. 2007, Dai et al. 2008, Osono 2008, Fang et al. 2013). Colletotrichum acutatum and C. gloeosporioides were recognised as frequently occurring endophytic species in Ca. japonica based on morphological characteristics (Osono 2008). Fang et al. (2013) also found that C. gloeosporioides was one of the dominant endophytic species in Ca. sinensis based on ITS sequence data. Other reports of endophytic isolates of Colletotrichum on Camellia were, however, only identified to genus level.

Because of the commercial yield losses experienced in tea plantations due to Colletotrichum infections, as well as the limited knowledge of their identity and endophytic growth in Camellia plants, accurate identification of the causal organisms is of extreme importance. Most of the recent taxonomic treatments have primarily focused on the study of different Colletotrichum species complexes, for example C. acutatum (Damm et al. 2012a), C. boninense (Damm et al. 2012b), C. caudatum (Crouch 2014), C. destructivum (Damm et al. 2014), C. gigasporum (Liu et al. 2014), C. gloeosporioides (Weir et al. 2012), C. graminicola (Crouch et al. 2009), and C. orbiculare (Damm et al. 2013). Robust identification of Colletotrichum species relies on multi-locus sequence data (Cai et al. 2009, Cannon et al. 2012, Weir et al. 2012, Damm et al. 2013, Liu et al. 2013a, Crouch 2014). However, previous phylogenetic studies have rarely included isolates from Camellia. Thus far only a few strains of C. boninense, C. fioriniae, C. lupini, and Glomerella cingulata ‘f. sp. camelliae’ from Camellia were included in multi-locus phylogenies (Damm et al. 2012a,b, Weir et al. 2012, Sharma et al. 2014). In contrast, most of the studies that focused on the identification of Colletotrichum species associated with Camellia were only based on host, morphology or ITS sequence data (Tai 1979, Alfieri et al. 1984, Copes & Thomson 2008, Thaung 2008, Fang et al. 2013, Guo et al. 2014). Published reports of C. acutatum and C. gloeosporioides on Camellia should therefore be interpreted with care. Furthermore, although C. camelliae is regarded as the causal agent of brown blight disease of tea, the taxonomic and phylogenetic status of this pathogen remains unresolved (Weir et al. 2012).

The aim of the present study was thus to investigate the taxonomic and phylogenetic diversity of Colletotrichum spp. associated with Ca. sinensis and other Camellia spp. based on sequence data of six loci (ACT, CAL, GAPDH, GS, ITS, TUB2). A further aim was to test the usefulness of the ApMat locus in resolving taxa in the C. gloeosporioides complex (Crouch et al. 2009, Rojas et al. 2010, Silva et al. 2012b, Doyle et al. 2013, Sharma et al. 2013a, 2014) in combination with the other loci listed above.

MATERIALS AND METHODS

Collection and isolates

Diseased and healthy leaves of tea plants (Ca. sinensis) and other Camellia spp. were collected from seven provinces in China (Fujian, Guizhou, Henan, Jiangxi, Sichuan, Yunnan, and Zhejiang). Plant pathogenic fungi were isolated from leaf spots using both single spore and tissue isolation methods. Single spore isolation following the protocol of Choi et al. (1999) was adopted for collections with visible foliar sporulation, while tissue isolation was used for sterile isolates. Fungal endophytes were isolated by cutting four fragments (4 mm²) per leaf from the apex, base and lateral sides, surface sterilised with 70 % ethanol for 1 min, 0.5 % NaClO for 3 min, 70 % ethanol for 1 min, rinsed in sterile water, and then transferred to quarter-strength potato dextrose agar (1/4 PDA; 9.75 g Difco PDA, 15 g Difco agar and 1 L distilled water). After 3–21 d, mycelial transfers were made from the colony periphery onto PDA. Colletotrichum colonies were primarily identified based on cultural characteristics on PDA, morphology of the spores, and ITS sequence data.

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 deposited in the China General Microbiological Culture Collection centre (CGMCC). A further seven isolates from Camellia originating from other countries including Indonesia, UK, and the USA used in this study were obtained from the culture collection of the International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand (ICMP) and the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (CBS).

Morphological analysis

Agar plugs (5-mm-diam) were taken from the periphery of actively growing cultures and transferred to the centre of 9-cm-diam Petri dishes containing PDA or synthetic nutrient-poor agar medium (SNA; Nirenberg 1976) amended with double-autoclaved stems of Anthriscus sylvestris placed onto the agar surface. Cultures were incubated at room temperature (c. 25 °C) for 7 d. Colony characters and pigment production on PDA were noted after 7 d. Colony colours were rated according to Rayner (1970). Colony diameters were measured after 7 and 10 d.

Conidia were taken from acervuli on PDA and mounted in clear lactic acid. Cultures were examined periodically for the development of ascomata. Ascospores were described from ascomata crushed in lactic acid. If a fungus was not sporulating on PDA, morphological characters were described from SNA or from inoculated stems of Anthriscus sylvestris. Hyphal appressoria were observed on the reverse side of colonies grown on SNA plates. At least 30 measurements per structure were noted and observed with a Nikon Eclipse 80i microscope using differential interference contrast (DIC) illumination. Descriptions and illustrations of taxonomic novelties were deposited in MycoBank (www.MycoBank.org; Crous et al. 2004).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from axenic cultures with a modified CTAB protocol as described in Guo et al. (2000). Seven loci including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), beta-tubulin (TUB2), glutamine synthetase (GS), calmodulin (CAL) and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) were amplified and sequenced using the primer pairs ITS1 + ITS4 (White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), ACT-512F + ACT-783R (Carbone & Kohn 1999), T1 + Bt-2b (Glass & Donaldson 1995, O’Donnell & Cigelnik 1997), GSF1 + GSR1 (Stephenson et al. 1997), CL1C + CL2C (Weir et al. 2012), and AMF1 + AMR1 (Silva et al. 2012b), respectively. PCR amplification protocols were performed as described by Liu et al. (2012), but the denaturing temperatures were adjusted to 52 °C for ITS, GAPDH, ACT, GS, CAL, and ApMat, and 55 °C for TUB2. Purification and sequencing of PCR amplicons were carried out by the SinoGenoMax Company, Beijing, China. DNA sequences generated with forward and reverse primers were used to obtain consensus sequences using MEGA v. 5.1 (Tamura et al. 2011). All novel sequences were deposited in NCBIs GenBank database (www.ncbi.nlm.nih.gov/; KJ954359–KJ955371, KM360143–KM360146, KM610172–KM610185, Table 1, 2), and the alignments and trees in TreeBASE (www.treebase.org/treebase-web/home.html; study S16761).

Table 1.

Strains of the C. gloeosporioides s.l. species studied in this paper with details about host and location, and GenBank accessions of the sequences generated.

Species	Accession numbera	Host	Locality	GenBank accessions
				ITS	GAPDH	ACT	TUB2	CAL	GS	ApMat
C. aenigma	ICMP 18608*	Persea americana	Israel	JX010244	JX010044	JX009443	JX010389	JX009683	JX010078	KM360143
	ICMP 18686	Pyrus pyrifolia	Japan	JX010243	JX009913	JX009519	JX010390	JX009684	JX010079
C. aeschynomenes	ICMP 17673, ATCC 201874*	Aeschynomene virginica	USA	JX010176	JX009930	JX009483	JX010392	JX009721	JX010081	KM360145
C. alatae	CBS 304.67, ICMP 17919*	Dioscorea alata	India	JX010190	JX009990	JX009471	JX010383	JX009738	JX010065	KC888932
	ICMP 18122	Dioscorea alata	Nigeria	JX010191	JX010011	JX009470	JX010449	JX009739	JX010136
C. alienum	ICMP 12071*	Malus domestica	New Zealand	JX010251	JX010028	JX009572	JX010411	JX009654	JX010101	KM360144
	ICMP 18621	Persea americana	New Zealand	JX010246	JX009959	JX009552	JX010386	JX009657	JX010075
	IMI 313842, ICMP 18691	Persea americana	Australia	JX010217	JX010018	JX009580	JX010385	JX009664	JX010074
	LC3114, LF322	Ca. sinensis, endophyte	China	KJ955131	KJ954832	KJ954411	KJ955279	KJ954684	KJ954982	KJ954545
C. aotearoa	ICMP 17324	Kunzea ericoides	New Zealan	JX010198	JX009991	JX009538	JX010418	JX009619	JX010109
	ICMP 18532	Vitex lucens	New Zealand	JX010220	JX009906	JX009544	JX010421	JX009614	JX010108
	ICMP 18537*	Coprosma sp.	New Zealand	JX010205	JX010005	JX009564	JX010420	JX009611	JX010113	KC888930
C. asianum	GM595, MTCC 11680	Mangifera indica	India	JQ894679	JQ894623	JQ894545	JQ894601	KC790789		JQ894554
	ICMP 18580, CBS 130418*	Coffea arabica	Thailand	FJ972612	JX010053	JX009584	JX010406	FJ917506	JX010096	FR718814
	IMI 313839, ICMP 18696	Mangifera indica	Australia	JX010192	JX009915	JX009576	JX010384	JX009723	JX010073
C. boninense	MAFF 305972, CBS 123755*	Crinum asiaticum var. sinicum	Japan	JQ005153	JQ005240	JQ005501	JQ005588	JQ005674
C. camelliae	CBS 125502	Camellia sp., pathogen	unknown	KJ955077	KJ954778	KJ954359		KJ954630	KJ954928
	ICMP 10643, LF897, LC3667	Camellia × williamsii	UK	JX010224	JX009908	JX009540	JX010436	JX009630	JX010119	KJ954625
	ICMP 10646, LF898, LC3668	Ca. sasanqua	USA	JX010225	JX009993	JX009563	JX010437	JX009629	JX010117	KJ954626
	ICMP 18542, LF899, LC3669	Ca. sasanqua	USA	JX010223	JX009994	JX009488	JX010429	JX009628	JX010118	KJ954627
	CGMCC 3.14924, LC1363	Ca. sinensis, pathogen	China	KJ955080	KJ954781	KJ954362	KJ955229	KJ954633	KJ954931	KJ954496
	CGMCC 3.14925, LC1364*	Ca. sinensis, pathogen	China	KJ955081	KJ954782	KJ954363	KJ955230	KJ954634	KJ954932	KJ954497
	CGMCC 3.14926, LC1365	Ca. sinensis, pathogen	China	KJ955082	KJ954783	KJ954364	KJ955231	KJ954635	KJ954933	KJ954498
	LC2944, LF152	Camellia sp., pathogen	China	KJ955090	KJ954791	KJ954372	KJ955239	KJ954643	KJ954941	KJ954506
	LC2962, LF170	Camellia sp., pathogen	China	KJ955091	KJ954792	KJ954373	KJ955240	KJ954644	KJ954942	KJ954507
	LC2998, LF206	Ca. sinensis, pathogen	China	KJ955094	KJ954795	KJ954376	KJ955243	KJ954647	KJ954945	KJ954510
	LC2999, LF207	Ca. sinensis, pathogen	China	KJ955095	KJ954796	KJ954377	KJ955244	KJ954648	KJ954946	KJ954511
	LC3000, LF208	Ca. sinensis, pathogen	China	KJ955096	KJ954797	KJ954378	KJ955245	KJ954649	KJ954947
	LC3001, LF209	Ca. sinensis, pathogen	China	KJ955097	KJ954798	KJ954379	KJ955246	KJ954650	KJ954948	KJ954512
	LC3002, LF210	Ca. sinensis, pathogen	China	KJ955098	KJ954799	KJ954380	KJ955247	KJ954651	KJ954949	KJ954513
	LC3004, LF212	Ca. sinensis, pathogen	China	KJ955099	KJ954800	KJ954381	KJ955248	KJ954652	KJ954950	KJ954514
	LC3005, LF213	Ca. sinensis, pathogen	China	KJ955100	KJ954801	KJ954382	KJ955249	KJ954653	KJ954951	KJ954515
	LC3006, LF214	Ca. sinensis, pathogen	China	KJ955101	KJ954802	KJ954383	KJ955250	KJ954654	KJ954952	KJ954516
	LC3007, LF215	Ca. sinensis, pathogen	China	KJ955102	KJ954803	KJ954384	KJ955251	KJ954655	KJ954953	KJ954517
	LC3008, LF216	Ca. sinensis, pathogen	China	KJ955103	KJ954804	KJ954385	KJ955252	KJ954656	KJ954954	KJ954518
	LC3014, LF222	Ca. sinensis, pathogen	China	KJ955104	KJ954805	KJ954386	KJ955253	KJ954657	KJ954955	KJ954519
	LC3015, LF223	Ca. sinensis, pathogen	China	KJ955105	KJ954806	KJ954387		KJ954658	KJ954956	KJ954520
	LC3017, LF225	Ca. sinensis, pathogen	China	KJ955106	KJ954807	KJ954388	KJ955254	KJ954659	KJ954957	KJ954521
	LC3018, LF226	Ca. sinensis, pathogen	China	KJ955107	KJ954808	KJ954389	KJ955255	KJ954660	KJ954958	KJ954522
	LC3019, LF227	Ca. sinensis, pathogen	China	KJ955108	KJ954809	KJ954390	KJ955256	KJ954661	KJ954959	KJ954523
	LC3054, LF262	Ca. sinensis, pathogen	China	KJ955110	KJ954811	KJ954391	KJ955258	KJ954663	KJ954961	KJ954525
	LC3057, LF265	Ca. sinensis, pathogen	China	KJ955111	KJ954812	KJ954392	KJ955259	KJ954664	KJ954962	KJ954526
	LC3070, LF278	Ca. sinensis, pathogen	China	KJ955112	KJ954813	KJ954393	KJ955260	KJ954665	KJ954963	KJ954527
	LC3071, LF279	Ca. sinensis, pathogen	China	KJ955113	KJ954814		KJ955261	KJ954666	KJ954964	KJ954528
	LC3076, LF284	Ca. sinensis, endophyte	China	KJ955114	KJ954815	KJ954394	KJ955262	KJ954667	KJ954965	KJ954529
	LC3089, LF297	Ca. sinensis, endophyte	China	KJ955115	KJ954816	KJ954395	KJ955263	KJ954668	KJ954966	KJ954530
	LC3091, LF299	Ca. sinensis, endophyte	China	KJ955116	KJ954817	KJ954396	KJ955264	KJ954669	KJ954967	KJ954531
	LC3092, LF300	Ca. sinensis, endophyte	China	KJ955117	KJ954818	KJ954397	KJ955265	KJ954670	KJ954968	KJ954532
	LC3095, LF303	Ca. sinensis, endophyte	China	KJ955118	KJ954819	KJ954398	KJ955266	KJ954671	KJ954969	KJ954533
	LC3096, LF304	Ca. sinensis, endophyte	China	KJ955119	KJ954820	KJ954399	KJ955267	KJ954672	KJ954970	KJ954534
	LC3100, LF308	Ca. sinensis, endophyte	China	KJ955120	KJ954821	KJ954400	KJ955268	KJ954673	KJ954971	KJ954535
	LC3101, LF309	Ca. sinensis, endophyte	China	KJ955121	KJ954822	KJ954401	KJ955269	KJ954674	KJ954972	KJ954536
	LC3102, LF310	Ca. sinensis, endophyte	China	KJ955122	KJ954823	KJ954402	KJ955270	KJ954675	KJ954973	KJ954537
	LC3103, LF311	Ca. sinensis, endophyte	China	KJ955123	KJ954824	KJ954403	KJ955271	KJ954676	KJ954974	KJ954538
	LC3107, LF315	Ca. sinensis, endophyte	China	KJ955124	KJ954825	KJ954404	KJ955272	KJ954677	KJ954975	KJ954539
	LC3109, LF317	Ca. sinensis, endophyte	China	KJ955126	KJ954827	KJ954406	KJ955274	KJ954679	KJ954977	KJ954540
	LC3111, LF319	Ca. sinensis, endophyte	China	KJ955128	KJ954829	KJ954408	KJ955276	KJ954681	KJ954979	KJ954542
	LC3112, LF320	Ca. sinensis, endophyte	China	KJ955129	KJ954830	KJ954409	KJ955277	KJ954682	KJ954980	KJ954543
	LC3113, LF321	Ca. sinensis, endophyte	China	KJ955130	KJ954831	KJ954410	KJ955278	KJ954683	KJ954981	KJ954544
	LC3116, LF324	Ca. sinensis, endophyte	China	KJ955132	KJ954833	KJ954412	KJ955280	KJ954685	KJ954983	KJ954546
	LC3117, LF325	Ca. sinensis, endophyte	China	KJ955133	KJ954834	KJ954413	KJ955281	KJ954686	KJ954984	KJ954547
	LC3123, LF331	Ca. sinensis, endophyte	China	KJ955134	KJ954835	KJ954414	KJ955282	KJ954687	KJ954985	KJ954548
	LC3128, LF336	Ca. sinensis, pathogen	China	KJ955135	KJ954836	KJ954415	KJ955283	KJ954688	KJ954986	KJ954549
	LC3129, LF337	Ca. sinensis, pathogen	China	KJ955136	KJ954837	KJ954416	KJ955284	KJ954689	KJ954987	KJ954550
	LC3130, LF338	Ca. sinensis, pathogen	China	KJ955137	KJ954838	KJ954417	KJ955285	KJ954690	KJ954988	KJ954551
	LC3131, LF339	Ca. sinensis, pathogen	China	KJ955138	KJ954839		KJ955286	KJ954691	KJ954989	KJ954552
	LC3142, LF350	Ca. sinensis, pathogen	China	KJ955139	KJ954840	KJ954418	KJ955287	KJ954692	KJ954990	KJ954553
	LC3143, LF351	Ca. sinensis, pathogen	China	KJ955140	KJ954841	KJ954419	KJ955288	KJ954693	KJ954991	KJ954554
	LC3147, LF355	Ca. sinensis, pathogen	China	KJ955141	KJ954842	KJ954420	KJ955289	KJ954694	KJ954992	KJ954555
	LC3148, LF356	Ca. sinensis, pathogen	China	KJ955142	KJ954843	KJ954421	KJ955290	KJ954695	KJ954993	KJ954556
	LC3158, LF367	Ca. sinensis, endophyte	China	KJ955144	KJ954845	KJ954423	KJ955292	KJ954697	KJ954995	KJ954558
	LC3173, LF383	Ca. sinensis, endophyte	China	KJ955147	KJ954848	KJ954425	KJ955295		KJ954998	KJ954560
	LC3269, LF491	Ca. sinensis, pathogen	China	KJ955150	KJ954851		KJ955297	KJ954702	KJ955001	KJ954562
	LC3270, LF492	Ca. sinensis, pathogen	China	KJ955151	KJ954852	KJ954428	KJ955298	KJ954703	KJ955002	KJ954563
	LC3274, LF496	Ca. sinensis, pathogen	China	KJ955153	KJ954854	KJ954430	KJ955300	KJ954705	KJ955004	KJ954564
	LC3279, LF501	Ca. sinensis, pathogen	China	KJ955154	KJ954855	KJ954431	KJ955301	KJ954706	KJ955005	KJ954565
	LC3282, LF504	Ca. sinensis, pathogen	China	KJ955155	KJ954856	KJ954432	KJ955302	KJ954707	KJ955006	KJ954566
	LC3319, LF541	Ca. sinensis, pathogen	China	KJ955160	KJ954861	KJ954436	KJ955307	KJ954712		KJ954571
	LC3322, LF544	Ca. sinensis, pathogen	China	KJ955161	KJ954862	KJ954437	KJ955308	KJ954713	KJ955011	KJ954572
	LC3323, LF545	Ca. sinensis, pathogen	China	KJ955162	KJ954863		KJ955309	KJ954714	KJ955012	KJ954573
	LC3328, LF550	Ca. sinensis, pathogen	China	KJ955163	KJ954864		KJ955310	KJ954715	KJ955013	KJ954574
	LC3330, LF552	Ca. sinensis, pathogen	China	KJ955164	KJ954865	KJ954438	KJ955311	KJ954716	KJ955014	KJ954575
	LC3335, LF557	Ca. sinensis, pathogen	China	KJ955165	KJ954866	KJ954439	KJ955312	KJ954717	KJ955015	KJ954576
	LC3350, LF572	Ca. sinensis, pathogen	China	KJ955166	KJ954867	KJ954440	KJ955313	KJ954718	KJ955016	KJ954577
	LC3352, LF574	Ca. sinensis, pathogen	China	KJ955167	KJ954868	KJ954441	KJ955314	KJ954719	KJ955017	KJ954578
	LC3355, LF577	Ca. sinensis, pathogen	China	KJ955168	KJ954869	KJ954442	KJ955315	KJ954720	KJ955018	KJ954579
	LC3367, LF589	Ca. sinensis, pathogen	China	KJ955170	KJ954871	KJ954444	KJ955317	KJ954722	KJ955020
	LC3374, LF596	Ca. sinensis, pathogen	China	KJ955173	KJ954874	KJ954447	KJ955320	KJ954725	KJ955023	KJ954582
	LC3379, LF601	Ca. sinensis, pathogen	China	KJ955174	KJ954875	KJ954448	KJ955321	KJ954726	KJ955024	KJ954583
	LC3385, LF607	Ca. sinensis, pathogen	China	KJ955178	KJ954879	KJ954451	KJ955325	KJ954730	KJ955028	KJ954586
	LC3387, LF609	Ca. sinensis, pathogen	China	KJ955179	KJ954880	KJ954452	KJ955326	KJ954731	KJ955029	KJ954587
	LC3389, LF611	Ca. sinensis, pathogen	China	KJ955180	KJ954881	KJ954453	KJ955327	KJ954732	KJ955030	KJ954588
	LC3395, LF617	Ca. sinensis, pathogen	China	KJ955181	KJ954882	KJ954454	KJ955328	KJ954733	KJ955031	KJ954589
	LC3398, LF620	Ca. sinensis, pathogen	China	KJ955182	KJ954883	KJ954455	KJ955329	KJ954734	KJ955032	KJ954590
	LC3401, LF623	Ca. sinensis, pathogen	China	KJ955183	KJ954884	KJ954456	KJ955330	KJ954735	KJ955033	KJ954591
	LC3403, LF625	Ca. sinensis, pathogen	China	KJ955185	KJ954886	KJ954458	KJ955332	KJ954737	KJ955035	KJ954593
	LC3408, LF630	Ca. sinensis, pathogen	China	KJ955186	KJ954887	KJ954459	KJ955333	KJ954738	KJ955036	KJ954594
	LC3469, LF694	Ca. sinensis, pathogen	China	KJ955204	KJ954905	KJ954474	KJ955350	KJ954755	KJ955054	KJ954610
	LC3488, LF715	Ca. sinensis, pathogen	China	KJ955206	KJ954907	KJ954476	KJ955352	KJ954757	KJ955056	KJ954612
	LC3492, LF720	Ca. sinensis, pathogen	China	KJ955208	KJ954909	KJ954478	KJ955354	KJ954759	KJ955058	KJ954614
	LC3506, LF734	Ca. sinensis, pathogen	China	KJ955209	KJ954910	KJ954479	KJ955355	KJ954760	KJ955059	KJ954615
	LC3513, LF741	Camellia sp., pathogen	China	KJ955210	KJ954911		KJ955356	KJ954761	KJ955060	KJ954616
	LC3514, LF742	Camellia sp., pathogen	China	KJ955211	KJ954912	KJ954480	KJ955357	KJ954762	KJ955061	KJ954617
	LC3515, LF743	Camellia sp., pathogen	China	KJ955212	KJ954913	KJ954481	KJ955358	KJ954763	KJ955062	KJ954618
	LC3516, LF744	Camellia sp., pathogen	China	KJ955213	KJ954914		KJ955359	KJ954764	KJ955063	KJ954619
	LC3561, LF789	Ca. sinensis, pathogen	China	KJ955217	KJ954918	KJ954485	KJ955363	KJ954768	KJ955067	KJ954621
	LC3562, LF790	Ca. sinensis, pathogen	China	KJ955218	KJ954919	KJ954486		KJ954769	KJ955068	KJ954622
C. clidemiae	ICMP 18658*	Clidemia hirta	USA, Hawaii	JX010265	JX009989	JX009537	JX010438	JX009645	JX010129	KC888929
	ICMP 18706	Vitis sp.	USA	JX010274	JX009909	JX009476	JX010439	JX009639	JX010128
C. cordylinicola	LC0886, ICMP 18579*	Cordyline fruticosa	Thailand	JX010226	JX009975	HM470235	JX010440	HM470238	JX010122	JQ899274
C. dianesei	CMM4083, MFLU 1300058*	Mangifera indica	Brazil	KC329779	KC517194	KC517298	KC517254	KC517209	KC430894
	CMM4088, MFLU 1300059	Mangifera indica	Brazil	KC329781	KC517162	KC517300	KC517255	KC517210	KC430900
	CMM4089, MFLU 1300060	Mangifera indica	Brazil	KC329783	KC517163	KC517302	KC517256	KC517211	KC430879
C. endophytica	MFLUCC 130417, LC1216	Pennisetum purpureum	Thailand	KC633853	KC832853	KC692467		KC810017
	MFLUCC 130418, LC0324*	Pennisetum purpureum	Thailand	KC633854	KC832854	KF306258		KC810018
	MFLUCC 130419, LC0327	Pennisetum purpureum	Thailand	KC633855	KC832846	KC692468		KC810016
C. fructicola	CBS 125395, ICMP 18645	Theobroma cacao	Panama	JX010172	JX009992	JX009543	JX010408	JX009666	JX010098
	CBS 238.49, ICMP 17921	Ficus edulis	Germany	JX010181	JX009923	JX009495	JX010400	JX009671	JX010090
	GM567, MTCC 11679	Mangifera indica	India	JQ894676	JQ894630	JQ894543	JQ894600	KC790787		JQ894576
	ICMP 18581, CBS 130416*	Coffea arabica	Thailand	JX010165	JX010033	FJ907426	JX010405	FJ917508	JX010095	JQ807838
	ICMP 18646, CBS 125397, MTCC 10906	Tetragastris panamensis	Panama	JX010173	JX010032	JX009581	JX010409	JX009674	JX010099
	LC2923, LF130	Ca. sinensis, pathogen	China	KJ955083	KJ954784	KJ954365	KJ955232	KJ954636	KJ954934	KJ954499
	LC2924, LF131	Ca. sinensis, pathogen	China	KJ955084	KJ954785	KJ954366	KJ955233	KJ954637	KJ954935	KJ954500
	LC2925, LF132	Ca. sinensis, pathogen	China	KJ955085	KJ954786	KJ954367	KJ955234	KJ954638	KJ954936	KJ954501
	LC2926, LF133	Ca. sinensis, pathogen	China	KJ955086	KJ954787	KJ954368	KJ955235	KJ954639	KJ954937	KJ954502
	LC3155, LF364	Ca. sinensis, endophyte	China	KJ955143	KJ954844	KJ954422	KJ955291	KJ954696	KJ954994	KJ954557
	LC3167, LF376	Ca. sinensis, endophyte	China	KJ955145	KJ954846		KJ955293	KJ954698	KJ954996	KJ954559
	LC3284, LF506	Ca. sinensis, pathogen	China	KJ955156	KJ954857	KJ954433	KJ955303	KJ954708	KJ955007	KJ954567
	LC3288, LF510	Ca. sinensis, pathogen	China	KJ955157	KJ954858		KJ955304	KJ954709	KJ955008	KJ954568
	LC3315, LF537	Ca. sinensis, pathogen	China	KJ955159	KJ954860	KJ954435	KJ955306	KJ954711	KJ955010	KJ954570
	LC3368, LF590	Ca. sinensis, pathogen	China	KJ955171	KJ954872	KJ954445	KJ955318	KJ954723	KJ955021	KJ954580
	LC3370, LF592	Ca. sinensis, pathogen	China	KJ955172	KJ954873	KJ954446	KJ955319	KJ954724	KJ955022	KJ954581
	LC3384, LF606	Ca. sinensis, pathogen	China	KJ955177	KJ954878	KJ954450	KJ955324	KJ954729	KJ955027	KJ954585
	LC3402, LF624	Ca. sinensis, pathogen	China	KJ955184	KJ954885	KJ954457	KJ955331	KJ954736	KJ955034	KJ954592
	LC3417, LF639	Ca. sinensis, endophyte	China	KJ955188	KJ954889	KJ954461	KJ955335	KJ954740	KJ955038	KJ954595
	LC3425, LF647	Ca. sinensis, endophyte	China	KJ955190	KJ954891	KJ954463	KJ955337	KJ954741	KJ955040	KJ954596
	LC3427, LF649	Ca. sinensis, endophyte	China	KJ955191	KJ954892	KJ954464	KJ955338	KJ954742	KJ955041	KJ954597
	LC3430, LF652	Ca. sinensis, endophyte	China	KJ955192	KJ954893	KJ954465	KJ955339	KJ954743	KJ955042	KJ954598
	LC3433, LF655	Ca. sinensis, endophyte	China	KJ955193	KJ954894	KJ954466	KJ955340	KJ954744	KJ955043	KJ954599
	LC3434, LF656	Ca. sinensis, endophyte	China	KJ955194	KJ954895	KJ954467	KJ955341	KJ954745	KJ955044	KJ954600
	LC3447, LF670	Ca. sinensis, endophyte	China	KJ955195	KJ954896		KJ955342	KJ954746	KJ955045	KJ954601
	LC3451, LF674	Ca. sinensis, endophyte	China	KJ955196	KJ954897		KJ955343	KJ954747	KJ955046	KJ954602
	LC3457, LF681	Ca. sinensis, endophyte	China	KJ955197	KJ954898	KJ954468	KJ955344	KJ954748	KJ955047	KJ954603
	LC3461, LF685	Ca. sinensis, pathogen	China	KJ955199	KJ954900		KJ955346	KJ954750	KJ955049	KJ954605
	LC3462, LF686	Ca. sinensis, pathogen	China	KJ955200	KJ954901	KJ954470	KJ955347	KJ954751	KJ955050	KJ954606
	LC3464, LF689	Ca. sinensis, pathogen	China	KJ955202	KJ954903	KJ954472		KJ954753	KJ955052	KJ954608
	LC3465, LF690	Ca. sinensis, pathogen	China	KJ955203	KJ954904	KJ954473	KJ955349	KJ954754	KJ955053	KJ954609
	LC3471, LF696	Ca. sinensis, pathogen	China	KJ955205	KJ954906	KJ954475	KJ955351	KJ954756	KJ955055	KJ954611
	LC3489, LF716	Ca. sinensis, endophyte	China	KJ955207	KJ954908	KJ954477	KJ955353	KJ954758	KJ955057	KJ954613
	LC3545, LF773	Ca. sinensis, endophyte	China	KJ955214	KJ954915	KJ954482	KJ955360	KJ954765	KJ955064	KJ954620
	LC3569, LF797	Ca. sinensis, pathogen	China	KJ955219	KJ954920	KJ954487	KJ955364	KJ954770	KJ955069	KJ954623
	LC3666, LF896, ICMP 18656	Ca. sinensis, pathogen	Indonesia	KJ955221	KJ954922	KJ954489	KJ955366	KJ954772	KJ955071	KJ954624
	LC3670, LF900, ICMP 10642	Camellia sp., pathogen	UK	KJ955225	KJ954926	KJ954492	KJ955370	KJ954776	KJ955075	KJ954628
C. fructivorum	Coll1092, BPI 884114, CBS 133135	Rhexia virginica	USA	JX145133			JX145184
	Coll1414, BPI 884103, CBS 133125*	Vaccinium macrocarpon	USA	JX145145			JX145196
C. gloeosporioides	IMI 356878, ICMP 17821, CBS 112999*	Citrus sinensis	Italy	JX010152	JX010056	JX009531	JX010445	JX009731	JX010085	JQ807843
	LC3110, LF318	Ca. sinensis, endophyte	China	KJ955127	KJ954828	KJ954407	KJ955275	KJ954680	KJ954978	KJ954541
	LC3312, LF534	Ca. sinensis, pathogen	China	KJ955158	KJ954859	KJ954434	KJ955305	KJ954710	KJ955009	KJ954569
	LC3382, LF604	Ca. sinensis, pathogen	China	KJ955176	KJ954877	KJ954450	KJ955323	KJ954728	KJ955026	KJ954584
	LC3686, LF916	Ca. sinensis, pathogen	China	KJ955226	KJ954927	KJ954493	KJ955371	KJ954777	KJ955076	KJ954629
C. grevilleae	CBS 132879, CPC 15481*	Grevillea sp.	Italy	KC297078	KC297010	KC296941	KC297102	KC296963	KC297033
C. henanense	LC3030, CGMCC 3.17354, LF238*	Ca. sinensis, pathogen	China	KJ955109	KJ954810	KM023257	KJ955257	KJ954662	KJ954960	KJ954524
	LC2820, LF24	Cirsium japonicum, pathogen	China	KM610182	KM610178	KM610172	KM610184	KM610176	KM610180	KM610174
	LC2821, LF25	Cirsium japonicum, pathogen	China	KM610183	KM610179	KM610173	KM610185	KM610177	KM610181	KM610175
C. horii	ICMP 17968	Diospyros kaki	China	JX010212	GQ329682	JX009547	JX010378	JX009605	JX010068
	NBRC 7478, ICMP 10492, MTCC 10841*	Diospyros kaki	Japan	GQ329690	GQ329681	JX009438	JX010450	JX009604	JX010137	JQ807840
C. jiangxiense	LC3266, CGMCC 3.17361, LF488	Ca. sinensis, pathogen	China	KJ955149	KJ954850	KJ954427		KJ954701	KJ955000	KJ954561
	LC3460, CGMCC 3.17362, LF684	Ca. sinensis, endophyte	China	KJ955198	KJ954899	KJ954469	KJ955345	KJ954749	KJ955048	KJ954604
	LC3463, CGMCC 3.17363, LF687*	Ca. sinensis, pathogen	China	KJ955201	KJ954902	KJ954471	KJ955348	KJ954752	KJ955051	KJ954607
C. kahawae subsp. ciggaro	ICMP 12952	Persea americana	New Zealand	JX010214	JX009971	JX009431	JX010426	JX009648	JX010126
	ICMP 18534	Kunzea ericoides	New Zealand	JX010227	JX009904	JX009473	JX010427	JX009634	JX010116	HE655657
	ICMP 18539*	Olea europaea	Australia	JX010230	JX009966	JX009523	JX010434	JX009635	JX010132
C. kahawae subsp. kahawae	IMI 319418, ICMP 17816*	Coffea arabica	Kenya	JX010231	JX010012	JX009452	JX010444	JX009642	JX010130	JQ894579
	CBS 982.69, ICMP 17915	Coffea arabica	Angola	JX010234	JX010040	JX009474	JX010435	JX009638	JX010125
	IMI 361501, ICMP 17905	Coffea arabica	Cameroon	JX010232	JX010046	JX009561	JX010431	JX009644	JX010127
C. melanocaulon	Coll126, BPI 884101, CBS 133123	Vaccinium macrocarpon	USA	JX145142			JX145193			JX145309
	Coll131, BPI 884113, CBS 133251*	Vaccinium macrocarpon	USA	JX145144			JX145195			JX145313
C. musae	CBS 116870, ICMP 19119, MTCC 11349*	Musa sp.	USA	JX010146	JX010050	JX009433	HQ596280	JX009742	JX010103	KC888926
	IMI 52264, ICMP 17817	Musa sapientum	Kenya	JX010142	JX010015	JX009432	JX010395	JX009689	JX010084
C. nupharicola	CBS 469.96, ICMP 17938	Nuphar lutea subsp. polysepala	USA	JX010189	JX009936	JX009486	JX010397	JX009661	JX010087
	CBS 470.96, ICMP 18187*	Nuphar lutea subsp. polysepala	USA	JX010187	JX009972	JX009437	JX010398	JX009663	JX010088	JX145319
	CBS 472.96, ICMP 17940	Nymphaea ordorata	USA	JX010188	JX010031	JX009582	JX010399	JX009662	JX010089
C. proteae	CBS 132882, CPC 14859*	Protea sp.	South Africa	KC297079	KC297009	KC296940	KC297101	KC296960	KC297032
	CBS 134301, CPC 14860	Protea sp.	South Africa	KC842385	KC842379	KC842373	KC842387	KC842375	KC842387
C. psidii	CBS 145.29, ICMP 19120*	Psidium sp.	Italy	JX010219	JX009967	JX009515	JX010443	JX009743	JX010133	KC888931
C. queenslandicum	ICMP 1778*	Carica papaya	Australia	JX010276	JX009934	JX009447	JX010414	JX009691	JX010104	KC888928
	ICMP 18705	Coffea sp.	Fiji	JX010185	JX010036	JX009490	JX010412	JX009694	JX010102
C. rhexiae	Coll1026, BPI 884112, CBS 133134*	Rhexia virginica	USA	JX145128			JX145179			JX145290
	Coll877, BPI 884110, CBS 133132	Vaccinium macrocarpon	USA	JX145157			JX145209			JX145302
C. salsolae	ICMP 19051*	Salsola tragus	Hungary	JX010242	JX009916	JX009562	JX010403	JX009696	JX010093	KC888925
C. siamense	DAR 76934, ICMP 18574	Pistacia vera	Australia	JX010270	JX010002	JX009535	JX010391	JX009707	JX010080
	GM018, MTCC 11672	Mangifera indica	India	JQ894653	JQ894624	JQ894533	JQ894594	KC790778
	GM057, MTCC 11590	Mangifera indica	India	JQ894658	JQ894620	JQ894534	JQ894590	KC790780		JQ894551
	GM172, MTCC 11591	Mangifera indica	India	JQ894662	JQ894621	JQ894535	JQ894591	KC790781		JQ894562
	GM385	Mangifera indica	India	JQ894668	JQ894626	JQ894536	JQ894596	KC790782		JQ894568
	GM390, MTCC 11677	Mangifera indica	India	JQ894670	JQ894627	JQ894537	JQ894597	KC790783		JQ894570
	GM473, MTCC 11589	Mangifera indica	India	JQ894673	JQ894622	JQ894539	JQ894592	KC790785		JQ894553
	GM529, MTCC 11592	Mangifera indica	India	JQ894675	JQ894629	JQ894540	JQ894599	KC790786		JQ894575
	GZAAS 5.09538	Murraya sp.	China	JQ247632	JQ247608	JQ247656	JQ247645	JQ247597	JQ247620
	ICMP 12567	Persea americana	Australia	JX010250	JX009940	JX009541	JX010387	JX009697	JX010076
	ICMP 18121	Dioscorea rotundata	Nigeria	JX010245	JX009942	JX009460	JX010402	JX009715	JX010092
	ICMP 18578, CBS 130417*	Coffea arabica	Thailand	JX010171	JX009924	FJ907423	JX010404	FJ917505	JX010094	JQ899289
	LC0148	Camellia sp., pathogen	China	KJ955078	KJ954779	KJ954360	KJ955227	KJ954631	KJ954929	KJ954494
	LC0149	Camellia sp., pathogen	China	KJ955079	KJ954780	KJ954361	KJ955228	KJ954632	KJ954930	KJ954495
	LC2931, CGMCC 3.17353, LF139	Camellia sp., pathogen	China	KJ955087	KJ954788	KJ954369	KJ955236	KJ954640	KJ954938	KJ954503
	LC2940, LF148	Camellia sp., pathogen	China	KJ955088	KJ954789	KJ954370	KJ955237	KJ954641	KJ954939	KJ954504
	LC2941, LF149	Camellia sp., pathogen	China	KJ955089	KJ954790	KJ954371	KJ955238	KJ954642	KJ954940	KJ954505
	LC2969, LF177	Camellia oleifera, pathogen	China	KJ955092	KJ954793	KJ954374	KJ955241	KJ954645	KJ954943	KJ954508
	LC2974, LF182	Camellia sp., endophyte	China	KJ955093	KJ954794	KJ954375	KJ955242	KJ954646	KJ954944	KJ954509
	LC3409, LF631	Ca. sinensis, pathogen	China	KJ955187	KJ954888	KJ954460	KJ955334	KJ954739	KJ955037
	MTCC 9660	Mangifera indica	India	JQ894649	JQ894619	JQ894532	JQ894589	KC790790		JQ894548
	NK24, MTCC 11599	Mangifera indica	India	JQ894681	JQ894632	JQ894546	JQ894602	KC790791		JQ894582
	NK28, MTCC 11593	Mangifera indica	India	JQ894687	JQ894633	JQ894547	JQ894603	KC790792
C. siamense (syn. C. hymenocallidis)	CBS 125378, ICMP 18642, LC0043	Hymenocallis americana	China	JX010278	JX010019	JX009441	JX010410	JX009709	JX010100	JQ899283
C. siamense (syn. C. jasmini-sambac)	CBS 130420, ICMP 19118	Jasminum sambac	Vietnam	HM131511	HM131497	HM131507	JX010415	JX009713	JX010105	JQ807841
C. siamense (syn. C. murrayae)	GZAAS 5.09506	Murraya sp.	China	JQ247633	JQ247609	JQ247657	JQ247644	JQ247596	JQ247621
C. temperatum	Coll1103, BPI 884098, CBS 133120	Vaccinium macrocarpon	USA	JX145135			JX145186			JX145297
	Coll883, BPI 884100, CBS 133122*	Vaccinium macrocarpon	USA	JX145159			JX145211			JX145298
C. theobromicola	MTCC 11350, CBS 124945, ICMP 18649*	Theobroma cacao	Panama	JX010294	JX010006	JX009444	JX010447	JX009591	JX010139	KC790726
C. theobromicola (syn. C. fragariae)	CBS 142.31, ICMP 17927, MTCC 10325	Fragaria × ananassa	USA	JX010286	JX010024	JX009516	JX010373	JX009592	JX010064	JQ807844
C. ti	ICMP 4832*	Cordyline sp.	New Zealand	JX010269	JX009952	JX009520	JX010442	JX009649	JX010123	KM360146
	ICMP 5285	Cordyline australis	New Zealand	JX010267	JX009910	JX009553	JX010441	JX009650	JX010124
C. tropicale	CBS 124949, ICMP 18653, MTCC 11371*	Theobroma cacao	Panama	JX010264	JX010007	JX009489	JX010407	JX009719	JX010097	KC790728
	MAFF 239933, ICMP 18672	Litchi chinensis	Japan	JX010275	JX010020	JX009480	JX010396	JX009722	JX010086
C. viniferum	GZAAS 5.08601, yg1*	Vitis vinifera cv. Shuijing	China	JN412804	JN412798	JN412795		JQ309639	JN412787
	GZAAS 5.08608, yg4	Vitis vinifera cv. Hongti	China	JN412802	JN412800	JN412793		JN412782	JN412784
C. xanthorrhoeae	BRIP 45094, ICMP 17903, CBS 127831*	Xanthorrhoea preissii	Australia	JX010261	JX009927	JX009478	JX010448	JX009653	JX010138	KC790689
	IMI 350817a, ICMP 17820	Xanthorrhoea sp.	Australia	JX010260	JX010008	JX009479		JX009652

^a AS, CGMCC: China General Microbiological Culture Collection; ATCC: American Type Culture Collection; BPI: U.S. National Fungus Collections, USA; BRIP: Plant Pathology Herbarium, Department of Employment, Economic, Development and Innovation, Queensland, Australia; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Working collection of Pedro W. Crous, housed at CBS, The Netherlands; DAR: Plant pathology Herbarium, Australia; GZAAS: Guizhou Academy of Agricultural Sciences Herbarium, China; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; LC: Working collection of Lei Cai, housed at CAS, China; LF: Working collection of Fang Liu, housed at CAS, China; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan; MFLUCC: Mae Fah Luang University Culture Collection, ChiangRai, Thailand; MTCC: Microbial type culture collection and gene bank, India; NBRC: NITE Biological Resource Centre, Japan.

* = ex-type culture. Strains/sequences studied in this paper are in bold font.

Table 2.

Strains of Colletotrichum excluded from the C. gloeosporioides species complex. Details are provided about host and location, and GenBank accessions of the sequences generated.

Species	Association numbera	Host	Locality	GenBank accessions
				ITS	GAPDH	ACT	TUB2
C. acutatum	CBS 112996, ATCC 56816*	Carica papaya	Australia	JQ005776	JQ948677	JQ005839	JQ005860
	CBS 979.69	Coffea arabica	Kenya	JQ948400	JQ948731	JQ949721	JQ950051
C. boninense	CBS 123755, MAFF 305972*	Crinum asiaticum var. sinicum	Japan	JQ005153	JQ005240	JQ005501	JQ005588
	CBS 128526, ICMP 18591	Dacrycarpus dacrydioides	New Zealand	JQ005162	JQ005249	JQ005510	JQ005596
	CBS 128547, ICMP 10338	Camellia sp.	New Zealand	JQ005159	JQ005246	JQ005507	JQ005593
	LC3422, CGMCC 3.14356,	Camellia sinensis, endophyte	China	KJ955189	KJ954890	KJ954462	KJ955336
	LF644
C. brasiliense	CBS 128501, ICMP 18607*	Passiflora edulis	Brazil	JQ005235	JQ005322	JQ005583	JQ005669
	CBS 128528, ICMP 18606	Passiflora edulis	Brazil	JQ005234	JQ005321	JQ005582	JQ005668
C. cliviae	CBS 125375*	Clivia miniata	China	JX519223	JX546611	JX519240	JX519249
	LC3546, CGMCC 3.17358,	Camellia sinensis, endophyte	China	KJ955215	KJ954916	KJ954483	KJ955361
	LF774
C. coccodes	CBS 369.75*	Solanum tuberosum	Netherlands	HM171679	HM171673	HM171667	JX546873
C. colombiense	CBS 129817	Passiflora edulis	Colombia	JQ005173	JQ005260	JQ005521	JQ005607
	CBS 129818*	Passiflora edulis	Colombia	JQ005174	JQ005261	JQ005522	JQ005608
C. constrictum	CBS 128504, ICMP 12941*	Citrus limon	New Zealand	JQ005238	JQ005325	JQ005586	JQ005672
C. dracaenophilum	CBS 118199*	Dracaena sanderana	China	JX519222	JX546707	JX519238	JX519247
C. fioriniae	CBS 119293	Vaccinium corymbosum	New Zealand	JQ948314	JQ948644	JQ949635	JQ949965
	CBS 128517*	Fiorinia externa	USA	JQ948292	JQ948622	JQ949613	JQ949943
	CBS 129948	Tulipa sp.	UK	JQ948344	JQ948674	JQ949665	JQ949995
	LC3381, CGMCC 3.17357, LF603	Camellia sinensis, pathogen	China	KJ955175	KJ954876	KJ954449	KJ955322
C. karstii	CBS 129824	Musa sp.	Colombia	JQ005215	JQ005302	JQ005563	JQ005649
	CBS 132134, CORCG6,	Vanda sp.	China	HM585409	HM585391	HM581995	HM585428
	CGMCC 3.14194*
	LC3108, LF316	Camellia sinensis, endophyte	China	KJ955125	KJ954826	KJ954405	KJ955273
	LC3168, LF377	Camellia sinensis, endophyte	China	KJ955146	KJ954847	KJ954424	KJ955294
	LC3210, LF421	Camellia sinensis, endophyte	China	KJ955148	KJ954849	KJ954426	KJ955296
	LC3272, LF494	Camellia sinensis, pathogen	China	KJ955152	KJ954853	KJ954429	KJ955299
	LC3357, LF579	Camellia sinensis, pathogen	China	KJ955169	KJ954870	KJ954443	KJ955316
	LC3560, LF788	Camellia sinensis, pathogen	China	KJ955216	KJ954917	KJ954484	KJ955362
	LC3570, CGMCC 3.17359,	Camellia sinensis, pathogen	China	KJ955220	KJ954921	KJ954488	KJ955365
	LF798
	MAFF 305973, ICMP 18598	Passiflora edulis	Japan	JQ005194	JQ005281	JQ005542	JQ005628
C. orchidophilum	CBS 632.80*	Dendrobium sp.	USA	JQ948151	JQ948481	JQ949472	JQ949802
C. phormii	CBS 118194*	Phormium sp.	Germany	JQ948446	JQ948777	JQ949767	JQ950097
	CBS 199.35	Phormium sp.	UK	JQ948447	JQ948778	JQ949768	JQ950098
C. rusci	CBS 119206*	Ruscus sp.	Italy	GU227818	GU228210	GU227916	GU228112
C. spaethianum	CBS 167.49*	Funkia sieboldiana	Germany	GU227807	GU228199	GU227905	GU228101
C. walleri	CBS 125472*	Coffea sp.	Vietnam	JQ948275	JQ948605	JQ949596	JQ949926
C. yunnanense	AS 3.9167, CBS 132135*	Buxus sp.	China	JX546804	JX546706	JX519239	JX519248
Monilochaetes infuscans	CBS 869.96*	Ipomoea batatas	South Africa	JQ005780	JX546612	JQ005843	JQ005864

^a AS, CGMCC: China General Microbiological Culture Collection; ATCC: American Type Culture Collection; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; LC: Working collection of Lei Cai, housed at CAS, China; LF: Working collection of Fang Liu, housed at CAS, China; MAFF: Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan.

* = ex-type culture. Strains/sequences studied in this paper are in bold font.

Phylogenetic analyses

Multiple sequence alignments were generated using MAFFT v. 7 (Katoh & Standley 2013), and if necessary, manually edited in MEGA v. 5.1. Bayesian analyses were performed on concatenated alignments using MrBayes v. 3.2.2 (Ronquist et al. 2012) as described by Crous et al. (2006) using nucleotide substitution models that were selected by MrModeltest v. 2.3 (Nylander 2004), with critical values for the topological convergence diagnostic set to 0.01. Maximum likelihood (ML) analyses were implemented using the CIPRES Science Gateway v. 3.3 (www.phylo.org), and the RAxML-HPC BlackBox was selected with default parameters. Six loci (ACT, CAL, GAPDH, GS, ITS, and TUB2) were concatenated for the multi-locus analysis of C. gloeosporioides s.l., while four loci (ACT, GAPDH, ITS, TUB2) were used for the multi-locus analysis of other Colletotrichum species. Due to the lack of available ApMat gene sequences of most of the recently identified Colletotrichum isolates, the ApMat locus could not be included in the concatenated alignment. Therefore, a single ApMat phylogeny was generated including sequences of 136 C. gloeosporioides s.l. isolates obtained from Camellia in this study, and 181 reference sequences that were retrieved from NCBI-GenBank. An additional phylogeny using a concatenated ApMat and GS sequence alignment was constructed which included 126 C. gloeosporioides s.l. isolates from Camellia and 33 reference isolates.

Genealogical concordance phylogenetic species recognition analysis

Phylogenetically related but ambiguous species were analysed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014). The PHI test was performed in SplitsTree4 (Huson 1998, Huson & Bryant 2006) in order to determine the recombination level within phylogenetically closely related species using a 6-locus concatenated dataset (ACT, CAL, GAPDH, GS, ITS, and TUB2). If the pairwise homoplasy index results were below a 0.05 threshold (Φ_w < 0.05), it was indicative for significant recombination present in the dataset. The relationship between closely related species was visualised by constructing a splits graph.

Pathogenicity

Koch’s postulates were conducted as described in Cai et al. (2009). Six Colletotrichum isolates were selected for pathogenicity tests: C. camelliae CGMCC 3.14925, C. henanense CGMCC 3.17354, C. jiangxiense CGMCC 3.17362 and CGMCC 3.17363, C. kahawae subsp. kahawae IMI 319418 and IMI 363578. Healthy leaves of intact 2-yr-old tea plants were washed with sterilised water, and then inoculated using the wound/drop and non-wound/drop inoculation methods. Plants inoculated with sterile water were used as control. The inoculated samples were incubated at room temperature in normal light regimes in the greenhouse for 14 d.

RESULTS

Isolates

In total, 144 Colletotrichum isolates were obtained from Camellia tissues from the main tea growing regions in China. Of these, 102 isolates were isolated from diseased tissues, and 42 from asymptomatic tissues (Table 1, 2).

Phylogenetic analyses of the combined datasets

Based on the BLAST search results of the NCBI database with the ITS sequences, all Colletotrichum isolates in this study were preliminarily allocated to species complexes: 141 iso-lates belonged to the C. gloeosporioides species complex, eight isolates belonged to the C. boninense species complex, one isolate belonged to C. acutatum species complex, and one isolate was identified as C. cliviae.

The 6-locus (ACT, CAL, GAPDH, GS, ITS, TUB2) phylogenetic analysis of the C. gloeosporioides species complex included 229 isolates from Camellia and other hosts, with C. boninense (CBS 123755) as the outgroup (see Fig. 1 for a version of this phylogeny with selected identical isolates removed; the complete alignment and tree, as Fig. S1, is available from TreeBASE). The dataset comprised 3 522 characters including the alignment gaps. For the Bayesian inference, a GTR+I+G model with inverse gamma-distributed rate was selected for ACT, HKY+G with gamma-distributed rates for CAL and ITS, GTR+G with gamma-distributed rates for GAPDH, GS, and TUB2. The maximum likelihood tree confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. Isolates from Camellia in the C. gloeosporioides complex clustered in seven clades (data present in TreeBASE as Fig. S1): one Camellia isolate clustered with the ex-type isolate of C. alienum, 32 isolates clustered with C. fructicola, four isolates clustered with C. gloeosporioides, 91 isolates clustered with C. camelliae (syn. Glomerella cingulata ‘f. sp. camelliae’), and eight isolates clustered with the ex-type isolates of C. siamense, C. dianesei, and C. melanocaulon in one clade. Three Camellia isolates formed a distinct clade (posterior probability = 1), most closely related to C. kahawae s.l. A simplified tree was subsequently generated by removing 87 isolates of C. camelliae and C. fructicola (Fig. 1).

Fig. 1.

Fifty percent majority rule consensus tree from a Bayesian analysis based on a 6-gene combined dataset (ACT, CAL, GAPDH, GS, ITS, TUB2) showing phylogenetic affinities of a reduced set of Colletotrichum isolates from Camellia isolated in this study with species of the C. gloeosporioides species complex. The RAxML bootstrap support values (ML > 50) and Bayesian posterior probabilities (PP > 0.95) are displayed at the nodes (ML/PP). The tree was rooted to C. boninense (CBS 123755). The scale bar indicates 0.9 expected changes per site. Ex-type cultures are emphasised in bold, and include the taxonomic name as originally described. Coloured blocks are used to indicate clades containing Chinese isolates from Camellia; stars indicate pathogens, squares indicate endophytes.

Fig. 2 shows the identity of the Camellia isolates that fell outside of the C. gloeosporioides species complex. The concatenated alignment (ACT, GAPDH, ITS, TUB2) contained 37 isolates, with Monilochaetes infuscans (CBS 869.96) as outgroup. The dataset comprised 1 559 characters including the alignment gaps. For the Bayesian inference, a HKY+G model with gamma-distributed rate was selected for ACT, HKY+I+G with inverse gamma-distributed rate for GAPDH, GTR+I+G with inverse gamma-distributed rates for ITS and TUB2. The maximum likelihood tree confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. Seven Camellia isolates clustered with the ex-type isolate of C. karstii, one isolate clustered with C. boninense, one isolate clustered with C. fioriniae and one isolate clustered with C. cliviae.

Fig. 2.

Fifty percent majority rule consensus tree from a Bayesian analysis based on a 4-gene combined dataset (ITS, GAPDH, ACT, TUB2) showing phylogenetic affinities of Colletotrichum isolates from Camellia with members of the Colletotrichum species outside of the C. gloeosporioides species complex. The RAxML bootstrap support values (ML > 50) and Bayesian posterior probabilities (PP > 0.95) are displayed at the nodes (ML/PP). The tree was rooted to Monilochaetes infuscans (CBS 869.96). The scale bar indicates 0.2 expected changes per site. Ex-type cultures are emphasised in bold. Coloured blocks are used to indicate clades containing Chinese isolates from Camellia; stars indicate pathogens, squares indicate endophytes.

The pathogenic and endophytic isolates of Colletotrichum studied here were labelled with stars and squares, respectively, on the multi-locus phylogenetic trees (Fig. 1, 2). Isolates from symptomatic Camellia leaves belong to eight clades, representing C. camelliae, C. fioriniae, C. fructicola, C. gloeosporioides, C. henanense, C. jiangxiense, C. karstii, and C. siamense. Isolates from asymptomatic tissues belong to nine clades representing C. alienum, C. boninense, C. camelliae, C. cliviae, C. fructicola, C. gloeosporioides, C. henanense, C. karstii, and C. siamense.

ApMat-based phylogenetic analysis

The phylogenetic analysis of the C. gloeosporioides species complex using the ApMat locus included 317 isolates from Camellia and other hosts (rooted with C. xanthorrhoeae), and 785 characters with alignment gaps were involved in the dataset. All isolates included in this analysis were separated into 15 main clades and 12 single-isolate lineages (see Fig. 3 for a cartoon version of this phylogeny; the complete alignment and tree, as Fig. S2, is available from TreeBASE). One of the clades is represented by an assemblage of more than one species, including C. fructivorum, C. jiangxiense, C. kahawae, C. rhexiae, and C. temperatum (Fig. 3, S2). Of these five species, C. fructivorum, C. rhexiae, and C. temperatum formed monophyletic species clades. However, strains from C. jiangxiense and C. kahawae were intermingled in one clade and the two species could not be differentiated from each other. The C. camelliae isolates were separated into two distinct clades, while the other species formed monophyletic clades.

Fig. 3.

Collapsed cartoon of the 50 % majority rule consensus tree from a Bayesian analysis based on the ApMat dataset showing phylogenetic affinities of Colletotrichum isolates from Camellia with members of the C. gloeosporioides species complex. Bayesian posterior probabilities values are displayed at the node. The tree was rooted to C. xanthorrhoeae (ICMP 17903). The scale bar indicates 0.6 expected changes per site. Ex-type cultures are emphasised in bold, and include the taxonomic name as originally described.

ApMat & GS-based phylogenetic analysis

Colletotrichum jiangxiense and C. kahawae subsp. kahawae cannot be separated on the basis of the ApMat locus. They are mainly distinguished from one another based on the GS gene (see also notes under C. jiangxiense); the two species formed distinct clades in the GS gene phylogeny (not shown). The potential of the concatenated ApMat and GS genes to serve as a barcode for the C. gloeosporioides species complex was demonstrated by re-constructing a phylogenetic tree using the sequences listed in Table 1 (Fig. 4). All species of the C. gloeosporioides species complex included in the analysis could be delimited clearly based on the concatenated ApMat & GS gene tree.

Fig. 4.

Collapsed cartoon of the 50 % majority rule consensus tree from a Bayesian analysis based on the combined ApMat and GS alignment showing phylogenetic affinities of Colletotrichum isolates from Camellia with species of the C. gloeosporioides species complex. Bayesian posterior probabilities values are displayed at the node. The tree was rooted to C. xanthorrhoeae (ICMP 17903). The scale bar indicates 0.5 expected changes per site. Ex-type cultures are emphasised in bold. Extremely long branches were halved in length (indicated with 2× above two diagonal lines) to better fit the tree to the page.

Pairwise homoplasy index (PHI) test

A pairwise homoplasy index (PHI) test using a 6-gene dataset (ACT, CAL, GAPDH, GS, ITS, TUB2) was further performed to determine the recombination level between C. jiangxiense and its phylogenetically closely related species, C. kahawae subsp. ciggaro and C. kahawae subsp. kahawae. Based on the result no significant recombination events could be detected between C. kahawae s.l. and C. jiangxiense (Φ_w = 1) (Fig. 5).

Fig. 5.

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

Pathogenicity

The tea plant leaves inoculated with a conidial suspension of Colletotrichum isolates from symptomatic tea leaves (C. camelliae CGMCC 3.14925, C. henanense CGMCC 3.17354, C. jiangxiense CGMCC 3.17363) developed typically brown lesions around the leaf wounds after 14 d (Fig. 6). The inoculated Colletotrichum isolates could be re-isolated from the periphery of these lesions, thereby fulfilling Koch’s postulates. Leaves of the control plants were inoculated with sterile water, and leaves inoculated with isolates of C. kahawae subsp. kahawae did not develop any symptoms after 14 d past inoculation (Fig. 6).

Fig. 6.

Pathogenicity test of selected isolates on tea plant leaves after 14 d. a. C. jiangxiense (CGMCC 3.17363); b, c. C. henanense (CGMCC 3.17354); d. C. kahawae subsp. kahawae (IMI 363578); e. C. camelliae (CGMCC 3.14925); f. control.

Taxonomy

Based on the multi-locus phylogenies (Fig. 1–4 and Fig. S1, S2 in TreeBASE), the 151 Colletotrichum isolates from Camellia sinensis and other Camellia spp. belonged to 11 species, including two species that proved to be new to science.

Colletotrichum alienum B. Weir & P.R. Johnst, Stud. Mycol. 73: 139. 2012

Description and illustrations — See Weir et al. (2012) and Liu et al. (2013b).

Material examined. CHINA, Jiangxi Province, Ganzhou, Yangling National Forest Park, on living leaf of Ca. sinensis, Apr. 2013, F. Liu, culture CGMCC 3.17355 = LC3114 = LF322.

Notes — Colletotrichum alienum was previously only known from Australia, New Zealand, Portugal, and South Africa (Weir et al. 2012, Liu et al. 2013b). In the present study, one endophytic isolate CGMCC 3.17355 from a tea leaf clustered together with the ex-type culture of C. alienum (ICMP 12071) in the multi-locus phylogenetic tree (Fig. 1); this is the first reported occurrence of C. alienum on Ca. sinensis and in China.

Both conidia and ascospores of the tea isolate (CGMCC 3.17355) are slightly shorter than that of the ex-type (ICMP 12071) of C. alienum (conidia 14.5 × 4.6 μm vs 16.5 × 5 μm, ascospores 16.3 × 4.4 μm vs 18.1 × 4.6 μm; Weir et al. 2012).

Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib., Mycoscience 44: 48. 2003

Description and illustrations — See Moriwaki et al. (2003) and Damm et al. (2012b).

Material examined. CHINA, Jiangxi Province, Ganzhou, Fengshan Mountain, on living leaf of Ca. sinensis, Sept. 2013, Y. Zhang, culture CGMCC 3.14356 = LC3422 = LF644.

Notes — The endophytic isolate (LF644) from a tea leaf eva-luated in this study was identified as C. boninense based on the multi-locus phylogenetic analyses (Fig. 2). This species was previously reported on Camellia sp. from New Zealand (Damm et al. 2012b).

Conidia of the tea isolate (CGMCC 3.14356) on PDA are wider, and the L/W ratio is smaller than that of the ex-type culture (CBS 123755) of C. boninense on Anthriscus stem and SNA (CGMCC 3.14356: 10–15 × 6.5–8 μm, mean = 13.7 × 7.3 μm, L/W ratio = 1.9 vs CBS 123755: on Anthriscus stem (9–)12–14.5(–16.5) × (4–)5.5–6.5 μm, av = 13.2 × 5.8 μm, L/W ratio = 2.3, on SNA (8.5–)11–14.5(–17.5) × (4–)5–6(–6.5) μm, av = 12.8 × 5.4 μm, L/W ratio = 2.4). Conidia of CBS 123755 often contain two large polar guttules, which were absent in the tea isolate.

Colletotrichum camelliae Massee, Bull. Misc. Inform. Kew 1899: 91. 1899. — Fig. 7

Fig. 7.

Colletotrichum camelliae (CGMCC 3.14925). a. Symptom on tea leaf; b, c. forward and reverse view of culture 7 d after inoculation; d. conidiophores; e, f, i. conidia; g, h. appressoria (b–f, i from PDA; g, h from SNA). — Scale bar: d–i = 10 μm.

= Glomerella cingulata ‘f. sp. camelliae’ Dickens & R.T.A. Cook, Pl. Pathol. 38: 85. 1989.

On PDA: Colonies 69–71 mm diam in 7 d, > 90 mm diam in 10 d, flat with entire edge, aerial mycelium white, cottony, sparse; reverse white at first, then grey to black at the centre. Conidiomata not observed, conidiophores formed directly on aerial mycelium, hyaline, septate. Conidiogenous cells hyaline, cylindrical, 16–42 × 1.5–4.5 μm. Conidia hyaline, smooth-walled, guttulate, cylindrical with obtuse ends, sometimes narrowed at the centre or towards the base, 9–25 × 3.5–7.5 μm, av ± SD = 15.5 ± 3.3 × 5.0 ± 0.9 μm, L/W ratio = 3.1. Appressoria irregularly shaped, clavate, crenate, lobed, brown to dark brown, solitary, branched, catenate, with age sometimes complex chlamydospore-like structures develop, 6.5–13.5 × 5.0–10.5 μm, av ± SD = 10.0 ± 1.8 × 7.5 ± 1.3, L/W ratio = 1.3.

Materials examined. CHINA, Fujian Province, Zhangzhou, on Ca. sinensis, Nov. 2012, L. Cai, culture LF214; Guizhou Province, Huishui District, on Ca. sinensis, 11 Nov. 2010, P. Tan (HMAS 243126 epitype designated here MBT178292, culture ex-epitype CGMCC 3.14925 = LC1364); ibid., HMAS 243127, culture CGMCC 3.14924 = LC1363; ibid., HMAS 243128, culture CGMCC 3.14926 = LC1365; Jiangxi Province, Yangling National Forest Park, on Ca. sinensis, Apr. 2013, F. Liu, culture LC3095 = LF303; ibid., culture LC3109 = LF317. – Sri Lanka, on leaves of Camellia sp., 8 Apr. 1899, J.C. Willis, K(M) 173540 holotype. – USA, South Carolina, on Ca. sasanqua, 1982, unknown collector, culture LC3668 = LF898 = ICMP 10646.

Notes — To our knowledge, the earliest known record of tea anthracnose was described in 1899 by Massee (in Willis 1899) from living leaves of Ca. sinensis from Sri Lanka. The holotype sample is preserved in K(M) 173540 and labelled C. camelliae (Fig. 8). Although it was subsequently synonymised with C. gloeosporioides (von Arx 1957), the name C. camelliae is still widely used in fungaria, websites, trade and semi-popular literature as the causal agent of the brown blight disease of tea plants (Weir et al. 2012). In 1989, Glomerella cingulata ‘f. sp. camelliae’ was proposed as the causal agent of disease on ornamental Ca. saluenensis hybrids, but without distinguishable morphological characteristics compared to G. cingulata (Dickens & Cook 1989). Weir et al. (2012) revealed G. cingulata ‘f. sp. camelliae’ to belong to the C. gloeosporioides complex. However, due to the lack of an ex-type culture of C. camelliae, the genetic relationship between C. camelliae and G. cingulata ‘f. sp. camelliae’ remained unresolved.

Fig. 8.

Holotype of C. camelliae (K (M) 173540). a. Label of the specimen; b. tea leaf with C. camelliae colonisation from above and below; c–g. conidia. — Scale bars: c–g = 10 μm.

We evaluated the holotype specimen of C. camelliae from K, but very few morphological characters could be observed on this old specimen, and DNA extraction was unsuccessful. Conidia on the holotype specimen are hyaline and cylindrical (Fig. 8), 14.5–20 × 4–6 μm, av ± SD = 17.2 ± 1.2 × 4.9 ± 0.4 μm. Conidial dimensions of isolates in this study on PDA (9–25 × 3.5–7.5 μm, av ± SD = 15.5 ± 3.3 × 5.0 ± 0.9 μm) are in accordance with the holotype specimen.

Several efforts to obtain a fresh culture from tea plants from Sri Lanka, the original location from where C. camelliae was reported, proved to be unsuccessful. However, we collected many anthracnose diseased samples in the tea fields from different provinces in China. Leaf lesions were dark brown and circular at first, then enlarged to become more irregular, with many of the lesions coalescing; raised black circular masses were found at the centre of lesions, bordered by a discoloured margin (Fig. 7a). Isolates from these samples clustered together with authentic isolates of G. cingulata ‘f. sp. camelliae’ (cited by Dickens & Cook 1989) in the 6-gene and ApMat phylogenetic trees (Fig. 1 and Fig. S2 in TreeBASE). Inoculations using conidial suspensions were performed on tea plants under controlled environmental conditions to test whether this fungus was the causal agent of tea anthracnose disease. The inoculations resulted in leaf infection of Ca. sinensis consistent with the original natural infections. Re-isolation and re-sequencing confirmed that the culture was identical to the one used for inoculation. No symptoms were produced in the negative control plants. A pathogenicity test with isolates of G. cingulata ‘f. sp. camelliae’ from ornamental Camellia on detached tea (Ca. sinensis) leaves was performed by Weir et al. (2012) and the isolates proved to be highly virulent. The Colletotrichum isolates from tea brown blight symptoms from India, showing affinities to G. cingulata ‘f. sp. camelliae’, were also pathogenic to detached tea leaves (Sharma et al. 2014). All the tests and analyses demonstrated that the isolates collected from typical brown blight symptoms on tea in the field and those from ornamental varieties are the same species. Since C. camelliae was published earlier than G. cingulata ‘f. sp. camelliae’ (1899 vs 1989), and there is no nomenclatural priority for formae speciales (Art. 4, http://www.iapt-taxon.org/nomen/main.php?page=art4), the name C. camelliae is adopted for the anthracnose pathogen of tea and is epitypified in this study, and G. cingulata ‘f. sp. camelliae’ is synonymised with C. camelliae.

Colletotrichum cliviae Y.L. Yang et al., Fung. Diversity 39: 133. 2009 — Fig. 9

Fig. 9.

Colletotrichum cliviae on Anthriscus stem (CGMCC 3.17358). a. Ascomata; b. ascospores; c, d. asci and ascospores. — Scale bar: b = 10 μm, scale bar of b applies to b–d.

On PDA: Colonies 65–69 mm diam in 7 d, > 90 mm diam in 10 d, flat with entire edge. Cultures on PDA and SNA are sterile, but a sexual morph developed on Anthriscus stem. Ascomata glo-bose, brown to black, covered by sparse and white aerial mycelium, outer wall composed of flattened angular cells. Asci cylindrical, 62–92 × 8–12 μm, 8-spored. Ascospores uni- or biseriately arranged, hyaline, aseptate, smooth-walled, allantoid, ellipsoidal or ovoid with rounded ends, 11–16.5 × 4–6.5 μm, av ± SD = 13.8 ± 1.6 × 5.8 ± 0.5 μm, L/W ratio = 2.4. No asexual morph was observed in this study. Yang et al. (2009) provided a description of the asexual morph of this species.

Material examined. CHINA, Guangxi Province, Guilin, on living leaf of Ca. sinensis, Sept. 2013, T.W. Hou, culture CGMCC 3.17358 = LC3546 = LF774.

Notes — Colletotrichum cliviae was reported to cause anthracnose diseases on Clivia miniata, Arundina graminifolia and Cymbidium hookerianum in China (Yang et al. 2009, 2011). The host range was recently extended to include Cattleya, Calamus thwaitesii, Phaseolus, and Saccharum (Sharma et al. 2013b). In the present study, a single isolate (CGMCC 3.17358) of Colletotrichum from a healthy tea leaf proved to belong to C. cliviae, but the asexual morph was not observed. Conversely, this is the first report of a sexual morph of C. cliviae, and the first report of this species on Ca. sinensis.

Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan, Fung. Diversity 39: 117. 2009

Basionym. Colletotrichum acutatum var. fioriniae Marcelino & Gouli, Myco-logia 100: 362. 2008.

Description and illustration — See Damm et al. (2012a).

Materials examined. CHINA, Jiangxi Province, Ganzhou, Yangling National Forest Park, on Ca. sinensis, Apr. 2013, F. Liu, culture CGMCC 3.17357 = LC3381 = LF603.

Notes — Colletotrichum fioriniae was previously reported from Ca. reticulata in Kunming, Yunnan Province and from Ca. sinensis in Fujian Province in China (Damm et al. 2012a, Liu 2013).

Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde, Fung. Diversity 39: 158. 2009 — Fig. 10

Fig. 10.

Colletotrichum fructicola on PDA (a, b, d, e from LC2923; c from LC3451). a. Acervulus; b, d. conidiophores; c. seta; e. conidia. — Scale bar: b = 10 μm, scale bar of b applies to b–e.

On PDA: Colonies 74–79 mm diam in 7 d, > 90 mm diam in 10 d, flat with entire edge, aerial mycelium dense, cottony, grey to dark grey in the centre, white at the margin; reverse greyish green with white halo. Chlamydospores not observed. Conidiomata acervular, only one seta was observed, brown, smooth-walled, 1-septate, 64 μm long, base inflated, 4 μm diam, tip more or less acute. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical or ampulliform, 7.5–18.5 μm, apex 1–3 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends rounded, 11.5–17.5 × 3–5.5 μm, av ± SD = 14.9 ± 1.3 × 4.4 ± 0.4 μm, L/W ratio = 3.4. Appressoria not observed.

Materials examined. CHINA, Guangxi Province, Guilin, on Ca. sinensis, Sept. 2013, T.W. Hou, culture LC3545 = LF773; ibid., culture LC3489 = LF716; Hangzhou, on Ca. sinensis, Oct. 2013, F. Liu, culture LC3569 = LF797; on Ca. sinensis, Sept. 2012, L. Cai, culture CGMCC 3.17352 = LC2923 = LF130; Jiangxi Province, Ganzhou, Fengshan Mountain, on Ca. sinensis, Sept. 2013, Y. Zhang, culture LC3462 = LF686; ibid., culture LC3451 = LF674; Yangling National Forest Park, on Ca. sinensis, Apr. 2013, F. Liu, culture LC3284 = LF506. – Indonesia, on Ca. sinensis, Jan. 1979, H. Semangun, culture LC3666 = LF896 = ICMP 18656. UK, on a shipment of Camellia flowers from New Zealand, on Camellia sp., 1982, staff of Ministry of Agriculture, Fisheries & Food, culture LC3670 = LF900 = ICMP 10642.

Notes — This study supplements the morphological characteristics of setae of C. fructicola that were not observed in the previous studies. Colletotrichum fructicola was reported to cause anthracnose diseases on several varieties of Ca. sinensis in many regions in Fujian Province, China (Liu 2013). In the present study, the species was found to be widely distributed throughout China, although there appears to be some variation in sequence data among isolates from Ca. sinensis. Conidia of the tea isolates (LC2923, av = 14.9 × 4.4 μm and LC3451, av = 15.03 × 4.35 μm) are longer than that of the ex-type (MFLU 090228, av = 11.53 × 3.55) of C. fructicola.

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., Atti Reale Ist. Veneto Sci. Lett. Arti., ser. 6, 2: 670. 1884 — Fig. 11

Fig. 11.

Colletotrichum gloeosporioides (LC3686). a. Acervulus; b. conidiophores; c. conidia; d, e. appressoria (a–c from PDA; d, e from SNA). — Scale bar: c = 10 μm, scale bar of c applies to b–e.

Basionym. Vermicularia gloeosporioides Penz., Michelia 2: 450. 1882.

On PDA: Colonies 56–58 mm diam in 7 d, > 90 mm diam in 10 d, flat with erose edge, scattered acervuli with orange conidial ooze near centre, fuscous black pigment near the edge; reverse honey with fuscous black near the edge. Chlamydospores not observed. Conidiomata acervular, conidiophores formed on a cushion of roundish and medium brown cells. Setae not observed. Conidiophores hyaline to pale brown, septate, branched. Conidiogenous cells hyaline, cylindrical to ampulliform, 5.5–17.5 μm, apex 1–2 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends bluntly rounded, 11–15.5 × 4.5–6 μm, av ± SD = 13.5 ± 1.2 × 5.5 ± 0.3 μm, L/W ratio = 2.5. Appressoria medium to dark brown, aseptate, solitary or in groups, variable in shape, circular, clavate, ellipsoidal or irregular in outline, crenate or slightly lobed at edge, 7.5–13.5 × 5–9.5 μm, av ± SD = 9.5 ± 1.4 × 6.5 ± 0.9 μm, L/W ratio = 1.5.

Materials examined. CHINA, Jiangxi Province, on Ca. sinensis, Sept. 2013, Y.H. Gao, culture CGMCC 3.17360 = LC3686 = LF916; Ganzhou, Yangling National Forest Park, on Ca. sinensis, Apr. 2013, F. Liu, culture LC3110 = LF318; ibid., culture LC3312 = LF534; ibid., culture LC3382 = LF604.

Notes — Colletotrichum gloeosporioides is listed as a pathogen of Camellia in Australia, Brazil, China, Hong Kong, Japan, and the USA (Farr & Rossman 2014). However, many of these reports probably refer to this species in its broader sense as a species complex and need to be further verified (Watson 1950, Shivas 1989, Osono 2008, Guo et al. 2014). For example, the anthracnose pathogen C. gloeosporioides was recently detected in 30–60 % of the Ca. sinensis fields in the Yellow Mountain region in China during 2011 to 2012 (Guo et al. 2014), the identification of which, however, was solely based on morphology and NCBI BLAST searches with ITS sequences, and was not based on the presently accepted classification system in Colletotrichum (Cannon et al. 2012). Colletotrichum gloeosporioides was also considered to be one of the dominant endophytic taxa of Camellia in the study of Fang et al. (2013) based on ITS analysis, the identification of which needs to be verified by multi-locus analysis. In our investigation, four isolates of C. gloeosporioides were associated with Camellia, confirming this species to occur on this host. However, C. gloeosporioides is not the dominant Colletotrichum species on Camellia spp. at the localities where we sampled.

Colletotrichum henanense F. Liu & L. Cai, sp. nov. — MycoBank MB809160; Fig. 12

Fig. 12.

Colletotrichum henanense (CGMCC 3.17354). a–c. Conidiophores; d, i. conidia; e–h. appressoria (a–d, i from PDA; e–h from SNA). — Scale bars: d, e = 10 μm, scale bar of d applies to a–d, i; scale bar of e applies to e–h.

Etymology. Named after the collection site, Henan province, China.

On PDA: Colonies 53–59 mm diam in 7 d, > 90 mm diam in 10 d, aerial mycelium pale olivaceous-grey to olivaceous-grey; reverse sulphur-yellow to straw with pale olivaceous-grey to iron-grey in the centre. Chlamydospores not observed. Conidiomata acervular, conidiophores formed on a cushion of roundish and medium brown cells. Setae not observed. Conidiophores hyaline to pale brown, septate, branched. Conidiogenous cells hyaline to pale brown, cylindrical to ovoid or ampulliform, 5.5–12.5 μm, apex 1–2 μm diam. Conidia hya-line, usually aseptate, sometimes becoming 1-septate with age, smooth-walled, cylindrical, both ends obtusely rounded, contents sometimes with guttulae, 8–17 × 3–5.5 μm, av ± SD = 12.5 ± 1.8 × 4.5 ± 0.6 μm, L/W ratio = 2.8. Appressoria single or in small groups, medium brown, outline mostly clavate or elliptical, rarely lobate, 7–14.5 × 5–9 μm, av ± SD = 11.2 ± 3.7 × 6.7 ± 2 μm, L/W ratio = 1.7.

Materials examined. CHINA, Henan Province, Xinyang, on Ca. sinensis, 23 Sept. 2012, M. Zhang & R. Zang (holotype HMAS 245381, culture ex-type CGMCC 3.17354 = LC3030 = LF238 = CSBX001); Beijing, Water Great Wall, on Cirsium japonicum, 2010, L. Cai, culture LC2820 = LF24; ibid., culture LC2821 = LF25.

Notes — The isolates of C. henanense isolated from tea plants and Cirsium japonicum formed a distinct clade that could be clearly distinguished from other species in the C. gloeo-sporioides species complex (Fig. 1). A BLASTn search of NCBI GenBank with the ITS sequence of CGMCC 3.17354 showed 99 % similarity to quite a number of sequences from isolates previously identified as C. gloeosporioides in other studies. The closest match in a BLASTn search in GenBank with the GAPDH sequence of CGMCC 3.17354 was GenBank JX009967 (99 % identity, 3 bp differences), the sequence generated from an authentic isolate of C. psidii CBS 145.29 (Weir et al. 2012), and with 98 % identity (5–6 bp differences) to some sequences of C. aotearoa, C. ti, and Glomerella cingulata ‘f. sp. camelliae’ isolates (Weir et al. 2012). The top 10 closest matches with the TUB2 sequence (with 97 % identity, 20–23 bp differences) were the isolates of C. aotearoa and C. kahawae subsp. ciggaro analysed in the study of Weir et al. (2012).

Colletotrichum jiangxiense F. Liu & L. Cai, sp. nov. — MycoBank MB809161; Fig. 13

Fig. 13.

Colletotrichum jiangxiense on PDA (CGMCC 3.17363). a, b. Conidiophores; c, d. conidia. — Scale bar: c = 10 μm, scale bar of c applies to a–d.

Etymology. Named after the collection site, Jiangxi Province, China.

On PDA: Colonies 50–53 mm diam in 7 d, > 90 mm diam in 10 d, flat with entire edge, aerial mycelium dense, cottony, white to grey, numerous small acervuli with orange conidial masses near the margin; reverse olivaceous with pale orange near the margin. Appressoria-like structures pale brown to brown, circular, ellipsoidal or irregular. Conidiomata acervular, conidiophores formed on a cushion of roundish and medium brown cells. Setae not observed. Conidiophores hyaline to pale brown, branched. Conidiogenous cells hyaline to pale brown, cylindrical, 11.5–20 μm, apex 1–2.5 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends bluntly rounded, or one end bluntly rounded and one end acutely rounded, 13–19 × 4–6 μm, av ± SD = 15.2 ± 1.0 × 5.2 ± 0.4 μm, L/W ratio = 2.9. Appressoria not observed.

Materials examined. CHINA, Jiangxi Province, Ganzhou, Fengshan Mountain, on Ca. sinensis, Sept. 2013, Y. Zhang (holotype HMAS 245382, culture ex-type CGMCC 3.17363 = LC3463 = LF687); ibid., culture CGMCC 3.17362 = LC3460 = LF684; Yangling National Forest Park, on Ca. sinensis, Apr. 2013, F. Liu, culture CGMCC 3.17361 = LC3266 = LF488.

Notes — Based on multi-locus sequence data (ACT, CAL, GAPDH, GS, ITS, TUB2), C. jiangxiense is phylogenetically closely related to the devastating coffee berry pathogen C. kahawae subsp. kahawae, and up to four other taxa, namely C. kahawae subsp. ciggaro, C. temperatum, C. fructivorum, and C. rhexiae (Fig. 1). All of the C. jiangxiense isolates differ from both C. kahawae subsp. kahawae and C. kahawae subsp. ciggaro by 1 bp change in CAL, 2 bp changes in ITS, and 17 bp changes and 1 bp indel in GS. Additionally, the 22 bp deletion in the GS sequence used to distinguish C. kahawae subsp. ciggaro from C. kahawae subsp. kahawae (Weir et al. 2012) is also lacking in the sequences of the C. jiangxiense isolates. Phylogenetic analyses based on single genes (except GS) could not clearly separate C. jiangxiense from the above listed species (results not shown). Comparisons of morphological and ecological characteristics were also made between these species. Conidia of the tea isolate (CGMCC 3.17363, av = 15.2 × 5.2 μm) are shorter than that of the ex-type culture (ICMP 18539, av = 17.8 × 5.1) of C. kahawae subsp. ciggaro. Colletotrichum kahawae subsp. kahawae is host-specific to Coffea and was confirmed causing no disease symptoms on Camellia sinensis by cross infection experiments (Fig. 6). In conclusion, the pathogenicity test, PHI test (Φ_w = 1) and phylogenetic analyses all suggested that C. jiangxiense is distinct from C. ka- hawae s.l.

The closest match in a BLASTn search with the ITS sequences of CGMCC 3.17363 was GenBank JN715848 (with 100 % iden-tity) from isolate R046 from a fruit of Rubus glaucus in Colombia, which was identified as C. kahawae subsp. ciggaro (Afanador-Kafuri et al. unpubl. data). Closest matches with the TUB2 sequence were GenBank KC297083 and KC297082 (with 100 % identity) from isolate CBS 115194 and CBS 112984 from Banksia sp., both of which are C. kahawae subsp. ciggaro (Liu et al. 2013b). The GAPDH blast result showed that the sequence of CGMCC 3.17363 was identical to those of the C. kahawae subsp. ciggaro isolates ICMP 18534 (GenBank JX009904) and ICMP 18544 (GenBank JX009920) (Weir et al. 2012), while CGMCC 3.17363 could be distinguished from ICMP 18534 in the multi-locus tree (Fig. 1).

Colletotrichum karstii Y.L. Yang et al., Cryptog. Mycol. 32: 241. 2011

Description and illustrations — See Yang et al. (2011) and Damm et al. (2012b).

Materials examined. CHINA, Hangzhou, on Ca. sinensis, Oct. 2013, F. Liu, culture CGMCC 3.17359 = LC3570 = LF798; on Ca. sinensis, Oct. 2013, F. Liu, culture LC3560 = LF788.

Notes — Colletotrichum karstii is a common and geographically diverse species, occurring on various host plants. It was previously reported to be pathogenic to Ca. sinensis in China (Liu 2013) and Camellia in Italy (Schena et al. 2013). Comparing it to the available TUB2 sequences from Camellia in Schena et al. (2013), 4 bp differences were detected between the Italian C. karstii and the Chinese isolates.

Colletotrichum siamense Prihast., L. Cai & K.D. Hyde, Fung. Diversity 39: 98. 2009 — Fig. 14

Fig. 14.

Colletotrichum siamense on PDA (CGMCC 3.17353). a. Acervulus; b, c, e. conidiophores; d. conidia; f–h. appressoria. — Scale bar: d = 10 μm, scale bar of d applies to b–h.

On PDA: Colonies 79 mm diam in 7 d, > 90 mm diam in 10 d, aerial mycelium white, cottony, sparse, surface of colony with numerous small acervuli with orange conidial ooze; reverse pale yellowish. Chlamydospores not observed. Conidiomata acervular, conidiophores formed on a cushion of roundish and medium brown cells. Setae not observed. Conidiophores hyaline, branched. Conidiogenous cells hyaline, cylindrical to ampulliform, 6.5–16 μm, apex 1–2 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends bluntly rounded, 12–15.5 × 4–5.5 μm, mean ± SD = 13.8 ± 0.9 × 4.7 ± 0.35 μm, L/W ratio = 2.9. Appressoria medium brown, aseptate, solitary, circular, clavate or ellipsoidal, 5.5–9.5 × 5–7.5 μm, mean ± SD = 7.5 ± 1.32 × 5.8 ± 0.7 μm, L/W ratio = 1.3.

Materials examined. CHINA, Sichuan Province, Chengdu Botanical Garden, on Ca. oleifera, Oct. 2012, F. Liu, culture LC2969 = LF177; on Camellia sp., Oct. 2012, F. Liu, culture LC2974 = LF182; ibid., culture CGMCC 3.17353 = LC2931 = LF139; Yunnan Province, Pu’er, on Camellia sp., 2010, D.M. Hu, culture LC0149 = PE007-2.

Notes — Conidiogenous cells of C. siamense were not well-illustrated in the original publication (Prihastuti et al. 2009), but are illustrated here based on our isolate from Camellia (Fig. 14). Colletotrichum melanocaulon was proposed as a novel species closely related to C. siamense based on the sequence data of ITS, TUB2, DNA lyase (APN2) and an intergenic spacer between the 3’ end of the DNA lyase and the mating type locus MAT1-2 (apn2mat/IGS) (Doyle et al. 2013). Since ACT, CAL, GAPDH and GS gene sequences of C. melanocaulon were unavailable, only ITS and TUB2 sequences of the ex-type culture (BPI 884101) were included in our genetic analysis. Another recently published new species C. dianesei (Lima et al. 2013), phylogenetically related to C. siamense, was also included in the study. The multi-locus phylogenetic analysis result showed that both C. melanocaulon and C. dianesei clustered together with the ex-type isolate of C. siamense (CBS 18578), and its synonyms C. murrayae (GZAAS 5.09506), C. jasmini-sambac (CBS 130420) and C. hymenocallidis (CBS 125378) (Fig. 1). As the ex-type of these species and isolates from tea plants formed a robust clade with high posterior probability (1, Fig. 1, and 0.96, Fig. 3), we suspect C. melanocaulon and C. dianesei to be synonyms of C. siamense. Further studies are needed to confirm if these taxa are synonymous, or if C. siamense is a species complex (Sharma et al. 2013a).

DISCUSSION

Colletotrichum species on Camellia

In this study, pathogenic and endophytic Colletotrichum isolates associated with Ca. sinensis and other Camellia spp. were allocated to different species complexes and further assigned to 11 species, including nine known and two new species. Furthermore, this study also represents the first report of C. alie-num, C. cliviae, C. jiangxiense, and C. henanense from tea plants. Six species were isolated from both symptomatic and asymptomatic leaves tissues, namely C. camelliae, C. fructicola, C. gloeosporioides, C. jiangxiense, C. karstii, and C. sia-mense. This indicates that they could switch their lifestyle from endophytic to plant pathogenic in nature, and provides additional support for the hypothesis that endophytes can be latent pathogens (Photita et al. 2001, Romero et al. 2001). Some Colletotrichum species were collected only once from this host; C. fioriniae and C. henanense were obtained from symptomatic tea leaves, while C. alienum, C. boninense and C. cliviae were only encountered as endophytes in tea plants. Previous pathogenicity tests showed that C. fructicola isolates from symptomless tissues could cause disease on Citrus fruits (Huang et al. 2013). Consequently, we hypothesise that endophytic species in Camellia could also be potential latent pathogens. Further investigations are therefore required to clarify the ecological relationships of the pathogenic and endophytic Colletotrichum species on Camellia.

Based on this study, C. camelliae is the dominant Colletotrichum species on Camellia in China and is probably host-specific to Camellia. These findings make C. camelliae an appropriate model for addressing questions of population structure and dispersal at broad geographical and landscape level. Knowledge of molecular demographic parameters, such as rates of gene flow, levels of species divergence and migration patterns between populations will elucidate the biogeographic history, and the evolutionary and adaptive mechanisms. Information on the genetic structure of the populations can also assist in the development of disease management strategies (Rampersad et al. 2013). Additional collections from Camellia growing regions across the world would therefore aid us to characterise the population structure of this important pathogen and to confirm whether this species is indeed the dominant Colletotrichum species globally.

Colletotrichum acutatum and C. gloeosporioides were previously reported as the dominant endophytic species in Camellia based on morphological characteristics or ITS sequence data (Osono 2008, Fang et al. 2013). However, we did not isolate any C. acutatum s.str. isolates in our study, and only a single isolate of C. fioriniae, belonging to the C. acutatum species complex, was obtained from symptomatic tissue. In addition, although the majority of strains from Camellia in this study belong to the C. gloeosporioides species complex, only four of them are C. gloeosporioides s.str., including three pathogenic and one endophytic isolates. This indicates that many of the previous identifications of Colletotrichum species on Camellia were probably incorrect.

Apart from the Colletotrichum species found in this study, Camellia spp. could also be infected or colonised by a few other species, i.e. C. lupini (Damm et al. 2012a), C. acutatum, C. car-veri, C. coccodes, and C. queenslandicum (syn. C. gloeosporioides var. minor, Weir et al. 2012) (Farr & Rossman 2014). These reports (except C. lupini), however, need to be verified based on the presently accepted classification system in Colletotrichum.

Combined use of ApMat and GS in the C. gloeosporioides species complex

The Apn2-Mat1 locus was introduced for differentiation of Colletotrichum species in the C. graminicola species complex by Crouch et al. (2009), while Rojas et al. (2010) applied it to the C. gloeosporioides species complex. Following this, a new marker in the intergenic region of APN2 and MAT1-2-1 was specifically designed to improve the systematics of the C. gloeosporioides species complex (Silva et al. 2012b), and the locus was renamed as ApMat, which has subsequently been used in molecular phylogenetic analyses of this group (Sharma et al. 2013a, 2014, Vieira et al. 2014).

In the study of Silva et al. (2012a), the ApMat locus proved to be the most informative marker compared to other standard markers, and could resolve species in the C. gloeosporioides species complex and provide a similar amount of information and support as the concatenated tree based on seven loci (ApMat, Apn25L, MAT5L, MAT1-2-1, ITS, β-tub2, GS). However, it is noteworthy that the sample size in their study was rather limited, including only 22 isolates belonging to six divergent species from Coffea. Subsequently, the ApMat marker was employed to analyse species in the C. gloeosporioides complex that are associated with Mangifera indica using a larger sample size, in which 39 Colletotrichum isolates were separated into nine lineages, namely C. fragariae, C. fructicola, C. jasmini-sambac, C. melanocaulon and five unnamed lineages (Sharma et al. 2013a). In that study, only 15 of the Colletotrichum isolates used in the ApMat gene analysis were also included in a multi-locus phylogenetic tree (ACT, CAL, CHS, GAPDH, ITS, TUB2) where they were separated into four clades corresponding to C. theobromicola, C. asianum, C. siamense and C. fructicola. However, no comparison was made between the results of the single-locus ApMat and the multi-locus phylogenetic analysis.

In order to determine if the ApMat sequences provide adequate phylogenetic information compared to that of a multi-locus dataset, we constructed both single-locus ApMat and combined 6-marker (ACT, CAL, GAPDH, GS, ITS, TUB2) trees using the same Colletotrichum isolates associated with Camellia collected in this study. All ApMat reference sequences used in Sharma et al. (2013a) were incorporated in our ApMat analysis, except for GenBank KC888927 from C. alienum isolate ICMP 12071 (incorrect sequence deposited by the original author). The ApMat sequence of isolate ICMP 12071 was re-sequenced and submitted to GenBank as GenBank KM360144 in this study. Our study demonstrated that 22 species (C. aenigma, C. aeschynomenes, C. alatae, C. alienum, C. asianum, C. aotearoa, C. camelliae, C. clidemiae, C. cordylinicola, C. fructicola, C. gloeosporioides, C. henanense, C. horii, C. musae, C. nupharicola, C. psidii, C. queenslandicum, C. salsolae, C. siamense, C. theobromicola, C. ti, and C. tropicale) could be clearly delimitated with ApMat (Fig. 3 and Fig. S2 in TreeBASE). Although C. fructivorum, C. jiangxiense, C. kahawae subsp. kahawae, C. rhexiae, and C. temperatum clustered together in one big clade, the species C. fructivorum, C. rhexiae, and C. temperatum could be delimitated by forming three small subclades with high posterior probabilities (Fig. S2 in TreeBASE). However, C. jiangxiense and C. kahawae subsp. kahawae could not be distinguished from each other. Furthermore, isolates of C. camelliae were separated into two subclades (Fig. 3 and Fig. S2 in TreeBASE). Although C. jiangxiense could be distinguished from C. kahawae s.l. by the GS marker, the other species in the C. gloeosporioides species complex could not be delimited very well, e.g. C. camelliae, C. fructicola, C. siamense, and C. queenslandicum (data not shown). This study demonstrates that the ApMat marker provides superior phylogenetic information compared to other used loci and can distinguish most species in the C. gloeo-sporioides species complex. A further phylogenetic analysis using the concatenated ApMat and GS alignment showed that all species could be delimited, including C. jiangxiense and C. kahawae subsp. kahawae. We therefore recommend a combination of ApMat and GS as an effective way of identifying species in the C. gloeosporioides species complex.

In the present study we mainly focused on the taxonomy and biodiversity of Colletotrichum species associated with tea plants in China as plant pathogens and/or endophytes. Further attention should be given to surveys from different geographical regions to help resolve the life cycles and ecology of these species, especially of C. camelliae. Because of the important commercial value of tea plantations, appropriate disease management strategies in tea plantations should also be developed to control infection by Colletotrichum species.