Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia.

Anthracnose of chili (Capsicum spp.) causes major production losses throughout Asia where chili plants are grown. A total of 260 Colletotrichum isolates, associated with necrotic lesions of chili leaves and fruit were collected from chili producing areas of Indonesia, Malaysia, Sri Lanka, Thailand and Taiwan. Colletotrichum truncatum was the most commonly isolated species from infected chili fruit and was readily identified by its falcate spores and abundant setae in the necrotic lesions. The other isolates consisted of straight conidia (cylindrical and fusiform) which were difficult to differentiate to species based on morphological characters. Taxonomic analysis of these straight conidia isolates based on multi-gene phylogenetic analyses (ITS, gapdh, chs-1, act, tub2, his3, ApMat, gs) revealed a further seven known Colletotrichum species, C. endophyticum, C. fructicola, C. karsti, C. plurivorum, C. scovillei, C. siamense and C. tropicale. In addition, three novel species are also described as C. javanense, C. makassarense and C. tainanense, associated with anthracnose of chili fruit in West Java (Indonesia); Makassar, South Sulawesi (Indonesia); and Tainan (Taiwan), respectively. Colletotrichum siamense is reported for the first time causing anthracnose of Capsicum annuum in Indonesia and Sri Lanka. This is also the first report of C. fructicola causing anthracnose of chili in Taiwan and Thailand and C. plurivorum in Malaysia and Thailand. Of the species with straight conidia, C. scovillei (acutatum complex), was the most prevalent throughout the surveyed countries, except for Sri Lanka from where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was also common in Indonesia, Sri Lanka and Thailand. Pathogenicity tests on chili fruit showed that C. javanense and C. scovillei were highly aggressive, especially when inoculated on non-wounded fruit, compared to all other species. The existence of new, highly aggressive exotic species, such as C. javanense, poses a biosecurity risk to production in countries which do not have adequate quarantine regulations to restrict the entry of exotic pathogens.


INTRODUCTION
Colletotrichum is one of the most important genera of plant pathogenic fungi with many of the 200 plus species known to cause disease in plant crops worldwide Marin-Felix et al. 2017). Colletotrichum species causing anthracnose are particularly important as post-harvest pathogens of fruit and vegetable crops growing in tropical and subtropical climates (Alahakoon et al. 1994;Rojas et al. 2010;Cannon et al. 2012;Damm et al. 2012aDamm et al. , 2012bUdayanga et al. 2013;Shivas et al. 2016;De Silva et al. 2017a).
Chili (Capsicum spp.) is an important vegetable crop in tropical and subtropical climates and the fresh or dried fruit is a major culinary ingredient in many cuisines. Anthracnose is a major disease of chili fruit causing significant yield loss as well as reducing the marketability of the fruit. Anthracnose of chili has been shown to be caused by 24 Colletotrichum species (Mongkolporn and Taylor 2018) reported from many countries including Australia (De Silva et al. 2017a), Brazil (de Oliveira et al. 2017, China (Diao et al. 2017), India , Indonesia (Voorrips et al. 2004), Korea (Kim et al. 1999), Malaysia (Noor and Zakaria 2018), Sri Lanka (Ranathunge et al. 2009), Thailand (Than et al. 2008) and the USA (Harp et al. 2008).
Colletotrichum species causing anthracnose of chili in Asia were previously identified as C. acutatum (straight conidia with acute ends), C. gloeosporioides (straight conidia with obtuse ends) and C. truncatum (falcate conidia) (Than et al. 2008, Mongkolporn et al. 2010. However, with the implementation of multigene phylogenetic analyses, C. acutatum was demonstrated to be a species complex (acutatum complex) composed of 34 closely related species (Marin-Felix et al. 2017), with seven species identified as causing anthracnose in chili (Mongkolporn and Taylor 2018). Similarly, C. gloeosporioides was shown to be a species complex of 38 closely related species (Marin-Felix et al. 2017), with nine species identified to cause anthracnose in chili (Mongkolporn and Taylor 2018). Morphological characters cannot differentiate many of the species with straight conidia, especially those in the acutatum and gloeosporioides complexes that are pathogens of chili, and thus multigene phylogenetic analyses are required for proper identification of these species (De Silva et al. 2017a).
The distribution of the Colletotrichum species that cause anthracnose of chili is quite variable across countries that produce this crop. For example, in Australia only six out of the 24 Colletotrichum pathogens of chili have been identified (De Silva et al. 2017a), in Thailand only three have been reported (Mongkolporn and Taylor 2018), and five from Malaysia (Noor and Zakaria 2018). In most of the previous reports the identification of species was based only on morphological data. Therefore, the status of the taxonomy of Colletotrichum spp. causing anthracnose in chili producing countries in Asia remains uncertain. Proper identification of these pathogens is important for mitigating the risk of incursion of new pathogens which if happens, may have devastating consequences for the local industries. In addition, accurate identification of the species is important for resistance breeding programs and in identifying the host-range of species. Several Colletotrichum species such as C. karsti, C. siamense and C. truncatum have broad host ranges (Cannon et al. 2012;Damm et al. 2012b). The main Colletotrichum species causing anthracnose in chili are known to be in the acutatum and gloeosporioides complexes. However, recently further species from the boninense and orchidearum complexes were implicated (Diao et al. 2017;Damm et al. 2019). Therefore, it is important to understand the taxonomy, diversity and pathogenicity of Colletotrichum species that infect chili and their distribution across countries.
The aims of the study were to (1) identify the Colletotrichum species with straight conidia (cylindrical and fusiform) causing anthracnose of chili in selected regions of Indonesia, Malaysia, Taiwan, Thailand and Sri Lanka, and (2) determine the pathogenicity of those species on chili.

Isolates
A total of 260 isolates associated with anthracnose disease symptoms on chili fruit and leaves were collected from chili producing countries in Asia: Indonesia, Malaysia, Taiwan, Thailand, and Sri Lanka (Table 1). Type specimens and ex-type cultures are deposited in the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (CBS), and in the University of Melbourne culture collection (UOM), Victoria, Australia. Fungal isolates were established from lesions on infected fruit and leaves using two methods. Surface sterilised (~1% ai sodium hypochlorite for 5 min) infected tissue (0.5 cm 2 ) was cultured on water agar (WA; Crous et al. 2009) and then after 2 to 3 d fungal hyphae were subcultured onto potato dextrose agar (PDA, Difco) and synthetic nutrient-poor agar (SNA, Nirenberg 1976) as described by De Silva et al. (2017a). Freshly collected fruit from field grown chili plants with typical anthracnose lesions was incubated for 1 to 2 d in a moist chamber until conidiomata appeared and then single spore isolation was performed according to Choi et al. (1999). Selected isolates were also cultured on oatmeal agar (OA; Crous et al. 2009) and malt extract agar (MEA) at 20°C under near UV light with a 12 h photoperiod for 10 d. Cultures were isolated and maintained either at the AQIS quarantine laboratory at the University of Melbourne or the Evolutionary Pathology Laboratory at the Westerdijk Fungal Biodiversity Institute, Netherlands (CBS).

Morphology
Cultures grown on PDA at 27°C were used for morphological analysis. Colony colour and texture were examined after 10 d, and colony growth rate calculated by measuring colony diameter 7 and 10 d after incubation.
Conidia from the conidiomata in culture were mounted in lactic acid and the length and width measured for 30 randomly selected conidia for each isolate, with the range and mean calculated. Size and shape of appressoria were determined on WA using a slide culture technique (Johnston and Jones 1997). Production of acervular conidiomata was observed on dried, sterilised chili peduncles inoculated with mycelia and incubated on WA and SNA media. Cultures were examined periodically for the development of perithecia. Ascospores were measured and described from perithecia squashed in lactic acid. Morphological characters were examined using a Leica DM6000 LED compound microscope with differential interference contrast (DIC) optics.

DNA extraction, PCR amplification and sequencing
The 260 Colletotrichum isolates were initially identified on the basis of culture characteristics on PDA (based on distinct morphotype groups), morphology of the spores, and/or internal transcribed spacer and intervening 5.8S nrDNA gene (ITS) sequence. A total of 115 isolates were identified as C. truncatum and the remaining 145 isolates with straight conidia were subsequently selected for multigene phylogenetic analyses. Genomic DNA was extracted from fresh mycelia grown on PDA using the DNeasy Plant Mini kit (QIAGEN, Australia), following the manufacturer's instructions. DNA quality was assessed on a 1.4% (w/v) agarose gel, quantified by comparing with a known amount of Lambda DNA/HindIII marker (Invitrogen, Australia), diluted to 2 ng/μL and then stored at _ 20°C until ready for PCR.
The PCR for each reaction was performed in a 2720 Thermal Cycler (Applied Biosystems) in a total volume of 25 μL, comprised of 1× PCR buffer, 0.2 mM dNTP, 0.4 μM of each primer, 2 mM MgCl 2 , 1 U Taq DNA polymerase (MangoTaq DNA polymerase; Bioline) and 6 ng template DNA and components were adjusted as required. PCR amplification protocols were performed as described by Damm et al. (2012aDamm et al. ( , 2012b and Silva et al. (2012), except for the annealing temperatures which were adjusted to 55°C for ITS, gapdh, tub2, 58°C for act, 60°C for gs and 62°C for ApMat.
All PCR products were purified with the QIAquick PCR Purification kit (QIAGEN, Australia), according to manufacturer's instructions. DNA sequence analysis of the PCR products was carried out at either the Australian Genome Research Facility (AGRF, Melbourne) or at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. The purified PCR products were sequenced in both forward and reverse directions, and the consensus sequences were obtained by alignment using Geneious Pro v. 11.1.4 (Kearse et al. 2012). The consensus sequences were deposited in GenBank (Table 2) and taxonomic novelties in MycoBank (Crous et al. 2004b). Sequences of each locus were assembled with MEGA v. 6 (Tamura et al. 2013). GenBank accession numbers of all the isolates used in the phylogenetic analyses are listed in Table 2.         C. siamense

Phylogenetic analyses
Gene sequences of each isolate were examined using Geneious Pro v. 11.1.4, aligned by CLUSTALW2 (Larkin et al. 2007) and edited manually where necessary. ITS and tub2 sequences of selected isolates representing all the species complexes were analysed to determine to which clade each isolate belonged, and an initial phylogenetic tree was produced with a maximum likelihood analysis (ML) as implemented in MEGA v. 6 with 1000 bootstrap replicates (data not shown). For isolates from the acutatum complex, concatenated datasets were generated comprising ITS, chs-1, act, gapdh, his3 and tub2 gene sequences. For isolates from the gloeosporioides complex, two concatenated datasets were generated comprising ITS, chs-1, act, gapdh and tub2 gene sequences, and comprising ApMat and gs gene sequences. For isolates from the boninense and orchidearum complexes concatenated datasets were generated comprising ITS, gapdh, act and tub2 gene sequences. Selected reference or ex-type strains from each complex (Table 2) were included in the analyses (Damm et al. 2012b(Damm et al. , 2019Marin-Felix et al. 2017;Weir et al. 2012). Further phylogenetic analyses were performed using MrBayes v. 3.2.6 (Ronquist et al. 2012) for Bayesian inference analyses (BI), and PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003) for parsimony analyses. For BI analyses, the best nucleotide substitution model for each locus was determined by MrModeltest v. 2.3 (Nylander 2004) (Table 3), and eight simultaneous MCMC chains were run for 1 bn generations. Trees were sampled every 100 generations for the acutatum, boninense and orchidearum complexes, and every 1000 generations for the gloeosporioides complex 2-gene alignment and every 10 generations for the gloeosporioides complex 5-gene alignment. The heating parameter was set to 0.2 and analyses stopped once the average standard deviation of split frequencies was below 0.01. The first 25% of trees, representing the burn-in phase of the analyses, were discarded and the remaining trees in each analysis were used to calculate posterior probabilities. The generated 50% majority rule consensus tree was viewed in TreeView v. 1.6.6 (Page 1996). A maximum parsimony (MP) analysis was performed on the multilocus alignments as well as for each gene separately with PAUP v. 4.0b10 (Swofford 2003) using the heuristic search option with 100 random sequence additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Gaps were treated as new character states and missing data as missing characters. Bootstrap support values were calculated based on 1000 bootstrap replicates. Statistical measures calculated included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) ( Table  3). Alignments and tree files are deposited in TreeBASE (accession https://www.treebase.org/treebase-web/home. html; study S23829).

Pathogenicity assay
Pathogenicity tests on chili fruit were conducted using only Colletotrichum isolates with straight conidia as previous studies had extensively studied the pathogenicity of C. truncatum in chili (Mongkolporn et al. 2010, Ranathunge et al. 2012). There were 15 representative isolates of C. scovillei from Indonesia, Thailand and Taiwan, 10 isolates of C. siamense from Indonesia and Thailand, and one isolate each from the other eight species with straight conidia. Detached mature red chili fruits (Capsicum annuum genotype Bangchang) were used for the pathogenicity assay as described by De Silva et al. (2017a). Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. Three replicate fruits were tested per isolate while experiments were carried out three times.
Data were analysed using the Mixed Procedure in SAS v. 9.4 by fitting the linear mixed model: where μ is the grand mean, S i is the fixed species effect, and R k , R k *S i , R k *I j (S i ) and e ijkl are respectively the random effects of replicate, replicate by species interaction, replicate by isolate within species interaction, and error. Separate analyses were done for wound and non-wound data as preliminary analysis showed there was significant species by wound interaction. Least squared means were estimated for each species and t-test carried out between each pair of means.

Isolates
The Colletotrichum isolates with falcate conidia and ITS sequences matching to those of the ex-type of C. truncatum were the most common (n = 115), representing 44% of all isolates. Colletotrichum truncatum was found in the collections from Indonesia, Malaysia, Sri Lanka and Thailand (Fig. 6). Colletotrichum truncatum isolates were not included in the collection from the World Vegetable Center in Taiwan as only the species with straight conidia were selected for identification. The remaining 56% of isolates (n = 145) were of species with straight conidia that mostly belonged to the acutatum and gloeosporioides complexes.

Phylogenetic analyses Acutatum complex
For the 69 isolates and 29 reference species in the acutatum complex, the phylogenetic analysis of the combined data sets using six genes (ITS, tub2, gapdh, chs-1, act   (Fig. 1). Overall, the species clades recognised received similar support values, although the association between species did not always receive similar support, e.g. the node linking C. paranaense and C. melonis (MP < 50% / PP = 0.99). The phylogenetic analyses of the acutatum complex identified C. scovillei as the most prevalent species in Indonesia, Malaysia, Thailand and Taiwan. However, C. scovillei was not isolated from Sri  Lanka. In addition, an isolate from Java in Indonesia (UOM 1115) clustered related to C. brisbanense (96% BS/ 1 PP; Fig. 1).

Boninense complex
For the four isolates and 10 reference species in the boninense complex the phylogenetic analyses of the combined data sets using five genes (ITS, gapdh, tub2, act and chs-1) with C. truncatum (CBS 151.35) as the outgroup comprised 24 isolates and 1867 characters including the alignment gaps and excluded characters (Fig. 2). The Bayesian analysis of the combined alignment, based on 432 unique site patterns (ITS: 42, gapdh: 130, tub2: 111, act: 94 and chs-1: 55) lasted 80,000 generations, resulting in 16,002 total trees of which 12,002 trees were used to calculate the posterior probabilities. The parsimony analysis yielded three equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 2). Overall, the nodes received similar support values, except for the subclustering of strains CBS 128545, CBS 128548 and CBS 129927 in the C. karsti clade (MP 67% / PP = 0.98).The phylogenetic analyses of the boninense complex identified the most prevalent species as C. karsti occurring only in Indonesia.

Gloeosporioides complex
For the 42 isolates and the 41 reference species in the gloeosporioides complex, two phylogenetic trees were constructed, one from the ApMat and gs sequence alignment and the second from the ITS, gapdh, act, tub2, chs-1 sequence alignment (Figs. 3 and 4). The analyses using the 5-gene alignment with C. theobromicola CBS 124945 as the outgroup (Fig. 4) (Fig. 4). Overall, the species clades recognised in this study received similar support values, except for the C. siamense clade (MP < 50% / PP < 0.91) and the C. fructicola clade (MP 57% / PP = 0.99).
The analysis using the ApMat and gs sequence alignment comprised of 92 isolates with C. theobromicola CBS 124945 as the outgroup (Fig. 3) and 1824 characters including the alignment gaps and excluded characters. The Bayesian analysis of the combined alignment, based on 952 unique site patterns (ApMat: 520, gs: 432) lasted 29,500,000 generations, resulting in 590,002 total trees, of which 442,502 trees were used to calculate the posterior probabilities. The parsimony analysis yielded 161 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 3). Overall, the species clades recognised in this study received similar support values, except for the C. siamense clade (MP = 92% / PP < 0.91).
Phylogenetic analyses of the gloeosporioides species complex identified 69% (29) of the chili fruit isolates as C. siamense. In the 2-gene tree a distinct subclade within the C. siamense clade formed with 100% bootstrap support which contained isolates from Chiang Mai in Thailand, and Gowa and Jeneponto in South Sulawesi of Indonesia (Fig. 3). These isolates from Indonesia and Thailand had very distinct sequences compared to the ex-type reference C. siamense strain (CBS 130417) with 28 bp difference in the gs gene and 25 bp difference in the ApMat gene. A significant sub-clade formed within C. siamense with full (100% BS/1 PP) support values in the 2-gene tree. The same isolates in the 5-gene tree did not show the same level of difference but showed a strong similarity between the C. siamense isolates. In the 2-gene tree there were also significant subclades of isolates associated with different geographical regions, in  Other species identified in the gloeosporioides complex included C. endophyticum and C. fructicola from Thailand, C. fructicola and C. tainanense sp. nov. from Taiwan, and C. tropicale and C. makassarense sp. nov. from Indonesia. Most of the identified species including the two new species were supported in distinct clades with significant bootstrap values in both the 5-gene and 2-gene trees (Figs. 3 and 4). However, due to a lack of sequence data of the ApMat gene for some reference strains, it was difficult to provide a good support for placement of some species such as C. endophyticum in the 2-gene trees. Three isolates (CPC 28607, UOM 1002, UOM 1003) collected from the Makassar region in Indonesia showed a close relationship to the reference species C. tropicale in the ApMat and gs tree (Fig. 3). Individual gene trees of ITS, act, tub2, chs-1 loci (data not shown) also supported these isolates as C. tropicale. Nevertheless, in the 5-gene tree a separate sub clade was formed with full support (100% BS/1 PP) different to the C. tropicale reference species (Fig. 4). In both trees, two isolates (UOM 1120, UOM 1119) collected from Tainan in Taiwan formed a significant distinct clade with full support (100% BS/1 PP) separate from C. salsolae.

Orchidearum complex
For the six isolates and nine reference species in the orchidearum complex the phylogenetic analysis of the combined data sets using four genes (ITS, gapdh, tub2 and act) with C dracaenophilum (CBS 118199) as the outgroup comprised 26 isolates and 1543 characters including the alignment gaps and excluded characters. The Bayesian analysis of the combined alignment, based on 221 unique site patterns (ITS: 34, gapdh: 55, act: 42, tub2: 90) lasted 275,000 generations, resulting in 5502 total trees of which 4128 trees were used to calculate the posterior probabilities. The parsimony analysis yielded 284 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 5). Overall, the species clades recognised in this study received similar support values, except for the C. plurivorum clade (MP = 64% / PP < 1) and the C. cliviicola clade (MP = 87% / PP < 1).  The phylogenetic analyses identified the isolates from Thailand and Malaysia as C. plurivorum. Five isolates collected from Thailand, including three taken from infected chili leaves from Chiang Rai and another two isolates collected from infected green chili fruit from Bangkok, formed a poorly supported subclade within C. plurivorum (Fig. 5).
Culture characteristics: Colonies on PDA 48-54 mm diam in 7 d (6.5-7.5 mm/d), flat with entire margin; surface covered with grey to olive-green short aerial mycelium, margin white to light grey, reverse mostly cream whitish, olivaceous grey to black in the centre. Yellow to orange acervular conidiomata. Colonies on SNA were 60-66 mm diam in 7 d (8-9.5 mm/d), flat with entire margin, hyaline to pale brown, surface covered with short grey aerial mycelium, reverse same colours. Orange acervular conidiomata at the centre of the culture. Colonies on OA were 55-60 mm diam in 7 d (7.8-8.5 mm/d), flat with entire margin; surface covered with cream to grey short aerial mycelium, margin white, reverse mostly light orange, with brown pigments. Orange acervular conidiomata. Colonies on MEA surface pale grey short aerial mycelium, reverse light orange.
Notes: The closest match in a blastn search with the gapdh sequence was GenBank JQ948617, C. sloanei strain IMI 364297 with 98% identity (4 bp differences), while the closest matches with the his3 sequence with 99% identity (2 bp differences) were GenBank JQ949279 C. indonesiense strain CBS 127551 and GenBank KJ947248 C. guajavae isolate OBP19.  Diagnosis: Colletotrichum makassarense is phylogenetically closely related to C. tropicale. Sequence data from ITS could not separate the two species, but they can be distinguished by all other genes tested and most effectively using ApMat (22 bp differences) and gs (18 bp differences) sequence data. Description: Sexual morph not observed. Asexual morph on OA. Vegetative mycelium consisting of hyaline, smooth-walled, septate, branched hyphae, 2-3 μm diam.
Culture characteristics: Colonies on PDA 45 mm diam after 7 d (6.5 mm/d), colonies flat, with moderate aerial mycelium, on OA surface smoke-grey. On PDA surface smoke-grey, reverse olivaceous grey. On MEA surface dirty white, reverse ochreous.
Culture characteristics: Colonies on PDA 45 mm diam after 7 d (6.5 mm/d), colonies flat, with moderate aerial mycelium. On OA surface pale mouse-grey. On PDA surface pale mouse-grey, reverse mouse-grey. On MEA surface pale mouse-grey, reverse olivaceous grey.
Notes: The closest match in a blastn search with the gapdh sequence with 99% identity (2 bp difference) was GenBank KC790761 Colletotrichum sp. strain MTCC 9664 while the closest match with the act sequence with 99% identity (2 bp difference) was GenBank KY995522 C. siamense strain LJDY1-2. The closest match with the tub2 sequence with 99% identity (7 bp difference) was GenBank MF143931 C. siamense strain 31-B-1.
Decription: Colonies on PDA 20-38 mm diam after 7 d (5-6.2 mm/d), flat with entire margin; surface covered with short light pink to orange aerial mycelium, turn grey with time, margin whitish to pale pink, reverse rosy buff, olivaceous grey to brown-grey in the centre; reverse orange to salmon, dark at the centre. Chlamydospores not observed. Conidiomata acervular, setae not observed, Conidiophores hyaline, septate, branched.
In Indonesia, C. scovillei was isolated from infected chili fruit in all surveyed regencies of South Sulawesi, except in Makassar, and in the field trial site in West Pairwise t-tests between species least square means, significant differences at alpha = 0.05 level are indicated by different letters Fig. 9 Prevalence of Colletotrichum species as a percentage of the total isolates collected in all regions and in Indonesia and Thailand specifically Java. Colletotrichum siamense was also isolated from throughout the region, from Gowa, Jeneponto and Makassar. The two new species, C. makassarense and C. javanense were isolated from Makassar and West Java, respectively.
In Thailand, C. scovillei was isolated mostly from the northern provinces of Chiang Mai and Chiang Rai, but was also obtained from infected chili fruit in a trial site of Kasetsart University in Nakhon Pathom. The Western provinces had a high incidence of C. siamense and one isolate of C. endophyticum was collected from Kanchana Buri. Colletotrichum plurivorum was isolated from chili leaves with necrotic lesions in Chiang Rai and from necrotic lesions of chili fruit found in a restaurant in Phaya Thai area of Bangkok. In addition, C. fructicola was identified from fruit collected from Chiang Mai.
In Taiwan, an isolate was identified as a new species, C. tainanense, collected from infected chili fruit in the Tainan province, and C. fructicola was identified from fruit in Hsinchu and Nantou, Taiwan. Colletotrichum plurivorum was also identified from a fruit collected in Johor, Malaysia.

Pathogenicity
All the Colletotrichum species caused anthracnose symptoms on wounded fruit, but there were significant differences in severity of the symptoms. On wounded fruit, isolates of C. scovillei and C. javanense showed the highest disease severity, producing large, necrotic lesions with mean lesion sizes 15.6-20.3 mm (Table 5). On non-wounded fruits, all species were less pathogenic, with mean lesion sizes less than 10 mm. Colletotrichum makassarense, C. tropicale and C. plurivorum, produced only very small lesions or no visible symptoms (mean lesion size < 1 mm) 10 d after inoculation of non-wounded fruits. Colletotrichum javanense and C. scovillei isolates were the most pathogenic in non-wounded fruit and produced lesions with mean sizes of 9.4 and 9.1 mm respectively. Lesions caused by C. scovillei were significantly larger than those caused by all other species except for C. javanense in wounded fruit and C. endophyticum in unwounded fruit. Significance of the pairwise t-tests is strongly dependent on the number of isolates of each species sampled, so the groupings shown in Table 5 do not simply change with the magnitude of the difference of the means; a large difference may not be significant if there are small sample sizes for both species while a smaller difference may be significant. In particular, comparisons involving C. javanense and C. endophyticum are very imprecise as only one isolate of each species was tested.

DISCUSSION
Colletotrichum isolates collected from infected fruit and leaf tissue of chili plants from Thailand, Indonesia, Taiwan, Sri Lanka, and Malaysia were allocated to different species complexes with 11 Colletotrichum species being identified and three new species described.
Colletotrichum truncatum was the most prevalent species of Colletotrichum causing anthracnose of chili in Asia, which supports previous reports of C. truncatum being widely distributed on chili fruit throughout Asia, Australia, and South America De Silva et al. 2017a;Diao et al. 2017;Mongkolporn and Taylor 2018). Colletotrichum truncatum has a broad host range infecting many crop species (https://nt.arsgrin.gov/fungaldatabases/; Ranathunge and Hewa Bajjamage 2016).
The remaining 56% of isolates with straight conidia belonged to the gloeosporioides, acutatum, boninense and orchidearum species complexes. Of these, C. scovillei, in the acutatum complex, was the most prominent species (35% of the total isolates) and was distributed in Indonesia, Malaysia, Thailand and Taiwan. However, C. scovillei was not identified in the survey of Sri Lanka, possibly because of the small number of samples assessed. Previous studies have also reported the predominance of C. scovillei in Asia and Brazil (Diao et al. 2017, Mongkolporn andTaylor 2018), but this species has not been identified in Australia (De Silva et al. 2017a) and hence remains an important biosecurity threat to the Australian capsicum/chili industry. Further surveys are required to confirm the presence of C. scovillei in Australia and Sri Lanka.
Colletotrichum siamense, in the gloeosporioides complex, was also prominent (11% of total isolates) in causing anthracnose of chili fruit in Indonesia, Sri Lanka and Thailand, but was not detected in Malaysia and Taiwan. Nevertheless, Noor and Zakaria (2018) reported the identification of C. siamense in Malaysia possibly due to a larger sampling size of infected chili across states in Malaysia. Within the C. siamense clade, small subclades were observed that coincided with different geographical regions from where the isolates were collected. Colletotrichum siamense has been reported to infect chili in Asia, Australia, Brazil, and is a common pathogen of many other plant species James et al. 2014;Liu et al. 2016aLiu et al. , 2016bde Oliveira et al. 2017;De Silva et al. 2017a;Diao et al. 2017;Suwannarat et al. 2017).
Colletotrichum siamense isolates from different countries appeared to show different morphological characters with varying growth rates and culture morphologies. This variability in morphological characters indicated that this taxon has high intra-specific diversity. The combined gene analyses of ApMat and gs sequences also supported the distinction of subclades within the C. siamense clade. In the last few years, there has been significant debate on whether C. siamense s. lat. should be separated into different species groups within the gloeosporioides complex, with the number of accepted species ranging from one to seven Udayanga et al. 2013). However, a recent case study concluded that C. siamense s. lat. was a single species rather than a species complex as no independent evolutionary lineages were found within this species (Liu et al. 2016a).
Colletotrichum tropicale is reported for the first time as causing anthracnose in Capsicum in Indonesia. Rojas et al. (2010) noted that C. tropicale was initially isolated from a wide range of hosts in forests in tropical America, from rotting fruit and as a leaf endophyte. Silva et al. (2017) recently reported C. tropicale causing chili anthracnose in Brazil. Colletotrichum tropicale was also reported from Japan, Panama, Thailand, and from other host species (Mongkolporn and Taylor 2018).
Colletotrichum fructicola is reported for the first time causing chili anthracnose in Thailand and Taiwan. Colletotrichum fructicola was previously reported to cause anthracnose in chili from India and China Diao et al. 2017). Prihastuti et al. (2009) originally isolated C. fructicola from coffee berries in Thailand, and then C. fructicola was reported as a leaf endophyte from several plants in South America Vieira et al. 2014) Colletotrichum fructicola has a wide host range and was reported by Weir et al. (2012) as a biologically and geographically diverse species.
All the species in the gloeosporioides complex were identified using combined multi-locus gene analyses, based on the ITS, gapdh, chs-1, act and tub2 genes, which showed higher diversity on chili than those in the acutatum, boninense and orchidearum species complexes. Phylogenetic tree provided good resolution of the species with high support values, which supported the species boundaries and identified the novel species.
Phylogenetic trees built from ApMat and gs gene sequences had similar topologies to the multigene phylogenetic tree confirming that the ApMat and gs loci were highly informative and that they distinguished most species in the gloeosporioides species complex (Silva et al. 2012;Sharma et al. 2013;Liu et al. 2015). However, some recent new species (Diao et al. 2017;Marin-Felix et al. 2017) were unable to be placed in the ApMat and gs phylogenetic tree due to the absence of the ApMat gene sequence data. Although there were reports that the gs gene alone is not a good marker for differentiating C. siamense isolates , these data showed multiple base pair differences in gene sequences of the gs loci of C. siamense species similar to the ApMat locus. In addition, the lack of noticeable subclading in C. siamense in the 5-gene tree compared to the ApMat and gs tree, confirmed that the ApMat and gs loci were more informative than the other five gene loci. The ApMat gene has been shown previously to improve the systematics of the gloeosporioides species complex, providing complementary phylogenetic information compared to other loci (Silva et al. 2012). Liu et al. (2015) also applied the ApMAT gene in a more recent molecular phylogenetic analyses of the species in this complex and discussed the merit of using ApMat and ApMat in combination with gs to resolve the phylogeny.
Although four isolates from Indonesia were identified as C. karsti in the boninense complex, they formed a subclade within the C. karsti species clade and had different conidial sizes to the ex-type strain of C. karsti (Yang et al. 2011), suggesting that these might be a new species. However, sufficient phylogenetic support was not observed in all the individual gene trees to justify the introduction of a novel species. Besides, Damm et al. (2012aDamm et al. ( , 2012b reported that the conidium size of C. karsti was quite variable. Colletotrichum karsti has been reported from China and India to cause anthracnose disease in Capsicum spp. (Liu et al. 2016b;Saini et al. 2016;Diao et al. 2017). Colletotrichum karsti has the widest known host range and distribution of all species in the boninense complex (Damm et al. 2012b). Most of the C. karsti strains had been isolated as endophytes but a few were derived from diseased plant tissues. This species has mostly been isolated from dicotyledonous plants, but some have occurred on monocotyledonous families, especially Orchidaceae and Musaceae (Damm et al. 2012b).
Colletotrichum plurivorum was identified for the first time causing anthracnose in Thailand and Malaysia. The five C. plurivorum isolates from chili in Thailand formed a distinct subclade with high support values within the C. plurivorum subclade, and separated from C. cliviicola (syn. C. cliviae, Damm et al. 2019). In addition, the C. plurivorum isolates formed a characteristic sexual morph in culture, which was not reported for C. cliviicola (Yang et al. 2009).
Recently, Damm et al. (2019) resolved the taxonomic placement of several Colletotrichum strains which did not belong to any of the accepted species complexes and assigned them to three new species complexes including the orchidearum complex. Recent studies in China and Brazil also identified multiple species belonging to these complexes, including C. brevisporum, C. cliviicola, C. liaoningense, and C. plurivorum that caused anthracnose disease in chili (Liu et al. 2016b;De Silva et al. 2017b;Diao et al. 2017). Colletotrichum plurivorum belongs to the orchidearum complex with many isolates reported to have a large host range (Damm et al. 2019). The type specimen was described as new from Coffea in Vietnam (Nguyen et al. 2010). Colletotrichum plurivorum was originally described as C. sichuanensis from Capsicum annuum in the Sichuan Province of China (Liu et al. 2016b). However, the name was invalid, because no holotype specimen was cited (Mongkolporn and Taylor 2018;Damm et al. 2019).
Pathogenicity tests of Colletotrichum spp. from chili showed that while all the species were pathogenic on chili fruits after wounding the fruit surface, most produced a low level of infection on non-wounded fruit. This illustrates the importance of the cuticle acting as a barrier to infection by Colletotrichum spp. (Auyong et al. 2015) and emphasises the need for informed and standardised inoculation techniques in pathogenicity assays. Some species such as C. tropicale, C. makassarense and C. plurivorum which produced a low level of infection in the assays on non-wounded fruit, may have a predominantly endophytic lifestyle then switch to a necrotrophic life style to complete their life-cycle (De Silva et al. 2017b). However, further pathogenicity tests on different chili cultivars and at different fruit maturity stages are necessary to comprehensively evaluate their pathogenicity. Pathogenicity testing of C. plurivorum on chili leaves and fruits showed that the isolates collected from Chiang Rai and Malaysia could infect leaves but not fruit (results not shown) and suggested they might be specialised leaf pathogens. In contrast, two isolates of C. plurivorum from Bangkok did not infect leaves but did infect wounded fruits. These results demonstrate the pathogenic variation that can exist within a single species. Mongkolporn et al. (2010) identified pathotypes of C. truncatum, C. scovillei (as C. acutatum) and C. siamense (as C. gloeosporioides) within isolates of each species from Thailand. Pathotypes were identified by inoculating wounded fruit of Capsicum baccatum and C. chinense genotypes. All the isolates identified as C. gloeosporioides and C. acutatum in Mongkolporn et al. (2010) were subsequently re-identified as C. siamense and C. scovillei, respectively except for isolate UOM 1137 (F5-2D), which was identified as C. endophyticum. The isolate UOM 1137 was pathogenic in both the wound and non-wound bioassays, and was classified in the most virulent C. siamense pathotype group (PCg1-R) in Mongkolporn et al. (2010). This contrasts with the study by  where C. endophyticum was described as an endophyte of Pennisetum purpureum. The severity of infection in chili may indicate that Capsicum annuum was the preferred host for C. endophyticum and P. purpureum was a less favoured host, where the pathogen infected but existed in an endophytic lifestyle. In addition, isolate UOM 1137 also has shorter spores than the type isolate of C. endophyticum, thus further isolates of this species need to be collected from chili plants and P. purpureum in Thailand to confirm taxonomy and pathogenicity.

CONCLUSIONS
Multigene phylogenetic analyses of Colletotrichum species causing anthracnose disease of Capsicum in Asia showed high species diversity with the identification of 11 different Colletotrichum species, including three novel species. Although C. siamense has been reported as infecting many plant species before, this was the first report of C. siamense causing anthracnose in chili in Indonesia and Sri Lanka. This was also the first report of C. fructicola infecting chili in Thailand and Taiwan. In addition, all three novel species were new additions to the Colletotrichum species causing anthracnose in chili. More surveys in countries in Asia and Oceania need to be conducted to identify the diversity and prevalence of species causing chili anthracnose. Understanding of the taxonomy and the pathogenicity of Colletotrichum spp. has great significance to fruit and vegetable industries, where there are serious biosecurity implications of incursion by exotic pathogens.