Molecular and phenotypic characterization of Colletotrichum species associated with anthracnose disease in peppers from Sichuan Province, China

The anthracnose caused by Colletotrichum species is an important disease that primarily causes fruit rot in pepper. Eighty-eight strains representing seven species of Colletotrichum were obtained from rotten pepper fruits in Sichuan Province, China, and characterized according to morphology and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence. Fifty-two strains were chosen for identification by phylogenetic analyses of multi-locus sequences, including the nuclear ribosomal internal transcribed spacer (ITS) region and the β-tubulin (TUB2), actin (ACT), calmodulin (CAL) and GAPDH genes. Based on the combined datasets, the 88 strains were identified as Colletotrichum gloeosporioides, C. siamense, C. fructicola, C. truncatum, C. scovillei, and C. brevisporum, and one new species was detected, described as Colletotrichum sichuanensis. Notably, C. siamense and C. scovillei were recorded for the first time as the causes of anthracnose in peppers in China. In addition, with the exception of C. truncatum, this is the first report of all of the other Colletotrichum species studied in pepper from Sichuan. The fungal species were all non-host-specific, as the isolates were able to infect not only Capsicum spp. but also Pyrus pyrifolia in pathogenicity tests. These findings suggest that the fungal species associated with anthracnose in pepper may inoculate other hosts as initial inoculum.

Morphological and cultural characteristics. Eighty-eight isolates were classified into six morphological groups according to morphological and cultural characteristics. Group 1 included 23 isolates fitting the description of the C. gloeosporioides complex, and Group 2 included 16 isolates fitting the description of C. fructicola. In addition, Group 3 consisted of 32 isolates matching the description of C. truncatum, Group 4 had seven isolates fitting the description of the C. acutatum complex, and Group 5 consisted of six isolates matching the description of C. brevisporum. Group 6 contained four isolates that did not fit the description of any currently known Colletotrichum species. Group 3 (C. truncatum) was the predominant group, accounting for 36.4% of the total isolates. A summary of the morphological data for the Colletotrichum species in Groups 1-6 is presented in Table 2.
Colony characteristics (Fig. 2): Distinct morphology on potato dextrose agar (PDA) was observed in each group after 7 days. The isolates from Group 1 produced pale yellowish colonies, with sparse white aerial mycelia. The reverse side of the colonies was white, and many bright orange conidial masses were observed near the inoculum point. The colonies produced by Group 2 isolates varied from white to black-green on PDA, with dense grey aerial mycelia and a few bright orange conidial masses near the inoculum point. The colonies produced by Group 3 isolates varied from pale grey to dark grey, with dense pale grey aerial mycelia and small black granules over the entire surface. The reverse side of the colonies was dark brown, and a few pale yellow conidial masses were observed near the inoculum point. The colonies produced by Group 4 isolates varied from white to pale orange, with dense white aerial mycelia, and the reverse side of the colonies was pale orange. Isolates belonging to Group 5 produced dark grey colonies with sparse grey aerial mycelia. The reverse side of the colonies was grey, and a few bright orange conidial masses were observed near the inoculum point, as well as some spots scattered over the colony surface. Lastly, the isolates from Group 6 produced pale grey colonies, with sparse white aerial mycelia. The colonies from Group 3, 4 and 5 were stable and unique, and the colonies from Group 2 were significantly different compared with those from the other groups under stable culture conditions. Growth rate ( Table 2): Group 4 exhibited a significantly different growth rate compared with the other five groups (P = 0.05). The isolates from Group 6 (6.1 ± 0.4 mm/day) grew the fastest, followed by those from Group 1 (5.6 ± 1.2 mm/day), Group 2 (5.9 ± 0.4 mm/day), Group 5 (5.3 ± 0.6 mm/day), Group 3 (4.5 ± 0.5 mm/day) and Group 4 (3.8 ± 0.4 mm/day).
Conidial morphology (Table 2 and Fig. 2): The following four types of conidia were observed: cylindrical (observed in Groups 1, 2 and 6), falcate (Group 3), fusiform (Group 4) and long cylindrical (Group 5). The conidial widths of Group 6 were significantly different from those of Groups 1 and 2; however, all of these groups had cylindrical conidia with obtuse to slightly rounded ends. The conidia produced by the Group 3 isolates were falcate, with gradual tapering towards each end. Group 4 produced fusiform conidia, whereas Group 5 produced long and cylindrical conidia, with obtuse to slightly rounded ends. The differences in the conidial shapes of Scientific RepoRts | 6:32761 | DOI: 10.1038/srep32761 Groups 3, 4 and 5 were very significant, allowing these groups to be easily distinguished from one another. Almost all of the conidia were aseptate, but they often developed a septum after germinating and forming appressoria.
Conidial appressorium morphology (Table 2 and Fig. 2): There was little distinction among the groups in terms of the sizes and shapes of conidial appressoria, except for Groups 4 and 6, which exhibited significant differences compared with the other groups. The conidial appressoria of Groups 1, 2, 3 and 5 varied from ovoid to slightly irregular in shape and from brown to dark black in colour. Group 4 produced grey, globular and smaller Type I symptoms were characterized by dark brown to black, sunken lesions with a slightly raised rim and many black acervuli on the surface, which produced dirty white conidial masses under humid conditions. (d-f) Type II symptoms included dark brown to black, sunken lesions with many black acervuli on the surface, which produced flesh pink, viscous conidial masses under humid conditions. (g-i) Type III symptoms included brown to light black to dark brown, sunken, lesions with orange conidial masses. conidial appressoria. Most of the conidial appressoria produced by Group 6 were irregular and pale brown to dark brown, with a crenate edge. Mycelial appressorium morphology (Table 2 and Fig. 2): The mycelial appressoria produced by the isolates of Groups 1 and 2 varied from ovoid, clavate and slightly irregular to irregular, smooth or slightly lobed, and they were light brown to brown in colour. The appressoria of Group 3 ranged from ovate, ellipsoidal or slightly irregular to irregular in shape, and they were smooth or lobate and brown to dark brown. The appressoria produced by Group 4 were globose or ovate to slightly irregular, and they were light brown to brown and smaller in size than those of the other groups. In addition, the appressoria produced by Group 5 varied from ovoid, clavate or slightly irregular to irregular in shape. They were smooth or slightly lobed and brown to dark brown and were sometimes black in the middle. Further, the appressoria of Group 6 were ellipsoidal or irregular, smooth or slightly lobed to strongly lobed, solitary or in chains, and light brown to brown in colour. Conidiophores (Fig. 2): The conidiophores of all groups were hyaline to pale brown, simple or septate, rarely branched, and smooth walled. Four types of conidiophores were observed: (i) nearly cylindrical, but narrower towards the end (as observed in Groups 1 and 2); (ii) cylindrical, with a truncate top (Groups 3 and 5); (iii) shortly clavate, nearly hyaline, with a cylindrical base, and obviously inflated, with gradually tapering towards the top (Group 4); and (iv) frequently produced by mycelia, cylindrical, with swollen ends (oblong) and slight narrowing in some areas (Group 6).
Setae: All isolates from Groups 3 and 5 and some isolates from Group 1 produced setae; in contrast, the isolates from all the other groups rarely produced setae. The setae were commonly smooth, septate, and light brown to dark brown in colour, base cylindrical to conical, and sometimes slightly inflated, and the tips were acute to roundish. No obvious differences in setal characteristics (shape and dimensions) were found among the different groups when grown on PDA.
Sclerotia and Ascomata: Most Group 5 isolates steadily produced a large amount of black solids that appeared similar to sclerotia and were round to irregular and semi-immersed. Conidial masses and setae sometimes formed on the black solids. On PDA, Group 6 isolates always produced ascomata in clusters, which were brown and globose to near globose and possessed a neck. The isolates from the other groups rarely produced ascomata, even in host tissues.
Multi-locus phylogenetic analysis was conducted among 87 strains, with Monilochaetes infuscans (CBS 869.96) used as an outgroup ( Table 3). The dataset for five genes (ITS, TUB2, ACT, GAPDH and CAL) contained 2,155 characters, including alignment gaps, of which 997 characters were parsimony-informative, 321 were parsimony-uninformative, and 837 were constant. This parsimony analysis resulted in the most parsimonious tree (TL = 2800, CI = 0.7257, RI = 0.9541, RC = 0.6924, and HI = 0.2743). The phylogram showed that the 52 pepper anthracnose isolates belonged to seven distinct clades. The isolates from Group 2 clustered with C. fructicola, those from Group 3 clustered with C. truncatum, those from Group 4 clustered with C. scovillei, and those from Group 5 clustered with C. brevisporum. The Group 1 isolates grouped with two clades; 4 isolates clustered with C. gloeosporioides, and the remaining isolates clustered with C. siamense (Fig. 4). After combining two phylograms (Figs 3 and 4), 8 and 16 strains were found to belong to C. gloeosporioides sensu stricto and C. siamense, respectively. The isolates from Group 6 were from an unknown species (Colletotrichum sp.). The submission number of the sequence alignment in TreeBASE is 18832.
Based on the description of the symptoms in pepper after inoculation, C. truncatum was determined to be the pathogen causing Type I symptom, characterized by copious black acervuli with seta and dirty white conidial masses produced on decaying tissues under humid conditions (Fig. 1a-c). C. scovillei induced Type III symptoms  Fig. 1g-i), and the other species caused Type II symptoms (Fig. 1d-f). Our results indicate that with the exception of C. truncatum and C. scovillei, it is difficult to differentiate among Colletotrichum species based solely on the symptom types in the field.

Discussion
The primary objective of this study was to identify the Colletotrichum species that are currently causing anthracnose disease in pepper grown in Sichuan Province, China. Based on the morphological characteristics and phylogenetic analysis, 88 isolates were identified as C. gloeosporioides sensu stricto (eight strains, 9.1%), C. siamense (16 strains, 18.2%), C. fructicola (15 strains, 17.0%), C. truncatum (32 strains, 36.4%), C. scovillei (seven strains, 8.0%), C. brevisporum (six strains, 6.8%) and C. sichuanensis (a new species, four strains, 4.5%). Additionally, C. gloeosporioides and C. siamense could only be distinguished by phylogenetic analyses and not by morphological   analyses. The morphological groupings based on colony characteristics, growth rate, conidial morphology, conidial appressorium morphology and mycelial appressorium morphology were almost completely consistent with the results of phylogenetic analysis derived from the molecular data.
In vitro culture-related characteristics were important for differentiating among Colletotrichum species 26 . C. truncatum, C. scovillei, C. brevisporum, C. sichuanensis isolates and some C. fructicola isolates with unique and relatively stable colonies could be easily distinguished. However, the colonies of C. gloeosporioides, C. siamense and some C. fructicola isolates overlapped in terms of their morphological characteristics, and phenotypic variations were identified among the species under different environmental conditions. The colony growth rate of C. scovillei was significantly slower than those of the species in the other groups. Previous studies have shown that C. acutatum can be differentiated from C. gloeosporioides based on its slower growth rate 30 . Than et al. 2 have also suggested that colony growth rates are important for distinguishing among C. gloeosporioides, C. truncatum and C. acutatum. In the present study, the slow growth of C. scovillei conformed to the characteristics of the C. acutatum complex. The observed differences in conidial size were significant, with the exception of the lengths and widths of Groups 1 and 2. Denoyes and Baudry 31 used conidial shape to differentiate among Colletotrichum species that are pathogenic to strawberries, although Cai et al. 25 and Crouch et al. 32 have suggested that conidial appressoria are taxonomically uninformative and of little use for species identification. In contrast, the conidial appressoria of C. scovillei could be easily distinguished from those of the other species examined in our study, in   33 . Similarly, Crouch et al. 32 have found that the shapes and sizes of mycelial appressoria in combination with the host range are useful for identifying grass-associated Colletotrichum species. We found that the mycelial appressoria produced by C. scovillei and C. brevisporum were typically smoother than those produced by the other species and that all C. truncatum and C. brevisporum isolates steadily produced setae.
In addition, C. gloeosporioides has been reported to produce setae occasionally or under certain conditions 34 , and many other Colletotrichum species are known to produce setae 3 . In the present study, the cultural characteristics, colony growth rate, conidial shapes and sizes, and conidial and mycelial appressoria were the primary features used for classification. Morphological examination was conducted to classify the 88 isolates into six groups, although our multi-locus phylogenetic analysis actually identified seven Colletotrichum species. Groups 2-6 contained different Colletotrichum species, and Group 1 consisted of two species: C. gloeosporioides and C. siamense. Thus, morphological criteria alone are not always sufficient for species identification 14 . Indeed, multi-locus phylogeny showed that the isolates with similar morphological characteristics belonged to the C. gloeosporioides, C. siamense and C. fructicola clades. Moreover, the C. gloeosporioides and C. siamense isolates could not be distinguished according to their morphological and cultural characteristics, indicating that multi-locus phylogenetic analysis is useful for differentiating among species in the Colletotrichum genus. Many investigators have suggested the use of multi-locus phylogenetic analysis to overcome the inadequacies of morphological criteria 3    Colletotrichum gloeosporioides was first described in citrus from Italy 40 . The name C. gloeosporioides represents both C. gloeosporioides sensu lato, which encompasses the entire species complex, and C. gloeosporioides sensu stricto 18 . C. gloeosporioides sensu lato consists of at least 22 species, including C. gloeosporioides, C. siamense, and C. fructicola 1,18,25,26,41 . C. siamense and C. fructicola were originally known as opportunistic pathogens of Coffea arabica berries in Thailand 26 , and both of these species are non-host-specific. C. fructicola has also been reported to be a pathogen causing pepper anthracnose in Thailand 16   C. siamense from chilli pepper in Thailand, the isolates belonging to C. siamense were identified as C. gloeosporioides in that study, and Weir et al. 18 later revised the classification. C. siamense has also been isolated from pepper in India. However, this species has not been reported to be a causative agent of pepper anthracnose in China. Therefore, this work is the first report of pepper anthracnose caused by C. siamense.
Colletotrichum truncatum, originally described on Phaseolus lunatus, was typified by Damm et al. 3 , and this species has been associated with anthracnose on legume crops and pepper, as well as on many other hosts 3,9,34 . The C. capsici isolate typified by Shenoy et al. 42 causes anthracnose in a wide range of hosts, including pepper and legume species 1,43,44 , and Damm et al. 3 synonymized the C. capsici taxon with C. truncatum on the basis of its multi-locus phylogeny and morphology. Regardless, not all researchers are in agreement with this viewpoint 1 .
Colletotrichum acutatum is widely known as a fruit rot pathogen in strawberry 2 , apple 45 , pepper 2,11 and grape 46 , and this fungus was first recorded in Australia on Carica papaya, Capsicum frutescens and Delphinium ajacis by Simmonds 30 . C. acutatum is also a species complex containing at least 14 species, including C. scovillei 47 . The ex-type strain of C. scovillei was initially identified as C. acutatum 48 , and Than et al. 2 also identified C. scovillei as C. acutatum on chilli pepper from Thailand. Although C. scovillei was identified as C. acutatum in these two papers, it was later revised by Damm et al. 47 . Kanto et al. 21 also isolated C. scovillei from sweet pepper in Japan. In our study, we only isolated C. scovillei belonging to C. acutatum sensu lato from the pepper fruits. Thus, the main species from the C. acutatum complex that is pathogenic to pepper in Sichuan Province might be C. scovillei rather than C. acutatum sensu stricto. To our knowledge, this work is also the first report of C. scovillei as a causative agent of pepper anthracnose in China.
Colletotrichum brevisporum has been recorded on Neoregelia sp. from Thailand, as well as on papaya fruits and Pandanus pygmaeus Thouars 35,49 . Yang 7 have also reported C. brevisporum on pepper from China. The conidial lengths of C. brevisporum in the present study were longer than those reported by Noireung et al. 35 , but they were consistent with those reported by Yang 7 .
The results of our phylogenetic analysis strongly support the Colletotrichum sichuanensis clade, which is closely related to C. cliviae. These two species have similar conidial shapes but different conidial sizes; C. sichuanensis has shorter conidia than C. cliviae (21.8 μ m), with a mean length of 16.7 μ m. C. sichuanensis also differs from C. cliviae with regard to colony colour. In addition, C. sichuanensis steadily produced ascomata on PDA, whereas the other species rarely produced ascomata. Further, C. sichuanensis grew more slowly in culture than C. cliviae (11.3-12.9 mm/day for C. sichuanensis compared with 15.2-16 mm/day for C. cliviae).
Given that they could infect not only Capsicum spp. but also Pyrus pyrifolia, all of the species isolated from pepper in our study were non-host-specific. In addition, C. scovillei was the most virulent species towards Capsicum spp. Tang 6 found that C. acutatum and C. truncatum were more virulent than C. gloeosporioides and that the C. acutatum incubation period was the shortest. Further, Than et al. 2,14 reported that C. acutatum was a very virulent species that could infect wound-resistant C. chinense PBC 932, whereas C. gloeosporioides and C. capsici (syn. C. truncatum) could not.
Colletotrichum acutatum 10 , C. truncatum 5 and C. boninense 19 have been previously reported in Sichuan; however, C. boninense was not isolated in our study; it is possible that this species was missed during sampling or isolation. In summary, C. siamense and C. scovillei are recorded for the first time as causing anthracnose in pepper from China. Additionally, we have identified one new species, which has been introduced as C. sichuanensis.

Methods
Collection and isolation. In 2012 and 2013, pepper fruits with anthracnose symptoms were collected from primary production areas in Sichuan Province, China. Tissues of approximately 5 mm in diameter were collected from the edges of lesions, surface-sterilized with 75% ethanol for 30 s and 1% NaClO for approximately 1 min, washed three times with sterile distilled water, and then dried on sterile filter paper. The treated tissues were plated on PDA supplemented with 50 mg l −1 streptomycin. The plates were incubated at 27 ± 1 °C for 5 days. Single-spore cultures were obtained for each Colletotrichum isolate according to the procedure described by Gong et al. 50 . The resulting strains were maintained on PDA slants at 4 °C for short-term storage and in 25% glycerol at − 70 °C for long-term storage.
Morphological and cultural characterization. Mycelial discs (5 mm diameter) were collected from actively growing areas near the growing edges of 5-day-old cultures, transferred to PDA and incubated at 27 °C in the dark for 10 days. Five replicates were employed. The colony diameter was recorded each day from two perpendicular cross-sections, and the colony characteristics were also recorded.
The sizes and shapes of conidia, asci and ascospores from each culture were recorded. The lengths and widths of 30 conidia, asci and ascospores were measured for each isolate.
Conidial appressoria were induced according to the method of Yang et al. 27 . Mycelial appressoria were produced using an improved slide culture technique, as described by Sutton 51 and Cai et al. 25 . One hundred microlitres of hot water agar (WA) was placed on a sterile slide. Mycelial plugs of approximately 2 mm in diameter were inoculated onto one-third of the WA and then incubated in a Petri dish with wet filter paper at 27 °C. After 5-7 days, agar pieces containing the inoculated plugs were gently removed with a scalpel, and the shapes and sizes of the appressoria that formed along the WA were then recorded.
Samples for microscopy were prepared using clear water or lactic acid and observed with a Carl Zeiss Axio Imager Z2 microscope(Germany) or a Nikon Eclipse 80i microscope(Japan) using differential interference contrast (DIC) illumination. DNA extraction. Fifty-two representative isolates were chosen according to the morphological and cultural characteristics and incubated on PDA at 27 °C for 7-10 days. Mycelia were scraped from the colony surface using Scientific RepoRts | 6:32761 | DOI: 10.1038/srep32761 a sterile medicine spoon. Total genomic DNA was extracted from the isolates using a modified protocol, as outlined by Guo et al. 52 .
PCR amplification and DNA sequencing. As an initial analysis of genetic diversity, the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified from the isolates in this study with the primers GDF/GDR 53 . Fifty-two isolates representing wide ranges of genetic diversity and geographic origins were selected for further investigation.
Phylogenetic analysis. Alignment of the GAPDH genes of all of the isolates was performed using Clustal X 59 . MEGA v. 5 was used to build a distance tree with the neighbour-joining (NJ) algorithm. The sequences were compared with those in the NCBI sequence database using the BLAST algorithm for approximate identification.
The sequences of the 52 isolates and the reference sequences obtained from GenBank (Table 3) were aligned using Clustal X. Then, a phylogenetic tree was constructed with the combined ITS, TUB2, ACT, GAPDH and CAL dataset.
Parsimony trees were inferred by PAUP v4.0b10 using a heuristic search option with 1,000 random sequence additions 60 . All gaps were treated as missing data. Max trees were unlimited, zero-length branches were collapsed, and all multiple parsimonious trees were saved. Clade stability was assessed by bootstrap (BT) analysis with 1,000 replicates. In addition, descriptive tree statistics, such as parsimony ( Pathogenicity tests. Pears were included in the pathogenicity tests for two main reasons: i) because peppers often are planted in pear orchards; and ii) to assess whether Colletotrichum species from pepper are host specific. Fruits of Capsicum annuum (Ca. annuum var. dactylus M and Ca. annuum L. var. conoides (Mill.) Irish) and Pyrus pyrifolia were surface-sterilized in 75% ethanol for 3 min and then rinsed three times in sterile distilled water. The fruits were stabbed lightly with a sterile needle, and a mycelial disc with a diameter of 5 mm from a 4-day-old colony obtained from an isolate grown on PDA at 27 °C was attached to each artificially wounded fruit. The PDA discs were covered with moistened cotton for 3 days. The cotton was then removed, and the fruits were incubated for 14 days in a growth chamber at 27 °C with a 12 h light/12 h dark cycle. Six replicates and an equal number of control fruits inoculated only with agar discs were included.