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Diverse Colletotrichum species cause anthracnose of tea plants (Camellia sinensis (L.) O. Kuntze) in China

Anthracnose caused by Colletotrichum is one of the most severe diseases that can afflict Camellia sinensis. However, research on the diversity and geographical distribution of Colletotrichum in China remain limited. In this study, 106 Colletotrichum isolates were collected from diseased leaves of Ca. sinensis cultivated in the 15 main tea production provinces in China. Multi-locus phylogenetic analysis coupled with morphological identification showed that the collected isolates belonged to 11 species, including 6 known species (C. camelliae, C. cliviae, C. fioriniae, C. fructicola, C. karstii, and C. siamense), 3 new record species (C. aenigma, C. endophytica, and C. truncatum), 1 novel species (C. wuxiense), and 1 indistinguishable strain, herein described as Colletotrichum sp. Of these species, C. camelliae and C. fructicola were the dominant species causing anthracnose in Ca. sinensis. In addition, our study provided further evidence that phylogenetic analysis using a combination of ApMat and GS sequences can be used to effectively resolve the taxonomic relationships within the C. gloeosporioides species complex. Finally, pathogenicity tests suggested that C. camelliae, C. aenigma, and C. endophytica are more invasive than other species after the inoculation of the leaves of Ca. sinensis.

has been reported inaccurate for interspecific relationship identifications 4,8,9,29 . Furthermore, these studies were also limited by the small area of the investigated regions and lacked information regarding the infected tea plant cultivar. For example, Guo et al. 3 investigated Colletotrichum only in yellow mountain tea plants in the Anhui province of China. Liu et al. 2 recently isolated Colletotrichum from healthy and diseased tissues of Camellia spp. from 7 provinces in China and from 3 other countries. However, their investigations included only partial areas of tea plant cultivation in China. Therefore, further study is necessary to identify Colletotrichum on Ca. sinensis in China to a species level by their morphological characteristics and multigene phylogenies.
To understand the diversity of Colletotrichum species on Ca. sinensis, we collected diseased leaves of tea plants from several of the major tea growing regions of China. After isolating and identifying Colletotrichum species, we summarized the Colletotrichum species associated with Ca. sinensis and their geographical distributions in China. We believe these results can provide phytopathologists and plant breeders with a reference for the prevention and control of anthracnose disease.

Results
Multilocus-based phylogenetic analysis. We collected 106 isolates of Colletotrichum spp. from diseased leaves of Ca. sinensis from the main tea growing regions in China and identified them based on phylogeny and morphological characteristics.
The phylogram in Fig. 1 shows the identified isolates in the C. gloeosporioides species complex. The combined aligned data matrix (ITS, ACT, GAPDH, CAL, CHS-1, TUB2, and GS) contained 152 sequences including the outgroup (C. boninense CBS 123755) and 3,879 characters including gaps. These species were determined from 81 isolates from Ca. sinensis plants in our study (the partly identical C. camelliae and C. fructicola isolates were removed from Fig. 1; the complete alignment and tree, shown in Fig. 1c, was available from TreeBASE). Of the 81 isolates, 2 clustered with C. aenigma ex-type culture, 33 clustered with C. camelliae, 3 clustered with C. endophytica, 34 clustered with C. fructicola, 8 clustered with C. siamense, 1 (SC3A3) was indistinguishable and named Colletotrichum sp., and 2 (JS1A32, JS1A44) did not cluster with any known species and formed a distinct clade with a high bootstrap support/posterior probability value (61/0.98).
The phylogram in Fig. 2 shows the isolates of Colletotrichum that were not in the C. gloeosporioides species complex. The combined aligned data matrix (ITS, ACT, GAPDH, TUB2, and CHS-1) contained 59 sequences including the outgroup (C. lindemuthianum CBS 144.31) and 1,928 characters including gaps. These data were determined from 25 isolates from Ca. sinensis in our study. The maximum likelihood tree (tree topology) had bootstrap support values greater than 50% and Bayesian posterior probability values of ≥ 0.95. The 25 isolates were grouped into 4 subclades: C. cliviae, C. fioriniae, C. karstii, and C. truncatum. Thirteen isolates clustered with the ex-type strain of C. karstii CORCG6, 1 isolate clustered with C. truncatum, 6 isolates clustered with C. fioriniae, and 5 isolates clustered with C. cliviae.
ApMat & GS-based phylogenetic analysis of the C. gloeosporioides species complex. Forty-seven strains of the C. gloeosporioides species complex isolated from tea plants and 35 reported strains were used for phylogenetic tree construction (rooted with C. xanthorrhoeae) based on ApMat and GS sequences. The dataset comprised 1,829 characters with alignment gaps (Fig. 3). All species could be separated with high support values, followed by multi-locus phylogenetic tree analysis, with the exception of isolates GX2A1 and GX2A3. These 2 isolates belonged to C. siamense rather than C. aeschynomenes (Fig. 1). Sequence alignment of ApMat and GS showed that the isolates GX2A1 and GX2A3 differed from C. aeschynomenes ICMP 17673 by only 5 bp in ApMat but by 19 bp in GS. The strain SC3A3 clustered with C. kahawae sensu lato.
Pathogenicity tests. Pathogenicity tests showed that C. aenigma CGMCC 3.17883, C. camelliae CGMCC 3.17884, and C. endophytica CGMCC 3.17887 have the typical brown lesions of anthracnose disease around wounded areas. Conidia from wounds were re-isolated and cultured on potato dextrose agar (PDA), which showed pathogenesis analogous to that of infected strains. However, wounded leaves of Ca. sinensis cv. Longjing 43 did not have obvious disease spots after inoculation with Colletotrichum sp. CGMCC 3.17890, C. siamense CGMCC 3.17892, or C. wuxiense CGMCC 3.17894 (see Supplementary Figs S4l, S9m and S10m). A possible explanation for the few disease spots caused by these strains is that the strains have weak virulence or lack necessary pathogenesis genes for Longjing 43 (the gene-for-gene hypothesis) 30,31 . Taxonomy. Based on their multi-locus phylogeny and morphological characteristics, the 106 isolates from Ca. sinensis were identified as 11 species of Colletotrichum (Figs 1-3), including 1 new species (named C. wuxiense), 3 new record species (C. aenigma, C. endophytica, and C. truncatum), 6 previously described species (C. camelliae, C. cliviae, C. fioriniae, C. fructicola, C. karstii, and C. siamense), and 1 indistinguishable strain (described as Colletotrichum sp. Description: Colonies on PDA were flat with entire edges, aerial mycelium sparse, cottony, pale white, scattered acervuli with yellow conidial mass near the center, pale white on reverse, had a growth rate of 11.4-12.3 mm per day at 25 °C after 5 days. Sexual morph was observed on PDA agar after 7 days. Ascomata globose, brown  Notes: Colletotrichum aenigma was first reported on Persea americana from Israel and has been subsequently reported on Pyrus pyrifolia from Japan 13 , Pyrus communis, Citrus sinensis, and Olea europaea from Italy 32 , Hylocereus undatus from Thailand 33 , Poplar sp. from China 22,34 , and Vitis vinifera from China 24 . However, none of these studies described the sexual morph and setae of this species. This is the first report of C. aenigma associated with anthracnose of Ca. sinensis in China and the first description of the morphological characteristics of its sexual morph and seta.
Colletotrichum camelliae Massee, Bull. Misc. Inform. Kew. 1899: 91. 1899. See Supplementary Fig. S2. Description: Colonies on PDA raised centers, aerial mycelium dense, cottony, iron-grey. Chlamydospores not observed, reverse buff, a growth rate of 11.9-12.9 mm per day at 25 °C after 5 days. Sexual morph was not observed. Chlamydospores black, hidden in medium. Only one acervular was observed, conidiophores and setae either directly formed from hyphae or on a cushion of roundish hyaline cells. Setae dark brown, smooth-walled, 1-2 septate, 56.  Notes: The morphological characteristics of Colletotrichum camelliae were systematically described in detail based on methodology used in previous studies 2 . Although C. camelliae had been described previously as the dominant Colletotrichum species on Camellia plants in China 2,13,18 , the characteristics of its seta had not been investigated. In the present study, the morphological characteristics of setae were described. Interestingly, 3 strains clustered with C. camelliae LF789 and formed a distinct subclade as shown in Fig. 1c. However, ApMat and GS-based phylogenetic analysis showed that these 3 strains were distinctly clustered with C. camelliae (Fig. 3). The PHI test also revealed significant genetic recombination levels between these 3 strains and C. camelliae, suggesting that they are conspecific. In addition, conidia and appressorium dimensions of the 2 strains from Jiangsu and Yunnan Province ( 35 Fig. S3.
Notes: Colletotrichum endophytica had been found only as an endophyte on Pennisetum purpureum 6 and on unknown wild fruit from Thailand 36 . In the present study, 3 pathogenic strains were isolated from diseased leaves of Ca. sinensis. This is the first report of C. endophytica causing anthracnose in Ca. sinensis and the first report of C. endophytica in China. Setae of C. endophytica are observed and described in this study for the first time.   Notes: Colletotrichum karstii is the most common Colletotrichum and is present in a wide range of hosts. It was previously reported to be pathogenic to Ca. sinensis from Fujian 28 and Zhejiang provinces 2 . In this study, species were isolated from the Fujian, Zhejiang, Yunnan, Jiangsu, and Hunan Provinces of China.
Notes: In the phylogenetic tree, strain SC3A3 appears as a sister clade between C. kahawae s. l. and C. jiangxiense (Fig. 1). Its sequences (ITS, ACT, GAPDH, CAL, CHS-1, and TUB2) were identical to those of ICMP 12952, but its GS sequence differed by 8 bp. The GS sequence of the SC3A3 strain also differed from that of the ex-type culture of C. jiangxiense (CGMCC 3.17363) by 25  Notes: Colletotrichum siamense was originally found on coffee berries from Thailand, has been found on various hosts, and now is considered a biologically and geographically diverse species 13,23,35,38 . A previous study reported that C. siamense caused anthracnose of several varieties of Ca. sinensis from many regions of China 28 . The species can be distinguished from other species in the C. gloeosporioides species complex through analysis of concatenated ApMat and GS sequences 2,17,18 . In the present study, multi-locus phylogenetic trees and morphological characterization were used to identify strains GX2A1 and GX2A3 as C. siamense (Fig. 1). However, in a phylogenetic tree constructed from ApMat and GS sequences, these 2 strains clustered with the ex-type strain of C. aeschynomenes ICMP 17673 rather than with C. siamense s. l. The ApMat sequences of the 2 strains (GX2A1 and GX2A3) were identical to those of ICMP 17673 with the exception of just 5 bp. Therefore, analysis of a single gene sequence or analysis of ApMat and GS gene sequences together were not sufficient to accurately separate the C. siamense.
Colletotrichum  Notes: Multi-locus (ITS, ACT, GAPDH, CAL, CHS-1, TUB2, and GS) phylogenetic tree analysis showed that Colletotrichum wuxiense is a sister clade of the closely related C. jiangxiense and C. kahawae s. l., and it clustered with C. camelliae (Fig. 1). C. wuxiense can be distinguished from these other species by the morphological features of its conidia, which are larger than those of these similar species 2,13 and are slightly curved. In addition, C. wuxiense can be directly separated from other species of the C. gloeosporioides species complex using its concatenated ApMat and GS gene tree (Fig. 3). A PHI test also showed that no significant recombination events between C. wuxiense and closely phylogenetically related species occurred.
Prevalence of Colletotrichum species. Of the 106 isolates of Colletotrichum, 33 isolates of C. camelliae were isolated from 14 provinces/cities, and 34 isolates of C. fructicola were isolated from 11 provinces/cities ( Table 1). Approximately 61.3% of the total isolates were harvested from tea-producing areas of China (Table 1, Fig. 6). These results suggest that C. camelliae and C. fructicola are the dominant species causing anthracnose of Ca. sinensis. Moreover, 7 of 11 species belonged to the C. gloeosporioides species complex (Figs 1 and 2, Table 1,  Supplementary Table S1).

Discussion
In this study, a total of 106 Colletotrichum isolates obtained from the diseased leaves of tea cultivars in China were identified as 11 species, of which 9 had been described previously, 1 was identified as a new species, and 1 was unidentifiable.
C. gloeosporioides was previously considered the dominant Colletotrichum species on tea plants in China 3 . In our study, C. camelliae and C. fructicola were the most prevalent species in China. These results were similar to those of Liu, et al. 2 . C. camelliae were collected from 14 out of 15 provinces of China (Table 1, Fig. 6, Supplementary Table S1). Nevertheless, 3 strains of C. camelliae (YN2A1, JS1A35, and SH1B4) formed a separate clade and were close to C. camelliae (CGMCC 3.14925) in the phylogenetic tree (Fig. 1). Moreover, conidiophores and setae of strain JS1A35 were directly produced from hyphae or on a cushion of roundish hyaline cells, rather than on aerial mycelium (CGMCC 3.14925). In addition, we also observed intraspecific differences in the colonial morphology and growth rate of C. camelliae isolates. Therefore, the genetic differentiation among the above isolates with different geographic distribution and morphology should be further clarified. C. fructicola, which was obtained from 11 provinces across China, was the second most prevalent species in our study (Table 1, Fig. 6, Supplementary Table S1). Liu 28 reported that this species could infect several varieties of Ca. sinensis in the Fujian Research on new record species of microorganisms in hosts can provide helpful information for understanding the interactions between hosts and microorganisms as well as their geographical distribution. Colletotrichum species could switch their lifestyle from endophytic to pathogenic, for which both internal and external environmental factors play important roles 5,40 . In our study, 3 new record species from Ca. sinensis were reported for the first time, including C. aenigma, C. endophytica and C. truncatum. C. endophytica was described as an endophyte or saprobe in Pennisetum purpureum and an unknown wild fruit 6,36 . Interestingly, our study showed that C. endophytica also can be a pathogen that infects Ca. sinensis (see Supplementary Fig. S3j). We speculated that there is a specific interaction between Ca. sinesis and C. endophytica 31,41 .
We collected a strain (SC3A3) that was not well distinguished and we classified it as Colletotrichum sp. using the multi-locus phylogenetic tree (Fig. 1). Its morphological characteristics were more similar to C. kahawae subsp. ciggaro than to C. jiangxiense (see Supplementary Fig. S4). Therefore, further studies are required to clarify the phylogenetic relationships among these species.
Previous studies indicated that Ca. sinensis can harbor various Colletotrichum species. C. acutatum 25 and C. gloeosporioides 42 were generally considered as dominantly endophytic. A few other species were considered as pathogenic or potentially pathogenic on Camellia, such as C. lupini, C. acutatum, C. carveri, C. coccodes and C. queenslandicum 11,13 . In this study, we collected the common species, such as C. cliviae, C. fioriniae, C. karstii, and C. siamense, as well as a novel species that was named C. wuxiense. However, C. acutatum and C. gloeosporioides were not the dominant species. Similar results were also reported by Liu,et al. 2 in the Colletotrichum classification study. These differences in dominant species identification may be caused by the variation of sampling range between studies.
In conclusion, we investigated the diversity of Colletotrichum species in tea plants in China and identified 11 species including 1 novel species. Moreover, we found that C. camelliae and C. fructicola are the dominant species in Ca. sinensis. Unfortunately, our study failed to characterize endogenetic Colletotrichum species due to the lack of healthy tissue collected from tea plants. In future studies, we will isolate endophytic Colletotrichum species from healthy tissues of tea plants and elucidate their geographical distribution, the evolutionary relationship between Colletotrichum and Ca. sinensis, and the differences in intraspecific virulence and morphology of C. camelliae.

Materials and Methods
Collection and isolation. Diseased leaves with visible anthracnose symptoms were collected from the following 15 provinces or cities of China: the provinces of Anhui, Fujian, Guangdong, Guangxi, Guizhou, Henan, Hunan, Hubei, Jiangsu, Jiangxi, Shaanxi, Sichuan, Yunnan, and Zhejiang and the city of Chongqing. Five randomly selected diseased leaves were sampled from each cultivar and region. Colletotrichum species were isolated by a single spore isolation technique as described by Cai,et al. 4 . Spore masses were picked off with a sterilized wire loop and suspended in sterilized water. The spore suspension was diluted to a reasonable concentration and spread onto the surface of PDA, followed by an incubation overnight at 25 °C. Single germinating spores were picked up with a sterilized needle and transferred to a new PDA plate. DNA extraction, PCR amplification, and sequencing. Fungal isolates were grown for 5-7 days on PDA. Mycelia were collected in a sterile centrifuge tube and stored at − 80 °C for DNA extraction. Total genomic DNA of the isolate was extracted using a Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Shanghai Company Limited, Shanghai, China) and stored at − 20 °C. The ribosomal internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), partial sequences of the chitin synthase 1 (CHS-1), calmodulin (CAL), glutamine synthetase (GS), mating type protein, and the Apn2-Mat1-2 intergenic spacer (ApMat) were amplified. The protocols for amplification and the PCR primers used in this study are listed in Supplementary Table S2. Each 50 μ L PCR mixture included 25 μ L of Premix Taq TM (Takara Biomedical Technology Company Limited, Beijing, China), 22 μ L of ddH 2 O, 1 μ L of each primer, and 1 μ L of genomic DNA. PCR purification and sequencing were performed by ShangHai Huagene Biotech Company Limited, Shanghai, China.
Phylogenetic analysis. The accession numbers of all sequences in this study were obtained from NCBI-GenBank and are listed in Supplementary Table S3. A phylogenetic tree was constructed using Multi-locus sequences. The dataset was assembled using MAFFT v. 7 43 and manually adjusted using MEGA v. 6.0 44 . All gaps were treated as missing data. Nucleotide substitution models were generated using MrModeltest v. 2.3 45 , and the GTR + I + G model with gamma-distributed rate was selected for constructing all phylogenic trees. A maximum likelihood phylogenetic analysis of the dataset was performed with RAxML 46 . Markov Chain Monte Carlo (MCMC) sampling was used to reconstruct phylogenies in Mrbayes v. 3.2 47 . Analyses of 6 MCMC chains based on the full dataset were run for 1 × 10 7 generations and sampled every 100 generations. The first 25% of the generations were discarded as burn-in. Morphological characterization. Mycelial discs (9 mm diameter) were taken from 5-day-old cultures, plated on PDA, and incubated at 25 °C in the dark. Daily growth rate was calculated after 5 days of growth and was based on values from three replicates. Colony characteristics were also recorded. Conidial, conidiophores, and appressoria characteristics were determined using methods described by Cai,et al. 4 . Additionally, appressoria were produced and measured using a slide culture technique and induced on synthetic nutrient-poor agar (SNA) medium. After 7 days, the shapes and sizes of 30 conidia, conidiophores, and appressoria were recorded (Eclipse 80i, Nikon, Japan).

Pathogenicity tests.
Inoculations were based on the method described by Liu, et al. 2 . Six strains were selected for pathogenicity testing: C. aenigma CGMCC 3.17883, C. camelliae CGMCC 3.17884, C. endophytica CGMCC 3.17887, Colletotrichum sp. CGMCC 3.17890, C. siamense CGMCC 3.17892, and C. wuxiense CGMCC 3.17894. Healthy and non-wounded mature tea leaves, collected from 5-year-old Ca. sinensis cv. Longjing 43 grown in a tea garden in Hangzhou, Zhejiang province, China, were washed with tap water and then disinfected in 1% sodium hypochlorite for 3 min. Disinfected mature leaves were washed three times with sterilized water and then dried on the benchtop. Using sterile needles, 20 μ L of conidial suspension (10 6 spores/mL) was added to three wounded leaves for each strain. Leaves inoculated with sterile water were used as controls. The inoculated samples were laid on plastic petri dishes 12 cm in diameter and cultured in a growth cabinet at 25 °C with a light cycle of 12 h fluorescent light and 12 h darkness for 14 d. Finally, conidia of each strain were collected from diseased leaves and cultured on a new PDA plate. They were then checked for morphological characteristics to confirm Koch's postulates 4 .
Genealogical concordance phylogenetic species recognition analysis. We used the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model, as described by Liu, et al. 2 , by performing a pairwise homoplasy index (Φ w , PHI) test to analyze related but ambiguous species in the phylogenetic tree. The PHI test was performed in Splits Tree 4 49,50 using 6-locus concatenated datasets (ITS, ACT, GAPDH, CAL, TUB2 and GS), and both the LogDet transformation and splits decomposition options were selected 51 . PHI results below a 0.05 threshold (Φ w < 0.05) were considered indicative of significant recombination in the dataset.
Prevalence of Colletotrichum species. The Isolation Rate (IR), calculated as IR% = (Cx/Ct) × 100, where Cx is the number of isolates of the same species and Ct was the total number of isolates 52 , was determined as a measure of the prevalence of Colletotrichum species in Ca. sinensis in China.