Research Article
Print
Research Article
Endophytic Colletotrichum (Sordariomycetes, Glomerellaceae) species associated with Citrus grandis cv. “Tomentosa” in China
expand article infoJia-Wei Liu‡, Ishara S. Manawasinghe‡, Xuan-Ni Liao‡, Jin Mao‡, Zhang-Yong Dong‡, Ruvishika S. Jayawardena§, Dhanushka N. Wanasinghe|, Yong-Xin Shu‡§, Mei Luo‡
‡ Zhongkai University of Agriculture and Engineering, Guangzhou, China
§ Mae Fah Luang University, Chiang Rai, Thailand
| Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China

Corresponding author: Ishara S. Manawasinghe ( ishara9017@gmail.com )

Corresponding author: Mei Luo ( 08luomei@163.com )

Academic editor: Malgorzata Ruszkiewicz-Michalska
© 2023 Jia-Wei Liu, Ishara S. Manawasinghe, Xuan-Ni Liao, Jin Mao, Zhang-Yong Dong, Ruvishika S. Jayawardena, Dhanushka N. Wanasinghe, Yong-Xin Shu, Mei Luo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Liu J-W, Manawasinghe IS, Liao X-N, Mao J, Dong Z-Y, Jayawardena RS, Wanasinghe DN, Shu Y-X, Luo M (2023) Endophytic Colletotrichum (Sordariomycetes, Glomerellaceae) species associated with Citrus grandis cv. “Tomentosa” in China. MycoKeys 95: 163-188. https://doi.org/10.3897/mycokeys.95.87121
Open Access

Abstract

Colletotrichum species are well-known plant pathogens, saprobes, endophytes, human pathogens and entomopathogens. However, little is known about Colletotrichum as endophytes of plants and cultivars including Citrus grandis cv. “Tomentosa”. In the present study, 12 endophytic Colletotrichum isolates were obtained from this host in Huazhou, Guangdong Province (China) in 2019. Based on morphology and combined multigene phylogeny [nuclear ribosomal internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), histone H3 (his3) actin (act), beta-tubulin (β-tubulin) and glutamine synthetase (gs)], six Colletotrichum species were identified, including two new species, namely Colletotrichum guangdongense and C. tomentosae. Colletotrichum asianum, C. plurivorum, C. siamense and C. tainanense are identified as being the first reports on C. grandis cv. “Tomentosa” worldwide. This study is the first comprehensive study on endophytic Colletotrichum species on C. grandis cv. “Tomentosa” in China.

Keywords

Chinese traditional medicinal plants, new ascomycete, phylogeny, six new host records, taxonomy, two new species

Introduction

Citrus grandis cv. “Tomentosa” is an important traditional medicinal plant which contains essential oils, flavonoids and polysaccharides. In traditional Chinese medicine, Citrus grandis cv. “Tomentosa” has been used for treatments due to its anti-inflammatory effect (Zhao et al. 2017). It has also been used in the treatment of coughs, asthma, food stagnation, vomiting and other symptoms (Peng et al. 2019). Current research on C. grandis cv. “Tomentosa” is still focused on medicinal components, with a relatively long timescale needed to accumulate the effective ingredient. It is likely that the endophytic community living inside the host affects the metabolites of the plant. Dai et al. (2017) found that nine species of Taxus endophytic fungi could produce paclitaxel. Hasan et al. (2022) found endophytic fungi, Penicillium crustosum from Annona muricata L. has anti-cancer activity against HeLa cells. Therefore, it is necessary to study the effects of the endophytic community associated with these traditional medicinal plants. The findings of this research can help in finding potential new natural medicines and form the basis for subsequent screening of strains.

Colletotrichum Corda (1831), belongs to Glomerellaceae (Sordariomycetes), which comprises plant pathogens, endophytes and saprobes on a wide range of hosts (Christy et al. 2020; Jayawardena et al. 2021). They are one of the most often isolated endophytic fungal groups encompassing a wide range of hosts. These endophytic Colletotrichum species have some advantages to the host, such as providing disease resistance, drought tolerance and promoting growth of the host (Hacquard et al. 2016; Dini-Andreote 2020). Endophytic species can also change their lifestyle and become pathogenic (Photita et al. 2004). Liu et al. (2022) accepted 280 Colletotrichum species, from which 23 species have been identified from Citrus spp. Therefore, studying diversity and clarifying taxonomic affinities of isolates can answer a range of important ecological and evolutionary questions. Although there have been several studies on Colletotrichum species associated with Citrus (Damm et al. 2012; Huang et al. 2013; Guarnaccia et al. 2017), there is still imprecise identification of endophytes of Colletotrichum species on C. grandis cv. “Tomentosa”.

Species delineation of Colletotrichum is challenging because there are few distinctive morphological characters available (Bhunjun et al. 2021). Colletotrichum is characterised as an intricate genus with 16 species complexes and 15 singleton species (Liu et al. 2022). Although host specificity was the most used character for identification in early studies, current taxonomic classifications and species delineations are based on morphology alongside multi-locus phylogeny (Bhunjun et al. 2021; Jayawardena et al. 2021; Liu et al. 2022). Phylogenetic analyses of Colletotrichum have been based on ITS, gapdh, chs-1, act and β-tubulin and multi-loci phylogeny. However, some complexes that cannot be distinguished by five loci required additional loci for identification (Bhunjun et al. 2021; Jayawardena et al. 2021; Liu et al. 2022). Therefore, the selection of gene combinations depends on the species complex (Jayawardena et al. 2021).

The objectives of this study were to isolate and identify the dominant endophytic Colletotrichum species associated with healthy C. grandis cv. “Tomentosa” in Huazhou, Guangdong, China. Morphology, molecular phylogeny and recombination analysis were used for the species characterisation. This resulted in two new species and six new host records. Detailed descriptions and coloured illustrations have been given for the novel taxa identified.

Materials and methods

Sample collection and isolation

Healthy leaves and twigs of Citrus grandis cv. “Tomentosa” were randomly collected from a Citrus orchard in Huazhou, Guangdong Province, China (21°66'N, 110°63'E). A total of 20 trees were randomly selected for the collection. Ten samples were collected from the upper, middle and lower parts of each plant. Asymptomatic samples were packed into zip-lock bags in a foam box with ice and were then brought to the plant pathology laboratory of Zhongkai University of Agriculture and Engineering where they were preserved at 4 °C before processing. Isolation was undertaken within 48 h after collection, following the procedure by Dong et al. (2021).

Endophytic fungi were isolated following the methods described by da Silva et al. (2020). The samples were initially washed with running tap water followed by sterile water. The leaves were cut into 3 mm × 3 mm segments, while the twigs were cut into 3 mm long pieces. Each piece was then surface sterilised by being dipped sequentially into 75% ethanol for 30 s, 2.5% NaClO (sodium hypochlorite) for 30–60 s (leaves for 30 s, twigs for 60 s), before being rinsed three times with sterilised water. They were then dried on sterilised filter paper. The cuttings were then placed on potato dextrose agar (PDA: 200 g potato, 20 g dextrose, 20 g agar per 1 litre of water). Plates were incubated at 25 °C with 12 h of dark and 12 h of fluorescent light. Pure cultures were cultured on PDA for 7 to 14 days at 25 °C. All the pure cultures obtained in this study were deposited in the Culture Collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). The living cultures (ex-type) of new species identified in this study were deposited in the Culture Collection of the Chinese Academy of Sciences (CGMCC, C. guangdongense for the holotype with CGMCC 3.24127 and C. tomentosae with CGMCC 3.24128). Herbarium materials as dry cultures of novel species were deposited in the Herbarium of Zhongkai University of Agriculture and Engineering (ZHKU). The strain numbers belonging to all isolates (from ZHKUCC 21-0095 to 21-0106 and 22-041 to 22-0042) for this study are presented in Appendix 1.

Morphological studies

For macro- and micro-morphological characterisation, 5 mm diameter agar plugs were cut from all the actively growing pure cultures on PDA and were then transferred on to new PDA. The colony diameter was measured daily for 5–9 d to determine the growth rate (mm/day) on the PDA at 25 °C under 12 h of dark and 12 h of fluorescent light. Appressoria formation was observed following Johnston and Jones (1997) and Cai et al. (2009). The cultures were incubated for 2–4 weeks and morphological characters (appressoria, ascomata, asci, ascospores, conidiophores and conidia) were observed. Macro-morphological characters were photographed using a SteREO Discovery.V20 (Zeiss, Germany) stereomicroscope. Fruiting bodies were cut into thin sections by a CM1860 freezing sliding microtome (LEICA, Germany). Digital images were captured with an Eclipse 80i photographic microscope (Nikon, Japan). Measurements were taken using NIS Elements BR 3.2 (Nikon, Japan). The mean values were calculated with their standard deviations (SDs).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from mycelium grown on PDA and incubated for approx. seven days at 25 °C using the CTAB method (Sun et al. 2009). The ITS region was amplified and sequenced. The resulting sequences were subjected to BLASTn searches in GenBank (https://blast.ncbi.nlm.nih.gov) to identify them to the genus level. Once the BLAST results had confirmed isolates as being Colletotrichum species, an additional six gene regions, namely gapdh, chs-1, his3, act, β-tubulin and gs, were amplified and sequenced. The PCR conditions for each primer pair are given below (Table 1). The amplicons were observed on 1% agarose electrophoresis gel and positive amplicons were sequenced by Tianyi Huiyuan Biotechnology Co., Ltd., Guangzhou, China. The initial sequence quality was checked using BioEdit v. 7.25 (Hall 2006). A total of 66 sequences generated in this study were submitted to GenBank (Appendix 1).

Table 1.
Download as
CSV
XLSX

Gene regions, respective primer pairs and PCR protocols used in the study.

Gene Primer pair Optimised PCR protocols References
ITS ITS1 94 °C: 5 min (94 °C: 30 s, 53 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min White et al. (1990)
ITS4
gapdh GDF 94 °C: 5 min (94 °C: 30 s, 60 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min Guerber et al. (2003)
GDR
chs-1 CHS-79F 94 °C: 5 min (94 °C: 30 s, 49 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min Carbone and Kohn (1999)
CHS-345R
his3 CYLH3F 94 °C: 5 min (94 °C: 30 s, 53 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min Crous et al. (2004)
CYLH3R
act ACT-512F 94 °C: 5 min (94 °C: 30 s, 54 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min Carbone and Kohn (1999)
ACT-783R
β-tubulin Bt2a 94 °C: 5 min (94 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 32 cycles, 72 °C: 10 min Glass and Donaldson (1995)
Bt2b
gs GSF1 94 °C: 5 min (94 °C: 30 s, 60 °C: 60 s, 72 °C: 1 min) × 35 cycles, 72 °C: 30 min Guerber et al. (2003)
GSR1

Phylogenetic analysis

For the phylogenetic analysis, reference sequences for Colletotrichum species and related taxa were obtained from NCBI GenBank (Appendix 1). Each locus was aligned together with the sequences obtained in the present study using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/) (Katoh et al. 2019). Alignments were checked and manually adjusted where necessary with BioEdit v. 7.25 (Hall 2006). Alignment results were automatically trimmed using the Trimal tool in PhyloSuite (v.1.2.1) (Zhang et al. 2020). Phylogenetic analyses were conducted according to Maximum Likelihood (ML) in RAxML (Silvestro and Michalak 2010), maximum parsimony (MP) in PAUP (v.4.0) (Swofford 2002) and Bayesian analyses (BP) in MrBayes (v. 3.1.2) (Ronquist and Huelsenbeck 2003). The final analyses of the Colletotrichum gloeosporioides complex were made using the concatenated dataset of act, chs-1, gapdh, ITS, β-tubulin and gs, following Liu et al. (2022). The other two complexes: Colletotrichum orchidearum complex and Colletotrichum magnum complex were analysed using act, chs-1, gapdh, his3, ITS and β-tubulin, following Liu et al. (2022).

In the MP analysis, ambiguous regions were excluded and gaps were treated as missing data. Tree stability was evaluated with 1,000 bootstrap replications. Zero-length branches were collapsed and all the parsimonious trees were saved. Tree parameters: tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI) were calculated. Kishino-Hasegawa tests (KHT) were conducted to evaluate the differences between the trees inferred as being under different optimality criteria (Kishino and Hasegawa 1989). MrModelTest v. 2.3 (Nylander 2004) was used to determine the evolutionary models for each locus to be used in Bayesian and Maximum Likelihood analyses. The Maximum Likelihood analyses were conducted using RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010). The GTR + I + G evolutionary model was employed with 1,000 non-parametric bootstrapping iterations. Bayesian analysis was performed in MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Posterior probabilities (PPs) were determined using Markov Chain Monte Carlo sampling (MCMC). Six simultaneous Markov chains were run for 108 generations, with sampling the trees at each 1000th generation. From the 10,000 trees obtained, the first 2,500 representing the burn-in phase were discarded. The remaining 7,500 trees were then used to calculate the posterior probabilities (BPs) in a majority rule consensus tree. Taxonomic novelties were submitted to the FacesofFungi database (Jayasiri et al. 2015) and Index Fungorum (http://www.indexfungorum.org). The final sequence alignments generated in this study were submitted to TreeBASE (http://www.treebase.org) under the submission ID 29668.

Pairwise homoplasy index (PHI) analysis

Recombination analyses were conducted to provide evidence for genetic distances for two new species identified, based on the phylogenetic analyses. The pairwise homoplasy index (Φw) (Bruen et al. 2006) was calculated in SplitsTree (version 4.1.4.4) using Kimura’s two-parameter (K2P) models for low genetic distance datasets. The standard deviation of split frequencies in the PHI test results (Φw) < 0.05 indicates significant recombination within the dataset.

Results

In total, 12 endophytic Colletotrichum strains were obtained: seven from leaves and five from twigs. Based on the initial species identification undertaken through BLASTn searches, taxa isolated in this study belonged to three species complexes, namely the C. gloeosporioides, C. magnum and C. orchidearum complexes.

Colletotrichum gloeosporioides complex

In the present study, eight Colletotrichum isolates were initially recognised as belonging to the C. gloeosporioides complex. Phylogenetic analyses of a combined act (1–281), chs-1 (282–573), gapdh (574–850), ITS (851–1384), β-tubulin (1385–1846) and gs (1847–2616) sequence alignment were conducted using 89 Colletotrichum strains. Colletotrichum boninense (ICMP 17904) and C. hippeastri (ICMP 17920) were used as outgroup taxa. The best-scoring MP tree is shown in Fig. 1. The dataset comprised 2,616 characters with 1,757 constant characters, 370 parsimony-informative and 489 parsimony-uninformative characters. The maximum number of trees generated was 1,000 and the most parsimonious trees had a length of 1,492 steps (CI = 0.707, RI = 0.848, RC = 0.600, HI = 0.293). The final ML tree topology was in line with the MP and BP trees. The best-scoring ML tree has a final likelihood value of −12,639.274168. The matrix consisted of 1,060 distinct alignment patterns, with 15.26% undetermined characters or gaps. For the Bayesian Inference, the TPM2uf+G model was selected for act, TIM1ef+G for chs-1, HKY+I for gapdh, TrNef+I+G for ITS, TIM3ef+G for β-tubulin and TVM+G for gs. In the phylogenetic analysis, three isolates (ZHKUCC 21-0103, ZHKUCC 21-0104 and ZHKUCC 22-0041) from this study developed a sister clade from other known species. The new species of C. tomentosae showed a close relationship to C. syzygicola (MFLUCC 10-0624) with 92% ML, 90% MP and 1.00 BP support. Three strains (ZHKUCC 21-0096, ZHKUCC 21-0097 and ZHKUCC 21-0098) from this study cluster together with C. siamense (ICMP 18578) with 0.99 BP support in the multi-locus phylogenetic tree. The strain ZHKUCC 21-0095 was clustered with C. asianum (ICMP 18580) with 100% ML, 100% MP and 1.00 BP in the phylogenetic tree. A single strain (ZHKUCC 21-0101) belongs to C. tainanense (CBS 143666) with 93% ML, 83% MP and 1.00 BP support. The PHI value indicates that there is no significant evidence for recombination amongst the species used in this analysis (p = 1.0) (Fig. 2). Based on this, we identified these isolates as novel Colletotrichum species. Species descriptions and illustrations of the new species, identified from the C. gloeosporioides complex, are presented below.

Figure 1. 

The most parsimonious tree of the gloeosporioides complex developed using combined act, chs-1, gapdh, ITS, β-tubulin and gs sequences. Colletotrichum boninense and C. hippeastri were used as outgroup taxa. Bootstrap values equal to or greater than 60% in MP and ML and BP equal to or greater than 0.95 are shown as MP/ML/BP above the respective node. The isolates belonging to the current study are given in blue for known species and new species are shown in red. Ex-type strains are noted with T.

Figure 2. 

Colletotrichum tomentosae (ZHKUCC 21-0103, holotype) A, B upper and reverse side of cultures on PDA seven days after inoculation C, D conidiophores with developing conidia E, F conidia G, H appressoria. Scale bars: 10 μm (C–H).

Taxonomy

Colletotrichum asianum Prihast., L. Cai & K.D. Hyde, Fungal Diversity 39: 96 (2009)

Index Fungorum Number: IF515408
Facesoffungi Number: FoF10689

Material examined

China, Guangdong Province, Huazhou, isolated from healthy twigs of Citrus grandis cv. “Tomentosa”, May 2019, Y.X. Shu, (dried culture ZHKU 21-0084); living culture ZHKUCC 21-095.

Notes

The single isolate (ZHKUCC 21-0095) obtained in this study clustered with the Colletotrichum asianum ex-type strain (ICMP: 1850) with 100% ML, 100% MP and 1.0 BP values (Fig. 1). Morphologically, the isolate obtained in this study is similar to those in the original description of C. asianum (Prihastuti et al. 2009). This is the first report of C. asianum on C. grandis cv. “Tomentosa”.

Colletotrichum siamense Prihast., L. Cai & K.D. Hyde, Fungal Diversity 39: 98 (2009)

Index Fungorum Number: IF515410
Facesoffungi Number: FoF03599

Material examined

China, Guangdong Province, Huazhou, isolated from healthy leaf of Citrus grandis cv. “Tomentosa”, May 2019, Y.X. Shu, (dried culture ZHKU 21-0085); living cultures ZHKUCC 21-0096, ZHKUCC 21-0097, ZHKUCC 21-0098).

Notes

Three isolates obtained in this study (ZHKUCC 21-0096–100) clustered with the ex-type strain of Colletotrichum siamense (ICMP: 18578) with 67% MP and 0.99 BP values (Fig. 1). Morphologically, the isolate obtained in this study is similar to those in the original description of C. siamense (Prihastuti et al. 2009). This is the first report of C. siamense on C. grandis cv. “Tomentosa”.

Colletotrichum tainanense de Silva, Crous & P.W.J. Taylor, IMA Fungus 10(1): 23 (2019)

Index Fungorum Number: IF827692
Facesoffungi Number: FoF10690

Material examined

China, Guangdong Province, Huazhou, isolated from healthy leaf of Citrus grandis cv. “Tomentosa”, May 2019, Y.X. Shu, (dried culture ZHKU 21-0086); living culture ZHKUCC 21-0101.

Notes

A single isolate obtained in this study (ZHKUCC 21-0101) clustered with the Colletotrichum tainanense (CBS 143666) ex-type strain with 93% ML, 83% MP bootstrap and 1.0 BP values (Fig. 1). Morphologically, the isolate obtained in this study is similar to those in the original description of C. tainanense (de Silva et al. 2019). To our knowledge, this is the first report of C. tainanense on C. grandis cv. “Tomentosa”.

Colletotrichum tomentosae J.W. Liu, Manawas. & M. Luo, sp. nov.

Index Fungorum Number: IF559482
Facesoffungi Number: FoF10692
Fig. 2

Etymology

The epithet refers to the cultivar of the host plant – Citrus grandis cv. “Tomentosa”.

Holotype

ZHKUCC 21-0103.

Description

Endophytic in C. grandis cv. “Tomentosa” leaf. Sexual morph: not observed. Asexual morph: Conidiophores 20–40 × 3–5 μm (x− = 29.8 ± 5.5 × 3.7 ± 0.6 μm, n = 30), hyaline, cylindrical, 1–3-celled, unbranched or branched at the base. Conidia 10–20 × 3–6 μm (x− = 12.5 ± 1.6 × 4.4 ± 0.6 μm, n = 50), 1–2-guttulate, aseptate, straight, hyaline, smooth-walled, middle part cylindrical both ends obtuse, middle part occasionally shrinkage or bulging. Appressoria 5–15 × 5–10 μm (x− = 10 ± 1.8 × 7 ± 1.5 μm, n = 50) solitary or in loose groups, light brown to medium brown, Ellipsoidal to subcircular or irregular-shaped.

Cultural characteristics

Colonies on PDA reach 70 mm diam. in seven days, with 10–11 mm/day (x− = 10 mm, n = 6) growth rate. Colonies flat with entire margin, floccose cottony, surface grey in the centre with glaucous margin. Reverse buff in the centre with off-white margin.

Material examined

China, Guangdong Province, Huazhou, isolated from a healthy leaf of Citrus grandis cv. “Tomentosa”, May 2019, Y.X. Shu, (dried cultures ZHKU 21-0088 holotype); ex-type culture ZHKUCC 21-0103 (= CGMCC 3.24128), ex-isotype ZHKUCC 21-0104, ZHKUCC 22-0041).

Notes

In the phylogenetic analysis of combined six genes, Colletotrichum tomentosae formed an independent clade (Fig. 1). This species is phylogenetically distinct from C. syzygicola. Morphologically, appressoria developed by C. syzygicola (DNCL021; Udayanga et al. (2013)) are longer than C. tomentosae (5–15 × 18–24 μm vs. 18–24 μm). Colletotrichum tomentosae has longer conidiophores (20–40 × 3–5 vs. 12–16 × 4–5 μm). This species can be distinguished from C. syzygicola by 32 nucleotide differences (1/511 in the ITS region, 2/229 in the gapdh region, 7/242 in the act region and 22/906 in the gs region). The PHI test revealed no significant evidence for a recombination (p = 1.0) event amongst C. syzygicola and its closely-related taxa (Fig. 3). Therefore, we have described this fungus as a novel species.

Figure 3. 

PHI analysis of combined ITS, gapdh, chs-1, act and β-tubulin sequence data. PHI test result (Φw) < 0.05 indicates significant recombination within the dataset.

Colletotrichum orchidearum complex

In the present study, a single isolate was recognised as belonging to the Colletotrichum orchidearum complex. The phylogenetic analysis of a combined ITS, gapdh, chs-1, his3, act and β-tubulin sequence alignment was constructed using 30 Colletotrichum strains. Colletotrichum magnum (CBS 519.97) and C. brevisporum (BCC 38876) were used as the outgroup. The best scoring MP tree is presented in Fig. 4. The dataset comprised 2,422 characters with 2,055 constant characters and 242 parsimony-informative and 125 parsimony-uninformative characters. The maximum number of trees generated was 1,000 and the most parsimonious trees had a length of 475 steps (CI = 0.874, RI = 0.904, RC = 0.790, HI = 0.126). The final ML tree topology was similar to the MP and BP trees. The best-scoring ML tree with a final likelihood value of – 6,065.417493 is shown in Fig. 4. The matrix comprised 479 distinct alignment patterns, with 10.74% of undetermined characters or gaps. The estimated base frequencies were as follows: A = 0.214401, C = 0.319513, G = 0.254583, T = 0.211503; substitution rates AC = 0.9523776, AG = 3.421321, AT = 0.568275, CG = 0.738898, CT = 6.093168, GT = 1.000000; gamma distribution shape parameter a = 0.814817. For the Bayesian Inference, the TPM1uf+I model was selected for act, GTR+I+G for chs-1, HKY+I for gapdh, TIM2+G for his3, TIM1+I for ITS and HKY+G for β-tubulin. In the phylogenetic analysis, isolates from this study clustered together with C. plurivorum. The species description and illustration are given below.

Figure 4. 

The most parsimonious tree for Colletotrichum orchidearum complex using a combined act, chs-1, gapdh, his3, ITS, and β-tubulin sequences. The tree is rooted to Colletotrichum brevisporum and C. magnum. Bootstrap support values equal to or greater than 60% in MP and ML and BP equal to or greater than 0.95 are shown as MP/ML/BP above the respective nodes. The isolates belonging to the current study is given in blue. Ex-type strains are noted with T.

Colletotrichum plurivorum Damm, Alizadeh & Toy. Sato, Studies in Mycology 92: 31 (2019)

Index Fungorum Number: IF824228
Facesoffungi Number: FoF10691

Material examined

China, Guangdong Province, Huazhou, isolated from healthy leaf of Citrus grandis cv. “Tomentosa”, May 2019, YX Shu, (dried culture ZHKU 21-0087), living culture ZHKUCC 21-0102.

Notes

A single isolate (ZHKUCC 21-0102) obtained in this study clustered with the ex-type strain of C. plurivorum (CBS 125474) with 99% ML, 97% MP and 1.0 BP support values (Fig. 4). Morphologically, the isolate obtained in this study is similar to those in the original description of C. plurivorum (Damm et al. 2019). Colletotrichum plurivorum was first introduced by Damm et al. (2019) as a pathogen on Capsicum annuum fruits and subsequently, has been reported as pathogens causing anthracnose or leaf spot diseases (Farr and Rossman 2022). This is the first report of C. plurivorum as an endophyte on Citrus grandis cv. “Tomentosa”.

Colletotrichum magnum complex

Three of our isolates were initially recognised as belonging to the Colletotrichum magnum species complex. The phylogenetic analysis of combined act, chs-1, gapdh, his3, ITS and β-tubulin sequence alignment was conducted using 17 Colletotrichum strains. Colletotrichum orchidearum (CBS 135131) and C. cliviicola (CBS 125375) were used as outgroup taxa. The best-scoring MP tree is given in Fig. 5. The dataset consisted of 2,296 characters with 2,013 constant characters and 196 parsimony-informative and 87 parsimony-uninformative characters. The maximum number of trees generated was 1,000 and the most parsimonious trees had a length of 350 steps (CI = 0.883, RI = 0.882, RC = 0.779, HI = 0.117). The final ML tree topology was similar to the MP and BP trees. The best-scoring ML tree had a −5198.901460 final likelihood value. The ML matrix comprised 258 distinct alignment patterns, with 6.18% undetermined characters or gaps. For the Bayesian Inference, the HKY model was selected for act, TIM2ef+G for chs-1, HKY+G for gapdh, TrN+G for his3, TIM1+I for ITS and TIM1+G for β-tubulin. In the phylogenetic analysis, isolates from this study developed to show the presence of an independent clade with high bootstrap and BP support. To confirm that these isolates belonged to novel species, the PHI index was calculated. The PHI test revealed no significant evidence for recombination (p = 1.0) amongst the taxon from this study and its closely-related taxa (Fig. 6).

Figure 5. 

The most parsimonious tree of the Colletotrichum magnum complex using combined act, chs-1, gapdh, his3, ITS and β-tubulin sequences. Colletotrichum cliviicola and C. orchidearum were used as outgroup taxa. Bootstrap support values equal to or greater than 60% in MP and ML and BP equal to or greater than 0.95 are shown as MP/ML/BP above the respective nodes. The isolates of the novel taxon described in the current study are highlighted in red. Ex-type strains are noted with T.

Figure 6. 

PHI analysis of combined act, chs-1, gapdh, his3, ITS and β-tubulin sequence data. A PHI test result (Φw) < 0.05 indicates significant recombination within the dataset.

Colletotrichum guangdongense J.W. Liu, Manawas. & M. Luo, sp. nov.

Index Fungorum Number: IF559483
Facesoffungi Number: FoF10693
Fig. 7

Etymology

The epithet refers to the Guangdong Province where the fungus was collected.

Holotype

ZHKUCC 21-0105

Description

Isolated from a Citrus grandis cv. “Tomentosa” twig. Sexual morph: not observed. Asexual morph. Conidiomata formed directly on hyphae, conidial masses abundant, coral. Setae pale to dark brown, smooth-walled, straight or flexuous, 2–4-septate, 60–136 μm long, basal cell cylindrical, 3.5–4.8 μm diam., tip more or less acute. Conidiophores 20–70 × 3–7 μm (x− = 39.1 ± 10.7 × 4.7 ± 0.7 μm, n = 50), cylindrical, hyaline, smooth-walled, 1–4-celled, unbranched or branched at the base. Conidia 14–22 × 3–7 μm (x− = 18.2 ± 1.6 × 4.9 ± 0.5 μm, n = 50), straight, hyaline and smooth-walled. Appressoria 7–12 × 5–10 μm (x− = 10.2 ± 1.8 × 7.3 ± 0.9 μm, n = 50), single, medium brown, round, oval to irregular in outline.

Figure 7. 

Colletotrichum guangdongense (ZHKUCC 21-0105, holotype) A, B upper and reverse sides of cultures on PDA seven days after inoculation C, D conidioma E, F setae G, H conidiophores I conidia J, K appressoria. Scale bars: 1 mm (C, D); 10 μm (E–K).

Cultural characteristics

Colonies on PDA reach 65 mm diameter after seven days, with 8–11 mm/day (x− = 10 mm, n = 6) growth rate. Colonies circular, slightly raised, flat, with pale coral red to light pink margin. Reverse dark vermillion to light ivory. Colonies on SNA flat, with entire margin, glaucous, reverse buff. Sporulates after 14 d on SNA.

Material examined

China, Guangdong Province, Huazhou, isolated from healthy twigs of Citrus grandis cv. “Tomentosa”, May 2019, Y.X. Shu (dried cultures ZHKU 21-0089 holotype); living cultures ZHKUCC 21-0105 (= CGMCC 3.24127) ex-type, ZHKUCC 21-0106 and ZHKUCC 22-0042 isotype).

Notes

In the phylogenetic analysis of combined act, chs-1, gapdh, his3, ITS and β-tubulin sequences, three isolates (ZHKUCC 21-0105, ZHKUCC 21-0106 and ZHKUCC 22-0042) obtained in this study developed a sister clade to Colletotrichum sp. MH0413 with 89% ML bootstrap, 60% MP bootstrap and 1.00 BP (Fig. 5). Colletotrichum guangdongense is also closely related to C. magnum (CBS 519.97) and C. panamense (CBS 125386). It can be distinguished from C. magnum (CBS 519.97) by having smaller conidia (10–20 × 4–6 μm vs. 17–24 × 3.5–5 μm) and longer conidiophores (20–70 μm vs. 20 μm) (Damm et al. 2019). Colletotrichum panamense (CBS 125386) has conidiophores shorter than C. guangdongense (30 μm vs. 20–70 μm). Colletotrichum guangdongense can be distinguished from C. magnum (CBS 519.97) also by 39 different nucleotides (4/538 in the ITS region, 9/204 in the gapdh region, 3/251 in the chs-1 region, 9/235 act, 5/431 tub2 and 9/403 his3) and from C. panamense (CBS 125386) by 39 different nucleotides (4/538 in the ITS region, 9/204 in the gapdh region, 3/251 in the chs-1 region, 9/235 act, 2/431 tub2 and 12/403 his3). The PHI test revealed no significant recombination event amongst C. guangdongense and its closely-related taxa (Fig. 6). Therefore, we have described this fungus as a novel species.

Discussion

In the present study, endophytic Colletotrichum species were isolated from Citrus grandis cv. “Tomentosa” in Guangdong Province, China. Guangdong Province has a mild subtropical monsoon climate with abundant rainfall and high average annual temperatures. Vigorous fruit trees provide suitable conditions for the colonisation of Colletotrichum species (Jayawardena et al. 2021). When the host is healthy, the endophyte has a symbiotic relationship with the host (Jayawardena et al. 2021). However, sometimes the interaction between the plant and the endophyte can switch from mutualistic to antagonistic or pathogenic (da Silva et al. 2020). Thus, the identification and characterisation of endophytic fungi are necessary. Based on the phylogenetic analysis using a combined seven loci (ITS, gapdh, chs-1, act, his3, tub2 and gs), 12 isolates from this study were identified as being six distinct species within the three Colletotrichum species complexes (Figs 1, 4, 5). These results included two new species, namely C. guangdongense, C. tomentosae and three new host records for C. asianum, C. plurivorum and C. tainanense. Colletotrichum siamense has also been identified and described as being associated with Citrus. The present study has re-affirmed that more than one Colletotrichum species can colonise a single host, which is consistent with the conclusion of Damm et al. (2012).

Species belonging to the C. gloeosporioides complex were often found as endophytes (Damm et al. 2012; Weir et al. 2012; Jayawardena et al. 2016). Here, we identified seven strains representing four species as endophytes from the C. gloeosporioides complex. Colletotrichum siamense was previously reported as an epiphyte and an endophyte associated with coffee berries in northern Thailand (Prihastuti et al. 2009) and tea plants in China (Liu et al. 2015). Colletotrichum siamense has also been reported as a pathogen of many plants (Liu et al. 2022). In the present study, this species was isolated from leaves. Liu et al. (2015) identified six species from symptomatic and asymptomatic leaf tissue, all of which belonged to the C. gloeosporioides species complex, namely C. camelliae, C. fructicola, C. gloeosporioides, C. jiangxiense and C. siamense, providing convincing evidence that these species could switch their lifestyle from endophytic to pathogenic. Therefore, further studies are necessary to understand the pathogenicity of these endophytic strains and the factors affecting these taxa becoming pathogenic on Citrus.

Colletotrichum species belonging to the C. magnum and C. orchidearum complexes were found on tropical or subtropical plants (Damm et al. 2019). It has been proposed that some of these species might be host- and region-specific (Damm et al. 2019). Colletotrichum plurivorum is widely distributed in several hosts and most of them are pathogens. This study is the first report of the species from Citrus. Here, we introduce a new taxon belonging to the C. magnum species complex. Whether it is host-specific or not needs further confirmation.

Endophytic fungal colonisation might vary in different tissues of the same plant (Taylor et al. 1999; Huang et al. 2015). Different fungal genera could have different tissue specificities and preferences. In the present study, endophytes were isolated from leaves and twigs. Additionally, there were higher numbers of Colletotrichum species from leaves in Citrus (Hakimeh et al. 2019) and some other plants like Dendrobium (Chen et al. 2011; Ma et al. 2018). Huang et al. (2015) and Dong et al. (2021) have observed that endophytic Diaporthe species are less abundant on leaves, whereas endophytic Colletotrichum species are abundantly isolated from the Dendrobium spp. leaves (Chen et al. 2011; Ma et al. 2018). These variations may be the result of differences in the tissue organisational structure, different nutrition contents of each tissue type or the lifestyle of each genus, locality or season (Zhou et al. 2014; Huang et al. 2015). To date, the reasons for these variations are not yet known.

Overall, in the present study, two novel endophytic Colletotrichum species have been described and illustrated. Our study is the first comprehensive study on endophytic Colletotrichum species associated with Citrus grandis cv. “Tomentosa”. Moreover, our molecular data and novel species introduced in this study contribute to understanding the diversity and biology of the genus Colletotrichum. These results provide an important resource and basis for plant pathologists and fungal taxonomists. However, future studies are necessary to understand the lifestyle changes of the endophytic taxa towards the pathogenicity, as well as the effects of fungus-related medicinal properties of Citrus grandis cv. “Tomentosa”.

Acknowledgements

We would like to thank Dr Shaun Pennycook, Nomenclature Editor of Mycotaxon, for his guidance on the species names. M. Luo would like to thank for the grant from the Guangdong Rural Science and Technology Commissioner project (KTP20210313) and the Research Project of Innovative Institute for Plant Health (KA21031H101). Z.Y. Dong would like to thank the Key Realm R & D Program of Guangdong Province (2018B020205003). R.S. Jayawardena would like to thank Thailand Science Research and Innovation, grant number 652A01003 entitled ‘Biodiversity, taxonomy, phylogeny and evolution of Colletotrichum on Avocado, Citrus, Durian and Mango in northern Thailand’. Ishara S Manawasinghe would like to thank the Research Project of the Innovative Institute for Plant Health (KA21031H101) and the project of the Zhongkai University of Agriculture and Engineering, Guangzhou, China (KA210319288).

References

  • Bhunjun CS, Phukhamsakda C, Jayawardena RS, Jeewon R, Promputtha I, Hyde KD (2021) Investigating species boundaries in Colletotrichum. Fungal Diversity 107(1): 107–127. https://doi.org/10.1007/s13225-021-00471-z
  • Chen J, Hu KX, Hou XQ, Guo SX (2011) Endophytic fungi assemblages from 10 Dendrobium medicinal plants (Orchidaceae). World Journal of Microbiology & Biotechnology 27(5): 1009–1016. https://doi.org/10.1007/s11274-010-0544-y
  • Christy JS, Balraj A, Agarwal A (2020) A Rare Case of Colletotrichum truncatum keratitis in a young boy with complete healing after medical treatment. Indian Journal of Medical Microbiology 38(3–4): 475–477. https://doi.org/10.4103/ijmm.IJMM_20_146
  • Corda ACI (1831) Die Pilze Deutschlands. In: Sturm J (Ed.) Deutschlands Flora in Abbildungen nach der Natur mit Beschreibungen. Sturm, Nürnberg 3: 33–64.
  • da Silva LL, Moreno HLA, Correia HLN, Santana MF, de Queiroz MV (2020) Colletotrichum: Species complexes, lifestyle, and peculiarities of some sources of genetic variability. Applied Microbiology and Biotechnology 104(5): 1891–1904. https://doi.org/10.1007/s00253-020-10363-y
  • Dai HY, He D, Liu MZ (2017) Progress and trends on researches of taxol-producing endophytic fungi. Western Forestry Science 46: 169–187. [in Chinese]
  • Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012) The Colletotrichum acutatum species complex. Studies in Mycology 73: 37–113. https://doi.org/10.3114/sim0010
  • Damm U, Sato T, Alizadeh A, Groenewald JZ, Crous PW (2019) The Colletotrichum dracaenophilum, C. magnum and C. orchidearum species complexes. Studies in Mycology 92(1): 1–46. https://doi.org/10.1016/j.simyco.2018.04.001
  • de Silva DD, Groenewald JZ, Crous PW, Ades PK, Nasruddin A, Mongkolporn O, Taylor PWJ (2019) Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia. IMA Fungus 10(1): 1–8. https://doi.org/10.1186/s43008-019-0001-y
  • Dong ZY, Manawasinghe IS, Huang YH, Shu YX, Phillips AJL, Dissanayake DJ, Hyde KD, Xiang MM, Luo M (2021) Endophytic Diaporthe associated with Citrus grandis cv. Tomentosa in China. Frontiers in Microbiology 11: e609387. https://doi.org/10.3389/fmicb.2020.609387
  • Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61(4): 1323–1330. https://doi.org/10.1128/aem.61.4.1323-1330.1995
  • Guerber JC, Liu B, Correll JC, Johnston PR (2003) Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95(5): 872–895. https://doi.org/10.1080/15572536.2004.11833047
  • Hacquard T, Kracher B, Hiruma K, Munch PC, Garrido-Oter G, Thon MR, Weimann A, Damm U, Dallery JF, Hainaut M, Henrissat B, Lespinet O, Sacristan S, Themaat EVL, Kemen E, McHardy AC, Schulze-Lefert P, O’Connell RJ (2016) Survival trade-offs in plant roots during colonization by closely related beneficial and pathogenic fungi. Nature Communications 7(1): e11362. https://doi.org/10.1038/ncomms11362
  • Hakimeh ZJ, Mohammad ATG, Heshmat R, Kaivan K (2019) Seasonal, tissue and age influences on frequency and biodiversity of endophytic fungi of Citrus sinensis in Iran. Forest Pathology 49(6): e12559. https://doi.org/10.1111/efp.12559
  • Hasan AEZ, Julistiono H, Bermawie N, Riyanti EI, Arifni FR (2022) Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells. Saudi Journal of Biological Sciences 29(8): e103354. https://doi.org/10.1016/j.sjbs.2022.103354
  • Huang F, Udayanga D, Wang XH, Hou X, Mei XF, Fu YS, Hyde KD, Li HY (2015) Endophytic Diaporthe associated with Citrus: A phylogenetic reassessment with seven new species from China. Fungal Biology 119(5): 331–347. https://doi.org/10.1016/j.funbio.2015.02.006
  • Jayasiri SC, Hyde KD, Ariyawansa HA, Bhat J, Buyck B, Cai L, Dai YC, Abd-Elsalam KA, Ertz D, Hidayat I, Jeeon R, Gareth Jones EB, Bahkali AH, Karunarathna SC, Liu JK, Luangsa-ard JJ, Lumbsch HT, Maharachchikumbura S, McKenzie EHC, Moncalvo JM, Ghobad-Nejhad M, Nilsson H, Pang KL, Pereira OL, Phillips A, Raspe O, Rollins AW, Romero AI, Etayo J, Sulcuk F, Stephenson S, Suetrong S, Taylor JE, Tsui C, Boonmee S, Dai D, Daranagama D, Dissanayake A, Ekanayaka A, Fryar S, Hongsanan S, Jayawardena R, Li W-J, Perera R, Phookamsak R, de Silva N, Thambugala K, Tian Q, Wijayawardene N, Zhao RL, Zhao Q, Kang JC, Promputtha I (2015) The faces of fungi database: fungal names linked with morphology, phylogeny and human impacts. Fungal Diversity 74(1): 3–18. https://doi.org/10.1007/s13225-015-0351-8
  • Jayawardena RS, Hyde KD, Damm U, Cai L, Liu M, Li XH, Zhang W, Zhao W, Yan J (2016) Notes on currently accepted species of Colletotrichum. Mycosphere: Journal of Fungal Biology 7(8): 1192–1260. https://doi.org/10.5943/mycosphere/si/2c/9
  • Jayawardena RS, Bhunjun CS, Gentekaki E, Hyde KD, Promputtha I (2021) Colletotrichum: Lifestyles, biology, morpho-species, species complexes and accepted species. Mycosphere: Journal of Fungal Biology 12(1): 519–669. https://doi.org/10.5943/mycosphere/12/1/7
  • Katoh R, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20(4): 1160–1166. https://doi.org/10.1093/bib/bbx108
  • Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Homeinoidea. Journal of Molecular Evolution 29(2): 170–179. https://doi.org/10.1007/BF02100115
  • Liu F, Weir BS, Damm U, Crous PW, Wang Y, Liu B, Wang M, Zhang M, Cai L (2015) Unravelling Colletotrichum species associated with Camellia, employing apmat and gs loci to resolve species in the C. gloeosporioides complex. Persoonia 35(1): 63–86. https://doi.org/10.3767/003158515X687597
  • Liu F, Ma ZY, Hou LW, Diao YZ, Wu WP, Damm U, Song S, Cai L (2022) Updating species diversity of Colletotrichum, with a phylogenomic overview. Studies in Mycology 101(1): 1–56. https://doi.org/10.3114/sim.2022.101.01
  • Ma X, Nontachaiyapoom S, Jayawardena RS, Hyde KD, Gentekaki E, Zhou S, Qian YX, Wen TC, Kang JC (2018) Endophytic Colletotrichum species from Dendrobium spp. in China and Northern Thailand. MycoKeys 43: 23–57. https://doi.org/10.3897/mycokeys.43.25081
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees, in Proceedings of the Gateway Computing Environments Workshop (GCE) 14 Nov 2010. Institute of Electrical and Electronics Engineers, New Orleans. https://doi.org/10.1109/GCE.2010.5676129
  • Peng Y, Hu MJ, Lu Q, Tian Y, He WY, Chen L, Pan SY (2019) Flavonoids derived from Exocarpium Citri Grandis inhibit LPS-induced inflammatory response via suppressing MAPK and NF-kB signalling pathways. Food and Agricultural Immunology 30: 564–580. https://doi.org/10.1080/09540105.2018.1550056
  • Prihastuti H, Cai L, Chen H, McKenzie EHC, Hyde KD (2009) Characterization of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Diversity 39: 89–109. https://doi.org/10.1016/j.riam.2009.11.001
  • Sun LF, Zhang YH, Pei KQ (2009) A rapid extraction of genomic DNA from fungi. Mycosystema 28: 299–302. [In Chinese]
  • Taylor JE, Hyde KD, Jones EBG (1999) Endophytic fungi associated with the temperate palm, Trachycarpus fortunei, within and outside its natural geographic range. The New Phytologist 142(2): 335–346. https://doi.org/10.1046/j.1469-8137.1999.00391.x
  • Weir BS, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115–180. https://doi.org/10.3114/sim0011
  • White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 18. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Zhang D, Gao F, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096
  • Zhao Y, Kao CP, Liao KC, Zhou X, Ho YL, Chang YS (2017) Chemical compositions, chromatographic fingerprints and antioxidant activities of Citri Exocarpium Rubrum (Juhong). Chinese Medicine 12: 1–6. https://doi.org/10.1186/s13020-017-0127-z
  • Zhou CR, Huang J, Li ZE, Li SB (2014) Diversity and tissue distribution of fungal endophytes in: An important south-China medicinal plant. China journal of Chinese materia medica 39: 3023–3029. https://doi.org/10.4268/cjcmm20141605

Appendix I

Table A1.
Download as
CSV
XLSX

Fungal isolates and sequences of molecular marker used in Colletotrichum phylogenetic analysis.

Species Culture Type Genbank accession number
ITS gapdh chs-1 his3 act tub2 gs
C. gloeosporioides complex
C. aenigma ICMP 18608 Holotype JX010244 JX010044 JX009774 - JX009443 JX010389 JX010078
ICMP 18686 JX010243 JX009913 JX009789 - JX009519 JX010390 JX010079
C. aeschynomenes ICMP 17673 Holotype JX010176 JX009930 JX009799 - JX009483 JX010392 JX009799
C. alatae ICMP 17919 Holotype JX010190 JX009990 JX009837 - JX009471 JX010383 JX010065
C. alienum ICMP 12071 Holotype JX010251 JX010028 JX009882 - JX009572 JX010411 JX010101
ICMP 18621 JX010246 JX009959 JX009755 - JX009552 JX010386 JX010075
C. aotearoa ICMP 18537 Holotype JX010205 JX010005 JX009853 - JX009854 JX010420 JX010113
C. arecicola CGMCC 3.19667 Holotype NR171191 - - - - - -
C. artocarpicola MFLUCC 181167 Holotype MN415991 - - - - - -
C. asianum ICMP 18580 Holotype JX010196 JX010053 JX009867 - JX009584 JX010406 JX010096
ICMP 18696 JX010192 JX009915 JX009753 - JX009576 JX010384 JX010073
ZHKUCC 21-0095 OL708418 OL855857 OL855867 - OL855877 OL855883 -
C. boninense ICMP 17904 JX010292 JX009905 JX009827 - JX009583 - -
C. camelliae CGMCC3 14925 Holotype KJ955081 KJ954782 MZ799255 - KJ954363 KJ955230 KJ954932
C. changpingense MFLUCC 150022 Holotype KP683152 KP852469 KP852449 - KP683093 KP852490 -
C. chrysophilum CMM 4268 Holotype KX094252 KX094183 KX094083 - KX093982 KX094285 KX094204
C. cigarro ICMP 18539 Holotype JX010230 JX009966 - - JX009523 - -
C. clidemiae ICMP 18658 Holotype JX010265 JX009989 JX009877 - JX009537 JX010438 JX010129
C. cobbittiense BRIP 66219 Holotype NR_163538 MH094133 MH094135 - MH094134 MH094137 -
C. conoides CAUG17 Holotype KP890168 KP890162 KP890156 - KP890144 KP890174 -
C. cordylinicola ICMP 18579 Holotype JX010226 JX009975 JX009864 - JX009586 JX010440 JX010122
LC0856 HM470246 HM470240 - - HM470234 HM470249 HM470243
C. endophytica CAUG28 KP145441 KP145413 KP145385 - KP145329 KP145469 -
MFLUCC 13-0418 Holotype KC633854 KC832854 - - KF306258 - -
C. fructicola LF130 KJ955083 KJ954784 - - KJ954365 KJ955232 -
CPC:28644 MH728811 MH707465 MH805851 - MH781481 MH846564 -
CPC:28645 MH728810 MH707466 MH805852 - MH781482 MH846565 -
CPC:30253 MH728817 MH707463 MH805846 - MH781476 MH846559 -
UOM 1138 MH728808 MH707468 MH805854 - MH781484 MH846567 -
C. fructicola ICMP 18581 Holotype JX010165 JX010033 JX009866 - JX009501 JX010405 JX010095
C. fructivorum CBS 133125 Holotype JX145145 MZ664047 MZ799259 - MZ664126 JX145196 -
C. gloeosporioides IMI 356878 JX010152 JX010056 JX009818 - JX009531 JX010445 JX010085
C. grevilleae CBS 132879 Holotype KC297078 KC297010 KC296987 - KC296941 KC297102 KC297033
C. grossum CAUG7 Holotype KP890165 KP890159 KP890153 - KP890141 KP890171 -
C. hebeiense MFLUCC 13-0726 Holotype KF156863 KF377495 KF289008 - KF377532 KF288975 -
C. hederiicola MFLU 150689 Holotype MN631384 - MN635794 - MN635795 - -
C. helleniense CPC:26844 KY856446 KY856270 KY856186 - KY856019 KY856528 -
C. henanense CGMCC 3.17354 Holotype KJ955109 KJ954810 MZ799256 - KM023257 KJ955257 KJ954960
C. hippeastri ICMP 17920 JX010293 JX009932 JX009838 - JX009485 - -
C. horii NBRC 7478 Holotype GQ329690 GQ329681 JX009752 - JX009438 JX010450 JN937000
C. hystricis CBS 142411 Holotype KY856450 KY856274 KY856190 - KY856023 KY856532 -
C. jiangxiense CGMCC 3.17363 Holotype KJ955201 KJ954902 - - KJ954471 KJ955348 KJ955051
C. kahawae ICMP 17816 JX010231 JX010012 JX009813 - JX009452 JX010444 JX010130
C. makassarense CPC:28612 Holotype MH728812 MH728820 MH805850 - MH781480 MH846563 MH748264
CPC:28555 MH728816 MH728822 MH805847 - MH781477 MH846560 MH748261
CPC:28556 MH728815 MH728821 MH805848 - MH781478 MH846561 MH748262
C. musae CBS:116870 Holotype JX010146 JX010050 JX009896 - JX009433 HQ596280 JX010103
CMM 4458 KX094249 KX094191 KX094080 - KX093967 KX094292 KX094234
C. nupharicola CBS 470.96 Holotype JX010187 JX009972 JX009835 - JX009437 JX010398 -
CBS 469.96 JX010189 JX009936 JX009834 - JX009486 JX010397 -
C. pandanicola MFLUCC 170571 Holotype MG646967 MG646934 MG646931 - MG646938 MG646926 -
C. perseae GA100 Holotype KX620308 KX620242 - - KX620145 KX620341 KX620275
C. proteae CBS 132882 Holotype KC297079 KC297009 KC296986 - KC296940 KC297101 KC297032
C. pseudotheobromicola MFLUCC 181602 Holotype MH817395 MH853675 MH853678 - MH853681 MH853684 -
C. psidii CBS 145.29 Holotype JX010219 JX009967 JX009901 - JX009515 JX010443 JX010133
C. queenslandicum ICMP 1778 Holotype JX010276 JX009934 JX009899 - JX009447 JX010414 JX010104
ICMP 18705 JX010185 JX010036 JX009890 - JX009490 JX010412 JX010102
C. rhexiae CBS 133134 Holotype NR_144797 MZ664046 MZ799258 - MZ664127 - -
C. salsolae ICMP 19051 Holotype JX010242 JX009916 JX009863 - JX009562 JX010403 JX010093
CBS 119296 JX010241 JX009917 JX009791 - JX009559 - -
C. siamense ICMP 18578 Holotype JX010171 JX009924 JX009865 - FJ907423 JX010404 JX010094
CPC:30210 MH707472 MH707453 MH805835 - MH781465 MH846548 MH748232
C. siamense CPC:30211 MH707473 MH707454 MH805836 - MH781466 MH846549 MH748233
CPC:30212 MH707474 MH707455 MH805837 - MH781467 MH846550 MH748234
CPC:30221 MH707475 MH707456 MH805838 - MH781468 MH846551 MH748235
CPC:30209 MH707471 MH707452 MH805834 - MH781464 MH846547 MH748231
ZHKUCC 21-0096 OL708414 OL855849 OL855859 - OL855869 OL855879 -
ZHKUCC 21-0097 OL708424 OL855852 OL855862 - OL855872 OL855881 -
ZHKUCC 21-0098 OL708423 OL855851 OL855861 - OL855871 OL855880 -
C. syzygicola DNCL021 Holotype KF242094 KF242156 - - KF157801 KF254880 KF242125
DNCL028 KF242095 KF242157 - - KF157802 KF254881 KF242126
DNCL018 KF242093 KF242155 - - KF157800 KF254879 KF242124
C. tainanense CBS 143666 Holotype MH728818 MH728823 MH805845 - MH781475 MH846558 MH748259
UOM 1119 MH728805 MH728819 MH805857 - MH781487 MH846570 -
ZHKUCC 21-0101 OL708421 OL855858 OL855868 - OL855878 OL855884 -
C. temperatum CBS 133122 Holotype MH877532 MZ664045 MZ799254 - MZ664125 - -
C. theobromicola CBS 124945 Holotype JX010294 JX010006 JX009869 - JX009444 JX010447 JX010139
C. ti ICMP 4832 Holotype JX010269 JX009952 JX009898 - JX009520 JX010442 JX010123
ICMP 5285 JX010267 JX009910 JX009897 - JX009553 JX010441 JX010124
C. tomentosae ZHKUCC 21-0103 CGMCC 3.24128 Dry culture: ZHKU 21-0088 Holotype OL708422 OL855850 OL855860 - OL855870 OL855887 ON315373
ZHKUCC 21-0104 OL708419 OL855856 OL855866 - OL855873 OL855888 ON315374
ZHKUCC 22-0041 ON303476 ON315382 ON315376 - ON315380 ON315378 ON315375
C. tropicale CBS 124946 KC566806 KC566660 KC566373 - KC566952 KC566228 -
CBS 124943 JX010277 JX010014 JX009868 - JX009570 - -
CBS 124949 Holotype JX010264 JX010007 JX009870 - JX009489 JX010407 JX010097
C. viniferum GZAAS 5.08601 Holotype JN412804 JN412798 - - JN412795 JN412813 -
C. wuxiense CGMCC 3.17894 Holotype KU251591 KU252045 KU251939 - KU251672 KU252200 KU252101
C. xanthorrhoeae BRIP 45094 Holotype JX010261 JX009927 JX009823 - JX009478 JX010448 JX010138
C. yulongense CFCC 50818 Holotype MH751507 MK108986 MH793605 - MH777394 MK108987 MK108988
C. magnum complex
C. brevisporum BCC 38876 Holotype JN050238 JN050227 MZ799287 MZ673841 JN050216 JN050244 -
CBS 129958 MG600763 MG600823 MG600870 MG600909 MG600967 MG601030 -
C. cacao CBS 119297 Holotype MG600772 MG600832 MG600878 MG600916 MG600976 MG601039 -
C. cliviicola CBS 125375 MG600733 MG600795 MG600850 MG600892 MG600939 MG601000 -
C. guangdongense ZHKUCC 21-0105 CGMCC 3.24127 Dry culture: ZHKU 21-0089 Holotype OL708415 OL855854 OL855864 ON315370 OL855875 OL855885 -
ZHKUCC 21-0106 OL708420 OL855855 OL855865 ON315371 OL855876 OL855886 -
ZHKUCC 22-0042 ON303474 ON315383 ON315377 ON315372 ON315381 ON315379 -
C. lobatum IMI79736 Holotype MG600828 MG600874 MG600912 MG600972 MG600972 MG601035 -
C. magnum CBS519.97 Holotype MG600769 MG600829 MG600875 MG600913 MG600973 MG601036 -
IMI391662 MG600771 MG600831 MG600877 MG600915 MG600975 MG601038 -
CAUOS2 MZ595839 MZ848400 OK236385 MZ673858 OK236387 MZ673960 -
C. merremiae CBS124955 Holotype MG600765 MG600825 MG600872 MG600910 MG600969 MG601032 -
C. okinawense MAFF240517 Holotype MG600767 MG600827 - - MG600971 MG601034 -
SB 08 MK830706 MK820658 - MK820660 MK820659 - -
C. orchidearum CBS135131 MG600738 MG600800 MG600855 MG600897 MG600944 MG601005 -
C. panamense CBS125386 Holotype MG600766 MG600826 MG600873 MG600911 MG600970 MG601033 -
Colletotrichum sp. MH0413 MZ595871 MZ664109 MZ799289 MZ673891 MZ664169 MZ673990 -
C. orchidearum complex
C. brevisporum BCC 38876 JN050238 JN050227 MZ799287 MZ673841 JN050216 JN050244 -
C. cattleyicola CBS 170.49 Holotype MG600758 MG600819 MG600866 MG600905 MG600963 MG601025 -
MAFF 238321 MG600759 - - - - MG601026 -
C. cliviicola CBS 133705 MG600732 MG600794 MG600849 MG600891 MG600938 MG600999 -
CBS 125375 Holotype MG600733 MG600795 MG600850 MG600892 MG600939 MG601000 -
C. magnum CBS519.97 MG600769 MG600829 MG600875 MG600913 MG600973 MG601036 -
C. monsterae LC13871 Holotype MZ595897 MZ664121 MZ799351 MZ673917 MZ664195 MZ674015 -
C. musicola CBS132885 Holotype MG600736 MG600798 MG600853 MG600895 MG600942 MG601003 -
CBS127557 MG600737 MG600799 MG600854 MG600896 MG600943 MG601004 -
C. orchidearum CBS135131 Holotype MG600738 MG600800 MG600855 MG600897 MG600944 MG601005 -
CBS136877 MG600739 MG600801 MG600856 MG600898 MG600945 MG601006 -
C. piperis IMI71397 Holotype MG600760 MG600820 MG600867 MG600906 MG600964 MG601027 -
C. plurivorum CBS125474 Holotype MG600718 MG600781 MG600841 MG600887 MG600925 MG600985 -
CBS132443 MG600719 MG600782 MG600842 MG600888 MG600926 MG600986 -
LC8240 MZ595848 MZ664113 MZ799291 MZ673868 MZ664146 MZ673969 -
LC8244 MZ595849 MZ772868 MZ799292 MZ673869 MZ664147 MZ673970 -
LC8322 MZ595853 MZ664114 MZ799293 MZ673873 MZ664151 MZ673974 -
LC8337 MZ595855 MZ664115 MZ799294 MZ673875 MZ664153 MZ673976 -
ZHKUCC 21-0102 OL708416 OL855874 OL855863 - OL855853 OL855882 -
C. reniforme LC8230 Holotype MZ595847 MZ664110 MZ799290 MZ673867 MZ664145 MZ673968 -
LC8248 MZ595850 MZ664111 MZ799295 MZ673870 MZ664148 MZ673971 -
C. sojae ATCC62257 Holotype MG600749 MG600810 MG600860 MG600899 MG600954 MG601016 -
CBS128510 MG600751 MG600812 MG600862 MG600901 MG600956 MG601018 -
LC8335 MZ595854 MZ664112 MZ799300 MZ673874 MZ664152 MZ673975 -
LC8492 MZ595858 MZ664116 MZ799301 MZ673878 MZ664156 MZ673979 -
C. syngoniicola LC8894 Holotype MZ595863 MZ664117 MZ799296 MZ673883 MZ664161 MZ673982 -
LC8895 MZ595864 MZ664118 MZ799297 MZ673884 MZ664162 MZ673983 -
LC8896 MZ595865 MZ664119 MZ799298 MZ673885 MZ664163 MZ673984 -
C. vittalense CBS126.25 MG600735 MG600797 MG600852 MG600894 MG600941 MG601002 -
CBS181.82 Holotype MG600734 MG600796 MG600851 MG600893 MG600940 MG601001 -
login to comment