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
﻿Endophytic Colletotrichum ( Sordariomycetes , Glomerellaceae ) species associated with Citrus grandis cv. “Tomentosa” in China
Chinese traditional medicinal plants,new ascomycete,phylogeny,six new host records,taxonomy,two new species,Fungi,Glomerellales,Glomerellaceae,Fungi,Glomerellales,Glomerellaceae,Fungi,Glomerellales,Glomerellaceae,Fungi,Glomerellales,Glomerellaceae,Fungi,Glomerellales,Glomerellaceae,Fungi,Glomerellales,Glomerellaceae
﻿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.
﻿Endophytic Colletotrichum ( Sordariomycetes , Glomerellaceae ) species associated with Citrus grandis cv. “Tomentosa” in China

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 Citrusgrandis 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 Colletotrichumguangdongense and C.tomentosae. Colletotrichumasianum, 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.

Citrusgrandis cv. “Tomentosa” is an important traditional medicinal plant which contains essential oils, flavonoids and polysaccharides. In traditional Chinese medicine, Citrusgrandis 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, Penicilliumcrustosum from Annonamuricata 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. ColletotrichumCorda (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.

Healthy leaves and twigs of Citrusgrandis 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.

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).

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).

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 Colletotrichumgloeosporioides 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: Colletotrichumorchidearum complex and Colletotrichummagnum 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.

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.

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.

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. Colletotrichumboninense (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.

Taxon classification Fungi Glomerellales Glomerellaceae Prihast., L. Cai & K.D. Hyde, Fungal Diversity 39: 96 (2009) 5C82DBB3-CFC0-54A4-8EC7-466A475C0433 Index Fungorum Number: IF515408 Facesoffungi Number: FoF10689

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

The single isolate (ZHKUCC 21-0095) obtained in this study clustered with the Colletotrichumasianum 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”.

Taxon classification Fungi Glomerellales Glomerellaceae Prihast., L. Cai & K.D. Hyde, Fungal Diversity 39: 98 (2009) 75590CA3-0B17-5518-A480-F26DF7E96487 Index Fungorum Number: IF515410 Facesoffungi Number: FoF03599

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

Three isolates obtained in this study (ZHKUCC 21-0096–100) clustered with the ex-type strain of Colletotrichumsiamense (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”.

Taxon classification Fungi Glomerellales Glomerellaceae de Silva, Crous & P.W.J. Taylor, IMA Fungus 10(1): 23 (2019) 9E6713DB-E25E-5111-A950-1835450F1E0C Index Fungorum Number: IF827692 Facesoffungi Number: FoF10690

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

A single isolate obtained in this study (ZHKUCC 21-0101) clustered with the Colletotrichumtainanense (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”.

Taxon classification Fungi Glomerellales Glomerellaceae J.W. Liu, Manawas. & M. Luo sp. nov. 57D630D6-F8DD-5470-8F0F-08F809AB3BCE Index Fungorum Number: IF559482 Facesoffungi Number: FoF10692 Fig. 2

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

ZHKUCC 21-0103.

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.

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.

China, Guangdong Province, Huazhou, isolated from a healthy leaf of Citrusgrandis 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).

In the phylogenetic analysis of combined six genes, Colletotrichumtomentosae 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). Colletotrichumtomentosae 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.

In the present study, a single isolate was recognised as belonging to the Colletotrichumorchidearum complex. The phylogenetic analysis of a combined ITS, gapdh, chs-1, his3, act and β-tubulin sequence alignment was constructed using 30 Colletotrichum strains. Colletotrichummagnum (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.

Taxon classification Fungi Glomerellales Glomerellaceae Damm, Alizadeh & Toy. Sato, Studies in Mycology 92: 31 (2019) D5DD5537-5639-565B-9521-9490D3BD34AC Index Fungorum Number: IF824228 Facesoffungi Number: FoF10691

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

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). Colletotrichumplurivorum was first introduced by Damm et al. (2019) as a pathogen on Capsicumannuum 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 Citrusgrandis cv. “Tomentosa”.

Three of our isolates were initially recognised as belonging to the Colletotrichummagnum species complex. The phylogenetic analysis of combined act, chs-1, gapdh, his3, ITS and β-tubulin sequence alignment was conducted using 17 Colletotrichum strains. Colletotrichumorchidearum (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).

Taxon classification Fungi Glomerellales Glomerellaceae J.W. Liu, Manawas. & M. Luo sp. nov. 43281A0E-8EE6-52A9-AAD1-3D9B8B8BF57D Index Fungorum Number: IF559483 Facesoffungi Number: FoF10693 Fig. 7

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

ZHKUCC 21-0105

Isolated from a Citrusgrandis 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.

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.

China, Guangdong Province, Huazhou, isolated from healthy twigs of Citrusgrandis 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).

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). Colletotrichumguangdongense 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). Colletotrichumpanamense (CBS 125386) has conidiophores shorter than C.guangdongense (30 μm vs. 20–70 μm). Colletotrichumguangdongense 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.

In the present study, endophytic Colletotrichum species were isolated from Citrusgrandis 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. Colletotrichumsiamense 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. Colletotrichumsiamense 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). Colletotrichumsiamense 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). Colletotrichumplurivorum 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 Citrusgrandis 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 Citrusgrandis cv. “Tomentosa”.