﻿Morphological and molecular phylogenetic analyses reveal three species of Colletotrichum in Shandong province, China

﻿Abstract Colletotrichum has numerous host range and distribution. Its species are important plant pathogens, endophytes and saprobes. Colletotrichum can cause regular or irregular depressions and necrotic lesions in the epidermal tissues of plants. During this research Colletotrichum specimens were collected from Mengyin County, Shandong Province, China. A multi-locus phylogenetic analysis of ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS sequence data combined with morphology, revealed a new species and two known species, viz. C.mengyinense sp. nov., C.gloeosporioides and C.pandanicola, belonging to the C.gloeosporioides species complex. The new species is described and illustrated in this paper and compared with taxa in the C.gloeosporioides species complex.


Introduction
Colletotrichum species (Glomerellaceae, Glomerellales) is one of the ten economically most important fungal plant pathogens worldwide (Dean et al. 2012). It was first observed by Tode (1790), who divided it into Vermicularia. Corda (1831) established Colletotrichum based on the characteristic of the conidiomata with setae in Vermicularia.

Isolation and morphological studies
The samples were collected from Mengyin County, Shandong Province, China. The strains of Colletotrichum were isolated from symptomatic leaves of Rosa chinensis and diseased fruit of Juglans regia using single spore and tissue isolation methods (Chomnunti et al. 2014). The spore suspension was obtained and spread onto PDA plate and incubated for one day under the biochemical incubator. After germination, the spores were transferred to a new PDA plate to obtain pure culture. Additionally, the surface sterilized plant tissue isolation was used to obtain sterile isolates from the host plant. About 25 mm 2 tissue fragments were taken from the margin of tissue lesions and surface sterilized by consecutively immersing in 75% ethanol solution for 60 s, 5% sodium hypochlorite solution for 30 s, and then rinsed in sterile distilled water for 60 s (Gao et al. 2013;Liu et al. 2015). The surface sterilized plant tissue was dried with sterilized paper and moved on the PDA plate ). All the PDA plates were incubated at biochemical incubator at 25 °C for 3-4 days, then hyphae were picked out of the periphery of the colonies and inoculated on to new PDA plates.
Following 5-14 days of incubation, morphological characters were recorded ). Photographs of the colonies were taken at 7 days and 14 days using a digital camera (Canon G7X). Micromorphological characters of colonies were observed using stereomicroscope (Olympus SZX10) and microscope (Olympus BX53), both fitted with high definition color digital cameras to photo document conidia and so on of fungal structures. All Colletotrichum strains were stored in 10% sterilized glycerin and sterile water at 4 °C for deep studies in the future. Every specimen was deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification
Genomic DNA was extracted from Colletotrichum fungal mycelia grown on PDA after 5-7 days, using a modified cetyltrimethylammonium bromide (CTAB) buffer, and then it was incubated at 65 °C for 30 min with occasional gentle inverting (Guo et al. 2000). Gene sequences were obtained from seven genes loci including the internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), partial chitin synthase 1 gene (CHS-1), partial actin gene (ACT), partial beta-tubulin gene (TUB2), partial calmodulin gene (CAL) and partial glutamine synthetase gene (GS) were amplified and sequenced using primers pairs (Table 1).
PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume which contained 12.5 μL 2× Taq Plus Master Mix II (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Tsingke, Qingdao, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL. PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The annealing temperature for each gene was 52 °C for ITS and GS, 59 °C for CAL, 60 °C for GAPDH, 58 °C for ACT and CHS-1, 55 °C for TUB2. The PCR products were visualized on 1% agarose electrophoresis gel. Sequencing was conducted by the Tsingke Company Limited (Qingdao, China) bi-directionally. Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 2).

Phylogenetic analyses
Novel sequences were generated from the nine strains in this study, and all reference available sequences of Colletotrichum species were downloaded from Gen-Bank. Multiple sequence alignments for ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS were constructed and carried out using the MAFFT v.7.11 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh et al. 2019) with the default settings, and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted individually for each locus and then as combined analyses of seven loci (ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian.
Inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) using RaxML-HPC2 on XSEDE (8.2.12) (Miller et al. 2012;Stamatakis 2014) and MrBayes on XSEDE (3.2.7a), respectively (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Ronquist et al. 2012). For ML analyses the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included seven parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 1000 generations. The burnin fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v. 1.4.4 (http:// tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0. New sequences generated in this study were deposited at GenBank (https://www.ncbi. nlm.nih.gov; Table 2).  Table 2. Species and GenBank accession numbers of DNA sequences used in this study with new sequences in bold.   Strains marked with "*" are ex-type or ex-epitype.

Phylogenetic analyses
Nine strains of Colletotrichum isolated from leaves of Rosa chinensis and fruit of Juglans regia in Mengyin County, Shandong Province, China, were grown in culture. Among the nine Colletotrichum isolates were identified a new species and two known species based on an analysis of combined ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS gene sequences composed of 69 isolates of C. gloeosporioides species complex and C. boninense (CBS 123755) as the outgroup taxon. A total of 3953 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1-619, GAPDH: 620-929, CHS-1: 930-1229, ACT: 1230-1542, TUB2: 1543-2288, CAL: 2289-3028, GS: 3029-3953. Of these characters, 2667 were constant, 674 were variable and parsimony-uninformative, and 612 were parsimony-informative.
The Bayesian analysis lasted 4,685,000 generations, resulting in 4686 total trees, of which 3515 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree. For the BI and ML analyses, HKY+G for GAPDH and ACT, SYM+I+G for ITS, K80+I+G for CHS-1, GTR+G for GS and CAL, HKY+I for TUB2 were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, the ML tree is presented (Fig. 1).
ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.90) are shown as first and second position above nodes, respectively. The 70 strains were assigned to 60 species clades based on the seven gene loci phylogeny (Fig. 1). The nine strains studied here represented a novel species and two known species. The new species of C. mengyinense showed a close relationship to C. fructicola (MFLU 090228) with full support (ML-BS: 100% and BYPP: 1). The strains SAUCC200952, SAUCC200954 and SAUCC201001 belong to C. gloeosporioides (IMI356878) with full support (ML-BS: 100% and BYPP: 1) by the multi-locus phylogeny. The strains SAUCC200204 and SAUCC201152 belong to C. pandanicola (MFLU 18-0003) with good support (ML-BS: 94% and BYPP: 0.99) by the multi-locus phylogeny.
Culture characteristics. Colonies on PDA flat with entire margin, aerial mycelium white, floccose cottony; surface and reverse grayish in the center and white margin. PDA attaining max 81 mm in diameter after 7 days, at 25 °C, growth rate 8.7-11.5 mm/day. Colonies on SNA sparse hyphae, slow growth.
Culture characteristics. Colonies on PDA flat with entire margin, aerial mycelium white or gray, floccose cottony; surface and reverse gray in the center and grayish margin. PDA attaining 69.3-75.6 mm in diameter after 7 days, at 25 °C, growth rate 9.9-10.8 mm/day. Colonies on SNA sparse hyphae, slow growth.

Discussion
In this study, the Colletotrichum specimens of diseased leaves and fruits were collected in Mengyin, Shandong Province, China. A temperate monsoon climate and an abundance of fruit trees provide the proper conditions for anthracnose propagation. As a result, 70 reference sequences (including an outgroup taxon: C. boninense CBS 123755) were selected based on BLAST searches of NCBI's GenBank nucleotide database and were included in the phylogenetic analyses (Table 2).
Phylogenetic analyses based on seven combined loci (ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS), as well as morphological characters of the asexual morph obtained in culture, were contributed to knowledge of the diversity of Colletotrichum species in Shandong Province. Based on a large set of freshly collected specimens from Shandong province, China, nine strains of Colletotrichum species were isolated from two host genera (Table 2). A new species is proposed: C. mengyinense. In a previous report, C. gloeosporioides has been isolated from Juglans regia (Zhu et al. 2014). Colletotrichum pandanicola was described from Pandanus sp. (Pandanaceae) in Thailand (Tibpromma et al. 2018) and C. pandanicola is first reported from Juglans regia in China. In this study, we described and illustrated C. gloeosporioides and C. pandanicola again.
Previously, species identification of Colletotrichum was largely referred to the hostspecificity and pure culture characteristics, leading to the chaos of names ). On the other hand, based on a polyphasic approach and known morphology, more than one species of Colletotrichum can colonize a single host, while one species can be associated with different hosts . It revealed diversity of Colletotrichum species from different hosts. Our study supported this result. For example, C. pandanicola (SAUCC200204 and SAUCC201152) and C. gloeosporioides (SAUCC200952, SAUCC200954 and SAUCC201001) were collected from Juglans regia. In addition, isolates of C. mengyinense were obtained from two hosts (Juglans regia and Rosa chinensis). The morphological descriptions and molecular data for species of Colletotrichum represent an important resource and basis for plant pathologists and fungus taxonomists.