Fresh healthy plant samples (leaves, stems and roots) of Nothapodytes pittosporoides were collected in Tongren City, Guizhou Province and Jishou City, Hunan Province, China. Materials were kept in zip-lock bags on ice. Fungal isolation was carried out within 24 hours of collection.

Each part of the plant was surface sterilised to eliminate epiphytic microorganisms. The samples were washed thoroughly in running tap water, followed by immersion in 70% (v/v) ethanol for 3 min to sterilise the surfaces, then rinsed with sterilised distilled water for 1 min. Samples were dried on sterilised filter paper and then placed in 3% hydrogen peroxide for 7 min, washed in sterilised distilled water and dried on a sterilised filter paper again. Each plant tissue was then cut into small cubes (0.5 × 0.5 cm) using a sterilised blade. The cubes were placed on potato dextrose agar (PDA) medium in Petri dishes containing with antibiotic (100 mg/l chloramphenicol) and incubated at 25 °C until fungal growth emerged from the plant segments. The endophytic fungi were isolated and sub-cultured on fresh PDA plates at 25 °C in darkness. Fungal isolates were stored on PDA and covered with sterilised water at 4 °C.

The type specimens are deposited in Guizhou Agricultural College (GACP), Guiyang, China. Ex-type living cultures are deposited at Guizhou Medical University Culture Collection (GMBC). Mycobank numbers are provided.

Genomic DNA was extracted from fresh fungal mycelia using the BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, Biomiga, USA), following the manufacturer’s instructions. DNA samples were stored at -20 °C until used for polymerase chain reaction (PCR). Four loci, rDNA regions of internal transcribed spacers (ITS), partial β-tubulin (TUB2), actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR with primers ITS1 (Gardes and Bruns 1993) + ITS4 (White et al. 1990), Bt-2a + Bt-2b (Glass and Donaldson 1995), ACT-512F + ACT-783R (Carbone and Kohn 1999) and GDF1 + GDR1 (Guerber et al. 2003), respectively. The components of a 50 µl volume PCR mixture were used as follows: 2.0 µl of DNA template, 1 µl of each forward and reverse primer, 25 µl of 2 × Easy Taq PCR Super Mix (mixture of Easy Taq TM DNA Polymerase, dNTPs and optimised buffer, Beijing Trans Gen Biotech Co., Chaoyang District, Beijing, China) and 19 µl sterilised water. PCR thermal cycle programmes for ITS and ACT gene amplification were provided as: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 50 s, elongation at 72 °C for 45 s and final extension at 72 °C for 10 min. The PCR thermal cycle programme for GAPDH gene amplification was provided as: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, elongation at 72 °C for 45 s and final extension at 72 °C for 10 min. The PCR thermal cycle programme for TUB2 gene amplification was provided as: initial denaturation 95 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, elongation at 72 °C for 45 s and final extension at 72 °C for 10 min. The quality of PCR products were checked with 1.5% agarose gel electrophoresis stained with ethidium bromide. PCR products were sent for sequencing to Sangon Co., Shanghai, China.

Sequence data of the four loci were blasted in the GenBank database and all top hits, including the corresponding type sequences, were retrieved (Table 1). Multiple sequence alignments for ITS, TUB2, ACT and GAPDH were constructed and carried out using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh and Standley 2013) with the default settings. Four datasets of ITS, TUB2, ACT and GAPDH of Colletotrichum spp. were combined and manually adjusted using BioEdit v.7.0.5.3 (Hall 1999), then assembled using SequenceMatrix1.7.8 (Vaidya et al. 2011). The final alignments contained 1593 characters with gaps, ITS with 522 sites, TUB2 with 510 sites, ACT with 269 sites and GAPDH with 292 sites. Fifty-four taxa and 1593 sites were used for phylogenetic analyses. Gaps were treated as missing data in maximum likelihood (ML), Bayesian Inference (BI) and parsimony trees. The phylogeny website tools “ALTER” (Glez-Peña et al. 2010) were used to convert the alignment file from Fasta to PhyLip file for RAxML analysis and Nexus for MrBayes. All loci were tested based on single maximum likelihood (ML) trees and Bayesian Inference (BI) methods.

Maximum Likelihood (ML) analysis was performed on the website of CIPRES Science Gateway v.3.3 (http://www.phylo.org/portal2/, Miller et al. 2010) using RAxML-HPC Blackbox version 8.2.10. All free model parameters were estimated by RAxML and ML estimate of 25 per site rate categories. Final ML searches were conducted using the GTRGAMMA model. Bootstrap Support values (BS) equal to or greater than 60% are given above each node (Fig. 1).

For Bayesian Inference (BI), a Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes 3.2.6 (Ronquist et al. 2012) for the combined sequence datasets. MrModeltest v.2.3 (Nylander 2004) was used to carry out the statistical selection of the best-fit model of nucleotide substitution. GTR+G model was selected for ITS, a GTR+I+G model for TUB2, a HKY+I+G model for ACT and GAPDH were incorporated into the analysis. Models of nucleotide substitution for each gene determined by MrModeltest v. 2.3 were included for each set of gene sequence data. Two runs were executed simultaneously for 1,000,000 generations and sampled every 100 generations. Of the trees, 25% were discarded as burn-in and the remaining trees were used to calculate the posterior probabilities. Convergence was assumed when the standard deviation of split sequences was less than 0.01. Phylogenetic trees were visualised using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/, Rambaut 2012). The final alignment was deposited in Treebase (http://www.treebase.org, submission number 23622).

Isolates were grown on PDA, water agar (WA) with bamboo and corn malt agar medium (CMA) for examination of morphological characters. Colonies were examined after 7, 14 and 21 d at 25 °C in darkness. The morphological characters of mycelia, conidiophores, conidiogenous cells and conidia were observed and photographed using a Nikon NI-SS microscope and processed with Adobe Photoshop CS3 Extended version 10.0 software (Adobe Systems, USA).

Four hundred and forty endophytic fungi were isolated from different parts of Nothapodytes pittosporoides in Jishou, Hunan Province and Tongren, Guizhou Province, belonging to twenty-four genera based on ITS sequences analysis. Colletotrichum was a common genus amongst the isolates. Herein, five endophytic taxa were isolated and identified as Colletotrichum of which GZU_HJ2_G2, GZU_HJ2_G3 and GZU_HJ2_G4 were isolated from roots and GZU_HJ3_J5 from stems of N. pittosporoides in Jishou, Hunan Province. GZU_TRJ1-37 was isolated from stems of N. pittosporoides in Tongren, Guizhou Province.

Phylogenetic analysis of four loci (ITS, GAPDH, ACT and TUB2) sequence datasets included 54 taxa, 1,593 positions including gaps (ITS: 1–522, TUB2: 523–1032, ACT: 1033–1301, GAPDH: 1302–1593) and Monilochaetes infuscans (CBS 869.96) was selected as the outgroup taxon. The 50% majority rule consensus Bayesian phylogram presented in Fig. 1 and the topology is recovered with the RAxML tree. Values of the Bayesian PP ≥ 0.70 from MCMC analyses and bootstrap support values of RAxML ≥ 90% are given on the branches.

Figure 1. 

Phylogram generated from Maximum Likelihood (RAxML) analysis based on combined ITS, ACT, TUB2 and GAPDH DNA sequence data of Colletotrichum. Bayesian Posterior Probabilities (BSPP) greater than 0.90 and Maximum Likelihood Bootstrap Support values (MLBS) greater than 70% are shown above branches. New isolates are in red. The tree is rooted with Monilochaetes infuscans CBS 869.96.

Representatives of complexes and species in Colletotrichum (Noireung et al. 2012; Tao et al. 2013; Liu et al. 2014; Jayawardena et al. 2016; Douanla-meli et al. 2017) are included in the phylogenetic analyses (Fig. 1). Four isolates, GZU_HJ2_G2, GZU_HJ2_G3, GZU_HJ2_G4 and GZU_HJ3_J5, were identified as distinct new species and are described as Colletotrichum jishouense sp. nov., and as C. tongrenense sp. nov., based on their morphology and molecular phylogeny.