Two new endophytic Colletotrichum species from Nothapodytespittosporoides in China

Abstract Two new endophytic species, Colletotrichumjishouensesp. nov. and. C.tongrenensesp. nov. were isolated from Nothapodytespittosporoides in Guizhou and Hunan provinces, China. Detailed descriptions and illustrations of these new taxa are provided and morphological comparisons with similar taxa are explored. Phylogenetic analysis with combined sequence data (ITS, GAPDH, ACT and TUB2) demonstrated that both species formed distinct clades in this genus. This is the first record of Colletotrichum species from N.pittosporoides in China.


Introduction
Nothapodytes pittosporoides (Oliv.) Sleum (Icacinacceae) has been used as Traditional Chinese Medicine (TCM) and is mainly distributed in southern China (Fang 1981). It is quickly gaining attention as the characteristic compounds of camptothecin and its derivatives (CIDs) in N. pittosporoides (Dong et al. 2015) are used as anti-cancer drugs in the world market (Demain and Vaishnav 2011). It is recognised that endophytes reside in the internal tissues of living plants and potentially have the capability to produce the same functional compounds as their hosts (Stierle et al. 1993(Stierle et al. , 1995Kusari et al. 2008;Bhalkar et al. 2016;Uzma et al. 2018). The endophytic fungi in N. pittosporoides were therefore studied for their secondary metabolites with pharmaceutical potential.
Endophytic fungi were isolated from different parts of Nothapodytes pittosporoides (Zhou et al. 2017;Qiao et al. 2018) collected from different sites. A high diversity of fungi were found, of which several species of Colletotrichum were isolated and identified.
Colletotrichum species are globally distributed and occur in various plants as endophytes (Tibpromma et al. 2018). Colletotrichum is the sole genus in the family Glomerellaceae (Glomerellales, Sordariomycetes, Wijayawardene et al. 2018) and was introduced by Corda (1831) with the type species C. lineola (Jayawardena et al. 2016, Wijayawardene et al. 2017. Recently, several studies have analysed this genus and these are summarised in Hyde et al. (2014), who accepted 163 names. Since this review, about 30 more species have been introduced (Baroncelli et al. 2017;Douanlameli et al. 2017;Jayawardena et al. 2017;Silva et al. 2018).
In this study, we introduce two novel species, C. jishouense sp. nov. and C. tongrenense sp. nov. isolated as endophytes from N. pittosporoides. These species are based on both morphological features and molecular sequence data evidence.

Sample collection
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.

Isolation and cultivation of fungal endophytes
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.

DNA extraction, PCR amplification, and sequencing
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 alignment and phylogenetic analyses
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).

Morphological analysis
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).

Sample collection and isolation
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.
Culture characteristics. Colonies on PDA, reaching 55-60 mm diam. in 14 days at 25 °C in darkness, circular, mycelium superficial and partially immersed, more or less planar, brown in the medium but covered with abundant, pale and lanose to cottony aerial mycelium, reverse greenish pale brown, margin entire and irregular.
Colonies on PDA at 25 °C reaching 45-55 mm diam. in 12 days in darkness, circular, more or less planar, surface dark brown, covered with abundant, pale grey, lanose to cottony aerial mycelium, margin smooth, entire and pale white. Reverse dark grey, margin pale white.
Cultures on CMA, 10-15 mm diam. in 21 days, covered with dark brown aerial mycelium, sparse, reverse light brown, margin irregular.

Discussion
Colletotrichum appears to have a wide host range and a geographic distribution , Hyde et al. 2014, Jayawardena et al. 2016). This study reports on five endophytic Colletotrichum isolates which were isolated from Nothapodytes pittosporoides. Two new species were introduced, named C. jishouense and C. tongrenense, respectively, based on morphological characters and multilocus (ITS, TUB2, ACT and GAPDH) phylogenetic analyses. The C. gigasporum species complex is associated with various host plants as pathogens and endophytes and also isolated from air and stored grain, indicating that the members are not host-specific and apparently have different life styles (Than et al. 2008, Jayawardena et al. 2016). The C. dracaenophilum species complex contains a few apparently host-specific species and these species seem to be uncommon (Damm et al. 2019). The complex includes C. coelogynes, C. dracaenophilum, C. excelsum-altitudinum, C. tropicicola and C. yunnanense. A further strain, C. tongrenense was identified to the C. dracaenophilum species complex in the study, based on the multilocus phylogeny and morphological features. Amongst them, C. excelsum-altitudinum was described from healthy leaves of Bletilla ochracea (Orchidaceae) in Guizhou, China (Tao et al. 2013.), C. tropicicola were described from leaves of Citrus maxima and Paphiopedilum sp. in Thailand and a further strain from C. sp. in Mexico (Noireung et al. 2012, Damm et al. 2019). The C. coelogynes strain CBS 132504 is an endophytic Colletotrichum isolate from both Dendrobium spp. in China (Yuan et al. 2009, Gao and Guo, unpublished data). C. yunnanense was described from healthy leaves of Buxus sp. in Yunnan, China (Liu et al. 2007).
Morphological features and genes sequence data are recognised as a basis for describing new species, but sometimes morphological features of Colletotrichum are not stable and may change under different growth conditions . DNA sequence comparison and multi-gene phylogenetic analyses can provide sufficient evidence to show distinct taxa (Jeewon and Hyde 2016). However, single gene data, including ITS, are usually insufficient for species identification in most of the Colletotrichum species complexes ). Multi-locus phylogenies are therefore necessary to describe Colletotrichum species (Jayawardena et al. 2016).
The composition of endophytic microorganisms may depend on the plant age, tissue, host type and time of isolation (Rosenblueth and Martinez-Romero 2006). The new species, Colletotrichum tongrenense lives in stems and C. jishouense lives in roots and stems of Nothapodytes pittosporoides. Nothing is known about their infection strategies on the host. It is also the first report of Colletotrichum species from N. pittosporoides. This study enriches the host diversity of Colletotrichum.