Three new Diaporthe species from Shaanxi Province, China

Abstract Diaporthe species (Sordariomycetes, Diaporthales) are often reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. In this study, Diaporthe specimens were collected from symptomatic twigs and branches at the Huoditang Forest Farm in Shaanxi Province, China. Identification was done using a combination of morphology and comparison of DNA sequence data of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions. Three new Diaporthe species are proposed: D. albosinensis, D. coryli and D. shaanxiensis. All species are illustrated and their morphology and phylogenetic relationships with other Diaporthe species are discussed.

Species identification in Diaporthe has traditionally been based on host association, morphology and culture characteristics (Mostert et al. 2001, Santos and Phillips 2009, Udayanga et al. 2011, resulting in the description of over 200 species (Hyde et al. 2020). Multiple species of Diaporthe can colonise a single host and one species can be associated with different hosts (Santos and Phillips 2009, Diogo et al. 2010, Santos et al. 2011, Gomes et al. 2013). In addition, considerable within-species variability of phenotypic characters has been reported (Rehner and Uecker 1994, Mostert et al. 2001, Udayanga et al. 2011). Thus, a polyphasic taxonomic approach, based on multilocus DNA data, morphology and ecology, has been increasingly employed for species boundaries in the genus Diaporthe (Gomes et al. 2013, Huang et al. 2013, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Du et al. 2016, Gao et al. 2016, Guarnaccia and Crous 2017, Long et al. 2019. Huoditang is located in the middle part of the southern slope of the Qinling Mountains at 33°18'~33°28'N, 108°21'~108°29'E. It belongs to the transitional zone of the northern subtropical and warm temperate zone in China. The terrain is complex and the climate is changeable (Zhang and Cao 2007). The plant communities are complex and, as a result, species diversity of fungi in the forest area is high (Zhang and Cao 2007). During trips to collect forest pathogens causing dieback in Shaanxi Province, cankered branches with typical Diaporthe fruiting bodies were investigated and sampled. The aim of the present study was to identify these fungi, based on modern polyphasic taxonomic concepts.

Isolates
Fresh specimens of Diaporthe were collected from symptomatic twigs or branches in Shaanxi Province (Table 1). Isolates were ob tained by removing a mucoid spore mass from conidiomata and spreading the suspen sion on the surface of 1.8% potato dextrose agar (PDA) in a 9 cm diam. Petri dish. Petri dishes were incubated at 25 °C until spores germinated. Single germinating conidia were transferred on to new PDA plates, which were kept at 25 °C in the dark. Specimens are deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC). Newly sequenced material is indicated in bold type. NA, not applicable.

Morphological analysis
Morphological observations of the asexual morph in the natural environment were based on features of the fruiting bodies produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Conidiomata from tree barks were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. The gross morphology of fruiting bodies was recorded using a Leica stereomicroscope (M205 FA). Fungal structures were mounted in clear lactic acid and micromorphological characteristics were examined at 1000× magnification using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Thirty measurements of each structure were determined for each collection. Colony characters and pigment production on PDA were noted after 10 d. Colony colours were described according to Rayner (1970).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using the CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). PCR amplifications of phylogenetic markers were done using the same primer pairs and conditions as in Yang et al. (2018). PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses
The quality of our amplified nucleotide sequences was checked and combined by Seq-Man v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Diaporthe , Long et al. 2019. Sequences were aligned using MAFFT v. 7.310 (http://mafft.cbrc.jp/alignment/server/index.html) (Katoh and Standley 2016) and manually corrected using Bioedit 7.0.9.0 (Hall 1999). The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion. Phylogenetic analyses of the combined gene regions were performed using Maximum-Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates. BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: S25522). The nucleotide sequence data of the new taxa have been deposited in GenBank (Table 1).

Phylogenetic analyses
The five-gene sequence dataset (ITS, cal, his3, tef1 and tub2) was analysed to infer the interspecific relationships within Diaporthe. The dataset consisted of 124 sequences including the outgroup, Diaporthella corylina (culture CBS 121124). A total of 2555 characters including gaps (505 for ITS, 513 for cal, 528 for his3, 475 for tef1 and 522 for tub2) were included in the phylogenetic analysis. The best nucleotide substitution model for ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was selected for both cal and tef1. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). Isolates from Shaanxi Province formed three individual clades representing three undescribed species. Etymology. Named after the host plant, Betula albosinensis, from which the holotype was collected.
Culture characters. Cultures incubated on PDA at 25 °C in the dark. Colony originally flat with white felted aerial mycelium, becoming light brown due to pigment formation, conidiomata irregularly distributed over agar surface, with yellowish conidial drops exuding from the ostioles.
Culture characters. Cultures incubated on PDA at 25 °C in the dark. Colony flat, felty with thick texture at the marginal area, with thin texture in the centre, producing beige pigment after 7-10 d. Aerial mycelium white, dense, conidiomata distributed in the centre, with translucent conidial drops exuding from the ostioles.
Culture characters. Cultures incubated on PDA at 25 °C in the dark. Colony originally flat with white fluffy aerial mycelium, becoming pale brown with pigment, with visible solitary conidiomata at maturity.

Discussion
In this study, an investigation of forest pathogens from Huoditang in Shaanxi Province was carried out and Diaporthe canker was observed as a common disease. Identification of our collections was conducted, based on isolates from fruiting bodies using five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters. Three new Diaporthe species were described. These are D. albosinensis sp. nov., D. coryli sp. nov. and D. shaanxiensis sp. nov.
Species delimitation of Diaporthe has improved considerably by using a combination of morphological, cultural, phytopathological and molecular phylogenetic analyses (Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Gao et al. 2017, Guarnaccia and Crous 2017, 2020, Long et al. 2019. As a result, many Diaporthe canker diseases and new species have been discovered and reported from all over the world and also in China. The descriptions and molecular data of Diaporthe species represent an important resource for plant pathologists, plant quarantine officials and taxonomists.