Diaporthe species in south-western China

Abstract Three strains of the genus Diaporthe were isolated from different plant hosts in south-western China. Phylogenetic analyses of the combined ITS, β-tubulin, tef1 and calmoudulin dataset indicated that these strains represented three independent lineages in Diaporthe. Diaporthe millettiaesp. nov. clustered with D. hongkongensis and D. arecae, Diaporthe osmanthisp. nov. grouped with D. arengae, D. pseudomangiferae and D. perseae and Diaporthe strain GUCC9146, isolated from Camellia sinensis, was grouped in the D. eres species complex with a close relationship to D. longicicola. These species are reported with taxonomic descriptions and illustrations.

Three strains of Diaporthe were isolated from different medicinal plants collected in Guizhou and Guangxi during a survey of fungal diversity in south-western China. All the strains produced conidiomata containing alpha-and beta-conidia, typical of Diaporthe. This paper describes these three collections using molecular evidence, based on the analysis of combined ITS, β-tubulin, tef1 and calmoudulin datasets, as Diaporthe millettiae sp. nov. and D. osmanthi sp. nov. and D. longicicola with a new host record from Camellia sinensis.

Isolation and morphological studies
The samples were collected from Guizhou and Guangxi provinces. The Diaporthe strains were isolated using the single-spore method (Chomnunti et al. 2014). Colonies, growing from single spores, were transferred to potato-dextrose agar (PDA) and incubated at room temperature (28 °C). Following 2-3 weeks of incubation, morphological characters were recorded as in Udayanga et al. (2011Udayanga et al. ( , 2015. Conidia and conidiophores were observed using a compound microscope (Olympus BX53). The holotype specimens are deposited in the Herbarium of Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Ex-type cultures are deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, China (GUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org) and Facesoffungi (http://www.facesoffungi.org).

DNA extraction and sequencing
Fungal cultures were grown on PDA medium until they nearly covered the whole Petri-dish (90 mm diam.) at 28 °C. Fresh fungal mycelia were scraped from the surface with sterilised scalpels. A BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416) was used to extract fungal genome DNA. DNA amplification was performed in a 25 μl reaction volume system which contained 2.5 μl 10 × PCR buffer, 1 μl of each primer (10 μM), 1 μl template DNA and 0.25 μl Taq DNA polymerase (Promega, Madison, WI, USA). Primers ITS4 and ITS5 (White et al. 1990) were used to amplify the ITS region. Three protein-coding gene fragments (β-tubulin, tef1 and calmoudulin) were amplified by the primers Bt2a/Bt2b (Glass and Donaldson 1995), CAL228F/CAL737R and EF1-728F/EF1-986R (Carbone and Kohn 1999). Gene sequencing was performed with an ABI PRISM 3730 DNA autosequencer using either a dRhodamine terminator or Big Dye Terminator (Applied Biosystems Inc., Foster 19 City, California). The sequences of both strands of each fragment were determined for sequence confirmation. The DNA sequences were submitted to GenBank and their accession numbers were provided in Table 1.

Phylogenetic analyses
DNA sequences from our three strains and reference sequences downloaded from GenBank (Dissanayake et al. 2017a, b), Guarnaccia et al. (2018) and Wanasinghe et al. (2018) were analysed by maximum parsimony (MP) and maximum likelihood (ML). Sequences were optimised manually to allow maximum alignment and maximum sequence similarity, as detailed in Manamgoda et al. (2012). MP analyses were performed in PAUP v. 4.0b10 (Swofford 2003), using the heuristic search option with 1,000 random taxa additions and tree bisection and re-connection (TBR) as the branch swapping algorithm. Maxtrees = 5000 was set to build the phylogenetic tree. The characters of the alignment document were ordered according to ITS+tef1+β-tubulin+CAL for GUCC9165 and GUCC9167 and tef1+β-tubulin for GUCC9146 with equal weight and gaps were treated as missing data. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. The resulting Phylip file was used to make ML and Bayesian trees by the CIPRES Science Gateway (https://www.phylo.org/portal2/ login.action) and RAxML-XSEDE with 1000 bootstrap inferences.
Habitat and distribution. Isolated from leaves of Millettia reticulata in China Etymology. Species epithet millettiae, referring to the host, Millettia reticulata from which the strain was isolated.
Habitat and distribution. Isolated from leaves of Osmanthus fragrans in China. Etymology. Species epithet osmanthi, referring to the host, Osmanthus fragrans from which our strain was isolated.

Diaporthe longicicola
Habitat and distribution. Isolated from leaves of Camellia sinensis in Duyun, Guizhou Province, China Notes. Phylogenetic analyses (Figures 1, 2) indicated that GUCC 9146 has a close relationship with D. longicicola, D. rosicola, D. eres and D. cotoneastri. Morphological comparison indicated that this strain was most similar to D. longicicola but not a related species by the width of alpha conidia and length of beta conidia (Udayanga et al. 2014;Gao et al. 2015).

Discussion
Phylogenetic analysis and morphology provide evidence for the introduction of Diaporthe millettiae and D. osmanthi as new species. In order to support the validity of these new species, we followed the guidelines of Jeewon and Hyde (2016) in comparing base pair differences (Suppl. material 1: Table S1). In accordance with Udayanga et al. (2014), we also believed that the ITS fragment was problematic for the D. eres species-complex. When not considering ITS, integration with morphological comparison was helpful and we concluded that GUCC 9146 is D. longicicola. Diaporthe longicicola was firstly reported on Lithocarpus glabra in Zhejiang Province, but our strain (GUCC 9146) was recovered from Camellia sinensis in Guizhou Province. Thus, this is the report of a new host and new location in China for D. longicicola.