﻿Four new species of Diaporthe (Diaporthaceae, Diaporthales) from forest plants in China

﻿Abstract Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. During trips to collect forest pathogens in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces in China, 16 isolates of Diaporthe were obtained from branch cankers and leaf spots. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef-1α) and β-tubulin (tub2) loci. Results revealed four new taxa, D.celticola, D.meliae, D.quercicola, D.rhodomyrtispp. nov. and two known species, D.eres and D.multiguttulata.

In China, the classification of Diaporthe has been progressing and the basis for the species identification is a combination of morphological, cultural and phylogenetical analyses (Huang et al. 2015;Gao et al. 2017;Guarnaccia and Crous 2017;Yang et al. 2017Yang et al. , 2018Yang et al. , 2020Yang et al. , 2021aManawasinghe et al. 2019;Jiang et al. 2021;Huang et al. 2021;Sun et al. 2021;Wang et al. 2021). The present study was conducted to identify Diaporthe species that cause dieback and leaf spot disease in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces based on modern taxonomic concepts.

Fungal isolation
From 2018 to 2020, sample collections have been ongoing in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces, China (Table 1). Collected samples were taken to the laboratory for isolation and photographed, documented and then kept at 4 °C for further study.
A total of 16 isolates from host material were obtained by removing a mucoid conidia mass from conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating conidium was removed and plated onto fresh PDA plates. Specimens were deposited in the Museums of the Beijing Forestry University (BJFC) and Central South University

Morphological and cultural characterization
Agar plugs (6 mm diam) were taken from the edge of actively growing cultures on PDA and transferred onto the centre of 9 cm diam Petri dishes containing 2% tap water agar supplemented with sterile pine needles (PNA) (Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 25 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation as described in recent studies (Gomes et al. 2013;Lombard et al. 2014). Colony characters and pigment production on PNA and PDA were noted in the 10-day culture. Colony features were rated according to the color charts of Rayner (1970). Cultures were examined periodically for the development of conidiomata. The microscopic examination was based on the morphological features of conidiomata obtained from the fungal growth mounted in clear lactic acid. At least 30 conidiomata and conidia were measured to calculate the mean size/length. Micro-morphological observations were done at ×1000 magnification using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature, and illustrations of taxonomic novelties were deposited at MycoBank (www.MycoBank. org) (Crous et al. 2004).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). For PCR amplifications of phylogenetic markers, five different primer pairs were used

Phylogenetic analyses
The quality of our amplified nucleotide sequences was checked and combined by Seq-Man v.7.1.0 and reference sequences (Table 1) were retrieved from the National Center for Biotechnology Information (NCBI), according to recent publications of the genus (Guo et al. 2020;Sun et al. 2021;Yang et al. 2021b). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and manually corrected using Bioedit 7.0.9.0 (Hall 1999). Phylogenetic analyses were carried out with maximum likelihood analysis   (ML), which was performed at the CIPRES web portal (Miller et al. 2010), 1000 rapid bootstrap replicates were run with GTRGAMMA model of nucleotide evolution. Bayesian inference analysis (BI) was performed in MrBayes v. 3.2.0 (Ronquist and Huelsenbeck 2003). 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. The best nucleotide substitution model for ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was selected for both cal and tef-1α. Phylogenetic trees were viewed in FigTree v1.4. The names of the isolates from the present study are marked in blue in the trees. Maximum likelihood bootstrap support values ≥ 75% (BT) are given at the nodes. Bayesian posterior probabilities ≥ 0.95 (PP) were thickened in the phylogenetic tree. Alignment and trees were deposited in TreeBASE (submission ID: S29621).

Diaporthe eres
Culture characters. Colony originally flat with white felty aerial mycelium, becoming auburn furcate mycelium with age, with irregular margin, conidiomata absent.
Morphologically, D. meliae can be distinguished from D. podocarpi-macrophylli by its longer conidiogenous cells (15-26.5 vs. 6-18 μm) and alpha conidia (8-9.5 vs. 3.5-8.5 μm) (Gao et al. 2017).  Notes. Diaporthe multiguttulata is characterised by ellipsoidal alpha conidia with one large guttulate, and was originally described as an endophyte from healthy branch of Citrus grandis in Fujian Province, China (Huang et al. 2015). Yang et al. (2021a) identified three isolates from Citrus maxima as D. multiguttulata based on DNA sequence data and confirmed from the morphological characters. In the present study, isolates (CFCC 53098, CFCC 53099, and CFCC 53100) from an additional specimen were observed and supplemented with beta conidia (Fig. 4j). Diagnosis. Distinguished from the phylogenetically closely-related species, D. biguttulata, by its filiform, eguttulate alpha conidia.
Culture characters. Colony at first white, becoming dark brown with age. Aerial mycelium white, dense, fluffy, with yellowish conidial drops exuding from the ostioles after 20 days.
Culture characters. Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium.

Discussion
In this study, investigations of forest pathogens in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces was carried out. Identification of our collections was conducted, based on isolates from fruiting bodies using five combined loci (ITS,cal,his3,, and tub2), as well as morphological characteristics. It includes Diaporthe eres and D. multiguttulata, as well as four new species named D. celticola, D. meliae, D. quercicola, and D. rhodomyrti. Diaporthe (Diaporthaceae, Sordariomycetes) are species-rich asexual taxa, which are common pathogens that cause a variety of diseases, including dieback, stem cankers, leaf spots, leaf and pod blights, fruit rots and seed decay (Uecker 1988;Rehner and Uecker 1994;Mostert et al. 2001;Thompson et al. 2001;Santos et al. 2011). Because many Diaporthe species have overlapping morphological traits, sequence data is essential to resolve this genus and introduce new species (Udayanga et al. 2014a). Combined gene sequence of ITS, cal, his3, tef-1α, and tub2 is the optimal combination for species delimitation (Santos et al. 2017). However, removing the ITS locus has little effect on reconstructed phylogenies, identifying the cal-his3-tef-1α-tub2 four loci tree as almost equivalent to the five loci phylogenetic tree.
Many confusions occur in species separation of Diaporthe eres complex with the lack of an ex-type culture or ex-epitype culture, although a broad species concept has historically been associated with D. eres (Udayanga et al. 2014b). Fan et al. (2018) demonstrated the effectiveness of three loci, including cal, tef-1α and tub2, for the identification of the D. eres complex in walnut trees. Similarly, Yang et al. (2018) also used three-locus sequences to identify D. eres species associated with different hosts in China, and Chaisiri et al. (2021) revealed the phylogenetic analysis from the combined dataset of cal, his3, tef-1α and tub2 was highly effective, but the ITS region impeded species delimitation, which conforms with Yang et al. (2018).
Recently, several studies have been conducted associated with various hosts in China. For instance, the research conducted by Guo et al. (2020) revealed six novel Diaporthe species that infect pears and are responsible for pear shoot canker. Sun et al. (2021) showed high species diversity of Diaporthe in tropical rain forests, with description of eight new species. Wang et al. (2021) represented the first characterization of Diaporthe species associated with peach constriction canker in China, and contributed useful data for practicable disease management. Yang et al. (2021b) identified two new species from Camellia oleifera, which is an important edible oil woody plant in southern China. This study also characterises the taxonomic and morphological diversity of Diaporthe spp. associated with different hosts, which indicated there is a potential of Diaporthe species remains to be discovered in China.