﻿Studies of Diaporthe (Diaporthaceae, Diaporthales) species associated with plant cankers in Beijing, China, with three new species described

﻿Abstract The genus Diaporthe (Diaporthaceae, Diaporthales) comprises endophytes, pathogens and saprophytes, inhabiting a wide range of woody hosts and resulting in serious canker disease. To determine the diversity of Diaporthe species associated with canker disease of host plants in Beijing, China, a total of 35 representative strains were isolated from 18 host genera. Three novel species (D.changpingensis, D.diospyrina and D.ulmina) and four known species (D.corylicola, D.donglingensis, D.eres and D.rostrata) were identified, based on morphological comparison and phylogenetic analyses using partial ITS, cal, his3, tef1-α and tub2 loci. These results provide an understanding of the taxonomy of Diaporthe species associated with canker diseases in Beijing, China.

The sexual morph of Diaporthe generally has immersed ascomata and erumpent pseudostroma with elongated perithecial necks. Asci are unitunicate and sessile producing hyaline ascospores (Udayanga et al. 2011). The asexual morph of Diaporthe can be identified by ostiolate conidiomata, cylindrical phialides and three types (alpha, beta and gamma) of conidia. All of the three types of conidia are aseptate and hyaline, but alpha conidia are fusiform, usually biguttulate; beta conidia are filiform, straight or more often hamate, lack guttules; gamma conidia are fusiform to subcylindrical, multiguttulate (Udayanga et al. 2011;Gomes et al. 2013).
In the past, species identification criteria in Diaporthe was largely based on host specificity and morphological features (Rehner and Uecker 1994;Santos et al. 2010;Dissanayake et al. 2020;Jiang et al. 2021b). However, many Diaporthe species have no obvious selectivity for hosts, for example, D. eres can infect more than 280 hosts (https://nt.ars-grin.gov/fungaldatabases; accessed on 23 Mar 2023). Additionally, although morphological characteristics were proved to be related to the DNA sequence of most Diaporthe species (Guo et al. 2020), many of them with similar morphology are still genetically distinct (Fan et al. 2018a;Jiang et al. 2021a, b). Therefore, it is unreliable for accurate identification when host specificity and morphological features were used alone (Udayanga et al. 2011(Udayanga et al. , 2014Gomes et al. 2013;Yang et al. 2018). Currently, molecular characteristics were proved to be relied on more heavily than morphology (Castlebury et al. 2003;Crous and Groenewald 2005;Udayanga et al. 2012). The taxonomy of Diaporthe species is resolved, based on polyphasic taxonomic concepts including multi-gene phylogenetic and morphological analyses (Udayanga et al. 2012;Fan et al. 2015;Guo et al. 2020;Gao et al. 2021;Jiang et al. 2021a). Five gene regions are used in phylogenetic analyses, including nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), translation elongation factor 1-α (tef1-α) and β-tubulin (tub2) (Dissanayake et al. 2020;Guo et al. 2020;Gao et al. 2021). The identification of Diaporthe species has significantly improved since the polyphasic taxonomic concept was applied, for example, 19 Diaporthe species were identified as pathogens associated with pear shoot canker, based on the five loci sequence data coupled with morphology (Guo et al. 2020). Additionally, some issues about species boundaries of the species complex in Diaporthe were also well resolved, such as the D. eres species complex being investigated and identified as a single species (Hilário et al. 2021;Norphanphoun et al. 2022).
Beijing is the capital city in China and is located in the northern part of the north China Plain. It has a temperate semi-humid monsoon climate, with more than 1,000 species of tree hosts (Ma et al. 1995;Liu et al. 2022). The pathogenic fungi of stem diseases in Beijing are diverse, especially Diaporthe. Diaporthe eres have been identified from Castanea Mollissima and an additional five hosts (Yang et al. 2018); two Diaporthe species were commonly isolated from Juglans mandshurica (Zhu et al. 2019); Diaporthe donglingensis, D. eres and D. huairouensis were confirmed as pathogens of Corylus heterophylla (Bai et al. 2022). During the investigation of plant pathogens in Beijing, branches with typical canker symptoms were collected and subsequently identified combining modern taxonomic concepts. The present study aims to reveal the taxonomy and systematics of Diaporthe species with detailed descriptions of novel species.

Collection, examination and isolation
Fresh specimens with typical ascomata/conidiomata were collected in the surveys of landscape plant canker in Beijing, China. Morphological features of the ascomata/conidiomata were determined by sectioning more than 30 fruiting bodies by hand vertically and horizontally under a stereomicroscope (M205 FA Leica). Over 50 asci/conidia were randomly selected to capture the micromorphological characteristics by using the compound microscope (DM2500 Leica) with differential interference contrast (DIC) optics. Isolates were obtained by cutting the mucoid asci/conidial mass with a sterile blade from the fruiting bodies to the surface of 1.8% potato dextrose agar (PDA) in a 9 cm Petri dish. Isolates were incubated at 25 °C until spores germinated. Hyphal tips were transferred to new PDA plates. The colour of the colony was assessed according to Rayner (1970). Axenic cultures were deposited in the China Forestry Culture Collection Centre (CFCC) and specimens were deposited in the Museum of Beijing Forestry University (BJFC).

DNA extraction and PCR amplification
The cetyltrimethylammonium bromide (CTAB) method was used to extract the genomic DNA when enough mycelium of each isolate had grown on PDA for about five days (Doyle and Doyle 1990). PCR amplifications of five genes (ITS,cal,his3, were done by the primer pairs and PCR conditions listed in Table 1. The five partial loci have the same PCR mixtures including 10 μl Mix (Promega), 7 μl double deionised water, 1 μl of each primer and 1 μl template DNA. All of the amplified DNA were sequenced by the Qingke Biotechnology (Beijing, China). SeqMan v. 7.1.0 was used to check and assemble sequences for each of the gene sequences. The sequence data have been deposited in GenBank and their accession numbers have been listed in Table 2.

Phylogenetic analyses
The sequences used in this study were aligned using MAFFT v. 6 (Katoh and Standley 2013) and corrected manually using MEGA v. 6.0 (Tamura et al. 2013). Reference sequences were obtained from the National Center for Biotechnology Information (NCBI), based on recent published literature associated with Diaporthe (Gao et al. 2021;Bai et al. 2022;Norphanphoun et al. 2022). The sequences of Diaporthella corylina (CBS 121124) were included as outgroups in the polygenic Diaporthe analyses. The alignment, based on combined five concatenated sequences, were concatenated and aligned to compare with other species in Diaporthe to infer the phylogenetic position using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.  Glass and Donaldson (1995) O' Donnell and Cigelnik (1997)  Maximum-likelihood (ML) analyses were conducted with 100 bootstrap support pseudoreplicates and the appropriate models for each gene using PhyML v. 3.0 (Guindon et al. 2010;Kozlov et al. 2019). Bayesian inference (BI) was conducted with a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). MrModeltest v. 2.3 was used to estimate the best fit evolutionary models for each partitioned locus following the Akaike Information Criterion (AIC) (Posada and Crandall 1998). Two MCMC chains were run from random trees for 1,000,000 generations and stopped when the average standard deviation of split frequencies fell below 0.01. Trees were sampled every 100 th generation, resulting in a total of 10,000 trees. For each analysis, the first 25% of the trees were discarded as the burn-in phase and the remaining 75% trees were assessed to calculate the posterior probabilities (BPP) (Rannala and Yang 1996)

Phylogenetic analyses
The concatenated sequences of five genetic regions (ITS,cal,his3, were analysed to infer the interspecific relationships within Diaporthe. The dataset consisted of 343 sequences including the outgroup, Diaporthella corylina CBS 121124. A total of 2,919 characters including gaps (547 for ITS, 578 for cal, 618 for his3, 619 for tef1-α and 557 for tub2) were included in the phylogenetic analysis. The topologies resulting from ML and BI analyses of the concatenated dataset were similar. ML bootstraps (ML BS ≥ 50%) and Bayesian posterior probabilities (BPP ≥ 0.95) have been shown above the branches (Fig. 1). In this study, 35   Etymology. Named after the place where it was first collected, Changping District, Beijing City.
Culture characteristics. Cultures on PDA initially white, growing slowly and entirely covering the 9 cm Petri dish after 14 days. The colonies flat, lacking aerial mycelium with an irregular edge. Conidiomata not observed on medium surface until 30 days.  ). Therefore, we described D. changpingensis as a novel species, based on morphology and sequence data.  (Gao et al. 2021). This species is similar to D. coryli in culture morphology, but it can be distinguished by its longer and thinner alpha conidia (11.0-16.5 × 2.0-3.5 vs. 11.5-13.0 × 3.0-3.5 µm) (Gao et al. 2021). The isolates in this study clustered with D. corylicola, while the phylogram supported it belonging to this species because of the identical DNA sequence. Etymology. Named after the host genus on which it was collected, Diospyros.
Culture characteristics. Colonies with felty aerial mycelium initially white, growing to 80 mm after 3 days, with a uniform texture and regular edge, becoming umber after 9 days. Conidiomata black, distributed randomly at the marginal area.
Notes. Diaporthe diospyrina and D. diospyricola were isolated from the same host genus Diospyros . Although D. diospyricola only has a sequence of the ITS locus, D. diospyrina can be distinguished from D. diospyricola by ITS (20/460). Morphologically, alpha conidia of D. diospyrina (7.5-9.0 μm) are longer than D. diospyricola (5.5-7.0 μm) . Therefore, the current two isolates (CFCC 58820 and 58821) were identified as a new species, D. diospyrina.  (Bai et al. 2022). Phylogenetically, isolates CFCC 58806 and 58807 clustered together with D. donglingensis with high statistical support (ML/BI = 100/1.00) (Fig. 1). Therefore, two isolates in this study were confirmed to be D. donglingensis.  Notes. Diaporthe eres was first described by Nitschke (1870) and isolated from Ulmus sp. in Germany. It is the most common species posing serious canker disease on diverse hosts (Gomes et al. 2013;Udayanga et al. 2014). In this study, 22 isolates were associated with canker diseases of 14 hosts genera including nine new host records in Beijing, China, which clustered in the D. eres species complex (Fig. 1). Therefore, these isolates were conformed to belong to D. eres, based on sequence data and morphology. Notes. Diaporthe rostrata was described as being associated with walnut dieback of Juglans mandshurica in China (Fan et al. 2015). The common symptom of this species was rostrate host tissue around the necks on infected branches (Fan et al. 2015). The current two isolates (CFCC 58843 and 58844) were identified as D. rostrata according to forming a fully supported clade with sequences from CFCC 50062, the ex-type of D. rostrata (ML/BI = 100/1.00). Etymology. Named after the host genus on which it was collected, Ulmus.
Culture characteristics. Cultures with felty aerial mycelium are initially white, growing slowly and entirely covering the 9 cm Petri dish after 8 days,  Notes. Diaporthe ulmina is associated with canker disease of Ulmus pumila. In this study, the isolates CFCC 58828 and 58829 formed a single-lineage clade with high support values (ML/BI = 100/1.00) and it appears to be most closely related to D. huairouensis (Fig. 1). Diaporthe ulmina differs from D. huairouensis isolated from Corylus heterophylla by host association (Bai et al. 2022). Phylogenetically, D. ulmina can be distinguished from D. huairouensis by base differences as follows: 16/466 for ITS, 4/420 for cal, 17/473 for his3, 34/329 for tef1-α and 10/420 for tub2 (Bai et al. 2022). Therefore, D. ulmina is described as a new species.

Discussion
The current study described three new species (D. changpingensis, D. diospyrina and D. ulmina) and four known species (D. corylicola, D. donglingensis, D. eres and D. rostrata), based on 35 isolates of Diaporthe in Beijing, China. The results indicate that Diaporthe species in Beijing are diverse and logical disease control strategies are required.
Since modern taxonomy approaches were applied, more than 40 novel species have been introduced in the recent five years (Fan et al. 2018b;Yang et al. 2018;Dissanayake et al. 2020;Guo et al. 2020;Hilário et al. 2020;Gao et al. 2021;Huang et al. 2021;Jiang et al. 2021b;Bai et al. 2022;Cao et al. 2022). Warmer climate and extensive application of chemicals in fungicides may lead to emergence of new species that are more resistant in northern China (Piao et al. 2010;Úrbez-Torres 2011;Manawasinghe et al. 2018;Jiang et al. 2022a, b). Diaporthe species pose a significant challenge to disease control due to their high species diversity and outstanding environmental adaptation.