﻿Two new species of Diaporthe (Diaporthaceae, Diaporthales) in China

﻿Abstract Species of Diaporthe have been reported as plant endophytes, pathogens and saprobes on a wide range of plant hosts. Strains of Diaporthe were isolated from leaf spots of Smilaxglabra and dead culms of Xanthiumstrumarium in China, and identified based on morphology and molecular phylogenetic analyses of combined internal transcribed spacer region (ITS), calmodulin (cal), histone H3 (his3), translation elongation factor 1-alpha (tef1) and β-tubulin (tub2) loci. As a result, two new species named Diaportherizhaoensis and D.smilacicola are identified, described and illustrated in the present study.


Introduction
Diaporthe (Diaporthaceae, Diaporthales) is a species-rich genus with its asexual morph previously known as Phomopsis (Rossman et al. 2007;Udayanga et al. 2011Udayanga et al. , 2012aUdayanga et al. , 2014aUdayanga et al. , 2015Dissanayake et al. 2017;Guarnaccia et al. 2018). The genus Diaporthe was established by Nitschke in 1870 and predates its sexual morph established in 1905, thus Diaporthe is recommended to be used for this genus following "one fungus one name" nomenclature (Nitschke 1870; Rossman et al. 2015).
The genus Diaporthe includes over 1000 epithets, mostly based on morphological characteristics and host associations (van der Aa et al. 1990;Santos et al. 2010;Guarnaccia et al. 2018). However, recent studies have shown that many species of Diaporthe are not host-specific, i.e., one species may infect more than one host species (Vrandecic et al. 2011;Bai et al. 2015;Zhang et al. 2018). And many Diaporthe species that are morphologically similar have proven to be genetically distinct (van Rensburg et al. 2006;Udayanga et al. 2011;Jiang et al. 2021b). Thus, polyphasic taxonomy is essential to identify and comprehensively characterize Diaporthe.
In previous studies, Smilax glabra and Xanthium strumarium have been reported as hosts of Diaporthe (Vrandecic et al. 2007(Vrandecic et al. , 2010Gao et al. 2013;Thompson et al. 2018). D. eres (= D. mahothocarpi) and D. lithocarpi were identified as the cause agents of leaf spot disease based on morphology and phylogenetics on S. glabra in China (Gao et al. 2013). D. helianthi and D. longicolla, pathogens of X. strumarium, have been collected from blighted stems and branches in Croatia (Vrandecic et al. 2007(Vrandecic et al. , 2010. D. pseudolongicolla (= D. novem) has been reported as a branch dieback agent in X. strumarium in Australia (Thompson et al. 2018).
In this study, we introduce two new species namely Diaporthe rizhaoensis and D. smilacicola, collected from diseased plant tissues in China. We further provide descriptions, illustrations, and DNA sequence-based phylogeny to verify identification and placement.

Isolation and morphological characterization
During 2021 and 2022, investigations were conducted to inspect for the presence of Diaporthe species associated with plant diseases in China. Leaves of Smilax glabra and culms of Xanthium strumarium showing typical symptoms of Diaporthe were collected. Infected tissues were cut into 0.5 × 0.5 cm pieces using a double-edge blade, and surface sterilized as follows. These sections underwent initial immersion for 2 min in 0.5% sodium hypochlorite, followed by 1 min in sterile distilled water, 2 min in 75% ethanol, and, finally, 1 min in sterile distilled water. The disinfected fragments were then plated onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) and malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, 15 g agar per L), and incubated at 25 °C to obtain the pure culture.
Species identification was based on morphological features of the new species produced on infected plant tissues and PDA plates. Conidiomata were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. Over 20 fruiting bodies were sectioned, and 50 conidia were selected randomly for measurement using Axio Imager 2 microscope (Zeiss, Oberkochen, Germany). Isolate characteristics incubated on PDA at 25 °C were observed and recorded at 7 days, including colony colour, texture and the arrangement of the conidiomata. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http:// www.cfcc-caf.org.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

DNA extraction, amplification and sequencing
Genomic DNA was extracted from the fresh mycelium harvested from PDA plates after 7 days using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced for all isolates. The BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the resulting sequences with those in GenBank. After confirmation of Diaporthe species, four additional partial loci, including calmodulin (cal), histone H3 (his3), partial translation elongation factor 1-alpha (tef1) and part of the beta-tubulin gene region (tub2) genes were amplified. The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 1. A PCR reaction was conducted in a 20 µL reaction volume, and the components were as follows: 1 µL DNA template (20 ng/µl), 1 µL forward 10 µM primer, 1 µL reverse 10 µM primer, 10 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg 2+ , Beijing TisingKe Biotech Co., Ltd., Beijing, China), and 7 µL sterile water. Amplifications were performed using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Strands were sequenced in both directions using PCR primers. All amplified PCR products were estimated visually 1.4% agarose gels stained with ethidium bromide and then PCR positive products were sent to Sangon Biotech (Shanghai) Co., Ltd., (Beijing, China) for sequencing.

Phylogenetic analyses
Sequences were edited and condensed with SeqMan v.7.1.0. The sequences generated in this study were supplemented with additional sequences obtained from GenBank (Table 2) based on blast searches and recent publications of the genus Diaporthe. The sequences were aligned with the MAFFT v.7 after which the alignments were manually corrected using MEGA v. 7.0. (Katoh and Toh 2010;Kumar et al. 2016). Phylogenetic analyses including Maximum Likelihood (ML) and Bayesian Inference (BI) methods were conducted for the single gene sequence data sets of the ITS, cal, his3, tef1 and tub2, and the combined data set of all five gene regions. ML analyses were conducted using RAxML-HPC BlackBox 8.2.10 on the CIPRES Science Gateway portal (https:// www.phylo.org) (Miller et al. 2012), employing a GTRGAMMA substitution model with 1000 bootstrap replicates (Stamatakis 2014). BI analyses were conducted using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v.3.0 (Ronquist and Huelsenbeck 2003). Two Markov chain Monte Carlo (MCMC) chains were run from a random starting tree for 1,000,000 generations, resulting in a total of 10,000 trees. The first 25% of trees sampled were discarded as burn-in and the remaining trees were used to calculate the posterior probabilities. Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7,500 trees. Phylogenetic trees were viewed with FigTree v. 1.4 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 2.

Discussion
Based on the morphology and the multi-locus phylogeny, six isolates from the present study can be recognized as two new species of Diaporthe, viz. D. rizhaoensis from dead culms of Xanthium strumarium and D. smilacicola from leaf spots of Smilax glabra.
Species identification in Diaporthe was primarily based on the assumption of hostspecificity, which has largely impeded the progress of establishing a proper taxonomy of Diaporthe (Gomes et al. 2013). More than one species of Diaporthe can often be recovered from a single host and one species was found to be associated with different host plants (Gomes et al. 2013;Gao et al. 2017;Guarnaccia and Crous 2017;Guo et al. 2020). For example, D. eres can infect blackberry (Vrandecic et al. 2011), pear (Bai et al. 2015), and jujube (Zhang et al. 2018); D. pometiae was isolated from Heliconia metallica and Persea americana ; D. melastomatis was collected from three hosts namely Camellia sinensis, Melastoma malabathricum and Millettia reticulata ; D. australiana, D. drenthii, D. macadamiae and D. searlei can cause diseases on macadamia in Australia and South Africa (Wrona et al. 2020) and seven endophytic Diaporthe species were discovered on Citrus trees (Huang et al. 2015). As was revealed in the present study, two additional species of Diaporthe were proposed from the host Smilax glabra and Xanthium strumarium. This study further demonstrates that host association is not a robust character to distinguish members of Diaporthe.
Recently, the species classification of Diaporthe has become more dependent on DNA sequence-based methods rather than traditional morphological characterization. (Udayanga et al. 2014a(Udayanga et al. , b, 2015Fan et al. 2015;Gao et al. 2017;Guarnaccia and Crous 2017;Guarnaccia et al. 2018;Hyde et al. 2018Hyde et al. , 2020Yang et al. 2018Yang et al. , 2020Yang et al. , 2021Long et al. 2019;Cao et al. 2022). The ITS sequence offers convincing proof for species demarcation and is recommended for identifying species boundaries in the genus Diaporthe Phillips 2009, 2011;Thompson et al. 2011). However, the intraspecific variation is even greater than the interspecific variation, which makes it difficult to identify Diaporthe species using the ITS sequence alone (Crouch et al. 2009). Considering this, concatenation of a five-loci dataset (ITS-tef1-tub2-cal-his3) was recommended as the best combination for species identification within the genus ( Two phylograms resulted from the present study also support the feasibility of the five loci data to separate species of Diaporthe.
The two newly introducing species could potentially be pathogens, because they were isolated from diseased plant tissues, and their pathogenicity should be evaluated in further studies. And, it is necessary to evaluate the effects of environmental conditions, such as temperature, pH, and carbon sources, on mycelium growth and pathogenicity.