Ya-Quan Zhu,Chun-Yan Ma,Han Xue,Chun-Gen Piao,Yong Li,Ning Jiang
﻿Two new species of Diaporthe ( Diaporthaceae , Diaporthales ) in China
Leaf spots,morphology,multi-gene phylogeny,taxonomy,Fungi,Diaporthales,Diaporthaceae,Fungi,Diaporthales,Diaporthaceae
﻿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 Smilax glabra and dead culms of Xanthium strumarium 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 Diaporthe rizhaoensis and D. smilacicola are identified, described and illustrated in the present study.
﻿Two new species of Diaporthe ( Diaporthaceae , Diaporthales ) in China

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.

Diaporthe (Diaporthaceae,Diaporthales) is a species-rich genus with its asexual morph previously known as Phomopsis (Rossman et al. 2007; Udayanga et al. 2011, 2012a, 2014a, 2015; Dissanayake 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 sexual morph of Diaporthe is characterized by immersed ascomata and an erumpent pseudostroma with single or multiple tapering perithecial necks. Asci are unitunicate, sessile and clavate to cylindrical. Ascospores are elliptical to fusiform, septate or aseptate, hyaline, biseriate to uniseriate in the ascus and sometimes have appendages (Udayanga et al. 2011; Senanayake et al. 2017, 2018). The asexual morph is characterized by black or dark brown conidiomata, with cylindrical phialides producing three types of aseptate and hyaline conidia (Type I: α-conidia, hyaline, fusiform, straight, guttulate or eguttulate, aseptate, smooth-walled; type II: β-conidia, hyaline, filiform, straight or hamate, aseptate, smooth-walled, eguttulate; type III: γ-conidia, rarely produced, hyaline, multiguttulate, fusiform to subcylindrical with an acute or rounded apex, while the base is sometimes truncate) (Udayanga et al. 2011; Gomes et al. 2013). Species of Diaporthe are widely distributed, and infect a broad plant host range, e.g., agricultural crops, forest trees, vegetables, and fruits (Farr et al. 2002a, b; Crous 2005; Rossman et al. 2007; Udayanga et al. 2011, 2012a, b, 2014a, b, 2015; Gomes et al. 2013; Du et al. 2016; Dissanayake et al. 2017; Guarnaccia and Crous 2017; Fan et al. 2018). As plant pathogens, Diaporthe spp. cause severe diseases, e.g., blights, cankers, decay, dieback, leaf spots and wilt of many economically important plants in genera Castanea, Citrus, Helianthus, Macadamia, Rosa, Vaccinium and Vitis, resulting in major losses (Thompson et al. 2011; Huang et al. 2015; Guarnaccia et al. 2018, 2020; Hilário et al. 2020; Wrona et al. 2020; Caio et al. 2021; Jiang et al. 2021a). 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 the present study, we have analyzed five-locus dataset of combined nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone (his3), translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2). To aid the identification of two new species, we followed Norphanphoun et al. (2022) for the taxonomic treatments of Diaporthe. Norphanphoun et al. (2022) clustered Diaporthe into 13 workable species complexes namely D.arecae, D.biconispora, D.carpini, D.decedens, D.eres, D.oncostoma, D.pustulata, D.rudis, D.scobina, D.sojae, D.toxica, D.varians and D.vawdreyi species complexes. In addition, nine species were retained as singletons, viz., D.acerina, D.acutispora, D.crataegi, D.multiguttulata, D.ocoteae, D.perjuncta, D.pseudoalnea, D.spartinicola and D.undulata based on multilocus phylogeny. In previous studies, Smilaxglabra and Xanthiumstrumarium have been reported as hosts of Diaporthe (Vrandecic et al. 2007, 2010; Gao 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, 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 Diaportherizhaoensis 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.

During 2021 and 2022, investigations were conducted to inspect for the presence of Diaporthe species associated with plant diseases in China. Leaves of Smilaxglabra and culms of Xanthiumstrumarium 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/).

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 Mg2+, 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.

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.

In the present study, we followed Norphanphoun et al. (2022) for the species complexes treatments of Diaporthe. Firstly, we conducted a genus tree including all species belonging to this genus according to Norphanphoun et al. (2022). After that, the phylogenetic analysis revealed that three isolates (CFCC 57562, CFCC 57563 and CFCC 57564) clustered in a distinct clade in the D.sojae species complex, and three isolates (CFCC 54582, CFCC 58764 and CFCC 58765) clustered in a distinct clade in the D.arecae species complex (Figs 1, 2). The combined sequence alignments of D.arecae species complex comprised 62 strains, with D.vawdreyi (BRIP 57887a) and D.biconispora (ZJUD62) as the outgroup taxa. The dataset comprised 2791 characters including alignment gaps (634 for ITS, 381 for tef1, 791 for tub2, 499 for cal and 486 for his3). The combined sequence alignments of D.sojae species complex comprised 111 strains, with D.aceris (LC8112) and D.alnea (CBS 146.46) as the outgroup taxa. The dataset comprised 2799 characters including alignment gaps (671 for ITS, 483 for tef1, 483 for tub2, 593 for cal and 569 for his3). The final maximum likelihood tree topology was similar to Bayesian analysis.

Taxon classification Fungi Diaporthales Diaporthaceae Y.Q. Zhu & Ning Jiang sp. nov. B0CFC039-E2A1-5035-8608-3273A2023E46 846816 Fig. 3

Named after the collection site of the type specimen, Rizhao City.

Conidiomata pycnidial, small, scattered, slightly erumpent through bark surface, nearly flat, discoid, with a solitary undivided locule, 150–400 μm diam. Conidiogenous cells 6.7–11.4 × 1.6–3.0 μm, hyaline, unbranched, densely aggregated, mostly ampulliform, guttulate, aseptate, straight or slightly curved, swelling at base, tapering towards apex. Beta conidia 12.9–23.4 × 1.1–2.1 μm (mean = 18.7 × 1.4 μm, n = 50), hyaline, filiform, straight or slightly curved, aseptate, base subtruncate, tapering towards the base. Alpha conidia and gamma conidia not observed. Sexual morph not observed.

Colonies on potato dextrose agar (PDA) flat, spreading, with flocculent aerial mycelium and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C; on malt extract agar (MEA) flat, spreading, with flocculent aerial mycelium and crenate edge, white, reaching a 90 mm diameter after 14 days at 25 °C, forming black conidiomata with black conidial masses; on synthetic low nutrient agar (SNA) flat, spreading, with flocculent aerial mycelium forming concentric rings and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C.

China, Shandong Province, Rizhao City, Wulian County, Zhongzhi Town, on dead culms of Xanthiumstrumarium, 5 May 2022, Ning Jiang & Chengbin Wang (holotype CAF 800069; ex-holotype culture CFCC 57562). Shandong Province, Rizhao City, Wulian County, Xumeng Town, on dead culms of Xanthiumstrumarium, 5 May 2022, Ning Jiang & Chengbin Wang (cultures CFCC 57563 and CFCC 57564).

Diaportherizhaoensis formed a distinct clade with high support (ML/BI = 100/1), and was close to D.guttulata and D.stewartia (Fig. 1). Diaportherizhaoensis is different from D.stewartia by host association (D.rizhaoensis on Xanthiumstrumarium vs. D.stewartia on Cosmosbipinnatus) (Harrison 1935; Dissanayake et al. 2020). In addition, D.guttulata and D.stewartia are only known in sexual morph. Moreover, Diaportherizhaoensis can be distinguished from D.guttulata (15/364 in cal, 5/428 in his3, 5/313 in tef1, and 1/408 in tub2) and D.stewartii (3/532 in ITS, 7/451 in cal, and 7/369 in tub2) by sequence data. Diaporthehelianthi, D.longicolla, D.pseudolongicolla (= D.novem) and D.rizhaoensis have been reported form the host Xanthiumstrumarium (Vrandecic et al. 2007, 2010; Petrović et al. 2018; Thompson et al. 2018). Morphologically, Diaporthehelianthi is a bit longer than D.rizhaoensis in the beta conidia, but not fully distinguished (Vrandecic et al. 2007, 2010). Morphology of D.longicolla and D.pseudolongicolla on Xanthiumstrumarium were not available. However, these four species are phylogenetically distinguished in the phylogram of D.sojae species complex (Fig. 1).

Taxon classification Fungi Diaporthales Diaporthaceae Y.Q. Zhu & Ning Jiang sp. nov. 1BC54B14-2E2C-5952-8A5A-3DC760DCB39F 846818 Fig. 4

Named after the host genus, Smilax.

Leaf spots subcircular to irregular, pale brown to brown, with dark brown margin. Conidiomata pycnidial, scattered, subglobose to globose, black, erumpent, exuding faint yellow translucent conidial droplets from central ostioles, 150–350 μm diam. Conidiogenous cells 11–16.2 × 1.8–2.4 μm, hyaline, phialidic, cylindrical, terminal, slightly tapering towards the apex. Alpha conidia 5.7–9.7 × 2.0–3.5 μm (mean = 7.8 × 2.6 μm, n = 50), hyaline, aseptate, smooth, guttulate, ellipsoidal to oblong ellipsoidal, with both ends obtuse. Beta conidia and gamma conidia not observed. Sexual morph not observed.

Colonies on PDA flat, with flocculent aerial mycelium and crenate edge, white to gray, reaching a 90 mm diameter after 14 days at 25 °C, forming black conidiomata with black conidial masses; on MEA flat, spreading, with flocculent aerial mycelium forming concentric rings, off-white to luteous, reaching a 90 mm diameter after 14 days at 25 °C; on SNA flat, spreading, with flocculent aerial mycelium forming concentric rings and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C.

China, Hunan Province, Changsha City, Changsha County, Kaihui Town, on leaf spots of Smilaxglabra, 2 November 2020, Ning Jiang (holotype CAF 800070; ex-holotype culture CFCC 54582). Hunan Province, Shaoshan City, on leaf spots of Smilaxglabra, 2 November 2020, Ning Jiang (cultures CFCC 58764 and CFCC 58765).

Three Diaporthe isolates representing D.smilacicola formed a well-supported clade (ML/BI = 100/1), and appear to be distinct from the other Diaporthe species phylogenetically (Fig. 2). Diaportheeres (= D.mahothocarpi), D.lithocarpi and D.smilacicola have been reported from the host S.glabra (Gao et al. 2013; Chaisiri et al. 2021). Morphologically, these three species are similar in conidial shape and size. However, Diaportheeres belongs to D.eres species complex, which is different from D.lithocarpi and D.smilacicola in D.arecae species complex. D.smilacicola is obviously different from D.lithocarpi based on sequence data (22/467 in ITS, 31/393 in cal, 52/317 in tef1, 19/420 in tub2) (Fig. 2).

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 Xanthiumstrumarium and D.smilacicola from leaf spots of Smilaxglabra. Species identification in Diaporthe was primarily based on the assumption of host-specificity, 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; Guarnaccia et al. 2018; 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 Heliconiametallica and Perseaamericana (Huang et al 2021); D.melastomatis was collected from three hosts namely Camelliasinensis, Melastomamalabathricum and Millettiareticulata (Sun et al. 2021); 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 Smilaxglabra and Xanthiumstrumarium. 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, b, 2015; Fan et al. 2015; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Hyde et al. 2018, 2020; Yang et al. 2018, 2020, 2021; Long 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 (Santos and 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 (Udayanga et al. 2014; Fan et al. 2018; Yang et al. 2018; Guo et al. 2020). 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.