Morphological and molecular identification of Diaporthe species in south-western China, with description of eight new species

Abstract Diaporthe species have often been reported as plant pathogens, endophytes and saprophytes, commonly isolated from a wide range of infected plant hosts. In the present study, twenty strains obtained from leaf spots of twelve host plants in Yunnan Province of China were isolated. Based on a combination of morphology, culture characteristics and multilocus sequence analysis of the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-α (TEF), β-tubulin (TUB), calmodulin (CAL), and histone (HIS) genes, these strains were identified as eight new species: Diaporthe camelliae-sinensis, D. grandiflori, D. heliconiae, D. heterostemmatis, D. litchii, D. lutescens, D. melastomatis, D. pungensis and two previously described species, D. subclavata and D. tectonendophytica. This study showed high species diversity of Diaporthe in tropical rain forests and its hosts in south-western China.


introduction
Diaporthe is a genus in the Diaporthaceae family (Diaporthales), with the asexual morph previously known as Phomopsis and type species Diaporthe eres Nitschke collected from Ulmus sp. in Germany (Nitschke 1870). Nevertheless, with the implementation of "one fungus one name" nomenclature, the generic names Diaporthe and Phomopsis are no longer used for both morphs of this genus, and Rossman et al. (2015) gave priority to the older name Diaporthe Nitschke over Phomopsis (Sacc.) Bubák because it was published first, encountered commonly in literatures and represents the majority of species. The sexual morph of Diaporthe is characterized by: immersed perithecial ascomata and an erumpent pseudostroma with more or less elongated perithecial necks; unitunicate clavate to cylindrical asci; fusoid, ellipsoid to cylindrical, septate or aseptate, hyaline ascospores, biseriately to uniseriately arranged in the ascus, sometimes having appendages (Udayanga et al. 2011;Senanayake et al. 2017Senanayake et al. , 2018. The asexual morph is characterized by ostiolate conidiomata, with cylindrical phialides producing three types of hyaline, aseptate conidia (Udayanga et al. 2011;Gomes et al. 2013): 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. The gamma conidia rarely produced and observed, those species  (Gomes et al. 2013; Guarnaccia and Crous 2017;Guo et al. 2020).
From previous studies, the methods of species identification and classification in genus Diaporthe were based on criteria such as morphological characters like the size and shape of ascomata (Udayanga et al. 2011) and conidiomata (Rehner and Uecker 1994). However, in recent studies, determining species boundaries only by morphological characters was demonstrated to be not always informative due to their variability under changing environmental conditions (Gomes et al. 2013). As for phylogenetic analysis for Diaporthe species, the use of a five-locus dataset (ITS-TUB-TEF-CAL-HIS) is the optimal combination for species delimitation as revealed by Santos et al. (2017). Thus, in recent years, many Diaporthe species have been described based on a polyphasic approach combined with morphological characterization and their host associations (Guarnaccia and Crous 2017;Gao et al. 2017;Yang et al. 2018Yang et al. , 2020Crous et al. 2020;Dayarathne et al. 2020;Guo et al. 2020;Hyde et al. 2020;Li et al. 2020;Zapata et al. 2020).
In this study, we propose eight novel species and two previously described species of Diaporthe, collected in Yunnan Province of China on twelve plant host genera, based on their morphological characters in culture, and molecular phylogenetic analysis.

Isolation and morphological studies
The leaves of samples were collected from Yunnan Province, China. Isolations from surface sterilized leaf tissues were conducted following the protocol of Gao et al. (2014). Tissue fragments (5 × 5 mm) were taken from the margin of leaf lesions and surfacesterilized by consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and finally rinsed in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and transferred on potato dextrose agar (PDA) in petri plates (Cai et al. 2009). All the PDA plates were incubated at biochemical incubator at 25 °C for 2-4 days, and hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates.
Following 2-3 weeks of incubation, photographs of the fungal colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Micromorphological characters were observed and documented in distilled water from microscope slides under Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both supplied with Olympus DP80 HD color digital cameras to photodocument fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Living strain cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).
PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume which contained 12.5 μL Green Taq Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Biosune, Shanghai, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL.
PCR parameters were as follows: 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Annealing temperature for each gene was 55 °C for ITS, 60 °C for TUB, 52 °C for TEF, 54 °C for CAL and 57 °C for HIS. The PCR products were visualized on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic analyses
Novel sequences generated from twenty strains in this study, and all reference available sequences of Diaporthe species downloaded from GenBank were used for phylogenetic analyses. Alignments of the individual locus were determined using MAFFT v. 7.110 by default settings (Katoh et al. 2017) and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of five loci (ITS, TUB, TEF, CAL and HIS regions). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) and MrBayes on XSEDE (3.2.7a) (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Ronquist et al. 2012), respectively. For ML analyses the default parameters were used and Isolates marked with "*" are ex-type or ex-epitype strains.
BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included five parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 500 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v.

Diaporthe grandiflori
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the Petri dish after 15 days kept in dark conditions at 25 °C, cottony with abundant aerial mycelium, white on surface side, white to grayish on reverse.
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized infected plant material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium abundant, cottony, white, dense in the center, sparse near the margin. White on surface side, white to tanned on reverse side.

Diaporthe litchii
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium abundant, white, cottony on surface, reverse white to pale brown with two concentric zonation.
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized infected plant material. Colonies on PDA cover the petri plate diameter after incubation for 15 days in dark conditions at 25 °C, initially white, becoming grayish, reverse pale brown, with concentric rings of dense and sparse hyphae, irregular margin, fluffy aerial mycelium. Pycnidia formed in 15 days.
Notes. From the phylotree, seen on Fig. 1 Etymology. Named after the host Melastoma malabathricum on which it was collected.
Diagnosis. Diaporthe melastomatis differs from D. parapterocarpi Crous in smaller α-conidia and the types of conidia.
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri diameter Notes. Diaporthe melastomatis is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (ML/ BI = 100/1). Diaporthe melastomatis is most closely related to D. parapterocarpi, but distinguished based on ITS and TUB loci from D. parapterocarpi by 32 nucleotides difference in the concatenated alignment, in which 20 are distinct in the ITS region, 12 in the TUB region. Morphologically, Diaporthe melastomatis differs from D. parapterocarpi in its smaller alpha conidia (5.5-8.5 × 1.7-2.5 vs. 8.0-10.0 × 2.5-3.0 μm). Furthermore, Diaporthe melastomatis can produce beta conidia, but D. parapterocarpi cannot . Etymology. Named after the host Elaeagnus pungens on which it was collected.
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the 3/4 of Petri dish diameter after incubation for 15 days in dark conditions at 25 °C, flat, cottony in the center with medium developed aerial mycelium, sparse in the outer region. With several concentric rings of dense and sparse hyphae, irregular margin, white on surface side, white to pale yellow and cinnamon speckle on reverse side.

Diaporthe tectonendophytica
Culture characteristics. Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C, aerial mycelium abundant, white to grayish on surface side, pale yellow on reverse with concentric zonation. Pycnidia are formed on 15 th day or later.
Notes. Diaporthe tectonendophytica was originally described from the asymptomatic branches of Tectona grandis in Thailand (Doilom et al. 2017). In the present study, two strains (SAUCC194.11 and SAUCC194.63) from symptomatic leaves of Elaeagnus conferta and Pometia pinnata were congruent with D. tectonendophytica based on morphology and DNA sequences data (Fig. 1). We therefore describe D. tectonendophytica as a known species for this clade.

Discussion
In the current study, 87 reference sequences (including an outgroup taxon) were selected based on BLAST searches of NCBIs GenBank nucleotide database and were included in the phylogenetic analyses (Table 1). Phylogenetic analyses based on five combined loci (ITS, TUB, TEF, CAL and HIS), as well as morphological characters of the non-sexual morph obtained in culture, contributed to knowledge of the diversity of Diaporthe species in Yunnan Province. Based on a large set of freshly collected specimens from Yunnan province, China, 20 strains of Diaporthe species were isolated from 12 host genera (Table 1). As a result, eight new species are proposed: Diaporthe camelliae-sinensis, D. grandiflori, D. heliconiae, D. heterostemmatis, D. litchii, D. lutescens, D. melastomatis, D. pungensis and two previously described species were described and illustrated, D. subclavata and D. tectonendophytica.