Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China

Abstract Species of Diaporthe have often been reported as plant pathogens, endophytes or saprobes, commonly isolated from a wide range of plant hosts. Sixteen strains isolated from species of ten host genera in Yunnan Province, China, represented three new species of Diaporthe, D. chrysalidocarpi, D. machili and D. pometiae as well as five known species D. arecae, D. hongkongensis, D. middletonii, D. osmanthi and D. pandanicola. Morphological comparisons with known species and DNA-based phylogenies based on the analysis of a multigene (ITS, TUB, TEF, CAL and HIS) dataset support the establishment of the new species. This study reveals that a high species diversity of Diaporthe with wide host ranges occur in tropical rainforest in Yunnan Province, China.


Introduction
The genus Diaporthe (Diaporthaceae Diaporthales) with asexual morphs previously known as Phomopsis spp. is based on the type species Diaporthe eres Nitschke (1870) from Ulmus sp. in Germany. Rossman et al. (2015) proposed to use the name Diaporthe over Phomopsis in the context of the one fungus -one name initiative, be-cause it was described first, is encountered commonly in literature and includes the majority of known species. The sexual morph of Diaporthe is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks; asci are unitunicate, clavate to cylindrical; and ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus, sometimes with appendages (Udayanga et al. 2011;Senanayake et al. 2017Senanayake et al. , 2018. The asexual morph is characterised by ostiolate pycnidia with cylindrical phialides often producing three types of hyaline, aseptate conidia called α-conidia, β-conidia and γ-conidia (Udayanga et al. 2011;Gomes et al. 2013). The α-conidia and β-conidia are produced frequently, but the γ-conidia are rarely observed (Gomes et al. 2013;Guarnaccia and Crous 2017;Guo et al. 2020).
In the past, methods of species identification of Diaporthe had previously been based only on host as well as morphological characters such as the size and shape of ascomata and conidiomata. Nowadays, molecular phylogenetic studies demonstrate that determining species boundaries only by morphological characters is not possible due to lack of host specificity and their variability under changing environmental conditions (Gomes et al. 2013). Phylogenetic analysis using a five-locus dataset (ITS-TUB-TEF-CAL-HIS) has been determined to be the optimal combination to identify species of Diaporthe species, as revealed by Santos et al. (2017). Many Diaporthe species are described based on a polyphasic approach together with morphological characterisation (Rehner and Uecker 1994;Udayanga et al. 2011;Gao et al. 2017;Guarnaccia and Crous 2017;Yang et al. 2018aYang 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).
The aim of this study was to explore the diversity of Diaporthe species from symptomatic leaves of plants in Yunnan Province. We present three novel species and five known species of Diaporthe, collected from species belonging to ten host genera, based on morphological characters and phylogenetic analysis.

Isolation and morphological studies
Leaves of samples were collected in 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 surface-sterilized by immersing them in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA) (Cai et al. 2009). PDA plates (90 mm) were incubated in an 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 colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Colour notations was done using the colour charts of Rayner (1970). Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both fitted with Olympus DP80 high definition colour digital cameras to document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher and type specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of 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 were 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 visualised 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 Gen-Bank (Table 1).

Phylogenetic analyses
Novel sequences generated from the sixteen strains in this study, and all reference 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 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. 1.4.2 (http://tree.bio.ed.ac.uk/ software/figtree) and edited with Adobe Illustrator CS5.1. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 1) and the alignments and trees were deposited in TreeBASE: S27479 (http://treebase.org/ treebase-web/home.html).

Phylogenetic analyses
Sixteen strains of Diaporthe isolated from plant hosts from Yunnan, China, were grown in culture and used for analyses of molecular sequence data. Diaporthe spp. were analysed by using multilocus data (ITS, TUB, TEF, CAL and HIS) from 115 isolates of Diaporthe spp. and Diaporthella corylina (CBS 121124) as the outgroup taxon. A total of 3005 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1-656, TUB: 657-1329, TEF: 1330-1860, CAL: 1861-2444,  Isolates marked with "*" are ex-type or ex-epitype strains.   HIS: 2445-3005. Of these characters, 1349 were constant, 453 were variable and parsimony-uninformative, and 1203 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for ITS, TUB, TEF and HIS, HKY+I+G for and CAL were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1). ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.90) are shown as first and second position above nodes, respectively. Based on the fivelocus phylogeny and morphology, nine isolates were assigned to five species, including Diaporthe arecae (1), D. hongkongensis (2), D. middletonii (4), D. osmanthi (1) and D. pandanicola (1), whereas seven isolates formed distinct well supported clades, which refer to novel species named D. chrysalidocarpi (2), D. machili (2) and D. pometiae (3), respectively. Figure 2 Subramanella arecae H.C. Srivast., Zakia & Govindar., in Srivastava, Banu and Govindarajan (1962 (Srivastava et al. 1962) and placed in Diaporthe by Gomes et al. (2013). The Diaporthe isolate from fruits of Citrus sp. (CBS 535.75) in Suriname was also placed in D. arecae by Gomes et al. (2013). In the present study, strain (SAUCC194.18) from symptomatic leaves of Persea americana was congruent with D. arecae based on morphology and DNA sequences data (Fig. 1). We therefore consider the isolated strain as D. arecae. Etymology. Named after the host genus on which it was collected, Chrysalidocarpus lutescens.
Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, growth rate 16.3-17.5 mm diam/day, aerial mycelium abundant, white on surface, reverse white to pale yellow, with an obvious concentric zonation, pycnidia forming after 15 days.

Diaporthe osmanthi
Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, growth rate 12.0-13.5 mm diam/day, cottony with abundant aerial mycelium, sparse at margin. With several concentric rings of dense hyphae, white on surface, white to pale brown on reverse.

Diaporthe pandanicola Tibpromma & K.D. Hyde, MycoKeys 33: 44 (2018)
Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, growth rate 12.8-15.0 mm diam/day, flat, cottony in centre, with aerial mycelium sparse toward margin, white on surface, white to pale yellow on reverse.   Notes. Diaporthe pandanicola was originally described by Tibpromma et al. (2018) on healthy leaves of Pandanus sp. (Pandanaceae) as an endophytic fungus. Our strain (SAUCC194.82) is closely related to Diaporthe pandanicola based on phylogenetic analyses (Fig. 1). The differences of nucleotides in the concatenated alignment (19/533 in the ITS region and 11/351 in the TUB region) are less than 3%. Morphologically, our strain produces alpha conidia, beta conidia and gamma conidia, while Diaporthe pandanicola did not sporulate. We therefore identify our strains as Diaporthe pandanicola. Etymology. Named after the host genus on which it was collected, Pometia pinnata.
Culture characteristics. Cultures incubated on PDA at 25 °C in darkness, growth rate 11.5-13.0 mm diam/day, cottony with abundant aerial mycelium, with a concentric zonation, white on surface, white to grayish on reverse.

Discussion
The Yunnan Province in southeastern China has a unique geography where three climatic regions meet: the eastern Asia monsoon region, the Tibetan plateau region, and the tropical monsoon region of southern Asia and Indo-China. The environment is conducive to growth of unusual microbial species. Species diversity in Yunnan Province is high compared to other parts of China.
Previously, species identification of Diaporthe relied on the assumption of hostspecificity, leading to the proliferation of names. The morphological characters of Diaporthe could be changeable, as most taxa in culture do not produce all spore states of the asexual (alpha, beta and gamma conidia) or the sexual morph (Gomes et al. 2013). Based on a polyphasic approach and morphology, more than one species of Diaporthe can colonize a single host, while one species can be associated with several hosts (Gomes et al. 2013;Gao et al. 2017;Guarnaccia and Crous 2017;Guarnaccia et al. 2018;Guo et al. 2020). These studies revealed a high diversity of Diaporthe species from different hosts. Our study supports this phenomenon. For example, Diaporthe arecae (SAUCC194.18) and D. pometiae (SAUCC194.19) were collected from Persea americana; In addition, isolates of D. middletonii were obtained from three hosts (Litchi chinensis, Lithocarpus craibianus, L. glaber). As for host specificity, in our study, four species of Diaporthe, D. machili (SAUCC194.69), D. middletonii (SAUCC194.27), D. osmanthi (SAUCC194.21), and D. pometiae (SAUCC194.72) were isolated from Litchi chinensis and Pometia pinnata belong to the Sapindaceae, and D. litchiicola also was reported from Litchi chinensis in Queensland (Tan et al. 2013); however, D. machili (SAUCC194.111) also was isolated from Machilus pingii (Lauraceae), D. middletonii (SAUCC194.45) from Lithocarpus glaber (Fagaceae), D. osmanthi (GUCC 9165) from leaves of Osmanthus fragrans (Oleaceae) (Long et al. 2019), and D. pometiae (SAUCC194.19 and SAUCC194.73) from Persea americana (Lauraceae) and Heliconia metallica (Musaceae). These results provide evidence that many species are able to colonise diverse hosts and several different species could co-occur on the same host. It seems obvious that specificity does not occur at the family level.
For the current study, sixteen strains isolated from ten host genera represented three new species and five known species, based on morphological characters and phylogenetic analyses of the five combined loci (ITS, TUB, TEF, CAL and HIS). The descriptions and molecular data for species of Diaporthe represent an important resource for plant pathologists, plant quarantine officials and taxonomists.