Unravelling Diaporthe Species Associated with Woody Hosts from Karst Formations (Guizhou) in China

Though several Diaporthe species have been reported in China, little is known about the species associated with nature reserves in Guizhou province. During a survey of fungi in six nature reserves in Guizhou province of China, thirty-one Diaporthe isolates were collected from different woody hosts. Based on morphology, culture characteristics and molecular phylogenetic analysis, these isolates were characterized and identified. Phylogenetic analysis of internal transcribed spacer region (ITS), combined with translation elongation factor 1-alpha (tef), β-tubulin (tub), calmodulin (cal) and histone H3 (his) gene regions identified five known Diaporthe species and seven distinct lineages representing novel Diaporthe species. The details of five known species: Diaporthe cercidis, D. cinnamomi, D. conica, D. nobilis and D. sackstonii are given and the seven new species D. constrictospora, D. ellipsospora, D. guttulata, D. irregularis, D. lenispora, D. minima, and D. minusculata are introduced with detailed descriptions and illustrations. This study revealed a high diversity of previously undescribed Diaporthe species associated with woody hosts in various nature reserves of Guizhou province, indicating that there is a potential of Diaporthe species remains to be discovered in this unique landform (Karst formations) in China. Interestingly, the five known Diaporthe species have been reported as pathogens of various hosts, and this could indicate that those newly introduced species in this study could be potentially pathogenic pending further studies to confirm.


Introduction
Diaporthe Nitschke (including the Phomopsis asexual morph) belongs to family Diaporthaceae, order Diaporthales and class Sordariomycetes [1][2][3] and its species are found worldwide on a diverse range of host plants as endophytes, pathogens and saprobes [4]. Rossman et al. [5] proposed the name Diaporthe over Phomopsis, as both names are well known amongst plant pathologists and subsequent studies have adopted the latter generic name [4,[6][7][8][9][10]. More than 1100 epithets for Diaporthe and 986 for Phomopsis are listed in Index Fungorum (2020) (http://www.indexfungorum.org/, accessed August 2020) with names often based on host association. Many Diaporthe species that are morphologically similar have proven to be genetically distinct [11,12], and several isolates formerly identified based on their hosts were shown to represent different taxa [1]. Diaporthe represents a highly complex genus containing numerous cryptic species. In recent studies, Diaporthe species have been distinguished mainly by their Table 1. Diaporthe species studied in this study ( Figure 1). Details of ex-type species introduced in this study are in bold.

Molecular Based Amplification
Fungal mycelium of 7 d old cultures was scraped for the extraction of genomic DNA using Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® ) following the manufacturer's protocol (Hangzhou, China). For the identification of Diaporthe specimens, the internal transcribed spacer region (ITS) was sequenced for all isolates and BLAST search (basic local alignment search tool) at GenBank was used to reveal the closest matching taxa. Besides ITS gene sequence data, translation elongation factor 1-alpha (tef ), β-tubulin (tub), calmodulin (cal) and histone H3 (his) gene regions were also employed to support the species identification. The ITS region was amplified using universal primers ITS1 and ITS4 [30]. The target region of the tef gene was amplified using primer pairs EF-728F and EF-986R [31]. A portion of the tub gene was amplified using the primers BT2a and BT2b [32], while the primer pair CAL228F and CAL737R was used to amplify the cal gene region [31]. The primers CYLH3F [33] and H3-1b [32] were used to amplify part of the his gene. The PCR reactions were accomplished in a Bio Rad C1000 thermal cycler. The amplification procedure was performed in a 50 µL reaction volume containing 5-10 ng DNA, 0.8 units Taq polymerase, 1X PCR buffer, 0.2 mM dNTP, 0.3 µm of each primer with 1.5 mM MgCl 2. Following the PCR amplification, products were visualized on 1% agarose gel under UV light using a Gel Doc TM XR Molecular Imager following ethidium bromide staining. PCR products were purified using minicolumns, purification resin and buffer according to the manufacturer's protocols (Amersham product code: 27-9602-01). Sequence analysis was carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).
Ambiguous regions in the MP alignment were excluded, and gaps were treated as missing data. The stability of the trees was evaluated by 1000 bootstrap replications. Branches of zero length were collapsed, and all multiple parsimonious trees saved. Statistics including tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI) were calculated. Differences between the trees inferred under different optimality criteria were evaluated using Kishino-Hasegawa tests (KHT) [52].
Bayesian analyses were performed in MrBayes v.3.0b4 [49] and posterior probabilities (PP) were determined by Markov Chain Monte Carlo sampling (MCMC). MrModeltest v. 2.3 [50] was used for the statistical selection of the best-fit model of nucleotide substitutions and was integrated into the analysis. Six simultaneous Markov chains were run for 10 6 generations; sampling the trees at every 100th generation. From the 10,000 trees obtained, the first 2000 representing the burn-in phase were discarded. The remaining 8000 trees were used for calculating posterior probabilities in the majority rule consensus tree.
The details of the fungal strains obtained in this study are listed in Table 1 with information of the type cultures and sequence data. Sequences generated in this study were deposited in GenBank (Table 1); alignments and trees were deposited in TreeBASE (www.treebase.org, study ID S27013). Reviewer access URL: http://purl.org/phylo/treebase/phylows/study/TB2:S27013?x-access-code=1369710211c386567d8b43ba36f49adf&format=html. Taxonomic novelties were submitted to the Faces of Fungi database [53], Index Fungorum (Index Fungorum 2020) and MycoBank (www. mycobank.org) [33].

Phylogenetic Analyses
Saprobic specimens sampled from numerous woody hosts in six nature reserves in the Karst region of Guizhou province, China resulted in the isolation of thirty-one isolates of Diaporthe (Table 1, Figure 1). The ITS gene was employed for the identification of all isolates to the genus level. The ITS, tef, tub, cal and his alignments (including the gaps) were determined to be approximately 570, 470, 450, 610 and 500 bp (base pair) in size, respectively. The combined ITS, tef, tub, cal and his sequences of Diaporthe contained data for 136 isolates, including the outgroup taxon Diaporthella corylina (CBS 121124). The analyses consisted of 31 isolates from this study ( Table 1) and 105 sequences (62 type species) originating from GenBank ( Table 2). Out of a total of 2594 characters in the MP analyses, 1079 were constant, and 269 were variable and parsimony uninformative. Ten most parsimonious trees resulted from the remaining 1246 parsimony-informative characters (TL = 7439, CI = 0.384, RI = 0.804, RC = 0.309, HI = 0.616). In the ML analyses, the best scoring RAxML tree ( Figure 1 The isolates obtained in this study were grouped into twelve clades. Three isolates were grouped with the ex-type of Diaporthe cercidis (CFCC 52565) while another three isolates were clustered with the ex-type of D. nobilis (CBS 587.79). In addition one isolate with D. cinnamomi (CFCC 52569), D. conica (CFCC 52571) and D. sackstonii (BRIP 54669b) respectively. Twenty-two isolates did not cluster with any known Diaporthe species; thus, seven novel species, Diaporthe constrictospora (2 isolates, Figure 2), Diaporthe ellipsospora (3 isolates, Figure 3), Diaporthe guttulata (2 isolates, Figure 4), Diaporthe irregularis (4 isolates, Figure 5), Diaporthe lenispora (3 isolates, Figure 6), Diaporthe minima (4 isolates, Figure 7) and Diaporthe minusculata (4 isolates, Figure 8) are determined to be new species based on the morphological and phylogenetic evidence ( Figure 1).

Morphology and Culture Characteristics
In this study, thirty-one Diaporthe isolates were obtained from decaying woody hosts from six nature reserves in Guizhou province, China ( Table 1). The Diaporthe isolates obtained in this study were further categorized based on morphological characteristics. Growth was rapid for all isolates grown on PDA, with mycelia covering the whole surface of the Petri dishes. Aerial mycelium was initially white and turned greyish after incubation in the dark at 25 • C for several days. All species exhibited phenotypic characteristics typical of the genus. The seven new species of Diaporthe described here are phylogenetically distinct from all previously described species for which sequence data are available. Table 2. GenBank accession numbers of species included in the phylogenetic analysis ( Figure 1). Ex-type/ex-epitype/ex-isotype/ex-neotype isolates are in bold.      Saprobic on decaying wood. Sexual morph: Ascomata 190-240 µm diam, black, globose to subglobose or irregular, clustered or solitary, deeply immersed in host tissue. Asci 40-48 µm × 9-11 µm (x = 43 × 8, n = 30), 8-spored, unitunicate, sessile, elongate to clavate. Ascospores 10-12 × 3-4 µm (x = 11 × 4, n = 50), hyaline, elongated to elliptical, two-celled, often 4-guttulate, with larger guttules at center and smaller ones at the ends. Asexual morph: Not observed.

Species
Culture characteristics: Colonies covering entire PDA Petri dishes after 10 d at 25 • C producing abundant white aerial mycelium, reverse fuscous black.   Notes: Two strains representing Diaporthe constrictospora cluster in a well-supported basal clade (ML/MP/BI = 100/100/1.0) and appear to be distinct from other Diaporthe species, and can be easily recognized by their distinctive phylogenetic placement (Figure 1). Since this species is not closely related to any Diaporthe species and we were unable to compare the nucleotide differences in the alignment. Diaporthe constrictospora is introduced as a phylogenetically distinct species (Figure 1).    Notes: Four isolates, representing Diaporthe irregularis, are retrieved in a well-supported clade (ML/MP/BI = 100/100/1.0) and appear to be distinct from other Diaporthe species phylogenetically (Figure 1). Since this species does not closely related to any particular Diaporthe species, we were unable to compare the nucleotide differences in the concatenated alignment. In addition, Diaporthe irregularis can be morphologically distinguished from other Diaporthe species based on the shape and the position of the ascomata.
Culture characteristics: Colonies covering entire PDA Petri dishes after 10 d at 25 • C producing abundant white aerial mycelium, reverse early yellow and turned to fuscous black. Notes: In the combined phylogenetic tree, Diaporthe lenispora groups in a distinct clade with maximum support (ML/MP/BI = 100/100/1.0) and it appears to be most closely related to D. vawdreyi (Figure 1). Diaporthe lenispora can be distinguished from D. vawdreyi based on ITS, tef and tub loci (19/539 in ITS, 56/467 in tef and 23/453 in tub), cal and his gene regions are unavailable for D. vawdreyi. We are not able to compare the morphology of D. lenispora and D. vawdreyi as the latter has no reported sexual morph [74].

Discussion
Based on the phenotypic characters and the multi-locus phylogeny, the 31 isolates obtained in this study can be recognized as twelve species. Among the five species are previously known and seven species are new to science. These newly discovered species are Diaporthe constrictospora, D. ellipsospora, D. guttulata, D. irregularis, D. lenispora, D. minima and D. minusculata. The other taxa are identified as Diaporthe cercidis [42], D. cinnamomi [42], D. conica [42], D. nobilis [4] and D. sackstonii [70]. Morphological characters of the known species isolated in this study were compared with their original descriptions. Phylogenetically, there were no significant base pair differences between these and their type based combined gene alignments.
A phylogenetic tree derived from an alignment of ITS sequences is beneficial as a guide for the identification of isolates of Diaporthe species [65,75]. ITS sequences offer convincing proof for species demarcation where a limited number of taxa are analyzed, such as species associated with the same host [62,64,76]. However, confusion arises when a large number of species from an extensive range of host species are examined. Santos et al. [77] proposed that tef is a superior phylogenetic marker in Diaporthe than ITS, and has been commonly used as the secondary locus for phylogenetic studies [8,10,64,75]. Gomes et al. [4] studied five loci from 95 species and stated that tef poorly distinguished species, and recommended that his and tub were suitable possibilities as subordinate phylogenetic markers to accompany the authorized fungi barcode: the internal transcribed spacer region (ITS). Dissanayake et al. [10] reviewed the genus Diaporthe and provided a checklist for 171 species with available molecular data (from culture and fruiting body) and a phylogenetic tree using four gene regions (ITS, tef, tub and cal). According to Santos et al. [16], incorporation of a five-loci dataset (ITS, cal, his, tef, tub) was recommended as the best combination for species identification within the genus and recent studies seems to favor the selection of four or five genes [13][14][15]33,[38][39][40][41][42][43][44]. Hence, the present study is conducted combining the five gene regions analyses of ITS, tef, tub, cal and his to reveal five known Diaporthe species and to assist in the introduction of seven new Diaporthe species.
Several studies have been conducted to reveal the association of Diaporthe species with various hosts in China. Huang et al. [60] revealed seven apparently undescribed endophytic Diaporthe species (Diaporthe biconispora, D. biguttulata, D. discoidispora, D. multigutullata, D. ovalispora, D. subclavata and D. unshiuensis) on Citrus. Gao et al. [63] identified four novel species (D. apiculata, D. compacta, D. oraccinii, D. penetriteum) and three known species (D. discoidispora, D. hongkongensis, D. ueckerae) associated with Camellia (tea). Gao et al. [78]  However, the identification of Diaporthe species associated with hosts in nature reserves in China has rarely been studied. Thus, an investigation of Diaporthe species was conducted and this provides the first molecular phylogenetic frame of Diaporthe diversity in six nature reserves in the Karst region of Guizhou province, combined with morphological descriptions.
Among the twelve species identified in this study, four species have been previously isolated from China. Yang et al. [42] introduced Diaporthe cercidis from twigs and branches of Cercis chinensis in Jiangsu Province, D. cinnamomi from symptomatic twigs of Cinnamomum sp. in Zhejiang Province and D. conica from symptomatic branches of Alangium chinense in Zhejiang Province. Diaporthe nobilis has been isolated from Camellia sinensis in Guizhou Province [25]. The other known species: D. sackstonii [70] has been isolated from petioles of sunflower plants (Helianthus annuus) inAustralia. Based on the percentage of occurrence, Diaporthe irregularis sp. nov (13%), D. minima sp. nov (13%), and D. minusculata sp. nov (13%) were categorized as being frequent. Diaporthe cinnamomi, D. conica and D. sackstonii were ranked as infrequent, since only one isolate has been isolated for each species. Interestingly, the type species of the genus, D. eres Nitschke [79] was not observed in our survey. This species is one of the frequent species in most of the studies and appears with 365 Fungus-Host combinations [80].
The discovery of these species of Diaporthe from different nature reserves in Guizhou province as well as worldwide occurrence shows the polyphagous and cosmopolitan behavior of species in this genus. Certainly, it is obvious that performing complementary studies based on sequencing five gene regions of Diaporthe species is essential in order to support reliable species identification. The descriptions and molecular data of Diaporthe species provided in this study would serve as a resource for plant pathologists, plant quarantine officials and taxonomists for better identification of Diaporthe and its species boundaries. Such studies are necessary to investigate this group of fungi in different unexploited biomes, to reveal the degree of diversity and to support more suitable control measures to prevent their dissemination. Importantly, based on the Diaporthe taxa identification in this study coupled with previous studies, it could be concluded that almost all the known species isolated (Diaporthe cercidis, D. cinnamomi, D. conica, D. nobilis and D. sackstonii) as saprobes in this study were pathogenic on various host plants [25,42,70]. This could indicate that the seven newly introducing species could potentially be pathogens even though they were isolated from decaying woody hosts, and their pathogenicity should be evaluated in further studies with more samples (from other kinds of habitats and hosts, as well as the different distributions and substrates). In the meantime, we provided the culture details and deposited them in publicly accessible culture collections for further evaluation or comparison of the life modes of these taxa.

Conclusions
We carried out fungal diversity investigations with large-scale sampling in the Karst region of southwestern China and this is the first report of Diaporthe species isolated from nature reserves in Karst region of Guizhou province, China. The identification of twelve Diaporthe species (five known species and seven new species) associated with saprobic woody hosts is documented.