Morphological and Phylogenetic Analyses Reveal Three New Species of Didymella (Didymellaceae, Pleosporales) from Jiangxi, China

Didymella contains numerous plant pathogenic and saprobic species associated with a wide range of hosts. Over the course of our mycological surveys of plant pathogens from terrestrial plants in Jiangxi Province, China, eight strains isolated from diseased leaves of four host genera represented three new species of Didymella, D. bischofiae sp. nov., D. clerodendri sp. nov., and D. pittospori sp. nov. Phylogenetic analyses of combined ITS, LSU, RPB2, and TUB2 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), revealed their taxonomic placement within Didymella. Both morphological examinations and molecular phylogenetic analyses supported D. bischofiae, D. clerodendri, and D. pittospori as three new taxa within Didymella. Illustrations and descriptions of these three taxa were provided, along with comparisons with closely related taxa in the genus.


Introduction
Didymella, the type genus of the family Didymellaceae, was introduced by Saccardo in 1880, with D. exigua as the type species, and later validated when a Latin diagnosis was provided [1,2].The genus was recently emended by Chen et al. [3,4], who gave a very detailed account of generic concepts.The sexual morphs of Didymella are mainly characterized by solitary or confluent, ostiolate pseudothecial ascomata with multi-layered, pseudoparenchymatous ascomatal walls and cylindrical to clavate or saccate, 8-spored, bitunicate asci with hyaline or brownish uniseptate (symmetrical or asymmetrical) or multiseptate ascospores.The asexual morphs of Didymella are mainly characterized by solitary or confluent, ostiolate or poroid, pycnidial conidiomata with multi-layered, pseudoparenchymatous conidiomatal walls, and phialidic, hyaline conidiogenous cells that produce smooth conidia, which are generally aseptate, variable in shape, hyaline or occasionally pigmented, and larger or septate in at least one species in older cultures.Unicellular chlamydospores are often present in pure culture [2][3][4][5][6][7].To date, 438 records of Didymella are listed in Species Fungorum [8], and most of them are usually found as saprobes from herbaceous and woody plants, but many are also important plant pathogens [3,4,9].
Jiangxi Province is located on the south bank of the middle and lower reaches of the Yangtze Riverin southern China.It lies at 24 • 29 ′ -30 • 04 ′ N and 113 • 34 ′ -118 • 28 ′ E and covers a total area of 166,900 km 2 with superior ecological environment, humid subtropical climate, and abundant plant resources, which provide favorable conditions for the survival and multiplication of various microbial species.During an investigation of the diversity of plant pathogens from terrestrial plants in Jiangxi Province, three interesting species of Didymella were collected from the symptomatic leaves of four host genera.Based on morphological and multi-loci (LSU, ITS, RPB2, and TUB2) phylogenetic analyses, they are proposed as new to science in the present study, and their names were registered in Index Fungorum [28].

Sample Collection and Fungal Isolation
Samples of diseased leaves were collected from botanical garden or conservation areas with rich plant resources in Jiangxi Province, China.Representative plants samples with leaf spots were placed in Ziploc™ bags, labeled, and returned to the laboratory.The strains from the collected diseased leaves were isolated and identified using a tissue separation method [29].Before isolation, the collected leaf samples were rinsed with running water, and several tissue pieces (5 mm × 5 mm) from the junction of diseased and healthy parts were selected for surface disinfection.The tissue pieces were disinfected with 75% ethanol for 45 s and 5% sodium hypochlorite for 30 s, rinsed 3 times with sterile water, dried with sterilized filter paper, transferred to the potato dextrose agar (PDA; 20% potato + 2% dextrose + 2% agar, w/v) plates [30], and then incubated at 25 • C in darkness for 3-5 days.The growing hyphae at the edge of the colony was inoculated onto new PDA plates for purification and morphological studies.

Morphological and Cultural Characterization
Each fungal isolate was removed to the new PDA, MEA, and OA plates and incubated at 25 • C in darkness.Their morphological characters were recorded after 7 days.Morphological characteristics of conidia on PDA were observed using an Olympus BX 53 light microscope and captured using the Olympus DP 27 digital camera (Olympus Optical Co., Tokyo, Japan) with a 40 × objective at the same background color and scale, and the sizes of conidia were randomly selected for measurement.All fungal strains were stored in 10% sterilized glycerin at 4 • C for further studies.The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).

DNA Extraction, PCR Amplification, and Sequencing
Fungal isolates were incubated on PDA plates at 25 • C for 7-14 days.The hyphae were scraped from the surface of colonies and transferred into 2 mL microcentrifuge tubes for genomic DNA extraction.DNA extraction was carried out using the Solarbio Fungi Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).To confirm the species, the regions (ITS, LSU, RPB2, and TUB2) of all fungal isolates were sequenced.A portion of the internal transcribed spacer region (ITS), large ribosomal subunit (LSU), β-tubulin (TUB2) regions, and the second largest subunit of RNA polymerase II (RPB2) genes were amplified using primer pairs ITS5/ITS4 [31], LR0R/LR7 [32], Bt2a/Bt2b [33], and dRPB2-5f/dRPB2-7r [34], respectively.The corresponding primer pairs and PCR processes are listed in Table 1.The total volume of the PCR reaction was 20 µL, including 10 µL of 2 × Power Taq PCR Master Mix, 0.8 µL of each the forward and reverse primer, 7.4 µL of double-distilled water (ddH2O), and 1 µL of DNA template.The PCR products were visualized on a 1% agarose gel electrophoresis and stained with ethidium bromide.Sequencing was performed bidirectionally by Beijing Tsingke Biotechnology Co., Ltd.(Beijing, China).Newly obtained sequences in this study were deposited in NCBI GenBank (www.ncbi.nlm.nih.gov,accessed on 21 November 2023; Table 2).

Phylogenetic Analyses
The newly generated sequences from this study were analyzed using other related sequences obtained from GenBank (Table 2).Sequences of the individual loci were initially aligned using MAFFTv.7 [35] on the online server (http://maffTh.cbrc.jp/alignment/server/, accessed on 18 December 2023) using default settings and manually corrected where necessary.Phylogenetic analyses were first conducted individually for each locus, and then for a combined analyses of four loci (ITS, LSU, TUB2, and RPB2).The ITS, LSU, RPB2, and TUB2 sequence data were concatenated by using the "Concatenate Sequence" function in Phylosuite software v1.2.1 [36], and absent sequences data in the comparisons were treated using the question mark and "-" as missing data.The concatenated aligned dataset was analyzed separately using maximum-likelihood (ML) and Bayesian inference (BI).The best evolutionary model for each alignment dataset was selected using Mod-elFinder [37] and incorporated into the analyses.Maximum-likelihood phylogenies were inferred using IQ-TREE [38] under an edge-linked partition model for 10,000 ultrafast bootstraps [39].The optima trees were inferred using the heuristic search option with 1000 random sequence additions.The best-fit model was TIM2e+I+G4 for ITS, LSU, TUB2, and RPB2 alignments.Based on the partition model (2 parallel runs, 2,000,000 generations), Bayesian inference phylogenies were inferred using MrBayes 3.2.6 [40], in which the initial 25% of sampled data was discarded as burn-in, and the best nucleotide substitution model for each locus was identified using ModelFinder of Phylosuite to be SYM+I+G4 for ITS and GTR+F+I+G4 for LSU, RPB2, and TUB2.The resulting trees were plotted using FigTree v.1.4.2 [36] (http://tree.bio.ed.ac.uk/software/figtree, accessed on 18 December 2023) and further edited in Adobe Illustrator 2021.

Molecular Phylogeny
Based on the sequence data of ITS, LSU, RPB2, and TUB2, the phylogenetic relationships of the eight strains of Didymella were analyzed using the regions of four genes of 118 strains representing 96 species in Didymellaceae.The combined data set (ITS:1-462, LSU:463-1189, RPB2:1190-1630, and TUB2:1631-1915) was composed of 477 distinct patterns, 341 parsimony informative sites, 61 singleton sites, and 1513 constant sites.A total of four single-locus data sets, ITS, LSU, RPB2, and TUB2, contained 54, 19, 171, and 97 parsimony informative sites, respectively.Epicoccum nigrum (CBS 173.73) and E. poae (LC 8160) served as outgroups.The phylogenetic reconstructions obtained from the combined dataset of maximum-likelihood and Bayesian inference analyses support largely similar topologies, and the best-scoring ML consensus tree (lnL = -14164.265)is shown in Figure 1

Taxonomy
Didymella bischofiae X.X.Luo, X.G.Zhang, and Jian Ma, sp.nov., Figure 2. Index Fungorum number: IF901249.Etymology: Referring to the host genus from which it was collected, Bischofia polycarpa.Culture characteristics: Colonies on PDA reaching 68-70 mm diam after 7 days at 25 • C, margin regular, aerial mycelium sparsely, flat, central pale olivaceous and white all around, reverse slightly pink, and abundant production of chlamydospores with growth.Colonies on MEA reaching 62-63 mm diam after 7 days at 25 • C, margin regular, the middle is pale brown and gradually becomes white around, covered with medium aerial mycelium, and reverse buff to white.Colonies on OA reaching 58-60 mm diam after 7 days at 25 • C, margin regular, pale olivaceous, and reverse concolorous.
Notes: Strains HJAUP 1776, HJAUP 1776b, and HJAUP 1776c are similar in morphological characteristics and have identical DNA sequences; form a single, high support clade (MLBS/BPP = 100/1.00,Figure 1); and, therefore, are identified as the same new species, Didymella bischofiae.The phylogenetic tree showed that the strains of D. bischofiae formed a distinct lineage sister to D. nigricans (CBS 444.81) in a fully supported clade (MLBS/BPP = 100/1.00,Figure 1).Didymella bischofiae is closely related to D. nigricans and has 8 bp differences in four loci from the latter.Morphologically, D. bischofiae clearly differed from D. nigricans which produce fewer chlamydospores, smaller conidiogenous cells (4-8 ×

Taxonomy
Didymella bischofiae X.X.Luo, X.G.Zhang, and Jian Ma, sp.nov., Figure 2.     Culture characteristics: Colonies on PDA reaching 68-70 mm diam after 7 days at 25 • C, margin regular, light brown in the middle, covered by white felt-like aerial hyphae, and white around; abundant production of pycnidia in the late growth stage.Colonies on MEA reaching 62-65 mm diam after 7 days at 25 • C, margin regular, covered by white felt-like aerial hyphae, and the back was buff.Colonies on OA reaching 57-59 mm diam after 7 days at 25 • C, margin regular, pale olivaceous, covered by a small amount of whitish aerial hyphae, and reverse pale olivaceous.
Culture characteristics: Colonies on PDA reaching 74-76 mm diam after 7 days at 25 • C, margin regular, aerial mycelium sparsely, flat, dark brown near the central zone and white around, and reverse concolorous; abundant production of pycnidia in the late growth stage.Colonies on MEA reaching 46-50 mm diam after 7 days at 25 • C, margin waved, light-brown in the middle, white around, covered by white felt-like aerial hyphae, and reverse buff.Colonies on OA reaching 62-65 mm diam after 7 days at 25 • C, margin regular, olivaceous, covered by a few whitish aerial hyphae, and reverse concolorous.

Discussion
There are many kinds of fungi in Jiangxi Province, and the fungal groups are complex.Relevant studies have shown that several mycological investigations are also constantly exploring and enriching the fungal diversity (e.g., [12,[43][44][45][46]).In this study, we isolated plant pathogens from diseased leaves of a wide range of plant hosts in Jiangxi Province, China.Based on the morphomolecular approach, three new species of Didymella, D. bischofiae, D. clerodendri, and D. pittospori, were introduced, which contributed to the supplementation of the diversity of this genus.
To date, studies conducted on Didymella have mainly focused on their alpha-taxonomy, and most species are considered to be saprobes or phytopathogens of woody and herbaceous hosts [4,9,13,28,42], whereas only a few species have been isolated from inorganic substrates, such as D. glomerata and D. pomorum from inorganic materials including asbestos, cement, paint, etc. [3,12,53].Recent studies also show that four didymella species, D. gardeniae, D. heteroderae, D. musae, and D. microchlamydospora, were found from human nail or cornea lesion [23], but there is no relevant data that support whether it has a direct relationship with the human disease.The genus Didymella is mainly recorded from China, Germany, India, Italy, The Netherlands, New Zealand, South Africa, and USA [3,4,7,12,21]; little published information is recorded in other regions [28].Scant attention has been accorded to the roles of their ecosystem function, substrate specificities, and fungal pathogenicity.Thus, the understanding of external factors that affect fungal lifestyles may have a significant impact on agricultural development, ecological environment, and human health, contributing significantly to the field of plant pathology and fungal taxonomy.

Figure 1 .
Figure 1.Phylogram of Didymellaceae based on concatenated ITS, LSU, TUB2, and RPB2 seq data.Significant MLBS/BPP support values above 80% and 0.80 are given at the nodes.The rooted to Epicoccum nigrum (CBS 173.73) and E. poae (LC 8160).Strains from the present stud indicated in red.Two branches were shortened according to the indicated multipliers to f page size, and these are indicated by the symbol (//).

Figure 1 .
Figure 1.Phylogram of Didymellaceae based on concatenated ITS, LSU, TUB2, and RPB2 sequence data.Significant MLBS/BPP support values above 80% and 0.80 are given at the nodes.The tree is rooted to Epicoccum nigrum (CBS 173.73) and E. poae (LC 8160).Strains from the present study are indicated in red.Two branches were shortened according to the indicated multipliers to fit the page size, and these are indicated by the symbol (//).

Table 1 .
Loci used in this study with the corresponding PCR primers and conditions.

Table 2 .
Species and GenBank accession numbers of the sequences used in the phylogenetic analyses.New sequences are in bold.