Three New Species of Microdochium (Sordariomycetes, Amphisphaeriales) on Miscanthus sinensis and Phragmites australis from Hainan, China

Species in Microdochium, potential agents of biocontrol, have often been reported as plant pathogens, occasionally as endophytes and fungicolous fungi. Combining multiple molecular markers (ITS rDNA, LSU rDNA, TUB2 and RPB2) with morphological characteristics, this study proposes three new species in the genus Microdochium represented by seven strains from the plant hosts Miscanthus sinensis and Phragmites australis in Hainan Island, China. These three species, Microdochium miscanthi sp. Nov., M. sinense sp. Nov. and M. hainanense sp. Nov., are described with MycoBank number, etymology, typification, morphological features and illustrations, as well as placement on molecular phylogenetic trees. Their affinity with morphologically allied and molecularly closely related species are also analyzed. For facilitating identification, an updated key to the species of Microdochium is provided herein.


Isolation and Morphology
Samples were collected from Hainan Province, China (108 • 37 -117 • 50 E, 3 • 58 -20 • 20 N). The strains of Microdochium were isolated from diseased leaves of Miscanthus sinensis and Phragmites australis using a tissue-isolation method [25]. Tissue fragments (5 × 5 mm) were taken from the margin of leaf lesions and surface-sterilized by immersing consecutively in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing in sterile distilled water for 1 min [26,27]. The sterilized leaf fragments were dried with sterilized paper towels and placed on potato-dextrose agar (PDA) [28]. All the plates were incubated in a biochemical incubator at 25 • C for 3-4 days, after which hyphae were picked out of the periphery of the colonies and transferred onto new PDA plates and oatmeal-agar (OA) [29] plates.
Pure cultures transferred to PDA and OA plates were incubated at 25 • C for 15 days and photographed twice at the 7th and 15th days using a Powershot G7X mark II digital camera. Macro-and micromorphological characteristics were observed using an Olympus SZX10 stereomicroscope and an Olympus BX53 light microscope, respectively. These two microscopes were both fitted with an Olympus DP80 high-definition color digital camera to photo-document fungal structures. All fungal strains were preserved at 4 • C in sterilized 10% glycerin for further studies. Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS) and Herbarium of the Department of Plant Pathology, Shandong Agricultural University, Taian, China (HSAUP).
Living 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/, accessed on 25 April 2022).
The PCR program consisted of an initial denaturation at 94 • C for 5 min, 35 cycles × [denaturation at 94 • C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 • C for 1 min] and a final elongation at 72 • C for 10 min. Annealing temperatures were 55 • C for ITS, 51 • C for LSU, 56 • C for RPB2 and 53 • C for TUB2. PCR products were visualized through 1% agarose-gel electrophoresis. Paired-end sequencing was conducted by Biosune Company Limited (Shanghai, China). Sequences were proofread for basic authenticity and reliability according to the five simple guidelines established by Nilsson et al. [35]. Consensus sequences were obtained using MEGA 7.0 [36]. All sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic Analyses
Twenty-eight new sequences were generated in this study, and available reference sequences of Microdochium species were retrieved from GenBank [2][3][4][5][6][7]. Four genetic markers (ITS, LSU, TUB2 and RPB2) were separately aligned using MAFFT v.7.110 (Osaka, Japan) [37]. Phylogenetic analyses were conducted individually for each marker at first and then for a combined dataset of the four genetic markers (Supplementary File S1).
Phylogenetic analyses were conducted with Bayesian inference (BI) and maximumlikelihood (ML) algorithms on the CIPRES Science Gateway portal (https://www.phylo. org/, accessed on 15 April 2022;) [38]. The BI ran with MrBayes on XSEDE v. 3.2.7a (Stockholm, Sweden) [39][40][41], and the ML ran with RAxML-HPC2 on XSEDE v. 8.2.12 (Heidelberg, Germany) [42]. The best evolutionary model for each partition was determined using MrModelTest v. 2.3 [43]. Default parameters were used for 1000 bootstrap ML analysis. In BI analysis, starting trees were random, and four MCMC chains ran simultaneously for five million generations. Trees were sampled once every 500 generations. These chains stopped when all convergences met and the standard deviation fell below 0.01. The burn-in fraction was set to 0.25 and Posterior Probabilities (PP) were determined from the remaining trees. All resulting trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 15 April 2022) and the layout of the trees was carried out with Adobe Illustrator CC 2019.

Phylogenetic Analyses
Seven Microdochium strains isolated from plant hosts were sequenced. Multilocus data (ITS, LSU, TUB2 and RPB2) were composed of 52 strains of Microdochium as ingroup and a strain CBS 204.56 of Idriella lunata as outgroup. A total of 2957 characters were fed to the phylogenetic analysis, viz. 1-573 (ITS), 574-1423 (LSU), 1424-2117 (TUB2), and 2118-2957 (RPB2). Of these characters, 2223, 97 and 637 were constant, variable parsimonyuninformative and parsimony-informative, respectively. For the BI and ML analyses, the evolutionary model of GTR+I+G was selected for ITS, TUB2 and RPB2, while SYM+I+G was selected for LSU ( Figure 1). The topology of the phylogenetic tree generated by the ML method was highly similar to that by BI, and therefore it was chosen to represent the evolutionary history of Microdochium.
The 59 strains are assigned to 29 species clades based on the four-marker phylogeny (  the first and second position, respectively. Strains marked with "*" are ex-types, ex-epitypes or holotypes. Strains from the current study are in red. The scale bar at the bottom middle indicates 0.08 substitutions per site.     Description-Colonies on PDA at 25 • C for 14 days attain 87.2-89.3 mm in diameter; when young, they are irregular in shape, dark green in the center and covered by white hyphae; when old, they are dark green overall, covered completely by white, lush, fluffy and beige hyphae. Mycelia are superficial and immersed, 1.3-2.3 µm wide, transparent, branched and diaphragmatic. Conidiophores are straight or slightly curved, produced from aerial hyphae, septate and often reduced to conidiogenous cells borne directly from hyphae. Conidiogenous cells are monoblastic, terminal, hyaline, smooth and cylindrical, 16.3-22.4 × 4.1-5.7 µm. Conidia are solitary, hyaline, spindle-shaped or cylindrical, 1-3-septate, 11.5-19.34 × 2.8-5.4 µm, 2-9 guttulate when mature and sometimes borne directly from hyphae. Chlamydospores were not observed. Sexual morphs unknown.

Microdochium miscanthi
Culture characteristics-Colonies on OA at 25 • C for 14 days, reach 86.4-88.9 mm in diameter; when young, they are circular gray in the center and wax yellow at the edge; when old, they have ravines, dense, yellow-brown overall and fluffy at the edge. Vegetative hyphae are transparent, branched and diaphragmatic.
Notes-Strains SAUCC211097 and SAUCC211098 are identified to the same species Microdochium sinense sp. nov. For details, refer to the notes for M. miscanthi.
Microdochium hainanense (Figure 4)      Notes-Strains SAUCC210781 and SAUCC210782 are identified to the same new species, M. hainanense. They share morphological characteristics, including culture characteristics, sporodochia and conidia. They are also identical in DNA sequences, gathering together with robust support values (MLBV 100% and BIPP 1.00, Figure 1

Key to the Species of Microdochium
Together with the three new species proposed in this study, we currently accepted a worldwide total of 47 species in the genus Microdochium. In order to facilitate identification in the future, a key to the species of Microdochium is provided herein, updating the key compiled 46 years ago [15]. Characteristics adopted in the key include perithecia, septa, asci, ascospores, conidiogenous cells, conidia and chlamydospores. with a typical tropical rainforest climate. This kind of environment is conducive to the growth of unusual microbial species, resulting in a high species diversity.
In order to accurately identify the species of Microdochium, molecular analysis is needed. In this study, the four genetic markers ITS, LSU, RPB2 and TUB2 were selected according to previous molecular studies of Microdochium. LSU provides enough information for the generic placement of Microdochium. Although any of the genetic markers ITS, TUB2 or RPB2 can be used for phylogenetic analysis at the species level in Microdochium (results not shown), TUB2 has more phylogenetic information, with longer distances between species and higher support values. This is consistent with previous studies on other xylariaceous genera [2,48,49].
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof8060577/s1, Supplementary File S1: The combined ITS, LSU, TUB2, and RPB2 multiple sequence alignment. Table S1: Specimens and GenBank accession numbers of DNA sequences used in this study.

Institutional Review Board Statement:
Not applicable for studies involving humans or animals.

Data Availability Statement:
The sequences from the present study were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 25 April 2022) and the accession numbers were listed in Table 1.

Conflicts of Interest:
The authors declare no conflict of interest.