Two New Species and a New Record of Microdochium from Grasses in Yunnan Province, South-West China

Microdochium species are frequently reported as phytopathogens on various plants and also as saprobic and soil-inhabiting organisms. As a pathogen, they mainly affect grasses and cereals, causing severe disease in economically valuable crops, resulting in reduced yield and, thus, economic loss. Numerous asexual Microdochium species have been described and reported as hyphomycetous. However, the sexual morph is not often found. The main purpose of this study was to describe and illustrate two new species and a new record of Microdochium based on morphological characterization and multi-locus phylogenetic analyses. Surveys of both asexual and sexual morph specimens were conducted from March to June 2021 in Yunnan Province, China. Here, we introduce Microdochium graminearum and M. shilinense, from dead herbaceous stems of grasses and report M. bolleyi as an endophyte of Setaria parviflora leaves. This study improves the understanding of Microdochium species on monocotyledonous flowering plants in East Asia. A summary of the morphological characteristics of the genus and detailed references are provided for use in future research.

Microdochium species have been collected worldwide, with more frequent collections in Europe and Asia. Where China stands out with the largest number of described species. They are frequently reported as phytopathogens [12], especially in grasses and cereals, causing severe diseases in economically valuable crops. Microdochium majus and M. nivale cause microdochium-patch (also known as pink snow mould or Fusarium patch) in wheat and barley [4,[16][17][18] and M. albescens causes rice leaf-scald [16], with a significant reduction in the crop yield. Tar spot disease, scald disease, root necrosis, and decay of grasses have been reported to be caused by species of Microdochium [11]. They have also been reported as saprobes on dead plants [4,[19][20][21][22] and as inhabiting rhizosphere soils [4,23] and some species have been reported as endophytes [24,25]. Moreover, Liu et al. [26] isolated M. lycopodinum and M. phragmitis from aquatic (marine) environments and salmon eggs. 2

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Microdochium has also been reported as beneficial to humans. Bioactive compounds of Microdochium species can be used against plant pathogens (i.e., Verticillium dahlia) [27]. Cyclosporine A, a bioactive compound that has the potential to control human and animal diseases, was isolated from M. nivale [28] and extracts of M. phragmitis were cytotoxic against human tumoral cell lines [29]. Thus, the biotechnological potential of Microdochium species should be explored from natural matrices and preserved for future research [30].
Hyde et al. [1] showed that the descriptive curve had not flattened, while Bhunjun et al. [31] showed that even speciose genera had many more new taxa to be described. In this study, we introduce two novel species, M. graminearum, M. shilinense, and a new record for M. bolleyi, isolated from grasslands in Kunming. This study had the following objectives: (1) to update the phylogenetic analysis of multigene sequence and refine the morphological characters of the genus and (2) to characterize these diverse isolates by incorporating morphological characteristics and molecular data. Microdochium species are either very important plant pathogens or non-pathogenic. This study provides information for future research on Microdochium and shows it is likely that many novel taxa are yet to be described.

Sample Collection, Isolation, and Identification
Litter and living grass samples were collected from Kunming, Yunnan Province, China, and brought to the laboratory for analysis. Specimens were examined using an Olympus SZ-61 dissecting microscope. Fungal fruiting structures were manually sectioned and mounted in water on a slide to observe their microscopic features. Pure cultures were obtained from litter samples via single spore isolation [32] and from living specimens by the tissue culture isolation method. In brief, leaf blades were cut into small pieces no larger than 1 cm in length, rinsed in sterile distilled water (SDW), and surface-sterilized with 75% ethanol for 3 min, 2.5% NaOCl solution for 0.5-5 min, rinsed in fresh SDW [12,33], blot-dried with sterile paper towels and, finally, cultured in potato dextrose agar (PDA) medium to obtain pure fungi [34]. Micro-morphological characteristics were examined using a Nikon ECLIPSE Ni compound microscope and photographed using a Canon EOS 600D digital camera fitted to the microscope. Photo plates were processed using Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA), and measurements of morphological structures were processed following the method described in Ren et al. [35]. The living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC), and the herbaria specimens were deposited in the herbarium of the Kunming Institute of Botany Academia Sinica (HKAS). The new taxa were registered in the Faces of Fungi [36], the Index Fungorum database (http://www.indexfungorum.org/, accessed on 9 September 2022) [13] and the database Fungi of the Greater Mekong Subregion (GMS Microfungi) [37].

DNA Extraction, PCR Amplification, and DNA Sequencing
Genomic DNA was extracted from 50 to 100 mg of axenic mycelium scraped from the edges of the culture grown on PDA at 28 • C for two weeks [38] using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China) following the manufacturer's protocol. Polymerase chain reaction (PCR) amplifications were carried out for the partial 28S large subunit nuclear ribosomal DNA (LSU), internal transcribed spacer region with intervening 5.8S nrRNA gene (ITS), partial beta-tubulin tub2, and partial RNA polymerase II second largest subunit (rpb2). The thermal conditions included initial denaturation at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 10 s, annealing temperatures listed in Table 1, elongation at 72 • C for 20 s, and final extension at 72 • C for 10 min. The total volume of PCR mixtures for amplification was 25 µL containing 8.5 µL ddH 2 O, 12.5 µL 2xF8 FastLong PCR MasterMix (Beijing Aidlab Biotechnologies Co., Ltd., Beijing, China), 2 µL of DNA template, and 1 µL of each forward and reverse primers (stock of 10 pM).

Phylogenetic Analyses
Representative Microdochium species used in the phylogenetic analyses were selected from recent studies [7][8][9][10][11][12] and the sequences downloaded from GenBank (https://www. ncbi.nlm.nih.gov/genbank/ (accessed on 30 August 2022)) ( Table 2). Individual alignments of LSU, ITS, tub2, and rpb2 sequences were aligned using MAFFT v. 7.475 [43], with default configurations, and trimmed with TrimAl v. 1.3 [44] via the web server Phylemon2 (http:// phylemon.bioinfo.cipf.es/utilities.html (accessed on 31 August 2022)). Individual datasets were concatenated into a combined dataset using BioEdit v. 7.0.5.3 [45]. The individual and combined datasets were subjected to maximum likelihood (ML) and Bayesian (BI) phylogenetic inference.  Maximum-likelihood (ML) analysis was performed using RaxML-HPC2 on XSEDE v. 8.2.10 [46] in CIPRES Science Gateway online platform [47], under the GTR+GAMMA model of nucleotide substitution, with 1000 bootstrapping replicates. The evolutionary model of nucleotide substitution for BI was selected independently for each locus using MrModeltest 2.3 [48]. Bayesian inference was conducted by MrBayes on XSEDE v. 3.2.7a in the CIPRES Science Gateway v. 3.3 [47], set with two runs and six simultaneous Markov chain Monte Carlo sampling (MCMC) chains for 2,000,000 generations, and the trees were sampled every 100th generation, for calculating the Bayesian posterior probabilities (BYPP). The first 25% of trees were considered burn-in and discarded. The MCMC heated chain "temperature" was set to the value of 0.15, and the run was stopped automatically when the average standard deviation of split frequencies reached 0.01.
Tree topologies generated in this study were visualized on FigTree v. 1.4.2 [49]. The phylogram was edited in Microsoft Office PowerPoint 2016 (Microsoft Inc., Redmond, WA, USA) and Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA). New sequences generated from the present study are deposited in GenBank ( Table 2).
The seven strains studied here represented three distinct clades ( Figure 1). The strains

Index Fungorum number: IF553309; Facesoffungi number: FoF12704
Etymology: Named refers to the location (Shilin Yi Autonomous County, China) from where the holotype was collected.
Culture characteristics: Ascospores germinated on PDA within 24 h at room temperature. Germ tube initially produced from the middle cell of the ascospore. Colonies on PDA reaching 50 mm diameter after four weeks at 25-27 • C, circular, slightly raised, smooth, fimbriate, filiform, floccose, white from above and yellowish from below.

Discussion
Grasses represent the plant family Poaceae and include over 10,000 species as herbaceous annuals, biennials, or perennial flowering plants [50]. They play a crucial role in

Discussion
Grasses represent the plant family Poaceae and include over 10,000 species as herbaceous annuals, biennials, or perennial flowering plants [50]. They play a crucial role in ecosystem functions such as undergrowth, weeds, or as the first members of food cycles [51]. Microfungi can occur on grasses as pathogens, endophytes, epiphytes, or saprobes. In many cases the anamorphs of these microfungi are reported as pathogenic on economically important grasses. Various authors have studied microfungi on grasses [50], and these studies indicated that they have a great diversity; however, there is a lack of information, especially from the Asian region. Therefore, it is important to collect microfungi on grasses in unexploited areas such as Yunnan province in China and assess their taxonomic placements, enabled by both morphological and molecular analyses. In the current study, we describe and illustrate two new species and one new record of microfungi on grasses, viz. Microdochium graminearum sp. nov., M. shilinense sp. nov., and M. bolleyi from Kunming, Yunnan, based on a biphasic approach (morphological plus molecular analyses) (Figures 1-4). Microdochium graminearum and M. shilinense are introduced with their sexual characteristics, whereas M. bolleyi is accounted for with its asexual morphological features.
Microdochium graminearum (HKAS 123200 and HKAS 123199) is introduced as a new species based on its distinct morphology and analysis of a combined LSU, ITS, tub2, and Phylogeny of a concatenated LSU-ITS-tub2-rpb2 sequence dataset depicts our M. bolleyi isolates as a monophyletic group (Figure 1). Morphologically, our specimens also have hyaline, smooth conidiogenous cells, and aseptate, hyaline, or ellipsoid conidia [23]. However, they differ slightly from CBS 540.92 in having cylindrical conidiogenous cells (12-14.6

Conclusions
In conclusion, we isolated seven fungi associated with Microdochium on grasses by single spore and tissue isolations. Based on morphology and phylogeny, they were identified as Microdochium graminearum sp. nov., M. shilinense sp. nov., and M. bolleyi. As many Microdochium species have been reported from China (Table 3), we believe that abundant Microdochium species will be discovered in future studies. Our results also highlight that Yunnan Province has not yet been properly studied and is an open field for new fungal discoveries.    Table 2).