Three New Species of Microdochium (Microdochiaceae, Xylariales) on Bambusaceae sp. and Saprophytic Leaves from Hainan and Yunnan, China

Species of the genus Microdochium (Microdochiaceae, Xylariales) have been reported from the whole world and separated from multiple plant hosts. The primary aim of the present study is to describe and illustrate three new species isolated from the leaf spot of Bambusaceae sp. and saprophytic leaves in Hainan and Yunnan provinces, China. The proposed three species, viz., Microdochium bambusae, M. nannuoshanense and M. phyllosaprophyticum, are based on multi-locus phylogenies from a combined dataset of ITS rDNA, LSU, RPB2 and TUB2 in conjunction with morphological characteristics. Descriptions and illustrations of three new species in the genus are provided.

Fungi associated with leaf spots were collected from Bambusaceae sp. and saprophytic leaves.Morphological characteristics were obtained by separation and purification.The sequences of four molecular markers, viz., the partial nuclear ribosomal large subunit (LSU), the ITS gene, the partial RNA polymerase II second-largest subunit (RPB2) and the partial β-tubulin gene (TUB2), were used in this study.We identify these fungi as three species of the genus Microdochium, proposed herein.

Sample Collection, Fungal Isolation and Morphology
Bambusaceae sp. and saprophytic specimens showing necrotic spots were collected during a series of field visits in Hainan and Yunnan provinces in China in 2022-2023.A specimen usually isolates multiple fungi, and we obtained a single colony through single spore isolation and tissue isolation methods [23].We removed fragments (4 × 4 mm) from the damaged edges of the leaves and disinfected the surface by continuously soaking them in 75% ethanol solution and rinsing them in sterile distilled water and 10% sodium hypochlorite solution, and then we rinsed them three times in sterile deionized water for 60 s, 45 s, 45 s and 30 s, respectively.We placed the processed fragments on sterile filter paper to absorb moisture, and then placed them on Potato Dextrose Agar (PDA potato: 200 g, dextrose: 15 g, agar: 15 g, distilled water 1 L and natural pH) or Oat Meal Agar (OA: oats: 30 g, agar: 15 g, distilled water: 1 L and natural pH) and incubated them at 24 • C for 3-5 days.Then, the agar portion containing fungal hyphae from the periphery of the colony was transferred to a new PDA plate and was photographed using a Sony ZV-E10L digital camera (Sony Group Corporation, Tokyo, Japan) on days 5, 10 and 15.Using an Olympus SZ61 stereo microscope and an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan), respectively, in conjunction with an Olympus DP73 and OPTIKA SC2000 high-definition color digital camera, the microscopic morphological characteristics of the structures produced in the culture were observed to capture and record fungal structures.All fungal strains were stored in 15% sterilized glycerol at 4 • C, with each strain stored in three tubes (2.0 mL tubes), for further research.Digimizer software (v5.6.0) was used for structural measurements, with 25 or more measurements taken for each character (conidiophores, conidiogenous cells, conidia and so on) [23].Specimens were deposited in the HSAUP and HMAS [23].Living cultures were deposited in the SAUCC [23].

DNA Extraction, PCR and Sequencing
Fungal DNA was extracted from the fresh mycelia grown on PDA or OA using a CTAB (cetyltrimethylammonium bromide) method and a kit method (OGPLF-400, Ge-neOnBio Corporation, Changchun, China) [24,25].Four molecular markers, including LSU, ITS, RPB2 and TUB2 gene, were amplified with the primer pairs listed in Table 1.An amplification reaction was carried out at 20 µL reaction volume, including 10 µL 2 × Hieff Canace ® Plus PCR Master Mix (Shanghai, China) (with dye) (Yeasen Biotechnology, Shanghai, China, Cat No. 10154ES03), 0.5 µL each of forward and reverse primer, and 0.5 µL template genomic DNA, adjusted to a total volume of 20 µL using distilled deionized water.Some 1% agarose gel and GelRed (TsingKe, Qingdao, China) were used to separate and purify the PCR product, and ultraviolet light was used to observed whether the fragment was consistent.Then a Gel Extraction Kit (Cat: AE0101-C) (Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China) was used for gel recovery.The PCR products were processed for purification and bidirectional sequencing by TsingKe Biological Technology, Qingdao, China.The raw data (trace data) were processed using MEGA v. 7.0 to obtained consistent sequences, including removing disordered peak sequences and complementary concatenation of forward and reverse sequences (ClustalW) [26].All sequences generated in this study were deposited in GenBank under the accession numbers in Table 2.

Phylogeny
The newly generated sequences in this study and closely associated sequences from Liu et al. [21] and Dissanayake et al. [22] were aligned using the MAFFT 7 online service with the default strategy and corrected manually using MEGA 7 [26,31].To determine the identity of the isolates at species level, phylogenetic analysis was first conducted separately for each marker, followed by a combination (ITS-LSU-RPB2-TUB2) (See Supplementary File S1).
Phylogenetic analysis of multi-labeled data was based on Bayesian inference (BI) and maximum likelihood (ML) algorithms.Firstly, MrModeltest v. 2.3 [32] was used determine the best evolutionary model for each partition under the Akaike Information Criterion (AIC), which was used to identified the best nucleotide substitution model settings prior to the BI analysis.Secondly, ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/,accessed on 30 October 2023) or offline software (ML was operated in RaxML-HPC2 on XSEDE v8.2.12, and BI analysis was operated in MrBayes v3.2.7a with 64 threads on Linux) [33][34][35][36][37]. Thirdly, for ML analyses, the default parameters were used and 1000 rapid bootstrap replicates were run with the GTR+G+I model of nucleotide evolution; BI analysis was performed using a fast bootstrap algorithm with an automatic stop option.Finally, all resulted trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 20 October 2023) or ITOL: Interactive Tree of Life (https://itol.embl.de/,accessed on 20 October 2023) [38], and the layout of the trees was produced in Adobe Illustrator CC 2019.

Phylogenetic Analyses
The comparison contained 58 isolates representing Microdochium and related taxa, and the used strains CBS 204.56 and CBS 177.57 of Idriella lunata were used as an outgroup.The final alignments consisting of 2945 characters were used for phylogenetic analyses, viz., 1-581 (ITS), 582-1418 (LSU), 1419-2258 (RPB2) and 2259-2945 (TUB2), including gaps.Of these characters, 2213 were constant, 68 were variable and parsimony-uninformative, and 664 were parsimony-informative.The topology of the ML tree was consistent with that of the Bayesian tree and was therefore considered to be representative of the evolutionary history of the genus Microdochium (Figure 1).The final ML optimization likelihood was −16,911.019760.The matrix had 681 distinct alignment patterns with 15.92% undetermined characters or gaps.Estimated base frequencies were as follows: A = 0.259370, C = 0.234527, G = 0.263997 and T = 0.242106; substitution rates were AC = 0.945501, AG = 4.719533, AT = 1.249092,CG = 0.946009, CT = 7.264699 and GT = 1.000000; and the gamma distribution shape parameter α = 0.124118.The Dirichlet base frequencies and the GTR+I+G evolutionary mode were used for ITS, K80+I+G was used for LSU, HKY+I+G was used for RPB2, and GTR+I was used for TUB2.MCMC analysis of these four tandem genes was performed over 245,000 generations in 4902 trees.The first 1224 trees representing the aging phase of the analysis were discarded, while the remaining trees were used to calculate the posterior probability in the majority-rule consensus tree (Figure 1; first value: PP > 0.80 shown).The alignment embodied a total of 848 unique site patterns (ITS: 237, LSU: 94, RPB2: 350 and TUB2: 167).
Culture characteristics-Cultures incubated on PDA at 25 • C in darkness, reaching 55-60 mm diam., had a growth rate of 3.9-4.3mm/day after 14 days, with moderate aerial mycelia, were gray white with regular margins, and the reverses were dark brown in the center and light brown to white at the edge.Cultures incubated on PDA at 25 • C in darkness, reaching 82-87 mm diam., had a growth rate of 5.8-6.2mm/day after 14 days, with moderate aerial mycelia on the surface, were gray white with regular margins, and the reverses were similar in color.
Culture characteristics-Cultures incubated on PDA at 25 • C in darkness, reaching 68-73 mm diam., had a growth rate of 4.8-5.2mm/day after 14 days, were creamy white to pale brown with regular margins, had moderate aerial mycelia, and the reverse was similar.Cultures incubated on OA at 25 • C in darkness, reaching 53-57 mm diam., with a growth rate of 3.7-4.1 mm/day, were creamy white with regular margins, had luxuriant aerial hyphae, and the reverse was similar.
Culture characteristics-Cultures incubated on PDA at 25 °C in darkness, reaching 68-73 mm diam., had a growth rate of 4.8-5.2mm/day after 14 days, were creamy white to pale brown with regular margins, had moderate aerial mycelia, and the reverse was similar.Cultures incubated on OA at 25 °C in darkness, reaching 53-57 mm diam., with a growth rate of 3.7-4.1 mm/day, were creamy white with regular margins, had luxuriant aerial hyphae, and the reverse was similar.
Culture characteristics-Cultures incubated on PDA at 25 • C in darkness, reaching 60.0-63.0mmdiam., had a growth rate of 4.3-4.5 mm diam/day after 14 days, were creamy white with regular margins, had luxuriant aerial hyphae, and the reverses were light brown in the center and white at the edge.Cultures incubated on PDA at 25 • C in darkness, reaching 62.0-68.0mm diam., had a growth rate of 4.4-4.8mm diam/day after 14 days, were creamy white with regular margins, had luxuriant aerial hyphae, and the reverses were similar.

Discussion
Microdochium was established by Syd.& P. Syd. in 1924; it has belonged to Microdochiaceae since 2016 (Microdochiacea was introduced by Hernández-Restrepo in 2016) [1,[9][10][11][12].Monographella Petr.was considered a sexual morph of the genus; "One Fungus = One Name" was proposed in The Amsterdam Declaration on Fungal Nomenclature in 2011, but the genus name of Microdochium was retained as the correct genus name [40].With the development of molecular techniques, Microdochium was divided into a new family called Microdochiaceae by Hernández-Restrepo et al. [8].Microdochiaceae is characterized by asexual morphs of polyblastic, sympodial or annellidic conidiogenous cells with hyaline conidia; the conidia come in a variety of shapes, i.e., cylindrical, fusiform, oval, rod-shaped, vertical or curved, truncated at the base and mostly rounded at the apex, and Monographella-like sexual morphs [8,21].
Microdochium species were reported as endophytes, plant pathogens and saprophytes.M. bolleyi was reported as a root endophyte and was proved to act against Gaeumannomyces graminis var.tritici, and it may be further used as a biocontrol agent [44].Most species of Microdochium were reported as plant pathogens, especially against economical cereal crops: for example, Microdochium albescens, M. oryzae isolated from Oryza sativa; M. majus, M. nivale, M. trichocladiopsis and M. triticicola isolated from Triticum aestivum; and so on [42].In reality, some species have not been regarded as plant pathogens; they are only isolated from leaf spot.However, the pathogenicity of this fungi has not been proven by Koch's Postulates.Generally, we believe that the type of fungus is an endophytic fungus associated with leaf spot.In addition, we should focus more on disease detection and biological control.

Conclusions
In this study, we isolated six strains associated with Microdochium on Bambusaceae sp. and saprophytic leaves by tissue isolation and single spore isolation from Hainan and Yunnan provinces in China.Based on morphology and phylogeny, six strains were identified as three new species, viz., Microdochium bambusae sp.nov., M. nannuoshanense sp.nov.and M. phyllosaprophyticum sp.nov.In the future, we firmly believe that Microdochium species will be isolated from more plants around the world.
Funding: This research was supported by National Natural Science Foundation of China (nos.32270024, U2002203 and 32370001).
Institutional Review Board Statement: Not applicable.

Figure 1 .
Figure 1.A maximum likelihood tree based on combined dataset of analyzed ITS, LSU, RPB2 and TUB2 sequence.Left, BIPP ≥ 0.80 and right, MLBV ≥ 70% are shown as BIPP/ML above the nodes.The branches BIPP/ML with 1/100 are indicated in bold.Ex-type cultures are indicated in bold face and marked with " T ".Strains from the present study are in red.The tree was rooted in Idriela lunata (CBS 204.56* and CBS 177.57).The yellow and green areas are used to distinguish different species.The scale bar at the bottom middle indicates 0.05 substitutions per site.

Figure 1 .
Figure 1.A maximum likelihood tree based on combined dataset of analyzed ITS, LSU, RPB2 and TUB2 sequence.Left, BIPP ≥ 0.80 and right, MLBV ≥ 70% are shown as BIPP/ML above the nodes.The branches BIPP/ML with 1/100 are indicated in bold.Ex-type cultures are indicated in bold face and marked with " T ".Strains from the present study are in red.The tree was rooted in Idriela lunata (CBS 204.56* and CBS 177.57).The yellow and green areas are used to distinguish different species.The scale bar at the bottom middle indicates 0.05 substitutions per site.

Figure 3 .
Figure 3. Microdochium nannuoshanense (holotype, HMAS 352652).(a) A leaf of Bambusaceae sp; (b) colonies on PDA from above and below after 14 days; (c) colonies on OA from above and below

Table 1 .
The PCR primers, sequence and cycles used in this study.

Table 2 .
GenBank accession numbers of the taxa used in phylogenetic reconstruction.
Notes: Ex-type or ex-epitype strains are marked with " T ", and the new species information described in this study is marked in bold.CBS: Westerdijk Fungal Biodiversity Institute; CGMCC: China General Microbiological Culture Collection; BRIP: Australian plant pathogen culture collection; LC: working collection of Dr. Lei Cai, housed at Institute of Microbiology, CAS, China; CPC: working collection of Pedro Crous maintained at the Westerdijk Institute.

Table 3 .
The asexual morphological characters of some Microdochium species.