Two new species of Microdochium from Indocalamus longiauritus in south-western China

Abstract Microdochium species have often been reported as plant pathogens and saprophytes and are commonly isolated from some diseased plant hosts. The primary aim of the present study was to describe and illustrate two new Microdochium species isolated from the leaf spot of Indocalamus longiauritus in Yunnan Province, China, namely Microdochium yunnanense and M. indocalami, spp. nov., based on their morphology and multilocus phylogenetic analyses of the combined ITS, LSU, TUB2, and RPB2. DNA sequence data indicate that six strains represent three independent groups from related and similar species in Microdochium. Microdochium indocalamisp. nov. clustered with M. fisheri, M. lycopodinum, M. rhopalostylidis, and M. phragmitis. Microdochium yunnanensesp. nov. grouped with M. bolleyi. In addition, the strain SAUCC1017 is recorded as an unidentified species in Microdochium. Descriptions and illustrations of the new species in the genus and Microdochium sp. indet. are provided.


Introduction
Microdochium is a genus in Microdochiaceae (Xylariales), which has been well-studied in recent years by Hernández-Restrepo et al. (2016), Zhang et al. (2017), Crous et al. (2018Crous et al. ( , 2019, and Marin-Felix et al. (2019) by incorporating morphological and molecular data with appropriate genes to resolve species limitations in the genus.

Isolation and morphological studies
The samples were collected from Yunnan Province, China. The strains of Microdochium were isolated from diseased or healthy leaves of Indocalamus longiauritus using single spore and tissue isolation methods (Chomnunti et al. 2014). Single spore isolation following the protocol of Choi et al. (1999) and Zhang et al. (2013) was adopted for collection with visible foliar sporulation. The spore suspension was obtained and spread onto potato dextrose agar (PDA) and incubated overnight under normal conditions. The germinated spores were then transferred to a new PDA plate to obtain a pure culture. Besides, the surface-sterilised plant tissue isolation was also used to obtain sterile isolates from plant host. Fungi were isolated by cutting eight fragments (5 × 5 mm) per leaf from the margin of leaf lesions and surface-sterilized by consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing in sterile distilled water for 1 min (Gao et al. 2014;Liu et al. 2015). The samples were dried with sterilized paper towels and placed on potato dextrose agar (PDA) (Cai et al. 2009). All the plates were incubated at biochemical incubator at 25 °C for 3-4 days, then hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates.
Following 2-3 weeks of incubation, morphological characters were recorded as by Hernández-Restrepo et al. (2016). Photographs of the colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and an Olympus BX53 microscope, both fitted with Olympus DP80 high definition color digital cameras to photo-document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher specimens were deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).
PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume, which contained 12.5 μL Green Taq Mix (vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Biosune, Shanghai, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL.
PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of 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 step at 72 °C for 10 min. Annealing temperature for each gene were 55 °C for ITS, 51 °C for LSU, 56 °C for RPB2 and 53 °C for TUB2. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic analyses
Novel sequences generated from the six strains in this study, and all reference available sequences of Microdochium species downloaded from GenBank (mostly used in Hernández-Restrepo et al. 2016;Zhang et al. 2017;Marin-Felix et al. 2019;Crous et al. 2018Crous et al. , 2019 were used for phylogenetic analyses. Alignments of the individual locus were determined using MAFFT v. 7.110 by default settings (Katoh et al. 2017) and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of four loci (ITS, LSU, TUB2, and RPB2 regions). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www. phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) and MrBayes on XSEDE (3.2.7a) (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Ronquist et al. 2012), respectively. For ML analyses the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included four parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 500 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS5.1. New sequences generated in this study were deposited at GenBank (https:// www.ncbi.nlm.nih.gov; Table 1), the alignments and trees were deposited in TreeBASE (http://treebase.org/treebase-web/home.html).

Phylogenetic analyses
Six Microdochium strains isolated from plant hosts were sequenced. Microdochium was analysed by using multilocus data (ITS, LSU, TUB2 and RPB2) composed of 50 isolates of Microdochium and Idriella lunata (CBS 204.56) as the outgroup taxon. A total of 3257 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1-572, LSU: 573-1429, TUB2: 1430-2395, RPB2: 2396-3257. Of these characters, 2019 were constant, 219 were variable and parsimony-uninformative, and 1019 were parsimony-informative. For the BI and ML analyses, GTR+I+G for LSU and RPB2, SYM+I+G for ITS, and GTR+G for TUB2 were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1). ML bootstrap support values (≥ 75%) and Bayesian posterior probability (≥ 0.95) are shown as first and second position above nodes, respectively. The 50 strains were assigned to 23 species clades based on the four gene loci phylogeny (Fig. 1) (CBS 285.71) with high support by the multi-locus phylogeny. From the tree (Fig. 1), strain SAUCC1017 formed a conspicuous branch independent from other Microdochium species, thus supporting the introduction of SAUCC1017 as an indeterminate species.  Description. Colonies on PDA attaining 46.1-51.2 mm in diameter after 7 days, formed a conspicuous concentric circle, periphery of aerial mycelium cottony, centre with scarce aerial mycelium, white initially, then becoming greyish sepia after 25 days. Some aerial hyphae aggregated and form a sporodochium within 15 days or longer. Mycelium composed of hyaline, immersed and superficial, smooth, branched, septate, 2.0-3.0 μm wide hyphae. Due to the soluble pigment secreted, reverse white to salmon. Conidiophores straight or slightly curved, aseptate, aggregated in the aerial mycelium, often reduced to conidiogenous cells borne directly from the hyphae. Conidiogenous cells terminal or intercalary, mono-or polyblastic, denticulate, smooth, hyaline, cylindrical, straight or bent, 11.0-28.3 × 1.5-2.9 μm. Conidia cylindrical, clavate to obovoid, 1-3-septate, 13.0-15.5 × 3.5-5.5 μm, base usually flattened 0.5-1.0 μm. Sometimes borne directly from the mycelial hyphae. Sexual morph: unknown.
Culture characteristics. Colonies on OA 58.1-61.5 mm in diameter after 15 days, entire, flat, white, lobate and radially margin, aerial mycelia cottony or sparse. Less exudate. Reverse white. Vegetative hyphae hyaline, abundant, branched, septate, thin-walled. Colonies on MEA 39.5-48.2 mm in diameter after 15 days, dense, initially white, becoming pale yellow, the centre of aerial mycelium cottony, periphery with scarce aerial mycelium, aerial mycelium formed a protuberance at center of colony.
Microdochium sp. indet. Figure 4 Description. Colonies on PDA attaining 73.9-80.4 mm in diameter after 15 days, felty to cottony, flat, margin entire or dentate, white, aerial mycelium abundant. Mycelium superficial, hyphae hyaline, septate, branched, smooth-walled. Reverse white to pale yellow, with yellow pigment produced with aging. Aerial hyphae aggregated to form numerous chlamydospores on agar surface. Chlamydospores thick-walled, terminal or intercalary, more frequently arranged in chains than clusters.

Discussion
Previous studies placed Microdochium in Amphisphaeriaceae (Parkinson et al. 1981;Samuels and Hallet 1983;von Arx 1984;Jaklitsch and Voglmayr 2012), which is a large heterogeneous family possessing pestalotiopsis-like asexual morphs characterised by holoblastic conidiogenous cells that produce septate, brown or hyaline conidia with appendages at both ends (Tanaka et al. 2011;Maharachchikumbura et al. 2014). Nevertheless, based on the results of phylogenetic analyses, Microdochium, Idriella, and Selenodriella were incorporated to a new family introduced as Microdochiaceae by Hernández-Restrepo et al. (2016), which is characterised by asexual morphs that produce polyblastic, sympodial or annellidic conidiogenous cells with hyaline conidia without appendages and sexual morphs that are monographella-like. In Microdochium, the color of conidiogenous cells is hyaline, and the shape of conidia seem to be taxonomic important feature. The conidial shape of Microdochium is more variable from cylindrical, fusoid or oblong, to lunate, straight or curved, with truncate bases and apices mainly rounded.
From the previous molecular studies of Microdochium (Jaklitsch and Voglmayr 2012;Jewell and Hsiang 2013;Zhang et al. 2015), the four gene regions (ITS, LSU, RPB2, TUB2) were chosen in this study. The LSU is informative enough for generic placement of Microdochium. The individual gene regions of ITS, TUB2, and RPB2 proved to be able to resolve species in Microdochium (results not shown). However, TUB2 was the more informative than other gene regions and showed longer distances between species and higher support values. This results in our study agree with previous studies in other xylariaceous genera (Hsieh et al. 2005;Laessøe et al. 2013;Hernández-Restrepo et al. 2016). By combining phylogenetic analysis and morphology, two species of Microdochium were delimited as new species, namely M. yunnanense sp. nov. and M. indocalami sp. nov. In order to support the validity of these new species, we followed the guidelines of Hernández-Restrepo et al. (2016).