Blastosporiumpersicolor gen. et sp. nov., a new helotialean fungus

Abstract A new genus and species, Blastosporiumpersicolor, is described and illustrated from leaves of mildewed tobacco. It is characterised by branched, septate hyphae from which arise macronematous, unbranched or spaced branched conidiophores and mono- or polyblastic conidiogenous cells that produced solitary and blastocatenate, obovoid, oblong, ellipsoidal, allantoid, broad fusiform to irregular, unicellular, hyaline conidia. The phylogenetic analyses, based on the combined sequence data from the small and large nuclear subunit ribosomal DNA (SSU and LSU), placed B.persicolor in the Leotiomycetes class, Helotiales order.


Introduction
The Kingdom Fungi contains a huge number of species, which continues to rise with more collections. With the advance in the studies of DNA sequence data, the fungal classification system has been updated over the years. Many described species obtained new taxonomic status after the molecular data and have been processed. Leotiomycetes is a large class in Ascomycota and has potential taxonomic value relating to the ecology and biology. The traditional classification of Leotiomycetes at high levels has experienced considerable challenges with the inclusion of the molecular techniques in systematics studies. For example, early research accepted five orders, 21 families and about 510 genera in the Leotiomycetes on the basis of both traditional classification and molecular phylogenetic studies (Eriksson 2005, Kirk et al. 2001), but a recent study reported a new classification of Leotiomycetes, including 11 orders, 44 families and about 590 genera (Wijayawardene et al. 2018) and this classification also lacks sufficient DNA sequence data. In Leotiomycetes, the order Helotiales, one of the largest non-lichen-forming ascomycetous groups, is composed of fungi of diverse morphology and ecology. Of these, members of the Helotiales thrive in various ecosystems and cover a broad range of niches and helotialean fungi have been found as plant pathogens, endophytes, nematode-trapping fungi, mycorrhizae, ectomycorrhizal parasites, fungal parasites, terrestrial saprobes, aquatic saprobes, root symbionts and wood rot fungi (Wang et al. 2006).
During a survey of fungi growing on mildewed tobacco leaves, an unknown fungus was found. Based on its morphological characters and DNA sequence data, it is proposed as a new asexual genus and species, Blastosporium persicolor.

Isolation and morphological study of strain
Samples of the mildewed tobacco leaves were collected from Xiamen Logistics Warehousing Center. Samples were preserved in zip-locked plastic bags, labelled and transported to the laboratory. The procedure was as follows: samples (5g) were placed in PDA liquid medium (200 g potato, 20 g glucose, 1000 ml distilled water), shaken at 140 rpm/min for 1 h and the filtrate was collected. The filtrate was coated on a CMA plate (20 g cornmeal, 10 g agar, 1000 ml distilled water) at 28 °C, supplemented with two antibiotics (penicillin G, 0.5 g/l; and streptomycin, 0.5 g/l; Gams et al. 1998). After 3-5 days, single colonies were isolated into pure culture, grown on potato dextrose agar plates (PDA). The characteristics of the colonies were from PDA, CMA and SNA (synthetic low nutrient agar). Microscopic characteristics were made from cultures growing on CMA after incubation at room temperature for one week.
The pure cultures and dried cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan).

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing
Pure cultures were grown on PDA for 5 days at 25 °C. Actively growing mycelium was scraped off the surface of a culture and transferred to 2 ml Eppendorf micro-centrifuge tubes. Total genomic DNA was extracted according to the procedures in Turner et al. (1997). Primers used for PCR amplification and sequencing of nucSSU rDNA, nucLSU rDNA and ITS rDNA were NS1-NS4, LROR-LR7 and ITS1-ITS4, respectively (White et al. 1990, Vilgalys andHester 1990). Detailed protocols and PCR conditions for the amplification were fully described by Su et al. (2015). PCR products were then purified using a commercial Kit (Bioteke Biotechnology Co, Ltd, China) and forward and reverse sequences with a LI-COR 4000L automatic sequencer, using a Thermo Sequenase-kit, as described by Kindermann et al. (1998). The sequences were deposited in the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Table 1.

Sequence alignment and phylogenetic analysis
Other fungal sequences were obtained from the GenBank nucleotide database. DNA sequence data were aligned using ClustalX 1.83 (Higgins 1994) with default parameters and the consensus sequences were manually adjusted and linked in BioEdit v.7.0 (Hall 1999). Manual gap adjustments were made to improve the alignment and ambiguously aligned regions were also excluded. Portions of the 5'-and 3'-ends of the nuclear small and large subunits ribosomal DNA (nucSSU and nucLSU) were excluded from all analyses and coded by a question mark (?). MrBayes (Ronquist and Huelsenbeck 2003) was used to calculate the SSU rRNA and LSU rRNA sequence-based Bayesian inference of the phylogeny tree, with the following parameters: ngen=1,000,000; samplefr=1,000; printfr=1,000. The GenBank accession numbers of sequences used in the phylogenetic analysis are shown in Table 1 including the classes of Leotiomycetes, Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Orbiliomycetes, Pezizomycetes and Sordariomycetes. Candida albicans (C.P. Robin) Berkhout (Saccharomycetes) was used as outgroup.

Sequence analyses
In BLAST searches, the ITS sequence B. persicolor, MH992518, had the highest similarity of 88% with Tetracladium and 87% with Chalara (Corda) Rabenh., both belonging to Leotiomycetes. Therefore, most sequences are mainly from Leotiomycetes in the dataset. The dataset comprised 57 taxa representing 7 classes, 11 orders, 22    (Figure 1). In this tree, the new genus is phylogenetically placed in the Leotiomycetes. This monophyletic group formed a close relationship with several genera, which are grouped in this class, e.g. Vibrissea flavovirens and Vibrissea truncorum (Vibrisseaceae), Cudonia circinans and Spathularia flavida (Cudoniaceae) that are grouped with the new genus in the same clade. Therefore, analysis of partial LSU and SSU nuc rDNA sequences placed the new genus in the Leotiomycetes. Additionally, the tree also supports the fact that the Helotiales is not monophyletic. Description. Mycelium partly superficial and partly immersed, composed of branched, septate, smooth, hyaline hyphae. Conidiophores macronematous or semimacronematous, erect or prostrate, smooth, hyaline, sometimes reduced to conidiogenous cells. Conidiogenous cells mono-and polyblastic, terminal, integrated or discrete, determinate, sometimes with sympodial elongations, smooth, hyaline. Conidia solitary or blastocatenate, acrogenous, unicellular, obovoid, oblong, ellipsoidal, allantoid, broad fusiform to irregular, smooth, hyaline.

Discussion
To determine the phylogenetic placement of this species, Blastosporium persicolor was analysed with species from 7 classes, Leotiomycetes, Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Orbiliomycetes, Pezizomycetes and Geoglossomycetes (Wang et al. 2006). By Bayesian analysis, the new genus was placed in the Helotiales, Leotiomycetes. In the tree, B. persicolor grouped with the Cudonia-Spathularia clade and Vibrissea clade, but the placement did not receive strong support. Therefore, we have temporarily designated this species as a new genus and family incertae sedis.
In the Helotiales, many genera, such as Bulgaria Fr. (Bulgariaceae), Rutstroemia P. Karst. (Rutstroemiaceae) and Hegermila Raitv. (Hyaloscyphaceae), were only observed as sexual morphs, but Neofabraea H.S. Jacks (Dermateaceae) and Articulospora Ingold (Helotiaceae) were observed as having asexual and sexual morphs (Chen et al. 2015, Wijayawardene et al. 2018, Wang et al. 2015a. In this study, we just observed the asexual morph of B. persicolor. Based on ITS sequence data, B. persicolor is 88% similar to the genus Tetracladium De Wild. (T. marchalianum De Wild. as the type species), which was placed in the Helotiales and family incertae sedis. Moreover, Blastosporium shares some morphological features with Tetracladium as pale yellow and compact colonies and hyphae branched, septate and hyaline and both Blastosporium and Tetracladium sporulated abundantly on natural substrates (Sati et al. 2009, Wang et al. 2015b). However, B. persicolor is obviously distinct from the genus Tetracladium by the size and shape of conidia.
By molecular phylogeny analysis, Blastosporium belongs to the order Helotiales that currently contains 27 families (Wijayawardene et al. 2018). Moreover, members of the Helotiales cover a broad range of niches, such as plant pathogens, endophytes and aquatic hyphomycetes. Blastosporium persicolor was discovered from mildewed tobacco; therefore, it may be a plant pathogen.