One new species and one new record of Zasmidium in China

Abstract Background Two hyphomycetous species were collected from leaves of Smilax china (Liliales, Smilacaceae) and Cremastra appendiculata (Asparagales, Orchidaceae). ITS barcoding indicated that they belong to the genus Zasmidium. New information Morphological data in combination with molecular phylogenetic analyses based on ITS, LSU and rpb2 confirmed that our Chinese strains represented a new species, Zasmidium liboense and a new record of Z. citri-griseum.


Introduction
The fungi of southern Asian are extremely diverse , Cheek et al. 2020. During a survey of fungal diversity in ornamental plants in south-western China from 2017 to 2019, more than 2000 strains were obtained, which represented asexual morphs of both Ascomycota and Basidiomycota. Some new taxa were previously described by our research group as pathogens or endophytes (e.g. Liang et al. 2018, Long et al. 2019, Sun et al. 2020).
The genus Zasmidium was established by Fries (1849) with Z. cellare (Pers.) Fr. as the type species. It is currently placed in the order Capnodiales within the Dothideomycetes . Arzanlou et al. (2007) showed that Zasmidium was the oldest name for Stenella-like hyphomycetes within Mycosphaerellaceae, which are characterised by conidiogenous loci and conidia with truncate hila (Bensch et al. 2012). Hence, many former Stenella species were transferred to Zasmidium (Braun et al. 2010, Kamal 2010. Up to now, the number of accepted species in the genus is about 150 . In this paper, we report on Zasmidium species found on medicinal plants in China. One new species (Zasmidium liboense) and one new Chinese record (Z. citri-griseum) are reported, based on evidence from morphology and molecular phylogeny.

Samples collection and fungal strains isolation
The samples were collected in Xishuangbanna City, Yunnan Province, China. In order to obtain pure cultures, diseased leaf pieces of Smilax china (Liliales, Smilacaceae) and Cremastra appendiculata (Asparagales, Orchidaceae) were surface-disinfected following the method of . The strains were isolated using the single-spore method (Chomnunti et al. 2014). Colonies growing from single spores were transferred to potatodextrose agar (PDA) and incubated at room temperature (28ºC). The holotype was deposited in the Herbarium of Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). The ex-type cultures were deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC) and the Mae Fah Luang University Culture Collection (MFLUCC) in Thailand.

Morphological description
Morphological culture characters were recorded after 2-3 weeks of growth on PDA. Microscopic slides were prepared in lactophenol. Light microscopy observations were made using a BX53 compound microscopy (Olympus, Tokyo, Japan) at 1000× magnification. The morphology was observed using a compound microscope (OLYMPUS BX53) showing all necessary details of morphology and ontogeny of reproductive propagules. Measurements were made of 30 structures for conidia, hila and conidiophores. The new species name was submitted to MycoBank (www.mycobank.org).

DNA extraction, PCR amplification and sequencing
Fungal cultures were grown on PDA at 28°C. When the whole Petri-dish (90 mm diam.) was nearly covered, fresh mycelia were scraped from the surface with sterilised scalpels. Genomic DNA was extracted using Fungus Genomic DNA Extraction Kit (Biomiga #GD2416, San Diego, California, USA) and following the manufacturer's instructions. PCR amplification of the internal transcribed spacer (ITS) region and the large subunit (LSU) of the ribosomal RNA gene was performed in a 25-μl reaction volume system as in Liang et al. (2018). Primers V9G andITS4 (White et al. 1990, de Hoog andvan den Ende 1998) were used to amplify the ITS and LSU1Fd and LR5 for the LSU (Vilgalys andHester 1990, Crous et al. 2009). In addition, one protein-coding gene fragment, RNA polymerase II second largest subunit (rpb2), was amplified with the primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999

Phylogenetic analyses
Our newly-generated sequences were aligned by locus with ex-type and other representative sequences of Zasmidium species, which were downloaded from GenBank (
Most of those, 64 species, produce larger conidia with more septa compared to  For strain GUCC 1507.3, our molecular phylogenetic data (Fig. 3, Table 2) showed that it was very close to Z. anthuriicola, Z. citri-griseum and Z. suregadae. Rbp2 sequences of strain GUCC 1507.3 and Z. citri-griseum (CBS 122455) are identical, but different from Z. anthuriicola (CBS 118742). Currently, no rpb2 sequences are available for Z. suregadae.
species of Zasmidium with a wider host range. Braun et al. (2014) listed hosts in the Fabaceae, Musaceae and Rutaceae. The host of our Chinese material, Cremastra appendiculata (Orchidaceae), represents a new host family for this fungus.