﻿Pleocatenatachiangraiensis gen. et. sp. nov. (Pleosporales, Dothideomycetes) from medicinal plants in northern Thailand

﻿Abstract Pleocatenata, a new genus, is introduced with its type species, Pleocatenatachiangraiensis, which was isolated from withered twigs of two medicinal plants, Clerodendrumquadriloculare (Blanco) Merr (Verbenaceae) and Tarennastellulata (Hook.f.) Ridl (Rubiaceae) in northern Thailand. The genus is characterized by mononematous, septate, brown or dark brown conidiophores, monotretic conidiogenous cells and catenate, obclavate, olivaceous to blackish brown conidia. Phylogenetic analysis of combined LSU, SSU, tef1-α, rpb2 and ITS sequence data showed Pleocatenata forms a distinct phylogenetic lineage in Pleosporales, Dothideomycetes. Therefore, we treat Pleocatenata as Pleosporales genera incertae sedis based on morphology and phylogenetic analyses. Descriptions and illustrations of the new taxa are provided, and it is compared with morphologically similar genera.


Introduction
Medicinal plants are a rich source of natural products with biological and chemical properties. They are used in health care or treatment of human ailments and have been used since prehistoric times worldwide (Rasool-Hassan 2012). Many fungi have been found on medicinal plants and are members of Dothideomycetes and Sordariomycetes (Bhagat et al. 2012;Long et al. 2019;Ma et al. 2019;Hyde et al. 2020;Tennakoon et al. 2021). They form important associations with medicinal plants and as pathogens or saprobes (Long et al. 2019;Tennakoon et al. 2021), sources of medicines (Strobel et al. 1993;Huang et al. 2008;Hyde et al. 2019), involved in nutrient recycling (Bonnardeaux et al. 2007) and some are used in biological control .
Pleosporales is the largest order in Dothideomycetes, which accounts for about a quarter of the class (Zhang et al. 2012;Hyde et al. 2013;Hongsanan et al. 2020a). They have a worldwide distribution with diverse lifestyles, including saprobes, pathogens of plants and humans, endophytes, epiphytes and hyperparasites (Ramesh 2003;Kirk et al. 2008;Zhang et al. 2012;Hyde et al. 2013;Sun et al. 2019;Ferdinandez et al. 2021). Many species in Alternaria Nees, Curvularia Boedijn and Corynespora Güssow, can invade medicinal plants and cause leaf spots and other diseases, as economically important plant pathogens (Mathiyazhagan et al. 2004;Abtahi and Nourani 2017;Zhang et al. 2020), and some also pose a threat to human health Iturrieta-González et al. 2020). Endophytes in Pleosporales also show important biocontrol value (Su et al. 2014;De Silva et al. 2019;Hyde et al. 2019), for example, an extract from Cochliobolus spicifer R.R. Nelson has mosquito-larvicidal activity (Abutaha et al. 2015).
During the examination of collections from medicinal plants in northern Thailand (Sun et al. 2021), two isolates representing a new species were obtained from Clerodendrum quadriloculare and Tarenna stellulata. Morphology and phylogenetic analyses confirmed that it was distinct in Pleosporales, but its familial placement was uncertain. Thus, we introduced a new genus, Pleocatenata (Pleosporales, genera incertae sedis) to accommodate the new species, P. chiangraiensis.

Collection, examination and isolation
The isolates used in this study were collected from decaying twigs of Clerodendrum quadriloculare and Tarenna stellulata from Mae Fah Luang University, Chiang Rai, Thailand during June to July 2020 in terrestrial habitat. The samples were packaged in envelopes and returned to the laboratory as described in Senanayake et al. (2020). The fruiting bodies on natural substrates were observed and photographed using a stereo-microscope (SteREO Discovery, V12, Carl Zeiss Microscopy GmBH, Germany). Morphological characters were observed using a Nikon ECLIPSE Ni compound microscope (Nikon, Japan) and photographed with a Nikon DS-Ri2 digital camera (Nikon, Japan). The Adobe Photoshop CS6 Extended v. 13.0 software was used to make photo-plates. Measurements were done with the Tarosoft (R) Image Frame Work software.
Single spore isolations were used to obtain pure cultures following the methods described by Senanayake et al. (2020). Germinated conidia were transferred to new potato dextrose agar (PDA) plates and incubated at 26 °C for four weeks. The pure cultures obtained were deposited in Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand. Herbaria materials were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand. Facesoffungi (FoF) and Index Fungorum numbers were acquired as described in Jayasiri et al. (2015) and Index Fungorum (2022).

DNA extraction, PCR amplification and sequencing
Fresh fungal mycelia grown on PDA medium for 4 weeks at 26 °C were scraped with a sterile scalpel. Genomic DNA was extracted from scraped mycelia using the BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, San Diego, California, USA) following the manufacture's protocol. Five genes were selected in this study: the 28S subunit rDNA (LSU), the 18S subunit rDNA (SSU), the internal transcribed spacers (ITS), the translation elongation factor 1 (tef1-α), and the RNA polymerase II subunit 2 (rpb2). Polymerase chain reaction (PCR) was carried out in 20 μL reaction volume which contained 10 μL 2 × PCR Master Mix, 7 μL ddH 2 O, 1 μL of each primer, and 1 μL template DNA. The PCR thermal cycle program and primers are given (Table 1). Purification and sequencing of PCR products were carried out at SinoGenoMax (Beijing) Co., China.
The BYPP analyses were performed in CIPRES (Miller et al. 2010) with MrBayes on XSEDE 3.2.7a (Ronquist et al. 2012). The best nucleotide substitution model for each data partition was evaluated by MrModeltest 2.2 (Nylander 2004). The substitution model GTR+I+G was decided for LSU, SSU, ITS, tef1-α and rpb2 sequences. The Markov chain Monte Carlo (MCMC) sampling approach was used to calculate posterior probabilities (PP) (Rannala and Yang 1996). Six simultaneous Markov chains were run for 10 million generations and trees were sampled every 1,000 th generation. The first 20% of trees, representing the burn-in phase of the analyses, were discarded and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree.  Table 2. Taxa of Pleosporales used in the phylogenetic analysis with the corresponding GenBank accession numbers. The newly generated strains are indicated in bold. N/A: Not available.  (Rambaut and Drummond 2008) and modified in Microsoft Office PowerPoint 2010 and converted to jpg file using Adobe Photoshop CS6 Extended 10.0 (Adobe Systems, San Jose, CA, USA). The new sequences derived from this study were deposited in GenBank. The final alignment and tree were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/ study/TB2:S29199).
Analyses of both ML and BYPP (not shown) yielded almost identical results, and the topology of the trees were similar to previous studies Hongsanan et al. 2020aHongsanan et al. , 2021. The combined analyses showed that two suborders Massarineae and Pleosporineae were well-supported and formed an upper clade in Pleosporales. Our two newly obtained fungal isolates (MFLUCC 21-0222 and MFLUCC 21-0223) clustered together and formed a distinct clade with maximum support (ML-BS = 100%, BYPP = 1.00) and they grouped with Amorosiaceae, Sporormiaceae and Sublophiostomataceae with weak support. Etymology. "Pleo-" an abbreviation of Pleosporales, the order in which this fungus is classified; "-catenata" refers to the catenate conidia of this fungus.
Type species. Pleocatenata chiangraiensis Y.R. Sun, Yong Wang bis & K.D. Hyde Notes. The morphology of Pleocatenata is distinguished from members in other families in Pleosporales by its tretic conidiogenous cells and catenate, euseptate conidia, and phylogenic analyses indicated it does not belong to any existing families. To avoid establishing a new family with only one species, Pleocatenata is introduced as a new genus and assigned to Pleosporales, genera incertae sedis. Pleocatenata is a monotypic genus reported from terrestrial habitats but without a known sexual morph. Further discovery of other species in Pleocatenata or phylogenetic related genera with supported monophyly will determine the familial level of Pleocatenata.  when mature, 5-8-euseptate, slightly constricted at septa, distal conidia rounded at apex, truncate at base, intercalary conidia truncate at both ends, with thickened and darkened scars at base or both ends, 34-70 μm long, 6.5-12 μm at the widest. Sexual morph: Unknown.
Culture characteristics. Conidia germinated on PDA within 12 hours at 26 °C. Germ tubes were produced from both ends. Colony reached 20-25 mm diameter after 4 weeks at room temperature on PDA media. Mycelia superficial, irregularly circular, entire edge, dark brown from above, black from below, pigment produced which turns the media reddish brown.
Material examined. Notes. Two isolates collected from different hosts share similar morphology and clustered together in the phylogenic tree. There are no base pair differences in LSU and tef1-α genes between these two isolates. One base pair and two base pair differences (without gaps) are observed in ITS and rpb2, respectively. Therefore, the two isolates MFLUCC 21-0222 and MFLUCC 21-0223 are identified as conspecific.
A recently introduced species, Corynespora sinensis Jian Ma, X.G. Zhang & R.F. Castañeda, resembles Pleocatenata in its unbranched, cylindrical conidiophores and monotretic, terminal conidiogenous cells that produce catenate, obclavate conidia . Morphologically, Corynespora sinensis is more similar to P. chiangraiensis than to the type species of Corynespora, C. cassiicola (Berk. & M.A. Curtis) C.T. Wei (Wei 1950). Since Corynespora (Corynesporascaceae, Pleosporales) is a polyphyletic genus (Schoch et al. 2009;Voglmayr and Jaklitsch 2017), and there is no available sequence data for C. sinensis, we presume that C. sinensis may belong to Pleocatenata. However, due to lack of molecular data, and since morphology-based classification is not reliable for many hyphomycetous genera (Shenoy et al. 2006;Su et al. 2016;Yang et al. 2018), we retain the current classification. Sequences of C. sinensis are needed to resolve its phylogenetic placement. Detailed morphological comparison among C. cassiicola, C. sinensis and P. chiangraiensis is provided (Table 3).