Research Article
Print
Research Article
Three novel species of Helminthosporium (Massarinaceae, Pleosporales) from China
expand article infoJingwen Liu‡, Yafen Hu‡, Xingxing Luo‡, Rafael F. Castañeda-Ruíz§, Jian Ma‡
‡ Jiangxi Agricultural University, Nanchang, China
§ Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro de Humboldt” (INIFAT), Havana, Cuba

Corresponding author: Jian Ma ( jxaumj@126.com )

Academic editor: Pedro Crous
© 2022 Jingwen Liu, Yafen Hu, Xingxing Luo, Rafael F. Castañeda-Ruíz, Jian Ma.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Liu J, Hu Y, Luo X, Castañeda-Ruíz RF, Ma J (2022) Three novel species of Helminthosporium (Massarinaceae, Pleosporales) from China. MycoKeys 94: 73-89. https://doi.org/10.3897/mycokeys.94.95888
Open Access

Abstract

Three new species of Helminthosporium, H. nabanhensis, H. sinensis and H. yunnanensis collected on dead branches of unidentified plants in Xishuangbanna, China, were proposed by morphological and molecular phylogenetic analysis. Phylogenetic analysis of the combined data of ITS-SSU-LSU-TEF1-RPB2 sequences was performed using Maximum-Likelihood and Bayesian Inference, although H. nabanhensis and H. sinensis lack the RPB2 sequences. Both molecular analyses and morphological data supported H. nabanhensis, H. sinensis and H. yunnanensis as three independent taxa within the Massarinaceae.

Keywords

asexual Ascomycota, hyphomycetes, lignicolous fungi, phylogenetic analysis, taxonomy

Introduction

Helminthosporium Link was originally erected by Link (1809) with H. velutinum as the type species, and was mainly characterized by macronematous, cylindrical, septate conidiophores with polytretic conidiogenous cells that producing solitary (rarely in short chains), acropleurogenous, clavate or obclavate, distoseptate conidia with a flat, ringed pore at the base (Ellis 1961, 1971; Luttrell 1964; Seifert et al. 2011). The genus became a repository for a large amount of species due to a lack of understanding of the generic concepts. To date, about 770 epithets for Helminthosporium are listed in Index Fungorum (2022), but most of these were not congeneric with the generic type in development of conidia and conidiophores. Ellis (1961) provided a review on Helminthosporium, and accepted ten species. Luttrell (1963, 1964) examined the type species and defined the generic concept, and Sivanesan (1987) transferred several unrelated pathogens of the Poaceae from Helminthosporium to the genera Cochliobolus (anamorph Bipolaris), Setosphaeria (anamorph Exserohilum) and Pyrenophora (anamorph Drechslera). Siboe et al. (1999) subsequently provided a synoptic table of the main morphological features that distinguish 27 accepted Helminthosporium species. Since then, 27 additional species have been described in the genus (Zhang et al. 2004, 2007, 2010; Shirouzu and Harada 2008; Zhang and Zhang 2009; Zhang and Sun 2010; Zhao and Zhao 2012; Wang et al. 2014; Tanaka et al. 2015; Zhu et al. 2016; Alves-Barbosa et al. 2017; Tian et al. 2017; Crous et al. 2018, 2019; Zhao et al. 2018; Boonmee et al. 2021; Chen et al. 2022). Voglmayr and Jaklitsch (2017) revealed the phylogenetic relationships of Corynespora, Exosporium and Helminthosporium species, synonymized Exosporium with Helminthosporium, and confirmed 17 species in Helminthosporium by morphological and molecular systematic analysis, but the generic concept has been widened by adding four Corynespora species that produce terminal, monotretic conidiogenous cells. So it is challenging to classify Corynespora and Helminthosporium species based on morphology alone because the distinction between monotretic vs. polytretic conidiogenous cells is the only character for separating Corynespora and Helminthosporium. Based on the records of Species Fungorum 2021, Konta et al. (2021) summarized the morphology, host information, locality, sequence data and related references of 216 Helminthosporium species reported worldwide. Unfortunately, sequence data for most species are unavailable, and only 27 species are represented by the DNA sequence in GenBank (Chen et al. 2022).

Helminthosporium is worldwide in distribution, usually found as a common saprobe on leaf or twig litter, but one specie, H. solani, is an economically important pathogen causing silver scurf disease in potatoes worldwide (Alcorn 1983; Voglmayr and Jaklitsch 2017; Boonmee et al. 2021). To date, only 28 species have been recorded in China, viz. H. aquaticum, H. bambusicola, H. cantonense, H. chengduense, H. chinense, H. citri, H. conidiophorellum, H. constrictum, H. corchori, H. dongxingense, H. guangxiense, H. hunanense, H. ipomoeae, H. juglandis, H. lablab, H. ligustri, H. marantae, H. multiseptatum, H. nanjingense, H. obpyriforme, H. oplismeni, H. ovoideum, H. piperis, H. pseudomicrosorum, H. rhodomyrti, H. sichuanense, H. subhyalinum and H. submersum (Zhang et al. 2004; Zhang and Zhang 2009; Zhang et al. 2010; Zhang and Sun 2010; Zhao and Zhao 2012; Wang et al. 2014; Zhu et al. 2016; Zhao et al. 2018; Chen et al. 2022).

Xishuangbanna lies on the northern edge of tropical Southeast Asia. It is located in the southwestern part of Yunnan Province, China. It covers 19,125 km2 and has a mountainous topography and humid tropical monsoon climate, with an average annual temperature of 19.3–23.9 °C, and an average annual precipitation of 1200–1800 mm. The primary forest vegetation types are tropical seasonal rain forest, tropical montane rain forest, evergreen broad-leaved forest, monsoon forest over limestone, and monsoon forest on river banks (Cao and Zhang 1997). Such conditions create a very wide range of habitats favoring the growth of various microbial species. During our continuing mycological surveys of saprobic microfungi from plant debris in this region, three interesting hyphomycetes with morphological features typical of Helminthosporium were collected on dead branches. Based on morphological data and multi-locus phylogenetic analysis, they were described as new to science in the present study.

Materials and methods

Sample collection, isolation and morphological studies

Samples of dead branches were collected from humid environments and river banks in the forest ecosystems of Xishuangbanna, Yunnan Province, China, and returned to the laboratory in Ziploc bags. Samples were processed and examined following the methods described in Ma et al. (2011). Fungi were mounted in a drop of lactic acid on microscope slides, and examined and photographed with an Olympus microscope (model BX 53), with a 100 × (oil immersion) objective at the same background color and scale. Adobe Photoshop 7.0 was used for image processing to assemble photographs into images. Single-spore isolations were made on potato dextrose agar (PDA) following Goh (1999). Colony colors were assessed according to the charts of Rayner (1970). All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fungal mycelia grown on PDA, using the Solarbio Fungi Genomic DNA Extraction Kit following the manufacturer’s protocol (Solarbio, China). The DNA amplification was performed by polymerase chain reaction (PCR) using the respective loci (ITS, SSU, LSU, TEF1, RPB2). Primer sets used for these genes were as follows: ITS: ITS5/ITS4 (White et al. 1990), SSU: 18S-F/18S-R, LSU: 28S1-F/28S3-R (Xia et al. 2017), TEF1: EF1-983F/EF1-2218R (Rehner 2001; Zhao et al. 2018) and RPB2: dRPB2-5f/dRPB2-7r (Voglmayr et al. 2016). The final volume of the PCR reaction was 25 μl, containing 1 μl of DNA template, 1 μl each of the forward and reverse primer, 12.5 μl of 2 × Power Taq PCR MasterMix and 9.5 μl of double-distilled water (ddH2O). The PCR thermal cycling conditions of ITS, SSU and LSU were initialized at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C, the TEF1 and RPB2 were initialized at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C. Annealing temperature was 60 °C for TEF1, 56 °C for RPB2. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and DNA sequencing were carried out at Beijing Tsingke Biotechnology Co., Ltd. China.

Sequence alignment and phylogenetic analysis

The newly generated sequences together with other sequences obtained from GenBank (Table 1) were initially aligned using MAFFTv.7 (Katoh and Standley 2013) on the online server (http://maffTh.cbrc.jp/alignment/server/), and optimized manually when needed. 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 five gene loci (ITS, LSU, SSU, TEF1 and RPB2). Five aligned data sets of ITS, LSU, SSU, TEF1 and RPB2 are concatenated using the concatenated sequence function of Phylosuite software v1.2.1 (Zhang et al. 2020a), and absent sequence data (i.e., ITS, LSU, SSU, TEF1 and RPB2 sequence data) in the alignments were treated with the question mark as missing data. Phylosuite software v1.2.1 (Zhang et al. 2020a) was used to construct the phylogenetic tree based on ITS, SSU, LSU, TEF1 and RPB2 sequence data. The concatenated aligned dataset was analyzed separately using Maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under Edge-linked partition model for 10000 ultrafast bootstraps (Hoang et al. 2017). The final tree was selected among suboptimal trees from each run by comparing the likelihood scores using the TIM2e+I+G4 for ITS+RBP2, TVMe+I+G4 for LSU+SSU, and TNe+R2 for TEF1 substitution model. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model (2 parallel runs, 2000000 generations), in which the initial 25% of sampled data were discarded as burn-in. The best-fit model was SYM+I+G4 for ITS+RBP2, LSU+SSU; SYM+G4 for TEF1. ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (Edge-linked) using BIC criterion. The trees were viewed in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and further edited in Adobe Illustrator 2021.

Table 1.
Download as
CSV
XLSX

Species and GenBank accession numbers of DNA sequences used in this study. New sequences are in bold.

 Taxon Strain Genbank accession numbers
SSU LSU ITS RPB2 TEF1
Byssothecium circinans CBS 675.92 GU205235 GU205217 OM337536 DQ767646 GU349061
Corynespora cassiicola CBS 100822 GU296144 GU301808 – GU371742 GU349052
Corynespora smithii L120 – KY984297 KY984297 KY984361 KY984435
Corynespora smithii L130 KY984419 KY984298 KY984298 KY984362 KY984436
Cyclothyriella rubronotata TR, CBS 121892 – KX650541 KX650541 KX650571 KX650516
Cyclothyriella rubronotata TR9, CBS 141486 KX650507 KX650544 KX650544 KX650574 KX650519
Helminthosporium aquaticum MFLUCC 15-0357, S-096HT KU697310 KU697306 KU697302 – –
Helminthosporium austriacum L132 HT, CBS 139924 KY984420 KY984301 KY984301 KY984365 KY984437
Helminthosporium austriacum L137 – KY984302 KY984302 KY984366 KY984438
Helminthosporium austriacum L169, CBS 142388 – KY984303 KY984303 KY984367 KY984439
Helminthosporium caespitosum L141 – KY984305 KY984305 KY984368 –
Helminthosporium caespitosum L151 – KY984306 KY984306 KY984369 –
Helminthosporium caespitosum L99 HT, CBS 484.77 KY984421 JQ044448 JQ044429 KY984370 KY984440
Helminthosporium chengduense UESTC 22.0024, CGMCC 3.23575 HT ON557757 ON557745 ON557751 ON563073 ON600598
Helminthosporium chengduense UESTC 22.0025 ON557756 ON557744 ON557750 ON563072 ON600597
Helminthosporium chiangraiense MFLUCC 21-0087 HT – MZ538538 MZ538504 – –
Helminthosporium chinense UESTCC 22.0026, CGMCC 3.23570 HT ON557760 ON557748 ON557754 – ON600601
Helminthosporium chlorophorae BRIP 14521 – – AF120259 – –
Helminthosporium dalbergiae H 4628, MAFF 243853 AB797231 AB807521 LC014555 – AB808497
Helminthosporium endiandrae CBS 138902, CPC 22194 HT – KP004478 KP004450 – –
Helminthosporium erythrinicola CBS 145569 HT – MK876432 NR_165563 MK876486 –
Helminthosporium genistae L128, CBS 139921 KY984422 KY984308 KY984308 KY984372 –
Helminthosporium genistae L129, CBS 139922 KY984423 KY984309 KY984309 KY984373 –
Helminthosporium genistae L142 ET, CBS 142597 – KY984310 KY984310 KY984374 –
Helminthosporium hispanicum L109 HT, CBS 136917 KY984424 KY984318 KY984318 KY984381 KY984441
Helminthosporium italicum MFLUCC 17-0241 – KY815015 KY797638 – KY815021
Helminthosporium juglandinum L118 HT, CBS 136922 – KY984321 KY984321 KY984384 KY984444
Helminthosporium juglandinum L97, CBS 136911 KY984425 KY984322 KY984322 KY984385 KY984445
Helminthosporium leucadendri CBS 135133, CPC 19345 HT – KF251654 KF251150 KF252159 KF253110
Helminthosporium livistonae CPC 32158, CBS 144413 HT – NG_064539 NR_160348 – –
Helminthosporium magnisporum H 4627, MAFF 239278, TS 33 HT AB797232 AB807522 AB811452 – AB808498
Helminthosporium massarinum KT 1564 HT, CBS 139690 AB797234 AB807524 AB809629 – AB808500
Helminthosporium massarinum KT 838EP, MAFF 239604 AB797233 AB807523 AB809628 – AB808499
Helminthosporium microsorum L94 KY984426 KY984327 KY984327 KY984388 KY984446
Helminthosporium microsorum L95 – KY984328 KY984328 KY984389 KY984447
Helminthosporium microsorum L96 ET, CBS 136910 KY984427 KY984329 KY984329 KY984390 KY984448
Helminthosporium nabanhensis HJAUP C2054 ET OP555400 OP555398 OP555394 – OP961931
Helminthosporium nanjingensis ZM020380 – – KF192322 – –
Helminthosporium oligosporum L106 – KY984330 KY984330 KY984391 KY984449
Helminthosporium oligosporum L92, CBS 136908 KY984428 KY984332 KY984332 KY984393 KY984450
Helminthosporium oligosporum L93ET, CBS 136909 – KY984333 KY984333 KY984394 KY984451
Helminthosporium quercinum L90 HT, CBS 136921 KY984429 KY984339 KY984339 KY984400 KY984453
Helminthosporium quercinum L91 – KY984340 KY984340 KY984401 KY984454
Helminthosporium sinensis HJAUP C2121 ET OP555399 OP555397 OP555393 – OP961932
Helminthosporium solani CBS 365.75 KY984430 KY984341 KY984341 KY984402 KY984455
Helminthosporium solani CBS 640.85 – KY984342 KY984342 KY984403 –
Helminthosporium submersum UESTCC 22.0021 ON557759 ON557747 ON557753 ON563075 ON600600
Helminthosporium submersum MFLUCC 16-1360 HT MG098796 MG098787 – – MG098586
Helminthosporium submersum MFLUCC 16-1290PT MG098797 MG098788 MG098780 MG098592 MG098587
Helminthosporium syzygii CBS 145570 HT – MK876433 NR_165564 MK876487 –
Helminthosporium tiliae L171 – KY984343 KY984343 KY984404 KY984456
Helminthosporium tiliae L88 ET, CBS 136907 KY984431 KY984345 KY984345 KY984406 KY984457
Helminthosporium tiliae L89 – KY984346 KY984346 KY984407 –
Helminthosporium velutinum H 4626, MAFF 243854 AB797240 AB807530 LC014556 – AB808505
Helminthosporium velutinum H 4739, MAFF 243855 AB797235 AB807525 LC014557 – AB808501
Helminthosporium velutinum L115, CBS 136924 – KY984347 KY984347 KY984408 KY984458
Helminthosporium velutinum L116 – KY984348 KY984348 KY984409 KY984459
Helminthosporium velutinum L117 – KY984349 KY984349 KY984410 KY984460
Helminthosporium velutinum L126 – KY984350 KY984350 KY984411 KY984461
Helminthosporium velutinum L127 – KY984351 KY984351 KY984412 KY984462
Helminthosporium velutinum L131 ET, CBS 139923 KY984432 KY984352 KY984352 KY984413 KY984463
Helminthosporium velutinum L98 KY984433 KY984359 KY984359 KY984417 KY984466
Helminthosporium velutinum yone 96, MAFF 243859 AB797239 AB807529 LC014558 – AB808504
Helminthosporium yunnanensis HJAUP C2071 ET OP555392 OP555396 OP555395 OP961934 OP961933
Massarina cisti CBS 266.62, JCM 14140 HT AB797249 AB807539 LC014568 FJ795464 AB808514
Massarina eburnea CBS 473.64 AF164367 GU301840 AF383959 GU371732 GU349040
Massarina eburnea H 3953, CBS 139697 AB521718 AB521735 LC014569 – AB808517
Periconia byssoides H 4600, MAFF 243872 AB797280 AB807570 LC014581 – AB808546
Periconia digitata CBS 510.77 AB797271 AB807561 LC014584 – AB808537
Periconia pseudodigitata KT 1395, CBS 139699, MAFF 239676 HT NG_064850 NG_059396 NR_153490 – AB808540
Pseudosplanchnonema phorcioides L16, CBS 122935 KY984434 KY984360 KY984360 KY984418 KY984467
Stagonospora paludosa CBS 135088, S601NT – KF251760 KF251257 KF252262 KF253207
Stagonospora perfecta KT 1726A, MAFF 239609 AB797289 AB807579 AB809642 – AB808555
Stagonospora pseudoperfecta KT 889, CBS 120236, MAFF 239607 HT AB797287 AB807577 AB809641 – AB808553
Stagonospora tainanensis KT 1866, MAFF 243860 AB797290 AB807580 AB809643 – AB808556

1“–”, sequence is unavailable. 2Strain with ET (epitype), HT (holotype), NT (neotype), and PT (paratype). 3Abbreviations: CBS: Central Bureau voor Schimmel cultures, Utrecht, The Netherlands; CGMCC: China General Microbiological Culture Collection Center; CPC: Collection of Pedro Crous housed at CBS; HJAUP: Herbarium of Jiangxi Agricultural University, Plant Pathology; MAFF: the National Institute of Agrobiological Sciences, Japan; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; UESTCC: The University of Electronic Science and Technology Culture Collection, Chengdu, China; ITS: Internal Transcribed Spacer; SSU: Small Subunit Ribosomal; LSU: Large Subunit Ribosomal; TEF1: Transcriptional Enhancer Factor 1-alpha; RPB2: The Second Largest Subunit of RNA Polymerase II; others are not registered abbreviations.

Results

Molecular phylogeny

Three new strains of Helminthosporium isolated from dead branches in Xishuangbanna, Yunnan Province, China, were grown in culture and used for analyses of molecular sequence data. ​Unfortunately, our two species, H. nabanhensis and H. sinensis lack the RPB2 sequences. Newly generated sequences were deposited in GenBank. Alignment has 75 sequences with 1511 total characters (The combined dataset, ITS:1–457, LSU:458–993, RBP2:994–1110, SSU:1111–1363, TEF1:1364–1511), 555 distinct patterns, 487 parsimony-informative, 89 singleton sites, 935 constant sites, and Cyclothyriella rubronotata (TR) and C. rubronotata (TR9) were regarded as an outgroup. Maximum likelihood and Bayesian Inference analyses of the combined dataset resulted in phylogenetic reconstructions with largely similar topologies, and bootstrap support values for Maximum likelihood higher than 90% and Bayesian posterior probabilities greater than 0.90 are given above the nodes. The best-scoring ML consensus tree (lnL = –10,686.191) with ultrafast bootstrap values from ML analyses and posterior probabilities from MrBayes analysis at the nodes are shown in Fig. 1. Helminthosporium nabanhensis form a distinct clade sister to H. chlorophorae with strong statistical support (ML/BI = 95/1.00); H. sinensis forms a high-support clade (ML/BI = 92/0.99) with the lineage consisting of H. nabanhensis and H. chlorophorae; H. yunnanensis is a sister to three different strains of H. austriacum with strong statistical support (ML/BI = 100/1.00).

Figure 1. 

Phylogram of Massarinaceae based on combined ITS, SSU, LSU, RPB2 and TEF1 sequences. The ML and BI bootstrap support values above 90% and 0.90 are shown at the first and second position, respectively. The tree is rooted to Cyclothyriella rubronotata (TR) and C. rubronotata (TR9). Strains from the current study are in red. Some branches were shortened according to the indicated multipliers.

Taxonomy

Helminthosporium nabanhensis Jing W. Liu & Jian Ma, sp. nov.

IndexFungorum No: 559980
Fig. 2

Etymology

Referring to the collecting site of Nabanhe Nature Reserve in Yunnan Province, China.

Holotypus

HJAUP M2054.

Description

Saprobic on dead branches. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on natural substrate effuse, scattered, hairy, brown to black. Mycelium partly superficial, partly immersed in the substratum, composed of branched, septate, pale brown to brown, smooth hyphae. Conidiophores macronematous, mononematous, solitary or in groups of 2–4, simple, occasionally branched, erect, straight or flexuous, cylindrical, smooth, 8–21-septate, brown to dark brown, paler towards the apex, with well-defined small pores at the apex and rarely laterally beneath the upper 1–3 septa, 365–557 × 6.5–13.5 μm. Conidiogenous cells polytretic, integrated, terminal and intercalary, cylindrical, brown, smooth, with noncicatrized, distinct pores. Conidial secession schizolytic. Conidia acropleurogenous, solitary, dry, obclavate, pale brown to brown, 3–6-distoseptate, smooth, straight or curved, wider below than apex, truncate and dark at base, apically rostrate and pale, guttulate when young, non-guttulate at maturity, 26.5–46.5 μm long, 6.5–10 μm wide, tapering to 3–3.5 μm wide near the apex, 3–6 μm wide at the basal scar.

Figure 2. 

Helminthosporium nabanhensis (HJAUPM2054, holotype) A, B culture on PDA from above and reverse C conidia D conidiophores with conidia E–G conidiophores with conidiogenous cells.

Cultural characteristics

Colony on PDA reaching 50–55 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregular circular, surface velvety, with white and denser mycelium at the center, becoming olivaceous and sparser towards the edge; reverse pale brown at the center, dark brown at the periphery.

Material examined

China, Yunnan Province: Xishuangbanna Dai Autonomous Prefecture, Nabanhe National Nature Reserve, on dead branches of an unidentified broadleaf tree, 12 July 2021, J.W. Liu, HJAUP M2054 (Holotype), ex-type living culture HJAUP C2054.

Notes

The phylogenetic tree shows that the strain of H. nabanhensis (HJAUP C2054) clusters with the ex-type strain of H. chlorophorae (BRIP 14521). The BLASTn analysis of ITS of our ex-type strain HJAUP C2054 showed 90% identity (425/471 bp, 10/471 gaps) with ex-type strain BRIP 14521 of H. chlorophorae. Moreover, H. nabanhensis morphologically differs from H. chlorophorae in bigger conidiophores (365–557 × 6.5–13.5 μm vs. 120–270 × 7–10 μm) occasionally branched, and smaller conidia (26.5–46.5 × 6.5–10 μm vs. 52–102 × 8–11 μm) with fewer septa (3–6 vs. 6–9), and from H. sichuanense (Zhang et al. 2004) in narrower conidiophores (6.5–13.5 μm vs. 14–25 μm) and smaller conidia (26.5–46.5 × 6.5–10 μm vs. 41–86 × 10–14 μm) with fewer septa (3–6 vs. 5–11).

Helminthosporium sinensis Jing W. Liu & Jian Ma, sp. nov.

IndexFungorum No: 559981
Fig. 3

Etymology

Referring to the country in which the fungus was collected.

Holotypus

HJAUP M2121.

Description

Saprobic on dead branches. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on natural substrate effuse, scattered, hairy, brown to black. Mycelium partly superficial, partly immersed in the substratum, composed of branched, septate, pale brown to brown, smooth hyphae. Conidiophores macronematous, mononematous, solitary or in groups of 2–4, simple, straight or flexuous, thick-walled, cylindrical, smooth, brown to dark brown, paler towards the apex, with well-defined small pores at the apex and rarely laterally beneath the upper 1–4 septa, 220–370 × 6–8.5 μm. Conidiogenous cells polytretic, integrated, terminal and intercalary, cylindrical, brown, smooth, with noncicatrized, distinct pores. Conidial secession schizolytic. Conidia acropleurogenous, solitary, rarely catenate, dry, obclavate, pale brown, 2–7-distoseptate, smooth, straight or curved, wider below than apex, truncate and dark at base, apically rostrate and pale, 37–60 μm long, 5.5–8.5 μm wide, tapering to 3–3.5 μm wide near the apex, 3–6 μm wide at the basal scar.

Figure 3. 

Helminthosporium sinensis (HJAUPM2121, holotype) A, B culture on PDA from above and reverse C conidia D, E conidiophores, conidiogenous cells and conidia F conidiophores.

Cultural characteristics

Colony on PDA reaching 30–37 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, pale brown, irregular circular, surface velvety, outermost layer gray; reverse dark brown, produces pale green pigment.

Material examined

China, Yunnan Province: Xishuangbanna Dai Autonomous Prefecture, Menghai County, Mengsong Township, on dead branches of an unidentified broadleaf tree, 13 July 2021, J.W. Liu, HJAUP M2121 (Holotype), ex-type living culture HJAUP C2121.

Notes

Phylogenetic analysis shows that the strain of H. sinensis (HJAUP C2121) forms an independent clade, and clusters with the strains of H. nabanhensis (HJAUP C2054) and H. chlorophorae (BRIP 14521). The BLASTn analysis of ITS of our ex-type strain HJAUP C2121 showed 89% identity (536/602 bp, 17/602 gaps) with ex-type strain HJAUP C2054 of H. nabanhensis, and showed 91% identity (430/471 bp, 13/471 gaps) with ex-type strain BRIP 14521 of H. chlorophorae. Moreover, H. sinensis differs from H. nabanhensis by its longer and narrower conidia (37–60 × 5.5–8.5 μm vs. 26.5–46.5 × 6.5–10 μm), and smaller conidiophores (220–370 × 6–8.5 μm vs. 365–557 × 6.5–13.5 μm), and from H. chlorophorae by its smaller conidia (37–60 × 5.5–8.5 μm vs. 52–102 × 8–11 μm) and longer and narrower conidiophores (220–370 × 6–8.5 μm vs. 120–270 × 7–10 μm), and from H. guangxiense (Zhang and Zhang 2009) in smaller conidiophores (220–370 × 6–8.5 μm vs. 330–850 × 14–25 μm) and smaller conidia (37–60 × 5.5–8.5 μm vs. 76–110 × 16–22 μm) with fewer septa (2–7 vs. 9–17). In addition, the conidia of H. sinensis are solitary or rarely catenate, whereas those of H. guangxiense, H. nabanhensis and H. chlorophorae are solitary.

Helminthosporium yunnanensis Jing W. Liu & Jian Ma, sp. nov.

IndexFungorum No: 559982
Fig. 4

Etymology

Referring to Yunnan province, where the type specimen was collected.

Holotypus

HJAUP M2071.

Description

Saprobic on dead branches. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on natural substrate effuse, scattered, hairy, brown to dark brown. Mycelium partly superficial, partly immersed in the substratum, composed of branched, septate, pale brown to brown, smooth hyphae. Conidiophores macronematous, mononematous, solitary or in groups of 2–4, simple, straight or flexuous, thick-walled, cylindrical, smooth, brown to dark brown, paler towards the apex, with one cylindrical, enteroblastic percurrent extension, and with well-defined small pores at the apex and rarely laterally beneath the upper 1–5 septa, 560–680 × 12.5–15.5 μm. Conidiogenous cells polytretic, integrated, terminal and intercalary, cylindrical, pale brown to brown, smooth, with noncicatrized, distinct pores. Conidial secession schizolytic. Conidia acropleurogenous, solitary, dry, obclavate, sigmoid, lunate or uncinate, pale brown, 4–7-distoseptate, smooth, straight or flexuous, wider below than apex, truncate and dark at base, apically rostrate and pale, 30.5–55.5 μm long, 9–11 μm wide, tapering to 2.5–3 μm near the apex, 3–7.5 μm wide at the basal scar.

Figure 4. 

Helminthosporium yunnanensis (HJAUPM2071, holotype) A, B culture on PDA from above and reverse C conidiophores with conidia D conidiogenous cells and conidia E conidia.

Cultural characteristics

Colony on PDA reaching 75–82 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregular circular, surface velvety, with brown and denser mycelium at the center, becoming white and sparser towards the edge; reverse pale brown at the center, with little black dots.

Material examined

China, Yunnan Province: Xishuangbanna Dai Autonomous Prefecture, Nabanhe National Nature Reserve, on dead branches of an unidentified broadleaf tree, 12 July 2021, J.W. Liu, HJAUP M2071 (Holotype), ex-type living culture HJAUP C2071.

Notes

Phylogenetic analysis shows that the strain of H. yunnanensis (HJAUP C2071) clustered together and formed a sister clade with three different strains of H. austriacum (L132, L137, L169) (Voglmayr and Jaklitsch 2017). The BLASTn analysis of H. yunnanensis (HJAUP C2071) and H. austriacum (L132HT) shows 97% identity (524/541, 4 gaps) using ITS, 99% identity (550/553, 2 gaps) using LSU, 99% identity (872/873, 1 gap) using SSU, 98% identity (738/752, no gap) using TEF1, and 98% identity (1077/1095, no gap) using RPB2. Helminthosporium yunnanensis morphologically differs from H. austriacum in wider conidiophores (560–680 × 12.5–15.5 μm vs. 275–700 × 7–11 μm) with one cylindrical, enteroblastic percurrent extension, and narrower conidia (30.5–55.5 × 9–11 μm vs. 35–48 × 13.7–16.5 μm), and from H. obpyriforme (Zhang and Zhang 2009) in bigger conidiophores (560–680 × 12.5–15.5 μm vs. 225–460 × 9.5–13 μm) and smaller conidia (30.5–55.5 × 9–11 μm vs. 47–74 × 14–19 μm) with fewer septa (4–7 vs. 5–9).

Discussion

The taxonomic history of the genus Helminthosporium is complex. To date, about 770 epithets for Helminthosporium are listed in Index Fungorum (2022), but most of these were not congeneric with the generic type. Konta et al. (2021) listed 216 Helminthosporium species based on records from Species Fungorum, but most species are identified based on morphological studies, and so far only 27 species are represented by a DNA sequence in GenBank (Voglmayr and Jaklitsch 2017; Boonmee et al. 2021; Chen et al. 2022). Morphological comparison is important for fungal identification, but species identification only based on morphological studies is not comprehensive. With the availability of supplementary sequence data for Helminthosporium species, the molecular phylogenetic analysis is being used to evaluate previously described Helminthosporium-like species by molecular methods. The introduction of a phylogenetic analysis of Helminthosporium led to a better improvement of the heterogeneity of the genus and further clarified the taxonomic status of Helminthosporium. Voglmayr and Jaklitsch (2017) revisited Corynespora, Exosporium and Helminthosporium, with phylogenetic and morphological analyses. Zhang et al. (2020b) transferred H. bigenum into a new genus Mirohelminthosporium K. Zhang, D.W. Li & R.F. Castañeda and replaced the illegitimate H. cylindrosporum Matsush. with H. matsushimae. Chen et al. (2022) suggested four Helminthosporium species, H. anomalum, H. asterinum, H. decacuminatum and H. gibberosporum to Bipolaris, Kirschsteiniothelia or Curvularia by performing blastn analysis. ​Furthermore, seven new species were described under the genus Helminthosporium by molecular methods (Crous et al. 2018, 2019; Zhao et al. 2018; Boonmee et al. 2021; Chen et al. 2022). Based on previous studies, we proposed three new species by morphological and molecular phylogenetic analysis.

Chen et al. (2022) described two new species, H. chengduense and H. chinense, based on combined ITS, LSU, SSU, TEF1 and RPB2 sequence data and morphological characters. Accordingly, we also used ITS, LSU, SSU, TEF1 and RPB2 for phylogenetic analysis and obtained high phylogenetic support, although our two species, H. nabanhensis and H. sinensis, lack the RPB2 sequences. They are considerably distinct from all other described Helminthosporium species by morphological characters and multi-locus phylogenetic analysis, so we are convinced that the newly introduced species are new to science.

Acknowledgements

This project was supported by the National Natural Science Foundation of China (Nos. 31970018, 32160006, 31360011).

References

  • Alcorn JL (1983) Generic concepts in Drechslera, Bipolaris and Exserohilum. Mycotaxon 17: 1–86.
  • Alves-Barbosa M, Costa PMO, Malosso E, Castañeda-Ruíz RF (2017) Two new species of Dictyosporium and Helminthosporium from the Brazilian Atlantic Forest. Nova Hedwigia 105(1–2): 65–73. https://doi.org/10.1127/nova_hedwigia/2017/0401
  • Boonmee S, Wanasinghe DN, Calabon MS, Huanraluek N, Chandrasiri SKU, Jones GEB, Rossi W, Leonardi M, Singh SK, Rana S, Singh PN, Maurya DK, Lagashetti AC, Choudhary D, Dai YC, Zhao CL, Mu YH, Yuan HS, He SH, Phookamsak R, Jiang HB, Martín P, Dueñas M, Telleria MT, Kałucka IL, Jagodziński AM, Liimatainen K, Pereira DS, Phillips AJL, Suwannarach N, Kumla J, Khuna S, Lumyong S, Potter TB, Shivas RG, Sparks AH, Vaghef N, Abdel-Wahab MA, Abdel-Aziz FA, Li GJ, Lin WF, Singh U, Bhatt RP, Lee HB, Nguyen TTT, Kirk PM, Dutta AK, Acharya K, Sarma VV, Niranjan M, Rajeshkumar KC, Ashtekar N, Lad S, Wijayawardene NN, Bhat DJ, Xu RJ, Wijesinghe SN, Shen HW, Luo ZL, Zhang JY, Sysouphanthong P, Thongklang N, Bao DF, Aluthmuhandiram JVS, Abdollahzadeh J, Javadi A, Dovana F, Usman M, Khalid AN, Dissanayake AJ, Telagathoti A, Probst M, Peintner U, Garrido-Benavent I, Bóna L, Merényi Z, Boros L, Zoltán B, Stielow JB, Jiang N, Tian CM, Shams E, Dehghanizadeh F, Pordel A, Javan-Nikkhah M, Denchev TT, Denchev CM, Kemler M, Begerow D, Deng CY, Harrower E, Bozorov T, Kholmuradova T, Gaforov Y, Abdurazakov A, Xu JC, Mortimer PE, Ren GC, Jeewon R, Maharachchikumbura SSN, Phukhamsakda C, Mapook A, Hyde KD (2021) Fungal diversity notes 1387–1511: Taxonomic and phylogenetic contributions on genera and species of fungal taxa. Fungal Diversity 111(1): 1–335. https://doi.org/10.1007/s13225-021-00489-3
  • Chen YP, Tian WH, Guo YB, Madrid H, Maharachchikumbura SSN (2022) Synhelminthosporium gen. et sp. nov. and two new species of Helminthosporium (Massarinaceae, Pleosporales) from Sichuan Province, China. Journal of Fungi 8(7): 712. https://doi.org/10.3390/jof8070712
  • Crous PW, Wingfield MJ, Burgess TI, Hardy GESJ, Gené J, Guarro J, Baseia IG, García D, Gusmão LFP, Souza-Motta CM, Thangavel R, Adamčík S, Barili A, Barnes CW, Bezerra JDP, Bordallo JJ, Cano-Lira JF, de Oliveira RJV, Ercole E, Hubka V, Iturrieta-González I, Kubátová A, Martín MP, Moreau P-A, Morte A, Ordoñez ME, Rodríguez A, Stchigel AM, Vizzini A, Abdollahzadeh J, Abreu VP, Adamčíková K, Albuquerque GMR, Alexandrova AV, Álvarez Duarte E, Armstrong-Cho C, Banniza S, Barbosa RN, Bellanger J-M, Bezerra JL, Cabral TS, Caboň M, Caicedo E, Cantillo T, Carnegie AJ, Carmo LT, Castañeda-Ruiz RF, Clement CR, Čmoková A, Conceição LB, Cruz RHSF, Damm U, da Silva BDB, da Silva GA, da Silva RMF, Santiago ALCMA, de Oliveira LF, de Souza CAF, Déniel F, Dima B, Dong G, Edwards J, Félix CR, Fournier J, Gibertoni TB, Hosaka K, Iturriaga T, Jadan M, Jany J-L, Jurjević Ž, Kolařík M, Kušan I, Landell MF, Leite Cordeiro TR, Lima DX, Loizides M, Luo S, Machado AR, Madrid H, Magalhães OMC, Marinho P, Matočec N, Mešić A, Miller AN, Morozova OV, Neves RP, Nonaka K, Nováková A, Oberlies NH, Oliveira-Filho JRC, Oliveira TGL, Papp V, Pereira OL, Perrone G, Peterson SW, Pham THG, Raja HA (2018) Fungal Planet description sheets: 716–784. Persoonia 40(1): 240–393. https://doi.org/10.3767/persoonia.2018.40.10
  • Crous PW, Carnegie AJ, Wingfield MJ, Sharma R, Mughini G, Noordeloos ME, Santini A, Shouche YS, Bezerra JDP, Dima B, Guarnaccia V, Imrefi I, Jurjević Ž, Knapp DG, Kovács GM, Magistà D, Perrone G, Rämä T, Rebriev YA, Shivas RG, Singh SM, Souza-Motta CM, Thangavel R, Adhapure NN, Alexandrova AV, Alfenas AC, Alfenas RF, Alvarado P, Alves AL, Andrade DA, Andrade JP, Barbosa RN, Barili A, Barnes CW, Baseia IG, Bellanger J-M, Berlanas C, Bessette AE, Bessette AR, Biketova AYu, Bomfim FS, Brandrud TE, Bransgrove K, Brito ACQ, Cano-Lira JF, Cantillo T, Cavalcanti AD, Cheewangkoon R, Chikowski RS, Conforto C, Cordeiro TRL, Craine JD, Cruz R, Damm U, de Oliveira RJV, de Souza JT, de Souza HG, Dearnaley JDW, Dimitrov RA, Dovana F, Erhard A, Esteve-Raventós F, Félix CR, Ferisin G, Fernandes RA, Ferreira RJ, Ferro LO, Figueiredo CN, Frank JL, Freire KTLS, García D, Gené J, Gęsiorska A, Gibertoni TB, Gondra RAG, Gouliamova DE, Gramaje D, Guard F, Gusmão LFP, Haitook S, Hirooka Y, Houbraken J, Hubka V, Inamdar A, Iturriaga T, Iturrieta-González I, Jadan M, Jiang N, Justo A, Kachalkin AV, Kapitonov VI, Karadelev M, Karakehian J, Kasuya T, Kautmanová I, Kruse J, Kušan I, Kuznetsova TA, Landell MF, Larsson K-H, Lee HB, Lima DX, Lira CRS, Machado AR, Madrid H, Magalhães OMC, Majerova H, Malysheva EF, Mapperson RR, Marbach PAS, Martín MP, Martín-Sanz A, Matočec N, McTaggart AR, Mello JF, Melo RFR, Mešić A, Michereff SJ, Miller AN, Minoshima A, Molinero-Ruiz L, Morozova OV, Mosoh D, Nabe M, Naik R, Nara K, Nascimento SS, Neves RP, Olariaga I, Oliveira RL, Oliveira TGL, Ono T, Ordoñez ME de M, Ottoni A, Paiva LM, Pancorbo F, Pant B, Pawłowska J, Peterson SW, Raudabaugh DB, Rodríguez-Andrade E, Rubio E, Rusevska K, Santiago ALCMA, Santos ACS, Santos C, Sazanova NA, Shah S, Sharma J, Silva BDB, Siquier JL, Sonawane MS, Stchigel AM, Svetasheva T, Tamakeaw N, Telleria MT, Tiago PV, Tian CM, Tkalčec Z, Tomashevskaya MA, Truong HH, Vecherskii MV, Visagie CM, Vizzini A, Yilmaz N, Zmitrovich IV, Zvyagina EA, Boekhout T, Kehlet T, Læssøe T, Groenewald JZ (2019) Fungal Planet description sheets: 868–950. Persoonia 42: 291–473. https://doi.org/10.3767/persoonia.2019.42.11
  • Ellis MB (1961) Dematiaceous hyphomycetes. III. Mycological Papers 82: 1–55.
  • Goh TK (1999) Single-spore isolation using a hand-made glass needle. Fungal Diversity 2: 47–63.
  • Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS (2017) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution 35(2): 518–522. https://doi.org/10.1093/molbev/msx281
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Konta S, Hyde KD, Karunarathna SC, Mapook A, Senwanna C, Dauner LAP, Nanayakkara CM, Xu JC, Tibpromma S, Lumyong S (2021) Multi-gene phylogeny and morphology reveal Haplohelminthosporium gen. nov. and Helminthosporiella gen. nov. associated with palms in Thailand and a checklist for Helminthosporium reported worldwide. Life 11(5): 454. https://doi.org/10.3390/life11050454
  • Link JHF (1809) Observationes in ordines plantarum naturales. Dissertatio 1. Magazin der Gesellschaft Naturforschender Freunde zu Berlin 3: 3–42.
  • Ma J, Wang Y, Ma LG, Zhang YD, Castañeda-Ruíz RF, Zhang XG (2011) Three new species of Neosporidesmium from Hainan, China. Mycological Progress 10(2): 157–162. https://doi.org/10.1007/s11557-010-0685-2
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Rayner RW (1970) A mycological colour chart. British Mycological Society. Commonwealth Mycological Institute.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Seifert K, Morgan-Jones G, Gams W, Kendrick B (2011) The genera of hyphomycetes. CBS Biodiversity Series 9: 1–997.
  • Shirouzu T, Harada Y (2008) Lignicolous dematiaceous hyphomycetes in Japan: Five new records for Japanese mycoflora, and proposals of a new name, Helminthosporium magnisporum, and a new combination, Solicorynespora foveolata. Mycoscience 49(2): 126–131. https://doi.org/10.1007/S10267-007-0399-8
  • Siboe GM, Kirk PM, Cannon PF (1999) New dematiaceous hyphomycetes from Kenyan rare plants. Mycotaxon 73: 283–302.
  • Sivanesan A (1987) Graminicolous species of Bipolaris, Curvularia, Exserohilum and their teleomorphs. Mycological Papers 158: 1–261. https://doi.org/10.2307/3759472
  • Tanaka K, Hirayama K, Yonezawa H, Sato G, Toriyabe A, Kudo H, Hashimoto A, Matsumura M, Harada Y, Kurihara Y, Shirouzu T, Hosoya T (2015) Revision of the Massarineae (Pleosporales, Dothideomycetes). Studies in Mycology 82(1): 75–136. https://doi.org/10.1016/j.simyco.2015.10.002
  • Tian Q, Li WJ, Hyde KD, Camporesi E, Bhat DJ, Chomnunti P, Xu JC (2017) Molecular taxonomy of five species of microfungi on Alnus spp. from Italy. Mycological Progress 17(1–2): 255–274. https://doi.org/10.1007/s11557-017-1336-7
  • Voglmayr H, Akulov OY, Jaklitsch WM (2016) Reassessment of Allantonectria, phylogenetic position of Thyronectroidea, and Thyronectria caraganae sp. nov. Mycological Progress 15(9): 921–937. https://doi.org/10.1007/s11557-016-1218-4
  • White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. Academic Press Inc, New York, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Xia JW, Ma YR, Li Z, Zhang XG (2017) Acrodictys-like wood decay fungi from southern China, with two new families Acrodictyaceae and Junewangiaceae. Scientific Reports 7(1): 7888. https://doi.org/10.1038/s41598-017-08318-x
  • Zhang M, Zhang TY (2009) Taxonomic studies of Helminthosporium from China 4. Six new species and a key to Helminthosporium from China. Mycotaxon 109(1): 399–413. https://doi.org/10.5248/109.399
  • Zhang M, Zhang TY, Wu WP (2004) Taxonomic studies of Helminthosporium from China II. Two new species in Sichuan Province. Junwu Xuebao 23(2): 179–182.
  • Zhang M, Zhang TY, Wu WP (2007) Taxonomic studies of Helminthosporium from China III. Three new species in Guangdong Province. Mycotaxon 99: 137–142.
  • Zhang M, Wu HY, Wang ZY (2010) Taxonomic studies of Helminthosporium from China 5. Two new species in Hunan and Sichuan Province. Mycotaxon 113(1): 95–99. https://doi.org/10.5248/113.95
  • Zhang D, Gao FL, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020a) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096
  • Zhang K, Zhang H, Li DW, Castañeda-Ruíz RF (2020b) Mirohelminthosporium gen. nov. for an atypical Helminthosporium species and H. matsushimae nom. nov. Mycotaxon 135(4): 777–783. https://doi.org/10.5248/135.777
  • Zhao GC, Zhao RL (2012) The Higher Microfungi from Forests of Yunnan Province. Yunnan Science and Technology Press, Kunming, 572 pp.
  • Zhao N, Luo ZL, Hyde KD, Su HY, Bhat DJ, Liu JK, Bao DF, Hao YE (2018) Helminthosporium submersum sp. nov. (Massarinaceae) from submerged wood in north-western Yunnan Province. China. Phytotaxa 348(4): 269–278. https://doi.org/10.11646/phytotaxa.348.4.3
  • Zhu D, Luo ZL, Baht DJ, McKenzie EC, Bahkali AH, Zhou DQ, Su HY, Hyde KD (2016) Helminthosporium velutinum and H. aquaticum sp. nov. from aquatic habitats in Yunnan Province, China. Phytotaxa 253(3): 179–190. https://doi.org/10.11646/phytotaxa.253.3.1
login to comment