Research Article |
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
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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.
asexual Ascomycota, hyphomycetes, lignicolous fungi, phylogenetic analysis, taxonomy
Helminthosporium Link was originally erected by
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 (
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 (
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
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 (
The newly generated sequences together with other sequences obtained from GenBank (Table
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 |
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.
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.
Referring to the collecting site of Nabanhe Nature Reserve in Yunnan Province, China.
HJAUP M2054.
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.
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.
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.
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 (
Referring to the country in which the fungus was collected.
HJAUP M2121.
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.
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.
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.
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 (
Referring to Yunnan province, where the type specimen was collected.
HJAUP M2071.
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.
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.
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.
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) (
The taxonomic history of the genus Helminthosporium is complex. To date, about 770 epithets for Helminthosporium are listed in
This project was supported by the National Natural Science Foundation of China (Nos. 31970018, 32160006, 31360011).