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Research Article
New Helminthosporium (Massarinaceae, Dothideomycetes) and Nigrospora (Incertae sedis, Sordariomycetes) species associated with walnut (Juglans regia L.) in China
expand article infoMengting Zou‡, Fatimah Al-Otibi§, Kevin David Hyde|§, Yong Wang‡, Xue-Jun Pan‡
‡ Guizhou University, Guiyang, China
§ King Saud University, Riyadh, Saudi Arabia
| Mae Fah Luang University, Chiang Rai, Thailand

Corresponding author: Yong Wang ( yongwangbis@aliyun.com )

Corresponding author: Xue-Jun Pan ( pxjun2050@aliyun.com )

Academic editor: Ajay Kumar Gautam
© 2024 Mengting Zou, Fatimah Al-Otibi, Kevin David Hyde, Yong Wang, Xue-Jun Pan.
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: Zou M, Al-Otibi F, Hyde KD, Wang Y, Pan X-J (2024) New Helminthosporium (Massarinaceae, Dothideomycetes) and Nigrospora (Incertae sedis, Sordariomycetes) species associated with walnut (Juglans regia L.) in China. MycoKeys 109: 265-284. https://doi.org/10.3897/mycokeys.109.133431
Open Access

Abstract

Six collections of ascomycetes were obtained from samples collected from dead branches and leaves of Juglans regia in Guizhou and Yunnan provinces, China. By incorporating multigene phylogenetic analysis (ITS, LSU, rpb2, SSU, tef1-α, tub2) supplemented by morphological data, we establish two novel species, namely Helminthosporium guizhouense and Nigrospora yunnanensis. In morphology, H. guizhouense can be distinguished from H. caespitosum by its narrower conidia (13–16 µm vs. 27.3–35.5 µm), and N. yunnanensis is characterized by black, globose conidia (16.2 × 14.4 µm). The phylogenetic results further substantiated them as novel taxa. The present study contributes to our comprehension of the range of fungi found in Juglans regia, thereby expanding our knowledge of the diversity of fungi within this host.

Key words

Ascomycota, morphology, new taxa, phylogeny, taxonomy

Introduction

Dothideomycetes and Sordariomycetes comprise plant pathogens, endophytes, and saprobes, and they can be identified by their distinct fruiting bodies (Marek et al. 2009; Wijayawardene et al. 2016; Hongsanan et al. 2020; Healy et al. 2022). They are widespread inhabitants of plant tissues including walnut (Juglans regia L.). Walnuts are a nutritious and health-beneficial drupaceous nut, globally recognized for their valuable properties (Caglarirmak 2003).

Helminthosporium (Massarinaceae, Pleosporales, Dothideomycetes) is a group of asexual Ascomycota proposed by Link (1809) with the type species H. velutinum. Most Helminthosporium species are saprobes that primarily inhabit various natural substrates, such as plant tissues, wood, bark, dung, and insects, and are also human pathogens (Luttrell 1964, Alcorn 1988; Konta et al. 2023; Hyde et al. 2023, 2024). About 781 taxa have been placed in Helminthosporium (http://www.indexfungorum.org, June.2024), but most Helminthosporium species differ from the generic type in the development of conidia and conidiophores and therefore are excluded from Helminthosporium. Furthermore, very few taxa have molecular data (Hyde et al. 2023). Very few instances of sexual morphs of Helminthosporium have been recorded, and the validity of most of these records is questionable as they have not been confirmed by sequence data (Voglmayr and Jaklitsch 2017).

Nigrospora (Apiosporaceae, Xylariales, and Sordariomycetes) was proposed by Zimmerman (1902) with the type species N. panici (Hyde et al. 2024). Initially, the characterization of Nigrospora species relied on morphological features, particularly large dark conidiospores. However, it was discovered that certain key morphological characteristics, such as the size of spores, were similar among species that are actually not closely phylogenetically related (Hao et al. 2020). To accurately identify different species, it is important to use a comprehensive approach that combines both morphological characteristics and phylogenetic analysis (Jayawardena et al. 2020, Maharachchikumbura et al. 2021). Wang et al. (2017) revised the classification of Nigrospora, increasing the known species from 15 to 27 by incorporating morphological and molecular data. Chethana et al. (2023) and Hyde et al. (2024) added records of further species. Wang et al. (2017) confirmed the placement of the genus in Apiosporaceae (Xylariales) based on multi-locus molecular phylogeny, including the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α), and b-tubulin (tub2) gene regions. This was confirmed by Samarakoon et al. (2021).

Southwest China is a biodiverse region. In this study, six isolates were collected from walnut leaves and dead tissues from Qianxi County, Guizhou Province, and Lincang City, Yunnan Province. This study aimed to determine the taxonomic status of the pathogenic species of walnut in Guizhou and Yunnan provinces through an analysis of both morphological and molecular characteristics. After conducting a multi-locus phylogenetic analysis and morphological examination, two new species, Helminthosporium guizhouense, and Nigrospora yunnanensis are identified and introduced.

Materials and methods

Sample collection, fungal strain isolation, and morphology

Samples exhibiting signs of disease were collected from walnuts in Qianxi County, Guizhou Province, and Lincang City, Yunnan Province, from 2023 to 2024. To establish uncontaminated cultures, disinfection processes were implemented on the sample surfaces (Zhang et al. 2020). Conidia were identified on the surface under a dissecting microscope. These conidia were aseptically extracted from the leaves using a sterilized needle and relocated to a sterile, water-filled drip board. The spores were then dispersed in sterile water, and a small quantity of the resulting spore suspension was absorbed and uniformly dispersed onto a potato dextrose agar (PDA) incorporated with streptomycin. After a 12-hour incubation period at 25 °C, individual germinated spores were selected and transferred to fresh PDA. Additionally, we prepared Malt Extract Agar (MEA) and Oatmeal Agar (OA) media for fungal growth. The cultures were subsequently maintained at room temperature (28 °C) for a duration of 10 days.

VHX-7000 (Keyence, Osaka, Japan), Fully-Integrated Head VHX-7100 (Keyence, Osaka, Japan), and High-Performance Camera VHX-7020 (Keyence, Osaka, Japan) dissecting microscopes were used as vehicles for observing the fungal colonies and fruiting bodies. The morphological characteristics of the fungi were studied and documented using a compound light microscope (Zeiss Scope 5) equipped with an attached camera (AxioCam 208 color). Morphological measurements of the new species’ features were taken using the ZEN 3.0 (blue edition) (Jena, Germany) software. All newly identified taxa have been registered in the Mycobank database (https://www.mycobank.org), accessed on 28 June 2024. For long-term conservation and research purposes, dried holotype specimens were preserved in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). The ex-type cultures have been deposited in the Departmental Culture Collection (GUCC).

DNA extraction and sequencing

Upon reaching the border of a 90 mm diameter Petri dish, a sterile scalpel was used to transfer mycelium into a 1.5 mL centrifuge tube for the extraction of genomic DNA. This extraction was performed using PrepMan Ultra Reagent (Applied Biosystems, CA, USA) in line with the manufacturer’s guidelines. The polymerase chain reaction (PCR) amplification was undertaken with a reaction volume of 25 µL. Primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990), dRPB2-5F/dRPB2-7cR (Voglmayr et al. 2016), NS1/NS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF-2 (O’Donnell et al. 1998, Carbone and Kohn 1999) were used to amplify the internal transcribed spacer regions (ITS), partial large subunit nrRNA (LSU) gene, partial DNA-directed RNA polymerase II second largest subunit (rpb2), 18S small subunit ribosomal RNA (SSU), partial beta-tubulin (tub2) gene, and translation elongation factor 1-alpha (tef1-α) gene sequence fragments, respectively.

The PCR thermal cycle program used for amplifying of ITS, LSU, rpb2, SSU, tub2, and tef1-α started with an initial denaturation at 95 °C for 5 minutes. This was followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for one minute each, and a final extension step at 72 °C lasting 10 minutes. Sangon Biotech (Chengdu, China) handled the purification and sequencing of PCR amplicons. Sequences meeting the quality criteria were submitted to GenBank, and their corresponding accession numbers are listed in Table 1, which also contains a complete list of all the strains utilized in this research.

Table 1.
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Species and GenBank accession numbers of DNA sequences used in in the phylogenetic analysis.

Species name Voucher specimens GenBank Accession numbers
ITS LSU rpb2 SSU tef1-α tub2
Byssothecium circinans CBS675.92 OM337536 GU205217 DQ767646 GU205235 – –
Haplohelminthosporium calami MFLUCC18-0074* MT928158 MT928156 – MT928160 – –
Helminthosporiella astilbacea COAD2126 MG668862 – – – – –
Helminthosporiella stilbacea MFLUCC15-0813* MT928159 MT928157 – MT928161 – –
Helminthosporiella stilbacea CPHmZC-01 KX228298 KX228355 – – – –
Helminthosporium maquaticum MFLUCC15-0357 = S-096* KU697302 KU697306 – KU697310 – –
Helminthosporium austriacum CBS139924 = L132* KY984301 KY984301 KY984365 KY984420 – –
Helminthosporium austriacum CBS14238 = L169 KY984303 KY984303 KY984367 – – –
Helminthosporium austriacum L137 KY984302 KY984302 KY984366 – – –
Helminthosporium caespitosum CBS484.77 = L99* JQ044429 JQ044448 KY984370 KY984421 – –
Helminthosporium caespitosum L141 KY984305 KY984305 KY984368 – – –
Helminthosporium caespitosum L151 KY984306 KY984306 KY984369 – – –
Helminthosporium chengduense UESTC22.0024 = YQ071048 = CGMCC ON557751 ON557745 ON563073 ON557757 – –
Helminthosporium chengduense UESTC22.0025 = YQ071047 ON557750 ON557744 ON563072 ON557756 – –
Helminthosporium chiangraiense MFLUCC21-0087* MZ538504 MZ538538 – – – –
Helminthosporium chlorophorae BRIP14521 AF120259 – – – – –
Helminthosporium dalbergiae MAFF243853 = H4628 = TS36 LC014555 AB807521 – AB797231 – –
Helminthosporium endiandrae CBS138902 = CPC22194* KP004450 KP004478 – – – –
Helminthosporium erythrinicola CPC35291 = CBS145569* NR_165563 MK876432 MK876486 – – –
Helminthosporium genistae CBS142597 = L142* KY984310 KY984310 KY984374 – – –
Helminthosporium genistae CBS139922 = L129 KY984309 KY984309 KY984373 KY984423 – –
Helminthosporium genistae CBS139921 = L128 KY984308 KY984308 KY984372 KY984422 – –
Helminthosporium guizhouense GUCC24-0011* PP915799 PP949847 PP947940 PP949912 – –
Helminthosporium guizhouense GUCC24-0012 PP915800 PP949848 PP947941 PP949913 – –
Helminthosporium guizhouense GUCC24-0013 PP915801 PP949849 PP947942 PP949914 – –
Helminthosporium hispanicum CBS136917 = L109* KY984318 KY984318 KY984381 KY984424 – –
Helminthosporium juglandinum CBS136922 = L118* KY984321 KY984321 KY984384 – – –
Helminthosporium juglandinum CBS136911 = L97 KY984322 KY984322 KY984385 KY984425 – –
Helminthosporium juglandinum CBS136912 = L101 KY984319 KY984319 KY984382 – – –
Helminthosporium juglandinum CBS136913 = L102 KY984320 KY984320 KY984383 – – –
Helminthosporium leucadendri CBS135133 = CPC19345* KF251150 KF251654 KF252159 – – –
Helminthosporium livistonae CPC32158 = CBS144413* NR_160348 NG_064539 – – – –
Helminthosporium magnisporum MAFF239278 = H4627 = TS33* AB811452 AB807522 – AB797232 – –
Helminthosporium massarinum CBS139690 = JCM13095 = MAFF239605 = KT1564* AB809629 AB807524 – AB797234 – –
Helminthosporium massarinum JCM13094 = MAFF239604 = KT838* AB809628 AB807523 – AB797233 – –
Helminthosporium microsorum CBS136910 = L96* KY984329 KY984329 KY984390 KY984427 – –
Helminthosporium microsorum L94 KY984327 KY984327 KY984388 KY984426 – –
Helminthosporium microsorum CBS136916 = L108 KY984323 KY984323 KY984386 – – –
Helminthosporium microsorum L95 KY984328 KY984328 KY984389 – – –
Helminthosporium nanjingensis HHAUF020380 = ZM020380 KF192322 – – – – –
Helminthosporium oligosporum CBS136909 = L93* KY984333 KY984333 KY984394 – – –
Helminthosporium oligosporum CBS136908 = L92 KY984332 KY984332 KY984393 KY984428 – –
Helminthosporium oligosporum L106 KY984330 KY984330 KY984391 – – –
Helminthosporium quercinum CBS136921 = L90* KY984339 KY984339 KY984400 KY984429 – –
Helminthosporium quercinum CBS112393 KY984334 KY984334 KY984395 – – –
Helminthosporium quercinum CBS136915 = L107 KY984336 KY984336 KY984397 – – –
Helminthosporium solani CBS365.75 KY984341 KY984341 KY984402 KY984430 – –
Helminthosporium solani CBS640.85 KY984342 KY984342 KY984403 – – –
Helminthosporium submersum MFLUCC16-1360* – MG098787 – MG098796 – –
Helminthosporium submersum MFLUCC16-1290PT MG098780 MG098788 MG098592 MG098797 – –
Helminthosporium submersum UESTCC22.0021 = Sara08_3 = CGMCC ON557753 ON557747 ON563075 ON557759 – –
Helminthosporium syzygii CPC35312 = CBS145570* NR_165564 MK876433 MK876487 – – –
Helminthosporium tiliae CBS136907 = L88* KY984345 KY984345 KY984406 KY984431 – –
Helminthosporium tiliae CBS136906 = L87 KY984344 KY984344 KY984405 – – –
Helminthosporium tiliae L171 KY984343 KY984343 KY984404 – – –
Helminthosporium velutinum CBS139923 = L131* KY984352 KY984352 KY984413 KY984432 – –
Helminthosporium velutinum L98 KY984359 KY984359 KY984417 KY984433 – –
Helminthosporium velutinum CBS136924 = L115 KY984347 KY984347 KY984408 – – –
Helminthosporium velutinum L116 KY984348 KY984348 KY984409 – – –
Helminthosporium velutinum L117 KY984349 KY984349 KY984410 – – –
Helminthosporium velutinum UESTCC22.0022 = BY14_2 = CGMCC3.23572 ON557755 ON557749 – ON557761 – –
Helminthosporium chinense UESTCC22.0026 = YQ071,005 = CGMCC3.23570* ON557754 ON557748 – ON557760 – –
Massarina cisti CBS266.62 = JCM14140* LC014568 AB807539 FJ795464 AB797249 – –
Massarina eburnea CBS473.64 OM337528 GU301840 GU371732 GU296170 – –
Massarina eburnea CBS139697 = JCM14422 = H3953 LC014569 AB521735 – AB521718 – –
Massarina pandanicola MFLUCC17-0596 = KUMCC17-0293* MG646958 MG646947 – MG646979 – –
Periconia pseudodigitata KT1395 = HHUF29370 = CBS139699 = JCM13166 = MAFF239676* NR_153490 NG_059396 – NG_064850 – –
Pseudodidymosphaeria spartii MFLUCC13-0273 KP325434 KP325436 – KP325438 – –
Pseudodidymosphaeria spartii MFLUCC14-1212 KP325435 KP325437 – KP325439 – –
Pseudosplanchnonema phorcioides L16 = CBS122935 KY984360 KY984360 KY984418 KY984434 – –
Pseudosplanchnonema phorcioides MFLUCC13-0533 = CGMCC3.17583 – KM875454 – KM875455 – –
Pseudosplanchnonema phorcioides MFLUCC13-0611 KP683375 KP683376 – KP683377 – –
Pseudosplanchnonema phorcioides MFLUCC14-0618 KP683372 KP683373 – KP683374 – –
Semifissispora natalis CPC25383 = CBS140659* KT950846 KT950858 – – – –
Semifissispora rotundata CBS172.93 = CPC549 KT950847 KT950859 – – – –
Semifissispora tooloomensis CBS143431 = CPC31680* NR_156674 NG_058526 – – – –
Stagonospora duoseptata CBS135093 = S618* KF251255 KF251758 KF252260 – – –
Stagonospora imperaticola MFLUCC15-0026 = ICMP21563* KY706143 KY706133 KY706149 KY706138 – –
Stagonospora multiseptata MFLUCC15-0449 = ICMP21562* NR_165854 NG_068239 – – – –
Stagonospora paludosa CBS135088* KF251257 KF251760 KF252262 – – –
Stagonospora perfecta KT1726A = JCM13099 = MAFF239609 AB809642 AB807579 – AB797289 – –
Stagonospora perfecta CBS135099 = S656* KF251258 KF251761 KF252263 – – –
Stagonospora pseudocaricis CBS135132 = S610* KF251259 KF251763 KF252265 – – –
Stagonospora pseudopaludosa CPC22654 = CBS136424* NR_137840 NG_058052 – – – –
Stagonospora pseudoperfecta CBS120236 = JCM13097 = MAFF239607* AB809641 AB807577 – AB797287 – –
Stagonospora tainanensis KT1866 = MAFF243860 AB809643 AB807580 – AB797290 – –
Stagonospora trichophoricola CBS136764 = D652* NR_156586 NG_058081 KJ869232 – – –
Stagonospora uniseptata CBS135090 = S611* KF251264 KF251767 KF252269 – – –
Stagonospora uniseptata S607 = CPC22151 KF251265 KF251768 KF252270 – – –
Stagonospora uniseptata S608 = CPC22150 KF251266 KF251769 KF252271 – – –
Suttonomyces clematidis MFLUCC14-0240 = GUCC18 – KP842917 – KP842920 – –
Suttonomyces rosae MFLUCC15-0051* MG828973 MG829085 – MG829185 – –
Synhelminthosporium synnematoferum UESTCC22.0023 = HLG072894 = CGMCC3.23574* ON557752 ON557746 ON563074 ON557758 – –
Apiospora malaysiana CBS 102053 KF144896 – – – KF145030 KF144988
Apiospora pseudoparenchymatica LC7234* KY494743 – – – KY705139 KY705211
Nigrospora aurantiaca CGMCC 3.18130* KX986064 – – – KY019295 KY019465
Nigrospora aurantiaca LC7034 KX986093 – – – KY019394 KY019598
Nigrospora bambusae CGMCC 3.18327* KY385307 – – – KY385313 KY385319
Nigrospora bambusae LC7245 KY385305 – – – KY385315 KY385321
Nigrospora brasiliensis CMM 1214* KY569629 – – – MK753271 MK720816
Nigrospora brasiliensis CMM 1217 KY569630 – – – MK753272 MK720817
Nigrospora camelliae-sinensis CGMCC 3.18125* KX985986 – – – KY019293 KY019460
Nigrospora chinensis LC6851 KX986049 – – – KY019450 KY019579
Nigrospora chinensis CGMCC 3.18127* KX986023 – – – KY019422 KY019462
Nigrospora covidalis CGMCC 3.20538* OK335209 – – – OK431485 OK431479
Nigrospora covidalis LC158337 OK335210 – – – OK431486 OK431480
Nigrospora endophytica URM8712 = A.R.M. 687 OM265226 – – – OP572415 OP572418
Nigrospora endophytica URM8462 = A.R.M. 973* OM265233 – – – OP572416 OP572420
Nigrospora falsivesicularis CGMCC 3.19678* MN215778 – – – MN264017 MN329942
Nigrospora falsivesicularis LC13553 MN215779 – – – MN264018 MN329943
Nigrospora globospora CGMCC 3.20539* OK335211 – – – OK431487 OK431481
Nigrospora globospora LC15839 OK335212 – – – OK431488 OK431482
Nigrospora gorlenkoana CBS 480.73* KX986048 – – – KY019420 KY019456
Nigrospora guangdongensis CFCC:53917* MT017509 – – – MT024493 MT024495
Nigrospora guilinensis LC7301 KX986063 – – – KY019404 KY019608
Nigrospora guilinensis CGMCC 3.18124* KX985983 – – – KY019292 KY019459
Nigrospora hainanensis CGMCC 3.18129* KX986091 – – – KY019415 KY019464
Nigrospora hainanensis URM8714 = A.R.M.967 OM265228 – – – OM642834 OM793057
Nigrospora hainanensis URM8715 = A.R.M.968 OM265229 – – – OM642835 OM793058
Nigrospora lacticolonia CGMCC 3.18123* KX985978 – – – KY019291 KY019458
Nigrospora lacticolonia URM8713 = A.R.M. 921 OM265227 – – – OM642833 OM642838
Nigrospora magnoliae MFLUCC 19–0112* MW285092 – – – – MW438334
Nigrospora manihoticola URM8461 = A.R.M. 645* OM265224 – – – OM914791 OM869479
Nigrospora musae CBS 319.34* KX986076 – – – KY019419 KY019455
Nigrospora musae LC6385 KX986042 – – – KY019371 KY019567
Nigrospora oryzae LC2724 KX985959 – – – KY019312 KY019486
Nigrospora oryzae LC4265 KX985994 – – – KY019335 KY019518
Nigrospora osmanthi CGMCC 3.18126* KX986010 – – – KY019421 KY019461
Nigrospora osmanthi LC4487 KX986017 – – – KY019438 KY019540
Nigrospora pernambucoensis URM8711 = A.R.M.651 OM265225 – – – OM914792 OM869480
Nigrospora pernambucoensis URM8463 = A.R.M. 974* OM265234 – – – OM914793 OM869481
Nigrospora philosophiae-doctoris CGMCC 3.20540* OK335214 – – – OK431490 OK431484
Nigrospora pyriformis CGMCC 3.18122* KX985940 – – – KY019290 KY019457
Nigrospora pyriformis URM8716 = A.R.M.970 OM265231 – – – OM513904 OM642839
Nigrospora rubi LC2698* KX985948 – – – KY019302 KY019475
Nigrospora saccharicola LC12057 MN215789 – – – MN264028 MN329952
Nigrospora saccharicola CGMCC 3.19362* MN215788 – – – MN264027 MN329951
Nigrospora sacchari-ofcinarum CGMCC 3.19335* MN215791 – – – MN264030 MN329954
Nigrospora sacchari-ofcinarum LC13531 MN215792 – – – MN264031 MN329955
Nigrospora singularis CGMCC 3.19334* MN215793 – – – MN264032 MN329956
Nigrospora singularis LC12068 MN215794 – – – MN264033 MN329957
Nigrospora sphaerica LC2839 KX985964 – – – KY019317 KY019491
Nigrospora sphaerica LC2840 KX985965 – – – KY019318 KY019492
Nigrospora sp. 1 LC2725 KX985960 – – – KY019313 KY019487
Nigrospora sp. 1 LC4566 KX986022 – – – KY019354 KY019545
Nigrospora sp. 2 LC6704 KX986047 – – – KY019373 KY019571
Nigrospora stoneae BRIP 75022a OR608744 – – – OR604065 OR604067
Nigrospora vesicularis LC0322 KX985939 – – – KY019296 KY019467
Nigrospora vesicularis CGMCC 3.18128* KX986088 – – – KY019294 KY019463
Nigrospora vesicularifera CGMCC 3.19333* MN215812 – – – MN264051 MN329975
Nigrospora vesicularifera URM8718 = A.R.M.975 OM265235 – – – OM513905 OM642840
Nigrospora yunnanensis GUCC24-0008* PP915796 – – – PP947933 PP947937
Nigrospora yunnanensis GUCC24-0009 PP915797 – – – PP947934 PP947938
Nigrospora yunnanensis GUCC24-0010 PP915798 – – – PP947935 PP947939
Nigrospora zimmermanii CBS 290.62* KY385309 – – – KY385311 KY385317
Nigrospora zimmermanii CBS 984.69 KY385310 – – – KY385316 KY385322

* = Type specimens. Our strains in this study were in bold.

Phylogenetic analyses

Reference sequences obtained from GenBank (Table 1) were utilized to assist with the phylogenetic analyses. Multiple sequence alignments were created using the online platform of MAFFT v.7.307 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2016). AliView (Larsson 2014) was utilized for manual refinement, with terminal ends and ambiguous regions of the alignment being manually excised. Phylogenetic analyses were performed using concatenated sequences from the six (ITS, LSU, rpb2, SSU, tub2, and tef1-α) through Maximum Likelihood (ML), and Bayesian Inference (BI) methodologies. The Maximum Likelihood analysis was executed on the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/) (Trifinopoulos et al. 2016). The models is: In Fig. 1, TIM2e+I+G4 for ITS, TIM2e+I+G4 for LSU, TN+F+I+G4 for rpb2 and K2P+G4 for SSU; In Fig. 2, TNe+I+G4 for ITS, TN+F+I+G4 for tef1-α, HKY+F+I+G4 for tub2. The number of bootstrap alignments is 1000 (Nguyen et al. 2015). Bayesian analysis (BI) was carried out using PhyloSuite v.1.2.2 as a tool (Zhang et al. 2019). In Fig. 1, SYM+I+G4 as the optimal model for ITS and LSU, HKY+F+I+G4 as the best-fit model for rpb2, K2P G4 as the optimal model for SSU; In Fig. 2, SYM+I+G4 as the optimal model for ITS, HKY+F+I+G4 as the best-fit model for tef1-α and tub2. Four chains were run for 10,000,000 generations and sampled every 500 generations. The initial 25% of the resulting trees were discarded as burn-in, and the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree. The final phylogenetic topology was visualized with FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and was modified in Microsoft Office PowerPoint 2019.

Figure 1. 

The maximum parsimony tree of 96 Helminthosporium taxa is based on ITS, LSU, rpb2, and SSU genes. The tree was rooted with Periconia pseudodigitata (KT1395). Bootstrap support values for ML greater than 75% and Bayesian posterior probabilities greater than 0.95 are given near nodes, respectively. The new isolates were in red. Ex-type strains were marked by T. The scale bar indicates 0.05 expected changes per site.

Figure 2. 

The maximum parsimony tree of 68 Nigrospora taxa is based on ITS, tef1-α, and tub2 genes. The tree was rooted with Apiospora malaysiana (CBS 102053) and A. pseudoparenchymatica (LC7234). Bootstrap support values for ML greater than 75% and Bayesian posterior probabilities greater than 0.95 are given near nodes, respectively. The new isolates were in red. Ex-type strains were marked by T. The scale bar indicates 0.08 expected changes per site.

Results

Phylogenetic analyses

For the Helminthosporium and related genera (Fig. 1), the phylogenetic trees accommodated 96 sequences listed in Table 1. The strains GUCC24-0011, GUCC24-0012, and GUCC24-0013 were characterized based on their molecular properties, specifically sequencing of the ITS, LSU, rpb2, and SSU genes regions. An outgroup consisting of the type strain KT1395 of Periconia pseudodigitata was also included in the study based on concatenated datasets, as shown in Table 1. The combined alignment consists of 5596 characters, including ITS (1766 characters), LSU (1648 characters), rpb2 (1114 characters), and SSU (1065 characters) regions. We constructed two phylogenetic trees: an ML tree and a BI tree. The ML tree was selected to represent the phylogenetic relationship of different Helminthosporium taxa (Fig. 1). Helminthosporium guizhouense (GUCC24-0011, GUCC24-0012, and GUCC24-0013) was found to be a sister taxon to H. caespitosum (CBS484.77, L141, and L151) with high support values from both ML and BI analyses (ML/BI: 100/1).

For the Nigrospora and related genera (Fig. 2), the phylogenetic trees accommodated 68 sequences listed in Table 1. The strains GUCC24-0008, GUCC24-0009, and GUCC24-0010 were characterized based on their molecular properties, and accurate sequencing of the ITS, tef1-α, and tub2 gene regions. Apiospora malaysiana (CBS 102053) and A. pseudoparenchymatica (LC7234) were selected as outgroups. The combined alignment consists of 1523 characters, including ITS (571 characters), tef1-α (562 characters), tub2 (385 characters) regions. We constructed two phylogenetic trees: an ML tree and a BI tree. The ML tree was selected to represent the phylogenetic relationship of different Nigrospora taxa (Fig. 2). Nigrospora yunnanensis (GUCC24-0011, GUCC24-0012, and GUCC24-0013) formed an independent branch without the DNA base differences in three loci supported by strong statistic data (ML/BI: 100/1) and were adjacent to the branch of N. falsivesicularis (CGMCC 3.19678 and LC13553), N. vesicularis (LC0322 and CGMCC 3.18128), N. aurantiaca (CGMCC 3.18130 and LC7034), N. stoneae (BRIP 75022a), N. lacticolonia (CGMCC 3.18123 and URM8713), N. osmanthi (CGMCC 3.18126 and LC4487), N. endophytica (URM8712 and URM8462), N. pernambucoensis (URM8711 and URM8463), and N. guilinensis (LC730 and CGMCC 3.18124) (ML/BI: 97/0.99).

Taxonomy

Helminthosporium guizhouense M.T. Zou & Yong Wang bis, sp. nov.

MycoBank No: 854537
Fig. 3a–r

Etymology

The name refers to Guizhou, the province where the fungus was collected.

Diagnosis

Helminthosporium guizhouense can easily be distinguished from H. caespitosum by its narrower conidia (13–16 µm vs. 27.3–35.5 µm).

Type

China • Guizhou Province, QianXi City; 26°56'11.58″N, 105°55'15.46″E; 1235 m; 24 January 2023; from rotten dead branch of Juglans regia, coll. M.T. Zou; HGUP24-0007 (holotype); ex-type culture GUCC23-0011 (ITS: PP915799, LSU: PP949847, rpb2: PP947940; SSU: PP949912).

Description on the natural substrate

Colonies hairy, brown, or blackish-brown, in groups. Mycelium partly immersed in the substratum, towards the surface forming stroma-like aggregations of light to brown pseudoparenchymatous cells.

Culture characteristics

Colony on PDA 25 mm diam after 2 weeks in an incubator under dark conditions at 28 °C, irregular circular, fat, raised, undulate, rough, with white and denser mycelium at the center, with white to deep-gray to creamy yellow, entire margin; reverse cream to yellow, with dark yellowish-brown spots. Teleomorph: Unknown. Anamorph: Conidiophores macronematous, erect, straight, or slightly curved, cylindrical, smooth, 171–718 μm long, 12–25 μm wide at the base, tapering to 7–13.5 μm near the apex, arising solitary or in fascicles from the stroma cells, erect, simple, straight or flexuous, thick-walled, brown to dark brown, with sympodial proliferation, 1–13-septate. Conidia 61–114 × 13–16 µm (x̄ = 85 × 18, n = 45), gradually tapering to 3–7 μm (x̄ = 5, n = 45) at the distal end, with a 4–10 μm (x̄ = 6, n = 42) wide, blackish-brown to black scars at the base, straight or flexuous, solitary, obclavate to rostrate, smooth-walled, hyaline, pale golden brown to brown, 8–12-distoseptate, with angular lumina; wall up to 6 µm thick.

Figure 3. 

Helminthosporium guizhouense sp. nov. (HGUP24-0008, holotype) on rotten dead branch of Juglans regia a–c colonies on the natural substrat; d, e culture on PDA after 2 weeks (d above e reverse) f conidiophore bases, stroma cells, and conidia l conidiophore m colony, conidiophores, and stroma cells n conidiophore g–k, o–r conidia. Scale bars: 1000 µm (a); 500 µm (b, c); 50µm (f–r).

Habit

Saprobic on decaying wood of Juglans regia.

Distribution

China, Guizhou Province, Qianxi City

Other material examined

China • Guizhou Province, Qianxi City; 105°92'E, 26°93'N; 1235 m; 24 January 2023; from rotten dead branch of Juglans regia, coll. M.T. Zou, HGUP24-0008 (holotype); living culture GUCC24-0011, GUCC24-0012, and GUCC24-0013.

Notes

Based on the multi-gene phylogenetic tree (Fig. 1), our strains are clustered in a distinct branch adjacent to the strain of Helminthosporium caespitosum (CBS 484.77). Topologically, there is a clear genetic distance between these taxa with ML-BS = 100%, BYPP = 1 support. When comparing the ITS, LSU, rpb2, and SSU nucleotides of H. guizhouense with H. caespitosum in the clade, there are 22 bp (0 gap) differences of 569 bp in ITS, 2 bp (0 gap) differences of 904 bp in LSU, and 38 bp (0 gap) differences of 401 bp in rpb2, and 4 bp (0 gap) differences of 1098 bp in SSU. Our collection of H. guizhouense (HGUP24-0008) differs significantly from the holotype of H. caespitosum (WU 38825 and WU 38826) (Voglmayr and Jaklitsch 2017) in the length of conidiophores (171–718 × 9.5–23 µm vs. 27–37 × 12.2–14.5 µm), the size of conidia (61–114 × 13–16 µm vs. 82–109 × 27.3–35.5 µm), the number of septa (8–12 vs. 6–10) and the wall thickness of angular lumina (6 μm vs. 8 μm). In addition, the colonies on the natural substrate of H. guizhouense are hairy, brown, or blackish brown, in groups, whereas H. caespitosum is dark-red-brown, scattered, or crowded. Through our analysis and classification process, we have identified these three strains as a new species Helminthosporium guizhouense.

Nigrospora yunnanensis M.T. Zou & Yong Wang bis, sp. nov.

MycoBank No: 854538
Fig. 4a–k

Etymology

The name refers to Yunnan, the province where the fungus was collected.

Diagnosis

Nigrospora yunnanensis is characterized by black, globose conidia (16.2 × 14.4 µm).

Type

China • Yunnan Province: Lincang City; 23°40'26.08"N, 99°56'47.70″E; 1900 m; 22 Dec 2023; on Juglans regia, coll. M.T. Zou; HGUP24-0007 (holotype); ex-type culture GUCC24-0008 (ITS: PP915796, tef1-α: PP947933, tub2: PP947937).

Culture characteristics

Colonies on PDA reaching 90 mm diam after ten days at 25 °C. The anterior surface and posterior surface are white, while the mycelium is thick and fluffy. Colonies on OA reach 90 mm diam. after ten days at 25 °C. The mycelium is circular, filiform, fluffy, while the surface and reverse are initially white, becoming gray to black, or black and producing a few black areas with age. Colonies on MEA reaching a diameter of 90 mm after ten days at 25 °C. The mycelium circular, filiform, thick, and fluffy. The surface and reverse are initially white, but they become gray to dark black with abundant black areas spreading from the periphery to the center as they age. Sexual morph undetermined. Asexual morph on OA: Hyphae 2.5–8 µm diam, smooth, hyaline to pale brown, branched, septate. Conidiophores smooth, hyaline to brown, branched, septate, sometimes reduced to conidiogenous cells. Conidiogenous cells (n = 30) 8–14 × 6–10 µm (av. = 10.4 × 8.2 µm), aggregated in clusters on hyphae, pale brown, subglobose to ampulliform. Conidia (n = 40) 14.5–18.5 × 11–17.5 µm (av. = 16.2 × 14.4 µm) solitary, globose to subglobose, black, shiny, smooth, aseptate.

Habitat

On Juglans regia.

Known distribution

China, Yunnan Province, Lincang city.

Additional material examined

China • Yunnan Province: Lincang city; 23°67'N, 99°94'E; 1900 m; 22 Dec 2023; on Juglans regia; coll. M.T. Zou; HGUP24-0007; living culture GUCC24-0008, GUCC24-0009, and GUCC24-0010.

Notes

Three isolates from walnut leaves were obtained in this study and clustered in a well-supported clade distinguished from other known species (Fig. 4). Nigrospora yunnanensis formed an independent branch. Morphological differences (Table 2) support that they belong to different taxa.

Figure 4. 

Nigrospora yunnanensis (GUCC23-0008) a–c culture characteristics on media after ten days (a on PDA b on CMA c on OA) d–j conidia attached to conidiogenous cells k coiled hyphae. Scale bars: 10 µm (d–k).

Table 2.
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XLSX

Comparison of conidia and conidiogenous cells of Nigrospora species related to this study.

Species Strain Conidia (µm) Conidiogenous cells (µm) Reference
Nigrospora endophytica URM8462 10–17.5 6.2–10 (Brito et al. 2023)
N. guilinensis CGMCC 3.18124 11.5–15 6–11 × 4–7.5 (Wang et al. 2017)
N. pernambucoensis URM8463 12.5–20 5–22.5 × 5–12.5 (Brito et al. 2023)
N. saccharicola CGMCC 3.19362 13.5–16.5 7.5–10.5 × 5–7.5 (Raza et al. 2019)
N. vesicularifera CGMCC 3.19333 11–19 7.5–10 × 12.5–15.5 (Raza et al. 2019)
N. yunnanensis GUCC24-0008 14.4 × 16.2 6–10 × 8–14 This study

Discussion

In the family Massarinaceae, along with Helminthosporium, there are ten other accepted genera: Byssothecium, Haplohelminthosporium, Helminthosporiella, Massarina, Mirohelminthosporium, Pseudodidymosphaeria, Pseudosplanchnonema, Semifissispora, Stagonospora, and Suttonomyces (Wijayawardene et al. 2022). Helminthosporium is polyphyletic, as confirmed by Konta et al. (2021), and its members were found mixed with other taxa of Byssothecium, Helminthosporiella, and Pseudosplanchnonema. According to a study by Chen et al. (2022), a new genus (Synhelminthosporium) was identified through morphological examination and multi-locus phylogenetic analyses. Most Helminthosporium species are saprobic, primarily found on woody plant materials. However, some are plant pathogens, and others thrive on fungi, particularly in Diaporthales, but the role of these Helminthosporium species on their fungal hosts is still uncertain (Voglmayr and Jaklitsch 2017).

Nigrospora belongs to the Apiosporaceae, and are endophytes, saprobes, and plant pathogens, causing harm to economically important plant species within both forestry and agricultural domains. Examples include N. oryzae causing panicle branch rot disease on Oryza sativa in China (Liu et al. 2021), N. sphaerica causing Leaf blight disease of Cacao in the Philippines (Villanueva et al. 2023), N. chinensis causing stem spot on dragon fruit in China (Guo et al. 2024). Nigrospora is also a human pathogen. N. oryzae and N. sphaerica can cause human corneal keratitis (Ananya et al. 2014; Takayama et al. 2024). In addition, N. yunnanensis was isolated from Juglans regia, which could potentially be a pathogen for the walnuts.

The nutritional benefits of walnut kernels are substantial, as they are rich in fat, protein, vitamins, and minerals, while also containing essential compounds such as flavonoids and phenolic acids (Caglarirmak 2003). Moreover, walnuts are hosts to multiple forms of microfungi, including pathogens, endophytes, and saprobes (Zhang et al. 2024). Given this, it is crucial to undertake a comprehensive study of the microfungi present on walnuts in previously unexplored regions, for instance, the provinces of Yunnan and Guizhou in China, and to perform a thorough taxonomic classification of these microorganisms.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research is supported by the following projects: National Natural Science Foundation of China (No. 31972222, 31660011), Guiyang Tobacco Science and Technology Project ([2019]2), Program of Introducing Talents of Discipline to Universities of China (111 Program, D20023), Guizhou Science, Technology Department of International Cooperation Base project ([2018]5806), the project of Guizhou Provincial Education Department ([2021]001), Guizhou Science and Technology Innovation Talent Team Project ([2020]5001), and Open Project of the Key Laboratory of Environment Friendly Management on Fruit and Vegetable Pests in North China (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Beijing Academy of Agricultural and Forestry [KFKT202301]. The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R114), King Saud University, Riyadh, Saudi Arabia.

Author contributions

Data curation: MZ. Formal analysis: FAO, KDH. Funding acquisition: XJP, YW. Supervision: XJP, YW. Writing – original draft: MZ. Writing – review and editing: FAO, XJP, YW, KDH.

Author ORCIDs

Fatimah Al-Otibi https://orcid.org/0000-0003-3629-5755

Kevin David Hyde https://orcid.org/0000-0002-2191-0762

Data availability

All of the data that support the findings of this study are available in the main text.

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