Abstract
Six collections of ascomycetes were obtained from samples collected from dead branches and leaves of Juglansregia 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 Helminthosporiumguizhouense and Nigrosporayunnanensis. 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 Juglansregia, 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 (Juglansregia 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, Helminthosporiumguizhouense, and Nigrosporayunnanensis 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.
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 | ||
| Byssotheciumcircinans | CBS675.92 | OM337536 | GU205217 | DQ767646 | GU205235 | – | – |
| Haplohelminthosporiumcalami | MFLUCC18-0074* | MT928158 | MT928156 | – | MT928160 | – | – |
| Helminthosporiellaastilbacea | COAD2126 | MG668862 | – | – | – | – | – |
| Helminthosporiellastilbacea | MFLUCC15-0813* | MT928159 | MT928157 | – | MT928161 | – | – |
| Helminthosporiellastilbacea | CPHmZC-01 | KX228298 | KX228355 | – | – | – | – |
| Helminthosporiummaquaticum | MFLUCC15-0357 = S-096* | KU697302 | KU697306 | – | KU697310 | – | – |
| Helminthosporiumaustriacum | CBS139924 = L132* | KY984301 | KY984301 | KY984365 | KY984420 | – | – |
| Helminthosporiumaustriacum | CBS14238 = L169 | KY984303 | KY984303 | KY984367 | – | – | – |
| Helminthosporiumaustriacum | L137 | KY984302 | KY984302 | KY984366 | – | – | – |
| Helminthosporiumcaespitosum | CBS484.77 = L99* | JQ044429 | JQ044448 | KY984370 | KY984421 | – | – |
| Helminthosporiumcaespitosum | L141 | KY984305 | KY984305 | KY984368 | – | – | – |
| Helminthosporiumcaespitosum | L151 | KY984306 | KY984306 | KY984369 | – | – | – |
| Helminthosporiumchengduense | UESTC22.0024 = YQ071048 = CGMCC | ON557751 | ON557745 | ON563073 | ON557757 | – | – |
| Helminthosporiumchengduense | UESTC22.0025 = YQ071047 | ON557750 | ON557744 | ON563072 | ON557756 | – | – |
| Helminthosporiumchiangraiense | MFLUCC21-0087* | MZ538504 | MZ538538 | – | – | – | – |
| Helminthosporiumchlorophorae | BRIP14521 | AF120259 | – | – | – | – | – |
| Helminthosporiumdalbergiae | MAFF243853 = H4628 = TS36 | LC014555 | AB807521 | – | AB797231 | – | – |
| Helminthosporiumendiandrae | CBS138902 = CPC22194* | KP004450 | KP004478 | – | – | – | – |
| Helminthosporiumerythrinicola | CPC35291 = CBS145569* | NR_165563 | MK876432 | MK876486 | – | – | – |
| Helminthosporiumgenistae | CBS142597 = L142* | KY984310 | KY984310 | KY984374 | – | – | – |
| Helminthosporiumgenistae | CBS139922 = L129 | KY984309 | KY984309 | KY984373 | KY984423 | – | – |
| Helminthosporiumgenistae | CBS139921 = L128 | KY984308 | KY984308 | KY984372 | KY984422 | – | – |
| Helminthosporiumguizhouense | GUCC24-0011* | PP915799 | PP949847 | PP947940 | PP949912 | – | – |
| Helminthosporiumguizhouense | GUCC24-0012 | PP915800 | PP949848 | PP947941 | PP949913 | – | – |
| Helminthosporiumguizhouense | GUCC24-0013 | PP915801 | PP949849 | PP947942 | PP949914 | – | – |
| Helminthosporiumhispanicum | CBS136917 = L109* | KY984318 | KY984318 | KY984381 | KY984424 | – | – |
| Helminthosporiumjuglandinum | CBS136922 = L118* | KY984321 | KY984321 | KY984384 | – | – | – |
| Helminthosporiumjuglandinum | CBS136911 = L97 | KY984322 | KY984322 | KY984385 | KY984425 | – | – |
| Helminthosporiumjuglandinum | CBS136912 = L101 | KY984319 | KY984319 | KY984382 | – | – | – |
| Helminthosporiumjuglandinum | CBS136913 = L102 | KY984320 | KY984320 | KY984383 | – | – | – |
| Helminthosporiumleucadendri | CBS135133 = CPC19345* | KF251150 | KF251654 | KF252159 | – | – | – |
| Helminthosporiumlivistonae | CPC32158 = CBS144413* | NR_160348 | NG_064539 | – | – | – | – |
| Helminthosporiummagnisporum | MAFF239278 = H4627 = TS33* | AB811452 | AB807522 | – | AB797232 | – | – |
| Helminthosporiummassarinum | CBS139690 = JCM13095 = MAFF239605 = KT1564* | AB809629 | AB807524 | – | AB797234 | – | – |
| Helminthosporiummassarinum | JCM13094 = MAFF239604 = KT838* | AB809628 | AB807523 | – | AB797233 | – | – |
| Helminthosporiummicrosorum | CBS136910 = L96* | KY984329 | KY984329 | KY984390 | KY984427 | – | – |
| Helminthosporiummicrosorum | L94 | KY984327 | KY984327 | KY984388 | KY984426 | – | – |
| Helminthosporiummicrosorum | CBS136916 = L108 | KY984323 | KY984323 | KY984386 | – | – | – |
| Helminthosporiummicrosorum | L95 | KY984328 | KY984328 | KY984389 | – | – | – |
| Helminthosporiumnanjingensis | HHAUF020380 = ZM020380 | KF192322 | – | – | – | – | – |
| Helminthosporiumoligosporum | CBS136909 = L93* | KY984333 | KY984333 | KY984394 | – | – | – |
| Helminthosporiumoligosporum | CBS136908 = L92 | KY984332 | KY984332 | KY984393 | KY984428 | – | – |
| Helminthosporiumoligosporum | L106 | KY984330 | KY984330 | KY984391 | – | – | – |
| Helminthosporiumquercinum | CBS136921 = L90* | KY984339 | KY984339 | KY984400 | KY984429 | – | – |
| Helminthosporiumquercinum | CBS112393 | KY984334 | KY984334 | KY984395 | – | – | – |
| Helminthosporiumquercinum | CBS136915 = L107 | KY984336 | KY984336 | KY984397 | – | – | – |
| Helminthosporiumsolani | CBS365.75 | KY984341 | KY984341 | KY984402 | KY984430 | – | – |
| Helminthosporiumsolani | CBS640.85 | KY984342 | KY984342 | KY984403 | – | – | – |
| Helminthosporiumsubmersum | MFLUCC16-1360* | – | MG098787 | – | MG098796 | – | – |
| Helminthosporiumsubmersum | MFLUCC16-1290PT | MG098780 | MG098788 | MG098592 | MG098797 | – | – |
| Helminthosporiumsubmersum | UESTCC22.0021 = Sara08_3 = CGMCC | ON557753 | ON557747 | ON563075 | ON557759 | – | – |
| Helminthosporiumsyzygii | CPC35312 = CBS145570* | NR_165564 | MK876433 | MK876487 | – | – | – |
| Helminthosporiumtiliae | CBS136907 = L88* | KY984345 | KY984345 | KY984406 | KY984431 | – | – |
| Helminthosporiumtiliae | CBS136906 = L87 | KY984344 | KY984344 | KY984405 | – | – | – |
| Helminthosporiumtiliae | L171 | KY984343 | KY984343 | KY984404 | – | – | – |
| Helminthosporiumvelutinum | CBS139923 = L131* | KY984352 | KY984352 | KY984413 | KY984432 | – | – |
| Helminthosporiumvelutinum | L98 | KY984359 | KY984359 | KY984417 | KY984433 | – | – |
| Helminthosporiumvelutinum | CBS136924 = L115 | KY984347 | KY984347 | KY984408 | – | – | – |
| Helminthosporiumvelutinum | L116 | KY984348 | KY984348 | KY984409 | – | – | – |
| Helminthosporiumvelutinum | L117 | KY984349 | KY984349 | KY984410 | – | – | – |
| Helminthosporiumvelutinum | UESTCC22.0022 = BY14_2 = CGMCC3.23572 | ON557755 | ON557749 | – | ON557761 | – | – |
| Helminthosporiumchinense | UESTCC22.0026 = YQ071,005 = CGMCC3.23570* | ON557754 | ON557748 | – | ON557760 | – | – |
| Massarinacisti | CBS266.62 = JCM14140* | LC014568 | AB807539 | FJ795464 | AB797249 | – | – |
| Massarinaeburnea | CBS473.64 | OM337528 | GU301840 | GU371732 | GU296170 | – | – |
| Massarinaeburnea | CBS139697 = JCM14422 = H3953 | LC014569 | AB521735 | – | AB521718 | – | – |
| Massarinapandanicola | MFLUCC17-0596 = KUMCC17-0293* | MG646958 | MG646947 | – | MG646979 | – | – |
| Periconiapseudodigitata | KT1395 = HHUF29370 = CBS139699 = JCM13166 = MAFF239676* | NR_153490 | NG_059396 | – | NG_064850 | – | – |
| Pseudodidymosphaeriaspartii | MFLUCC13-0273 | KP325434 | KP325436 | – | KP325438 | – | – |
| Pseudodidymosphaeriaspartii | MFLUCC14-1212 | KP325435 | KP325437 | – | KP325439 | – | – |
| Pseudosplanchnonemaphorcioides | L16 = CBS122935 | KY984360 | KY984360 | KY984418 | KY984434 | – | – |
| Pseudosplanchnonemaphorcioides | MFLUCC13-0533 = CGMCC3.17583 | – | KM875454 | – | KM875455 | – | – |
| Pseudosplanchnonemaphorcioides | MFLUCC13-0611 | KP683375 | KP683376 | – | KP683377 | – | – |
| Pseudosplanchnonemaphorcioides | MFLUCC14-0618 | KP683372 | KP683373 | – | KP683374 | – | – |
| Semifissisporanatalis | CPC25383 = CBS140659* | KT950846 | KT950858 | – | – | – | – |
| Semifissisporarotundata | CBS172.93 = CPC549 | KT950847 | KT950859 | – | – | – | – |
| Semifissisporatooloomensis | CBS143431 = CPC31680* | NR_156674 | NG_058526 | – | – | – | – |
| Stagonosporaduoseptata | CBS135093 = S618* | KF251255 | KF251758 | KF252260 | – | – | – |
| Stagonosporaimperaticola | MFLUCC15-0026 = ICMP21563* | KY706143 | KY706133 | KY706149 | KY706138 | – | – |
| Stagonosporamultiseptata | MFLUCC15-0449 = ICMP21562* | NR_165854 | NG_068239 | – | – | – | – |
| Stagonosporapaludosa | CBS135088* | KF251257 | KF251760 | KF252262 | – | – | – |
| Stagonosporaperfecta | KT1726A = JCM13099 = MAFF239609 | AB809642 | AB807579 | – | AB797289 | – | – |
| Stagonosporaperfecta | CBS135099 = S656* | KF251258 | KF251761 | KF252263 | – | – | – |
| Stagonosporapseudocaricis | CBS135132 = S610* | KF251259 | KF251763 | KF252265 | – | – | – |
| Stagonosporapseudopaludosa | CPC22654 = CBS136424* | NR_137840 | NG_058052 | – | – | – | – |
| Stagonosporapseudoperfecta | CBS120236 = JCM13097 = MAFF239607* | AB809641 | AB807577 | – | AB797287 | – | – |
| Stagonosporatainanensis | KT1866 = MAFF243860 | AB809643 | AB807580 | – | AB797290 | – | – |
| Stagonosporatrichophoricola | CBS136764 = D652* | NR_156586 | NG_058081 | KJ869232 | – | – | – |
| Stagonosporauniseptata | CBS135090 = S611* | KF251264 | KF251767 | KF252269 | – | – | – |
| Stagonosporauniseptata | S607 = CPC22151 | KF251265 | KF251768 | KF252270 | – | – | – |
| Stagonosporauniseptata | S608 = CPC22150 | KF251266 | KF251769 | KF252271 | – | – | – |
| Suttonomycesclematidis | MFLUCC14-0240 = GUCC18 | – | KP842917 | – | KP842920 | – | – |
| Suttonomycesrosae | MFLUCC15-0051* | MG828973 | MG829085 | – | MG829185 | – | – |
| Synhelminthosporiumsynnematoferum | UESTCC22.0023 = HLG072894 = CGMCC3.23574* | ON557752 | ON557746 | ON563074 | ON557758 | – | – |
| Apiosporamalaysiana | CBS 102053 | KF144896 | – | – | – | KF145030 | KF144988 |
| Apiosporapseudoparenchymatica | LC7234* | KY494743 | – | – | – | KY705139 | KY705211 |
| Nigrosporaaurantiaca | CGMCC 3.18130* | KX986064 | – | – | – | KY019295 | KY019465 |
| Nigrosporaaurantiaca | LC7034 | KX986093 | – | – | – | KY019394 | KY019598 |
| Nigrosporabambusae | CGMCC 3.18327* | KY385307 | – | – | – | KY385313 | KY385319 |
| Nigrosporabambusae | LC7245 | KY385305 | – | – | – | KY385315 | KY385321 |
| Nigrosporabrasiliensis | CMM 1214* | KY569629 | – | – | – | MK753271 | MK720816 |
| Nigrosporabrasiliensis | CMM 1217 | KY569630 | – | – | – | MK753272 | MK720817 |
| Nigrosporacamelliae-sinensis | CGMCC 3.18125* | KX985986 | – | – | – | KY019293 | KY019460 |
| Nigrosporachinensis | LC6851 | KX986049 | – | – | – | KY019450 | KY019579 |
| Nigrosporachinensis | CGMCC 3.18127* | KX986023 | – | – | – | KY019422 | KY019462 |
| Nigrosporacovidalis | CGMCC 3.20538* | OK335209 | – | – | – | OK431485 | OK431479 |
| Nigrosporacovidalis | LC158337 | OK335210 | – | – | – | OK431486 | OK431480 |
| Nigrosporaendophytica | URM8712 = A.R.M. 687 | OM265226 | – | – | – | OP572415 | OP572418 |
| Nigrosporaendophytica | URM8462 = A.R.M. 973* | OM265233 | – | – | – | OP572416 | OP572420 |
| Nigrosporafalsivesicularis | CGMCC 3.19678* | MN215778 | – | – | – | MN264017 | MN329942 |
| Nigrosporafalsivesicularis | LC13553 | MN215779 | – | – | – | MN264018 | MN329943 |
| Nigrosporaglobospora | CGMCC 3.20539* | OK335211 | – | – | – | OK431487 | OK431481 |
| Nigrosporaglobospora | LC15839 | OK335212 | – | – | – | OK431488 | OK431482 |
| Nigrosporagorlenkoana | CBS 480.73* | KX986048 | – | – | – | KY019420 | KY019456 |
| Nigrosporaguangdongensis | CFCC:53917* | MT017509 | – | – | – | MT024493 | MT024495 |
| Nigrosporaguilinensis | LC7301 | KX986063 | – | – | – | KY019404 | KY019608 |
| Nigrosporaguilinensis | CGMCC 3.18124* | KX985983 | – | – | – | KY019292 | KY019459 |
| Nigrosporahainanensis | CGMCC 3.18129* | KX986091 | – | – | – | KY019415 | KY019464 |
| Nigrosporahainanensis | URM8714 = A.R.M.967 | OM265228 | – | – | – | OM642834 | OM793057 |
| Nigrosporahainanensis | URM8715 = A.R.M.968 | OM265229 | – | – | – | OM642835 | OM793058 |
| Nigrosporalacticolonia | CGMCC 3.18123* | KX985978 | – | – | – | KY019291 | KY019458 |
| Nigrosporalacticolonia | URM8713 = A.R.M. 921 | OM265227 | – | – | – | OM642833 | OM642838 |
| Nigrosporamagnoliae | MFLUCC 19–0112* | MW285092 | – | – | – | – | MW438334 |
| Nigrosporamanihoticola | URM8461 = A.R.M. 645* | OM265224 | – | – | – | OM914791 | OM869479 |
| Nigrosporamusae | CBS 319.34* | KX986076 | – | – | – | KY019419 | KY019455 |
| Nigrosporamusae | LC6385 | KX986042 | – | – | – | KY019371 | KY019567 |
| Nigrosporaoryzae | LC2724 | KX985959 | – | – | – | KY019312 | KY019486 |
| Nigrosporaoryzae | LC4265 | KX985994 | – | – | – | KY019335 | KY019518 |
| Nigrosporaosmanthi | CGMCC 3.18126* | KX986010 | – | – | – | KY019421 | KY019461 |
| Nigrosporaosmanthi | LC4487 | KX986017 | – | – | – | KY019438 | KY019540 |
| Nigrosporapernambucoensis | URM8711 = A.R.M.651 | OM265225 | – | – | – | OM914792 | OM869480 |
| Nigrosporapernambucoensis | URM8463 = A.R.M. 974* | OM265234 | – | – | – | OM914793 | OM869481 |
| Nigrosporaphilosophiae-doctoris | CGMCC 3.20540* | OK335214 | – | – | – | OK431490 | OK431484 |
| Nigrosporapyriformis | CGMCC 3.18122* | KX985940 | – | – | – | KY019290 | KY019457 |
| Nigrosporapyriformis | URM8716 = A.R.M.970 | OM265231 | – | – | – | OM513904 | OM642839 |
| Nigrosporarubi | LC2698* | KX985948 | – | – | – | KY019302 | KY019475 |
| Nigrosporasaccharicola | LC12057 | MN215789 | – | – | – | MN264028 | MN329952 |
| Nigrosporasaccharicola | CGMCC 3.19362* | MN215788 | – | – | – | MN264027 | MN329951 |
| Nigrosporasacchari-ofcinarum | CGMCC 3.19335* | MN215791 | – | – | – | MN264030 | MN329954 |
| Nigrosporasacchari-ofcinarum | LC13531 | MN215792 | – | – | – | MN264031 | MN329955 |
| Nigrosporasingularis | CGMCC 3.19334* | MN215793 | – | – | – | MN264032 | MN329956 |
| Nigrosporasingularis | LC12068 | MN215794 | – | – | – | MN264033 | MN329957 |
| Nigrosporasphaerica | LC2839 | KX985964 | – | – | – | KY019317 | KY019491 |
| Nigrosporasphaerica | 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 |
| Nigrosporastoneae | BRIP 75022a | OR608744 | – | – | – | OR604065 | OR604067 |
| Nigrosporavesicularis | LC0322 | KX985939 | – | – | – | KY019296 | KY019467 |
| Nigrosporavesicularis | CGMCC 3.18128* | KX986088 | – | – | – | KY019294 | KY019463 |
| Nigrosporavesicularifera | CGMCC 3.19333* | MN215812 | – | – | – | MN264051 | MN329975 |
| Nigrosporavesicularifera | URM8718 = A.R.M.975 | OM265235 | – | – | – | OM513905 | OM642840 |
| Nigrosporayunnanensis | GUCC24-0008* | PP915796 | – | – | – | PP947933 | PP947937 |
| Nigrosporayunnanensis | GUCC24-0009 | PP915797 | – | – | – | PP947934 | PP947938 |
| Nigrosporayunnanensis | GUCC24-0010 | PP915798 | – | – | – | PP947935 | PP947939 |
| Nigrosporazimmermanii | CBS 290.62* | KY385309 | – | – | – | KY385311 | KY385317 |
| Nigrosporazimmermanii | 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 Periconiapseudodigitata (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 Apiosporamalaysiana (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 Periconiapseudodigitata 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). Helminthosporiumguizhouense (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. Apiosporamalaysiana (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). Nigrosporayunnanensis (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.
8CFDB1A2-8D26-5A26-9B65-8FAFD9804B6D
854537
Figure 3.
Helminthosporiumguizhouense sp. nov. (HGUP24-0008, holotype) on rotten dead branch of Juglansregiaa–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).
Etymology.
The name refers to Guizhou, the province where the fungus was collected.
Diagnosis.
Helminthosporiumguizhouense 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 Juglansregia, 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.
Habit.
Saprobic on decaying wood of Juglansregia.
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 Juglansregia, 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 Helminthosporiumcaespitosum (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 Helminthosporiumguizhouense.
. Nigrospora yunnanensis
M.T. Zou & Yong Wang bis sp. nov.
A666839F-A87E-5F0C-BC50-27AE196CA3ED
854538
Figure 4.
Nigrosporayunnanensis (GUCC23-0008) a–c culture characteristics on media after ten days (a on PDAb on CMA c on OA) d–j conidia attached to conidiogenous cells k coiled hyphae. Scale bars: 10 µm (d–k).
Etymology.
The name refers to Yunnan, the province where the fungus was collected.
Diagnosis.
Nigrosporayunnanensis 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 Juglansregia, 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 Juglansregia.
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 Juglansregia; 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). Nigrosporayunnanensis formed an independent branch. Morphological differences (Table 2) support that they belong to different taxa.
Table 2.
Comparison of conidia and conidiogenous cells of Nigrospora species related to this study.
| Species | Strain | Conidia (µm) | Conidiogenous cells (µm) | Reference |
|---|---|---|---|---|
| Nigrosporaendophytica | 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 Oryzasativa 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 Juglansregia, 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.
Supplementary Material
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
Funding Statement
This research is supported by the following projects: National Natural Science Foundation of China (No. 31972222, 31660011), 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 ([2020]001), and Guizhou Science and Technology Innovation Talent Team Project ([2020]5001). The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R114), King Saud University, Riyadh, Saudi Arabia.
Contributor Information
Yong Wang, Email: yongwangbis@aliyun.com.
Xue-Jun Pan, Email: pxjun2050@aliyun.com.
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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Data Availability Statement
All of the data that support the findings of this study are available in the main text.