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Research Article
Three new endophytic Apiospora species (Apiosporaceae, Amphisphaeriales) from China
expand article infoXiao-Ni Yan‡, Chu-Long Zhang‡
‡ Zhejiang University, Hangzhou, China

Corresponding author: Chu-Long Zhang ( clzhang@zju.edu.cn )

Academic editor: Chayanard Phukhamsakda
© 2024 Xiao-Ni Yan, Chu-Long Zhang.
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: Yan X-N, Zhang C-L (2024) Three new endophytic Apiospora species (Apiosporaceae, Amphisphaeriales) from China. MycoKeys 105: 295-316. https://doi.org/10.3897/mycokeys.105.122583
Open Access

Abstract

Apiospora species are widely distributed fungi with diverse lifestyles, primarily functioning as plant pathogens, as well as exhibiting saprophytic and endophytic behaviors. This study reports the discovery of three new species of Apiospora, namely A. gongcheniae, A. paragongcheniae, and A. neogongcheniae, isolated from healthy Poaceae plants in China. These novel species were identified through a multi-gene phylogenetic analysis. The phylogenetic analysis of the combined ITS, LSU, tef1, and tub2 sequence data revealed that the three new species formed a robustly supported clade with A. garethjonesii, A. neogarethjonesii, A. setostroma, A. subrosea, A. mytilomorpha, and A. neobambusae. Detailed descriptions of the newly discovered species are provided and compared with closely related species to enhance our understanding of the genus Apiospora.

Key words

Apiospora, Ascomycota, endophyte, phylogeny, taxonomy

Introduction

Apiospora is an important genus of fungal Sordariomycetes, that produces a basauxic, arthrinium-like conidiogenesis (Hyde et al. 2020). The family Apiosporaceae was established to accommodate the genus Apiospora with the special conidiogenesis (Hyde et al. 1998). Over time, the membership of Apiosporaceae has undergone several revisions. It presently comprises several genera of fungi with similar morphology, including Apiospora, Arthrinium, Nigrospora, and Neoarthrinium (Wang et al. 2017; Pintos and Alvarado 2021; Jiang et al. 2022).

Within the family Apiosporaceae, Apiospora is closely related to Arthrinium and they were once considered as two life stages of a single taxon (Ellis 1965; Crous and Groenewald 2013; Réblová et al. 2016; Jiang et al. 2019). Morphologically, Apiospora and Arthrinium lack clear diagnostic features, although species of Arthrinium often produce conidia of various shapes (Minter and Cannon 2018; Pintos and Alvarado 2021), while most species of Apiospora have rounded lenticular conidia (Li et al. 2023; Liao et al. 2023). Ecologically, most sequenced collections of Arthrinium were found on Cyperaceae or Juncaceae in temperate, cold, or alpine habitats, while those of Apiospora were mainly collected on Poaceae, as well as various other plant host families, in a wide range of habitats, including tropical and subtropical regions (Dai et al. 2016; Jiang et al. 2018; Wang et al. 2018; Feng et al. 2021; Tian et al. 2021; Kwon et al. 2022; Monkai et al. 2022). With the addition of molecular evidence and the expansion of the sample, the latest phylogenetic analysis suggests that Arthrinium s. str. and Apiospora represent independent lineages within Apiosporaceae (Pintos and Alvarado 2021). Consequently, most species of Arthrinium have been reclassified under Apiospora. Furthermore, Pintos and Alvarado defined the exact identity of Apiospora montagnei (the type species of Apiospora) and delineated the phylogenetic boundaries of Apiospora (Pintos and Alvarado 2022).

Currently, there are 176 records in Apiospora (Index Fungorum; http://www.indexfungorum.org/; accessed on 8 Mar 2024). These fungi primarily act as plant pathogens, causing diseases in a wide range of host plants. For example, A. arundinis is the causal agent for several important plant diseases, such as kernel blight of barley (Martínez-Cano et al. 1992), brown culm streak of Phyllostachys praecox (Chen et al. 2014), moldy sugarcane (Liao et al. 2022), and leaf spot on Polygonatum cyrtonema (Gong et al. 2023). A. marii causes dieback of olive trees (Gerin et al. 2020), while A. kogelbergense leads to blight of Bambusa intermedi (Yin et al. 2020). Whereas, many Apiospora species are saprophytes, such as A. acutiapica (Senanayake et al. 2020), A. garethjonesii (Dai et al. 2016), A. magnispora (Zhao et al. 2023), A. sasae (Crous et al. 2021), and A. thailandicum (Dai et al. 2017). In addition, certain Apiospora species are reported as endophytes with wide host range, including bamboo (Wang et al. 2018), Camellia sinensis (Wang et al. 2018), Wurfbainia villosa (Liao et al. 2023), and even hive-stored pollen (Zhao et al. 2018).

Endophytic fungi exhibit rich diversity and play a significant role in the ecosystem. In a previous study, we collected and isolated endophytic fungi from healthy Poaceae plants in China (Liu et al. 2021). In this study, three new endophytic species of Apiospora were identified and described based on morphological characteristics and a multi-gene phylogenetic analysis, utilizing a dataset comprising the combined nuclear ribosomal DNA internal transcribed spacer (ITS), nuclear ribosomal DNA large subunit (LSU), the translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) sequences.

Materials and methods

Fungal isolation

In the present work, Poaceae plant samples were collected from three locations in China: Xilingol Grassland National Nature Reserve in Inner Mongolia, Xishuangbanna, Naban River Watershed National Nature Reserve in Yunnan province, and Baishanzu National Nature Reserve in Zhejiang province (Liu et al. 2021). To isolate endophytic Apiospora strains, healthy tissues of asymptomatic plants were first disinfected for 3 min in 75% ethanol and 10 min in 1% sodium hypochlorite, followed by three washes in sterile distilled water. The disinfected tissues were excised, and then incubated on malt extract agar (MEA) medium at 25 °C. Subsequently, the growing hyphae were transferred to potato dextrose agar (PDA) medium to obtain pure cultures.

All strains of Apiospora were stored in the Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou, China. In addition, the holotype and ex-type culture were deposited in the Guangdong Microbial Culture Collection Center (GDMCC). Fungal names were registered in the Fungal Names, one of the recognised repositories of fungal taxonomy (https://nmdc.cn/fungalnames/).

Morphological study

Morphological descriptions were recorded on PDA and MEA. The morphological characteristics of the colonies were captured with a digital camera (Canon EOS700D). The fungal structures were observed and photographed using a stereomicroscope (Leica S9D) and a Leica DM2500 microscope equipped with differential interference contrast (DIC). Measurements of conidiogenous cells and conidia were reported as follows: a-b × c-d (mean, n), where “a” and “c” represent the minimum values, “b” and “d” represent the maximum values, and the mean value and number of measurements (n) are shown in parentheses (Wang et al. 2018).

DNA extraction, PCR amplification and sequencing

Fresh fungal mycelia from pure cultures grown on PDA at 25 °C for 5–7 d were used for DNA extraction. Genomic DNA was extracted following the method as described in Chi et al. (2009).

Polymerase chain reaction (PCR) amplification was applied to amplify four gene fragments, including ITS, LSU, tef1, and tub2. The primer pairs were used: ITS1/ITS4 for ITS (White et al. 1990), LR0R/LR5 for LSU (Rehner and Samuels 1995), EF1-728F/EF2 for tef1 (O’Donnell et al, 1998; Carbone and Kohn 1999), and T1/Bt2b for tub2 (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997). PCR program for ITS amplification was conducted with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min. The annealing temperatures were adjusted to 56 °C for LSU, tef1, and tub2.

PCR was performed using a Veriti Thermal Cycler (Waltham, MA, USA). Amplification reactions contained 10 μL of 2× Taq Plus Master Mix II (Vazyme, Nanjing, China), 0.8 μL of each primer (10 μM) (Sunya, Hangzhou, China), 0.8 μL of DNA template, and double-distilled water to reach a total volume of 20 μL. Purification and sequencing of PCR products were performed by Sunya Biotechnology Company (Hangzhou, China). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.
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Species of Apiosporaceae used in the phylogenetic analyses. Notes: Strains in this study are marked in bold. “T” indicates a type culture. NA = not available.

Species Strain Numbers Host and Substrates Locality GenBank accession numbers
ITS LSU tef1 tub2
Apiospora acutiapica KUMCC 20-0209 Bambusa bambos China MT946342 MT946338 MT947359 MT947365
Apiospora acutiapica KUMCC 20-0210 T Bambusa bambos China MT946343 MT946339 MT947360 MT947366
Apiospora adinandrae SAUCC 1282B-1 T Diseased leaves of Adinandra glischroloma China OR739431 OR739572 OR753448 OR757128
Apiospora adinandrae SAUCC 1282B-2 Diseased leaves of Adinandra glischroloma China OR739432 OR739573 OR753449 OR757129
Apiospora agari KUC21333, SFC20161014-M18 T Agarum cribrosum South Korea MH498520 MH498440 MH544663 MH498478
Apiospora aquatic MFLU 18-1628, S-642 T Submerged wood China MK828608 MK835806 NA NA
Apiospora arctoscopi KUC21331, SFC20200506-M05 T Eggs of Arctoscopus japonicus South Korea MH498529 MH498449 MN868918 MH498487
Apiospora arundinis CBS 124788 Living leaves of Fagus sylvatica Switzerland KF144885 KF144929 KF145017 KF144975
Apiospora arundinis LC4951 Dichotomanthes tristaniicarpa China KY494698 KY494774 KY705097 KY705168
Apiospora aseptata KUNCC 23-14169 T Living roots of Dicranopteris pedata China OR590341 OR590335 OR634949 OR634943
Apiospora aurea CBS 244.83 T Air Spain AB220251 KF144935 KF145023 KF144981
Apiospora balearica CBS 145129, AP24118 T Poaceae plant Spain MK014869 MK014836 MK017946 MK017975
Apiospora bambusicola MFLUCC 20-0144 T Schizostachyum brachycladum Thailand MW173030 MW173087 MW183262 NA
Apiospora bawanglingensis SAUCC BW0444 T Leaves of Indocalamus longiauritus China OR739429 OR739570 OR753446 OR757126
Apiospora biserialis CGMCC 3.20135 T Bamboo China MW481708 MW478885 MW522938 MW522955
Apiospora camelliae-sinensis CGMCC 3.18333, LC5007 T Camellia sinensis China KY494704 KY494780 KY705103 KY705173
Apiospora camelliae-sinensis LC8181 Brassica rapa China KY494761 KY494837 KY705157 KY705229
Apiospora cannae ZHKUCC 22-0139 Leaves of Canna sp. China OR164902 OR164949 OR166286 OR166322
Apiospora cannae ZHKUCC 22-0127 T Leaves of Canna sp. China OR164901 OR164948 OR166285 OR166321
Apiospora chiangraiense MFLUCC 21-0053 T Dead culms of bamboo Thailand MZ542520 MZ542524 NA MZ546409
Apiospora chromolaenae MFLUCC 17-1505 T Chromolaena odorata Thailand MT214342 MT214436 MT235802 NA
Apiospora cordylinae GUCC 10026 Cordyline fruticosa China MT040105 NA MT040126 MT040147
Apiospora cordylinae GUCC 10027 T Cordyline fruticosa China MT040106 NA MT040127 MT040148
Apiospora coryli CFCC 58978 T Dead plant culms of Corylus yunnanensis China OR125564 OR133586 OR139974 OR139978
Apiospora coryli CFCC 58979 T Dead plant culms of Corylus yunnanensis China OR125565 OR133587 OR139975 OR139979
Apiospora cyclobalanopsidis CGMCC 3.20136 T Cyclobalanopsidis glauca China MW481713 MW478892 MW522945 MW522962
Apiospora cyclobalanopsidis GZCC 20-0103 Cyclobalanopsidis glauca China MW481714 MW478893 MW522946 MW522963
Apiospora dendrobii MFLUCC 14-0152 T Roots of Dendrobium harveyanum Thailand MZ463151 MZ463192 NA NA
Apiospora dematiacea KUNCC 23-14202 T Living stems of Dicranopteris ampla China OR590346 OR590339 OR634953 OR634948
Apiospora descalsii CBS 145130 T Ampelodesmos mauritanicus Spain MK014870 MK014837 MK017947 MK017976
Apiospora dichotomanthi CGMCC 3.18332, LC4950 T Dichotomanthes tristaniicarpa China KY494697 KY494773 KY705096 KY705167
Apiospora dichotomanthi LC8175 Dichotomanthes tristaniicarpa China KY494755 KY494831 KY705151 KY705223
Apiospora dicranopteridis KUNCC23-14171 T Living stems of Dicranopteris pedata China OR590342 OR590336 OR634950 OR634944
Apiospora dicranopteridis KUNCC23-14177 Roots of Dicranopteris pedata China OR590343 OR590337 OR634951 OR634945
Apiospora dongyingensis SAUCC 0302 T Leaves of bamboo China OP563375 OP572424 OP573264 OP573270
Apiospora dongyingensis SAUCC 0303 Leaves of bamboo China OP563374 OP572423 OP573263 OP573269
Apiospora elliptica ZHKUCC 22-0131 T Dead stems of unknown plant China OR164905 OR164952 OR166284 OR166323
Apiospora elliptica ZHKUCC 22-0140 Dead stems of unknown plant China OR164906 OR164953 NA OR166324
Apiospora endophytica ZHKUCC 23-0006 T Living leaves of Wurfbainia villosa China OQ587996 OQ587984 OQ586062 OQ586075
Apiospora endophytica ZHKUCC 23-0007 Living leaves of Wurfbainia villosa China OQ587997 OQ587985 OQ586063 OQ586076
Apiospora esporlensis CBS 145136 T Phyllostachys aurea Spain MK014878 MK014845 MK017954 MK017983
Apiospora esporlensis UNIPAMPA010 Living leaves of the Antarctic Hairgrass Deschampsia antarctica Antarctica MN947641 genome genome genome
Apiospora euphorbiae IMI 285638b Bambusa sp. Bangladesh AB220241 AB220335 NA AB220288
Apiospora fermenti KUC21288, SFC20140423-M86 Seaweeds South Korea MF615230 NA MH544668 MF615235
Apiospora fermenti KUC21289 T Seaweeds South Korea MF615226 MF615213 MH544667 MF615231
Apiospora gaoyouensis CFCC 52301T Phragmites australis China MH197124 NA MH236793 MH236789
Apiospora gaoyouensis CFCC 52302 Phragmites australis China MH197125 NA MH236794 MH236790
Apiospora garethjonesii GZCC 20-0115 Dead culms of bamboo China MW481715 MW478894 MW522947 NA
Apiospora garethjonesii KUMCC 16-0202, JHB004, HKAS 96289 T Dead culms of bamboo China KY356086 KY356091 NA NA
Apiospora garethjonesii SICAUCC 22-0027 Bamboo China ON228603 ON228659 NA ON237651
Apiospora garethjonesii SICAUCC 22-0028 Bamboo China ON228606 ON228662 NA ON237654
Apiospora gelatinosa GZAAS 20-0107 Bamboo China MW481707 MW478889 MW522942 MW522959
Apiospora gelatinosa HKAS 11962 T Bamboo China MW481706 MW478888 MW522941 MW522958
Apiospora globosa KUNCC 23-14210 T Living stems of Dicranopteris linearis China OR590347 OR590340 OR634954 NA
Apiospora gongcheniae GDMCC 3.1045, YNE00465 T Living stems of Oryza meyeriana subsp. granulata China PP033259 PP033102 PP034683 PP034691
Apiospora gongcheniae YNE00565 Living stems of Oryza meyeriana subsp. granulata China PP033260 PP033103 PP034684 PP034692
Apiospora guangdongensis ZHKUCC 23-0004 T Living leaves of Wurfbainia villosa China OQ587994 OQ587982 OQ586060 OQ586073
Apiospora guangdongensis ZHKUCC 23-0005 Living leaves of Wurfbainia villosa China OQ587995 OQ587983 OQ586061 OQ586074
Apiospora guiyangensis HKAS 102403 T Dead culms of Poaceae China MW240647 MW240577 MW759535 MW775604
Apiospora guiyangensis KUNCC 22-12539 Poaceae plant China OQ029540 OQ029613 OQ186444 OQ186446
Apiospora guizhouensis CGMCC 3.18334, LC5322 T Air in karst cave China KY494709 KY494785 KY705108 KY705178
Apiospora guizhouensis LC5318 Air in karst cave China KY494708 KY494784 KY705107 KY705177
Apiospora hainanensis SAUCC 1681 T Leaves of bamboo China OP563373 OP572422 OP573262 OP573268
Apiospora hainanensis SAUCC 1682 Leaves of bamboo China OP563372 OP572421 OP573261 OP573267
Apiospora hispanica IMI 326877 T Beach sands Spain AB220242 AB220336 NA AB220289
Apiospora hydei CBS 114990 T Culms of Bambusa tuldoides China KF144890 KF144936 KF145024 KF144982
Apiospora hydei LC7103 Leaves of bamboo China KY494715 KY494791 KY705114 KY705183
Apiospora hyphopodii JHB003, HKAS 96288 Bamboo China KY356088 KY356093 NA NA
Apiospora hyphopodii MFLUCC 15-003 T Bambusa tuldoides Thailand KR069110 NA NA NA
Apiospora hyphopodii SICAUCC 22-0034 Bamboo China ON228605 ON228661 NA ON237653
Apiospora hysterina AP12118 Phyllostachys aurea Spain MK014877 KM014844 MK017953 MK017982
Apiospora hysterina AP29717 Phyllostachys aurea Spain MK014875 MK014842 MK017952 MK017981
Apiospora hysterina ICPM 6889 T Bamboo New Zealand MK014874 MK014841 MK017951 MK017980
Apiospora iberica CBS 145137, AP10118 T Arundo donax Portugal MK014879 MK014846 MK017955 MK017984
Apiospora intestine CBS 135835 Gut of grasshopper India KR011352 MH877577 KR011351 KR011350
Apiospora intestine MFLUCC 21-0052 T Dead culms of bamboo Thailand MZ542521 MZ542525 MZ546406 MZ546410
Apiospora italic CBS 145138, AP221017 T Arundo donax Italy MK014880 MK014847 MK017956 MK017985
Apiospora italic CBS 145139 Phragmites australis Spain MK014881 MK014848 NA MK017986
Apiospora jatrophae CBS 134262, MMI00052 T Living Jatropha podagrica India JQ246355 NA NA NA
Apiospora jiangxiensis CGMCC 3.18381, LC4577 T Maesa sp. China KY494693 KY494769 KY705092 KY705163
Apiospora jiangxiensis LC4578 Camellia sinensis China KY494694 KY494770 KY705093 KY705164
Apiospora kogelbergensis CBS 113332 Cannomois virgata South Africa KF144891 KF144937 KF145025 KF144983
Apiospora kogelbergensis CBS 113333 T Dead culms of Restionaceae South Africa KF144892 KF144938 KF145026 KF144984
Apiospora koreanum KUC21332, SFC20200506-M06 T Eggs of Arctoscopus japonicus South Korea MH498524 MH498444 MH544664 MH498482
Apiospora koreanum KUC21348 Eggs of Arctoscopus japonicus South Korea MH498523 NA MN868927 MH498481
Apiospora lageniformis KUC21686 T Culms of Phyllostachys nigra Korea ON764022 ON787761 ON806626 ON806636
Apiospora lageniformis KUC21687 Culms of Phyllostachys nigra Korea ON764023 ON787764 ON806627 ON806637
Apiospora locuta-pollinis LC11683 T Brassica campestris China MF939595 NA MF939616 MF939622
Apiospora longistroma MFLUCC 11-0479 Dead culms of bamboo Thailand KU940142 KU863130 NA NA
Apiospora longistroma MFLUCC11-0481 T Dead culms of bamboo Thailand KU940141 KU863129 NA NA
Apiospora lophatheri CFCC 58975 T Diseased leaves of Lophatherum gracile China OR125566 OR133588 OR139970 OR139980
Apiospora lophatheri CFCC 58976 T Diseased leaves of Lophatherum gracile China OR125567 OR133589 OR139971 OR139981
Apiospora machili SAUCC 1175A-4 T Diseased leaves of Machilus nanmu
of Machilus nanmu 		China OR739433 OR739574 OR753450 OR757130
Apiospora machili SAUCC 1175 Diseased leaves of Machilus nanmu
of Machilus nanmu 		China OQ592560 OQ615289 OQ613333 OQ613307
Apiospora magnispora ZHKUCC 22-0001 T Dead stems of Bambusa textilis China OM728647 OM486971 OM543543 OM543544
Apiospora malaysiana CBS 102053 T Macaranga hullettii Malaysia KF144896 KF144942 KF145030 KF144988
Apiospora marianiae AP18219 T Dead stems of Phleum pratense Spain ON692406 ON692422 ON677180 ON677186
Apiospora marii CBS 497.90 T Beach sands Spain AB220252 KF144947 KF145035 KF144993
Apiospora marinum KUC21328, SFC20140423-M02 T Seaweeds South Korea MH498538 MH498458 MH544669 MH498496
Apiospora mediterranea IMI 326875 T Air Spain AB220243 AB220337 NA AB220290
Apiospora minutispora 1.70E-042 T Mountain soils South Korea LC517882 NA LC518889 LC518888
Apiospora montagnei AP19421 Arundo micrantha Spain ON692418 ON692425 ON677183 ON677189
Apiospora montagnei AP301120, CBS 148707, PC:0125164 T Arundo micrantha Spain ON692408 ON692424 ON677182 ON677188
Apiospora mori MFLUCC 20-0181 T Dead leaves of Morus australis China MW114313 MW114393 NA NA
Apiospora mori NCYUCC 19-0340 Dead leaves of Morus australis China MW114314 MW114394 NA NA
Apiospora mukdahanensis MFLUCC 22-0056 T Dead leaves of bamboo Thailand OP377735 OP377742 NA NA
Apiospora multiloculata MFLUCC 21-0023 T Dead culms of Bambusae Thailand OL873137 OL873138 NA OL874718
Apiospora mytilomorpha DAOM 214595 T Dead blades of Andropogon sp. India KY494685 NA NA NA
Apiospora neobambusae CGMCC 3.18335, LC7106 T Leaves of bamboo China KY494718 KY494794 KY806204 KY705186
Apiospora neobambusae LC7107 Leaves of bamboo China KY494719 KY494795 KY705117 KY705187
Apiospora neobambusae LC7124 Leaves of bamboo China KY494727 KY494803 KY806206 KY705195
Apiospora neochinensis CFCC 53036 T Fargesia qinlingensis China MK819291 NA MK818545 MK818547
Apiospora neochinensis CFCC 53037 Fargesia qinlingensis China MK819292 NA MK818546 MK818548
Apiospora neogarethjonesii KUMCC 18-0192, HKAS 102408 T Dead culms of Bambusae China MK070897 MK070898 NA NA
Apiospora neogongcheniae GDMCC 3.1047, YNE01248 T Living stems of Poaceae plant China PP033263 PP033106 PP034687 PP034695
Apiospora neogongcheniae YNE01260 Living stems of Poaceae plant China PP033264 PP033107 PP034688 PP034696
Apiospora neosubglobosa JHB 006 Bamboo China KY356089 KY356094 NA NA
Apiospora neosubglobosa JHB 007 T Bamboo China KY356090 KY356095 NA NA
Apiospora obovata CGMCC 3.18331, LC4940 T Lithocarpus sp. China KY494696 KY494772 KY705095 KY705166
Apiospora obovata LC8177 Lithocarpus sp. China KY494757 KY494833 KY705153 KY705225
Apiospora oenotherae CFCC 58972 Diseased leaves of Oenothera biennis China OR125568 OR133590 OR139972 OR139982
Apiospora oenotherae LS 395 Diseased leaves of Oenothera biennis China OR125569 OR133591 OR139973 OR139983
Apiospora ovate CBS 115042 T Arundinaria hindsii China KF144903 KF144950 KF145037 KF144995
Apiospora pallidesporae ZHKUCC 22-0129 T Dead wood of unknown host China OR164903 OR164950 NA NA
Apiospora pallidesporae ZHKUCC 22-0142 Dead wood of unknown host China OR164904 OR164951 NA NA
Apiospora paragongcheniae GDMCC 3.1046, YNE00992 T Living stems of Poaceae plant China PP033261 PP033104 PP034685 PP034693
Apiospora paragongcheniae YNE01259 Living stems of Poaceae plant China PP033262 PP033105 PP034686 PP034694
Apiospora paraphaeosperma MFLUCC 13-0644 T Dead culms of bamboo Thailand KX822128 KX822124 NA NA
Apiospora paraphaeosperma KUC21488 Culms of bamboo Korea ON764024 ON787763 ON806628 ON806638
Apiospora phragmitis CPC 18900 T Phragmites australis Italy KF144909 KF144956 KF145043 KF145001
Apiospora phyllostachydis MFLUCC 18-1101 T Phyllostachys heteroclada China MK351842 MH368077 MK340918 MK291949
Apiospora piptatheri CBS 145149, AP4817A T Piptatherum miliaceum Spain MK014893 MK014860 MK017969 NA
Apiospora piptatheri SAUCC BW0455 Diseased leaves of Indocalamus longiauritus China OR739430 OR739571 OR753447 OR757127
Apiospora pseudomarii GUCC 10228 T Leaves of Aristolochia debilis China MT040124 NA MT040145 MT040166
Apiospora pseudohyphopodii KUC21680 T Culms of Phyllostachys pubescens Korea ON764026 ON787765 ON806630 ON806640
Apiospora pseudohyphopodii KUC21684 Culms of Phyllostachys pubescens Korea ON764027 ON787766 ON806631 ON806641
Apiospora pseudoparenchymatica CGMCC 3.18336, LC7234 T Leaves of bamboo China KY494743 KY494819 KY705139 KY705211
Apiospora pseudoparenchymatica LC8173 Leaves of bamboo China KY494753 KY494829 KY705149 KY705221
Apiospora pseudorasikravindrae KUMCC 20-0208 T Bambusa dolichoclada China MT946344 NA MT947361 MT947367
Apiospora pseudosinensis CPC 21546 T Leaves of bamboo Netherlands KF144910 KF144957 KF145044 MN868936
Apiospora pseudosinensis SAUCC 0221 Leaves of bamboo China OP563377 OP572426 OP573266 OP573272
Apiospora pseudospegazzinii CBS 102052 T Macaranga hullettii Malaysia KF144911 KF144958 KF145045 KF145002
Apiospora pterosperma CBS 123185 Machaerina sinclairii New Zealand KF144912 KF144959 NA KF145003
Apiospora pterosperma CPC 20193, CBS 134000 T Lepidosperma gladiatum Australia KF144913 KF144960 KF145046 KF145004
Apiospora pusillispermum KUC21321 T Seaweeds South Korea MH498533 MH498453 MN868930 MH498491
Apiospora pusillispermum KUC21357 Seaweeds South Korea MH498532 NA MN868931 MH498490
Apiospora qinlingensis CFCC 52303 T Fargesia qinlingensis China MH197120 NA MH236795 MH236791
Apiospora qinlingensis CFCC 52304 Fargesia qinlingensis China MH197121 NA MH236796 MH236792
Apiospora rasikravindrae LC8179 Brassica rapa China KY494759 KY494835 KY705155 KY705227
Apiospora rasikravindrae MFLUCC 21-0051 Dead culms of bamboo Thailand MZ542523 MZ542527 MZ546408 MZ546412
Apiospora sacchari CBS 372.67 Air Not mentioned KF144918 KF144964 KF145049 KF145007
Apiospora sacchari CBS 664.74 Soils under Calluna vulgaris Netherlands KF144919 KF144965 KF145050 KF145008
Apiospora saccharicola CBS 191.73 Air Netherlands KF144920 KF144966 KF145051 KF145009
Apiospora saccharicola CBS 831.71 Not mentioned Netherlands KF144922 KF144969 KF145054 KF145012
Apiospora sargassi KUC21228 T Sargassum fulvellum South Korea KT207746 KT207696 MH544677 KT207644
Apiospora sargassi KUC21232 Seaweeds South Korea KT207750 NA MH544676 KT207648
Apiospora sasae CPC 38165, CBS 146808 T Dead culms of Sasa veitchii Netherlands MW883402 MW883797 MW890104 MW890120
Apiospora septata CGMCC 3.20134, CS19-8 T Bamboo China MW481711 MW478890 MW522943 MW522960
Apiospora septata GZCC 20-0109 Bamboo Food China MW481712 MW478891 MW522944 MW522961
Apiospora serenensis IMI 326869 T Excipients, atmosphere and home dust Spain AB220250 AB220344 NA AB220297
Apiospora setariae CFCC 54041 T Decaying culms of Setaria viridis China MT492004 NA MW118456 MT497466
Apiospora setariae MT492005 Setaria viridis China MT492005 NA MW118457 MT497467
Apiospora setostroma KUMCC 19-0217 Dead branches of bamboo China MN528012 MN528011 MN527357 NA
Apiospora sichuanensis HKAS 107008 T Dead culms of Poaceae China MW240648 MW240578 MW759536 MW775605
Apiospora sorghi URM 93000, URM 7417 T Sorghum bicolor Brazil MK371706 NA NA MK348526
Apiospora sphaerosperma CBS 114314 Leaves of Hordeum vulgare Iran KF144904 KF144951 KF145038 KF144996
Apiospora sphaerosperma CBS 114315 Leaves of Hordeum vulgare Iran KF144905 KF144952 KF145039 KF144997
Apiospora stipae CPC 38101, CBS 146804 T Dead culms of Stipa gigantea Spain MW883403 MW883798 MW890082 MW890121
Apiospora subglobosa MFLUCC 11-0397 T Dead culms of bamboo Thailand KR069112 KR069113 NA NA
Apiospora subrosea CGMCC 3.18337, LC7292 T Leaves of bamboo China KY494752 KY494828 KY705148 KY705220
Apiospora subrosea LC7291 Leaves of bamboo China KY494751 KY494827 KY705147 KY705219
Apiospora taeanense KUC21322T Seaweeds South Korea MH498515 NA MH544662 MH498473
Apiospora taeanense KUC21359 Seaweeds South Korea MH498513 NA MN868935 MH498471
Apiospora thailandica MFLUCC 15-0199 Dead culms of bamboo Thailand KU940146 KU863134 NA NA
Apiospora thailandica MFLUCC 15-0202 T Dead culms of bamboo Thailand KU940145 KU863133 NA NA
Apiospora tropica MFLUCC 21-0056 Dead culms of Bambusoideae Thailand OK491657 OK491653 NA OK560922
Apiospora wurfbainiae ZHKUCC 23-0008 T Wurfbainia villosa China OQ587998 OQ587986 OQ586064 OQ586077
Apiospora wurfbainiae ZHKUCC 23-0009 Wurfbainia villosa China OQ587999 OQ587987 OQ586065 OQ586078
Apiospora vietnamensis IMI 99670 T Citrus sinensis Vietnam KX986096 KX986111 NA KY019466
Apiospora xenocordella CBS 478.86 T Soils from roadway Zimbabwe KF144925 KF144970 KF145055 KF145013
Apiospora xenocordella CBS 595.66 Soils Austria KF144926 KF144971 NA NA
Apiospora xishuangbannaensis KUMCC 21-0695 T Rhinolophus pusillus China ON426832 OP363248 OR025969 OR025930
Apiospora xishuangbannaensis KUMCC 21-0696 Rhinolophus pusillus China ON426833 OP363249 OR025970 OR025931
Apiospora yunnana DDQ 00281 Phyllostachys nigra China KU940148 KU863136 NA NA
Apiospora yunnana MFLUCC 15-1002 T Phyllostachys nigra China KU940147 KU863135 NA NA
Apiospora yunnanensis ZHKUCC 23-0014 T Dead stems of grass China OQ588004 OQ587992 OQ586070 OQ586083
Apiospora yunnanensis ZHKUCC 23-0015 Dead stems of grass China OQ588005 OQ587993 OQ586071 OQ586084
Arthrinium austriacum GZU 345004 Carex pendula Austria MW208928 NA NA NA
Arthrinium austriacum GZU 345006 Carex pendula Austria MW208929 MW208860 NA NA
Arthrinium caricicola CBS 145127, AP23518 Carex ericetorum China MK014871 MK014838 MK017948 MK017977
Arthrinium caricicola CBS 145903, CPC33297 T Dead and attached leaves Germany MN313782 MN317266 NA MN313861
Arthrinium crenatum AG19066, CBS 146353 T Carex sp. France MW208931 MW208861 MW221917 MW221923
Arthrinium curvatum AP25418 Leaves of Carex sp. China MK014872 MK014839 MK017949 NA
Arthrinium japonicum IFO 30500 Carex despalata Japan AB220262 AB220356 NA AB220309
Arthrinium japonicum IFO 31098 Leaves of Carex despalata Japan AB220264 AB220358 NA AB220311
Arthrinium luzulae AP7619-3 Luzula sylvatica Spain MW208937 MW208863 MW221919 MW221925
Arthrinium morthieri GZU 345043 Cyperaceae carex Austria MW208938 MW208864 MW221920 MW221926
Arthrinium phaeospermum AP25619, CBS 146355 Poaceae plant Norway MW208943 MW208865 NA NA
Arthrinium puccinioides CBS 549.86 Lepidosperma gladiatum Germany AB220253 AB220347 NA AB220300
Arthrinium sporophleoides GZU 345102 Carex firma Austria MW208944 MW208866 NA MW221927
Arthrinium sporophleum AP21118, CBS 145154 Dead leaves of Juncus sp. Spain MK014898 MK014865 MK017973 MK018001
Nigrospora guilinensis CGMCC 3.18124, LC 3481 T Camellia sinensis China KX985983 KX986113 KY019292 KY019459
Nigrospora guilinensis LC 7301 Stems of Nelumbo sp. China KX986063 NA KY019404 KY019608
Nigrospora hainanensis CGMCC 3.18129, LC 7030 T Leaves of Musa paradisiaca China KX986091 KX986112 KY019415 KY019464
Nigrospora hainanensis LC 6979 Leaves of Musa paradisiaca China KX986079 NA KY019416 KY019586
Nigrospora pyriformis CGMCC 3.18122, LC 2045 T Citrus sinensis China KX985940 KX986100 KY019290 KY019457
Nigrospora pyriformis LC 2688 Lindera aggregata China KX985941 NA KY019297 KY019468
Nigrospora vesicularis CGMCC 3.18128, LC 7010 T Leaves of Musa paradisiaca China KX986088 KX986099 KY019294 KY019463
Nigrospora vesicularis LC 0322 Unknown host plant Thailand KX985939 NA KY019296 KY019467
Neoarthrinium lithocarpicola CFCC 54456 T Lithocarpus glaber China ON427580 ON427582 NA ON456914
Neoarthrinium lithocarpicola CFCC 55883 Lithocarpus glaber China ON427581 ON427583 NA ON456915
Neoarthrinium trachycarpi CFCC 53038 Trachycarpus fortune China MK301098 NA MK303396 MK303394
Neoarthrinium trachycarpi CFCC 53039 Trachycarpus fortune China MK301099 NA MK303397 MK303395
Sporocadus trimorphus CFCC 55171 Rose China OK655798 OK560389 OL814555 OM401677
Sporocadus trimorphus ROC 113 Rose China OK655799 OK560390 OL814556 OM401678

Phylogenetic analyses

The quality of obtained sequences was assessed using Chromas v.2.6.6 and the sequences were assembled using SeqMan v.7.1.0. The reference sequences were retrieved from GenBank. All sequences, including the reference sequences, were aligned in batches with MAFFT (Katoh and Standley 2013), manually correcting the resulting alignment by MEGA v.11.0.13 where necessary. A single alignment was made using ITS, LSU, tef1 region including partial exon 4 and partial exon 5 (the largest exon), tub2 region including exon 2, exon 3, and partial exon 4. Then phylogenetic analyses were conducted using partial sequences of the above four loci. The sequences were trimmed and concatenated, and subsequent phylogenetic analyses were performed in PhyloSuite platform (Zhang et al. 2020). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (Edge-unlinked) using the BIC criterion. Maximum likelihood (ML) phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under Edge-linked partition model for 5000 ultrafast (Minh et al. 2013) bootstraps. Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model, in which the initial 27% of sampled data were discarded as burn-in. The resulting phylogenetic tree was visualized in FigTree v1.4.3. (http:/tree.bio.ed.ac.uk/software/figtree/) with maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 0.90, as shown at the nodes. The phylogram was edited in Adobe Illustrator v.27.5 (Adobe Systems Inc., USA). All GenBank accession numbers of sequences used in this study are provided in Table 1.

Results

Phylogeny

The combined ITS, LSU, tef1, and tub2 dataset encompassed 215 strains, including six newly sequenced strains, with Sporocadus trimorphus CFCC 55171 and ROC 113 serving as the outgroup taxa, and representative species of Arthrinium, Nigrospora, and Neoarthrinium as the sister groups. The multi-locus sequence dataset comprised 2,081 characters, including gaps, with the following character ranges: ITS (1-352), LSU (353-1149), tef1 (1150-1775), and tub2 (1776-2081). The topologies of phylogenetic trees generated by ML and BI analyses were congruent, and the BI tree with MLBP and BIPP is presented in Fig. 1.

Figure 1. 

Phylogenetic tree of Apiospora based on the combined ITS, LSU, tef1, and tub2 sequences alignment. Maximum likelihood bootstrap proportions ≥70% (left) and Bayesian inference posterior probability ≥0.90 (right) are indicated at nodes (MLBP/BIPP). Sporocadus trimorphus (CFCC 55171 and ROC 113) are chosen as the outgroup taxa. The novel species from this study are highlighted in red.

The phylogenetic analysis revealed that the species of Apiospora, Arthrinium, Nigrospora, and Neoarthrinium formed four well-supported distinct lineages. Within the genus Apiospora, the 187 strains, encompassing six newly sequenced strains, formed twelve well-supported major clades. The six endophytic strains clustered within one of the major clades H, along with A. garethjonesii, A. neogarethjonesii, A. setostroma, A. subrosea, A. mytilomorpha, and A. neobambusae. Concurrently, the six endophytic strains segregated into three independent clades with robust supported values, indicating the presence of three novel species. These novel taxa are formally described herein and assigned the new names A. gongcheniae, A. paragongcheniae, and A. neogongcheniae.

Taxonomy

Apiospora gongcheniae C. L. Zhang, sp. nov.

Fungal Names: FN 571885
Fig. 2

Etymology

Named after Prof. Gongchen Wang in recognition of her significant contribution to the fields of mycology and plant pathology in China.

Type

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 22°04'N, 100°32'E, on the stems of Oryza meyeriana subsp. granulata, Aug 2015, J.J. Chen, YNE00465 (holotype GDMCC 3.1045, stored in a metabolically inactive state); ex-type culture YNE00465.

Description

Asexual morph : Hyphae hyaline, branched, septate, smooth, 1.1–2.6 μm diameter (mean = 1.7 μm, n = 30). Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline to pale brown, erect, verrucose, cylindrical with tiny denticles, clustered in groups, sometimes aggregated in clusters on hyphae or sporodochia, 3.5–9.4 × 1.9–5.2 μm (mean = 5.6 × 3.1 μm, n = 30). Conidia pale brown to dark brown, smooth, granular, globose to subglobose in surface view, lenticular to side view with a pale longitudinal germ slit, with obvious central basal scar, 8.0–17.0 × 6.8–16.1 μm (mean = 13.6 × 11.6 μm, n = 50). Sexual morph: Undetermined.

Figure 2. 

Apiospora gongcheniae (YNE00465, ex-type culture) a colonies after 7 d at 25 °C on PDA b colonies after 7 d at 25 °C on MEA c conidiomata on MEA d-g conidiogenous cells giving rise to conidia h–i conidia with pale germ slit. Scale bars: 500 μm (e); 10 μm (f–k).

Culture characteristics

On PDA, colonies flat, cottony, dense, margin circular, greyish, reverse light orange, covering the 90 mm plate after 7 days at 25 °C. On MEA, colonies dusty pink, dense, covering the 90 mm plate after 7 days at 25 °C. Conidiomata black, globose, abundant, attach to surface of substrate, forming on PDA and MEA after 7–10 days.

Additional specimens examined

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 22°04'N, 100°32'E, on the stems of Oryza meyeriana subsp. granulata, Aug 2015, J.J. Chen, YNE00565.

Note

Phylogenetic analyses confirmed that A. gongcheniae formed an independent clade, exhibiting a close evolutionary relationship with A. garethjonesii, A. neogarethjonesii and A. subrosea. Based on a BLASTN search of the GenBank database, it was found that A. paragongcheniae shares high similarities with the following strains: A. garethjonesii strain HKAS 96289 (93.76% in ITS, 99.81% in LSU), strain GZCC 20-0115 (93.76% in ITS, 99.24% in LSU, 94.06% in tef1), strain SICAUCC 22-0027 (93.76% in ITS, 99.81% in LSU, 94.51% in tub2), strain SICAUCC 22-0028 (93.76% in ITS, 99.81% in LSU, 93.63% in tub2); A. subrosea strain CGMCC 3.18337 (96.94% in ITS, 99.42% in LSU, 93.47% in tef1, 91.87% in tub2), strain LC7291 (90.09% in ITS, 99.41% in LSU, 93.47% in tef1, 91.87% in tub2); and A. neogarethjonesii strain HKAS 102408 (92.86% in ITS, 99.82% in LSU). The tef1 and tub2 sequence data are currently unavailable for A. neogarethjonesii to compare with A. gongcheniae.

As a synopsis of the morphological characteristics presented in Table 2, A. gongcheniae differs from A. garethjonesii and A. neogarethjonesii in having smaller conidia (8.0–17.0 × 6.8–16.1 μm, mean = 13.6 × 11.6 μm) compared to A. garethjonesii (surface view: 16–19 µm diam, side view: 17–22 µm diam) and A. neogarethjonesii (20–35 × 15–30 µm, mean = 28.5 × 25.6 µm). Additionally, A. gongcheniae exhibits shorter conidiogenous cells (3.5–9.4 × 1.9–5.2 μm, mean = 5.6 × 3.1 μm) in contrast to A. garethjonesii (6–19 × 3–5 µm, mean = 11 × 4 µm) and A. neogarethjonesii (10–48 × 4–5.5 µm, mean = 35.4 × 4.3 µm). While A. gongcheniae shares a similar size range for conidia and conidiogenous cells with A. subrosea, it is distinguished by A. gongcheniae having conidia featuring a central basal scar and cylindrical conidiogenous cells with tiny denticles. Based on molecular and morphological evidence, we propose A. gongcheniae as a new species.

Table 2.
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Synopsis of morphological characteristics of related Apiospora species. Notes: ND = Not determined.

Strains Apiospora garethjonesii (D.Q. Dai & H.B. Jiang) Pintos & P. Alvarado (2021) A. neogarethjonesii (D.Q. Dai & K.D. Hyde) Pintos & P. Alvarado (2021) A. subrosea (M. Wang & L. Cai) Pintos & P. Alvarado (2021) A. neobambusae Pintos & P. Alvarado (2021) (=Arthrinium bambusae M. Wang & L. Cai (2018)) A. gongcheniae A. paragongcheniae A. neogongcheniae
Host / Substrate Dead culms of bamboo Dead culms of bamboo Leaves of bamboo Leaves of bamboo Stems of Oryza meyeriana subsp. granulata Stems of unidentified Poaceae plant Stems of unidentified Poaceae plant
Known lifestyle Saprobe Saprobe Endophyte Endophyte Endophyte Endophyte Endophyte
Asci 125–154 × 35–42 μm (x– = 139 × 38 μm, n = 20), 8-spored 95–125 × 20–25 μm (x– = 97.6 × 21.3 μm, n = 20), 8-spored ND ND ND ND ND
Ascospores 30–42 × 11–16 μm (x– = 39 × 13 μm, n = 20), 2-seriate, 1-septate, ellipsoidal 25–30 × 9.5–11 μm (x– = 29.1 × 10.3 μm, n = 20), 2-seriate, overlapping, 1-septate, ellipsoidal, 3–10 µm wide ND ND ND ND ND
Conidiomata Black, with hair-like setae Black, ellipsoid to irregular, coriaceous Black, irregular Black, irregular Black, globose, abundant, attach to the surface of the substrate Black, globose to irregular shape, sparse, semi-immersed in the substrate ND
Conidiophores Reduced to conidiogenous cells 4.5–6 × 3.5–4.5 µm (x– = 5.4 × 4.3 µm, n = 20), cylindrical, aseptate Hyaline to pale brown, smooth, erect or ascending, simple, flexuous, subcylindrical, clustered in groups, aggregated in brown sporodochia, up to 20 µm long, 2–4.5 µm width Reduced to conidiogenous cells Reduced to conidiogenous cells Hyaline, erect, basauxic, doliiform, subspherical to barrel-shaped, aggregated in clusters on pale brown sporodochia, sometimes reduced to conidiogenous cells, 12.2–35.1 × 2.1–8.8 μm (x– = 24.5 × 4.3 μm, n = 30) ND
Conidiogenous cells Hyaline to pale brown, smooth, ampulliform, aggregated in black sporodochia, (5−) 6–19 (−20) µm × (2−) 3–5 (−7) µm (x– = 11 µm × 4 µm, n = 20) Basauxic, cylindrical, discrete, smooth-walled, 10–48 × 4–5.5 µm (x– = 35.4 × 4.3 µm, n = 20) Pale brown, smooth, doliiform to subcylindrical, 3.0–6.5 × 2.0–5.0 µm (x– = 4.7 ± 1.2 × 3.7 ± 0.9, n = 30) Hyaline to pale brown, erect, aggregated in clusters on hyphae, smooth, doliiform to ampulliform, or lageni-form, 4.0–12.0 × 3.0–7.0 µm (x– = 6.6 ± 1.8 × 4.8 ± 0.9, n = 30) Hyaline to pale brown, erect, verrucose, cylindrical with tiny denticles, clustered in groups, sometimes aggregated in clusters on hyphae or sporodochia, 3.5–9.4 × 1.9–5.2 μm (x– = 5.6 × 3.1 μm, n = 30) Hyaline, ampulliform, doliiform to clavate, verrucose, 5.0–13.1 × 2.1–6.0 μm (x– = 8.2 × 3.9 μm, n = 30) ND
Conidia (14–)16–19 (–20) µm diam, brown, smooth, granular, globose to subglobose in surface view, and (16−) 17–22 (−23) µm diam, with pale equatorial slit in side view Dark brown, globose to subglobose, smooth-walled, with a truncate basal scar, 20–35 × 15–30 µm (x– = 28.5 × 25.6 µm, n = 20) Pale brown to dark brown, smooth, globose to subglobose or ellipsoidal, 12.0–17.5 × 9.0–16.0 µm (x– = 14.9 ± 1.4 × 11.8 ± 1.8, n = 50) Olivaceous to brown, smooth to finely roughened, subglobose to ellipsoid, 11.5–15.5 × 7.0–14.0 µm (x– = 13.2 ± 0.8 × 11.4 ± 1.2, n = 50) Pale brown to dark brown, smooth, granular, globose to subglobose in surface view, lenticular to side view with a pale longitudinal germ slit, with obvious central basal scar, 8.0–17.0 × 6.8–16.1 μm (x– = 13.6 × 11.6 μm, n = 50) Pale brown to dark brown, smooth to granular, subglobose to oval, occasionally swollen into pyriform to reniform, with a pale longitudinal germ slit in side view, 8.2–18.7 × 6.4–13.4 μm (x– = 12.4 × 10.0 μm, n = 50) ND
References (Dai et al. 2016; Feng et al. 2021) (Hyde et al. 2020) (Wang et al. 2018) (Wang et al. 2018) This study This study This study

Apiospora paragongcheniae C. L. Zhang, sp. nov.

Fungal Names: FN 571886
Fig. 3

Etymology

Named after its phylogenetic close related to A. gongcheniae.

Type

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 22°04'N, 100°32'E, on the stems of unidentified Poaceae plant, Sep 2016, J.J. Chen, YNE00992 (Holotype GDMCC 3.1046, stored in a metabolically inactive state); ex-type culture YNE00992.

Description

Asexual morph : Hyphae hyaline, branched, septate, smooth, 1.1–2.2 μm diameter (mean = 1.6 μm, n = 30). Conidiophores hyaline, erect, basauxic, doliiform, subspherical to barrel-shaped, aggregated in clusters on pale brown sporodochia, sometimes reduced to conidiogenous cells, 12.2–35.1 × 2.1–8.8 μm (mean = 24.5 × 4.3 μm, n = 30). Conidiogenous cells hyaline, ampulliform, doliiform to clavate, verrucose, 5.0–13.1 × 2.1–6.0 μm (mean = 8.2 × 3.9 μm, n = 30). Conidia pale brown to dark brown, smooth to granular, subglobose to oval, occasionally swollen into pyriform to reniform, with a pale longitudinal germ slit in side view, 8.2–18.7 × 6.4–13.4 μm (mean = 12.4 × 10.0 μm, n = 50). Sexual morph: Undetermined.

Culture characteristics

On PDA, colonies flat, rounded, initially white, becoming yellowish-white, with sparse aerial mycelia, mycelium partly immersed in the medium, covering the 90 mm plate after 6 days at 25 °C. On MEA, colonies white, more abundant aerial mycelia, covering the 90 mm plate after 6 days at 25 °C. Conidiomata black, globose to irregular shape, sparse, solitary, semi-immersed in the substrate, observed on MEA after 21–30 days.

Additional specimens examined

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 21°10'N, 99°55'E, on the stems of unidentified Poaceae plant, Oct 2018, X.X. Feng, YNE001259.

Note

Phylogenetic analyses confirmed that A. paragongcheniae formed an independent clade, exhibiting a close evolutionary relationship with A. subrosea, A. neobambusae and A. neogarethjonesii. Based on a BLASTN search of the GenBank database, it was found that A. paragongcheniae shares high similarities to the following strains: A. subrosea strain CGMCC 3.18337 (98.05% in ITS, 99.23% in LSU, 95.93% in tef1, 93.63% in tub2), strain LC7291 (98.05% in ITS, 99.22% in LSU, 95.93% in tef1, 93.63% in tub2); A. neobambusae strain CGMCC 3.18335 (98.05% in ITS, 100% in LSU, 97.13% in tef1, 93.48% in tub2), strain LC7107 (98.03% in ITS, 100% in LSU, 94.44% in tef1, 93.48% in tub2), strain LC7124 (98.05% in ITS, 100% in LSU, 96.82% in tef1, 93.47% in tub2); and A. neogarethjonesii strain HKAS 102408 (95.43% in ITS, 99.63% in LSU). The tef1 and tub2 sequence data are currently unavailable for A. neogarethjonesii to compare with A. paragongcheniae.

As a synopsis of morphological characteristics presented in Table 2, A. paragongcheniae distinguishes itself from A. neobambusae, A. neogarethjonesii, and A. subrosea in the shapes and sizes of its conidia. The conidia of A. paragongcheniae range from subglobose to oval, occasionally swollen into pyriform to reniform shapes, measuring 8.2–18.7 × 6.4–13.4 μm. This contrasts with A. neobambusae (subglobose to ellipsoid, 11.5–15.5 × 7.0–14.0 µm), A. neogarethjonesii (globose to subglobose, 20–35 × 15–30 µm), and A. subrosea (globose to subglobose or ellipsoidal, 12.0–17.5 × 9.0–16.0 µm). Furthermore, A. paragongcheniae exhibits elongated conidiogenous cells (5.0–13.1 × 2.1–6.0 μm, mean = 8.2 × 3.9 μm) compared to A. neobambusae (4.0–12.0 × 3.0–7.0 µm, mean = 6.6 × 4.8 μm) and A. subrosea (3.0–6.5 × 2.0–5.0 µm, mean = 4.7 × 3.7 μm). Additionally, A. paragongcheniae exhibits shorter conidiogenous cells (5.0–13.1 × 2.1–6.0 μm) compared to A. neogarethjonesii (10–48 × 4–5.5 µm). Moreover, these species differ in the morphology of their conidiophores. A. paragongcheniae displays hyaline, basauxic, doliiform, subspherical to barrel-shaped conidiophores, whereas A. neogarethjonesii has shorter conidiophores, and A. subrosea has hyaline to pale brown, simple, subcylindrical conidiophores. Notably, the conidiophores of A. neobambusae have reduced to conidiogenous cells.

Figure 3. 

Apiospora paragongcheniae (YNE00992, ex-type culture) a colonies after 7 d at 25 °C on PDA b colonies after 6 d at 25 °C on MEA c conidioma on MEA d–i conidiogenous cells giving rise to conidia j–o conidia. Scale bars: 500 μm (c); 10 μm (d–o).

Apiospora neogongcheniae C. L. Zhang, sp. nov.

Fungal Names: FN 571887
Fig. 4

Etymology

Named after its phylogenetic close related to A. gongcheniae.

Type

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 21°10'N, 99°55'E, on the stems of unidentified Poaceae plant, Oct 2018, X.X. Feng, YNE01248 (holotype GDMCC 3.1047, stored in a metabolically inactive state); ex-type culture YNE01248.

Description

Asexual morph : Hyphae hyaline, branched, septate, smooth, 1.0–2.5 μm diameter (mean = 1.5 μm, n = 30). Conidia not observed. Chlamydospores single, terminal, globose, rare. Sexual morph: Undetermined.

Culture characteristics

On PDA, colonies flat, rounded, initially white, becoming yellowish-white, cottony, with moderate aerial mycelia, covering the 90 mm plate after 7 days at 25 °C. On MEA, colonies white, dense aerial mycelia, forming multiple circles around the center, covering the 90 mm plate after 7 days at 25 °C. Conidiomata were not observed.

Additional specimens examined

China, Yunnan Province: Xishuangbanna, Naban River Watershed National Nature Reserve, 21°10'N, 99°55'E, on the stems of unidentified Poaceae plant, Oct 2018, X.X. Feng, YNE001260.

Note

Phylogenetic analyses confirmed that A. neogongcheniae formed an independent clade, exhibiting a close evolutionary relationship with A. garethjonesii, A. neogarethjonesii and A. subrosea. Based on a BLASTN search of the GenBank database, it was found that A. neogongcheniae shares high similarities with the following strains: A. garethjonesii strain HKAS 96289 (94.88% in ITS, 100% in LSU), strain GZCC 20-0115 (94.88% in ITS, 99.41% in LSU, 96.67% in tef1), strain SICAUCC 22-0027 (94.88% in ITS, 100% in LSU, 96.69% in tub2), strain SICAUCC 22-0028 (94.88% in ITS, 100% in LSU; 96.79% in tub2); A. subrosea strain CGMCC 3.18337 (98.35% in ITS, 99.80% in LSU, 94.61% in tef1, 94.99% in tub2), strain LC7291 (91.41% in ITS, 99.80% in LSU, 94.38% in tef1, 94.99% in tub2); and A. neogarethjonesii strain HKAS 102408 (93.97% in ITS, 100% in LSU). The tef1 and tub2 sequence data are currently unavailable for A. neogarethjonesii to compare with A. neogongcheniae.

Due to the absence of sexual and asexual sporulation characters in A. neogongcheniae, a comparison of its culture characteristics with those of A. garethjonesii, A. neogarethjonesii and A. subrosea was conducted. On PDA, A. neogongcheniae exhibits a yellowish-white surface and reverse color, whereas A. garethjonesii displays a white surface with a reddish reverse, A. neogarethjonesii shows a white to black surface coloration, and A. subrosea presents a light pink surface with a peach-puff reverse. Phylogenetically, A. neogongcheniae strains YNE01248 and YNE01260 form a distinct branch with 99% MLBP and 0.95 BIPP. Therefore, we propose A. neogongcheniae as a novel species.

Figure 4. 

Apiospora neogongcheniae (YNE01248, ex-type culture) a colonies after 7 d at 25 °C on PDA b colonies after 7 d at 25 °C on MEA c colonies after 7 d at 25 °C on SNA d colonies after 7 d at 25 °C on PDA with rice leaves e colonies after 7 d at 25 °C on MEA with rice leaves f colonies after 7 d at 25 °C on SNA with rice leaves g–h chlamydospores. Scale bars: 20 μm.

Discussion

In the present study, three new species of endophytic Apiospora were examined: A. gongcheniae, A. paragongcheniae, and A. neogongcheniae, all of them isolated from the stems of Poaceae plants in Yunnan province of China. According to morphological and molecular identification, the taxonomic position of the three new species was verified.

The generic circumscription of Apiospora was primarily defined through phylogenetic analysis, given the limited morphological characteristics of Apiospora and Arthrinium. The results of a multi-locus phylogenetic analysis in this study, utilizing a combined dataset of ITS, LSU, tef1, and tub2 sequences, supported the previous classification that Apiospora and Arthrinium are distinct lineages rather than synonyms (Pintos and Alvarado 2021). Unlike the six major clades identified in a previous study (Pintos and Alvarado 2022), the current study revealed twelve major clades with robust support through the phylogenetic analysis of 114 Apiospora species, including all known species with available sequences. Apiospora minutispora (Das et al. 2020) and Apiospora marianiae AP18219 (Pintos and Alvarado 2022) were not classified within these twelve major clades due to their representation by a single record. The delineation of most Apiospora species into major clades remained consistent across both studies. Notably, A. garethjonesii, A. neogarethjonesii, A. neobambusae, A. mytilomorpha, A. subrosea, and A. setostroma clustered together in a strongly supported major clade H, aligning with findings from previous studies (Crous et al. 2021; Monkai et al. 2022; Pintos and Alvarado 2022; Liao et al. 2023; Liu et al. 2024). Within this major clade, three distinct clades representing three new species were identified (Fig. 1). A. gongcheniae is distinguished from A. garethjonesii by 34/545 nucleotides in the ITS sequences, from A. neogarethjonesii by 39/546, and from A. subrosea by 13/425. A. paragongcheniae is distinguished from A. subrosea by 10/512, from A. neobambusae by 10/512, and from A. neogarethjonesii by 24/525 nucleotides in the ITS sequences. A. neogongcheniae is distinguished from A. garethjonesii by 28/547, from A. neogarethjonesii by 34/547, and from A. subrosea by 7/425 nucleotides in the ITS sequences.

Apiospora exhibits ecological diversity, as evidenced by its wide host ranges. Most reported Apiospora species show a host preference within the Poaceae family, as noted by Monkai et al. (Monkai et al. 2022). Our new species were also found growing on plant hosts of the Poaceae family. Specifically, A. gongcheniae was discovered on the stems of Oryza meyeriana subsp. granulata, a member of the plant family Poaceae. The other two new species, A. paragongcheniae and A. neogongcheniae, were found on the stems of unidentified Poaceae plants. Their close relatives, A. garethjonesii, A. neogarethjonesii, A. neobambusae, and A. subrosea, were found on bamboo plants. Most Apiospora species exhibit saprobic and endophytic lifestyles, which are likely associated with the prevalence of Apiospora (Liao et al. 2023). Our new species occurred as endophytic fungi. Further investigation into endophytic Apiospora species will significantly enhance the diversity within the Apiospora genus.

Morphological characteristics, including asexual and sexual structures, serve as a fundamental basis for fungal systematics and phylogenetic studies, playing a vital role in the comprehensive examination of fungi. However, many endophytes do not form distinct asexual and sexual structures, as observed in A. neogongcheniae in this study, posing challenges in determining their taxonomic status based on morphological features. Recent advances in fungal taxonomy and phylogeny have provided new insights into many species with limited morphological features. Future taxonomic efforts necessitate the integration of morphological traits with molecular evidence to elucidate the natural and stable phylogenetic relationships among Apiospora species and their related Arthrinium species.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was financed by the National Natural Science Foundation of China (Grant No. 31870010).

Author contributions

Xiao-Ni Yan: Investigation, data curation, formal analysis and writing-original draft. Chu-Long Zhang: Conceptualization, methodology, validaiton, formal analysis, supervision, writing-review & editing, funding acquistition.

Author ORCIDs

Xiao-Ni Yan https://orcid.org/0009-0009-9984-3617

Chu-Long Zhang https://orcid.org/0000-0001-5180-0348

Data availability

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

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