Three new endophytic Apiospora species (Apiosporaceae, Amphisphaeriales) from China

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.

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 Apiosporamontagnei (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 Phyllostachyspraecox (Chen et al. 2014), moldy sugarcane (Liao et al. 2022), and leaf spot on Polygonatumcyrtonema (Gong et al. 2023). A.marii causes dieback of olive trees (Gerin et al. 2020), while A.kogelbergense leads to blight of Bambusaintermedi (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), Camelliasinensis (Wang et al. 2018), Wurfbainiavillosa (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.

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
Apiosporaacutiapica	KUMCC 20-0209	Bambusabambos	China	MT946342	MT946338	MT947359	MT947365
Apiosporaacutiapica	KUMCC 20-0210 T	Bambusabambos	China	MT946343	MT946339	MT947360	MT947366
Apiosporaadinandrae	SAUCC 1282B-1 T	Diseased leaves of Adinandraglischroloma	China	OR739431	OR739572	OR753448	OR757128
Apiosporaadinandrae	SAUCC 1282B-2	Diseased leaves of Adinandraglischroloma	China	OR739432	OR739573	OR753449	OR757129
Apiosporaagari	KUC21333, SFC20161014-M18 T	Agarumcribrosum	South Korea	MH498520	MH498440	MH544663	MH498478
Apiosporaaquatic	MFLU 18-1628, S-642 T	Submerged wood	China	MK828608	MK835806	NA	NA
Apiosporaarctoscopi	KUC21331, SFC20200506-M05 T	Eggs of Arctoscopusjaponicus	South Korea	MH498529	MH498449	MN868918	MH498487
Apiosporaarundinis	CBS 124788	Living leaves of Fagussylvatica	Switzerland	KF144885	KF144929	KF145017	KF144975
Apiosporaarundinis	LC4951	Dichotomanthestristaniicarpa	China	KY494698	KY494774	KY705097	KY705168
Apiosporaaseptata	KUNCC 23-14169 T	Living roots of Dicranopterispedata	China	OR590341	OR590335	OR634949	OR634943
Apiosporaaurea	CBS 244.83 T	Air	Spain	AB220251	KF144935	KF145023	KF144981
Apiosporabalearica	CBS 145129, AP24118 T	Poaceae plant	Spain	MK014869	MK014836	MK017946	MK017975
Apiosporabambusicola	MFLUCC 20-0144 T	Schizostachyumbrachycladum	Thailand	MW173030	MW173087	MW183262	NA
Apiosporabawanglingensis	SAUCC BW0444 T	Leaves of Indocalamuslongiauritus	China	OR739429	OR739570	OR753446	OR757126
Apiosporabiserialis	CGMCC 3.20135 T	Bamboo	China	MW481708	MW478885	MW522938	MW522955
Apiosporacamelliae-sinensis	CGMCC 3.18333, LC5007 T	Camelliasinensis	China	KY494704	KY494780	KY705103	KY705173
Apiosporacamelliae-sinensis	LC8181	Brassicarapa	China	KY494761	KY494837	KY705157	KY705229
Apiosporacannae	ZHKUCC 22-0139	Leaves of Canna sp.	China	OR164902	OR164949	OR166286	OR166322
Apiosporacannae	ZHKUCC 22-0127 T	Leaves of Canna sp.	China	OR164901	OR164948	OR166285	OR166321
Apiosporachiangraiense	MFLUCC 21-0053 T	Dead culms of bamboo	Thailand	MZ542520	MZ542524	NA	MZ546409
Apiosporachromolaenae	MFLUCC 17-1505 T	Chromolaenaodorata	Thailand	MT214342	MT214436	MT235802	NA
Apiosporacordylinae	GUCC 10026	Cordylinefruticosa	China	MT040105	NA	MT040126	MT040147
Apiosporacordylinae	GUCC 10027 T	Cordylinefruticosa	China	MT040106	NA	MT040127	MT040148
Apiosporacoryli	CFCC 58978 T	Dead plant culms of Corylusyunnanensis	China	OR125564	OR133586	OR139974	OR139978
Apiosporacoryli	CFCC 58979 T	Dead plant culms of Corylusyunnanensis	China	OR125565	OR133587	OR139975	OR139979
Apiosporacyclobalanopsidis	CGMCC 3.20136 T	Cyclobalanopsidisglauca	China	MW481713	MW478892	MW522945	MW522962
Apiosporacyclobalanopsidis	GZCC 20-0103	Cyclobalanopsidisglauca	China	MW481714	MW478893	MW522946	MW522963
Apiosporadendrobii	MFLUCC 14-0152 T	Roots of Dendrobiumharveyanum	Thailand	MZ463151	MZ463192	NA	NA
Apiosporadematiacea	KUNCC 23-14202 T	Living stems of Dicranopterisampla	China	OR590346	OR590339	OR634953	OR634948
Apiosporadescalsii	CBS 145130 T	Ampelodesmosmauritanicus	Spain	MK014870	MK014837	MK017947	MK017976
Apiosporadichotomanthi	CGMCC 3.18332, LC4950 T	Dichotomanthestristaniicarpa	China	KY494697	KY494773	KY705096	KY705167
Apiosporadichotomanthi	LC8175	Dichotomanthestristaniicarpa	China	KY494755	KY494831	KY705151	KY705223
Apiosporadicranopteridis	KUNCC23-14171 T	Living stems of Dicranopterispedata	China	OR590342	OR590336	OR634950	OR634944
Apiosporadicranopteridis	KUNCC23-14177	Roots of Dicranopterispedata	China	OR590343	OR590337	OR634951	OR634945
Apiosporadongyingensis	SAUCC 0302 T	Leaves of bamboo	China	OP563375	OP572424	OP573264	OP573270
Apiosporadongyingensis	SAUCC 0303	Leaves of bamboo	China	OP563374	OP572423	OP573263	OP573269
Apiosporaelliptica	ZHKUCC 22-0131 T	Dead stems of unknown plant	China	OR164905	OR164952	OR166284	OR166323
Apiosporaelliptica	ZHKUCC 22-0140	Dead stems of unknown plant	China	OR164906	OR164953	NA	OR166324
Apiosporaendophytica	ZHKUCC 23-0006 T	Living leaves of Wurfbainiavillosa	China	OQ587996	OQ587984	OQ586062	OQ586075
Apiosporaendophytica	ZHKUCC 23-0007	Living leaves of Wurfbainiavillosa	China	OQ587997	OQ587985	OQ586063	OQ586076
Apiosporaesporlensis	CBS 145136 T	Phyllostachysaurea	Spain	MK014878	MK014845	MK017954	MK017983
Apiosporaesporlensis	UNIPAMPA010	Living leaves of the Antarctic Hairgrass Deschampsiaantarctica	Antarctica	MN947641	genome	genome	genome
Apiosporaeuphorbiae	IMI 285638b	Bambusa sp.	Bangladesh	AB220241	AB220335	NA	AB220288
Apiosporafermenti	KUC21288, SFC20140423-M86	Seaweeds	South Korea	MF615230	NA	MH544668	MF615235
Apiosporafermenti	KUC21289 T	Seaweeds	South Korea	MF615226	MF615213	MH544667	MF615231
Apiosporagaoyouensis	CFCC 52301T	Phragmitesaustralis	China	MH197124	NA	MH236793	MH236789
Apiosporagaoyouensis	CFCC 52302	Phragmitesaustralis	China	MH197125	NA	MH236794	MH236790
Apiosporagarethjonesii	GZCC 20-0115	Dead culms of bamboo	China	MW481715	MW478894	MW522947	NA
Apiosporagarethjonesii	KUMCC 16-0202, JHB004, HKAS 96289 T	Dead culms of bamboo	China	KY356086	KY356091	NA	NA
Apiosporagarethjonesii	SICAUCC 22-0027	Bamboo	China	ON228603	ON228659	NA	ON237651
Apiosporagarethjonesii	SICAUCC 22-0028	Bamboo	China	ON228606	ON228662	NA	ON237654
Apiosporagelatinosa	GZAAS 20-0107	Bamboo	China	MW481707	MW478889	MW522942	MW522959
Apiosporagelatinosa	HKAS 11962 T	Bamboo	China	MW481706	MW478888	MW522941	MW522958
Apiosporaglobosa	KUNCC 23-14210 T	Living stems of Dicranopterislinearis	China	OR590347	OR590340	OR634954	NA
Apiosporagongcheniae	GDMCC 3.1045, YNE00465 T	Living stems of Oryzameyerianasubsp.granulata	China	PP033259	PP033102	PP034683	PP034691
Apiosporagongcheniae	YNE00565	Living stems of Oryzameyerianasubsp.granulata	China	PP033260	PP033103	PP034684	PP034692
Apiosporaguangdongensis	ZHKUCC 23-0004 T	Living leaves of Wurfbainiavillosa	China	OQ587994	OQ587982	OQ586060	OQ586073
Apiosporaguangdongensis	ZHKUCC 23-0005	Living leaves of Wurfbainiavillosa	China	OQ587995	OQ587983	OQ586061	OQ586074
Apiosporaguiyangensis	HKAS 102403 T	Dead culms of Poaceae	China	MW240647	MW240577	MW759535	MW775604
Apiosporaguiyangensis	KUNCC 22-12539	Poaceae plant	China	OQ029540	OQ029613	OQ186444	OQ186446
Apiosporaguizhouensis	CGMCC 3.18334, LC5322 T	Air in karst cave	China	KY494709	KY494785	KY705108	KY705178
Apiosporaguizhouensis	LC5318	Air in karst cave	China	KY494708	KY494784	KY705107	KY705177
Apiosporahainanensis	SAUCC 1681 T	Leaves of bamboo	China	OP563373	OP572422	OP573262	OP573268
Apiosporahainanensis	SAUCC 1682	Leaves of bamboo	China	OP563372	OP572421	OP573261	OP573267
Apiosporahispanica	IMI 326877 T	Beach sands	Spain	AB220242	AB220336	NA	AB220289
Apiosporahydei	CBS 114990 T	Culms of Bambusatuldoides	China	KF144890	KF144936	KF145024	KF144982
Apiosporahydei	LC7103	Leaves of bamboo	China	KY494715	KY494791	KY705114	KY705183
Apiosporahyphopodii	JHB003, HKAS 96288	Bamboo	China	KY356088	KY356093	NA	NA
Apiosporahyphopodii	MFLUCC 15-003 T	Bambusatuldoides	Thailand	KR069110	NA	NA	NA
Apiosporahyphopodii	SICAUCC 22-0034	Bamboo	China	ON228605	ON228661	NA	ON237653
Apiosporahysterina	AP12118	Phyllostachysaurea	Spain	MK014877	KM014844	MK017953	MK017982
Apiosporahysterina	AP29717	Phyllostachysaurea	Spain	MK014875	MK014842	MK017952	MK017981
Apiosporahysterina	ICPM 6889 T	Bamboo	New Zealand	MK014874	MK014841	MK017951	MK017980
Apiosporaiberica	CBS 145137, AP10118 T	Arundodonax	Portugal	MK014879	MK014846	MK017955	MK017984
Apiosporaintestine	CBS 135835	Gut of grasshopper	India	KR011352	MH877577	KR011351	KR011350
Apiosporaintestine	MFLUCC 21-0052 T	Dead culms of bamboo	Thailand	MZ542521	MZ542525	MZ546406	MZ546410
Apiosporaitalic	CBS 145138, AP221017 T	Arundodonax	Italy	MK014880	MK014847	MK017956	MK017985
Apiosporaitalic	CBS 145139	Phragmitesaustralis	Spain	MK014881	MK014848	NA	MK017986
Apiosporajatrophae	CBS 134262, MMI00052 T	Living Jatrophapodagrica	India	JQ246355	NA	NA	NA
Apiosporajiangxiensis	CGMCC 3.18381, LC4577 T	Maesa sp.	China	KY494693	KY494769	KY705092	KY705163
Apiosporajiangxiensis	LC4578	Camelliasinensis	China	KY494694	KY494770	KY705093	KY705164
Apiosporakogelbergensis	CBS 113332	Cannomoisvirgata	South Africa	KF144891	KF144937	KF145025	KF144983
Apiosporakogelbergensis	CBS 113333 T	Dead culms of Restionaceae	South Africa	KF144892	KF144938	KF145026	KF144984
Apiosporakoreanum	KUC21332, SFC20200506-M06 T	Eggs of Arctoscopusjaponicus	South Korea	MH498524	MH498444	MH544664	MH498482
Apiosporakoreanum	KUC21348	Eggs of Arctoscopusjaponicus	South Korea	MH498523	NA	MN868927	MH498481
Apiosporalageniformis	KUC21686 T	Culms of Phyllostachysnigra	Korea	ON764022	ON787761	ON806626	ON806636
Apiosporalageniformis	KUC21687	Culms of Phyllostachysnigra	Korea	ON764023	ON787764	ON806627	ON806637
Apiosporalocuta-pollinis	LC11683 T	Brassicacampestris	China	MF939595	NA	MF939616	MF939622
Apiosporalongistroma	MFLUCC 11-0479	Dead culms of bamboo	Thailand	KU940142	KU863130	NA	NA
Apiosporalongistroma	MFLUCC11-0481 T	Dead culms of bamboo	Thailand	KU940141	KU863129	NA	NA
Apiosporalophatheri	CFCC 58975 T	Diseased leaves of Lophatherumgracile	China	OR125566	OR133588	OR139970	OR139980
Apiosporalophatheri	CFCC 58976 T	Diseased leaves of Lophatherumgracile	China	OR125567	OR133589	OR139971	OR139981
Apiosporamachili	SAUCC 1175A-4 T	Diseased leaves of Machilusnanmu of Machilusnanmu	China	OR739433	OR739574	OR753450	OR757130
Apiosporamachili	SAUCC 1175	Diseased leaves of Machilusnanmu of Machilusnanmu	China	OQ592560	OQ615289	OQ613333	OQ613307
Apiosporamagnispora	ZHKUCC 22-0001 T	Dead stems of Bambusatextilis	China	OM728647	OM486971	OM543543	OM543544
Apiosporamalaysiana	CBS 102053 T	Macarangahullettii	Malaysia	KF144896	KF144942	KF145030	KF144988
Apiosporamarianiae	AP18219 T	Dead stems of Phleumpratense	Spain	ON692406	ON692422	ON677180	ON677186
Apiosporamarii	CBS 497.90 T	Beach sands	Spain	AB220252	KF144947	KF145035	KF144993
Apiosporamarinum	KUC21328, SFC20140423-M02 T	Seaweeds	South Korea	MH498538	MH498458	MH544669	MH498496
Apiosporamediterranea	IMI 326875 T	Air	Spain	AB220243	AB220337	NA	AB220290
Apiosporaminutispora	1.70E-042 T	Mountain soils	South Korea	LC517882	NA	LC518889	LC518888
Apiosporamontagnei	AP19421	Arundomicrantha	Spain	ON692418	ON692425	ON677183	ON677189
Apiosporamontagnei	AP301120, CBS 148707, PC:0125164 T	Arundomicrantha	Spain	ON692408	ON692424	ON677182	ON677188
Apiosporamori	MFLUCC 20-0181 T	Dead leaves of Morusaustralis	China	MW114313	MW114393	NA	NA
Apiosporamori	NCYUCC 19-0340	Dead leaves of Morusaustralis	China	MW114314	MW114394	NA	NA
Apiosporamukdahanensis	MFLUCC 22-0056 T	Dead leaves of bamboo	Thailand	OP377735	OP377742	NA	NA
Apiosporamultiloculata	MFLUCC 21-0023 T	Dead culms of Bambusae	Thailand	OL873137	OL873138	NA	OL874718
Apiosporamytilomorpha	DAOM 214595 T	Dead blades of Andropogon sp.	India	KY494685	NA	NA	NA
Apiosporaneobambusae	CGMCC 3.18335, LC7106 T	Leaves of bamboo	China	KY494718	KY494794	KY806204	KY705186
Apiosporaneobambusae	LC7107	Leaves of bamboo	China	KY494719	KY494795	KY705117	KY705187
Apiosporaneobambusae	LC7124	Leaves of bamboo	China	KY494727	KY494803	KY806206	KY705195
Apiosporaneochinensis	CFCC 53036 T	Fargesiaqinlingensis	China	MK819291	NA	MK818545	MK818547
Apiosporaneochinensis	CFCC 53037	Fargesiaqinlingensis	China	MK819292	NA	MK818546	MK818548
Apiosporaneogarethjonesii	KUMCC 18-0192, HKAS 102408 T	Dead culms of Bambusae	China	MK070897	MK070898	NA	NA
Apiosporaneogongcheniae	GDMCC 3.1047, YNE01248 T	Living stems of Poaceae plant	China	PP033263	PP033106	PP034687	PP034695
Apiosporaneogongcheniae	YNE01260	Living stems of Poaceae plant	China	PP033264	PP033107	PP034688	PP034696
Apiosporaneosubglobosa	JHB 006	Bamboo	China	KY356089	KY356094	NA	NA
Apiosporaneosubglobosa	JHB 007 T	Bamboo	China	KY356090	KY356095	NA	NA
Apiosporaobovata	CGMCC 3.18331, LC4940 T	Lithocarpus sp.	China	KY494696	KY494772	KY705095	KY705166
Apiosporaobovata	LC8177	Lithocarpus sp.	China	KY494757	KY494833	KY705153	KY705225
Apiosporaoenotherae	CFCC 58972	Diseased leaves of Oenotherabiennis	China	OR125568	OR133590	OR139972	OR139982
Apiosporaoenotherae	LS 395	Diseased leaves of Oenotherabiennis	China	OR125569	OR133591	OR139973	OR139983
Apiosporaovate	CBS 115042 T	Arundinariahindsii	China	KF144903	KF144950	KF145037	KF144995
Apiosporapallidesporae	ZHKUCC 22-0129 T	Dead wood of unknown host	China	OR164903	OR164950	NA	NA
Apiosporapallidesporae	ZHKUCC 22-0142	Dead wood of unknown host	China	OR164904	OR164951	NA	NA
Apiosporaparagongcheniae	GDMCC 3.1046, YNE00992 T	Living stems of Poaceae plant	China	PP033261	PP033104	PP034685	PP034693
Apiosporaparagongcheniae	YNE01259	Living stems of Poaceae plant	China	PP033262	PP033105	PP034686	PP034694
Apiosporaparaphaeosperma	MFLUCC 13-0644 T	Dead culms of bamboo	Thailand	KX822128	KX822124	NA	NA
Apiosporaparaphaeosperma	KUC21488	Culms of bamboo	Korea	ON764024	ON787763	ON806628	ON806638
Apiosporaphragmitis	CPC 18900 T	Phragmitesaustralis	Italy	KF144909	KF144956	KF145043	KF145001
Apiosporaphyllostachydis	MFLUCC 18-1101 T	Phyllostachysheteroclada	China	MK351842	MH368077	MK340918	MK291949
Apiosporapiptatheri	CBS 145149, AP4817A T	Piptatherummiliaceum	Spain	MK014893	MK014860	MK017969	NA
Apiosporapiptatheri	SAUCC BW0455	Diseased leaves of Indocalamuslongiauritus	China	OR739430	OR739571	OR753447	OR757127
Apiosporapseudomarii	GUCC 10228 T	Leaves of Aristolochiadebilis	China	MT040124	NA	MT040145	MT040166
Apiosporapseudohyphopodii	KUC21680 T	Culms of Phyllostachyspubescens	Korea	ON764026	ON787765	ON806630	ON806640
Apiosporapseudohyphopodii	KUC21684	Culms of Phyllostachyspubescens	Korea	ON764027	ON787766	ON806631	ON806641
Apiosporapseudoparenchymatica	CGMCC 3.18336, LC7234 T	Leaves of bamboo	China	KY494743	KY494819	KY705139	KY705211
Apiosporapseudoparenchymatica	LC8173	Leaves of bamboo	China	KY494753	KY494829	KY705149	KY705221
Apiosporapseudorasikravindrae	KUMCC 20-0208 T	Bambusadolichoclada	China	MT946344	NA	MT947361	MT947367
Apiosporapseudosinensis	CPC 21546 T	Leaves of bamboo	Netherlands	KF144910	KF144957	KF145044	MN868936
Apiosporapseudosinensis	SAUCC 0221	Leaves of bamboo	China	OP563377	OP572426	OP573266	OP573272
Apiosporapseudospegazzinii	CBS 102052 T	Macarangahullettii	Malaysia	KF144911	KF144958	KF145045	KF145002
Apiosporapterosperma	CBS 123185	Machaerinasinclairii	New Zealand	KF144912	KF144959	NA	KF145003
Apiosporapterosperma	CPC 20193, CBS 134000 T	Lepidospermagladiatum	Australia	KF144913	KF144960	KF145046	KF145004
Apiosporapusillispermum	KUC21321 T	Seaweeds	South Korea	MH498533	MH498453	MN868930	MH498491
Apiosporapusillispermum	KUC21357	Seaweeds	South Korea	MH498532	NA	MN868931	MH498490
Apiosporaqinlingensis	CFCC 52303 T	Fargesiaqinlingensis	China	MH197120	NA	MH236795	MH236791
Apiosporaqinlingensis	CFCC 52304	Fargesiaqinlingensis	China	MH197121	NA	MH236796	MH236792
Apiosporarasikravindrae	LC8179	Brassicarapa	China	KY494759	KY494835	KY705155	KY705227
Apiosporarasikravindrae	MFLUCC 21-0051	Dead culms of bamboo	Thailand	MZ542523	MZ542527	MZ546408	MZ546412
Apiosporasacchari	CBS 372.67	Air	Not mentioned	KF144918	KF144964	KF145049	KF145007
Apiosporasacchari	CBS 664.74	Soils under Callunavulgaris	Netherlands	KF144919	KF144965	KF145050	KF145008
Apiosporasaccharicola	CBS 191.73	Air	Netherlands	KF144920	KF144966	KF145051	KF145009
Apiosporasaccharicola	CBS 831.71	Not mentioned	Netherlands	KF144922	KF144969	KF145054	KF145012
Apiosporasargassi	KUC21228 T	Sargassumfulvellum	South Korea	KT207746	KT207696	MH544677	KT207644
Apiosporasargassi	KUC21232	Seaweeds	South Korea	KT207750	NA	MH544676	KT207648
Apiosporasasae	CPC 38165, CBS 146808 T	Dead culms of Sasaveitchii	Netherlands	MW883402	MW883797	MW890104	MW890120
Apiosporaseptata	CGMCC 3.20134, CS19-8 T	Bamboo	China	MW481711	MW478890	MW522943	MW522960
Apiosporaseptata	GZCC 20-0109	Bamboo Food	China	MW481712	MW478891	MW522944	MW522961
Apiosporaserenensis	IMI 326869 T	Excipients, atmosphere and home dust	Spain	AB220250	AB220344	NA	AB220297
Apiosporasetariae	CFCC 54041 T	Decaying culms of Setariaviridis	China	MT492004	NA	MW118456	MT497466
Apiosporasetariae	MT492005	Setariaviridis	China	MT492005	NA	MW118457	MT497467
Apiosporasetostroma	KUMCC 19-0217	Dead branches of bamboo	China	MN528012	MN528011	MN527357	NA
Apiosporasichuanensis	HKAS 107008 T	Dead culms of Poaceae	China	MW240648	MW240578	MW759536	MW775605
Apiosporasorghi	URM 93000, URM 7417 T	Sorghumbicolor	Brazil	MK371706	NA	NA	MK348526
Apiosporasphaerosperma	CBS 114314	Leaves of Hordeumvulgare	Iran	KF144904	KF144951	KF145038	KF144996
Apiosporasphaerosperma	CBS 114315	Leaves of Hordeumvulgare	Iran	KF144905	KF144952	KF145039	KF144997
Apiosporastipae	CPC 38101, CBS 146804 T	Dead culms of Stipagigantea	Spain	MW883403	MW883798	MW890082	MW890121
Apiosporasubglobosa	MFLUCC 11-0397 T	Dead culms of bamboo	Thailand	KR069112	KR069113	NA	NA
Apiosporasubrosea	CGMCC 3.18337, LC7292 T	Leaves of bamboo	China	KY494752	KY494828	KY705148	KY705220
Apiosporasubrosea	LC7291	Leaves of bamboo	China	KY494751	KY494827	KY705147	KY705219
Apiosporataeanense	KUC21322T	Seaweeds	South Korea	MH498515	NA	MH544662	MH498473
Apiosporataeanense	KUC21359	Seaweeds	South Korea	MH498513	NA	MN868935	MH498471
Apiosporathailandica	MFLUCC 15-0199	Dead culms of bamboo	Thailand	KU940146	KU863134	NA	NA
Apiosporathailandica	MFLUCC 15-0202 T	Dead culms of bamboo	Thailand	KU940145	KU863133	NA	NA
Apiosporatropica	MFLUCC 21-0056	Dead culms of Bambusoideae	Thailand	OK491657	OK491653	NA	OK560922
Apiosporawurfbainiae	ZHKUCC 23-0008 T	Wurfbainiavillosa	China	OQ587998	OQ587986	OQ586064	OQ586077
Apiosporawurfbainiae	ZHKUCC 23-0009	Wurfbainiavillosa	China	OQ587999	OQ587987	OQ586065	OQ586078
Apiosporavietnamensis	IMI 99670 T	Citrussinensis	Vietnam	KX986096	KX986111	NA	KY019466
Apiosporaxenocordella	CBS 478.86 T	Soils from roadway	Zimbabwe	KF144925	KF144970	KF145055	KF145013
Apiosporaxenocordella	CBS 595.66	Soils	Austria	KF144926	KF144971	NA	NA
Apiosporaxishuangbannaensis	KUMCC 21-0695 T	Rhinolophuspusillus	China	ON426832	OP363248	OR025969	OR025930
Apiosporaxishuangbannaensis	KUMCC 21-0696	Rhinolophuspusillus	China	ON426833	OP363249	OR025970	OR025931
Apiosporayunnana	DDQ 00281	Phyllostachysnigra	China	KU940148	KU863136	NA	NA
Apiosporayunnana	MFLUCC 15-1002 T	Phyllostachysnigra	China	KU940147	KU863135	NA	NA
Apiosporayunnanensis	ZHKUCC 23-0014 T	Dead stems of grass	China	OQ588004	OQ587992	OQ586070	OQ586083
Apiosporayunnanensis	ZHKUCC 23-0015	Dead stems of grass	China	OQ588005	OQ587993	OQ586071	OQ586084
Arthriniumaustriacum	GZU 345004	Carexpendula	Austria	MW208928	NA	NA	NA
Arthriniumaustriacum	GZU 345006	Carexpendula	Austria	MW208929	MW208860	NA	NA
Arthriniumcaricicola	CBS 145127, AP23518	Carexericetorum	China	MK014871	MK014838	MK017948	MK017977
Arthriniumcaricicola	CBS 145903, CPC33297 T	Dead and attached leaves	Germany	MN313782	MN317266	NA	MN313861
Arthriniumcrenatum	AG19066, CBS 146353 T	Carex sp.	France	MW208931	MW208861	MW221917	MW221923
Arthriniumcurvatum	AP25418	Leaves of Carex sp.	China	MK014872	MK014839	MK017949	NA
Arthriniumjaponicum	IFO 30500	Carexdespalata	Japan	AB220262	AB220356	NA	AB220309
Arthriniumjaponicum	IFO 31098	Leaves of Carexdespalata	Japan	AB220264	AB220358	NA	AB220311
Arthriniumluzulae	AP7619-3	Luzulasylvatica	Spain	MW208937	MW208863	MW221919	MW221925
Arthriniummorthieri	GZU 345043	Cyperaceaecarex	Austria	MW208938	MW208864	MW221920	MW221926
Arthriniumphaeospermum	AP25619, CBS 146355	Poaceae plant	Norway	MW208943	MW208865	NA	NA
Arthriniumpuccinioides	CBS 549.86	Lepidospermagladiatum	Germany	AB220253	AB220347	NA	AB220300
Arthriniumsporophleoides	GZU 345102	Carex firma	Austria	MW208944	MW208866	NA	MW221927
Arthriniumsporophleum	AP21118, CBS 145154	Dead leaves of Juncus sp.	Spain	MK014898	MK014865	MK017973	MK018001
Nigrosporaguilinensis	CGMCC 3.18124, LC 3481 T	Camelliasinensis	China	KX985983	KX986113	KY019292	KY019459
Nigrosporaguilinensis	LC 7301	Stems of Nelumbo sp.	China	KX986063	NA	KY019404	KY019608
Nigrosporahainanensis	CGMCC 3.18129, LC 7030 T	Leaves of Musaparadisiaca	China	KX986091	KX986112	KY019415	KY019464
Nigrosporahainanensis	LC 6979	Leaves of Musaparadisiaca	China	KX986079	NA	KY019416	KY019586
Nigrosporapyriformis	CGMCC 3.18122, LC 2045 T	Citrussinensis	China	KX985940	KX986100	KY019290	KY019457
Nigrosporapyriformis	LC 2688	Linderaaggregata	China	KX985941	NA	KY019297	KY019468
Nigrosporavesicularis	CGMCC 3.18128, LC 7010 T	Leaves of Musaparadisiaca	China	KX986088	KX986099	KY019294	KY019463
Nigrosporavesicularis	LC 0322	Unknown host plant	Thailand	KX985939	NA	KY019296	KY019467
Neoarthriniumlithocarpicola	CFCC 54456 T	Lithocarpusglaber	China	ON427580	ON427582	NA	ON456914
Neoarthriniumlithocarpicola	CFCC 55883	Lithocarpusglaber	China	ON427581	ON427583	NA	ON456915
Neoarthriniumtrachycarpi	CFCC 53038	Trachycarpusfortune	China	MK301098	NA	MK303396	MK303394
Neoarthriniumtrachycarpi	CFCC 53039	Trachycarpusfortune	China	MK301099	NA	MK303397	MK303395
Sporocadustrimorphus	CFCC 55171	Rose	China	OK655798	OK560389	OL814555	OM401677
Sporocadustrimorphus	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 Sporocadustrimorphus 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). Sporocadustrimorphus (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.

3CB66A0C-DA1A-52F5-B42B-C85EE95BDB90

Fungal Names: FN 571885

Fig. 2

Figure 2.

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

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 Oryzameyerianasubsp.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.

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 Oryzameyerianasubsp.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.

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

Strains	Apiosporagarethjonesii (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) (=Arthriniumbambusae 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 Oryzameyerianasubsp.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.

4B35590E-B30E-5BC6-8608-CEE8CD1E6423

Fungal Names: FN 571886

Fig. 3

Figure 3.

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

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.

. Apiospora neogongcheniae

C. L. Zhang sp. nov.

658C79C8-D19C-517C-BED6-D851C9B7EDF9

Fungal Names: FN 571887

Fig. 4

Figure 4.

Apiosporaneogongcheniae (YNE01248, ex-type culture) a colonies after 7 d at 25 °C on PDAb colonies after 7 d at 25 °C on MEAc 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.

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.

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. Apiosporaminutispora (Das et al. 2020) and Apiosporamarianiae 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 Oryzameyerianasubsp.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.

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

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.