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Research Article
Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China
expand article infoRongyu Liu‡§, Duhua Li‡, Zhaoxue Zhang‡, Shubin Liu‡, Xinye Liu§, Yixin Wang§, Heng Zhao|, Xiaoyong Liu§, Xiuguo Zhang§‡, Jiwen Xia‡, Yujiao Wang§
‡ Shandong Agricultural University, Taian, China
§ Shandong Normal University, Jinan, China
| Beijing Forestry University, Beijing, China

Corresponding author: Jiwen Xia ( xiajiwen1@126.com )

Corresponding author: Yujiao Wang ( 18354285903@163.com )

Academic editor: Xinlei Fan
© 2023 Rongyu Liu, Duhua Li, Zhaoxue Zhang, Shubin Liu, Xinye Liu, Yixin Wang, Heng Zhao, Xiaoyong Liu, Xiuguo Zhang, Jiwen Xia, Yujiao Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Liu R, Li D, Zhang Z, Liu S, Liu X, Wang Y, Zhao H, Liu X, Zhang X, Xia J, Wang Y (2023) Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China. MycoKeys 95: 27-45. https://doi.org/10.3897/mycokeys.95.96400
Open Access

Abstract

The genus Apiospora includes endophytes, pathogens and saprobes, with a wide host range and geographic distribution. In this paper, six Apiospora strains isolated from diseased and healthy tissues of bamboo leaves from Hainan and Shandong provinces in China were classified using a multi-locus phylogeny based on a combined dataset of ITS, LSU, tef1 and tub2, in conjunction with morphological characters, host association and ecological distribution. Two new species, Apiospora dongyingensis and A. hainanensis, and a new record of A. pseudosinensis in China, are described based on their distinct phylogenetic relationships and morphological analyses. Illustrations and descriptions of the three taxa are provided, along with comparisons with closely related taxa in the genus.

Keywords

Apiospora dongyingensis, Apiospora hainanensis, Ascomycota, bamboo, taxonomy

Introduction

Apiospora Sacc., the type genus of Apiosporaceae K.D. Hyde, J. Fröhl., Joanne E. Taylor & M.E. Barr, was introduced by Saccardo with A. montagnei Sacc. as the type species (Saccardo 1875). The sexual morphs of Apiospora are characterized by multi-locular perithecial stromata with hyaline ascospores surrounded by a thick gelatinous sheath (Dai et al. 2016, 2017; Pintos and Alvarado 2021). The asexual morphs of Apiospora are characterized by their basauxic conidiogenesis, and globose to subglobose conidia, which are usually lenticular or obovoid in the side view, and pale brown to brown in color (Kunze 1817; Hyde et al. 1998; Dai et al. 2016). Most species of Apiospora are quite similar to each other in morphology, thus it is difficult to distinguish them without molecular phylogenetic data.

Until the studies of Pintos and Alvarado (2021) and Jiang et al. (2022a), the closely related genera Apiospora, Arthrinium Kunze and Neoarthrinium Ning Jiang were considered a single taxon because of their similar morphological characteristics, especially the basauxic conidiogenesis. However, the conidia of Apiospora and Neoarthrinium are more or less rounded in the face view and lenticular in the side view, whereas the conidia of Arthrinium are variously shaped (angular, curved, fusiform, globose, polygonal, navicular). In addition, the conidiophores of several Arthrinium and Neoarthrinium species have thick blackish septa, which are rarely observed in Apiospora (Pintos and Alvarado 2021; Tian et al. 2021; Jiang et al. 2022a). Apiospora species have a worldwide distribution and can be found on various hosts, while Arthrinium species are rarely found in tropical and subtropical habitats and commonly occur on Cyperaceae Juss. and Juncaceae Juss. (Ramos et al. 2010; Dai et al. 2017; Wang et al. 2018; Hyde et al. 2020; Pintos and Alvarado 2021; Tian et al. 2021). Four Neoarthrinium species have been discovered on four hosts from three distantly related host plant families in China, Colombia and Great Britain (Jiang et al. 2022a). Most Apiospora species are associated with plants as endophytes, pathogens or saprobes (Agut and Calvo 2004; Dai et al. 2016, 2017; Tian et al. 2021). Some species are economically important plant pathogens, for example, A. arundinis causes bamboo brown culm streak, chestnut leaf spot and barley kernel blight (Martínez-Cano et al. 1992; Chen et al. 2014; Jiang et al. 2021), while A. sacchari causes damping-off of durum wheat (Mavragani et al. 2007). Some species have also been isolated from lichens, air, soil, seaweeds and animal tissues, and a few species are human pathogens which can cause cutaneous infections (Tian et al. 2021).

The aim of this study was to explore the diversity of Apiospora species in symptomatic and asymptomatic bamboo leaves collected in Hainan and Shandong provinces (China). We describe two newly discovered species, Apiospora dongyingensis and A. hainanensis, and a new record of A. pseudosinensis in China based on phylogenetic data and morphology.

Materials and methods

Isolation and morphological studies

The samples were collected at the Diaoluoshan National Nature Reserve, Hainan Province, and the Dongying Botanical Garden, Shandong Province (China). The strains of Apiospora were isolated from single spores and fungal tissue obtained from diseased and healthy bamboo leaves following the methods described by Chomnunti et al. (2014). Sampled spores were suspended in sterile distilled water, spread onto potato dextrose agar (PDA) plates, and incubated for one day at 25 °C. After germination, the spores were transferred to a new PDA plate to obtain a pure culture. Additionally, about 25 mm2 tissue fragments were taken from the margin of leaf lesions and their surface sterilized by consecutive immersions in a 75% ethanol solution for 60 s, 5% sodium hypochlorite solution for 30 s, and then rinsed in sterile distilled water for 60 s (Mu et al. 2021). The surface sterilized plant tissue was dried with sterilized paper and moved on the PDA plates. All the PDA plates were incubated at 25 °C for 3–4 days in darkness, and then hyphae were picked out of the periphery of the colonies and grown on new PDA plates (Jiang et al. 2022b).

After 7 days of incubation, the morphological characters of the colonies were recorded on PDA with a digital camera (Canon G7X). Morphological descriptions were based on cultures sporulating on water agar (WA). The size of the conidiogenous cells and conidia were shown as minimum-maximum. Color notations were done using the color charts of Rayner (1970). The micro-morphological characters of the colonies were studied using a stereomicroscope (Olympus SZX10) and a microscope (Olympus BX53), both fitted with high-definition color digital cameras. Grown cultures of Apiospora were stored in 10% sterilized glycerin and sterile water at 4 °C for further studies in the future. All specimens were deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). DNA sequences of four different loci were obtained, including the nrDNA internal transcribed spacer regions 1 and 2 with the intervening 5.8S subunit (ITS), a partial sequence of the large subunit nrDNA subunit (LSU), a partial sequence of the translation elongation factor 1-alpha gene (tef1), and a partial sequence of the beta-tubulin gene (tub2). They were all amplified with the primer pairs and polymerase chain reaction (PCR) program listed in Table 1.

Table 1.
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Gene regions and respective primer pairs used in the study.

Locus PCR primers PCR: thermal cycles: (Annealing temperature in bold) Reference
ITS ITS5/ITS4 (94 °C: 30 s, 55 °C: 30 s, 72 °C: 45 s) × 29 cycles White et al. 1990
LSU LR0R/LR5 (94 °C: 30 s, 48 °C: 50 s, 72 °C: 1 min 30 s) × 35 cycles Vilgalys and Hester 1990; Cubeta et al. 1991
tef1 EF1-728F/EF2 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles O’Donnell et al. 1998; Carbone and Kohn 1999
tub2 Bt-2a/Bt-2b (95 °C: 30 s, 56 °C: 30 s, 72 °C: 1 min) × 35 cycles Glass and Donaldson 1995

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions contained 12.5 μL 2× Taq Plus Master Mix II (Vazyme, Nanjing, China), 1 μL of each forward and reverse primers (10 μM) (Tsingke, Qingdao, China), 1 μL of template genomic DNA, and distilled deionized water to a total volume of 25 μL. The PCR products were visualized on 1% agarose electrophoresis gels. Bi-directional sequencing was conducted by the Tsingke Company Limited (Qingdao, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 2).

Table 2.
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Isolates and GenBank accession numbers used in the phylogenetic analyses.

Species Isolate/Strain Host/Substrate Origin GenBank accession numbers
ITS LSU tef1 tub2
Apiospora acutiapica KUMCC 20-0210 (Type) Bambusa bambos China MT946343 MT946339 MT947360 MT947366
A. agari KUC21333 (Type) Agarum cribrosum Korea MH498520 MH498440 MH544663 MH498478
A. aquatica S-642 (Type) Submerged wood China MK828608 MK835806 NA NA
A. arctoscopi KUC21331 (Type) Egg of Arctoscopus japonicus Korea MH498529 MH498449 MN868918 MH498487
A. arundinis CBS 124788 Living leaves of Fagus sylvatica Switzerland KF144885 KF144929 KF145017 KF144975
A. aurea CBS 244.83 (Type) Air Spain AB220251 KF144935 KF145023 KF144981
A. balearica CBS 145129 (Type) Undetermined Poaceae Spain MK014869 MK014836 MK017946 MK017975
A. biserialis CGMCC 3.20135 (Type) Bamboo China MW481708 MW478885 MW522938 MW522955
A. camelliae-sinensis LC5007 (Type) Camellia sinensis China KY494704 KY494780 KY705103 KY705173
A. chiangraiense MFLUCC21-0053 (Type) Dead culms of bamboo Thailand MZ542520 MZ542524 NA MZ546409
A. chromolaenae MFLUCC 17-1505 (Type) Chromolaena odorata Thailand MT214342 MT214436 NA NA
A. cordylines GUCC 10027 (Type) Leaves of Cordyline fruticosa China MT040106 NA MT040127 MT040148
A. cyclobalanopsidis CGMCC 3.20136 (Type) Cyclobalanopsidis glauca China MW481713 MW478892 MW522945 MW522962
A. descalsii CBS 145130 (Type) Ampelodesmos mauritanicus Spain MK014870 MK014837 MK017947 MK017976
A. dichotomanthi LC4950 (Type) Dichotomanthus tristaniaecarpa China KY494697 KY494773 KY705096 KY705167
A. dongyingensis SAUCC 0302 (Type) Leaf of bamboo China OP563375 OP572424 OP573264 OP573270
SAUCC 0303 Leaf of bamboo China OP563374 OP572423 OP573263 OP573269
A. esporlensis CBS 145136 (Type) Phyllostachys aurea Spain MK014878 MK014845 MK017954 MK017983
A. euphorbiae IMI 285638b Bambusa sp. Bangladesh AB220241 AB220335 NA AB220288
A. fermenti KUC21289 (Type) Seaweed Korea MF615226 MF615213 MH544667 MF615231
A. gaoyouensis CFCC 52301 (Type) Phragmites australis China MH197124 NA MH236793 MH236789
A. garethjonesii JHB004 (Type) Culms of dead bamboo China KY356086 KY356091 NA NA
A. gelatinosa HKAS 111962 (Type) Culms of dead bamboo China MW481706 MW478888 MW522941 MW522958
A. guiyangensis HKAS 102403 (Type) Dead culms of Poaceae China MW240647 MW240577 MW759535 MW775604
A. guizhouensis LC5322 (Type) Air in karst cave China KY494709 KY494785 KY705108 KY705178
A. hainanensis SAUCC 1681 (Type) Leaf of bamboo China OP563373 OP572422 OP573262 OP573268
SAUCC 1682 Leaf of bamboo China OP563372 OP572421 OP573261 OP573267
A. hispanica IMI 326877 (Type) Maritime sand Spain AB220242 AB220336 NA AB220289
A. hydei CBS 114990 (Type) Culms of Bambusa tuldoides China KF144890 KF144936 KF145024 KF144982
A. hyphopodii MFLUCC 15-0003 (Type) Dead culms of bamboo Thailand KR069110 NA NA NA
A. hysterina ICPM 6889 (Type) Bamboo New Zealand MK014874 MK014841 MK017951 MK017980
A. iberica AP10118 (Type) Arundo donax Portugal MK014879 MK014846 MK017955 MK017984
A. intestini CBS 135835 (Type) Gut of grasshopper India KR011352 KR149063 KR011351 KR011350
A. italica CBS 145138 (Type) Arundo donax Italy MK014880 MK014847 MK017956 MK017985
A. jatrophae CBS 134262 (Type) Jatropha podagrica India JQ246355 NA NA NA
A. jiangxiensis LC4577 (Type) Maesa sp. China KY494693 KY494769 KY705092 KY705163
A. kogelbergensis CBS 113333 (Type) Dead culms of Restionaceae South Africa KF144892 KF144938 KF145026 KF144984
A. koreana KUC21332 (Type) Egg of Arctoscopus japonicus Korea MH498524 MH498444 MH544664 MH498482
A. locuta-pollinis LC11683 (Type) Brassica campestris China MF939595 NA MF939616 MF939622
A. longistroma MFLUCC 11-0481 (Type) Culms of decaying bamboo Thailand KU940141 KU863129 NA NA
A. malaysiana CBS 102053 (Type) Macaranga hullettii stem colonised by ants Malaysia KF144896 KF144942 KF145030 KF144988
A. marianiae AP18219 (Type) Dead stems of Phleum pratense Spain ON692406 ON692422 ON677180 ON677186
A. marii CBS 497.90 (Type) Atmosphere, pharmaceutical excipients, home dust and beach sands Spain MH873913 KF144947 KF145035 KF144993
A. marina KUC21328 (Type) Seaweed Korea MH498538 MH498458 MH544669 MH498496
A. mediterranea IMI 326875 (Type) Air Spain AB220243 AB220337 NA AB220290
A. minutispora 17E-042 (Type) Soil South Korea LC517882 NA LC518889 LC518888
A. montagnei AP301120 (Epitype) Arundo micrantha Spain ON692408 ON692424 ON677182 ON677188
AP19421 Arundo micrantha Spain ON692418 ON692425 ON677183 ON677189
CPC 18900 Culms of Phragmites australis Italy KF144909 KF144956 KF145043 KF145001
A. mori MFLU 18-2514 (Type) Dead leaves of Morus australis China MW114313 MW114393 NA NA
A. multiloculata MFLUCC 21-0023 (Type) Dead culms of Bambusae Thailand OL873137 OL873138 NA OL874718
A. mytilomorpha DAOM 214595 (Type) Dead blades of Andropogon sp. India KY494685 NA NA NA
A. neobambusae LC7106 (Type) Leaf of bamboo China KY494718 KY494794 KY806204 KY705186
A. neochinense CFCC 53036 (Type) Fargesia qinlingensis China MK819291 NA MK818545 MK818547
A. neogarethjonesii HKAS 102408 (Type) Dead culms of Bambusae China MK070897 MK070898 NA NA
A. neosubglobosa JHB007 (Type) Bamboo China KY356090 KY356095 NA NA
A. obovata LC4940 (Type) Lithocarpus sp. China KY494696 KY494772 KY705095 KY705166
A. ovata CBS 115042 (Type) Arundinaria hindsii China KF144903 KF144950 KF145037 KF144995
A. paraphaeosperma MFLUCC13-0644 (Type) Dead clumps of Bambusa sp. Thailand KX822128 KX822124 NA NA
A. phyllostachydis MFLUCC 18-1101 (Type) Phyllostachys heteroclada China MK351842 MH368077 MK340918 MK291949
A. piptatheri CBS 145149 (Type) Piptatherum miliaceum Spain MK014893 MK014860 MK017969 NA
A. pseudomarii GUCC 10228 (Type) Leaves of Aristolochia debilis China MT040124 NA MT040145 MT040166
A. pseudoparenchymatica LC7234 (Type) Leaf of bamboo China KY494743 KY494819 KY705139 KY705211
A. pseudorasikravindrae KUMCC 20-0208 (Type) Bambusa dolichoclada China MT946344 NA MT947361 MT947367
A. pseudosinensis CPC 21546 (Type) Leaf of bamboo Netherlands KF144910 KF144957 KF145044 MN868936
A. pseudosinensis SAUCC 0221 Leaf of bamboo China OP563377 OP572426 OP573266 OP573272
SAUCC 0222 Leaf of bamboo China OP563376 OP572425 OP573265 OP573271
A. pseudospegazzinii CBS 102052 (Type) Macaranga hullettii stem colonized by ants Malaysia KF144911 KF144958 KF145045 KF145002
A. pterosperma CPC 20193 (Type) Lepidosperma gladiatum Australia KF144913 KF144960 KF145046 KF145004
A. pusillisperma KUC21321 (Type) Seaweed Korea MH498533 MH498453 MN868930 MH498491
A. qinlingensis CFCC 52303 (Type) Fargesia qinlingensis China MH197120 NA MH236795 MH236791
A. rasikravindrae LC5449 Soil in karst cave China KY494713 KY494789 KY705112 KY705182
A. sacchari CBS 212.30 Phragmites australis UK KF144916 KF144962 KF145047 KF145005
A. saccharicola CBS191.73 Air Netherlands KF144920 KF144966 KF145051 KF145009
A. sargassi KUC21228 (Type) Sargassum fulvellum Korea KT207746 KT207696 MH544677 KT207644
A. sasae CBS 146808 (Type) Dead culms of Sasa veitchii Netherlands MW883402 MW883797 MW890104 MW890120
A. septata CGMCC 3.20134 (Type) Bamboo China MW481711 MW478890 MW522943 MW522960
A. serenensis IMI 326869 (Type) Food, pharmaceutical excipients, atmosphere and home dust Spain AB220250 AB220344 NA AB220297
A. setariae CFCC 54041 (Type) Decaying culms of Setaria viridis China MT492004 NA NA NA
A. sichuanensis HKAS 107008 (Type) Dead culms of Poaceae China MW240648 MW240578 MW759536 MW775605
A. sorghi URM 93000 (Type) Sorghum bicolor Brazil MK371706 NA NA MK348526
A. sphaerosperma CBS114314 Leaf of Hordeum vulgare Iran KF144904 KF144951 KF145038 KF144996
A. stipae CBS 146804 (Type) Dead culm of Stipa gigantea Spain MW883403 MW883798 MW890082 MW890121
A. subglobosa MFLUCC 11-0397 (Type) Dead bamboo culms Thailand KR069112 KR069113 NA NA
A. subrosea LC7292 (Type) Leaf of bamboo China KY494752 KY494828 KY705148 KY705220
A. thailandica LC5630 Rotten wood China KY494714 KF144970 KY705113 KY806200
A. vietnamensis IMI 99670 (Type) Citrus sinensis Vietnam KX986096 KX986111 NA KY019466
A. xenocordella CBS 478.86 (Type) Soil from roadway Zimbabwe KF144925 KF144970 KF145055 KF145013
A. yunnana MFLUCC 15-0002 (Type) Decaying bamboo culms China KU940147 KU863135 NA NA
Arthrinium caricicola CBS 145127 Carex ericetorum China MK014871 MK014838 MK017948 MK017977

Notes: Strains in this study are marked in bold. NA = not available.

Phylogenetic analyses

Newly generated ITS, LSU, tef1 and tub2 sequences from the six strains studied were aligned with all reference sequences of Apiospora and related species available in GenBank using the MAFFT v.7.11 online software (http://mafft.cbrc.jp/alignment/server/, Katoh et al. 2019) with the default settings, manually correcting the resulting alignment where necessary. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were conducted individually on each locus (ITS, LSU, tef1 and tub2) and on a combined dataset including all of them. The best-fitting evolutionary model of each partition was determined using MrModeltest v. 2.3 (Nylander 2004). ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) using RaxML-HPC2 on XSEDE (8.2.12) (Miller et al. 2012; Stamatakis 2014) and MrBayes on XSEDE (3.2.7a), respectively (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012). For ML analyses the default parameters were used, while BI was carried out using a Markov chain Monte Carlo (MCMC) algorithm. BI analyses included four MCMC chains and were run for 5,000,000 generations until the average standard deviation of split frequencies was below 0.01 with trees saved every 1000 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting 50% majority-rule tree was plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0.

Results

Phylogenetic analyses

Among the six strains of Apiospora isolated from the samples studied, two new species were discovered, and another one found for the first time in China after the combined analysis of ITS, LSU, tef1 and tub2 DNA sequences from 89 isolates of Apiospora plus Arthrinium caricicola Kunze & J.C. Schmidt (CBS 145127) as the outgroup taxon.

A total of 2241 characters including gaps were compared in the phylogenetic analysis, viz. ITS: 1–706, LSU: 707–1513, tef1: 1514–1932, tub2: 1933–2241. Of these characters, 1436 were constant, 271 were variable and parsimony-uninformative, and 534 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G was selected for all partitions.

The BI analysis reached the established convergence after 3935000 generations, resulting in 39351 sampled trees, of which 29514 trees were used to calculate the posterior probabilities. The ML tree topology agreed with that obtained from the BI analysis, and therefore, only one tree (the ML) is presented (Fig. 1). The four strains (SAUCC 0302, SAUCC 0303, SAUCC 1681 and SAUCC 1682) studied in the present work represent two independent clades, interpreted as newly discovered independent species. These are described below and accommodated under the new names Apiospora dongyingensis and A. hainanensis. Another two strains (SAUCC 0221 and SAUCC 0222) clustered with A. pseudosinensis (CPC 21546) with full support (MLBS: 100% and BYPP: 1), and are therefore considered no different from this species.

Figure 1. 

Phylogram of Apiospora based on combined ITS, LSU, tef1 and tub2 genes. ML bootstrap support values (MLBS ≥ 50%) and Bayesian posterior probability (BYPP ≥ 0.90) are shown as first and second position above nodes, respectively. Strains from this study are shown in red, ex-type or ex-epitype cultures are indicated in bold face. Some branches were shortened according to the indicated mulipliers.

Taxonomy

Apiospora dongyingensis R.Y. Liu, J.W. Xia & X.G. Zhang, sp. nov.

MycoBank No: 846065
Fig. 2

Etymology

Named after Dongying City (China) where the type was collected.

Type

China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, holotype HSAUP 0302, ex-type living culture SAUCC 0302.

Description

Asexual morph : On WA, hyphae 1.3–3.6 μm diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggregated in clusters on hyphae, hyaline to pale brown, smooth, branched, 8.2–13.9 × 4.2–8.2 μm, mean ± SD: 9.6 ± 1.6 × 6.7 ± 1.1 μm (n = 40). Conidia globose, subglobose to lenticular, with a longitudinal germ slit, occasionally elongated to ellipsoidal, brown to dark brown, smooth to finely roughened, 8.0–16.5 × 5.5–9.0 μm, mean ± SD: 9.4 ± 1.9 × 7.3 ± 1.0 μm, L/W = 1.3–1.9 (n = 40). Sexual morph: Undetermined.

Figure 2. 

Apiospora dongyingensis (SAUCC 0302, ex-holotype culture) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 μm (e–h).

Culture characteristics

Colonies on PDA flat with entire margin, aerial mycelium white to gray, floccose cottony; surface and reverse gray in the center and grayish margin. PDA attaining 78.5–86.5 mm in diameter after 7 days at 25 °C, growth rate 11.0–12.5 mm/day.

Additional specimen examined

China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, paratype HSAUP 0303, ex-paratype living culture SAUCC 0303.

Notes

Apiospora dongyingensis is closely related but phylogenetically distinct from A. camelliae-sinensis (M. Wang, F. Liu & L. Cai) Pintos & P. Alvarado and A. cyclobalanopsidis (Y. Feng & Jian K. Liu) X.G. Tian & Tibpromma (Fig. 1). A. dongyingensis differs from A. camelliae-sinensis by 18 nucleotides (13/518 in ITS, 2/804 in LSU, 2/374 in tef1 and 1/265 in tub2) and A. cyclobalanopsidis by 58 nucleotides (17/518 in ITS, 4/799 in LSU, 26/377 in tef1 and 11/266 in tub2). Morphologically, it differs from A. camelliae-sinensis and A. cyclobalanopsidis in its conidia (globose, subglobose to lenticular, 8.0–16.5 × 5.5–9.0 μm in A. dongyingensis vs. globose to subglobose, 9.0–13.5 × 7.0–12.0 μm in A. camelliae-sinensis and surface view globose to ellipsoid, 8–12 μm long and side view lenticular, 10–14 μm long in A. cyclobalanopsidis; Wang et al. 2018; Feng et al. 2021; Pintos and Alvarado 2021; Tian et al. 2021).

Apiospora hainanensis R.Y. Liu, J.W. Xia & X.G. Zhang, sp. nov.

MycoBank No: 846066
Fig. 3

Etymology

Named after Hainan Province (China) where the type was collected.

Type

China, Hainan Province: Diaoluoshan National Nature Reserve, on diseased leaves of bamboo, 23 June 2021, R.Y. Liu, holotype HSAUP 1681, ex-type living culture SAUCC 1681.

Description

Asexual morph : On WA, hyphae 1.2–3.4 μm diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggregated in clusters on hyphae, hyaline to pale brown, smooth, branched, 6.4–8.8 × 5.2–7.1 μm, mean ± SD: 7.9 ± 1.1 × 6.1 ± 0.9 μm (n = 40). Conidia globose, subglobose to lenticular, with a longitudinal germ slit, occasionally elongated to ellipsoidal, brown to dark brown, smooth to finely roughened, 5.5–8.5 × 5.0–7.5 μm, mean ± SD: 6.8 ± 0.9 × 6.7 ± 0.7 μm, L/W = 1.0–1.1 (n = 40). Sexual morph: Undetermined.

Culture characteristics

Colonies on PDA flat with entire margin, aerial mycelium white to grey, floccose cottony; reverse white to pale honey colored. PDA attaining 77.5–85.5 mm in diameter after 7 days at 25 °C, growth rate 10.5–12.5 mm/day.

Figure 3. 

Apiospora hainanensis (SAUCC 1681, ex-holotype culture) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 μm (e–h).

Additional specimen examined

China, Hainan Province: Diaoluoshan National Nature Reserve, on diseased leaves of bamboo, 23 June 2021, R.Y. Liu, paratype HSAUP 1682, ex-paratype living culture SAUCC 1682.

Notes

The two strains (SAUCC 1681 and SAUCC 1682) of A. hainanensis clustered together with significant support in an isolated branch basal to A. sacchari and related species of the phaeospermum clade (Pintos and Alvarado 2022; Fig. 1). Other species in a more or less similar phylogenetic position include A. septata (Y. Feng & Jian K. Liu) X.G. Tian & Tibpromma, A. piptatheri (Pintos & P. Alvarado) Pintos & P. Alvarado, A. longistroma (D.Q. Dai & K.D. Hyde) Pintos & P. Alvarado, A. pseudospegazzinii (Crous) Pintos & Alvarado and A. fermenti (S.L. Kwon, S. Jang & J.J. Kim) S.L. Kwon & J.J. Kim. Morphologically, it differs from A. septata, A. piptatheri, A. longistroma, A. pseudospegazzinii and A. fermenti in its conidia (globose, subglobose to lenticular, 5.5–8.5 × 5.0–7.5 μm in A. hainanensis vs. surface view globose to ellipsoid, 8–13 μm long and side view lenticular, 8–14 μm long in A. septata, globose to ellipsoidal, 6–8 × 3–5 μm in A. piptatheri, asexual morph undetermined in A. longistroma, surface view globose, 7–9 μm diam. and side view lenticular, 5–6 μm diam. in A. pseudospegazzinii, surface view globose to elongate ellipsoid, 7.5–9 × 7–9 μm and side view lenticular, 6–7 μm diam. in A. fermenti; Crous and Groenewald 2013; Dai et al. 2016; Pintos et al. 2019; Feng et al. 2021; Kwon et al. 2021, 2022; Pintos and Alvarado 2021; Tian et al. 2021).

Apiospora pseudosinensis (Crous) Pintos & P. Alvarado, Fungal Systematics and Evolution 7: 207. (2021)

Fig. 4

≡ Arthrinium pseudosinense Crous, in Crous & Groenewald, IMA Fungus 4(1): 148 (2013).

Description

Asexual morph : On WA, hyphae 1.1–2.9 μm diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggregated in clusters on hyphae, hyaline to pale brown, smooth, branched, 9.4–11.0 × 6.1–8.8 μm, mean ± SD: 10.4 ± 0.7 × 7.7 ± 1.1 μm (n = 40). Conidia globose, subglobose to lenticular, with a longitudinal germ slit, occasionally elongated to ellipsoidal, brown to dark brown, smooth to finely roughened, 7.5–11.5 × 7.0–9.5 μm, mean ± SD: 10.1 ± 1.3 × 8.3 ± 0.6 μm, L/W = 1.1–1.3 (n = 40). Sexual morph: Undetermined.

Culture characteristics

Colonies on PDA flat with irregular margin, aerial mycelium white to pale yellow, floccose cottony; reverse pale yellow to yellow. PDA attaining 69.5–78.5 mm in diameter after 7 days at 25 °C, growth rate 9.5–11.5 mm/day.

Figure 4. 

Apiospora pseudosinensis (SAUCC 0221) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 μm (e–h).

Specimens examined

China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 15 July 2022, R.Y. Liu, HSAUP 0221, living culture SAUCC 0221; China, Hainan Province: Diaoluoshan National Nature Reserve, on diseased leaves of bamboo, 29 June 2021, R.Y. Liu, HSAUP 0022, living culture SAUCC 0022.

Notes

Apiospora pseudosinensis was originally described from bamboo leaves collected in the Utrecht Botanical Garden of the Netherlands (Crous and Groenewald 2013; Pintos and Alvarado 2021). In the present study, DNA sequences obtained from two strains (SAUCC 0221 and SAUCC 0222) collected also from bamboo leaves, were not significantly different from those of A. pseudosinensis (Fig. 1). Morphologically, our strains were similar to the original description (conidia 8–10 × 7–10 μm diam. in surface view, 7–8 μm diam. in side view). We therefore consider the newly found strains as A. pseudosinensis (Crous and Groenewald 2013; Pintos and Alvarado 2021).

Discussion

The family Apiosporaceae was proposed to accommodate genera with apiosporous hyaline ascospores and a basauxic, Arthrinium-like conidiogenesis (Hyde et al. 1998). Crous and Groenewald (2013) synonymized Apiospora with Arthrinium on the basis of the one fungus-one name policy (Hawksworth et al. 2011). Crous and Groenewald (2013) also resolved the genetic identity of multiple species of Arthrinium (= Apiospora), analysing ex-type collections, and confirmed that most species occur in Poaceae (R.Br.) Barnh. hosts, although some were known from many other plant host families. However, with the aid of additional genetic data from the type species of Arthrinium, Ar. caricicola, Apiospora and Arthrinium were separated again as two distinct genera (Pintos and Alvarado 2021). Arthrinium species have variously shaped conidia and inhabit Cyperaceae and Juncaceae in temperate, cold or alpine habitats. Most Apiospora species have rounded/lenticular conidia and inhabit mainly Poaceae (and many other host plant families) in a wide range of habitats, including tropical and subtropical regions (Pintos and Alvarado 2021; Samarakoon et al. 2022). An epitype for the type species of Apiospora, A. montagnei, was recently proposed by Pintos and Alvarado (2022).

There are many Apiospora species found on bamboos across the world (Table 2). Bamboos (Poaceae) are distributed in tropical and subtropical to mild temperate regions, with the heaviest concentration and largest number of species in China. Due to their abundance and economic importance, it is of great significance to study and identify the fungi growing on bamboo (Feng et al. 2021). In the present study, two new species (Apiospora dongyingensis and A. hainanensis) are introduced, and another one (A. pseudosinensis) is reported for the first time in China. All of them were collected from bamboo leaves and described based on their phylogenetic data and morphological characters. The descriptions and molecular data for species of Apiospora represent an important resource for understanding the diversity of bamboo fungi.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31900014, U2002203 and 31750001).

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Supplementary material

Supplementary material 1 

Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China

Rongyu Liu, Duhua Li, Zhaoxue Zhang, Shubin Liu, Xinye Liu, Yixin Wang, Heng Zhao, Xiaoyong Liu, Xiuguo Zhang, Jiwen Xia, Yujiao Wang

Data type: phylogenetic

Explanation note: The combined ITS, LSU, tef1 and tub2 genes.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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