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22 October 2021

Occurrence and Morpho-Molecular Identification of Botryosphaeriales Species from Guizhou Province, China

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,
and
1
School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 611731, China
2
Institute of Crop Germplasm Resources, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
3
Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
4
Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
J. Fungi2021, 7(11), 893;https://doi.org/10.3390/jof7110893
This article belongs to the Special Issue Fungal Biodiversity and Ecology 2.0
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Abstract

Botryosphaeriales is an important order of diverse fungal pathogens, saprobes, and endophytes distributed worldwide. Recent studies of Botryosphaeriales in China have discovered a broad range of species, some of which have not been formerly described. In this study, 60 saprobic isolates were obtained from decaying woody hosts in southwestern China. The isolates were compared with other species using morphological characteristics, and available DNA sequence data was used to infer phylogenetic analyses based on the internal transcribed spacer (ITS), large subunit rRNA gene (LSU), and translation elongation factor 1-α (tef) loci. Three novel species were illustrated and described as Botryobambusa guizhouensis, Sardiniella elliptica, and Sphaeropsis guizhouensis, which belong to rarely identified genera within Botryosphaeriaceae. Botryobambusa guizhouensis is the second species identified from the respective monotypic genus. The previously known species were identified as Aplosporella hesperidica, Barriopsis tectonae, Botryosphaeria dothidea, Diplodia mutila, Di. neojuniperi, Di. pseudoseriata, Di. sapinea, Di. seriata, Dothiorella sarmentorum, Do. yunnana, Lasiodiplodia pseudotheobromae, Neofusicoccum parvum, Sardiniella celtidis, Sa. guizhouensis, and Sphaeropsis citrigena. The results of this study indicate that numerous species of Botryosphaeriales are yet to be revealed in southwestern China.

1. Introduction

The Botryosphaeriales are among the most widespread, common, and important fungal pathogens of woody plants. Many are known to exist as endophytes in healthy plant tissues and also as saprobes in dead tree materials. This fungal order has gone through significant revisions, and several new families, genera, and species have been introduced or synonymized over the last decade, mainly on the basis of combined morphological and multiple gene sequence data [,,,,,,,,]. Schoch et al. [] introduced the order Botryosphaeriales to accommodate the single family Botryosphaeriaceae. In the “Outline of Ascomycetes” [], nine families (Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae, Saccharataceae, Septorioideaceae, Endomelanconiopsidaceae, and Pseudofusicoccaceae) were recognized in Botryosphaeriales. Phillips et al. [] revised the order and accepted Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae, and Saccharataceae, while Endomelanconiopsidaceae, Pseudofusicoccaceae, and Septorioideaceae were considered as synonyms of Botryosphaeriaceae, Phyllostictaceae, and Saccharataceae, respectively. We followed this last taxonomical revision in our study.
Presently, the order Botryosphaeriales comprises 33 genera [,]. Alanomyces and Aplosporella are the only two genera accepted within the family Aplosporellaceae. The family Botryosphaeriaceae currently comprises 22 genera: Alanphillipsia, Barriopsis, Botryobambusa, Botryosphaeria, Cophinforma, Diplodia, Dothiorella, Endomelanconiopsis, Eutiarosporella, Lasiodiplodia, Macrophomina, Marasasiomyces, Mucoharknessia, Neodeightonia, Neofusicoccum, Neoscytalidium, Oblongocollomyces, Phaeobotryon, Sakireeta, Sardiniella, Sphaeropsis, and Tiarosporella [,]. Melanopsaceae accommodates only one genus, Melanops, which was supported by several phylogenetic analyses [,,], while Phyllostictaceae includes two genera, Phyllosticta and Pseudofusicoccum [,,]. Planistromellaceae was revised to accommodate Kellermania and Umthunziomyces []. Saccharataceae comprises three genera, Pileospora, Saccharata, and Septorioides [,].
Botryosphaeriales species cause blight, canker, dieback, and fruit rots on a variety of woody perennials globally [,]. In China, infections related to Botryosphaeriales have been described on numerous hosts such as grapes [,,], Caragana arborescens [], Cercis chinensis [], Eucalyptus [], Chinese hackberry [], blueberry [,], forest trees [,], and various other woody hosts. Hence, the aim of this study was to characterize the Botryosphaeriales taxa associated with woody hosts in southwestern China based on morphology, DNA sequence data, and phylogeny.

2. Materials and Methods

2.1. Collection of Specimens, Isolation, Morphology, and Culture Characteristics

From 2017 to 2019, specimens were collected in field investigations of numerous decomposing woody hosts in Fanjing mountain (Tongren District), Forest Park (Chishui District), Huaxi wetland park, Xiaochehe wetland park (Guiyang District), Maolan natural reserve (Libo District), Suiyang broad water nature reserve, and Xingyi Wanfenglin in the Karst region of Guizhou province (Table 1). Samples were placed into ziplock plastic bags, relevant data (location, date, etc.) were documented, and samples were taken into the laboratory.
Table 1. Botryosphaeriales species isolated and characterized in this study. The details of the type species of novel taxa are given in bold. N/A: No sequence available.
Morphological observations of conidiomata or ascostromata were carried out using a Motic SMZ 168 series stereomicroscope and photographed using a Nikon E80i microscope-camera system. Tarosoft® Image Framework was used to measure morphological characters as in Liu et al. [], and images included in figures were processed with Adobe Photoshop cs v. 5. To isolate single spores, the procedure according to Chomnunti et al. [] was followed. Spores germinated on water agar (WA) for 12–24 h were examined and then transferred to potato dextrose agar (PDA) media (OXOID CM0139). Obtained pure cultures were incubated at 25 °C for two weeks, and colony characteristics and morphology of fungal structures were examined for a total of 60 isolates. According to Rayner [], colony color was inspected after 5–10 days of progression on PDA at 25 °C. Approximately 20 ascomata/conidiomata, 25 asci, and 50 conidia/ascospores were measured to obtain the mean size/length. Shape, color, and presence or absence of the mucous sheath of conidia/ascospores were also documented.
Herbarium specimens were deposited at the HKAS (Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica Kunming, China) and GZAAS (Herbarium of Guizhou Academy of Agricultural Sciences, Guiyang, China), while living cultures were deposited in the CGMCC (China General Microbiological Culture Collection Center in Beijing, China) and GZCC (Guizhou Culture Collection in Guiyang, China) (Table 1).

2.2. DNA Extraction and Molecular Based Amplification

About 10 mg of aerial mycelia were scraped from 5 day-old isolates grown on PDA medium at 25 °C for the extraction of total genomic DNA using an Extraction Kit of Biospin Fungus Genomic DNA (BioFlux®, Hangzhou, China) according to the manufacturer’s protocol (Hangzhou, China). For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced for all isolates. The BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 14 August 2020) was used to compare the resulting sequences with those in GenBank. After confirmation of Botryosphaeriales species, two additional gene regions coding for translation elongation factor 1-α (tef) and large subunit rRNA gene (LSU) were sequenced as in Dissanayake et al. []. The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 2. A Bio-Rad C1000 thermal cycler was used to conduct the PCR reactions. The resulting PCR products were visualized on a 1% agarose gel stained with ethidium bromide under UV light by a Gel DocTM XR Molecular Imager (Bio-Rad, USA). All positive amplicons were sequenced by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).
Table 2. Details of gene regions, respective primer pairs, primer sequences, and PCR conditions used in the study.

2.3. Sequence Alignment and Phylogenetic Analyses

Sequence quality was assured by inspecting the chromatograms using BioEdit v. 5 []. Sequences were obtained with both forward and reverse primers, and consensus sequences were obtained using DNAStar v. 5.1 (DNASTAR, Inc.). The sequence data generated in this study have been deposited in GenBank (Table 1).
Reference sequences of ITS, tef, and LSU were retrieved from NCBI GenBank, referring to recent publications [,,,,] (Table 3) to conduct phylogenetic analyses. The reference sequences were aligned with the sequences obtained in this study (Table 1) using MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 22 December 2020) [], then manually adjusted, and phylogenetic relationships were inferred with maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) using procedures provided in detail by Dissanayake et al. []. An overview phylogenetic tree for the order Botryosphaeriales was constructed using ITS, LSU, and tef sequence data as some families in Botryosphaeriales (except Botryosphaeriaceae) comprise only ITS and LSU sequences. Separate phylogenetic trees of the diverse genera (Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, and Neofusicoccum) were constructed by combining ITS and tef sequences.
Table 3. Botryosphaeriales taxa used in the phylogenetic analysis. The culture collection accession number for the type strains are given in bold. N/A: No sequence available.
Alignments generated in this study were submitted to TreeBASE (https://treebase.org/treebase-web/home.html, accessed on 18 August 2021). The submission numbers and reviewer access URL for each alignment are provided in Table 4. Taxonomic novelties were submitted to the Faces of Fungi database [] and Index fungorum (http://www.indexfungorum.org, accessed on 5 August 2021). New species were established based on the guidelines provided by Jeewon and Hyde [].
Table 4. Alignment details and comparison of MP, ML, and BI analyses results of each phylogenetic tree constructed in this study.

3. Results

3.1. Phylogenetic Analyses

Sixty isolates obtained from various decaying woody hosts in various locations in Guizhou province were primarily recognized by colony characteristics, such as abundant greenish black aerial mycelia on PDA medium. The ITS gene region sequences compared with those in GenBank using the BLAST tool exhibited 95–99% similarity to known Botryosphaeriales species, and these closely related known species were included in the phylogenetic analysis. All details of the alignments (ITS, LSU, tef alignment of the overview phylogenetic tree for the order Botryosphaeriales and ITS and tef alignments for the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, and Neofusicoccum) are provided in Table 4. The best-scoring RAxML tree (Figure 1) is presented as the MP and BI methods produced trees with topologies similar to those of ML.
Figure 1. Maximum likelihood tree based on analysis of combined ITS, LSU, and tef sequences of selected Botryosphaeriales taxa. Bootstrap values of ML, MP >75% are shown near nodes and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species, and novel taxa are in red. Ex-type strains are in bold. The tree is rooted to Lecanosticta acicola (LNPV252). The scale bar represents the expected number of nucleotide substitutions per site.
Six different phylogenetic trees were constructed for the 60 isolates obtained in this study. Twelve isolates (20% of total isolates) were treated together in an overview phylogenetic tree and seven of them did not cluster with any known Botryosphaeriales species, thus, three novel species were identified based on the morphological and phylogenetic evidence (Figure 1). In this phylogeny, the isolates obtained in the study were clustered with Aplosporella hesperidica (Figure 2), Barriopsis tectonae (Figure 3), Botryobambusa guizhouensis sp. nov. (Figure 4), Sardiniella celtidis (Figure 5), Sardiniella elliptica sp. nov. (Figure 6 and Figure 7), Sardiniella guizhouensis (Figure 8), Sphaeropsis citrigena, and Sphaeropsis guizhouensis sp. nov (Figure 9).
Twenty-three isolates (38.3% of total isolates) belong to the genus Botryosphaeria, and all of them clustered with B. dothidea (Figure 10). Six isolates (10% of total isolates) belong to the genus Diplodia and were identified as Di. mutila, Di. neojuniperi, Di. pseudoseriata, Di. sapinea, and Di. seriata (Figure 11). Two isolates (3.3% of total isolates) were identified as species of Dothiorella (Do. sarmentorum and Do. yunnana, Figure 12), while five isolates (8.3% of total isolates) belong to the genus Lasiodiplodia (L. pseudotheobromae, Figure 13). All twelve isolates (20% of total isolates) of the genus Neofusicoccum were identified as N. parvum (Figure 14).

3.2. Taxonomy

Aplosporella hesperidica Speg., Anal. Soc. cient. argent. 13: 18 (1882) (Figure 2).
Index Fungorum number: IF218239; Facesoffungi number: FoF07830.
Saprobic on decaying wood. Sexual morph: Not observed. Asexual morph: Conidiomata 220–360 × 420–610 µm ( x ¯ = 320 × 550 µm, n = 20), solitary, dark brown, immersed to semi-immersed, erumpent, multiloculate, locules separated by pale brown textura prismatica. Ostiole 60–80 µm diam., central. Peridium 75–150 µm (6–10 cell-layers), outer layers composed of dark brown textura angularis, becoming hyaline towards the inner region. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 6–11 × 2.5–3 µm ( x ¯ = 8 × 2.5 µm, n = 20), holoblastic, hyaline, cylindrical to doliiform, smooth-walled, proliferating percurrently with 1–3 annellations near the apex. Paraphyses 35–95 × 4–8 µm ( x ¯ = 60 × 5 µm, n = 20), wide at the base, 1–3 µm wide in the upper part, hyaline, smooth-walled, septate, branched below. Conidia 17–25 × 10–18 µm ( x ¯ = 20 × 12 µm, n = 50), aseptate, initially hyaline, smooth-walled, broadly ellipsoidal to subcylindrical, with rounded ends, becoming dark brown (black in mass), prominently verruculose before discharge from pycnidia.
Culture characteristics: Conidia germinate on WA within 12 h at room temperature. Colonies on PDA after five days at 25 °C become olivaceous to grey-olivaceous in the center, olivaceous-buff to greenish-olivaceous towards the margin. Aerial mycelium appressed, floccose, white to smoke grey. Colonies flat with undulate edge, 38 mm diameter after two days, reaching the edge of the Petri dish within 10 days.
Material examined: China, Guizhou province, Tongren District, Fanjing mountain, on decaying woody host, July 2018, Y. Y. Chen, GZAAS 19-1814, living culture GZCC 19-0095.
Notes: Our sample morphologically lines up with the description of Aplosporella hesperidica provided by Spegazzini [] as it has erumpent, black conidiomata and brown, smooth-walled, oblong conidia with overlapping spore dimensions of 22–25 × 9–11 µm. It is identical to A. hesperidica based on morphology and phylogeny (Figure 1). This is the first time A. hesperidica has been reported in China.
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Figure 2. Aplosporella hesperidica (GZAAS 19-1814). (a–c) Conidiomata on host surface. (d,e) Vertical hand sections of multiloculate conidiomata. (f,g) Developing conidia attached to conidiogenous cells. (h) Hyaline immature conidium. (i–k) Mature brown conidia. (l,m) Five-day-old culture on PDA (OXOID CM0139) from above and below. Scale bars: (d,e) = 100 μm, (f–k) = 10 μm.
Barriopsis tectonae Doilom, L.A. Shuttlew., and K.D. Hyde, Phytotaxa 176: 84 (2014) (Figure 3).
Index Fungorum number: IF808202; Facesoffungi number: FoF09644.
Saprobic on decaying wood. Sexual morph: Ascostromata 286–420 × 258–349 μm ( x ¯ = 350 × 318 µm, n = 20), black, immersed, aggregated or clustered, scattered, multi-loculate, composed of one or up to three globose ascomata in each ascostroma, erumpent through the bark at maturity, discoid to pulvinate or hemispherical, discrete or wide-spreading. Peridium composed of several layers of dark brown-walled cells of textura angularis. Pseudoparaphyses 2.5–3.5 μm wide, hyphae-like, septate, embedded in a gelatinous matrix. Asci 107–154 × 34–41 ( x ¯ = 129 × 36 µm, n = 25), eight-spored, bitunicate, fissitunicate, clavate to sub-clavate, broad, with a short pedicel and apically rounded with an ocular chamber. Ascospores 31–34 ×14–15 µm ( x ¯ = 32 × 15 µm, n = 50), biseriate, brown to dark brown, aseptate, ellipsoid-oval, inequilateral, slightly curved, widest in the median, ends rounded, light brown in the center, smooth or verrucose, without a gelatinous sheath. Asexual morph: Not observed.
Culture characteristics: Ascospores germinate on WA within 18 h. Colonies growing on PDA reach 2 cm diameter after five days at 25 °C. Effuse, velvety with entire to slightly undulate edge. Blackish green to black.
Material examined: China, Guizhou province, Libo District, Maolan natural reserve, on decaying woody host, July 2017, Y. Y. Chen, GZAAS 19-1985, living culture GZCC 19-0266.
Notes: In the phylogenetic analysis, an isolate obtained in this study (GZCC 19-0266) was grouped with the ex-type isolate of Barriopsis tectonae (Figure 1) with absolute bootstrap support (ML/MP/BI = 100/100/1.0). This isolate is morphologically similar to Ba. tectonae as of the report by Doilom et al. [] with erumpent, black ascostromata and overlapping biseriate, brown, aseptate, ellipsoid, thick-walled conidia with overlapping spore dimensions of 29–33 × 13–15 μm ( x ¯ = 30 × 14 μm, n = 30). It is identical to Ba. tectonae based on morphology and phylogeny (Figure 1). We therefore identify our isolate as Ba. tectonae based on phylogenetic analyses, and the isolate is introduced here as a new locality record from Guizhou province, China. This is the first time Ba. tectonae has been reported in China.
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Figure 3. Barriopsis tectonae (GZAAS 19-1985). (a,b) Appearance of ascomata on wood. (c) Horizontal section of the ascomata. (d,e) Vertical section of ascomata. (f) Peridium. (g,h) Immature asci. (i) Immature and mature asci. (j) Mature asci. (k) Pseudoparaphyses. (l–p). Brown ascospores. Scale bars: (d) = 50 μm, (e) = 100 μm, (f) = 50 μm, (g–j) = 20 μm, (k–p) = 10 μm.
Botryobambusa guizhouensis Y.Y. Chen, A. J. Dissanayake, and Jian K. Liu., sp. nov (Figure 4).
Index Fungorum number: IF558473; Facesoffungi number: FoF09645.
Etymology: Name refers to the location where the fungus was collected, Guizhou, China.
Holotype: HKAS 112600.
Saprobic on a decaying bamboo. Sexual morph: Ascostromata 218–340 × 210–420 μm ( x ¯ = 275 × 345 μm, n = 20), black, immersed or erumpent, gregarious, uniloculate, locules globose to subglobose, coriaceous. Peridium 36–60 μm, comprises several layers of cells textura angularis, broader at the base, outer layers dark to dark brown and thick-walled, inner layers hyaline and thin-walled. Asci 78–115 × 12–16 μm ( x ¯ = 94.5 × 14.5 μm, n = 25), eight-spored, bitunicate, fissitunicate, clavate to cylindro-clavate, usually wider at the apex, pedicellate, apically rounded with well-developed ocular chamber. Ascospores 13–22 × 8–11 μm ( x ¯ = 17.5 × 9 μm, n = 50), uniseriate at the base or irregularly biseriate, hyaline, aseptate, ellipsoidal to obovoid, thick-walled, surrounded by distinctive structured mucilaginous sheath. Asexual morph: Not observed.
Culture characteristics: Ascospores germinate on WA within 24 h. Colonies growing on PDA reach a 5 cm diameter after five days at 25 °C. Fast growing; white in the first few days, become grey to green-black after one week. Reverse grey to black, flattened, fairly dense, surface smooth with crenate edge.
Material examined: China, Guizhou province, Chishui District, Forest Park, on decaying bamboo, July 2019, Y. Y. Chen, 171, (HKAS 112600, holotype); ex-type living culture CGMCC 3.20348; ibid., (GZAAS 20-0718, paratype), living culture GZCC 19-0734.
Notes:Botryobambusa guizhouensis formed a distinct clade with absolute support (ML/MP/BI = 100/100/1.0) and differed from its closely related species Bo. fusicoccum in the concatenated alignment by 7/680 bp in ITS, 4/803 bp in LSU, and 5/479 bp in tef. Morphologically, Bo. guizhouensis differs from Bo. fusicoccum in having longer asci (94.5 × 14.5 μm vs. 60 × 15.5 μm) and larger ascospores (17.5 × 9 μm vs. 11.5 × 6 μm).
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Figure 4. Botryobambusa guizhouensis (HKAS 112600, holotype). (a,b) Appearance of ascomata on bamboo. (c) Vertical section of ascomata. (d,e) Immature asci. (f,g) Immature and mature asci. (h–j) Hyaline, aseptate ascospores enclosed with mucilaginous sheath. Scale bars: (c) = 50 μm, (d–g) = 100 μm, (h,i) = 20 μm, (j) = 10 μm.
Sardiniella celtidis Dissan., Camporesi and K.D. Hyde, Fungal Divers 87: 12 (2017) (Figure 5).
Index Fungorum number: IF552896; Facesoffungi number: FoF02732.
Saprobic on a decaying host. Sexual morph: Ascostromata 210–300 × 275–340 μm ( x ¯ = 270 × 310 μm, n = 20), dark brown to black, globose, immersed in the substrate, partially erumpent at maturity, ostiolate. Ostiole circular, central. Peridium 48–72 μm thick, composed of dark brown thick-walled cells of textura angularis, becoming thin-walled and hyaline towards the inner region. Pseudoparaphyses 3–5 μm wide, thin-walled, hyaline. Asci 62–90 × 26–34 μm ( x ¯ = 78 × 30 μm, n = 20), four- to eight-spored, bitunicate, cylindric-clavate, endotunica thick-walled, with a well-developed ocular chamber. Ascospores 19–27 × 15–18 μm ( x ¯ = 23 × 16 μm, n = 50), 1-septate, irregularly biseriate, dark brown, oblong to ovate, widest in center, straight, moderately thick-walled, surface smooth. Asexual morph: Not observed.
Culture characters: Ascospores germinate on WA within 24 h. Colonies on PDA reach a 2 cm diameter after five days at 25 °C. Mycelium velvety and moderately fluffy with an irregular margin, surface initially white and later turning dark olivaceous from the middle of the colony and dark grey in reverse.
Material examined: China, Guizhou province, Guiyang city, Xingyi Wanfenglin, on decaying woody host, June 2019, Y.Y. Chen, GZAAS 19-1967, living culture GZCC 19-0248.
Notes: Our sample is phylogenetically identical to Sardiniella celtidis (Figure 1). Only the asexual morph of Sa. celtidis was provided when Hyde et al. [] introduced this species. Here, we provide the sexual morph of Sa. celtidis.
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Figure 5. Sardiniella celtidis (GZAAS 19-1967). (a,b) Appearance of ascomata on decaying wood. (c) Peridium. (d,e) Vertical section of ascomata. (f) Immature ascus. (g,h) Mature asci. (i) Pseudoparaphyses. (j–l) Brown, one-septate ascospores. (m,n) Five-day-old culture on PDA (OXOID CM0139) from above and below. Scale bars: (c–e) = 100 μm, (f–l) = 20 μm.
Sardiniella elliptica Y.Y. Chen, A. J. Dissanayake, and Jian K. Liu., sp. nov (Figure 6 and Figure 7).
Index Fungorum number: IF558474; Facesoffungi number: FoF09646.
Etymology: Named referring to the shape of the conidiospores.
Holotype: HKAS 112594.
Saprobic on decaying host. Sexual morph: Ascostromata 280–390 × 295–340 μm ( x ¯ = 340 × 310 μm, n = 20), dark brown to black, globose, submerged in the substrate, partially erumpent at maturity, ostiolate. Ostiole circular, central. Peridium 30–48 μm thick, composed of dark brown thick-walled cells of textura angularis, becoming thin-walled and hyaline towards the inner region. Pseudoparaphyses 2–3 μm wide, thin-walled, hyaline. Asci 71–93 × 19–24 μm, ( x ¯ = 86 × 22 μm, n = 25), 4(–8)-spored, bitunicate, cylindric-clavate, endotunica thick-walled, with a well-developed ocular chamber. Ascospores 26–33 × 9–12 μm ( x ¯ = 29 × 11 μm, n = 50), irregularly biseriate, initially hyaline and becoming dark brown, oblong to ovate, widest in center, straight, moderately thick-walled, surface smooth. Asexual morph: Appearing as subepidermal black spots on the substrate with black margins, with circular sunken perforation through the bark. Conidiomata 190–240 × 274–310 µm ( x ¯ = 220 × 290 µm, n = 20), pycnidial, immersed, arranged singly or in small groups within the bark, globose to subglobose, dark brown to black, solitary or gregarious. Ostiole central. Peridium 27–35 µm thick, comprising dark brown to hyaline, multi-layered (3–5 layered), thick-walled cells of textura angularis. Conidiogenous cells lining the inner surface of the conidioma, hyaline, short obpyriform to subcylindrical. Conidia 25–32 × 10–13 µm ( x ¯ = 28 × 12 µm, n = 50), ellipsoid to obovoid, immature conidia hyaline, mature conidia becoming medium to dark brown.
Culture characters: Colonies on PDA reaching a 70 mm diameter after five days at 25 °C. Mycelium velvety and moderately fluffy with an irregular margin. Surface initially white and later turning dark olivaceous from the middle of the colony and dark grey in reverse.
Material examined: China, Guizhou province, Guiyang District, Huaxi wetland park, on decaying woody host, April 2017, Y.Y. Chen, 18-76 (HKAS 112594, holotype); ex-type living culture CGMCC 3.20349; ibid., Libo District, Maolan natural reserve, July 2017, 19-120 (GZAAS 19-1855, paratype), living culture GZCC 19-0262; ibid., Xingyi Wanfenglin, June 2019, 19-96 (GZAAS 19-1838, paratype), living culture GZCC 19-0245.
Notes: Three isolates of Sardiniella elliptica clustered together with Sa. celtidis, Sa. guizhouensis, and Sa. urbana and formed a well-supported clade representing the genus Sardiniella; thus, it can be recognized as a distinct lineage within Sardiniella. Sardiniella elliptica can be distinguished from the above closely related species based on ITS and tef loci for Sa. celtidis (5/680 bp in ITS, 21/479 bp in tef) and Sa. urbana (5/680 bp in ITS, 8/803 bp in LSU, 27/479 bp in tef). In addition, Sa. elliptica can be morphologically distinguished from other known Sardiniella species based on its aseptate mature ascospores.
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Figure 6. Sardiniella elliptica (HKAS 112594, holotype, sexual morph). (a,b) Appearance of ascomata on decaying wood. (c) Vertical section of ascomata. (d) Peridium. (e) Pseudoparaphyses. (f) Immature and mature asci. (g) Immature ascus. (h,i) Mature asci. (j–l) Immature hyaline ascospores. (m–o) Mature, brown, aseptate ascospores. Scale bars: (c) = 100μm, (d) = 50 μm, (e–o) = 10 μm.
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Figure 7. Sardiniella elliptica (GZAAS 19-1838, asexual morph). (a–c) Conidiomata on host surface. (d) Vertical section of conidiomata. (e) Peridium. (f–h) Conidiogenous cells and developing conidia. (i–l) Immature, hyaline conidia. (m) Mature, brown conidia. Scale bars: (d) = 100 μm, (e) = 20 μm, (f–m) = 10 μm.
Sardiniella guizhouensis Y.Y. Chen, and Jian K. Liu, Phytotaxa 508: 190 (2021) (Figure 8).
Index Fungorum number: IF558352; Facesoffungi number: FoF09647.
Saprobic on decaying wood. Sexual morph: Not observed. Asexual morph: Conidiomata 180–245 × 275–395 µm ( x ¯ = 229 × 330 µm, n = 20), immersed, arranged singly or in small groups within the bark, globose to subglobose, dark brown to black, solitary or gregarious. Ostiole central. Peridium 22–34 µm thick, outer layer composed of pigmented thick-walled cells of textura angularis, inner layer composed of hyaline thin-walled cells of textura angularis (three- to five-layered). Conidiogenous cells 6–11 × 6–7 µm ( x ¯ = 8.5 × 6.5 μm, n = 25), lining the inner surface of the conidioma, hyaline, short obpyriform to subcylindrical. Conidia 21–28 × 10–14 µm ( x ¯ = 24.5 × 12.5 μm, n = 50), ellipsoid to obovoid, immature conidia hyaline, mature conidia becoming medium to dark brown.
Culture characteristics: Conidia germinating on WA within 18 h and producing germ tubes from each septum. Colonies growing on PDA, reaching a diameter of 4 cm after five days at 25 °C, effuse, velvety, with entire to slightly undulate edge. The early stage of the white, later green.
Material examined: China, Guizhou province, Libo District, Maolan natural reserve, July 2017, GZAAS 19-1948, living culture GZCC 19-0229.
Notes:Sardiniellaguizhouensis was introduced by Chen et al. [] with both sexual and asexual morphs. One isolate obtained in this study clustered with the ex-type of Sa. guizhouensis (CGMCC 3.19222) in the phylogenetic analyses of combined ITS, LSU, and tef sequence data (Figure 1). We identified our collection as Sa. guizhouensis based on morphology and phylogeny.
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Figure 8. Sardiniella guizhouensis (GZAAS 19-1809, asexual morph). (a,b) Conidiomata on host surface. (c) Horizontal section of conidiomata. (d) Vertical section of conidiomata. (e–i) Conidiogenous cells and developing conidia. (j–n) Hyaline conidia. Scale bars: (d) = 100 μm, (e–n) = 10 μm.
Sphaeropsis guizhouensis Y.Y. Chen, A. J. Dissanayake, and Jian K. Liu., sp. nov (Figure 9).
Index Fungorum number: IF558475; Facesoffungi number: FoF09648.
Etymology: Name refers to the location where the fungus was collected, Guizhou, China.
Holotype: HKAS 112084.
Saprobic on decaying wood. Sexual morph: Ascostromata 132–185 × 122–165 µm ( x ¯ = 152 × 145 µm, n = 20), initially immersed under host epidermis, becoming semi-immersed to erumpent, breaking through cracks in bark, gregarious and fused, uniloculate, globose to subglobose, membraneous, ostiolate. Ostiole 43–52 μm high, 30–42 μm wide, central, papillate, pale brown, relatively broad, periphysate. Peridium 28–44 μm wide, broader at the base, comprising several layers of relatively thick-walled, dark brown to black-walled cells arranged in a textura angularis. Pseudoparaphyses hyphae-like, numerous, embedded in a gelatinous matrix. Asci 67–101 × 19–23 µm ( x ¯ = 89 × 20 μm, n = 25), eight-spored, bitunicate, fissitunicate, clavate to cylindro-clavate, sometimes short pedicellate, mostly long pedicellate, apex rounded with an ocular chamber. Ascospores 20–23 × 7.8–8.3 µm ( x ¯ = 22 × 8 μm, n = 50), overlapping uniseriate to biseriate, hyaline, aseptate, ellipsoidal to obovoid, slightly wide above the center, minutely guttulate, smooth-walled. Asexual morph: Not observed.
Culture characteristics: Ascospores germinating on PDA within 18 h. Germ tubes produced from both ends of the ascospores. Fast growing; fimbriate, flat or effuse, dense, convex with papillate surface, reaching the edge of the Petri dish after seven days.
Material examined: China, Guizhou province, Libo District, Maolan natural reserve, on decaying woody host, July 2017, Y.Y. Chen, GZAAS4 (HKAS 112084, holotype); ex-type living culture CGMCC 3.20352; ibid., Xingyi Wanfenglin, on decaying woody host, June 2019, Y.Y. Chen, (GZAAS 19-2892, paratype), living culture GZCC 19-0273.
Notes:Sphaeropsis guizhouensis formed a distinct clade (Figure 1) and is phylogenetically distinct from Sp. eucalypticola (MFLUCC 11-0579) in a clade with absolute support (ML/MP/BI = 100/100/1.0). Sphaeropsis guizhouensis can be distinguished from Sp. eucalypticola based on ITS (3/680 bp), LSU (1/803 bp), and tef (25/479 bp). Sphaeropsis guizhouensis differs from Sp. eucalypticola in having smaller asci (89 × 20 μm vs. 106 × 29 μm) and ascospores (22 × 8 μm vs. 30 × 12 μm).
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Figure 9. Sphaeropsis guizhouensis (HKAS 112084, holotype). (a–c) Appearance of ascomata on wood. (d,e) Vertical section of ascomata. (f) Peridium. (g–j) Immature and mature asci. (k) Pseudoparaphyses. (l–p) Hyaline mature ascospores. Scale bars: (d,e) = 50 μm, (f) = 20 μm, (g–p) = 10 μm.
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Figure 10. Maximum likelihood tree based on analysis of combined ITS and tef sequences of Botryosphaeria. Bootstrap support values of ML, MP > 75% are shown near the nodes, and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species. Ex-type strains are in bold. The tree is rooted to Macrophomina phaseolina (CBS 227.33).
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Figure 11. Maximum likelihood tree based on analysis of combined ITS and tef sequences of Diplodia. Bootstrap support values of ML, MP > 75% are shown near the nodes, and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species. Ex-type strains are in bold. The tree is rooted to Lasiodiplodia theobromae (CBS 164.96).
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Figure 12. Maximum likelihood tree based on analysis of combined ITS and tef sequences of Dothiorella. Bootstrap support values of ML, MP > 75% are shown near the nodes, and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species. Ex-type strains are in bold. The tree is rooted to Neofusicoccum parvum (CMW9081).
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Figure 13. Maximum likelihood tree based on analysis of combined ITS and tef sequences of Lasiodiplodia. Bootstrap support values of ML, MP > 75% are shown near the nodes, and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species. Ex-type strains are in bold. The tree is rooted to Diplodia mutila (CBS 112553).
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Figure 14. Maximum likelihood tree based on analysis of combined ITS and tef sequences of Neofusicoccum. Bootstrap support values of ML, MP > 75% are shown near the nodes, and branches in bold indicate BI probabilities >0.95. Isolates obtained in this study are in blue for known species. Ex-type strains are in bold. The tree is rooted to Dothiorella viticola (CBS 117009).

4. Discussion

This study reports the largest collection of Botryosphaeriales isolates from Guizhou province, China, and is the first attempt to characterize Botryosphaeriales species from various nature reserves in the province. Three new species were described in three rarely observed genera: Botryobambusa (Bo. guizhouensis), Sardiniella (Sa. elliptica), and Sphaeropsis (Sp. guizhouensis). These three genera have few species and have been sporadically isolated worldwide. Apart from these novel species, 15 known species—Aplosporella hesperidica, Barriopsis tectonae, Botryosphaeria dothidea, Diplodia mutila, Di. neojuniperi, Di. pseudoseriata, Di. sapinea, Di. seriata, Dothiorella sarmentorum, Do. yunnana, Lasiodiplodia pseudotheobromae, Neofusicoccum parvum, Sardiniella celtidis, Sa. guizhouensis, and Sphaeropsis citrigena—were isolated and included in their respective phylogenies.
The genus Aplosporella consists of plant pathogens, saprobes, and endophytes. Though 300 epithets of Aplosporella are registered in Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, accessed in 23 August 2021), only 12 species are accepted within this genus (www.botryosphaeriales.org). Aplosporella hesperidica has been reported in several Asian countries [,,,,,,], but this is the first time it has been identified in China.
Five species are accepted in the genus Barriopsis [,,,]. Barriopsis tectonae was introduced by Doilom et al. [] from a dead Tectona grandis branch collected in Thailand. So far, this species has been reported only from Thailand (http://nt.arsgrin.gov/fungaldatabases/, accessed on 25 September 2021), and here we provide a new country report of Ba. tectonae (Figure 3) based on sexual morphological characteristics and molecular evidence.
Botryobambusa is a monotypic genus (www.botryosphaeriales.org). Liu et al. [] introduced and compared Botryobambusa with other existing genera in Botryosphaeriales. It is thus far only identified from bamboo in Thailand. In this study, two isolates obtained from decaying bamboo in Forest Park, Chishui District in Guizhou province were identified as a novel species (Bo. guizhouensis). The sexual morph of Bo. guizhouensis was distinguished from Bo. fusicoccum by its larger asci and ascospores as well as by molecular phylogeny. Our study shows that the genus can be clearly discriminated from the morphologically similar genus Botryosphaeria by its velvety, hyaline, and sheathed ascospores. Phylogenetically, these two genera are clearly distinct lineages.
Linaldeddu et al. [] introduced the genus Sardiniella by denoting Sa. urbana as the type species. Hyde et al. [] introduced the second species in the genus, Sa. celtidis, while Chen et al. [] introduced the third species, Sa. guizhouensis, reporting a sexual morph of the genus for the first time. In this study, another new species (Sa. elliptica) is described and assigned to the genus with details provided for a previously known species (Sa. celtidis). With morphological and molecular support, here we present the sexual morph report for Sa. elliptica; a newly introduced species in this study (Figure 6 and Figure 7). So far, Sardiniella species are known only from Italy and China (http://nt.arsgrin.gov/fungaldatabases/, accessed on 25 September 2021).
Though more than 630 names exist in Sphaeropsis (Index Fungorum, August 2021), only five species are currently accepted [,]. In this study, two isolates obtained from decaying woody hosts in Guizhou province were identified as a novel species, Sp. guizhouensis. The sexual morph of Sp. guizhouensis (Figure 9) is distinguished from the other species in this genus by ascospore dimensions. Another previously known species Sp. citrigena was also isolated and included in the phylogenetic analysis.
Botryosphaeria dothidea (Figure 10) and Neofusicoccum parvum (Figure 14) were the most isolated species in this study, consistent with some prior studies [,,,,], which indicates the ability of species in these genera to inhabit a variety of plant species and geographic areas globally. Certain Diplodia species occupy extensive host ranges, such as Di. seriata which has been documented on more than 250 hosts [,]. In this study, we isolated five Diplodia species (Figure 11): Di. mutila, Di. neojuniperi, Di. pseudoseriata, Di. sapinea, and Di. seriata. Our study revealed two previously known Dothiorella species (Figure 12), Do. sarmentorum and Do. yunnana, for the first time from Guizhou province. Lasiodiplodia pseudotheobromae is a common and cosmopolitan species on diverse host plants and has been reported from various localities globally. This study revealed five saprobic L. pseudotheobromae isolates (Figure 13) in Guizhou province.
Members of Botryosphaeriales signify a rising risk to agricultural crops and urban and natural forest ecosystems in China. Collecting and identifying Botryosphaeriales isolates from various hosts and locations is required to describe and understand these species. The occurrence and significance of Botryosphaeriales species in various nature reserves has not been investigated at a larger scale so far in Guizhou province. Hence, in this study, we provided a larger collection of Botryosphaeriales isolates and identify them to species level by both morphology and phylogeny. Further studies are needed to explore and gather data on their occurrence, as precise data of the causal agents is essential.

5. Conclusions

We carried out fungal diversity investigations at a large scale in southwestern China and here we provided a report of Botryosphaeriales species isolated from various woody hosts in Guizhou province, China. The identification of 18 Botryosphaeriales species (15 known species and three new species) associated with saprobic woody hosts was revealed.

Author Contributions

Conceptualization, J.-K.L. and Y.-Y.C.; methodology, Y.-Y.C. and A.J.D.; formal analysis, Y.-Y.C. and A.J.D.; resources, Y.-Y.C. and J.-K.L.; data curation, A.J.D., R.C., and J.-K.L.; writing—original draft preparation, A.J.D.; writing—review and editing, A.J.D. and J.-K.L.; supervision, J.-K.L.; project administration, J.-K.L.; funding acquisition, J.-K.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study is supported by the Joint Fund of the National Natural Science Foundation of China and the Karst Science Research Center of Guizhou province (Grant No. U1812401).

Acknowledgments

We would like to thank Shaun Pennycook (Landcare Research-Manaaki Whenua, New Zealand) for advising on the new fungal names. A.J.D. acknowledges the University of Electronic Science and Technology of China for financial support.

Conflicts of Interest

The authors declare no conflict of interest.

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