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

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.


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 [1][2][3][4][5][6][7][8][9]. Schoch et al. [10] introduced the order Botryosphaeriales to accommodate the single family Botryosphaeriaceae. In the "Outline of Ascomycetes" [7], nine families (Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae, Saccharataceae, Septorioideaceae, Endomelanconiopsidaceae, and Pseudofusicoccaceae) were recognized in Botryosphaeriales. Phillips et al. [3] 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 [3,9]. Alanomyces and Aplosporella are the only two genera accepted within the family Aplosporellaceae. The Morphological observations of conidiomata or ascostromata were carried out using a Motic SMZ 168 series stereomicroscope and photographed using a Nikon E80i microscopecamera system. Tarosoft ® Image Framework was used to measure morphological characters as in Liu et al. [23], and images included in figures were processed with Adobe Photoshop cs v. 5. To isolate single spores, the procedure according to Chomnunti et al. [24] 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 [25], 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).

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. [5]. 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 Doc TM XR Molecular Imager (Bio-Rad, USA). All positive amplicons were sequenced by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).

Sequence Alignment and Phylogenetic Analyses
Sequence quality was assured by inspecting the chromatograms using BioEdit v. 5 [29]. 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 [3][4][5][6]9] (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) [30], 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. [31]. 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.             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 [32] 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 [33].

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.
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. (Figures 6 and 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).  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 of the order Botryosphaeriales (Figure 1). In this phylogeny, each of the isolates obtained in this study were clustered with Aplosporella hesperidica, Barriopsis tectonae, Sphaeropsis citrigena, Sardiniella celtidis, and Sa. guizhouensis. Seven isolates did not cluster with any known Botryosphaeriales species, thus, three novel species (Botryobambusa guizhouensis, Sardiniella elliptica, and Sphaeropsis guizhouensis) were identified based on the morphological and phylogenetic evidence (Figure 1).
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.
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.
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.
Notes: Sardiniella guizhouensis was introduced by Chen et al. [37] 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.
Five species are accepted in the genus Barriopsis [35,[43][44][45]. Barriopsis tectonae was introduced by Doilom et al. [35] 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. [1] 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. [46] introduced the genus Sardiniella by denoting Sa. urbana as the type species. Hyde et al. [36] introduced the second species in the genus, Sa. celtidis, while Chen et al. [37] 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 (Figures 6 and 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 [2,3]. 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 [47][48][49][50][51], 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 [3,5]. 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.

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.