Morpho-Phylogenetic Evidence Reveals Novel Species and New Records of Botryosphaeriaceae in China and Thailand

Species in the Botryosphaeriaceae are common plant pathogens, endophytes, and saprobes found on a variety of mainly woody hosts. Botryosphaeriaceae is a high-profile fungal family whose genera have been subjected to continuous revisions in recent years. Surveys conducted during 2019 and 2020 on several decaying woody hosts (from dead arial twigs, branches, stems, bark, and seed pods) in China and Thailand revealed a high diversity of Botryosphaeriaceae fungi. Identification of 16 Botryosphaeriaceae isolates was carried out based on both morphological characteristics and phylogenetic analyses of combined ITS, LSU, tef1-α, and tub2 sequence data. Four novel species (Dothiorella ovata, Do. rosacearum, Do. septata, and Lasiodiplodia delonicis) and seven previously known species (Botryosphaeria fujianensis, Diplodia mutila, Di. seriata, L. crassispora, L. mahajangana, Macrophomina euphorbiicola and Sphaeropsis eucalypticola) were identified while new hosts and geographical records were reported. This study indicates that the fungal family Botryosphaeriaceae seems to be common and widespread on a broad range of hosts in China and Thailand.

During an investigation of Botryosphaeriaceae diversity in China and Thailand, a collection of 16 Botryosphaeriaceae-like isolates was obtained from several arial parts of the decaying woody hosts.A multi-gene phylogeny based on combined ITS, LSU, tef1-α, and tub2, coupled with morphological comparisons, was carried out to confirm the taxonomic placement.Additionally, this study extended the taxonomic framework of Botryosphaeriaceae by discovering new species, new host records, and new geographical records in China and Thailand.

Specimen Collection, Examination, and Single Spore Isolation
Samples of decaying woody plants were collected from Chiang Rai and Chiang Mai Provinces in Thailand and from Sichuan Province in China between June 2019 and November 2020.Samples were brought to the laboratory by placing them in brown craft paper bags, and the sampling information (date, host, place, GPS, etc.) was recorded.
Morphological observations of fungal structures were made using a LEICA EZ4 dissecting microscope following the method described in Chomnunti et al. [19].The fungal structures were transferred to a small drop of double distilled water on a clean slide and covered with a glass coverslip.Photomicrographs of the fungal specimens were captured using a Nikon ECLIPSE Ni compound microscope fitted with a Nikon DS-Ri2 digital camera.Macro-morphological structures were photographed with a Discovery V.8 stereo microscope fitted with a CARL ZEISS Axio Cam ERc5S microscope camera.All measurements were made with the Tarosoft (R) Image Frame Work (IFW) program [20], and the images were processed with Adobe Photoshop CC extended version 21.1.2.
Single spore isolations were carried out as described by Chomnunti et al. [19], and fruiting body contents were transferred to a drop of sterile water on a sterilized spot plate.This spore suspension was spread over the Petri dishes containing potato dextrose agar (PDA) and incubated at 25 • C for 12 to 24 h.Germinated spores were transferred onto fresh PDA media plates.These culture plates were incubated at 25 • C in incubators, and colony characteristics were observed and recorded after one week following the method described in Rayner [21].A total of 56 isolates have been obtained, among which 16 isolates belong to Botryosphaeriaceae.In this study, we focus only on the fungal taxa of Botryosphaeriaceae.To induce sporulation, cultures were transferred onto fresh PDA media plates using sterile toothpicks or pine needles.The induction results were observed after incubating under near-ultraviolet light for 14-30 d at 25 • C.
Herbarium specimens were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and Guizhou Academic of Agriculture Sciences (GZAAS), China.Axenic cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and Guizhou Culture Collection (GZCC).In deciding whether we have new species, we followed the papers of Chethana et al. [22] and Pem et al. [23].The descriptions are added to the GMS database [24].

DNA Extraction, PCR Amplification and Sequencing
In a sterile environment, fungal mycelium (about 50-100 mg) was scraped using a sterilized toothpick from the colonies grown on PDA media at 25 • C for 2 weeks and then transferred to sterilized 1.5 mL microcentrifuge tubes and maintained at −20 • C for long term storage.Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) was used to extract DNA according to the manufacturer's instructions.The amplifications were performed in a 25 µL reaction volume containing 8.5 µL ddH 2 O, 12.5 µL 2 × PCR Master Mix (Green) (TsingKe Co., Beijing, China), 2 µL of DNA template, and 1 µL of each primer.The genes, primers, and amplification conditions used in this study are listed in Table 1.The PCR products were analyzed by 1.2% agarose gels containing the Safeview DNA stain and sent to Tsingke Biotechnology Co., Ltd.(Chengdu, China) for sequencing.

Sequence Alignment and Phylogenetic Analysis
Sequences generated in this study were checked and assembled using BioEdit v.7.0.9 [29] to assure the sequence quality.The closest taxa to the strains obtained in this study were determined with standard nucleotide BLAST searches in NCBI (http://www.ncbi.nlm.nih.gov/,accessed on 20 July 2022).According to the BLAST results and previous literature, appropriate sequences were determined and downloaded from GenBank to construct phylogenetic analysis.Two phylogenetic trees were constructed, one for the whole family Botryosphaeriaceae (Figure 1) and the other for the genus Dothiorella (Figure 2).Details of the isolates used in this study are listed in Table 2, where two strains of Pseudofusicoccum adansoniae (CBS 122055, CBS 122056) and Neofusicoccum parvum (CBS 110301, CMW 9081) were selected as the outgroup taxa for Botryosphaeriaceae analyses and Dothiorella analysis respectively.The sequences were aligned using MAFFT v.7 online (https://mafft.cbrc.jp/alignment/server/,accessed on 1 August 2023) and AliView [30], and the results were checked using BioEdit [29] and manually edited where necessary.The concatenation of different genes was conducted using SequenceMatrix 1.8 [31].The NEXUS and Phylip files for phylogenetic analyses were obtained using AliView [30].Phylogenetic analyses of the combined sequence data were performed using maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) methods, as detailed in Dissanayake et al. [32].The best model of evolution was determined using MrModeltest v2 [33].The ML analysis was accomplished using RAxML GUI v. 1.3.1 [34], the MP analysis was performed using PAUP v.4.0b10 [35], and the BI analysis was conducted in MrBayes v 3.2.6 [36].Phylogenetic trees were visualized with FigTree v.1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 11 August 2023) and further edited in Adobe Illustrator 2020 (Adobe Systems Inc., Lehi, UT, USA).The final alignment was submitted to Figshare (https://figshare.com, at https://doi.org/10.6084/m9.figshare.24187500,accessed on 22 September 2023).and maximum parsimony (MP) equal to or greater than 75% are given near the nodes.Branches with Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are thickened.The new isolates obtained in this study are indicated in red, and ex-type strains are in bold.The tree is rooted to Neofusicoccum parvum (CBS 110301, CMW 9081).The combined ITS, LSU, tef1-α, and tub2 sequence dataset of Botryosphaeriaceae analysis comprises 89 taxa, including two outgroup taxa.The aligned dataset comprised 2241 characters (ITS: 1-540; LSU: 541-1394; tef1-α: 1395-1793; tub2: 1794-2241) including gaps.The maximum parsimonious dataset consisted of 2241 variable characters, of which 1470 were constant, 655 were parsimony-informative, and 115 were parsimony-uninformative.The MP analysis resulted in a tree length of 2775 steps [consistency index (CI) = 0.444, retention index (RI) = 0.843, relative consistency index (RC) = 0.374, homoplasy index (HI) = 0.556].The RAxML analysis of the combined data set yielded a best-scoring tree (Figure 1) with a final ML optimization likelihood value of −17,381.405163.The matrix had 1017 distinct alignment patterns, with 25.04% of undetermined characters or gaps.Estimated base frequencies were: A = 0.218861, C = 0.278646, G = 0.277782, T = 0.224710; substitution rates AC = 1.166395,AG = 2.628375, AT = 1.104924,CG = 1.478465,CT = 4.949967, GT = 1.000000; gamma distribution shape parameter (alpha) = 0.207919.The maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods (BI) for phylogenetic analyses resulted in trees with similar topologies.According to the results of phylogenetic analysis, 16 isolates obtained in this study were grouped into 11 clades and located in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Macrophomina and Sphaeropsis (Figure 1).
The phylogenetic analysis for the genus Dothiorella was carried out, and a phylogenetic tree combining ITS, LSU, tef1-α, and tub2 sequence data was also constructed (Figure 2).This dataset included 53 ingroup taxa and two outgroup taxa.The aligned dataset comprised 2062 characters (ITS: 1-514; LSU: 515-1331; tef1-α: 1332-1630; tub2: 1631-2062) including gaps.The maximum parsimonious dataset consisted of 2066 variable characters, of which 1637 were constant, 328 were parsimony-informative, and 101 were parsimonyuninformative.The MP analysis resulted in a tree length of 1223 steps [consistency index (CI) = 0.517, retention index (RI) = 0.760, relative consistency index (RC) = 0.393, homoplasy index (HI) = 0.483].In the ML analyses, the best-scoring RAxML tree with a final likelihood value of −9705.573604 is presented.The matrix had 606 distinct alignment patterns, with 25.62% of undetermined characters or gaps.Estimated base frequencies were: A = 0.215885, C = 0.284638, G = 0.269261, T = 0.230216; substitution rates AC = 1.152119,AG = 2.141304, AT = 1.169569,CG = 1.149449,CT = 5.009593, GT = 1.000000; gamma distribution shape parameter (alpha) = 0.143638.The maximum likelihood (ML), maximum parsimony (MP), and Bayesian methods (BI) for phylogenetic analyses resulted in trees with similar topologies, and the result of ML analysis is shown in Figure 2. Phylogenetic results showed that the isolates obtained in this study were nested within the genus Dothiorella and grouped into three clades as distinct species.Notes: The phylogenetic results (Figure 1) showed that our newly obtained isolate clustered together with Botryosphaeria fujianensis, and we identified it as B. fujianensis.There was only the asexual morph provided when Chu et al. [37] described this species, and we illustrate the sexual morph of B. fujianensis and reported it as the new record from Thailand in this study.Notes: The phylogenetic results (Figure 1) showed that our newly obtained isolate clustered together with Botryosphaeria fujianensis, and we identified it as B. fujianensis.There was only the asexual morph provided when Chu et al. [37] described this species, and we illustrate the sexual morph of B. fujianensis and reported it as the new record from Thailand in this study.
Diplodia mutila Fries in Montagne, Ann.Sci.nat., sér.21: 302 [38] Figure 4.   Notes: We identified our isolate as Diplodia mutila based on morphology and phylogeny.This is the first record of Di. mutila found on Prunus persica in China.
Dothiorella ovata N. Notes: Dothiorella ovata is nested in between Do. albiziae, Do. septata and Do.thailandica but can be recognized as a distinct lineage (Figure 2).Morphologically, Do. ovata is similar to Do. septata and Do.albiziae, which in having oblong to ovoid, hyaline conidia becoming brown and one septate at maturation.However, Do. ovata differs from Do. septata and Do.albiziae by its slightly constricted septum and the larger conidia.In addition, a comparison of tef1-α sequences data of Do. ovata and Do.albiziae showed that there are 14 bp (base pair) differences (of 252 bp including the gaps), while Do.ovata and Do.thailandica showed 18 bp differences (of 302 bp including the gaps).Therefore, we introduce Do. ovata as a new species.Dothiorella rosacearum N. Wu, A.J. Dissanayake & Jian K. Liu, sp.nov. Figure 7. Index Fungorum number: IF900579; Facesoffungi number: FoF14259.Etymology: Referring to the host family Rosaceae on which the type specimen was collected.
Holotype: MFLU 23-0014.Notes: The phylogenetic result (Figure 2) showed that two isolates of Dothiorella rosacearum constitute a distinct lineage but claded closer to Do. brevicollis, Do. diospyricola, Do. lampangensis, Do. longicollis, Do. obovata and Do.tectonae.A comparison of ITS sequences data between Do. rosacearum and Do.tectonae showed that there are 19 bp of 539 base pairs differences (including the gaps).In addition, the shortly raised irregular striations can be found on the conidia of Do. tectonae, while no striations were observed in Do. rosacearum.Therefore, Do. rosacearum is a morphologically and phylogenetically distinct species and herein introduced as a new species.Dothiorella septata N. Wu, A.J. Dissanayake & Jian K. Liu, sp.nov.), oblong to ovoid with a broadly rounded apex, initially hyaline to yellowish and aseptate, becoming brown to dark brown and one septate at maturation, slightly constricted at the septum, smooth-walled, without a mucilaginous sheath.
Culture characteristics: Conidia germinating on PDA within 12 h.Colonies are fast growing on PDA, reaching 90 mm diam.after 5-6 days at 20-23 Notes: The phylogenetic results (Figure 2) showed that our isolates clustered with Do. ovata and formed a sister group.A comparison of ITS and tef1-α nucleotides shows that Do. septata is significantly different from its sister species, Do. ovata by 7/569 bp (1.2%) in ITS and 13/303 bp (4.3%) in tef1-α.In the phylogenetic analysis, these two species formed two distinct clades in Dothiorella.Morphologically, there are several differences in conidial morphology between these two species.Considering the morpho-molecular data, we introduced Do. septata as a new species.Notes: The phylogenetic results (Figure 2) showed that our isolates clustered Do. ovata and formed a sister group.A comparison of ITS and tef1-α nucleotides s that Do. septata is significantly different from its sister species, Do. ovata by 7/569 bp ( in ITS and 13/303 bp (4.3%) in tef1-α.In the phylogenetic analysis, these two sp formed two distinct clades in Dothiorella.Morphologically, there are several differenc   Culture characteristics: Conidia germinating on PDA within 24 h.Colonies are fast growing on PDA at 20-25 • C, becoming ash-grey on the surface after 7 days, the reverse is pale grey to grey, and finally black after two weeks, felt-like, sparse, aerial, surface smooth with a crenate edge, filamentous.
Material examined: Thailand, Chiang Mai, Amphoe Mae Taeng, Tambon Cho Lae,  Culture characteristics: Conidia germinating on PDA within 24 h.Colonies are fast growing on PDA, reaching 90 mm diam.after 5 days at 20-25 • C, becoming ash-grey on the surface after one week, with the reverse side of the colonies pale grey to grey, and finally black after two weeks, felt-like, sparse, aerial, surface smooth with crenate edge, filamentous.
Notes: The phylogenetic tree based on ITS, LSU, tef1-α, and tub2 sequence data showed that the new species Lasiodiplodia delonicis (Figure 1) is supported by an absolute bootstrap support (ML/MP/BI = 100/100/1.0).Morphologically, L. delonicis is distinct from other Lasiodiplodia species by its thicker conidial wall and larger conidia.Additionally, conidia of L. delonicis are hyaline throughout the life cycle.
Macrophomina euphorbiicola A.R. Notes: Macrophomina euphorbiicola was introduced by Machado et al. [43].Due to the previous cultures failing to sporulate, comparison with the type species was not possible.The phylogenetic analysis showed that our isolate was nested within M. euphorbiicola and claded closer to M. pseudophaseolina (Figure 1).We, thus, identify the new collection as M. euphorbiicola.

Discussion
Studies on Botryosphaeriaceae, dealing with the phylogenetic traits and morphology of isolates associated with various hosts, have increased in recent years, enabling the worldwide identification of taxa at the species level [2,5,18,[45][46][47][48][49].In this study, 16 Botryosphaeriaceae isolates were obtained from several decaying woody hosts (dead arial twigs, branches, stems, bark, and seed pods) in southwestern China and northern Thailand, and they were identified as 11 species based on a polyphasic approach of morphological features and molecular phylogeny.These species included Botryosphaeria fujianensis, Diplodia mutila, Di. seriata, Dothiorella ovata, Do. rosacearum, Do. septata, Lasiodiplodia crassispora, L. delonicis, L. mahajangana, Macrophomina euphorbiicola and Sphaeropsis eucalypticola.Of these, Do. ovata, Do. rosacearum, Do. septata and L. delonicis are introduced as novel species, and the remaining seven species were identified as new hosts or new geographical records.All species collected in this study are saprophytic on the host.It should be noted that even though sporulation was induced on sterile toothpicks or pine needles on PDA, the respective asexual morph or sexual morph was not observed.Thus, the fungal identification and classification in this study are based on their morphological characteristics of either asexual or sexual morphs and the phylogenetic analysis results.
Macrophomina and Sphaeropsis are two of the least common genera in the family Botryosphaeriaceae.Five species are validly known in Macrophomina, among which M. phaseolina and M. euphorbiicola were introduced as pathogens [43,[50][51][52].In this study, the asexual morph of M. euphorbiicola was collected from Plukenetia volubilis in northern Thailand.Due to the previous cultures failing to sporulate, the morphology of M. euphorbiicola has not been described [43].Hence, we provide the first detailed description and illustration of M. euphorbiicola for the first time and also report it as a new record from Plukenetia volubilis in Thailand.Sphaeropsis was typified with S. visci by Saccardo [53] with 632 records in Index Fungorum (Accessed July 2023), and only eight species are recognized with accessible cultures so far [16,44].In this study, one previously known species, S. eucalypticola, was collected from Tectona grandis in Thailand and reported as a new host record.Sphaeropsis eucalypticola has also been reported on Bauhinia purpurea and Eucalyptus sp. in Thailand [4,54].Mapook et al. [55] identified S. chromolaenicola from Chromolaena odorata in Thailand.However, the remaining members of the genus have not been found on any host in Thailand.
This study revealed two previously known Diplodia species, Di. mutila and Di.seriata from Sichuan province.It is worth noting that similarly to our collection of Di. mutila (from Prunus persica) and Di.seriata (from Wisteria sinensis), Li et al. [49] also found these two species from dead branches of Camellia oleifera, and another two Diplodia species (Di.acerigena and Di.pistaciicola) in Sichuan province.Diplodia species mainly occur on woody hosts, causing rots, cankers, shoot and tip blight [11,[56][57][58][59]. Thus, the discovery and in-depth research of this genus are conducive to the protection of woody plants and the maintenance of greater economic benefits.
Dothiorella was the most frequently isolated genus in this study, as seven Dothiorella isolates were obtained from decaying woody hosts in Chiang Mai Province, Thailand.Dothiorella was introduced by Saccardo [53] with Do. pyrenophora as the type species, and presently, only 38 species are accepted in this genus based on phylogenetic analyses [16,18,49].Zhang et al. [48] made a systematic revision of Dothiorella by synonymizing 15 known species, which reduced the number of Dothiorella members and established a more stable systematic relationship.Most of the members of the genus Dothiorella were rarely collected in Thailand in the past; however, there have been many reports of Dothiorella species being collected in Thailand in recent years [60][61][62][63].We speculate that this may be due to random sampling.In this study, three new species Do. ovata, Do. rosacearum and Do.septata are introduced based on morphological features (asexual morphs) and phylogenetic evidence.
Lasiodiplodia was formally established by Clendenin [64] with L. tubericola Ellis and Everhart (=L.theobromae) [4] as the type species.So far, 37 ex-type/isotype/neotype species entries have been accepted and uploaded to the Botryosphaeriales website [16,65,66].It is worth noting that most of the species were introduced as asexual morphs of Lasiodiplodia, and only a few species of sexual morph have been found in nature, such as L. gonubiensis, L. lignicola and L. theobromae [44,67,68].The three Lasiodiplodia species collected in this study were all asexual morphs and collected from woody plants.Among them, L. crassispora and L. mahajangana were previously known species, and L. delonicis was introduced as a new species.Lasiodiplodia crassispora was first introduced by Burgess et al. [41] based on distinctive morphological characters and phylogenetic analyses.Zhang et al. [48] synonymized L. pyri under L. crassispora.In this study, L. crassispora was collected from decaying wood.Though the species has been found in several countries, such as Australia, Brazil, Namibia, Senegal, and Venezuela [41,48], this is the first time L. crassispora has been reported in Thailand.We collected L. mahajangana from Dipterocarpus retusus in this study.Zhang et al. [48] synonymized L. caatinguensis, L. curvata, L. exigua, L. irregularis, L. macroconidia, and L. pandanicola under L. mahajangana, thus expanding the host range and geographical distribution of this species.Interestingly, the conidia of L. mahajangana are straight or curved, and its conidia morphology is more special compared with other species of Lasiodiplodia [42,48].At the same time, this study also collected a new species, L. delonicis, from a fallen pod of Delonix regia.In addition, mature conidia with longitudinal striations of Lasiodiplodia is one of its distinguishing features from Diplodia [44].However, it has been observed that if the asexual stage is produced on culture, the conidia often have obvious longitudinal striations, while the asexual stage produced in nature has less distinct or absent longitudinal striations [61,[68][69][70].This inference can be found in previous reports and this study.The reason for this phenomenon might be due to the variations in the environment in which the fungi grow.Thus, it is important to collect more fresh specimens to verify this observation.
Botryosphaeria fujianensis was introduced as a pathogen-causing stem canker of blueberry in Fujian province, China [37], whereas our species was isolated from dead twigs of Tectona grandis (Lamiaceae) in Chiang Mai Province, Thailand.As this is the first record of B. fujianensis isolated from Thailand, we suspect that it might be found on more hosts in the future.
With the increased number of studies of Botryosphaeriaceae based on morphology, ecology, and DNA-based phylogeny, more new species and records are constantly being discovered [7,[71][72][73][74].However, there are still many aspects needed to clarify this fungal family, such as specifying species from environmental samples, resolving the opportunistic pathogenic nature, and defining species boundaries.The results of this study indicate that there is still much potential for Botryosphaeriaceae members to be discovered in China and Thailand.As members of the Botryosphaeriaceae family represent a growing threat to agricultural crops and urban and natural forest ecosystems [75][76][77][78], this finding raises questions about the origin, introduction, and pathway of these fungi as well as underlining the need to develop suitable actions to limit their further spread.

Figure 1 .
Figure1.Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined ITS, LSU, tef1-α, and tub2 sequence data for selected closely related genera within the family Botryosphaeriaceae.Bootstrap values for maximum likelihood (ML) and maximum parsimony (MP) equal to or greater than 75% are given near the nodes.Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are denoted in thickened branches.The strain numbers are given after the species names, and ex-type strains are indicated in bold.The newly generated isolates of this study are in red.The tree is rooted with two isolates of Pseudofusicoccum adansoniae (CBS 122055, CBS 122056).

Figure 1 .
Figure1.Phylogenetic tree generated from maximum likelihood (ML) analysis based on combined ITS, LSU, tef1-α, and tub2 sequence data for selected closely related genera within the family Botryosphaeriaceae.Bootstrap values for maximum likelihood (ML) and maximum parsimony (MP) equal to or greater than 75% are given near the nodes.Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are denoted in thickened branches.The strain numbers are given after the species names, and ex-type strains are indicated in bold.The newly generated isolates of this study are in red.The tree is rooted with two isolates of Pseudofusicoccum adansoniae (CBS 122055, CBS 122056).

Figure 2 .
Figure 2. Phylogenetic tree generated from the maximum likelihood (ML) analysis based on combined ITS, LSU, tef1-α, and tub2 sequence data of Dothiorella.Bootstrap values for maximum likelihood (ML)

Table 1 .
Primers and PCR protocols used in this study.

Table 2 .
Taxa Names, Strain or Specimen numbers, and corresponding GenBank accession numbers of the taxa used for the phylogenetic studies.The newly generated sequences are indicated in red, and ex-type strains are indicated in bold.
• C. Sparse, aerial, filamentous, smooth with a crenate edge, white in first few days, becoming grey after one week, and after 2 weeks, becoming black.