﻿Botryosphaerialean fungi associated with woody oil plants cultivated in Sichuan Province, China

﻿Abstract Woody oil plants are important economic trees which are widely cultivated and distributed throughout China. Surveys conducted during 2020 and 2021 on several woody oil plantations from five regions of Sichuan Province, China, revealed a high diversity of Botryosphaerialean fungi. The identification of 50 botryosphaeriaceous isolates was carried out based on both morphology and multi-gene phylogenetic analysis of internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (tef1) and β-tubulin gene (tub2). This allowed the identification of twelve previously known Botryosphaeriales species: Aplosporellaprunicola, A.ginkgonis, Barriopsistectonae, Botryosphaeriadothidea, Bo.fabicerciana, Diplodiamutila, Di.seriata, Dothiorellasarmentorum, Neofusicoccumparvum, Sardiniellaguizhouensis, Sphaeropsiscitrigena, and Sp.guizhouensis, and four novel species belonging to the genera Diplodia and Dothiorella, viz. Di.acerigena, Di.pistaciicola, Do.camelliae and Do.zanthoxyli. The dominant species isolated across the surveyed regions were Botryosphaeriadothidea, Sardiniellaguizhouensis and Diplodiamutila, representing 20%, 14% and 12% of the total isolates, respectively. In addition, most isolates were obtained from Pistaciachinensis (14 isolates), followed by Camelliaoleifera (10 isolates). The present study enhances the understanding of Botryosphaeriales species diversity on woody oil plants in Sichuan Province, China.


Introduction
Botryosphaeriaceae is a diverse group of fungi that includes endophytes, saprobes and plant pathogens . They have broad host ranges, and are widely distributed in tropical and temperate regions (Batista et al. 2021). Botryosphaeriaceae was introduced by Theissen and Sydow (1918) to accommodate three genera Botryosphaeria, Dibotryon and Phaeobotryon. Botryosphaeriales was proposed to include the single family, Botryosphaeriaceae, based on multi-gene phylogeny (Schoch et al. 2006). Up to date, six families and 32 genera are accepted in Botryosphaeriales, while Botryosphaeriaceae is known to be the largest monophyletic family, including 22 genera and more than 200 species (https://botryosphaeriales.org/, accessed on 15 th April 2023).
The members of Botryosphaeriaceae have been taxonomically characterized based on both sexual and asexual morphs. The production of large, ovoid to oblong, typically hyaline, aseptate ascospores, which may become brown and septate with age, as well as bitunicate asci within unilocular or multilocular botryose ascomata known as pseudothecia is typical to the sexual state (Sivanesan 1984;Phillips et al. 2005). The asexual states of Botryosphaeriaceae exhibit a wide range of conidial morphologies; for example, its conidia can be thin-walled and hyaline, or thickwalled and pigmented, aseptate or 1-2-septate (Phillips et al. 2005). Additonaly, the spermatial states were also frequently observed in Botryosphaeriaceae species, which produced unicellular, hyaline, allantoid to rod-shaped spermatia on culture. Botryosphaeriaceae species are significantly different from other fungi in that the color of its aerial hyphae, changing from gray to black with age on 2% potato dextrose agar (PDA), which can be used for the rapid determination of botryosphaeriaceous fungi.
Woody oil plants are economically important as they are used for the production of cooking and industrial oil. Recently, many Botryosphaeriaceae species have been frequently reported on woody oil plants. Diplodia olivarum was first reported from rotting olive drupes in Italy (Lazzizera et al. 2008) and later it was reported as associated with declining Prunus dulcis trees in Spain (Gramaje et al. 2012). Diplodia insularis was isolated from branch canker of Pistacia lentiscus in Italy (Linaldeddu et al. 2015). Dothiorella gregaria was isolated from the stems with asymptomatic of Zanthoxylum bungeanum in China (Li et al. 2016b). Botryosphaeria dothidea, Diplodia mutila, Di. seriata, Dothiorella iberica, Do. omnivora, Do. sarmentorum, Lasiodiplodia citricola, L. pseudotheobromae, L. theobromae, Neofusicoccum mediterraneum, N. nonquaesitum, N. parvum, N. ribis, N. vitifusiforme, and Neoscytalidium dimidiatum have been reported as pathogens of English walnut (Juglans regia L.) in California (Chen et al. 2014), Chile (Jimenez Luna et al. 2022), China (Li et al. 2016a;Zhang et al. 2017), Iran Panahandeh et al. 2019), South Africa (Cloete et al. 2011), Spain (Gramaje et al. 2012) and USA (Chen et al. 2014). However, very little is known about the Botryosphaerialean species occurring on native woody oil plants in China. Hence, the aim of this study was to gain a more comprehensive understanding of the diversity of Botryosphaeriaceae species associated with common woody oil plants grown in Sichuan Province, China.

Isolates and morphology
The isolates in this study were collected from the woody oil tree plantations in Sichuan Province during the period of 2020 and 2021. The hosts include Acer truncatum, Camellia oleifera, Idesia polycarpa, Olea europaea, Paeonia suffruticosa, Pistacia chinensis, Vernicia fordii and Zanthoxylum bungeanum. The samples were collected from decayed stems, branches and twigs of woody oil trees. Mature fruiting bodies were selected for fungal isolation and for morphological observations under stereo microscope Motic SMZ 168 series. Measurements were made with Tarosoft Image Frame Work program v. 0.9.7 (Liu et al. 2010). Thirty conidia/ascospores were measured per isolate, and 10-30 measurements were taken of other morphological structures. At least 20 conidia/ ascospores were used to calculate the average length/width ratio (L/W). Single spore isolation was conducted in accordance with the methods described in Chomnunti et al. (2014). Germinated spores were individually placed on PDA plates and grown at 25 °C in daylight.
Herbarium specimens were stored in the herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS) and duplicated at Herbarium, University of Electronic Science and Technology (HUEST), Chengdu, China. Living cultures were deposited at China General Microbiological Culture Collection Centre (CGMCC), Beijing, China and duplicated at the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China. MycoBank numbers were registered as outlined in MycoBank (http://www.MycoBank.org. Accessed on 11 th November 2022).

DNA extraction, PCR amplification and sequencing
The total genomic DNA was extracted from 7day-old isolates grown on 2% PDA median at 25 °C, using the EZ geneTM fungal gDNA kit (GD2416), following the manufacturer's instructions and protocols. Partial gene sequences were determined for the internal transcribed spacer 1 and 2 including the intervening 5.8S nrDNA gene (ITS), the nuclear ribosomal 28s large subunit (LSU), the translation elongation factor 1-alphagene (tef1), and the β-tubulin gene (tub2). The primers used for amplification are ITS5/ITS4 for ITS (White et al. 1990), LR0R/LR5 for LSU (Vilgalys and Hester 1990), EF1-728F/EF1-986R for tef1 (Carbone and Kohn 1999) and Bt2a/Bt2b for tub2 (Glass and Donaldson 1995). Polymerase chain reaction (PCR) amplification conditions were followed as of . PCR products were sent for sequencing at Beijing Tsingke Biological Engineering Technology and Services Co. Ltd. (Beijing, P.R. China). All newly generated sequences are deposited in GenBank, and the obtained accession numbers are listed in Table 1.
ML analyses was performed using RAxML (Stamatakis 2006). The tree search included 1,000 non-parametric bootstrap replicates and the best scoring tree was selected from suboptimal trees under the GTRGAMMA substitution model. Maximum  (Nylander 2004) was used to determine the best nucleotide substitution model for each data partition. The Markov Chain Monte Carlo (MCMC) sampling approach was used to calculate the posterior probabilities (PP) (Rannala and Yang 1996). Bayesian analyses of four simultaneous Markov chains were run for 10,000,000 generations with trees sampled every 1,000 th generations. The first 20% of trees, representing the burn-in phase of the analyses, were discarded, and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. PP values equal or greater than 0.95 are marked near each node.

Phylogenetic analyses
A concatenated dataset of ITS and tef1 was used to determine the phylogenetic position of Aplosporellaceae and Botryosphaeriaceae isolates obtained in this study. Combined sequences of ITS and tef1 were used for the analyses of Botryosphaeria, while ITS, tef1 and tub2 were used for the analyses of Diplodia and Dothiorella isolates. All details of the alignments are provided in Table 2.
Three individual phylogenetic trees were constructed for the genera Botryosphaeria, Diplodia and Dothiorella. Twelve isolates belonged to the genus Botryosphaeria and ten of them were nested with Bo. dothidea, while the remaining two isolates clustered with Bo. fabicerciana (Fig. 2). Another twelve isolates were treated in Diplodia and seven isolates were clustered with two known species of Diplodia (Di. mutila and Di. seriata, Fig. 3). The other five isolates did not cluster with any previously known Diplodia species, thus, two novel species were preliminarily identified based on phylogenetic evidence. Eight isolates were nested within Dothiorella and six isolates of them were occupied in the basal position of the Dothiorella tree and formed two well-supported subclades, representing two new species. The other two isolates were nested within the Do. sarmentorum isolates (Fig. 4). Phylogram generated from RAxML analysis based on combined ITS and tef1 sequence data of Botryosphaeriaceae and Aplosporellaceae isolates. The tree was rooted to Lecanosticta acicula (LNPV 252). The ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in red and ex-type strains are marked in bold.  Figure 2. Phylogram generated from RAxML analysis based on combined ITS and tef1 sequence data of Botryosphaeria isolates. The tree was rooted to Barriopsis iraniana (IRAN1448C and IRAN1449C). The ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in red and ex-type strains are marked in bold. The ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in red and ex-type strains are marked in bold.  The ML (≥ 75%) and BI (≥ 95%) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in red and ex-type strains are marked in bold.   Description. Saprobic on decaying branches of Zanthoxylum bungeanum. Sexual morph: Not observed. Asexual morph: Coelomycetous, Conidiomata 558-657 × 216-241 μm (x -= 235.5 × 228.5 μm, n = 10), immersed, partially erumpent when mature, multilocular, locules separated by pale brown cells of textura angularis. Peridium 65-106 μm wide, wall 6-10 cell-layers thick, outer layers composed of 3-4 layers of pale brown cells of textura globulosa, intermediate layers composed of dark brown cells of textura angularis, becoming pale brown towards the inner region. Ostiole 138-171 μm diam., central. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 12-13 × 7.5-8 μm (x -= 12.5 × 8 μm, n = 20), holoblastic, hyaline, cylindrical to doliiform, smooth-walled. Conidia 17-20 × 6.5-7.5 μm (x -= 18.5 × 7 μm, n = 30), L/W ratio = 2.5, ellipsoidal to subcylindrical, with both ends rounded, initially hyaline, becoming dark brown, aseptate.

D o t h i o r e l l a t e c t o n a e
Culture characteristics. Colonies on PDA developing dense aerial mycelium with age, becoming white to gray-brown at the surface, and whitish to yellowish brown at the reverse, producing a brown pigment, with sinuate edges.
Culture characteristics. Colonies on PDA after 7 d, becoming pale olivaceousgray to olivaceous-black at the surface, and olivaceous black at the reverse, with irregular edges.   Damm et al. (2007) in having immersed to erumpent, multilocular conidiomata and brown, smooth-walled, ovoid to oblong conidia. The strain UESTCC 22.0090 is phylogenetically and morphologically similar to A. yalgorensis and A. prunicola, however, A. yalgorensis can be distinguished from other Aplosporella species by its pitted conidial walls. Thus, the strain UESTCC 22.0090 was identified as A. prunicola based on current evidence. This is the first time A. prunicola is reported from Zanthoxylum bungeanum in China. Etymology. The epithet ''acerigena'' refers to the host genus Acer, on which the holotype was collected.
Culture characteristics. Colonies on PDA initially olivaceous buff in the center of the colony and white at the edge, becoming olivaceous within 7 d on the surface, with smooth edge.
Materials examined. China, Sichuan Province, Jiangyou City, Shuanghe County, 31°54'10"N, 104°55'57"E, elevation 657 m, on dead branches of Camellia oleifera, Notes. The phylogenetic tree show that six strains isolated from Camellia oleifera, Olea europaea and Vernicia fordii nested with Diplodia mutila (CBS 112553) with a moderate bootstrap support (ML/BI 86%/1). Diplodia mutila, the type of the genus, is a well-known and most commonly reported species. It has been recorded mainly from woody substrates, and it is known from more than 50 hosts (Batista et al. 2021). Morphologically, one of the isolates obtained in this study UESTCC 22.0068 shares similar conidia shape and size with Di. mutila, but hardly observed the mature conidia with septa. We identify these taxa as Di. mutila based on morphology and phylogeny evidences. This is the first report of Di. mutila, isolated from Camellia oleifera, Olea europaea and Vernicia fordii. Etymology. The epithet ''pistaciicola'' refers to the host genus Pistacia, on which the holotype was collected.
Culture characteristics. Conidia germinate on PDA within 12 h. 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 colony is white, later turning dark olivaceous and dark gray in reverse.
Culture characteristics. Ascospores germinate on PDA within 12 h. Colonies growing on PDA, reaching a diameter of 4 cm after five days at 25 °C, effuse, velvety, with entire to slightly undulate edge.
Notes. The morphology of the taxa isolated from decaying woody oil plants is similar to Diplodia seriata. In the multi-gene phylogenetic analysis, our new collection clustered with the ex-type strain of Di. seriata (CBS 112555) with strong bootstrap support. Diplodia seriata has been isolated from a wide range of hosts (121 species) and has a worldwide distribution (reported in 46 countries) (Batista et al. 2021). This is the first report of Di. seriata isolated from Camellia oleifera. Etymology. The epithet ''camelliae'' refers to the host genus Camellia, on which the holotype was collected.

Sardiniella guizhouensis
Culture characteristics. Conidia germinate on PDA within 12 h. Colonies growing on PDA, reaching a diameter of 7 cm after five days at 25 °C, effuse, velvety, with entire to slightly undulate edge. Surface initially white and later turning dark olivaceous from the surrounding of the colony and dark gray in reverse.
Material examined. Notes. Seven isolates of our collection are morphologically similar to the original description of Sardiniella guizhouensis . The multi-gene phylogenetic analysis showed that the newly obtained isolates clustered together with ex-type of Sa. guizhouensis (CGMCC 3.19222) and this is the first report of Sa. guizhouensis from Pistacia chinensis. Notes. The phylogenetic tree shows that two isolates of Sphaeropsis from our collection clustered together with the ex-type strain of Sp. citrigena (ICMP 16812) with high bootstrap support (ML/BI 100%/1). Sphaeropsis citrigena was first described as Phaeobotryosphaeria citrigena by Phillips et al. (2008), later transferred to Sphaeropsis based on morphological and phylogenetic analyses ). The new collection (UESTCC 22.0105) isolated from Camellia oleifera resembles Sp. citrigena isolated from Citrus sinensis  in the shape of asci and ascospores, though their asci are somewhat smaller than those of Sp. citrigena (93.5-107 × 28.5-33 μm vs. 180-230 × 35-43 μm). In addition, there are no base pair differences in ITS and tef1 sequences of these two strains. We, thus, identify the new collection as Sp. citrigena and this is the first record of Sp. citrigena from Camellia oleifera.   (CGMCC 3.20352) in the phylogenetic analyses of combined ITS and tef1 sequence data with high bootstrap support. A comparison of ITS and tef1 shows that there are no base pair differences between the isolates of UESTCC 22.0104 and CGMCC 3.20352. The new collection is morphologically similar to Sp. guizhouensis, with immersed to erumpent, black ascostromata and biseriate, aseptate, ellipsoid to obovoid, thick-walled conidia. In addition, ascospores become brown and septate when aged. Considering similar morphology and strong molecular evidence, we identify UESTCC 22.0104 as Sp. guizhouensis and this is the first record of Sp. guizhouensis on Camellia oleifera.

Discussion
In this study, 48 Botryosphaeriaceae isolates and two Aplosporellaceae isolates were obtained from woody oil plants in Sichuan Province, China, and they were identified as 16 species based on morphological characters and multi-gene phylogenetic analyses. These species included Aplosporella prunicola, A. ginkgonis, Barriopsis  Descriptions, illustrations and notes were provided for 13 species, and only sequences data were provided for the remaining three species viz. Barriopsis tectonae, Botryosphaeria dothidea and Bo. fabicerciana due to low specimen quality.
According to previous studies, Barriopsis tectonae, Sardiniella guizhouensis, Sphaeropsis citrigena and Sp. guizhouensis have limited geographical distribution. So far, Barriopsis tectonae has been reported from China, Thailand and South Africa (Doilom et al. 2014;Dissanayake et al. 2021). Sardiniella guizhouensis and Sphaeropsis guizhouensis were only found in China while Sp. citrigena was isolated from China, Colombia and New Zealand. It's worth noting that most of the species obtained from this study were also reported previously from Guizhou province . Earlier studies have shown that the distribution of Botryosphaeriaceae species is influenced by the climate condition (Úrbez-Torres et al. 2006;Pitt et al. 2010;Li et al. 2020;Vivas et al. 2021). Thus, we speculate that the adjacent geographical location and similar climatic conditions may be important reasons for the similarity of fungal species isolated from the Sichuan and Guizhou provinces.
The remaining Botryosphaeriaceae species identified in this study are all wellknown and reported from various geographic regions. Botryosphaeria dothidea, Di. seriata and Ne. parvum are recognized to be globally distributed while Di. mutila and Do. sarmentorum are founded only in the temperate and Mediterranean areas. In addition, these species have a broad host range. Batista et al. (2021) reported Neofusicoccum parvum from 223 hosts, B. dothidea from 403 hosts and Di. seriata from 121 hosts. Diplodia mutila and Di. seriata have previously been reported on Olea europaea in Uruguay (Hernández-Rodríguez et al. 2022). Botryosphaeria dothidea was recently isolated from diseased Camellia oleifera in China (Hao et al. 2022). In this study, Bo. dothidea, Di. mutila and Ne. parvum occurred on most of the woody oil plants species we examined. However, some common genera e. g. Lasiodiplodia, Neodeightonia and Phaeobotryon have never been collected from this group of hosts (Fig. 19). The absence of these genera from there is likely a sampling effect.
Aplosporella (Aplosporellaceae) was introduced by Spegazzini (1880) with A. chlorostroma as the genetic type. In a previous study, Aplosporella represents anamorph lineage within the Botryosphaeriaceae. Slippers et al. (2013) later proposed the family Aplosporellaceae to accommodate Aplosporella and Bagnisiella. Aplosporella species are infrequently isolated in China. Aplosporella ginkgonis, isolated from Gansu Province, was first descripted by Du et al. (2017) while Aplosporella macropycnidia was reported in Yunnan Province. Subsequently, Jiang et al. (2021) isolated a new collection of A. prunicola. However, other species have not been recorded in China. Our study revealed new host records of A. ginkgonis and A. prunicola. Though the phylogenetic analyses indicated that A. yalgorensis and A. prunicola have a low genetic divergence (Taylor et al. 2008, in this study), A. yalgorensis is still considered as a different species as it differs from other Aplosporella species (including A. prunicola) by its pitted conidial walls.
Though there are more than 1,000 Diplodia epithets listed in Index fungorum (www. Index Fungorum. Accessed in November 2022), presently only 30 species are accepted in this genus based on phylogenetic analyses (Slippers et al. 2017;Wu et al. 2021). Holomorphic species in Diplodia are Di. tsugae, Di. seriata, Di. mutila and Di. sapinea. This study revealed two previously known Diplodia species, Di. mutila and Di. seriata, and two new species, Di. acerigena and Di. pistaciicola. Among them, Di. acerigena is a holomorphic species, as its sexual stage was observed on the dead branches of Acer truncatum, and the asexual stage produced on culture (PDA). However, the sexual morph of Di. mutila and Di. pistaciicola, as well as the asexual morph of Di. seriata have not been observed on woody oil plants.
Dothiorella was established by Saccardo with Do. pyrenophora as the type species (Saccardo 1880). Recently, Dothiorella encountered a series of revisions as many species in this genus have been reduced to synonymy, such as Do. americana, Do. eriobotryae and Do. iberica Zhang et al. 2021). So far, 31 species are valid in Dothiorella. Most of the species were reported as the asexual morph of Dothiorella and the sexual stage is rarely founded on nature (Dissanayake et al. 2016). Phillips et al. (2013) initiated a link of asexual-sexual morph for Do. sarmentorum, Do. iberica and Do. vidmadera. However, the latter two species were synonymized under Do. sarmentorum (Zhang et al. 2021). In this study, two new species Do. camelliae and Do. zanthoxyli are introduced based on their sexual morphs as well as strong molecular evidences. Besides, new collections of Do. sarmentorum is reported on Pistacia chinensis for the first time.
Multiple molecular systematic studies, mainly of pathogenic fungi of woody plants Slippers et al. 2013;Dissanayake et al. 2021;Zhang et al. 2021), have generated a robust phylogeny for Botryosphaeriaceae. However, the classification and identification of some species in this family remains a major challenge, due to the reasons 1) With the increase of the number of Botryosphaeriaceae species, morphological feature of inter-genera and inter-species is vague, 2) Some species occurred as asexual morph on nature and it is difficult to establish the link of asexual and sexual morph, 3) In general, Botryosphaeriaceae species do not show an obvious host specialization, while some populations displayed a certain degree of host association. Thus, the traditional host-based classification system made taxonomic position confusion of some species. Therefore, collection of more fresh specimens is very important for better understanding the life cycle of Botryosphaeriaceae species, their host range (e. g. native plants) and potential pathogenicity.