﻿Morphology and molecular analyses reveal three new species of Botryosphaeriales isolated from diseased plant branches in China

﻿Abstract The Botryosphaeriales represents an ecologically diverse group of fungi, comprising endophytes, saprobes, and plant pathogens. In this study, taxonomic analyses were conducted based on morphological characteristics and phylogenetic analyses of multi-gene sequence data from four loci (ITS, LSU, tef1-α, and tub2). Thirteen isolates obtained from Beijing and Yunnan Province were identified as seven species of Botryosphaeriales, including Aplosporellajaveedii, Dothiorellaalpina, Phaeobotryonaplosporum and Ph.rhois, and three previously undescribed species, namely Aplosporellayanqingensis, Dothiorellabaihuashanensis, and Phaeobotryonplatycladi. Additionally, the new records of Dothiorellaalpina from the host species Populusszechuanica, Phaeobotryonaplosporum from Juglansmandshurica, and Phaeobotryonrhois from Populusalbavar.pyramidalis are included.

Aplosporellaceae was introduced by Slippers et al. (2013) to accommodate two genera viz. Aplosporella and Bagnisiella Speg. However, Slippers et al. (2013) suggested that Aplosporella and Bagnisiella should be synonymized based on their close phylogenetic relationships and their remarkably similar multiloculate sporocarps. Ekanayaka et al. (2016) agreed with this and provided evidence that the sexual morph of Aplosporella thailandica Ekanayaka, Dissanayaka, Q. Zhao & K.D. Hyde resembles Bagnisiella. Phillips et al. (2019) formally placed Bagnisiella as a synonym of Aplosporella. Sharma et al. (2017) introduced Alanomyces Roh. Sharma in Aplosporellaceae based on four loci phylogeny. Therefore, two genera (Alanomyces and Aplosporella) can be accepted in Aplosporellaceae. The morphological characters of Aplosporellaceae are multiloculate ascostromata, septate pseudoparaphyses, aseptate and ellipsoid to ovoid ascospores, and ellipsoid to subcylindrical and hyaline to pigmented conidia Phillips et al. 2019).
Botryosphaeriaceae was introduced by Theissen and Sydow (1918) for three genera (Botryosphaeria, Phaeobotryon, and Dibotryon Theiss. & Syd.). Over the years the family and genera have undergone several taxonomic revisions and updates. Currently, the Botryosphaeriaceae has approximately 100 verified species in 24 genera, according to DNA sequence data Slippers et al. 2013;Yang et al. 2017;Xiao et al. 2021;Zhang et al. 2021). Botryosphaeria has uniloculate and clustered ascostromata and septate pseudoparaphyses (Phillips et al. 2019). In the phylogenetic tree of Botryosphaeriaceae, hyaline or colored conidia or ascospores are distributed randomly . A large number of new species have been described in recent years, which indicated that the diversity of Botryosphaeriaceae was worthy of further exploration (Bezerra et al. 2021;Zhang et al. 2021;Sun et al. 2022).
With the modern taxonomic approaches applying, more than 30 novel species have been identified in the last five years Rathnayaka et al. 2022;Sun et al. 2022;Wang et al. 2023). Considering the important economic status of Botryosphaeriales, a survey to explore more hidden species of Botryosphaeriales was considered imperative. Thus, a survey on the diversity of Botryosphaeriales on diseased branches was conducted in Beijing and Yunnan Province from 2021 to 2022. In this study, we introduce three new species, in which Aplosporella yanqingensis and Phaeobotryon platycladi were collected from Platycladus orientalis and Dothiorella baihuashanensis were collected from Juniperus chinensis in China. Moreover, the newly discovered Dothiorella alpina from Populus szechuanica, Phaeobotryon aplosporum from Juglans mandshurica, and Ph. rhois from Populus alba var. pyramidalis are featured.

Fungal isolation
Fresh specimens (woody branches and twigs with canker or dieback symptoms) were randomly collected in Beijing and Yunnan Province from the summer of 2021 to the autumn of 2022. The specimens were packed in kraft paper bags and transferred to the laboratory for fungal isolation following Jiang et al. (2022). Isolates were obtained by removing the spore mass from conidiomata to sterilised distilled water using sterilised needle, and generating single spore colonies on potato dextrose agar (PDA: 200 g potatoes, 20 g dextrose, 20 g agar per L) at 25 °C in the dark. After three to five days, hyphal tips were transferred to new PDA plates twice to obtain a pure culture. The cultures are deposited in the China Forestry Culture Collection Center (CFCC; http:// www.cfcc-caf.org.cn/), and the specimens in this study are deposited in the Museum of the Beijing Forestry University (BJFC).

Morphology
Morphological observations were conducted based on conidiomata produced on infected plant tissues. The conidiomata were manually sectioned using a double-edged blade and examined under a dissecting microscope for macroscopic and microscopic characterization, while conidiomata structure and size were imaged with a Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). Conidia and other microstructures were selected randomly for observation using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DSRi2 high-definition colour camera with differential interference contrast (DIC). Fifty conidia were measured per species, and 30 measurements were taken of other morphological structures. Colony characters i.e. colours and texture on PDA and MEA (malt extract agar; 30 g malt extract, 5 g mycological peptone, 15 g agar per L) at 25 °C were observed and noted over 14 days. The colony colours were determined based on the colour charts of Rayner (1970).

DNA extraction, amplification and sequencing
The fresh mycelium from PDA was scraped and put it in a 1.5 mL centrifuge tube for genomic DNA extraction which used the modified CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced using the primer pairs ITS1/ ITS4 (White et al. 1990) for all isolates. The BLAST tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi) was used to compare the resulting sequences with those in GenBank. After confirmation to the genus level, additional partial loci were amplified, including the nuclear ribosomal large subunit (LSU), the partial translation elongation factor 1-alpha (tef1-α), and partial beta-tubulin (tub2) using the primer pairs LR0R/LR5 (Vilgalys and Hester 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The additional combination of T1 and Bt2b (Glass and Donaldson 1995;O'Donnell and Cigelnik 1997) was used in case of amplification failure of the primer Bt2a and Bt2b. The genes used in different genera and the amplification conditions are listed in Table 1. The PCR mixture for all regions consisted of 1 µL DNA template, 1 µL each 10 µM primer, 10 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg 2+ , Beijing TisingKe Biotech Co., Ltd., Beijing, China), and 7 µL sterile water. PCR products were electrophoresed in 1% agarose gel and the DNA was sequenced by the SinoGenoMax Company Limited (Beijing, China). The forward and reverse reads were edited and assembled with Seqman v. 7.1.0 in the DNASTAR Lasergene core suite software (DNASTAR Inc., Madison, Wisconsin USA). All sequences generated in this study were submitted to GenBank (Suppl. material 1).

Phylogenetic analyses
The sequences obtained in this study were supplemented with additional sequences obtained from GenBank (Suppl. material 1) based on BLAST searches and from relevant published literature on the related genera (Bezerra et al. 2021;Wijayawardene et al. 2021;Xiao et al. 2021;Zhang et al. 2021;Peng et al. 2023). The individual data-sets of each gene region were aligned separately using MAFFT v. 6.0 (Katoh and Standley 2013) and trimmed at both terminal ends in MEGA v. 6.0 (Tamura et al. 2013). Maximum Likelihood (ML) analyses were conducted for the single gene sequence data sets (ITS and tef1-α regions for Aplosporella; ITS, tef1-α, and tub2 regions for Dothiorella; ITS, LSU, and tef1-α regions for Phaeobotryon). Then the combined data set of each genus of all gene regions were used for multi-gene phylogenetic analyses including Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Alanomyces indica (CBS 134264), Lasiodiplodia americana (CFCC 50065), and Alanphillipsia aloeicola (CBS 138896) were selected as the outgroup taxa for Aplosporella, Dothiorella, and Phaeobotryon analyses respectively.  quist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 1,000,000 generations, resulting in a total of 10,000 trees. The first 25% of trees sampled were discarded as the burn-in phase of each analysis. The posterior probabilities (BPP) were calculated from the remaining trees (Rannala and Yang 1996). Phylogenetic trees were shown using FigTree v .1.4.4 (Rambaut 2018) and processed by Adobe Illustrator 2019.

Phylogenetic analyses
The BLAST results indicated that the 13 isolates in this study resided in Aplosporella, Dothiorella, and Phaeobotryon. Datasets for the three genera, the number of characters of each gene with gaps and the substitution models used for BI analyses are provided in Table 2. The topologies of BI analyses did not significantly differ from the ML analyses.

Species of Dothiorella
Three isolates clustered in two clades for the individual genes (ITS, tef1-α, and tub2), as well as the combined gene dataset (Fig. 2). In ML analysis based on the combined gene dataset, the matrix had 478 distinct alignment patterns. Estimated base frequencies are as follows:

Species of Phaeobotryon
Five isolates clustered into three clades for the individual genes (ITS, LSU, and tef1-α), as well as the combined gene dataset (Fig. 3). In ML analysis based on the combined gene dataset, the matrix had 223 distinct alignment patterns. Estimated base frequencies are as

Taxonomy
Based on DNA sequences and morphology, seven species belonging to three genera were identified. Of these, Aplosporella javeedii, Dothiorella alpina, Phaeobotryon aplosporum, and Ph. rhois are known species. The remaining three species are identified as new species (Aplosporella yanqingensis, Dothiorella baihuashanensis, and Phaeobotryon platycladi) and described below. Collect information and notes of all seven species were provided. Etymology. Named after the collection site of the type specimen, Yanqing District in Beijing City.
Culture characters. Colonies on PDA spreading, covering a 90 mm plate after 14 days at 25 °C, upper white to pale grey, reverse buff to dark grey. Colonies on MEA spreading, covering a 90 mm plate after 14 days at 25 °C, uniform with appressed aerial mycelium and crenate edge, upper white to pale grey, reverse honey to dark grey. Notes. The isolates CFCC 58549 and 58788 in this study formed a distinct linage in the phylogenetic trees of each individual gene (ITS, tef1-α, and tub2) and the combined gene dataset (Fig. 2). They were isolated from the branches Juniperus chinensis. Dothiorella iberica was also recorded to host genus Juniperus . However, these two species are not closely related in our phylogenetic analysis. Notes. Phaeobotryon aplosporum was first discovered from Rhus typhina and Syzygium aromaticum (Pan et al. 2019). It can be distinguished from other species in Phaeobotryon by its aseptate conidia (Pan et al. 2019). In this study, the conidia formed on the specimen BJFC CF20230112 are dark brick when mature, aseptate, (16.5-20.0 × 6.0-9.0 µm (av. ± S.D. = 18.3 ± 1.1 × 7.5 ± 0.8 µm), which overlap with the morphological characteristics described by Pan et al. (2019). Phylogenetically, the isolates CFCC 58596 and 58784 were clustered in a clade with Ph. aplosporum with high statistical support (ML/BI = 99/1). Therefore, the isolates CFCC 58596 and 58784 are identified as Ph. aplosporum. The current study extends its host range to Juglans mandshurica.   Notes. Phaeobotryon platycladi is monophyletic with Ph. cupressi in the phylogenetic tree without a significant statistical support. Conidial sizes of the two species overlap, but there are differences in 6/488 in ITS region, 3/556 in LSU region, and 18/293 in tef1-α gene with gaps. Notes. Phaeobotryon rhois was first discovered on Rhus typhina distributed in Ningxia Province, China (Fan et al. 2015). Pan et al. (2019) reported this species from Dioscorea nipponica, Platycladus orientalis and Rhamnus davurica in Beijing, China. The current study extends its host range to Populus alba var. pyramidalis.

Discussion
In this study, a total of 13 isolates are identified as seven species of Botryosphaeriales, including three new species (Aplosporella yanqingensis, Dothiorella baihuashanensis, and Phaeobotryon platycladi) and four known species (A. javeedii, Do. alpina, Ph. aplosporum, and Ph. rhois). All three new species were isolated from coniferous trees: A. yanqingensis and Ph. platycladi from Platycladus orientalis and Do. baihuashanensis from Juniperus chinensis. Furthermore, the new records of Do. alpina from the host species Populus szechuanica, Ph. aplosporum from Juglans mandshurica, and Ph. rhois from Populus alba var. pyramidalis are included.
The fungi of Botryosphaeriales play various ecological roles, such as saprotrophs, endophytes, or plant pathogens (Phillips et al. 2005(Phillips et al. , 2008Luque et al. 2016). Some fungi exhibit strong pathogenicity, leading to severe diseases in different parts of various plants, such as Botryosphaeria dothidea, which can cause apple ring rot of stems and fruits (Zhang et al. 2016b), as well as poplar cankers (Li et al. 2019), and the dieback and leaf spot diseases of Euonymus japonicus (Lin et al. 2023). Sometimes their ecological roles change, such as Diplodia sapinea, which is both an endophytic and a plant pathogenic fungus . In this article, all species were isolated from diseased plant tissues, and their pathogenicity remains to be verified.
In this study, both Dothiorella and Phaeobotryon belong to Botryosphaeriaceae. Slippers et al. (2013) mentioned that some morphological features within Botryosphaeriaceae are not always stable, such as pigment production of conidia. These features might have already existed before the diversification of the group and have undergone further changes later . In this study, only aseptate conidia were observed in Phaerobotryon platycladi, and they may become pigmented with age. Moreover, whether septate or not seem to be an unstable characteristic throughout the genus Phaerobotryon. Phillips et al. (2013) mentioned that in most cases, the conidia of Phaeobotryon have two septa when mature. However, both the Phaeobotryon aplosporum observed in this study and the one described by Pan et al. (2019) have pigmented but without septa. Phaeobotryon rhoinum also shows pigmented and aseptate conidia (Daranagama et al. 2016). Other species of Phaeobotryon with pigmented and septate conidia are either saprobic or pathogenic, but Ph. aplosporum and Ph. rhoinum are both pathogenic (Rathnayaka et al. 2023). The phylogenetic state analysis of the trophic pattern, conidial colour, and separation of Botryosphaeriales conducted by Rathnayaka et al. (2023) indicate that this may correspond to nutritional mode.