Discovery of Three Novel Cytospora Species in Thailand and Their Antagonistic Potential

: During an ongoing research survey of saprobic fungi in Thailand, four coelomycetous strains were isolated from decaying leaves in Chiang Mai and Phitsanulok Provinces. Morphological characteristics demonstrated that these taxa are typical of Cytospora in forming multi-loculate, entostromatic conidiomata, branched or unbranched conidiophores, with enteroblastic, phialidic conidiogenous cells and hyaline, allantoid, aseptate conidia. Multiloci phylogeny of ITS, LSU, ACT, RPB2, TEF1- α and TUB2 conﬁrmed these taxa are distinct new species in Cytospora in Cytosporaceae (Diaporthales, Sordariomycetes), viz., Cytospora chiangmaiensis , C. phitsanulokensis and C . shoreae . Cytospora chiangmaiensis has a close phylogenetic relationship with C . shoreae , while C. phitsanulokensis is sister to C. acaciae . These three novel species were also preliminary screened for their antagonistic activity against ﬁve plant pathogenic fungi: Colletotrichum fructicola , Co. siamense , Co. artocarpicola , Co. viniferum and Fusarium sambucinum . Cytospora shoreae and C. phitsanulokensis showed >60% inhibition against Co. viniferum and F. sambucinum, while C. chiangmaiensis had moderate inhibition activity against all pathogens.

During a survey of saprobic fungi in Thailand, three Cytospora species were collected and isolated. Based on morphological characteristics and phylogenetic analyses, our strains were identified as new species of Cytospora. In addition, these fungi were tested in vitro as the first step for screening new biocontrol agents against pathogenic fungi.

Collection and Isolation of Fungi
Decaying leaves were collected from disturbed forests in Chiang Mai and Phitsanulok Provinces in Thailand. The forests were disturbed due to the utilization of bioresources by the local communities. Samples were kept in plastic bags with labels of location, date, host and collector details before being taken to laboratory for morphological observation. We followed Senanayake et al. [28] for single spore isolation by using potato dextrose agar (PDA) and incubating at room temperature (28 • C). Pure cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and type specimens were deposited in the Herbarium of Mae Fah Luang University (MFLU). New taxa were registered in Faces of Fungi [29] and Index Fungorum databases [19].

Morphological Observation
Conidiomata on host surface were examined using a Motic SMZ 168 Series stereo microscope (Motic Incorporation Ltd., Hong Kong). Hand-sectioning of conidiomata was carried out and the sections were mounted on a slide with a drop of distilled water. Morphological characteristics including structure and size of stromata, ectostromatic disc, ostioles as well as shape and size of conidiogenous cells, conidiophores and conidia were observed and photographed using a Nikon ECLIPSE 80i compound microscope equipped with a Canon EOS 600D digital camera. Microscopic elements were measured using the Tarosoft (R) Image Frame Work program. The measurements of each structure were represented as minimum value-maximum value (x = sum of all measurements/n, n = number of measurements). The figures were processed using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, San Jose, CA, USA).

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was extracted from fresh mycelium which was grown on PDA for 1-2 weeks using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China) following the manufacturer's protocol. The amplification of specific ribosomal DNA regions was carried out using two gene regions, including the internal transcribed spacers region of ribosomal DNA (ITS) [30] and the partial 28S large subunit nuclear ribosomal DNA (LSU) [31], and four protein coding gene regions: the RNA polymerase II second largest subunit (RPB2) [32], α-actin (ACT) [33], the translation elongation factor 1-α (TEF1-α) [33] and beta-tubulin (TUB2) [34]. The final volume of PCR mixtures was 25 µL, including 8.5 µL ddH 2 O, 12.5 µL 2× PCR MasterMix (TIANGEN Co., Beijing, China), 2 µL DNA template and 1 µL of each forward and reverse primer. The PCR primers and conditions for each gene regions are described in Table 1. The purification and sequencing of PCR products were conducted by TsingKe Company (Kunming, China).  [34] ACT (512F/783R) [33] An initial denaturation step of 5 min at 96 • C, followed by 35 cycles of 40 s at 95 • C, 30 s at 58 • C and 1 min at 72 • C, and a final extension step of 5 min at 72 • C RPB2 (fRPB2-5F/fRPB2-7cR) [32] An initial denaturation step of 5 min at 95 • C, followed by 40 cycles of 1 min at 95 • C, 1 min at 52 • C and 90 s at 72 • C, and a final extension step of 10 min at 72 • C
Maximum likelihood (ML) analysis was performed by RAxML-HPC2 (v.8.2.12) on XSEDE implemented in the CIPRES Science Gateway web server (http://www.phylo.org accessed on 1 August 2021; [37] using 1000 rapid bootstrap replicates and the GTR + GAMMA + I substitution model. Maximum parsimony (MP) analysis was generated by PAUP (Phylogenetic Analysis Using Parsimony) v.4.0b10 [38] using the heuristic search option and 1000 random sequence additions. The branch-swapping was analysed using tree-bisection reconnection (TBR) algorithm. Maxtrees was set up at 1000 and all characters were unordered and of equal weight. The branches of zero length were collapsed and gaps were treated as missing data. All multiple and equally parsimonious trees were saved. The stability of the most parsimonious tree was evaluated by a bootstrap analysis with 1000 replicates, each with 100 replicates of random stepwise addition of taxa.  [39]. GTR + I + G was selected as the best-fitting model for LSU, RPB2, TEF-α and TUB2 datasets, SYM + I + G for the ITS dataset and HKY + I + G for the ACT dataset. BI analysis was conducted by Markov chain Monte Carlo sampling (BMCMC) to assess posterior probabilities (PP) [40,41] using MrBayes v3.1.2 [42]. Six simultaneous Markov chains were run for random trees for 10,000,000 generations, and trees were sampled every 1000th generation. The effective sampling sites (ESS) of initial trees were checked using the Tracer v. 1.6 [43]. The first 10% of generated trees were discarded, and the remaining trees were used to calculate posterior probabilities (PP) in the majority rule consensus tree (the standard deviation of split frequency lower than 0.01). Bootstrap support values for ML and MP equal to or greater than 60% and Bayesian posterior probabilities (PP) equal to or greater than 0.95 were given above the nodes in the phylogenetic tree (Figure 1).

Genealogical Concordance Phylogenetic Species Recognition Analysis
New species and phylogenetically related species were analyzed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by conducting a pairwise homoplasy index (PHI) test as described by Bruen et al. [45] and Quaedvlieg et al. [46]. The PHI test was conducted in SplitsTree4 [47,48] to examine the recombination level within phylogenetically closely related species using a six-locus concatenated dataset (ITS, LSU, ACT, RPB2, TEF1-α and TUB2). The significant recombination in the dataset was indicated by PHI value below 0.05 (Φw < 0.05). The results were visualized by generating a split graph, using both the LogDet transformation and split decomposition options.

Preliminary Screening of Antagonistic Activity against Fungal Pathogens
The fungal pathogens, Colletotrichum artocarpicola (MFLUCC 18-1167), Co. fructicola (MFLUCC 18-1160), Co. siamense (MFLUCC 18-1162), Co. viniferum (MFLUCC 18-1179) and Fusarium sambucinum (MFLUCC 17-1056) were obtained from MFLUCC and used for the antagonistic activity test. Fungal isolates were screened using in vitro dual culture assays for their ability to suppress the mycelial growth of fungal pathogens. An antagonism test was performed with 10-day-old cultures of pathogens and new strains. Fresh cultures (pathogens and our strains) 10 days after incubation were used for the antagonism test. A fungal pathogen disc (5 mm) was placed 3 cm from the margin of the PDA plate (9 cm in diam.). An antagonist fungus disc (5 mm) was also placed in a similar manner but on the direct opposite of the pathogen disc. The plate was incubated at room temperature (28 • C) for 10 days. Plates inoculated with a fungal pathogen in the absence of an antagonistic fungus were used as negative controls. The assay was replicated three times. Observations were carried out for the 3rd, 5th, 7th and 10th days. Clear inhibition zone was recorded and the percentage inhibition in mycelial growth was calculated using the following formula [49]: I% = [(R1-R2)/R1] × 100, where I% = the percentage inhibition, R1 = the radial growth of test pathogen in a control plate and R2 = the radial growth of test pathogen in the direction of antagonistic fungus. Data were statistically analyzed with ANOVA using SPSS version 22 (SPSS, Inc., Chicago, IL, USA). Tukey's HSD test was used to determine the significant differences between treatments at p ≤ 0.05. In the phylogenetic analyses (Figure 1), two new species, Cytospora chiangmaiensis (MFLUCC 21-0049) and Cytospora shoreae (MFLUCC 21-0047, MFLUCC 21-0048), clustered in a monophyletic lineage but well separated branch with strong bootstrap support (94% ML/99% MP/1.00 PP). Another new species, Cytospora phitsanulokensis (MFLUCC 21-0046), formed an independent branch adjacent to C. acaciae, C. magnoliae and C. italica with high bootstrap support (100% ML, 100% MP, 1.00 PP; Figure 1).

Phylogenetic Analyses
A pairwise homoplasy index (PHI) test revealed no significant recombination event between Cytospora phitsanulokensis and the closely related taxa, C. acaciae, C. italica and C. magnoliae (Figure 2). There was also no significant recombination among Cytospora chiangmaiensis, C. shoreae, C. thailandica, C. diopuiensis and C. xinglongensis (Figure 2). This evidence supports that they are different species. The significant recombination between two strains of Cytospora shoreae (MFLUCC 21-0047 and MFLUCC 21-0048) indicate that they are conspecific (Figure 3).    Etymology: Name reflects the locality, Chiang Mai Province, Thailand, where the holotype was collected.

Cytospora shoreae
Culture characteristics: Conidia germinating on PDA within 24 h germ tubes produced from both poles. Colonies on PDA reached at 6 cm diam. after 7 days at 28 • C, irregular in shape, surface slightly rough, effuse, slightly raised, with undulate margin, medium dense, pale brown to white, in reverse pale yellowish to white. Notes: Cytospora shoreae formed a sister clade to C. chiangmaiensis with 94% ML, 99% MP and 1.00 PP statistical support (Figure 1). Cytospora chiangmaiensis and C. shoreae share similar morphology in the size and characteristics of conidiogenous cells and conidia. However, C. chiangmaiensis has circular shaped conidiomata. Cytospora shoreae has larger, flask-shaped conidiomata. The single gene comparison of ACT, RPB2, TEF1-α and TUB2 showed that there are significant nucleotide differences (more than 1.5%) between Cytospora chiangmaiensis and two strains of C. shoreae (MFLUCC 21-0047 and MFLUCC 21-0048; Table  3) and this provides evidence that they are different species [50]. Cytospora shoreae differs from C. lumnitzericola and C. platycladi by its longer conidia [1,5] (Table 4). Cytospora shoreae is distinguished from C. thailandica by having longer conidiogenous cell and conidia [5] ( Table 4). Cytospora shoreae is distinct from C. xinglongensis in having longer conidiogenous cells and shorter conidia [16] (Table 4). Cytospora shoreae, strains MFLUCC 21-0047 and MFLUCC 21-0048 showed similar characteristics of conidiomata, conidiogenous cells, conidia and culture characteristics, with no significant differences. They were also collected from the same site with the notes of different hosts, where the holotype was isolated from Shorea sp. and the paratype was isolated from unidentified host. The single gene comparison of ITS, LSU, ACT, RPB2, TEF1-α and TUB2 showed that there is no significant difference between two strains of Cytospora shoreae (MFLUCC 21-0047 and MFLUCC 21-0048; Table 3), and this confirms that these two strains are the same species [50].  Figure 6. Etymology: Name reflects the locality, Phitsanulok Province, Thailand, where the holotype was collected.
Culture characteristics: Conidia germinating on PDA within 24 h germ tubes produced from both poles. Colonies on PDA reached at 8 cm diam. after 7 days at 28 • C, circular in shape, effuse, slightly raised, with entire margin, medium dense, floccose, white, in reverse pale yellowish to white.
Notes: Phylogenetically, Cytospora phitsanulokensis forms a distinct lineage and is closely related to C. acaciae, C. magnoliae and C. italica with 100% ML, 100% MP and 1.00 PP statistical support ( Figure 1). Cytospora phitsanulokensis differs from C. italica by its larger conidiomata, shorter conidiogenous cell and longer conidia [51] (Table 4). Complete descriptions of C. acaciae and C. magnoliae were not available for morphological comparison [11,52]. Adam et al. [11] reported that Cytospora acaciae produces phialides with a long narrow channel of apical pores with lipid globules at one end of conidium; however, these characteristics were not observed in C. phitsanulokensis.

Discussion
This study provides taxonomic novelties of Cytospora species discovered from Thailand and their antagonistic activities against fungal pathogens. We show that six-locus phylogeny (ITS, LSU, ACT, RPB2, TEF1-α and TUB2) facilitates species delineation in Cytospora which is consistent with previous studies [1,3,17,18]. In addition, Cytospora chiangmaiensis and C. shoreae are phylogenetically closely related to C. diopuiensis, which was also collected from Chiang Mai, Thailand [2], indicating the close geographical relationship of these taxa. Based on the Fungus-Host USDA database [53], two new species, Cytospora chiangmaiensis and C. shoreae, are recorded for the first time on Shorea sp. (Dipterocarpaceae). Cytospora phitsanulokensis collected from Phitsanulok, Thailand, is phylogenetically closely related to C. acaciae, C. italica and C. magnoliae. The GCPSR analyses provided evidence for Cytospora phitsanulokensis as a separate species. However, the presence of recombination among strains of C. acaciae, C. italica and C. magnoliae shown in the spilt graph ( Figure 2) may be caused by the lack of some gene regions in the dataset. Thus, more strains and new sequence data of C. italica and C. magnoliae should be obtained to better confirm their taxonomic placements in the C. acaciae clade.
Cytospora species are discovered in woody substrates such as bark, branches and twigs [1]. It should be noted that the stromata of Cytospora were only detected on vein and petioles, but not on leaf lamina for all our samples. Adams et al. [11] mentioned that the formation of stroma of Cytospora could possibly decrease in leaves compared to bark. Cytospora species are known as saprobes on dead plants and are one of the important plant pathogens causing dieback and canker diseases on a wide range of hosts [1][2][3]5,9,[16][17][18]23]. Considering that the species number of Cytospora is ever-increasing and related taxonomic knowledge continues to expand [1], more extensive sampling of both fresh and dead plant samples in selected hosts and locations should be implemented to improve and stabilize the identification and classification of Cytospora species. In addition, Cytospora are interesting fungal taxa for screening potential biological activities [24,25,27]. Previous studies reported novel bioactive compounds produced by Cytospora spp. [24,25,27,54]; however, species identification of these potential strains was not established. The taxonomic classification and phylogenetic relationships can be used as important tools for the screening of biologically active strains and their biological activities for further applications [55,56].
In this study, we identified new Cytospora species and demonstrated that different strains and/or species can inhibit fungal pathogens. However, our study only conducted a preliminary screening of Cytospora that can inhibit fungal pathogens, using the dual culture method. Although our new species were identified as saprobes based on their occurrence on decaying leaf substrates, their pathogenicity should be clarified, as this genus is well known for pathogens. Therefore, to confirm whether our Cytospora strains are true saprobes, further research is needed. Further studies will be carried out in the near future to check the pathogenicity of the new Cytospora species, elucidate the biology of these fungi and screen secondary metabolites.