Species diversity of Pleosporalean taxa associated with Camellia sinensis (L.) Kuntze in Taiwan

Pleosporales species are important plant pathogens, saprobes, and endophytes on a wide range of economically important plant hosts. The classification of Pleosporales has undergone various modifications in recent years due to the addition of many families described from multiple habitats with a high level of morphological deviation. Numerous asexual genera have been described in Pleosporales that can be either hyphomyceteous or coelomycetous. Phoma- or coniothyrium-like species are common and have been revealed as polyphyletic in the order Pleosporales and linked with several sexual genera. A total of 31 pleosporalean strains were isolated in different regions of Taiwan between 2017 and 2018 from the leaves of Camellia sinensis plants with symptoms of leaf spot disease. These strains were evaluated morphologically and genotypically using multi-locus sequence analyses of the ITS, LSU, SSU, rpb2, tef1 and tub2 genes. The results demonstrated the affiliation of these strains with the various families in Pleosporales and revealed the presence of one new genus (Neoshiraia) and eight new species (Alloconiothyrium camelliae, Amorocoelophoma camelliae, Leucaenicola camelliae, L. taiwanensis, Neoshiraia camelliae, N. taiwanensis, Paraconiothyrium camelliae and Paraphaeosphaeria camelliae). Furthermore, to the best of our understanding, Didymella segeticola, Ectophoma pomi and Roussoella mexican were reported for the first time from C. sinensis in Taiwan.

phylogeny of pleosporales based on concatenated alignment of six molecular markers (itS, LSU, SSU, rpb2, tef1 and tub2). The dataset consisted of 5,229 characters (ITS 422, LSU 945, SSU 1,034, rpb2 1,080, tef1 938 and tub2 810). A best scoring RAxML tree is shown in Fig. 1, with the likelihood value of − 79,379.027274. The Bayesian analysis resulted in 90,000 trees after 90,000,000 generations. The first 18,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining trees were used for calculating Bayesian posterior probability (PP) in the majority rule consensus tree. All methods resulted in largely the same topology with high support for most branches in the ML and BI analyses and with similar overall topologies of family and genus level relationships in agreement with previous work based on ML and BI analyses [1][2][3][4][5]10,18,19 . A total of 229 strains representing 42 families including the 31 strains generated in the present study were included in the final Pleosporales alignment.
The family Shiraiaceae was resolved into four distinct clades (A, B, C and D). Shiraia (Clade A) includes the generic type of the genus, S. bambusicola, and putatively named Shiraia species strains JP256, JP93, JP7, and JP232; the new genus Neoshiraia (Clade D) with two species N. camelliae sp. nov and N. formosanum sp. nov. However, putatively named Shiraia species strains JP119, JP151 (Clade B) and JP185 (Clade C) formed two distinct clades basal to the Neoshiraia clade.
Didymella segeticola (NTUCCH 17-004) isolated in this study clustered in a well-supported clade with another isolate of D. segeticola (CGMCC 3.17498) that was used by Chen et al. 18 to describe the species, therefore confirming the identification of the studied species. In addition, Ectophoma pomi (NTUCC 17-034) isolated from C. sinensis formed a highly supported clade with isolate CBS 267.92 of E. pomi that was examined by Valenzuela-Lopez et al. 5 to describe the taxon, thus verifying the identification of the studied species. Furthermore, Pyrenochaetopsis americana (NTUCC 17-033) used in this study grouped in a well-supported clade with the type strain of P. americana (UTHSC D116-225T) that was used by Valenzuela-Lopez et al. 5 to describe the species, hence validating the identification of the studied species.
The family Bambusicolaceae was composed of three clades, which correspond to the genera Bambusicola, Leucaenicola and Palmiascoma. For both ML and BI, and with both single locus and concatenated datasets, the four strains of Leucaenicola (NTUCC 18-093-1 to NTUCC 18-093-4) formed a distinct clade with high statistical support that was sister to the clade representing L. aseptata (MFLUCC 17-2423). Therefore, the new lineage is introduced here as a new species, L. camelliae sp. nov. Finally, the two strains of Leucaenicola (NTUCC 18-094-1 and NTUCC 18-094-2) isolated in the present study formed a basal terminal clade in Leucaenicola with both single locus and concatenated datasets. Thus, this new lineage is presented here as the new species L. taiwanensis sp. nov.
The family Thyridariaceae, which was highly supported in both ML and BI analyses, resulted in two clades that we identify as the genera Thyridaria and Roussoella. R. mexicana NTUCC 18-099-1, NTUCC 18-099-2 and NTUCC 18-099-3 in this study grouped in a well-supported clade with R. mexicana (CPC 25355) that was used by Crous et al. 20 to introduce the species, therefore confirming the identification. The family Amorosiaceae grouped into four terminal clades, which correspond to known genera Angustimassarina, Amorocoelophoma, Amorosia, and Neothyrostroma. For both ML and BI, the three strains of Amorocoelophoma (NTUCC 18-097-1, NTUCC 18-097-2 and NTUCC 18-097-3) formed a distinct clade with high statistical support and sister to the clade representing A. cassiae (MFLUCC 17-2283) in both single locus and concatenated datasets. Thus, the novel lineage is presented here as A. camelliae sp. nov. phylogeny of the family Didymosphaeriaceae based on the concatenated alignment of four molecular markers (itS, LSU, SSU, and tub2). The dataset in this analysis consisted of 2,970 characters (ITS 525, LSU 923, SSU 1,025, and tub2 497). The Bayesian analysis resulted in 10,000 trees after 10,000,000 generations. The first 20% of trees, representing the burn-in phase of the analysis, were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus tree. A best scoring RAxML tree is shown in Fig. 2, with a likelihood of − 15,447.350059. All methods resulted in the same topology    Etymology The name reveals the fact that species in this genus are comparable to, but dissimilar from those in the genus Shiraia.
A new genus is introduced here in Shiraiaceae to accommodate two coelomycetous species isolated from C. sinensis in Taiwan. Shiraia Henn. can be distinguished from Neoshiraia in having fusiform, muriform, asymmetrical, hyaline to light brown conidia whereas Neoshiraia possesses hyaline, aseptate, obovoid to ellipsoidal conidia. Moreover, Shiraia-like species are either parasitic or endophytic on bamboo, while Neoshiraia species are reported on leaf lesions of C. sinensis (tea).
Notes In the present study we introduce another Alloconiothyrium species, A. camelliae from C. sinensis in Taiwan. Alloconiothyrium aptrootii can be distinguished from A. camelliae due to pycnidial or eustromatic conidiomata consisting of complexes with several cavities and covered by grey mycelium, discrete, broadly ampulliform, holoblastic, annellidic conidiogenous cells often with an elongated neck showing several distinct percurrent proliferations and verruculose conidia. In contrast, A. camelliae has uni-loculate, globose to subglobose conidiomata, ampulliform to doliiform or cylindrical conidiogenous cells and smooth-walled conidia 25,26 .
In a recent study Crous et al. 27 introduced a species having coniothyrium-like morphology and cautiously classified it as A. encephalarti. However, in our multi-gene phylogeny, the type strain of A. encephalarti (CBS 146012) clustered in the family Didymosphaeriaceae but was separated from the generic clade of Alloconiothyrium and other genera of the family in a clade with relatively low statistical support (Fig. 2B). Therefore, generic placement of the tentatively named A. encephalarti strain in Didymosphaeriaceae remains unresolved. However, both A. aptrootii and A. camelliae can be easily distinguished from A. encephalarti by the shape and the sizes of the conidiogenous cells and conidia.

Discussion
Recently, large number of fungal species have been identified through phylogenetic studies based on DNA sequence data. These studies have helped scientists to understand the cryptic nature of many pathogenic fungal groups classified in the order Pleosporales such as Alternaria 10,30 , Phoma 25 , Coniothyrium 25 , Paraconiothyrium 25,29 and Paraphaeosphaeria 25,26,29 . Furthermore, these studies suggest that multi-gene phylogenies together with phenotypic features are necessary to identify the species/genus of fungal pathogens classified in Pleosporales 10,25,30 . Fungal disease development and weather conditions are highly correlated and this relationship is regularly accounted for in disease management and forecasting 31 . However, disease severity and the epidemiology of fungal pathogens can vary over time due to climatic change 31 . In recent years, the general climate patterns have been drastically changed mainly due to global warming, resulting in major effects on natural ecosystems 32 . As a direct consequences of this scenario, threats from unpredicted incidences of plant diseases have increased [33][34][35] .
Camellia sinensis (tea) is an evergreen shrub that is widely cultivated throughout tropical and subtropical regions of Asia and Africa. Tea is reported to have a wide range of beneficial physiological and medicinal effects [36][37][38][39] . Several fungal pathogens and endophytes are associated with the tea plant and fungal pathogens cause a significant threat to tea leaves [40][41][42] . Brown blight and leaf blotch caused by Colletotrichum species, grey blight caused by pestalotiopsis-like species, blister blight (Exobasidium vexans Massee), twig die-back and stem canker (Macrophoma theicola Petch) are common fungal diseases affecting tea plantations in major tea growing countries 12 . However, the global species diversity of Pleosporalean taxa associated with C. sinensis have received limited attention.
In the present study, we identified one genus and eight species associated with leaf spots of C. sinensis in Taiwan that are new to science. Apart from the novel species introduced, D. segeticola, E. pomi and Roussoella mexican were identified for the first time as foliar pathogens of C. sinensis. Our results can be used to develop a system to characterize and identify pleosporalean species in common practice and discover potential biocontrol agents either to cure or minimize the damage done by phytopathogenic pleosporalean taxa linked with tea leaves in Taiwan.

Materials and methods
Sample collection and isolation. The field survey was carried out in seven organic and three commercial tea fields, and three tea research stations (Nov. 2017 to Sep. 2018) in the nine main tea cultivation provinces (Taipei, New Taipei, Taoyuan, Yilan, Hsinchu, Nantou, Chiayi, Hualien, Pingtung and Miaoli) in Taiwan. Diagnostic samples were obtained from infected tealeaves displaying leaf spots, characterized by tiny black spots of less than 1 mm diam., accompanied by chlorosis and browning, and resulting in defoliation. A single conidium isolation was done following the method described in Ariyawansa et al. 43 in plant tissues where spore masses were formed (diseased leaves) that were obtained from the tea fields.
Morphological examination. Morphological descriptions were made from isolates cultured on 2% potato dextrose agar (PDA; DIFCO) following the method described in Ariyawansa et al. 2 . Preparations for microscopy were made in distilled water, checked with an Olympus BX51 microscope with differential interference contrast (DIC) illumination and at least 30 measurements per structure were noted 2 . Voucher specimens were deposited in the herbarium of the Department of Plant Pathology and Microbiology, National Taiwan University Herbarium (NTUH) 2 . Living cultures are stored at the Department of Plant Pathology and Microbiology, National Taiwan University Culture Collection (NTUCC) 2 . Taxonomic descriptions and nomenclature details were deposited in MycoBank.  ITS  ITS5: GGA AGT AAA AGT CGT AAC AAGG   44,45   ITS4: TCC TCC GCT TAT TGA TAT GC   LSU  LROR: ACC CGC TGA ACT TAAGC   44,45   LR5: TCC TGA GGG AAA CTTCG   SSU  NS1: GTA GTC ATA TGC TTG TCT  isolates grown for 7 d on PDA using the Bioman Fungus Genomic DNA Extraction Kit (BIOMAN) following the manufacturer's protocol (Bioman Scientific Co., Ltd). PCR amplification was done following the methods described in Ariyawansa et al. 1 The PCR reactions for amplification of the internal transcribed spacer (ITS) regions 44 , were executed under standard conditions 45,46 . PCR conditions for amplification of the partial SSU (small subunit of the nrRNA gene) and LSU (large subunit of the nrRNA gene) followed the protocol of White et al. 47 . Amplification of partial tub2 (β-tubulin), rpb2 (partial RNA polymerase II second largest subunit gene) and tef1 (partial translation elongation factor 1-α gene) followed the procedures of Woudenberg et al. 30 and Ariyawansa et al. 1,2 . Primer sets used for these genes are in Table 1.
Agarose gels (1.5%) stained with SYBR safe DNA gel stain (BIOMAN) were used to visualise the PCR products. PCR products were purified and sequenced by Genomics (New Taipei, Taiwan) using the Sanger sequencing method. SeqMan Pro v.8.1.3 (Lasergene, DNASTAR, Inc., Madison, WI, USA) was used to obtain consensus sequences from sequences generated using forward and reverse primers. Newly obtained sequences were deposited at NCBI GenBank under the accession numbers provided in Supplementary Table S1.
Sequence alignment and phylogenetic analysis. MAFFT v. 6.864b was used to produce multiple sequence alignments (https ://mafft .cbrc.jp/align ment/serve r/index .html). MEGA v. 5 50 was used to check alignments visually and adjust manually where required. All introns and exons were aligned individually. Previously published sequences [1][2][3][4][5]10,15,18,19 were obtained from GenBank and are listed in Supplementary Table S1. Single alignments for each locus and the combined gene datasets were analysed using different tree inference methods. Two different datasets were organized to infer two phylogenies. The first tree focused on phylogenetic placement of the new genus and species introduced in this study in the Pleosporales. The second tree was used to determine the evolutionary placement of Didymosphaeria and allied taxa within the family Didymosphaeriaceae.
MrModeltest v. 2.3 51 with the Akaike Information Criterion (AIC) implemented in PAUP v. 4.0b10 was used to determine evolutionary models for each locus individually. RAxML-HPC2 on XSEDE (v 8.2.8) 52 with default parameters and bootstrapping with 1,000 replicates was used to conduct maximum likelihood (ML) analysis and was executed in the CIPRES webportal 53 . Another analysis of the same dataset was performed using Bayesian Inference (BI) as implemented in MrBayes ver. 3.1.2 54 . The number of generations was set at 10 million and the run was stopped automatically when the average standard deviation of split frequencies fell below 0.01. Trees were saved every 1,000 generations. The MCMC heated chain was set with a "temperature" value of 0.15. The distribution of log-likelihood scores was checked with Tracer v 1.5 to determine the stationary phase for each search and to decide if extra runs were required to achieve convergence 10,55 . All sampled topologies below the asymptote (20%) were discarded as part of a burn-in procedure, and the remaining trees were used to calculate posterior probabilities in the majority rule consensus tree.
For the concatenated gene analyses the topologies of the trees inferred for single genes were evaluated visually to confirm that the overall tree topology of the single locus datasets were comparable to each other and to that of the tree acquired from the concatenated dataset alignment. ML bootstrap values (MLBS) equal to or greater than 70% and Bayesian posterior probability (PP) equal to or greater than 0.95 are given at each node (Figs. 1,2). Nodes with bootstrap support (BS) lower than 70% or PP lower than 0.95 were considered unresolved. Phylogenetic trees and data files were viewed in MEGA v. 5 50