Discovery of Cytospora species associated with canker disease of tree hosts from Mount Dongling of China

Abstract Members of Cytospora encompass important plant pathogens, saprobes and endophytes on a wide range of woody hosts with a worldwide distribution. In the current study, we obtained seven representative isolates from six tree hosts of Betulaceae, Juglandaceae, Rosaceae, Tiliaceae and Ulmaceae in Mount Dongling of China. Based on morphological comparison and phylogenetic analyses using partial ITS, LSU, act, rpb2, tef1-α and tub2 gene sequences, we identified two known species (Cytospora leucostoma and C. pruinopsis) and two novel species (C. coryli and C. spiraeicola). These results represent the first study on Cytospora species associated with canker disease from Mount Dongling of China.


Introduction
The genus Cytospora was established by Ehrenberg (1818) and belongs to Cytosporaceae, Diaporthales, Sordariomycetes (Wijayawardene et al. 2018, Fan et al. 2020. It is characterised by single or labyrinthine of pycnidial locules, filamentous conidiophores (enteroblastic and phialidic conidiogenous cells) producing hyaline, allantoid conidia in the asexual morph; diaporthalean-like perithecia, clavate to elongate obovoid asci with four or eight hyaline, allantoid ascospores in the sexual morph (Spielman 1983, 1985, Adams et al. 2005. Species of Cytospora contain important pathogens that cause stem canker and dieback disease on more than 100 species of woody and coniferous plants, thereby causing severe commercial and ecological damage and significant losses worldwide (Sinclair et al. 1987, Adams et al. 2005, 2006, Fan et al. 2014a, b, 2015a, b, Lawrence et al. 2018, Pan et al. 2018, Zhu et al. 2018a, Zhang et al. 2019. Previous Cytospora species and their related sexual morphs viz. Leucostoma, Valsa, Valsella and Valseutypella were listed by old fungal literature without any living culture and sufficient evidence for their identification (Fries 1823, Saccardo 1884, Kobayashi 1970, Barr 1978, Sutton 1980, Gvritishvili 1982, Spielman 1983, 1985. Adams et al. (2005) revised the genus Cytospora from Eucalyptus with 28 species and accepted all sexual genera combined under Valsa, either as subgenera or species without additional infrageneric rank. Following the single-name for pleomorphic taxa, Cytospora (1818), the older asexual typified name was proposed as the recommended name against Valsa (1849), the younger sexual typified name (Fan et al. 2015a, b, Rossman et al. 2015. Currently, 388 species epithets of Cytospora have been recorded in Index Fungorum (2020) (accessed 2 January 2020). However, Kirk et al. (2008) estimated approximately 110 species, but most of them lack herbarium materials, ex-type cultures and DNA sequence data.
Species identification criteria of Cytospora were previously carried out by the hostbased method and morphology in China; however, these bases are unreliable due to the uninformative illustrations and descriptions, weak host specificity and overlapping morphological characteristics (Teng 1963, Tai 1979, Wei 1979). Recent studies have been able to use multiphase approaches to solve the taxonomy of Cytospora (Fan et al. 2014a, b, 2015a, b, Yang et al. 2015, Lawrence et al. 2016, Norphanphoun et al. 2017, Pan et al. 2018, Zhu et al. 2018a, Zhang et al. 2019. Fan et al. (2020) summarised 52 species of Cytospora associated with canker and dieback disease in China, using a six gene matrix (ITS,LSU,act,rpb2,, of which 13 species were newly introduced.
Mount Dongling has high plant diversity in western Beijing, including more than 1,000 tree hosts (Ma et al. 1995). As more plant species were recorded in this region, the exploration of fungal diversity gradually increased as most fungi are often linked to particular host plants as pathogens or endophytes. Species of Alternaria, Diaporthe, Leptostroma, Pestalotiopsis and Phoma were the most commonly isolated endophytes from Pinus tabuliformis and later, an additional 38 endophytic taxa were identified from Acer truncatum from Mount Dongling (Guo et al. 2008, Sun et al. 2011. Further, pathogens belonging in Botryosphaeriales have been identified from Mount Dongling, including five species from Aplosporella, Botryosphaeria and Phaeobotryon (Zhu et al. 2018b). Zhu et al. (2019) subsequently introduced six species of diaporthalean fungi residing in four families (viz. Diaporthaceae, Erythrogloeaceae, Juglanconidaceae and Melanconidaceae) from Mount Dongling. For the current understanding, many common host plants represent high fungal diversity causing canker and dieback disease in Mount Dongling. Juglans mandshurica and J. regia (Juglandaceae) were infected by Botryosphaeria dothidea (Botryosphaeriaceae), Diaporthe eres, D. rostrata (Diaporthaceae) and Juglanconis oblonga (Juglanconidaceae). Rhus typhina (Anacardiaceae) was infected by Aplosporella ginkgonis, A. javeedii (Aplosporellaceae), Phaeobotryon rhois and P. rhoinum (Botryosphaeriaceae). Quercus mongolica (Fagaceae) was infected by Dendrostoma donglinensis (Erythrogloeaceae) (Zhu et al. 2018b(Zhu et al. , 2019. During the course of cognitive practices to investigate forest pathogens that cause canker or dieback disease in Mount Dongling of China, seven Cytospora strains were obtained from six unrelated hosts, i.e. Corylus mandshurica (Betulaceae), Juglans mandshurica (Juglandaceae), Prunus sibirica, Spiraea salicifolia (Rosaceae), Tilia nobilis (Tiliaceae) and Ulmus pumila (Ulmaceae). Phylogenetic analyses inferred from combined ITS, LSU, act, rpb2, tef1-α and tub2 gene regions were conducted to provide a multi-gene phylogeny for Cytospora, based on a large set of freshly collected specimens in Mount Dongling of China. Thus, the current study aims to clarify the systematics and taxonomy of Cytospora species with detailed descriptions and illustrations and compare it to known species in the genus.

Sampling and isolation
Seven infected branches of six hosts were collected from Mount Dongling of China (Table 1). Sampled trees expressed general symptoms and signs of canker diseases including elongate, slightly sunken and discoloured areas in the bark, several prominent dark sporocarps immersed in bark, erumpent through the surface of bark when mature (Fig. 1). A total of seven isolates was established by removing a mucoid spore mass from conidiomata or ascomata of fresh material, spreading the suspension on the surface of 1.8 % potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. Single germinating spores were transferred on to fresh PDA plates. Specimens and isolates were deposited in the Key Laboratory for Silviculture and Conservation of the Ministry of Education in Beijing Forestry University (BJFU) and at the working Collection of X.L. Fan (CF), housed at the BJFU. Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
Species identification was based on morphological features of the ascomata or conidiomata from infected host materials and micromorphology, supplemented by cultural characteristics. Microscopic photographs (structure and size of stromata; structure and size of ectostromatic disc and ostioles) were captured using a Leica stereomicroscope (M205 FA) (Leica Microsystems, Wetzlar, Germany). Microscopic observations (shape and size of conidiophores, asci and conidia/ascospores) were determined under a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination. The Nikon software NIS-Elements D Package v. 3.00, Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. More than 10 conidiomata/ascomata, 10 asci and 30 conidia/ascospores were measured by Nikon software NIS-Elements D Package v. 3.00 to calculate the mean size/length and respective standard deviations (SD). Colony diameters were measured and the colony features were described using the colour charts of Rayner (1970).

DNA extraction, PCR amplification and sequencing
Fungal mycelium grown on the cellophane of PDA was scraped for the extraction of genomic DNA following a modified CTAB method (Doyle and Doyle 1990). The primers and PCR conditions are listed in Table 2. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). The DNA sequences, obtained from forward and reverse primers, were combined using SeqMan v. 7.1.0 in the DNASTAR Lasergene Core Suite software (DNASTAR Inc., Madison, WI, USA).

Phylogenetic analyses
The current isolates were initially identified as Cytospora species, based on both morphological observations and BLAST results. To clarify their further phylogenetic position, an analysis, based on the combined six genes (ITS, LSU, act, rpb2, tef1-α and tub2), was performed to compare Cytospora species from the current study with other strains in GenBank. Diaporthe vaccinii was selected as the outgroup in all analyses. Subsequent alignments for each gene were generated using MAFFT v.7 (Katoh and Standley 2013) and manually adjusted using MEGA v. 6 (Tamura et al. 2013).
Ambiguously aligned sequences were excluded from analysis. Reference sequences were selected, based on ex-type or ex-epitype sequences available from recently published literature (Fan et al. 2014a, b, 2015a, b, 2020, Yang et al. 2015, Lawrence et al. 2016, Norphanphoun et al. 2017, Zhu et al. 2018a, Zhang et al. 2019    A partition homogeneity test (PHT) with heuristic search and 1,000 replicates was performed using PAUP v.4.0b10 to test the discrepancy amongst the ITS, LSU, act, rpb2, tef1-α and tub2 sequence datasets in reconstructing phylogenetic trees. MP analysis was performed using a heuristic search option of 1,000 random-addition sequences with a tree bisection and reconnection (TBR) branch swapping algorithm (Swofford 2003). The branches of zero length were collapsed and all equally parsimonious trees were saved. Clade stability was assessed with a bootstrap analysis of 1,000 replicates (Hillis and Bull 1993). Other parsimony scores, such as tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC), were calculated (Swofford 2003). ML analysis was performed with the GTR + G + I model of site substitution following recent studies (Zhu et al. 2018a), including estimation of gamma-distributed rate heterogeneity and a proportion of invariant sites using PhyML v. 3.0 (Guindon et al. 2010). The branch support was evaluated with a bootstrapping method of 1,000 replicates (Hillis and Bull 1993). BI analysis was performed using a Markov Chain Monte Carlo (MCMC) algorithm with Bayesian posterior probabilities (Rannala and Yang 1996). A nucleotide substitution model was estimated by MrModeltest v.2.3 (Posada and Crandall 1998) and a weighted Bayesian analysis was considered. Two MCMC chains were run from random trees for 1,000,000 generations and trees were sampled each 100 generations. The first 25% of trees were discarded as the burn-in phase of each analysis and the posterior probabilities (BPP) were calculated to assess the remaining trees (Rannala and Yang 1996). The branch support from MP and ML analysis was evaluated with a bootstrapping (BS) method of 1,000 replicates (Hillis and Bull 1993). Phylograms were plotted in Figtree v. 1.4.4 (http://tree.bio. ed.ac.uk/software/figtree) and edited in Adobe Illustrator CS6 v.16.0.0 (https://www. adobe.com/cn/products/illustrator.html). Novel sequences, generated in the current study, were deposited in GenBank (Table 1) and the aligned matrices, used for phylogenetic analyses, were submitted in TreeBASE (www.treebase.org; study ID S25564).

Cytospora pruinopsis
Culture characteristics. Cultures are white, growing up to 4 cm in diam. with irregular margin after 3 days, covering the 9 cm Petri dish after 6 days, becoming vinaceous buff to hazel after 7-10 days. In reverse, the cultures are the same as the upper colour after 3 days, becoming isabelline to umber after 7-10 days. Colonies are felty with a heterogeneous texture, lacking aerial mycelium.

Discussion
In the present study, seven specimens were collected from symptomatic branches and twigs associated with canker disease. Four Cytospora species were isolated from six tree hosts of Betulaceae, Juglandaceae, Rosaceae, Tiliaceae and Ulmaceae, which include two known species (Cytospora leucostoma and C. pruinopsis) and two novel species (C. coryli and C. spiraeicola). This study represents an investigation of Cytospora species associated with canker disease in Mount Dongling of China and included a comprehensive analysis of DNA sequence data to compare the novelties with known Cytospora species.
In a previous study, Zhu et al. (2018a) described Cytospora spiraeae from Spiraea salicifolia in Gansu Province of China during an investigation of forest pathogens of three hosts. Compared to the new species Cytospora spiraeicola, C. spiraeae has larger perithecia (270-400 vs. 210-250 µm) in diam. and shorter ascospores (7-8 × 2.5-3.5 × 8.5-12 vs. 2-2.5 µm). These morphological deviations are in line with the combined phylogenetic analyses which resolved C. spiraeicola as a unique lineage, highly supported. Besides this, the only strain of C. coryli, closely related to C. euonymicola and C. gigalocus, was distinguished by its different size of multiple locules and conidia (Fan et al. 2015a(Fan et al. , 2020.
This study focused on Cytospora species in Mount Dongling of Beijing (China), which is considered as an attractive location with a high richness of fungal species (Guo et al. 2008, Zhu et al. 2018b. We hope that the descriptions and molecular data of Cytospora in this study could provide a resource for future studies in this genus and lay the foundation for the future canker disease caused by Cytospora species.