High Diversity of Cytospora Associated With Canker and Dieback of Rosaceae in China, With 10 New Species Described

Cytospora canker is a destructive disease of numerous hosts and causes serious economic losses with a worldwide distribution. Identification of Cytospora species is difficult due to insufficient phylogenetic understanding and overlapped morphological characteristics. In this study, we provide an assessment of 23 Cytospora spp., which covered nine genera of Rosaceae, and focus on 13 species associated with symptomatic branch or twig canker and dieback disease in China. Through morphological observation and multilocus phylogeny of internal transcribed spacer (ITS), large nuclear ribosomal RNA subunit (LSU), actin (act), RNA polymerase II subunit (rpb2), translation elongation factor 1-α (tef1-α), and beta-tubulin (tub2) gene regions, the results indicate 13 distinct lineages with high branch support. These include 10 new Cytospora species, i.e., C. cinnamomea, C. cotoneastricola, C. mali-spectabilis, C. ochracea, C. olivacea, C. pruni-mume, C. rosicola, C. sorbina, C. tibetensis, and C. xinjiangensis and three known taxa including Cytospora erumpens, C. leucostoma, and C. parasitica. This study provides an initial understanding of the taxonomy of Cytospora associated with canker and dieback disease of Rosaceae in China.


INTRODUCTION
Many species of Rosaceae plants have economic value, which are important parts of the greening of urban parks and widely cultivated around the world. Many species are also famous fresh and dried fruits with excellent varieties. However, most Rosaceae plants are under serious disease of dieback and stem canker caused by Cytospora species, which have contributed to a severe reduction in yield and quality. A study to clarify the pathogens causing stem canker and dieback of Rosaceae plants is urgently needed.
Cytospora was first introduced by Ehrenberg (1818), which is one of the most important pathogenic fungi of hardwoods and coniferous trees in the world (Adams et al., 2005;Fan et al., 2020). About 150 species epithets of Cytospora are associated with dieback and stem canker on over 130 species of woody hosts (Spielman, 1985;Adams et al., 2005;Kirk et al., 2008;Fan et al., 2020). Over 660 species epithets of Cytospora have been described in Index Fungorum (2020). However, most of these were regarded as synonyms, and most descriptions were unable to identify them accurately (Adams et al., 2005). Leucostoma, Valsa, Valsella, and Valseutypella have been treated as the synonyms of Cytospora (Rossman et al., 2015). The traditional identification of Cytospora species was based heavily on their host affiliations; nevertheless, a single species of Cytospora may occur on a different host, and more than one Cytospora species may be isolated from a single host (Adams et al., 2005;Wang et al., 2011;Ariyawansa et al., 2015;Fan et al., 2015a,b;Hyde et al., 2016;Lawrence et al., 2018). Accurate identification needs additional informative morphological observation and multilocus phylogeny to test the relationship among species (Adams and Taylor, 1993;Harrington and Rizzo, 1999;Adams et al., 2002). Adams et al. (2005) introduced 28 species of Cytospora from Eucalyptus by morphology and phylogeny using ITS sequence. A total of 144 strains from Iran represented 20 species of Cytospora based on ITS phylogeny (Fotouhifar et al., 2010). Later, many species were described based on multilocus phylogeny in recent studies (Fan et al., 2014a(Fan et al., ,b, 2015aYang et al., 2015;Lawrence et al., 2017Lawrence et al., , 2018Zhu et al., 2018Zhu et al., , 2020Jiang et al., 2020;Shang et al., 2020). Fan et al. (2020) provided an assessment of 52 species of Cytospora in China using a six-locus phylogeny [internal transcribed spacer (ITS), large nuclear ribosomal RNA subunit (LSU), actin (act), RNA polymerase II subunit (rpb2), translation elongation factor 1-α (tef1-α), and beta-tubulin (tub2)]. However, most boundaries of known Cytospora species are tentative and indistinct due to the overlapped morphological characteristics, poor condition of multilocus phylogeny (only ITS is available for most species), and the shortage of fresh collected specimens. Thus, a geography-or host-centered strategy to define species of Cytospora using multiphase approaches has been proposed .
In this study, a total of 29 strains of Cytospora were isolated from symptomatic hosts of Rosaceae in China. The objectives were to (1) define the species of Cytospora associated with canker and dieback disease of Rosaceae, with illustrations and descriptions; (2) supplement a multi-gene DNA dataset of Cytospora, including ITS, LSU, act, rpb2, tef1-α, and tub2.

Sample Collection and Isolation
Fresh specimens of Cytospora canker disease symptoms were collected from infected branches or twigs of five host genera of Rosaceae (11 host species) during collecting trips in China (Supplementary Table S1). The symptoms of Cytospora dieback disease included wilting and killing of twigs and branches, which commenced at the tips and progressed downward to the larger branches, inducing wood lesions and canker formation. Cytospora canker disease represents slightly sunken and discolored areas in the bark, diseased inner bark, and the bark above the infected cambium may appear yellow, brown, reddish brown, gray, or black, becoming watery and odorous as the tissues deteriorate. Several prominent dark sporocarps immersed in the bark, erumpent through the surface of bark when mature (Figures 1, 2). The occurrence of canker diseases of Cytospora in Rosaceae is widespread, which could cause a large area of death in several apple orchards (Figure 2). A total of 29 strains were isolated by removing a mucoid spore mass from conidiomata and/or ascomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) in a Petri dish, and incubating at 25 • C for up to 24 h. Single-germinating conidia were transferred onto fresh PDA plates. All specimens are deposited at the Museum of the Beijing Forestry University (BJFC) and the working Collection of X.L. Fan (CF)

Morphology
Observation and description of Cytospora species were based on morphological characteristics of the fruiting bodies produced on infected host materials including arrangement and size of stromata; presence and absence of a conceptacle; size, color, and shape of discs; and number and diameter of ostioles per disc. The morphological characteristics were determined under a Leica stereomicroscope (M205). Micro-morphological observations include size and shape of conidiophores and conidia determined under a Nikon Eclipse 80i microscope. Over 30 conidiomata were sectioned, and 50 conidia were selected randomly for measurement. Incubation was done on PDA at 25 • C in darkness, and colony diameters and colors were recorded and described after 1 or 2 weeks according to the color charts of Rayner (1970). Adobe Bridge CS v.6 and Adobe Photoshop CS v.5 were used for the manual editing. Taxonomic novelties were deposited in MycoBank (Crous et al., 2004).

DNA Extraction and PCR Amplification
Genomic DNA was extracted from mycelium cultured on PDA with cellophane for 3 days using the modified CTAB method (Doyle and Doyle, 1990). The extracted DNA was estimated visually by electrophoresis in 1% agarose gels, and band intensity was compared with a DNA maker, 1 kbp (Takara Biotech). The qualities of DNA were measured with NanoDrop TM 2000 (Thermo, United States). The internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (White et al., 1990). The large nuclear ribosomal RNA subunit (LSU) region was amplified with primers LROR and LR7 (Vilgalys and Hester, 1990). The actin (act) region was amplified with primers ACT-512F and ACT-783R (Carbone and Kohn, 1999). The RNA polymerase II subunit (rpb2) region was amplified with primers RPB2-5F and fRPB2-7cR (Liu et al., 1999). The translation elongation factor 1-α (tef1-α) gene was amplified with primers EF-688F and EF-1251R (Carbone and Kohn, 1999). The betatubulin (tub2) gene was amplified with Bt-2a and Bt-2b (Glass and Donaldson, 1995). The PCR amplicons were electrophoresed in 2% agarose gels. DNA sequencing was carried out using an ABI PRISM R 3730XL DNA Analyzer with BigDye R Terminater Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). DNA sequences generated by the forward and reverse primers were used to  obtain consensus sequences using Seqman v.9.0.4 (DNASTAR Inc., Madison, WI, United States).

Phylogenetic Analyses
To infer a preliminary phylogenetic relationship for the new sequences, the first alignment based on ITS sequence data was performed using MAFFT v.6 (Katoh and Standley, 2013) and edited manually using MEGA v.6.0 (Tamura et al., 2013). Some characters were excluded from both ends of the alignments to approximate the size of our sequences to those included in the dataset. A second alignment was performed based on a combined six concatenate sequences (ITS, LSU, act, rpb2, tef1α, and tub2). For individual datasets, sequences were aligned using MAFFT v.6 and edited manually using MEGA v.6.0 and some characters were excluded from both ends of the alignments. A partition homogeneity test (PHT) with heuristic search and 1,000 homogeneity replicates was performed using PAUP v.4.0b10 to test the discrepancy among the six-gene dataset in reconstructing phylogenetic trees. The sequences of Diaporthe vaccinii (CBS 160.32) was included as outgroup in all analyses. The phylogenetic analyses for all the datasets were run using PAUP v.4.0b10 for maximum parsimony (MP) (Swofford, 2003), MrBayes v.3.1.2 for Bayesian inference (BI) (Ronquist and Huelsenbeck, 2003), and RAxML-NG v.0.9.0 for maximum likelihood (ML) (Kozlov et al., 2019). Trees were visualized using FigTree v. 1.3.1 (Rambaut and Drummond, 2010). MP analysis was performed using a heuristic search (1,000 bootstraps) (Hillis and Bull, 1993), with random sequence addition as option to stepwise addition (1,000 replicates and one tree held at each addition step), and maxtrees limited to 200 by replicate. The tree bisection and reconnection (TBR) algorithm was selected (Swofford, 2003). The branches of zero length were collapsed using the command minbrlen, and all equally most parsimonious trees were saved. Other parsimony scores such as tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC) were calculated to describe tree statistics (Swofford, 2003). The branch supports of MP were evaluated with a bootstrapping (BS) method of 1,000 replicates (Hillis and Bull, 1993). For ML and BI analyses, the best-fit evolutionary models for each partitioned locus were estimated by MrModeltest v.2.3 following the Akaike Information Criterion (AIC) (Posada and Crandall, 1998). ML analysis was performed with RAxML-NG 1 (Kozlov et al., 2019). The bootstrap was used with 100 replicates and the appropriate models for each gene. BI analysis was done by a Markov chain Monte Carlo (MCMC) algorithm with Bayesian posterior probabilities (BPP) (Rannala and Yang, 1996). Two MCMC chains were run from random trees for 10 million generations, and trees were sampled each 100th generation. The first 25% of the trees were discarded as the burn-in phase of each analysis; branches with significant BPP were calculated to assess the remaining trees (Rannala and Yang, 1996). Phylograms are shown using Figtree v.1.3.1 (Rambaut and Drummond, 2010). All sequences from this study data were deposited in GenBank. The ITS and multi-gene sequence alignment files were deposited in TreeBASE 2 (accession number: S25903).

RESULTS
A total of 29 Cytospora isolates from Rosaceae hosts were collected in China. Following alignment, the ITS sequence data comprised 248 Cytospora in group taxa with a total of 629 characters including gaps, of which 365 characters were constant, 63 variable characters were parsimony uninformative, and 201 characters were variable and parsimony informative. To clarify the phylogenetic position of these Cytospora species, a multi-locus analysis (ITS, LSU, act, rpb2, tef1-α, and tub2) is presented in Figure 3. The final analysis combined sequence data of six genes composed of 218 Cytospora ingroup taxa with a total of 3,712 characters including gaps, of which 2,037 characters were constant, 202 variable characters were parsimony uninformative, and 1,473 characters were variable and parsimony informative. MP analysis generated 200 equally parsimonious trees each with similar clade topologies, and one of which is presented in Figure 3 (TL = 9,782, CI = 0.303, RI = 0.803, RC = 0.244). For ML and BI analyses, the best-fit model of nucleotide evolution was deduced on the AIC (ITS and LSU: GTR, act: TVM, rpb2 and tef1-α: TrN, and tub: HKY). ML method and Bayesian analyses were in agreement and no difference from the MP tree. The MP bootstrap supports (MP-BS) and ML bootstrap (ML-BS) equal to or above 70% were shown in branches in Figure 3. The branches with significant Bayesian posterior probabilities (BPP) equal to or above 0.95 are thickened in the phylograms.
Culture characteristics: Cultures on PDA are initially white, fast growing, and covering the 9-cm Petri dish after 3 days, becoming cinnamon to fawn after 30 days. The colonies are flat and with a uniform texture, conidiomata sparse and distributed irregularly on the medium surface.
Habitat and distribution: Known only on Prunus armeniaca from the type locality.
Habitat and distribution: Known on Cotoneaster sp. from the type locality and an additional locality in the Tibet Autonomous Region. Notes: Cytospora cotoneastricola is described as being associated with canker disease of Cotoneaster sp. in China. Cytospora tibetensis and C. ochracea are associated with the same host. Morphologically, C. cotoneastricola is distinguished from C. tibetensis by having multi-locules and larger size of conidia (13.0-14.0 × 2.5-3 vs. 5.0-5.5 × 1.5-2 µm). Cytospora cotoneastricola also has obvious central column and larger conidia than C. ochracea (13.5-14.0 × 2.5-3 vs. 8.5-9.0 × 1.5-2.5 µm). This species needs to be re-collected from Cotoneaster sp. in Tibet of China, as presently no living culture is available.  (Norphanphoun et al., 2017). Afterward, it was reported on Prunus padus in China . This fungus can be identified by its black-discoid conidiomata with long ostiolar necks, producing elongate-allantoid conidia (6.4-6.7 × 1.3-1.4 µm) (Norphanphoun et al., 2017). Combined morphology and the DNA sequence data of our strain, which was collected from dead branches of Prunus padus belongs to this species.
Cytospora leucostoma (Pers.) Sacc.,  Notes: Cytospora leucostoma is a common species associated with stem canker diseases of woody plants of Rosaceae in China . This species has obvious black conceptacle, numerous locules, which were subdivided frequently by invaginations with independent walls, and hyaline, allantoid, aseptate conidia with the size of 4.5-5.5 × 1-1.5 µm. In a recent study, Cytospora donetzica has been treated as the synonym of C. leucostoma based on Fan et al. (2020). Etymology: Named after the host genus on which it was collected, Malus spectabilis 'Royalty'.
Culture characteristics: Cultures on PDA are initially white, growing up to 6.5 cm after 3 days, entirely covering the 9-cm Petri dish and becoming buff after 7 days. The colonies ultimately are gray olivaceous and flat with a uniform texture. Conidiomata are randomly distributed on medium surface.
Habitat and distribution: Known only on Malus spectabilis 'Royalty' from the type locality.
Culture characteristics: Cultures on PDA are initially white, growing up to 5.5 cm after 3 days, becoming ochreous in center after 7 days, deepened in later stage gradually. Colonies are tight, thin with a uniform texture, lacking aerial mycelium. Conidiomata are randomly distributed on medium surface.
Habitat and distribution: Known only on Cotoneaster sp. from the type locality.
Culture characteristics: Cultures on PDA are initially white and become olivaceous buff, growing fast and entirely covering the 9-cm Petri dish after 3 days, becoming olivaceous gray and slight helical after 30 days. The colonies are flat and with a uniform texture. Conidiomata are randomly distributed on medium surface, extruding a pale white conidial mass.
Habitat and distribution: Known on Cotoneaster sp. and Prunus spp. from the type locality and an additional locality in Xinjiang Uygur Autonomous Region.  Notes: Cytospora olivacea is associated with canker disease of Prunus spp. in China. It has multiple locules with black conceptacle, which is commonly discovered in Cytospora spp., while the molecular phylogenies show a clearly different position from all other strains included in this study. Therefore, we describe this species as novel based on morphology and combined sequence data of six genes.
Culture characteristics: Cultures on PDA are initially white, growing up to 8.5 cm after 3 days and entirely covering the 9-cm Petri dish after 7 days, becoming buff but white mostly. The colonies are flat with a uniform texture, becoming effuse on the surface, without aerial mycelium, conidiomata are randomly distributed on medium surface.
Habitat and distribution: Known from only Malus sp. in China and Russia. Notes: Cytospora parasitica was introduced by Ariyawansa et al. (2015) relating to canker disease of Malus pumila. Morphologically, our isolates are similar to C. parasitica in having multi-loculate pycnidial conidiomata, producing black area on bark, having smooth-walled, elongate-allantoid, aseptate conidia, whereas the size of our conidia differs from those isolates (5.5-6.0 × 1-2 vs. 6.5-8.0 × 1.3-1.5 µm) . Ma et al. (2018) reported this species from the same host plant Malus pumila in Xinjiang of China, which is similar with C. parasitica in the current study. Etymology: Named after the host genus on which it was collected, Prunus mume.
Culture characteristics: Cultures on PDA are initially white, growing fast and entirely covering the 9-cm Petri dish after 3 days, becoming pale yellow after 7 days. The colonies are flat with a uniform texture, conidiomata were randomly distributed on medium surface.
Habitat and distribution: Known on Prunus mume and Prunus armeniaca from the type locality.
Additional Notes: Cytospora pruni-mume is associated with canker disease of Prunus mume. The molecular phylogenies show a position clearly distinct from all other strains included in this study (Figure 3). Therefore, we describe this species as a new species. Etymology: Named after the host genus on which it was collected, Rosa sp.
Habitat and distribution: Known only on Rosa sp. from the type locality.
Cytospora Etymology: Named after the host genus on which it was collected, Sorbus tianschanica.
Habitat and distribution: Known only on Sorbus tianschanica from the type locality.
Phylogenetic analyses based of combined six sequences data indicates that these species form two single lineages, separate from each other with high bootstrap support (Supplementary Figure S1 and Figure 3). Cytospora tibetensis is thus here considered as a novel species. This species needs to be re-collected from Cotoneaster sp. in Tibet of China, as presently no living culture is available.
Descriptions: Symptoms appeared as elongate or circular, dark, slightly sunken, dehiscent lesions on the twigs or branches, with discolored bark above the infected cambium. Asexual morph: Conidiomata pycnidial, immersed in bark, erumpent through the surface of bark when mature, erumpent, discoid, with large multiple locules, 965-1,410 µm (x = 1,145 µm, n = 30) in diam. Conceptacle absent. Ectostromatic disc dark brown to black, Notes: Cytospora japonica has been reported in China from twigs and branches of Prunus cerasifera by Fan et al. (2020). This species was introduced as a common pathogen in Rosaceae host (Tai, 1979). It is characterized by discoid to conoid conidiomata with hyaline, allantoid, aseptate conidia (6.5-8.5 × 1.5-2 µm) as well as numerous locules, which arranged circularly or irregularly with common walls .
Cytospora leucosperma ( (Teng, 1963;Tai, 1979;Zhuang, 2005). Infected branches collected from Tilia were regarded as the neotype (Urban, 1957;Spielman, 1985), but no living culture and DNA sequence data are available at present. C. leucosperma is similar to C. mali from Malus spp., leading to confusion in both morphology and molecular data (Wang et al., 2011).
The plants of Rosaceae are important ecological and economic tree species in China. However, the current study indicates that the incidence of Cytospora species is serious and have different symptoms in various hosts, including 20 host species of nine genera in Rosaceae, i.e., Cotoneaster, Crataegus, Malus, Prunus, Pyrus, Rosa, Sibiraea, Sorbus, and Spiraea. The result coincides with previous reports that widely extended Cytospora species have been identified to occur in many host species (Adams et al., 2005;Fan et al., 2014aFan et al., , 2015aAriyawansa et al., 2015;Liu et al., 2015;Maharachchikumbura et al., 2015Maharachchikumbura et al., , 2016Hyde et al., 2016;Li et al., 2016) in the current study. Six Cytospora species recovered from diverse Prunus species in California was reported by Lawrence et al. (2018), which are Cytospora amygdali, C. californica, C. eucalypti, C. longispora, C. plurivora, and C. sorbicola. The current results also supplement seven different Cytospora species afflicted Prunus host plants in China. The comparison shows that the species occurrence may be related by geographical and environmental factors, rather than the taxa actually being host specific. Cytospora species and accumulation of DNA dataset are required to expand our understanding of their host range and distribution. Furthermore, Cytospora cotoneastricola, C. ochracea, and C. tibetensis were all collected from Cotoneaster sp., which indicates that the same host could be infected by more than one species. Stevens (1919) summarized the symptoms and species of Rosaceae infected by Cytospora, whereas these reports lacked molecular data. Only a few relative taxonomic studies of Cytospora canker or dieback disease from the plants of Rosaceae were reported, such as Cytospora chrysosperma, C. cincta, C. leucostoma, and C. schulzeri (Mehrabi et al., 2011). Moreover, the host specificity and pathogenicity of many Cytospora species are poorly known. In the current study, Cytospora leucostoma is a common species associated with stem canker diseases of woody plants of Rosaceae, mainly Prunoideae host plants, and C. mali, C. parastica, and C. schulzeri are the common species collected from apple trees.
In China, Cytospora species from cankered apple and pear bark were examined and compared with morphology and ITS sequence data (Wang et al., 2007(Wang et al., , 2011. The species identity of the pathogen of Valsa (now Cytospora) canker on pear tree was determined through a combined study of ITS sequence data and cultural characteristics of isolates from apple trees and pear trees in China (Zhang et al., 2007). Ma et al. (2018) clarified and illustrated C. parasitica from the Malus sp. using the ITS, LSU, and tef1-α regions. Fan et al. (2020) summarized 52 species of Cytospora and recommended the dataset of ITS, LSU act, rpb2, tef1-α, and tub2 gene regions. At present, China is a hot place to study these taxa as many species of Cytospora are isolated from important hosts such as Rosaceae. Thus, further studies are required to discover the species of Cytospora in China.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the MK672943-MK672956, MK672958-MK672985,