Identification of six Cytospora species on Chinese chestnut in China

Abstract Chinese chestnut (Castanea mollissima) is an important crop tree species in China. In the present study, Cytospora specimens were collected from Chinese chestnut trees and identified using molecular data of combined ITS, LSU, ACT and RPB2 loci, as well as morphological features. As a result, two new Cytospora species and four new host records were confirmed, viz. C. kuanchengensissp. nov., C. xinglongensissp. nov., C. ceratospermopsis, C. leucostoma, C. myrtagena and C. schulzeri.

Cytospora (Cytosporaceae, Diaporthales) is a widely distributed genus worldwide, occurring on a broad range of hosts (Sarma and Hyde 2001, Yang et al. 2015, Lawrence et al. 2017, Norphanphoun et al. 2017, 2018, Wijayawardene et al. 2018, Jayawardena et al. 2019, Phookamsak et al. 2019, Fan et al. 2020. Some species can cause severe canker diseases on woody trees, such as Cytospora chrysosperma, which is a commom pathogen on the commercial tree genera, Populus and Salix (Fan et al. 2014b, Zhang et al. 2014, Kepley et al. 2015, Wang et al. 2015. Host affiliation was considerd as the main evidence for separating species in Cytospora before DNA sequences were used; however, morphology combined with phylogeny has revealed many cryptic species. For example, 28 Cytospora species were discovered from Eucalyptus from South Africa (Adams et al. 2005) and six from apple trees in Iran (Mehrabi et al. 2011), three from Chinese scholar tree (Fan et al. 2014a), four from walnut tree (Fan et al. 2015a), six from anti-desertification plants in China (Fan et al. 2015b) and two from grapevine in North America (Lawrence et al. 2017). Several recent studies discovered new species of Cytospora using multiphasic analyses (Lawrence et al. 2018, Norphanphoun et al. 2017, 2018, Senanayake et al. 2017, 2018, Pan et al. 2018, Zhang et al. 2019. During our investigations of chestnut disease in China from 2016 to 2019, diseased branches with typical Cytospora fruiting bodies were discovered and collected ( Fig. 1). In the present study, Cytospora species from Castanea mollissima were identified using a combined method of morphology and phylogeny.

Sample collections and isolations
Chinese chestnut has a wide distribution in China. In the present study, we surveyed Hebei, Shaanxi and Shandong Provinces from 2016 to 2019. Dead and dying branches with typical Cytospora fruiting bodies were collected and packed in paper bags. Isolates were obtained by removing the ascospores or conidial masses from the fruiting bodies on to clean PDA plates and incubating at 25 °C until spores germinated. Single germinated spores were transferred on to the new PDA plates and incubated at 25 °C in the dark. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC) and axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
Observation and description of Cytospora species from Castanea mollissima was based on fruiting bodies formed on tree barks. Ascomata and conidiomata from tree barks were sec- tioned by hand using a double-edged blade and strctures were observed under a dissecting microscope. At least 10 conidiostromata/ascostromata, 10 asci and 50 conidia/ascospores were measured to calculate the mean size and standard deviation. Measurements are reported as maximum and minimum in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements is given in parentheses (Voglmayr et al. 2017). Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination. Introduction of the new species, based on molecular data, follow the recommendations of Jeewon and Hyde (2016).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from young mycelium growing on PDA plates following Doyle and Doyle (1990). PCR amplifications were performed in a DNA Engine Pelti-er Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer pair ITS1/ITS4 (White et al. 1990) was used to amplify the ITS region. The primer pair LR0R/LR5 (Vilgalys and Hester 1990) was used to amplify the LSU region. The primer pair ACT512F/ACT783R (Carbone and Kohn 1999) was used to amplify ACT gene. The primer pair dRPB2-5f/dRPB2-7r (Voglmayr et al. 2016) was used to amplify the RPB2 gene. The polymerase chain reaction (PCR) assay was conducted as described in Fan et al. (2020). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses
The preliminary identities of the isolates sequenced were obtained by conducting a standard nucleotide BLAST search using ITS, LSU, ACT and RPB2. Then all Cytospora isolates were selected to conduct phylogenetic analyses, based on sequence datasets from Fan et al. (2020). Diaporthe vaccinia (CBS 160.32) in Diaporthaceae was selected as the outgroup taxon. All sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for Maximum Parsimony (MP) analysis (Swofford 2003) and PhyML v. 3.0 for Maximum Likelihood (ML) analysis (Guindon et al. 2010).
MP analysis was run using a heuristic search option of 1000 search replicates with random-additions of sequences with a tree bisection and reconnection algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated using a bootstrapping method of 1000 replicates (Hillis and Bull 1993). Phylograms were shown using FigTree v. 1.4.3 (Rambaut 2016). Novel sequences, generated in the current study, were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (accession number: S25160).
Culture characters. On PDA at 25 °C in darkness. Cultures are initially white, becoming olivaceous buff in centre after 7 d and finally olivaceous at 30 d. The colony is flat, thin with a felt and tight texture in centre. Pycnidia distributed irregularly on medium surface.
Culture characters. On PDA at 25 °C in darkness. Cultures are white. The colony is flat, thin with a uniform texture, lacking aerial mycelium. Pycnidia distributed uniformly on medium surface.

Discussion
In the present study, an important fruit tree species, Castanea mollissima was investigated and Cytospora canker was found as a commom disease in plantations in Hebei Province. Identification was conducted based on 13 isolates from fruiting bodies using both morphological and molecular methods. As a result, six Cytospora species were confirmed. Cytospora kuanchengensis and C. xinglongensis are introduced as new species, C. ceratospermopsis, C. leucostoma, C. myrtagena and C. schulzeri are firstly reported on Castanea mollissima.
These six chestnut Cytospora species can be easily distinguished using DNA sequences of single ITS sequence or combined sequences of ITS, LSU, ACT and RPB2 ( Fig. 2; Suppl. material 1: Fig. S1). In addition, colonies on PDA and MEA of these six species are also different (Fig. 9). Cytospora xinglongensis never produce fruiting bodies on PDA or MEA, while the other five species form conidiomata in one month (Fig. 9). Morphologically, Cytospora xinglongensis has obviously longer conidia than others. However, the conidial dimension can hardly distinguish C. ceratospermopsis, C. kuanchengensis, C. leucostoma, C. myrtagena and C. schulzeri.
Dar and Rai reported Cytospora diseases on Castanea sativa in India, causing perennial cankers on stems and branches (Dar and Rai 2014). The Cytospora isolates were identified mainly based on ITS sequence data, which were introduced as a new species named Cytospora castaneae (wrongly wrriten as Cytospora castanae in the original paper) (Dar and Rai 2014). However, further study is required to confirm the species position within the genus, including detailed morphogical features and sequences of high quality. Cytospora canker is a common disease on chestnut trees, but there are few formal reports. In China, this disease is known amongst phytopathologists, but no-one conducted accurate identifications. Hence, this paper is the first formal report of Cytospora chestnut canker in China. From our investigations of chestnut diseases in China, Cytospora species are closely associated with canker diseases in chestnut plantations. In most cases, they infect twigs or small branches, causing necrotic lesions (Fig. 1A), finially forming fruiting bodies on dead tissues (Fig. 1D). However, Cytospora myrtagena was discovered on stems of a 15-year-old chestnut tree, causing typical Cytospora canker symptoms. More works should be conducted on the newly emerging pathogens from several aspects.
As the species concept of Cytospora has been improved a lot by using molecular data (Yang et al. 2015, Lawrence et al. 2017, Norphanphoun et al. 2017, 2018, Jaya-wardena et al. 2019, Fan et al. 2020), many Cytospora canker diseases and new species have been discovered and reported in recent years. Further studies are, however, now required to confirm their pathogenicity.