First Report of Leaf Spot Caused by <i>Didymella segeticola</i> on <i>Rosa</i> <i>roxburghii</i> in China

Rosa roxburghii Tratt, known as Cili in China, is a fruit crop that grows in the mountains of southwest China at altitudes of 500 - 2500 m, especially in Guizhou province (Huang et al. 2022). In July 2021, leaf spot symptoms were observed on approximately 20 to 30% of R. roxburghii plants in a field of 6,000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou Province, China. Severe leaf spot can lead to excessive leaf drop, significantly weakening the tree and adversely affecting its growth and fruit quality, which in turn can result in reduced or even lost harvests. The symptoms appeared as irregular brown spots (0.5 to 9.5 mm), which could coalesce when densely clustered and could lead to yellowing of the leaves in severe cases. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees. Symptomatic leaves were washed with sterile distilled water and then portions of the tissue (0.5×0.5cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol, 2 min with 3% NaOCl, washed three times with sterilized distilled water), the leaves were dried and placed flat on potato dextrose agar (PDA) and left for 3-4 days incubated at 25°C (Fang, 2007). From this process, three isolates, denoted as F3-Y-21, F3-Y-22 and F3-Y-23, were obtained through single spore isolation, all displaying identical morphology. Subsequently, isolate F3-Y-21 was selected for further study. The colonies had dense aerial hyphae, initially white and later turning gray near the colony center when cultured on PDA at 28℃. Pycnidia were dark, spherical or flat spherical, and 42.2 to 52.6 μm × 51.5 to 55.2 µm in diameter (n = 50). Conidia were oval, smooth, aseptate, usually guttulate, and the size was 3.0 to 4.6 μm × 2.3 to 2.8 µm (n = 50). These morphological attributes were consistent with the description of Didymella segeticola (Chen et al. 2015). The isolate F3-Y-21 was confirmed to be D. segeticola by amplification and sequencing of the rDNA internal transcribed spacer region (ITS; primers ITS5/ITS4), large subunit ribosomal RNA gene (LSU; primers LROR/LR5), beta-tubulin gene (TUB2; primers Bt2a/Bt2b), and RNA polymerase II second largest subunit gene (RPB2; primers RPB2-5F2/fRPB2-7cR) (Liu et al. 1999; Suwannarachetal. 2019). Sequences from PCR amplification were deposited in GenBank under accessions PP159078 (ITS), PP159081 (LSU), PP178656 (TUB2), and PP178653 (RPB2). BLASTn searches of the sequences in GenBank revealed 100.00% identity of ITS (486/486 bp), 100.00% identity of LSU (574/574 bp), 98.93% identity of TUB2 (277/280 bp), and 99.05% identity of RPB2 (838/846 bp) with those sequences of D. segeticola CGMCC 3.17489 (accessions KP330443, KP330455, KP330399, and KP330414, respectively). A phylogenetic tree was constructed by MEGA7.0 using the maximum likelihood method. The isolate F3-Y-21 clustered in the same branch with D. segeticola. To assess its pathogenicity, a pot assay was conducted. Twelve leaves of three healthy R. roxburghii plants were spray-inoculated with a spore suspension (106 spores/ml), and an additional three plants were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The experiment was repeated three times. After 7 days, the inoculated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The pathogen was reisolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, LSU, TUB2 and RPB2), and the reisolated pathogen was identical to D. segeticola, thus fulfilling Koch's postulates. Similar results were obtained from three replications of the pathogenicity test. To our knowledge, this is the first report of leaf spot diseases of R. roxburghii plants caused by D. segeticola in China, although it has been previously reported to cause diseases on other hosts in China (Guo et al. 2020). It provides a theoretical basis for the detection and prevention of R. roxburghii leaf spot disease.