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First Report of Round Leaf Spot on Sophora tonkinensis Caused by Didymella glomerata in China.

The root of Sophora tonkinensis Gagnep is an important medicinal material in China. An unknown foliar disease, first observed In July 2018, occurred over 240 ha of S. tonkinensis (totally cultured 600 ha) in Guangxi, China, in December 2019. The initial symptoms on leaf were seen as small, tawny spots (0.5 to 1.5 mm in diam.). As the disease progressed, the lesions enlarged into grayish and dark brown concentric rings (5 to 10.0 mm in diam.) resulting in black protuberances in the center of the spots. Severe infections would adversely affect plant growth, and cut the production by 30-40%. Symptomatic leaves were sampled and the surface was sterilized with 75% ethanol for 30 s and then soaked in 0.1% HgCl2 for 2 min. After three washes with sterile distilled water, the samples were dried, placed aseptically onto potato dextrose agar (PDA) plates, and incubated at 28°C. Three days later, the isolates were placed on new PDA medium for subsequent purification and sporulation. The fungus, SDG-1, was recovered from 85% of the total 40 isolates. Its colonies were whitish initially and then became olive green 7 days after incubation at 28 °C. The pycnidias were globose to subglobose, initially brown and darken at maturity, 85 to 300 × 70 to 280 μm in size. The conidia were colorless, single-celled, rounded to ellipsoidal and 3.5 to 6.6 × 1.5 to 3.8 μm. The chlamydospores were multicellular and brick trellised, usually forming branched or unbranched chains, light to dark brown in color and measured 28.5 to 44.5 × 8.2 to 16.5 μm. The morphological characteristics were consistent with Didymella glomerata. The rDNA-ITS, β-tubulin and actin of strain SDG-1 were PCR amplified, and the DNA sequencing results were almost 100% identical to those of D. glomerata, respectively (GenBank database accession numbers MN 435377, MN447333 and MN447334, respectively). The multi-locus phylogenetic analysis was carried out with the obtained sequences, which revealed that the isolates clustered within D. glomerata with the similarity of 100% (Fig.3). Therefore, based on the morphology and phylogenetic tree, strain SDG-1 was identified as D. glomerata. For pathogenicity tests, the S. tonkinensis was cultured for two years, and the conidial suspension of SDG-1 (1 × 106 conidia /mL) was prepared by harvesting conidia from a 10-day-old culture on PDA. Conidia were sprayed onto the healthy leaves of S. tonkinensis for co-culture, while the control group was sprayed with sterile distilled water. Each experiment was performed three times. All plants were covered with plastic bags for 3 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 7 days after the leaves were inoculated with spores, which were identical to those observed in the field. The pathogenic fungus was re-isolated from the infected leaves and identified as previously described. The control leaves remained symptomless during the pathogenicity tests. According to the previous literature, D. glomerata is a plant pathogenic fungus with a wide host range, which can damage up to 100 species of woody and herbaceous plants (Aghapour et al. 2009, Lahoz et al. 2007). To our knowledge, this is the first report of D. glomerata causing round leaf spot on S. tonkinensis in China.