First Report of <i>Botrytis cinerea</i> Causing Leaf Spot on <i>Tinospora sagittata</i> in China

Tinospora sagittata (Oliv.) Gagnep. (Tinosporeae) has been used in traditional Chinese medicine in the Chinese pharmacopoeia. In October of 2023, a leaf spot on T. sagittata with incidence of about thirty percent was observed in an herbal nursery at Yunyang District of Shiyan City, Hubei province in China (110°49' 28''E, 32°46'14''N). Initial symptoms included irregular chlorosis, necrotic lesions and grey mold layer with dark brown borders appearing on the leaf tips and margins. After 10 days or so, the lesions spread to cover the entire leaf, with further development leading to leaf desiccation and death. For isolating the pathogens, 6 leaves with disease symptoms were randomly collected and 12 pieces (5mm2) including the diseased and healthy tissues were firstly sterilized with 75% ethanol, then were sterilized with sodium hypochlorite. After rinsing with sterile water three times, samples were plated onto potato dextrose agar (PDA) at 25°C in the dark for 7 days. Twelve isolates (TSJG1-12) with similar morphology were obtained after 7 days, from which TSJG2 was selected as a representative isolate for further study. The initial colony was gray white, and the mycelium grew vigorously. In the later stage, black irregular sclerotia started to form after 10 day incubation on PDA media. Conidia were 7.24-14.36×6.10-10.42 μm (n=50), hyaline, ovoid or ellipsoid in shape. The strain TSJG2 was tentatively identified as Botrytis cinerea based on the morphological characteristics (Ellis 1971). Genomic DNA was extracted from its mycelia, using a commercial DNA extraction kit (Tsingke Biotechnology Co., Ltd, Wuhan, China). Four genes encoding the internal transcribed spacer region of rDNA (ITS), β-tubulin (TUB2), heat shock protein (HSP60), RNA polymerase subunit II second largest subunit (RPB2) were amplified specific primers ITS1/ITS4, bt2a/bt2b, HSP60-F/HSP60-R and RPB2-F/RPB2-R respectively (Aktaruzzaman et al., 2022; Lu et al., 2023). The sequences were generated and deposited into GenBank with accession numbers PP425840 (527bp), PP471570 (430bp), PP476363 (1031bp), PP442152 (1224bp) for ITS, TUB2, HSP60 and RPB2. The sequences showed 99-100% similarity to the reference strain of Botrytis cinerea (ITS TUB, HSP60 and RPB2 sequence accession numbers MT495453.1, ON238899.1, MF468109.1 and MH479932.1, respectively). Based on morphological and molecular features, the strain TSJG2 was identified as Botrytis cinerea. The suspension with a concentration of 1x106 spores/mL was inoculated on 10 unwounded 1-year-old leaves of T. sagittata. Sterile water was inoculated on 10 healthy plant leaves as control. All treated plants were placed in a greenhouse at of 22℃with high humidity 75%. All inoculated leaves showed chlorosis and black-brown necrotic lesions on leaves after 5 days, whereas control leaves were asymptomatic. Pathogens were reisolated from the diseased leaves and identified as B. cinerea based on its morphological and molecular characteristics, conforming Koch's postulates. B. cinerea has been reported to cause grey mold on the leaves of Rhizoma paridis (You et al., 2014) and strawberry (Marin et al., 2022), but it has not been reported on the leaves of T. sagittata. This is the first to report the B. cinerea causing the leaf spot disease on the T. sagittata in China.