First Report of Sclerotinia Blight Caused by Sclerotinia sclerotiorum on Calendula officinalis in China.

Abstract

Calendula officinalis is widely cultivated in China for its medicinal and ornamental value. In December 2023, symptoms of an unknown sclerotinia-like disease were observed on C. officinalis plants grown on the campus of Huanggang Normal University in the Huanggang area of Hubei, China (30°44'91″ N, 114°92'31″E). Affected leaves and petioles exhibited chlorosis, with internal rotting initiating from the leaf lamina. Dense white mycelial mats developed on infected tissues, particularly leaves and stems. Leaf tips progressively turned grayish, curled, and withered, while petioles displayed grayish discoloration and necrosis, ultimately causing corolla drooping. Severely infected plants eventually died, with approximately 40% of 1,000 monitored plants exhibiting these symptoms. Diseased tissues were excised and immersed in a sodium hypochlorite solution (1%) for approximately 10 s, then washed in sterilized water. Small pieces of affected leaves, petioles, and stems were successfully cultured on potato dextrose agar (PDA) medium, incubated at 25°C. Twenty Sclerotium-like isolates were obtained through mycelial tip isolation. Isolate HGNU2023-P1 displayed white, tightly adherent aerial mycelium and produced 10~20 irregular sclerotia (3.65 ± 0.8 mm in size, mean ± SD, n = 100) near the edge of the petri dishes after 15 d of incubation at 25°C under alternating 12-hour light/dark cycles. For molecular identification, DNA from two representative isolates (HGNU2023-P1 and HGNU2023-S1) were used to amplify the internal transcribed spacer (ITS) region with primers ITS1/ITS4, the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene with primers G3PDHfor/G3PDHrew (Zhang et al. 2021), and the heat shock protein 60 (HSP60) gene with primers HSP60for/HSP60rew (Yin Fuqiang et al. 2024). The gene products were sequenced at Wuhan JinKairui Biological Co., Ltd., and the obtained sequences were submitted to the NCBI database under accession numbers PV330002, PV330003, and PV344707 to PV344710. The sequences exhibited 99%~100% identity with those of Sclerotinia sclerotiorum in GenBank. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA11 based on concatenated ITS, G3PDH, and HSP60 sequences. Pathogenicity of the isolate was evaluated on 18 two-month-old C. officinalis plants. Three plants were inoculated with 5-mm-diameter mycelial plugs of HGNU2023-P1 grown on PDA for 3 days, while control plants received sterile PDA plugs, with three replicates. All seedlings were incubated at 25°C and 90% relative humidity. Within 2 dpi, inoculated plants developed symptoms consistent with field observations, whereas controls remained asymptomatic. S. sclerotiorum was consistently reisolated from decayed plant tissue, fulfilling Koch's postulates. S. sclerotiorum is known to severely threaten numerous Asteraceae hosts (Underwood et al. 2022). This is the first report of S. sclerotiorum causing Sclerotinia blight on Calendula officinalis in China. This report will help identify disease symptoms in the field and provide a basis for determining the occurrence, distribution, and control of the disease.