First Report of Botrytis cinerea Causing Gray Mold on Tea (Camellia sinensis) in China.

Abstract

Tea (Camellia sinensis (L.) O. Kuntze) is an important economic crop cultivated in China. In March 2019, leaf blight symptoms were observed on tea trees (C. sinensis cv. Fuding-dabaicha) in Enshi City (30º02'66" S, 109º01'56" W), Hubei Province (Fig. 1A). The disease occurred on the senescent flowers and mature leaves. The average disease incidence was approximately 10% of plants in the surveyed 30 ha tea garden. Initial symptoms consisted of brown lesions, which expanded and became covered with gray mold, followed by abscission. To identify the pathogen, 20 symptomatic leaves were collected and 0.5 cm2 diseased leaf pieces were excised and surface sterilized by immersion in 1.5% sodium hypochlorite (NaOCl) for 2 min, and rinsed three times in sterilized distilled water. The leaf pieces were allowed to dry, placed on potato dextrose agar (PDA) plates, and incubated at 20°C under 12h fluorescent light. The fungus formed gray to grayish brown colonies (Fig. 1B) and produced conidia after 1 week. The isolate was purified by single spore isolation. The conidia were one-celled, ellipsoid or ovoid, almost colorless, with a size range of 5.3 to 10.24 × 5.2 to 8.1 μm (n = 50) (Fig. 1C). The sclerotia produced in culture were black, round or irregular in shape and 1.3 to 2.2 × 2.0 to 3.3 mm (average 1.8 × 2.3 mm) in size. Morphological characteristics of these isolates matched the description of Botrytis spp. (Hong et al. 2001). Representative isolate LCHM was selected for molecular identification based on DNA sequencing of the ITS region of rDNA and three nuclear protein-coding genes (G3PDH, HSP60 and RPB2) (Staats et al. 2005). BLAST analysis showed that isolate LCHM (GenBank Acc. Nos. MN448502, MN448500, MN433708 and MN448501 for ITS, G3PDH, HSP60 and RPB2, respectively) shared 99 to 100% identity with B. cinerea (GenBank Acc. Nos. MH316147.1, MG846500.1, MG846504.1 and MG846510.1, respectively), which suggested that isolate LCHM belongs to B. cinerea. This identification was further confirmed by phylogenetic analysis based on combined DNA sequence data of G3PDH, HSP60 and RPB2 (Fig. 2). For the pathogenicity test, needle-wounded, attached leaves on 1-year-old C. sinensis cv. Fuding-dabaicha plants were inoculated with mycelial plugs (6 mm diameter from 2-day-old PDA cultures) or a conidial suspension (20 μL, 1×106 conidia/ml in half strength potato dextrose broth) of isolate LCHM on three plants per treatment (six leaves). The control treatments were also wounded, but only treated with agar plugs or half strength PDB. All inoculated plants and controls were incubated in a growth chamber (20°C, 90 ± 10% RH). Leaves inoculated with mycelial plugs, whether wounded or not, showed brown necrotic lesions around the agar plugs after 2 days (Fig. 1D, E), whereas conidial inoculations of wound sites showed necrotic lesions after 6 days (Fig. 1 F, G). Non-wounded leaves inoculated with conidia and all the control treatments remained symptomless. B. cinerea was reisolated from the inoculated leaves and isolates were morphologically similar to the original cultures. Gray mold of C. sinensis caused by B. cinerea has been recorded in Japan, Turkey, and Brazil (Hamaya 1981, Aziz and Harun 2010, Pereira and Mio, 2020). To our knowledge, this is the first report of gray mold caused by B. cinerea on C. sinensis in China. B. cinerea may cause economic losses of tea, therefore, more surveys in other tea-growing regions should be done to specifically search for this disease.