First Report of Target Leaf Spot on Flue-Cured Tobacco by Rhizoctonia solani AG-3 in Sichuan, China

Tobacco is an important commercial crop in Sichuan Province in China. In early July 2018, widespread (more than 500-ha) leaf-spot symptoms were observed with a general incidence of 10 to 30% on many tobacco-growing farms in Gulin and Xuyong counties of Luzhou City in Sichuan Province in southwest China, resulting in a huge economic loss to the local farmers. Infected leaves appeared as water-soaked spots (2 mm) and then expanded to 2- to 3-cm dark brown lesions on the middle to lower leaves. Lesions exhibited concentric rings with a tear in necrotic center and margin and resulted in a shot-hole appearance. The symptomatic leaf tissues selected from Luzhou City were cut into 4- to 5-mm squares, surface sterilized in 75% ethanol for 30 s, disinfested with 0.5% NaOCl for 30 s, rinsed three times with sterile distilled water, and then placed on the surface of a half-pot of potato dextrose agar (PDA) at 25°C. A total of 12 isolates were obtained and identified as Rhizoctonia solani Kühn on the basis of mycelial characteristics: septate hyphae constricted at the junction of hyphae, hyphal branching at approximately right angles, with 5.8 to 10.3 μm diameters (Meyer et al. 1990). To further confirm the identity of isolates, LZ03 isolate was selected at random. The sequence of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA from LZ03 isolate was amplified by polymerase chain reaction assay using universal primers ITS1 and ITS4 (White et al. 1990). The sequence of LZ03 isolate (GenBank accession no. MH714869) had 99% sequence identity with R. solani AG-3 (HQ241274.1). Twelve isolates were respectively confront cultured on a slide with R. solani standard strains AG-1-IA, AG-2-1, AG-3, AG-4-HG II, AG-5, AG-6GV, AG-8, and AG-9. The mycelium fusion observation showed that all the isolates belonged to the anastomosis group 3 (AG-3). On the basis of morphological characteristics and nucleotide homology, all isolates were identified as R. solani AG-3. Koch’s postulates were fulfilled on healthy, potted tobacco plants (cv. NC89) using five isolates in a greenhouse at 25°C, 90% relative humidity, and a 12-h light/dark photoperiod. Ten potted plants were inoculated on the leaves by placing a 5-mm-diameter mycelial plug (3-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten plants were inoculated with PDA plugs as controls. Three to 5 days after inoculation, all inoculated leaves developed water-soaked spots similar to those observed on naturally infected leaves in the field, whereas the control plants remained asymptomatic. Fungi isolated from the inoculated leaves exhibited morphological characteristics identical to R. solani AG-3, and no fungus was isolated from the control plants. To our knowledge, this is the first report of tobacco target leaf spot caused by R. solani AG-3 in Sichuan Province of China. The same disease was reported in 2012 (Wu et al. 2012) and also found in Yunnan, China, in 2017 (Xu et al. 2018). Sichuan Province has the third largest planting area of flue-cured tobacco in China, and this disease appears to be a serious risk for future tobacco production.