First report of AG-2-2 IIIB causing Rice sheath blight in China.

Rice (Oryza sativa) is an important crop worldwide, rice is susceptible to many pathogens, one of the most significant being Rice Sheath Blight, caused by Rhizoctonia solani. This disease initially produces cloudy spots on the leaf sheaths and later affects grain filling, resulting in yield losses of over 45%（Chen et al. 2013） when severe. In many southern rice-growing areas of China, the impact of this disease has risen to become the most damaging of the three major rice diseases (Margani et al. 2018). In July 2023, In Yongfu County, Guangxi (110.022°E, 25.010°N), symptoms of rice sheath blight were observed. The leaf sheaths were affected, with small, water-soaked, dark green spots with indistinct edges appearing near the water surface. These spots gradually expanded into elliptical or cloud-like lesions. Eventually, the center of the lesions turned straw-yellow to grayish-white, while the edges turned brown to dark brown. Often, several lesions merged into large cloud-like patches. Fifteen symptomatic sheaths were collected disinfecting pieces of necrotic tissue with 3% NaClO for 1.5 minutes, followed by 75% alcohol for 1 minute. The pieces were then rinsed with sterile distilled water, subsequently plated on Potato Dextrose Agar in Petri dishes, and incubated at 28°C in the dark. One isolate was obtained from each diseased plant using the hyphal tip method. (Feng et al. 2008). Isolates were obtained and displayed initially white mycelium and gradually turned brown after three to four days. Septate hyphae were 4.27 to 10.73 μ m (average 6.41 μ m) in diameter and branched at Right angle or acute angle with a constriction at the origin of the branch point. Staining with 1% safranin O and 3% KOH solution (Bandoni 1979) revealed multinucleated cells (three to nine nuclei per cell, n = 144). In summary, these characteristics were consistent with the description of Rhizoctonia solani Kühn (Meyer et al. 1990). The anastomosis group (AG) was confirmed by selecting three representative isolates (GL-Q-10, GL-Q-13, GL-Q-15) for molecular identification. The target DNA was extracted using Chelex-100. The internal transcribed spacer (ITS) region was amplified and sequenced with primers ITS1 and ITS4. The sequences were deposited in GenBank (ITS, PQ047154, PQ047150, and PQ047151 The base pairs are respectively 713bp, 715bp and 776bp, respectively). Upon searching GenBank, accession number MT385836 was found (Zhou et al. 2021), which has a similarity of 99.15% with PQ047154, 98.87% with PQ047150, and 99.30% with PQ047151. Phylogenetic tree analysis based on ITS sequences showed that the isolates clustered monophyletically with strains of R. solani AG-2-2 IIIB. The fusion group of the strain is verified by the shape and color of its mycelial growth on PDA at 35°C, enabling the distinct differentiation of AG-2-2 IIIB from AG-2-2 IV in terms of both morphology and coloration.（Aktaruzzaman et al. 2019） Pathogenicity tests involved culturing the pathogenic bacteria on PDA for 7-10 days, Then, 10 healthy rice plants (greenhouse potted rice variety Dian Heyou 615) were selected at the heading stage, and 5 plants were inoculated on the leaf sheaths with 5 strains of 5 mm fungus cake with pathogenic bacteria and 5 plants without pathogenic bacteria (The rice soil was disinfected), wrapped in cotton for moisture retention. All plants were sealed in transparent plastic bags and incubated in a greenhouse at 30 °C for 7-15 days, with daily moisturizing using sterile distilled water (Humidity control at 70%). Seven days postinoculation, Those containing pathogenic bacteria have symptoms of rice sheath blight, No symptoms were detected on control plants. Rhizoctonia solani AG-2-2 IIIB was re-isolated from the inoculated plants as previously described, thus fulfilling Koch's postulates. The pathogenicity tests were repeated three times. At present, Rhizoctonia solani AG-2-2 IIIB is primarily pathogenic in plants such as sugar beet and beans. It has only been reported in Japan and other countries to cause rice disease (Engelkes et al. 1996; Kenji Inagaki et al. 2004), and Rhizoctonia solani AG-2-2 IIIB has never been reported in China to cause disease in rice. To our knowledge, this study is the first to identify Rhizoctonia solani AG-2-2 IIIB causing rice sheath blight in China. This finding will aid further research on rice sheath blight defense strategies and contribute to the development of better management practices for this disease.