Occurrence of Rhizoctonia solani AG-5 Causing Root Rot on Astragalus mongholicus in Northwestern China
Authors: Xu Sheng Jun , Jing Zhuo Qiong , Guo Zhi Jie 
Published: January 2025

Astragalus mongholicus is a perennial Chinese medicinal herb in the family Leguminosae widely cultivated in China. In September 2023, A. mongholicus plants in a field in Weiyuan County, Gansu Province, showed symptoms of circular or irregular brown, sunken and necrotic lesions, multiple lesions coalesced, and brown longitudinal cracks in the roots. An investigation was performed in a 1998 m2 field with a root rot incidence of 10%, and the severity ranged from 5 to 60% of root surface area. Five rotted root samples were collected. Fragments of symptomatic roots were surface sterilized with 75% ethanol for 10 s, 2% sodium hypochlorite for 2 min, washed three times with sterilized distilled water, and then blotted dry on sterile filter paper. Fragments were placed on potato dextrose agar (PDA) medium and incubated at 25 ± 1°C in darkness for 5 days and five isolates were purified by transferring single hyphal tips to new PDA plates. Isolates WY23AM01 and WY23AM05, two of five isolates with similar morphology, were randomly selected for morphological and molecular identification and pathogenicity tests. Colonies of the fungus were white initially, then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 20 days of incubation at 25 ± 1°C in the dark. Genomic DNA from each of the two isolates was extracted, and the internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences of isolates WY23AM01 and WY23AM05 were deposited in GenBank (PQ362092 and PQ362093). Phylogenetic analyses were performed by the maximum likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani using MEGA 10.0. The phylogenetic tree grouped the two isolates within the R. solani AG-5 clade with high bootstrap support (99%). PCR analysis was performed with 21 primers specifically designed to detect individual anastomosis groups or anastomosis subgroups of R. solani (Carling et al., 2002; Misawa and Kurose, 2019; Misawa et al., 2020; Okubara et al., 2008). Among the 21 specific primer pairs, only AG-5 specific primer amplified the fungal DNA, indicating that the two isolates belonged to the R. solani AG-5. For pathogenicity tests, two isolates were grown individually on sterile wheat kernels at 25 ± 1°C for 10 days. Certified pathogen-free seedling stage roots were grown in the plastic pot filled with the commercially available sterilized horticultural soil and inoculated by burying twenty infested wheat kernels in the soil adjacent to the roots. Control plants were inoculated with sterile wheat kernels using the same procedure. Each pot had three roots, and each treatment had 12 pots as replicates. All plants were placed in a greenhouse with 60% relative humidity and a 12h/12h light/dark photoperiod at 15 to 30°C. Sixty days after inoculation, typical root rot symptoms developed on all inoculated plants, similar to symptoms observed in the field, whereas control plants remained symptomless. The pathogenicity test was performed three times and symptoms were similar to those observed in the field. Finally, fungal isolates were reisolated from the symptomatic roots of all inoculated plants and identified as R. solani AG-5 by molecular analysis as the isolates used for inoculation, thus fulfilling Koch’s postulates. To our knowledge, this is the first confirmed report of R. solani AG-5 causing root rot on A. mongholicus in China. Our findings improve knowledge about R. solani AGs occurring in A. mongholicus fields in China and broadens the host range of R. solani AG-5. Due to serious damages caused by this disease in recent years in China, further studies should be conducted to investigate the diversity, prevalence, disease control measures and fungicide sensitivity of AGs distributed in the main A. mongholicus-producing areas in China.