Occurrence of Rhizoctonia solani AG-2-1 and R. solani AG-4 HG-Ⅱ Causing Black Root on Cauliflower in Gansu Province, China

Cauliflower (Brassica oleracea var. botrytis) is a major cultivated crop of the summer vegetables on the plateau of western China, which not only plays a crucial role in the summer vegetable supply, but also serves as a core cash crop for local vegetable farmers. In April 2024, symptoms of blackening, constriction, and white mold under moist conditions were observed on roots and stems of cauliflower seedlings grown in plug trays in Yuzhong County, Gansu Province. A survey was conducted in five commercial seedling greenhouses covering 25 plug trays per greenhouse (72 seedlings per tray), with the disease incidence of seedlings ranging from 10% to 30% across the five surveyed greenhouses, which represents a substantial threat to seedling production and causes economic losses to local growers. Twelve disease samples with typical symptoms were collected by 2-3 samples randomly selected from each of the five greenhouses. Symptomatic fragments (5 mm × 2 mm) were cut from the junction between healthy and diseased tissues of roots and crowns of cauliflower seedlings and surface sterilized with 75% ethanol for 15 s, 2% sodium hypochlorite for 60 s, rinsed three times with sterile distilled water, and then blotted dry on sterile filter paper. These fragments were placed on potato dextrose agar (PDA) medium and incubated at 25 ± 1°C in darkness for 5 days and twelve isolates were purified by transferring single hyphal tips to new PDA plates. Fungal colonies were white initially, then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 15-30 days of incubation at 25 ± 1°C in the dark. Genomic DNA from each isolate was extracted, and the ITS region was amplified and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences of the 12 isolates were deposited in GenBank (PX677427 to PX677438). A total of 21 pairs of specific primers targeting different anastomosis groups (AGs) of Rhizoctonia solani were employed to perform PCR amplification for the genomic DNA of the 12 isolates (Carling et al., 2002; Misawa and Kurose, 2019; Misawa et al., 2020; Okubara et al., 2008). PCR amplification with specific primers revealed that four of the 12 isolates were identified as R. solani AG-2-1, and the remaining eight isolates were confirmed to be R. solani AG-4 HG-Ⅱ. Phylogenetic analyses of the 12 isolates were conducted using the maximum likelihood method based on ITS sequences with reference to known AGs of R. solani using MEGA Ⅹ. The phylogenetic tree grouped the 12 isolates within R. solani AG-2-1 and R. solani AG-4 HG-Ⅱ clade with high bootstrap support (99%), respectively. For pathogenicity tests, two representative isolates YZ24SH01 and YZ24SH03 were cultured individually on sterile wheat kernels at 25 ± 1°C for 10 days. Seeds of the pathogen-free cauliflower cv. Qingxi D60 were sown in 50-well tray filled with sterilized soil. For each isolate, eight-week-old seedlings were inoculated by digging soil and burying three infested wheat kernels in soil adjacent to the roots. Control seedlings were inoculated with sterile wheat kernels using same procedure. Each treatment had 30 seedlings as replicates. All seedlings were placed in a greenhouse with 12h/12h light/dark photoperiod at 15 to 30°C. Thirty days after inoculation, symptoms of blackening, constriction, and slight white mold under moist conditions were observed on roots and basal stems of all seedlings inoculated with wheat diseased grains, which was consistent with the typical symptoms in the surveyed greenhouse, whereas control seedlings remained symptomless. Pathogenicity test was performed three times and 100% seedling infection was observed in all three independent trials, and symptoms were similar to those observed in seedling greenhouses. Finally, fungal isolates were reisolated from the symptomatic roots of all inoculated seedlings and confirmed as R. solani AG-2-1 and R. solani AG-4 HG-Ⅱ via ITS sequencing and AG‑specific PCR, matching original inocula, respectively, thus fulfilling Koch’s postulates. To the best of our knowledge, this is the first confirmed report of R. solani AG-2-1 and R. solani AG-4 HG-Ⅱ causing black root on cauliflower in Gansu Province, China, which is consistent with the reports of R. solani AG-4 HG-Ⅰ infecting white cabbage (Saygi et al. 2020) and AG-1-ⅠB infecting B. oleracea var. acephala (Benli et al. 2021). These results suggest that different AGs of R. solani have a broad host range among B. oleracea varieties, posing a potential threat to the production of cruciferous vegetables in western China. Further studies are needed to investigate the prevalence, disease control measures and fungicide sensitivity of R. solani AGs distributed in summer vegetables producing areas on the plateau of western China. This study clarifies the causal agents of cauliflower black root in Gansu, providing a basis for targeted disease management and safeguarding the regional vegetable industry.