First Report of Root and Bulb Rot Caused by Rhizoctonia solani AG-6 on Lanzhou Lily (Lilium davidii var. unicolor) in China.

Abstract

Lanzhou lily (Lilium davidii var. unicolor) is the only famous sweet lily variety that has high edible, medicinal and ornamental value in China, which is mostly planted in the middle areas of Gansu Province in China. In recent years, severe yellowing and wilting of leaves, stem wilt, root and bulb rot symptoms were observed on Lanzhou lily in Qilihe District, Lanzhou, which has resulted in serious loss of bulb production. From June to August 2022, a survey of Lanzhou lily disease was carried out in Xiguoyuan and Weiling township of Qilihe District, Lanzhou. Typical symptoms of root and bulb rot were observed in Lanzhou lily fields. The disease incidence was estimated up to 30%. Fragments of symptomatic roots and bulbs 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 PDA medium and incubated at 25 ± 1°C in darkness for 5 days and 2 isolates were purified by the single-tip culture. Colonies of the fungus were white initially, and then turned light brown to brown, raised, and with entire or undulate edges. Sclerotia were brown and produced on PDA after 25 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 strains QLH22LD01 and QLH22LD02 were deposited in GenBank (OR710804 and OR710805). Phylogenetic analyses were performed using the Maximum Likelihood method with ITS sequences for anastomosis groups (AG) of Rhizoctonia solani. The phylogenetic tree grouped the two isolates within the R. solani AG-6 clade with high bootstrap support (100%). 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-6 specific primer amplified the fungal DNA, indicating that the two isolates tested belonged to the R. solani AG-6. Therefore, these two strains were identified as R. solani AG-6. For pathogenicity tests, two isolates were grown individually on sterile wheat kernels at 25 ± 1°C for 14 days. Certified pathogen-free Lanzhou lily bulbs were grown in the plastic pot filled with the sterilized soil. Fifteen 2- week-old plants were inoculated by digging the soil and burying ten infested wheat kernels in the soil adjacent to the roots. Control plants were inoculated with sterile wheat kernels using the same procedure. All plants were placed in a greenhouse with a 12h/12h light/dark photoperiod at 15 to 30°C. Fifty days after inoculation, typical root and bulb rot symptoms developed on all inoculated plants, similar to symptoms observed in the field, whereas control plants remained symptomless. Pathogenicity test was performed three times with similar to symptoms observed in the field. Finally, the fungi were reisolated from the symptomatic plants and identified 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-6 causing root and bulb rot on Lanzhou lily in China. Our findings improve knowledge about R. solani AGs occurring in Lanzhou lily fields in China. 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 Lanzhou lily-producing states in China.