关键词: 白菜, 立枯丝核菌融合群, 实时荧光定量PCR, 检测

Abstract:

【Objective】 The objective of this study is to identify the pathogen causing cabbage died in Gansu Province, develop a molecular method for identification, and to provide a reference for prevention and control of the disease.【Method】Diseased samples collected from Gansu Province were purified and cultured, and isolates were characterized by colony morphology, it was initially identified the pathogen as Rhizoctonia solani. For accurate identification, the internal transcribed spacer region of the ribosomal DNA (rDNA-ITS) and translation elongation factor 1-alpha (TEF-1α) of isolates were amplified and sequenced. MEGA 7.0 was used to draw the phylogenetic tree of strains and other related sequences. Molecular identification of the pathogen was carried out by PCR amplification using a specific primer pair F-RS/R-RS. Pathogenicity on 20 cabbage cultivars was verified by spray inoculation and irrigating roots treatment. Based on the conserved region of rDNA-ITS, a pair of specific primer was designed and the SYBR Green I real-time PCR reaction system was established. The specificity, sensitivity and repeatability of conventional PCR and real-time PCR were also evaluated, and R. solani AG3-AG11, binulceate Rhizoctonia AG-A, Fusarium spp., Pythium spp. were used as control fungi. The established system was used to detect R. solani in the cabbage and rhizosphere soils. 【Result】 A total of 86 strains of R. solani were isolated. The results of conventional PCR specific test showed that most strains belonged to AG-2-1 (68/86) and others were AG-1-IB (18/86). The phylogenetic analyses showed that 50 strains were divided into AG-2-1 and AG-1-IB, and the homology of each strain in two groups was 100%. Pathogenicity test results showed that the two anastomosis groups could both cause lesions on stems, and AG-2-1 was not pathogenic to the leaves of Jinwawa and Xiaohuangbao. The pathogenicity of AG-1-IB was slightly stronger than that of AG-2-1. The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. solani strains, but not from the DNA of other fungal strains by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 8.41×10 ⁵copies/μL plasmid, while the sensitivity of real-time PCR was 8.41×10 ³copies/μL, which increased two orders of magnitude. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.9983 and with high amplification efficiency (91%). The method could successfully detect the pathogens in cabbage and rhizosphere soils collected in fields. For the indoor potted experiments, the detection results of real-time PCR of infected cabbage samples inoculated after different days showed that the stems and rhizosphere soils had the largest amount of R. solani on the 5th day after inoculation. 【Conclusion】 Through molecular biological identification and pathogenicity test of pathogen of cabbage died, it was confirmed that the causal agent of cabbage died in Gansu were R. solani AG-2-1 and AG-1-IB. The developed real-time PCR system for R. solani has strong specificity and high sensitivity, it can realize the detection and monitoring of R. solani in plant and soil, and can provide technical support for early warning and management of diseases.

Key words: cabbage, anastomosis groups of Rhizoctonia solani, real-time fluorescent quantitative PCR, detection