关键词: 黄瓜, 多主棒孢, AS-real-time PCR, 抗药性, 琥珀酸脱氢酶B亚基, 啶酰菌胺

Abstract:

【Objective】 The objective of this study is to establish a rapid and efficient allele specific real-time PCR (AS-real-time PCR) method for quantitative detection of the H278R mutation in the SdhB associated with Corynespora cassiicola in cucumber, and to verify the effect. 【Method】 A total of 24 single conidial strains of C. cassiicola were isolated from Daxing, Beijing. The method of mycelial growth inhibition was used to determine the EC₅₀ value to boscalid. Mycelium growth rate, spore outputs and pathogenicity of 4 susceptible (S) and 8 resistant (R) strains were measured in vitro. Primer pair Cc-SdhB-F/R was used to detect the sequence of SdhB and the base change of SdhB. Based on the sequencing results in SdhB by Cc-SdhB-F/R primers, specific primer pair B-H278R-2F/2R14 and internal primers B-H278R-TY-F/R were designed. An AS-real-time PCR reaction system was established and optimized. The specificity, sensitivity and melt curve of the system were also evaluated. DNA and spore suspension containing different proportions of H278R mutation were detected with the optimized reaction system, respectively. 【Result】 The sensitive frequency of C. cassiicola to boscalid was 66.67% in 24 strains and the EC₅₀ values ranged from 0.057 to 0.563 μg·mL ^-1. The resistance frequency of C. cassiicola to boscalid was 33.33% and the EC₅₀ values ranged from 5.395 to 11.710 μg·mL ^-1. Resistant and susceptible strains only significantly differed in terms of mycelium growth rate, and there was a significant negative correlation between the rate of mycelial growth and EC₅₀ values, but no significant difference in spore outputs and pathogenicity. Sequencing analysis showed that all the resistant strains carried SdhB-H278R mutation. The primers B-H278R-2F/2R14 were of great specificity, the specific fragment was amplified from the DNA of H278R strains, but not from the DNA of other fungal strains. The sensitivity of ordinary AS-PCR was 91 pg·μL ^-1, while that of AS-real-time PCR was 9.1 pg·μL ^-1, which was 10 times higher than that of ordinary AS-PCR. The standard curve established by AS-real-time PCR showed a fine linear relationship between ΔCT value and lg of H278R frequency, the correlation coefficient of the standard curve was 0.9857 and with high amplification efficiency (92.59%). The absorption peak of melting curve was single. The internal reference primers B-H278R-TY-F/R and the specific primer B-H278R-2F/2R14 had a single specific peak at 87.81℃and 91.62℃, respectively. The result of mixtures DNA and mixtures spore suspension to verify the standard curve showed a fine linear relationship between expected percentage and detected percentage (R ²=0.9998 and R ²=0.9922). As the proportions of H278R mutation in DNA and spore suspension decreased, the accuracy of the system gradually increased, and the detection limit was 5%. 【Conclusion】 An efficient, quantitative, and rapid AS-real-time PCR detection system was established for the detection of SdhB-H278R mutation site, which can provide a theoretical basis for the SDHIs resistance management.

Key words: cucumber, Corynespora cassiicola, AS-real-time PCR, resistance, SdhB, boscalid