Abstract: 【Objective】 Bacterial wilt of plants, caused by Ralstonia solanacearum, is one of the most devastating soil-borne diseases in the worldwide and severely restricts production of economically important crops. Simple and sensitive detection assay is the basis for effective prevention and control. The objective of this study is to establish a rapid and specific method for detection of R. solanacearum using an isothermal method known as loop-mediated isothermal amplification (LAMP), to make it possible for researchers and technical staff achieve simple detection of this pathogen. 【Method】 Four specific LAMP primers were designed to target the lpxC of R. solanacearum using online design software Primer Explorer Version 4.0, the inner primers are FIP (5′-TACGCCGTTTCATCGGCCAGGTACACGGCGCACAAGT-3′) and BIP (5′-ATCGTCACGTTCGACAAGGTGGAATGCCG GCTGCAACTG-3′) , the outer primers are F3 (5′-CCTGTACGTGGTCGGCTAT-3′) and B3 (5′-ACCGCAACACGGGATCA-3′). Single-factor experiments were conducted to optimize the parameters of the reaction system, the reaction temperatures were set ranging from 60 to 65℃, the concentrations of Mg²⁺ were set ranging from 2 to 12 mmol·L^-1, the concentration ratios of inner and outer primers were set ranging from 2﹕1 to 12﹕1. The specificity of LAMP was tested by using 24 strains of R. solanacearum isolated from different hosts and 5 different strains of non R. solanacearum (Ralstonia mannitolilytica, Ralstonia pickettii, Enterobacter sp., Acidovorax citrulli, Burkhoderia cepacia), of which 3 species are closely related to R. solanacearum and the others are common bacteria in nature. The sensitivities of LAMP and PCR for detecting R. solanacearum were compared by using ten-fold serially diluted DNA of GMI1000 as templates (including original DNA, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, and 10⁷ times of diluent). The LAMP method was used to detect the mixture of potato tissue and strain Po41, mixture of ginger tissue and strain Z-Aq-1, also to detect the wilted tomato plant inoculated with R. solanacearum strain Po82 and the health one. Furthermore, the samples of potatoes which may be infected by R. solanacearum were detected by LAMP method. The results of LAMP could be observed by the magnesium pyrophosphate precipitate produced during the reaction, or by the color changing after adding SYBR Green Ⅰ, the positive samples were green and negative ones were orange. 【Result】 The LAMP assay for rapidly and specifically detecting R. solanacearum was established. In this reaction system, the reacting temperature was determined as 63℃ and the concentrations of Mg²⁺ was 6 mmol·L^-1, the concentration ratios of inner and outer primers was 8﹕1 (1.6﹕0.2 μmol·L^-1). The result of specificity test showed that only the reaction liquids with the DNA of R. solanacearum change green, which indicated this method had a good specificity. Sensitivity experiments indicated that LAMP could detect original DNA, 10¹, 10², 10³, 10⁴, and 10⁵ times of diluent, the sensitivity was 1.42 pg which is 10 times higher than conventional PCR. Also this assay could quickly and accurately detect R. solanacearum from plant tissue suspension, diseased plants as well as infected potato tubers sampled in the field. 【Conclusion】 The LAMP assay established in this study had advantages of high sensitivity, specificity, efficiency and low cost over traditional methods and conventional PCR, the reaction results could be directly observed by naked eyes. All the characteristics of LAMP made it suitable to be widely used in field and grass-roots units.

Key words: Ralstonia solanacearum, detection, LAMP