Detection of Indian isolates of soil borne and tuber borne Ralstonia solanacearum (Smith) infecting Potato (Solanum tuberosum L.) through LAMP assay

Abstract

Abstract Bacterial wilt caused by Ralstonia solanacearum, is one of the most notorious plant diseases of potato and causes severe yield losses globally. R. solanacearum produce fluidal white colored colonies with a light pink centre on CPG medium. Bacterial wilt pathogen is a soil as well as tuber borne, so earlier detection of bacterial wilt pathogen is necessary to take up timely management practices. Traditional methods used for detection of R. solanacearum are labor-intensive, expensive, time-consuming and can be carried out only in well-equipped laboratories. Hence, the present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for R. solanacearum, as an alternative approach for quick and efficient detection of the bacterial wilt. LAMP reaction mix was optimized by adjusting the concentrations of MgSO4, dNTPs, betaine, time, temperature etc. The optimum temperature and time for the detection of R. solanacearum was 65°C for 50 minutes. The positive samples showed colour change from violet and reconfirmed in 2% agarose gel electrophoresis which produced ladder like bands. The LAMP assay developed was highly specific to detect R. solanacearum from other bacteria and sensitive with a lowest detection limit of 10 pg/µl of template DNA. The developed LAMP assay was validated with R. solanacearum isolates, infected stem, tubers and soil and also it was capable of detecting latent infection of R. solanacearum in seed tubers. Hence, LAMP assay protocol provides a rapid, specific and sensitive tool for the latent detection of R. solanacearum in seed potato tubers.