Loop-Mediated Isothermal Amplification assays for rapid detection of Ralstonia solanacearum species complex and Phylotype I in solanaceous crops

Abstract

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt diseases, which affect a wide range of plant hosts, including Solanaceae, and is of major economic importance. Point-of-care (POC) techniques like LAMP (loop-mediated isothermal amplification) allow the rapid and sensitive detection of plant pathogens and can be deployed directly in the field. For fast and reliable diagnosis on-site, we optimized a RSSC-LAMP assay and developed a real-time LAMP assay targeting the phylotype I. Both LAMPs were highly specific, yielding negative results for a wide collection of non-target strains and positive results for all target strains, except for two particular mulberry strains for the phylotype I assay. These results were supported by an extensive in silico analysis performed on 6,105 genomes of Burkholderiaceae. The two LAMP assays displayed high sensitivity on pure suspensions with a detection limit at 103 CFU/ml and 104 CFU/ml for phylotype I and RSSC, respectively. These thresholds correspond to theoretical quantities of as low as 5 CFU and 50 CFU per reaction. Two simplified extraction methods were successfully used to detect the pathogen from different Solanaceae samples. We demonstrated that LAMP assays were operable on solanaceous crops during field surveys and varietal evaluation trials as rapid POC diagnostics tools, which can improve the disease management and control of RSSC.