Development and evaluation of PCR primers for specific detection of Ralstonia pseudosolanacearum (RSSC phylotype I)

Abstract

Ralstonia pseudosolanacearum, a member of the Ralstonia solanacearum species complex (RSSC), is a soil-borne phytopathogen causing bacterial wilt in economically important crops such as tomato, eggplant, and pepper, leading to substantial yield losses. Early detection of this pathogen is essential for better disease management. The present study, conducted during 2021 to 2024 at the Defence Institute of Bioenergy Research, Haldwani Uttarakhand, was intended to design and evaluate polymerase chain reaction (PCR) primer pairs specific to R. pseudosolanacearum (RSSC Phylotype I). A total of nine primer pairs, including two previously reported primer sets (759/760 and OL1/Y2), were assessed for their specificity and amplification efficiency using DNA from multiple R. pseudosolanacearum isolates, non-RSSC bacterial strains, and soil metagenome samples. Among them, two novel primer pairs, pme F/R and J21535 F/R, produced clear and consistent amplification in R. pseudosolanacearum strains while showing no cross-reactivity with non-target organisms or environmental DNA. The primers demonstrated high specificity and sensitivity, confirming their suitability for reliable detection of the pathogen under laboratory conditions. These findings highlight the diagnostic potential of the pme F/R and J21535 F/R primer sets for specific identification of R. pseudosolanacearum from suspected bacterial isolates. Further validation across diverse hosts, geographical locations, and environmental conditions is recommended to establish their robustness for routine investigation and diagnostics.