Molecular characterisation of Ralstonia solanacearum (Smith) Yabuuchi et al causing bacterial wilt in solanaceous vegetables

Abstract

Bacterial wilt incited by Ralstonia solanacearum is one of the most devastating diseases of solanaceous vegetable crops in Kerala. Crop losses due to the incidence of this disease may go upto 100 per cent. Existence of different strains, races and biovars has been responsible for breaking down of resistance of varieties evolved through breeding programmes. In view of wide variability, a study was undertaken to characterise the isolates of R. solanacearum collected from three different agro climatic zones ofKerala at molecular level. Nine isolates of R. solanacearum collected from three different locations from brinjal, chilli and tomato were used in the study. These were isolated, purified and maintained in sterile distilled water at room temperature. Inoculation techniques were standardised in brinjal, chilli and tomato plants for assessing the virulence and aggressiveness of the isolates. Virulence and aggressiveness of the isolates were studied on respective host plants and found them highly varying. Vellanikkara and Kumarakom isolates could cross inoculate, whereas Ambalavayal isolates did not. The isolates were characterised by various cultural, morphological and biochemical tests and the variability among them was studied. Biovars, III and IlIA and races, 1 and 3 were identified among the isolates. The isolates were resistant to ampicillin and sensitive to chloramphenicol. Plasmid DNA profile of the isolates were studied and no difference was found in the plasmid DNA profile of the nine isolates. Polymorphism among the isolates was studied using RAPD with ten decamer primers. RAPD profiles exhibited great diversity among biovars III and IlIA as well as among race 1 isolates. Race 3 isolates were less polymorphic with certain primers tested. OPF8 yielded a unique band specific to race 3 isolates. Dendrogram obtained from the pooled data of RAPD profiles also showed high genetic similarity between race 3 isolates. Dendrogram obtained from the pooled data of RAPD profiles also showed high genetic similarity between race 3 isolates. Restriction analysis could not characterise the isolates since no banding pattern was obtained with restricted DNA. No hybridization signal was detected after Southern hybridization in RFLP. Curing of plasmid DNA at high temperatures was found unsuccessful. Plasmid profiles of both mucoid and non-mucoid colonies were compared to assess the role of plasmid in EPS production and the plasmid could be observed in both types of colonies. In the latter, a reduction in size of the plasmid was noticed. Thus the study revealed that great diversity existed among strains of R. solanacearum at different locations of Kerala when molecular techniques, especially RAPD was used as a tool.