Cultural, Physiological, Biochemical and Molecular Characterization of Ralstonia solanacearum Causing Bacterial Wilt of Tomato in Kerala, India

Abstract

Bacterial wilt is a destructive vascular disease, especially seen in solanaceous vegetable crops, caused by Ralstonia solanacearum. It affects tomato and other crops particularly in tropical and subtropical regions. This study aimed to isolate and characterize R. solanacearum from bacterial wilt infected tomato plants collected from Kottarakara, Kollam district of Kerala, India. The infected plants exhibited characteristic symptoms, including drooping of young leaves, green wilting and complete plant death. Infected plants showed vascular browning and bacterial ooze streaming from the cut ends of the stem confirmed the presence of the pathogen. The bacterium was cultured on different media such as nutrient agar (NA), King’s B agar (KB), casamino acid peptone glucose agar (CPG) and triphenyl tetrazolium chloride agar (TTC) and showed difference in colony colour, margin and texture. On TTC medium, the virulent colonies exhibited fluidal, creamy white bordered margin with pink centre, indicative for excessive exopolysaccharide (EPS) production. Further, physiological tests confirmed its gram negative (G-ve) nature and showed positive for catalase, oxidase and levan production. The bacterial isolate tested positive for all biochemical tests, except for citrate utilization and phenylalanine deamination.  Biovar determination of the isolate confirmed it as, R. solanacearum biovar 3. To best of our knowledge, this is the first detailed characterization on the biovar of R. solanacearum infecting tomato plants in Kerala.  Molecular characterization using 16S rRNA and phylogenetic analysis further confirmed the identity of R. solanacearum at the genetic level. The sequence was deposited in the NCBI GenBank under the accession number PV022497. These findings demonstrated the effectiveness of the integrated diagnosis approach, including symptomatology, cultural, physiological, biochemical and molecular methods for accurate identification and characterization of R. solanacearum. Understanding the pathogenic and virulence traits of R. solanacearum offers insights for the development of resistant cultivars and specific management strategies in tomato cultivation.