Isolation, Characterization and Use of Bacteriophages Against Bacterial Wilt of Brinjal [Ralstonia solanacearum (SMITH) Yabuuchi et al.]
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Date
2017-10
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University of Agricultural Science, Dharwad
Abstract
A major constraint in brinjal production is bacterial wilt disease caused by Ralstonia solanacearum. An attractive option is the use of bacteriophages, (viruses) that specifically kill bacteria. The present investigation on isolation and characterization of bacteriophages against bacterial wilt of brinjal was carried out at Department of Plant Pathology, University of Agricultural Sciences, Dharwad during 2016-17. Five isolates of R. solanacearum from brinjal were collected during survey covering four districts of northern Karnataka, cultured and confirmed for their biochemical and molecular identity using specific primer pairs Rs-sp and fliC which showed their genetic uniformity. The bacteriophages were isolated from raw water of municipal sewage treatment plant, pond and river water, rhizosphere and phylloplane samples of brinjal plant. The plaque forming units (PFU) assay was conducted to understand plaque morphology. Among the nine phage isolates, three phages against R. solanacearum viz., Rsab1, Rsab2 and Rsab3, having clear plaques were obtained with titre value of 2.40 x 105, 2.60 x 105 and 0.50 x 105 PFU ml-1and diameter of 12.10, 3.01 and 8.20 mm, respectively. From host range analysis, phages were found to be non-effective against other tested bacteria Escherichia coli, Staphylococcus aureus, Pseudomonas fluorescens, Xanthomonas axonopodis pv. citri and X. a. pv. punicae and have a broad lytic activity on R. solanacearum isolates. The chloroform sensitivity test showed that the phages were insensitive to the chloroform. The genetic diversity of isolated phages was confirmed by Randomly Amplified Polymorphic DNA (RAPD). In vitro UV sensitivity test showed pregelatinised corn flour (1%) as phage formulation. In rhizosphere stability studies, persistance of formulated phages was observed up to 16th day. In glasshouse studies, sole application of phage mixture on one month old seedling was found better in comparison to standard chemical check (Copper oxy Chloride (0.3 %) + Streptocycline (500 ppm)).