Protocol for Isolation and Identification of Agrobacterium Isolates from Stone Fruit Plants and Sensitivity of Native A. tumefaciens Isolates against Agrocin Produced by A. radiobacter Strain K84

Abstract

Agrobacteria from rhizosphere soil and galls developed on stone fruit plants, were isolated on D1 medium. Agrobacterium isolates, belonging to biovar-1, developed glistening, transparent, convex colonies with cherry red centre at maturity on medium 1A and biovar-2 developed typical transparent to red coloured colonies on 2E. Single cell colonies of Agrobacterium took red colour when streaked on yeast extract mannitol agar supplemented with Congo red. Pathogenic Agrobacterium isolates (A. tumefaciens) developed galls whereas non-pathogenic isolates (A. radiobacter) failed to develop galls on stems of artificially inoculated 4-week old potted tomato plants. Agrocin production was detected in A. radiobacter isolates without and after exposure to chloroform. The results on number of flagella and their arrangement confirmed by scanning electron microscopy revealed that individual cells of all the native isolates possess single flagellum at their sub-polar ends. The isolate showed luxurious growth on Hofer’s alkaline, produced ketolactose and showed no acid production with bromothymol blue. All the native A. tumefaciens isolates were sensitive to agrocin produced by strain K84 which indicated their nopaline utilizing behaviour. All native Agrobacterium isolates showed 97–100 % sequence homology with A. radiobacter and A. tumefaciens.