Immunodiagnosis of bacterial wilt pathogen using polyclonal antiserum to Ralstonia solanacearum

A Direct antigen coating (DAC) immune assay was developed for detection of virulent strains of Ralstonia solanacearum, which indicates the presence or absence of bacteria in the plant and soil. A simple strategy was used for the production of polyclonal antiserum for the detection of virulent strains of Ralstonia. We identified EPS as major virulence factor. So both whole bacterial cells of Ralstonia fixed with glutaraldehyde and the purified extracellular polysaccharides were the two antigens used for immunization to produce a high titre polyclonal antiserum. The type of antigen used for immunization significantly influenced the specificity of polyclonal antibodies produced. The rabbit immunized using glutaraldehyde- killed bacterial cells (Ag1) produced polyclonal antibodies with higher specificity than that immunized using the extracellular polysaccharides (Ag2). EPS could be a weak immunogen when compared to whole cells. Both the antiserum produced successfully detected original bacterial culture of different concentrations as well as natural infection of Ralstonia in infected tomato, brinjal and chilli crops and infected soil at 1:8000 dilutions. The sensitivityof detectionof R. solanacearum in different samples has indicated that high absorbance in root samples followed by ooze and soil in all the hosts tested such as chilli, brinjal and tomato. The sensitivity assay has also indicated that pure culture, showed the higher detection limit, up to 1012 cells per ml as compared to the detection limits of plant samples and soil samples. Thus this is a semiquantitative method and is proportional to the pathogen concentration.