Development of Elisa for Detection of Clavibacter Xyli Susp. Xyli, The Causal Agent of Ratoon Stunting Disease in Sugarcane

Abstract

Rabbits and sheep were immunized with cultured Clavibacter xyli susp. xyli (Cxx) fixed by heat, gluteraldehyde or formalin, respectively. Antigen (1 x 10⁹ cells/ml) was administrated by multiple injections on the back and intramuscularly in the hind quarter of the animal. Bleeds were evaluated for the presence of antibodies against Cxx (anti-Cxx) in an indirect ELISA assay. Polystyrene beads coated with fixed bacteria directly or in conjunction with poly-l-l-lysine were used as a solid phase. Second antibodies labelled with alkaline phosphatase served as a tracer. This assay was reliable enough for discrimination of normal and immune bleeds. The highest anti-Cxx response was observed when a heat-fixed preparation of Clavibacter xyli susp. xyli was used as the antigen. Formalin-fixed antigen generated a very low anti-Cxx titer. The test for Cxx detection features a double antibody sandwich ELISA: sheep anti-Cxx coated beads as a solid phase and rabbit anti-Cxx conjugated with alkaline phosphatase as a tracer. Preliminary results indicate that ELISA is capable of detecting Cxx in sap from major U.S. sugarcane varieties with ratoon stunting disease. Experiments are now in progress in order to optimize the assay conditions, eliminate non-specific binding caused by plant sap, and to improve the sensitivity of quantitative ELISA.