Development of a sensitive ELISA using three monoclonal antibodies against lipopolysaccharides to detect Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato and pepper

Abstract

Bacterial spot, caused by Xanthomonas campestris pv. vesicatoria (Doidge 1920) Dye 1978 (Xcv), is responsible for severe production losses in tomato and pepper. Three specific monoclonal antibodies (MABs), 7AH10, 5HB3, and 4AD2, which are reactive to lipopolysaccharides of the bacteria, were tested in three kinds of enzyme-linked immunosorbent assays (ELISAs). The most sensitive ELISA was a competitive one able to detect populations of Xcv as low as 10³–10⁴ cfu/ml. Bacterial growth in artificially inoculated tomato and pepper plants could be successfully monitored, and assays on field-collected samples reconfirmed the diagnostic utility of the MABs. Studies on the accessibility of the MABs to their lipopolysaccharide epitopes confirmed that sample preparation can be improved to further increase the sensitivity of the assay. Immunoblotting of differential extracts of the bacterial cells and immunoelectron microscopy demonstrated that the epitopes were present throughout the cell wall and involved in complexes that can be loosened through the action of proteinase K or lysozyme.