Abstract
Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all commonA.citrullistrains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting allA.citrullistrains. We used a high-throughput bead array technique to screen and characterizeA.citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against fiveA.citrullistrains (PSA, KK9, SQA, SQB and P) and the closely related bacterium,Delftia acidovorans. Three MAbs exhibiting different binding patterns toA.citrulliwere used to develop an ELISA-based method called double antibody pairs sandwich ELISA (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16A.citrullistrains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5x10(5)to 1x10(6)CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5x10(4)to 1x10(7)CFU/mL and 5x10(4)to 5x10(5)CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of allA.citrullistrains. The method can be applied to seed testing prior to planting as well as to routine field inspections.
