Development of Polyclonal Rabbit Serum-Based ELISA for Detection of Pectobacterium carotovorum subsp. carotovorum and its Specificity against other Causing Soft Rot Bacteria

The soft rot bacteria Pectobacterium and Dickeya species cause economically important diseases on potato crops. Specific and efficient detection methods are essential to investigate the ecology and pathogenesis of the soft rot bacteria as well as in seed certification programmes. Polyclonal antibodies against potato soft rot bacterium; Pectobacterium carotovorum subsp. carotovorum were generated in female New Zealand white rabbits. Determination of the optimum period to collect the antiserum (As) using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed that, the first collecting date 7 days after the last injection was the best followed by the second collecting date 14 days in both killed (As1) and live (As2) bacterial antisera. The best positive values of antiserum titer were obtained with As1 (7 days) up to dilution of 1:1.28×104 then As1 (14 days) 1:6.4×103 and As2 (7 or 14 days) 1:8×102. Efficiency of the antiserum to comparing among six isolates causing soft rot, Pectobacterium carotovorum subsp. carotovorum (PCCS63) which used for antisera production, P. carotovorum subsp. carotovorum (IPO1949), P. carotovorum subsp. atrosepticum (1007), P. carotovorum subsp. wasabiae (ipp041), Dickeya dianthicola (IPO2114) and Dickeya chrysanthemi (DSM4610) revealed that, our polyclonal antiserum had a high sensitivity to react with P. carotovorum subsp. carotovorum isolates PCCS63 and IPO1949 than with D. chrysanthemi DSM4610 isolate and the least coupling was found with P. carotovorum subsp. wasabiae ipp041 and Dickeya dianthicola IPO2114 isolates. The produced antiserum was more sensitive to detect P. carotovorum subsp. carotovorum isolates than the other soft rot bacteria.