Development and evaluation of polyclonal antibodies for detection of Pythium aphanidermatum and Fusarium oxysporum in ginger

Abstract

Polyclonal antibodies against Pythium aphanidermatum and Fusarium oxysporum proteins were developed for the detection of rhizome rot in ginger using serological assays. Under optimal experimental conditions, the detection limit of P. aphanidermatum by indirect ELISA was 10 µg/ml with a linear working range from 5 to 100 µg/ml (R2 = 0.994). In case of F. oxysporum, the linear working range was 5–100 µg/ml (R2 = 0.991) and the limit of detection was 25 µg/ml. The developed antibodies showed the highest titer in ELISA at 1:2000 dilutions. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis identified proteins ranging from molecular weights 15–97 kDa and 14–116 kDa of P. aphanidermatum and F. oxysporum isolates, respectively. In Western blot analysis, the developed antisera gave positive reactions against the isolated antigens of the fungi. The antibodies revealed immune-reactive bands of molecular weights 59 and 65 kDa in P. aphanidermatum and 44 and 75 kDa in F. oxysporum. The results suggest that the developed antibodies could be successfully applied for the specific immunodetection of P. aphanidermatum and F. oxysporum at an early stage of rhizome rot disease.

Keywords

• ginger
• rhizome rot
• pythium aphanidermatum
• fusarium oxysporum
• polyclonal antibodies
• sds-page
• elisa
• western blot