Development of a real time immuno-PCR assay for the detection of pea root rot causal agent, Aphanomyces euteiches

Abstract

Effective measures to detect Aphanomyces euteiches, one of the devastating pathogen of pea, are lacking. An indirect ELISA and a real time immuno-PCR (RT-iPCR) assay for the timely and sensitive detection of A. euteiches were developed using antiserum specific to oospores of A. euteiches isolates. RT-iPCR was 100 times more sensitive over the linear working range than indirect ELISA. To assess the performance of the RT-iPCR assay, non-infested soils representing a range of soil textures were collected from Alberta fields. An extraction protocol was developed by spiking the soils with A. euteiches oospores and the RT-iPCR assay was used to quantify oospores in the soil extracts. The method performed well at the concentration 100 oospores/g and above. A rolled towel bioassay and root PCR were performed on infested field samples which showed a good agreement with our RT-iPCR results. Thus, RT-iPCR may be an invaluable tool for field diagnostics.