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Development of a TaqMan PCR assay for quantification of Aphanomyces euteiches from field soils

S. Chatterton, A. Erickson, and S. Banniza. Development of a TaqMan PCR assay for quantification of Aphanomyces euteiches from field soils. Annual Meeting of the Plant Pathology Society of Alberta, Edmonton AB, November 7 – 9, 2016. (Invited feature oral presentation)

Abstract

Aphanomyces root rot, caused by Aphanomyces euteiches Drechs., was first detected in pea fields in Saskatchewan and Alberta in 2012 and 2013, respectively, and can cause significant crop loss in both provinces. The only management strategy is field avoidance by extended rotations away from susceptible host crops (peas, lentil and alfalfa). To develop a DNA-based quantification tool to determine Aphanomyces root rot risk of field soils, a specific and sensitive molecular detection protocol from soil is first required. To achieve this, primer and hydrolysis probe sets were designed following bioinformatics analysis of the ITS region of related oomycetes and tested against known A. euteiches isolates. The best assay was then tested against non-target fungi including Aphanomyces, Fusarium and Pythium spp. The qPCR assay detected all A. euteiches positives, but at later cycles, also amplified Aphanomyces cochlioides, a closely related species of A. euteiches. All other non-target species were not amplified. The assay was then tested against oospore soil dilution curves prepared in grey and brown soil types. Detection limit in soil was 100 oospores/g of soil, which is also the threshold level for disease development. Future testing will focus on evaluating soil samples from known infected fields to determine the inoculum potential of field soils and validate the A. euteiches infection curve.

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