Abstract Detail

Host/Plant Pathogen Interactions and Plant Health Management

Reich, Jonathan [1], Alexander, Trevor [2], Chatterton, Syama [3].

Development of a multiplex qPCR TaqMan assay for the quantification of airborne spores of Botrytis cinerea and Sclerotinia sclerotiorum.

Fungal pathogens Botrytis cinerea Pers.:Fr. and Sclerotinia sclerotiorum (Lib.) de Bary have a highly homologous genetic code, which makes them difficult to distinguish from each other using molecular techniques. Previously published primer sets for S.sclerotiorum have not always reliably differentiated it from B. cinerea or other Sclerotinia species. The goal of this research was to develop a specific, sensitive primer set for S. sclerotiorum that could be used for quantitative real-time PCR (qPCR) on field aerosol samples. Six primer and probe sets were designed that targeted the intergenic spacer (IGS) region of the ribosomal DNA and genes of several hypothetical proteins. Primers passing initial screening were tested against 34 fungal species and field isolates of S. sclerotiorum and B. cinerea. All primers targeting hypothetical proteins amplified both S. sclerotiorum and S. trifoliorum. The primer/probe set, SsIGS_2, targeting the IGS region was highly specific to S. sclerotiorum, but also faintly amplified Penicillium aurantiogriseum and Leptosphaeria maculans. Sensitivity tests were run with this primer set, which was able to reliably detect 0.04 pg target DNA. SsIGS_2 was then multiplexed with a previously published primer set for B. cinerea in a TaqMan assay; both retained high sensitivity and were able to amplify as little as 0.07 pg of target DNA. This assay is currently being used to analyze aerosol samples collected in the 2014 growing season to quantify the seasonal discharge patterns of ascospores and conidia in seed alfalfa fields of southern Alberta.

1 - University of Lethbridge, Geography, 4401 University Dr W, Lethbridge, AB, T1K 6T5, Canada
2 - Agriculture and Agri-Food Canada, Ruminant Health, 5403 - 1 Avenue South, P.O. Box 3000, Lethbridge, AB, T1J 4B1, Canada
3 - Agriculture and Agri-Food Canada, Sustainable Production Systems, 5403 - 1 Ave. South, P.O. Box 3000, Lethbridge, AB, T1J 4B1, Canada

Keywords:
TaqMan qPCR
Botrytis cinerea
Sclerotinia sclerotiorum
Disease forecasting.

Presentation Type: Oral Paper:Papers for Topics
Session: 55
Location: Salon 2/The Shaw Conference Centre
Date: Tuesday, July 28th, 2015
Time: 3:30 PM
Number: 55002
Abstract ID:726
Candidate for Awards:CPS Best Student Presentation Awards