Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of Aphanomyces cochlioides in naturally infested soil samples
Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R2 > 0.98, p < 0.001) with pathogen inoculum density was shown, demonstrating the suitability for monitoring soil samples. The limit of detection (LOD) was evaluated in several different soil types and varied between 1 and 50 oospores/g soil, depending on clay content. Soils with a high LOD were characterised as having a low clay content and high content of sand. Varying levels of the A. cochlioides target sequence were detected in 20 of the 61 naturally infested soil samples. Discrepancies between the bioassay and the qPCR assay were found in soils from low- and medium-risk fields. However, the qPCR diagnostic assay provides a potentially valuable new tool in disease risk assessment, enabling sugar beet growers to identify high-risk fields.
KeywordsAphanomyces cochlioides Real-time PCR Diagnostics Soil-borne plant pathogen Sugar beet root rot
The authors wish to thank the Swedish Farmers’ Foundation for Agricultural Research, the Faculty of Natural Resources and Agricultural Sciences at the Swedish University of Agricultural Sciences, Nordic Beet Research foundation and Eurofins Agro Testing Sweden AB for funding. Thanks to Dr. Fredrik Heyman, Dr. Eva Blixt and Prof. Christina Dixelius for kindly providing some of the fungal isolates. Also, thanks to Dr. Katarzyna Marzec-Smith and Kristina Nordström for assistance with the soil DNA extractions and qPCR analyses and thanks to Lotta Eriksson for assistance with the bioassay. Thanks to Dr. Ann-Charlotte Wallenhammar and Prof. Christina Dixelius for critically reading of the manuscript. Finally, thanks to Dr. Björn Gustavsson for providing some of the soil samples.
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