Abstract
BackgroundPesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance.Methodology/Principal FindingsWe demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k’ at the inflexion point based on a four parameter sigmoid curve. Our results show that k’ is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k’ has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k’, allowing the combination of samples across multiple runs in a single analysis.Conclusions/SignificanceThe transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally
Highlights
Insecticide resistance has long been a problem of agriculture but has risen in prominence since the introduction of synthetic organic insecticides in the 1950’s [1]
The threat remains despite the introduction of new transgenic cotton varieties and Integrated Pest Management (IPM)
One such compound is pirimicarb (Pirimor), an insecticide that is highly effective at killing aphids but not the desirable and beneficial predatory species associated with aphids [2]
Summary
Insecticide resistance has long been a problem of agriculture but has risen in prominence since the introduction of synthetic organic insecticides in the 1950’s [1]. While the use of toxins remains a fundamental method of pest control, resistance will continue to threaten sustainable agriculture. The threat remains despite the introduction of new transgenic cotton varieties and Integrated Pest Management (IPM). This is because transgenics, such as Bt-cotton, rely on toxins and so expose pests to high selection for resistance. IPM systems favour the use of more selective compounds, thereby narrowing the range of chemicals used. One such compound is pirimicarb (Pirimor), an insecticide that is highly effective at killing aphids but not the desirable and beneficial predatory species associated with aphids [2]. Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance
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