Combatting insect pests in major Brazilian Cropping Systems through novel Biotech Approaches

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Crop production is the main driver of the Brazilian economy, both for the home-market and for export, with an estimated harvest of 232 million tons of grains and 1.48 million tons of cotton for 2016/2017. However, productivity is severely constrained: (i) as a direct result of the development of resistance in insect pest populations to current control methods, and consequently (ii) by lack of effective control measures and implementation of IPM (Integrated Pest Management) strategies. This proposal seeks to address these major constraints on agricultural productivity through the development of novel, efficacious and safe biopesticides, which can be used as part of an IPM system. For Phase 1 of the project two target insect pests have been selected for study, the native species Spodoptera frugiperda (armyworm), and an invasive pest species, Helicoverpa armigera (cotton bollworm). These species are highly polyphagous and are not only major pests of soybean, but also of other crops important to the Brazilian economy, including maize and cotton, causing an estimated damage of R$ 2.5 billion in the absence of effective crop protection measures. Both pest species have evolved high levels of resistance to synthetic pesticides previously used for their control and are now evolving resistance to Bt-expressing transgenic crops. Our overall strategy is two-fold: (i) to develop highly effective and safe biopesticides and; (ii) to better understand the bases of resistance to currently deployed pest-control strategies with a view to mitigating/controlling these effects in the future. For pest control, we will use RNA interference-based technology to inactivate key genes both in the pest insect itself, and to target microbial symbionts which are critical to insect development and survival. This technology has the potential to be highly specific, reducing/eliminating potential non-target effects seen with the use of synthetic pesticides. Initially we will target the voltage-gated ion channels (VGICs), which are known targets for many synthetic chemistries. In parallel, we will design and produce novel biopesticides using species-specific dsRNAs against targets in the two pests selected by in silico approaches for preliminary toxicity testing. In Phase 1 we will also generate metatranscriptomic data of gut associated microbials of the target insects. These data will be used to design novel biopesticides which affect obligate insect symbionts, using antisense gene regulators targeted to essential genes in symbionts, as 'proof of concept'. To address pesticide resistance, we will also use in silico approaches to identify suitable molecular targets in selected insect pests with known resistance to conventional pesticides; we will test their sensitivity to dsRNA targeted to the VGICs compared to pesticide-sensitive strains. In Phase 2 of the project, we will extend the pest range to include a major virus vector, whitefly (Bemisia tabaci). We will also investigate the potential of using symbionts to deliver the dsRNA targeted to the insect pests (paratransgenesis), as an alternative to targeting symbionts. To extend work on pesticide resistance, recent evidence from the consortium has shown that insecticide-resistant strains of S. frugiperda carry insecticide-degrading bacteria in their gut microbiota, which can contribute to the evolution of resistance. As a 'proof of concept' we will target these microbes using RNAi, to determine whether removing them restores pesticide-susceptibility in this major crop pest. A limitation of using RNAi to control insect pests relates to the sensitivity of the dsRNA to nucleases within the insect gut. We have recently demonstrated that chimeric protein PTD-DRBD combined with dsRNA forms a ribonucleoprotein particle, which is more stable to nucleases, leading to greater gene knockdown and greater toxicity to the insect. We therefore propose to compare the efficacy of the modified dsRNA to the native dsRNA.