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

农作物生产是巴西经济的主要驱动力，无论是国内市场还是出口市场，2016/2017年预计收获2.32亿吨谷物和148万吨棉花。然而，生产力受到严重限制：（i）由于昆虫害虫种群对当前控制方法产生抗性的直接结果，以及因此（ii）缺乏有效的控制措施和IPM（综合害虫管理）战略的实施。该提案旨在通过开发新型、有效和安全的生物农药来解决这些对农业生产力的主要限制，这些生物农药可用作IPM系统的一部分。在项目的第一阶段，选择了两种目标害虫进行研究，即本地物种草地贪夜蛾（粘虫）和入侵害虫物种棉铃虫（棉铃虫）。这些物种是高度杂食性的，不仅是大豆的主要害虫，而且也是对巴西经济重要的其他作物，包括玉米和棉花的主要害虫，在缺乏有效作物保护措施的情况下，估计造成25亿雷亚尔的损失。这两种害虫物种已经进化出对以前用于控制它们的合成农药的高水平抗性，并且现在正在进化出对表达Bt的转基因作物的抗性。我们的总体战略是双重的：（i）开发高效和安全的生物农药;（ii）更好地了解目前部署的害虫控制策略的抗性基础，以期在未来减轻/控制这些影响。在害虫防治方面，我们将使用基于RNA干扰的技术来检测害虫本身的关键基因，并靶向对昆虫发育和生存至关重要的微生物共生体。这项技术具有高度特异性的潜力，可以减少/消除使用合成农药可能产生的非目标效应。最初，我们将靶向电压门控离子通道（VGIC），这是许多合成化学的已知目标。与此同时，我们将设计和生产新的生物农药，使用物种特异性的双链RNA对目标的两个害虫选择在计算机方法进行初步毒性测试。在第1阶段，我们还将生成目标昆虫肠道相关微生物的元转录组学数据。这些数据将被用来设计新的生物农药，影响专性昆虫共生体，使用反义基因调节器针对共生体中的必需基因，作为“概念的证据”。为了解决农药抗性问题，我们还将使用计算机模拟方法在已知对传统农药具有抗性的选定昆虫害虫中鉴定合适的分子靶标;我们将测试它们对靶向VGIC的dsRNA的敏感性，并与农药敏感菌株进行比较。在该项目的第二阶段，我们将扩大害虫范围，包括一种主要的病毒载体烟粉虱（Bemisia tabaci）。我们还将研究使用共生体来递送靶向昆虫害虫的dsRNA（parattransgenesis）的潜力，作为靶向共生体的替代方案。为了扩展对杀虫剂抗性的研究，最近来自该联盟的证据表明，耐除草剂的S。草地贪夜蛾在其肠道微生物群中携带降解草甘膦的细菌，这可能有助于抗药性的进化。作为“概念验证”，我们将使用RNAi靶向这些微生物，以确定去除它们是否会恢复这种主要作物害虫的农药敏感性。使用RNAi控制昆虫害虫的局限性与双链RNA对昆虫肠道内核酸酶的敏感性有关。我们最近证明，嵌合蛋白PTD-DRBD与dsRNA结合形成核糖核蛋白颗粒，其对核酸酶更稳定，导致更大的基因敲除和对昆虫更大的毒性。因此，我们建议将修饰的dsRNA的功效与天然dsRNA进行比较。

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.