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

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年的谷物收成估计为2.32亿吨，棉花收成为148万吨。然而，生产力受到严重制约：(1)由于害虫种群对当前防治方法产生抵抗力的直接结果，以及(2)缺乏有效的控制措施和实施综合害虫管理(IPM)战略。这项建议寻求通过开发新型、有效和安全的生物杀虫剂来解决这些对农业生产力的主要制约因素，这些杀虫剂可用作综合防治系统的一部分。在该项目的第一阶段，选择了两种目标害虫进行研究，即本地物种果蝇(粘虫)和入侵物种棉铃虫(棉铃虫)。这些物种高度多食性，不仅是大豆的主要害虫，也是对巴西经济重要的其他作物，包括玉米和棉花，在缺乏有效的作物保护措施的情况下，估计造成25亿雷亚尔的损失。这两种害虫都对以前用于控制它们的合成农药产生了高水平的抗药性，现在对表达Bt的转基因作物也产生了抗药性。我们的总体战略有两个方面：(1)开发高效和安全的生物杀虫剂；(2)更好地了解对目前部署的虫害防治战略产生抗药性的基础，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干扰的技术来灭活害虫本身的关键基因，并针对对昆虫发育和生存至关重要的微生物共生体。这项技术有可能具有高度的特异性，减少/消除使用合成杀虫剂可能产生的非靶标影响。首先，我们将目标是电压门控离子通道(VGIC)，这是许多合成化学的已知目标。同时，我们将使用针对两种害虫的物种特异性dsRNA来设计和生产新型生物杀虫剂，这些靶标是通过电子方法选择的，用于初步毒性测试。在第一阶段，我们还将生成目标昆虫肠道相关微生物的元转录数据。这些数据将被用来设计影响专性昆虫共生体的新型生物杀虫剂，使用针对共生体中必要基因的反义基因调节器作为“概念证明”。为了解决杀虫剂抗药性问题，我们还将使用电子计算机方法，在已知对常规杀虫剂具有抗药性的选定害虫中确定合适的分子靶标；我们将测试它们对针对VGICs的dsRNA的敏感性，并与对农药敏感的菌株进行比较。在该项目的第二阶段，我们将扩大害虫的范围，以包括一种主要的病毒媒介--烟粉虱。我们还将研究利用共生体传递针对害虫(副转基因)的dsRNA作为靶向共生体的替代方案的可能性。为了扩大对杀虫剂耐药性的研究，该联盟最近的证据表明，耐杀虫剂的果蝇菌株在其肠道微生物群中携带杀虫剂降解菌，这可能有助于耐药性的演变。作为一个“概念证明”，我们将使用RNAi针对这些微生物，以确定移除它们是否会恢复这种主要作物害虫的杀虫剂敏感性。使用RNAi控制害虫的局限性与dsRNA对昆虫肠道内的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，对核酸酶更稳定，导致更大的基因敲除和对昆虫的更大毒性。因此，我们建议比较修饰的dsRNA和天然的dsRNA的效果。

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.