Cloning and characterization of the

Use of botanical insecticides for sustainable agriculture: Future perspectives

In the last century, the banana crop and industry experienced dramatic losses due to an epidemic of Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f.sp. cubense (Foc) race 1. An even more dramatic menace is now feared due to the spread of Foc tropical race 4. Plant genetic resistance is generally considered as the most plausible strategy for controlling effectively such a devastating disease, as occurred for the first round of FWB epidemic. Nevertheless, with at least 182 articles published since 1970, biological control represents a large body of knowledge on FWB. Remarkably, many studies deal with biological control agents (BCAs) that reached the field-testing stage and even refer to high effectiveness. Some selected BCAs have been repeatedly assayed in independent trials, suggesting their promising value. Overall under field conditions, FWB has been controlled up to 79% by using Pseudomonas spp. strains, and up to 70% by several endophytes and Trichoderma spp. strains. Lower biocontrol efficacy (42-55%) has been obtained with arbuscular mycorrhizal fungi, Bacillus spp., and non-pathogenic Fusarium strains. Studies on Streptomyces spp. have been mostly limited to in vitro conditions so far, with very few pot-experiments, and none conducted in the field. The BCAs have been applied with diverse procedures (e.g., spore suspension, organic amendments, bioformulations, etc.) and at different stages of plant development (i.e., in vitro, nursery, at transplanting, post-transplanting), but there has been no evidence for a protocol better than another. Nonetheless, new bioformulation technologies (e.g., nanotechnology, formulation of microbial consortia and/or their metabolites, etc.) and tailor-made consortia of microbial strains should be encouraged. In conclusion, the literature offers many examples of promising BCAs, suggesting that biocontrol can greatly contribute to limit the damage caused by FWB. More efforts should be done to further validate the currently available outcomes, to deepen the knowledge on the most valuable BCAs, and to improve their efficacy by setting up effective formulations, application protocols, and integrated strategies.

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Biological Control Agents Against Fusarium Wilt of Banana

Plant growth-promoting bacterial endophytes as biocontrol agents of pre- and post-harvest diseases: Fundamentals, methods of application and future perspectives

Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

https://journals.asm.org/doi/pdf/10.1128/AEM.00579-19

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith).

Bacillus thuringiensis (Bt) are Gram-positive bacteria that produce different insecticidal proteins, named Cry, Vip, and Cyt, during the sporulation phase of growth. Here we will describe each one of these classes of protein, their mechanism of action, and their three-dimensional structure if it is available. We will also describe the different strategies that have been used to find novel insecticidal genes that could be used in biological control of insect pests as well as the strategies to evolve known genes to produce proteins with improved toxicity against selected insect pests. These novel strategies include site-directed mutagenesis and domain swapping among different Cry toxins where novel hybrid proteins containing domains or loop regions from different Cry proteins were constructed, resulting in improved toxicity against selected insect pests. Finally we will describe high-throughput systems that have been used to evolve Cry toxins in vitro. Overall, Bt toxins represent one of the most successful strategies for the biocontrol of insect pests.

Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action

Cell lines as models for the study of Cry toxins from Bacillus thuringiensis

Bacillus thuringiensis (Bt) bacteria synthesize different insecticidal proteins named Cry, Vip, and Cyt that are able to kill different insect orders, or nematodes. These proteins have been extensively used in insect control practices in agriculture as sprays or expressed in genetically modified plants. This chapter reviews the mechanism of action of these Bt proteins. In addition, genetic engineering has proven to help in developing novel biotechnological applications of these toxins, such as Cry1AMod toxins, which are active against insects that have developed resistance to Cry toxins; and zymogen-like Vip2 proteins, which can be used to control specific insect pests. Overall, the great diversity of Bt toxins represents a broad opportunity to control the most important insect pests that affect different crops, as well as vectors of human diseases such as malaria and dengue, and to design strategies to manage the development of insect resistance to Bt toxins.

Janette Onofre

Papers

Insecticidal Proteins from Bacillus thuringiensis and Their Mechanism of Action

Cell lines as models for the study of Cry toxins from Bacillus thuringiensis

Mechanism of action of Bacillus thuringiensis insecticidal toxins and their use in the control of insect pests

Identification of Aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta.

Oligomerization is a key step for Bacillus thuringiensis Cyt1Aa insecticidal activity but not for toxicity against red blood cells.