Nucleopolyhedrovirus coocclusion technology: a new concept in the development of biological insecticides

Nucleopolyhedroviruses (NPV, Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems.This review was funded by the Ministerio de Economía y Competitividad, Spain, project number AGL2017-83498-C2-1-R and previous projects AGL2014-57752-C2-1-R, AGL2011-30352-CO2-01, AGL2008-05456-C03-01, AGL2005-07909-CO3-01, and AGL2002-04320-C02-01

Genomic Sequences of Five Helicoverpa armigera Nucleopolyhedrovirus Genotypes from Spain That Differ in Their Insecticidal Properties.

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has proved effective as the basis for various biological insecticides. Complete genome sequences of five Spanish HearNPV genotypes differed principally in the homologous regions (hrs) and the baculovirus repeat open reading frame (bro) genes, suggesting that they may be involved in the phenotypic differences observed among genotypes.The Departamento de Innovación, Empresa y Empleo, Gobierno de Navarra (project IIQ14065.RI1), funded the study. M.A. received a fellowship from CSIC.Peer Reviewe

Diversity of Iberian nucleopolyhedrovirus wild-type isolates infecting Helicoverpa armigera (Lepidoptera: Noctuidae)

Larvae of the tomato fruitworm, Helicoverpa armigera, were surveyed for nucleopolyhedrovirus (NPV) infection (referred to as HearNPV) in three different locations from the Iberian Peninsula: Olivenza and Toledo in Spain and the Oeste region in Portugal. Twenty HearNPV isolates were obtained from single field-collected larval cadavers. Restriction endonuclease (REN) profiles of the collected isolates with BglII and PstI allowed identification of six different H. armigera single-embedded NPV strains in Spain (referred to as HearSP3, HearSP4, HearSP5, HearSP6, HearSP7, and HearSP8) and two in Portugal (referred to as HearPT1 and HearPT2). No strains were shared by isolates from different geographical regions except HearSP5, which was found in isolates from Olivenza and Toledo. Cluster analysis based on the restriction fragment length polymorphisms of these strains in relation to two previously identified strains from Badajoz (HearSP1) and Cordoba (HearSP2) in Spain, showed no correlation among the strains and their geographical origin. The biological activity of HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, and HearPT2 was compared in terms of pathogenicity (50% lethal concentration, LC50) and virulence (mean time to death). HearPT2 and HearSP7 were significantly more pathogenic than HearSP2, with LC50 values 2.8 and 2.6-fold higher than the latter, respectively, on H. armigera second instars. HearSP4 and HearPT2 killed larvae significantly faster than HearSP8, whereas HearSP2, HearSP7, and HearPT1 showed intermediate mean time to death value

Study of the bacillus thuringiensis Cry1Ia protein oligomerization promoted by midgut brush border membrane vesicles of lepidopteran and coleopteran insects, or cultured insect cells

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities, the State Research Agency of Spain and the European FEDER founds (Refs. AGL2015-70584-C2 and RTI2018-095204-B-C21), and by the Generalitat Valenciana (GVPROMETEOII-2015-001). M. Domínguez received a predoctoral fellowship from the Universidad Pública de Navarra, Spain