Open Access
The Vip3Ag4 insecticidal protoxin from bacillus thuringiensis adopts a tetrameric configuration that is maintained on proteolysis
(MDPI, 2017) Palma Dovis, Leopoldo; Scott, David J.; Harris, Gemma; Din, Salah-Ud; Williams, Thomas L.; Roberts, Oliver J.; Young, Mark T.; Caballero Murillo, Primitivo; Berry, Colin; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.
Open Access
Vip3C, a novel class of vegetative insecticidal proteins from Bacillus thuringiensis
(American Society for Microbiology, 2012) Palma Dovis, Leopoldo; Hernández Rodríguez, C.; Maeztu Martínez, Mireya; Hernández Martínez, Patricia; Ruiz de Escudero Fuentemilla, Íñigo; Escriche, Baltasar; Muñoz Labiano, Delia; Rie, Jeroen van; Ferré, Juan; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm2.
Open Access
Determinant factors in the production of a co-occluded binary mixture of Helicoverpa armigera alphabaculovirus (HearNPV) genotypes with desirable insecticidal characteristics
(Public Library of Science, 2016) Arrizubieta Celaya, Maite; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, IIQ14065:RI1
A co-occluded binary mixture of Helicoverpa armigera nucleopolyhedrovirus genotypes HearSP1B and HearLB6 at a 1:1 ratio (HearSP1B+HearLB6) was selected for the development of a virus-based biological insecticide, which requires an efficient large-scale production system. In vivo production systems require optimization studies in each host-virus pathosystem. In the present study, the effects of larval instar, rearing density, timing of inoculation, inoculum concentration and temperature on the production of HearSP1B+HearLB6 in its homologous host were evaluated. The high prevalence of cannibalism in infected larvae (40–87%) indicated that insects require individual rearing to avoid major losses in OB production. The OB production of recently molted fifth instars (7.0 x 109 OBs/larva), combined with a high prevalence of mortality (85.7%), resulted in the highest overall OB yield (6.0 x 1011 OBs/100 inoculated larvae), compared to those of third or fourth instars. However, as inoculum concentration did not influence final OB yield, the lowest concentration, LC80 (5.5 x 106 OBs/ml), was selected. Incubation temperature did not significantly influence OB yield, although larvae maintained at 30°C died 13 and 34 hours earlier than those incubated at 26°C and 23°C, respectively. We conclude that the efficient production of HearSP1B+HearLB6 OBs involves inoculation of recently molted fifth instars with a LC80 concentration of OBs followed by individual rearing at 30°C.
Open Access
Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV): natural occurrence and efficacy as a biological insecticide on young banana plants in greenhouse and open-field conditions on the Canary Islands
(Public Library of Science, 2017) Fuentes Barrera, Ernesto Gabriel; Hernández Suárez, Estrella; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Chrysodeixis chalcites, an important pest of banana crops on the Canary Islands, is usually controlled by chemical insecticides. The present study aimed to evaluate the efficacy of the most prevalent isolate of the Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV, Baculoviridae) as a biological insecticide. Overall the prevalence of ChchNPV infection in C. chalcites populations was 2.3% (103 infected larvae out of 4,438 sampled), but varied from 0±4.8% on Tenerife and was usually low (0±2%) on the other islands. On Tenerife, infected larvae were present at 11 out of 17 plantations sampled. The prevalence of infection in larvae on bananas grown under greenhouse structures was significantly higher (3%) than in open-field sites (1.4%). The ChchNPV-TF1 isolate was the most abundant and widespread of four genetic variants of the virus. Application of 1.0x109 viral occlusion bodies (OBs)/l of ChchNPV-TF1 significantly reduced C. chalcites foliar damage in young banana plants as did commonly used pesticides, both in greenhouse and open-field sites. The insecticidal efficacy of ChchNPV-TF1 was similar to that of indoxacarb and a Bacillus thuringiensis (Bt)- based insecticide in one year of trials and similar to Bt in the following year of trails in greenhouse and field crops. However, larvae collected at different time intervals following virus treatments and reared in the laboratory experienced 2±7 fold more mortality than insects from conventional insecticide treatments. This suggests that the acquisition of lethal dose occurred over an extended period (up to 7 days) compared to a brief peak in larvae on plants treated with conventional insecticides. These results should prove useful for the registration of a ChchNPV-based insecticide for integrated management of this pest in banana crops on the Canary Islands.
Open Access
Anticarsia gemmatalis nucleopolyhedrovirus from soybean crops in Tamaulipas, Mexico: diversity and insecticidal characteristics of individual variants and their co-occluded mixtures
(Florida Entomological Society, 2018) Ángel, Christian del; Lasa, Rodrigo; Rodríguez del Bosque, Luis A.; Mercado, Gabriel; Beperet Arive, Inés; Caballero Murillo, Primitivo; Williams, Trevor; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
In 1999, Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) was introduced into a major soybean-growing region in Tamaulipas, Mexico, for control of its lepidopteran host, Anticarsia gemmatalis Hilbner (Lepidoptera: Noctuidae). The virus introduction proved to be highly successful in controlling this agronomically important pest. In order to determine the genotypic diversity and insecticidal traits of Mexican AgMNPVs, we obtained 30 field-collected isolates from Tamaulipas State. Five distinct variants (genotypes 1-5) were identified from plaques replicated in A. gemmatalis larvae by examination of restriction profiles using HindIII. Initial screening indicated that none of the variants, or co-occluded mixtures of variants in different proportions, was more pathogenic than the 30 field isolates mixture or a reference variant from Brazil (AgMNPV-2D). Mean occlusion body production also was similar among genotype variants, the mixture of 30 field isolates and AgMNPV-2D treatments, but was significantly reduced in 1 co-occluded mixture. Speed of kill also was similar among variants (except genotype 1) and their mixtures. Lethal concentration metrics indicated that these results were unlikely due to selection of variants with reduced pathogenicity during the plaque purification process. We conclude that the mixture of 30 field isolates most likely would prove suitable for use as a biological insecticide in the soybean-growing region of Mexico.
Open Access
A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus population from the Canary Islands is genotypically structured to maximize survival
(American Society for Microbiology, 2013) Bernal Rodríguez, Alexandra; Simón de Goñi, Oihane; Williams, Trevor; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua, IIQ14065:RI1
A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1- ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands.
Open Access
Genetic variation and biological activity of two closely related alphabaculoviruses during serial passage in permissive and semi-permissive heterologous hosts
(MDPI, 2019) Belda García, Isabel María; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Phylogenetic analyses suggest that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) may be strains of the same virus species. Most of the studies comparing their biological activities have been performed in their homologous hosts. A comparison of host range and stability in alternative hosts was performed. The host range of these viruses was compared using high concentrations of inoculum to inoculate second instars of six species of Lepidoptera. One semi-permissive host (Spodoptera littoralis) and one permissive host (S. exigua) were then selected and used to perform six serial passages involving a concentration corresponding to the ~25% lethal concentration for both viruses. Restriction endonuclease analysis showed fragment length polymorphisms in every hostvirus system studied. In S. littoralis, serial passage of MbMNPV resulted in decreased pathogenicity and an increase in speed-of-kill, whereas no significant changes were detected for HearMNPV with respect to the initial inoculum. In contrast, both viruses showed a similar trend in S. exigua. These results highlight the low genetic diversity and a high phenotypic stability of HearMNPV with respect to the original inoculum after six successive passages in both insect hosts. This study concludes that host-baculovirus interactions during serial passage are complex and the process of adaptation to a novel semi-permissive host is far from predictable.
Open Access
Selection of a nucleopolyhedrovirus isolate from Helicoverpa armigera as the basis for a biological insecticide
(Wiley, 2014-05-01) Arrizubieta Celaya, Maite; Williams, Trevor; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Background: the cotton bollworm, Helicoverpa armigera, is an insect that causes damage in a wide range of crops in Spain.Seven isolates of H. armigera single nucleopolyhedrovirus (HearSNPV) from the Iberian Peninsula were subjected to molecularand biological characterization and compared with a Chinese genotype (HearSNPV-G4). Results: the estimated sizes of the Iberian genomes varied between 116.2 and 132.4 kb, compared to 131.4 kb of theHearSNPV-G4 reference genome. Phylogenetic analysis based on the lef-8, lef-9 and polh genes revealed that the Iberianstrains were more closely related to one another than to other HearSNPV isolates. Occlusion body (OB) concentration-mortalityresponses (LC 50 values) did not differ signiﬁcantly among Iberian isolates when tested against a Helicoverpa armigera colonyfrom Oxford (UK). Despite being the fastest killing isolate, HearSNPV-SP1 was as productive as isolates with lower virulence,with an average yield of 3.1 × 109 OBs larva−1 . OBs of HearSNPV-SP1 and HearSNPV-G4 were similarly pathogenic against arecently established colony from southern Spain, although HearSNPV-SP1 was faster killing than HearSNPV-G4 against a rangeof instars. Conclusion: the insecticidal properties of HearSNPV-SP1 mean that this strain is likely to prove useful as the basis for abiological insecticide for control of Helicoverpa armigera in Spain.
Open Access
Occlusion body pathogenicity, virulence and productivity traits vary with transmission strategy in a nucleopolyhedrovirus
(Elservier, 2011-10-25) Cabodevilla de Andrés, Oihana; Ibáñez Elosua, Itxaso; Simón de Goñi, Oihane; Murillo Pérez, Rosa; Caballero Murillo, Primitivo; Williams, Trevor; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The prevalence of sublethal infections of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) was quantified in natural populations of S. exigua in Almería, Spain, during 2006 and 2007. Of 1045 adults collected, 167 (16.1%) proved positive for viral polyhedrin gene transcripts by RT-PCR. The prevalence of covert infection varied significantly according to sex and sample date. Of 1660 progeny of field-collected insects, lethal disease was observed in 10¿33% of offspring of transcript-positive females and 9¿49% of offspring of transcript-negative females. Isolates associated with vertically transmitted infections were characterized by restriction endonuclease analysis using BglII or EcoRV and compared with isolates originating from greenhouse soil-substrate believed to be horizontally transmitted. Insects from a sublethally infected Almerian colony were between 2.3-fold and 4.6-fold more susceptible to infection than healthy insects from a Swiss colony, depending on isolate. Horizontally transmitted isolates were significantly more pathogenic than vertically transmitted isolates in insects from both colonies. Mean speed of kill in second instars (Swiss colony) varied between isolates by >20 h, whereas mean occlusion body (OB) production in fourth instars (Swiss colony) varied by 3.8-fold among isolates. Intriguingly, all three horizontally transmitted isolates were very similar in speed of kill and OB production, whereas all three vertically transmitted isolates differed significantly from one another in both variables, and also differed significantly from the group of horizontally transmitted isolates in speed of kill (one isolate) or both variables (two isolates). We conclude that key pathogenicity and virulence traits of SeMNPV isolates vary according to their principal transmission strategy.
Open Access
Chrysodeixis chalcites, a pest of banana crops on the Canary Islands: incidence, economic losses and current control measures
(Elsevier, 2018-03-05) Fuentes Barrera, Ernesto Gabriel; Hernández Suárez, Estrella; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Chrysodeixis chalcites is an emergent pest in bananas (Musa acuminata Colla) grown on the Canary Islands. Feeding damage to leaves and fruit and the control measures targeted at this pest were evaluated over a two-year period (2013–2014). The prevalence of infestations (42–100%) on the islands was similar during the two years of the study. Mean foliar damage (1.5–7.3% depending on island) and fruit damage (1.0–5.7%) detected in field surveys varied significantly across islands, plantation aspect (north- or south-facing) and season. Fruit damage was not correlated with foliar damage (P > 0.05). The weight of C. chalcites damaged bananas varied significantly (0.2–4.2% of harvested fruit) across islands, particularly in the spring. Overall, 3155 tonnes of bananas/yr are likely discarded due to C. chalcites damage, representing 1.5% of annual production or 2.68 million €/yr. The most frequently used pesticide was indoxacarb, usually applied on three occasions per crop cycle, for which the cost of control measures would average 240 €/ha per crop cycle. The direct damage that C. chalcites causes to banana fruit results in significant economic losses in addition to the direct costs of pesticide based control measures. Effective and sustainable control strategies are required against this pest.