DSpace Repository :: Browsing by Author "Caballero Murillo, Primitivo"

Browsing by Author "Caballero Murillo, Primitivo"

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Open Access
Analagous population structures for two alphabaculoviruses highlight a functional role for deletion mutants
(American Society for Microbiology, 2012) Serrano García, Amaya; Williams, Trevor; Simón de Goñi, Oihane; López Ferber, Miguel; Caballero Murillo, Primitivo; Muñoz Labiano, Delia; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations.
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
Bacillus thuringiensis Cyt proteins as enablers of activity of Cry and Tpp toxins against Aedes albopictus
(2023) Lai, Liliana; Villanueva, Maite; Muruzabal Galarza, Ane; Fernández González, Ana Beatriz; Unzue Pozas, Argiñe; Toledo Arana, Alejandro; Caballero Murillo, Primitivo; Caballero Sánchez, Carlos; Institute for Multidisciplinary Research in Applied Biology - IMAB
Aedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.
Open Access
Bacillus thuringiensis toxins: an overview of their biocidal activity
(MDPI, 2014) Palma Dovis, Leopoldo; Muñoz Labiano, Delia; Berry, Colin; Murillo Martínez, Jesús; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities.
Open Access
Bacmid expression of granulovirus enhancin En3 accumulates in cell soluble fraction to potentiate nucleopolyhedrovirus infection
(MDPI, 2021) Ricarte Bermejo, Adriana; Simón de Goñi, Oihane; Fernández González, Ana Beatriz; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.
Open Access
Baculovirus expression and functional analysis of Vpa2 proteins from Bacillus thuringiensis
(MDPI, 2020) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Fernández González, Ana Beatriz; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee.
Open Access
Biotechnological development of a new bioinsecticide based on a Helicoverpa armigera nucleopolyhedrovirus from Spain
(2015) Arrizubieta Celaya, Maite; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena
El taladro del tomate, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), es una de las principales plagas polífagas de la Península Ibérica. El nucleopoliedrovirus simple de H. armigera (HearSNPV) es un método eficaz para el control de dicha especie. En esta tesis, se evaluó la diversidad genotípica de dos aislados españoles del HearSNPV con el objetivo de seleccionar una mezcla de genotipos con mejores características insecticidas. La caracterización biológica reveló que la mezcla co-ocluida de dos genotipos (HearSP1B:LB6), en proporción 1:1, presenta propiedades insecticidas mejoradas, por lo que fue seleccionada como materia activa de un nuevo bioinsecticida. Con el objetivo de detectar los cambios genéticos responsables de estas diferencias en el fenotipo, se realizó la secuenciación completa del genoma de 5 genotipos. Las mayores diferencias entre todos estos genotipos se localizan en las hrs y en los genes bro. Además, se identificaron mutaciones puntuales en genes implicados en la replicación del ADN, la transcripción viral, o genes estructurales, que podrían ser responsables de la reducida producción de OBs de los genotipos de HearSP1 o el aumento de la patogenicidad de HearSP1B. También, se identificaron diversas mutaciones localizadas en los genes iap-2, iap-3 y hoar que podrían estar relacionadas con la estrategia de transmisión o con la capacidad para establecer infecciones encubiertas en el insecto huésped. Con el objetivo de ampliar el espectro de huéspedes de HearSNPV se aplicó la tecnología de co-oclusión de baculovirus para obtener muestras de OBs en las que se encontrasen co-envueltos HearSNPV y HearMNPV, para obtener una mezcla con las características insecticidas deseables de HearSNPV y el amplio espectro de huéspedes de HearMNPV. Cuando larvas de H. armigera fueron infectadas primero con HearMNPV y 12 o 24 horas más tarde con HearSNPV, los genomas de ambos se co-ocluyeron en los OBs en la misma proporción (1:1). Sin embargo, la mezcla co-envuelta no presentó mejores características fenotípicas, pero aumentó el espectro de huéspedes de HearSNPV, ya que este virus fue capaz de entrar e infectar a especies no susceptibles como S. frugiperda y M. brassicae. Con el fin de optimizar las condiciones para la producción masiva del virus, se evaluó el efecto de varios factores sobre la producción de cuerpos de oclusión (OBs). Los resultados obtenidos mostraron que la mayor producción de OBs se consigue inoculando larvas L5 recién mudadas con la CL80, e incubando las larvas individualizadas a 30ºC. La eficacia y la persistencia en campo de un formulado sencillo del nuevo bioinsecticida se compararon con las de varios insecticidas comerciales en cultivos de tomate en invernadero y en campo abierto. Dicho formulado protegió al cultivo con una eficacia similar a la de los insecticidas comerciales Dursban®, Turex® y Spintor®. En cambio, su persistencia fue mayor que la de los insecticidas comerciales, aunque las diferencias fueron más notables en los cultivos de invernadero. Toda esta información ha sido objeto de una solicitud de patente (P201430956), y constituye la base para el desarrollo de un nuevo bioinsecticida. Este nuevo bioinsecticida es una herramienta muy útil para el establecimiento de una agricultura sostenible en los cultivos de tomate en la Península Ibérica, ya que puede ser incluido en programas de Manejo Integrado de Plagas.
Embargo
Captura de CO2 del aire mediante cultivo de Bacillus altitudinis en biorreactores
(2024) Gómez Borobia, Miguel; Baztán Valencia, Fernando; Caballero Murillo, Primitivo; Escuela Técnica Superior de Ingeniería Agronómica y Biociencias; Nekazaritzako Ingeniaritzako eta Biozientzietako Goi Mailako Eskola Teknikoa
En este trabajo se sientan las bases para la creación de una empresa biotecnológica, cuyo objetivo es ofrecer al mundo soluciones altamente efectivas para luchar contra la contaminación del aire por CO2 y hacer frente a uno de los mayores desafíos de nuestro tiempo, el cambio climático. A lo largo del trabajo se detalla la tecnología en la que se basa la empresa, que consiste en el cultivo en biorreactor de una cepa de Bacillus altitudinis mejorada genéticamente para aumentar su tasa de fijación de CO2. El producto principal de la empresa consiste en un biorreactor de gran tamaño, en el que tendrá lugar el cultivo de la cepa de Bacillus altitudinis y que será colocado en lugares estratégicos, en los que haya un elevado nivel de CO2. El aire contaminado se hará pasar por el biorreactor a través del cultivo bacteriano, de forma que se captura el CO2 que contiene y el aire purificado se libera de nuevo al exterior. Por otro lado, se detallan los puntos clave en un proyecto de emprendimiento, como son, la descripción de la idea de negocio, la propuesta de valor, el estudio y delimitación de la oferta, así como, el análisis y comprensión de la demanda y la fijación de los segmentos objetivo.
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
Chrysodeixis chalcites nucleopolyhedrovirus: a useful component for IPM programs on the Canary Islands banana crops
(2017) Fuentes Barrera, Ernesto Gabriel; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Hernández Suárez, Estrella; Producción Agraria; Nekazaritza Ekoizpena
La platanera (Musa acuminata Colla) es uno de los principales cultivos de las Islas Canarias, representando el 30% de la producción agrícola total del archipiealago. Dicho cultivo se realiza bajo invernaderos de malla principalmente en las vertientes cálidas del sur de las islas, y al aire libre en las frías vertientes del norte. Por ello, los cultivos bajo malla presentan mayores poblemas fitosanitaríos. Chrysodeixis chalcites (Esper, 1789) (Lepidoptera: Noctuidae) ha sufrido cambios importantes en su hábitos alimenticios. Actualmente se alimenta principalmente de los frutos de platanera, produciendo dafios en la epidermis que reducen claramente su valor comercial. El control de dicha plaga implica el uso repetido de un reducido número de materias activas, lo que favorece la aparición de resistencia, y por lo tanto disminuyendo su efectividad. Además, desde el año 2014 la gestión integrada de plagas (GIP) es de obligado cumplimiento en los sistemas de cultivo españoles. El objetivo de esta tesis ha sido abordar varios algunos aspectos importantes para la implementación de un programa de GIP efectivo en los cultivos de platanera de las Islas Canarias, España. La toma de decisiones efectiva en GIP se basa en la comprensión de las relaciones que se producen entre el número de individuos plaga, la respuesta de las plantas a los daños producidos por dichos individuos y las pérdidas económicas resultantes. Por ello, en la presente tesis se estimó en primer lugar la incidencia y el daño alimenticio producido por C. chalcites, así como las pérdidas de producción debidas al daño directo a la fruta y al coste indirecto derivado de la compra y aplicación de insecticidas. La prevalencia de infestaciones varió entre el 42 y 100% y fue similar en tos dos años de prospecciones. El daño foliar medio (1.5-7.3%) y el daño en fruta (1.0-5.7%) varió significativamente en las islas según el tipo de plantación (vertientes orientadas al norte o al sur) y estación. En general, los daños resultaron similares entre los dos tipos de plataciones, excepto en Gran Canaria y Tenerife, donde se registaron más daños foliares y en fruto, respectivamente , en la vertiente sur. Los daños también fueron similares entre las estaciones, Jo que indica que C. chalcites está presente en el cultivo durante todo el año. El peso de plátanos dañados respecto a la fruta cultivada varió significativamente entre las islas, desde el 0,2% en Tenerife hasta el 4,2% en El Hierro, siendo este daño especialmente importante en primavera. Dicho periodo es el más susceptible para determinar la cantidad y calidad de la fruta cosechada, ya que coincide con el desarrollo del fruto tras la floración. En total se perdieron unas 3.155 toneladas de platano/año, lo que representa el 1,5% de la producción anual (2,68 millones de euros/afio). Además, los costes de control con indoxacarb, el insecticida más utilizado (73%), supondrían unos 240€/ha por ciclo de cultivo. Dado que su uso continuado probablemente favorezca el desarrollo de resistencia, se necesitan nuevos insecticidas para rotar con los pocos productos autorizados . Entre ellos el nucleopoliedrovirus de C. chalcites, ChchNPV (Género Alphabaculovirus, Baculoviridae) resulta ser un insecticida seguro, eficiente y sostenible. El siguiente objetivo fue evaluar el uso potencial de ChchNPV como un nuevo agente de control biológico en GTP. Para ello, primero se estimó Ja prevalencia y diversidad genética de las variantes de ChchNPV presentes en las poblaciones naturales de C. chalcites en Canarias y después se evaluó la eficacia insecticida del aislado más prevalente en comparación con dos insecticidas usados frecuentemente, indoxacarb y Bacillus thuringiensis (Bt), a pequeña escala en plantas jóvenes de platanera en condiciones de invernadero y aire libre. En general, la prevalencia de infección por ChchNPV fue del 2,3%, siendo de 0-13,8% en El Hierro, 0-5,6% en La Palma, 0-4,8% en Tenerife, 0-1% en La Gomera y 0% en Gran Canaria. En plantaciones bajo invernadero, la prevalencia de infección fue el doble (3%) que al aire libre (l,4%). ChchNPV-TF1 fue la variante más abundante (82%) y extendida, por lo que se seleccionó para los ensayos de campo. La aplicación 109 cuerpos de oclusión (OBs)/I de ChchNPV-TF1 redujo significativamente la densidad larvaria y el daño foliar en plantas jóvenes de platanera, al igual que los productos convencionales indoxacarb y Bt. Sin embargo, la mayor mortalidad producida por ChchNPV-TF1 en las larvas recolectadas a lo largo del tiempo sugiere que adquisición de una infección letal ocurre durante un periodo de tiempo más extendido, en comparación con el breve periodo que manifiestan las larvas tratadas con los insecticidas convencionales. Estos resultados indican una mayor persistencia de ChchNPV-TF1 en la p!anta de platanera. El último objetivo de este trabajo abordó la evaluación de la eficacia de ChchNPV-TF1 para proteger los frutos de platanera de los daños producidos por C. chalcites en invernaderos de malla en plantaciones comerciales, durante un ciclo de cultivo completo en comparación con el tratameitno convencional. En los ensayos de 2014, el daño foliar fue similar entre las dos estrategias de control en las tres islas, mientras que el daño en fruto en las parcelas tratadas con ChchNPV-TF1 fue sorprendentemente mayor en Tenerife, pero similar en Gran Canaria y La Palma. En Tenerife y Gran Canaria Ja mortalidad larvaria fue mayor en la parcela tratada con ChchNPV-TFl, mientras en La Palma la baja incidencia no permitió determinar la mortalidad larvaria. En un segundo ensayo en Tenerife en 2015 los tratamientos con virus se aplicaron varios meses antes del dasarrollo del fruto, y esta vez ChchNPV-TF1 proporcionó un control efectivo, similar al proporcionado por los insecticidas convencionales. En conclusión, este nuevo agente de control biológico resulta útil para ser incluido en la gestión integrada de C. chalcites en cultivos de platanera de Canarias. Los resultados de esta tesis forman parte de un trabajo más amplio que tiene como objetivo definir los umbrales de daño de C. chalcites en cultivos de platanera y el uso potencial de este nuevo insecticida biológico.
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
Co-infection with iflaviruses influences the insecticidal properties of Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies: implications for the production and biosecurity of baculovirus insecticides
(Public Library of Science, 2017) Carballo Palos, Arkaitz; Murillo Pérez, Rosa; Jakubowska, Agata; Herrero, Salvador; Williams, Trevor; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Biological insecticides based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can efficiently control S. exigua larvae on field and greenhouse crops in many parts of the world. Spanish wild populations and laboratory colonies of S. exigua are infected by two iflaviruses (SeIV-1 and SeIV-2). Here we evaluated the effect of iflavirus co-infection on the insecticidal characteristics of SeMNPV occlusion bodies (OBs). Overall, iflavirus coinoculation consistently reduced median lethal concentrations (LC50) for SeMNPV OBs compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV was similar in the presence or absence of the iflaviruses. A reduction of the weight gain (27%) associated with iflavirus infection resulted in a 30% reduction in total OB production per larva. Adult survivors of SeMNPV OB inoculation were examined for covert infection. SeMNPV DNA was found to be present at a high prevalence in all SeIV-1 and SeIV-2 coinfection treatments. Interestingly, co-inoculation of SeMNPV with SeIV-2 alone or in mixtures with SeIV-1 resulted in a significant increase in the SeMNPV load of sublethally infected adults, suggesting a role for SeIV-2 in vertical transmission or reactivation of sublethal SeMNPV infections. In conclusion, iflaviruses are not desirable in insect colonies used for large scale baculovirus production, as they may result in diminished larval growth, reduced OB production and, depending on their host-range, potential risks to non-target Lepidoptera.
Open Access
Coocclusion of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV): pathogenicity and stability in homologous and heterologous hosts
(MDPI, 2022) Arrizubieta Celaya, Maite; Simón de Goñi, Oihane; Ricarte Bermejo, Adriana; López Ferber, Miguel; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Gobierno de Navarra / Nafarroako Gobernua
Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nu-cleopolyhedrovirus (HearSNPV) is a different virus species with a broader host range. This study aimed to examine the consequences of coocclusion of HearSNPV and HearMNPV on the patho-genicity, stability and host range of mixed-virus occlusion bodies (OBs). HearSNPV OBs were approximately 6-fold more pathogenic than HearMNPV OBs, showed faster killing by approximately 13 h, and were approximately 45% more productive in terms of OB production per larva. For coocclusion, H. armigera larvae were first inoculated with HearMNPV OBs and subsequently inoculated with HearSNPV OBs at intervals of 0-72 h after the initial inoculation. When the interval between inoculations was 12-24 h, OBs collected from virus-killed insects were found to comprise 41¿57% of HearSNPV genomes, but the prevalence of HearSNPV genomes was greatly reduced (3- 4%) at later time points. Quantitative PCR (qPCR) analysis revealed the presence of HearSNPV genomes in a small fraction of multinucleocapsid ODVs representing 0.47¿0.88% of the genomes quan-tified in ODV samples, indicating that both viruses had replicated in coinfected host cells. End-point dilution assays on ODVs from cooccluded mixed-virus OBs confirmed the presence of both viruses in 41.9¿55.6% of wells that were predicted to have been infected by a single ODV. A control exper-iment indicated that this result was unlikely to be due to the adhesion of HearSNPV ODVs to HearMNPV ODVs or accidental contamination during ODV band extraction. Therefore, the dispar-ity between the qPCR and end-point dilution estimates of the prevalence of mixed-virus ODVs likely reflected virus-specific differences in replication efficiency in cell culture and the higher in-fectivity of pseudotyped ODVs that were produced in coinfected parental cells. Bioassays on H. armigera, Spodoptera frugiperda and Mamestra brassicae larvae revealed that mixed-virus OBs were capable of infecting heterologous hosts, but relative potency values largely reflected the proportion of HearMNPV present in each mixed-virus preparation. The cooccluded mixtures were unstable in serial passage; HearSNPV rapidly dominated during passage in H. armigera whereas HearMNPV rapidly dominated during passage in the heterologous hosts. We conclude that mixed-virus coocclusion technology may be useful for producing precise mixtures of viruses with host range properties suitable for the control of complexes of lepidopteran pests in particular crops, although this requires validation by field testing.
Open Access
Coping with environmental eukaryotes; identification of Pseudomonas syringae genes during the interaction with alternative hosts or predators
(MDPI, 2018) Dorati, Federico; Barrett, Glyn A.; Sánchez Contreras, María; Murillo Martínez, Jesús; Caballero Murillo, Primitivo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g., water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.
Open Access
Deletion genotypes reduce occlusion body potency but increase occlusion body production in a Colombian Spodoptera frugiperda nucleopolyhedrovirus population
(Public Library of Science, 2013) Barrera Cubillos, Gloria Patricia; Williams, Trevor; Villamizar, Laura; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures cooccluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.
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
Development of a new bioinsecticide based on a Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islands
(2014) Bernal Rodríguez, Alexandra; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena
Chrysodeixis chalcites (Lepidoptera: Noctuidae) es una plaga importante que causa valiosos daños económicos en los cultivos de platanera de las Islas Canarias. El control efectivo de esta plaga con insecticidas químicos requiere muchas aplicaciones, aumentando los costes de producción, lo que puede derivarse en riesgos ambientales graves, y la acumulación de residuos químicos que dificultan la comercialización del plátano. En estos casos, una de las alternativas más realistas para el control seguro y eficaz de la plaga la constituyen los bioinsecticidas basados en microorganismos entomopatógenos incluidos los baculovirus. En condiciones naturales las poblaciones de C. chalcites se ven afectadas por un Alphabaculovirus (Baculoviridae) llamado C. chalcites nucleopoliedrovirus (ChchSNPV). El objetivo de esta tesis doctoral ha consistido en abordar algunos de los desarrollos biotecnológicos necesarios para la obtención de un nuevo bioinsecticida basado en un ChchSNPV autóctono de las Islas Canarias. Una buena parte de los resultados de esta tesis forman parte del contenido de una solicitud de patente para el desarrollo de un nuevo bioinsecticida (P201330487). Este bioinsecticida, además de ser el agente de control biológico más efectivo para el control de esta plaga en la actualidad, es una herramienta muy útil para la implantación de programas de protección integrada de cultivos y el establecimiento de una agricultura sostenible.
Open Access
Discriminación de Euplectrus plathypenae en larvas de Spodoptera frugiperda infectadas con SfMNPV
(2016) Domínguez Salvatierra, Javier; Caballero Murillo, Primitivo; Murillo Pérez, Rosa; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena
La alta incidencia natural de los parasitoides Chelonus insularis y Euplectrus plathypenae y el uso potencial del SfNPV en el control de Spodoptera frugiperda en diversas regiones de América Latina hacen que sea altamente probable la ocurrencia de diversos escenarios de competencia interespecífica. Dado que las probabilidades de que ocurran escenarios de competencia dependen de la capacidad de discriminación de las hembras del parasitoide, se evalúa esta característica en las hembras de E. plathypenae entre larvas sanas e infectadas por virus SfNPV. En los bioensayos de discriminación en los que se ofrecían larvas infectadas en distintos momentos del desarrollo de la infección respecto a larvas sanas del mismo tamaño, se observó que el porcentaje de parasitismo en larvas infectadas es similar al obtenido en larvas sanas, en las primeras 24 h del desarrollo de la infección viral, mientras que se reduce a la mitad en un periodo más avanzado de la infección, comprendido entre las 96 y las 144 h tras la inoculación del SfNPV. Esto sugiere que las hembras del parasitoide prefieren larvas sanas en los casos en los que la infección en la larva está suficientemente avanzada como para impedir el correcto desarrollo de la descendencia del parasitoide.
Open Access
Domain shuffling between Vip3Aa and Vip3Ca: chimera stability and insecticidal activity against European, American, African, and Asian pests
(MDPI, 2020) Gomis Cebolla, Joaquín; Santos, Rafael Ferreira dos; Wang, Yueqin; Caballero Sánchez, Javier; Caballero Murillo, Primitivo; He, Kanglai; Jurat Fuentes, Juan Luis; Ferré, Juan; Institute for Multidisciplinary Research in Applied Biology - IMAB
The bacterium Bacillus thuringiensis produces insecticidal Vip3 proteins during the vegetative growth phase with activity against several lepidopteran pests. To date, three different Vip3 protein families have been identified based on sequence identity: Vip3A, Vip3B, and Vip3C. In this study, we report the construction of chimeras by exchanging domains between Vip3Aa and Vip3Ca, two proteins with marked specificity differences against lepidopteran pests. We found that some domain combinations made proteins insoluble or prone to degradation by trypsin as most abundant insect gut protease. The soluble and trypsin-stable chimeras, along with the parental proteins Vip3Aa and Vip3Ca, were tested against lepidopteran pests from different continents: Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Helicoverpa armigera, Mamestra brassicae, Anticarsia gemmatalis, and Ostrinia furnacalis. The exchange of the Nt domain (188 N-terminal amino acids) had little effect on the stability and toxicity (equal or slightly lower) of the resulting chimeric protein against all insects except for S. frugiperda, for which the chimera with the Nt domain from Vip3Aa and the rest of the protein from Vip3Ca showed a significant increase in toxicity compared to the parental Vip3Ca. Chimeras with the C-terminal domain from Vip3Aa (from amino acid 510 of Vip3Aa to the Ct) with the central domain of Vip3Ca (amino acids 189–509 based on the Vip3Aa sequence) made proteins that could not be solubilized. Finally, the chimera including the Ct domain of Vip3Ca and the Nt and central domain from Vip3Aa was unstable. Importantly, an insect species tolerant to Vip3Aa but susceptible to Vip3Ca, such as Ostrinia furnacalis, was also susceptible to chimeras maintaining the Ct domain from Vip3Ca, in agreement with the hypothesis that the Ct region of the protein is the one conferring specificity to Vip3 proteins.
Open Access
Draft genome sequence of Bacillus cereus CITVM-11.1, a strain exhibiting interesting antifungal activities
(Karger, 2018) Caballero Sánchez, Javier; Peralta, C.; Molla, A.; Valle, Eleodoro E. del; Caballero Murillo, Primitivo; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Bacillus cereus is a gram-positive, spore-forming bacterium possessing an important and historical record as a human-pathogenic bacterium. However, several strains of this species exhibit interesting potential to be used as plant growth-promoting rhizobacteria. Here, we report the draft genome sequence of B. cereus strain CITVM-11.1, which consists of 37 contig sequences, accounting for 5,746,486 bp (with a GC content of 34.8%) and 5,752 predicted protein-coding sequences. Several of them could potentially be involved in plant-bacterium interactions and may contribute to the strong antagonistic activity shown by this strain against the charcoal root rot fungus, Macrophomina phaseolina. This genomic sequence also showed a number of genes that may confer this strain resistance against several polluting heavy metals and for the bioconversion of mycotoxins.