Arrizubieta Celaya, Maite

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https://academica-e.unavarra.es/handle/2454/48614

Last Name

Arrizubieta Celaya

First Name

Maite

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Producción Agraria

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1175052

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8668

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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.
Open Access
Genomic sequences of five Helicoverpa armigera nucleopolyhedrovirus genotypes from Spain that differ in their insecticidal properties
(American Society for Microbiology, 2015) Arrizubieta Celaya, Maite; Simón de Goñi, Oihane; Williams, Trevor; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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.
Open Access
A novel binary mixture of Helicoverpa armigera single nucleopolyhedrovirus genotypic variants has improved insecticidal characteristics for control of cotton bollworms
(American Society for Microbiology, 2015) 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
The genotypic diversity of two Spanish isolates of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics for control of the cotton bollworm. Two genotypic variants, HearSP1A and HearSP1B, were cloned in vitro from the most pathogenic wild-type isolate of the Iberian Peninsula, HearSNPV-SP1 (HearSP1-wt). Similarly, six genotypic variants (HearLB1 to -6) were obtained by endpoint dilution from larvae collected from cotton crops in southern Spain that died from virus disease during laboratory rearing. Variants differed significantly in their insecticidal properties, pathogenicity, speed of kill, and occlusion body (OB) production (OBs/larva). HearSP1B was ~3-fold more pathogenic than HearSP1-wt and the other variants. HearLB1, HearLB2, HeaLB5, and HearLB6 were the fastest-killing variants. Moreover, although highly virulent, HearLB1, HearLB4, and HearLB5 produced more OBs/ larva than did the other variants. The co-occluded HearSP1B:LB6 mixture at a 1:1 proportion was 1.7- to 2.8-fold more pathogenic than any single variant and other mixtures tested and also killed larvae as fast as the most virulent genotypes. Serial passage resulted in modified proportions of the component variants of the HearSP1B:LB6 co-occluded mixture, suggesting that transmissibility could be further improved by this process. We conclude that the improved insecticidal phenotype of the HearSP1B:LB6 co-occluded mixture underlines the utility of the genotypic variant dissection and reassociation approach for the development of effective virus-based insecticides.
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
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
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.
Restricted
Caracterización biológica y molecular de un nucleopoliedrovirus aislado de larvas de Trichoplusia ni del insectario de la UPNA
(2006) Arrizubieta Celaya, Maite; Muñoz, Delia; Escuela Técnica Superior de Ingenieros Agrónomos; Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoa; Producción Agraria; Nekazaritza Ekoizpena