Open Access
Draft genome sequences of two bacillus thuringiensis strains and characterization of a putative 41.9-kDa insecticidal toxin
(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; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 μg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.
Open Access
A novel use of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) as inoculative agent of baculoviruses
(Wiley, 2023) Gutiérrez Cárdenas, Oscar Giovanni; Adán, Ángeles; Medina, Pilar; Caballero Murillo, Primitivo; Garzón, Agustín; Muñoz Labiano, Delia; Institute for Multidisciplinary Research in Applied Biology - IMAB
Background: Alphabaculoviruses are Lepidoptera-specific virulent pathogens that infect numerous pests, including the Spodoptera complex. Due to their low environmental persistence, the traditional use of Alphabaculoviruses as bioinsecticides consist in high-rate spray applications with repeated treatments. Several abiotic and biotic factors can foster its dispersion, promoting their persistence in the agroecosystem. Amongst biotic factors, predatory arthropods can disperse the viruses by excretion after preying on infected individuals. Therefore, this study focused on promoting predator's ingestion of nucleopolyhedrovirus (NPV)-treated diets, and the later exposition of the insect host to leaf surfaces contaminated with predator excreta. The virus–host–predator system studied was Spodoptera littoralis nucleopolyhedrovirus (SpliNPV), Spodoptera littoralis (Boisduval) and Nesidiocoris tenuis (Reuter). The infective potential of N. tenuis feces and the retention time of SpliNPV were assessed under laboratory conditions after feeding on treated diets (sucrose solution and Ephestia kuehniella eggs). Results: Mortality of S. littoralis larvae was lower via N. tenuis excretion than in positive control (spray application) in the first infection cycle, together with a delay in host death. In the second infection cycle, both SpliNPV-treated diets triggered 100% mortality. Both diets allowed the transmission of SpliNPV, with a faster excretion via sucrose solution compared to E. kuehniella eggs. SpliNPV remained in N. tenuis digestive tract and was viable after excretion at least for 9 days for both diets. Conclusions: This study demonstrated the potential of the predator N. tenuis as inoculative agent of baculoviruses, representing a new alternative that, along with inundative applications, might contribute to improve pest management strategies.
Open Access
Insecticidal traits of variants in a genotypically diverse natural isolate of anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV)
(MDPI, 2023) Parras-Jurado, Ana; Muñoz Labiano, Delia; Beperet Arive, Inés; Williams, Trevor; Caballero Murillo, Primitivo; Institute for Multidisciplinary Research in Applied Biology - IMAB
Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype.
Open Access
Complete genome sequence of five Chrysodeixis chalcites nucleopolyhedrovirus genotypes from a Canary Islands isolate
(American Society for Microbiology, 2013-10-24) Bernal Rodríguez, Alexandra; Williams, Trevor; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Simón de Goñi, Oihane; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The Chrysodeixis chalcites single nucleopolyhedrovirus (ChchSNPV) infects and kills C. chalcites larvae, an important pest of banana crops in the Canary Islands. Five genotypes present in the most prevalent and widespread isolate in the Canary Islands were sequenced, providing genetic data relevant to the genotypic and phenotypic diversity of this virus.
Open Access
Molecular and insecticidal characterization of a novel cry-related protein from bacillus thuringiensis toxic against Myzus persicae
(MDPI, 2014) Palma Dovis, Leopoldo; Muñoz Labiano, Delia; Berry, Colin; Murillo Martínez, Jesús; Escudero, Iñigo de; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect pests.
Open Access
Sequence comparison between three geographically distinct Spodoptera frugiperda multiple nucleopolyhedrovirus isolates: detecting positively selected genes
(Elsevier, 2011-01-14) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Beperet Arive, Inés; Muñoz Labiano, Delia; López Ferber, Miguel; Caballero Murillo, Primitivo; Williams, Trevor; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The complete genomic sequence of a Nicaraguan plaque purified Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genotype SfMNPV-B was determined and compared to previously sequenced isolates from United States (SfMNPV-3AP2) and Brazil (SfMNPV-19). The genome of SfMNPV-B (132,954 bp) was 1623 bp and 389 bp larger than that of SfMNPV-3AP2 and SfMNPV-19, respectively. Genome size differences were mainly due to a deletion located in the SfMNPV-3AP2 egt region and small deletions and point mutations in SfMNPV-19. Nucleotide sequences were strongly conserved (99.35% identity) and a high degree of predicted amino acid sequence identity was observed. A total of 145 open reading frames (ORFs) were identified in SfMNPV-B, two of them (sf39a and sf110a) had not been previously identified in the SfMNPV-3AP2 and SfMNPV-19 genomes and one (sf57a) was absent in both these genomes. In addition, sf6 was not previously identified in the SfMNPV-19 genome. In contrast, SfMNPV-B and SfMNPV-19 both lacked sf129 that had been reported in SfMNPV-3AP2. In an effort to identify genes potentially involved in virulence or in determining population adaptations, selection pressure analysis was performed. Three ORFs were identified undergoing positive selection: sf49 (pif-3), sf57 (odv-e66b) and sf122 (unknown function). Strong selection for ODV envelope protein genes indicates that the initial infection process in the insect midgut is one critical point at which adaptation acts during the transmission of these viruses in geographically distant populations. The function of ORF sf122 is being examined.
Open Access
Improved photocatalytic and antibacterial performance of Cr doped TiO2 nanoparticles
(Elsevier, 2021) Gómez Polo, Cristina; Larumbe Abuin, Silvia; Gil Bravo, Antonio; Muñoz Labiano, Delia; Rodríguez Fernández, L.; Fernández Barquín, Luis; García-Prieto, Ana; Fernández-Gubieda, María Luisa; Muela, Alicia; Zientziak; Institute for Advanced Materials and Mathematics - INAMAT2; Ciencias
The effect of Cr and N doping in the adsorption capacity, photocatalytic properties and antibacterial response of TiO2 anatase nanoparticles is analyzed. The nanoparticles (N-TiO2, Cr-TiO2 and Cr/N-TiO2) were prepared by the sol-gel method. The structural (X-ray diffraction and TEM) and magnetic (SQUID magnetometry) characterization confirms the nanosized nature of the anatase nanoparticles and the absence of secondary phases. The enhancement of the adsorption capacity of the dye (methyl orange) on the surface of the catalysts for the Cr and Cr/N doped samples, together with the redshift of the UV-Vis absorbance spectra promote a high photocatalytic performance under visible light in these nanocatalysts. The culturability and viability of the Escherichia coli DH5α in a medium supplemented with the nanoparticles was characterized and compared with the evolution under visible light (both without and with nanoparticles). The results show that Cr-TiO2 nanoparticles under visible light display antibacterial activity that cannot be accounted by the toxicity of the nanoparticles alone. However the antibacterial effect is not observed in N-TiO2 and Cr/N-TiO2. The differences in the electrostatic charge (isoelectric point) and the degree of nanoparticle dispersion are invoked as the main origins of the different antibacterial response in the Cr-TiO2 nanoparticles.
Open Access
Remarkably efficient production of a highly insecticidal Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) isolate in its homologous host
(Wiley, 2018-01-03) Bernal Rodríguez, Alexandra; Simón de Goñi, Oihane; Williams, Trevor; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Background: a Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islands (ChchNPV-TF1) has proved to be effective for control of Chrysodeixis chalcites on banana crops. Commercialization of this virus as a bioinsecticide requires an efficient production system. Results: the sixth instar (L6) was the most suitable for virus production, producing 1.80 × 1011 occlusion bodies (OB)/larva and showed a lower prevalence of cannibalism (5.4%) than fourth (L4) or fifth (L5) instars. Inoculation of L6 at 24 h post molting produced six times more OB (5.72 × 1011 OB/larva) than recently molted L6 larvae (1.00 × 1011 OB/larva). No significant differences were recorded in mean time to death (165–175 h) or OB production per larva (3.75 × 1011 to 5.97 × 1011) or per mg larval weight (1.30 × 1011 to 2.11 × 109), in larvae inoculated with a range of inoculum concentrations (LC50–LC90). Groups of infected L6 larvae reared at a density of 150 larvae/container produced a greater total number of OBs (8.07 × 1013 OB/container) than lower densities (25, 50 and 100 OB/container), and a similar number to containers with 200 inoculated larvae (8.43 × 1013 OB/container). Conclusion: the processes described here allow efficient production of sufficient OBs to treat ∼ 40 ha of banana crops using the insects from a single container.
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
The parasitoid hyposoter didymator can transmit a broad host range baculovirus in a two host system
(MDPI, 2023) Morel, Ariel; Leigh, Brendan; Muñoz Labiano, Delia; Caballero Murillo, Primitivo; Medina, Pilar; Dáder, Beatriz; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura
Hyposoter didymator (Thunberg) (Hymenoptera: Ichneumonidae) and baculovirus (BV) might be used jointly to provide effective control of the Spodoptera genus. The literature has mostly covered the safe compatibility between natural enemies and BV-based insecticides, but research on the potential dispersal of BV by natural enemies is lacking. Thus, the goal of this manuscript was to ascertain if H. didymator was able to disperse the broad host range of Autographa californica nucleopolyhedrovirus (AcMNPV) to Spodoptera littoralis and Spodoptera exigua in choice and non-choice conditions and whether the preference of the parasitoid by one of these noctuids could mediate this dispersion. It was previously needed to improve the rearing of the parasitoid in the laboratory, concerning the optimal host age and length of parasitization, parasitoid competition, and influence of parasitization on the longevity of females. The best rearing conditions for S. littoralis are collective parasitization of mature L3 larvae for 24 h, after at least one day of copulation. Hyposoter didymator transmits AcMNPV to both lepidopterans, but its efficiency is mediated by host preference and the pathogenicity of the BV in each host. In this particular case, H. didymator as well as AcMNPV showed a clear preference towards S. exigua.