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
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
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
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
Spodoptera frugiperda multiple nucleopolyhedrovirus as a potential biological insecticide: genetic and phenotypic comparison of field isolates from Colombia
(Elsevier, 2011-04-24) Barrera Cubillos, Gloria Patricia; Simón de Goñi, Oihane; Villamizar, Laura; Williams, Trevor; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Thirty-eight isolates of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), collected from infected larvae on pastures, maize, and sorghum plants in three different geographical regions of Colombia, were subjected to molecular characterization and were compared with a previously characterized Nicaraguan isolate (SfNIC). Restriction endonuclease analysis (REN) using six different enzymes showed two different patterns among Colombian isolates, one profile was particularly frequent (92%) and was named SfCOL. The physical map of SfCOL was constructed and the genome was estimated to be 133.9 kb, with few differences in terms of number and position of restriction sites between the genomes of SfNIC and SfCOL. The PstI-K and PstI-M fragments were characteristic of SfCOL. These fragments were sequenced to reveal the presence of seven complete and two partial ORFs. This region was collinear with SfMNPV sf20–sf27. However, two ORFs (4 and 5) had no homologies with SfMNPV ORFs, but were homologous with Spodoptera exigua MNPV (se21 and se22/se23) and Spodoptera litura NPV (splt20 and splt21). Biological characterization was performed against two different colonies of S. frugiperda, one originating from Colombia and one from Mexico. Occlusion bodies (OBs) of the SfCOL isolate were as potent (in terms of concentration–mortality metrics) as SfNIC OBs towards the Mexican insect colony. However, SfCOL OBs were 12 times more potent for the Colombian colony than SfNIC OBs and three times more potent for the Colombian colony than for the Mexican colony. SfCOL and SfNIC showed a slower speed of kill (by ∼50 h) in insects from the Colombian colony compared to the Mexican colony, which was correlated with a higher production of OBs/larvae. SfCOL is a new strain of SfMNPV that presents pathogenic characteristics that favor its development as the basis for a biopesticide product in Colombia.
Open Access
Gender-mediated differences in vertical transmission of a nucleopolyhedrovirus
(Public Library of Science, 2013) Virto Garayoa, Cristina; Zárate Chaves, Carlos Andrés; López Ferber, Miguel; Murillo Pérez, Rosa; Caballero Murillo, Primitivo; Williams, Trevor; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H=)6healthy females (HR); ii) infected males (I=)6healthy females (HR); iii) healthy males (H=)6infected females (IR) and iv) infected males (I=)6infected females (IR). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Malemediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virusbased insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.
Open Access
Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization
(American Society for Microbiology, 2014) Beperet Arive, Inés; Irons, Sarah L.; Simón de Goñi, Oihane; King, Linda A.; Williams, Trevor; Possee, Robert D.; López Ferber, Miguel; Caballero Murillo, Primitivo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses—AcMNPV and SfMNPV—but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses.
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
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
Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses
(Elsevier, 2012-10-21) Simón de Goñi, Oihane; Palma Dovis, Leopoldo; Williams, Trevor; López Ferber, Miguel; Caballero Murillo, Primitivo; Producción Agraria; Nekazaritza Ekoizpena; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A–I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366–27,225) and 60 bp (119,759–119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.