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Bacillus thuringiensis is a Gram-positive and sporulated bacterium exhibiting insecticidal activity against a wide range of insects.3 During sporulation, this bacterium produces a number of different proteins forming crystalline inclusions adjacent to the spores (parasporal crystals). Among these insecticidal proteins, the most abundant are those commonly known as Cry (Crystal) proteins, which are responsible for exerting a toxic activity (upon ingestion) against insects of different species.5 For this reason, B. thuringiensis has proved to be the most efficient and used bioinsecticide to date.2 However, Spodoptera cosmioides, Spodoptera eridania and Agrotis sp. (Lepidoptera) are species that are not yet controlled by some transgenic crops (e.g. Intacta RR2Pro soybean). Thus, in an attempt to enlarge the host spectrum of this bacterium it is necessary to search for novel strains. In this work we show a sporulated B. thuringiensis Bt-UNVM_84 strain exhibiting a number of rare amorphous to spherical crystal combinations, whereas sporulated B. thuringiensis strain Bt-UNVM-94 showed quasi symmetric bipyramidal parasporal crystals, by using scanning electron microscopy (SEM) (Fig. 1). Strains Bt-UNVM_84 and Bt-UNVM_94 were isolated from Oncativo (Córdoba, Argentina) and Cululú (Santa Fe, Argentina), respectively. The insecticidal activity of these different B. thuringiensis strains is currently under investigation. Each strain was grown in liquid CCY sporulation medium6 for ∼48 h (150 rpm) until no vegetative cells were observed under a light microscope. The presence of parasporal crystals was first determined using Coomassie blue stained slides1 (1000×) under a Nikon E100 light microscope and confirmed later by a Nikon Ti-Eclipse phase contrast microscope (1000×) (data not shown). For the SEM analysis, aliquots of 1 ml were centrifuged for 5 minutes (16,000 g) at room temperature. Each pellet was washed three times with sterile distilled water and fixed with 100 μl 4% formaldehyde. Each fixed preparation was then sent to Centro Integral de Microscopía Electrónica (CIME ? CONICET ? UNT) for SEM examination. Fil: Peralta, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Sauka, Diego Herman. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marozzi, Antonela Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Patagonia Norte. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede San Martín de Los Andes-inibioma | Universidad Nacional del Comahue. Centro Regional Universitario Bariloche. Instituto de Investigaciones En Biodiversidad y Medioambiente. Subsede San Martín de Los Andes-inibioma.; Argentina Fil: del Valle, Eleodoro Eduardo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Agropecuarias del Litoral. - Universidad Nacional del Litoral. Instituto de Ciencias Agropecuarias del Litoral.; Argentina Fil: Palma, Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina

The Bacillus cereus group includes eight species: Bacillus anthracis, B. cereus, Bacillus cytotoxicus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis and Bacillus toyonensis, which are highly related at the phylogenetic level. In this work, we performed the isolation and characterization of a novel Bacillus sp. strain exhibiting parasporal crystals with insecticidal activity that was initially classified as a Bacillus thuringiensis strain. Its genome encoded three genes showing homology to known pesticidal proteins from B. thuringiensis and Lysinibacillus sphaericus proteins: Cry7Ga1 (a crystal protein), a Mpp2Aa3 (ETX/Mtx2 family) homolog and a mosquitocidal-like protein (NPP1). However, since its genome sequence shared > 98% ANI with several Bacillus toyonensis genomes, the strain has subsequently been renamed as Bacillus toyonensis biovar Thuringiensis and designated Bto-UNVM_94. Bioassays demonstrated that this novel strain exhibited toxicity against Cydia pomonella (Lepidoptera: Tortricidae) and Anthonomus grandis (Coleoptera: Curculionidae), a low toxicity against Aedes aegypti (Diptera: Culicidae) and Alphitobius diaperinus (Coleoptera: Tenebrionidae) whereas no toxicity was shown to the free-living nematode Panagrellus redivivus (Rhabditida: Panagrolaimidae).

Bacillus thuringiensis is a gram-positive and spore-forming bacterium that synthesizes a wide diversity of proteins with insecticidal activity and which has demonstrated its potential and safety as a biocontrol agent for more than four decades. However, several susceptible insect species have been reported for evolving resistance, which demands screening for strains exhibiting novel insecticidal properties. In this work, we performed the genome sequence analysis and the insecticidal characterization of B. thuringiensis strain Bt-UNVM_94 isolated from Argentina. This strain produced quasi symmetric bipyramidal parasporal crystals as shown using Scanning Electron Microscopy. Its genomic sequence harbours one coding sequence showing homology to the crystal toxin Cry7Ga2 and another, with similarity to the Mpp2Aa3 (Mtx2) toxin. Known Cry7A and Cry7B are known to be active against some coleopteran and lepidopteran larvae, respectively. However, bioassays performed with spore-crystal mixtures of strain Bt-UNVM_94 exhibited dual toxicity with 50% and 91% mortality against Cydia pomonella (Lepidoptera: Tortricidae) and Anthonomus grandis (Coleoptera: Curculionidae), respectively. No toxicity was detected against the free-living nematode Panagrellus redivivus (Rhabditidae: Panagrolaimidae). This strain also showed no PCR amplification of the type I b-exotoxin thuE gene, consistent with the absence of mortality in b-exotoxin bioassays with Musca domestica (Diptera: Muscidae). Screenings of novel B. thuringiensis strains may provide toxins with novel insecticidal properties that can be used to suppress insect resistance to the most used B. thuringiensis-crops in the field.

Entomopathogenic nematodes belonging to the genus Steinernema are able to infest and kill insect hosts in association with their resident, entomopathogenic symbiont bacteria in the gram-negative genus Xenorhabdus (Enterobacteriaceae). However, only a few species of Xenorhabdus have been isolated from their hosts and their insecticidal properties reported. Here we performed the genome sequence analysis of 14 Xenorhabdus strains isolated from Steinernema nematodes in Argentina, able to kill 6th instar Galleria mellonella (Lepidoptera) larvae. The 14 draft genome sequences encoded 110 putative insecticidal toxins (Tc, Pir and Mcf toxin homologs) plus other virulence factors with similarity to putative nematicidal toxins and chitinases. The genome sequences of strains Flor, 5, PSL, Reich, 42, Vera, M, 18, Cul, DI, 12, 38, 3 and ZM exhibited 4, 9, 2, 10, 9, 5, 7, 10, 10, 7, 3, 18, 8 and 8 putative insecticidal toxin genes, respectively. Strains 5, Reich, 42, 18, DI, 38, 3 and ZM carried their predicted insecticidal toxin genes arranged into putative pathogenicity islands whereas strains Flor, PSL, Vera, M, Cul and 12) showed them spread thorough different contigs. Multigene phylogenetic analysis and average nucleotide identity (ANI) calculations were also performed and allowed the identification of three strains (PSL, Reich and 12) that should be considered as novel Xenorhabdus genomospecies. In this work, we provide a dual insight into the diversity of the species belonging to the Xenorhabdus genus and into their predicted insecticidal toxin repertory, which is currently under investigation.

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. This research was funded by the Spanish Ministry project AGL2017-83498-C2-1-R.

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.

Brevibacillus laterosporus is a globally ubiquitous, spore forming bacterium, strains of which have shown toxic activity against invertebrates and microbes and several have been patented due to their commercial potential. Relatively little is known about this bacterium. Here, we examined the genomes of six published and five newly determined genomes of B. laterosporus, with an emphasis on the relationships between known and putative toxin encoding genes, as well as the phylogenetic relationships between strains. Phylogenetically, strain relationships are similar using average nucleotide identity (ANI) values and multi-gene approaches, although PacBio sequencing revealed multiple copies of the 16S rDNA gene which lessened utility at the strain level. Based on ANI values, the New Zealand isolates were distant from other isolates and may represent a new species. While all of the genomes examined shared some putative toxicity or virulence related proteins, many specific genes were only present in a subset of strains., Highlights • We examined genomes of 11 Brevibacillus laterosporus, a bacterium which is antagonistic to invertebrates and/or microbes • Multiple phylogenetic methods showed New Zealand isolates more distant than all other isolates • Each genome could contain 11–13 copies of the 16S rDNA gene, some of which were not identical • Many putative toxin encoding genes were present in the genomes, but the toxin complement varied from isolate to isolate • Variation in occurrence of toxin-encoding genes indicates the potential to find strains with new combinations of activities

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.

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., This research was supported by the Spanish Ministry of Science and Innovation (grant reference AGL2009-13340-C02) and by the Universidad Pública de Navarra (PhD contract awarded to Leopoldo Palma).

As biological control agents take an expanding share of the pesticides market and the production of insect-resistant crops increases, it is essential to understand the structure and function of the active agents, the invertebrate-active toxins that are the fundamental ingredients of these control systems. The potential for these agents in industry, agriculture and medicine necessitates a thorough investigation of their activity. Fil: Palma, Leopoldo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Berry, Colin. Cardiff University; Reino Unido

The advance of no-till management systems in agriculture favored the increase of Diloboderus abderus (Sturm) populations in most of the cultivated areas in South America. Damage to wheat crops (Triticum aestivum Linnaeus) is caused by the larvae, commonly known as white grubs, which consume seeds and roots, weakening or killing the plants. Entomopathogenic nematodes (EPNs) are insect parasites usually used as biological control agents of larvae of Scarabaeidae species. However, a great variability in pathogenicity and virulence against different white grubs has been observed among EPN species and isolates of a single species. The aim of this work was to evaluate the pathogenicity of Steinernema rarum (de Doucet) CUL isolate, Heterorhabditis bacteriophora (Poinar) SMC isolate and their symbiotic bacteria against D. abderus larvae. Wheat plants were grown in plastic pots under greenhouse conditions and one larva (L1, L2 or L3) of D. abderus was placed in each pot and then inoculated with 1000 EPN IJs. H. bacteriophora caused 95 and 45% mortality to L1 and L2, respectively. No L3 mortality caused by the studied isolates was observed. Field experiments were conducted in wheat plots in 2013 and 2014. Treatments consisted of surface application of S. rarum or H. bacteriophora (2.5 × 109 IJs/ha) and chlorpyrifos; control treatments contained only distilled water. Evaluations were made at day 0, 30 and 60 after treatments by randomly selecting eight sites from each plot and determining the number of D. abderus L2. Larval populations were reduced in plots treated with H. bacteriophora and chlorpyrifos. Larval populations were reduced in plots treated with H. bacteriophora and chlorpyrifos. The plots treated with S. rarum did not show differences in the insect population with respect to the control. EPN symbiotic bacteria were injected into the hemocoel of each larval stage to determine virulence against D. abderus. D. abderus larval mortality caused by symbiotic bacteria was significantly influenced by bacterial concentration and larval stage. H. bacteriophora showed a greater reproductive capacity than S. rarum. We conclude that H. bacteriophora SMC have potential for use as biological control agents of D. abderus first and second-instar larvae. Fil: del Valle, Eleodoro Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Frizzo, Laureano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Lax, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Bonora, José Sebastián. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Palma, Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Bernardi Desch, Nahuel Pablo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Pietrobón, Marianela. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: Doucet, Marcelo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina

The Vip3 proteins produced during vegetative growth by strains of the bacterium Bacillus thuringiensis show insecticidal activity against lepidopteran insects with a mechanism of action that may involve pore formation and apoptosis. These proteins are promising supplements to our arsenal of insecticidal proteins, but the molecular details of their activity are not understood. As a first step in the structural characterisation of these proteins, we have analysed their secondary structure and resolved the surface topology of a tetrameric complex of the Vip3Ag4 protein by transmission electron microscopy. Sites sensitive to proteolysis by trypsin are identified and the trypsin-cleaved protein appears to retain a similar structure as an octomeric complex comprising four copies each of the ~65 kDa and ~21 kDa products of proteolysis. This processed form of the toxin may represent the active toxin. The quality and monodispersity of the protein produced in this study make Vip3Ag4 a candidate for more detailed structural analysis using cryo-electron microscopy.

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. © 2014 by the authors; licensee MDPI, Basel, Switzerland., This research was supported by the Spanish Ministry of Science and Innovation (grant ref. AGL2009-13340-C02) and by the Universidad Pública de Navarra (P hD contract awarded to L.P.).

The antifungal activity of Bacillus spp.strains has been already reported against a different number of phytopathogenicfungi3. Some species belonging to thisgenus (i.e. B. subtilis, B. pumilus and B. licheniformis) are considered GRAS (generally regarding as safe) microorganisms and exhibit potential to enhance plant growth by the production of an amplenumber of beneficial substances1,portraying these species as an environmentally friendly alternative tochemical fungicides. Macrophomina phaseolina (Tassi) Goid is a wide range soil-bornephytopathogenic fungus causing charcoal rot to several significant plant crops in Argentina (i.e. soybean, common bean,peanuts and corn)5,6 and is also capableto affect more than five hundred plant species worldwide4. Its ability to infect these hostplants is commonly correlated to the production of viable sclerotia, anon-sporic resistance structure constituted by a mass of densely packed hyphae, in the field5. In this work, we report theisolation of a Bacillus sp. strain 11.1 obtained from soil samples from Córdoba Province,Argentina2exhibiting a strongantagonistic activity against M. phaseolina. In order to analyse thepotential antagonistic effect of strain 11.1, both microorganisms were grown on dual culture platescontained potato dextrose agar (PDA) and tryptone soya agar (TSA) (1:1 v/v) and incubated for 48-72hours at 28ºC (Figure 1). An important and irreversible inhibition of the fungal growth was showed (Figure1A). In addition, the fungal mycelium located at the edge of the inhibited region was analysed bymicroscopic examination. The most evident antagonistic effect over the fungus was depicted by the lack ofblack pigmentation and the reduced size of formed sclerotiain comparison with the control (Figure 1B and C). Fil: Felipe, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Palma, Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Yaryura, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90 kDa) was processed to an approximately 70 kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM)., esearch at University of Valencia was supported by grants AGL2012-39946-C02-01 and AGL2015-70584-C2-1-R (MINECO/FEDER, EU) and by grants from the Generalitat Valenciana (ACOMP/2011/094, PROMETEO 2011/044 and GVPROMETEOII-2015-001). Research at the Universidad Pública de Navarra was supported by a grant from the MINECO/FEDER, EU (AGL2015-70584-C2-R). JGC was recipient of a PhD grant from the Spanish Ministry of Economy and Competitivity (grant ref. BES-2013-065848).

Here, we report the draft genome sequence of Photorhabdus luminescensstrain DSPV002N, which consists of 177 contig sequences accounting for 5,518,143 bp, with a GC content of 42.3% and 4,701 predicted protein-coding genes (CDSs). From these, 27 CDSs exhibited significant similarity with insecticidal toxin proteins from Photorhabdus luminescenssubsp. laumondii TT01., This research was supported by the Spanish Ministry of Economy and Competitiveness (grant AGL2015-70584-C2-2-R). We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z) for the generation of sequencing data.

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. © 2011 Elsevier Inc., This study received financial support from a Navarre Government Project (Resolution 228/2008) awarded to O.S.

10 p., 3 tables, 4 figures and references, 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., This work was supported by the Grant AGL200507909-C03-01. TW acknowledges support from CONACYT infrastructure Grant I0-110/162/10.

We present the draft whole-genome sequence of the entomopathogenic Bacillus pumilus 15.1 strain that consists of 3,795,691 bp and 3,776 predicted protein-coding genes. This genome sequence provides the basis for understanding the potential mechanism behind the toxicity and virulence of B. pumilus 15.1 against the Mediterranean fruit fly. Fil: García Ramón, Diana Carolina. Universidad de Granada; España Fil: Palma, Leopoldo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Berry, Colin. Cardiff University; Reino Unido Fil: Osuna, Antonio. Universidad de Granada; España Fil: Vilchez, Susana. Universidad de Granada; España

Ekino et al. [1] reported the cloning and characterization of a novel cytotoxic protein (Parasporin-5) produced by Bacillus thuringiensis strain A1100. The 33.8-kDa inactive precursor protein exhibited strong cytocidal activity upon proteinase K activation against several mammalian (cancer) cell lines, and showed slight homology with Cry and aerolysin-type -pore-forming toxins. Most research concerning parasporins has mainly been performed in order to demonstrate their use as potential therapeutic agents against cancer, but they are lacking additional research supporting the absence of activity against invertebrates; especially, taking into account that these toxins are not expected to evolve to kill cancer cells. Therefore, it is reasonable to think that they should have another (unknown) target in nature. Despite the fact that in this work, the authors demonstrated the toxic activity of this protein against several types of cancer cells, further complementary studies against a minimum number of insects would be of great interest in order to determine the potential insecticidal activity of this protein and understand its natural role. For example, Palma and collaborators [2] reported the molecular and insecticidal characterization of a novel Cry-related protein closely related to parasporins 2 and 4, (Cry41Aa1 and Cry41Ab1). This protein did not show any toxic activity against five species of Lepidoptera but, after more extensive testing, this protein was found to demonstrate a specific toxic activity against the green-peach aphid Myzus persicae. Nowadays, parasporin proteins are commonly known in the literature to be produced by “non-insecticidal” B. thuringiensis strains and because they exhibit significant and preferential cytocidal activity against cancer cells of various origins [3]. However, the absence of insecticidal activity deserves to be more deeply investigated since a single B. thuringiensis toxin has shown to have a narrow host range while, in general, they are active against a wide range of invertebrates [4,5]. Therefore, the determination of the activity against a minimum number of insects (preferably from different taxonomic orders) is highly desirable and might change the non-insecticidal concept we currently have about parasporins proteins produced by non-insecticidal B. thuringiensis strains. Fil: Palma, Leopoldo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

The use of chemical pesticides revolutionized agriculture with the introduction of DDT (Dichlorodiphenyltrichloroethane) as the first modern chemical insecticide. However, the effectiveness of DDT and other synthetic pesticides, together with their low cost and ease of use, have led to the generation of undesirable side effects, such as pollution of water and food sources, harm to non-target organisms and the generation of insect resistance. The alternative comes from biological control agents, which have taken an expanding share in the pesticide market over the last decades mainly promoted by the necessity to move towards more sustainable agriculture. Among such biological control agents, the bacterium Bacillus thuringiensis (Bt) and its insecticidal toxins have been the most studied and commercially used biological control agents over the last 40 years. However, some insect pests have acquired field-evolved resistance to the most commonly used Bt-based pesticides, threatening their efficacy, which necessitates the immediate search for novel strains and toxins exhibiting different modes of action and specificities in order to perpetuate the insecticidal potential of this bacterium. Fil: Peralta, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Palma, Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina

We report here the complete annotated 6,510,053-bp draft genome sequence of Bacillus thuringiensis serovar tolworthi strain Na205-3, which is toxic for Helicoverpa armigera . This strain potentially contains nine insecticidal toxin genes homologous to cry1Aa12 , cry1Ab1 , cry1Ab8 , cry1Ba1 , cry1Af1 , cry1Ia10 , vip1Bb1 , vip2Ba2 , and vip3Aa6 .

Key virus traits such as virulence and transmission strategies rely on genetic variation that results in functional changes in the interactions between hosts and viruses. Here, comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host– virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates had a similar genome size and content. Complete genome analyses of single nucleotide polymorphisms and insertions/deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus–host cell interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150) or secondary infections (ME35) and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbour high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest that the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways., This study received financial support from the Spanish Ministry for Science and Technology (AGL2005-07909-CO3-01, AGL2008-05456- C03-01). O. C. received a Predoctoral Fellowship from the Spanish Ministry of Education and Culture. J. T. and E. A. H. are supported by the European Research Council (grant 205206 GENOVIR).

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., This research was supported by the Spanish Ministry of Science and Innovation (Grant Ref. AGL2009-13340-C02) and by the Universidad Pública de Navarra (contract awarded to L.P.). We thank Trevor Williams for his kind revision of the manuscript., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

The biological control of agricultural pests by using differententomopathogenic microorganisms has been significantlyincreased in the last years, due to the need of reducing theamount of chemical insecticides in agricultural ecosystems. Fil: Palma, Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina Fil: del Valle, Eleodoro Eduardo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

A Spanish Bacillus thuringiensis strain collection was screened for the presence of vip genes. One hundred strains from a Canary Island collection were screened for vip1 and vip2 genes and 7% contained potentially novel vip1 and vip2-like genes, as indicated by the low degree of similarity with previously known vip1 and vip2 genes. Four hundred strains from a collection originating from the Spanish mainland were screened for vip3 genes and 14.5% of them contained potentially novel vip3-like genes. Reconstruction of the full-length vip sequences could only be achieved for two vip3 gene variants encoding 789 and 787 amino acid proteins that were designated as Vip3Aa45 and Vip3Ag4, respectively. These proteins showed 82% pairwise identity between them and differed from Vip3Aa1 in the putative signal peptide, two specific proteolytic processing sites and the 66-kDa insecticidal fragment. The purified proteins were tested against nine lepidopteran pest species and displayed toxicity, expressed as mean lethal concentration, for five of them. The two toxins were highly toxic for Lobesia botrana (~1-2μg/ml) and Spodoptera littoralis (~20ng/cm2), moderately toxic for Spodoptera exigua (~100-300ng/cm2), and varied greatly in their toxicity for Mamestra brassicae or Chrysodeixis chalcites, with high toxicity for Vip3Aa45 in M. brassicae (~40ng/cm2) and for Vip3Ag4 in C. chalcites (~45ng/cm2). © 2013 Elsevier Inc., We acknowledge the technical support of N. Gorría and I. Ibañez for their help in insect rearing. This research was supported by the Spanish Ministry of Science and Innovation (grant Ref. AGL2009-13340-C02) and by the Universidad Pública de Navarra (PhD contract awarded to L.P.). We acknowledge the review of the manuscript by Colin Berry and Trevor Williams.