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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.

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).

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

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 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).

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.

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

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 .

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).