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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

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.

We report the complete sequence and analysis of pFR260, a novel megaplasmid of 260,595 bp from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. It carries 7 insecticidal genes: 3 cry8 copies previously reported, 2 vip1, and 2 vip2. Also, it carries a gene encoding a putative atypical Cry protein. These genes are arranged in a region of approximately 105 kbp in size with characteristics of a pathogenicity island with a potential coleopteran-specific insecticide profile. DNA strand composition asymmetry, as determined by GC skew analysis, and the presence of a Rep protein involved in the initiation of replication suggest a bidirectional theta mechanism of replication. In addition, many genes involved in conjugation and a CRISPR-Cas system were detected. The pFR260 sequence was deposited in GenBank under accession number KX258624.

Phosphate-solubilizing strain INTA 293-1 from a soil sample of Corrientes province (Argentina) was identified as Bacillus thuringiensis based on its faculty to produce crystalline inclusions during sporulation and 16S rDNA sequence similarity. INTA 293-1 was found to produce bipyramidal and cuboidal crystals, containing major proteins of ca. 130 and 65 kDa respectively, resembling those present in Lepidoptera- and Diptera-active strains. Accordingly, INTA 293-1 showed insecticidal activity against lepidopteran and dipteran larvae. No insecticidal activity nor evident effects on the normal development of coleopteran larvae or nematodes were observed. Screening of cry, cyt and vip genes showed that INTA 293-1 contains cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa genes. Furthermore, this new entomopathogenic strain of B. thuringiensis was able to solubilize different sources of inorganic phosphates in liquid media following similar solubilization kinetics. INTA 293-1 was effective in solubilizing hydroxyapatite, patagonic phosphate rock, and to a lesser extent the aluminium phosphate. The differential behaviour between them may be explained by differences in INTA 293-1 survival. Phosphate solubilization was associated with pH reduction. This study reveals a little-explored aspect of B. thuringiensis and paves the way for the development of new agrobiological products with multiple modes of action.

We found and characterized two cry8 genes from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. These genes, cry8Kb3 and cry8Pa3, are located in a tandem array within a 13,200-bp DNA segment sequenced from a preparation of total DNA. They encode 1,169- and 1,176-amino-acid proteins, respectively. Both genes were cloned with their promoter sequences and the proteins were expressed separately in an acrystalliferous strain of B. thuringiensis leading to the formation of ovoid crystals in the recombinant strains. The toxicity against larvae of Anthonomus grandis Bh. (Coleoptera: Curculionidae) of a spore-crystal suspension from the recombinant strain containing cry8Pa3 was similar to that of the parent strain INTA Fr7-4.

Insecticidal activity of Bacillus thuringiensis is attributed largely to the crystal proteins. These were found with specific toxic activity against insects in different orders. A novel cry8 gene from B. thuringiensis strain INTA Fr7-4 from Argentina was characterized. The encoded protein, Cry8Qa2, is 1184 amino acids long and its sequence is more similar to those of Cry8F class. We cloned and expressed the protein in an acrystalliferous strain of B. thuringiensis using two differentially regulated promoters. The recombinant strains produced ovoid crystals with low toxicity against larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae). The morphology and insecticidal properties of these crystals resembled those produced by the native strain INTA Fr7-4.

Some Bacillus thuringiensis strains secrete type I β-exotoxin, which is a non-specific insecticidal and thermostable adenine nucleoside oligosaccharide. Toxicity bioassays and HPLC are traditional methods for detecting β-exotoxin. With the aim of establish a first rapid approach for prediction of type I β-exotoxin production, two PCR-based methods were successfully evaluated in B. thuringiensis strains and native isolates. In order to validate a reliable technology, results obtained by this method were correlated with that obtained from Musca domestica bioassays. Fil: Sauka, Diego Herman. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Perez, Melisa Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Lopez, Nanci N.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Onco, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Berretta, Marcelo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Benintende, Graciela Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina

The objective of this work was to establish and test the induced-feeding bioassay in order to detect Bacillus thuringiensis insecticidal activity against Epilachna paenulata (Coleoptera: Coccinellidae). Larvae were induced to swallow high concentrations of spore-crystal suspensions of seven exotic and 30 Argentine B. thuringiensis strains. The great majority of strains showed no toxicity to E. paenulata larvae, and observed mortality was lower than 30%. Induced-feeding bioassay is feasible, and should be used for prospecting strains that produce right combinations of Cry proteins needed to an efficient pest control.