Thuringiensis Kurstaki HD1 Research Articles

Synergic effect between Bacillus thuringiensis Kurstaki HD1 δ-endotoxins and Melia azedarach (Meliacea) methanolic extract against the larvae of Thaumetopoea pityocampa Den. & Schiff. (Lepidoptera, Notodontidae)

Synergic effect between <i>Bacillus thuringiensis</i> Kurstaki HD1 δ-endotoxins and <i>Melia azedarach</i> (Meliacea) methanolic extract against the larvae of <i>Thaumetopoea pityocampa</i> Den. &amp; Schiff. (Lepidoptera, Notodontidae)

Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests

AbstractIn this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae.

Toxicity, activation process, and histopathological effect of Bacillus thuringiensis vegetative insecticidal protein Vip3Aa16 on Tuta absoluta.

Tuta absoluta is a destructive moth of Solanaceae plants and especially tomatoes. Here, we considered the entomopathogenic activity of the Bacillus thuringiensis Vip3Aa16 protein heterologously produced by Escherichia coli against T. absoluta. Purified Vip3Aa16 showed lower lethal concentration 50 % against third instar larvae (Toxin/tomato leaf) (335 ± 17 ng/cm(2)) compared to that of B. thuringiensis kurstaki HD1 δ-endotoxins (955 ± 4 ng/cm(2)) (P < 0.05). Action mode examination showed that Vip3Aa16 (88 kDa) was more sensitive to proteolysis activation by the chymotrypsin than the trypsin or the larvae gut soluble proteases, yielding derivative proteins essentially of about 62 and 33 kDa. The gut-soluble proteases could contain trypsin-like enzymes implicated in Vip3Aa16 activation since the proteolysis was inhibited using specific protease inhibitors. Additionally, we showed that the histopathological effect of Vip3Aa16 on T. absoluta larva midguts consisted on a microvillus damage and an epithelial cell rupture.

Isolation and characterization of native Bacillus thuringiensis isolates from Syrian soil and testing of their insecticidal activities against some insect pests

Bacillus thuringiensis was detected in 12.5% of soil samples collected from different regions in Syria and 25 B. thuringiensis isolates were found to be highly toxic to larvae of Ephestia kuehniella Zeller, Phthorimaea operculella Zeller, and Cydia pomonella L. (Lepidoptera), but not to the larvae of Culex quinquefasciatus (Diptera). Light microscopy investigation showed the presence of bipyramidal and cuboidal parasporal bodies produced by these isolates. The existence of different cry genes in the tested isolates was studied using a PCR strategy with a set of general primers recognizing some of the cry genes reported in the relevant literature. Primers corresponding to 2 types of cry genes (cry1 and cry2) successfully amplified DNA in all of the tested isolates. Moreover, the proteins encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. The 50% lethal concentration of the spore-crystal mixture of the 25 isolates against E. kuehniella larvae varied from 8.4 to 97.6 µg g^{-1}. A comparison of the LC_{50} values of the tested isolates with those of the reference strains B. thuringiensis kurstaki HD-1 and HD-73 (20.8 and 46 µg g^{-1}, respectively) showed that some of these isolates have a higher toxicity potential. Moreover, flagellar serotyping revealed that 4 isolates, which were among the most toxic, belonged to serotype kurstaki. This study constitutes the first isolation and characterization of local B. thuringiensis isolates in Syria. Some of these isolates exhibit toxic potential and, therefore, could be adopted for future applications to control some important insect pests.

Development of a Cost Effective Medium for Production of Bacillus Thuringiensis Bioinsecticide Using Food Barley

Development of a Cost Effective Medium for Production ofBacillus ThuringiensisBioinsecticide Using Food BarleyGenetically manipulated food barley,Hordeum vulgareL. var.valfajr, was compared to commercial-grade starch as carbon sources in groundnut and soybean based media for supporting growth, sporulation and delta-endotoxin production byBacillus thuringiensisBerlinerB. thuringiensiskurstaki HD-1 and a promising newly isolatedB. thuringiensisstrain (referred to as BTA) were used. MgCl2, CaCl2, and MnCl2were used as trace-elements. Culture media were compared in shaken flasks and then in 5 l Fermentors. Biomass, delta-endotoxin levels and the number of spores as colony forming units (CFU) were evaluated. For each of the two strains, biomass and delta-endotoxin synthesis were not significantly different in soybean-based media after substitution starch by barley flour. There were significant differences between the two strains with respect to biomass and toxin production. Evaluated costs of media preparation showed that food barley is an economical alternative to commercial-grade starch in the production of BTA bioinsecticide.

Occupational exposure to airborne Bacillus thuringiensis kurstaki HD1 and other bacteria in greenhouses and vegetable fields

When microorganisms are used for pest control in vegetable production, the active organisms become part of the microbiota growers are exposed to. The aim of this study was to quantify vegetable growers' exposure to the bacterial strain Bacillus thuringiensis kurstaki strain HD1 (termed HD1) from the biocontrol agent Dipel®, and other airborne mesophilic bacteria. Personal (n=102) and stationary (n=43) measurements of exposure were performed in greenhouses and open fields. Air samples were analysed by plate counts, and total counts with a microscope. Isolates resembling HD1 were identified by PCR analysis. HD1-like bacteria were only detected in environments where Dipel® was used. In a greenhouse with Dipel® treated tomato plants, the growers' exposure to airborne HD1-like bacteria reached 5300 cfu/m3 and 1400 cfu/m3 during harvest and clearing of old plants, respectively. In untreated greenhouses, the highest concentration of total mesophilic bacteria, 1,100,000 cfu/m3, was detected in a cucumber greenhouse. The median concentrations of mesophilic bacteria in tomato greenhouses were significantly lower than the median concentrations in cucumber greenhouses. There was no significant difference in exposure to mesophilic bacteria in tomato greenhouses and in vegetable fields. We found that greenhouse workers, especially in cucumber production, were exposed to high concentrations of total bacteria. Thus, the already present airborne bacteria in greenhouses might have a greater influence on growers' health than applied biocontrol strains. However, further studies are needed to establish an occupational threshold limit for airborne bacteria and to secure a healthy working environment for vegetable growers.

Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains

The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.

Detection ofBacillus thuringiensis kurstakiHD1 on cabbage for human consumption

The objectives of the study were to develop a specific procedure for quantification and identification of Bacillus thuringiensis kurstaki HD1, which is used as a biopesticide, and to quantify its presence in different kinds of cabbage for human consumption. We found that B. thuringiensis kurstaki HD1 can be distinguished from other B. thuringiensis strains by its unique random amplification of polymophic DNA-PCR pattern with the OPA9 primer and the presence of the flagellin genes, as detected by the primers FLAB1 and FLAB2. We detected from one to 100 Bacillus cereus-like bacteria in 10 batches of five different cabbage products for consumption. As many as 73 out of 134 isolates (53.7%) were identical with B. thuringiensis kurstaki HD1. The results show that B. thuringiensis kurstaki HD1 from biopesticides can be found in vegetables for human consumption.

Production of Bacillus thuringiensis biopesticides using wastewater sludge as a raw material: effect of inoculum and sludge solids concentration

Studies were conducted to optimise inoculum quantity and total solids concentration for the production of Bacillus thuringiensis (Bt) based biopesticides using wastewater sludge as a raw material in shake flasks and 15 l fermenters. The sludge used was a mixture of primary and secondary sludges. Two different Bt strains were employed; Bacillus thuringiensis kurstaki HD-1 (Btk) and a new strain (Bta) isolated from sewage sludge. Preparation of inoculum in the same sludge as that used for the production medium resulted in higher cell and spore counts, specific growth rate and entomotoxicity value. The final cell count and entomotoxicity achieved at the end of fermentation depended on the amount of inoculum used. A cell count of 2.2×10 9 and 3.6×10 9 CFU/ml was observed at 2% (v/v) and 3% (v/v) inoculum volume, respectively. A lower cell count was recorded at 1, 4 and 5% (v/v) inoculum concentration. Bioassay results also revealed that a 2%(v/v) inoculum furnished higher entomotoxicity (12 900 IU/μl) against spruce budworm. The sludge solids concentration significantly influenced growth and entomotoxicity yield. Solids concentration of 46 g/l using Btk resulted in decreased cell concentration (5.4×10 8 CFU/ml), spore concentration (4.8×10 8 CFU/ml) and low entomotoxicity (9743 IU/μl). The sporulation rate was 89%. The same phenomena was observed when a lower sludge solids concentration (10 g/l) was used, resulting in cell and spore concentrations of 4.3×10 8 and 3.9×10 8 CFU/ml, respectively, and potency of 8231 IU/μl. The optimum total solids concentration was 26 g/l, which resulted in an improved potency of 12 970 IU/μl, cell and spore concentrations of 5.0×10 9 and 4.8×10 9 CFU/ml, respectively, and a sporulation rate of 96%. A similar trend of growth and entomotoxicity at different sludge solids concentration was also observed with new Bt strain isolated from sewage sludge. The fermenter results showed that one of the reasons for inferior entomotoxicity at higher sludge solids was oxygen transfer limitation.

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.

Analysis of common antigen of flagella inBacillus cereusandBacillus thuringiensis

Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus. This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis.

The parasporal inclusion of Bacillus thuringiensis subsp. shandongiensis: Characterization and screening for insecticidal activity

The parasporal body of Bacillus thuringiensis subsp. shandongiensis was characterized in terms of its structure, protein composition, and toxicological properties against several types of insects. The crystals of B. thuringiensis shandongiensis appear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, tritration curve analysis, and SDS-PAGE of the solubilized crystals. A second protein of ca. 60 kDa is present in trace amounts and appears to be associated with a small bar-shaped inclusion. The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological cross reactivity. Its N-terminal amino acid sequence differed from that of other B. thuringiensis crystal proteins. The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars ( B. thuringiensis israelensis and B. thuringiensis kurstaki HD-1) and one nontoxic serovar ( B. thuringiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDa B. thuringiensis shandongiensis protein, the B. thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B. thuringiensis kurstaki P1 proteins. In contrast to most B. thuringiensis serovars, B. thuringiensis shandongiensis crystals did not dissolve at pH 12. Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presence of 25 m m dithiothreitol. The protein pattern of dissolved and undissolved B. thuringiensis shandongiensis crystals after trypsin treatment did not differ from the controls, as observed by SDS-PAGE. Therefore, under the conditions of this study, the results suggest that trypsin sites were not available on the 144-kDa protein. Crystals were nontoxic to larvae of the lepidopterans Trichoplusia ni, Spodoptera exigua, and Helicoverpa virescens or to the mosquitoes Aedes aegypti and Culex quinquefasciatus.

Effect of Bacillus thuringiensis Berliner on prepupal and pupal stages of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

The prepupal stage of Spodoptera littoralis was affected by Bacillus thuringiensis kurstaki HD-1 (Dipel) at high concentration (5%), when sprayed or kept in soil treated with the pathogen. The emerged moths showed a short longevity associated with low egg production and low fertility. Increase in malformed individuals was observed. Treatment of the pupae of S. littoralis with B. thuringiensis during different ages showed an increased effect when treatment was applied, immediately after pupation.

Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715.

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

Effects of the three proteases from gut juice of the silkworm, Bombyx mori, on the two morphologically different inclusions of .DELTA.-endotoxin produced by Bacillus thuringiensis kurstaki HD-1 strain.

Three proteases designated P-I, P-II, and P-III were separated from larval gut juice of the silkworm, Bombyx mori, and the effects of these proteases on the two morphologically different inclusions produced by Bacillus thuringiensis kurstaki HD-1 strain were investigated. Bipyramidal inclusions were solubilized by each protease at pH 10.2, and proteins with a molecular weight of about 60,000 were produced, while cuboidal inclusions were resistant to the proteases. Protease P- III appeared to be most important in the dissolution of the bipyramidal inclusions. Based on the affinity to various synthesized substrates, P-III was considered to be a chymotrypsin-like protease. After dissolution of inclusions by P-III, dissolved and undissolved fractions were separated, and the insecticidal activity of each fraction was tested. Results indicate that bipyramidal inclusions are preferentially toxic to the silkworm and the cuboidal ones carry the mosquitocidal activity.