Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

It has been reported that C-type lectins(CTLs),which are pattern recognition receptors of the insect innate immunity response,may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in insects.However,to date,which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not clear.In this study,33 CTL genes were identified from S.exigua.Based on the number of carbohydrate-recognition domains(CRDs)and the domain architectures,they were classified into three groups:(1)nineteen CTL-S(single-CRD),(2)eight immulectin(dual-CRD)and(3)six CTL-X(CRD with other domains).RT-qPCR analysis revealed that expression levels of SeCTL-S15,IML-4 and CTL-X6 were upregulated after challenge with Bt and Cry1Ab.Tissue and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva,respectively.Knockdown of SeCTL-S15 significantly increased Bt susceptibility,as indicated by reduced survival and larval weight.These results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in S.exigua.Our results provide new insights into CTL function in insects.

Animal Safety Test of Bacillus thuringiensis BT Protein

[Objectives]To determine the biological safety of BT protein from Bacillus thuringiensis(Bt)fermentation broth to mammals at high doses.[Methods]Healthy mice were randomly divided into 4 groups with 10 mice in each group.The experimental groups were fed with Bt fermentation supernatant at 0.2,0.6 and 1.0 mL/kg,respectively,once a day for 7 consecutive days.The blank control group was fed normally without intragastric administration.[Results]There was no significant difference in blood routine and blood biochemical analysis between the experimental group and the control group.After intragastric administration,the mice were dissected,and no obvious pathological changes were found;the heart,liver,spleen,lung and kidney were taken to make tissue sections,and no pathological changes were found by microscopic observation.[Conclusions]Mice can tolerate high doses of BT protein from B.thuringiensis fermentation broth.

Degradation of N-Acyl Homoserine Lactone Quorum Sensing Signals by Bacillus thuringiensis AHL Lactonase

Bacterial cells rely on signaling molecules to communicate with others from the same species and induce certain genes in a process known as quorum sensing (QS). A common molecule is N-acyl homoserine lactone (AHL) which is responsible for the expression of virulence and other factors that allow the organisms to compete in a given environment. On the other hand, other bacteria produce certain enzymes such as AHL-lactonase that break down AHL molecules and prevent gene expression of these factors. The aim of this work was to examine the level of degradation of AHL molecules by AHL-lactonase in 62 Bacillus thuringiensis (Bt) strains isolated from Middle Tennessee, Mississippi, and Alabama. N-hexanoyl-homoserine lactone (C6-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL), which cause Chromobacterium violaceum (CV026) to produce a purple pigment were tested at different concentrations to view the Bt lactonase activity. In addition, PCR was used to test for the presence of the lactonase gene. The results showed that among the 62 Bt strains, there were 58 that possessed the AHL-lactonase (aiiA) gene and 48 strains were able to degrade C6-HSL. At high concentrations of AHL, only 13 strains were able to completely degrade C6-HSL. In addition, degradation of 3-oxo-C6-HSL was weak compared to C6-HSL. The results also revealed that AHL lactonase was thermostable, and it was concluded that the level of degradation varies in Bt strains. Only 13 of the strains studied have potent inhibitory activity against C6-HSL, which may be good to be used in field applications to control agricultural pest.

Screening on Synergist of Bacillus thuringiensis Wettable Powder

[Objective] This study aimed to screen the best synergistic material for Bt wettable powder and evaluate their synergistic effect. [Method] The synergism of six different kinds of additives for Bacillus thuringiensis wettable powder （Bt WP） on the 2^nd instar larvae of Plutella xylostella was tested by method of leaf dipping in labora- tory. [Result] The mixtures of Bt with 0.1% ZnCl2, 0.5% ZnCl2, 1.0% ZnCl2, 1.0% MgCI2, 0.5% boric acid, 1.0% boric acid, 0.5% citric acid or 1.0% citric acid all ex- hibited synergistic effect, in which the synergistic effect of mixture containing 0.5% boric acid was the highest, with 17.2 synergistic ratio; followed by the mixture containing 1.0% ZnCl2, with 15.6 synergistic ratio. Moreover, addition of 0.5% boric acid could shorten the median lethal time of Bt wettable powder by about 10 h. After the mixtures of Bt with 0.5% boracic acid or 1.0% ZnCl2 was stored for 15 d at room temperature, toxicities of the two mixtures did not change significantly. [Conclusion] Boracic acid as the synergist of Bt wettable powder could not only increase insecti- cidal effect of Bt, but also accelerate its insecticidal rate. So, boracic acid could improve the disadvantages of Bt wettable powder such as poor insecticidal effect and slow insecticidal speed in a certain degree.

Isolation and Biological Determination of Bacillus thuringiensis with High Toxicity in Soil of Changbai Mountain Area

[Objective] This study was conducted to isolate and screen new Bacillus thuringiensis （Bt） strains against Lepidoptera insecticides. [Method] Bt strains were isolated from soil of Changbai Mountain area by temperature screening method, and highly-toxic Bt stains were then selected by biological determination and toxicity de- termination. [Result] From 150 soil samples, 18 Bt isolates were isolated, with an average isolation rate of 12.0%. Specifically, the isolation rate from mountain field was 8.5 %, and the isolation rate from farmland was 16.2%. The results of activity determination showed that there were17, 5 and 4 strains showing lethality rate over 90% against Plutel/a xylosrel/a, Spodoptera litura and Spodptera exigua, respectively, and among them, strain YNI-1 exhibited high activity against all the 3 kinds of in- sects. The results of toxicity determination showed that strain YNl-lhad the best fast-acting property against S.litura and S. exigua; strains with high toxicity against S. exigua were YN6-2〉YN1-1〉YN4-2〉YN2-6 sequentially; and toxicity of strains against S. /itura was in order of YN4-4〉YN1-1 〉YN6-1 〉YN4-1 〉YN2-1. [Conclusion] According to activity determination and toxicity determination, strain YN1-1 was screened as the target strain with wide spectrum, fast-acting property and high toxi- city.

Evidence for Positive Darwinian Selection of Vip Gene in Bacillus thuringiensis

Vegetative insecticidal proteins （VIPs）, produced during the vegetative stage of their growth in Bacillus thuringiensis, are a group of insecticidal proteins and represent the second generation of insecticidal trans-genes that will complement the novel δendotoxins in future. Fewer structural and functional relationships of Vip proteins are known in comparison with those of δ-endotoxins. In this study, both the maximum-likelihood methods and the maximum parsimony based sliding window analysis were used to evaluate the molecular evolution of Vip proteins. As a result, strong evidence was found that Vip proteins are subject to the high rates of positive selection, and 16 sites are identified to be under positive selection using the Bayes Empirical Bayesian method. Interestingly, all these positively selected sites are located from site-705 to site-809 in the C-terminus of the Vip proteins. Most of these sites are exposed and clustered in the loop regions when mapped onto its computational predicted secondary tertiary and a part of the tertiary structure. It has been postulated that the high divergence in the C-terminal of Vip proteins may not result from the lack of functional constraints, but rather from the rapid mutation to adapt their targeted insects, driven by positive selection. The potential positive selection pressures may be an attempt to adapt for the ＂arm race＂ between Vip proteins and the targeted insects, or to enlarge their target＇s host range. Sites identified to be under positive selection may be related to the insect host range, which may shed a light on the investigation of the Vip proteins＇ structural and functional relationships.

A Fragment of Cadherin-Like Protein Enhances Bacillus thuringiensis Cry1B and Cry1C Toxicity to Spodoptera exigua(Lepidoptera:Noctuidae)

Cry toxins produced by Bacillus thuringiensis（Bt） are effective biological insecticides against certain insect species.In this study,bioassay results indicated that Cry1B and Cry1C were toxic to Spodoptera exigua.We also identified a cadherin-like gene in S.exigua that could enhance the toxicity of Cry1B and Cry1C.The cadherin-like gene identified from the larvae midgut tissue was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of cDNA ends（RACE）.The full-length cDNA of the gene consisted of 5 220 bp encoding 1 740 amino acid with a predicted molecular mass of 196 kD.BLAST search analysis showed that the predicted amino acid sequence had a high sequence identity to the published sequences of cadherin-like proteins from other Lepidoptera insects.Spatial expression of the cadherin-like gene detected by qRT-PCR analysis revealed that the cadherin-like gene was mainly present in the gut of 4th instar larvae and during different life stages.The results suggested that the commercial development of this synergist has the potential to enhance Cry1B and Cry1C toxicity against Lepidoptera insects.

Adsorption and Insecticidal Activity of Toxin from Bacillus thuringiensis on Rectorite

The adsorption and desorption of the toxin from Bacillus thuringiensis strain WG-001 on rectorite were studied at different toxin and/or rectorite concentrations, pH values and temperatures. The insecticidal activity of the adsorbed toxin was evaluated by determining the lethal concentration to kill 50% of the larvae of Heliothis armigera （LC50）. The adsorption of the toxin on rectorite in sodium carbonate buffer （pH 9） reached equilibrium within 0.5-1.0 h and the adsorption isotherm of the toxin followed the Langmuir equation （R^2 〉 0.99）. In the pH range from 9 to 11 （carbonate buffer）, the adsorbed toxin decreased with increasing pH. The adsorption amounts decreased with increasing rectorite：toxin ratio. The adsorption was not significantly affected by the temperature between 10 and 50 ℃. The X- ray diffraction analysis indicated occurrence of the intercalation of the rectorite by the toxin. The infrared absorption spectrum showed that the binding of the toxin did not alter its structure. The LC50 wlues of the adsorbed toxin were smaller than those of the free toxin. The rectorite protected the toxin from ultraviolet irradiation damage. The desorption of the adsorbed toxin in water ranged from 37.5% to 56.4% and from 27.4% to 41.8% in a carbonate buffer. The desorption percentage also decreased with increasing rectorite：toxin ratio.

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Protein Characteristics and Field Efficacy of YN1-1, a Highly Virulent Strain of Bacillus thuringiensis

[Objective] This study aimed to investigate the biological characteristics of a Bacillus thuringiensis strain YNI-1, which has high virulence to Lepidoptera spp. [Method] The crystal protein of YNI-1 was analyzed by SDS-PAGE, and its indoor and field efficacy for Lepidoptera spp. was investigated. [Result] The parasporal crystal of YNI-1 has a diamond-like structure. The molecular weight of the original toxin protein is 136 kDa. After trypsin treatment, the original toxin protein was hy- drolyzed into active toxin protein with molecular weight of 63 kDa. For Plutella xy- Iostella and Pieris rapae, the indoor efficacy of B. thuringiensis was better than that of commercial B. thuringiensis （WP）. In view of field efficacy, rate of YNI-1 strain was higher than that of commercial B [Conelusion] YNI-1 strain has excellent development potential. the insects reduced thuringiensis （WP）.

Homology Modeling of Mosquitocidal Cry30Ca2 of Bacillus thuringiensis and Its Molecular Docking with N-acetylgalactosamine

Abstract Objective To investigate the theoretical model of the three-dimensional structure of mosquitocida Cry3OCa2 and its molecular docking with N-acetylgalactosamine. Methods The theoretical model of Cry30Ca2 was the Cry4Ba. Docking studies were performed N-acetylgalactosamine on the putative receptor. predicted by homology modeling on the structure of to investigate the interaction of Cry3OCa2 with Results Cry3OCa2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60A. Domain I is a helix bundle, Domain Ⅱ consists of three antiparallel β-sheets, Domain Ⅲ is composed of two β-sheets that adopt a 13-sandwich fold. Residue 32111e in loop1, residues 342Gin 343Thr and 345Gin in loop2, residue 393Tyr in loop3 of Cry3OCa2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand. Conclusion The 3D structure of Cry3OCa2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain Ⅱ are responsible for the interactions with N-acetylgalactosamine.

Toxicity studies for indigenous Bacillus thuringiensis isolates from Malang city,East Java on Aedes aegypti larvae

Objective:To investigate the toxicity of indigenous Bacillus thuringiensis（B.thuringiensis）isolates from Malang City for controlling Aedes aegypti（Ae.aegypti）larvae.Methods:Soil samples were taken from Purwantoro and Sawojajar sub-districts.Bacterial isolation was performed using B.thuringiensis selective media.Phenotypic characteristics of the isolates were obtained with the simple matching method.The growth and prevalence of spores were determined by the Total Plate Count method,and toxicity tests were also performed on the third instar larval stage of Ae.aegypti.The percentage of larval mortality was analysed using probit regression.The LC50was analysed by ANOVA,and the Tukey HSD interval was 95%.Results:Among the 33 selected bacterial isolates,six were obtained（PWR4-31,PWR4-32,SWJ4-2b,SWJ4-4b,SWJ-4k and SWJ5-1）that had a similar phenotype to reference B.thuringiensis.Based on the dendrogram,all of the bacterial isolates were 71%similar.Three isolates that had a higher prevalence of reference B.thuringiensis were PWR4-32,SWJ4-4b and SW5-1,of which the spore prevalence was 52.44%,23.59%,34.46%,respectively.These three indigenous isolates from Malang City successfully killed Ae.aegypti larvae.The PWR4-32 isolates were the most effective at killing the larvae.Conclusions:Six indigenous B.thuringiensis isolates among the 33 bacterial isolates found in the Sawojajar and Purwantoro sub-districts were toxic to the third instar larvae of Ae.aegypti.The PWR4-32 isolates were identical to tbe reference B.thuringiensis and had 88%phenotype similarity.The PWR4-32 isolates had the highest spore prevalence（52.44%）,and the early stationary phase occurred at 36 h.The PWR4-32 isolates were the most effective at killing Ae.aegypti larvae(LC50-72 h=2.3×108 cells/mL).

Complete genome sequence of Bacillus thuringiensis Bt185, a potential soil insect biocontrol agent

Bacillus thuringiensis Bt185 and its insecticidal spectrum-expanded engineering strains are considered as potential biocontrol agents to soil insect Holotrichia parallela,Holotrichia oblita or Anomala corpulenta.Here we reported the complete genome of strain Bt185,it harbors eight plasmids,and plasmid p BT1850294 carries three cry8 genes.

The use of Bacillus thuringiensis on Forest Integrated Pest Management

Bacillus thuringiensis is a major microbial insecticide and a source of genes encoding several proteins toxic to insects. In this paper the authors g ive a brief summary of Bacillus thuringiensis used on the integrated pest manage ment in forestry. The derivatives of Bt strain HD1 subsp kurstaki have been wide ly used to control the forest pests such as the gypsy moth (Lymantria dispar), s pruce budworm (Choristoneura fumiferana), the pine processionary moth (Thaumetop oea pityocampa), the European pine shoot moth (Rhyacionia buoliana) and the nun moth (Lymantria monacha). Some progresses of transferring and expressing Bt toxi n gene in forest trees are offered with a discussion on the limits and future pr ospects of using Bt products in forestry.

Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

Plant biotechnology:a case study of Bacillus thuringiensis(Bt) and its application to the future of genetic engineered trees

Agricultural productivity may be raised in a sustainable way by many different technologies such as biological fertilizers, soil and water conservation, biodiversity conservation, improved pest control, and changes in land ownership and distribution. Of these measures, biotechnology applications probably hold the most promise in augmenting conventional agricultural productivity, because biotechnology applications give not only the need to increase production, but also protect the environment and conserving natural resources for future generations. Biotechnology applications will have the possibilities to increase productivity and food availability through better agronomic performance of new varieties, including resistance to pests; rapid multiplication of disease-free plants; ability to obtain natural plant products using tissue culture; diagnosis of diseases of plants and livestock; manipulation of reproduction methods increasing the efficiency of breeding; and the provision of incentives for greater participation by the private sector through investments. Insect resistance through the transfer of a gene for resistance fromBacillus thuringiensis (Bt) is one of the most advanced biotechnology applications already being commercialized in many parts of the world. This paper reviews the development and the status ofBt technology and application ofBt transgenic plants in current agriculture, and discusses specific issues related to the transfer of the technology to the future of genetic engineered trees with emphasis on conifers. Key words Agricultural productivity - Bacillus thuringiensis - Genetic engineering - Insect resistance - Trees CLC number Q812 - S763.306 Document code A Biography: Tang Wei (1964-), male, Ph. Doctor, Research associate, Department of Biology, Howell Science Complex, East Carelina University, Greenville, NC 27858-4353, USA.Responsible editor: Chal Ruihai

Does cotton bollworm show cross-resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab? A mini review

Since 1996, transgenic Bacillus thuringiensis(Bt) cotton has been commercially grown in numerous countries in an effort to stem the losses caused by key lepidopteran pests. However, the development of pest resistance to Bt toxins has jeopardized the continued utilization of Bt cotton. As a strategy designed to circumvent the development of resistance, Bt cotton varieties expressing two or more toxins targeting the same pest have been introduced. Nevertheless, from the perspective of long-term planting of Bt cotton, the potential risk of cross-resistance to these Bt toxins is a threat that cannot be ignored. In this paper, we review current research(including that based on the analysis of protein binding sites and resistance genes) on the resistance of cotton bollworm(Helicoverpa armigera) to the Bt toxins Cry1 Ac and Cry2 Ab and the interrelationship between these toxins. On the basis of existing evidence, we assume that the actions of Cry1 Ac and Cry2 Ab against cotton bollworm are not completely independent, and then propose the "resistance-associated gene mutation potential hypothesis". Although the mechanisms underlying the resistance of pests to Bt toxins are yet to be comprehensively elucidated, this hypothesis could undoubtedly have important implications for adopting "pyramid" strategy in the future. Further research is recommended to devise strategies to retard the development of H. armigera resistance to Bt cotton, either using different Bt toxins or their various combinations.

Penetration of a Single Domain of Bacillus thuringiensis Cry1Ie-Domain I to a Lipid Membrane In vitro

Domain I of the activated Crystal protein from Bacillus thuringiensis has a seven a-helix bundle structure, which is responsible for membrane channel formation in its insecticidal mechanism. Crylle is toxic to Asian corn borer, Ostrinia furnacalis （Guen6e）, and plays important roles in insect biological control. The domain I from Crylle has been expressed and purified in its normal conformation, as embedded in the full length homologous toxin structure. The membrane insertion ability of this single domain was compared with the full length homologous toxin using a monolayer insertion experiment. The results indicated that the Crylle-domain I had the ability to insert into the lipid monolayer, and this ability is greater than that of the IE648 toxin. However, the state of insertion is not stable and remains for only a short period of time. The Crylle-domain I plays no role in receptor binding as it had a nonspecific binding with the brush border membrane vesicles of the Asian corn borer.

Biodegradation of Kirkuk light crude oil by <i>Bacillus thuringiensis</i>, Northern of Iraq

This study was conducted to identify the viability of Bacillus thuringiensis bacterial on a bio-degradation process for Kirkuk light crude oil. The viable count of Bacillus thuringiensis showed great capability on the biodegradation of crude oil. These bacteria exhibit the ability to dismantle crude oil through clear emulsion layer of crude oil. And they have a good efficiency to dismantle hydrocarbon compounds by 80%, and total biomass reaches to 5 g/l, while the amount of emulsion reaches to 2.3 g/l. For more evidences on the biodegradation action of Bacillus thuringiensis which have been supported by using the technology of gas-Chromatography which confirms the occurring of biodegradation process. The visual examination of gas-Chromatography shows the disappearance of a number of chemicals, as well as decrease in peak area for some material.

Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.