Bacillus thuringiensis (Bt) is a naturally occurring bacterium common in soils throughout the world and belongs to the group Bacillus cereus sensu lato (Huang et al., 2001). Bt is from the family of Bacillaceae which encompasses two genus divisions, namely Clostridium and Bacillus. Hence, Bt is a ubiquitous bacteria with Gram- positive, spore-forming, rod-shaped in nature and is approximately 1 µm in width and 5 µm in length (Konecka et al., 2006). Asokan et al., 2013 used soil samples of Great Nicobar Islands to isolate new Bt strains, where no collection has been characterized previously. Lee et al., 2012 isolated B.thuringiensis from forests soils in Korea. The results suggest that forest areas in Korea are a rich source of B. thuringiensis and need to be further explored to discover novel B. thuringiensis isolates. Konecka et al., 2012 Isolated B. thuringiensis strains from soil and water. Baig and Mehnaz, 2010 isolated 31 Bt strains from Arabian Sea sedimentary rocks. PCR approach was used to analyze the presence of different crystal toxin encoding genes with six pairs of universal primers that could detect the cry1, cry4, cry7, cry8, cry9, and cry10 genes. Strains containing
In 1996, crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins were introduced to the agricultural market. The acreage on which these crops are grown has increased by 18 million ha in the last 10 years. The terrestrial and aquatic fate of transgenic Bt proteins are key parameters governing exposure of non-target organisms in the environment. Because of potential non-target effects on terrestrial and aquatic organisms, it is important to accurately quantify Bt protein exposure with environmental fate studies. However, conflicting results have been found for terrestrial fate studies, and there is almost no information on the aquatic fate of transgenic Bt proteins. The various results seen in the terrestrial fate studies are likely in part due to differences in the analytical methods used. Currently, there is no reliable and accurate analytical technique for quantifying transgenic Bt proteins from environmental matrices with high recovery efficiency. The goals of this study were to improve the extraction of Bt proteins from environmental matrices. A series of spike-and-recovery experiments using the lepidopteran-active Bt Cry1F and the coleopteran- active Bt Cry3Bb1 were conducted to determine the best extraction method for these two proteins. A differential extraction was found for these two classes of proteins, with the best- available buffer for Cry1F being a biomimetic buffer and the best-available buffer for Cry3Bb1 being a high-salt, high-pH buffer. Despite a good extraction efficiency of Cry1F from soil, the biomimetic buffer was not able to extract any Cry1F from the soil of field plots containing Herculex 1 corn. The results from this study indicate a need for extraction buffers that can overcome the adsorption of Bt proteins to surface-active particles in soil. Several solid-phase extraction (SPE) methods were attempted for cleanup and concentration of Bt proteins from environmental matrices. Although good retention to the SPE tubes was achieved using carbon-18 and strong-anion-exchange cartridges, an efficient method for eluting Bt proteins from the tubes was not achieved.
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Mosquitoes are the important vectors of several human diseases such as malaria, filariasis, dengue fever, and yellow fever causing serious health problems to world human populations. At present, mosquito-borne diseases are a greater threat in tropical and subtropical countries . The resurgence of these diseases is due to the increase in mosquito breeding places. These disease-causing insects are generally controlled by conventional insecticides . Further, the indiscriminate use of neurotoxic insecticides caused various environmental toxic problems to non-target organisms and insecticidal resistance . Globally, there are conscientious efforts to overcome these problems, and great emphasis is placed recently on eco-friendly and economically viable methodologies for insect control. An alternative approach for mosquito control is the use of natural products such as plant and microorganisms. Bacillus thuringiensis (Bt) is a soil-dwelling, Gram-positive, rod-shaped, and spore-forming bacterium that produces crystal (Cry) proteins that are toxic to insect species among the orders Lepidoptera , Diptera, and Coleoptera [4-7]. The lethality of Bt to the insects is mainly attributed by the Cry and cytolytic protein (Cyt) proteins, which was produced during the growth cycle [4,8]. These Bt strains are found in two different states as either vegetative and can be found as spores. These endospores are resistant to any environmental stress. They are metabolically inactive and a resting form of the bacterium which is a completely different structural form, chemical composition and enzymatic constitution from its vegetative state. The insecticidal activities of the various toxins are differing, but they are considered to be harmless to higher organisms including humans . The Cry proteins are protoxins which can be converted to active toxins on ingestion by a susceptible insect . The microbial insecticides have significant importance in the pest management. Further, when compared with chemical pesticides, these
The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin’s effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.
Strains of Bacillus thuringiensis (Bt) are known to produce crystalline proteins (δ-endotoxins) concomitantly with sporulation during their stationary phase of growth, which are demonstrated as lethal to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes. Upon ingestion, the δ-nascent endotoxin is an inactive protoxin complex of (Cry alone or Cry and Cyt toxins together) high molecular mass, which is cleaved upon ingestion into the active component proteins at the high alkaline environments in the digestive tract of these agricultural pests. Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation techniques in com- mercial media, but recently many workers have used solid-state fermentation strategy for the enhanced production of Bt-toxin at low cost. Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin (thuringiensin), which may be harmful to humans. Moreover, resistance to Bt developed in various target pest is yet another concern for Bt-industry. Following a brief introduction, this review addresses various toxins produced by various strains of Bt, Bt production media and media formulations with emphasis to solid-state fermenta- tion, general structure of Cry toxin, its mode of action, target pests, bioassay, resistance to Bt toxins and resistance management. Briefly, this review would provide the readers an overview on the general aspects of Bt toxin, its general structure and mechanism of action.
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Microbial bio-pesticides are eco-friendly and target spe- cific alternates to hazardous synthetic pesticides (Kumar and Singh 2015; Majeed et al. 2017). Worldwide, an an- nual increase of 10% in the use of bio-pesticides has been estimated and among them, approximately 90% formula- tions are derived from Bacillus thuringiensis (Bt) (Kumar and Singh 2015; Osman et al. 2015). However, there is a great potential to look for the indigenous strains and iso- lates of Bt and characterize their pathogenicity and tox- icity against insect pest species (Sree and Varma 2015).
ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host’s death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentia- tion course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet- uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment.
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Bacillus thuringiensis is a gram positive rod shaped, aerobic spore forming soil bacterium, which produces crystalline insecticidal proteins within the cytoplasm at the time of sporulation. These insecticidal crystal proteins are highly toxic to various insects belong to Lepidopteron Coleopterus and Dipteran families. Isolation and characterization to obtain efficient Lepidopteran specific Bacillus thuringiensis as bio-control agent from different soil samples. Out of 50 soil samples collected, only 20 samples were used for the isolation. Sodium acetate selection method was used and the results were positive for presence of Bacillus thuringiensis bacteria. More than one method or biochemical test is used for isolate Bacillus thuringiensis strains from different soil samples. The isolates which are positive for crystal protein production were invariable endospore formers but the morphology of the crystal protein inside. These isolated Bacillus thuringiensis can be used in future for the transformation techniques as a biopesticide and may be on the control of Teak defoliator.
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ABSTRACT Understanding the genetic basis of host shifts is a key genomic ques- tion for pathogen and parasite biology. The Bacillus cereus group, which encom- passes Bacillus thuringiensis and Bacillus anthracis, contains pathogens that can infect insects, nematodes, and vertebrates. Since the target range of the essential virulence factors (Cry toxins) and many isolates is well known, this group presents a powerful system for investigating how pathogens can diversify and adapt to phylogenetically distant hosts. Specialization to exploit insects occurs at the level of the major clade and is associated with substantial changes in the core genome, and host switching between insect orders has occurred repeatedly within subclades. The transfer of plasmids with linked cry genes may account for much of the adaptation to particular insect orders, and network analysis implies that host specialization has produced strong associations between key toxin genes with similar targets. Analysis of the dis- tribution of plasmid minireplicons shows that plasmids with orf156 and orf157, which carry genes encoding toxins against Lepidoptera or Diptera, were contained only by B. thuringiensis in the specialized insect clade (clade 2), indicating that tight genome/plasmid associations have been important in adaptation to invertebrate hosts. Moreover, the accumulation of multiple virulence factors on transposable ele- ments suggests that cotransfer of diverse virulence factors is advantageous in terms of expanding the insecticidal spectrum, overcoming insect resistance, or through gains in pathogenicity via synergistic interactions between toxins.
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In the histological study the transverse section of both the organs liver and kidney were observed. In case of hematoxylene eosin staining of liver the sections containing cysts were observed under 400X(40*10) magnification. The liver hepatocyte cells observed after the treatment of Bt protein in the H/E staining showed some structural deformities. The kidney sections observed under microscope for Bromophenol blue and PAS staining didn’t show any significant difference. Total protein content was estimated from tissue homogenate mainly for liver & kidney, isolated from control & Bt cry protein treated rat by lowry method, and it was observed increased level of total protein in liver & kidney tissue in Bt fed rat as compared to the control rat. In this study elevated levels of protein content in case liver tissue observed in treated animal as compared to control rat & showing the P value is less than 0.05(P<0.05),indicating there was a significant difference. Further we measured the amount of bilirubin is formed in case of liver tissue. The breakdown of old red blood (RBCs) in the body produces bilirubin, which thus travels to the liver is stored in the bile duct.RBCs have a lifespan of around 120 days & renew continuouslly. In my case Bt cry protein treated rats showing gradual decrease in bilirubin content in case of liver compaired to control group & it gives the P value less than the 0.05,thus it can be concluded that there was a significant difference affecting the normal functioning of liver. The fluctuation observed in values of treated animal may be due to variation of waste product excretion by treated animals.
Abstract— Cotton in Eswatini contributes 2.1 % of the country’s Gross Domestic Product owing to low cotton yield due to high pest pressure. Eswatini farmers grow Alba QM 301 a conventional non Bt variety which is affected by bollworm. Cotton is no longer profitable and farmers are quitting the industry, yet it is the only source of livelihood in drought prone areas of Eswatini . Countries lik e India and South Africa have replaced conventional cotton with high yielding Bt or genetically modified cotton.The study analyses yield and adaptation of Bt cotton under rain fed condition.Bt cotton hybrid was evaluated under field condition for adaptation and yield performance in 2016 and 2017 season. Two Bt cotton varieties JKCH 1947 Bt and JKCH 1050 Bt were tested against the local variety Alba Plus QM 301 and JKC 724 both Non Bt (NBt).JKCH 1947 recorded significantly higher seed cotton yield per ha of 3070 k g/ha on the first year. It was closely followed by JKCH 1050 with a yields of 2955 k g/ha.The number of boll per plant was also significant higher compared the control. Alba Plus QM 301 and JKC 724 both Non Bt (NBt) recorded the lower yields of 2066 and 821 k g/ha respectively, under the same condition with less number of bolls per plant. Similar observations were recorded on the second year, JKCH1947 and JKCH 1050 recording 1765k g/ha and 1865k g/ha respectively. A similar trend was observed onthe number of bolls per plant,higher number of bolls were recorded in JKCH 1050 Bt followed by JKCH 1947 Bt. Alba Plus QM 301 NBt and JKC 724 NBt recorded fewer boll in both years. All varieties showed good adaptability to local environment with good plant stand. Keywords— Bt cotton, rain fed conditions, seed cotton.
In previous studies, Cry4, Cry10, Cry11 and Cyt proteins were established as the prin- cipal virulence factors of Cuban native B. thu- ringiensis isolates (8, 9). Nevertheless we as- sociated the reduction of the Cry and Cyt tox- ins under then effect of temperature with lack of toxicity of U81 isolate (25–35 °C) and worse efficiency of A21 and A51 isolates. Taking in- to account that Cyt proteins potentiate the ac- tion of Cry (7), the loss of these proteins may be the cause of the significant reduction in the toxicity and efficiency. We also demonstrated reduction of Cry and Cyt proteins at 40 °C but no bioassays were performed. We speculate that in natural breeding sites the toxicity of biolarvicides fail by the loss of these proteins at temperatures over 40 °C. This result should be taken into account for the preservation of B. thuringiensis aqueous formulations.
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The amount of Cry1Ac δ -endotoxin in transgenic Bacillus thuringiensis Berliner (Bt) or Bollgard cotton varies among commercial cultivars. These differences in expression have been correlated with survival levels in Lepidop- tera, indicating that all Bollgard cultivars do not provide the same level of control. The objective of this study was to determine if differences in overall expression among commercial cultivars of Bollgard cotton were under simple genetic control. These ﬁndings could inﬂuence the way breeders select cultivars by evaluating for efﬁcacy in insect control in addition to agronomic traits. Two sets of crosses were made in the greenhouse with cultivars that express the endotoxin at high and low levels. The parents and F 1 and F 2 gen-
The protein profi le of Bt-S84-13a determined through SDS-PAGE did not show any correlation with the δ-endotoxin gene detected, which was cry4. The absence of correlation between protein profi le and cry gene pattern was also reported by Armengol et al. (2007). These variations could be caused by proteins that are coded by new gene types or that the primers did not amplify the genes. It is also possible that the detected genes may code for some non- active or low expressed proteins (Armengol et al., 2007). Despite being characterized with the dipteran cry4 gene, yet Bt-S84-13a did not show its predicted insecticidal activity. The lack of correspondence between δ-endotoxins genotype of Bt-S84-13a and its biological activity may be due to various factors, for example, the production of inactive crystal proteins whereby the gene that synthesize the crystal proteins could be under the control of a weak promoter (Masson et al., 1998; Ferrandis et al., 1999; Armengol et al., 2007). Some cry genes may be also expressed in low levels which may lead to the low expression of the respective Cry proteins. Porcar and Juárez-Pérez (2003) mentioned that the detection of a cry gene by PCR has no direct proof of its level of expression, but variation in the expression level of individual cry genes could weaken the correlation between cry gene content and toxicity. Therefore, it is very common that strains differ greatly in their insecticidal effi cacy although they carried the same δ-endotoxin genes, owing to differences in the level of cry genes expression.
Conventional breeding for resistance against these pests has not yielded the desired results. Consequently, they are managed mainly with insecticides in Ghana and many other cotton-growing areas in West Africa [2–4]. Globally, cotton is responsible for about 16–25% of all chemical insecticides used in agriculture, which is more than what is used for any other single crop [6, 7, 10]. Although most of these insecticides are effective for control, they pose health hazards to farmers who use them and also contaminate the environment. Moreover, they are expensive and their indiscriminate use can cause emergence of resistant biotypes in insect populations resulting in control failures such as those reported for pyrethroid insecticide use in parts of West Africa [5, 8, 9]. In an effort to scale down on insecticide use, amidst fears for environmental contamination and insect resistance build-up, genetic modification of plants for resistance to insect pests has been found to be a better and environ- mentally friendlier alternative . Genetic modification with the soil-borne bacterium Bacillus thuringiensis (Bt) Berliner has been used to control insect pests in several crops [11–13]. Genetically modified cotton contains the Bt gene(s) that produce(s) toxins or bio-pesticides inside the plant to offer protection against insects. It has spe- cific activity against lepidopteran insects such as the boll- worm complex due to specific receptors and conditions in the caterpillar’s gut that allow activation of the Bt crys- tal proteins [14, 15]. The objective of the present study was to evaluate the field efficacy of the genetically modi- fied Bollgard II (BG II) cotton, FK 95 BG II for control of cotton bollworms in Ghana.
Background: Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an impor- tant destructive disease of pine forests worldwide. In addition to behaving as a plant-parasitic nematode that feeds on epithelial cells of pines, this pest relies on fungal associates for completing its life cycle inside pine trees. Manipulating microbial symbionts to block pest transmission has exhibited an exciting prospect in recent years; however, trans- forming the fungal mutualists to toxin delivery agents for suppressing PWN growth has received little attention. Results: In the present study, a nematicidal gene cry5Ba3, originally from a soil Bacillus thuringiensis (Bt) strain, was codon-preferred as cry5Ba3Φ and integrated into the genome of a fungus eaten by PWN, Botrytis cinerea, using Agrobacterium tumefaciens-mediated transformation. Supplementing wild-type B. cinerea extract with that from the cry5Ba3Φ transformant significantly suppressed PWN growth; moreover, the nematodes lost fitness significantly when feeding on the mycelia of the cry5Ba3Φ transformant. N-terminal deletion of Cry5Ba3Φ protein weakened the nematicidal activity more dramatically than did the C-terminal deletion, indicating that domain I (endotoxin-N) plays a more important role in its nematicidal function than domain III (endotoxin-C), which is similar to certain insecticidal Cry proteins.
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Im Rahmen ihrer langjährigen Tätigkeit als wissenschaftliche Mitarbeiterin des Instituts für Biologischen Pflanzenschutz des Julius Kühn-Instituts, Bundesforschungsinstitut für Kulturpflan- zen hielt die Autorin anlässlich der gemeinsamen Lehrveranstal- tung „Biologischer und Integrierter Pflanzenschutz“ mit dem Fachbereich Biologie der Technischen Universität Darmstadt in den Jahren 2008 und 2009 je eine Vorlesung über „Bacillus thuringiensis“. Die Inhalte dieser Vorlesungen wurden in Form der vorliegenden Broschüre aufgearbeitet.
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Hilbeck et al.  described a structured, stepwise selec- tion procedure for the identification of organism groups potentially suitable as test species for the assessment of GMP. Applying this procedure for the receiving environ- ments of Germany, larvae of the dipteran family Sciari- dae were identified as a potential test taxon because of their ecological relevance in Central European agricul- tural soils [8, 33]. Similar to other soil dwelling dipteran larvae, Sciaridae can reach high numbers in species and individuals in agricultural fields in the temperate regions of Central Europe  and participate in organic matter decomposition. A comprehensive appraisal of the ecolog- ical importance and ecosystem function of soil dwelling Diptera, including Sciaridae, is given by Frouz . Of the worldwide 1700 species , approximately 340 spe- cies occur in Germany . Whereas Lycoriella castane- scens (Diptera: Sciaridae) has been listed as a promising candidate species , no culture of L. castanescens was available at the start of the study. However, for another sciarid species, Bradysia impatiens, a culture existed at the Julius-Kühn-Institute (JKI) in Kleinmachnow (Ger- many). Büchs [6, 7] performed tests with B. impatiens and Bacillus thuringiensis (Bt) maize and confirmed the exposure of the larval stage to the Bt toxin from MON810 maize. Bradysia spp. are found in agricultural fields at abundances similar to those of L. castanescens [8, 33], but no published records of the species B. impatiens from agricultural fields exist . For the test development, B. impatiens was used as there is no reason to believe that both sciarid species differ in terms of susceptibility to GMP material. Both are destruents of soil organic matter, feed on fungi and have similar life history parameters [4, 9, 24, 29].
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The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.
Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B.