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Braz. j. biol ; 81(4): 872-880, Oct.-Dec. 2021. tab, graf
Article in English | LILACS | ID: biblio-1153450


Abstract Bacillus thuringiensis is the most commonly used entomopathogen in the control of Aedes aegypti, which is a vector for different etiological agents that cause serious infections in humans. Several studies aim to isolate strains of this bacterium from different environments, with the perspective of selecting isolates with larvicidal activity for mosquitoes. Aiming at the insecticidal action of B. thuringiensis, the present study aimed to prospect B. thuringiensis of restinga and mangrove soils from the state of Maranhão, Brazil, with toxic potential for use in the biological control of Ae. aegypti. Bioassays were performed to determine the entomopathogenic activity of the bacilli against Ae. aegypti and lethal concentrations (LC50 and CL90) were estimated after the tests. Polymerase Chain Reaction and SDS-PAGE techniques were performed to verify the gene and protein content of the isolates, respectively. The soil of the mangrove and restinga ecosystems showed potential for obtaining B. thuringiensis. This isolate, in addition to having proteins with molecular mass similar to the toxins Cry and Cyt, also presented several diptera-specific genes cry and cyt, demonstrating that it has high potential to be used in the biological control of Ae. aegypti.

Resumo Bacillus thuringiensis é o entomopatógeno mais utilizado no controle do Aedes aegypti, vetor de diferentes agentes etiológicos que causam infecções graves em humanos. Diversos estudos têm como objetivo isolar cepas dessa bactéria de diferentes ambientes, com a perspectiva de selecionar isolados com atividade larvicida para mosquitos. Visando a ação inseticida de B. thuringiensis, o presente estudo teve como objetivo prospectar B. thuringiensis de solos de restinga e mangue do estado do Maranhão, Brasil, com potencial tóxico para uso no controle biológico de Ae. aegypti. Bioensaios foram realizados para determinar a atividade entomopatogênica do bacilo contra Ae. aegypti e as concentrações letais (CL50 e CL90) foram estimadas após os testes. As técnicas de Reação em Cadeia da Polimerase e SDS-PAGE foram realizadas para verificar o conteúdo de genes e proteínas dos isolados, respectivamente. Os solos dos ecossistemas de mangue e restinga apresentaram potencial para obtenção de B. thuringiensis. O isolado BtMA-750, obtido a partir da amostra de solo da restinga, foi interessantemente distinguido por sua alta toxicidade para Ae. aegypti. Este isolado, além de apresentar proteínas com massa molecular semelhante às toxinas Cry e Cyt, apresentou também diversos genes díptero-específicos cry e cyt, demonstrando que tem alto potencial para ser usado no controle biológico de Ae. aegypti.

Humans , Animals , Bacillus thuringiensis/genetics , Aedes , Brazil , Pest Control, Biological , Ecosystem , Mosquito Vectors , Larva
Braz. j. biol ; 81(1): 114-124, Feb. 2021. tab, graf
Article in English | LILACS | ID: biblio-1153325


Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Humans , Animals , Bacillus thuringiensis/genetics , Aedes , Insecticides , Culicidae , Pest Control, Biological , Ecosystem , Mosquito Vectors , Larva
Braz. arch. biol. technol ; 64: e21200480, 2021. graf
Article in English | LILACS | ID: biblio-1285546


Abstract Coccidiosis, a disease caused by the parasitic Eimeria spp., affects birds of all ages, particularly young birds more intensely. Infected poultry presents significant economic losses. Bacillus thuringiensis var israelensis (Bti) is a Gram-positive, spore-forming bacterium that produces proteins with high specific parasiticidal activity against various orders of parasites. Thus, the aim of the present study was to evaluate the parasiticidal potential of Bti in quails that were naturally infected with Eimeria bateri. Twenty 12-week-old male quails (Coturnix coturnix coturnix), naturally infected with Eimeria bateri, were randomly divided into two groups of 10 birds: Bti treated and control. The treated group was supplemented with Bti (1×108 spores∙g-1) in the feed, while; the control group received the same feed without Bti. To evaluate the occurrence of oocysts, samples of feces were collected every week for four weeks. Significant (P < 0.05) oocysts reductions of 56.64% and 94.51% were noted in the Bti treated group at 2nd and 4th week of study, respectively. The Bti supplementation may contribute to the reduction of oocysts in quails and environmental contamination. Bacillus thuringiensis var israelensis appeared to be a promising complementary alternative in E. bateri control.

Bacillus thuringiensis , Pest Control, Biological , Coccidiosis , Eimeria
Chinese Journal of Biotechnology ; (12): 782-791, 2020.
Article in Chinese | WPRIM | ID: wpr-826898


L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Aminobutyrates , Bacillus thuringiensis , Candida , Escherichia coli , Formate Dehydrogenases , Metabolism , Leucine Dehydrogenase , Metabolism , Threonine , Metabolism , Threonine Dehydratase , Metabolism
Arq. Inst. Biol ; 87: e0202019, 2020. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1118082


Induced resistance emerges as an alternative method for controlling plant diseases. This study aimed to evaluate the efficacy of biotic and abiotic resistance inducers for controlling white rust in rocket (Eruca sativa), as well as biochemical changes (peroxidase) and fitness costs. The experiments were developed with the abiotic inducers acibenzolar-S-methyl (ASM) (12.5, 25, and 50 mg ai L-1) and citrus biomass (CB) (0.1, 0.25, and 0.5%), as well as with the biotic ones Saccharomyces cerevisiae (25 mg mL-1), Bacillus thuringiensis (25 mg p.c. mL-1), Saccharomyces boulardii (25 mg mL-1), and phosphorylated mannan oligosaccharide (PMO) (0.25%), in preventive and curative interventions. Fungicide mancozeb (1.6 g ai L-1), Bordeaux mixture (1%), and water were the control treatments. Leaf samples were collected 3, 7, 11, 15, and 19 days after the treatments to determine peroxidases and assess the severity and production. Concerning abiotic inducers, all doses of ASM and CB 0.5% (preventive) and CB 0.25% (curative) reduced the severity of white rust, whereas, among biotic inducers, only PMO applied preventively controlled the disease. Peroxidase activity was higher for CB 0.25% and ASM 50 mg L-1. Bordeaux mixture induced higher peroxidase activity.(AU)

A indução de resistência surge como um método alternativo para o controle de doenças em plantas. O objetivo deste trabalho foi avaliar a eficiência de indutores de resistência bióticos e abióticos no controle de ferrugem branca em rúcula (Eruca sativa), bem como alterações bioquímicas (peroxidase) e o impacto na produção. Foram desenvolvidos experimentos com os indutores abióticos acibenzolar-S-metil (ASM) (12,5; 25 e 50 mg i.a. L-1) e biomassa cítrica (BC) (0,1; 0,25 e 0,5%), e os bióticos Saccharomyces cerevisiae (25 mg p.c. mL-1), Bacillus thuringiensis (25 mg p.c. mL-1), Saccharomyces boulardii (25 mg p.c. mL-1) e manano-oligossacarídeo fosforilado (MOF) (0,25%), sendo aplicados preventiva e curativamente. Como controles foram utilizados o fungicida mancozeb (1,6 g i.a. L-1), calda bordalesa (1%) e água. Amostras de folhas foram coletadas aos 3, 7, 11, 15 e 19 dias após os tratamentos para a determinação de peroxidases, e avaliações de severidade e produção. Entre os indutores abióticos, todas as doses de ASM e BC 0,5% (preventivamente) e BC 0,25% (curativamente) reduziram a severidade da ferrugem branca, enquanto entre os indutores bióticos, apenas o MOF aplicado preventivamente, controlou a doença. A atividade de peroxidase foi superior para BC 0,25% e ASM 50 mg L-1. O tratamento com calda bordalesa também incrementou a atividade de peroxidase.(AU)

Plant Diseases , Saccharomyces , Bacillus thuringiensis , Peroxidase , Brassicaceae , Efficiency
Braz. arch. biol. technol ; 63: e20180428, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132198


Abstract Development of transgenic Bt crops with stable and high level of Bt protein expression over generations under different environmental conditions is critical for successful deployment at field level. In the present study, progenies of transgenic cotton Coker310 event, CH12 expressing novel cry2AX1 gene were evaluated in T3 generation for stable integration, expression and resistance against cotton bollworm, Helicoverpa armigera. The cry2AX1 gene showed stable inheritance and integration in the T3 progeny plants as revealed by PCR and Southern blot hybridization. The expression of Cry2AX1 protein on 90 days after sowing (DAS) was in the range of 1.055 to 1.5 µg/g of fresh leaf tissue except one plant which showed 0.806 µg/g of fresh leaf tissue and after 30 days (i.e., on 120 DAS) three plants recorded in between 0.69 to 0.82 µg/g and other plants are in range of 0.918 to 1.058 µg/g of fresh leaf tissue. Detached leaf bit bioassay in T3 progeny on 110 DAS recorded mortality of 73.33 to 93.33 per cent against H. armigera and severe growth retardation in surviving larvae. These results indicate that the expression of chimeric cry2AX1 is stable and exhibits insecticidal activity against H. armigera in T3 progeny of CH12 event of transgenic cotton.

Animals , Bacillus thuringiensis/pathogenicity , Pest Control, Biological/methods , Gossypium/genetics , Endotoxins/genetics , Moths , Plant Diseases/prevention & control , Plants, Toxic , Biological Assay , Plants, Genetically Modified
Chinese Journal of Biotechnology ; (12): 1511-1519, 2019.
Article in Chinese | WPRIM | ID: wpr-771778


With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.

Animals , Bacillus thuringiensis , Bacterial Proteins , Cryptochromes , Metabolism , Endotoxins , Hemolysin Proteins , Moths , Pest Control, Biological , Plants, Genetically Modified
Arq. Inst. Biol ; 86: e0492017, 2019. ilus, tab
Article in English | LILACS, VETINDEX | ID: biblio-1024585


The present study had the objective of evaluating the longevity of A. mellifera workers fed on a diet incorporating commercial entomopathogens, Beauveria bassiana, and Bacillus thuringiensis. It also aimed at verifying possible morphological alterations in the midgut. To this purpose, the entomopathogens used were B. bassiana (Product A) (5.0 × 1011 viable, B. thuringiensis (Product B) (2.5 × 109 viable spores.g-1), and B. thuringiensis (Product C) (1.0 × 109 viable spores.g-1); and two controls: T1: sterilized distilled water, and T2: sterilized distilled water + Tween 80® (0.01%). For the bioassays, 2 mL of each treatment were incorporated into Candy paste. For each treatment, 80 bees were individually in flat bottom glass tubes (2.5 cm Ø) covered with voile, containing a piece of cotton soaked in water and Candy paste. These tubes were stored in a B.O.D (30 ± 2°C, R.H 70% ± 10%, 12 h), and mortality was evaluated every six hours, for 10 days. Soon after verifying mortality, two bees per treatment were selected for the removal of their midgut. Midgut samples were processed using standard methodology for Scanning Electron Microscopy (SEM). It was verified that products A, B, and C reduced the longevity of bees when compared to T1 and T2 controls. In the qualitative analyses carried out using SEM, it was not possible to observe external or internal morphological alterations to midgut tissues. Although products A, B, and C cause a reduction in longevity, their presence was not verified when tissues were analyzed using SEM.(AU)

No presente trabalho objetivou-se avaliar a longevidade de operárias de A. mellifera alimentadas com dieta incorporada com os entomopatógenos comerciais Beauveria bassiana e Bacillus thuringiensis, e verificar possíveis alterações morfológicas em seu mesêntero. Para isso, os entomopatógenos utilizados foram B. bassiana (Produto A) (5,0 × 1011 conídios viá, B. thuringiensis (Produto B) (2,5 × 109 esporos viáveis.g-1), B. thuringiensis (Produto C) (1,0 × 109 esporos viáveis.g-1); e dois controles: T1: água destilada esterilizada e T2: água destilada esterilizada + Tween 80® (0,01%). Para os bioensaios, 2 mL de cada tratamento foram incorporados à pasta Cândi. Para cada tratamento, 80 abelhas foram acondicionadas, individualmente, em tubos de vidro de fundo chato (2,5 cm Ø), cobertos com voile, contendo um pedaço de algodão embebido em água e pasta Cândi. Os tubos contendo as abelhas foram acondicionados em B.O.D (30 ± 2°C, U.R. 70% ± 10%, 12 h), e a mortalidade foi avaliada a cada seis horas, durante 10 dias. Logo após a verificação da mortalidade, foram separadas duas abelhas por tratamento para a retirada do mesêntero. Essas amostras foram processadas em metodologia padrão para Microscopia Eletrônica de Varredura (MEV). Verificou-se que os produtos A, B e C reduziram a longevidade das abelhas quando comparados aos controles T1 e T2. Nas análises qualitativas realizadas com MEV, não foi possível observar alterações morfológicas externas ou internas nos tecidos do mesêntero. Apesar dos produtos A, B e C causarem redução na longevidade, sua presença não foi verificada quando os tecidos foram analisados por MEV.(AU)

Bees , Longevity , Bacillus thuringiensis , Beauveria
Rev. Soc. Bras. Med. Trop ; 52: e20190135, 2019. graf
Article in English | LILACS | ID: biblio-1041502


Abstract INTRODUCTION: Musca domestica is resistant to many insecticides; hence, biological control is a suitable alternative. METHODS: We evaluated the lethality of strain Btk176 towards the larval and adult M. domestica and the histopathological effects in the larvae midgut. RESULTS: We observed 99% larval and 78.9% adult mortality within 48 hours of spore ingestion (dosage, 2.4×108 CFU/ml). The histopathological effects were consistent with cytotoxicity. PCR analysis showed the presence of the cry1Ba gene. Transmission electron microscopy revealed a bipyramidal parasporal body. Thurigiensin activity was not detected. CONCLUSIONS: The serovar, Btk176 might be a potential biocontrol agent for houseflies.

Animals , Bacillus thuringiensis , Bacterial Toxins/pharmacology , Houseflies/drug effects , Insecticides/pharmacology , Larva/drug effects , Colony Count, Microbial , Pest Control, Biological/methods , Reproducibility of Results , Analysis of Variance , Microscopy, Electron, Transmission , Exotoxins
São Paulo; s.n; s.n; 2019. 89 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-1048888


Dados de vigilância epidemiológica apontam uma crescente associação entre o consumo de hortaliças e surtos de origem alimentar. São inúmeras as fontes de contaminação aos quais os vegetais estão sujeitos ao longo da cadeia produtiva. Estudos sugerem que práticas agrícolas, como o uso de adubo constituído por esterco animal e água de irrigação não tratada, podem aumentar o risco de contaminação por micro-organismos patogênicos. Com as restrições ao uso de pesticidas sintéticos no sistema orgânico de produção agrícola, agentes de controle biológico, como Bacillus thuringiensis (Bt) desempenham um importante papel para a garantia da produtividade. No entanto, recentemente, a segurança do uso de Bt passou a ser questionada em função da possibilidade de produzir enterotoxinas. Este estudo teve por objetivos levantar dados sobre práticas adotadas no cultivo de hortaliças orgânicas no Estado de São Paulo, Brasil e sobre as características microbiológicas de fertilizantes, água de irrigação, água de lavagem e alfaces nas etapas pré e pós-colheita, assim como avaliar a persistência e interações entre Bt e Salmonella em hortaliças, visando contribuir para avaliações de risco microbiológico mais adequadas. Na primeira parte do estudo, dez propriedades de cultivo orgânico certificadas foram visitadas para a obtenção de dados sobre práticas adotadas e para a coleta de amostras para análise microbiológica. As amostras foram submetidas à enumeração e identificação (gênero e espécie) de Enterobacteriaceae; pesquisa de Salmonella spp. por método convencional e qPCR; e enumeração de coliformes totais e Escherichia coli nas amostras de água. Na segunda fase da pesquisa, avaliou-se a persistência e as interações entre Bt e Salmonella Montevideo no pré e pós-colheita de espinafres. Por fim, bactérias epifíticas isoladas de hortaliças foram testadas quanto a capacidade de inibir bactérias do grupo Bacillus cereus e cepas de Salmonella enterica. As contagens de Enterobacteriaceae variaram de <1 a 7,2 ± 0,1 log UFC/g nos fertilizantes, de 4,1 ± 0,3 a 5,6 ± 0,3 log UFC/g nas alfaces coletadas nos canteiros, de 2,9 ± 0,6 a 5,3 ± 0,5 log UFC/g nas alfaces lavadas, de <1 a 3,5 ± 0,1 log UFC/mL nas amostras de água de irrigação e de <1 a 3,0 ± 0,3 log UFC/mL nas amostras de água de lavagem. Salmonella não foi isolada por cultivo em placa, mas foi detectada por qPCR em uma amostra de alface orgânica lavada. Utilizando MALDI-TOF MS, 45 espécies pertencentes a 24 gêneros bacterianos foram identificadas na cadeia produtiva de hortaliças orgânicas. Bt foi capaz de persistir nas folhas de espinafre nas etapas pré e pós-colheita e afetou a persistência de Salmonella durante o cultivo, mas não durante o armazenamento pós-colheita a 12 ºC. Não foi observada tendência de germinação dos esporos de Bt após a aplicação nos espinafres, reduzindo assim a possibilidade de multiplicação e produção de enterotoxinas. A bactéria epifítica Pseudomonas chlororaphis, isolada de hortaliça, foi capaz de inibir membros do grupo Bacillus cereus, incluindo cepas patogênicas e Bt em testes in vitro, sugerindo uma barreira biológica para o controle da multiplicação destes micro-organismos. Este estudo traz importantes informações sobre a segurança microbiológica de hortaliças orgânicas e de práticas agrícolas, evidenciando a importância de boas práticas para a promoção do alimento seguro. Os resultados constituem uma importante contribuição para o desenvolvimento de modelos de avaliação de risco microbiológico e prevenção de surtos de origem alimentar

Epidemiological surveillance data indicate a growing association between vegetable consumption and food-borne outbreaks. There are numerous sources of contamination to which plants are subjected throughout the production chain. Studies suggest that agricultural practices such as the use of manure fertilizer and untreated irrigation water may increase the risk of contamination by pathogenic microorganisms. With the restrictions on the use of synthetic pesticides in the organic farming system, biological control agents such as Bacillus thuringiensis (Bt), play an important role in ensuring productivity. However, the safety of Bt has recently been questioned due to the possibility of producing enterotoxins. This study aimed to gather information about the agricultural practices employed in the organic vegetables production fields and the microbiological characteristics of fertilizer, irrigation water, wash water, and lettuces in pre and post-harvest stages, and to evaluate the persistence and interactions between Bt and Salmonella on leafy greens, aiming to contribute for more adequate microbiological risk assessments. In the first part of the study, ten certified organic farms were visited to collect data on the farming practices and for collection of samples for microbiological evaluations. The samples were submitted to Enterobacteriaceae enumeration and identification (genus and species); Salmonella spp. by conventional method and qPCR; and enumeration of total coliforms and Escherichia coli in water samples. In the second part of the study, the persistence and interaction between Bacillus thuringiensis subsp Aizawai (Bt) and Salmonella Montevideo in the pre and post-harvest of spinach were evaluated. Finally, epiphytic bacteria isolated from vegetables were tested for their ability to inhibit growth of Bacillus cereus group members and Salmonella strains. Enterobacteriaceae counts ranged from <1 to 7.2 ± 0.1 log CFU/g in fertilizers, from 4.1 ± 0.3 to 5.6 ± 0.3 log CFU/g in lettuces collected from the fields, from 2.9 ± 0.6 to 5.3 ± 0.5 log CFU/g in washed lettuces, <1 to 3.5 ± 0.1 log CFU/mL in irrigation water and <1 to 3.0 ± 0.3 log CFU/mL in wash water. Salmonella was not isolated by plating but it was detected by qPCR in one sample of washed organic lettuce. Using MALDI-TOF MS, 45 species belonging to 24 bacterial genera were identified in the organic vegetable production chain. Bt was able to persist on pre and post-harvest of spinach and affected Salmonella persistence during cultivation, but not during the storage at 12 ºC. Bt spores showed no tendency to germinate during pre-harvest of spinach, thus reducing the probability of growth and production of enterotoxins. The epiphytic bacterium Pseudomonas chlororaphis isolated from one vegetable sample was able to inhibit members of the Bacillus cereus group, including pathogenic strains and Bt in in vitro tests, suggesting a biological barrier to control the multiplication of these microorganisms. These studies provide important information about the microbiological safety of organic vegetables and agricultural practices, highlighting the importance of good practices for the promotion of safe food. These data are fundamental for the development of microbiological risk assessment models and prevention of foodborne outbreaks

Vegetables/adverse effects , Agricultural Cultivation , Biological Control Agents/analysis , Salmonella , Bacillus thuringiensis/classification , Enterobacteriaceae , Food, Organic/microbiology
Biomédica (Bogotá) ; 38(supl.2): 95-105, ago. 2018. tab, graf
Article in English | LILACS | ID: biblio-974011


Introduction: Aedes (Stegomyia) aegypti is the vector for dengue, chikungunya, and Zika arboviruses. Bti-CECIF is a bioinsecticide designed and developed in the form of a solid tablet for the control of this vector. It contains Bacillus thuringiensis var. israelensis (Bti) serotype H-14. Objective: To evaluate under semi-field and field conditions the efficacy and residual activity of Bti-CECIF tablets on Aedes aegypti larvae in two Colombian municipalities. Materials and methods: We tested under semi-field conditions in plastic tanks (Rotoplast™) four different Bti doses (0.13, 0.40, 0.66 and 0.93 mg/L) in the municipality of Apartadó, department of Antioquia, to assess Bti-CECIF efficacy (percentage of reduction of larval density) and the residual activity in water tanks containing A. aegypti third-instar larvae. The efficacy and residuality of the most lethal dose were subsequently evaluated under field conditions in cement tanks in the municipality of San Carlos, department of Córdoba. Results: Under semi-field conditions, the highest tested dose exhibited the greatest residual activity (15 days) after which larval mortality was 80%. Under field conditions, the highest tested Bti-CECIF doses showed 100% mortality and exhibited a residual activity of seven days in 90% of the tanks. Conclusion: Bti-CECIF tablets effectively controlled A. aegypti larvae under field conditions for up to seven days post-treatment.

Introducción. Aedes (Stegomyia) aegypti es el vector de los arbovirus del dengue, el chikungunya y el Zika. Para el control de este vector, se diseñó y desarrolló un bioinsecticida en presentación de tableta sólida, el Bti-CECIF, que contiene Bacillus thuringiensis var. israelensis (Bti) de serotipo H-14. Objetivo. Evaluar en condiciones de 'semicampo' y de campo, la eficacia y la actividad residual de las tabletas de Bti-CECIF en larvas de A. aegypti en dos municipios colombianos. Materiales y métodos. En el municipio de Apartadó, departamento de Antioquia, se probaron bajo condiciones de 'semicampo' en tanques de plástico de 250 l (Rotoplast™) cuatro dosis diferentes de Bti (0,13, 0,40, 0,66 y 0,93 mg/l) para evaluar la eficacia del Bti-CECIF (porcentaje de reducción de la densidad larvaria) y la actividad residual en tanques de agua que contenían larvas de tercer estadio de A. aegypti. La eficacia y el efecto residual de la dosis más letal fueron posteriormente evaluadas en tanques de cemento bajo condiciones de campo en el municipio de San Carlos, departamento de Córdoba. Resultados. Bajo condiciones de 'semicampo', la mayor dosis probada exhibió la mayor actividad residual (15 días), después de lo cual la mortalidad de las larvas fue de 80 %. Bajo condiciones de campo, la máxima dosis probada de Bti-CECIF mostró una mortalidad de 100 % y exhibió una actividad residual de siete días en el 90 % de los tanques. Conclusión. Las tabletas Bti-CECIF controlaron eficazmente A. aegypti en condiciones de campo durante siete días a partir de su aplicación.

Bacillus thuringiensis , Aedes , Chikungunya virus , Colombia , Dengue , Disease Vectors , Zika Virus
Rev. méd. hondur ; 86(1/2): 7-10, ene-. jul. 2018.
Article in Spanish | LILACS | ID: biblio-1007044


Introducción: El dengue es la enfermedad viral transmitida por mosquitos con la propagación más rápida en el mundo. En Honduras constituye un importante problema de salud pública debido a su alta incidencia. Bacillus thuringiensis var israelensis (Bti) es un agente de control biológico aplicado en el control de vectores, se ha utilizado recientemente como parte de la estrategia contra el Aedes aegypti en Honduras. El presente estudio tiene como objetivo evaluar la susceptibilidad de la larva de Ae. aegypti a Bti en Tegucigalpa, Honduras para el año 2014. Métodos: Una muestra de 960 larvas de Ae. aegypti se recogieron de recipientes de almacenamiento en viviendas de las colonias La Cañada y Nueva Suyapa, ubicados en Tegucigalpa, Honduras. Los bioensayos se realizaron de acuerdo con las directrices emitidas por la Organización Mundial de la Salud y la Red Latinoamericana de Control de Vectores. Se llevaron a cabo 12 bioensayos por cada concentración estudiada. Se utilizaron cuatro concentraciones de Bti: 0,5 mg/L, 1 mg/L, 2,5 mg/L y 5 mg/L. La mortalidad se registró a las 24 h. Resultados: Se encontraron valores de mortalidad larvaria de 98.734 % para la dosis de 0.5 mg/L; 99.375 % para 1 mg/L; 100 % para 2,5 mg/L y 100 % para 5 mg/l. Conclusiones: Bti ha probado ser efectivo contra las larvas de Ae. aegytpi, se recomienda su evaluación sistemática y de manera periódica para mejorar su aplicación y posible desarrollo de resistencia...(AU)

Animals , Bacillus thuringiensis , Aedes , Dengue/complications , Vector Control
Chinese Journal of Biotechnology ; (12): 1809-1822, 2018.
Article in Chinese | WPRIM | ID: wpr-776288


Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Animals , Bacillus thuringiensis , Bacterial Proteins , Metabolism , Bombyx , CD13 Antigens , Metabolism , Cadherins , Metabolism , Endotoxins , Metabolism , Hemolysin Proteins , Metabolism , Larva
Arq. Inst. Biol ; 85: e0132018, 2018. ilus, tab
Article in English | LILACS, VETINDEX | ID: biblio-999204


Bacillus thuringiensis subsp. israelensis has been used to control the Aedes aegypti (Diptera: Culicidae) mosquito larvae, the vector of virus diseases such as dengue, Chikungunya and Zika fever, which have become a major public health problem in Brazil and other tropical countries since the climate favors the proliferation and development of the transmitting vector. Because B. thuringiensis has shown potential for controlling insects of the Diptera order, this work aimed at testing the Bacillus thuringiensis subsp. thuringiensis strain T01-328 and its proteins Cry2Aa and Cry2Ab for control A. aegypti and at comparing the results to the B. thuringiensis subsp. israelensis specific dipteran strain. To this end, bioassays using spore-crystal of both strains, and Cry2Aa and Cry2Ab proteins from the heterologous expression in Escherichia coli, were performed against A. aegypti larvae. The results showed that the B. thuringiensis thuringiensis ­T01-328 has insecticidal activity against the larvae, but it is less toxic than B. thuringiensis subsp. israelensis. Cry2Aa and Cry2Ab proteins expressed heterologously were effective for controlling A. aegypti larvae. Therefore, the results indicate that the Cry2Aa and Cry2Ab proteins of the B. thuringiensis thuringiensis T01-328 can be used as an alternative to assist in the control of A. aegypti.(AU)

Bacillus thuringiensis subsp. israelensis vem sendo empregada no controle do díptero Aedes aegypti, vetor do vírus causador de doenças como dengue, febre Chikungunya e Zika, que se tornou um dos grandes problemas de saúde pública no Brasil e em outros países de clima tropical, que favorece a proliferação e o desenvolvimento do transmissor. Em virtude do potencial de B. thuringiensis no controle de dípteros, a proposta deste trabalho foi testar as proteínas Cry2Aa e Cry2Ab da linhagem de Bacillus thuringiensis subsp. thuringiensis T01-328 no controle de A. aegypti, em comparação à linhagem díptero específica B. thuringiensis subsp. israelensis. Para tanto, foram realizados bioensaios com larvas de A. aegypti com o esporo-cristal de ambas as linhagens, bem como com as proteínas Cry2Aa e Cry2Ab com expressão heteróloga em Escherichia coli. A linhagem B. thuringiensis thuringiensis T01-328 apresentou atividade inseticida contra as larvas, porém foi menos tóxica que a B. thuringiensis subsp. israelensis. As proteínas Cry2Aa e Cry2Ab expressas de forma heteróloga foram eficazes no controle de A. aegypti. Os resultados obtidos sugerem as proteínas Cry2Aa e Cry2Ab da linhagem B. thuringiensis thuringiensis T01-328 como alternativas para contribuir no controle do A. aegypti.(AU)

Bacillus thuringiensis , Pest Control, Biological , Aedes/microbiology , Dengue , Disease Vectors , Chikungunya Fever , Zika Virus Infection
Arq. Inst. Biol ; 85: e0052018, 2018. tab
Article in English | LILACS, VETINDEX | ID: biblio-999052


The entomopathogen Bacillus thuringiensis (Bt) is widely used as one of the ingredients in pest control formulations, but researches conducted on its effect on non-target organisms are still in the nascent stage. This investigation aimed to uncover if Bt treated with Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and pupae could affect the biological variables of Podisus nigrispinus (Hemiptera: Pentatomidae) and Palmistichus elaeisis (Hymenoptera: Eulophidae), all of which established natural enemies of leaf defoliator caterpillars in the eucalyptus culture. Larvae of T. molitor were fed on wheat bran containing different concentrations of B. thuringiensis (0.00; 0.25; 0.50; 1.00; 2.00 and 4.00 g Agree/kg bran). When the larvae attained size of about 2 cm, they were used as prey for P. nigrispinus (Bioassay I), and their pupae used as hosts for P. elaeisis (Bioassay II). Only the biological variables oviposition period and egg numbers by posture of the predator P. nigrispinus were altered. The biological variables of P. elaeisis were not altered, since it was possible to use these control methods within the integrated pest management.(AU)

O entomopatógeno Bacillus thuringiensis é amplamente empregado no controle de pragas, porém estudos de seu efeito sobre organismos não alvo ainda são incipientes. Com isso, objetivou-se neste trabalho avaliar se larvas de Tenebrio molitor (Coleoptera: Tenebrionidae), tratadas com Bacillus thuringiensis, podem afetar as variáveis biológicas de Podisus nigrispinus (Hemiptera: Pentatomidae) e Palmistichus elaeisis (Hymenoptera: Eulophidae), importantes inimigos naturais de lagartas desfolhadoras na cultura do eucalipto. Larvas de T. molitor foram alimentadas com farelo de trigo contendo diferentes concentrações de Bacillus thuringiensis (0,00; 0,25; 0,50; 1,00; 2,00 e 4,00 g de Agree®/kg farelo). Quando as larvas atingiram em média 2 cm de comprimento, foram usadas como presa para P. nigrispinus (bioensaio I) e as pupas como hospedeiro para P. elaeisis (bioensaio II). As variáveis biológicas período de oviposição e número de ovos por postura do predador P. nigrispinus foram alteradas. Já as variáveis biológicas de P. elaeisis não foram modificadas, sendo possível o uso conjunto desses métodos de controle no manejo integrado de pragas.(AU)

Tenebrio , Bacillus thuringiensis , Pest Control , Eucalyptus , Hemiptera , Insecticides
Comun. ciênc. saúde ; 28(1): 58-63, jan. 2017. ilus
Article in Portuguese | LILACS | ID: biblio-972636


Duas tecnologias alternativas para o controle de Aedes aegypti foram avaliadas: a aplicação espacial de larvicida biológico-Bti em potenciais criadouros peridomiciliares, e a liberação de machos estéreis para inviabilização reprodutiva das fêmeas do mosquito. As ações foram realizadas pelos Agentes dos Serviços de Saúde em 15 vilas da Ilha de Fernando de Noronha, e em uma área (900 imóveis) no bairro da Várzea/Recife/Pernambuco. A efetividade dos métodos foi avaliada por indicadores entomológicos,estimados pela presença, quantidade e viabilidade de ovos do mosquito, coletados em armadilhas, e por marcadores genéticos. A aplicação de Bti, com atomizador costal, ocorreu a cada 30 dias em ambas as áreas. Uma redução importante e sustentável da população de A. aegypti,por este método, foi alcançada em 2015/2016 na Várzea e, em 2016, na Ilha, onde a remoção de 18 toneladas de resíduos sólidos em 2015 contribuiu possivelmente para os resultados. Machos esterilizados com radiação gama foram produzidos em massa no laboratório e liberados em uma das vilas da Ilha. A análise espaço-temporal dos indicadores, de dez/2015a ago/2016, revelou redução expressiva da densidade populacional do mosquito e da diversidade genética da população local. Ambas as abordagens parecem ter reduzido o contato homem-vetor e os riscos de transmissão de arboviroses na Ilha, apesar da elevada competência vetorial da população local do mosquito para os vírus Zika e Dengue. Os métodos testados se mostraram eficientes e passíveis de serem integradas às ações do SUS voltadas ao controle de A. aegypti.

Two alternative technologies were evaluated for Aedes aegypti control:the spraying of a biological larvicide (Bti) in potential peridomiciliarybreeding sites and the release of sterile males to promote reproductionblockage in wild females. Actions were carried out by Agents of theHealth Services, in 15 villages of the Fernando de Noronha Island and in 900 properties from the district of Várzea, Recife-PE. The effectiveness of both methods was evaluated by entomological indicators, estimatedby the presence, quantity and viability of eggs from the mosquito collectedin traps and through genetic markers. Bti was delivered by backpacksprayer every 30 days in both areas. A significant and sustainablereduction of the A. aegypti population as a result of this technique wasachieved in 2015/2016 in Várzea and in 2016 in the Island, where it wasstrengthened by the removal of 18 tons of solid waste in 2015. Malessterilized with gamma radiation were mass-produced in the laboratoryand released in one village of the Island. The spatiotemporal analysis ofthe indicators, from Dec/2015 to Aug/2016, revealed a significant reductionin mosquito density, which impacted on the genetic diversity of thelocal population. Both approaches seem to have reduced human-vectorcontact and the risk of arbovirus transmission in the Island, althoughlocal mosquito population presented high vector competence to Zikaand Dengue virus. These methods were efficient and could be integratedinto SUS actions directed to A. aegypti control.

Humans , Bacillus thuringiensis , Vector Control , Aedes/immunology , Pest Control, Biological , Entomology
Biosci. j. (Online) ; 32(6): 1522-1536, nov./dec. 2016. tab, ilus
Article in English | LILACS | ID: biblio-965794


Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) affects diverse crops of great economic interest, for instance, it can cause severe yield losses in maize, rice and sorghum. In this study, a selection and characterization of Bacillus thuringiensis (BERLINER, 1911) isolates with a high insecticidal activity against S. frugiperda was performed. Fifty-two crystal-forming B. thuringiensis isolates that were identified from 3384 Bacillus-like colonies were examined and screened by PCR for the presence cry genes (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 and cry2Ab). Four isolates that showed high toxicity towards S. frugiperda were shown to harbor cry2 genes. The crystals were analyzed by electron microscopy and showed bipyramidal and cuboidal shapes. Furthermore, these four isolates had lethal concentration (LC50) values of 44.5 ng/cm2 (SUFT01), 74.0 ng/cm2 (SUFT02), 89.0 ng/cm2 (SUFT03) and 108 ng/cm2 (SUFT 04) to neonate S. frugiperda larvae. An ultrastructural analysis of midgut cells from S. frugiperda incubated with the SUFT01 spore-crystal complex showed disruptions in cellular integrity and in the microvilli of the midgut columnar cells. The isolates characterized in this work are good candidates for the control of S. frugiperda, and could be used for the formulation of new bioinsecticides.

Spodoptera frugiperda (SMITH, 1797) (Lepidoptera: Noctuidae) afeta diversas culturas de grande interesse econômico, por exemplo, pode causar severas perdas em milho, arroz e sorgo. Neste estudo, foi realizada uma seleção e caracterização de isolados de Bacillus thuringiensis (BERLINER, 1911) com elevada atividade inseticida contra S. frugiperda. Cinquenta e dois isolados formadores de cristal B. thuringiensis que foram identificados a partir de 3384 colônias foram examinados e testados por PCR para a presença dos genes cry (cry1, cry1Aa, cry1Ab, cry1Ac, cry1D, cry2 e cry2Ab). Quatro isolados que apresentaram alta toxicidade contra S. frugiperda foram mostrados para abrigar os genes cry2. Os cristais foram analisados por microscopia eletrônica e mostraram formas bipiramidais e cúbicas. Os valores da concentração letal (CL50) destes quatro isolados foram de 44,5 ng / cm2 (SUFT01), 74,0 ng / cm2 (SUFT02), 89,0 ng / cm2 (SUFT03) e 108 ng / cm2 (suft 04) para larvas recém-eclodidas de S. frugiperda. Uma análise ultra-estrutural das células do intestino médio de S. frugiperda incubadas com complexo esporo-cristal do isolado SUFT01 mostrou rupturas na integridade celular e microvilosidades das células cilíndricas do intestino médio. Neste estudo, o alto nível de atividade inseticida de isolados os torna excelentes candidatos para o controlo de S. frugiperda, e pode proporcionar alternativas no controle destas populações de pragas, bem como a formação de novos bioinsecticidas.

Bacillus thuringiensis , Spodoptera , Insecticides , Lepidoptera
Braz. j. microbiol ; 47(3): 597-602, July-Sept. 2016. graf
Article in English | LILACS | ID: lil-788966


ABSTRACT In this study, the cry1Ab gene of previously characterized and Lepidoptera-, Diptera-, and Coleoptera-active Bacillus thuringiensis SY49-1 strain was cloned, expressed and individually tested on Ephestia kuehniella (Lepidoptera: Pyralidae) and Plodia interpunctella (Lepidoptera: Pyralidae) larvae. pET-cry1Ab plasmids were constructed by ligating the cry1Ab into pET28a (+) expression vector. Constructed plasmids were transferred to an Escherichia coli BL21 (DE3) strain rendered competent with CaCl2. Isopropyl β-D-1-thiogalactopyranoside was used to induce the expression of cry1Ab in E. coli BL21(DE3), and consequently, ∼130 kDa of Cry1Ab was obtained. Bioassay results indicated that recombinant Cry1Ab at a dose of 1000 µg g-1 caused 40% and 64% mortality on P. interpunctella and E. kuehniella larvae, respectively. However, the mortality rates of Bt SY49-1 strains' spore-crystal mixture at the same dose were observed to be 70% on P. interpunctella and 90% on E. kuehniella larvae. The results indicated that cry1Ab may be considered as a good candidate in transgenic crop production and as an alternative biocontrol agent in controlling stored product moths.

Animals , Bacillus thuringiensis/physiology , Bacterial Proteins/genetics , Gene Expression , Insect Control , Endotoxins/genetics , Hemolysin Proteins/genetics , Bacillus thuringiensis/ultrastructure , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Insect Control/methods , Cloning, Molecular , Endotoxins/metabolism , Endotoxins/toxicity , Hemolysin Proteins/metabolism , Hemolysin Proteins/toxicity , Insecticides , Larva , Moths/drug effects
Electron. j. biotechnol ; 19(3): 54-57, May 2016. ilus
Article in English | LILACS | ID: lil-787008


Background: Bacillus thuringiensis Cry toxins bind with different insect midgut proteins leading to toxin oligomerization, membrane insertion and pore formation. However, different Cry toxins had been shown to readily form high molecular weight oligomers or aggregates in solution in the absence of receptor interaction. The role of Cry oligomers formed in solution remains uncertain. The Cry9A proteins show high toxicity against different Lepidoptera, and no-cross resistance with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in solution mediated by disulfide bonds, according to SDS-PAGE analysis under non-reducing and reducing conditions. However, oligomerization is not observed if Cry9Aa655 is activated with trypsin, suggesting that cysteine residues, C14 and C16, located in the N-terminal end that is processed during activation participate in this oligomerization. To determine the role of these residues on oligomerization and in toxicity single and double alanine substitution were constructed. In contrast to single C14A and C16A mutants, the double C14A-C16A mutant did not form oligomers in solution. Toxicity assays against Plutella xylostella showed that the C14A-C16A mutant had a similar insecticidal activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa formed in solution in the absence of receptor binding are not related with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in oligomerization in solution. These aggregate forms are not related to the mode of action of Cry9Aa leading to toxicity.

Bacillus thuringiensis , Bacterial Proteins , Insecticides , Biological Assay , Disulfides , Electrophoresis, Polyacrylamide Gel , Bacillus thuringiensis Toxins , Mutation
Journal of the Egyptian Society of Parasitology. 2016; 46 (1): 81-92
in English | IMEMR | ID: emr-180162


The histochemical effects of the lethal concentration that kills 50% of larvae [LC[50]] of three biological agents, abamectin, Bacillus thuringiensis and spinosad on the carbohydrates [polysaccharides], proteins, nucleic acids and lipids content of the midgut and fat bodies of Culex pipiens 2[nd] instar larvae were studied. The results showed that the three tested compounds reduced the carbohydrates [polysaccharides], proteins, RNA synthesis and lipids content after 72 hours of treatment where abamectin was the most effective followed by Bacillus thuringiensis then spinosad

Biological Products , Ivermectin , Bacillus thuringiensis , Macrolides , Drug Combinations , Mosquito Control/methods , Pest Control, Biological/methods